Neuroendocrinology, Volume 182: Pathological Situations and Diseases

PROGRESS IN BRAIN RESEARCH

VOLUME 182

NEUROENDOCRINOLOGY: PATHOLOGICAL

SITUATIONS AND DISEASES

EDITED BY

LUCIANO MARTINI Department of Endocrinology,

University of Milano, Milano, Italy

GEORGE P. CHROUSOS First Department of Pediatrics

Athens University Medical School, Athens, Greece

FERNAND LABRIE Molecular Endocrinology

Laval University, Quebec City, Canada

KAREL PACAK Section on Medical Neuroendocrinology

NICHD-NIH, Bethesda, MD, USA

DONALD W. PFAFF Laboratory of Neurobiology and Behavior,

Rockefeller University, New York, NY, USA

AMSTERDAM – BOSTON – HEIDELBERG – LONDON – NEW YORK – OXFORD

PARIS – SAN DIEGO – SAN FRANCISCO – SINGAPORE – SYDNEY – TOKYO

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK 360 Park Avenue South, New York, NY 10010-1710 First edition 2010 Copyright � 2010 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-444-53616-7 ISSN: 0079-6123 For information on all Elsevier publications visit our website at elsevierdirect.com

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List of Contributors

K. Ashida, Department of Endocrinology and Diabetes Mellitus, School of Medicine, Fukuoka University, Fukuoka, Japan H.A. Bimonte-Nelson, Department of Psychology, Arizona State University, Tempe, AZ, USA R. Diaz Brinton, Departments of Pharmacology & Pharmaceutical Sciences, Biomedical Engineering and Neurology, University of Southern California, Los Angeles, CA, USA H.S. Chahal, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK G.P. Chrousos, Division of Endocrinology, Diabetes and Metabolism, First Department of Pediatrics, University of Athens, “Agia Sophia” Children’s Hospital, Goudi, Athens, Greece J.A. Cidlowski, Molecular Endocrinology Group, Laboratory of Signal Transduction, NIEHS, NIH, DHHS, Research Triangle Park, NC, USA A. Colao, Department of Molecular & Clinical Endocrinology and Oncology, “Federico II” University of Naples, Naples, Italy C. Conrad, Department of Psychology, Arizona State University, Tempe, AZ, USA M.C. De Martino, Department of Internal Medicine, Division of Endocrinology, Erasmus Medical Center, Rotterdam, The Netherlands A. Faggiano, Department of Molecular & Clinical Endocrinology and Oncology, “Federico II” University of Naples, Naples, Italy W. Fan, Division of Endocrinology and Metabolism, School of Medicine, University of California, San Diego, La Jolla, CA, USA A.B. Grossman, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK V.W. Henderson, Departments of Health Research & Policy (Epidemiology) and of Neurology & Neurological Sciences, Stanford University, Stanford, CA, USA L.J. Hofland, Department of Internal Medicine, Division of Endocrinology, Erasmus Medical Center, Rotterdam, The Netherlands C. Kanaka-Gantenbein, Division of Endocrinology, Diabetes and Metabolism, First Department of Pediatrics, University of Athens, “Agia Sophia” Children’s Hospital, Goudi, Athens, Greece V. Kantorovich, Division of Endocrinology and Metabolism, Department of Internal Medicine, College of Medicine, University of Arkansas for Medical Sciences, Arkansas Cancer Research Center, AR, USA B. Kola, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK M. Korbonits, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK F. Labrie, Research Center in Molecular Endocrinology, Oncology and Human Genomics, Laval University and Laval University Hospital Research Center (CRCHUL), Québec, Canada S.W.J. Lamberts, Department of Internal Medicine, Division of Endocrinology, Erasmus Medical Center, Rotterdam, The Netherlands C.T. Lim, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK v

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S. Melmed, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA L. Min, Department of Medical Biochemistry, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan S. Molatore, Institute of Pathology, Helmholtz Zentrum München-German Research Center for Environmental Health, Neuherberg, Germany H. Nawata, Graduate School of Medical Science, Kyushu University; Fukuoka Prefectural University, Tagawa City, Fukuoka, Japan M. Nomura, Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan K. Pacak, Section on Medical Neuroendocrinology, Reproductive and Adult Endocrinology Program, NICHD, NIH, Bethesda, MD, USA N.S. Pellegata, Institute of Pathology, Helmholtz Zentrum München-German Research Center for Environmental Health, Neuherberg, Germany P. Pervanidou, Developmental and Behavioral Pediatrics Unit, First Department of Pediatrics, Athens University Medical School, “Agia Sophia” Children’s Hospital, Goudi, Athens, Greece R. Pivonello, Department of Molecular & Clinical Endocrinology and Oncology, “Federico II” University of Naples, Naples, Italy S. Sakka, Division of Endocrinology, Diabetes and Metabolism, First Department of Pediatrics, University of Athens, “Agia Sophia” Children’s Hospital, Goudi, Athens, Greece L.K. Smith, Molecular Endocrinology Group, Laboratory of Signal Transduction, NIEHS, NIH, DHHS, Research Triangle Park, NC, USA A. Tahir, Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK T. Watanabe, Department of Internal Medicine, Nakatsu Municipal Hospital, Nakatsu, Oita, Japan T. Yanase, Department of Endocrinology and Diabetes Mellitus, School of Medicine, Fukuoka University, Fukuoka, Japan R. Yu, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA

“en guise d’introduction …’’

As presented to me by Luciano Martini, Neuroendocrinology: volume 181 the normal neuroendocrine system and volume 182 pathological situations and diseases will be two virtual volumes of over 500 virtual pages written by a group of distinguished colleagues all well qualified, many of these friends of old. It is not my intention to present or discuss in any shape or form the enormous sum that these few lines will precede and accompany. I would need proficiency in several foreign languages, foreign to me certainly. I am referring to the languages of molecular biology and the immensely intellectual country they come from and serve. Reading the list of titles of the many chapters, these two volumes will be made of, is in itself the description of what happened to that field of knowledge since the word neuro-endocrinology (with the hyphen) was coined and used for the first time in 1946 as the title of the also (already) enormous book (1106 pages) Traité de Neuro-endocrinologie by Gustave Roussy and Michel Mosinger. With the well-established knowledge of neurotransmitters as small molecules (acetylcholine, catechola­ mines, etc.) between nerve endings (synapses) (Sherrington, von Euler, etc.) and that of hormones (coining of the word by Starling in 1904 for secretin out of extracts of duodenal tissue), the concept of – and the word – neurosecretion appeared in the 1940s with the stunning images by the Scharrers (Ernst and Berta) of protein granules (Gomori stain) in neuronal cell bodies and moving along with axoplasmic flow. In vertebrates, that was essentially and originally dealing with observations in the hypothalamus and the posterior pituitary. But similar images were also found in invertebrates, leading to a major extension of the concept. And when the interest started about the mechanisms involved in the control of regulation of the anterior pituitary functions, involving both hypothalamic centres and unusual capillary connections, the very concept of specific molecules of neuronal origin travelling to the pituitary became the question of the day (see Geoffrey W. Harris’s Neural Control of the Pituitary Gland, 1955), which was eventually answered after over 17 years of research with the characterization of all the suspected releasing factors plus an unexpected inhibitory factor, somatostatin, all first characterized in extracts of hypothalamic tissues. So far, a rather linear way of thinking, so to speak. But all along and more and more intriguing were the observations generated by the molecular biology approach I mentioned above, that followed: syntheses of analogues of the original sequences with agonist or antagonist activities, recognition of multiple receptors for each and all of these peptides, cDNA cloning of all, etc. So much so that the conclusion was reached that each and all of these ligands and their receptors were actually quite ubiquitous and functional throughout the organ­ ism and not only located in the hypothalamus and other classical structures of the nervous system.

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One remarkable example, out of a large library of the same: presence of the full CRH system – peptides, mRNAs, receptors, binding proteins … in adipose tissues (e.g. Seres, J., Bornstein, S. R., Seres, P., Willenberg, H. S., Schulte, K. M., Scherbaum, W.A., et al. (2004). Corticotropin-releasing hormone system in human adipose tissue. Journal of Clinical Endocrinology and Metabolism, 89, 965– 970). Also, that several of these peptides and their chemistry are involved in psychological events normal and abnormal is another of these now unquestionable conclusions as discussed in several chapters here; David DeWied (1926–2004) was a precursor. Let me close here these simple opening lines for what will be major reference volumes – if the word may apply to a virtual entity. We do indeed live in a world where even the virtual is real … Roger Guillemin, MD Nobel Laureate The Salk Institute for Biological Studies, La Jolla, California, United States of America

L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 1

Glucocorticoid-induced apoptosis of healthy and malignant lymphocytes Lindsay K. Smith and John A. Cidlowski
Molecular Endocrinology Group, Laboratory of Signal Transduction, NIEHS,

NIH, DHHS, Research Triangle Park, NC, USA

Abstract: Glucocorticoids exert a wide range of physiological effects, including the induction of apoptosis in lymphocytes. The progression of glucocorticoid-induced apoptosis is a multi-component process requiring contributions from both genomic and cytoplasmic signaling events. There is significant evidence indicating that the transactivation activity of the glucocorticoid receptor is required for the initiation of glucocorticoid-induced apoptosis. However, the rapid cytoplasmic effects of glucocorticoids may also contribute to the glucocorticoid-induced apoptosis-signaling pathway. Endogenous glucocorticoids shape the T-cell repertoire through both the induction of apoptosis by neglect during thymocyte maturation and the antagonism of T-cell receptor (TCR)-induced apoptosis during positive selection. Owing to their ability to induce apoptosis in lymphocytes, synthetic glucocorticoids are widely used in the treatment of haematological malignancies. Glucocorticoid chemotherapy is limited, however, by the emergence of glucocorticoid resistance. The development of novel therapies designed to overcome glucocorticoid resistance will dramatically improve the efficacy of glucocorticoid therapy in the treatment of haematological malignancies. Keywords: glucocorticoid; apoptosis; lymphocyte; haematological malignancy; glucocorticoid resistance

environmental stress, nociception and emotion. Stimulation of hypothalamic corticotropin-releasing hormone (CRH) secretion prompts the release of adreno-corticototropic hormone (ACTH) from the pituitary, which induces glucocorticoid synth­ esis within the zona fasiculata of the adrenal cortex. Glucocorticoids auto-regulate their secretion though negative feedback inhibition of CRH and ACTH synthesis and release. In humans, cortisol is the predominant circulating glucocorticoid. Once in circulation, natural glucocorticoids are predominately bound to corticosteroid-binding globulin (CBG). Due to their lipophilic nature,

Introduction Glucocorticoids are a class of essential stressinduced steroid hormones regulating a variety of cardiovascular, metabolic, homeostatic and immunological functions. Endogenous glucocorticoids are synthesized and secreted under the control of the hypothalamic–pituitary–adrenal axis in response to stressors including


Corresponding author. Tel.: (919)541-1564; Fax: (919)541-1367; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82001-1

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of physiological actions. For example, in the liver and adipose tissue, glucocorticoids positively regulate metabolism through the stimulation of gluconogenesis and lipolysis, respectively. Con­ versely, in the immune compartment, glucocorti­ coids are largely inhibitory, causing immune suppression through the induction of apoptosis and cell cycle arrest and the inhibition of inflam­ mation via the repression of pro-inflammatory cytokines (Fig. 1) (Hermoso and Cidlowski, 2003; Rhen and Cidlowski, 2005). Given their broad bioavailability and diverse physiological effects, synthetic glucocorticoids are among the most commonly prescribed drugs for

endogenous glucocorticoids are widely bioavail­ able and easily cross the cell membrane via pas­ sive diffusion (Hermoso and Cidlowski, 2003; Rhen and Cidlowski, 2005). Glucocorticoids exert their physiological effects through the ubiquitously expressed glucocorticoid receptor (GR), a member of the nuclear hormone receptor super family of ligand-activated tran­ scription factors. Upon ligand binding, the GR translocates to the nucleus where it activates or represses the transcription of glucocorticoidresponsive genes. Due to the broad distribution of both glucocorticoids and their cognate recep­ tors, glucocorticoid signaling exerts a wide range

Cerebrum Emotion via limbic system

Cytokines

Thalamus Nociceptive pathways

Hypothalamus CRH

Cerebellum

Anterior pituitary Dorsal root ganglion

Pain receptors

Adrenal gland

Ventral root

ACTH Spleen

Cortisol

Lymph nodes

Thymus

Sympathetic

ganglion

Leukocytes Immune system

Adipose tissue

Cardiovascular system Liver Musculoskeletal system

Fig. 1. Pleitrophic effects of glucocorticoids in responsive tissues. Endogenous glucocorticoids are generated in response to various stressors, including emotion and nociception. The subsequent physiolgical actions of glucocorticoids in responsive tissues are denoted as stimulatory (dashed line) or inhibitory (dotted line); (adapted from Rhen and Cidlowski, 2005).

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the treatment of inflammatory disorders, autoim­ mune diseases and sepsis. They are also a main­ stay in the treatment of haematological malignancies. Numerous high-affinity synthetic glucocorticoids are clinically available, including prednisone and dexamethasone. However, pro­ longed use of these compounds is complicated by numerous deleterious side effects such as osteo­ porosis, hypertension, psychosis, Cushing’s syndrome and leucopenia (Rhen and Cidlowski, 2005). Glucocorticoid use in chemotherapy is lim­ ited by the development of glucocorticoid resis­ tance. Glucocorticoid resistance in leukemia and lymphoma is correlated with a poor prognosis (Dordelmann et al., 1999; Irving et al., 2005; Riml et al., 2004; Schmidt et al., 2004). The mechanisms governing glucocorticoid resistance in these malignancies are an area of considerable interest to both the scientific and medical commu­ nities. This chapter will address the role of gluco­ corticoids in the induction of apoptosis of healthy and malignant lymphocytes as well as the molecu­ lar determinants of glucocorticoid resistance in haematological malignancies.

Glucocorticoid-induced apoptosis of lymphoid cells

region houses the activating function (AF-1) transactivation domain (amino acids 77–262), which interacts with the basal transcription machinery in order to induce transcription (Wright et al., 1993). The central DBD is encoded by exons 3–4 and mediates receptor binding to glucocorticoid response elements (GREs) within the promoters of responsive genes. The DBD also consists of two conserved zinc fingers, which facil­ itate interaction with nuclear factor-kB (NF-kB) and AP-1 transcription factor (first zinc finger) as well as receptor dimerization (second zinc finger) (Heck et al., 1994; Liden et al., 1997; Tao et al., 2001). The region between the two zinc fingers houses a nuclear export signal (NES) (Black et al., 2001; Miesfeld et al., 1987; Tao et al., 2001). A hinge region adjacent to the DBD contains a nuclear localization signal at amino acids 491–498 (Freedman and Yamamoto, 2004). Finally, exons 5–9 encode the C-terminal LBD. This region is responsible for ligand binding and cofactor binding, and also contains a weak AF-2 transactivation domain (Bledsoe et al., 2002; Dahlman-Wright et al., 1992; Hollenberg et al., 1987; Schaaf and Cidlowski, 2002b) (Fig. 2). Following translation, the mature GR resides in the cytosol complexed with an hsp90 dimer, a p23 stabilizing protein and a variety of co-chaperones (Cheung and Smith, 2000; Pratt and Toft, 1997).

Glucocorticoid receptor Structure

Expression

The GR is a member of the type I nuclear hor­ mone receptor super family. Members of this super family are characterized by the formation of homodimers and the presence of three distinct functional domains: the C-terminal ligand-binding domain (LBD), the internal zinc-finger DNAbinding domain (DBD) and the N-terminal transactivation domain (NTD) (Escriva et al., 2004; Escriva et al., 1997; Giguere et al., 1986; Laudet et al., 1992; Weinberger et al., 1985). The GR gene (NR3C1) is located on chromosome 5q31.3 and encodes nine exons (Theriault et al., 1989). Exon 1 represents the 50 -untranslated region while exons 2–9 are protein coding (Duma et al., 2006). Exon 2 encodes the majority of N-terminal domain. This

The predominant GR expressed in human tissues is the full-length GRa isoform (Pujols et al., 2002). However, there are numerous additional GR pro­ tein isoforms generated from the single GR gene via alternative splicing and the use of alternative translation initiation sites (Fig. 3). Alternative splicing of GR pre-mRNA generates five distinct GR protein isoforms, namely GRa, GRb, GRg, GR-A and GR-P. Of these, GRa and GRb are the most widely expressed. These two receptor iso­ forms differ in their carboxyl termini due to the use of alternative splicing sites within exon 9 (Hollenberg et al., 1985). GRa is produced from the splicing of exon 8 to the proximal end of exon 9, thus generating a 777-amino acid protein. Exon

4 Domain structure of GRα protein DNA binding Hinge domain

N-Terminal domain (NTD) AF-1 1

77

DBD 282

421

488 527

Ligand-binding domain (LBD) NLS

AF-2 777

Fig. 2. Domain structure of human glucocorticoid receptor (GR) protein. The GR contains three major functional regions: the N-terminal transactivation domain, the central DNA-binding domain and the C-terminal ligand-binding domain.

9 contributes an additional 50 residues to the LBD of the GRa receptor. GRb is created from the splicing of exon 8 to the distal end of exon 9, generating a shorter protein of 742 residues, including 15 unique C-terminal residues contribu­ ted by exon 9. This splice site is predominantly found in humans and has not been identified in the mouse, explaining the lack of GRb expression in mice (Otto et al., 1997). The 15 C-terminal residues contributed by exon 9 render GRb unable to bind glucocorticoids or transactivate glucocorticoid-responsive promoters (Oakley et al., 1999; Yudt et al., 2003). Initially, GRb was described solely as a dominant negative inhibitor of GRa transactivation (Bamberger et al., 1995; Oakley et al., 1999; 1996). This inhibition occurs through both direct interaction with GRa and competitive recruitment of transcriptional coacti­ vators (de Castro et al., 1996). Deletion of the 15 unique C-terminal amino acids rendered GRb unable to repress GRa transactivation (Oakley et al., 1996). In contrast to earlier findings, recent studies have described a direct transcriptional role for GRb. For example, human glucocorticoid receptor b (hGRb), expressed in the absence of hGRa, possesses intrinsic transcriptional activity. Furthermore, GRb can selectively bind the GR antagonist RU-486 and this binding diminishes its intrinsic transcriptional activity (Lewis-Tuffin et al., 2007). More recently, Kelly et al. found that GRb is capable of repressing transcription from the cytokine interleukin (IL)-5 and (IL)-13 promoters via recruitment of histone deactylase I (Kelly et al., 2008). The GRg splice variant harbours an additional three base insertion in the DBD, resulting in the

insertion of an arginine residue between the two zinc fingers, thus reducing its transcriptional capa­ city. The GRg isoform is largely expressed in lym­ phocytes (3.8–8.7% of total GR mRNA) (Rivers et al., 1999). However, the evaluation of GRg protein expression in lymphocytes is hampered by the absence of a specific GRg antibody. The GR-A splice variant lacks exons 5–7, resulting in a truncated LBD and impaired transactivation activity. Finally, the GR-P splice variant lacks exons 8 and 9, resulting in a truncated LBD lacking the ability to bind glucocorticoids (Moalli et al., 1993). Additional GR isoforms are generated through the use of alternative translation initiation sites. These isoforms were first identified by immuno­ blotting as a GRa doublet migrating at 94 and 91 kDa (Lu and Cidlowski, 2005; Yudt and Cidlowski, 2001). The existence of additional GRa isoforms was confirmed by the identification of an in-frame internal translation initiation site at methionine 27. Use of this site is responsible for the generation of the 91-kDa species of GRa, now termed GRa-B (Yudt and Cidlowski, 2001). Further scanning for internal translational start sites revealed the presence of six additional GRa translational isoforms: GRa-C1, C2, C3, D1, D2 and D3 (Lu and Cidlowski, 2005). These iso­ forms differ only in the length of their N-termini. All bind glucocorticoids with similar affinity, but possess unique gene expression profiles and transcriptional activities. For example, GRa-C3 is the most transcriptionally active, even enhan­ cing the basal transcriptional activity of GRa-A (Lu and Cidlowski, 2005). Conversely, the GRaD isoform is the least transcriptionally active and

5 The glucocorticoid receptor: one gene yields many proteins Chromosome 5

GR gene 1

2

3 4

5

6 7

8

9α

9β

(a) GR precursor mRNA

(b) GR splice variants

1 1

777

GRα

742

GRβ

778

GRγ

R453 1 Δ Exons 5–7 1

592 Δ Exons 8–9

GR-A

1

676

GR-P

(c) GRα translational isoforms

777 A

1 27

B

86

C1

90

C2

98

C3

316

D1

331

D2

336

D3

Fig. 3. The glucocorticoid receptor (GR): one gene yields many proteins. (a and b) The hGR pre-mRNA undergoes alternative splicing, generating the dominant GRa isoform and the lower abundance GRb, GRg, GR-A and GR-P isoforms. (c) The GRa isoform is also subject to alternative translation initiation, giving rise to translational isoforms differing in the length of the N-terminal domain.

unable to induce apoptosis in a stably overexpressing osteosarcoma cell line (Lu et al., 2007). Given the distinct actions of these diverse GR

protein isoforms, alterations in their intracellular ratios may influence the lymphoid response to glucocorticoids.

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GR expression is under the control of three distinct promoters: 1A, 1B and 1C. Alternative promoter usage results in transcripts bearing exons 1A, 1B and 1C. Alternative splicing of these exons leads to the generation of nine alter­ native exon 1 splice variants (1A, 1B, 1C, 1D, 1E, 1F, 1H, 1I and 1J) (Breslin et al., 2001; Presul et al., 2007; Turner and Muller, 2005). Further splicing of exons 1A and 1C yields exons 1A1, 1A2 and 1A3 and exons 1C1, 1C2 and 1C3, respectively (Breslin et al., 2001; Turner and Muller, 2005). These assorted transcripts all encode the same protein; however, selective promoter usage may influence downstream splicing and translation initiation events as well as GR protein levels and, ultimately, responsiveness to glucocorticoid treatment (Breslin et al., 2001; Pedersen et al., 2004; Presul et al., 2007; Russcher et al., 2007).

Glucocorticoid receptor signaling Genomic effects In the cytoplasm, unliganded GR exists as a multiprotein heterocomplex associated with an Hsp90 dimer, a p23 stabilizing protein and various immu­ nophilin chaperone proteins (Cheung and Smith, 2000; Pratt et al., 1996). Upon ligand binding, the GR heterocomplex undergoes a conformational change, releasing the GR from cytoplasmic sequestration, promoting receptor homodimeriza­ tion and nuclear translocation of the GR homodimer (Davies et al., 2002; Elbi et al., 2004; Freedman and Yamamoto, 2004; Hager et al., 2004; Nagaich et al., 2004). In the nucleus, the activated GR homodimer binds specific DNA ele­ ments or GREs within the promoter regions of glucocorticoid-responsive genes. The consensus GRE is composed of two hexamer half-sites sepa­ rated by three random nucleotides. A majority of GREs contain the hexamer half-site sequence TGTTCT. The number and location of these GREs influences the intensity of the transcrip­ tional response (Freedman and Luisi, 1993). Once bound to the GRE, the GR homodimer recruits transcriptional coactivators as well as the basal transcriptional machinery to the

transcription start site. These co-activators include cAMP response element-binding (CREB)-bind­ ing protein (CBP), steroid receptor co-activator-1 (SRC-1), GR-interacting protein-1 (GRIP-1), p300 and SWI/SNF (Adcock, 2001; Rogatsky et al., 2002; Wallberg et al., 2000). These co-activators induce histone acetylation, thus allowing for transactivation of glucocorticoidresponsive genes. The activated GR homodimer is also capable of gene repression. GR can directly interact with DNA via negative GREs within the promoter regions of target genes (Dostert and Heinzel, 2004; Sakai et al., 1988). This interaction inhibits the transcription of genes associated with these promoters. Promoters with described nGREs include the corticotropin-releasing factor (CRF), osteocalcin and prolactin promoters (Drouin et al., 1993; Malkoski and Dorin, 1999; Meyer et al., 1997). Alternatively, GR can regulate tran­ scription independent of GR binding through direct interaction with other nuclear transcription factors such as NF-�B, AP-1, STAT5 and STAT3, thus modulating the transcription of genes under the control of these transcription factors (Fig. 4) (McKay and Cidlowski, 2000; Ray and Prefontaine, 1994; Scheinman et al., 1995; Stocklin et al., 1996; Yang-Yen et al., 1990; Zhang et al., 1997).

Cytoplasmic effects GR signaling has been reported to induce rapid effects in the cytoplasm within minutes of ligand binding. For example, upon ligand binding, Src kinase is released from the cytosolic GR hetero­ complex, resulting in lipocortin-1 activation and inhibition of arachidonic acid release. The activa­ tion of Src required ligand-bound GR, but was independent of transactivation (Croxtall et al., 2000). Furthermore, glucocorticoids have long been known to alter cytoplasmic ion content, causing rapid alterations in calcium, sodium and potassium concentrations (Bortner et al., 1997; McConkey et al., 1989a, 1989b). Glucocorticoids also cause rapid increases in the mitochondrial production of reactive oxygen species, ceramide

7 Glucocorticoid signaling

Glucocorticoids

Extracellular Space

Cytoplasm

p23 HSP90

p23

GR

0

P9

HS

HSP9

0

GR

Gene regulation Nucleus Induction

Repression

GILZ

GR

Osteocalcin

GR

GRE

nGRE

P

GR

Stat5 IGF-1

GR p65 p50

IL-1β

P

STAT

NF-κB

Fig. 4. Mechanisms of glucocorticoid-regulated gene expression. Ligand binding liberates the glucocorticoid receptor (GR) from cytosolic sequestration, leading to rapid nuclear translocation and homodimerization. In the nucleus, GR can activate gene transcription through direct binding to GREs in the DNA or through stimulatory interactions with transcription factors such as STAT5. GR can also repress gene expression by directly interacting with nGREs in the DNA, or through inhibitory protein–protein interactions with transcription factors including NF-kB.

and hydrogen peroxide, and the lysosomal release of cathepsin B (Cifone et al., 1999; Tonomura et al., 2003; Wang et al., 2006a; Zamzami et al., 1995). Interestingly, glucocorticoids have been reported

to induce the translocation of GR to the mitochon­ dria in both thymocytes and lymphoma cells (Sio­ nov et al., 2006). This mitochondrial GR may mediate mitochondrial production of reactive

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oxygen species and ceramide as well as the rapid calcium mobilization following glucocorticoid treat­ ment (Gavrilova-Jordan and Price, 2007). Recently, membrane-bound GR (mGR) has been suggested in T lymphocytes. Glucocorticoid treatment inhibits TCR signaling via the mGR. This inhibition occurs through the disruption of the lymphocyte-specific protein tyrosine kinases Lck and Fyn. These kinases, anchored to Hsp90, are components of the TCR-linked mGR–multi­ protein complex. Glucocorticoid treatment results in the rapid dissociation of Lck and Fyn from the multi-protein complex, leading to reduced phos­ phorylation of Lck/Fyn substrates and impaired initiation of TCR signaling. This diminished TCR signaling suppresses downstream cytokine synthesis, cellular migration and proliferation of T-lymphocytes (Lowenberg et al., 2005, 2006).

Glucocorticoid-induced apoptosis Genomic signaling The progression of glucocorticoid-induced apop­ tosis is a multi-faceted process requiring contribu­ tions from both genomic and cytoplasmic signaling events. Genomic events alter the protein content of the cell, creating an environment favorable to the execution of the apoptotic pathway. There is significant evidence indicating that the transacti­ vation activity of the GR is required for the initia­ tion of glucocorticoid-induced apoptosis. For example, glucocorticoid-induced apoptosis of lym­ phocytes does not progress in the presence of actinomycin D or cycloheximide, indicating a requirement for de novo transcription and transla­ tion in the execution of the apoptotic cascade (Cifone et al., 1999; Mann and Cidlowski, 2001; Mann et al., 2000; McConkey et al., 1989b; Wang et al., 2006b). The finding that an activation-defi­ cient GR mutant possessing unaltered transre­ pression capability fails to initiate glucocorticoidinduced apoptosis further supports this observa­ tion (Ramdas and Harmon, 1998). Finally, thymo­ cytes isolated from a knock-in mouse harbouring a point mutation presumably preventing receptor dimerization, and thus GR transactivation, also

failed to undergo glucocorticoid-induced apopto­ sis (Reichardt et al., 1998). Numerous laboratories have performed gen­ ome-wide microarray analysis in order to identify genes differentially regulated during glucocorti­ coid-induced apoptosis (Medh et al., 2003; Schmidt et al., 2006; Thompson and Johnson, 2003; Thompson et al., 2004; Tissing et al., 2007; Wang et al., 2003a; Yoshida et al., 2002). How­ ever, to date, only a few genes have been assigned a functional role in the regulation of glucocorti­ coid-induced apoptosis. Most notably, the expres­ sion of the pro-apoptotic BH3-only Bcl-2 family member Bim is induced by glucocorticoid treat­ ment in murine lymphoma cell lines, human leu­ kemic cell lines, mouse primary thymocytes and human primary chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) samples (Distelhorst, 2002; Iglesias-Serret et al., 2007; Schmidt et al., 2004, 2006; Wang et al., 2003a). The mechanism of induction is likely indir­ ect, as there is no GRE in the promoter region of the Bim gene (Bouillet et al., 2001; Wang et al., 2003a). One potential mechanism of Bim induc­ tion is via the induction of the Fox03A/FKHRL1 transcription factor, which is up-regulated by glu­ cocorticoids (Dijkers et al., 2000; Planey et al., 2003). A more recent study found that the activity of the serine/threonine kinase GSK3 is a key med­ iator of glucocorticoid-induced Bim up-regulation (Nuutinen et al., 2009). The induction and activa­ tion of Bim leads to downstream activation of the apoptotic mediators Bax and Bak (Kim et al., 2009). Once activated, these mediators mediate the destabilization of the mitochondrial mem­ brane potential, a hallmark of the intrinsic mito­ chondrial apoptosis pathway (Kim et al., 2009). The up-regulation of Bim is likely an important mediator of glucocorticoid-induced apoptosis. For example, thymocytes from homozygous Bim knock-out mice exhibit decreased sensitivity to glucocorticoid-induced apoptosis (Bouillet et al., 1999). Furthermore, in vitro studies utilising shRNA or siRNA targeting the various Bim tran­ scripts confirm a substantial role for Bim induction in the progression of glucocorticoid-induced apop­ tosis (Abrams et al., 2004; Lu et al., 2006; Ploner et al., 2008).

9

The role of glucocorticoid-induced leucine zip­ per (GILZ) is an area of expanding interest in the study of glucocorticoid-induced apoptosis. GILZ was first identified as a glucocorticoid-responsive gene by a systemic screen for genes responsive to glucocorticoids in the thymus (D’Adamio et al., 1997). Due to the presence of three GREs in the GILZ promoter, the glucocorticoid induction of GILZ expression is direct and robust (Wang et al., 2004). The strongest evidence that GILZ may mediate glucocorticoid-induced apoptosis is provided by studies of GILZ transgenic mice. In these mice, the GILZ transgene is specifically targeted to the T-cell compartment. Primary thymocytes from these mice were resistant to TCR-induced apoptosis. However, they exhibited augmented glucocorticoid-induced apoptosis due to reduced expression of the Bcl-2 family member, Bcl-XL, as well as increased activation of caspases 8 and 3 (Delfino et al., 2004). GILZ also mediates glucocorticoid-induced cell cycle arrest through direct interaction with and inhibition of the prolif­ erative Ras and Raf oncogenes (Ayroldi et al., 2007). In addition to Bim and GILZ, glucocorticoids rapidly transactivate the stress gene dexametha­ sone-induced gene 2 (Dig2) in murine lymphoma cell lines. Interestingly, Dig2 overexpression reduced the sensitivity of these cells to glucocorti­ coid-induced apoptosis, suggesting a pro-survival function for this gene (Wang et al., 2003b). Gran­ zyme A is up-regulated following glucocorticoid treatment in B-ALL cells. Pharmacological inhibi­ tion of granzyme A blunted the apoptotic response, indicating that this enzyme is an effector of glucocorticoid-induced apoptosis (Yamada et al., 2003). T-cell death-associated gene (TDAG8) is rapidly induced by glucocorti­ coids in thymocytes. Thymocytes from TDAG8 transgenic mice exhibited increased activation of caspases 3, 8 and 9 following glucocorticoid expo­ sure (Tosa et al., 2003). However, thymocytes from TDAG8-deficient mice remained sensitive to glucocorticoid-induced apoptosis, suggesting a minor role for TDAG8 in glucocorticoid-induced apoptosis (Radu et al., 2006). Glucocorticoid exposure represses the pro-survival oncogene c­ myc in human leukemic CEM cells (Wang et al.,

2003a). Furthermore, glucocorticoids repress the glycolytic hexokinase II enzyme (Tonko et al., 2001). Hexokinase II acts as a stabilizer of the mitochondrial voltage-dependent anion channel (VDAC). Interestingly, hexokinase II overexpres­ sion abrogated glucocorticoid-induced apoptosis via inhibition of mitochondrial destabilization (Sade et al., 2004). In summary, glucocorticoids regulate the transcription of several genes, the expression of which influences cellular progression through the glucocorticoid-induced apoptotic pathway.

Cytoplasmic signaling Glucocorticoids cause rapid and sustained increases in cytosolic calcium concentrations in thymocytes, lymphoma cells and B lymphoblasts (Bian et al., 1997; Distelhorst and Dubyak, 1998; Hughes et al., 1997; Lam et al., 1993; McConkey et al., 1989b; Orrenius et al., 1991). Buffering of cytosolic calcium or culture in calcium-free media prevented glucocorticoidinduced apoptosis of primary thymocytes (McConkey et al., 1989b). Interestingly, phar­ macological inhibition of the calcium-binding protein calmodulin inhibited DNA fragmentation without interfering with cytosolic calcium increase, indicating that calmodulin mediates the downstream apoptotic effect of glucocorticoid-induced calcium mobilization (Dowd et al., 1991). However, the precise role of calcium mobilization in glucocorticoid-induced apoptosis remains controversial. A subsequent study reported that chelation of intracellular cal­ cium inhibits DNA fragmentation, but not glucocor­ ticoid-induced apoptosis in primary thymocytes (Iseki et al., 1993). Thus, calcium mobilization is likely required for endonuclease activation, but not other aspects of glucocorticoid-induced apoptosis. Glucocorticoids also cause a net potassium efflux in thymocytes and CEM T-ALL cells (Benson et al., 1996; Bortner and Cidlowski, 2000). This potassium loss enhances apoptosis in thymocytes and normal intracellular potassium levels inhibit DNA fragmentation and caspase-3 activation in lymphocytes (Bortner et al., 1997;

10

Hughes et al., 1997). Furthermore, pharmacologi­ cal inhibition of plasma membrane potassium channels in primary thymocytes effectively inhib­ ited glucocorticoid-induced apoptosis through the prevention of cytosolic potassium loss and inhibi­ tion of mitochondrial membrane destabilization (Dallaporta et al., 1999). Glucocorticoids (ranging from 107 to 1012 M) induce a rapid increase in intracellular ceramide concentrations in primary thymocytes within 15 min of treatment (Cifone et al., 1999). This increase is receptor dependent, as determined through co-treatment with the GR antagonist RU-486 (Cifone et al., 1999). Furthermore, phar­ macological inhibition of ceramide biosynthesis prevented apoptosis in glucocorticoid-treated thymocytes (Cifone et al., 1999). The generation of reactive oxygen species is also dramatically increased in response to glucocorticoids (Zamzami et al., 1995). This increase accompanies a decrease in the mRNA levels of anti-oxidant enzymes and contributes to glucocorticoid-induced cell death (Baker et al., 1996). Furthermore, co-treatment with an exogenous thiol anti-oxidant inhibits gluco­ corticoid-induced apoptosis (Tonomura et al., 2003). Moreover, in the absence of oxygen, cell death is prevented, indicating that the generation of reactive oxygen species is an important step in the progression of glucocorticoid-induced apopto­ sis (Torres-Roca et al., 2000). Finally, glucocorti­ coids induce a rapid translocation of GR to the mitochondria in glucocorticoid-sensitive cells (Sio­ nov et al., 2006). Overexpression of a GR species specifically targeted to the mitochondria is suffi­ cient to induce apoptosis, perhaps through the mediation of mitochondrial reactive oxygen species and ceramide production as well as the rapid cal­ cium mobilization following glucocorticoid expo­ sure (Sionov et al., 2006). These rapid cytoplasmic effects are likely participants in the complex, multi­ component glucocorticoid-induced apoptosis-sig­ naling pathway.

Execution of glucocorticoid-induced apoptosis The glucocorticoid-induced apoptosis pathway culminates in the activation of the caspase

cascade. Caspases are a family of proteases that cleave substrates at aspartate residues, mediating the dramatic morphological and biochemical changes occurring during apoptosis (Thornberry and Lazebnik, 1998). Studies utilising broad cas­ pase inhibitors have found that glucocorticoidinduced apoptosis requires caspase activation (Bellosillo et al., 1997; Hughes and Cidlowski, 1998; Sarin et al., 1996; Weimann et al., 1999). Caspase activation occurs through two broadly conserved pathways: the extrinsic and intrinsic apoptosis pathways. In the extrinsic pathway, ligands such as Fas or tumor necrosis factor (TNF) bind cognate receptors on the cell surface, initiating the caspase cascade via activation of caspase 8 (Ashkenazi and Dixit, 1998). The sec­ ond pathway, the intrinsic or mitochondrialmediated pathway, involves disruption of the mitochondrial membrane by the pro-apoptotic Bcl-2 family members Bim, Bax and Bak, leading to the release of cytochrome c and apoptotic peptidase-activating factor 1 (Apaf-1) and subse­ quent activation of caspase 9 (Saleh et al., 1999). Both pathways culminate in the activation of downstream of effector caspases (caspases 3, 7 and 6) (Green, 2000). There is evidence suggesting that glucocorti­ coid-induced apoptosis proceeds via the intrinsic mitochondrial pathway. For example, thymocytes from Apaf-1 and caspase 9 deficient mice exhibit reduced sensitivity to glucocorticoid-induced apoptosis (Hakem et al., 1998; Kuida et al., 1998; Yoshida et al., 1998). Therefore, in a simplified model of glucocorticoid-induced apoptosis, gluco­ corticoid exposure leads to the regulation of genes involved in the initiation of apoptosis, namely the pro-apoptotic Bcl-2 family member, Bim. Bim transactivation leads to the activation of down­ stream apoptotic mediators, Bax and Bak. Upon activation, Bax and Bak mediate the disruption of the mitochondrial membrane potential and the subsequent release of Apaf-1 and cytochrome c into the cytosol. The mitochondria is also respon­ sible for some of the rapid, glucocorticoidmediated cytoplasmic effects including calcium mobilization and the production of reactive oxygen species and ceramide, all of which may contribute to the progression of glucocorticoid­

11

induced apoptosis. Apaf-1 and cytochrome c release leads to the activation of caspase 9, the subsequent activation of downstream effector cas­ pases and the execution of glucocorticoid-induced apoptosis (Fig. 5).

Glucocorticoid-induced apoptosis of healthy lymphocytes In the highly coordinated process of T-lymphocyte development, bone marrow progenitors migrate to the thymus where they undergo a transition to double negative (CD48) immature thymocytes (Radtke et al., 2004). Following random rearran­ gement of the TCR a and b genes, cells become double positive (CD4þ8þ) and undergo selection based on the specificity of the TCR for self-pep­ tides bound to major histocompatibility complex (MHC)-encoded molecules (Kisielow and von Boehmer, 1995). Double positive thymocytes with TCRs bearing low avidity for self-antigen/ MHC undergo ‘death by neglect’. Thymocytes with TCRs bearing high avidity for self-antigen/ MHC undergo TCR activation-induced apoptosis (negative selection). Only cells bearing TCRs with intermediate avidity for self-antigen/MHC are res­ cued from the neglect or negative selection cell death programmes. These cells undergo positive selection, becoming either CD4þ or CD8þ cells. Upon maturation, single positive T cells enter the periphery (Huesmann et al., 1991). How the TCR induces cell death during neglect and negative selection while simulta­ neously rescuing cells from cell death during posi­ tive selection is an area of considerable research. A role for systemic glucocorticoids in the regula­ tion of thymocyte development was first suggested in 1924, when it was demonstrated that bilateral adrenalectomy leads to thymic hypertrophy (Jaffe, 1924). It has been proposed that glucocorticoids interact with TCR signaling in a relationship termed ‘mutual antagonism’. The first evidence in support of the mutual antagonism model reported that stimulation of the TCR protected T cells from glucocorticoid-induced apoptosis. Conversely, glucocorticoids prevented TCR activation-induced cell death in the same model

system, thus coining the phrase ‘mutual antagon­ ism’ (Iwata et al., 1991; Zacharchuk et al., 1990). In this model, glucocorticoids are key modulators of the ‘death by neglect’ of low avidity TCRbearing thymocytes (Zilberman et al., 1996, 2004). In addition, glucocorticoids interfere with the TCR-induced death signaling in cells with intermediate avidity TCRs, allowing these cells to escape TCR-induced apoptosis and undergo positive selection (Iwata et al., 1991; Zacharchuk et al., 1990). However, the TCR activationinduced apoptotic signaling in double positive thy­ mocytes bearing high avidity TCRs overwhelms glucocorticoid antagonism, allowing for the dele­ tion of these cells during negative selection (Ashwell et al., 2000). TCR signaling activates extracellular signalrelated kinase (ERK) signaling (Tsitoura and Rothman, 2004). ERK is responsible for rapid phosphorylation and inactivation of Bim, a key modulator of the glucocorticoid-induced apoptotic pathway (Ley et al., 2003). Therefore, TCR may antagonize glucocorticoid signaling via ERK acti­ vation and Bim inactivation. Alternatively, gluco­ corticoid signaling results in the rapid dissociation of the lymphocyte-specific kinases LCK and FYN from the TCR complex, thus inactivating TCR signaling (Lowenberg et al., 2006). There­ fore, one potential mechanism of glucocorticoid antagonism of TCR is through disruption of the TCR complex. In summary, in this model of mutual antagonism, glucocorticoids act as a rheostat, modulating the threshold for TCR acti­ vation-induced apoptosis during thymocyte selection. In vitro evidence for the mutual antagonism model was generated by experiments in fetal thy­ mic organ culture (FTOC). Pharmacological inhibition of glucocorticoid production or gluco­ corticoid signaling sensitized cells to TCR-induced apoptosis, ultimately altering the T-cell repertoire (Vacchio et al., 1998, 1994). Furthermore, in vivo knock-down of GR in the T-cell compartment dramatically reduced thymus size due to a decrease in the number of double positive and single positive thymocytes. Thus, the absence of glucocorticoid signaling in vivo sensitized double positive thymocytes to negative selection (King

12 Glucocorticoid-induced apoptosis Plasma membrane Glucocorticoid Cytoplasm

Apoptosis Ceramide

p23

ROS

90

p23

P HS

HSP90

Ca2+

HSP90

Effector caspases (3, 6, 7)

GR Destablization Apaf1 GR

Active caspase 9

VDAC Nucleus

GR

Cyto c Activation Bax/Bak

Bim

Fig. 5. Glucocorticoid-induced apoptosis signaling cascade. In this abbreviated model, glucocorticoids regulate the expression of apoptosis-effector genes, namely the pro-apoptotic Bcl-2 family member, Bim. Bim transactivation leads to the activation of downstream apoptotic mediators, Bax and Bak. Upon activation, Bax and Bak mediate disruption of the mitochondrial membrane potential and the subsequent release of cytochrome c and Apaf-1 into the cytosol. Apaf-1 and cytochrome c release leads to the activation of caspase 9, the subsequent activation of downstream effector caspases and the execution of glucocorticoid-induced apoptosis. The mitochondria is also responsible for some of the rapid, glucocorticoid-mediated cytoplasmic effects including calcium mobilization and the production of reactive oxygen species and ceramide, all of which may contribute to the progression of glucocorticoid-induced apoptosis.

et al., 1995). However, more recent studies of T-cell specific GR knock-out mice detected no abnormality in thymocyte development (Baumann et al., 2005; Brewer et al., 2003). Conversely, additional studies modulating GR expression in the thymus report dramatic alterations in thymic cellularity and thymocyte maturation (Jondal et al., 2004; King et al., 1995; Pazirandeh et al., 2002). Therefore, the precise role of glucocorticoids in thymocyte development remains controversial.

Glucocorticoid-induced apoptosis of haematological malignancies The therapeutic potential of synthetic glucocorti­ coids for the treatment of haematological malig­ nancies was first suggested in 1943, when the administration of hydroxycorticosterone resulted in apoptosis of malignant lymphocytes in the mouse (Dougherty and White, 1943; Heilman FR, 1944). Subsequently, synthetic glucocorti­ coids were implemented in the clinical treatment

13

of leukemia and lymphoblastomas (Pearson, 1949; Stickney, 1950). Today, glucocorticoids are central in the treatment of haematological malig­ nancies and are utilized as adjuvants in a majority of chemotherapeutic regimens. Chemotherapy of haematological malignancies consists of three phases: induction of remission, intensification (or consolidation) and maintenance of remission. The induction phase involves the rapid destruc­ tion of malignant cells in order to achieve remis­ sion. Remission is defined as the presence of fewer than 5% malignant cells in the bone marrow and periphery. The intensification phase utilizes chemotherapy to further reduce tumor burden and may consist of bone marrow stem cell transplantation. Finally, maintenance of remission utilizes combination therapies to main­ tain remission and prevent relapse (Hoffbrand et al., 2001). This section will review the efficacy and limitations of synthetic glucocorticoids in the treatment of individual haematomalignancies.

Glucocorticoid therapy of acute lymphoblastic leukemia ALL is characterized as an accumulation of bone marrow-derived malignant, immature lympho­ cytes. ALL is predominantly a disease of children and adolescents, with this age group comprising two-thirds of the 4000 cases diagnosed annually in the United States (Pui and Evans, 1998). Histori­ cally, the use of prednisone monotherapy in the induction phase was efficacious, resulting in remission of 45–65% of the primary childhood ALL cases reported (Hyman et al., 1959). Today, prednisone is widely used in conjunction with the alkylating agent vincristin, the topoi­ somerase II inhibitor anthracycline and catalytic asparaginase in the induction phase of ALL che­ motherapy. This combination regimen induces complete remission in 98% of children and 85% of adults (Pui and Evans, 2006). Following the induction of remission, prednisone is commonly utilized in the maintenance therapy of ALL (Gokbuget and Hoelzer, 2006). The response to prednisone therapy is a strong predictor of prog­ nosis in infant ALL. Infants with a poor response

following a 7-day course of prednisone are less responsive to conventional chemotherapy and require more intensive induction therapies (Dordelmann et al., 1999). Furthermore, early response to combined chemotherapy is also a predictor of outcome in childhood ALL (Schrappe et al., 1996). Therefore, the prednisone response in childhood and infant ALL is currently a prognostic factor utilized in the adaptation of chemotherapy treatment protocols.

Glucocorticoid therapy of chronic lymphoblastic leukemia CLL is characterized as a malignant neoplastic proliferation of the B-cell (common) or T-cell (rare) compartment. CLL is the most commonly diagnosed lymphoproliferative disorder of the Western world (30% of cases) and is primarily an adult disease, with a majority of cases occurring in individuals over 50 (Rozman and Montserrat, 1995). Historically, synthetic glucocorticoids were not added to the conventional chemotherapy regi­ men of CLL. This exclusion was based on the observations of clinical studies performed in the 1970s and 1980s, which found that the addition of prednisone to the conventional CLL regimen con­ ferred no additional benefit (Pettitt, 2008). How­ ever, in the late 1990s an in vitro study of cultured CLL cells from patients with relapsed or resistant disease found that these cells were sensitive to apoptosis induced by high doses of the synthetic glucocorticoid methylprednisolone (Bosanquet et al., 1995). Subsequent clinical trials of highdose methylprednisolone (HDMP) in CLL patients achieved a response rate of 43% (Bosan­ quet et al., 1995). Further trials have been per­ formed in CLL patients with primary or relapsed/ resistant disease utilizing combination chemother­ apy consisting of HDMP and the chimeric mono­ clonal CD20 antibody, rituximab. This study observed a response rate of 93% for primary CLL and 83% for relapsed/refractory CLL (Cas­ tro et al., 2009). The success of HDMP therapy may be due to the fact that glucocorticoid-induced apoptosis is p53 independent. Accordingly, in a separate trial, CLL patients with mutations in the

14

p53 tumor suppressor responded well to HDMP treatment (Thornton et al., 2003). Currently, the use of HDMP in conjunction with conventional chemotherapy or rituximab is gaining popularity in the treatment of CLL.

Glucocorticoid therapy of multiple myeloma Multiple myeloma (MM) is characterized by an increase of monoclonal plasma cells in the bone marrow, resulting in anaemia, hypercalcaemia and renal failure. MM accounts for 10% of all haematomalignancies (Kyle and Rajkumar, 2008). Use of alkylating agent melphalan in con­ junction with the synthetic glucocorticoid predni­ sone has been the foundation of first-line MM chemotherapy for decades. The overall response rate for this regimen was 50% (Rajkumar et al., 2002). Recently, the FDA approved the use of a more aggressive combination chemotherapy con­ sisting of the anti-angiogenic drug thalidomide and high doses of the synthetic glucocorticoid dexamethasone (Thal/Dex). Randomized trial of this induction regimen resulted in a response rate of 63% (Rajkumar et al., 2006). The Thal/ Dex or Thal/Dex/melphalan regimen is currently the most commonly prescribed remission induc­ tion regimen for the treatment of MM (Kyle and Rajkumar, 2008). Therefore, synthetic glucocor­ ticoids remain a cornerstone in the chemother­ apy of MM.

Glucocorticoid therapy of Hodgkin's and non-Hodgkin's lymphoma Hodgkin’s lymphoma, or Hodgkin’s disease, is characterized by the orderly spread of malignant lymphocytes through the lymphatic system and the presence of multi-nucleated lymphocytes known as Reed–Sternberg cells (Kuppers et al., 2002). Historically, Hodgkin’s disease has been treated with a combination chemotherapy consist­ ing of mechlorethamine, vincristine, procarbazine and prednisone (MOPP), resulting in remission induction of 80% of patients (DeVita et al.,

1980). However, the MOPP programme has recently been replaced in favour of more tailoured chemotherapy regimens. These regimens include the Stanford V (doxorubicin, bleomycin, vinblastine, vincristine, mechlorethamine, etoposide and prednisone) and the BEACOPP (doxorubicin, bleomycin, vincristine, cyclophosphamide, procar­ bazine, etoposide and prednisone), both of which incorporate the synthetic glucocorticoid predni­ sone (Evens et al., 2008). However, the impor­ tance of synthetic glucocorticoids in the remission induction of Hodgkin’s disease is unclear as the gold standard chemotherapy regi­ men, ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine), does not include a synthetic glucocorticoid (Evens et al., 2008). Nevertheless, the Stanford V and BEACOPP regimens repre­ sent important glucocorticoid-inclusive treatment alternatives in the chemotherapy of Hodgkin’s disease. Non-Hodgkin’s lymphoma is comprised of a diverse group of haematomalignancies character­ ized by the absence of Reed–Sternberg cells. Induction chemotherapies vary depending on the type of lymphoma; however, the first-line che­ motherapy regimen in a majority of lymphomas consists of cyclophosphamide, vincristine, doxor­ ubicin and prednisone (CHOP). This combined chemotherapy results in remission induction of 50–60% of primary disease (Fisher et al., 1993). The remaining patients either fail to respond or have relapsed. Recent studies find that these patients may be rescued by a salvage therapy con­ sisting of dexamethasone, etoposide, ifosfamide and cisplatin (DVIP), especially when this therapy is combined with stem cell transplantation (Lazar et al., 2009).

Limitations of glucocorticoid chemotherapy Given their pleiotrophic physiological effects, the prolonged use of glucocorticoids in chemotherapy is complicated by numerous injurious side effects including muscle wasting, osteoporosis in adults, inhibition of longitudinal bone growth in children and increased susceptibility to opportunistic infec­ tions (Rhen and Cidlowski, 2005). These side

15

effects are dose and duration dependent. For example, cancer patients receiving high dose glu­ cocorticoid therapy (more than 400 mg dexa­ methasone) and prolonged therapy (more than 3 weeks) reported a 76 and 75% incidence of toxi­ city, respectively (Weissman et al., 1987). Glucocorticoids promote the degradation of muscle protein to free amino acids, resulting in muscle wasting (Mitch, 2000). In children, gluco­ corticoids induce apoptosis and inhibit prolifera­ tion of chondrocytes, the cells responsible for longitudinal bone growth (Chrysis et al., 2005). In adults, glucocorticoids induce apoptosis of mature osteoblasts and increase the bone resorp­ tion activity of osteoclasts, leading to the onset of osteoporosis (Chrysis et al., 2005; Rhen and Cidlowski, 2005). Glucocorticoids are immuno­ suppressive due to their interference with the NF-�B and AP-1 signaling pathways. These tran­ scription factors are key modulators of the inflammatory response and their repression results in decreased expression of inflammatory mediators including TNFa, IL-1b and numerous inflammatory cytokines (Rhen and Cidlowski, 2005). The glucocorticoid-induced immunosup­ pression renders patients vulnerable to opportu­ nistic infections such as oral candidiasis, an infection common in patients undergoing longterm glucocorticoid therapy (Walsh and Avashia, 1992). Another limitation of glucocorticoid che­ motherapy is the emergence of glucocorticoidresistant clonal populations during prolonged glucocorticoid therapy, glucocorticoid resistance upon relapse and the existence of inherently resis­ tant haematomalignancies. Leukemias of the mye­ logenous lineage are often innately resistant to glucocorticoid therapy (Zwaan et al., 2000). Furthermore, patients with relapsed ALL exhibit increased resistance to glucocorticoid therapy (Schrappe et al., 2000). Glucocorticoid resistance in these cancers is associated with a poor prog­ nosis (Dordelmann et al., 1999; Hongo et al., 1997; Kaspers et al., 1997). Therefore, a more compre­ hensive understanding of the factors governing glucocorticoid resistance in haematomalignancies may improve the efficacy of glucocorticoid therapy.

Mechanisms of glucocorticoid resistance in haematomalignancies Altered expression of glucocorticoid receptor isoforms There is compelling clinical and empirical evi­ dence suggesting that altered expression of GR isoforms contributes to the varied responses of haematomalignancies to glucocorticoid therapy. For example, increased expression of the ‘domi­ nant-negative’ GRb isoform has been reported in several haematomalignancies. Under normal con­ ditions, GRb is expressed at extremely low levels (0.16% in healthy lymphocytes) (Honda et al., 2000). However, the levels of GRb expression are increased in T-ALL CEM cells (0.22%), primary ALL patient samples (0.5–1.2%) and glucocorticoid-resistant CLL (Haarman et al., 2004; Shahidi et al., 1999). Interestingly, the ability of prednisone to induce apoptosis of pediatric ALL cells is inversely correlated with GRb levels (Koga et al., 2005). Furthermore, human neutro­ phils exhibit increased GRb levels and decreased glucocorticoid responsiveness, and the in vitro transfection of mouse neutrophils with GRb conferred partial glucocorticoid resistance (Strickland et al., 2001). However, the functional significance of increased GRb expression in haematomalignancies remains controversial, as other studies of GRb in primary cells from ALL patients have not reported a correlation between GRb expression and glucocorticoid resistance (Haarman et al., 2004; Tissing et al., 2005a). In the absence of conclusive evidence, the gen­ eration of a GRb transgenic mouse model would be of great value in determining the precise role of GRb in the development of glucocorticoid resistance. Altered expression of the additional GR iso­ forms may also contribute to glucocorticoid resis­ tance. For example, elevated levels of GRg mRNA are associated with glucocorticoid resistance in a study of primary versus relapsed ALL samples (Haarman et al., 2004). Several studies have iden­ tified increased expression of GR-P or GR-A mRNA in glucocorticoid-resistant malignancies (de Lange et al., 2001; Krett et al., 1995; Moalli

16

et al., 1993). However, these findings are contra­ dicted by more recent studies of patient-derived primary ALL cells (Tissing et al., 2005a). Further­ more, altered ratios of GR translational isoform expression may influence glucocorticoid respon­ siveness. For example, overexpression of the GRa-D isoform, the least transcriptionally active sub-type, confers resistance to glucocorticoidinduced apoptosis in osteosarcoma cells (Lu et al., 2007). Future studies investigating the complement of various GR translational isoforms in glucocorti­ coid-resistant patient samples would clarify the role of these variants in the development of glucocorti­ coid resistance. Finally, alternative GR promoter usage has been associated with glucocorticoid resistance. For example, exon 1A contains a GRE, which promotes the auto-induction of GR transcription upon glucocorticoid treatment (Geng and Vedeckis, 2004). This auto-induction has been associated with increased responsiveness to gluco­ corticoid treatment (Purton et al., 2004). Further­ more, cell-type-specific expression of GR promoter transcripts has been reported in a vari­ ety of haematomalignant cell lines (Breslin et al., 2001; Geng and Vedeckis, 2004; Nunez and Vedeckis, 2002; Pedersen and Vedeckis, 2003). This variable expression of GR promoter tran­ scripts has been correlated with the diverse responsiveness of haematologic malignancies to glucocorticoid therapy (Breslin et al., 2001; Purton et al., 2004). However, other reports contradict these findings and cite no differences in GR pro­ moter usage or differential induction of various GR promoter transcripts in glucocorticoid-resis­ tant primary ALL cells (Tissing et al., 2006).

Glucocorticoid-induced alterations in GR expression In lymphoid cells sensitive to glucocorticoids, GR auto-induction in response to steroid treatment is a common observation. This auto-regulatory action can be attributed to the presence of a GRE in the GR promoter region. GR auto-induc­ tion has been observed in the glucocorticoid-sen­ sitive CEM T-ALL cell lines, primary ALL cells

and immature thymocytes (Barrett et al., 1996; Tissing et al., 2006). Accordingly, the extent of GR auto-induction following glucocorticoid treat­ ment has been directly correlated with the degree of cell death in glucocorticoid-sensitive cells (Geley et al., 1996). However, the importance of GR auto-induction in glucocorticoid-induced apoptosis is challenged by studies of primary ALL xenografts in immune-deficient mice and primary thymocytes. Glucocorticoid-sensitive xenografts contained high levels of basal GR and failed to auto-induce GR upon hormone treat­ ment. Furthermore, glucocorticoid-sensitive pri­ mary thymocytes expressing high-basal GR levels also undergo apoptosis in the absence of auto-induction (Oldenburg et al., 1997). These results suggest that in sensitive cells with high basal GR expression, GR auto-induction is not required to induce apoptosis (Bachmann et al., 2007). However, GR auto-induction is essential for apoptosis in cells harbouring low basal levels of GR (Miller et al., 2007; Ramdas et al., 1999; Riml et al., 2004). Conversely, in cells resistant to glucocorticoids, homologous down-regulation of GR following glucocorticoid treatment is a common observa­ tion. For example, in leukemias of myelogenous origin, GR is down-regulated by glucocorticoids, perhaps accounting for their inherent resistance to glucocorticoid therapy (Kfir et al., 2007). Further­ more, homologous GR down-regulation is an indi­ cator of poor prognosis in ALL (Bloomfield et al., 1981; Pui and Costlow, 1986). Therefore, pro­ longed glucocorticoid therapy, in certain cellular contexts, may result in the emergence of glucocor­ ticoid-resistant cells harbouring low levels of basal GR expression. Homologous down-regulation of GR may employ multiple mechanisms including diminished promoter activity or destabilization of mRNA or protein expression. Homologous downregulation of GR may be due to decreased activity of the GR promoter. However, use of a hetero­ logous promoter to drive GR expression found that the repressive action of glucocorticoids on GR expression remained intact. Further in vitro studies determined that the region of GR encoded by amino acids 550–697 is essential to the homo­ logous down-regulation of GR (Alksnis et al.,

17

1991; Burnstein et al., 1990, 1994). Additionally, decreased GR mRNA stability may contribute to homologous down-regulation. For example, the presence of AUUUA motifs in the 30 -UTR of GR might be involved in the destabilization of GR mRNA. These motifs are common RNAbinding protein motifs and the action of these RNA binding proteins may contribute to the destabilization of GR mRNA (Chen and Shyu, 1995; Schaaf and Cidlowski, 2002a; Shaw and Kamen, 1986). Finally, homologous down-regula­ tion could be due to decreased GR protein stabi­ lity. Studies have indicated that the proteosome complex degrades the GR. Pre-treatment of GR overexpressing cells with the proteosomal inhibi­ tor MG-132 impedes homologous down-regula­ tion of GR protein (Wallace and Cidlowski, 2001). Furthermore, GR contains a conserved proline, glutamic acid, serine and threonine (PEST) degradation motif, also contributing to the destabilization of GR protein (Wallace and Cidlowski, 2001). Additionally, non-coding microRNAs have recently been identified as negative regulators of gene expression through transla­ tional repression of target mRNAs. Hormonal induction of specific microRNAs targeting GR is an attractive model of homologous GR down-reg­ ulation. However, microRNAs targeting GR have only been identified in neurons and their function in the regulation of GR mRNA translation in the lymphoid compartment remains unaddressed (Vreugdenhil et al., 2009). The precise role of these proposed mechanisms in the homologous down-regulation of GR observed in glucocorti­ coid-resistant haematomalignancies remains unclear and additional experiments are war­ ranted. Furthermore, studies of primary ALL xenografts in immune-deficient mice found that the glucocorticoid-resistant tumors possessed suf­ ficient basal levels of GR and did not undergo homologous GR down-regulation upon steroid treatment (Bachmann et al., 2007). These obser­ vations suggest that in these neoplasms, glucocor­ ticoid resistance occurs downstream of GR. Clearly, the precise mechanisms governing gluco­ corticoid resistance in haematomalignancies are divergent and cell-type specific. However, for many haematomalignant cell types, the

directionality of GR expression in response to glucocorticoid treatment modulates the efficacy of glucocorticoid therapy.

Mutations in the glucocorticoid receptor Numerous mutations affecting GRa signaling have been identified in laboratory-derived gluco­ corticoid-resistant leukemic cell lines. For exam­ ple, the CEM T-ALL cell line harbours a mutation in the GR LBD (L753F) that hinders GR ligand binding and subsequent transactivation (Hillmann et al., 2000). Accordingly, the T-ALL 6TG1.1 cell line bearing this mutation is resistant to glucocorticoid-induced cell death (Liu et al., 1995; Powers et al., 1993). Additional mutations in the GR LBD, including the G679S, F737L, I559N, V571A, D641V, V729I and I747M also impair the transcriptional activity of GR (Charmandari et al., 2004). Furthermore, a R477H mutation in one allele of the GR gene inhibits DNA binding in glucocorticoid-resistant Jurkat T-ALL cells. This mutation renders the GR unable to transactivate, thus eliminating GR auto-induction and contribut­ ing to glucocorticoid resistance in these cells (Riml et al., 2004). However, the majority of studies in patient-derived samples have failed to associate glucocorticoid resistance with mutations in the GR. Mutations in the GR were absent in both glucocorticoid-resistant primary cells derived from ALL patients or glucocorticoid-resistant pri­ mary ALL xenografts (Bachmann et al., 2007; Beesley et al., 2009; Tissing et al., 2005b). There­ fore, there are key differences in the mechanisms of glucocorticoid resistance in long-term cultured cells and patient-derived samples and the lesions responsible for impaired GR signaling and gluco­ corticoid resistance in vitro do not contribute to the development of glucocorticoid resistance in vivo.

Aberrant expression of Bcl-2 Aberrant expression of the anti-apoptotic Bcl-2 protein is frequently reported in B-cell follicular lymphomas. In these lymphomas, translocation of

18

Bcl-2 places this oncongene adjacent to the immu­ noglobulin heavy chain locus, resulting in rampant expression of the Bcl-2 fusion gene. This fusion gene is identified in over 80% of B-cell lympho­ mas and Bcl-2 transgenic mice develop diffuse large-cell lymphomas at old age (Cleary et al., 1986; McDonnell et al., 1989; McDonnell and Korsmeyer, 1991; Tsujimoto and Croce, 1986). Bcl-2 functions as a stabilizer of the mitochondrial membrane, thereby preventing the loss of mito­ chondrial membrane potential and the release of cytochrome c and Apaf-1 in response to glucocor­ ticoids (Susin et al., 1999). In addition, the phos­ phorylation of Bcl-2 at specific residues is required for the establishment of glucocorticoid resistance in cultured B lymphocytes (Huang and Cidlowski, 2002). A number of in vitro studies have found that Bcl-2 overexpression confers varying degrees of resistance to glucocorticoid-induced apoptosis, while in vivo knock-down of Bcl-2 expression sen­ sitizes haematopoietic cells to glucocorticoidinduced apoptosis, supporting its role as an impor­ tant mediator of glucocorticoid resistance in haematomalignancy (Kamada et al., 1995; Kfir et al., 2007; Memon et al., 1995; Nakayama et al., 1993; Veis et al., 1993a, 1993b). In addition, the in vitro and in vivo manipulation of the Bcl-2 family members Bcl-XL and Mcl-1 also alters the efficacy of glucocorticoid-induced apoptosis (Opferman et al., 2003; Wei et al., 2006). How­ ever, defining the role of these two family mem­ bers in the development of haematomalignancy and their contribution to glucocorticoid resistance requires further study.

Failure to induce Bim expression It is well established that the induction of Bim expression is important for glucocorticoid-induced apoptosis. Lymphocytes derived from Bim transgenic mice exhibit reduced sensitivity to glucocorticoid-induced apoptosis (Bouillet et al., 1999). Furthermore, inhibition of Bim expression via siRNA and shRNA inhibits caspase-3 activa­ tion and glucocorticoid-induced apoptosis in ALL cell lines (Abrams et al., 2004). A recent study of ALL xenografts in immune-deficient mice

revealed that the resistant xenografts all failed to induce Bim expression upon glucocorticoid treat­ ment (Bachmann et al., 2007). This was not due to lack of transactivation activity, as these xenografts were able to induce the expression of GILZ, sug­ gesting that the failure of glucocorticoid-resistant xenografts to induce Bim is downstream of transactivation. Several mechanisms of glucocorticoidinduced Bim expression have been proposed including (1) transactivation of Bim by the gluco­ corticoid-responsive transcription factors Foxo3A/ FKHRL1, RUNX3 and E2F1 (Dijkers et al., 2000; Yamamura et al., 2006; Zhao et al., 2005), (2) release of Bim from cytoskeletal sequestration (Puthalakath et al., 1999) and (3) repression of Src-mediated inhibitory phosphorylation of Bim (Ley et al., 2003). Therefore, targeting one or more of the pathways governing glucocorticoidinduced Bim expression may prove a beneficial in the development of therapies to overcome glu­ cocorticoid resistance in haematomalignancies.

Interactions with the kinome Glucocorticoid signaling involves complex com­ munication between multiple signaling pathways. For example, the mitogen-activated protein (MAP) kinases ERK and c-Jun N-terminal kinase (JNK) inhibit glucocorticoid-induced apoptosis, while p38 stimulates glucocorticoid-induced apop­ tosis in the CEM leukemic cell line (Miller et al., 2007; Wada and Penninger, 2004). Glucocorticoidresistant CEM clones possess high constitutive JNK activity and glucocorticoid stimulation results in increased ERK activity accompanied by the weak induction of p38 (Miller et al., 2007). The cAMP-driven protein kinase A (PKA) pathway also contributes to glucocorticoid-induced apopto­ sis. Stimulation of PKA with forskolin inhibits JNK signaling and sensitizes resistant CEM cells to glucocorticoid-induced apoptosis by increasing the transcriptional activity of GR (Medh et al., 1998). These results suggest that the GR and PKA pathways exert cooperative effects on GRmediated gene transcription. Additionally, inhibi­ tion of the mammalian target of rapamycin (mTOR)-signaling pathway with rapamycin

19

inhibits JNK signaling, sensitizing CEM and MM cell lines as well as primary MM cells to glucocor­ ticoid-induced apoptosis. This increased sensitiv­ ity is associated with decreased expression of cyclin D2 and the anti-apoptotic protein survivin. The PI3K–AKT pathway has also been shown to prevent glucocorticoid-induced cell death through the inhibitory phophorylation of key apoptotic mediators including Bcl-2-associated death pro­ moter (BAD), caspase 9, Fox03A/FKHRL and CREB (Maddika et al., 2007). Furthermore, the AKT-mediated phospho-inhibition of Fox03A/ FKHRL inhibits the transcription of Bim and con­ tributes to glucocorticoid resistance (Maddika et al., 2007). Finally, Src kinase is released from Hsp90 sequestration upon glucocorticoid treat­ ment and pharmacological inhibition of Src activa­ tion ameliorates glucocorticoid resistance in MM cells (Ishikawa et al., 2003). Given the intricate cross-talk between various kinase pathways and GR signaling, the modulation of the kinome is a compelling avenue in the development of novel therapies to ameliorate glucocorticoid resistance in haematomalignancies.

Novel therapies targeting glucocorticoid resistance Targeting the kinome Previous studies have shown that MAP kinase signaling modulates the response of various leu­ kemic cell lines to glucocorticoids (Miller et al., 2007, 2005). Direct pharmacological inhibition of JNK and ERK signaling sensitizes resistant cells to glucocorticoid-induced apoptosis (Miller et al., 2007). A more recent study of five diverse gluco­ corticoid-resistant haematomalignant cell lines confirmed that inhibition of ERK and JNK signal­ ing results in glucocorticoid sensitivity (Garza et al., 2009). This increased susceptibility to gluco­ corticoid-induced cell death was directly corre­ lated with increases in total and phospho-GR as well as Bim. Conversely, pharmacological inhibi­ tion of p38, one of the kinases responsible for GR phosphorylation at serine 211, protects leukemic cells from glucocorticoid-induced apoptosis

(Miller et al., 2005). Mutation of this residue abro­ gates glucocorticoid-induced apoptosis, suggesting that p38-mediated GR phosphorylation at site 211 is a key component in the glucocorticoid-induced apoptosis signaling cascade (Miller et al., 2005). Indirect inactivation of JNK signaling via the mTOR inhibitor rapamycin also sensitizes resis­ tant cells to glucocorticoid-induced apoptosis through the relief of JNK-mediated phosphoinhibition of Bim (Miller et al., 2007; Stromberg et al., 2004). Additional studies have found that mTOR inhibition leads to decreases in anti-apop­ totic Mcl-1 protein expression, further contribut­ ing to the sensitization potential of mTOR inhibitors (Wei et al., 2006). Encouragingly, mTOR inhibition sensitizes inherently resistant MM cells and primary MM xenografts to gluco­ corticoid-induced apoptosis and glucocorticoid sensitivity persists despite the addition of exogen­ ous survival signals, suggesting that rapamycin inhibition of mTOR signaling may be a suitable option for the circumvention of glucocorticoid resistance in vivo (Miller et al., 2007; Stromberg et al., 2004). Stimulation of the cAMP PKA signaling path­ way with forskolin sensitizes glucocorticoidresistant ALL cells to apoptosis (Miller et al., 2007). Furthermore, recent studies have found that inhibiting the activity of three cAMP phosphodiesterases (PDEs) overexpressed in glucocorticoid-resistant CEM cells (PDE3, PDE4 and PDE7) ameliorates glucocorticoid resistance in these cells (Dong et al., 2009). Activation PI3K– AKT signaling is correlated with the expansion of glucocorticoid-resistant MM cells in the bone marrow (Hideshima et al., 2007). Accordingly, inhibition of AKT signaling with perifosine sensitizes resistant MM cells to glucocorticoidinduced apoptosis through the reduced expression survival factors including IL-6 and survivin (Hideshima et al., 2007). Currently, a phase III clinical trial is evaluating the efficacy of perifosine and dexamethasone combined chemotherapy in the treatment of drug-resistant MM (Richardson, 2009). In addition, the efficacy of glucocorticoid treatment combined with JNK/ERK inhibitors or mTOR inhibitors is being evaluated clinically in order to overcome the dilemma of glucocorticoid

20

resistance in haematological malignancies (Davies et al., 2007; Mita et al., 2008).

future therapies interfering with glucocorticoidinduced Noxa down-regulation may improve the efficacy of glucocorticoid therapy in childhood ALL.

Targeting Bcl-2 family members Recently, small-molecule BH3 mimics have been developed to impede the anti-apoptotic effects of Bcl-2 family members. These molecules directly interact with anti-apoptotic Bcl-2 proteins at their BH3-binding groove, eliminating their ability to sequester pro-apoptotic BH3 domain Bcl-2 family members (Kang and Reynolds, 2009). For instance, the BH3 mimic gossypol (AT-101) binds directly to Bcl-2 and inhibits its function. Co-treat­ ment with gossypol potentiates dexamethasoneinduced cell death in glucocorticoid-resistant MM cell lines and patient-derived primary cells (Kline et al., 2008). The Bcl-2-specific BH3 mimic ABT­ 737 enhanced dexamethasone-induced cell death via the mitochondrial pathway in five of seven ALL cell lines evaluated (Kang and Reynolds, 2009). Furthermore, addition of the Bcl-2-specific BH3 mimic TW-37 to the CHOP regimen enhanced apoptosis in diffuse large-cell lymphoma xenografts (Mohammad et al., 2007). In addition to Bcl-2-targeted BH3 mimics, the Mcl-1-specific inhibitor obatoclax also enhanced glucocorticoidinduced apoptosis (Trudel et al., 2007). Co-admin­ istration of obatoclax and dexamethasone over­ came glucocorticoid resistance in 15 of 16 patientderived MM primary cell lines. In these cells, Mcl-1 inhibition was associated with an increase in Bim expression. Finally, anti-sense antagonism of Bcl-2 with the G3139 oligonucleotide resulted in decreased Bcl-2 protein expression and an enhanced response to dexamethasone/thalidomide chemotherapy in phase II clinical trials (Badros et al., 2005). The efficacy of G3139/dexamethasone combined therapy in the treatment of MM is cur­ rently being evaluated in phase III clinical trials (Kang and Reynolds, 2009). Interestingly, glucocorticoid monotherapy results in the decreased expression of the pro­ apoptotic Bcl-2 family member Noxa in pediatric ALL samples. Conditional overexpression of Noxa in CEM ALL cells accelerated glucocorticoidinduced apoptosis (Ploner et al., 2009). Therefore,

Concluding remarks Glucocorticoids exert pleiotrophic physiological effects, including the induction of apoptosis in the lymphocyte compartment. Glucocorticoid effects are mediated through the GR, a ligand-activated transcription factor. GR transactivation is required for the induction of apoptosis, primarily, the induction of Bim expression is important for the apoptotic activity of glucocorticoids. However, glucocorticoid-induced apoptosis is a highly coordi­ nated, multi-component process. The rapid cytoplasmic effects of glucocorticoids may also con­ tribute to the progression of apoptosis. However, the precise role of these cytoplasmic effects in the advancement of glucocorticoid-induced cell death in lymphocytes requires further investigative scru­ tiny. Endogenous glucocorticoids shape the T-cell repertoire through the induction of apoptosis by neglect as well as the antagonism of TCR-induced apoptosis during positive selection. Owing to their ability to induce apoptosis in lymphocytes, synthetic glucocorticoids are widely used in the treatment of haematological malignancies. Gluco­ corticoid chemotherapy is limited by the emergence of glucocorticoid-resistant clonal populations follow­ ing prolonged glucocorticoid therapy, glucocorticoid resistance upon relapse and the existence of inher­ ently resistant haematomalignancies. The mechan­ isms involved in the development of glucocorticoid resistance are complex and cell-type specific. Altered expression ratios of GR isoforms, homologous down-regulation of GR, the inability to autoinduce GR, mutations in the GR, dysregulation of Bcl2 family members, the failure to induce Bim and interactions with the kinome may all contri­ bute to the formation of glucocorticoid resistance in haematomalignancies. The development of novel therapies to overcome glucocorticoid resistance in haematomaligancy, including specific targeting of the kinome and Bcl-2 family members, will dramatically improve the efficacy of

21

glucocorticoid therapy in the treatment of haema­ tological malignancies.

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 2

Impact of the hypothalamic–pituitary–adrenal/ gonadal axes on trajectory of age-related cognitive decline Cheryl D. Conrad
and Heather A. Bimonte-Nelson Department of Psychology, Arizona State University, Tempe, AZ, USA

Abstract: Life expectancies have increased substantially in the last century, dramatically amplifying the proportion of individuals who will reach old age. As individuals age, cognitive ability declines, although the rate of decline differs amongst the forms of memory domains and for different individuals. Memory domains especially impacted by aging are declarative and spatial memories. The hippocampus facilitates the formation of declarative and spatial memories. Notably, the hippocampus is particularly vulnerable to aging. Genetic predisposition and lifetime experiences and exposures contribute to the aging process, brain changes and subsequent cognitive outcomes. In this review, two factors to which an individual is exposed, the hypothalamic–pituitary–adrenal (HPA) axis and the hypothalamic–pituitary–gonadal (HPG) axis, will be considered regarding the impact of age on hippocampal-dependent function. Spatial memory can be affected by cumulative exposure to chronic stress via glucocorticoids, released from the HPA axis, and from gonadal steroids (estrogens, progesterone and androgens) and gonadotrophins, released from the HPG axis. Additionally, this review will discuss how these hormones impact age-related hippocampal function. We hypothesize that lifetime experiences and exposure to these hormones contribute to the cognitive makeup of the aged individual, and contribute to the heterogeneous aged population that includes individuals with cognitive abilities as astute as their younger counterparts, as well as individuals with severe cognitive decline or neurodegenerative disease. Keywords: spatial memory; declarative memory; hippocampus; stress; glucocorticoid; estrogen; proges­ terone; testosterone; menopause; ovariectomy percentage is expected to substantially increase to 20% by the year 2020 due to the aging of the ‘baby boomer’ generation (U.S. Census Bureau, 2007). As individuals age, it is well documented that some memory loss is observed (Erickson and Barnes, 2003; Tulving and Craik, 2000). However, age influences some forms of learning and mem­ ory differently than others, with those mediated by the hippocampus and frontal cortex particu­ larly susceptible to age-related disruption.

Introduction: rationale for studying aging and hippocampal function In the United States, the proportion of the population that is over 65 is increasing. Today, about 12% of the population is over 65 and this


Corresponding author.

Tel.: þ480-965-7761; Fax: 480-965-8544; E-mail: [email protected] and [email protected]

DOI: 10.1016/S0079-6123(10)82002-3

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Learning and novel memory formation allows adaptability in an organism. It allows acquisition and updating of knowledge and skills within dif­ ferent and overlapping neurobiological domains (Kausler, 1994): strangers become familiar friends, new facts are learned and skill-sets for playing sports, musical instruments, cooking or computer programs are acquired. Within the specific domain of spatial navigation, an individual learns to navi­ gate through a novel environment so that a route to the target eventually becomes familiar, and cues in the environment form associations to help with overall navigation. For a human, this allows one to learn to find their way to a new coffee shop, and, after learning occurs, to navigate to this new location whether one starts the journey from home or the grocery store. For an animal, this allows it to learn the route to a new food source or reward, regardless of its starting loca­ tion. While progression into old age is sometimes associated with a decreased proficiency in learning or plasticity, learning still occurs within many types of memory functions and cognitive domains. Indeed, decades of research have resulted in the general consensus that not all domains of learning and memory are equally affected by aging. Nor­ mally aged humans usually retain the knowledge of how to drive an automobile and brush their teeth (non-declarative memories) and typically remember the name of the President (semantic memories, Balota et al., 2000; Erickson and Barnes, 2003), but demonstrate an age-associated decline in the recall of personal experiences and events, a type of memory referred to as episodic declarative memory, which shows greater ageassociated decline than semantic memory (Balota et al., 2000; Erickson and Barnes, 2003). It is gen­ erally thought that by the fifth decade of life in humans, learning occurs more slowly for difficult tasks such as the declarative memory delayed recall task (Albert, 2002; Albert et al., 1987). Per­ haps most considerably altered with age is spatial learning and memory, which depends upon medial temporal lobe structures such as the hippocampus, and involves the ability to navigate effectively through an environment, acquiring, integrating and retaining environmental features such as land­ marks and other prominent cues (Barnes, 1998).

Since the historic work of Tolman, learning to navigate through a new environment has been referred to as the formation of a cognitive map (Tolman, 1948). Additionally, working memory, requiring manipulation of information kept ‘on­ line’, is intimately related to neocortex, frontal cortex more specifically, and is also affected by normal aging revealing a memory decline that becomes more severe as task difficulty increases (Balota et al., 2000). The hippocampus facilitates the formation of a cognitive map and declarative memories, as well as the knowledge of facts and their relationships (Eichenbaum, 2000; O’Keefe and Nadel, 1978), and the neocortex is the loca­ tion of long-term memory storage (Eichenbaum, 2000). Notably, both the hippocampus and the frontal cortex appear to be particularly vulnerable to age-related changes (Burke and Barnes, 2006). Spatial learning and memory requires an animal to navigate through space to locate a goal and can be readily tested in rodent models. Many experi­ mental tests of rodent spatial memory aim to assess spatial working memory, a form of shortterm memory which requires a subject to retain spatial information which must be updated and is useful for only a short period of time (trial-specific information, Baddeley and Hitch, 1974). In gen­ eral, working memory is distinguished from refer­ ence memory, which is necessary to remember information that remains constant over time (task-specific information, for discussion, see Olton et al., 1979). Rodent studies evaluating aging agree with human studies that spatial navi­ gation decreases in normally aged individuals, for both spatial working and reference memory, and that both elderly humans and aged rodents show individual variability in age-related cognitive change (Kausler, 1994). Many researchers study­ ing aging effects on learning and memory divide aged rodent subjects into impaired and nonimpaired groups to optimize differences in various physiological measurements (Armstrong et al., 1993; Croll et al., 1998; Diana et al., 1995; Fischer et al., 1991; Hasenohrl et al., 1997; Lindner et al., 1996; Nilsson and Gage, 1993; Quirion et al., 1995; Rapp and Gallagher, 1996; Sugaya et al., 1998). While the operational definitions for these classi­ fications are not homogeneous amongst studies, as

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some classify age impairment as those scores that are beyond the mean of the young group by one standard deviation (Croll et al., 1999, 1998), two standard deviations (Armstrong et al., 1993; Fischer et al., 1991; Nilsson and Gage, 1993; Quirion et al., 1995) or outside the range of the young (Rapp and Gallagher, 1996; Sugaya et al., 1998), they all concur that individual expression of age-related memory change exhibits a large range of variability. How that age-related variability in cognition comes about is a topic of intense inquiry. The progression of age-related decline in hip­ pocampal-dependent spatial function is embedded within the context of an individual’s life experi­ ences and exposures. These experiences and expo­ sures span a range of an infinite number of individual and interactive factors, including, but not limited to, genetic predisposition, early orga­ nizational effects of stress and gonadal hormones, enriching experiences, dietary factors (e.g. choline exposure), stress history and activational actions of stress and gonadal hormones. For the purpose of this review, we will focus on the life experiences of the cumulative exposure to stress and gonadal hormones in the context of activational effects in the adult. The underlying theme and main tenet driving discussion are the links amongst aging, gonadal hormones and cognition, with the hypoth­ esis that cumulative stress impacts the trajectory of age-related cognitive changes, with a poten­ tially interactive effect with gonadal hormones.

Aging influences on hippocampal plasticity Age increases risk for Alzheimer's disease and depression A misconception regarding the natural progres­ sion of cognitive function across the lifespan is that cognitive decline is synonymous with neuro­ nal loss. This perception is partially driven by epidemiological data showing that neuron loss occurs in many neurodegenerative disorders, especially those with dementia, with age increas­ ing risk. Alzheimer’s disease (AD) is the cause of about 70% of dementia cases, and studies report an alarming 5–10% prevalence in persons over 65

and 47–50% prevalence for those over 85 (Drachman, 2006). AD is a devastating disease character­ ized by cognitive decline, with initial deficits most pronounced in working memory and visuospatial functioning (Tulving and Craik, 2000). Function­ ing progressively worsens so that all effective cog­ nitive functioning is lost by the latter stage. Unfortunately, AD has no cure and there are scarce drug treatment options. However, aging and AD are not synonymous. While most studies concur that brains unaffected by neurodegenera­ tive disease will show some age-related memory loss, defining the boundaries between normal agerelated changes and neuropathologies has proven to be difficult. To date, age is recognized as the strongest risk factor for developing sporadic AD (Drachman, 2006; Yankner et al., 2008), but AD is not simply ‘accelerated’ aging (Drachman, 2006; Terry, 2006; Yankner et al., 2008). One recent hypothesis regarding the transition to sporadic AD suggests an accumulation of multiple and varied agerelated changes that burden the brain in a summative as well as interactive fashion (Drachman, 2006). Therefore, understanding and deciphering factors affecting the trajectory of age-related changes seen in normal aging may eventually lead to further understanding of neurodegenerative processes such as those seen in AD. Older adults are also at risk for cognitive decline associated with major affective disorder, or commonly discussed as depression. According to the American Psychiatric Association’s Diag­ nostic and Statistical Manual of Mental Disorders, 4th Edition Text Revision (DSM-IV-TR), depres­ sion is characterized by changes in mood, appetite, weight, and increased anhedonia, as well as impaired cognitive function and disrupted sleep, motivation and hypothalamic–pituitary–adrenal (HPA) axis activity. Following teenagers, adults over the age of 65 have the highest rate of suicide, identifying this as a significant public health issue and a major cause of death worldwide (Carballo et al., 2008; WHO, 2008). Moreover, the risk for depression substantially increases in women and those with certain genetic predispositions (i.e. polymorphisms for the gene that transports sero­ tonin) (Altemus, 2006; Caspi et al., 2003; Kessler et al., 1994), suggesting important gonadal/sex

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hormone influences for depression. Finally, potent environmental risk factors include lifetime expo­ sure to stress (Caspi et al., 2003; Levinson, 2006; Nestler et al., 2002), especially when initiated dur­ ing childhood (Andersen and Teicher, 2008). Therefore, genetics factors, including sex, confer a risk to developing depression, but environmen­ tal factors, such as stress, also play an important role. The HPA axis may contribute to AD onset, pro­ gression and accelerated aging (Phillips et al., 2006). Studies investigating AD-related disorders (i.e. dementia of the Alzheimer type) or depression show evidence for dysregulated HPA axis activity or elevated glucocorticoids, steroids released by the adrenal glands in response to stressors (Csernansky et al., 2006; Davis et al., 1986; Gomez et al., 2006; Magri et al., 2006; Rasmuson et al., 2002; Rubinow et al., 1984; Swaab et al., 2005; Umegaki et al., 2000; Weiner et al., 1997). In some cases, greater symp­ tom severity correlates with more HPA axis dysre­ gulation (Dong and Csernansky, 2009; Miller et al., 1998) and decreased hippocampal volume (de Leon et al., 1988; Keller et al., 2006; Magri et al., 2006; O’Brien et al., 1996). Nonetheless, there are some longitudinal data that fail to support the interpreta­ tion that HPA axis dysregulation corresponds with severity of AD symptoms (Swanwick et al., 1998) or depression (Adler and Jajcevic, 2001; O’Brien et al., 2004). It is intriguing that one report indicated that higher HPA activity predicts rapid disease progres­ sion as opposed to the disease state per se (Cser­ nansky et al., 2006). Moreover, evidence that disease etiology differs in young and aged subjects is revealed by findings that older subjects are inclined to show hyper- or hypo-HPA axis activity with depressive symptoms (Bremmer et al., 2007; Chida and Hamer, 2008). These seemingly mixed outcomes may be a consequence of older indivi­ duals having increased risk(s) for other conditions (i.e. cardiovascular disease, cancer, to name a few, Evans et al., 2005; Everson-Rose et al., 2004; Gump et al., 2005; Penninx et al., 1998; Wassertheil-Smol­ ler et al., 2004), which could further moderate HPA axis activity with AD or depressive symptoms. As previously noted, genetic and environmental factors increase the risk for developing AD or depression (Caspi et al., 2003; de Geus et al., 2007), with a

history of chronic stress being particularly impor­ tant (Hammen et al., 2009; Paykel, 2003; Weber et al., 2008). Therefore, young and aged individuals may both carry genetic predispositions, but the aged population has lifetime experiences that can further moderate risk for developing AD and depression, with a history of chronic stress and HPA axis activity playing a prominent role.

Why investigate hippocampal structural changes beyond neuronal loss? The mammalian brain shows considerable plasti­ city, especially in regions related to learning and memory such as the hippocampus and neocortex. Indeed, the hippocampus contains one of the rare sites for neurogenesis that persists long after early development (Eriksson et al., 1998; Kaplan and Hinds, 1977; Kornack and Rakic, 1999). However, the hippocampus is also susceptible to neurode­ generation. The pathological hallmarks of AD include presence of neurofibrillary tangles and amyloid plaques, which occur to a lesser degree in the normal aging brain and are particularly pervasive within the hippocampus and prefrontal cortex (PFC) (Roth et al., 1966; Yankner et al., 2008). Importantly, the hippocampus and neocor­ tex of the AD brain show extensive neuronal loss (Gomez-Isla et al., 1996; Price et al., 2001; West et al., 1994), whereas accounts of neuronal loss with normal aging are mixed. Early research reported age-related loss of neurons in the hippo­ campus (Landfield et al., 1981; West et al., 1994). A recently published study quantifying magnetic resonance imaging (MRI) scans from over 2200 healthy persons showed the largest age-related changes in brain volume after age 50, with the frontal and temporal lobes showing the largest decreases (~12% and ~9%, respectively), with modest age-related alterations elsewhere (DeCarli et al., 2005; Raz and Rodrigue, 2006), which corroborates other reports (Miyahira et al., 2004); however, brain volume changes do not necessarily indicate neuronal loss. Studies report­ ing neuronal counts suggest decreases in neuronal number with normal aging in the hippocampus and the subiculum (Simic et al., 1997; West,

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1993), and that PFC neuron death is related to working memory decline in aged monkeys (Smith et al., 2004). However, other reports sug­ gest that the majority of neuron loss during nor­ mal aging occurs in the neocortex, and not the hippocampus (Drachman, 2006), or that neither cortex nor hippocampus express neuronal loss with normal aging (Peters et al., 1998; Terry, 2006), even when age-related memory deficits are found (Gallagher et al., 2003). Most studies do agree, however, that while brains of normally aging humans may not show neuron loss, AD brains consistently do (Hof and Morrison, 2004). To summarize, the literature consistently shows age-related changes in function, such as spatial memory, but the effects on brain structure and micro-structure during aging are equivocal. Age-related cognitive decline may involve neo­ plastic changes within the brain, such as altera­ tions in dendritic structure, spine number and spine shape. Such changes would be consistent with studies showing that aging is not necessarily synonymous with neuronal loss. Stereological counting methods that allow for unbiased quanti­ fication of cell numbers show that hippocampal neuron numbers can remain relatively constant with aging, even when aged subjects are further categorized into cognitively impaired and cogni­ tively unimpaired based on performance assess­ ments from a spatial navigation task (Rapp and Gallagher, 1996). Indeed, the majority of studies evaluating synaptic alterations (e.g. synaptic den­ sity as revealed via synaptophysin evaluations) in healthy individuals show an age-related decline, resulting in a potentially interesting dissociation between synapse loss and neuron loss, if the latter in fact does not occur (Hof and Morrison, 2004; Terry, 2006). Evidence from updated stereology techniques show that hippocampal synapse num­ ber decreases with the progression from young adulthood to normal aging, and that synapse num­ ber in AD hippocampi is fewer than in those of the normally aged (Bertoni-Freddari et al., 2003). Moreover, factors influencing synaptic transmis­ sion, including the reductions mentioned above in synaptic density, as well as in dendritic spines and number and length of dendrites, have been noted (Esiri, 2001). These findings indicate that

many age-related alterations may involve neoplas­ tic changes that are flexible and dynamic. Studies investigating changes in brain structure of patients suffering from depression suggest that dynamic and reversible alterations may be impor­ tant. Individuals with depression show decreased volumes in the hippocampus (Bremner et al., 2000; Feldmann et al., 2007; Janssen et al., 2004; MacMaster et al., 2008; Neumeister et al., 2005; Saylam et al., 2006; Sheline et al., 2003; 1999; Sheline et al., 1996) and PFC (Bremner, 2002; Coryell et al., 2005; Rajkowska et al., 1999). While neuron death received early attention (Sapolsky, 2000), permanent neuronal loss was inconsistent with the observation that post-mor­ tem hippocampal tissue from patients diagnosed with depression showed reductions in neuropil without neuron loss (Stockmeier et al., 2004). Although cell loss was found in some tissue of patients with depression (Rajkowska, 2000), a likely interpretation is that cell loss represents a small proportion of the etiology underling depres­ sion. Moreover, signs that volumetric changes can be dynamic are revealed from studies showing that antidepressant treatment increases volumes of the hippocampus and PFC (Neumeister et al., 2005; Sheline et al., 2003; Vermetten et al., 2003), and hippocampal volumes increase in depressed patients who are in remission (Frodl et al., 2008). Indeed, patients on long-term antidepressant treatment may be protected against hippocampal volume decreases from cumulative depressive epi­ sodes (Sheline et al., 2003). Consequently, some structural changes occurring in depression most likely include reversible and/or dynamic processes without necessarily involving neuronal loss.

Stress via the HPA axis and gonadal steroid influences on the hippocampus Dendritic retraction and changes in synapse num­ ber/shape may provide a putative mechanism through which hippocampal-dependent cognitive decline occurs with normal aging. Dendritic retrac­ tion involves the pruning of dendrites within the hippocampus, a phenomenon that essentially ren­ ders the hippocampus less sensitive to converging

36

afferents, which in turn could modulate cognition. Importantly, hippocampal dendritic retraction does not represent neuronal loss per se, but a reduction in the neuropil that contains primarily dendrites and a small proportion of glial processes (Tata and Anderson, 2009), perhaps via decreases in cytoskeletal proteins (Cereseto et al., 2006). Chronic stress acting through elevated glucocorti­ coids reliably produces hippocampal synapse loss (Tata et al., 2006) and dendritic retraction (Conrad et al., 1999b, 2007; Fuchs et al., 2001; Kole et al., 2004; Magariños and McEwen, 1995a, 1995b; Magariños et al., 1996; McKittrick et al., 2000; Watanabe et al., 1992b), which can be detected by functional magnetic resonance imaging (fMRI) without necessarily producing noticeable changes in forebrain volume and body weight (Lee et al., 2009). Dendritic retraction is most pronounced within the CA3 region of the hippocampus (Con­ rad, 2006), perhaps reflecting this region’s high sensitivity to stress or glucocorticoids. It is noted that other hippocampal regions can express dendri­ tic retraction when chronic stress or glucocorticoid elevations are severe or persistent (Lambert et al., 1998; Sousa et al., 2000). Importantly, stress-pro­ duced hippocampal dendritic retraction is highly dynamic, demonstrating complete reversal to nonstress control levels following the termination of the stressor and with sufficient time for recovery (Conrad et al., 1999b; Sousa et al., 2000; Vyas et al., 2004). Moreover, antidepressant treatment can prevent stress-induced hippocampal dendritic retraction (Luo and Tan, 2001; Magariños et al., 1999; Watanabe et al., 1992a) and volumetric reductions (Czéh et al., 2001), even when neuro­ genesis is blocked (Bessa et al., 2009). Therefore, stress- or glucocorticoid-induced hippocampal den­ dritic retraction provides a model to study neoplas­ tic changes that are consistent with the types of structural brain dynamics observed in normal aging and depression. Gonadal hormones, and especially estrogens, have been demonstrated to have significant effects on hippocampal morphology. Estrogens were first shown to influence brain regions historically known for roles in reproduction, such as the hypothala­ mus, in rodents (McEwen, 1981; Parsons et al., 1982; Pfaff and McEwen, 1983) and then also

found to alter regions involved with cognition (Foy, 2001; Terasawa and Timiras, 1968). Subse­ quent studies extended these findings to show that estrogens altered neuron morphology by increasing hippocampal CA1 spine density in rats (Gould et al., 1990; Silva et al., 2000; Woolley and McEwen, 1992, 1993). Dendritic spines have long been theorized to be a structural component of memory (Geinisman, 2000; Kasai et al., 2003; Moser, 1999; Nimchinsky et al., 2002; Sorra and Harris, 2000), leading to wide-reaching implications regarding estrogen’s influence on cognitive func­ tion. This tenet is supported by recent work show­ ing that spatial working and reference memory performance is enhanced after 17b-estradiol injec­ tions (McLaughlin et al., 2008; Sandstrom and Williams, 2001, 2004) within the timeframes corre­ sponding to 17b-estradiol-induced increases in hip­ pocampal dendritic spines (Gould et al., 1990; Silva et al., 2000; Woolley and McEwen, 1992, 1993) and other markers of synaptic plasticity (Foy, 2001). Stress and glucocorticoids can also modify spine density and shape within the hippocampus (Diamond et al., 2006; Fuchs et al., 2006; Komatsu­ zaki et al., 2005; McLaughlin et al., 2009; 2005; Shors et al., 2001; Sunanda et al., 1995), with 17b-estradiol, and even cholesterol, the precursor to estrogens, protecting against stress-induced CA3 dendritic retraction in females (McLaughlin et al., 2010). Consequently, morphological alterations in neuronal dendritic structure and synapses may con­ tribute to and/or underlie functional outcomes on hippocampal-dependent cognition.

Influence of the HPA axis on hippocampaldependent learning and memory: inverted U-shaped function Spatial learning and memory How glucocorticoids are thought to influence spa­ tial learning is consistent with the original report by Yerkes and Dodson (1908), stating that perfor­ mance declines in challenging tasks when arousal falls along the extremes of being too low or high (Fig. 1A). However, the manner by which gluco­ corticoids alter spatial memory are complex, and

37 A.

Yerkes–Dodson Law (Hebbian version) Optimal arousal Optimal performance

Strong

Impaired performance because of strong anxiety

Performance

Increasing attention and interest

Weak Low

B.

High

Arousal Yerkes–Dodson Law (Original version)

Strong

Simple task Focused attention, flashbulb memory fear conditioning Difficult task Impairment of: Divided attention, working memory, Decision-making and multi-tasking

Performance

Weak Low

Arousal

High

Fig. 1. Representation of cognitive performance based on: (A) Hebbian version of the Yerkes–Dodson law and (B) the actual findings from Yerkes and Dodson (1908). (A) Cognitive performance is curvilinear with arousal with optimal performance occurring when arousal is not too low or high. (B) Under very simple learning conditions, performance is linear with arousal until a plateau is reached, while complex learning conditions show curvilinear influences with arousal as shown in (A). (From Diamond et al. (2007) with permission.)

involve ascertaining when glucocorticoids are ele­ vated (or absent) relative to the learning processes of acquisition, consolidation and retrieval, and whether the learning event is intrinsically aversive (for review, see Conrad, 2005; Diamond et al., 2007; Sandi and Pinelo-Nava, 2007). For the pur­ pose of this review, the focus will be on whether glucocorticoids are elevated upon the start of the acquisition phase in spatial tasks because gluco­ corticoid elevations often precede learning events in many of the clinical populations discussed in aging, as well as AD and depression. With regard to the type of task, spatial tasks are intrinsically more difficult to perform than simple passive

avoidance or fear conditioning. Spatial tasks require the integration of spatial relationships and demonstrate a non-linear learning function with arousal. In contrast, passive avoidance and fear conditioning motivate subjects by incorporat­ ing aversive stimuli, such as footshock, and exhibit a linear learning function with arousal (Fig. 1B). This review will focus on spatial learning in water or land-navigation tasks that require the integra­ tion of multiple cues to navigate using allocentric strategies for optimal performance (Paul et al., 2009). In healthy young adults, many studies show that the HPA axis has a non-linear relationship with

38

hippocampal-dependent spatial learning, with very low or high corticosterone levels impairing spatial learning and moderate corticosterone levels being optimal. In rodents, the removal of glucocorticoids via adrenalectomy (ADX) impairs spatial learning on the water maze (Oitzl and de Kloet, 1992; Roozendaal et al., 1996) and spatial recognition on the Y-maze (Conrad et al., 1999a; 1997). At the other end of the spectrum, subjects administered with stress levels of glucocorticoids or glucocorticoid receptor agonists exhibit impaired spatial learning on the Y-maze (Conrad et al., 1999a, 1997). In young rats, basal levels of glucocorticoids during the morning positively cor­ relate with spatial learning (Yau et al., 1995). Basal glucocorticoid levels averaged about 2 mg/dl, which represent non-stress levels, reveal­ ing that when glucocorticoids increase within this range, spatial memory improves. Exogenously manipulating glucocorticoid levels in juvenile ground squirrels also show that very high or low glucocorticoids impairs spatial memory, while moderate glucocorticoid levels provide the best spatial performance (Mateo, 2008). The context by which stress or glucocorticoids influence spatial learning and memory is also critical (Yang et al., 2003), as elegantly demonstrated in a study that compared the exposure of rats to a context with a predator and comparing the outcome to context containing a sexually receptive conspecific female (Woodson et al., 2003). In both situations, gluco­ corticoid levels were similarly elevated; however, only the predator exposure effectively and signifi­ cantly impaired spatial memory and showed a sig­ nificant positive correlation between corticosterone levels with spatial memory deficit (Woodson et al., 2003). Moderate corticosterone replacement in animals with their adrenals removed to eliminate the source of glucocorticoids improves spatial memory deficits caused by ADX (Conrad and Roy, 1993, 1995). Work investigating long-term potentiation (LTP) or primed burst potentiation (PBP), processes that are believed to underlie forms of learning and memory (Bliss and Collingridge, 1993; Heynen et al., 1996), cor­ roborate the curvilinear effects of stress and glu­ cocorticoids on hippocampal plasticity (Avital et al., 2006; Diamond et al., 1992; 2005; Joéls,

2006; Kerr et al., 1994). Taken together, these studies demonstrate that elevated glucocorticoids are necessary, but not sufficient to modulate spa­ tial learning and memory.

Influence of chronic stress and glucocorticoids on spatial learning and memory in the young adult The majority of studies show that chronic stress impairs spatial learning and memory in young adults under a variety of manipulations, proce­ dures, durations and assessment. Spatial learning and/or memory using the land radial-arm maze, the land Y-maze or water mazes is worsened fol­ lowing systematic, chronic exposure to the same (homotypic) immobilization stressor over many days to months (Conrad et al., 1996; Kitraki et al., 2004; Luine et al., 1994; Moosavi et al., 2007; Radecki et al., 2005; Srikumar et al., 2006; Sunanda et al., 2000; Venero et al., 2002; Wright and Conrad, 2005), social disruption (Aleisa et al., 2006; Alzoubi et al., 2009; Gerges et al., 2004; Krugers et al., 1997, Bodnoff et al., 1995; Srivar­ eerat et al., 2009), intruder paradigms (Ohl and Fuchs, 1999; Ohl et al., 2000; Touyarot et al., 2004), loud noise (Manikandan et al., 2006) or cold water immersion (Nishimura et al., 1999). Chronic unpredictable stressors, or other forms of heterotypic stressors, also reveal detri­ mental outcomes on spatial learning and memory when using the land radial-arm maze (Park et al., 2001; Zoladz et al., 2008), the land Y-maze (Orsetti et al., 2007) and the Morris water maze (Cerqueira et al., 2007; Song et al., 2006; Sousa et al., 2000) The aforementioned studies suggest that a vari­ ety of chronic stress durations can lead to similar detrimental outcomes on spatial ability in young adults, but there are some important considera­ tions. Both the elevation of glucocorticoids and a compromised hippocampus are hypothesized to underlie the chronic stress-induced spatial learn­ ing and memory deficits. Nearly all of the chronic stress paradigms have demonstrated that they can successfully elevate glucocorticoids. However, hippocampal dendritic retraction requires time to develop with chronic stress. Although the CA3

39

region of the hippocampus is most prone to exhi­ biting atrophied dendrites, it has a relatively slow time course of development: In response to wire mesh restraint, CA3 dendritic retraction occurs following 6 h/day of restraint for 21 days, but not with shorter durations of 2 h/day for 10 or 21 days, nor with wire mesh restraint for 6 h/day for 10 days (Luine et al., 1996; McLaughlin et al., 2007). Importantly, chronic stress conditions that impair spatial memory also produce CA3 dendritic retraction within the same subjects (Fig. 2). In the other chronic stress paradigms, hippocampal dendritic retraction may show a different develop­ mental time course, depending upon the severity of the stressor. For example, CA3 dendritic retrac­ tion can be found in as little as 6 days following activity stress combined with food restriction (Lambert et al., 1998), 10 days after complete immobilization for 2 h/day (Vyas et al., 2002) and 14 days following continual competition for resources (Vyas et al., 2002). Therefore, chronic stress alters hippocampal dendritic structure slowly and different types of stressors can have their own time course of development for impos­ ing structural alterations on hippocampal dendrites.

Role of CA3 dendritic retraction on the hippocampus As found with glucocorticoid elevations, the pre­ sence of CA3 dendritic retraction reveals suscept­ ibility to impaired spatial memory, but is insufficient alone. When early work reported that chronic stress or glucocorticoids potentially harmed the hippocampus (Sapolsky, 1992; Sapolsky and Pulsinelli, 1985), subsequent research followed to investigate the functional out­ comes on spatial ability, but with mixed results. Some studies found 3 weeks to nearly 3 months of chronic glucocorticoid treatment impaired spa­ tial ability in young adults (Bardgett et al., 1994; Dachir et al., 1993; Endo et al., 1996; Luine et al., 1993; McLay et al., 1998), whereas others found no effect (Bodnoff et al., 1995; Clark et al., 1995). The duration of glucocorticoid exposure may be impor­ tant because 3–4 weeks of glucocorticoid

treatment did not alter spatial ability unless gluco­ corticoid treatment continued for another month (Coburn-Litvak et al., 2003). One interpretation is that corticosterone-produced structural alterations within the hippocampus were not evident in the shorter (3–4 weeks) durations of glucocorticoid exposure. However, a study found that chronic glucocorticoid treatment failed to impair spatial memory despite confirmation from the same rats that CA3 dendritic retraction occurred (Conrad et al., 2007). Therefore, the presence of CA3 den­ dritic retraction does not necessarily indicate impaired hippocampal function as measured by spatial navigation. Given that CA3 dendritic retraction can exist without necessarily showing negative conse­ quences on spatial ability, then what role, if any, does CA3 dendritic retraction have in hippocam­ pal function? We hypothesize that the presence of CA3 dendritic retraction indicates susceptibility to compromised function, as well as to metabolic and neurotoxic challenges. Evidence that CA3 dendri­ tic retraction compromises hippocampal function comes from work investigating strategy use and from studies manipulating glucocorticoid levels during behavioral assessment. Several reports show that chronic stress shifts the strategy used during learning. In one study, chronically stressed rats exhibited impaired spatial recognition memory, a hippocampal-dependent task, but the same rats navigated quite well under conditions that were independent of the hippocampus (Wright and Conrad, 2005). In a direct test of which strategy was favoured, chroni­ cally stressed mice implemented a hippocampalindependent stimulus–response strategy significantly more than did the non-stressed controls, which favoured a hippocampal-dependent spatial strategy 100% of the time (Schwabe et al., 2008). Moreover, humans who self-reported a history of chronic stress also favoured stimulus–response strategies more than did subjects reporting a low chronic stress history (Schwabe et al., 2008). These findings were corroborated by another study showing that chronic stress has a tendency to impair learning on a spatial T-maze, but not on a response version (Sadowski et al., 2009). Current thinking is that chronic stress biases behavioral

40 Control

2 h–10 days

2 h–21 days

Basal

Apical

A.

6 h–10 days

6 h–21 days

B. Novel Other

Arm entries in first minute

2.0

1.5

1.0

0.5

0.0

Control

2 h–10 days

2 h–21 days

6 h–10 days

6 h–21 days

Fig. 2. Effects of different durations of chronic restraint stress on hippocampal CA3 dendritic retraction and hippocampaldependent spatial recognition memory on the Y-maze. (A) Chronic stress produced dendritic retraction in the apical CA3 region following 6 h/day for 21 days of restraint, but failed to produced CA3 apical dendritic retraction at a shorter restraint duration (2 h/day for 10 or 21 days) or for the same duration each day, but fewer days (6 h/day/10 days). (B) The status of the CA3 apical dendritic arbors corresponded to the performance on the hippocampal-dependent Y-maze task: chronic restraint for 6 h/day for 21 days impaired spatial recognition of the novel arm compared to the other arm (6 h–21 days), but spatial recognition memory was intact in the other conditions as rats entered the novel arm significantly more than the other arm.
p < 0.05 for novel vs. other for control, 2 h–10 days, 2 h–21 days and 6 h–10 days. (Reprinted from McLaughlin et al. (2007), with permission from Elsevier.)

strategies towards habit (Dias-Ferreira et al., 2009), or processes that rely heavily upon the striatum rather than the hippocampus.

Therefore, chronic stress compromises spatial ability through influences that may include hippocampal CA3 dendritic retraction, but

41

facilitates neural networks that underlie response/habit that likely involve striatal net­ works (for review, see Conrad, 2010). Manipulations of glucocorticoid levels during spatial navigation has demonstrated that chroni­ cally stressed subjects with a compromised hip­ pocampus are capable of spatial learning and memory. Rats that were chronically stressed by restraint for 6 h/day for 21 days, a procedure and timeframe that reliably produces hippocam­ pal CA3 dendritic retraction (Conrad, 2006), showed spatial memory deficits as expected (Wright et al., 2006). However, chronically stressed rats that were injected once on the day of spatial assessment with metyrapone, to attenuate stress levels of glucocorticoids, showed functional spatial abilities despite condi­ tions that produced hippocampal CA3 dendritic retraction (Fig. 3, Wright et al., 2006). Another report found that rats with chemical lesions tar­ geting the CA3 region also showed spatial def­ icits that were prevented with a single injection of metyrapone on the training day and deficits, and then were reinstated with a single corticos­ terone injection (Roozendaal et al., 2001). These studies reveal that manipulations that compromise the CA3 region of the hippocam­ pus can negatively impact spatial ability, but a compromised CA3 region alone is not sufficient because spatial ability can be modified further by the presence or absence of glucocorticoids during spatial learning and/or recall. In addition to compromising spatial ability, hip­ pocampal CA3 dendritic retraction is thought to enhance susceptibility to metabolic challenges. Nearly 25 years ago, glucocorticoid hypersecre­ tion was hypothesized to endanger the hippocam­ pus to contribute to age-related cognitive decline (Sapolsky, 1992; Sapolsky et al., 1986). Key con­ cepts were that glucocorticoid elevations exacer­ bated damage to the hippocampus caused by neurotoxicity or ischemic challenges as compared to the effects with glucocorticoid elevations or the challenges when presented alone (Sapolsky, 1985b; Sapolsky and Pulsinelli, 1985). Repeated exposure to glucocorticoids was proposed to down-regulate hippocampal glucocorticoid recep­ tors, which would hinder HPA axis regulation and

potentiate glucocorticoid elevations with age, and hence, the potential for hippocampal damage (Sapolsky et al., 1983, 1984a, 1984b). Enthusiasm for this hypothesis began to wane when prolonged glucocorticoid elevations failed to consistently produce hippocampal cell loss in adults (Bodnoff et al., 1995; Coburn-Litvak et al., 2004; Leverenz et al., 1999; Müller et al., 2001; Sousa et al., 1998) and evidence suggested that non-human primates expressed insufficient numbers of glucocorticoid receptors to effectively mediate ‘glucocorticoid toxicity’ (Sánchez et al., 2000). Recent findings, however, suggest that hippocampal CA3 neurons can be harmed without concurrent glucocorticoid elevations during the neurotoxic challenge, pro­ vided that stress- or glucocorticoid-induced CA3 dendritic retraction was present during the chal­ lenge (Conrad et al., 2004, 2007). Consequently, hippocampal neurons expressing dendritic retrac­ tion are susceptible to damage beyond the window of when glucocorticoids are elevated. Since stress­ or glucocorticoid-induced CA3 dendritic retrac­ tion is reversible (Conrad et al., 1999b; Sousa et al., 2000; Vyas et al., 2004), then CA3 neurons can recover to their pre-stress/glucocorticoid con­ dition when a metabolic or neurotoxic incident fails to occur. Therefore, CA3 dendritic retrac­ tion conveys susceptibility to neuronal loss, but is not damage per se, which contributed to a revised hypothesis, called the ‘Glucocorticoid Vulnerability’ hypothesis (Conrad, 2008), and provides insight into differences in susceptibility to age-related cognitive decline.

Model for chronic stress and CA3 dendritic retraction in spatial ability Under chronic stress conditions that produce CA3 dendritic retraction, we hypothesize that the hip­ pocampus will be susceptible to spatial memory deficits following chronic glucocorticoid eleva­ tions at levels normally not disruptive to spatial memory in non-stressed controls. Chronically stressed subjects respond to a novel spatial mem­ ory testing paradigm with slightly potentiated glucocorticoid levels. Under these conditions that are combined with a compromised hippocampus

42 A. Percentage of arm entries in first minute

80 Novel Other 60

40

20

0

Total serum corticoterone (µg/100 ml)

B.

CV

CM35

CM75

SV

SM35

35

SM75 Train Test

30 25 20 15 10 5 0

CV

CM35

CM75

SV

SM35

SM75

Fig. 3. Effects of a single metyrapone injection on the day of spatial recognition memory assessment in chronically stressed rats. (A) Rats that were chronically stressed by restraint (6 h/day/21 days) and then tested on the Y-maze showed impaired spatial recognition memory (SV), but those chronically stressed rats given a single injection of metyrapone (75 mg/kg) to attenuate stress-induced release of glucocorticoids prior to the Y-maze showed functional spatial recognition memory (SM75). Spatial recognition memory of controls (CV, CM35, CM75) was unaltered by metyrapone at either dose (
p < 0.05 for novel arm entries vs. other arm entries). (B) Total serum corticosterone levels were measured after the first training trial (Train) and the second testing trial (Test) in a separate cohort of rats. While the corticosterone levels of the chronically stressed rats were higher than those of controls (‡ = significant main effect of stress), metyrapone injections dose-dependently lowered corticosterone in both controls and chronically stressed rats (
p < 0.05 for CM75 and SM75 compared to the remaining groups given vehicle or 35 mg/kg). Moreover, the corticosterone levels of the chronically stressed rats injected with vehicle or 35 mg/kg of metyrapone (SV, SM35) were statistically similar to the controls injected with vehicle or 35 mg/kg of metyrapone (CV, CM35). Therefore, these data support the interpretation that both corticosterone levels and brain sensitivity to corticosterone contribute to outcomes on spatial recognition memory. (From Wright et al. (2006), with permission from Wiley-Blackwell Publishing).

expressing CA3 dendritic retraction, chronically subjects will express spatial learning and memory impairments under conditions that would

normally facilitate optimal spatial abilities in non-stressed controls (Fig. 4). This hypothesis assumes that the spatial navigation paradigm is

43 A. Effect of acute stress on spatial ability in normal controls Acute stress

Hippocampus

Spatial ability (solid line in graph ‘C’)

HPA axis B. Effect of acute stress on spatial ability after chronic stress Hippocampus w/ CA3 dendritic retraction

Acute stress

Spatial ability (dotted line in graph ‘C’)

HPA axis C. Non-linear function for glucocorticoids and spatial ability

Spatial ability

Optimal

Poor ADX Basal Moderate

High

Glucocorticoid levels Fig. 4. Model for chronic stress actions via elevated glucocorticoids influencing spatial ability. (A) Without a chronic stress history, an acute stressor triggers the HPA axis and elevates glucocorticoids, which could feedback to influence the hippocampus and its functions, such as spatial ability. The hippocampus also provides negative feedback on the HPA axis. The up and down arrows illustrate the reciprocal relationship between the hippocampus and the HPA axis. Under these conditions, glucocorticoids influence spatial ability by the inverted U-shaped function, and illustrated by the solid curve in graph C. (B) Following chronic stress, the hippocampus is compromised and exhibits CA dendritic retraction, along with other neurochemical and neuromorphological changes. Consequently, the hippocampus has poor regulation of the HPA axis, indicated by the dashed arrow, whereas the HPA axis and adrenals are over-active, indicated by the two large upward pointing arrows, creating an imbalance. Under these chronic stress conditions, a change in the sensitivity of the hippocampus to novel stress and glucocorticoids is hypothesized to narrow the inverted U-shaped function, as illustrated by the dotted curve in graph C. (C) Consequently, glucocorticoid elevations that optimize spatial ability in non-stressed animals will impair spatial ability in those following chronic stress: compare the curves under the line thin downward arrow. The thick arrowhead illustrates that at low glucocorticoid levels, functional spatial ability is possible following chronic stress.

not overtly arousing or aversive, as would be found in fear conditioning or water maze paradigms, conditions that activate the amygdalar regions and can mask hippocampal function (Conrad, 2006; McLaughlin et al., 2009), perhaps by shifting to well-learned behaviors (Dias-Ferreira et al., 2009). However, when subjects are navigating in a spatial environment under non-threatening conditions, then spatial abilities are hypothesized to differ between controls and

chronically stressed subjects based upon HPA activity and a compromised hippocampus. There are several lines of evidence supporting the interpretation that both the HPA axis and hippocampus contribute to impaired spatial navi­ gation in chronically stressed subjects. Even when rats are tested on a relatively benign spatial navi­ gation task that taps into rats’ innate tendency to explore novelty, chronically stressed rats release higher levels of glucocorticoids than the controls

44

(Fig. 3B). Prior work showing that chronically stressed rats release more glucocorticoids in response to a novel stressor compared to a famil­ iar stressor supports these findings (Bhatnagar et al., 2002; Dallman, 2007; Dallman et al., 2000; Herman et al., 2005). For spatial assessment on the Y-maze, the repeated, same stressor (homo­ typic) is the daily restraint and the novel stressor (heterotypic) is the Y-maze. Consequently, chronically stressed rats release higher levels of glucocorticoids in the Y-maze compared to con­ trols, even though the Y-maze is a relatively benign task that does not require water or food deprivation, or water escape. However, glucocor­ ticoid levels alone do not directly correspond with spatial performance, as tested on the Ymaze. Indeed, we found that circulating glucocor­ ticoid levels released during Y-maze exploration in the chronically stressed rats injected with the moderate dose of metyrapone (35 mg/kg), were statistically similar to the glucocorticoid levels in the controls injected with either vehicle or 35 mg/ kg of metyrapone. In spite of comparable circu­ lating glucocorticoid levels, the controls showed optimal spatial ability whereas the chronically stressed rats injected with the moderate metyra­ pone dose did not (Wright et al., 2006). More­ over, biologically active levels of circulating glucocorticoids were similar among controls and chronically stressed rats (Wright et al., 2006). That circulating glucocorticoid levels did not dif­ fer among groups, while spatial maze perfor­ mance did, supports the hypothesis that changes in brain sensitivity to glucocorticoids contribute to the spatial ability outcomes in chronically stressed rats. How the hippocampus and the HPA axis contri­ bute to spatial ability following chronic stress is illustrated in Fig. 4B. The compromised hippocam­ pus expressing CA3 dendritic retraction is unable to regulate the HPA axis effectively, as illustrated by the downward dotted arrow, and the hippocam­ pus is susceptible to glucocorticoid elevations (indicated by two solid upward arrows targeting the hippocampus). Consequently, the influence between the hippocampus and the HPA axis is no longer balanced, which we hypothesize is revealed by a change in sensitivity of the hippocampus to

novel stress and glucocorticoid elevations. The consequence of the enhanced hippocampal sensi­ tivity to glucocorticoids and glucocorticoid hyper­ secretion is that levels of glucocorticoids that optimize spatial ability in controls lead to impaired spatial ability in chronically stressed rats. Specifi­ cally, chronic stress is hypothesized to narrow the inverted U-shaped function for glucocorticoid effects on spatial ability, by shifting the descending right limb of the inverted U-shaped function to the left to represent an enhanced sensitivity to gluco­ corticoid levels (Fig. 4C). Consequently, moderate levels of glucocorticoids that do not impair spatial ability in controls are predicted to do so following chronic stress conditions that produce CA3 dendritic retraction.

The influence of stress and the HPA axis on age-related cognitive decline The aging population adds another dimension underlying cognitive function as cognitive deficits progressively worsen, with hippocampal-dependent functions being particularly vulnerable (Barnes’, 1979; Fischer et al., 1992; Gallagher and Pelleymounter, 1988b; Geinisman et al., 1995; Ingram, 1988; Luine et al., 1990; Moss et al., 1988; Winocur and Gagnon, 1998). Despite the increased risk for cognitive decline with age, many older indi­ viduals show cognitive performance similar to younger counterparts (Gage et al., 1984; Gallagher and Pelleymounter, 1988a; Issa et al., 1990; Mar­ kowska et al., 1989; Matzel et al., 2008). Indeed, cognitive decline represents the increased variability of the aged population’s performance (Matzel et al., 2008; Rapp and Amaral, 1992), and findings such as these contributed to the characterization that aging may represent a continuum of cognitive decline (Brayne and Calloway, 1988). Understanding why some individuals are able to successfully retain their cognitive abilities with age, whereas others do not, will be essential for facilitating healthy aging. Many factors can contribute to non-optimal aging, and HPA axis dysregulation may be an important component underlying unsuccessful aging and related cognitive declines. As described earlier, a critical risk factor for poor cognitive

45

function associated with aging is life-long expo­ sure to stressful events (McEwen, 1999; Pardon and Rattray, 2008). An individual exposed to a chronically stressful situation will likely suffer poor cognitive outcomes, but can potentially recover quickly when the stressful situation ends. Indeed, some chronic stress situations can opti­ mize cognitive ability later in life, as illustrated by mild stress during the postnatal period in rat (Meaney et al., 1988), while other postnatal manipulations accelerate cognitive decline (Brunson et al., 2005). Importantly, these stressful episodes do not occur in isolation, as they are overlaid upon the individual’s genetic predisposi­ tion and other mitigating factors. As examples, an individual’s cardiovascular status (Raz et al., 2005), caloric intake and/or nutrition (Baran et al., 2005; Joseph et al., 2009), immune function (Segerstrom and Miller, 2004) and aerobic activ­ ity/environmental enrichment (Cotman et al., 2007; Winocur, 1998; Wright and Conrad, 2008) can all impact how chronic stress could influence brain health (Conrad et al., 2009; Raz and Rodrigue, 2006). Moreover, aged individuals have an accumulation of a lifetime of experiences that could further alter the progression of brain aging. This concept has been described as ‘allostatic load’, and refers to the price the body pays for adapting to adverse situations (McEwen, 2000a, 2000b, 2000c; Stewart, 2006). Essentially, the body alters itself to compensate for the immediate threat, but with potentially debilitating long-term ramifications. To illustrate how chronic stress could influence brain health when additional risk factors are involved, the example of hypertension will be dis­ cussed. Undiagnosed hypertension increases the risk of structural alterations in the brain (den Hei­ jer et al., 2005; Raz et al., 2003a, 2003b; Raz et al., 2003c). If this hypertensive person is concurrently experiencing chronic stress, then the stressinduced morphological alterations within the hip­ pocampus could become exacerbated. A severe outcome may even involve cell loss as we recently found that chronic stress increases susceptibility to hippocampal damage (Conrad et al., 2004, 2007). This susceptibility corresponds to brain regions that show stress-induced dendritic retraction and

is not likely attributed to acute elevations of glu­ cocorticoids (Conrad et al., 2004, 2007). While acute stress or glucocorticoid elevations can exacerbate hippocampal damage caused by neu­ rotoxic or metabolic insults (Sapolsky, 1985a, 1985b; Sapolsky and Pulsinelli, 1985), as proposed in the ‘Glucocorticoid Cascade Hypothesis’ (Sapolsky et al., 1986), the current findings suggest that glucocorticoid elevations are not required to confer susceptibility to damage. Instead, suscept­ ibility is revealed by conditions surrounding dendritic retraction, which persists for many days or longer than the hours that glucocorticoids are elevated from a stress episode. Consequently, individuals with a history of stressful life events have broader window by which opportunistic challenges could shift the reversible morpho­ logical changes into permanent hippocampal damage and continued HPA dysfunction. The ‘Glucocorticoid Vulnerability Hypothesis’, recognizes the distinction between glucocorticoids facilitating dendritic retraction without needing to be elevated to create a susceptible brain to poten­ tial damage (Conrad, 2008). Aged individuals are particularly susceptible to alterations in brain health following chronic stress, based upon neuromorphological and cognitive evidence. As eloquently stated by Burke and Barnes (2006), neuronal loss does not have a sig­ nificant role in age-related cognitive decline, but rather, small region-specific changes in dendritic branching and spine density may be critical. The normal aging hippocampus appears to maintain or even increase dendritic complexity in CA1 (Hanks and Flood, 1991; Turner and Deupree, 1991), CA3 (Flood et al., 1987) and dentate gyrus (Flood, 1993), while dendritic complexity decreases within the PFC (de Brabander et al., 1998; Grill and Riddle, 2002; Markham and Juraska, 2002). More­ over, hippocampal spine density does not appear to change with age in the CA1 (Markham et al., 2005) or dentate gyrus (Curcio and Hinds, 1983). However, the aged hippocampus shows regionspecific decreases in synapse number at the mole­ cular layer of the dentate gyrus (Bondareff and Geinisman, 1976; Geinisman et al., 1977), decreases in the number of perforated synapses (Geinisman et al., 1986) and increases in calcium

46

conductance (Foster and Norris, 1997; Landfield, 1988). Indeed, the hippocampus of aged rats with cognitive deficits is unable to update encoding information when environmental cues are manipulated, demonstrating reduced plasticity (Tanila et al., 1997) and that chronic stress can accelerate this process (Kerr et al., 1991). These findings show that brain plasticity and function is already reduced in the aged brain, which can be hindered further by an additional mitigating factor, such as chronic stress. Studies investigating the effects of chronic stress or glucocorticoids on spatial learning show that the aged hippocampus is at a disadvantage. When young and middle-aged rats were directly compared, exposure to glucocorticoids for 3 months impaired spatial learning in middle-aged rats without affecting the performance of the younger cohorts (Bodnoff et al., 1995). Chronic stress accelerated spatial cognitive decline in aged rats (18 months), when 4 weeks of unpredict­ able stress was initiated at 12 months of age and then administered sporadically thereafter (Borcel et al., 2008). Spatial memory positively correlated with neurogenesis in the dentate gyrus (Borcel et al., 2008) and glucocorticoid exposure deter­ mined extent of age-related cognitive decline (Heffelfinger and Newcomer, 2001; Hibberd et al., 2000; Montaron et al., 2006). Moreover, chronic stress or glucocorticoids predicted hippo­ campal atrophy and hippocampal-dependent memory deficits in aged humans (Lupien et al., 1998), and facilitated cognitive decline in middleaged rats (Arbel et al., 1994), including those that were predisposed for high reactivity to novelty (Sandi and Touyarot, 2006). A distinction between how chronic stress influences the learn­ ing of new information and retrieving previously learned information is illustrated by a study that trained rats on a water maze, then administered corticosterone for several weeks, followed by assessment of retention (Hebda-Bauer et al., 1999). Under these conditions, corticosterone facilitated retention in the very old rats (31 months), which is consistent with the interpreta­ tion that chronic stress via the HPA axis shifts behavior towards habits: aged rats may be predis­ posed to shift towards well-learned behaviors

given their reduced hippocampal plasticity. These studies demonstrate a relationship among gluco­ corticoid exposure, hippocampal plasticity and cognitive ability with aging. The synopsis of these findings is that while the aged population consists of a heterogeneous popu­ lation, the aged brain and especially the aged hip­ pocampus will have difficulty rebounding from chronic stress and elevated glucocorticoids. The aged hippocampus is capable of plasticity, but its baseline differs compared to the young hippocam­ pus. Consequently, the inverted U-shaped relation­ ship between glucocorticoids and cognition described for the young may already be narrowed for the aged, perhaps revealing a function that is closer to the dashed curve than the solid one (Fig. 4C). The aged population may have more difficulty recovering from chronic stress than the young, which would provide an even larger window of opportunity for vulnerability. One’s life experi­ ences and exposures to stress, and the resulting effects on the HPA axis, impact the trajectory of aging. This results in an aged population composed of a diverse group of individuals with varied cogni­ tive and brain aging outcomes.

HPG axis and aging: influences on hippocampaldependent functions The HPG axis, age and spatial memory: overarching relationships Several decades of research have converged to indicate that gonadal hormones are potent mod­ ulators of brain structure and function, including in brain regions known to be intimately linked to learning and memory. Many of these cognitive brain regions are sensitive to changes as aging ensues. As represented in Fig. 5, there is a tertiary model representing interactions between aging, spatial learning and memory, and gonadal hor­ mones wherein research within each domain has identified strongly supported tenets: (1) gonadal hormones change with age, (2) age influences spa­ tial learning and memory and (3) gonadal hor­ mones influence spatial learning and memory. While it is clear these individual tenets are strongly

47

ac Im p

cts

Gonadal hormones

Stress and Resulting HPA Change

pa Im

ts

Age

Memory Impact

Fig. 5. Tertiary model representing interactions among aging, spatial learning and memory, and gonadal hormones. Research within each point has been strongly supported by empirical data: (1) aging impacts gonadal hormone milieu, (2) aging impacts spatial learning and memory and (3) gonadal hormones impact spatial learning and memory. Stress can impact trajectory of change across time for each of these three points. How these three tenets interact with each other, and with glucocorticoid release due to stress, is a key area of research that will yield insight into life experiences and exposures that influence age-related brain and cognitive change to ultimately impact phenotype of the aged individual.

supported by empirical data, a fundamental ques­ tion in aging research regarding interactions has yet to be clearly answered. How do gonadal hormone changes that occur with age relate to memory changes that occur with age? There is also evidence that stress can impact trajectory of change across time for each of these three points, as discussed and exemplified above. How these three tenets interact with each other, and with glucocorticoid release and HPA axis alterations due to stress, is an impor­ tant area of research that will yield insight into life experiences and exposures that influence agerelated brain and cognitive change to ultimately impact phenotype of the aged individual. Below we discuss each of these three tenets in turn, with examples of interactions presented when available from the literature.

Reproductive senescence Menopause, occurring typically in the fifth decade of life, is characterized by loss of ovarian-derived

circulating hormones, including estrogen and pro­ gesterone (Timaras et al., 1995). The majority of women undergo menopause not from oophorect­ omy (i.e. surgical removal of the ovary), but as a transitional hormone loss following age-related alterations of the hypothalamus, pituitary and ovary, ultimately resulting in follicular depletion (Timaras et al., 1995). Early in the aging process, neuronal changes in the hypothalamus are hypothesized to initiate transition into reproduc­ tive decline, leading to reproductive senescence (Downs and Wise, 2009). The ultimate hormone profile of the older reproductively senescent female rat and woman differ, limiting the use of ovary-intact female rat as an optimal model of human menopause when testing cognition. None­ theless, the rodent model provides exciting, illu­ minating insights to understand mechanisms of menopause itself since there are some commonal­ ities regarding reproductive physiology (Downs and Wise, 2009). As aging ensues in women, estro­ gen and progesterone decline due to decreased ovarian follicular reserves (Timaras et al., 1995). Thus, ovarian follicle depletion ultimately causes hormone loss during menopause. In contrast, the aging rat undergoes estropause, a persistent estrus state due to chronic anovulation rendering intermediate estrogen levels, or a pseudopreg­ nant/persistent diestrus state characterized by high progesterone levels due to increased ovula­ tion and corpora lutea (Meites and Lu, 1994). These changes in ovarian-derived hormone release in the rat are primarily due to hypothala­ mic/pituitary axis alterations (Meites and Lu, 1994). Thus, the primary mechanism that ulti­ mately results in reproductive senescence and cir­ culating hormone alterations in the woman is ovarian follicle depletion, while in the rat it is the hypothalamic–pituitary axis.

Aging effects on spatial learning and memory One of the most consistent findings in the animal cognition literature is that aged rodents exhibit poor scores on working and/or reference memory tasks that require spatial navigation compared to young counterparts. The majority of the studies

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testing age-related changes in spatial cognition used male rodents, with studies reporting agerelated memory decline on a multitude of tasks using various protocols and procedures (Arendash et al., 1995; Barnes et al., 1980; Beatty et al., 1985; Bond et al., 1989; Chrobak et al., 1995; Frick et al., 1995; Kadar et al., 1994; Lebrun et al., 1990; Lindner et al., 1992; Noda et al., 1997; Pitsikas and Algeri, 1992; Rapp et al., 1987; Shukitt-Hale et al., 2004; Stewart et al., 1989; Wallace et al., 1980; Wellman and Pelleymounter, 1999; Wyss et al., 2000). In the last decade, there have been increased efforts to study the effects of age-related spatial cognitive decline in female rodents, with the driving force of many studies being interest in the relationships with concurrent gonadal hor­ mone change. Aged females show spatial working memory deficiency compared to young females (Bimonte et al., 2003; Bimonte-Nelson et al., 2003b, 2004; Kobayashi et al., 1988; Luine et al., 1990), with age-related changes in spatial memory likely related to circulating ovarian hormone levels. For example, spatial reference memory decline on the Morris maze emerged by 12–16 months of age, when ovarian hormone levels start to change and estropause ensues (Markowska, 1999; Talboom et al., 2008). When evaluating the vast cognitive aging litera­ ture in the rodent, age-related changes in spatial learning and memory are multi-dimensional and complex, with, for example, age-related changes being dependent upon which phase of learning is being tested and the demand level of the task. Age-related cognitive deficits seem to be greatest during task acquisition, as young and aged rodents eventually reach comparable asymptotic levels on spatial working and reference memory tasks when given extended training (Ikegami, 1994; Rapp et al., 1987). This effect may interact with degree of cognitive impairment in the aged animal at the onset of testing sessions (Ikegami, 1994). It is noted that age-related deficits have been observed after spatial task acquisition as well, with the greatest age-associated cognitive decrements seen when spatial memory demand is high. In humans, age-related deficits are exacerbated with greater working memory complexity, an effect shown on multiple tasks and within non-spatial

(e.g. verbal) and spatial domains (Salthouse et al., 1989). In animals, age-related spatial deficits become more pronounced as memory demand increases. This has been shown for age-associated interference-related deficits (Lebrun et al., 1990) and for memory capacity deficits (Aggleton et al., 1989; Bimonte et al., 2003; Bimonte-Nelson et al., 2003b; 2004). For example, in the spatial water radial-arm maze task, as trials progress within any given session, animals need to hold a greater number of items of information in spatial working memory. Young and aged female rats did not differ in the ability to handle an increasing spatial working memory load during the initial portion of testing (Fig. 6A, Bimonte et al., 2003). However, as testing progressed across days young animals learned to handle more spatial working memory information. This resulted in a significant learning curve for young animals on all trials, whether spatial working memory load was low, moderate or high. In contrast, aged animals exhibited a sig­ nificant learning curve only on the earliest trials, when spatial working memory load was low. As spatial working memory load increased aged rats had difficulty remembering which arms they had visited within a session, and they could not learn to handle this increasing amount of information across testing sessions. Aged rats also made dis­ proportionately more errors on the latter trials, when spatial working memory load was highest. Thus, aged female rats exhibited progressive per­ formance deterioration as the number of items to be remembered, or working memory load, increased (Fig. 6B). There is other evidence that aged animals have difficulty sustaining successful performance as other types of memory demands increase, such as when a delay is imposed between trials. Such findings that aged rats exhibit delay-dependent deficiencies in performance is typically interpreted as a faster rate of forgetting (Beatty et al., 1985; Dunnett et al., 1988; Winocur, 1986; Zornetzer et al., 1982), although not all studies find age differences in rates of forgetting, which may be related to overtraining or animals reaching criter­ ion performance (Beatty et al., 1985; Wallace et al., 1980; Willig et al., 1987). This latter point is underscored by research in humans touching on

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the boundaries between what is, and what is not, normal cognitive aging. For example, research indicates that if younger and older people learn information to the same level, even though the older individuals may take longer to get to this point, the older people will not forget the informa­ tion more rapidly (Albert, 2002; Albert et al., 1987). Thus, while there are clear age-related changes in learning and memory in humans and animal models, there is great variability across individuals. Moreover, decrements associated with aging are typically seen regarding only certain types of information, with spatial memory one of the most robust deficits noted, as well as an age-related selective vulnerability to acquisition and demand task components.

Gonadal hormones and spatial learning and memory: general context and clinical implications By the beginning of the 20th century, specific ‘internal secretions’, now referred to as steroid hormones, were known to be chemical media­ tors of the phenotype (Adler, 1981). Steroid hormones released from the gonads have since been shown to be important for not just classical reproductive actions (Beach, 1947), but also for providing neural plasticity and influencing brain functions such as learning and memory. In humans and animal models, estrogens, proges­ terone and testosterone have each been shown to impact spatial cognition. To evaluate steroid hormone levels and cognitive effects in humans, researchers have been creative in their assess­ ments and have reported effects: across meno­ pause transition stages (Luetters et al., 2007), with sex-change operations and concomitant sex hormone treatment (Gomez-Gil et al., 2009), and before versus after hormone therapy treatment in surgically menopausal women (Sherwin, 2006). To test the cognitive effects of steroid hormones in rodent or monkey models, the traditional procedure is to remove the source of major endogenous synthesis and release, the testes in the male (gonadectomy, or GDX) or the ovaries in the female (ovariect­ omy, or OVX), and give the exogenous steroid

of question as a treatment regimen. Over the last decade, both the human and animal litera­ ture evaluating the potential influence of gona­ dal hormones on brain health and function during aging have increased in breadth and depth. Much of this is because of the recent intense discussion and debate about whether hormone therapies impact normal aging or AD, emanating in part from reports including a meta­ analysis that estrogen-containing hormone therapies decrease the risk of AD by 29% (Yaffe et al., 1998), that placebo-controlled stu­ dies showed that estrogens improved memory or dementia scores in female AD patients (Asthana et al., 2001, 1999; Ohkura et al., 1994, 1995), that menopause might exacerbate age-related cogni­ tive changes in several domains, including visuospatial abilities (Halbreich et al., 1995), and then more recently, the outcome of the Women’s Health Initiative (WHI) studies show­ ing null or detrimental effects on cognition or dementia from the most commonly used hor­ mone therapies (for discussion, see Sherwin and Henry, 2008). Further, the clinical implica­ tions and health-related importance of under­ standing the effects of ovarian hormone loss and replacement has been underscored by reali­ zation that life expectancy of women has increased from an average of 54 years in 1900, to recent values estimating expectancy to about 80 years (Singh et al., 1996). Since age of spon­ taneous menopause has remained stable, women are now living approximately one-third of their lives in a hypo-estrogenic menopausal state (Amundsen and Diers, 1970, 1973; Sherwin, 2003). The emerging findings in the literature have been illuminating, diverse and exciting, showing that multiple parameters impact the extent, and even the direction, of cognitive effects of gonadal hormones, underscoring the impact of gonadal hormone effects on brain function and its rich plasticity. The discussion here will not exhaustively cover this immense and growing literature, and will be limited to points necessary to provide context for the dis­ cussion of gonadal hormone effects on cognitive aging, with special emphasis on the spatial domain.

50 A. Aged animals cannot learn to handle an increasing spatial working memory load, while young animals can. 5 Aged Young

Working memory errors

4

3 High memory Load block 2

1

Moderate memory Load block

Low memory Load block 12

11

10

9

8

7

6

5

4

3

2

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Day of maze testing

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Aged rats

3 Young rats

2 1

gh Hi

M

od er a

te

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0 Lo

Working memory errors

B. Aged animals exhibit an impaired ability to handle an increasing spatial working memory load.

Working memory load Fig. 6. (legend continued)

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HPG axis and spatial learning and memory: estrogens and progesterone Accumulating evidence supports the interpreta­ tion that ovarian hormone loss contributes to cog­ nitive decline in women. Clinical findings show women exhibit cognitive decline after surgical menopause (Farrag et al., 2002; Phillips and Sherwin, 1992; Sherwin, 1988), including on global cognitive function as tested 3 or 6 months postsurgery (Farrag et al., 2002). Further, surgically menopausal women exhibit lower memory scores relative to naturally menopausal women, and age of oopherectomy (surgical ovary removal in the woman) and greater years since surgery correlated with poorer performance (Nappi et al., 1999). Pre­ clinical rodent model evaluations also show that ovarian hormone loss can induce cognitive changes, an effect depending on many factors, including age (Bimonte-Nelson et al., 2006; Markowska and Savonenko, 2002; Savonenko and Markowska, 2003; Talboom et al., 2008). Sur­ gical ovarian hormone loss (e.g. OVX) produces cognitive decrements in young adult female rats (Bimonte and Denenberg, 1999; Daniel et al., 1999; El-Bakri et al., 2004; Feng et al., 2004; Gibbs and Johnson, 2008; Talboom et al., 2008), while enhancing cognition in old age (discussed in more detail below; Bimonte-Nelson et al., 2003b, 2004; Braden et al., 2010). To date, only four studies evaluated OVX effects on maze learning and memory in middle-aged rats (Bimonte-Nelson et al., 2006; Markowska and Savonenko, 2002; Savonenko and Markowska, 2003; Talboom

et al., 2008). In middle-aged females 12–16 months-old, OVX did not impact spatial reference memory (Bimonte-Nelson et al., 2006; Markowska and Savonenko, 2002; Talboom et al., 2008) or spatial working memory (Markowska and Savonenko, 2002; Savonenko and Markowska, 2003). However, spatial working memory deficits were detected in 17-month-old OVX rats following high-demand time-delayed memory retention tests (Markowska and Savonenko, 2002). These data suggest that OVXrelated memory changes in middle-age may become evident when working memory demands are more challenging. In this regard, we previously demonstrated that OVX alters memory in both young and old rats (Bimonte and Denenberg, 1999; Bimonte-Nelson et al., 2003b, 2004; Braden et al., 2010), effects which were more pronounced as working load increased by escalating the num­ ber of items to remember. Thus, elevating work­ ing memory demand either by extending time delays to challenge retention, or by increasing the number of items to remember, allows a broader scope of evaluations to realize OVXinduced memory changes across the ages. It is noted that exacerbated deficits in aged animals are also seen with a higher memory demand, as described above, further indicating that incremen­ tal alterations in task demand could yield insights into changes that may not be seen otherwise. In the rodent, transitional hormone loss can be induced via the industrial chemical 4-vinylcyclo­ hexene diepoxide (VCD), which produces follicu­ lar depletion by selectively destroying primordial

Fig. 6. (A) Mean number of spatial working memory errors committed by Aged and Young rats averaged into the Low (Trials 2 and 3), Moderate (Trials 4 and 5) and High (Trials 6–8) working memory load blocks. Also depicted is the linear trend for each of the working memory load blocks, for each group. Both Young and Aged groups exhibited significant linear trends on the Low working memory load block (regression equations: Aged: y = 0.042x þ 0.600, Young: y = 0.058x þ 0.639), while only the Young group showed a significant linear trend on Moderate and High working memory load blocks (ys = 0.058x þ 1.12 and 0.176x þ 3.76, respectively). These data suggest that while both Young and Aged animals could learn to handle a low spatial working memory load, the young animals could also learn to handle moderate and high spatial working memory loads, while the aged animals could not. (B) Number of spatial working memory errors + SE on Low (Trials 2 and 3), Moderate (Trials 4 and 5) and High (Trials 6–8) load trial blocks, averaged across the latter days of testing, for Aged and Young rats. As trials increased, the number of elements of information to be remembered increased. The significant Age × Trial block interaction reflects that Aged animals committed disproportionately more errors on the latter trials, when the spatial working memory load was highest.

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and primary follicles via acceleration of the nat­ ural atresia process, resulting in hormone profiles more similar to naturally menopausal women ver­ sus OVX (Mayer et al., 2004; Springer et al., 1996; Timaras et al., 1995). Data from work using this novel menopause model, like data from work using the OVX model, have indicated that ovarian hormone loss impacts cognition. VCD-induced transitional menopause impaired learning of a spatial recent memory task, and transitional menopause before OVX was better for spatial memory than an abrupt loss of hormones via OVX only (Acosta et al., 2009a). These results correspond with findings from Rocca et al. (2007) showing women that had undergone oopherectomy prior to menopause onset had elevated cognitive impairment risk compared to age-matched women without oopherectomy. Col­ lectively, the findings suggest that initiation of transitional menopause before surgical ovary removal might benefit mnemonic function and could obviate some negative cognitive conse­ quences of surgical menopause alone, and that surgical menopause may be worse for cognition than transitional menopause. Future studies addressing this important question of cognitive effects of type of ovarian hormone loss, combined with evaluation of potential interactions between type of ovarian loss and subsequent hormone therapy, should yield valuable insight and new interpretations of variables impacting cognitive outcome due to ovarian hormone loss and therapy in menopausal women. Estrogens are a class of hormones including 17b-estradiol, estrone and estriol; 17b-estradiol is the most potent naturally circulating estrogen, followed by estrone and estriol, in order of receptor affinity (Kuhl, 2005; Sitruk-Ware, 2002). To date, 17b-estradiol has been the pri­ mary estrogen used to test cognitive effects of hormone therapy in the animal model. The majority of studies evaluating activational effects of estrogens on a zero-level circulating hormone background (i.e. OVX animals) for spatial learn­ ing and memory have been performed in young rodents, with many showing enhancements due to treatment (Bimonte and Denenberg, 1999; Daniel et al., 1997, 1999; Dohanich et al., 1994;

Galea et al., 2001; Holmes et al., 2002; Luine et al., 2003, 1998; Marriott and Korol, 2003; McLaughlin et al., 2008; Packard and Teather, 1997; Sandstrom and Williams, 2001; Singh et al., 1996). Studies in young OVX monkeys have shown no benefits for specific aspects of learning and memory (Lacreuse and Herndon, 2003; Voytko, 2000), but benefits due to 17b­ estradiol treatment for certain measures such as visuospatial attention (Voytko, 2002). The context of studies experimentally evaluat­ ing effects of estrogens and progestins in aging animal models is usually to understand whether estrogen-containing hormone therapies impact cognition within this older cohort, so that findings might be eventually translated to understand and optimize hormone therapies given to women (e.g. see Daniel, 2006; Frick, 2009). Since the first controlled clinical evaluation showing that 17b-estradiol injections given to a 75-year-old women enhanced memory (Caldwell and Watson, 1952), there have been numerous stu­ dies showing cognitive decline after ovarian hor­ mone loss, and enhancement after treatment with various types of preparations containing estro­ gens, in menopausal women (for review, see Sherwin, 2006). Premarin, the most commonly prescribed hormone therapy given to women (Hersh et al., 2004), is conjugated equine estro­ gens (CEEs), which contains the sulphates of at least 10 estrogens, is over 50% estrone sulphate, 20–25% equilin sulphate and has only trace amounts of 17b-estradiol; after metabolism, the resulting biologically active circulating hormones are primarily estrone and, after estrone’s conversion, 17b-estradiol, as well as equilin (Bhavnani, 2003; Sitruk-Ware, 2002). It is hypothesized that these three metabolites are pri­ marily responsible for the estrogenic effects of CEE (Sitruk-Ware, 2002), although there are other estrogens and related metabolites present that could initiate effects on their own; these hor­ mones include, but are not limited to D8,9 Dehydro-estrone, dihydroequilin-17b and equile­ nin (Bhavnani, 2003; Kuhl, 2005). CEE-contain­ ing therapy improved memory via self-report (Campbell and Whitehead, 1977), case studies (Ohkura et al., 1995) and randomized

53

psychometric evaluations (Kantor et al., 1973). Yet, findings evaluating global cognitive function in the large placebo-controlled WHI Memory Study (WHIMS), conducted by the National Institutes of Health, showed an increase in prob­ able dementia risk and no effect on mild cognitive impairment in women 65 years or older taking the combination therapy CEE plus the synthetic pro­ gestin, medroxyprogesterone acetate (MPA, Shu­ maker et al., 2003). CEE alone showed a non­ significant increase in incidence of probable dementia and mild cognitive impairment (Espeland et al., 2004; Shumaker et al., 2004). An ancillary study to the WHI testing more specific cognitive functions, the WHI Study of Cognitive Aging (WHISCA), reported that CEE plus MPA therapy had a negative effect on verbal memory and a trend for positive effects on figural memory in women 65 and over free of probable dementia (Resnick et al., 2006). Most recently, the WHIMS-MRI study found that CEE use with or without MPA was associated with small but measurable atrophy in the frontal cortex and hippocampus (Resnick et al., 2009). At the pre­ sent time, the studies in women have little con­ sensus, although new studies will be crucial to the understanding and clarification of the complex effects of ovarian hormone loss and hormone therapies. Identifying the effects of the various components of hormone therapies, including detailed evaluations using basic science and sys­ tem approaches, is the optimal approach to con­ verge the many findings that appear contradictory. In fact, as new data emerge it may become clear that the cognitive effects of hor­ mone therapy are not contradictory at all. Rather, they may be dependent on numerous variables not yet taken into account in many studies. One way to further our understanding of gonadal hor­ mone effects on spatial cognition is by using ani­ mal models. For example, female animal models can provide insight into the cognitive effects of ovarian hormones, allowing evaluative changes in cognition due to ovarian hormone withdrawal and treatment, while enabling experimental con­ trol not possible in clinical research. With this in mind, we now ask, do aged animals show cognitive enhancements after treatment with

estrogens? The answer to this question is vague, ‘Yes, but …’ Indeed, effects depend on a multi­ tude of factors. It has become clear that the cog­ nitive effects of estrogens are rich with complexity, and that they have a multi-dimensional nature that we are just starting to understand. In addition to age, estrogenic effects appear to be influenced by innumerable factors including, but not limited to, timing of hormone administration relative to hor­ mone loss, dose, mode of treatment and whether progestins are given concurrently. Two of the most interesting questions driving much of the newer animal research in the field of estrogenic actions on brain health during aging have been spawned from the WHIMS findings. Women who participated in the WHIMS were between 65- and 79-years-old, and many had experienced ovarian hormone deprivation for a substantial amount of time before receiving CEE-containing treatment (Shumaker et al., 1998). This presents the intriguing question of whether the older age of the women, and/or whether the extended window of time from menopause (which occurs in the 50s, on average), impacted outcome. Data collected within the last few years indicate that whether cognitive benefits of estrogen ther­ apy are realized is influenced by the delay between OVX and hormone treatment, with limited benefits seen when there is an extended window between ovarian hormone loss and 17b­ estradiol treatment. This has been seen in the rat on tests of spatial memory, whereby 17b-estradiol replacement initiated immediately after OVX enhanced spatial memory performance in mid­ dle-aged rats, but imparted no benefit when given 5 months after OVX (Daniel et al., 2006). These behavioral findings correspond with neuro­ chemical data from both young and middle-aged rats showing that 17b-estradiol treatment given immediately after OVX increased choline acetyltransferase (ChAT) levels in the hippocampus, while this increase was not seen when initiated 5 months after OVX; it is noteworthy that this pattern was opposite of that seen in PFC (Bohacek et al., 2008). There may also be a critical window for the well-established findings that 17b-estradiol regulates dendritic spines in the hippocampus (Woolley, 2000), as a 10-week

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delay after OVX decreased the effectiveness of 17b-estradiol to increase CA1 apical spine density as compared to treatment given immediately, in young rats (McLaughlin et al., 2008). In the mon­ key, the important question of a critical window for cognitive efficacy of estrogens has not yet been directly addressed, but there is evidence that the surgically menopausal monkey is still sensitive to estrogenic effects in old age even if there is a substantial window after ovarian hormone loss. Indeed, benefits due to treatment with estrogens were seen when treatment was initiated immedi­ ately (Voytko et al., 2008; 2009) within 30 weeks (Rapp et al., 2003), or even when given 10–16 years (Lacreuse et al., 2002) after OVX, although it is noted this latter study used a within-subjects repeated measures design of placebo or estrogenic treatments, and effects were transiently expressed, somewhat limiting interpretation of this specific critical window question since there were windows of absence and exposure across the timeframes of the study. A number of studies evaluated estrogenic effects on spatial ability in middle-aged or older female rodents, most using 17b-estradiol (Bimonte-Nelson et al., 2006; Foster et al., 2003; Frick et al., 2002; Gibbs, 2000; Luine and Rodri­ guez, 1994; Markham et al., 2002; Markowska and Savonenko, 2002; Savonenko and Markowska, 2003; Talboom et al., 2008; Ziegler and Gallagher, 2005), or the synthetic or semi-synthetic estradiol preparations ethinyl estradiol or estradiol cypio­ nate, in monkeys (Lacreuse et al., 2002; Rapp et al., 2003). In general, studies indicate that 17b-estradiol treatment can enhance performance on cognitive tests when treatment is initiated dur­ ing middle-age or in old age, as seen in rodents (Aenlle et al., 2009; Foster et al., 2003; Frick et al., 2002; Gibbs, 2000; Markham et al., 2002; Markowska and Savonenko, 2002; Talboom et al., 2008) and non-human primates (Lacreuse et al., 2002; Rapp et al., 2003; Voytko et al., 2008), although the effect may be transient in monkeys for visual recognition, with benefits seen 12 weeks following treatment initiation, but not another 12 weeks later when tested 24 weeks after treatment initiation (Voytko et al., 2008). A similar transient effect of ethinyl estradiol

treatment was seen in surgically menopausal aged monkeys for spatial delayed recognition, a test that shows hippocampal lesion (Beason-Held et al., 1999) and age-related (Moss et al., 1997) impairments, with benefits seen for months 2–4, but not 6–8, of the study (Lacreuse et al., 2002). There is also new evidence from our laboratory that both tonic and cyclic CEE, at doses relevant to what women take as hormone therapy, can enhance spatial memory and retention, and pro­ tect against cholinergic challenge on spatial tasks in middle-aged OVX rats (Acosta et al., 2009b; Engler-Chiurazzi et al., in press). Age-related changes in responsiveness to treat­ ment with estrogens have been shown for spatial cognition in studies directly testing multiple ages after 17b-estradiol as compared to vehicle control treatment. Aged OVX rats were not responsive to the 17b-estradiol treatment regimen that effec­ tively enhanced spatial reference memory in young and middle-aged OVX rats (Talboom et al., 2008), concurring with age-related interac­ tions with 17b-estradiol replacement for spatial memory shown by others, described below (Foster et al., 2003). Why, then, have some studies shown that aged female rodents can exhibit cognitive enhancements in response to 17b-estradiol treat­ ment? For example, 17b-estradiol injections enhanced spatial reference memory in 27–28­ month-old ovary-intact mice (Frick et al., 2002). The difference in results may relate to type of 17b-estradiol administration as cyclic versus tonic, as priming with cyclic 17b-estradiol­ enhanced responsiveness to tonic 17b-estradiol in older OVX rats (Markowska and Savonenko, 2002). Whether 17b-estradiol replacement improves performance of aged animals may also relate to memory type and demand. Working memory enhancements due to tonic 17b-estradiol treatment have been reported in aged rats, although this effect is more pronounced when memory demand is high (Gibbs, 2000; Luine and Rodriguez, 1994). We and others have shown 17b­ estradiol-induced spatial working memory improvements in young OVX rats as well, an effect most pronounced when spatial working memory demand is high (Bimonte and Denenberg, 1999), which depends on

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administered 17b-estradiol dose (Bimonte and Denenberg, 1999; Daniel et al., 1997; Holmes et al., 2002; Sandstrom and Williams, 2001). Further, a higher supraphysiological 17b-estradiol dose may be necessary to enhance spatial refer­ ence memory retention in rats approaching old age (Foster et al., 2003). These findings corre­ spond with data showing that higher serum levels of exogenous 17b-estradiol treatment correlates with better spatial reference memory performance in young and middle-aged OVX rats (Talboom et al., 2008). It also appears that sensitivity and responsiveness to ovarian hormone loss does not predict sensitivity and responsiveness to 17b-estra­ diol treatment. Indeed, Talboom et al. (2008) found that young animals were responsive to both ovarian hormone removal and replacement, middle-aged animals were not responsive to ovar­ ian hormone removal but were responsive to estrogen replacement and aged animals were not responsive to ovarian hormone removal or repla­ cement for the test trials for spatial reference memory. Estrogens’ effects on cognition may be impacted by the presence of progesterone, sug­ gesting, again, that gonadal hormone effects on cognition are multi-dimensional and complex. Dis­ cussion regarding, and research testing, the com­ bined regimen of estrogens plus progestins has increased as of late, mostly due to the findings that menopausal women taking CEE alone did not differ significantly from those taking placebo for dementia diagnoses (Shumaker et al., 2004), while, in contrast, twice as many women receiving CEE plus the synthetic progestin MPA were diag­ nosed with dementia as compared to the placebo group, a significant effect (Shumaker et al., 2003). Of note, women with a uterus that are taking estrogens must include progestin in their regimen because of increased risk of endometrial hyper­ plasia associated with unopposed estrogen treat­ ment (Smith et al., 1975). We have shown that progesterone abolished the 17b-estradiol-induced benefits on the spatial reference memory Morris maze in middle-aged rats (Bimonte-Nelson et al., 2006). Accordingly, progesterone plus 17b-estra­ diol injections impaired performance on the spa­ tial reference memory Morris water maze, while

17b-estradiol or progesterone treatment alone did not influence performance, and this pattern of effects was not seen on the non-spatial Morris maze suggesting this combination treatment has specific effects within the spatial domain (Chesler and Juraska, 2000). These effects may not trans­ late to working memory tasks, however, as pro­ gesterone treatment enhanced 17b-estradiol’s effects on a delayed-match-to-position spatial T-maze (Gibbs, 2000). Progesterone alone has been associated with detrimental cognitive effects, in both clinical and preclinical studies. The ‘maternal amnesia’ phe­ nomenon in pregnant women is hypothesized to result from high circulating progesterone levels during late pregnancy (Brett and Baxendale, 2001). In healthy women, a large oral progester­ one dose is detrimental to memory (Freeman et al., 1992). High circulating progesterone levels are also observed in most rats following estro­ pause (Lu et al., 1979), and many endocrine stu­ dies have shown that aged female rats enter a pseudopregnant estropause state, whereby pro­ gesterone values become significantly elevated but estradiol levels remain relatively unchanged (Huang et al., 1978; Wise and Ratner, 1980). This pseudopregnant state has in fact been associated with poorer spatial cognition (Warren and Juraska, 2000). Correspondingly, one study has shown that in young, cycling rats spatial maze performance was worse during the proestrus phase, when estrogen and progesterone levels are at their highest, and best during the estrus phase, when estrogen and progesterone are at their lowest (Warren and Juraska, 1997) (but see Berry et al., 1997; Stackman et al., 1997). OVX in aged rats improves cognition (Bimonte-Nelson et al., 2003b), which is likely related to progester­ one removal, since progesterone administration has large detriments on spatial working and refer­ ence memory, reversing the beneficial effects of OVX (Bimonte-Nelson et al., 2004). This effect was recently found in our laboratory using the synthetic progestin MPA, contained in the com­ monly used hormone therapy Prempro, as well. MPA impaired spatial memory retention and exa­ cerbated overnight forgetting on a spatial task (Braden et al., 2010). Noting the same pattern

56

seen with detrimental effects of aging, and bene­ ficial effects of 17b-estradiol treatment, progester­ one and MPA supplementation had the most marked performance effects on the water radialarm maze at the highest working memory load, with progestin-treated aged OVX rats showing disproportional impairments as working memory load reached its highest demand (Bimonte-Nelson et al., 2004; Braden et al., 2010).

HPG axis and spatial learning and memory: testosterone Similar to the postulated relationship between estrogen loss and memory decline in aging women (Sherwin, 1988), recent studies suggest that a decline in testosterone levels is related to age-asso­ ciated memory changes in men (Tan, 2001). In men, testosterone levels decline slowly with age and to only about 40% lower than levels seen in younger men, as compared to the more drastic loss of estro­ gens and progesterone seen in women after meno­ pause (Davidson et al., 1983; Hijazi and Cunningham, 2005). One of the most striking rela­ tionships regarding this research area was discov­ ered in the last decade, with reports that lower testosterone levels are linked with a higher risk of AD (Hogervorst et al., 2001; Moffat et al., 2002, 2004; Rosario et al., 2004), and that, for example, lower serum testosterone levels are seen in male AD patients versus controls (Hogervorst et al., 2001). This work spawned many hypothesis-driven stu­ dies targeting the question of whether testoster­ one enhances cognition in AD patients or in individuals with normal age-associated memory impairment. Relationships between endogenous testoster­ one levels and cognition have been observed in younger and older individuals, with, in general, the strongest relationships seen in the older popu­ lation in retrospective and randomized treatment studies. Testosterone was related to cognitive per­ formance in young men and women, and spatial ability was related to the change in seasons in accordance with seasonal alterations in testoster­ one levels (Kimura and Hampson, 1994; Neave et al., 1999; Silverman et al., 1999). Retrospective

studies have suggested there is a relationship between a greater age-related cognitive decline and lower bioavailable testosterone levels (Barrett-Connor et al., 1999; Moffat et al., 2002; Yaffe et al., 2002). In general, in older men, higher endogenous testosterone levels have been linked to better cognitive function, while an inverted Ushaped dose–response relationship between circu­ lating testosterone and cognition has also been noted, with most beneficial effects with moderate circulating testosterone levels (Barrett-Connor et al., 1999; Gouchie and Kimura, 1991; Yaffe et al., 2002). One of the most convincing pieces of evidence that testosterone effects on cognition hold to an inverted U-shaped function for older men was recently reported. This randomized, pla­ cebo-controlled study evaluating healthy older men found that weekly testosterone injections resulting in moderate increases in serum testoster­ one or its metabolites yielded enhanced spatial and verbal memory, while the testosterone injec­ tions resulting in relatively smaller, or relatively larger, testosterone increases yielded no signifi­ cant change (Cherrier et al., 2007). Of note, the moderate increases due to the testosterone treat­ ment that enhanced memory resulted in circulat­ ing levels that were normal to high normal levels seen in young men, and the large increases due to the testosterone treatment that did not enhance memory were pushed into the supraphysiological range. Work evaluating endogenous levels also suggest a non-linear U-shaped function for testos­ terone levels and spatial ability with moderate levels optimal, as lower relative levels of salivary testosterone in men, and higher relative levels of salivary testosterone in women, were related to the highest performance scores for spatial ability (Gouchie and Kimura, 1991). It is possible that accumulating data will continue to support this inverted U-shaped quadratic relationship, and will help to synthesize the range of findings includ­ ing, for example, newer work showing that agerelated declines in endogenous testosterone levels did not directly correlate with age-related cogni­ tive declines in spatial abilities, such as that seen on the mental rotation test (Martin et al., 2008). As well, the first study testing the effects of testosterone supplementation in monkeys was

57

published this year, and found that supraphysiolo­ gical testosterone levels did not yield benefits on spatial cognition in young male monkeys; whether lower physiological levels within the postulated optimal range of the inverted U-function would have yielded benefits is yet to determined (Lacreuse et al., 2009). Placebo-controlled testosterone treatment stu­ dies have reported enhanced spatial cognition in healthy older men (Cherrier et al., 2001, 2007; Gray et al., 2005; Janowsky et al., 1994), an effect not seen in younger men (Bhasin et al., 2001). Men with AD or mild cognitive impairment who received testosterone supplementation showed improved spatial cognition (Cherrier et al., 2005; Tan and Pu, 2003), although benefits of testoster­ one replacement were not seen in a pilot study evaluating old men with early- to mild-cognitive impairment and pre-treatment low levels of bioa­ vailable testosterone (Kenny et al., 2004). Since testosterone can be converted to either dihydrotestosterone (DHT), which binds to androgen receptors, or to estrogen via the aroma­ tase enzyme, testosterone’s mnemonic effects could be due to either DHT or estrogen (Becker, 1995). In fact, 80% of circulating estradiol is not of testicular origin in men; it is from aromatization of testosterone occurring in the periphery or brain areas, including the hippocampus (Becker, 1995; Naftolin, 1994). However, recently, an elegant study showed that the spatial memory benefits of testosterone were seen in men even when aroma­ tization to 17b-estradiol was pharmacologically blocked, and blood levels confirmed that the tes­ tosterone plus aromatase inhibitor reduced 17b­ estradiol levels by 50%, thereby indicating the testosterone-induced spatial memory improve­ ments occurred in the absence of concomitant 17b-estradiol increases (Cherrier et al., 2005). However, this does not preclude interactions between testosterone and estrogens for spatial cognitive performance. We have shown that tes­ tosterone supplementation given to gonad-intact aged male rats enhanced learning on a spatial working and reference memory task, and improved the ability to handle an increasing spa­ tial working memory load (Bimonte-Nelson et al., 2003a). These effects may be due to an interaction

between estrogens and testosterone; DHT, which is not converted to estrogens, had no effect on spatial maze scores in aged male rats (BimonteNelson et al., 2003a). Accordingly, others have shown enhanced spatial memory retention after testosterone, but not DHT, treatment in aged mice (Benice and Raber, 2009). Of note, in the Bimonte-Nelson et al. (2003a) study, the vehicletreated gonad-intact aged sham rats exhibited elevated serum 17b-estradiol levels and showed compromised maze performance. Conversely, testosterone treatment decreased serum 17b­ estradiol levels and improved maze performance. These combined findings suggest that testosterone did not initiate its effects by increasing serum 17b-estradiol levels, and that the group of aged rats that exhibited the best performance, the tes­ tosterone-treated group, showed relatively higher testosterone and lower 17b-estradiol circulating levels. Similarly, in older men, better cognitive performance was related to higher testosterone and lower 17b-estradiol levels (Barrett-Connor et al., 1999) and testosterone treatment improved spatial cognition in older men, while at the same time it decreased 17b-estradiol levels (Janowsky et al., 1994). It is clear that testosterone can impact spatial cognition, and that these effects may involve estrogenic interactions.

HPG axis and spatial learning and memory: gonadotrophins While it is well established that the gonadotropins follicle stimulating hormone (FSH) and lutenizing hormone (LH) are involved in regulating repro­ ductive functions via negative and positive feed­ back loops, it is becoming increasingly apparent that gonadotropins might, directly or indirectly, impact cognitive function as well, including within the spatial domain. Although there have been few links between FSH and cognition (e.g. Acosta et al., 2009a; Luetters et al., 2007), there is strong evidence that LH is related to cognition, with perhaps the strongest evidence from the neurode­ generative disease literature (Webber et al., 2007). Supporting plausibility of LH effects on the brain and spatial cognition, the highest density of LH

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receptors in the brain are found in the hippocam­ pus (Lei et al., 1993; Zhang et al., 1999), a region intimately involved in spatial learning and mem­ ory and affected by aging and AD. Further, LH can cross the blood–brain barrier (Lukacs et al., 1995). In a recent study by our laboratory evaluating VCD-induced follicular depletion and OVX effects on cognition in the middle-aged rat model, there was a very clear inverted U-shaped function for serum LH and number of spatial memory errors, with highest and lowest levels associated with the best performance, an effect not seen with FSH (Acosta et al., 2009a). This relationship with LH became apparent in scatterplots including all treatment groups so that the range of values across groups could be noted. Indeed, this range was broad, because, as expected, OVX increased LH levels due to a lack of ovarian hormone negative feedback after ovarian hormone loss, while Sham Control ani­ mals showed LH values in the relatively lower range. When LH levels ranged from ~0 to 2 ng/ ml, higher LH levels correlated with worse maze performance to reveal a positive relationship with errors. However, when LH levels ranged from ~2 to 10 ng/ml, higher LH levels correlated with bet­ ter maze performance to reveal a negative rela­ tionship with errors. When the groups were put in the same scatterplot the effect was a striking inverted U-shaped function (Fig. 7). This pattern was seen for multiple measures, including spatial working and reference memory. While limitations exist in interpreting this relationship between LH and memory scores in this study because LH levels were confounded by group membership, this quadratic relationship is nonetheless striking, especially given the increasing evidence that LH levels are linked to cognition and pathologies associated with neurodegenerative disorders (Webber et al., 2007). Other studies report higher LH levels are related to better cognitive perfor­ mance, similar to the effects seen in our OVX animals. Tonic treatment with LH-releasing hor­ mone, elevating LH concentrations to OVX levels, enhanced performance on visual discrimi­ nation in young rats (Nauton et al., 1992) and enhanced non-spatial working memory in aged

rats (Alliot et al., 1993). That higher LH levels were associated with better memory in these stu­ dies is likely related to LH levels being increased to that of OVX animals. On the other hand, cor­ responding with the Acosta et al. (2009a) findings in ovary-intact animals that higher LH levels correlated with worse cognitive performance, in ovary-intact aged female mice, experimentally induced LH reductions decreased amyloid-b concentrations and enhanced cognition, while LH increases promoted biochemical brain changes consistent with AD, although none of these studies correlated circulated LH levels with memory scores in individual animals (Bowen et al., 2004; Casadesus et al., 2007; 2006). Also, men and women with AD had higher circulating LH levels than controls (Bowen et al., 2004; Short et al., 2001). In sum, over the last decade, there is increasing evidence that LH levels are related to cognition and possibly pathologies associated with neurodegenerative diseases such s AD. The grow­ ing literature evaluating the relationship between LH and cognition suggests that it may subserve an inverted U-shaped function, with an intermediate level resulting in optimal brain function. This is an important area that will require further study, with results possibly revealing important media­ tors of cognitive function.

Summary and conclusion Cognitive function is multi-dimensional and com­ plex, with hippocampal-dependent spatial mem­ ory being particularly susceptible to age-related decrements, especially when task demand or load is elevated. The presence of age-related memory changes are some of the most consistent seen in the human and animal literature, although there is variability in age-related decrements across indi­ viduals, with the boundaries between normal and pathological age-associated impairments just beginning to be defined. The neurobiology under­ lying age-related spatial memory deficits are not likely caused by neuronal loss, although age increases the risk for brain damage attributed to AD and other disorders. Age-related spatial mem­ ory decrements most likely engage potentially

Distance swum on the spatial reference memory morris maze (higher distance scores reflects poorer performance)

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Fig. 7. Findings from a study assessing the cognitive effects of two experimentally induced forms of ovarian hormone loss, VCD-induced follicular depletion and surgical menopause (OVX), in the middle-aged rat model (Acosta et al., 2009a). There was an inverted U-shaped function for serum LH and spatial memory, with the highest and lowest LH levels associated with the best performance. Regression analysis indicated that in rats without ovaries (OVX and VCD followed by OVX), higher LH was associated with better spatial reference memory as tested on the Morris maze (A, with less distance swum associated with better performance), and the water radial-arm maze (B, with fewer errors associated with better performance). In rats with ovaries (SHAM and VCD), higher LH was associated with worse spatial working memory as tested on the water radial-arm maze (C). Graphically, the significant regressions for these analyses are shown as solid lines; the dashed lines of the other groups are shown for comparison purposes to aid interpretation. The inverted U-shaped, quadratic relationship with LH became apparent in scatterplots including all treatment groups so that the range of values across groups could be noted. When LH levels ranged from ~0 to 2, the relationship was positive with higher LH levels associated with worse maze performance. When LH levels ranged from ~2 to 10, the relationship was negative with higher LH levels associated with better maze performance. It is noteworthy that this pattern was seen for multiple domains of spatial memory, including both spatial working and spatial reference memory.

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reversible neoplastic changes. Morphological changes could include dendritic reorganization and alterations in spine density and/or even spine shape, which could have far-reaching effects on neuronal function without obvious cell loss. Hormones from both the HPA and the HPG axes can substantially alter spatial ability. Chronic stress, via elevated glucocorticoids, attenuates hippocam­ pal plasticity by causing substantial hippocampal CA3 dendritic retraction, which is proposed to impair spatial memory and increase hippocampal vulnerability to metabolic challenges in the young individual. In the aged individual, chronic stressinduced CA3 dendritic retraction is hypothesized to become more severe or slower to recover than in the young adult. Consequently, the inverted U-shaped relationship between glucocorticoids and spatial ability described for the young may be narrowed following chronic stress, which may also represent the process that occurs during aging. Moreover, the window by which opportunistic chal­ lenges can potentially trigger hippocampal neuronal death may be broadened in the aged because of the relative slow rate at which the neoplastic changes may recover from chronic stress. Gonadal hormones are potent modulators of brain structure and function, including in brain regions well documented to modulate spatial learn­ ing and memory. Many of these cognitive brain regions are affected by aging. The gonadal hor­ mones estrogens, progesterone and testosterone each impact spatial learning and memory, and there are some indications that aging alters respon­ sivity or sensitivity to these hormones for spatial cognition and postulated underlying neurobiological mechanisms. We propose a tertiary model repre­ senting interactions between aging, spatial learning and memory and gonadal hormones given that: (1) gonadal hormones change with age, (2) aging impacts spatial learning and memory and (3) gona­ dal hormones impact spatial learning and memory. While these individual tenets are driven and sup­ ported by empirical data, a fundamental question in aging research regarding interactions of these three points has yet to be clearly answered. It is, however, clear that the lifetime experiences, exposures and differences in hormonal milieu from the HPA and HPG axes across individuals create an aged

population that is heterogeneous with diverse cog­ nitive and brain aging outcomes. This tremendous brain plasticity can be interpreted within a frame­ work of allowing one the ability to ‘optimize their aging’. By aligning scientific discoveries with clinical interpretations, we can maximize opportunities for interventions so that individuals can optimize their potential for brain health as aging ensues, even in the context of lifetime experiences and stressor exposures.

Acknowledgements The authors gratefully acknowledge the following individuals for their constructive feedback: Jazmin Acosta, Blair Braden, Elizabeth Engler-Chiurazzi and Joshua Talboom.

Abbreviations AD ADX CEE ChAT DHT fMRI FSH GDX HPA HPG LH LTP MPA MRI OVX PBP PFC VCD WHI WHIMS WHISCA

Alzheimer’s disease adrenalectomy conjugated equine estrogen choline acetyltransferase dihydrotestosterone functional magnetic resonance imaging follicle stimulating hormone gonadectomy hypothalamic–pituitary– adrenal hypothalamic–pituitary– gonadal lutenizing hormone long-term potentiation medroxyprogesterone acetate magnetic resonance imaging ovariectomy primed burst potentiation prefrontal cortex 4-vinylcyclohexene diepoxide Women’s Health Initiative Women’s Health Initiative Memory Study Women’s Health Initiative Study of Cognitive Aging

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 3

Menopause and mitochondria: Windows into Estrogen effects on Alzheimer’s disease risk and therapy Victor W. Henderson1,
and Roberta Diaz Brinton2 1

Departments of Health Research & Policy (Epidemiology) and of Neurology & Neurological Sciences, Stanford

University, Stanford, CA, USA

2 Departments of Pharmacology & Pharmaceutical Sciences, Biomedical Engineering and Neurology, University of

Southern California, Los Angeles, CA, USA

Abstract: Metabolic derangements and oxidative stress are early events in Alzheimer’s disease pathogenesis. Multi-faceted effects of estrogens include improved cerebral metabolic profile and reduced oxidative stress through actions on mitochondria, suggesting that a woman’s endogenous and exogenous estrogen exposures during midlife and in the late post-menopause might favourably influence Alzheimer risk and symptoms. This prediction finds partial support in the clinical literature. As expected, early menopause induced by oophorectomy may increase cognitive vulnerability; however, there is no clear link between age at menopause and Alzheimer risk in other settings, or between natural menopause and memory loss. Further, among older post-menopausal women, initiating estrogen-containing hormone therapy increases dementia risk and probably does not improve Alzheimer’s disease symptoms. As suggested by the ‘critical window’ or ‘healthy cell’ hypothesis, better outcomes might be expected from earlier estrogen exposures. Some observational results imply that effects of hormone therapy on Alzheimer risk are indeed modified by age at initiation, temporal proximity to menopause, or a woman’s health. However, potential methodological biases warrant caution in interpreting observational findings. Anticipated results from large, ongoing clinical trials [Early Versus Late Intervention Trial with Estradiol (ELITE), Kronos Early Estrogen Prevention Study (KEEPS)] will help settle whether midlife estrogen therapy improves midlife cognitive skills but not whether midlife estrogen exposures modify latelife Alzheimer risk. Estrogen effects on mitochondria adumbrate the potential relevance of estrogens to Alzheimer’s disease. However, laboratory models are inexact embodiments of Alzheimer pathogenesis and progression, making it difficult to surmise net effects of estrogen exposures. Research needs include better predictors of adverse cognitive outcomes, biomarkers for risks associated with hormone therapy, and tools for monitoring brain function and disease progression. Keywords: Alzheimer’s disease; estrogen; hormone therapy; memory; menopause; mitochondria


Corresponding author. Tel.: 1-605-723-5456; Fax: 1-605-725-6951; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82003-5

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Introduction Gonadal steroid hormones – which include estro­ gens (e.g. 17b-estradiol and estrone), androgens (e.g. testosterone and dihydrotestosterone) and progestagens (e.g. progesterone) – modulate cognitive function and non-reproductive beha­ viors in a variety of mammalian species, including humans. With respect to cognitive aging and dementia, estrogens are of greatest interest to scientists and clinical investigators, because of the striking change in estrogenic hormonal milieu associated with the menopause (Burger et al., 1999) and because estrogen-containing hormone therapy remains among the most commonly prescribed medications. Steroid hormone receptors function as intracel­ lular transcription factors. After ligand activation, they translocate to the cell nucleus, where they bind response elements on the genome to modu­ late expression of target genes. Estrogen, andro­ gen and progesterone receptors are found in human brain, where they are expressed on sub-sets of neurons and glia in topographic distri­ butions unique to each receptor and receptor sub-type. The two types of classic intranuclear recep­ tors for estrogen are estrogen receptor alpha (ERa) and estrogen receptor beta (ERb). They are encoded by different genes on sepa­ rate chromosomes and are expressed on glia and neurons in brain areas involved with cogni­ tive function. A number of ERa and ERb splice variants have been identified in human brain, which are area specific (Taylor et al., 2009) and whose expression may be modified by Alzheimer’s disease (Ishunina and Swaab, 2009). Forebrain cholinergic neurons of the nucleus basalis, believed to play important roles in memory and attention, express ERa (Shughrue et al., 2000). ERb is the predominant estrogen receptor in the neocortex, and both receptor types are expressed on pyramidal neu­ rons and dentate granule cells in the hippocam­ pus (González et al., 2007), an archicortical structure critical to memory encoding. In the mitochondria, estrogen receptors play a pivotal role in regulating energy expenditures and

protecting against oxidative stress (Brinton, 2008; Simpkins et al., 2009). As discussed below, mitochondrial actions provide a model for gauging relations among menopause, estro­ gen exposures and Alzheimer’s disease. Estro­ gen receptors are also associated with the plasma membrane, where G-protein-coupled receptors may serve to regulate intracellular signaling cascades and to mediate rapid effects that do not require genomic activation (Pross­ nitz and Maggiolini, 2009; Raz et al., 2008).

Alzheimer’s disease Dementia can be defined as a decline in cogni­ tive skills that substantially interferes with occupational activities, social activities or inter­ personal relationships. Decline affects more than a single cognitive domain, usually memory plus at least one other area of mental function­ ing. An estimated 24 million people have dementia, and this number is expected to double over the next 20 years (Ferri et al., 2005). Alzheimer’s disease, by far the most common cause of dementia occurring later in life, affects an estimated 2.5–4.5 million older Americans (Hebert et al., 2003; Plassman et al., 2007). More women than men have Alzheimer’s disease, in part because there are more women than men in the oldest segment of the popula­ tion. Studies from Europe (Launer et al., 1999) but not from the United States (Edland et al., 2002) suggest that Alzheimer incidence is increased among women compared to men. Cognitive decline in Alzheimer’s disease begins insidiously and worsens gradually over a period of a decade or more. The earliest manifestation is usually impairment in episodic memory (Small et al., 2000). This form of memory is reflected in one’s ability to learn information and then to recall this information after some interval of time, be it minutes, hours or days. Recollection is explicit (conscious) rather than implicit (without conscious awareness). A decline in episodic memory occurs in dementing disorders other than Alzheimer’s disease, but deficits are less often an early

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or prominent feature. Over time, many Alzheimer patients show behavioral symptoms such as apathy or depression, and they inevitably evince deficits in other cognitive domains. Pathological features of Alzheimer’s disease include neurofibrillary tangles and neuritic pla­ ques. Tangles are intraneuronal inclusions formed of paired helical filaments. These in turn are com­ posed largely of a hyperphosphorylated form of tau, a microtubule-associated protein. Plaques, which are extracellular structures, typically consist of a core of b-amyloid protein, dystrophic nerve processes and activated microglia. Astrocytes are distributed circumferentially around this core. Plaques are associated with a robust chronic inflammatory response (Schwab and McGeer, 2008). Soluble b-amyloid oligomers are neurotoxic (De Felicea et al., 2008), but amyloid in plaque cores is sequestered in an inert b-pleated sheet configuration. Gross cerebral atrophy becomes apparent dur­ ing the course of Alzheimer’s disease. Atrophy is preceded by regional metabolic decline, as demonstrated by positron emission tomography (PET) imaging of the resting brain using a radio­ labeled glucose analogue, 18F-fluorodeoxyglucose (FDG). As revealed by FDG–PET, metabolism in Alzheimer’s disease is typically reduced in neocor­ tical association areas of the parietal and temporal lobes, hippocampus and cortex of the posterior cingulate gyrus (Mosconi et al., 2008).

Estrogens and the brain During a woman’s reproductive years, estrogens (predominately b-estradiol, but also estrone) and progesterone are produced cyclically by develop­ ing ovarian follicles. Menstrual irregularity and fluctuating hormone levels begin on an average about 2 years before the final menstrual period, which is the defining event of the natural meno­ pause. Mean levels of estradiol and estrone fall during this transitional stage, reaching a nadir about 2 years after the final menstrual period (Burger et al., 1999). A distinction is sometimes made between neu­ rosteroids (steroid hormones synthesized within

the central nervous system) and neuroactive steroids (steroid hormones that affect neuronal function independently of origin) (Baulieu, 1997). Estradiol, although clearly neuroactive, was originally not classified as a neurosteroid. It was believed that the central nervous system estrogens were derived solely from steroids pro­ duced in peripheral tissues and circulated to the brain. More recently, however, it has been appre­ ciated that some neurons synthesize estradiol directly from cholesterol, and there may be local effects on synaptic plasticity and other neuronal functions (Hojo et al., 2008). Progesterone is also a neurosteroid (Baulieu, 1997). Thus, effects of menopause on brain activities modulated by ovar­ ian steroids may be less precipitous or calamitous than sometimes envisioned.

Effects on metabolism Metabolic derangements in Alzheimer’s disease brain are evident on FDG-PET images long before the onset of cognitive impairment (Mosconi et al., 2009). Estrogen effects on mitochondrial function seem particularly germane to this reduc­ tion. Energy demands of the healthy brain are extraordinarily high. The brain represents only about 2% of body weight yet consumes 20% of body energy. Energy production is a key function of mitochondria. An outer membrane encloses an intermembrane space, and a folded inner membrane surrounds the mitochondrial matrix (Kroemer and Reed, 2000). These organelles are found in the neuron soma, dendrites, axons and nerve terminals. The density of these orga­ nelles varies within compartments of a single neuron and among different neuronal types (Dubinsky, 2009). In addition to their role in energy generation, mitochondria are also involved in free radical formation, protection against oxidative stress and the determination of cell death through apoptosis. Under normal circumstances, glucose is the near-obligatory substrate for energy required to maintain ionic gradients across cell membranes, to synthesize neurotransmitters and to fuel other metabolic activities in the brain. For each mole

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of glucose oxidized to carbon dioxide and water, about 30 moles of adenosine triphosphate (ATP) are ultimately generated during a set of sequential reactions in the cell cytoplasm and mitochondria (Rich, 2003). As the initial step, blood-borne glucose crosses the blood–brain endothelial bar­ rier to enter astrocytes and neurons. This process of facilitated transport, which is mediated by specific glucose transporters and associated with an increase in insulin growth factor-1 expression, is modulated by estradiol (Cheng et al., 2001; Shi and Simpkins, 1997). Within the cytosol, glucose is converted to pyruvate in the familiar biochemical sequence known as glycolysis. Pyruvate is then decarboxylated, yielding acetyl-coenzyme A, which enters the Krebs tricarboxylic acid cycle. Tricarboxylic acid cycle reactions take place in the matrix of the mitochondrion. Most ATP is generated through the associated process of oxi­ dative phosphorylation coupled to an electron transport chain, whose enzyme complexes are embedded within the inner membrane of the mitochondrion. ERb is found within mitochondrial matrix (Yang et al., 2004), and many of the genes regu­ lated by this receptor sub-type, in contradistinc­ tion to those regulated by ERa, are involved with mitochondrial electron transport and remediation of oxidative stress (O’Lone et al., 2007). Key gly­ colytic enzymes – hexokinase, phosphofructokinase and pyruvate kinase – are up-regulated by estra­ diol during the generation of pyruvate from glucose (Kostanyan and Nazaryan, 1992). Sub­ units of the pyruvate dehydrogenase complex, which regulate the generation of acetyl-coenzyme A from pyruvate, are similarly up-regulated (Nilsen et al., 2007). In addition, estradiol increases activity of complex IV sub-units in the electron transport chain and of ATP synthetase (Nilsen et al., 2007), the final steps in the oxidative phosphorylation of adenosine diphosphate to make ATP. These estrogen effects involve proteins encoded by the nuclear genome (for oxi­ dative phosphorylation) and by the mitochondrial genome (for the electron transport chain). Lasercapture micro-dissection of neurons from autopsy brains confirms under-expression of key sub-units of the electron transport chain in Alzheimer’s

disease patients compared to healthy controls (Liang et al., 2008). Reductions are particularly evident in brain regions where glucose metabo­ lism is known to be diminished in this disorder (posterior cingulum, middle temporal gyrus and the CA1 region of the hippocampus). Functional magnetic resonance imaging shows that short-term use of an estrogen can enhance regional blood flow as cognitive tasks are per­ formed (Joffe et al., 2006; Shaywitz et al., 1999). In this setting, blood flow change is thought to parallel metabolic change. In a 2-year longitudinal study of healthy post-menopausal women, region­ al blood flow was assessed with 15O-PET during performance of memory tasks (Maki and Resnick, 2000). In comparisons between long-term hor­ mone therapy users and women not currently using hormone therapy, the pattern of change in brain activation differed; differences generally reflected increased activation among hormone users in hippocampus and other temporal lobe regions involved with memory. Functional neuroimaging studies also confirm estrogen effects on glucose utilization. In one study of post-menopausal women, acute estra­ diol infusion increased FDG-PET activity in portions of the right frontal cortex and right hippocampus. Path analysis suggested that estradiol had enhanced connectivity within a pre-frontal-hippocampal circuit (Ottowitz et al., 2008). Another small study compared FDG­ PET metabolism between healthy hormone users and non-users (mean age 65 years) (Ras­ gon et al., 2005). Although there were no base­ line metabolic differences between the two groups, 2 years later women not using hor­ mones showed significant declines in FDG­ PET glucose utilization in one of two defined regions of interest (the posterior cingulate but not the lateral temporal cortex). Declines in the same regions were not significant for the hor­ mone users. Another investigative group com­ pared regional metabolism in healthy older women (including both hormone users and non-users) and women with Alzheimer’s disease (mean age about 74 years) (Eberling et al., 2000). Regional metabolic rates were lowest in the Alzheimer group, highest in the hormone user

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group and intermediate for healthy women not using hormone therapy. A follow-up report found a greater metabolic rate in the inferior frontal cortex and temporal cortex among hormone users compared to non-users (mean age 67 years) (Eber­ ling et al., 2004). Among older women with Alzheimer’s disease, spinal fluid concentrations of estradiol are reported to correlate with cerebral glucose metabolism in the left hippocampus but not other brain areas (Scho¨ nknecht et al., 2003). These data, although primarily observational and based on small sample sizes, imply that estrogen-containing hormone therapy improves brain bioenergetics among older post-menopausal women who in most instances have presumably used hormone therapy over long periods of time. Randomized clinical trial data coupled with cogni­ tive outcomes would provide more direct support for this inference, but longer term consequences of hormone therapy are more feasibly studied within an observational framework than with an experimental design.

Other effects A variety of other estrogen effects are potentially relevant to Alzheimer pathogenesis. Mitochon­ drial enzymes within the tricarboxylic acid cycle and the electron transport chain are capable of transferring electrons to oxygen, generating super­ oxide anions. During the vital process of oxidative phosphorylation, reactive oxygen species are thus generated as a by-product of ATP formation. They arise from enzymatic reactions in the mitochondrial outer membrane, inner membrane and matrix. These compounds damage cellular proteins, lipids and nucleic acids. Metabolically active tissues, such as neural tissues, are more vulnerable to oxidative stress. Indeed, oxidative stress is an early event in Alzheimer pathogenesis (Nunomura et al., 2001). Mitochondria are increasingly recognized as key determinants of cell survival or death. A num­ ber of mitochondrial systems are involved in detoxifying reactive oxygen species (Andreyev et al., 2005), and estradiol can reduce free radical formation (Nilsen et al., 2007). Anti-oxidant

effects of estrogens are well documented in sev­ eral model systems (Dykens et al., 2003; Moos­ mann and Behl, 1999). Programmed cell death, or apoptosis, is activated by a wide range of signals and can be initiated through an intrinsic pathway involving release of cytochrome c and other pro­ teins from the mitochondrial membrane space (Kroemer and Reed, 2000). Estradiol increases expression of B-cell lymphoma (Bcl) anti-apopto­ tic proteins Bcl-2 and Bcl-XL (Garcia-Segura et al., 1998; Nilsen and Diaz Brinton, 2003; Pike, 1999), located mainly in the outer membrane, rendering neurons less vulnerable to apoptosis. Calcium sequestration within mitochondria in response to estradiol reduces neuronal vulnerabil­ ity to glutamate excitotoxicity (Brewer et al., 2006; Nilsen and Diaz Brinton, 2003), another potential trigger for apoptosis. Anti-inflammatory actions of estrogens (Pozzi et al., 2006) would be expected to reduce oxidative stress, although estrogen actions are pro-inflammatory as well as anti-inflammatory (Zegura et al., 2003; Hu et al., 2006). Mitochondrial dysfunction is associated with accelerated formation of b-amyloid (Busciglio et al., 2002), although the mechanism is not known. b-Amyloid also contributes to mitochon­ drial toxicity (Lustbader et al., 2004). It is recog­ nized that estradiol speeds up trafficking of the amyloid precursor protein within the Golgi apparatus, reducing the generation of b-amyloid from its precursor (Greenfield et al., 2002). Ovariectomy leads to accumulation of b-amyloid in brains of wild-type laboratory animals (Beach, 2008; Petanceska et al., 2000) and trans­ genic mice that express features of Alzheimer’s disease (Carroll et al., 2007; Zheng et al., 2002). Concentrations of b-amyloid are lowered by treatment with estradiol. Estrogens modulate several neurotransmitter systems. Effects on cholinergic neurons are par­ ticularly relevant to memory and dementia. Magnocellular cholinergic neurons of the basal forebrain nuclei project widely to the hippocam­ pus and neocortex. These neurons express estrogen receptors (Shughrue et al., 1998). They are also selectively vulnerable to neurofi­ brillary tangle formation during the course of Alzheimer’s disease (Rasool et al., 1986) in a

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manner correlated with tangle density in other brain areas (Samuel et al., 1991). In the labora­ tory, estradiol administration after ovariectomy elevates choline acetyltransferase activity, a mar­ ker of acetylcholine synaptic activity, in the basal forebrain and in cortical projection areas (Luine, 1985; Ping et al., 2008; Yamamoto et al., 2007). Estrogen actions on basal forebrain cholinergic neurons mediate physiological effects of estro­ gen on the hippocampus (Rudick et al., 2003) and on performance enhancement on certain kinds of learning tasks (Gibbs, 2002; Gibbs et al., 2009; Markowska and Savonenko, 2002).

Implications for Alzheimer’s disease Given multi-faceted effects of estrogen – includ­ ing improved metabolic profile, lower oxidative stress, reduced b-amyloid formation and enhanced cholinergic transmission – endogenous estrogen and exogenous exposures in the form of estrogen-containing hormone therapy would be expected to influence Alzheimer pathogenesis and symptoms. Predictions include the following: Early menopause should be attended by greater risk of Alzheimer’s disease later in life; estrogen therapy should improve symptoms of Alzheimer’s disease; and hormone therapy should reduce Alzheimer risk. Because episodic memory impairment is a recognized risk factor for Alzheimer’s disease (Elias et al., 2000), it might also be predicted that natural menopause would be attended by memory decline and that hormone therapy would benefit memory perfor­ mance in women without dementia. As consid­ ered below, some of these predictions find support in the clinical data, but others are not supported at all.

Early menopause and cognitive risk In the following discussion, early menopause is used in a general way to describe menopause occurring before the mean age of natural menopause, about 51 years. Little research regarding cognitive out­ comes has included women with premature

menopause, usually defined as menopause occur­ ring before 40 years of age. Statements on early menopause or statements regarding younger age of hormone therapy initiation should, therefore, not be generalized to women with premature menopause. If exposures to endogenous estrogens reduce the risk of developing Alzheimer’s disease, then early menopause should be associated with elevated risk. There is partial support for this prediction. The largest group of women undergoing early meno­ pause are those whose menopause is induced by oophorectomy. By definition, surgical menopause occurs prior to when natural menopause would have otherwise occurred. In a large case–control study from Olmstead County, Minnesota, oophor­ ectomy was associated with elevated risk later in life of cognitive impairment or dementia (relative risk 1.5, 95% confidence interval 1.1–1.9) (Rocca et al., 2007). Risk in this study increased with younger age at the time of surgery. When compared to risks of women not undergoing surgical menopause, bilat­ eral oophorectomy before age 43 years was asso­ ciated with a relative risk of 1.7, between the ages of 43 and 48 with the same risk (namely 1.7), and after age 48 years with a relative risk (1.1) similar to that of the reference group. Early menopause is also associated with increased Alzheimer’s disease risk in women with Down’s syndrome, a chromosomal disorder where pathological features of Alzheimer’s disease appear at an unusually early age. In a community-based sample of women aged 40–60 years, the risk of Alzheimer’s disease for women undergoing menopause before the age of 46 years was 2.7 (95% confidence interval 1.2–5.9) times that of women experiencing later menopause (Schupf et al., 2003). Similar findings are reported in Down’s syndrome cohorts from Ireland and the Nether­ lands, where early age at menopause was signifi­ cantly associated with younger age at diagnosis of dementia (Coppus et al., 2009; Cosgrave et al., 1999). The predicted association between menopause age and Alzheimer’s risk, however, is challenged by findings from several cohorts of older women, where no significant relations between age at menopause and Alzheimer risk were observed (Baldereschi et al., 1998; Paganini-Hill and

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Henderson, 1996; Roberts et al., 2006; Tang et al., 1996). In the Leisure World retirement commu­ nity, for example, when compared to women reporting menopause before age 45 years, the relative risk of Alzheimer’s disease for women undergoing menopause between ages 45 and 54 years was 1.0, and for women older than 54 years, risk was 1.2 (test for trend p = 0.6) (Paganini-Hill and Henderson, 1996).

Hormone therapy and symptoms of Alzheimer’s disease Early, very small open-labelled studies of hormone use among women with Alzheimer’s disease raised the hope that estrogen treatment might improve dementia symptoms (e.g. Fillit et al., 1986; Honjo et al., 1989; Ohkura et al., 1994). This expectation has since been assessed in randomized placebocontrolled, double-blind trials (Asthana et al., 1999, 2001; Henderson et al., 2000; Honjo et al., 1993; Mulnard et al., 2000; Rigaud et al., 2003; Wang et al., 2000; Zhang et al., 2006) (Table 1). Most blinded trials have been relatively small and of relatively short duration. Most, but not all, suggest no cognitive, functional or global benefit. In particular, the largest, longest trial reported no benefit of hormone therapy in this setting (Mulnard et al., 2000), and a recent systematic review concluded that hormone therapy is not indicated for cognitive improvement or main­ tenance in women with Alzheimer’s disease (Hogervorst et al., 2009).

Hormone therapy and risk of Alzheimer’s disease Many, but not all, case–control and cohort stu­ dies have associated hormone therapy use with lower risks of Alzheimer’s disease. Modest but significant protective associations are reported from Leisure World (Paganini-Hill and Hender­ son, 1996), northern Manhattan (Tang et al., 1996), the Baltimore Longitudinal Study of Aging (Kawas et al., 1997),and Cache County, Utah (Zandi et al., 2002). Meta-analyses suggest overall risk reductions of about a third

(Hogervorst et al., 2000). This estimate, if valid, has obvious public health implications. Disappointing clinical trial results reported from the Women’s Health Initiative Memory Study (WHIMS) (Shumaker et al., 2004) chal­ lenge a sanguine interpretation of the observa­ tional findings on hormone therapy and Alzheimer risk. WHIMS was designed as an ancil­ lary study embedded within the Women’s Health Initiative clinical trials. WHIMS eligibility was restricted to participants who were at least age 65 years. The primary outcome was incident dementia, and a total of 108 women developed dementia during the parallel WHIMS trials (Table 2). Half of the dementia cases were attrib­ uted to Alzheimer’s disease, but separate out­ comes were not reported for this diagnosis. In the estrogen–progestin trial of women with a uterus, the relative risk of dementia for women who were assigned to hormone treatment was double that of women assigned to placebo (Shumaker et al., 2003). In the estrogen-alone trial of women with prior hysterectomy, the rela­ tive risk for women assigned to estrogen was also increased, but not significantly so (Shumaker et al., 2004). Why do WHIMS findings not support observa­ tional inferences of estrogen protection against Alzheimer’s disease? A common explanation is that observational findings are methodologically flawed by difficulty to resolve biases (Henderson, 2006). Prior to hormone therapy initiation, hor­ mone users are often healthier than non-users and are more likely to engage in health-promoting behaviors (Matthews et al., 1996). These differ­ ences might reduce Alzheimer risk independently of hormone use (healthy user bias). Recall bias in some reports might also contribute to apparent benefit (Petitti et al., 2002). A second candidate for the discrepancy con­ cerns generalization of WHIMS outcomes to the broader population of women likely to consider hormone therapy (Henderson, 2006). This consid­ eration is relevant if key characteristics of WHIMS participants differed from those of women in observational studies and, equally important, if effects of hormone therapy on dementia risk are modified by these

Table 1. Randomized, double-blind, placebo-controlled trials of hormone therapy in women with Alzheimer’s disease

Reference

Age mean (years)

Menopause typea

Number

Duration

Primary cognitive outcome

Functional outcomeb

Global outcomeb

Asthana et al. (1999) Henderson et al. (2000) Mulnard et al. (2000) Wang et al. (2000) Asthana et al. (2001) Rigaud et al. (2003)e Zhang et al. (2006)f

79 78 75 72 80 76 55

Natural Both Surgical Natural Both Both Not stated

12 42 120 50 20 117 41

8 weeks 16 weeks 12 months 12 weeks 8 weeks 28 weeks 16 weeks

þEstrogenc NS NS NS þEstrogenc NS þEstrogen

– NS NS – NS NS þEstrogen

– NS NSd NS NS NS NSf

Trials with a duration of at least 1 month and an objective measure of cognitive outcome. Active treatment was with oral conjugated equine estrogens (Henderson et al., 2000; Honjo et al., 1993; Mulnard et al., 2000; Wang et al., 2000; Zhang et al., 2006), oral estradiol (Rigaud et al., 2003) or transdermal estradiol (Asthana et al., 1999; 2001). Women in Rigaud et al. (2003) randomized to estradiol also received oral progesterone. þEstrogen = significant difference in favour of active treatment with an estrogen. NS = non-significant probability p < 0.05. a Natural menopause is inferred from statements that all participants underwent Papanicolaou examinations; surgical menopause is based on hysterectomy status. b Functional outcomes assess activities of daily living; global outcomes reflect overall change. c No cognitive outcome was defined as primary; results favoured women in the estrogen group on a sub-set of cognitive tasks. d No difference on the primary global outcome (Clinical Global Impression of Change); significant difference favoured placebo on the Clinical Dementia Rating scale. e Women in both groups received a cholinesterase inhibitor. f Participants were younger than 65 years of age; dosing schedules differed for hormone (once daily) and vitamin B1 placebo (3 times daily), implying the possibility of unblinding. Betweengroup comparisons were not provided; presented data imply significant differences favouring conjugated equine estrogens for cognition (revised Hasegawa Dementia Scale) and activities of daily living but probably not for global performance (Functional Activities Questionnaire).

85 Table 2. Randomized, double-blind, placebo-controlled trials of hormone therapy in older women without dementia: dementia outcomes in the Women’s Health Initiative Memory Study

Reference Shumaker et al. (2003)

Shumaker et al. (2004)

Age range (years)

Menopause typea

Number

Duration (years)

Number of events

Hazard ratio (95%

confidence interval)

Probability

65–79

Surgical

4532

4.1

61

2.1 (1.2–3.5)

0.01

65–79

Natural

2947

5.2

47

1.5 (0.8–2.7)

0.18

Active treatment was with conjugated equine estrogens (0.625 mg/d) plus medroxyprogesterone acetate (2.5 mg/d) in a continuous combined oral formulation (Shumaker et al., 2003) or conjugated equine estrogens alone (Shumaker et al., 2004). a Surgical menopause based on hysterectomy status; in the parent Women’s Health Initiative, 41% of women with hysterectomy reported bilateral oophorectomy (Stefanick et al., 2003).

characteristics. One especially salient difference is the age when hormone therapy was initiated and used. Vasomotor symptoms are most preva­ lent near the time of menopause, and most hor­ mone therapy is prescribed to reduce these bothersome symptoms. Hormone use in observa­ tional studies thus tended to be at a younger age in closer proximity to the menopause. Hormone initiation (or re-initiation among prior hormone users) in WHIMS occurred at age 65 years or older.

Atherosclerosis as a model for the critical window hypothesis Is it reasonable to consider that effects of estrogen therapy might be modified by age, proximity to menopause (i.e. duration of ovarian hormone deprivation) or some health-related factor asso­ ciated with age? One potential mechanism for a so-called ‘critical window’ (Resnick and Hender­ son, 2002) or ‘timing’ (Clarkson and Mehaffey, 2009) effect is the down-regulation of estrogen receptors after a prolonged period of ligand depri­ vation (Toran-Allerand, 2000). Prolonged hypo­ estrogenemia might also alter the number or type of estrogen receptor, or its splice variants, leading to a different effect when estrogen expo­ sures recur. A somewhat different model for the critical window hypothesis – one tied more closely to health factors – relates to atherosclerosis, an

age-associated pathological alteration chiefly affecting walls of large elastic and muscular arteries. Atherosclerosis is an inflammatory, pro­ liferative lesion, which, when advanced, includes endothelial disruption, extracellular lipid deposits, lipid-laden macrophages, an acute-phase inflam­ matory response, smooth muscle proliferation, disruption of the intercellular matrix and collagen deposition (Stary et al., 1992). Experimental and clinical data indicate that an exogenous estrogen could help prevent atherosclerosis when adminis­ tered during one temporal window (younger age with less atherosclerosis, close to menopause) but have no effect or be deleterious when adminis­ tered during another window of time (older age with more atherosclerosis, remote from meno­ pause). In vitro, a similar phenomenon has been proposed as the ‘healthy cell bias’ hypothesis of estrogen action, suggesting that healthy neurons respond differently to an estrogen than stressed neurons (Chen et al., 2006). Genomic and non-genomic effects of estradiol on vascular endothelial and smooth muscle cells promote endothelial restoration after vascular injury and enhance vasodilation (Mendelsohn and Karas, 1999). These salubrious effects are antagonized by 27-hydroxycholesterol, a choles­ terol metabolite found in atherosclerotic lesions (Umetani et al., 2007). In animal models, estradiol can inhibit new fatty deposits without inhibiting progression of established vascular lesions (Rosenfeld et al., 2002); this protective effect may depend on an intact vascular endothelium

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(Hanke et al., 1999). In a primate model, large artery atherosclerosis is reduced when estrogens are given immediately after ovariectomy but not when treatment is delayed (Clarkson and Mehaf­ fey, 2009). Indeed, rupture of an established atherosclerotic plaque is more likely, rather than less likely, in the presence of estrogens. In humans, oral estradiol reduces progression of sub-clinical atherosclerosis in healthy post-meno­ pausal women (mean age 62 years) (Hodis et al., 2001) but has no effect on atherosclerosis progres­ sion in women of about the same age with estab­ lished coronary artery disease (Hodis et al., 2003). In the Women’s Health Initiative clinical trials, women who initiated hormone therapy closer to menopause tended to have less coronary heart dis­ ease risk compared with increased risk among women more distant from menopause (Rossouw et al., 2007). Among surgically menopausal partici­ pants in the Women’s Health Initiative, prior use of hormone therapy close to the time of bilateral oophorectomy was associated with a lower preva­ lence of coronary artery calcification, a risk factor for coronary heart disease (Allison et al., 2008).

Critical window and cognitive outcomes The atherosclerosis model might be directly rele­ vant to Alzheimer’s disease pathogenesis. Risk factors for Alzheimer’s disease and vascular disease overlap substantially (Stampfer, 2006), and cerebrovascular disease potentiates clinical manifestations of Alzheimer neuropathology (Schneider et al., 2007). However, here is no direct evidence that adverse vascular consequences of hormone therapy led to dementia in the WHIMS trials (Coker et al., 2009). Clinical evidence supporting the critical window hypothesis for cognitive outcomes remains limited. In randomly selected households of women over age 60 years, self-reported early initiation of hor­ mone therapy was associated with better perfor­ mance on some cognitive tasks, whereas initiation in late post-menopause was associated with worse performance (MacLennan et al., 2006). Follow-up

analysis of participants in studies of hormone ther­ apy for osteoporosis found that middle-age women randomly assigned to hormone treatment for 2 or 3 years were less likely to be cognitively impaired after a mean interval of 11 years than women in placebo groups (Bagger et al., 2005). Use of hor­ mone therapy at a younger age, i.e. past use but not current use, was associated with reduced Alzheimer risk in the Cache County cohort (Zandi et al., 2002). In the Multi-Institutional Research in Alzheimer Genetic Epidemiology (MIRAGE) study, hormone therapy was associated with reduced Alzheimer risk among younger, but not older, post-menopausal women (Henderson et al., 2005); hormone use necessarily occurred at a younger age among younger women. In animal models of learning, specific effects of estrogens depend on the behavioral paradigm, animal age, interval between ovariectomy and estrogen replacement and mode administration (e.g. Markowska and Savonenko, 2002; Savonenko and Markowska, 2003; Zurkovsky et al., 2007). Given these experimental complex­ ities, it is interesting that behavioral enhancement in some paradigms is observed only if an estrogen is initiated soon after ovariectomy (Daniel et al., 2006; Gibbs, 2000), as predicted by the critical window hypothesis. Menopause and episodic memory Women cannot be randomly ‘assigned’ to undergo menopause, and thus it is obvious that cognitive consequences of the natural menopause in humans cannot be studied experimentally. That is not to say that important questions cannot be rigorously addressed and reasonably answered. Further, cognitive consequences of hormone ther­ apy can be addressed experimentally in rando­ mized clinical trials, although such trials are more feasible for short-term than long-term outcomes. One emerging conclusion from cohorts of midlife women seems to be that cognitive function is not substantially impacted by the natural meno­ pause, despite pronounced changes in hormonal milieu during this midlife transition. At least, no

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important short-term decline is readily discernible. It is more difficult to know whether potential cognitive consequences might become evident only some decades later. Many women complain of forgetfulness around the time of the menopausal transition (Mitchell and Woods, 2001). Because of the association between episodic memory loss and Alzheimer’s disease, this symptom is of course worrisome. However, forgetfulness is a common symptom at other ages as well, and self-reported poor memory is often more strongly linked to low mood than to objective loss of memory per­ formance (Weber and Mapstone, 2009). Crosssectional and longitudinal findings from midlife cohorts in Australia, the United Kingdom, Taiwan, Sweden and the United States are con­ sistent in suggesting that the natural menopausal transition probably has no important effect on episodic memory or on other cognitive skills (Fuh et al., 2006; Henderson et al., 2003; Herlitz et al., 2007; Kok et al., 2006; Luetters et al., 2007). Analyses from the multi-ethnic Study of Women’s Health Across the Nation (SWAN) sample suggests a mild learning deficit during the menopausal transition compared to pre­ menopause, inferred from annual trends in prac­ tice effects (Greendale et al., 2009). However, this small reduction in practice effect was not statistically significant, and there was no reduc­ tion in practice effect when midlife women prior to entering the menopausal transition were com­ pared to women in the early post-menopause. Clinical relevance may confined to the transition per se, a time when estrogen levels are charac­ terized by large variability (Burger et al., 1999).

Hormone therapy and episodic memory Because cognitive outcomes of estrogen-contain­ ing hormone therapy could vary depending on age of initiation or use, the following discussion, which emphasizes findings from randomized clinical trials, separates hormone use by midlife women and by older post-menopausal women. Age 65 years is often taken as a convenient dividing line for this purpose.

Midlife women without dementia Two small, short-term randomized clinical trials in women with surgical menopause suggest that estradiol improves verbal episodic memory when initiated in this setting (Sherwin, 1988; Phillips and Sherwin, 1992). After natural menopause, however, randomized clinical trials in midlife women have generally not reported significant effects of hormonal treatment (reviewed by Henderson and Sherwin, 2007). Treatment durations have been relatively short and sample sizes relatively small. The largest trial involved 180 post-menopausal women aged 45–55 years randomized to conjugated equine estrogens combined with medroxypro­ gesterone acetate, or placebo. Four months of treatment provided no benefit for memory or other cognitive skills (Maki et al., 2007). Two much larger clinical trials currently in progress, the KEEPS (ClinicalTrials.gov identifier NCT00154180) and the ELITE (NCT00114517) will provide clearer evidence regarding cogni­ tive outcomes in this age group after treatment with oral estradiol (ELITE), oral conjugated equine estrogens (KEEPS) and transdermal estradiol (KEEPS). There are several possibilities for differences in study outcomes when trials after surgical menopause are compared to trials after natural menopause (Henderson and Sherwin, 2007). One is that differences are due to chance. Another possibility is reporting bias; results of a small trial with a significant outcome are more likely to be submitted and accepted for publica­ tion than findings from a small trial where between-group comparisons are not significant. An interesting possibility concerns the younger age of women in the two studies of surgical menopause (mean ages of 45 and 48 years) (Phillips and Sherwin, 1992; Sherwin, 1988), compared to women studied after natural meno­ pausal. Other considerations are the prompt initiation of treatment at the time of bilateral oophorectomy, the particular hormone formula­ tion used in these surgical menopause trials and unique physiological changes associated with surgical menopause.

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The hormone formulation is probably not key, even though biological effects of estradiol differ from those of other estrogens and biological effects of progesterone differ from those of syn­ thetic progestins (Brinton et al., 1997; Nilsen and Brinton, 2003). Although surgically menopausal women in these two trials were treated with high doses of parenteral estradiol, similar findings regarding verbal memory in younger women are reported with the addition of a standard dose of oral conjugated equine estrogens after pharmaco­ logical suppression of ovarian function (Sherwin and Tulandi, 1996). With respect to the hormone milieu, natural menopause is attended, of course, by loss of ovarian estrogens and progesterone. In addition to loss of these gonadal steroids, surgical menopause is also accompanied by reduced levels of testosterone (Davison et al., 2005). After nat­ ural menopause, testosterone is derived in part from androgen precursors produced by residual ovarian stromal cells, and hormonal losses are

thus exacerbated among surgically menopausal women. A potential modulating role for testoster­ one is plausible but remains to be explored fully.

Older women without dementia Although clinical trial findings on hormone ther­ apy are limited for middle-age women, more sub­ stantial data exist for older women (Henderson and Sherwin, 2007). Findings from larger rando­ mized trials of late post-menopausal women are summarized in Table 3. The table separates hor­ mone effects on episodic memory tasks from hor­ mone effects on other types of cognitive tasks, because of the important relation between mem­ ory decline and Alzheimer’s disease. As shown in this table, randomized assignment to hormone therapy in these trials did not notably improve episodic memory and did not show consistent effects in other areas.

Table 3. Large randomized, double-blind, placebo-controlled trials of hormone therapy in older women without dementia: cognitive outcomes

Reference

Age mean or range (years)

Menopause typea

Number

Duration

Episodic memory

Other cognitive outcomes

Grady et al. (2002)b Rapp et al. (2003)d Espeland et al. (2004)d Viscoli et al. (2005)b Almeida et al. (2006) Resnick et al. (2006)d Yaffe et al. (2006) Resnick et al. (2009)d

67 65–79 65–79 70 74 71 67 74

Both Natural Surgical Both Surgical Natural Natural Surgical

1063 4381 2808 461 115 1416 417 886

4 years 4 years 5 years 3 years 5 months 4 years 2 years 6 years

NS – – NS NS Variablee NS NS

Most NSc NS NS NS NS NS NS Most NSf

Trials with sample size of at least 100, mean age of at least 60 years, trial duration of at least 1 month and an objective measure of cognitive outcome.

Active treatment was with conjugated equine estrogens (Espeland et al., 2004; Grady et al., 2002; Rapp et al., 2003; Resnick et al., 2006, 2009), oral

estradiol (Almeida et al., 2006; Viscoli et al., 2005), or very low-dose transdermal estradiol (Yaffe et al., 2006). Some women randomized to an estrogen

also received a progestagen (medroxyprogesterone acetate) (Rapp et al., 2003; Resnick et al., 2006).

NS = non-significant probability p > 0.05.

a Surgical menopause based on hysterectomy status.

b Participants had coronary heart disease (Grady et al., 2002) or cerebrovascular disease (Viscoli et al., 2005).

c Significant difference in verbal fluency favoured the placebo group; other cognitive outcomes did not differ.

d Women’s Health Initiative Memory Study of women with (Rapp et al., 2003; Resnick et al., 2006) or without (Espeland et al., 2004; Resnick et al.,

2009) a uterus. Rapp et al. (2003) and Espeland et al. (2004) report global cognition on the Modified Mini-Mental State examination. In the Women’s Health Initiative Study of Cognitive Aging, Resnick et al. (2006, 2009) report more detailed cognitive analyses on sub-sets of women included in reports of Rapp et al. (2003) and Espeland et al. (2004). e Based on annual rates of change, significant differences on verbal memory favoured the placebo group, and significant differences on non-verbal memory favoured the hormone group. f Significant differences on a mental rotation task 3 years after treatment randomization favoured the placebo group, but thereafter the estrogen group showed greater improvement over time.

89

Inferences and conclusions It is difficult to disentangle the relation between menopause – a normal, universal midlife event for women – and Alzheimer’s disease, a common late-life disorder affecting both women and men. We begin with a physiological process character­ ized by loss of ovarian hormone production and end with a series of clinical events that are in some instances difficult to reconcile with each other and with experimental findings from the basic laboratory. This focussed review has emphasized laboratory effects of estrogens acting on and through mitochondria, recognizing that a number of relevant actions involve other biolo­ gical targets. The clinical tableau seems to be the following: Early menopause induced by oophorectomy (sur­ gical menopause) may increase cognitive vulner­ ability (Phillips and Sherwin, 1992; Rocca et al., 2007; Sherwin, 1988), but in other settings there is no clear link between menopause age and Alzhei­ mer risk (Baldereschi et al., 1998; Paganini-Hill and Henderson, 1996; Roberts et al., 2006; Tang et al., 1996). Estrogen therapy initiation probably does not improve Alzheimer’s disease symptoms (Table 1); one of the small trials reporting cogni­ tive benefit to women with Alzheimer’s disease involved only participants younger than 65 years (Zhang et al., 2006). Importantly, hormone ther­ apy initiated at an older age is linked to increased – not decreased – dementia risk (Table 2). Whether effects of hormone therapy on Alzhei­ mer risk are modified by age at initiation, or by prolonged use after early initiation, is implied by some observational results but is not answered with certainty by current evidence. WHIMS find­ ings of increased dementia risk with late life initia­ tion counsel caution in interpreting the observational data. Further, contrary to prediction, natural meno­ pause is not attended by persistent memory decline across the menopause transition, and hor­ mone therapy does not appear to boost memory performance, certainly not when initiated during the late post-menopause (Table 3). Results from the ELITE and KEEPS trials will inform us whether similar results are expected from

hormone use initiated at younger ages, and estro­ gen effects on memory after early menopause merit further study. Despite disappointing outcomes in short- and medium-term clinical trials of estrogens (Tables 1–3), other clinical data raise the possibi­ lity that long-term outcomes might differ. With respect to dementia, increasing duration of hor­ mone use by healthy women is associated with decreasing risk of Alzheimer’s disease (PaganiniHill and Henderson, 1996), although prolonged use is not associated with better cognition when initiated at older ages (Kang et al., 2004). Some observational studies imply that hormone users who develop Alzheimer’s disease experience milder symptoms than women who develop Alzheimer’s disease but are not taking hormones (Doraiswamy et al., 1997; Henderson et al., 1994). Hormone use by these patients likely began years prior to the onset of overt dementia, a situation different from that of initiating ther­ apy after the onset of cognitive impairment. Long-term hormone users, however, are often relatively healthier than never users and former users (reflecting healthy user bias and compli­ ance bias), and any inference concerning longterm estrogen use, Alzheimer symptom ameliora­ tion or other cognitive benefit is at best speculative. The important role of estrogens on brain metabolism and the relation between metabolic decline and Alzheimer’s disease risk suggest a role for FDG-PET as a surrogate marker, for example, after the randomized assignment of an estrogen to recently menopausal women. The predicted response would be an increase in resting brain metabolism, in comparison to pla­ cebo. According to the critical window or healthy cell hypothesis, no discernible response or even a reduced metabolic response would be expected in older post-menopausal women remote from the menopause. This prediction for older women will be more difficult to assess experimentally, given accumulating evidence that hormone initiation in this age group is attended by competing health risks (Anderson et al., 2004; Rossouw et al., 2002; Shumaker et al., 2004).

90

An important consideration, but still not yet well investigated, is that neuronal synthesis of estradiol and other neurosteroids might moder­ ate central nervous system consequences of menopausal loss. In a small post-mortem series, significant concentrations of estradiol were identified in brains of post-menopausal women, suggesting a role for local neuronal production (Bixo et al., 1995). In the hippocampus, neuronal metabolism after menopause may be augmented by locally synthesized estrogens (Ishunina et al., 2007). Nevertheless, regional brain estradiol levels appeared to reflect peripheral concentrations, sug­ gesting that brain levels also depend in part on ovarian production (Bixo et al., 1995). In the pre-clinical laboratory, healthy cellular and animal models acutely exposed to toxic insults provide an inexact model of human neurodegen­ eration, where the process is one of incremental damage over a period of years (Brinton, 2008). A related difficulty in translating laboratory findings to a clinical arena is that sex steroid effects are complex, affect brain functions directly as well as indirectly through actions on non-neural tissues and are almost certainly modified by a variety of health-related and age-related factors (Table 4). Net effects on Alzheimer’s disease risk or on memory performance are therefore difficult to predict. Estrogen effects on mitochondrial function are likely to decrease risk for Alzheimer’s disease. Other actions – including increased levels of

inflammatory proteins and proteins that play roles in coagulation (Katayama et al., 2009) – may affect cognition adversely, leading to out­ comes different from those anticipated on the basis of simpler in vitro experiments and in vivo models. These actions may account, for example, for increased incidence of ischaemic stroke reported in some trials of hormone therapy (Rossouw et al., 2007). Outcomes could also vary according to estrogen type (Brinton et al., 1997), dose (Chen et al., 2006), dosing schedule (Mar­ kowska and Savonenko, 2002) and route of administration (Zegura et al., 2003). Finally, in the laboratory (e.g. Rosario et al., 2006; Tanapat et al., 2005) and perhaps in the clinic (Kang et al., 2004; Rice et al., 2000), nervous system effects of an estrogen differ from those of an estrogen com­ bined with a progestagen. The challenges are daunting but not unsolvable. As suggested by participants in a National Insti­ tute on Aging workshop on estrogen and the aging female brain (Asthana et al., 2009), there remains pressing need for pre-clinical and clinical research on the relation between the menopausal transition and midlife exposures to estrogens, progestagens and related compounds, and on risks for age-asso­ ciated cognitive disorders such as Alzheimer’s dis­ ease. Research needs include better predictors of adverse cognitive outcomes, biomarkers for risks associated with hormone therapy and tools for monitoring brain function and disease progression.

Table 4. Potential factors contributing to net estrogen effects on Alzheimer’s disease Specific effects Different estrogens have different neural and non-neural effects Different progestagens have different neural and non-neural effects Class effects Wide-ranging effects on neural tissues Wide-ranging effects on non-neural tissues that affect brain function Effects modified by dose, dosing schedules (e.g. continuous or sequential) and route (e.g. oral or transdermal) Effects modified by progestagens and androgens Effects modified by age at time of hormone exposure or by timing with respect to menopause (critical window hypothesis) Effects modified by other physiological parameters (e.g. presence of atherosclerosis) (healthy cell bias hypothesis, critical window hypothesis) Menopausal hormonal losses mitigated by local effects of neurosteroids

91

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 4

DHEA, important source of sex steroids in men and even more in women Fernand Labrie
Research Center in Molecular Endocrinology, Oncology and Human Genomics, Laval University and

Laval University Hospital Research Center (CRCHUL), Qu�ebec, Canada

Abstract: A major achievement from 500 million years of evolution is the establishment of a high secretion rate of dehydroepiandrosterone (DHEA) by the human adrenal glands coupled with the indroduction of menopause which stops secretion of estrogens by the ovary. Cessation of estrogen secretion at menopause eliminates the risks of endometrial hyperplasia and cancer which would result from non-opposed estrogen stimulation during the post-menopausal years. In fact, from the time of menopause, DHEA becomes the exclusive and tissue-specific source of sex steroids for all tissues except the uterus. Intracrinology, a term coined in 1988, describes the local formation, action and inactivation of sex steroids from the inactive sex steroid precursor DHEA. Over the past 25 years most, if not all, the genes encoding the human steroidogenic and steroid-inactivating enzymes have been cloned and sequenced and their enzymatic activity characterized. The problem with DHEA, however, is that its secretion decreases from the age of 30 years and is already decreased, on average, by 60% at time of menopause. In addition, there is a large variability in the circulating levels of DHEA with some post-menopausal women having barely detectable serum concentrations of the steroid while others have normal values. Since there is no feedback mechanism controlling DHEA secretion within ‘normal’ values, women with low DHEA will remain with such a deficit of sex steroids for their remaining lifetime. Since there is no other significant source of sex steroids after menopause, one can reasonably believe that low DHEA is involved, in association with the aging process, in a series of medical problems classically associated with post-menopause, namely osteoporosis, muscle loss, vaginal atrophy, fat accumulation, hot flashes, skin atrophy, type 2 diabetes, memory loss, cognition loss and possibly Alzheimer’s disease. A recent randomized, placebo-controlled study has shown that all the signs and symptoms of vaginal atrophy, a classical problem recognized to be due to the hormone deficiency of menopause, can be rapidly improved or corrected by local administration of DHEA without systemic exposure to estrogens. In addition, the four domains of sexual dysfucntion are improved. For the other problems of menopause, although similar large scale, randomized and placebo-controlled studies usually remain to be performed, the available evidence already strongly suggests that they could be improved, corrected or even prevented by exogenous DHEA.
Corresponding author. Tel.: 418-652-0197; Fax: 418-651-1856; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82004-7

97

98

In men, the contribution of adrenal DHEA to the total androgen pool has been measured at 40% in 65–75-year-old men. Such data stress the necessity of blocking both the testicular and adrenal sources of androgens in order to achieve optimal benefits in prostate cancer therapy. On the other hand, the comparable decrease in serum DHEA levels observed in both sexes has less consequence in men who continue to receive a practically constant supply of testicular sex steroids during their whole life. In fact, in men, the appearance of hormone-deficiency symptoms common to women is observed at a later age and with a lower degree of severity. Consequently, DHEA replacement has shown much more easily measurable beneficial effects in women. Most importantly, despite the non-scientific and unfortunate availability of DHEA as a food supplement in the United States, a situation that discourages rigorous clinical trials on the crucial physiological and therapeutic role of DHEA, no serious adverse event related to DHEA has ever been reported in the world literature (thousands of subjects exposed) or in the monitoring of adverse events by the FDA (millions of subjects exposed), thus indicating, as expected from its known physiology, the excellent safety profile of DHEA. With today’s knowledge, one can reasonably suggest that DHEA offers the promise of a safe and efficient replacement therapy for the multiple problems related to hormone deficiency after menopause without the risks associated with estrogen-based or any other treatments. Keywords: DHEA; intracrinology; menopause; osteoporosis; vaginal atrophy; type 2 diabetes; Alzheimer’s disease

Introduction The unique physiological importance of DHEA in men was first recognized in the early 1980s when it was discovered that after complete medical castration achieved with gonadotropin-releasing hormone (GnRH) agonists in men suffering from prostate cancer (Labrie et al., 1980), 40–50% of active androgens were left in the prostate, thus indicating an important extratesticular source of androgens (Labrie et al., 1985). When pooling data obtained by various laboratories around the world, values of residual intraprostatic dihydrotestosterone (DHT) range from 25 to 50% for an average of 40% of DHT left in the prostate after castration (Labrie et al., 2009b). That originally surprising observation is now well explained by the transformation of DHEA into androgens and/or estrogens by specific steroidogenic enzymes in each cell type in each peripheral target tissue according to the process of intracrinology, an expression coined in 1988 (Labrie, 1991; Labrie et al., 1988; Labrie et al., 2005; Luu-The and Labrie, 2010). While the supply of sex steroids from DHEA decreases in both men and women in a comparable fashion from the age of 30 years, men receive a

practically continuous supply of testosterone, estrone (E1) and estradiol (E2) from the testicles during their whole life (Fig. 1) while, in women, E2 secretion by the ovaries stops at menopause (Fig. 2). Consequently, after menopause, all estrogens and androgens are derived from DHEA which has already decreased by an average of 60% at time of menopause (Labrie et al., 2006) and continues to decrease thereafter (Fig. 3), with some women hav­ ing barely detectable serum levels (Labrie, 2010). Since DHEA is the only source of sex steroids after menopause, it is reasonable to believe that such a decrease in DHEA-derived sex steroid availability, coupled with aging, is at least partially responsible for the numerous symptoms of hormone deficiency observed after menopause. These pertain to vaginal atrophy, bone loss, fat accumulation, type 2 diabetes, skin atrophy, cognition problems, memory loss and possibly Alzheimer’s disease (Labrie, 2007). A major problem with DHEA, the only source of sex steroids after menopause, is that there is no feedback control of its secretion. In other words, there is no endogenous mechanism in either women or men to increase DHEA secretion when serum DHEA concentrations become low. Consequently, the only possibility to correct a

99 A.

Women

B.

GnRH

GnRH CRH

Anterior pituitary

E2

Men

LH

ACTH

Cortisol

Ovary

Adrenal

Estradiol (E2)

CRH

Anterior pituitary

DHEA

Testosterone

LH

ACTH

Adrenal

Testis

Testosterone

DHEA

Intracrinology

E2 Testo DHT Peripheral target tissues

Cortisol

Intracrinology

E2

Testo

DHT

Peripheral target tissues

Fig. 1. (A) Schematic representation of the role of the ovarian and adrenal sources of sex steroids in pre-menopausal women. (B) Schematic representation of the role of testicular and adrenal sources of sex steroids in men. ACTH, adrenocorticotropin; CRH, corticotropin-releasing hormone; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; E2, 17b-estradiol; LH, luteinizing hormone; GnRH, gonadotropin-releasing hormone.

clinically significant lack of DHEA availability in post-menopausal women is to administer exogen­ ous DHEA in order to replace the amount of DHEA missing in these women. Such a replace­ ment with DHEA can thus improve or even make the symptoms of hormone deficiency disappear as observed recently in women treated for vaginal atrophy, a classical consequence of hormone defi­ ciency during post-menopause (Buster, 2009; Labrie et al., 2009a, 2009b, 2009c). In fact, the progressive and variable fall in serum DHEA starting in the thirties has been associated with a series of medical problems including cardiovascu­ lar mortality (Barrett-Connor et al., 1986), malig­ nancy (Ebeling and Koivisto, 1994), osteoporosis

(Sambrook et al., 1992) and a series of other med­ ical problems to be discussed later. Due to the marked decrease in the serum levels of DHEA starting in the thirties, a relatively long series of clinical studies have administered DHEA to women and men in order to correct various symptoms of hormone deficiency (Tables 1 and 2). One should be careful, however, about going as far as considering DHEA as the response to all problems of aging or a ‘fountain of youth’. Unfortunately, for more than 15 years, DHEA has been available over the counter and/or on the Internet, especially in the United States. This uncontrolled availability of DHEA raises serious concerns about self-administration of a series of

100 Postmenopause

New findings intracrinology GnRH CRH –

ACTH Intracrinology

DHEA E2

Adrenal

Cortisol Aldosterone

DHT

Peripheral tissue

Fig. 2. Schematic representation of the unique source of sex steroids in post-menopausal women, namely adrenal DHEA. At menopause, the secretion of E2 by the ovaries ceases. Consequently, after menopause, all estrogens and practically all androgens are made locally from DHEA in peripheral target intracrine tissues. The amount of sex steroids made in peripheral target tissues depends upon the level of the steroid-forming enzymes specifically expressed in each tissue.

Serum DHEA

30

Serum DHEA-S

15

25

12

20

(μmol/l)

(nmol/l)

9 15

6 10

3

5

0

0

20

40

60

80

20

40

60

80

Age (years) Fig. 3. Effect of age on DHEA and DHEA-S levels in women. The graph shows the serum concentrations of (A) DHEA and (B) DHEA-S in women aged 20–80 years. Data are expressed as means + SEM. DHEA, dehydroepiandrosterone; DHEA-S, DHEA sulphate (Labrie, 2007).

DHEA formulations not submitted to the quality control process of pharmaceuticals required by regulatory agencies, with the result that the

quantity and quality of the DHEA available in these formulations is very uncertain (Parasram­ puria et al., 1998).

101 Table 1. Summary of the different studies performed with DHEA in women

Authors

Daily dose

Findings

Negative effects

Abrahamsson and Hackl (1981)

200 mg 3 months n = 17 50 mg 4 months placebo/cross-over n = 24 Adrenal insufficiency 50 mg, placebo 3 months n = 30 Perimenopausal 50 mg ! 600 mg 6 months n = 23 400 mg, n = 14 600 mg, n = 3 Systemic lupus erythematosus 50 mg 4 weeks n = 22 Pilot study 50 mg, placebo 12 months n = 70

Decreased serum LH

No

Improved mood, well-being and increased frequency of sexual thoughts and interest

No

No significant effect on mood, perimenopausal symptoms, cognition, memory or well-being

No

Improvement Acne

No

Hot flash score decreased by 50%

No

Increase in BMD at femoral neck and Ward’s triangle in 60–69-year-old women and increased radius BMD in 70–79-year­ old women Improved libido and sexual function, skin hydration, epidermal thickness, pigmentation and increased sebum secretion Trend for decreased body fat. Improved life satisfaction

No

Improved quality of life, social functioning and general health perception

No

Leptin decreased Osteocalcin slightly increased No change in glucose, insulin, body mass index or exercise capacity Insulin sensitivity increased

No

No significant effect on BMD. Insulin sensitivity improved. Triglycerides decreased.

No

Arlt et al. (1999)

Barnhart et al. (1999)

Barry et al. (1998)

Barton et al. (2006)

Baulieu et al. (2000)

Bilger et al. (2005)

Brooke et al. (2006a, 2006b)

Callies et al. (2001)

Casson et al. (1995)

Casson et al. (1998)

50 mg, placebo 12 months n=5 Hypopituitarism 50 mg, placebo 6 months followed by 6 months open n = 26 Hypopituitarism 50 mg 4 months n = 24 Adrenal insufficiency 50 mg, placebo 3 weeks n = 11 25 mg, placebo 6 months n=7

No

No

(Continued)

102 Table 1. (Continued ) Authors

Daily dose

Findings

Chang et al. (2002); Genelabs Briefing Document, 2001 – Taiwan Same study

200 mg 6 months n = 61 ! 58 completed Systemic lupus erythematosus

Diamond et al. (1996); Labrie et al. (1997) Same study

4–6 g of 10% DHEA cream, placebo/cross-over 12 months n = 15

Dhatariya et al. (2005)

50 mg, placebo/cross-over 3 months n = 28 Adrenal insufficiency 175 mg 8 days n = 5 women þ 3 men Hypercholestorelemia 50 mg 6 months n = 31 25 mg 12 months n = 20

Triglycerides decreased Mild acne Improved flare Acne and seborrhoea No hirsutism Infection and headache decreased Sub-cutaneous fat decreased Increase in femoral muscle area Decreased serum insulin and glucose Increased BMD of total hip Vaginal maturation stimulated Sebum production increased Well-being and energy improved Fasting insulin and glucagon lowered Increased insulin sensitivity Decreased total, HDL and LDL cholesterol Total cholesterol decreased

Felt & Starka (1966)

Genazzani et al. (2001)

Genazzani et al. (2003); Genazzani et al. (2006) Same study

Gebre-Medhin et al. (2000)

Gordon et al. (2002)

50 –200 mg 3 months n = 5(50 mg), n = 4(200 mg) Addison’s disease 50 mg 12 months n = 61 Anorexia nervosa

Gurnell et al. (2008)

50 mg, parallel group 12 months n = 30 Addison’s disease

Hackbert and Heiman (2002)

300 mg Acute study n = 16 50 mg 3 months n = 24 Addison’s disease

Hunt et al. (2000)

Negative effects No

No

No

No

Osteocalcin increased Levels of GH and IGF-1 increased

No

Improved vasomotor symptoms No change in endometrial thickness The response to ACTH was increased, thus neutralizing the effect of age on adrenal enzymatic activities HDL and LDL cholesterol decreased Lean mass increased

No

No

Hip BMD increased Bone markers improved Improvement of psychological parameters Increased muscle mass and strength Improved health-related quality of life No change in fat mass lipids Increased BMD at femoral neck Increased lean body mass No change in cognitive function Mental and physical arousal to erotic video improved

No

Mood and fatigue improved No significant effect on cognitive or sexual function Significantly improved self-esteem Mild acne and facial hair growth

No

No

No

(Continued )

103 Table 1. (Continued ) Negative effects

Authors

Daily dose

Findings

Igwebuike et al. (2008)

50 mg, placebo 3 months n = 17

No

Jankowski et al. (2006)

50 mg 12 months n = 34

Johannsson et al. (2002)

20 or 30 mg, placebo 6 months, followed by 6 months DHEA in placebo group n = 38 Pituitary deficiency 25 or 50 mg every 2 or 3 days 3–29 months n=4 Hereditary angioedema 6g of 0.1 to 2.0% DHEA cream, placebo 13 weeks n = 60 4g of 10% DHEA gel or cream or 100 mg oral 14 days n = 36 6.5 ! 23.4 mg intravaginal, placebo, 7 days n = 30 Vaginal atrophy 3g of 0.3% DHEA cream, placebo, 12 months n = 73

Body fat decreased Skeletal muscle weight significantly increased when DHEA was added to exercise Increased BMD at total hip, trochanter, femoral shaft and lumbar spine No significant effect on fat or fat-free mass and handgrip strength Sexual function, mood and behavior improved Appearance of normal skin and/or pubic and/or axillary hair Increased muscle strength Dramatic improvement in clinical state

DHEA metabolism following percutaneous administration Modulation of several genes involved in collagen biosynthesis Bioavailability and metabolism of DHEA after oral and percutaneous administration

No

Metabolism of DHEA after intravaginal administration Beneficial effects against vaginal atrophy

No

Metabolism of DHEA following longterm percutaneous administration Prevention of the appearance of new skin wrinkles Stimulation of sebaceous gland activity Metabolism of DHEA following 12-week intravaginal administration Reversal of all the symptoms and signs of vaginal atrophy Improvement of all four domains of sexual dysfunction and libido Insulin sensitivity improved HDL cholesterol increased LDL cholesterol and triglycerides decreased Glucose tolerance unchanged Fat mass decreased Total cholesterol decreased No change in glucose Fat mass decreased

No

Koo et al. (1983)

Labrie et al. (2007a) Calvo et al. 2008 Same study Labrie et al. (2007b)

Labrie et al. (2008a, 2008b)

Labrie et al. (2008c) Labrie et al. unpublished

Labrie et al. (2009a, 2009b, 2009c)

3.25 ! 13 mg intravaginal, placebo, 3 months n = 163 Vaginal atrophy

Lasco et al. (2001)

25 mg, placebo 12 months n = 20

Libe et al. (2004)

50 mg 4 months n=7 Addison’s disease

No

No

No

No

No

No

No

(Continued)

104 Table 1. (Continued ) Authors

Daily dose

Findings

Negative effects

Lovas et al. (2003)

25 mg, parallel group 9 months n = 19 Adrenal failure 50 mg 3 months n = 17

No statistically significant effect.

No

Insulin sensitivity and percentage of body fat unchanged Improved physical and psychological well-being No significant change in libido Muscle strength unchanged Lean body mass increased No significant effect on fat mass Insulin resistance decreased Total and HDL cholesterol decreased

No

No change in ‘insulin action’ Increased lean body mass’.

No

Improved mental health-related quality of life and McCoy’s sex scale No significant effect on bone density

No

No effect on hand grip and knee muscle strength. Muscle area at thigh unchanged. Acne in 41% at both doses versus 19% in controls HDL cholesterol decreased with 200 mg Hirsutism in 11 and 7.8% at 100 and 200 mg doses and 4.1% in placebo

No

Mild acne in 33% and hirsutism in 16% versus 14 and 2% in controls No therapy withdrawal required HDL cholesterol, triglycerides and C3 complement decreased Myalgia and stomatitis decreased Increased lumbar and spine BMD Some subjective improvement but not in tests of neurological dysfunction

No

Improved Kupperman score – quality of life

No

Improved Kupperman score – quality of life Vasomotor and psychological symptoms

No

Morales et al. (1994)

Morales et al. (1998)

Mortola and Yen (1990)

Nair et al. (2006) Basu et al. (2007) Same study Nordmark et al. (2005)

Percheron et al. (2003)

Petri et al. (2002)

Petri et al. (2004)

Roberts and Fauble (1990)

Stomati et al. (1999)

Stomati et al. (2000)

100 mg 6 months n = 10 1600 mg, placebo 28 days n=6 50 mg, placebo 23 months n = 27 20–30 mg, placebo 6 months followed by 6 months open n = 91 Systemic lupus erythematosus 50 mg, placebo 12 months n = 70 ! 51 (end) 100 mg n = 63 ! 6 (end) 200 mg n = 64 ! 47 (end) 7–9 months Systemic lupus erythematosus 200 mg 12 months n = 189 ! 124 (end) Systemic lupus erythematosus

90 ! 180 mg 6 months n=9 Multiple sclerosis 50 mg (DHEA-S) 3 months n=8 50 mg 6 months n = 31

No

No

No

No

(Continued )

105 Table 1. (Continued ) Negative effects

Authors

Daily dose

Findings

Suh-Burgmann et al. (2003)

150 mg, intravaginal n = 12 for 3 months n = 7 for 6 months 50 mg, placebo/cross-over 4 months n = 16 Hypopituitarism 200 mg 3–6 months n = 10 Systemic lupus erythematosus 100 mg, placebo 3 months, n = 14 Followed by 50–200 mg 3 months, open, n = 21 Systemic lupus erythematosus 200 mg, placebo 6 months (n = 50 ! 34) 12 months, n = 21, 29 months, n = 21 200 mg, placebo 6 months n=9 Systemic lupus erythematosus 50 mg 6 months n = 18 50 mg 6 months n = 28 50 mg, placebo 12 months n = 58 50 mg 6 months n = 17 100 mg 12 months n=8 100 mg 3 months n = 36

DHEA promotes regression of low-grade cervical lesions

No

Depression score and health perception slightly improved

No

Possible improvement

No

Improved flare Modest benefits

No

Improvement of lupus Mild acne and hirsutism

No

Improvement of lupus

No

Improved muscle mass and strength and lumbar BMD

No

Decreased visceral and subcutaneous fat Sensitivity to insulin increased

No

Lumbar spine BMD increased

No

Serum IGF-1 increased

No

No significant effect on knee muscle strength

No

Total cholesterol decreased Flow-mediated dilatation and lower Doppler velocimetry in brachial artery increased Endothelium-dependent, cutaneous blood flow increased Tendency to increased well-being

No

Suggestion of anti-depressant effect

No

Van Thiel et al. (2005)

van Vollenhoven et al. (1994)

van Vollenhoven et al. (1995)

van Vollenhoven et al. (1998)

van Vollenhoven et al. (1999)

Villareal et al. (2000)

Villareal and Holloszy (2004)

Von Muhlen et al. (2008)

Yen et al. (1995)

Yen et al. (1995)

Williams et al. (2004)

Wolf et al. (1997)

Wolkowitz et al. (1997)

50 mg 2 weeks n = 15 30–90 mg 4 weeks n=3

No

106 Table 2. Summary of the different studies performed with DHEA in men Authors

Dose

Findings

Serious side effects

Arlt et al. (2001)

50 mg 4 months n = 22 50 mg placebo n = 70 n = 70 50 mg placebo 6 months followed by 6 months open n = 18 Hypopituitarism 175 mg 8 days n = 5 women þ 3 men Hypercholestorelemia 100 mg, placebo 3 months n = 39 25 mg 12 months n = 10 50 mg, placebo 12 months n = 24 Addison’s disease 50 mg 3 months n = 15 Addison’s disease 50 mg 12 months n = 35 25 mg 3 months 24 men with hypercholesterolemia 50 mg 5 months n=9 25 or 50 mg every 2 or 3 days 3–29 months n=4 Hereditary angioedema 50 mg 4 months n = 13 Addison’s disease

No significant effect

No

No significant change of BMD Skin hydration and pigmentation improved

No

No statistically significant effect

No

Total cholesterol decreased

No

No significant change in body composition Total and HDL cholesterol decreased

No

Improvement in mood, fatigue and joint pain

No

BMD of femoral neck improved Total body and truncal lean mass improved No significant change in fat mass or lipids Well-being partially improved Mood and fatigue improved No significant effect on cognitive or sexual function

No

BMD increased at total hip, trochanter and shaft No significant effect on fat or fat-free mass

No

Glucose decreased but not insulin Insulin sensitivity improved and plasminogen activator inhibitor type 1 decreased

No

Stimulation of immune function

No

Dramatic improvement in clinical state

No

Total cholesterol decreased No significant change in glucose or insulin Fat mass decreased

No

Baulieu et al. (2000)

Brooke et al. (2006a), JCEM

Felt and Starka (1966)

Flynn et al. (1999)

Genazzani et al. (2004)

Gurnell et al. (2008)

Hunt et al. (2000)

Jankowski et al. (2006)

Kawano et al. (2003)

Khorram et al. (1997)

Koo et al. (1983)

Libe et al. (2004)

No

(Continued )

107 Table 2. (Continued ) Authors

Dose

Findings

Serious side effects

Morales et al. (1994)

50 mg 3 months n = 13 100 mg 6 months n=9 75 mg, placebo 23 months n = 29 1600 mg 28 days n=5 50 mg, placebo 12 months n = 70 ! 60 (end) 90 ! 180 mg 6 months n=9 Multiple sclerosis 100 mg 6 months n = 86 Osteoporosis 50 mg, placebo 4 months n = 15 Hypopituitarism 50 mg, placebo 6 months n = 28 50 mg 12 months n = 55 1600 mg 4 weeks n=8 50 mg 2 weeks n = 25 30–90 mg 4 weeks n=3 50 mg 6 months n = 13 100 mg 12 months n=8

Improved physical and psychological well-being Insulin sensitivity and percentage of body fat not significantly changed Muscle strength increased Fat mass decreased No significant effect on BMD Increase in femoral neck BMD No significant change in insulin action

No

Decreased total and LDL cholesterol Decreased body fat No significant change in insulin sensitivity No effect on handgrip and knee muscle strength Muscle area at thigh not significantly changed

No

Some subjective improvement but not in tests of neurological dysfunction

No

BMD increased at lumbar spine and femoral neck

No

Depression score and health perception slightly improved

No

Decrease in visceral and sub-cutaneous fat Sensitivity to insulin increased

No

No significant effect on BMD or bone marker CTX

No

No significant effect

No

No significant effect on cognitive functions

No

Suggestion of antidepressant effect

No

Serum IGF-1 increased

No

Increased knee muscle strength

No

Morales et al. (1998)

Nair et al. (2006) Basu

et al. (2007)

Same study

Nestler et al. (1988)

Percheron et al. (2003)

Roberts and Fauble

(1990)

Sun et al. (2002)

Van Thiel et al. (2005)

Villareal and Holloszy

(2004)

Von Muhlen et al.

(2008)

Welle et al. (1990)

Wolf et al. (1997)

Wolkowitz et al.

(1997)

Yen et al. (1995)

Yen et al. (1995)

No

No

No

108

On the other hand, it should be mentioned that many studies reported as negative do not have the statistical power to reach any conclu­ sion. One has always to consider that data which do not reach the statistical level of significance should not be interpreted as demonstration of an absence of effect. Critical evaluation must always be made of the design of the study, the precision of the parameters used to assess the effect(s) of treatment, the true placebo component, the population studied and, most importantly, the duration of treatment and the number of subjects investigated. As can be seen in Tables 1 and 2, the majority of studies examined the effect of DHEA in a too small number of subjects and/ or the duration of treatment was too short to truly assess the potential changes induced by DHEA. This chapter is an opportunity to briefly mention the beneficial effects of DHEA already observed in the literature and, as expected from the physiological mechanisms involved, to realize that no significant negative effect has ever been observed in any study reported, despite the high doses sometimes used. This lack of significant side effects could, up to an unknown extent, be related to the fact that a natural saturation mechanism limits the transformation of DHEA into andro­ gens and/or estrogens in post-menopausal women at a serum DHEA concentration of about 7 ng/ml, a value well within the physiologi­ cal range (Labrie et al., 2006).

Intracrinology Changes in DHEA secretion with age The foetal adrenal gland secretes high levels of DHEA which is transformed in the placenta into the E2 required for maintenance of pregnancy (Chakravorty et al., 1999). Secretion of DHEA then declines to very low levels immediately after parturition (Parker, 1991). Strong DHEA secretion resumes during the pre-pubertal years at about 8–10 years of age, thus permitting the growth of pubic and axillary hair, a physiological phenomenon called adrenarche (Parker et al.,

1978). After a peak in the early twenties, serum DHEA [and DHEA sulphate (DHEA-S)] levels then decline at the rate of about 5% per year starting in the early thirties (Orentreich et al., 1984) (Fig. 3). The decline in serum DHEA levels in both women and men is highly variable and is driven by unknown factors.

Intracrine system It is remarkable that man, in addition to posses­ sing very sophisticated endocrine and paracrine systems, has largely invested in sex steroid forma­ tion in peripheral tissues (Labrie, 1991; Labrie et al., 1997; Labrie et al., 1985, 1988) (Figs. 1, 2 and 4). The ovaries and testicles have traditionally been considered the exclusive sources of estro­ gens and androgens in women and men, respec­ tively. For example, the fall in serum E2 to extremely low levels at menopause coupled with the beneficial effects of exogenous estrogens on menopausal symptoms (Archer et al., 1999) has focussed the efforts of hormone replacement ther­ apy almost exclusively on various forms of estro­ gens to compensate for the cessation of estrogen secretion by the ovaries. In men, on the other hand, the 95% (or more) fall in serum testoster­ one induced by castration and the clinical benefits of this partial elimination of androgens in men with advanced prostate cancer (Huggins and Hodges, 1941) have led the urological community to erroneously believe that castration eliminates 95% (or more) of androgens and that castration alone is an acceptable treatment for prostate cancer. The scientific findings recently provided by intra­ crinology are quite different (Labrie, 1991; Labrie et al., 1985; Bélanger et al., 1989). For example, since ovarian estrogen secretion ceases at meno­ pause, the exclusive role of peripheral estrogen formation in post-menopausal women is clearly demonstrated, as mentioned above, by the obser­ vation of the major benefits of aromatase inhibitors in advanced breast cancer in post-menopausal women (Goss et al., 2003; Howell et al., 2005; Mouridsen et al., 2003; Nabholtz et al., 2000) as

109

well as by the findings of a 76% decrease in breast cancer incidence in post-menopausal osteoporotic women who received the selective estrogen recep­ tor modulator (SERM) raloxifene for 3 years (Cummings et al., 1999). These effects of estro­ gen blockade are the consequence of the rela­ tively high levels of estrogens which are made locally by intracrine mechanisms in breast can­ cer tissue after menopause (Poortman et al., 1983). In men, on the other hand, the finding that 25–50% of androgens are left in the prostate after castration (Bélanger et al., 1989; Labrie et al., 1985; Mostaghel et al., 2007; Nishiyama et al., 2004) explains why the addition of a pure (non-steroidal) anti-androgen to castration achieves a more complete blockade of androgens and has been the first treatment shown to prolong life in prostate cancer (Caubet et al., 1997; Labrie et al., 1982; Labrie et al., 1985; Labrie et al., 2005; Prostate Cancer Triallists’ Collaborative Group, 2000). The androgens remaining at relatively high levels after castration also explain why com­ bined androgen blockades or the blockade of the androgens of both testicular and adrenal origins at start of treatment can provide cure for most patients when the treatment is started at the loca­ lized stage of the cancer (Akaza, 2006; Labrie et al., 2002; Ueno et al., 2006), thus clearly demon­ strating the major role of extratesticular andro­ gens or intracrinology in men. Transformation of the adrenal precursor steroid DHEA into androgens and/or estrogens in periph­ eral target tissues depends upon the levels of expression of the various steroidogenic and meta­ bolizing enzymes in each cell of these tissues. This situation of a high secretion rate of adrenal pre­ cursor sex steroids in men and women is thus completely different from all animal models used in the laboratory (namely rats, mice, guinea pigs and all others except monkeys), where the secre­ tion of sex steroids takes place exclusively in the gonads (Bélanger et al., 1989; Labrie et al., 1985, 1988, 1997). The androgens testosterone and DHT as well as E2 made in peripheral tissues from DHEA of adrenal origin exert their action locally in the same cells where their synthesis takes place

(Fig. 4). This sophisticated mechanism permits to maintain biologically active levels of intracellular estrogens and/or androgens in specific tissues in need of these sex steroids while the same steroids leak in the blood at very low levels, thus sparing the other tissues from a potentially negative influ­ ence. Following their local formation and immedi­ ate availability for local intracellular action, testosterone and DHT (the most active natural androgen) and E2 are inactivated and transformed in the same cells into water-soluble glucuronide or sulphate derivatives which can then diffuse quan­ titatively into the general circulation where they can be measured by mass spectrometry (Labrie et al., 2006) before their elimination by the kidneys. In women, as mentioned earlier, the role of the adrenal precursor steroid DHEA in the periph­ eral formation of sex steroids is even more impor­ tant than in men. In fact, in men, sex steroid secretion by the testicles continues at an almost constant and high level through life while, in women, estrogen secretion by the ovaries comple­ tely ceases at menopause, thus leaving the adre­ nals as the exclusive source of sex steroids (Fig. 2). Accordingly, the best estimate is that the intracrine formation of estrogens in peripheral tissues in women accounts for 50% of all estro­ gens before menopause and 100% after meno­ pause (Labrie et al., 2003). It should also be noted that the importance of the intracrine formation of androgens and estro­ gens extends to non-malignant diseases such as acne, seborrhoea, hirsutism and androgenic alo­ pecia as well as to osteoporosis and vaginal atrophy (Cusan et al., 1994; Labrie et al., 1997; Labrie et al., 2009a, 2009b, 2009c). Most tissues possess, at various levels, a battery of steroido­ genic enzymes that can transform DHEA.

In men, 40% of total androgens are made from DHEA While the serum levels of testosterone are reduced by 97.4% following castration in 69–80­ year-old men (Labrie et al., 2009b), the sum of the metabolites of androgens, the only accurate and

110

Steroidogenic enzymes adrenal – intracrine tissues Exogenous DHEA

Adrenal

Cholesterol

DHEA-S

P450scc

Sult

P450c17

PREG 3β-HSD

DHEA

20OH-PROG

P450c11β

17-OH-PROG

4-DIONE 17β-HSD 2, 10 5α-red 1, 2, 3

ALDOSTERONE

TESTO 5α-red 1, 2, 3

17β-HSD 5, 15

A-DIONE

P450c11β

DHT 17β-HSD 2, 9, 10 3α-HSD

3α-HSD Aromatase

CORTICO STERONE

3β-HSD

17β-HSD 5

11-DEOXY CORTISOL

11-deoxycorTicosterone

5-DIOL 17β-2, 4

3β-HSD

P450c21

P450c21

P450c11α

17β-HSD 1

P450c17

3β-HSD P450c17

PROG 20α-HSD

17-OH-PREG

17β-HSD 5, 15

ADT

CORTISOL

E1

3α-DIOL

Aromatase

17β-HSD 2, 9, 10, 14 17β-HSD 1, 7, 12

E2

17β-HSD 2, 4, 8, 14

Adrenal

Intracrine tissues

Fig. 4. Schematic representation of the adrenal and intracrine steroidogenic pathways. DHEA, dehydroepiandrosterone; DHEA-S, DHEA sulphate; DHT, dihydrotestosterone; HSD, hydroxysteroid dehydrogenase.

valid parameter of total androgenic activity mea­ surable in the circulation (Labrie et al., 2006), is only reduced by 58.9% (Labrie et al., 2009b), thus indicating that a very important proportion (41.1%) of androgens remains in men after com­ plete elimination of testicular androgens. Such data are in close agreement with the concentration of intraprostatic DHT which shows that, on aver­ age, 39% of DHT is left in the prostate after castration in various studies, namely 45% (Labrie et al., 1985), 51% (Bélanger et al., 1989), 25% (Nishiyama et al., 2004) and 35% (Mostaghel et al., 2007) (see Fig. 4 in Labrie, this volume, Chapter 14).

Comparable amounts of sex steroids of adrenal origin are made in men and women With the knowledge of the major importance of androgens of adrenal origin in men, it is of interest to compare the data mentioned above for men

with the serum levels of the same steroids mea­ sured in intact post-menopausal women. As can be seen in Fig. 5A and B, the serum levels of testosterone and of the total androgen metabolites are almost superimposable in castrated men and post-menopausal women of comparable age. Most interestingly, it can be observed that the serum levels of estrone sulphate (E1S) are also compar­ able (Fig. 5C). It could also be seen that the serum levels of E1 and E2 are also comparable, thus indicating that similar amounts of estrogens of adrenal origin are found in both men and women (Labrie et al., 2009b). The above-summarized data show that ~40% of androgens are made in peripheral tissues in the absence of testicles in 69–80-year-old men. Since serum DHEA decreases markedly with age start­ ing in the thirties (Labrie et al., 2005), and testi­ cular androgen secretion decreases only slightly, it is most likely that androgens of adrenal origin have an even greater relative and absolute impor­ tance at younger ages. The same conclusion

111

applies to women with respect to the androgens synthesized from DHEA.

Women synthesize 40–50% as much androgens as men The data summarized above show that post-meno­ pausal women synthesize androgens and estrogens in quantities similar to castrated men of compar­ able age (Fig. 5). The 50–60% higher androgen formation in men is essentially attributable to the androgens of testicular origin.

High circulating levels of DHEA and menopause are unique to women As mentioned above, protection of the endome­ trium is the most obvious reason why evolution over 500 million years has succeeded in building an intracrine system unique to the human species and able to protect women from systemic expo­ sure to estrogens after menopause. It is remark­ able that while the steroidogenic enzymes appeared ~500 million years ago with the verte­ brates, it is only about 50 million years ago that

3 A.

the adrenals of primates gained the property to secrete large amounts of DHEA (Baker, 2004). DHEA becomes the exclusive source of sex steroids at menopause Recently, it has been more evident that DHEA of adrenal origin became practically the only source of sex steroids in women with the appearance of meno­ pause which corresponds to the arrest of significant sex steroid secretion by the ovaries. It took more than 500 million years of evolution to separate the role of gonadal and DHEA-derived sex steroids, thus per­ mitting women to be free during all their post-meno­ pausal years from the negative systemic effects of estrogens and benefit from a strictly local formation and action of sex steroids made according to the specific age-related needs of each cell type in each tissue by the process of intracrinology (Labrie, 1991). Systemic estrogens are not physiological after menopause Since all women are no more exposed to systemic estrogens after menopause, it is reasonable to believe that the non-physiological situation created

40 B.

250

C.

200

30 2 pg/ml

ng/ml

ng/ml

150 20

100

1 10

0

Castrated men

Post-meno­ pausal women

Testosterone

0

50

Castrated men

Post-meno­ pausal women

ADT – G + 3α – diol – 3G + 17G

0

Castrated men

Post-meno­ pausal women

Estrone sulphate

Fig. 5. Comparison of the serum concentrations of testosterone (A), total androgenic pool (sum of ADT-G, 3a-diol-3G and 17G) (B) and E1S (C) in castrated 69–80-year-old men (n = 34) and intact 55–65-year-old post-menopausal women (n = 377) (Labrie et al., 2006; Labrie et al., 2009b).

112

by the administration of estrogens could be respon­ sible, up to an unknown extent, for the side effects reported in women receiving traditional estrogen replacement therapy (ERT) and HRT (estrogen þ progestin replacement therapy) (Beral, 2003; Beral et al., 2005; Grodstein et al., 2006; Hsia et al., 2006; Pines et al., 2007; Riman et al., 2002; Rossouw et al., 2002; Ruttimann, 2008). The recent observations indicating the risks associated with exogeneous estrogens as replacement therapy should normally help to focus our attention on DHEA, the only physiological source of sex steroids after menopause (Labrie, 2010), and better understand the mechan­ ism of action of DHEA and its preventive and therapeutic roles.

suffering from vaginal atrophy symptoms (~75% of women) and those without symptoms (~25%) is not related to estrogens. In fact, the only remaining hormonal difference between these two groups of women is the difference in the availability of DHEA, the exclusive source of sex steroids (Fig. 2). In addition to markedly decreasing with age (Fig. 3), the serum levels of DHEA are highly variable with some women having barely detectable levels while others have values up to 9–10 ng/ml (Labrie et al., 2006, 2010) (Fig. 5).

High variability of serum DHEA

It should be mentioned that saturation of the enzymatic systems which transform DHEA into active androgens and/or estrogens is observed at serum DHEA levels of about 7 ng/ml, thus pro­ tecting women against potential excess levels of sex steroids (Labrie et al., 2007a) (Fig. 6). We believe that the presence of this natural saturation

An important observation is that despite the arrest of estrogen secretion in all women at time of meno­ pause, not all post-menopausal women suffer from menopausal symptoms. Consequently, the hormo­ nal difference between post-menopausal women

Rapid saturation of the enzymatic mechanisms transforming DHEA into sex steroids: Prevention of overexposure and potential side effects

Highly variable serum levels of DHEA Premenopausal Women (n = 47)

10

Postmenopausal Women (n = 442)

DHEA (ng/mL)

8

6

4

2

0 30

40

50

60

70

Age (Years) Fig. 6. Illustration of the wide variability of serum DHEA levels in normal women aged 30–40 years and 50–75 years. Data are presented individually as well as means and 5–95% centiles (Labrie, 2010).

113

of the enzymatic mechanism makes practically impossible to administer a dose of DHEA which could lead to excess tissue exposure to estrogens and/or androgens. The fact that no serious adverse event has ever been reported with exo­ genous DHEA (Tables 1 and 2) is likely to be explained, up to an unknown extent, by this selfprotecting mechanism. In addition, when replace­ ment with DHEA is limited to physiological amounts of this tissue-specific sex steroid prehor­ mone free of intrinsic sex steroid activity, no adverse effect can be logically expected. In fact, all our pharmacokinetic studies per­ formed in post-menopausal women indicate that doses of prasterone much larger than those used in our efficacy studies could only have a limited effect on global sex steroid formation (Labrie et al., 2007a; Labrie et al., 2007b; Labrie et al., 2008c). As illustrated in Fig. 7, it would be extremely difficult or most likely impossible to administer a dose of DHEA intravaginally (or otherwise) able to increase the serum levels of

the androgen metabolites above the normal post­ menopausal range.

Changes in serum DHEA following treatment with DHEA are overestimates of the true changes in sex steroid formation Before looking at efficacy data, it is important to mention that recent information has shown that following administration of DHEA, the observed changes in serum DHEA are at least twice as high as the ‘true’ changes in sex steroid forma­ tion (Labrie et al., 2008c). This effect is even more pronounced when comparing serum levels of DHEA with ‘true’ levels of estrogens. This observation is of major importance in analyzing the data from DHEA studies where, almost always, changes in serum DHEA are used as the parameter of reference to assess the choice of dose and the efficacy of replacement therapy. Consequently, the dose of DHEA used is

123.6 1.0% DHEA

ADT−G + 3α−DIOL−3G + 3α−DIOL−17G (ng/ml)

50

2.0% DHEA

95th Centile 40

30

0.3 % DHEA

20 0.1 % DHEA

(Basal

post-menopausal)

10

5th Centile 0 0

5

10 DHEA (ng/ml)

Pre-meno­ pausal women

Fig. 7. Effect of increasing serum concentrations of DHEA induced by twice daily percutaneous administration of 3 g of 0% (placebo), 0.1, 0.3, 1.0 or 2.0% DHEA cream on the sum of the serum levels of the androgen metabolites androsterone glucuronide (ADT-G), androstane-3a, 17b-diol-3 glucuronide (3a-diol-3G) and androstane-3a, 17b-diol-17 glucuronide (3a-diol-17G) expressed in ng/ml (Labrie et al., 2007a).

114

frequently too low compared with the additional intra-tissular sex steroid levels needed to show efficacy.

Tissue-specific effects of DHEA reported in the literature Limitations of the available clinical data As mentioned above and indicated by Arlt and Allolio (2003), a problem with the available clin­ ical DHEA studies (Tables 1 and 2) is that a large proportion of these studies are underpowered, thus not contributing to increasing our knowledge in the field. On the contrary, many of these studies add confusion by stating that DHEA has no effect on such and such parameter(s) while, in fact, the study performed did not have the statistical power needed to reach any valid conclusion: A common problem is that a lack of statistical power leads to the erroneous conclusion that there is no effect of DHEA because the p value does not reach <0.05. Such wrong interpretations are frequent in clinical trials. The most damaging example of such a misinterpretation of statistics which had and con­ tinues to have serious negative therapeutic conse­ quences is a study in advanced prostate cancer comparing early versus late combined androgen blockade (Eisenberger et al., 1998): With a p value of 0.14 on overall survival, the authors incorrectly concluded that the addition of an anti-androgen to castration had no effect on survival while the correct interpretation should have been that the ‘study shows that the addition of the antiandrogen to castration at start of treatment has a 86% chance of being superior to castration alone at start of treatment followed by addition of the anti-androgen later at time of progression’. More­ over, all causes of death were included, thus decreasing the specific effect of treatment on pros­ tate cancer and the anti-androgen was added at time of progression in patients who had started with castration alone, thus further decreasing the difference between the two groups. This erro­ neous conclusion has been unanimously followed by the entire Western Urology Community with the consequence that androgen blockade in

prostate cancer remains generally limited to castration, thus leaving in the prostate cancer 40–50% of androgens which continue to stimulate prostate cancer to proliferate, metastasize and become resistant to further treatment, thus nega­ tively affecting the lives of millions of prostate cancer patients worldwide. As stated by Arlt and Allolio (2003), ‘the scientific community needs the results from well-designed, adequately powered and large scale studies to securely establish the role of DHEA in the standard replacement regimen for adrenal insuf­ ficiency’. We need to add that such rigorous data are required for a much larger population, namely for the majority of women who suffer from menopausal symptoms during their whole post-menopausal years without an available treatment free of the risks of the side effects of estrogens. “Lacking a pharmaceutical sponsor to usher it through the arduous process of FDA approval, DHEA drifted for years in the netherworld of health care products” (Spark, 2002). Most unfortu­ nately, passage of the 1994 Dietary Supplement and Health Education Act which classified DHEA as a ‘dietary supplement’ to be sold as an over-the-counter health care product has facilitated the promotion of DHEA without rigorous scientific evidence as a ‘fountain of youth able to avoid most or all problems of aging’. It is extremely difficult to understand how DHEA, a compound scientifically established as precursor of all sex steroids, could still be classified as a food supplement in 2010. This is certainly a good example where the available scientific knowledge has been put aside to meet the financial wishes of a few citizens at the expense of the quality of health care of the whole popula­ tion. Considering its role, DHEA should be treated as a pharmaceutical product and should be sub­ mitted to the proper regulations in order to ensure quality, efficacy and safety.

Uncertain quality of DHEA formulations A major problem with DHEA is the uncertainty about the available formulations. In fact, very few, if any (except in some clinical trials), DHEA pre­ paration meets the ICH/FDA pharmaceutical

115 160

149.5

140

% Label claim

120 100

91.3 94.3

80

69.3

95.3 96.3 96.8 98.6 98.9

104.2

74.5 76.2 77.3

60 40 20 0

0

0

0

4

7

8

3

1

9

13

12

15

14

2

11

5

6

16

10

Product identification number Fig. 8. Analysis results as a percentage of the label claim for content on dehydroepiandrosterone (DHEA) dietary supplement products. Error bars indicate SDs; Products 7 and 8 claimed to contain naturally occurring DHEA with no specific amount specified (Parasrampuria et al., 1998).

guidelines. In fact, when DHEA was analyzed from 17 different formulations, 3 formulations contained no detectable DHEA while most tablets/capsules had 59–82% of the amount indicated on the label while one tablet had 149% of the amount of DHEA stated (Fig. 8) (Parasrampuria et al., 1998). While, as mentioned above, the quantity of DHEA, an easily measur­ able parameter, in commercially available pre­ parations is unlikely to be correct (Fig. 8), it is even less likely that the quality of the DHEA formulations meets the ICH/FDA guidelines with any impurity at <0.1% and total impurities at <0.5% (ICH pharmaceutical grade).

Exogenous DHEA is more efficacious in women than men Although some beneficial effects of DHEA have been reported in men (Table 2), the effects are of much greater amplitude in women, especially after menopause (Table 1). As can be seen in Fig. 5, the serum levels of testosterone and total androgen metabolites as well as E1S are almost super­

imposable between intact post-menopausal women and castrated men of comparable age, thus indicating, as mentioned above, that the adre­ nals of men and women provide the same amount of androgens and estrogens. Accordingly, similar serum levels of E1 and E2 are also found between post-menopausal women and castrated men of similar age (Labrie et al., 2009a). However, while women have no other significant source of sex steroids after menopause, the important source of sex steroids provided by the testicles decreases only slightly with age in men, thus explaining why women have more severe symptoms of hormone deficiency during their post-menopausal years than men of the same age. Consequently, it is normal that women show more responsiveness to treatment with DHEA. The relative importance of the gonads and DHEA as providers of sex steroids in men and women is presented in Fig. 9. As can be seen in this figure, the testis remains an important source of both androgens and estrogens during the whole life of men while the contribution of DHEA pro­ gressively decreases. In women, the situation is very different since the contribution of the ovary

116

DHEA and vaginal atrophy

Change of values (%) with age at 30 years % A.

Androgens Estrogens

100 From DHEA 50

From testis 0 30

40

50

60

70

80

% B. 100 From ovary

50 From DHEA

0 30

40

50 60 Age (years)

70

80

Fig. 9. Estimate of the contribution of the gonads and DHEA to the total pool of androgens and estrogens in men (A) and women (B) with age. Serum androsterone glucuronide (ADT-G) is used as representing total androgens while estrone sulphate (E1S) is used to estimate total estrogens. The effect of castration in men and menopause (and ovariectomy) in women permits to separate the contribution of the gonads. The data used are from Labrie et al. (2006), Labrie et al., (2009b) and Labrie et al., unpublished data. Values at 30 years are taken as 100%.

stops at menopause, thus leaving only DHEA as source of sex steroids for all post-menopausal years. This distribution is based upon the serum levels of androsterone glucuronide (ADT-G) used as an estimate of androgens while E1S serves as a parameter of estrogenic activity (Labrie et al., 2006; Labrie et al., 2009b and unpublished data). The distribution is expressed as the percentage of change from the age of 30 years taken as 100% of total sex steroid exposure. From the serum levels of ADT-G and E1S, women have ~50% as much androgens as men while the opposite is found for estrogens where men are exposed to ~50% as much estrogens as women.

General Vaginal dryness is found in 75% of post­ menopausal women (NAMS, 2007). However, only 10–20% of symptomatic women suffering from vaginal atrophy seek medical treatment for a variety of reasons, most commonly the fear of estrogen-related side effects (Barbaglia et al., 2009). There is thus a clear medical need and a major opportunity to remove the fear of breast cancer associated with today’s estrogen­ based therapies while improving the quality of life of 75–80% of women who are presently left with the problems of vaginal atrophy for a large proportion of their lifetime. Vaginal atro­ phy is a typical example of hormone deficiency in post-menopausal women (Table 3). Since DHEA is practically the exclusive source of androgens in women, it is of interest to see in Fig. 10 the potential sites of action of androgens. In fact, the total pool of androgens in women decreases in parallel with serum DHEA, thus indicating a major lack of androgens after meno­ pause (Fig. 3; Labrie et al., 2006).

Intravaginal estrogen formulations increase serum estrogen levels Although intravaginal formulations were devel­ oped to avoid systemic exposure to estrogens, a series of studies have unanimously demonstrated that all intravaginal estrogen formulations cause Table 3. Hormone-related menopausal symptoms Menopausal symptoms Vaginal atrophy Osteoporosis Hot flashes Loss of muscle mass Fat accumulation Type 2 diabetes High cholesterol Skin atrophy Loss of libido Sexual dysfunction

117

Sites of androgen (DHEA) action Brain

(Libido, hot flashes,

memory, cognition ...)

Skin Breast

Blood vessels Heart

Fat tissues Bones

Muscles

Vagina Glucose and insulin metabolism

Fig. 10. Schematic representation of the sites of androgen action in women. Tissue-specific action from sex steroids made locally from DHEA avoids exposure of the other tissues to estrogens and androgens, thus eliminating the negative effects of systemic exposure reported for standard hormonal therapy.

important increases in serum estrogen levels measured directly by radioimmunoassay or through their systemic effects (Rioux et al., 2000; Kendall et al., 2006; Labrie et al., 2009a). In fact, serum E2 levels are increased five-fold following administration of a 25 mg E2 pill (Vagi­ fem, Novo Nordisk, Princeton, NJ) or 1 g of 0.625 mg conjugated estrogen cream (Premarin, Wyeth Laboratories, Collegeville, PA) (Labrie et al., 2009a) (Fig. 11). These findings were obtained using mass spectrometry, the most accurate and precise technology. Such results indicate that the effects of estrogens applied locally in the vagina are unlikely to be limited to the vagina and that systemic activity should be expected (Kendall et al., 2006; Rioux et al., 2000).

What is vaginal atrophy? Vaginal atrophy is characterized by a decrease in the thickness and function of the three layers of the vagina. The most common symp­ toms of vaginal atrophy are dryness, irritation, burning, itching, inflammation, bleeding, vagi­ nal and urinary infections as well as pain

during intercourse frequently leading to decreased libido and sexual dysfunction. Vagi­ nal atrophy affects 50% of post-menopausal women from 50 to 60 years of age and 72% of women aged 70 years and older.

Correction of vaginal atrophy with intravaginal DHEA Vaginal atrophy is the first clear example of the high efficacy and safety of DHEA to treat sex steroid deficiency at post-menopause (Labrie et al., 2009c, 2009b). In agreement with previous phase II data (Labrie et al., 2008b; Labrie et al., 1997), it can be seen in Fig. 12 that at the standard 12-week time interval, 0.5% DHEA caused a 45.9 + 5.31% (p < 0.0001 vs. placebo) decrease in the percentage of parabasal cells, a 6.8 + 1.29% (p < 0.0001 vs. placebo) increase in superficial cells, a 1.3 + 0.13 unit (p < 0.0001 vs. placebo) decrease in vaginal pH and a 1.5 + 0.14 score unit (p < 0.0001) decrease in the severity of the most bothersome symptom. Similar changes were seen in vaginal secretions, colour, epithelial sur­ face thickness and epithelial integrity (Labrie et al., 2009a).

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E2 Day 7

Estradiol (pg/ml)

82

0.5% Prasterone

Vagifem

Premarin

30

160

Pre-menopause

30

25

25

20

20

15

15 10

10 Post-menopause

5

5 0

0 0

4

8

12

16

20

24

Hours post-dosing

55–65 30–35­ -year year -old -old

Fig. 11. Changes in serum estradiol (E2) over the 24-hour period following the seventh daily intravaginal administration of 0.5% DHEA (Prasterone, VaginormTM), 25 mg E2 (Vagifem) and 0.625 mg conjugated estrogens (Premarin cream) (Labrie et al., 2008a, 2008b; Labrie et al., 2009a).

Day 1 12 Weeks

70

7

10

vs baseline NS p = 0.83

3

vs placebo p < 0.0001

vs placebo

p = 0.0002

60

vs placebo p = 0.0033

8 50

6

2

6

40 30

vs placebo p < 0.0001

vs placebo p < 0.0001

4 vs placebo p < 0.0001

20

vs baseline NS

p = 0.194

1

5

2

10

0 0.5% DHEA

% Parabasal cells

0

4

0 Placebo

Placebo

0.5% DHEA

% Superficial cells

Placebo

0.5% DHEA pH

Placebo

0.5% DHEA

Pain at sexual activity

Fig. 12. Effect of daily intravaginal application of 0.5% dehydroepiandrosterone (DHEA; Prasterone) for 12 weeks on the percentage of vaginal parabasal cells (A), percentage of superficial cells (B), vaginal pH (C) and pain at intercourse (D) (Labrie et al., 2009b) in post-menopausal women. Data are expressed as means + SEM; the p values are compared with placebo for the DHEA-treated groups or with baseline for the placebo groups.

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Beneficial effects of intravaginal DHEA on sexual dysfunction In addition to the effects of intravaginal DHEA on the symptoms and signs of vaginal atrophy, a timeand dose-dependent improvement of the four domains of sexual dysfunction was observed. At the 12-week time interval, the 1.0% DHEA dose led, compared with placebo, to 49% (p = 0.0061) and 23% (p = 0.0257) improvements of the desire domains in the Menopause-Specific Quality of Life (MENQOL) and Abbreviated Sex Function (ASF) questionnaires, respectively. Compared with placebo, the ASF arousal/sensation domain was improved by 68% (p = 0.006), the arousal/lubrication domain by 39% (p = 0.0014), orgasm by 75% (p = 0.047) and dryness during intercourse by 57% (p = 0.0001) (Labrie et al., 2009c). Similar effects were observed at the 0.25% and 0.5% DHEA doses. This effect observed at doses which did not affect serum steroids which all remain well within normal post-menopausal values. Such data indicate that combined androgenic/ estrogenic stimulation in the three layers of the vagina exerts important beneficial effects on sexual function in women without systemic action on the brain and other extravaginal tissues.

No significant change in serum estrogens or androgens following intravaginal administration of DHEA

endometrium is the universally recognized obser­ vation that endometrial atrophy is found in all post-menopausal women despite the fact that almost all post-menopausal women have relatively high serum DHEA levels, albeit reduced com­ pared to pre-menopause (Fig. 6). Considering the major concern related to the wellrecognized stimulatory effect of estrogens on endo­ metrial proliferation with the accompanying risk of endometrial carcinoma (Friedl and Jordan, 1994; Parazzini et al., 1991), an endometrial biopsy was performed before starting treatment and after 12 months of daily administration of 4–6 g of a 10% DHEA cream in 15 women where average serum DHEA was increased at least 10-fold to 31 ng/ml (Labrie et al., 1997). As illustrated in representative Fig. 13, the endometrial atrophy seen at the start of treatment remained unaffected by 12 months of per­ cutaneous DHEA administration where at least a 10-fold increase in serum DHEA was observed com­ pared to the serum concentrations seen in women receiving daily administration of 0.5% DHEA ovules (Labrie et al., 1997; Labrie et al., 2008a, 2008b). Moreover, in the ERC-210 study where 163 women received DHEA administered intravag­ inally for 12 weeks, no change in the atrophic endo­ metrium was seen at the end of study. In agreement with these data, no change in endometrial thickness was found in 35 women who received daily 50 mg DHEA orally for 6 months (Stomati et al., 2000).

Most importantly, no significant leakage of the active sex steroids into the circulation takes place with VAGINORMTM, thus explaining the highly benefi­ cial effects observed in the vagina in the absence of significant changes in circulating estrogens or andro­ gens (Labrie et al., 2009a, 2009b, 2009c). In fact, as mentioned above, the active steroids are inactivated locally before being released as inactive metabolites into the general circulation from which they are eliminated by the kidneys and liver.

No effect of DHEA on the endometrium The most direct and undisputable proof of the absence of stimulatory effect of DHEA on the

Fig. 13. Atrophic endometrium after 12 months of DHEA treatment in a representative 65-year-old woman (×100). (Labrie et al., 1997).

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These findings of an absence of effect of DHEA on the endometrium are explained by the absence in the normal human endometrium of the enzymes, especially aromatase, needed to trans­ form DHEA into estrogens (Baxendale et al., 1981; Bulun et al., 2005) (see Table 1 in Luu-The and Labrie, this volume). In agreement with the atrophy of the endometrium which follows cessa­ tion of ovarian estrogen secretion at menopause and the absence of effect of DHEA on the endo­ metrium (Labrie et al., 1997; Labrie et al., 2009c), no transformation of androstenedione, the steroid made directly from DHEA by 3b-hydroxysteroid dehydrogenase, (Luu-The et al., 1989) into E1 (aromatase activity) has been found in normal human endometrial or myometrial tissue (Baxen­ dale et al., 1981), thus confirming previous obser­ vations (Gurpide and Welch, 1969; Reed et al., 1979). Both steroidogenic acute regulatory protein (StAR) and aromatase are either absent or barely detectable in normal human endometrium – the lack of both enzymes preventing local estrogen formation (Gurates et al., 2002; Noble et al., 1996; Noble et al., 1997; Sun et al., 2003; Tsai et al., 2001).

DHEA and bone A predominant role of androgens in bone phy­ siology is well documented (Davis et al., 1995; Labrie et al., 1997; Martel et al., 1998; Miller et al., 2002; Need et al., 1987; Raisz et al., 1996; Savvas et al., 1988; ). Most interestingly, andro­ gens increase bone mass by stimulation of bone formation (Kenny and Raisz, 2002; Liegibel et al., 2002). As shown in Tables 1 and 2, quite a few studies have shown a stimulatory effect of DHEA on bone mineral density (BMD), especially in post-menopausal women (Baulieu et al., 2000; Gordon et al., 2002; Gurnell et al., 2008; Jan­ kowski et al., 2006; Labrie et al., 1997; Nair et al., 2006; Petri et al., 2004; Sun et al., 2002; Villareal et al., 2000; von Muhlen et al., 2008). In men, however, due probably to a lower degree of sex steroid deficiency, significant effects on BMD

have been observed less frequently (Baulieu et al., 2000; Gurnell et al., 2008; Jankowski et al., 2006; Nair et al., 2006; Sun et al., 2002) while no signifi­ cant effects were also reported. Other studies have shown a positive correlation between BMD and serum DHEA levels (Gordon et al., 2002; Miller et al., 2002; Nordin et al., 1985; Sambrook et al., 1992; Taelman et al., 1989; Takayanagi et al., 2002). In order to avoid the limitations of standard ERT or HRT, we have studied the effect of DHEA administration to 60–70-year-old women for 12 months on BMD, parameters of bone formation and turnover, serum lipids, glu­ cose and insulin, adipose tissue mass, muscle mass, energy and well-being as well as on vagi­ nal cytology and endometrial histology (Dia­ mond et al., 1996; Labrie et al., 1997). DHEA was administered percutaneously to avoid first passage of the steroid precursor through the liver. We have thus evaluated the effect of chronic replacement therapy with a daily relatively high dose of 4–6 g of a 10% DHEA cream applied once daily for 12 months in 60–70-year-old women (n = 15). BMD was increased by 2.3% at the lumbar spine level (p < 0.05) (Labrie et al., 1997). These changes in BMD were accompanied by significant decreases at 12 months of 38% and 22% in urinary hydroxy­ proline and in plasma bone alkaline phospha­ tase, respectively (all p < 0.05). An increase of 135% over control (p < 0.05) in plasma osteo­ calcin was concomitantly observed, thus sug­ gesting a stimulatory effect of DHEA on bone formation. In another 12-month study, daily oral treatment with 50 mg DHEA increased BMD at femoral neck and Ward’s triangle in 60–69-year-old women and increased radius BMD in 70–79-year-old women (Baulieu et al., 2000). In a prospective randomized, placebo-controlled study performed in 70 women and 70 men aged 60–88 years with low serum DHEA-S levels and receiving the daily oral dose of 50 mg DHEA for 12 months (Jankowski et al., 2006), BMD was significantly increased in men at total hip (1%),

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trochanter (1.2%) and femur shaft (1.2%). In women, in addition to the sites with significant changes observed in men, lumbar spine BMD was increased by 2.2% while no effect was seen at that site in men. The effect on total hip, tro­ chanter and shaft BMD was larger in women (1.1–1.4%) than men (0.6–1.1%). Most interestingly, while bisphosphonates, SERMs, estrogens and calcitonin only slow down bone loss, DHEA, through its conversion into androgens, stimulates bone formation (Lab­ rie et al., 1997). In fact, DHEA, through the activation of both the androgen (AR) and estro­ gen (ER) receptors present in the osteoblast population, increases trabecular thickness, corti­ cal thickness and cortical density (Moverare et al., 2003). As preclinical support, recent data using AR knock-out (AR KO) mice have shown that AR is necessary not only for cortical but also for trabecular bone development (Kawano et al., 2003; Venken et al., 2006). The beneficial effects of DHEA in rodents on bone mass (Mar­ tel et al., 1998; Turner et al., 1990) are well documented.

DHEA and muscle and lean body mass A characteristic of menopause and aging is a pro­ gressive increase in fat mass and loss of muscle mass (Visser et al., 2003). The loss of muscle mass is paralleled by a loss of muscle function due to a progressive shift from anabolic to catabolic meta­ bolism resulting in a reduced capacity to synthe­ size proteins and repair muscle damage (Proctor et al., 1998). Since 40–50% of androgens in 60–70-year-old men originate from adrenal DHEA (Labrie et al., 2009b), it is reasonable to believe that adrenal DHEA has an importance comparable to testicular testosterone in the control of muscle mass and strength in men. In women, on the other hand, where the ovary secretes negligible amounts of androgens and DHEA is practically the only source of androgens, the impact of DHEA on muscle development and strength is of even greater importance.

There is no doubt that androgens play the pre­ dominant role in muscle growth, development and function. Androgens are well known to increase muscle mass in normal men (Bhasin et al., 1996; Bhasin et al., 2001b), this effect being related to the ban of androgens by the International Olym­ pic Committee. In fact, the major form of sports doping remains androgenic-anabolic abuse. At suitable doses, exogenous androgens enhance muscle mass and strength in all men and women athletes (Handelsman, 2006). As a result, since the early 1970s, exogenous androgens have been banned for men and women in sports. The marked decline in serum DHEA in aging women and men (Fig. 3) has led to the suggestion that a series of changes associated with aging, including loss of muscle mass and strength, may be due to declining DHEA with age (Labrie et al., 1998; Lamberts, 2003). The beneficial effects of DHEA in rodents on body composition are well known (Han et al., 1998; Tagliaferro et al., 1986). Several age-related changes observed in men, especially loss of muscle and bone mass, as well as sexual dysfunction and increase in fat mass are similar to those observed in androgen deficiency (Matsumoto, 2002; Morley and Perry, 2003). Based on cross-sectional data, maximal muscle strength at the age of 70 years is 30–50% of peak muscle strength found at the age of 30 years (Kallman et al., 1990; Murray et al., 1985). The age-associated muscle strength loss seems to be correlated with a reduced cross-sectional area of the muscles (Kallman et al., 1990; Larsson et al., 1979). Age-related sarcopenia increases the risk of falls, fractures, disability and life-threatening complications (Evans, 1997; Frontera et al., 2000; Hughes et al., 2001; Hughes et al., 2002; Iannuzzi-Sucich et al., 2002; Melton et al., 2000b; Melton et al., 2000a). Following studies where apparently too low doses of testosterone were used (Elashoff et al., 1991), a series of recent studies have unequivocally demon­ strated a dose–response stimulatory effect of andro­ gens on muscle size and strength (Bhasin et al., 1996; Bhasin et al., 1997; Bhasin et al., 2001a; Bhasin et al., 2005; Bross et al., 1998; Storer et al., 2003; Woodhouse et al., 2003). Bhasin et al. (2005)

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have compared the efficacy of increasing doses of testosterone on androgen-sensitive parameters in 60–75-year-old and 19–35-year-old men. All men were treated with a GnRH agonist to eliminate endogenous and variable levels of testicular andro­ gens. The weekly doses of testosterone enanthate were 25, 50, 125, 300 and 600 mg for 20 weeks. The effects observed in both young and old men were dose related. The increases in fat-free mass and muscle strength were correlated with the testoster­ one dose and were not different in old and young men. The best tolerance was achieved with the 125 mg dose, a dose giving high normal serum tes­ tosterone levels, low levels of adverse effects and an increase in fat-free mass and muscle strength. (Bhasin et al., 2005). The effects of androgens on the muscle are well recognized in hypogonadal men(Bhasin et al., 1997; Snyder et al., 2000) and men receiving glucocorticoid therapy (Crawford et al., 2003). In a study of 558 men aged 20–95 years, serum DHEA-S was found to be an independent predictor of muscle strength and mass in men aged 60–79 years (Valenti et al., 2004). These results are in agreement with another study showing a correlation between serum DHEA-S and muscle power (Bonnefoy et al., 2002; Kostka et al., 2000). The administration of a daily dose of 50 or 100 mg DHEA for 6 or 12 months, respectively, improved knee extension strength in older men (Yen et al., 1995). No significant effect, however, was found following the administration of DHEA in 60–80-year-old women but the number of sub­ jects was small. Muscle mass increase following DHEA administration has been observed by Yen et al. (1995), Diamond et al. (1996), GebreMedhin et al. (2000), Gordon et al. (2002), Johannsson et al. (2002), Morales et al., (1998), Villareal et al. (2000), Johannsson et al. (2002) and Yen et al. (1995) while others found no sig­ nificant effect (Callies et al., 2001; Percheron et al., 2003; Yen et al., 1995) in women. In a small size study where 17 women received 50 mg DHEA per day for 12 weeks plus exercise training compared to exercise training alone (14 women), body fat was decreased and skeletal weight was increased significantly when DHEA was

combined to exercise (Igwebuike et al., 2008). Lean body mass has been reported to be increased by DHEA treatment (Diamond et al., 1996; Gebre-Medhin et al., 2000; Gurnell et al., 2008; Morales et al., 1998; Nair et al., 2006; Villareal et al., 2000). Postural imbalance and falls are increasingly associated with hip fractures during aging (Cummings and Nevitt, 1989). In fact, it is esti­ mated that 80% of fractures in the elderly occur in the absence of peripheral osteoporosis (Siris et al., 2004). Such data stress the major impor­ tance of preventing falls in older adults by main­ taining muscle mass and strength (Chang et al., 2004). A large proportion of fractures thus result from falls due to loss of muscle mass and strength which should be preventable, up to an unknown extent, by appropriate DHEA replacement.

DHEA and insulin sensitivity Aging is well known to be associated with insulin insensitivity (Davidson, 1979). A deterioration of the glucose tolerance has been found even in the absence of fasting hyperglycemia (Defronzo, 1979). It is useful to remember that a close association is well recognized between type 2 diabetes and cardi­ ovascular disease (CVD) (Defronzo, 1979). Increased insulin sensitivity following DHEA administration has been observed in many studies in women (Casson et al., 1995; Casson et al., 1998; Dhatariya et al., 2005; Diamond et al., 1996; Kawano et al., 2003; Lasco et al., 2001; Mortola and Yen, 1990; Villareal and Holloszy, 2004). In a study performed in post-menopausal women who received a DHEA cream for 12 months, insulin resistance was found to be decreased (Diamond et al., 1996). Similarly, following daily administra­ tion of 50 mg DHEA for 6 months in 65–70-year­ old men and women, the responsiveness of serum insulin to the glucose tolerance test was decreased by 13% with no change in the glucose response, thus leading to a 34% improvement in the insulin sensitivity index following DHEA administration (Villareal and Holloszy, 2004).

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At the daily dose of 50 mg, DHEA has been found to have beneficial effects on glucose metabolism in middle-age post-menopausal women (Casson et al., 1995). Daily oral adminis­ tration of 25 mg DHEA to hypercholesterolemic men for 3 months caused a 30% decrease in basal serum glucose with no significant change in insulin levels (Kawano et al., 2003). DHEA has been observed to induce hypogly­ caemia and anti-diabetic effects in animal models (Coleman et al., 1982; Coleman et al., 1984; McIntosh and Berdanier, 1991; Mukasa et al., 1998). In other studies, no effect was seen on fasting glucose, insulin or glucose/insulin ratio (Callies et al., 2001; Christiansen et al., 2005; Liu and Dillon, 2002). Other preclinical data indicate that some of the anti-diabetic effects of DHEA could be caused by inhibition of the amplification of the local action of glucocorti­ coids by type I 11b-HSD in adipose tissue (Apostolova et al., 2005).

DHEA and lipids Following administration of various doses of DHEA for variable periods of time, small but significant decreases in total and high-density lipoprotein (HDL) cholesterol have been reported (Arlt et al., 1999; Barnhart et al., 1999; Mortola and Yen, 1990; Nestler et al., 1988; Petri et al., 2002; Petri et al., 2004) while, in other studies, low-density lipoprotein (LDL) cholesterol was also decreased in addition to total and HDL cholesterol (Dhatariya et al., 2005; Gebre-Medhin et al., 2000) (Table 1). A small decrease in serum HDL cholesterol has been reported in previous studies with DHEA administered at the daily dose of 50 mg (Arlt et al., 1999; Barnhart et al., 1999; Hunt et al., 2000), 4–6 g 10% DHEA cream (Labrie et al., 1997), 1600 mg (Mortola and Yen, 1990) or 25 mg (Casson et al., 1998) while, in other stu­ dies, no significant effect was seen at 50 mg/day (Barnhart et al., 1999; Morales et al., 1994; Villareal et al., 2000) or 25 mg/day (Kawano et al., 2003; Lovas et al., 2003).

DHEA, contrary to estrogens, does not increase triglycerides (Diamond et al., 1996). In fact, a decrease in triglycerides is often seen with DHEA (Dhatariya et al., 2005; Chang et al., 2002; Lasco et al., 2001). Increased HDL and decreased LDL cholesterol have also been reported (Lasco et al., 2001) while a decrease in total cholesterol only has been reported (Libe et al., 2004; Wil­ liams et al., 2004). DHEA administration in post­ menopausal women has also been reported to decrease serum Apolipoprotein A and increase HDL cholesterol (Casson et al., 1998; Morales et al., 1998). DHEA has been found to decrease serum Lp(A) (Barnhart et al., 1999), an effect which should be beneficial for CVD (Lobo, 1991). The decrease in triglycerides and HDL choles­ terol levels under the influence of androgens has been reported to result from increased hepatic lipase activity which results in increased clear­ ance of HDL (Haffner et al., 1983; Hazzard et al., 1984; Kantor et al., 1985). The increased reverse cholesterol transport (removal of choles­ terol from peripheral tissues via increased HDL clearance) seems responsible for the decreased HDL and triglyceride levels rather than decreased HDL production (Wu & von Eckardstein, 2003). The relatively small (when present) inhibitory effect of DHEA on total cholesterol, HDL cholesterol and sometimes LDL cholesterol could also involve the effects of DHEA-derived androgens on hepatic lipase activity, thus impairing hepatic cholesterol forma­ tion (Tan et al., 1998). The consensus is that DHEA has only small and no clinically significant effects on lipids (Arlt et al., 1998; Gebre-Medhin et al., 2000; Gurnell et al., 2008; Lasco et al., 2001; Lovas and Husebye, 2008; Morales et al., 1998; Poretsky et al., 2006).

DHEA and obesity Decreased fat mass in clinical studies following DHEA administration has been reported by Diamond et al. (1996), Villareal and Holloszy (2004), Libe et al. (2004), Nair et al. (2006),

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Morales et al. (1998) and Igwebuike et al. (2008). In one study, anthropometric measurements showed no change in body weight but a 9.8% decrease in sub-cutaneous skin-fold thickness at 12 months (p < 0.05) (Diamond et al., 1996). A mechanisms of down-regulation of adiposity by DHEA could be through the decrease of peroxisome proliferator­ activated receptor gamma (PPARg) in adipocytes (Kajita et al., 2003). An inverse relationship between DHEA and abdominal obesity in men is well recognized (Tchernof and Labrie, 2004).

DHEA and the breast Women with elevated androgen levels, whether endogenous or exogenous, experience breast atro­ phy consistent with the notion that androgens, per se, are anti-proliferative for the breast (Wierman et al., 2006). Another strong argument against a potential positive correlation between androgen levels and breast cancer is provided by the polycystic ovary syndrome, a situation character­ ized by an androgen excess in which the relative risk (RR) of breast cancer is decreased to 0.52 (Gammon and Thompson, 1991; Wierman et al., 2006). The best demonstration of the inhibitory role of endogenous androgens on the proliferation of the normal epithelial cells of the mammary gland in the primate has been obtained in the Rhesus monkey where physiological levels of exogenous testosterone completely blocked the stimulatory effect of E2 on mammary cell proliferation (Dimitrakakis et al., 2003). It is worth mention­ ing that female athletes as well as transsexuals taking androgens show atrophy of breast gland­ ular tissue (Dimitrakakis et al., 2003; Labrie et al., 2005). In the breast, ERa stimulates several breast cancer-related genes, an effect which is antago­ nized by ERb (Bentov and Casper, 2008; Williams et al., 2008). In the Rhesus monkey, addition of testosterone to estrogen increases breast ERb, markedly reduces the ERa/ERb ratio and decreases mammary epithelium prolif­ eration and MYC gene expression (Dimitrakakis et al., 2003).

All the clinical and most preclinical data show that the administration of androgens inhibits proliferation of the normal mammary gland and breast cancer (Labrie et al., 2003; Ulrich, 1939). The first observation of an inhibitory effect of androgens in women with breast cancer was made by Ulrich (1939). In fact, as reviewed by Labrie et al. (2003), a series of clinical observa­ tions have shown that androgens such as testos­ terone, calusterone and other anabolic steroids have an efficacy comparable to that achieved with other types of endocrine manipulations; however, because of its virilizing effects, andro­ gen therapy has been replaced by tamoxifen, a better-tolerated compound. Nevertheless, andro­ gens and DHEA, acting through the AR, have been shown, in the vast majority of studies, to inhibit estrogen-stimulated proliferation of human breast cancer cell lines in vitro and in vivo as xenografts (for review, see Labrie et al., 2003). Preclinical and clinical data clearly indicating the inhibitory effect of DHEA on mammary gland and breast cancer can be found in the following reviews: Labrie et al. (2003), Labrie et al. (2006) and Labrie (2006, 2007 and 2008).

DHEA and CVD There is undisputed evidence that androgens have beneficial effects on CVD in men (Alexan­ dersen et al., 1996; Anker et al., 1997; Beer et al., 1996; Hak et al., 2002). This is in agreement with the observation that high serum DHEA is associated with decreased deaths and CVD (Alexandersen et al., 1996). Such data obtained in men suggest that similar benefits of androgens should be found in women. In fact, there is no reason to believe that sex steroids have different basic effects in women and men. After all, women have 40–50% as much androgens as men (Labrie et al., 2009b). Clinical trials suggest that testosterone replace­ ment therapy in men may help testosteronedeficient men with angina (English et al., 2000; Malkin et al., 2004), congestive cardiac failure

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(Malkin et al., 2006; Pugh et al., 2004) and type 2 diabetes (Kapoor et al., 2006; Kapoor et al., 2005). Moreover, in the human, data indicate that DHEA inhibits atherosclerosis (Eich et al., 1993; Hayashi et al., 2000; Komesaroff, 2008; Kurzman et al., 1998), reduces cardiovascular risk markers (Beer et al., 1996; Mortola and Yen, 1990) and improves endothelial function (Kawano et al., 2003; Williams et al., 2004). A protective role of DHEA against atherosclerosis has also been observed in primates (ChristopherHennings et al., 1995) and is particularly well known in rabbits (Eich et al., 1993; Gordon et al., 1988). Low serum DHEA-S has been found to be positively associated with the incidence of cardio­ vascular events (Mitchell et al., 1994), the extent (Herrington et al., 1990) as well as the incidence (Herrington et al., 1996) of angiographic coronary stenosis, thus suggesting a protective role of DHEA-S on CVD. Moreover, low serum testos­ terone has been associated with an increased risk of coronary artery disease in men (Turhan et al., 2007) while low DHEA levels have been reported to predispose to earlier death from CVD (BarrettConnor et al., 1986; Tivesten et al., 2009; Ohlsson et al., 2010). Since the assays so far used to measure serum testosterone in women are not reliable due to their lack of specificity and sensitivity at the low serum testosterone levels found in both pre- and post-menopausal women (Labrie et al., 2006), no reliable epidemiological data are available to assess the role of androgens on CVD in women, despite the efforts made (Bell et al., 2007; Liu et al., 2001; Sowers et al., 2005; Sutton-Tyrrell et al., 2005; Wild, 2007). The technical limitations of the testosterone assays have thus precluded the performance of reliable clinical trials on the association of serum androgens and CVD in women. DHEA administration over 3 months increased endothelium-mediated vascular reactivity in both large and small blood vessels, with no change in blood pressure and a decrease in total cholesterol (Williams et al., 2004). Such data indicate the peripheral vasculature as a potential site of

DHEA action and suggest a mechanism by which DHEA may contribute to the improvement of CVD, possibly playing a role in the observed decrease in CVD in men with high serum DHEA­ DHEA-S. DHEA has been found to increase endothelial cell proliferation in vitro and improve endothelial function in vivo in post-menopausal women by mechanisms apparently independent from AR and ER. DHEA increased flow-mediated dilata­ tion and laser Doppler velocimetry (Williams et al., 2004). It is possible that DHEA is acting in endothelial cells through interaction with a specific receptor described in bovine endothelial cells (Liu and Dillon, 2002), although a variety of actions of estrogens and androgens on the vas­ cular endothelium have been described and could well account for the effects observed with DHEA (Chou et al., 1996; White et al., 1997; Williams et al., 2004). In fact, the effects observed in vitro with DHEA in bovine endothe­ lial cells mimic the responses seen with estrogens and androgens (Singh et al., 2000; Zhu et al., 1999). DHEA has been found to act as a survival factor for endothelial cells in protecting them against apoptosis (Liu et al., 2007). In fact, in endothelial cells, DHEA increases nitric oxide secretion, thus protecting endothelial cells against apoptosis (Ling et al., 2002; Simoncini et al., 2003). This anti-atherogenic action of DHEA may be related to a reduction of programmed cell death in the endothelium (Simoncini and Genazzani, 2007). A recent controlled trial with a transdermal patch containing 300 mg testosterone daily in women with androgen deficiency due to hypopi­ tuitarism has shown positive effects on bone den­ sity, body composition and neurobehavioral functions without increasing markers of CVD (Miller et al., 2006; Miller et al., 2007).

DHEA and the brain In addition to the traditional symptoms of menopause (Raven and Hinson, 2007), the

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DHEA decline with age has been linked to loss of memory and cognitive function (Flood and Roberts, 1988; Grimley Evans et al., 2006; Panjari and Davis, 2007). Although the cogni­ tive effects of systemic estrogens are controver­ sial, there is a consensus that endogenous gonadal steroids, namely estrogens, androgens and progesterone, have important effects on the brain, especially in women at the time around menopause (Sherwin, 2003; Yaffe et al., 2000; Yaffe et al., 2002). A role of DHEA has been proposed in the etiology and treatment of neuronal damage induced by Alzheimer’s disease (Simpkins et al., 1997; Weill-Engerer et al., 2002; Yau et al., 2003). The hippocampus is a brain region involved in learning, cognition and memory. This brain area shows pronounced changes dur­ ing aging and in Alzheimer’s disease (Beck and Handa, 2004). Estrogens and DHEA which can form estrogens locally in the brain have been shown to enhance memory and learning functions (Foy, 2001; McEwen et al., 1995; Vallee et al., 2001). Studies have shown that DHEA-S can influence brain function and posi­ tively affect memory mood and energy and indirectly physical activity (Hunt et al., 2000; Huppert and van Nickerk, 2001; Wolkowitz et al., 1999). DHEA is recognized in the brain as a neuro­ steroid (Baulieu and Robel, 1998; Roberts et al., 1987). In addition to DHEA reaching the brain from the circulation, DHEA can be synthesized locally (Compagnone et al., 1995; Corpechot et al., 1981; Robel and Baulieu, 1995). DHEA from both adrenal and local sources can then be converted in the brain into estrogens and/or androgens modulating neuronal activity (Steckelbroeck et al., 2002; Zwain and Yen, 1999). ER, AR and progesterone receptor have been identified in several areas of the brain in women (Stomati et al., 1999) and astrocytes from the hypothalamus have been shown to be able to metabolize DHEA into estrogens and androgens by intracrine mechanisms (Zwain and Yen, 1999). The reported actions of DHEA in the brain include increased neuronal excitability,

g-aminobutyric acid (GABA) type A receptor antagonistic properties and changes in synaptic transmission in the hippocampus (Meyer et al., 1999; Roberts et al., 1987). Whether the potential cognitive effects of DHEA are exerted through interaction with a neuronal receptor or via intra­ cellular conversion into androgens and/or estro­ gens by the same intracrine mechanisms operating in peripheral tissues is not yet known but is of major interest. In addition to the interac­ tion of androgens and estrogens made from DHEA with their own receptors, DHEA could act directly with N-methyl-D-aspartate (NMDA) receptors (Bergeron et al., 1996) as well as GABA and sigma (Majewska et al., 1990) receptors. In aging women and men, circulating levels of gonadal steroids have been positively asso­ ciated with cognitive performance (Yaffe et al., 2000; Yaffe et al., 2002). In laboratory animals, androgens and estrogens both significantly increase cognitive performance (Bimonte-Nel­ son et al., 2003; Dohanich, 2002). Moreover, DHEA has been found to improve memory in mice (Flood et al., 1992; Schlegel et al., 1985). In the ovariectomized rat, daily sub-cutaneous administration of 1 mg DHEA for 2 days increased CA1 synapse density by more than 50% compared to vehicle control (Hajszan et al., 2004). These data are consistent with the hypothesis that DHEA treatment is cap­ able of reversing the decline in hippocampal spine synapse density observed after loss of ovarian estrogen secretion. The blockade of the effect of DHEA on hippocampal synapse density by the aromatase inhibitor letrozole indicates that this particular effect of DHEA is mediated by aromatization of DHEA into estrogens. Pre-treatment with DHEA-S can prevent or reduce the neurotoxic actions in the hippocam­ pus of the glutamate agonists NMDA both in vivo and in vitro, a-amino-3-hydroxy-5-methyl­ 4-isoxazolepropionic acid (AMPA) in vitro and kainic acid in vitro (Kimonides et al., 1998). Similarly, in vitro, 10 nM DHEA was active against NMDA-induced toxicity in hippocampal cultures. In vivo, serum DHEA levels comparable

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to those found in young adults protected CA1/2 neurons against unilateral infusions of NMDA. Since glutamate has been suggested to play a role after cerebral ischaemia and other neural insults (Choi, 1988; Simon et al., 1984), these results led the authors to suggest that decreased DHEA levels may contribute to the higher vulnerability of the aging or stressed human brain to such damage (Kimonides et al., 1998). DHEA has also been shown to reduce damage due to anoxia (Li et al., 2001) and glutamate-induced toxicity (Kimonides et al., 1998). DHEA has been found to have beneficial effects on brain functions, including memory, in experimental animals (Flood and Roberts, 1988; Melchior and Ritzmann, 1994; Young et al., 1991). In fact, DHEA administration has been observed to improve learning potential and memory in aging mice (Flood et al., 1988; Flood and Roberts, 1988; Roberts et al., 1987). In the human, tests of long-term memory have been improved by DHEA administration (Barrett-Connor and Edelstein, 1994). In addi­ tion, the oral administration of 25 mg DHEA per day for 12 months in aging males with partial androgen deficiency improved mood and fatigue in addition to joint pain (Genazzani et al., 2004). Serum cortisol has been reported to increase with aging (Laughlin and Barrett-Connor, 2000). The increase in the serum cortisol/DHEA ratio has been suggested as being involved in the impairment of cognitive functions in the elderly (Kalmijn et al., 1998). Since serum DHEA decreases markedly with age, the serum cortisol/DHEA increases proportionally. In fact, several studies have suggested a positive corre­ lation between cortisol plasma levels and mem­ ory disturbance (Swaab et al., 2005). Moreover, patients affected by Alzheimer’s disease have high plasma cortisol levels (Swaab et al., 2005). On the other hand, DHEA administration has been found to decrease serum cortisol levels (Genazzani et al., 2003; Stomati et al., 2000). Moreover, an anti-depressant effect of DHEA has also been described (Wolkowitz et al., 1999).

DHEA and sexual function As mentioned above, intravaginal administration of DHEA has important beneficial effects on the four domains of sexual dysfunction in post-meno­ pausal women suffering from vaginal atrophy (Labrie et al., 2009c). The effects were observed without significant changes in serum estrogens or androgens, thus indicating a strictly local effect (Fig. 11). Studies have also been performed using oral DHEA. In a placebo-controlled study per­ formed in 70 post-menopausal women, daily treatment with 50 mg DHEA for 12 months, improved libido and sexual function (Baulieu et al., 2000). With the same DHEA dose administered for 4 months in another placebocontrolled trial performed in 24 women, increased frequency of sexual thoughts and interest was reported (Arlt et al., 1999). In an acute study performed in 16 women who received 300 mg of DHEA orally, greater mental and physical sexual arousal to an erotic video was reported (Hackbert and Heiman, 2002). Not all studies have reported a significant effect of DHEA on sexual function. For exam­ ple, no significant effect was found when DHEA was administered at the 50 mg daily dose for 3 months to 17 women and 13 men, respectively (Morales et al., 1994). Improved physical and psychological well-being was how­ ever observed. In another small size trial, no significant effect on sexual dysfunction was observed (Hunt et al., 2000). Since it is now understood that serum testosterone does not reflect the total androgen pool (Labrie et al., 2006), it is not surprising that serum testoster­ one needs to be increased to supraphysiological levels to improve sexual function since the serum levels represent only a small fraction of total androgens, which are almost all made intracellularly. Based upon the observation that the pool of androgens in women is 40% of that in men, serum testosterone levels of 1.37 ng/ml would be needed to cause a 50% increase in the total androgen pool in women aged 60–70-year-old (Labrie et al., 2009b; Shif­ ren et al., 2000).

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DHEA and the skin DHEA has been shown to have important effects on the skin of aged individuals, the most salient of which is an increase in sebum production (Labrie et al., 1997). The index of sebum secretion was 79% increased after 12 months of DHEA therapy with a return to pre-treatment values 3 months after cessation of treatment. This effect has been shown in another study performed in women, par­ ticularly those aged >70 years who are physiologi­ cally hyposeborrheic (Baulieu et al., 2000). The DHEA-induced increase in sebum production is probably due to the fact that the sebaceous glands contain all the steroidogenic enzymes necessary to catalyze the transformation of DHEA into the androgen DHT, this androgen being the main stimulator of sebaceous gland activity (Labrie et al., 2000; Labrie et al., 2003). Apart from sebum production, other beneficial effects of DHEA on the skin have been noticed. Dermatological aspects of DHEA administration have been evaluated in some detail in a study where male and female subjects between the ages of 60 and 79 years were orally administered 50 mg DHEA, once daily for 1 year (Baulieu et al., 2000). Skin surface hydration significantly increased for the whole DHEA-treated popula­ tion examined after 12 months of treatment. Skin surface hydration is considered a real ben­ efit for the skin, especially in aged individuals since in these subjects the dryness makes the skin rough. DHEA also significantly decreased facial skin pigmentation (yellowness) for the whole population. This decrease was more pro­ nounced in women aged >70 years who are more concerned by age-related pigment changes. The two other components of skin colour remained stable during the duration of the study (i.e. lightness and redness).

Anti-carcinogenic activity of DHEA The absence in rodent peripheral tissues of the uridine diphosphate glucuronosyltransferases responsible, in large part, for the inactivation of estrogens and androgens suggests that in the

presence of comparable serum concentrations of DHEA following its exogenous administration, the intracellular levels of sex steroids should be much higher in rodents than humans due to the absence of peripheral inactivation of active sex steroids in rodents. Accordingly, the finding of an anti-carcinogenic action of DHEA in the rodent very strongly indicates the absence of car­ cinogenic potential of DHEA in the human where lower intratissular concentrations of DHEAderived steroids should be found due to the high rate of their inactivation in the human. DHEA is well known to exert anti-proliferative effects (Ho et al., 2008). Much evidence has been obtained on the preclinical chemopreventive effi­ cacy of DHEA (reviewed in Schwartz et al., 1992; Levi et al., 2001; Elmore et al., 2001). In fact, a long series of studies have shown that DHEA, instead of being carcinogenic as shown for estro­ gens, exerts an opposite effect, namely an anticarcinogenic effect (Boone et al., 1992; Ratko et al., 1991; Kelloff et al., 1996; Kelloff et al., 1994; White et al., 1998). Among these, it has been shown that oral DHEA administration in rats and mice inhibits the development of sponta­ neous breast cancer and various chemically induced tumors of the colon (Nyce et al., 1984; Steele et al., 1994), lung (Schwartz et al., 1981; Schwartz and Tannen, 1981), breast (Ratko et al., 1991), liver (Garcea et al., 1987; Moore et al., 1986; Simile et al. 1995; Weber et al., 1988) and prostate (Christov et al., 2004; Steele et al., 1994). DHEA has also been found to inhibit che­ mically induced tumors of the thyroid, skin and lymphatic tissue as well as the formation of preneoplastic liver foci and hemangiosarcomas of the liver (Green et al., 2001; Levi et al., 2001; Schwartz and Pashko, 1993; Schwartz et al., 1986; Schwartz et al., 1992). It was also shown that DHEA confers significant protection against prostate carcinogen­ esis (Rao et al., 1999; Weber et al., 1988). DHEA has also been shown to prevent tumorigenesis in p53 knock-out mice (Hursting et al., 1995). DHEA and its analogue 8354 have shown inhibitory effects similar to DHEA on the devel­ opment of mammary gland carcinogenesis (McCormick et al., 1996; Ratko et al., 1991; Schwartz et al., 1989; Steele et al., 1994), including

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spontaneous breast cancer in the mouse (Schwartz, 1979; Schwartz et al., 1981). We have also observed the inhibitory effects of androgens and DHEA on dimethylbenzanthra­ cene (DMBA)-induced mammary carcinoma in the rat (Dauvois et al., 1989; Li et al., 1993; Luo et al., 1997; Luo et al., 1997) as well as on human breast cancer ZR-75-1 xenografts in nude mice (Couillard et al., 1998; Dauvois et al., 1991). Topi­ cal application of DHEA inhibits the development of DMBA-induced and tetradecanoylphorbol acetate (TPA) promoted skin papillomas and car­ cinomas in the mouse (Pashko et al., 1985; Pashko et al., 1984). The anti-carcinogenic effect of DHEA in breast cancer can be logically explained by the predomi­ nant formation of androgens over estrogens from DHEA in breast tissue, thus providing a higher influence of androgens which are well-known inhi­ bitors of mammary gland proliferation in vitro, as well as in vivo in animal models and in women (Labrie et al., 2003). In fact, DHEA has been identified as one of the promising chemoprotec­ tive agents by the US National Cancer Institute (NCI), Division of Cancer Prevention and Con­ trol. DHEA is part of the ~30 promising com­ pounds across a group of agents chosen by the NCI for clinical chemoprevention trials (Kelloff et al., 1996). It can be mentioned that low DHEA levels have been reported to predispose to certain cancers (Gordon et al., 1993; Gordon et al., 1991; Regelson and Kalimi, 1994) including an increased risk of breast (Zumoff et al., 1981) and bladder (Gordon et al., 1991) cancers. In addition to its conversion into androgens, other proposed mechanisms are possibly involved. These pertain to inhibition of glucose-6-phosphate dehydrogenase, 3-hydroxy-3-methylglutaryl CoA reductase, glucose metabolism or mitochondrial gene expression (Ho et al., 2008; Pascale et al., 1995; Tian et al., 1998).

DHEA and increased longevity/decreased mortality There are many data associating low serum DHEA with increased risk of death in men,

including CVD (Barrett-Connor et al., 1986; Maggio et al., 2007; Trivedi and Khaw, 2001; Tivesten et al., 2009; Ohlsson et al., 2010). In the Rancho Bernardo Study, a correlation is found between higher serum DHEA-S levels and low cardiovascular risk in men aged 50 years or older (Barrett-Connor et al., 1986). The same correla­ tion has been observed in young men (SlowinskaSrzednicka et al., 1991). The PAQUID study has shown higher mortality in men having lower serum DHEA (Berr et al., 1996; Mazat et al., 2001). Low testosterone, in addition, has been reported to be a predictor of mortality in male veterans (Shores et al., 2006). Low serum DHEA-S was also associated with higher mortality in the InCHIANTI study (Invec­ chiare in Chianti, aging in the Chianti area) (Maggio et al., 2007). When adding one or two other anabolic hormone(s) (such as testosterone, DHEA-S or IGF-1), the risk of mortality increases with the number of anabolic hormones from 1.47 to 1.85 and to 2.29 (Maggio et al., 2007). Ageassociated decline in serum anabolic hormones thus appears a strong independent predictor of mortality in older men (Maggio et al., 2007). In fact, the decrease in serum testosterone, DHEA-S and insulin-like growth factor (IGF-1) may be associated with a decrease in muscle mass, increase in fat deposition, development of insulin resistance and other medical conditions which affect mortality (Anker et al., 1997; Anker et al., 1997). In a recent study performed in 2644 men of median age of 75 years followed for 4.5 years during which 328 deaths occurred, low serum DHEA and DHEA-S levels have been found to be associated with 49% and 47% increased risk of all causes of deaths when the 25% of men having the lowest levels of serum DHEA were compared with the rest of the cohort [hazard ratio (HR) of 1.49 with 95% confidence interval (CI) of 1.18–1.88]. When deaths from ischaemic heart disease was considered, the 25% of men having lowest DHEA and DHEA-S levels had 72% and 61% higher risks of cardiovascular death (n = 73 deaths, HR = 1.94 with a 95% con­ fidence interval of 1.20–3.13) (Ohlsson et al., 2010). The corresponding increased risks of

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death from ischemic heart disease were 94% and 75%, respectively, for low DHEA and DHEA-S. It is of interest that 11.5% of all deaths and 11.6% of cardiovascular deaths which occurred during the 4.5-year follow-up period could have been prevented if the men having serum DHEA in the low quartile had serum DHEA in the other quartiles with higher serum DHEA. This increased risk with low serum DHEA was inde­ pendent from traditional risk factors for CVD. In men, there are also studies reporting no signifi­ cant correlation (Barrett-Connor and GoodmanGruen, 1995; Feldman et al., 2001; Kahonen et al., 2000; Legrain et al., 1995; Tilvis et al., 1999) between serum DHEA and mortality. Since approximately equal amounts of andro­ gens in men are of testicular origin (directly reflected by serum testosterone), it is pertinent to remember that in a prospective populationbased study of 794 men aged 50–91 years who were followed for an average of 11.8 years, 538 deaths occurred. In that study, men having serum testosterone concentrations in the lowest quartile (<2.41 ng/ml) were 40% more likely to die during the next 20 years compared to the remaining men with higher serum testosterone levels (Laughlin & Barrett-Connor, 2000). In fact, low serum tes­ tosterone was associated with increased risk of CVD and respiratory disease. As a possible explanation for the association between anabolic hormones and longevity, treat­ ment with DHEA is well known to reduce experi­ mental atherosclerosis (Alexandersen et al., 1999; Arad et al., 1989; Eich et al., 1993; Gordon et al., 1988). These beneficial effects of DHEA on ather­ osclerosis may be related to the described effects of DHEA which improves endothelial functions (Liu and Dillon, 2002, 2004; Simoncini et al., 2003; Yorek et al., 2002) and exerts anti-inflammatory (Altman et al., 2008; Barkhausen et al., 2006; Chen and Parker, 2004; Dillon, 2005; Gutierrez et al., 2007) and anti-oxidative effects (Altman et al., 2008; Khalil et al., 1998; Poynter and Daynes, 1998; Yorek et al., 2002). Approximately 30% of men aged 60 years and older have low serum testosterone (Harman et al., 2001) which is accompanied by low bone and muscle mass, increased fat

mass (especially central adiposity), low energy and impaired physical sexual and cognitive func­ tion (Laughlin et al., 2008). Prospective cohort studies have shown low testosterone as being associated with type 2 diabetes (Ding et al., 2006), depressive illness (Shores et al., 2004) and Alzheimer’s disease (Hak et al., 2002; Mof­ fat et al., 2002). In the United States alone, 300 000 women have bilateral oophorectomy at time of hyster­ ectomy to prevent the risk of developing of ovarian cancer (Keshavarz et al., 2008), while another 300 000 US women undergo bilateral ovariectomy for a benign condition every year (Melton et al., 1991; Rocca et al., 2006). While there is much evidence in men that low serum testosterone and/or DHEA is associated with increased mortality, especially CVD, it has been found that women who underwent bital­ eral oophorectomy before age 45 years experi­ enced an increased mortality associated with CVD compared with referent women (HR, 1.44; 95% CI, 1.01–2.05; p = 0.04) (Rivera et al., 2009). Early analyses of the Nurses’ Health Study, has indicated that women with oophorectomy between ages 40 and 44 years had double risk of myocardial infraction (RR, 2.2; 95% CI, 1.2 – 4.2) (Colditz et al., 1987). In a Dutch study, delaying oophorectomy was found to be associated with lower long-term mortality (HR, 0.96; 95% CI, 0.91–0.99 per year of delayed oophorectomy) and mortality due to coronary heart disease (HR, 0.92; 95% CI, 0.87–0.98 per year of delayed oophorect­ omy (Ossewaarde et al., 2005). It has also been found that oophorectomy after the age of 50 years increased the risk of a first myocar­ dial infarction (RR, 1.4; 95% CI, 1.0–2.0) (Falkeborn et al., 2000). Moreover, in a meta­ analysis of observational studies, oophorectomy more than doubled the risk of CVD (RR, 2.62; 95% CI, 2.05–3.35) (Atsma et al., 2006). On the other hand, it has been found that ovarian conservation improved survival in women younger than 65 years at time of oophorectomy (Parker et al., 2005). In the Nurses’ Health Study with over 24 years of follow-up, the multi-variable HRs for

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women who had bilateral oophorectomy com­ pared to ovarian conservation were 1.12 (95% CI, 1.03–1.21) for total mortality, 1.17 (95% CI, 1.02–1.35) for fatal and non-fatal coronary heart disease, 1.14 (95% CI, 0.98–1.33) for stroke and 1.26 (95% CI, 1.02 – 1.56) for lung cancer. Since it was estimated that for 2005, coronary heart disease was responsible for 326 900 deaths while stroke was the cause of 86 900 deaths per year (Kung et al., 2008) among US women (Parker and Manson, 2009), the impact of oophorectomy on mortality in women could be very significant. “With an approximate 35­ year life expectancy after oophorectomy, one additional death would be expected for every nine oophorectomies performed. Furthermore, prophylactic oophorectomy did not improve survival at any age” (Parker et al., 2009). It has been recently observed that circulating DHEA, the precursor of practically all estro­ gens and androgens after menopause, is 18% lower in oophorectomized post-menopausal women aged 42–74 years compared to intact women of the same age (Labrie et al. unpub­ lished data). These data could suggest that lower serum DHEA in oophorectomized women is involved in the increased mortality described above. Low circulating DHEA would then have the same negative impact on cardiovascular mortality in women as already well recognized in men.

Safety profile of DHEA No serious adverse event related to DHEA has ever been reported in any published data describing DHEA administration in women or men (Tables 1 and 2; Allolio, Arlt, & Hahner, 2007). The only side effects reported, despite the high doses frequently used, in a small pro­ portion of women are mild facial acne, increased sebum production and mild changes in hair growth which, usually, do not require withdrawing from study (Allolio et al., 2007; Hunt et al., 2000; Labrie et al., 1997). The incidence of these mild side effects is usually

reported at about 50% of the same rate observed in placebo-treated subjects (Genelabs, Briefing document, 19 April 2001 FDA Arthri­ tis Advisory Committee). The higher sebum production is welcome in post-menopausal women who frequently complain of dry skin while some regrowth of pubic and axillary hair is generally considered positive. In two Genelabs’ placebo-controlled trials and the open-label extension study which followed completion of the double-blind studies, 387 women have received DHEA, usually at the daily 200 mg dose for at least 6 months, 242 for  12 months, 138 for  18 months and 36 for  24 months. Six hundred and forty one women have been exposed to DHEA (Briefing document FDA, Arthritis Advisory Committee, 19 April 2001). The patients in the Genelabs’ studies were suffering from systemic lupus erythematosus (SLE) and, in a large proportion, were already treated with relatively high doses of glucocorti­ coids. In 200 mg DHEA-treated patients, a sig­ nificant increase versus placebo was observed for acne (36.0 vs. 15.2%) and hirsutism (14.2 vs. 2.3%). The relatively high incidence of acne in both the placebo- and DHEA-treated groups could be related to the fact that many of these patients were taking glucocorticoids. It is of interest that 64 and 53% of the incidence of acne and hirsutism, respectively, were reported during the first 182 days of treatment compared to during the later interval between 183 and 547 days. Hematuria (3.6 vs. 0.4%) and increased crea­ tinine (2.4 vs. 0%) occurred only in a small number of patients and were not associated with renal dysfunction. The clinical significance of these changes seen in patients with SLE probably directly affecting the kidney was reported of unknown significance (Genelabs, Briefing document, 19 April 2001 FDA Arthri­ tis Advisory Committee). That these low fre­ quency side effects were associated with SLE and/or glucocorticoids is supported by the absence of such findings in any other study. There were no significant differences between treatment groups in haematology or liver

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function parameters. Myalgia reported in 30.9% of placebo patients was decreased to 22.2 and 21.7% (both p < 0.01 vs. placebo) in women with SLE treated with the 100 and 200 mg DHEA doses, respectively. Nasal ulcers, joint disorders, rash lupus erythematosus and anor­ exia were less frequent in women treated with 200 mg DHEA (n = 253) compared to placebo (n = 256) (p < 0.01 for all). It is also pertinent to mention the Memorandum from Claudia B. Karkowski dated 12 March 2001. This document from the FDA provides an over­ view of post-marketing adverse events reported in association with the use of DHEA, where much higher doses or oral DHEA have been used for long time periods. These cases were retrieved from the Adverse Event Reporting System (AERS), Center for Food Safety and Applied Nutrition (CFSAN)’s post-marketing database, and the med­ ical literature. The conclusion was that no clear safety signals were identified with this product. This served as a companion document to the review by Parivash Nourjah, Ph.D., entitled Epidemiologic evidence of DHEA in the etiology of neoplasia which focussed in a review of the lit­ erature for any published epidemiologic studies that examined cancer risk associated with exogenous DHEA administration. The primary objective in this review was to determine if there were any case report in our post-marketing databases of the medical literature of neoplasia in association with the use of DHEA. The same single case of worsening of metastatic prostate cancer was described (Jones et al., 1997). In the memorandum (20 February 2001) of Parivash Nourjah, Division of Post-marketing Drug Risk Assessment 1, HFD-430, it was concluded that ‘no meaningful conclusion about the association of exogenously administered DHEA and cancer risk can be made based on these epidemiological studies of endogenous levels of DHEA’. ‘Of the 65 cases identified in the AERS data­ base, SCSAN ARMS database, and the medical literature, there was only one report of neoplasia’, namely, as mentioned above, a case of worsening of prostate cancer in a man with metastatic pros­ tate cancer who received 200–700 mg DHEA daily for the treatment of anaemia unresponsive to

erythropoietin. ‘This was already summarized in Dr. Nourjah’s review’. ‘His blood cells increased during DHEA eliminating his need for transfu­ sions. However, the patient began to develop facial numbness, increase in prostate size and dif­ ficulty voiding. His PSA (prostatic-specific anti­ gen) levels increased to greater than 10 000 ng/ml (2726 ng/ml prior to DHEA). DHEA was discon­ tinued and DES was initiated with improvement in symptoms and decrease in PSA. Although he exhibited a positive dechallenge after discontinua­ tion of DHEA, his improvement may have been due to treatment with DES (Jones et al., 1997). There was no report of neoplasia in women. The recent elucidation of the physiological role of DHEA, especially in post-menopausal women, can explain the good safety profile of the com­ pound. In fact, DHEA is a physiological and essential precursor of sex steroids which are required, to various degrees, for the normal func­ tioning of most if not all organs in the human. Since there is no feedback mechanism to increase DHEA secretion in subjects with low DHEA (Fig. 6) suffering from the resulting hormone defi­ ciency symptoms (Table 3), the only physiological means of providing the missing amount of DHEA is local or systemic administration of the sex ster­ oid precursor. Due to the normal saturation mechanisms mentioned above (Fig. 7) and the relatively small bioavailable DHEA from local or systemic administration compared to the rela­ tively high amounts already present in normal women, it is unlikely that any significant side effect will ever be seen with reasonable DHEA replacement therapy using high-quality pharma­ ceutical grade product. Most importantly, the intracellular action of DHEA with no significant release of estrogens eliminates the risks asso­ ciated with estrogen-based replacement therapies, including the increased risk of breast cancer. Treatment with DHEA simply mimics the physio­ logical situation after menopause where systemic exposure to estrogens has ceased following 500 million years of evolution and DHEA becomes the tissue-specific provider of sex steroids with no systemic exposure to the active compounds which are inactivated locally before being released in the circulation for elimination.

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 5

Neuroendocrinology of post-traumatic stress disorder Panagiota Pervanidou1,� and George P. Chrousos2 1

Developmental and Behavioral Pediatrics Unit, First Department of Pediatrics, Athens University Medical School,

“Agia Sophia” Children’s Hospital, Goudi, Athens, Greece

2 Division of Endocrinology, Diabetes and Metabolism, First Department of Pediatrics, University of Athens, “Agia

Sophia” Children’s Hospital, Goudi, Athens, Greece

Abstract: Dysregulation of the stress system, including the hypothalamic–pituitary–adrenal (HPA) axis and the locus caeruleus/norepinephrine–sympathetic nervous system (SNS), is involved in the pathophysiology of post-traumatic stress disorder (PTSD), an anxiety disorder that develops after exposure to traumatic life events. Neuroendocrine studies in individuals with PTSD have demonstrated elevated basal cerebrospinal fluid corticotropin-releasing hormone concentrations and contradictory results from peripheral measurements, exhibiting low 24 hours excretion of urinary free cortisol, low or normal circulating cortisol levels or even high plasma cortisol levels. The direction of HPA axis activity (hyper-/or hypo-activation), as evidenced by peripheral cortisol measures, may depend on variables such as genetic vulnerability and epigenetic changes, age and developmental stage of the individual, type and chronicity of trauma, co-morbid depression or other psychopathology, alcohol or other drug abuse and time since the traumatic experience. On the other hand, peripheral biomarkers of the SNS activity are more consistent, showing increased 24 h urinary or plasma catecholamines in PTSD patients compared to control individuals. Chronically disturbed hormones in PTSD may contribute to brain changes and further emotional and behavior symptoms and disorders, as well as to an increased cardiometabolic risk. Keywords: hypothalamic–pituitary–adrenal (HPA) axis; locus caeruleus; norepinephrine; sympathetic nervous system; psychiatric disorders; co-morbid disorders

of the 20th century, he formulated the hypothesis of the overwhelming impact of a traumatic experience on the psychologic and physical health of an individual, by damaging the person’s ability to adequately cope with the situation (Chrousos 2009; Freud, 1973; Pervanidou and Chrousos, 2007). Later, during the 1930s and 1940s, Hans Selye conceptualized the physiologic adaptive response to stress as a process called ‘The General

Adaptation Syndrome’ (Chrousos and Gold,

Trauma and stress: a historical perspective The concept of ‘psychological trauma’, as an individual’s experience of a perceived life threat caused by an external life event, is attributed to Sigmund Freud. In the second and third decades �

Corresponding author.

Tel.: 0030-210-746-74-57; Fax: 0030-210-77-59-167; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82005-9

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1992). Selye described the crucial role of the hypo­ physis and the adrenal cortex in the stress response and named the stress-causing agent ‘stressor’. He also differentiated the negative sta­ tus of ‘distress’ with positive ‘eustress’ conditions. Though trauma is not only an ‘extreme form of stress’ and although the stress model has become much more complex overtime, both psychologic and physiologic causes of trauma and stress con­ tribute to the understanding of post-traumatic stress disorder (PTSD), as an entity of behavior, emotional and physiologic responses to perceived stress.

Concepts and current definition of PTSD Since the 1980s, concepts of trauma had focused on the fear evoked by trauma and the anxiety resulting from and related to that fear. The cur­ rent conceptualization of PTSD has its origins in Vietnam combat veterans; however, nowadays, in addition to the large number of soldiers experi­ encing combats around the word, societies also face high rates of exposure to traumatic life events, such as terrorism, assaults, rapes, child­ hood maltreatment and accidents. Most indivi­ duals are able to cope with stressors; however, a percentage of individuals fails to recover and exhibits abnormal and prolonged behavior and physical manifestations, expressed most com­ monly as PTSD. PTSD emerged as a clinical diagnosis in 1980 on the DSM-IV and is classified as an anxiety disorder. The term describes a syn­ drome of distress that develops after exposure to events or circumstances that involved actual death or injury or a threat to the physical integ­ rity of the individual or others and that evoked intense fear, helplessness or horror. PTSD includes the following clustering of symptoms: (1) re-experience of the initial trauma via intru­ sive memories and/or dreams about the event, feeling as if the trauma was continuing to occur and intense distress on exposure to cues that recall the event. In young children, repetitive play or trauma-specific re-enactment may occur in which themes or aspects of the trauma are

expressed. (2) Avoidance of stimuli associated with the trauma and numbing of overall respon­ siveness. (3) Symptoms of excessive arousal, including insomnia, angry outbursts, hypervigi­ lance, exaggerated startle response and difficulty concentrating. PTSD lasts at least 4 weeks, as opposed to acute stress disorder that begins within 1 month of the event and lasts from 2 days to 4 weeks (Diagnostic and Statistical Man­ ual of Mental Disorders-DSM IV, 1994).

Pathophysiology of symptom clustering in PTSD Clinical manifestations of PTSD reflect stressrelated changes in neurobiological structures and systems of the organism, induced by severe external stressors. Inevitably, the stress system has become the target of neurobiological research in PTSD. The stress system, including the hypothalamic–pituitary–adrenal (HPA) axis and the locus caeruleus/norepinephrine–sympa­ thetic nervous system (LC/NE–SNS), is critical in the physiologic response of the organism to stressors. Normally, activation of the stress sys­ tem leads to behavior and physical adaptive changes that improve an organism’s ability to survive. However, excessive and prolonged acti­ vation of the stress system, might lead to longterm behavior and physical complications (Chrousos and Gold, 1992). In addition to altera­ tions in specific neuroendocrine parameters, neu­ roanatomical structures such as the hippocampus, amygdalae and the prefrontal cortex are impli­ cated in PTSD pathophysiology. Findings are summarized below.

Hypothalamic–pituitary–adrenal axis: a summary of findings The HPA axis has been the main focus of neu­ roendocrine research in PTSD. Exposure to stress is associated with secretion of corticotropinreleasing hormone (CRH) by parvicellular neu­ rons in the hypothalamic paraventricular nucleus. CRH is released into the hypothalamo-pituitary

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portal circulation and stimulates the secretion of adrenocorticotropin (ACTH) by the anterior pituitary. ACTH, in turn, stimulates the release of glucocorticoids from the adrenal cortex. Corti­ sol, as the final peripheral molecule, exerts its actions on metabolism, immunity and brain func­ tions. Increased and prolonged production of CRH and cortisol explain many of the behavior, circulatory, metabolic and immune manifestations of syndromes associated with chronic stress, such as PTSD. However, repeated or chronic stress might also result in hypo-activation of the HPA axis, likely reflecting a compensatory physiologic adaptation (Charmandari et al., 2005; Chrousos and Gold, 1992). Neuroendocrine studies of the HPA axis in adults with PTSD have demonstrated that: 1. Basal cerebrospinal fluid (CSF) CRH levels are elevated, as indicated mainly by two studies, the first using a single lumbar puncture and the second serial CSF sampling (Baker et al., 1999; Bremner et al., 1997). 2. Urinary cortisol levels were reported to be low in the majority of studies; however, other studies demonstrated variable results: no differences or more rarely increased urinary cortisol excretion compared to control subjects (Mason et al., 1986; Yehuda et al., 1990, 1995; Yehuda, 2001). 3. Plasma cortisol levels provide also inconsistent findings in PTSD; however, a large study of more than 2000 Vietnam trauma victims reported low cortisol concentrations (Boscarino, 1996). 4. Consecutive blood sampling in PTSD patients showed reduced cortisol concentrations several times during the circadian cycle, mainly in the late evening and early morning hours compared to the non-PTSD group (Yehuda et al., 1994). A cortisol rhythm study indicated that cortisol levels in PTSD subjects were comparable at their peak to the non-PTSD group, but lower at the nadir (Yehuda, 2001). A more recent study in PTSD survivors, examined years after the trauma, showed an altered circadian rhythm in salivary cortisol (Yehuda et al., 2005).

5. No detectable differences in ACTH levels have been reported in PTSD patients compared to non-PTSD controls (Yehuda et al., 2004). 6. An increased number of lymphocyte glucocorticoid receptors (GRs) has been reported in patients with PTSD compared to normal controls (Yehuda et al., 1991). 7. Enhanced cortisol suppression, as reflected by lower cortisol levels after the dexamethasone (Dex) suppression test, has been noted in PTSD patients compared to the non-PTSD controls (Yehuda et al., 1993). However, a recent meta-analysis of 37 studies and 828 people with PTSD (Meewisse et al., 2007) found no differences in cortisol levels between individuals with PTSD and controls. This meta­ analysis revealed some interesting results in the sub-group analyses: Although no differences were found for morning samples, people with PTSD had lower afternoon levels of cortisol than con­ trols. Gender was also an important variable, since women with PTSD had highly significant lower cortisol levels than controls. Examining the type of trauma, only the sub-group with physical or sexual abuse had significantly lower cortisol levels than controls, whereas this finding was not con­ firmed for other types of trauma. Despite the limitation of differences in data collection and assessment, this meta-analysis clearly reveals the complexity and heterogeneity of the neuroendo­ crine concomitants of the disorder.

The sympathetic nervous system The catecholamines – norepinephrine (NE), epi­ nephrine (E) and dopamine (DA) – comprise a family of neurotransmitters, derived from the amino acid tyrosine. NE is one of the key neuro­ transmitters of the central nervous system (CNS) and autonomic stress responses. NE is produced in neurons of the locus caeruleus (LC), projecting to various brain regions, including the prefrontal cor­ tex, amygdalae, hippocampus and hypothalamus. In this central circuit, NE interacts with CRH to increase fear conditioning and encoding of

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emotional memories. In the periphery, stress leads to release of NE and E from the adrenal medulla, resulting in an adaptive alarm reaction of the organism. Hyperarousal and re-experience, expressed with increased heart rate, blood pres­ sure, skin conductance and an exaggerated startle response, are two main – and diagnostic – clusters of symptoms in PTSD (O’Donnell et al., 2004; Southwick et al., 1999a). Furthermore, emotional stressful stimuli may enhance memory encoding in individuals suffering from PTSD, resulting in defective consolidation of memory for the stress­ ful event, and leading to the development and maintenance of intrusive thoughts, images, flash­ backs and repetitive nightmares (Cahill, 1997; Southwick et al., 2002). Consequently, NE has been a key candidate in studying the pathophy­ siology of PTSD. Indeed, the most consistent finding in PTSD neuroendocrine studies is increased noradrener­ gic activity, both centrally (Geracioti et al., 2001) and peripherally (Yehuda, 2001; Yehuda et al., 1992; Southwick et al., 1999b) in adults. Similarly, children with PTSD and post-traumatic sympto­ matology have been reported to have increased peripheral sympathetic nervous system (SNS) activity (Delahanty et al., 2005; De Bellis et al., 1994; Pervanidou et al., 2007a) Urinary catecho­ lamine excretion is higher in PTSD patients than control subjects or subjects with other psychiatric disorders (Kosten et al., 1987; Mason et al., 1988; Pitman and Orr, 1990; Yehuda et al., 1992). Few studies examined plasma NE levels: mean plasma NE and 3-methoxy-4-hydroxyphenylglycol, a metabolite of NE, concentrations were higher in war veterans with PTSD than in veterans with PTSD and co-morbid depression, patients with major depressive disorder (MDD) and healthy volunteers (Yehuda et al., 1998). Other studies have shown elevated plasma NE concentrations (Blanchard et al., 1991; Murburg et al., 1995; Yang, 1998) in adults with PTSD compared to controls, while CSF NA concentrations were also reported high in PTSD subjects (Geracioti et al., 2001). Furthermore, decreased platelet a2 receptor binding suggests NE hyperactivity in PTSD (Vermetten and Bremner, 2002; Strawn and Geracioti, 2008).

Longitudinal interactions between the HPA axis and the SNS in PTSD development and maintenance Although a variety of abnormalities in the HPA axis have been reported in PTSD patients, the longitudinal course and interaction between this axis and the arousal/sympathetic system have rarely been described. Our study (Pervanidou et al., 2007a, 2007b) investigated the longitudinal neuroendocrine changes in relation to the devel­ opment and maintenance of PTSD diagnosis in children and adolescents after motor vehicle acci­ dents. We compared a group that developed PTSD 1 month after the accident (30% of the population), with the group that did not develop PTSD but had experienced the traumatic event, and a control group without exposure to accident. We further studied the sub-group (15%) that developed PTSD at month 1 and maintained PTSD diagnosis at month 6, in comparison to those that did not develop PTSD at month 1, nor at month 6. Evening (9.00 p.m.) salivary cor­ tisol in the aftermath of the trauma was higher, and circadian rhythm was more disturbed, in those children that later developed PTSD com­ pared to those that did not develop PTSD (Fig. 1). Evening salivary cortisol and morning interleukin-6 were both predictive of PTSD development at month 6. Plasma NE concentra­ tions did not differ among groups after the acci­ dent and they were not predictive of later PTSD development (Pervanidou et al., 2007b). The PTSD group exhibited higher NE concentrations compared to the other two groups at both months 1 and 6. Furthermore, NE became gradually greater (from month 1 to month 6) within the PTSD group. Interestingly, the non-PTSD group followed a similar pattern of gradual elevation of NE within the group, at a lower setting, showing that the maintenance of sub-threshold PTSD symptoms is also related to NE elevations. As evening cortisol concentrations and circadian rhythm normalized in the PTSD subjects at month 6, NE elevations became greater (Figs. 1 and 2) (Pervanidou et al., 2007a). This could be the effect of lifting a cortisol-mediated noradre­ nergic system restraint. Clinical symptoms

153 B.

PTSD (1 and 6 months)

A.

*

Salivary cortisol (µg/dl) month 1

Salivary cortisol (µg/dl) time 0

Non-PTSD (1 and 6 months) 0.5

Control

0.4 * 0.3 * 0.2 0.1

8

12

15

18

PTSD (1 and 6 months) Non-PTSD (1 and 6 months)

0.5

Control

0.4

0.3 0.2 0.1

8

21

12

15

18

21

Time of the day (h)

Time of the day (h)

Salivary cortisol (µg/dl) month 6

C. PTSD (1 and 6 months)

0.5

Non-PTSD (1 and 6 months) Control

0.4

0.3 0.2

0.1

8

12

15

18

21

Time of the day (h) Fig. 1. Circadian salivary cortisol rhythm immediately (A) and 1 (B) and 6 (C) months after a motor vehicle accident in the longitudinal PTSD group (subjects who had the diagnosis at month 1 and maintained it through month 6, N = 8) and non-PTSD (subjects without PTSD at both months 1 and 6, N = 23). Age- and BMI-matched control values are included. (Adapted from Pervanidou et al. (2007a).)

observed in PTSD patients may thus be attributed to cortisol decrease that fails to shut down the catecholaminergic response in limbic structures, leading to PTSD development and maintenance through time. This study hypothesizes that in individuals exposed to traumatic stress, an initial increase in cortisol levels is followed by a state of low basal cortisol levels, as time passes from the traumatic

event. At the same time, and interacting with the HPA axis, a progressive elevation of NE is noted in those individuals that continue to exhibit PTSD symptoms (Fig. 3). It seems that a longitudinal interaction of peripheral measures of the sympathetic systems and the HPA axis characterizes those that develop and maintain the disorder. Thus, low cortisol levels, together with high NE concentrations, may be the end stage of the

154

*

* Plasma norepinephrine (pg/ml)

500

*

400

300

Salivary cortisol (µg/dl) 21.00 h

PTSD (1 and 6 months)

600

Non-PTSD (1 and 6 months)

0.3

0.2

0.1

200 6 1 0 Months after the accident

0

6 1 Months after the accident

Fig. 2. Morning plasma NE and evening salivary cortisol concentrations immediately, and 1 and 6 months after a motor vehicle accident in subjects who developed PTSD at month 1 and maintained it through month 6 (N = 8) and those without PTSD at months 1 and 6 after the accident, respectively (N = 23). (Adapted from Pervanidou et al. (2007a).)

Post exposure to traumatic PTSD life event initiation

PTSD maintenance

Chronic PTSD

Norepinephrine Cortisol Normal values

for cortisol

Normal values for norepinephrine Time (months) Fig. 3. A simplified figure showing the proposed longitudinal divergence of cortisol and norepinephrine responsible for PTSD maintenance in a proportion of individuals with PTSD. Only the peripheral measures of the two main axes (HPA and SNS) are mentioned here: Exposure to traumatic stress, in the absence of previous trauma in the majority of typical individuals, results in immediate elevation of cortisol in the periphery. PTSD by definition begins at least 1 month later, and cortisol levels are found still high or normal while NE is gradually elevated. Progressive divergence of cortisol and NE concentrations over time may be the underlying pathophysiologic mechanism leading to PTSD maintenance. Low cortisol and high NE is a common finding in chronic adult PTSD and may represent the natural history of the disorder, as well as a biologic risk factor for further PTSD vulnerability. (Adapted from Pervanidou (2008), with permission from the Publisher.)

155

disorder in adults with chronic PTSD. These long­ itudinal data may also explain some of the discre­ pancies in the stress hormone findings between sub-groups of individuals with PTSD.

Other neurotransmitters and neuropeptides Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine neurotransmitter whose biological roles include regulation of sleep, appetite, sexual behavior, aggression, impulsivity and neuroendo­ crine control. Serotonin interacts with both axes of the stress system, is implicated in the patho­ physiology of mood and anxiety disorders, and may contribute to the pathophysiology of PTSD. There is some evidence of altered 5-HT neuro­ transmission in PTSD, including decreased serum concentrations of 5-HT, decreased density of pla­ telet 5-HT uptake (Vermetten and Bremner, 2002); however, no differences were found in PTSD individuals, using positron emission tomo­ graphy imaging (Bonne et al., 2005). It is believed that altered 5-HT may partly contribute to PTSD symptoms, and this is supported by the efficacy of selective serotonin re-uptake inhibitors in patients with PTSD. Gamma-aminobutyric acid (GABA) is a neuro­ transmitter with inhibitory role in the CNS. It acts on GABAA receptors which are components of the GABAA/benzodiazepine (BZ) receptor com­ plex, and may be involved in the pathophysiology of PTSD, as evidenced by decreased platelet BZbinding sites in patients with PTSD as well as decreased BZ receptor binding in the hippocam­ pus, cortex and thalamus of PTSD patients (Gavish et al., 1996; Geuze et al., 2008).

PTSD and the brain: neuroanatomical findings There is evidence for alterations in brain anatomi­ cal structures and function in patients with PTSD or exposure to chronic stress. Brain regions vul­ nerable to stress and altered in patients with PTSD, include the hippocampus, amygdala and prefrontal cortex. These regions participate in the stress system, and mediate adaptation to

stress. Imaging techniques that have been used in the study of PTSD include magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS) and functional MRI. The majority of MRI studies in PTSD have focused on volumetric changes of the hippocampus. This brain region is critical in memory processing and fear condition­ ing, and is involved in the pathogenesis of symp­ toms of persistent re-experience of the traumatic event. The hippocampus represents one of the most plastic regions of the brain. Prolonged expo­ sure to stress and high levels of glucocorticoids damages the hippocampus, leading to reduction in dendritic branching, loss of dendritic cells and impairment in neurogenesis (Heim and Nemeroff, 2009). MRI studies have demonstrated smaller hippocampal volumes in Vietnam veterans with PTSD and patients with chronic PTSD compared to control individuals (Bremner, 2007). It has been hypothesized, however, that hippocampal volume reduction may represent a pre-existing vulnerabil­ ity factor, rather than a consequence of exposure in extreme stress in individuals with PTSD. Stu­ dies using proton MRS observed reduced levels of N-acetyl aspartate in the hippocampus of adults with PTSD (Rauch et al., 2006). Functional neu­ roimaging studies have shown that patients with PTSD exhibit deficits in hippocampal activation during a verbal declarative memory task (Fran­ cati, 2007). The amygdala is another brain structure, involved in emotional processing and in the acqui­ sition of the fear response in PTSD. Functional studies have shown hyper-responsivity of the amygdala during the presentation of traumatic scripts and other reminders of the trauma in patients with PTSD (Liberzon and Sripada, 2008). The medial prefrontal cortex (mPFC) is connected with the amygdala, where it exerts inhi­ bitory control on the stress response and the fear reaction. Individuals with PTSD show decreased volumes of the frontal cortex, including the ante­ rior cingulate cortex (Rauch et al., 2003). Func­ tional imaging studies have found decreased activation of the mPFC in PTSD individuals in response to stimuli such as traumatic scripts, com­ bat pictures and sounds (Bremner et al., 1999; Britton et al., 2005).

156

Understanding diversity in PTSD neuroendocrinology Diverse patterns of cortisol secretion, as a per­ ipheral biomarker of HPA axis function, have been reported in individuals with PTSD, while markers of the SNS activity are more consis­ tent, showing high plasma or urine catechola­ mines concentrations. The most prevalent finding among adults with chronic PTSD is the hypo-active HPA axis with the hyperactive LC/ NE–SNS. Indeed, it has been suggested that exaggerated catecholamine secretion without the concurrent regulatory effect of accompany­ ing cortisol elevations during stress could result in defective consolidation of traumatic memories in the brain and subsequent PTSD symptoma­ tology (Pitman et al., 1993). However, fewer studies, especially those in younger age groups, report normal or high cortisol levels in the per­ iphery (Pervanidou, 2008). The following para­ meters seem to contribute to diversity of PTSD neuroendocrine findings among studies (Table 1).

Genetic vulnerability Lifetime prevalence of exposure to traumatic life events is estimated between 40 and 90% in the general population; however, the overall lifetime

Table 1. Parameters affecting the direction of HPA axis in PTSD 1. Biological vulnerability • Genetic polymorphisms

• Epigenetic changes

2. Early experiences

• Previous trauma • Environment 3. Nature of the trauma

• Single, acute, • Chronic, repeated 4. Developmental stage, age and gender of the individual 5. Time since the trauma 6. Co-morbidity (anxiety and depression)

prevalence of PTSD is between 7 and 12% (Breslau, 2001). Family and twin studies have investigated the contribution of genetic variants, in combination with external traumatic experi­ ences and other environmental factors, in the development and resilience of PTSD. Similarly to other mental disorders, heritability of PTSD should be viewed as polygenic, meaning that dif­ ferent genes interact or play an additional role in the onset of the disorder (Broekman et al., 2007). Given the complexity of PTSD as an entity, genetic research has focused on the endopheno­ type traits underlying the clinical manifestations of the disorder. These traits are neuroanatomical, biochemical, endocrine cognitive and neuropsy­ chological. Endophenotypes in PTSD include the dysregulation of the HPA axis, the hyperar­ ousal (linked to the SNS), and psychological vari­ ables, such as the memory problems (Segman and Shalev, 2003). Genes associated with the basic biochemical, endocrine and neuroanatomical characteristics of the disorder include the serotonin transporter, the dopamine transporter, the GR, the glucocor­ ticoid action regulating genes (FKBP5), the GABA (A) receptor, the apolipoprotein E, the brain-derived neurotrophic factor and the neuro­ peptide Y (Amstadter et al., 2009; Broekman et al., 2007). Recently, the cannabinoid receptor gene (CNR1) was also associated with attention deficit hyperactivity disorder and PTSD (Lu et al., 2008).

Epigenetic changes Epigenetic alterations, resulting from environ­ mental factors, can modify gene expression in a potentially transmissible manner. Although no current findings suggest a clear epigenetic mod­ ification related to PTSD, a number of studies indicate that adverse early experiences contri­ bute to PTSD biological vulnerability. These studies include data on stress-related gene expression or effects of in utero stress on the neurobiology of PTSD (Yehuda and Bierer, 2009). Stress in utero, through fetal program­ ming of the HPA axis, may contribute to

157

PTSD vulnerability in the offspring. In mothers exposed to the 11th September attacks during pregnancy, maternal PTSD was associated with awakening and bedtime salivary cortisol in their babies. Furthermore, lower cortisol values were noted in the infants of PTSD mothers versus non-PTSD (Yehuda et al., 2005). There is also evidence suggesting that prenatal stress, through elevations of glucocorticoid levels, influences fetal brain development and pro­ grammes the HPA axis (Seckl, 2004). Reduced activity of placental 11-b-hydroxycorticosteroid dehydrogenase type 2, the enzyme that cata­ lyses the conversion of maternal cortisol to cor­ tisone, may result in an increased exposure of the foetus to glucocorticoids and may contribute to HPA axis programming (Seckl, 2004).

Early life experiences Early postnatal environment and especially the interaction between the infant and the mother represents a critical life period associated with emotional and cognitive development (Fernald and Gunnar 2009). The quality of mother– infant interaction and, in general, the infant’s early experiences are key determinants of ‘pro­ gramming’ the infant’s brain to be more resili­ ent or vulnerable to stress-related disorders in adult life (Korosi and Baram, 2009). Adverse conditions during early life represent risk factors for stress-related disorders, such as depression and PTSD. There is an abundant pre-clinical literature describing the enduring effects of early stress exposure on HPA regula­ tion (Pryce et al., 2005). In studies of rodents and non-human primates, adverse environments have been linked to abnormal (both exagger­ ated or blunted) patterns of HPA axis reactivity (Sanchez et al., 2001). Clinical studies in adults with adverse early life experiences have also reported alterations of HPA axis reactivity. In a recent study of phenotypically healthy adults without current psychiatric disorders (Carpenter et al., 2009), a history of self-reported childhood emotional abuse, independently diminished cor­ tisol response in the Dex/CRH test. These data

suggest that dampened cortisol reactivity (which is considered to be a risk factor for the devel­ opment of PTSD) may be a consequence of childhood emotional abuse that is cumulative over time, representing a potential endophe­ notype related to biological vulnerability to PTSD.

Previous trauma exposure and time since the traumatic event Prior traumatization is a risk factor pre-disposing to the development of PTSD, in a cumulative manner. From a neuroendocrine perspective, exposure to previous trauma may contribute to altered HPA axis responses to a new stressor, and the low cortisol levels observed in adults with chronic PTSD (Resnick et al., 1995; Weems and Carrión, 2009). This view hypothesizes that an initial increase in cortisol levels after exposure to the traumatic event is followed by a state of low basal cortisol levels, as time passes from the trau­ matic event. Indeed, a recent study demonstrated that cortisol and PTSD symptoms were signifi­ cantly positively correlated in children having recently experienced trauma, but negatively in children with distal trauma (Weems and Carrión, 2009). Consequently, low cortisol levels reported shortly after the trauma in adult studies, may be the long-term effect of previous trauma expo­ sure, rather than a biologic factor responsible per se for the subsequent development of PTSD (Pervanidou, 2008; Resnick et al., 1995). In contrast to adult studies, where low cortisol levels found shortly after an acute stressor are predictive of PTSD development (Delahanty et al., 2000), high cortisol levels are most fre­ quently found to be predictive of PTSD in chil­ dren (Delahanty et al., 2005). Children are, in general, less likely to have experienced prior traumas, than adults, and less likely to exhibit co-morbid disorders or alcohol or drug abuse. This might be the explanation of high cortisol levels reported more frequently in pediatric than in adult PTSD studies.

158

Type of stressor/trauma (acute vs. chronic)

Conclusions

Acute stressors, such as accidents and earth­ quakes, and chronic trauma, such as child mal­ treatment, are qualitative different. Child maltreatment, defined as physical and/or sexual abuse or neglect is a chronic and complex stres­ sor. Child maltreatment most often co-exists with other psychosocial risk factors, such as low socio-economic status, unemployment, par­ ental stress, drug and alcohol abuse. Further­ more, parental psychopathology in chronically maltreated children, may represent an addi­ tional innate biological risk factor. In contrast, accidents, or physical disasters are single trau­ mas, potentially producing distinct clinical symptoms and neuroendocrine alterations. Terr differentiated single from repeated traumas and suggested that greater dissociation was related to repeated trauma (Pervanidou and Chrousos, 2007).

Chronic alteration of stress hormones in PTSD can increase the risk of an individual to develop metabolic abnormalities and cardiovascular dis­ eases. In addition to adverse health behaviors related to PTSD symptomatology, biological path­ ways may also link PTSD to metabolic syndrome manifestations and cardiovascular disease. Incor­ porating recent neurobiological information into clinical research will assist researchers and clini­ cians in capturing more of the complexity and heterogeneity of trauma and human post-trau­ matic stress reactions, leading to more effective prevention and intervention strategies.

Co-morbid disorders PTSD, as many other psychiatric disorders, is often co-morbid with other disorders, such as major depressive disorder (MDD), generalized anxiety disorder and addictive disorders, as well as with a number of clinical manifestations, such as depressive symptoms or symptoms of dissocia­ tion (Kar and Bastia, 2006). The direction of the HPA axis activity in individuals with more than one psychiatric diagnoses is consequently influ­ enced by a variety of symptoms and disorders. The impact of co-morbidity on neuroendocrine factors is highlighted in one of the first neuroen­ docrine studies examining maltreated children: In this study in a day camp, maltreated children with internalizing problems demonstrated higher morning, afternoon and average daily cortisol levels, while non-maltreated boys with externa­ lizing problems were more likely not to show the expected diurnal decrease in cortisol (Cicchetti and Rogosch, 2001). This study clarifies the diversity of neuroendocrine patterns, depending on the type of maltreatment and the concurrent psychopathology in childhood.

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright � 2010 Elsevier B.V. All rights reserved.

CHAPTER 6

Assisted reproduction and its neuroendocrine impact on the offspring Christina Kanaka-Gantenbein
, Sophia Sakka and George P. Chrousos Division of Endocrinology, Diabetes and Metabolism, First Department of Pediatrics, University of Athens, “Agia Sophia” Children's Hospital, Goudi, Athens, Greece

Abstract: Assisted reproductive technologies (ARTs) have been widely used during the last three decades and progressively more children are born with the help of such methods. There is now evidence that ARTs may be associated with slight epigenetic modifications in the expression of several genes that could have a long-term impact on the health of the offspring. Also, a clear association between such techniques and genomic imprinting abnormalities has been reported. The neuroendocrine impact of ART on the offspring includes slight elevations of systolic blood pressure (SBP) and diastolic blood pressure (DBP), as well as increased circulating triglyceride concentrations, in children born after ART, especially in those with rapid catch-up growth in weight during early childhood. However, the postnatal growth of most children after ART is normal and no increased incidence of the full metabolic syndrome has been observed in these children and adolescents. Moreover, the pace and timing of puberty of such children is normal and no increased incidence of premature adrenarche could be discerned in ART children in the absence of restricted fetal growth. Finally, a slight modification of the set point of thyroid stimulating hormone sensitivity was observed in ART children, without an apparent impact on thyroid hormone secretion. This has been attributed to epigenetic changes. Questions remain to be answered regarding the future reproductive capacity of children born after ART, as well as their cardiovascular risk in later adult life. Long-term prospective studies should be performed to provide robust evidence. Keywords: IVF; ART offspring; children; genomic imprinting; growth; metabolic syndrome; puberty; adrenarche; hyperthyrotropinaemia

treatment to help to obtain children. ART is comprised mainly by classic in vitro fertilization (IVF), that first led to a living child in 1978, and the intracytoplasmic sperm injection (ICSI) procedure, that was introduced in 1992, primar­ ily to treat male infertility (Wilkins-Haug, 2008). Nowadays, ARTs account for 1–3% of births in industrialized countries and it is estimated that more than two million babies have been

delivered using ART (Shiota and Yamada,

Introduction In developed countries, sub-fertility has become an increasing problem and progressively more sub-fertile or infertile couples are offered assisted reproductive technologies (ART)


Corresponding author.

Tel.: þ306932-416686; Fax: þ30-210-7779919; E-mail: [email protected] or [email protected]

DOI: 10.1016/S0079-6123(10)82006-0

161

162

2009). However, although classic IVF started 30 years ago, long-term detailed systematic prospective studies of IVF offspring have been scarce (Grace and Sinclair, 2009; Van der Lende et al., 2000). Most studies have been re-assuring concerning the safety of drugs used during ART for women’s health (Kanakas and Mantzavinos, 2006), nevertheless, some epidemiologic studies have suggested an excess occurrence of malformations and other peri­ natal complications in babies conceived by ART (Basatemur and Sutcliffe, 2008; Grace and Sinclair, 2009). Specifically, slight increases in the frequency of congenital malformations, most commonly of the urogenital tract, especially in pre-term, low birth weight infants, twins and triplets (Hansen et al., 2005), as well as a significant increase in cerebral palsy have been reported, although no differences in the overall psychomotor development from naturally conceived children have been noted (Bonduelle et al., 2005; Knoester et al., 2007; Middelburg et al., 2008; Ponjaert-Kristoffersen et al., 2005). Infants born by ART are at a higher risk of pre-term delivery and intrauterine growth res­ triction (IUGR), born small for gestational age (SGA) (Bergh et al., 1999). Being SGA is well known to pre-dispose to adult disease and to be associated with an unfavourable cardiometabolic profile in later life (Barker et al, 1993; Saenger et al., 2007; Gluckman et al., 2008). This alone could raise concern about the long-term health issues of children born after ART and may explain the recently observed poor cardiometabolic out­ come of IVF children (Ceelen et al., 2008a). How­ ever, the latter might also be the result of the in vitro manipulations of the blastocyst and/or an adverse intra-tubal and/or intrauterine environ­ ment. This is in agreement with Barker’s fetal origin of adult disease hypothesis, which postu­ lates development of epigenetic and constitutional changes occurring in prenatally distressed indivi­ duals (Gluckman et al., 2008; Kanaka-Gantenbein et al., 2003). By its nature, assisted reproduction is usually associated with increased parental stress com­ pared with spontaneous conception (Ardenti et al., 1999; Sanders and Bruce, 1999). Indeed, women undergoing IVF treatment are often

anxious and/or depressed because of their infer­ tility and the uncertainties of the treatment they are exposed to (Smeenk et al., 2005). The state of distress in sub-fertile women is confirmed by elevations in the circulating levels of stressrelated hormones compared to those of fertile controls (Czemiczky et al., 2000), while mater­ nal anxiety before IVF treatment has been posi­ tively correlated with urinary adrenaline levels during treatment (Smeenk et al., 2005). Although it is still debated whether stress influ­ ences the outcome of ART, it is proposed that reducing stress should be the first treatment option to be offered in assisted reproductive programmes (Campagne, 2006; Facchinetti et al., 2004), since it is well established that glucocorticoids, by stimulating hepatic gluco­ neogenesis, inhibiting insulin actions on skeletal muscle and potentiating its actions on adipose tissue, are the main mediators of the poor longterm outcome of an adverse intrauterine envir­ onment, ultimately promoting sarcopenia and visceral adiposity of the offspring and the occur­ rence of the metabolic syndrome (MS) in later life (Bjorntorp, 2001; Chrousos, 2000; Wadhwa, 2005). This chapter summarizes current knowledge on the neuroendocrine manifestations and health issues of children born after ARTs.

Assissted reproductive techniques and imprinting disorders of the offspring There is increasing evidence for an association between ART and imprinting disorders, such as the Beckwith–Wiedemann or Angelmann syndromes, mostly in children born after ICSI (Lidegaard et al., 2005, 2006; Ludwig et al., 2006), but also less frequently after classic IVF (Manipalviratn et al., 2009). These associations have led to great concern in the international litera­ ture whether ART may increase the risk of genomic imprinting disorders and possibly other harmful epigenetic changes (Bowdin et al., 2007; Laprise, 2009; Thompson et al., 2002). Genomic imprinting is a process of chemical modification of nucleotides in which only one

163

allele of a specific gene is functioning, while the other allele is silenced based on the parent of origin. DNA methylation and histone modifica­ tion are two major mechanisms for genetic imprinting in mammals, including humans (Manipalviratn et al., 2009; Morison et al., 2005). Germ cells have the ability to erase imprinting marks during their development in embryonic life. During the erasure process, there is marked gen­ ome-wide demethylation, which is completed by embryonic day 12–13 in both male and female mice (Reik and Walter, 2001; Schaefer et al., 2007). After the erasure of the imprinting marks, germ cells re-establish de novo imprinting marks according to their sex. The re-establishment pro­ cess begins in germ lines of both sexes at late fetal stages and continues after birth (Brandeis et al., 1993). ARTs, which generally involve stimulation of oocyte growth and retrieval of oocytes directly from the ovary prior to ovulation, could theoreti­ cally disrupt the imprinting process in oocytes, because these cells do not complete the re-methy­ lation process until just before or after ovulation. Unlike oocytes, sperm cells complete their imprint­ ing process earlier in their development, so ART is unlikely to affect imprinting in sperm (Allegrucci et al., 2005; Hartmann et al., 2006). Furthermore, embryo culture media have been shown to influ­ ence the imprinting status of some imprinted genes (Khosla et al., 2001). Moreover, hormonal hyper­ stimulation of the ovary is a common practice in ART to help obtain multiple oocytes for fertiliza­ tion and improve pregnancy success and recent studies have demonstrated that this treatment may alter the imprinting status of some genes (Santos et al., 2010; Sato et al., 2007). Therefore, recent studies on IVF in animals focused on the epigenetic modifications that may occur during ARTs, which might cause alterations in gene expression (Niemitz and Feinberg, 2004). Indeed, changes in epigenetic programming during the pre-implantation period could be a potential mechanism for alterations in the growth, development and metabolism of IVF children, as will be discussed in the following sections of this chapter. In conclusion, several case reports, case series and cohort studies have suggested an association

between ART use and offspring born with an imprinting disorder, mainly Beckwith–Wiedemann and Angelmann syndromes, specifically because of hypomethylation of the maternal allele. It remains to be determined whether the underlying infertility etiology, the hormonal stimulation used for ovulation induction, the IVF/ICSI procedure per se or a combination thereof is the true cause for the increased prevalence of imprinting disor­ ders following ART. However, since the absolute incidence of imprinting disorders is small (<1:12,000 births), only a long-term systematic registry of all imprinting disorders observed in children could provide the real incidence of such disorders in ART children as opposed to that of spontaneously conceived children (Manipalviratn et al., 2009). Moreover, all indices of epigenetic modifications in gene expression, in general, in ART children should be carefully monitored and documented.

Assisted reproduction and neuroendocrine impact on the postnatal growth of the offspring Infants born after ART are often born prema­ turely and have a low birth weight for their gesta­ tional age, characterized, thus, as SGA. Up to 85% of SGA newborns may show catch-up growth up to the age of 2–3 years and enter thereafter into normal for the race and sex growth curves (Saenger et al., 2007). Several research groups investigated the natural course of intrauterine and postnatal growth of children conceived by ART, as summarized in Table 1 (Ceelen et al., 2007a; Ceelen et al., 2009; Kai et al., 2006; Miles et al., 2007; Sakka et al., 2009a). Kai et al. exam­ ined both infants as well as children born after classic IVF, ICSI and naturally conceived (NC) controls. There were no differences in length at 3 months, 18 months or 36 months of age between children conceived by ICSI, or IVF and naturally conceived ones and postnatal growth was similar in all the three groups studied. As far as weight is concerned, 3-months-old singleton IVF or ICSI boys were slightly heavier than age-matched spon­ taneously conceived controls. At a cross-sectional study of 5-year-olds IVF, ICSI and NC children,

164 Table 1. Comparison of reported neuroendocrine findings in children born after ARTs in comparison to naturally conceived controls Characteristic

ART versus controls

References

Growth Birthweight

#

Gestational age Gestational age Postnatal growth Postnatal growth IGF-I in childhood IGFBP3 in childhood

#   "  

Bergh et al. (1999), Miles et al. (2007), Ceelen et al. (2007a), Sakka et al. (2009a) Ceelen et al. (2007a), Sakka et al. (2009a) Miles et al. (2007) Kai et al. (2006), Ceelen et al. (2007a, 2007b, 2008a), Sakka et al. (2009a) Miles et al. (2007) Kai et al. (2006), Sakka et al. (2009a) Kai et al. (2006)

Cardiometabolic status Systolic BP Diastolic BP Fasting blood glucose Fasting blood glucose Triglycerides Triglycerides HDL HDL Adiponectin Leptin HsCRP IL-6

" " "  # " "     

Ceelen et al.( 2008a), Sakka et al. (2009a)

Ceelen et al. (2008a), Sakka et al. (2009a)

Ceelen et al. (2008a)

Miles et al. (2007), Sakka et al. (2009a)

Miles et al. (2007)

Sakka et al. (2009a)

Miles et al. (2007)

Sakka et al. (2009a)

Sakka et al., 2009a

Sakka et al. (2009a)

Sakka et al. (2009a)

Sakka et al. (2009a)

Puberty Timing and progression



Ceelen et al. (2008b)

Precocious adrenarche DHEA-S DHEA-S

" ", only in SGA-ART

Ceelen et al. (2008b) Sakka et al. (2009a)

Thyroid function TSH T3, T4

" 

Sakka et al. (2009b) Sakka et al. (2009b)

Abbreviations/explanations of symbols: ART, assisted reproductive technologies; IGF-I, insulin-like growth factor I; IGFBP3, insulin-like growth factor binding protein 3; BP, blood pressure; HDL, high-density lipoprotein; IL-6, high-sensitivity interleukin-6; hsCRP, high-sensitivity C-reactive protein; SGA, small for gestational age; DHEA-S, dehydroepiandrosterone-sulphate; TSH, thyroid stimulating hormone; T3, triiodothyronine; T4, thyroxine; ", increased; #, reduced; , similar; no statistical difference.

there were no more differences among the three groups studied in height or weight. Since postnatal growth is largely influenced by the availability of insulin growth factor-I (IGF-I), the levels of IGF-I and its major binding protein during postnatal life, insulin-like growth factor binding protein 3 (IGFBP3), were investigated in all groups (ICSI, IVF and NC). At 3 months of age, serum IGF-I levels were significantly lower in singleton ICSI boys and singleton IVF girls than those of naturally conceived ones. However, at 5 years of age, no more differences were observed

among the three groups in either IGF-I or IGFBP3 levels (Kai et al., 2006). However, in a later study by Miles et al. (2007), who examined 6-year-old children conceived either by classic IVF or naturally, higher insulinlike growth factor-II (IGF-II) and IGF-I to IGFBP3 ratios were observed in the IVF group than in the control group and IVF children had even a better growth than NC ones. In our own study, comprising 106 children (48 boys, 58 girls) conceived by classic IVF, aged 8.8 + 2.9 years and 68 (33 boys, 35 girls) age-matched naturally

165

conceived controls, we found similar IGF-I levels in both IVF children and the age-matched controls (Sakka et al., 2009a). Ceelen et al. (2007a, 2007b, 2008a), as well as our group (Sakka et al., 2009a) have demon­ strated the same re-assuring results of a normal postnatal growth of children born after ART, since children examined at the age of 12 + 2.6 years by Ceelen et al., (2007a, 2009) and 8.8 + 2.9 years by us, respectively, had similar height and weight achieved to age-matched con­ trols. Moreover, no difference in the prevalence of obesity, or in mean body mass index standard deviation score (BMI SDS) between IVF children and naturally conceived ones was detected (Kai et al., 2006; Miles et al., 2007; Ceelen et al., 2007a, 2007b; Sakka et al., 2009a). However, in a very recent study, Ceelen et al. (2009) investigated the evolution of early infancy growth data of children conceived after ART in comparison to spontaneously conceived ones from sub-fertile couples. Growth data from birth to 4 years of age were available for 392 children (n = 193 IVF, n = 199 control) and were used to study the early postnatal growth. The authors found significantly lower weight, height and BMI SDSs at 3 months, and weight SDS at 6 months of age in IVF children than in controls. Later on, IVF children demonstrated a greater gain in weight SDS (p < 0.001), height SDS (p = 0.013) and BMI SDS (p = 0.029) during late infancy (3 months to 1 year) than controls. Therefore, late infancy growth velocity of IVF children was significantly higher than that of controls. In conclusion, several publications reporting postnatal growth of ART children in comparison to NC controls provide conflicting results on their early infancy growth, some of them reporting higher body weight (Kai et al., 2006), and others reporting lower body weight in ART infants than spontaneously conceived controls (Ceelen et al., 2009). However, height seems to be less affected and most studies agree that ART infants and chil­ dren demonstrate height within the normal range for their target height and within the normal growth curves for their sex and race. Furthermore, data on IGF-I concentrations and availability in IVF children versus controls have reported lower,

higher or similar levels in IVF children in compar­ ison to controls. One has to bear in mind that even in the case of significant group differences, the reported differences are subtle and may not have any clinical significance in the individual case. Therefore, parents of ART children as well as health professionals taking care of these children should be reassured that their postnatal growth is expected to be normal. However, special attention should be paid to prevent rapid catch-up growth in weight, especially during early childhood, in the light of data of an increased future cardiometa­ bolic risk, as will be discussed in the next section.

Assisted reproduction and cardiometabolic risk of the offspring Since children born after ART have a higher inci­ dence of premature birth and are frequently born SGA, one would expect an increased risk for poor cardiometabolic outcome, according to the ‘fetal origin of adult diseases’. Furthermore, since ART is associated with increased incidence of genomic imprinting errors and DNA hypomethylation in the offspring, it is important to examine whether ART per se may permanently influence the neu­ roendocrine status of the offspring even if the off­ spring is born appropriate for gestational age (AGA). Animal studies have shown that condi­ tions during ARTs may interfere with normal pro­ gramming of early development with subsequent postnatal developmental consequences, including aberrant cardiovascular physiology (Lonergan et al., 2006; Watkins et al., 2007). Several research groups have therefore focused on the cardiometa­ bolic profile of children born after ART, as sum­ marized in Table 1 (Ceelen et al., 2008a; Miles et al., 2007; Sakka et al., 2009a) and on the impact of postnatal catch-up growth on the cardiometa­ bolic risk (Ceelen et al., 2009). Miles et al. (2007), studied prepubertal IVF children and found higher high-density lipoprotein (HDL) levels and lower triglyceride levels than controls, reporting thus a more favourable lipid profile in IVF offspring compared to naturally conceived children (Miles et al., 2007) and provid­ ing reassuring data regarding the long-term health

166

95% CISBP SDS

p = 0.005 0,5

0,0

–0,5 p = 0.01 p=0.001 SGA-IVF

AGA-IVF

controls

95% CI DBP SDS

1,5 p = 0.003

1,0

0,5

0.0 p = 0.03

–0,5 SGA-IVF

AGA-IVF

controls

65 95% CI TGL [mg/dl]

outcome of ART children. However, more recent studies that focused on the body composition and cardiometabolic profile of IVF children, including both pre-pubertal and pubertal children, reported both a higher peripheral body fat and higher blood pressure (BP) levels in ART children than in naturally conceived controls (Ceelen et al., 2007b, 2008a, 2008b; Sakka et al., 2009a). Specifically, both the group of Ceelen et al. (2007b, 2008a) as well as our group (Sakka et al., 2009a) examined the SBP and DBP, the height SDS, the weight SDS and BMI SDS of children conceived by classic IVF and compared them to the same parameters in spontaneously conceived age- and BMI-matched controls. Both research groups found a significantly higher percentage of lower birth weight, birth weight SDS and gesta­ tional age in IVF children than controls. More­ over, both research groups found higher systolic and diastolic BP measurements in IVF children than in controls, irrespective of them being born SGA or AGA, pointing to the ART per se also being a pre-disposing factor for unfavourable cardiometabolic outcome above and beyond being born SGA rather than AGA (Fig. 1). Although both studies demonstrated only subtle BP elevation in IVF children, BP is known to track from childhood into adult life and the increased BP after IVF may be amplified with age (Law et al., 1993). Regarding glucose metabolism and insulin resistance markers, in the study of Ceelen et al. (2008a) higher fasting blood glucose levels were found in their IVF cohort than in the controls, although fasting insulin levels and height, weight and BMI were similar in their study groups. On the contrary, in our study and in that of Miles et al. (2007), similar fasting glucose and insulin levels, as well as insulin resistance markers were found in the two cohorts (Miles et al., 2007; Sakka et al., 2009a). In our study, we also investigated lipid metabolism, as well as the presence of MS in IVF versus control children and observed higher triglycerides levels in the IVF cohort (Fig. 1). The criteria we used to diagnose the MS were accord­ ing to Weiss et al. slightly modified (2004). Since we did not have data on a 2-h glucose tolerance test, we used fasting glucose to insulin ratio

60

55

50

45

p = 0.031 IVF

control

Fig. 1. Comparison of systolic (SBP SDS) (upper panel) and diastolic (middle panel) blood pressure (DBP SDS) between SGA–IVF children, AGA–IVF children and controls, as well as of triglycerides (TGL) (lower panel) between IVF children and controls. Abbreviations: SDS, standard deviation score; SGA, small for gestational age; AGA, appropriate for gestational age; IVF, in vitro fertilization. (Reproduced with permission from Sakka et al. (2009a).)

(FGIR) according to Silfen et al. (2001) to demon­ strate insulin resistance. The children were classi­ fied as having the MS if they met three or more of

167

the following criteria for age, sex and ethnic group: • BMI >95th percentile (Chiotis et al., 2004) • Triglycerides >95th percentile (Schulpis and Karikas, 1998) • HDL <5th percentile (Schulpis and Karikas, 1998) • BP >95th percentile • FGIR <7 (Silfen et al., 2001) for age, race and sex. In our study, the increase of BP and triglycer­ ide concentrations, which are early markers of the MS, in the IVF group, could be explained by the fact that SGA children are generally at increased risk to develop MS (Barker et al., 1993; Kanaka-Gantenbein et al., 2003) and IVF chil­ dren are more frequently born SGA than spon­ taneously conceived ones. However, after sub­ dividing our IVF cohort into SGA and AGA sub-groups, both SBP and DBP remained signifi­ cantly elevated in the SGA–IVF and the nonSGA–IVF sub-groups, compared to controls, suggesting that antenatal stress or pre-implanta­ tion manipulations in IVF-conceived children may be an independent risk factor for the earlier occurrence of arterial hypertension in this popu­ lation, irrespectively of them being born SGA or not. Our results suggest that the above metabolic alterations are not merely the result of IUGR, but may also be due to the periconceptual manip­ ulation of the blastocysts, as also suggested by Ceelen et al. (2008a). The discrepancy between our results and those of Ceelen et al. (2008a) from those of Miles et al. (2007), who found higher HDL and lower trigly­ cerides levels in IVF children than controls, can be explained by the fact that the study by Miles et al. (2007) enrolled only pre-pubertal IVF children. It is possible that the metabolic derangements we observed became evident as children entered into puberty, a stage of inherent insulin resistance. Furthermore, as already reported, in a very recent study by Ceelen et al. (2009), who examined the impact of rapid weight gain during the early infan­ tile or childhood period on BP elevation, a weight gain during early childhood (1–3 years) was

related to BP in IVF children but not in controls, suggesting that rapid catch-up growth in weight during early childhood is associated with a poorer cardiometabolic profile. Many recent studies have shown that altera­ tions of the non-traditional metabolic risk factors leptin and adiponectin accompany pediatric MS and correlate with traditional cardiovascular risk factors and chronic inflammation markers, such as high-sensitivity C-reactive protein (hsCRP) and plasma interleukin-6 (IL-6) concentrations (Gualillo et al., 2007; Retnakaran et al., 2006). There is a clear association of low adiponectin and high leptin levels with cardiovascular risk factors and adiponectin was suggested to be a very early predictor of MS in children (Ko¨ rner et al., 2007). Furthermore, research has eluci­ dated that low-grade systemic inflammation may underlie the clustering of cardiovascular risk fac­ tors in obesity. In obese adults and children, hsCRP is elevated and an elevation of IL-6 is also observed in obese children (Ko¨ rner et al., 2007; Ronti et al., 2006). In our study, we found no significant differences in leptin, adiponectin, hsCRP or IL-6 levels between IVF children and controls, even when children were sub-divided according to their gender, pubertal stage, weight for gestational age and whether they were single­ tons or twins/triplets, suggesting that, despite the BP elevations and increased triglyceride concen­ trations, no major adiposity and inflammatory status markers are present in young IVF children (Sakka et al., 2009a). Adipocytokines are associated with intrauterine growth and are thought to play an important role in linking poor fetal growth to subsequent devel­ opment of adult diseases (Briana and MalamitsiPuchner, 2009). Therefore, we analysed leptin, adiponectin, as well as hsCRP and interleukin-6 (IL-6), separately in AGA and SGA IVF children; however, there were still no significant differences between the two groups, showing that SGA had no effect on the results. The only significant dif­ ference observed in singleton, pubertal boys was a higher level of IL-6 and hsCRP in IVF compared to spontaneously conceived children. This might reveal an early effect of puberty on low-grade inflammation markers. A lack of such a difference

168

in pubertal girls may be explained by sexual dimorphism, since gender dimorphism has already been reported regarding the circulating levels of hsCRP and IL-6 (Chapman et al., 2009; Khera et al., 2005). Moreover, in our study, we found that polycystic ovary syndrome (PCOS) was present only in mothers of IVF children, who also had a greater prevalence of gestational diabetes mellitus (GDM). Since PCOS is associated with a higher prevalence of insulin resistance and MS (Salehi et al., 2004; Schroder et al., 2004) and a strong hereditary component is present for both PCOS (Govind et al., 1999) and insulin resistance and obesity (Pankow et al., 2004), while an increased incidence of type 2 diabetes, obesity and cardio­ vascular alterations have been associated to in utero exposure to increased glucose concentra­ tions (Simeoni and Barker, 2009), we examined whether IVF children had higher incidence of glu­ cose metabolism abnormalities, but found no dif­ ferences. Furthermore, by using maternal PCOS, BMI, age at conception and presence of GDM, as confounding factors in the multivariate analysis of BP elevation, we found that the differences between the two groups were still significant, which excludes these factors as a cause for the BP elevation observed in IVF children (Sakka et al., 2009a). In conclusion, subtle BP and triglyceride elevations in IVF-conceived children, but no dif­ ferences in the occurrence of the MS, the presence of cardiometabolic and inflammatory risk factors, such as leptin, adiponectin, hsCRP and IL-6, or insulin resistance markers, in IVF children com­ pared to naturally conceived ones were observed. Furthermore, in the light of the recent publication by Ceelen et al. (2009) that rapid catch-up growth in weight in early childhood is positively corre­ lated to higher SBP and DBP, special emphasis should be placed on the avoidance of rapid catch­ up growth in weight in order to avoid a poor cardiometabolic outcome in later life. Healthcare professionals taking care of such children should be aware of this association. Further pro­ spective, longitudinal follow-up studies in IVF children are necessary to confirm these observa­ tions, to determine the natural history of the

metabolic profile of ART offspring and to deter­ mine whether and when they will develop the full MS.

Assisted reproduction and the neuroendocrine impact on the pubertal timing of the offspring Although ARTs has been utilized during the last three decades and many ART children have in the meanwhile entered puberty or reached adulthood, the sexual maturation of IVF children has only scarcely been examined (Ceelen et al., 2008b; Rojas-Marcos et al., 2005). An association between prenatal development and timing and progression of puberty in humans has been suggested over the last decade (Hokken-Koelega, 2002; van Weissenbruch and Delamarre-Van de Waal, 2006) and periconcep­ tual events, such as IVF, may have an impact on the hypothalamic–pituitary–gonadal axis. More­ over, SGA infants are at increased risk of pre­ mature adrenarche, which can, in turn, be considered as a forerunner of adult disease (Ibanez et al., 2000) and may predispose both to the PCOS (Ibanez et al., 1998), as well as the MS (Utriainen et al., 2007). In the study by Ceelen et al. (2008b) a total population of 233 IVF children (115 boys, 118 girls) aged 8–18 years, each with an age- and sexmatched comparison control, were studied and pubertal Tanner stages, age at menarche as well as menstrual cycles were recorded. Pubertal tim­ ing and age at menarche, as well as menstrual cycles were not different between IVF children and controls (Table 1). However, in the pubertal subpopulation, a higher bone age to chronological age (BA/CA) ratio and a larger BA – CA differ­ ence were observed in IVF-conceived girls than in controls. The authors also demonstrated a higher dehydroepiandrosterone-sulphate (DHEA-S) and higher luteinizing hormone (LH) level in IVF-conceived girls than in controls. However, hormonal determinations were not reported within a specific phase of their menstrual cycle and IVF-conceived children were not sub-divided into SGA or AGA ones, in order to elucidate

169

whether the DHEA-S elevation was more exag­ gerated in those born SGA, as repeatedly reported in the literature (Ibanez et al., 2000; Saenger and Dimartino-Nardi, 2001). In our study, we wanted to investigate whether periconceptual and intrauterine stress due to the ART procedure and the increased stress of the pregnant woman might also promote a precocious or early adrenarche in ART offspring (Sakka et al., 2009a). However, there was no significant difference either in the occurrence of precocious or early adrenarche or in the levels of DHEA-S between our IVF children as a whole and controls. Since precocious adrenarche has been reported in girls with restricted fetal growth, we subsequently sub-divided our IVF girls cohort into those born SGA versus AGA and found that our SGA–IVF sub-group presented significantly higher DHEA-S levels than both our AGA–IVF subgroup and our control group, while no difference was observed in DHEA-S levels between AGA–IVF and controls, confirming that SGA is associated with an exag­ gerated adrenarche, while ART per se does not drive higher DHEA-S levels if not accompanied by fetal growth restriction (Table 1). We found similar follicle-stimulating hormone and LH levels for both IVF children and controls, in both the pre-pubertal and the pubertal sub-group. Prolac­ tin levels, however, were higher in our SGA–IVF sub-group of prepubertal girls than the AGA–IVF ones. In conclusion, timing of pubertal onset as well as pace of progression of puberty seems to be normal in ART children. Furthermore, age at menarche as well as menstrual cycles are similar in ART girls and NC controls. As far as premature adrenarche or DHEA-S elevation is concerned, SGA–IVF children seem to have an exaggerated adrenarche, while AGA–IVF children do not have significant differences in comparison to agematched NC controls. The reproductive capacity of children born after ART is an unexamined issue and whether the genetic basis of the infertility of their parents or the circumstances of the ARTs utilized may have had an impact on their own future reproductive capacity should be examined prospectively.

Assisted reproduction and neuroendocrine impact on the hypothalamic–pituitary–thyroid axis of the offspring We conducted the first study investigating thyr­ oid function in 4–14-year-old IVF children and found significantly higher thyroid stimulating hormone (TSH) levels – albeit within the normal range – in the total cohort of IVF children than in naturally conceived controls and a higher inci­ dence of euthyroid hyperthyrotropinaemia (EH) among IVF children than normally conceived controls (Sakka et al., 2009b). These differences could not be attributed to an autoimmune aetiol­ ogy, either of the children themselves, or through passive passage of thyroid autoantibodies from their mothers, since thyroid autoantibodies were absent in all children studied and the children were all older than 6-months-old, excluding thus maternal thyroid autoantibodies as a plausible explanation. Furthermore, these children had no history of congenital hypothyroidism or maternal thyroiditis. Recently, it has become clear that the early embryonic environment might permanently alter the settings of various hormonal axes with changes that may persist into later life (KanakaGantenbein et al., 2003). Low birth weight and short birth length, short height during early child­ hood and low BMI during late childhood have been shown to characterize women who develop spontaneous hypothyroidism in adult life (Kajantie et al., 2006). Also, hyperthyrotropinae­ mia has been reported in SGA children in the context of multiple hormonal resistance (Radetti et al., 2004). However, when the effect of birth weight, gestational age, SGA–AGA status on the occurrence of EH in our cohort of IVF children was studied, no significant differences were found. Furthermore, the high incidence of the EH in IVF children could not be attributed to their sex, age or pubertal status, nor to being the first, second or third child of the family. Triplets appeared to have significantly more often EH compared to single­ tons and twins. A previous study showed a more than three-fold higher frequency of congenital hypothyroidism in twins than in the general popu­ lation (Olivieri et al., 2007), but no such evidence

170

exists for acquired sub-clinical hypothyroidism in multiple pregnancies. Hypothyroidism predisposes to hypertension, dyslipidaemia and disorders of carbohydrate metabolism (Duntas, 2002). A higher prevalence of sub-clinical hypothyroidism has also been reported in patients with MS than in normal con­ trols (Uzunlulu et al., 2007). In addition, as already reported in the previous section, recent studies in IVF children showed significant increases of arterial BP, as well as a higher amount of adipose tissue (Ceelen et al., 2007a, 2007b, 2008a) in comparison to age-matched sponta­ neously conceived controls and our own data con­ firm higher levels of BP and triglycerides in IVF children than controls, although no differences in the indices of low-grade inflammation, such as IL­ 6 or hsCRP, nor of adipokines, such as adiponec­ tin or leptin, were observed between IVF children and controls (Sakka et al., 2009a). These cardio­ metabolic alterations in IVF children might be partly attributed to a higher occurrence of sub­ clinical hypothyroidism. However, in our popula­ tion no differences in the metabolic profile between IVF children with or without EH and controls were found. Higher BMI SDS has been associated with TSH elevation (Iacobellis et al., 2005). Nevertheless, no difference was found in BMI SDS of IVF children with or without EH and controls, ruling out a bias attributable to higher BMI SDS of IVF children with TSH elevation (Sakka et al., 2009b). A plausible explanation for the higher incidence of EH among IVF children is the possible epigenetic alterations that might occur during pre-implantation manipulations. As already reported, there is now substantial evidence sug­ gesting that ART pregnancies are associated with altered outcomes in fetal and neonatal develop­ ment, mainly due to epigenetic modifications of gene expression (Lawrence and Moley, 2008; Lidegaard et al., 2006). Thus, the EH observed in our IVF population may be the result of epige­ netic alteration in the set point of TSH sensitivity, reflecting a mild TSH resistance. In conclusion, there is now evidence of an increased incidence of EH in IVF children com­ pared to controls, not attributable to birth weight,

gestational age, SGA–AGA status, breast feeding duration, sex, current age, BMI SDS or pubertal status of IVF children, nor to BMI, thyroiditis, PCOS, GDM, parity, arterial hypertension or smoking of their mothers. These findings further underline the importance of continuous monitor­ ing of endocrine axes of IVF children and high­ light once again the important impact of epigenetic modification of gene expression in the offspring of ART.

Assisted reproductive technologies and overall health of the offspring ART has been widely used during the last three decades and several papers dealing with the over­ all health outcome of the offspring have been published recently. It seems that ART, either because of hormonal overstimulation of the oocyte, the artificial culture media, the cause of the infertility per se or any combination thereof, may lead to slight epigenetic modifications in the expression of several genes that may have some impact on the health of the offspring. Slight elevations of the SBP and DBP and the circulating concentrations of the triglycerides have been observed in IVF children, especially in those with rapid catch-up growth in weight during early childhood and special attention should be paid to preventing rapid weight gain during early life in order to minimize future car­ diometabolic risk. However, the postnatal growth of most ART children is normal and no increased incidence of the full MS was observed in these children and adolescents. Moreover, the pace and timing of puberty of ART children was nor­ mal and no increased incidence of premature adrenarche, especially in AGA-born IVF chil­ dren, could be discerned. Finally, a slight modifi­ cation of the set point of TSH sensitivity was observed, without a change in thyroid hormone concentrations. Questions remain to be answered concerning the future reproductive capacity of children born after ART, as well as their future cardiovascular risk in later adult life. Long-term prospective studies should be performed to pro­ vide clear answers.

171

Abbreviations AGA ART

BA BMI BP CA DBP DHEA-S EH FGIR GDM HDL hsCRP ICSI IGFBP3 IGF-I IGF-II IL-6 IUGR IVF LH MS NC PCOS SBP SC SDS SGA FSH T3 T4 TSH

appropriate for gestational age assisted reproduction/ reproductive technique/ technology bone age body mass index blood pressure chronological age diastolic blood pressure dehydroepiandrosterone­ sulphate euthyroid hyperthyrotropinaemia fasting glucose insulin ratio gestational diabetes mellitus high -density lipoprotein high sensitivity C-reactive protein intracytoplasmic sperm injection insulin-like growth factor binding protein 3 insulin-like growth factor-I insulin-like growth factor-II Interleukin-6 intrauterine growth retardation in vitro fertilization luteinizing hormone metabolic syndrome naturally conceived polycystic ovary syndrome systolic blood pressure spontaneously conceived standard deviation score small for gestational age follicle stimulating hormone triiodothyronine thyroxine thyroid stimulating hormone

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 7

Sex hormone and neuroendocrine aspects of the metabolic syndrome Hajime Nawata1,
, Tetsuhiro Watanabe2, Toshihiko Yanase3, Masatoshi Nomura4,

Kenji Ashida3, Liu Min5 and WuQiand Fan6

1

Graduate School of Medical Science, Kyushu University and Fukuoka Prefectural University,

Tagawa City, Fukuoka, Japan

2 Department of Internal Medicine, Nakatsu Municipal Hospital, Nakatsu, Oita, Japan

3 Department of Endocrinology and Diabetes Mellitus, School of Medicine, Fukuoka University, Fukuoka, Japan

4 Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu

University, Fukuoka, Japan

5 Department of Medical Biochemistry, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan

6 Division of Endocrinology and Metabolism, School of Medicine, University of California, San Diego, La, Jolla, CA, USA

Abstract: We discuss the recent advances in the knowledge that the sex steroids testosterone (T), estradiol and dehydroepiandrosterone sulphate (DHEA-S) are involved in the development of visceral obesity and of the metabolic syndrome. Cross talk between leptin and the androgen receptor (AR) in the hypothalamus as well as the peripheral conversion of DHEA and T to estrone, estradiol and dihydrotestosterone (DHT) in adipocytes and hepatocytes play important roles in the metabolic syndrome in men. Finally, we discuss the development of new drugs, selective AR modulators, for treating the metabolic syndrome in men. Keywords: sex steroids (testosterone, estradiol, DHEA-S); visceral fat obesity; metabolic syndrome; selective androgen receptor modulator (SARM) cause cardiovascular diseases. This condition is known as the metabolic syndrome. Clinical and experimental animal studies have clearly showed that sex steroids play an important role in the regulation of fat mass in men. Testos­ terone (T) and adrenal androgen dehydroepian­ drosterone sulphate (DHEA-S) can be regarded as prohormones. T is converted to dihydrotestos­ terone (DHT) or estradiol, while DHEA-S is con­ verted to T or estradiol in the target tissue. These steroid hormones are closely involved in the meta­ bolism, accumulation and distribution of lipids in adipose tissues and the liver. The brain is also an important target for sex steroids and cross talk with peptide hormones can

Introduction The accumulation of fat in the abdominal cavity (visceral obesity) is associated with an increased risk of cardiovascular disease. Many epidemiological studies have established that a cluster of factors, including hypertension, dyslipidaemia (elevated levels of triglyceride and low level of high-density lipoprotein) and impaired glucose tolerance in individuals with visceral obesity and insulin resistance




Corresponding author. Tel.: þ0947-42-2118; Fax: þ0947-42-6171;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82007-2

175

176

regulate food intake and energy expenditure. Here, we discuss the current knowledge of the mechanisms involved in the metabolic syndrome, particularly visceral obesity, focusing on the role of sex steroids.

Brain and androgen activity: cross talk between leptin signalling and androgen receptor in the hypothalamus Age-dependent or other pathologically hypogonadal men have a significantly higher fat mass, which can be reversed by T administration. By contrast, the suppression of plasma T in healthy young men increases the per cent fat mass and decreases lipid oxidation rates and resting energy expenditure. Androgens exert their biological action via the androgen receptor (AR). AR gene variations were reported to be independently correlated with body fat content, leptin and insulin levels, and are strongly associated with central obesity indices in older males. Androgen and the AR play important roles in metabolic regulation. Low androgen–AR signalling tone or AR gene abnormalities are associated with the development of metabolic syndrome in males. We previously showed that AR-null male (ARL-/Y) (ARKO) mice developed late-onset obesity, exhib­ ited low spontaneous activity and showed decreased overall oxygen consumption ratio and lipolytic activ­ ity compared with wild-type ARX/Y mice (Fan et al., 2005). Although ARL-/Y mice are obese and have elevated plasma leptin levels, they consume an amount of food similar to their wild-type ARX/Y mice. This suggests that the ARL-/Y mice do not adequately respond to the increased production of leptin with reduced food intake. Thus, they are lep­ tin resistant. That study also demonstrated that AR signalling has a negative effect on adiposity in males and enhances lipolytic activity in adipose tissue. We have also demonstrated that whole-body inac­ tivation of AR significantly decreased spontaneous locomotor activity and the energy consumption ratio. We hypothesized that the mechanisms may involve the central nervous system (CNS). The androgen–AR system may affect metabolism at both the peripheral and CNS levels (Fan et al., 2005). The hypothalamus is rich in receptors for gona­ dal steroids.

The AR is highly expressed in various hypotha­ lamic nuclei, particularly the SNC, VWH, arcuate (ARC) and PMV nuclei, in male wild-type mice, but not in female mice (Fig. 1). By contrast, ARKO mice did not express the AR in the hypothalamus (Fan et al., 2008). In addition to the AR, the hypothalamus also expresses the androgen-activating enzyme 5a­ reductase, which converts T to the most potent natural androgen, DHT. Thus, the hypothalamus can locally activate androgen and, at the same time, expresses cognate receptors in specific nuclei. The functional relevance of the hypothalamic androgen–AR system is assumed to be that the system contributes to the control of the hypothalamic– pituitary–gonadal axis by feedback regulation. We previously demonstrated the anatomic rela­ tionships between AR and functional leptin recep­ tor at the neuronal level in the hypothalamus. In addition to the pronounced overlapping hypotha­ lamic distribution patterns, AR and leptin recep­ tor (OBRB) co-reside in the ARC neurons, one of the most important nuclei for the central action of leptin (Figs. 1, 2) (Fan et al., 2008). Leptin, which is secreted from adipocytes, is a key regulator of energy homeostasis and adipos­ ity. It acts directly on the hypothalamus and other regions of the brain to suppress food intake and increase energy expenditure. The leptin receptor, which is highly expressed in the hypothalamus, activates the tyrosine kinase Jak2 upon ligand binding. Activated Jak2 phosphorylates itself and residues Tyr1138 within the leptin receptor. Tyr1138 phosphorylation recruits and activates the transcription factor STAT3. Activated STAT3 undergoes homodimerization and nuclear translocation, which eventually leads to transcriptional regulation of key hypothalamic neuropeptides that are leptin responsive. Leptin­ activated STAT3 suppresses the expression of orexigenic agouti-related protein and neuropep­ tide Y, and concurrently stimulates the expression of the anorexigenic pro-opiomelanocortin in the ARC nucleus. STAT3 activation is believed to be essential for leptin regulation of food intake, energy expenditure and, ultimately, adiposity. In addition to morphological considerations, we found that the AR can enhance leptin-induced

177 A.

B.

m

f

m ARKO

SCN

B:–0.46 mm

VMH, DMH, ARC B:–1.90 mm

PMV

B:–2.46 mm

Funct AR Reported -ional (m) OBRB OBRB SCN

+

+

±

ARC

+

+

+

VMH

+

+

+

DMH

+

+

+

LH

±

+

+

PVH

±

+

+

PMV

+

+

±

m Fig. 1. Endogenous AR expression in the hypothalamus of adult mice. (A) Immunohistochemistry photomicrographs from three matched series (ARX/Y (m), ARX/X (f), and ARL-/Y (ARKO) mice) of coronal brain sections showing the expression of AR in the hypothalamus of 20-week-old mice. B, Bregma; 3v, third ventricle. Scale bar, 100 mm. (B) A comparison of the hypothalamic distribution of the AR and the OBRB (leptin receptor) in male mice. The hypothalamic distributions of OBRB are either from the literature (reported OBRB) or were studied by staining for nuclear STAT3 at 30 min after icv leptin at a dose of 30 ng/g body weight (functional OBRB). þ, high expression; +, low expression.

STAT3 transactivation, which is the most important leptin signalling pathway in the CNS that regulates metabolic homeostasis. Furthermore, AR-null male mice show impaired responses to central administration of leptin in terms of appetite suppression and body weight loss. The impaired responsiveness to leptin is evident before the onset of obesity and suggests that it is a direct result of AR inactivation rather than secondary to increased adiposity. It is interesting to note that women have higher serum leptin levels and higher pulse amplitudes of leptin secretion from adipose tissue than men. This sex difference may be explained by findings that estrogens induce leptin production, whereas androgens suppress it. Based on the finding that AR, which substantially potentiates leptin– STAT3 signalling, is expressed more abundantly in the male hypothalamus, we speculated that males may actually need a lower physiological concentration of leptin. We have described the highlights of the potential involvement of the androgen–AR system in

metabolic homeostasis at the level of the CNS by interacting with leptin signalling, at least in male mice. AR–leptin interactions in the hypothalamus may also provide new insights into the reproduction– metabolism integration. Obesity and hypogonadism are strongly related. Leptin is essential to correct gonadal function and reproduction because ob/ob and db/db mice are infertile and hypogonadal, and leptin restores fertility in ob/ob mice. Leptin regulates the hypothalamus–pituitary–gonadal axis at both the central and gonadal levels, regulates gonadotropin secretion in the hypothalamus, stimulates LH and FSH in pituitary gonadotropes and is implicated in gonadal steroidogenesis. The present study suggests the presence of bidirectional communication between the leptin and androgen signalling pathways. Functional AR in the male brain may assist in leptin signalling and therefore contribute to the regulation of metabolism by lep­ tin, as well as other physiological processes. Because a sufficient level of androgen is required to maintain normal AR and 5a-R expression in the

178 A.

Anti-AR-Alexa 488

20 µm

Anti-OBRB-Alexa 546

20 µm

B.

Anti-AR-Alexa 488

20 µm

Merge

20 µm

Normal IgG-Alexa 546

20 µm

Merge

20 µm

Fig. 2. Co-expression of AR and OBRB (leptin receptor) in ARC neurons. (A) Hypothalamic sections (bregma, 1.90 mm) of 20-week-old male mice were double stained for AR and OBRB, which were labelled with Alexa 488 and Alexa 546, respectively. Channels for Alexa 488 (AR), Alexa 546 (OBRB), and merged are shown individually. The merged channel is amplified to better indicate co-residence. (B) Omitting the anti-OBRB antibody prevented OBRB staining, indicating its specificity. Scale bar, 20 mm.

brain, a functional testis might affect central leptin signalling via the production of androgen.

Dehydroepiandrosterone and its anti-metabolic activity Dehydroepiandrosterone (DHEA) and DHEA-S are steroids that are abundantly produced by the adrenal gland. The serum concentrations of DHEA and DHEA-S increase during adrenarche but decrease steadily after puberty (Nawata et al., 2004). Although DHEA and DHEA-S have few intrinsic androgenic actions, they have recently

attracted widespread attention due to their beneficial anti-metabolic effects. We have previously demonstrated the beneficial anti-obesity (Taniguchi et al. 1995), anti-diabetic, anti-atherosclerotic, anti-dementia (neurosteroid) and anti-osteoporotic effects of DHEA. Interestingly, Roth et al. reported that calorie restriction in rhesus monkeys decreases the rate of mortality. They reported that low body tem­ perature, low serum insulin and high serum DHEA-S concentrations are good markers for longevity. They also compared the survival rates of healthy men in a 25-year longitudinal study of aging in Baltimore and found that men

179

with high concentrations of serum DHEA-S lived longer than men with low concentrations. High serum DHEA-S concentrations were shown to be a good biomarker for longevity in men. Therefore, DHEA appears to be important for longevity. Previous studies have shown an inverse rela­ tionship between serum DHEA-S with the extent of atherosclerosis in humans and a significant anti-atherosclerotic effect of exogenous adminis­ tration of DHEA in cholesterol-fed rabbits. Our in vitro data suggest that one mechanism for the anti-atherogenic action of DHEA involves the inhi­ bitory effect of DHEA on the accumulation of cholesteryl ester in macrophages (Taniguchi et al., 1996). In our previous study, an age-related signifi­ cant decrease in bone mineral density (BMD) was observed in post-menopausal women over the age of 50 years. A significant positive corre­ lation between BMD and serum DHEA-S was found in 127 post-menopausal women, but no correlation was seen between BMD and serum estradiol. In a sub-set analysis, serum DHEA-S and serum estrone were positively correlated with BMD in post-menopausal women aged between 50 and 70 years, suggesting that adrenal androgens are converted to estrogens in peripheral tissues, including osteoblasts, and may contribute to the maintenance of BMD in post-menopausal women (Nawata et al., 2002;, Yanase et al., 2003). In fact, we showed that osteoblasts express aromatase activity and are therefore able to convert DHEA to T and estro­ gens (Nawata et al., 1995). DHEA plays a biological role in many organs and has three possible mechanisms of action. One is as a specific receptor, although no recep­ tor for DHEA has yet been identified. The sec­ ond is the conversion of DHEA into a more active sex steroid such as T or estradiol in per­ ipheral tissues, after which it is bound to the AR or estrogen receptor (ER). This mechanism is known as an ‘intracrine action’ (Labrie et al., 1995). The last possibility is that the hydropho­ bic DHEA molecule may affect cell functions after binding to macromolecules such as enzyme proteins.

We have focused on identifying the DHEA target genes in humans. We found that treating activated human T-lymphocytes with DHEA increased the high-affinity binding of DHEA to the cytosolic fractions of human T-lymphocytes. We then constructed a subtraction cDNA library and selected 400 clones for subsequent nucleotide sequencing, and performed reverse transcriptionpolymerase chain reaction screening using specific primers to isolate cDNA clones that were more abundantly expressed after co-treatment with DHEA. We identified a novel dual specific mitogenactivated protein kinase (MAPK) phosphatase enhanced by DHEA, named DDSP. DDSP is highly homologous to leukocyte protein tyrosine phosphatase (LC-PTP) (Fig. 3A) and binds speci­ fically to p38 MAPK but not to Erk or JNK by immunoprecipitation (Fig. 3B). Thus, DDSP is a p38-specific MAPK phosphatase. Although

A MAP kinase phosphatase enchanced by DHEA (DDSP) M 1

282 293

DDSP

Alu

M M M 1 22 49

LC-PTP

331

RR

360 (A)n

ERK -binding p38-binding

B

(A)n

PTP signature motif

Specific binding to p38 MARK α−Erk α−JNK2 α−p38

IP DDSP DDSP ERK JNK2 p33

α−flag − − − −

+ − − −

− + − −

+ + − −

− − + −

+ − + −

− − − +

+ − − +

Fig. 3. (A). Schematic of comparison of the DDSP structure with the LC-PTP structure. In DDSP, the aa residues required for ERK binding are lacking. The C-terminal 11 aa residues of DDSP are different from those of LC-PTP. The box represents the PTP/DSP central catalytic domain. (B). Immunoco­ precipitation of DDSP with ERK, p38 or JNK. NIH3T3 cells transfected with a plasmid expressing flag-tagged DDSP were treated with NaCl (for p38 and JNK) or with PMA (for ERK). The endogenous ERK, p-38 or JNK in the whole-cell lysates from the treated cells was immunoprecipitated with an antiERK, anti-p38 or anti-JNK antibody, respectively, and then Western blotting was performed using an anti-flag antibody. DDSP specifically interacts with the endogenous phosphorylated p38-MAPK.

180

LC-PTP mRNA is not induced by several steroids, DDSP mRNA is specifically induced by DHEA in human T-cells (Ashida et al., 2005). Recent studies have revealed that p38 MARK plays important roles in adipocyte differentiation and adipocyte leptin production, and may be involved in obesity. Therefore, it is of great interest to elucidate the roles of DDSP as a target molecule for DHEA with respect to obesity. The ubiquitous expression of DDSP in human tissues observed in our earlier studies prompted us to hypothesize that the broad range of physiological effects of DHEA may be conveyed, at least in part, by DDSP. We explored the physiological roles of DDSP, particularly in the development of obe­ sity, by generating transgenic mice (DDSP-Tg) whose DDSP expression is driven by the CAG promoter, a modified chicken b-actin promoter with a CMV-IE enhancer. We found that DDSPTg mice were resistant to obesity induced by high-fat/high-sucrose diet. Db/db mice carry a mutation in the leptin recep­ tor and are widely used as an animal model of obesity and diabetes. Therefore, it is of interest to elucidate whether DDSP-Tg can protect against obesity in db/db mice, we generated db/db mice hemizygous for the DDSP transgene (DDSP-Tg db/db). These db/db mice bearing the DDSP-Tg were protected from obesity at 6 weeks of age (p < 0.01) when fed a normal chow diet. Of note, there was no significant difference in food intake between the DDSP-Tg db/db and db/db mice. Our observation in DDSP-Tg mice suggest cri­ tical roles for DDSP in the generation of obesity through the p38 MAPK pathway. Since DDSP is one of the candidate molecules for DHEA stimu­ lation, the anti-obesity effect of DHEA may be partly explained by DDSP induction. Thus, DDSP may offer a novel therapeutic target for obesity. DHEA and DHEA-S are synthesized in the zona reticularis of the adrenal gland and are con­ verted to sex steroids, T or estradiol, in the per­ ipheral tissues, with increases in the synthesis of IGF-I and a decrease in the synthesis of 11b­ hydroxysteroid dehydrogenase type 1 (11b­ HSD1). 11b-HSD1 is also a target of DHEA. Taken together, DHEA has a number of effects,

which include promoting a sense of well-being, improving cognitive function, improving skin status and promoting sexual activity, in addition to its anti-inflammatory, anti-atherosclerotic, anti-osteoporotic, anti-insulin resistance and anti­ obesity effects (Nawata et al., 2002).

Roles of T and estradiol in the metabolic syndrome in men There is a striking difference in body fat distribu­ tion between men and women. Men tend to accu­ mulate adipose tissue in the abdomen while women tend to accumulate fat in the gluteal– femoral region. Furthermore, the visceral accumu­ lation of abdominal adipose tissue is more pro­ nounced in men than in women. Abdominal obesity is usually associated with low plasma T levels in cross-sectional and longitudinal studies of men with late-onset hypogonadism (partial androgen deficiency). Visceral fat accumulation measured by computed tomography (CT) is sig­ nificantly correlated with low plasma T concentra­ tions and low plasma sex hormone binding globulin (SHBG) levels (Blouin et al. 2008). Recently, a cross-sectional study evaluated the prevalence of metabolic syndrome in men with pro­ static cancer (PCa) undergoing long-term androgen deprivation therapy (ADT). Interestingly, more than half (55%) of the men in the ADT group had metabolic syndrome, compared with 22 and 20% in the non-ADT and control groups, respectively. Cardiovascular disease has recently become one of the most common causes of mortality in men with PCa undergoing long-term ADT. Therefore, it is thought that the use of ADT may trigger the development of metabolic syndrome, which may accelerate atherosclerosis and ultimately cardiovas­ cular disease (Shahani et al. 2008). Compared with men, it is generally believed that abdominal obesity in women is associated with high plasma T levels. This is based on the observation that women with polycystic ovary syn­ drome often show hyperandrogenism along with visceral obesity, insulin resistance and hyperinsu­ linaemia (Dunaif 1997). Of note, ~50% of women with polycystic ovary syndrome are obese with

181

extensive visceral adipose tissue accumulation, suggesting that elevated androgen levels might increase the fat mass in women (Dunaif et al. 1997). Visceral fat is also increased in femaleto-male transsexuals under androgen treatment, with supraphysiological plasma T levels. Thus, plasma T, within the physiological range, is associated with a good metabolic profile. Con­ versely, hypogonadal men with late-onset hypogo­ nadism, men with PCa undergoing long-term ADT, women with polycystic ovary syndrome showing hyperandrogenism and female-to-male transsexuals treated by supraphysiological T dis­ play increased visceral fat accumulation and increased risk for cardiovascular disease. Human as well as animal data indicate that T plays an important role in the regulation of body fat distribution. Low plasma T levels in men and T excess in women are generally associated with visceral obesity. At physiological concentrations, T mostly decreases fat mass with possible regionspecific effects. The effects of T on adipose tissue function may be mediated through a reduction in adipogenesis. Local T and estrogen synthesis may also be involved in the regulation of body fat distribution. Peripheral DHEA and T metabolism are posi­ tively associated with visceral obesity in men. Furthermore, numerous steroid-converting enzymes, including aromatase (Nishi et al., 2001), are present in subcutaneous and visceral adipose tissue in both sexes. T may regulate adipose tissue metabolism in men either directly by stimulation of the AR or indirectly by aromatization of androgens into estrogens and, thereafter, by stimulation of the ERs. Clinical case reports have documented disturbances of adipose tissue metabolism, including obesity, in aromatase-deficient and estrogen-resistant men. Studies using ER-inacti­ vated male mice have shown that ERa- but not ERb-inactivated mice are obese, indicating that ERa activation results in reduced fat mass in men. Adult males with aromatase deficiency were reported to show undetectable plasma levels of estrogen, normal to high levels of T and gonado­ tropins, and their body mass index was in the

overweight range (i.e. 25–30 kg/m2) with abdom­ inal adipose tissue accumulation. The plasma levels of triglycerides were generally elevated, with low high-density lipoprotein cholesterol levels (Jones et al., 2006). Lipid-associated parameters improved after estradiol treatment. However, the results were variable, probably as a consequence of the varying treatment regimens or differences in genetic back­ ground. Hepatic steatosis was reported in two aromatase-deficient men, and was associated with an enlarged liver and significantly elevated liver enzyme levels. An elevated HOMA-IR, a marker for insulin resistance, indicated insulin resistance in one of these men, and another was diagnosed with type 2 diabetes mellitus with elevated fasting glucose and insulin levels. Estradiol treatment improved insulin sensitivity in both of these men. These data clearly demonstrate that the loss of estrogen in men results in the development of features of the metabolic syndrome including abdominal obesity, elevated blood lipids and insu­ lin resistance. The relationship between estrogen deficiency and the metabolic syndrome in males might involve increased body weight with the con­ sequent development of insulin resistance. Male patients with aromatase deficiency are typically of tall stature with delayed skeletal maturation and epiphyseal closure, have eunuchoid body propor­ tions and osteoporosis with bone pain. These findings clearly indicate that estrogen plays en essential role in male physiology. Accord­ ingly, the androgen to estrogen ratio might be more important than the individual actions of each hormone in a tissue- and sex-specific manner. Although previous studies have investigated the effect of androgen on adipose tissue metabolism using aromatizable T, which results in the activation of ER and AR, the specific role of AR activation in the regulation of adipose tissue metabolism in men has remained unclear. The interpretations of the effect of AR activa­ tion, or mixed AR/ER activation, on fat mass are conflicting. Therefore, one study determined the direct effect of AR activation on fat mass by treating orchidectomized mice with the non-aromatizable androgen DHT. In that study,

182

significant increase in lean body mass, a decrease in adipose tissue and an increase in muscle strength (Saad et al., 2009). However, T replacement therapy in elderly men is often limited because of potential side effects, which include hyper-stimulation of the prostate, increased hematocrit, liver dysfunction and sleep apnea syndrome. In addition, exacerbation of sub­ clinical benign prostatic hypertrophy and/or PCa has long been claimed. This provides the strategy for developing molecules with marked tissue specificity for selected indications. The nuclear receptor superfamily comprises a large and diverse group of eukaryotic transcrip­ tion factors that control many biological functions through the regulation of specific genes. The AR is involved in ontogeny, differentiation and regen­ eration, endocrine disruption, androgen insuffi­ ciency syndrome (Adachi et al., 2000; Nawata et al., 2002) and obesity (Fig. 4). The AR has a common structure, consisting of transcription activation domains and a DNA-bind­ ing domain, which targets the receptor to specific DNA sequences known as hormone response

AR activation induced obesity and altered lipid metabolism in the orchidectomized mice (Move­ rare-Skrtic et al., 2006). Thus, one may speculate that AR antagonists might be useful in the treat­ ment of obesity in men.

The androgen receptor as a target for drug development for the metabolic syndrome: selective androgen receptor modulators Plasma T levels decline progressively with aging in men. The decline in bioavailable T is asso­ ciated with changes in body composition (distri­ bution of fat), including reduced energy expenditure, BMD, muscle strength and sexual function, in addition to depressed mood. This T-deficient state in aging males is often called late-onset hypogonadism. T replacement therapy using injectable, oral and transdermal prepara­ tions is widely available to treat a variety of male disorders. Several clinical studies have already shown that supplementation of T at physiologic doses in elderly men results in a

Co-activator ANT-1 (JBC 2002)

SRC-1/NCoA-1, TIF2/GRIP-1,

ACTR/AIB1/RAC3, CBP/p300, P/CAF,

TRAP220, DRIP, RIP140, TRIP1/SUG1,

ARA70, SRA

co-repressor

Androgen receptor

p160

SMRT,

ON EX pre-mRNA FoxH1 (JBC 2005) N-CoR

CBP/p300 EX

ON

Basic transcription machinary

histone

histone

ARE

Target gene mRNA

(1) Ontogeny, differentiation and regeneration

(2) Endocrine disruptor

(3) Androgen insensitivity syndrome (co-activator disease)

(4) Androgen and obesity (metabolic syndrome)

Fig. 4. Structure of AR and specific regulation by co-activators and co-repressor and gene regulation of specific functions.

183

elements. There are two transcription activation domains; the activation function-1 (AF-1) in the N-terminal region and the activation function-2 (AF-2) in the C-terminal region. The AF-2 domain is a ligand-binding domain, and is rela­ tively conserved among nuclear receptors. The AF-1 domain differs widely, and is important for the tissue-specific action of nuclear hormone receptors. Upon ligand binding to the receptor, the receptor changes in structure, translocates from the cytoplasm to the nucleus and then binds to the promoter region of the target gene. Co­ regulator proteins bind to the nuclear receptors in a promoter- and cell-specific manner. There are two types of co-regulators, co-activators such as CBP/p300, p160 family SRC 1/NcoA1, TIF and ANT-1, which activate transcription, and co­ repressors such as NcoR, SMRT and FoxH1, which repress transcription (Fig. 4) (Tomura et al., 2001). These co-regulators operate together by form­ ing an enormous protein complex. A complex composed mainly of CBP/p300 and the p160 system has histone acetyltransferase (HAT) activity that acetylates a basic amino acid of the histone protein and alters the chromatin struc­ ture. This facilitates transcription factor recruit­ ment to DNA and promotes transcription. On the other hand, the co-repressor SMRT/Ncor complex represses transcription activity by cou­ pling with the nuclear receptor that is unbound to the ligand and recruits the histone deacetyla­ tion enzyme, which has an opposing effect to HAT on the promoter. Therefore, it is specu­ lated that, once a ligand binds to the nuclear receptor, the co-repressor complex dissociates from the receptor and the co-activators are recruited to the promoter. Differences in the structure of the ligand for the same nuclear receptor induce different conforma­ tional changes in the nuclear receptor. These different conformational changes are responsible for the tissue-specific recruitment of co-activators or co-repressors and induce stimula­ tory or inhibitory effects on the cell in a cell- and tissue-specific manner. According to this theory, selective ER modula­ tors for the ER and selective progesterone

receptor modulators (Chwalisz et al. 2005) for the progesterone receptor are already available. A selective androgen receptor modulator (SARM) may have the potential to maintain or improve muscle strength and BMD, and to improve body composition, mental health, libido and sexual function in elderly men, without con­ comitant deleterious effects on the prostate. In fact, several groups have developed a series of SARMs that exhibit weaker effects on the pros­ tate while maintaining their effects on muscle, bone and male contraception. However, SARM compounds exhibiting strong metabolic effects with limited prostatic effects have not yet been developed. To evaluate this, we used a new two-step screening method. After first screening negative compounds for the stimulation of prostate-specific antigen (PSA) expression levels in PCa cells, we selected uncoupling protein-1 (UCP-1) as a sec­ ondary screening marker to identify chemicals that may have beneficial effects on energy expen­ diture or lipid metabolism (Min et al., 2009). We have previously reported that male ARKO mice show late-onset obesity, partly because of decreased energy expenditure, which may be associated with a dramatic decrease in UCP-1 expression in the white adi­ pose tissue of these mice. We also noted that the AR, on ligand binding, directly activates UCP-1 transcription. These findings prompted us to consider UCP-1 as a second screening marker to identify SARMs that possess metabolic effects. By examining their effect on these two specific markers, PSA and pre-adipocyte UCP-1, we searched for candidate chemicals with SARM activities from a large number of syn­ thetic AR ligands and DHEA derivatives. Using this strategy, we newly identified a syn­ thetic AR ligand, called SARM42 (S42; TZP­ 3157 TZCL050709) (Fig. 5). The results of the whole-cell binding assay using COS-7 cells exogenously expressing var­ ious steroid receptors indicated that S42 speci­ fically binds to AR and the progesterone receptor. When orchidectomized Sprague–Dawley rats were administered with S42 for 3 weeks, the muscle weight of the levator ani was

184 A.

SARM-42 (TZP-3157 TZCL050709)

O

C21H28O

Molecular weight: 296.46

B. 120 B/B0(3H-R1881, dpm) (×100)

110 100 90 80

R1881

70

DHT

60

Flutamide

50

S42

40 30 20 10 0 –12 –11 –10

–9

–8

–7

–6

–5

–4

–3

–2

[Competitor] (Log M) Fig. 5. Structure of S42 (TZP-3157 TZCL050709) and whole-cell binding studies of R1881, DHT, hydroxyflutamide and S42. (A) Structure of S42. (B) The whole-cell binding studies were conducted in AR-transfected COS-7 cells. Different concentrations of compound were co-incubated with 1 nM [3H]R1881 in serum-free phenol red-free medium for 4 h. Cells were lysed with 400 ml CelLytic M Cell Lysis Reagent, and the radioactivity was counted. The cell number was normalized with bicinchonininc acid by measuring protein levels. S42 showed binding to AR with a similar or slightly weaker potency (IC50 = 365 nM) compared with that of hydroxyflutamide.

increased as markedly as that induced by DHT, but the weight of the prostate did not increase at any doses of S42, whereas DHT did increase the weight of the prostate (Fig. 6). The plasma gonadotropin and adiponectin levels were downregulated by DHT, but unaffected by S42. Of particular interest, S42 was found to dramatically decrease the plasma triglyceride level without affecting the plasma cholesterol level. Insulin resistance and hyperinsulinemia are often associated with hypertriglyceridaemia because of the enhanced synthesis of very-low­

density lipoprotein triglyceride in these states, mainly via the activation of SREBP-1c in liver. Lipogenic gene expression in the liver and adipocytes is regulated by several transcription factors such as SREBP-1c. SREBP-1c has been shown to directly activate the expression of more than 30 genes involved in the biosynthesis of cholesterol, fatty acid, triglycerides and phos­ pholipids. CPT-1 is a key enzyme involved in carnitine-dependent transport across the mito­ chondrial inner membrane, and its deficiency decreases the rate of fatty acid b-oxidation.

185 Prostate weight

250

## 168.6**

200 150

132.5 b

10.2**

50

11.1**

13.3**S42

0 0 1 Dose (mg/kg)

DHT

## 209.8**

200 150

114.4

100 1.0**

50

10

Sham

Levator ani weight

D. ## 120.6**

300 ## 93.8

250 200

## 211.4**

250

b

1.0**

1.2**

S42

0 Sham

C. Levator ani weight (mg/100 g B.W.)

DHT

100

PSA mRNA in prostate 300

## 216.9*

Relative mRNA level of PSA/Gapdh

300

B.

DHT

73.1

150

b

S42

41.6** # 59.1

100

66.2

50 0

0 1 Dose (mg/kg)

10

Myostatin mRNA in levator ani 1.5

Relative mRNA level of myostatin/Gapdh

Prostate weight (mg/100 g B.W.)

A.

1.0

1.2

1.0

# 0.56*

# 0.57* DHT ## 0.29** S42

0.5 # 0.26* 0.0

Sham

0 1 Dose (mg/kg)

10

Sham

0 1 Dose (mg/kg)

10

Fig. 6. S42 had no virilizing activity but had an anabolic action in orchiectomized rats over 3 weeks of treatment. (A) The prostate weight increased with DHT treatment but not with S42 treatment. (B) PSA mRNA level was dramatically decreased by castration compared with the sham operation and was dose dependently increased by DHT treatment, and there was no change in PSA mRNA levels in the S42-treated groups. (C) DHT and S42 increased the weight of levator ani (skeletal muscle). (D) DHT reduced myostatin mRNA level by about 40%, whereas S42 more strongly reduced the level to about 70% as compared with the sham group.
p < 0.05,

p < 0.01.
,

: sham vs. orchiectomy group. # p < 0.05, ## p < 0.01. #, ##: vehicle vs. treatment group.

As expected, S42 decreased the mRNA level of SREBP-1c and FAS, which suppresses lipo­ genesis, and increased the mRNA level of CPT­ 1, which activates lipolysis in the liver and in visceral fat (Fig. 7). Insulin receptor substrates (IRSs) are a family of proteins that are phos­ phorylated by the activated insulin receptor. The deletion of IRS-2 in the mouse hypothala­ mus and in b cells was recently shown to cause a type 2 diabetes-like syndrome. IRS-2 also promotes b-cell growth and survival. In our study, S42 dramatically increased the transcrip­ tional level of IRS-2 in the liver in contrast to DHT. Because SREBP-1c is the upstream

suppressor of IRS-2, S42-induced suppression of SREBP-1c expression is closely linked to an increase in IRS-2 mRNA expression in the liver. Therefore, S42 may exert its insulinsensitizing effect in the liver through the upregulation of IRS-2. Taken together, S42 acts as an AR agonist in muscle and as an AR antagonist in the prostate, pituitary gland and liver, accompanying its bene­ ficial effects on lipid metabolism. In summary, S42 is a promising new SARM that could be used for the treatment of metabolic syn­ drome, without exerting adverse effects on the prostate.

186

4

##

**

3.2

DHT

3

2

1.0

0.71

1

0.82

0.57 b S42

0 shem

D. Relative mRNA level of SREBP1c/Gapdh

**

0 1 Dose (mg/kg)

2.2 3

1.8 DHT 1.0

1.0

1

S42 0.5

0.8

0 shem

0 1 Dose (mg/kg)

##

8.0** 10

10

##

7.1** DHT

5

1.0

0.9

0.7

0.5 S42

b

0 shem

4

C.

15

10

SREBP-1c mRNA in rat visceral fat

2

FAS mRNA in rat liver

Relative mRNA level of CPT1/Gapdh

3.4

Relative mRNA level of FAS/Gapdh

##

5

B.

E.

0 1 Dose (mg/kg)

3.5* 2.3

DHT

3

2

1.0

1

0.5

0.3

S42 0.6

0 shem

0 1 Dose (mg/kg)

10

a

b

#

** 3.2

4

2

1.0

S42

1.4 0.4 DHT

1.3

0

F. ##

4

4.2 *

6

shem

FAS mRNA in rat visceral fat 5

CPT-1 mRNA in rat liver

10

Relative mRNA level of CPT1/Gapdh

SREBP-1c mRNA in rat liver

Relative mRNA level of FAS/Gapdh

Relative mRNA level of SREBP1c/Gapdh

A.

0 1 Dose (mg/kg)

10

CPT-1 mRNA in rat visceral fat 4

*

2.6 3

2

2.0

S42 a

1.7

DHT 0.8

1.7 1.0

1

0 shem

0 1 Dose (mg/kg)

10

Fig. 7. S42 decreases the plasma triglyceride level through the SREBP-1c and CPT-1 pathway in the liver and visceral fat of ORX rats. (A–C) SREBP-1c (A), FAS (B) and CPT-1 (C) mRNA levels in the liver of orchiectomized rats after treatment with DHT or S42 for 3 weeks. (D–F) SREBP-1c (D), FAS (E) and CPT-1 (F) mRNA levels in the visceral fat of orchiectomized rats after treatment with DHT or S42 for 3 weeks. The reduced expression of SREBP-1c and FAS and increased expression of CPT-1 in both tissues may contribute to the decreased plasma triglyceride levels with S42 treatment.

187

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Movérare-Skrtic, S., Venken, K., Andersson, N., Lindberg, M. K., Svensson, J., Swanson, C., et al. (2006) Dihydrotes­ tosterone treatment results in obesity and altered lipid meta­ bolism in orchidectomized mice. Obesity, 14, 662–672. Nawata, H., Adachi, M., & Yanase, T. (2002) Androgen insensitivity by coactivator abnormality.Clinical Pediatric Endocrinology, 11, 17–24. Nawata, H., Tanaka, S., Tanaka, S., Takayanagi, R., Sakai, Y., Yanase, T., et al. (1995) Aromatase in bone cell: Association with osteoporosis in postmenopausal women. Journal of Steroid Biochemistry and Molecular Biology, 53, 165–174. Nawata, H., Yanase, T., Goto, K., Okabe, T., & Ashida, K. (2002) Mechanism of action of anti-aging DHEA-S and the replacement of DHEA-S. Mechanism of Ageing and Development, 123, 1101–1106. Nawata, H., Yanase, T., Goto, K., Okabe, T., Nomura, M., Ashida, K., et al. (2004) Adrenopause. Hormone Research, 62(Suppl. 3), 110–114. Nishi, Y., Yanase, T., Mu, Y., Oba, K., Ichino, I., Saito, M., et al. (2001) Establishment and characterization of a steroi­ dogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor. Endocrinology, 142, 437–445. Saad, F., & Gooren, L. (2009) The role of testosterone in the metabolic syndrome: A review. Journal of Steroid Biochem­ istry and Molecular Biology, 114, 40–43. Shahani, S., Braga-Basaria, M., & Basaria, S. (2008) Androgen deprivation therapy in prostate cancer and metabolic risk for atherosclerosis. The Journal of Clinical Endocrinology and Metabolism, 93, 2042–2049. Taniguchi, S., Yanase, T., Haji, M., Ishibashi, K., Takaya­ nagi, R., & Nawata, H. (1995) The antiobesity effect of dehydroepiandrosterone in castrated or noncastrated obese Zucker male rats. Obesity Research, 3(Suppl. 5), 639S–643S. Taniguchi, S., Yanase, T., Kobayashi, K., Takayanagi, R., & Nawata, H. (1996) Dehydroepiandrosterone markedly inhi­ bits the accumulation of cholesteryl ester in mouse macro­ phage J774-1 cells. Atherosclerosis, 126, 143–154. Tomura, A., Goto, K., Morinaga, H., Nomura, M., Okabe, T., Yanase, T., et al. (2001) The subnuclear three-dimensional image analysis of androgen receptor fused to green fluores­ cence protein. The Journal of Biological Chemistry, 276, 28395–28401. Yanase, T., Suzuki, S., Goto, K., Nomura, M., Okabe, T., Takayanagi, R., et al. (2003) Aromatase in bone: roles of Vitamin D3 and androgens. Journal of Steroid Biochemistry and Molecular Biology, 86, 393–397.

L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 8

Ghrelin’s role as a major regulator of appetite and its other functions in neuroendocrinology Chung Thong Lim, Blerina Kola, Márta Korbonits and Ashley B. Grossman
Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and

Dentistry, Queen Mary University of London, London, UK

Abstract: Ghrelin is a circulating growth-hormone-releasing and appetite-inducing brain-gut peptide. It is a known natural ligand of the growth hormone secretagogue receptor (GHS-R). Ghrelin is acylated on its serine 3 residue by ghrelin O-acyltransferase (GOAT). The acylation is essential for its orexigenic and adipogenic effects. Ghrelin exerts its central orexigenic effect through activation of various hypothalamic and brain stem neurons. Several new intracellular targets/mediators of the appetite-inducing effect of ghrelin in the hypothalamus have recently been identified, including the AMP-activated protein kinase, its upstream kinase calmodulin kinase kinase 2, components of the fatty acid pathway and the uncoupling protein 2. The ghrelin/GOAT/GHS-R system is now recognised as a potential target for the development of anti-obesity treatment. Ghrelin regulates the function of the anterior pituitary through stimulation of secretion not only of growth hormone, but also of adrenocorticotrophin and prolactin. The implication of ghrelin and its receptor in the pathogenesis of the neuroendocrine tumors will also be discussed in this review. Keywords: ghrelin; GOAT; GHS-R; appetite; pituitary tumors

Introduction

used by growth-hormone-releasing hormone and somatostatin (Leontiou et al., 2007). Howard et al. (1996) cloned the orphan growth hormone secre­ tagogue receptor (GHS-R), which is a G-protein­ coupled receptor that is mainly expressed in the hypothalamus and the pituitary. It was hypothesized that there is an endogenous ligand that binds GHS-R and stimulates the release of growth hormone (GH). Kojima et al. (1999) successfully purified (from the stomach) the endogenous hormone that binds to GHS-R. This hormone was shown to stimulate the release of GH and was thus named ‘ghrelin’, after ghre,

which is the Proto-Indo-European root of the

The growth hormone secretagogue (GHS) con­ cept was first introduced in 1978 when a group of synthetic compounds derived from met-enkephalin was found to have growth-hormo­ ne-releasing activities (Momany et al., 1981); since then, various other GHSs have been discovered (Leontiou et al., 2007). These GHSs have all been shown to act through a different receptor to that


Corresponding author.

Tel.: þ44-20-7601-8343; Fax: þ44-20-7601-8505; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82008-4

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word ‘grow’. The discovery of ghrelin is therefore an example of ‘reverse pharmacology’, meaning that the synthesis of artificial compounds led to the cloning of a natural receptor and then finally to the discovery of the natural ligand. Following its discovery, the physiological and pathophysiological functions of ghrelin have been studied extensively (Table 1). Both ghrelin and its receptor are strongly conserved during evolution, supporting the notion that the GHS­ R and its natural ligand play a fundamentally important role in biology (Palyha et al., 2000). At least two major neuroendocrine functions of ghrelin have been described as signalling through the GHS-R in the brain: the stimulation of GH release and the regulation of appetite and energy balance. Ghrelin has other endocrine effects such as the stimulation of prolactin (PRL) and of adre­ nocorticotrophin (ACTH) secretion, inhibition of the pituitary–gonadal axis at both central and peripheral levels, and modulation of pancreatic exocrine and endocrine functions (Broglio et al., 2003; Korbonits et al., 2004; Leite-Moreira and Soares, 2007; Leontiou et al., 2007; Lorenzi et al., 2009; Van Der Lely et al., 2004). Ghrelin is also known to influence cardiac function, control gas­ tric motility and acid secretion, affect sleep dura­ tion, memory, learning and behavior, modulate pulmonary and immune functions, regulate cell proliferation and stimulate bone formation (Asakawa et al., 2001a; Carlini et al., 2008;

Charoenthongtrakul et al., 2009; Fukushima et al., 2005; Hattori et al., 2001; Korbonits et al., 2004; Leite-Moreira and Soares, 2007; Leontiou et al., 2007; Trudel et al., 2002; van der Lely et al., 2004; Weikel et al., 2003). Interestingly, ghrelin is also expressed in a variety of tumors of the neu­ roendocrine system, and its endocrine, paracrine and autocrine effects seem to play a role in the tumorigenic process (Leontiou et al., 2007).

The structure and expression of ghrelin Ghrelin is a 28-amino acid hormone and is synthe­ sized mainly in the endocrine cells of the gastric mucosa (Gnanapavan et al., 2002). It also has lowlevel widespread expression throughout the body and has been detected immunohistochemically in the hypothalamus, pituitary, small intestine, pan­ creas and the immune system, and also in most of the normal human tissues examined (Korbonits and Grossman, 2004; Rindi et al., 2004). The ghre­ lin gene is located at chromosome 3p25-26 and the main mRNA codes for the 117-amino acid pre­ proghrelin, which is then enzymatically cleaved into proghrelin and obestatin. Obestatin is thought to be a peptide with anorexigenic activ­ ities opposing those of ghrelin, but its central or peripheral effects on energy balance were not shown in lean, obese, fed or fasted rodents (Castaneda et al., 2010; Chartrel et al., 2007;

Table 1. Several neuroendocrine and non-neuroendocrine physiological roles of ghrelin Physiological roles of ghrelin

Key references

Stimulates GH, ACTH and PRL release

Kojima et al. (1999), Date et al. (2000), Wren et al. (2000), Arvat et al. (2000), Ghigo et al. (2001), Korbonits et al. (2004) Wren et al. (2000), Tschop et al. (2000), Wren et al. (2001), Korbonits et al. (2004) Broglio et al. (2003), Lorenzi et al. (2009) Asakawa et al. (2001), Weikel et al. (2003), Carlini et al. (2008) Trudel et al. (2002), Charoenthongtrakul et al. (2009) van der Lely et al. (2004), Leite-Moreira and Soares (2007) Fukushima et al. (2005) Hattori et al. (2001), Korbonits et al. (2004), van der Lely et al. (2004), LeiteMoreira and Soares (2007) Tschop et al. (2000), Korbonits et al. (2004) Korbonits et al. (2004), van der Lely et al. (2004), Leite-Moreira and Soares (2007), Leontiou et al. (2007)

Stimulates appetite Negatively influences the pituitary–gonadal axis Influences sleep, memory, learning and behavior Controls gastric motility and secretion Influences cardiovascular functions Increases bone formation Modulates pulmonary and immune functions Induces adiposity Regulates cell proliferation

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Gourcerol et al., 2007; Nogueiras et al., 2007). Proghrelin is then processed into ghrelin and a C-terminal polypeptide. Ghrelin has a unique fatty acid modification on its N-terminal end (Ser3) that is necessary for its biological activity and which is conserved across species (Kojima et al., 1999). However, the major­ ity of circulating ghrelin actually lacks this acyl group, and is known as des-acyl or des-octanoyl ghrelin. Des-acyl ghrelin has been shown to have various metabolic effects although its role in appe­ tite regulation remains unclear (Higgins et al., 2007). We have shown that des-acyl ghrelin does not have an effect on hypothalamic AMP-acti­ vated protein kinase (AMPK) activity (Kola et al., 2005), supporting the human data of a lack of effect of des-acyl ghrelin on appetite (Neary et al., 2006). Recently, ghrelin O-acyltransferase (GOAT), which is a porcupine-like enzyme belonging to the super-family of membranebound O-acyltransferase 4 (MBOAT4; Gualillo et al., 2008; Yang et al., 2008), has been identi­ fied as the enzyme that acylates the Ser3 on ghrelin with octanoate (Yang et al., 2008). All enzymes of this super-family have several membrane-spanning regions, share a region of detectable sequence similarity and are able to transfer organic acids, typically long-chain fatty acids, to hydroxyl groups in membraneembedded substrates. Human GOAT is expressed in the stomach and pancreas (Gutier­ rez et al., 2008). It can acylate ghrelin with other types of fatty acids besides octanoate, such as acetic and tetradecanoic acid (Gutierrez et al., 2008) whereas the murine GOAT can only spe­ cifically bind octanoate to the Ser3 of ghrelin (Hofmann, 2000). The ghrelin–GOAT system has been shown to alert the central nervous system to the presence of dietary calories (Kirch­ ner et al., 2009).

The growth hormone secretagogue receptor The GHS-R is encoded by a single gene located at chromosome 3q26.2 (McKee et al., 1997). The GHS-R has two variants, presumably as a result

of alternate processing of a pre-mRNA, GHS-R1a and GHS-R1b. GHS-R1a is a type 1a G-protein­ coupled receptor of 366 amino acids with seven transmembrane domains and a molecular mass of approximately 41kDa. It was shown to transduce the GH-releasing effect of synthetic GHSs as well as ghrelin, and also plays a role in neuroendocrine and appetite-stimulating activities centrally. GHS­ R1a is predominantly expressed in the pituitary as well as in several nuclei of the brain, particularly the arcuate nucleus (ARC), the ventromedial nucleus (VMN) and the paraventricular nuclei (PVN) of hypothalamus (Guan et al., 1997). It is also expressed at much lower levels in the thyroid gland, pancreas, spleen, myocardium and adrenal gland (Gnanapavan et al., 2002). GHS-R1b is a non-spliced variant with wide expression throughout the body (Gnanapavan et al., 2002) and consists of 289 amino acids with only five transmembrane domains. The biological function of GHS-R1b remains unclear. GHS-R1b does not bind ghrelin or other GHSs, but it was shown to have a counter-regulatory attenuating role on GHS-R1a signalling, possibly via the formation of heterodimers with GHS-R1a (Chan and Cheng, 2004).

Ghrelin and the regulation of appetite One of the most important established roles of ghrelin is the regulation of appetite and energy homeostasis (Kojima et al., 2004; Korbonits et al., 2004; Tschop et al., 2000). Intense interest in ghrelin rose when it was demonstrated that its concentration in human plasma rise during fasting and fall post-prandially (Cummings, 2006; Cum­ mings et al., 2001), making ghrelin known as the ‘hunger hormone’, while its levels rise pre-pran­ dially and fall post-prandially, supporting the hypothesis that ghrelin is a physiological feeding initiator (Cummings, 2006; Cummings et al., 2001). Ghrelin levels also rise with voluntary meal initiation, in the absence of time- and foodrelated cues (Cummings, Frayo et al., 2004). In animal models, both central and peripheral administration of ghrelin causes hyperphagia and an increase in body weight (Tschop et al., 2000).

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Intracerebroventricular infusion of acylated ghrelin in rats and peripheral administration of ghrelin to humans cause an increase in food intake (Kamegai et al., 2001; Small and Bloom, 2004; Wren et al., 2001). Peripheral ghrelin translates information about nutrients and gut to the brain to determine short-term appetite and long-term body weight regulation (Cummings and Shannon, 2003; Kojima et al., 1999). Ghrelin levels are upregulated under negative energy balance condi­ tions and consequently can act as a signal for food intake on the hypothalamus. Ghrelin reaches the brain through general circulation, crossing the blood–brain barrier and via the stimulation of vagal afferent nerves (Date et al., 2002; Venkova and Greenwood-Van Meerveld, 2008). Ghrelin is also synthesized locally in the hypothalamus, where it exerts paracrine effects (Korbonits and Grossman, 2004). In the hypothalamus, ghrelin acts mainly by binding to its receptors in areas that are impor­ tant for appetite regulation, namely the ARC, PVN, dorsomedial region, central nucleus of amygdala and the nucleus of solitary tract (Mano-Otagiri et al., 2006; Olszewski et al., 2003). The ARC neurons are the main regulator of appetite and energy balance in the hypothalamus (Kohno et al., 2003). The ARC neurons contain the orexigenic neuropeptide Y (NPY) and agoutirelated peptide (AgRP), and anorexigenic pep­ tides cocaine amphetamine-related transcript (CART) and pro-opiomelanocortin (POMC) con­ taining neurons. Ghrelin activates arcuate NPY neurons and inhibits the POMC and CART neu­ rons, leading to an increase in appetite and food intake (Andrews et al., 2008; Chen et al., 2004; Cowley et al., 2003). Interestingly, NPY knockout (NPY–/–) mice showed only a slight decrease in food intake and co-administration of ghrelin with AgRP did not produce a synergistic effect on feeding (Shrestha et al., 2006). In agreement with this, AgRP–/– or NPY–/– embryo or neonate mice were shown to be still susceptible to the effects of ghrelin (Chen et al., 2004; Tschop et al., 2002; Qian et al., 2002), whereas the double knockout mice were not (Chen et al., 2004). However, loss of either or both of these sets of neurons in adult animals

causes a significant reduction in body weight and appetite (Bewick et al., 2005; Gropp et al., 2005; Luquet et al., 2005; Ste Marie et al., 2005). Activation of AgRP by ghrelin will also antagonize a-melanocyte-stimulating hormone (a-MSH) at the melanocortin receptors (MC) 3 and 4, which are the main MC receptors in the brain (Tolle and Low, 2008). This will lead to an increase in food intake. ARC neurons also project to other hypo­ thalamic nuclei, including the orexigenic orexin­ containing neurons in the lateral hypothalamic area, to stimulate appetite (Toshinai et al., 2003). The PVN additionally receives projections from the anorexigenic POMC neurons as well as from the orexigenic NPY/AgRP neurons. It is proposed that ghrelin might stimulate appetite by antagoniz­ ing the inhibitory tone of the POMC neurons via the release of GABA from NPY/AgRP projections in the PVN (Cowley et al., 2003). Recently, it has been shown that the effects of ghrelin on fatty acid synthase (FAS) expression (see later) and feeding were inhibited by a decreased in AMPK activity in the VMN (Lopez et al., 2008). This indicates that modulation of hypothalamic fatty acid metabolism specifically in the VMH in response to ghrelin is a physiolo­ gical mechanism that controls feeding.

The hypothalamic intracellular signalling pathway responsible for the orexigenic effects of ghrelin Considering several important recent publications that have identified intracellular ghrelin targets in the hypothalamus, we have hypothesized that a plausible pathway via which ghrelin could explicit its orexigenic effect is the following: ghre­ lin – GHS-R - Ca2þ - endocannabinoids - canna­ binoid receptor type 1 (CB1) - Ca2þ - calmodulin kinase kinase – AMPK - malonyl coenzyme A (malonyl-CoA) - carnitine palmitoyl transferase 1 (CPT1) - b-oxidation - reactive oxygen species (ROS) – uncoupling protein 2 (UCP2) - NPY/ AgRP - food intake (Fig. 1; Kola and Korbonits, 2009). It can be seen that the enzyme AMPK is pivotal in this process.

193

Ghrelin Cannabinoids GHS-R1a

CB1

G

FAS

Ca2+

ACC

Malonyl-CoA

Palmitate

Palmitoyl-CoA

CPT1 CaMKK2 ROS UCP2 P

α

γ

NPY/AgRP neuron

β AMPK

β-oxidation

(mitochondria)

POMC neuron

Food intake

Fig. 1. Schematic diagram (modified with permission from Kola and Korbonits, 2009) showing the pathway in which ghrelin stimulates appetite via AMPK (GHS-R1a, growth hormone secretagogue receptor type 1a; CB1, cannabinoid receptor type 1; CaMKK, calmodulin kinase kinase; AMPK, AMP-activated protein kinase; ACC, acetyl-coenzyme A carboxylase; malonyl-CoA, malonyl coenzyme A; FAS, fatty acid synthase; CPT1, carnitine palmitoyl transferase 1; ROS, reactive oxygen species; UCP2, uncoupling protein 2; NPY, neuropeptide Y; AgRP, agouti-related peptide; POMC, pro-opiomelanocortin).

AMPK is a highly conserved enzyme, which is identified as one of the key players in the regulation of appetite and energy metabolism (Minokoshi et al., 2004). AMPK activity is regulated by change in the cellular AMP/ATP ratio. A rise in the AMP/ ATP ratio will activate the enzyme and upon activation, AMPK switches on ATP-producing catabolic processes and switches off ATP-consum­ ing anabolic processes (Kola et al., 2006; Lim et al., 2009). AMPK is activated by phosphorylation on the Thr172 residue by upstream kinases LKB1, calmodulin kinase kinase 2 (CaMKK2), transform­ ing growth factor-b-activated kinase or ataxia-tel­ angiectasia mutated (ATM) gene (Fu et al., 2008; Hawley et al., 2005; Lim et al., 2009; Momcilovic et al., 2006; Suzuki et al., 2004; Woods et al., 2005). Increased AMP does not directly promote phos­ phorylation of Thr172 by upstream kinases but rather inhibits the dephosphorylation of AMPK by protein phosphatase-2C-a (Lim et al., 2009; Sanders et al., 2007). Recently, a new model of AMPK regulation has been identified. Cell death-

inducing DFFA-like effector A, or Cidea, has been shown to form a complex with the b-subunit of AMPK, eliciting an ubiquination-mediated degra­ dation of AMPK (Qi et al., 2008). More recently, kinase suppressor of Ras 2 (KSR2) has also been identified as an essential regulator of AMPK activ­ ity (Costanzo-Garvey et al., 2009). KSR2 promotes phosphorylation and thus activation of AMPK. AMPK mediates the orexigenic and metabolic effects of several metabolic hormones and elements such as leptin, adiponectin, cannabinoids, insulin, glucose, glucocorticoids, thyroid hormone, a-lipoic acid and ciliary neutrophic factor (Kola et al., 2006; Kubota et al., 2007; Lim et al., 2009). We have shown that hypothalamic AMPK activity was stimulated with both intraperitoneal (ip) and intracerebroventricular (icv) administration of ghrelin in rats as well as in mice (Kola et al., 2005, 2008), suggesting that AMPK plays a role in med­ iating part of ghrelin’s orexigenic effect. This was recently confirmed in an elegant study from Lopez and colleagues with the use of compound C, an

194

inhibitor of AMPK, and the use a-1/a-2 dominant negative AMPK adenoviruses in the presence of which ghrelin failed to elicit an increase in food intake (Lopez et al., 2008).

Ghrelin affects AMPK via its upstream kinase CaMKK2 Recently, CaMKK2, which is regulated by intra­ cellular Ca2þ levels, has been shown to regulate hypothalamic NPY and to mediate the activation of hypothalamic AMPK by ghrelin (Anderson et al., 2008; Sleeman & Latres, 2008). CaMKK2 is expressed throughout the brain. The mRNA for CaMKK2 was found to be highly expressed in the ARC at levels 20- to 40-fold higher than in other hypothalamic areas (Anderson et al., 2008). CaMKK2 knockout mice (CaMKK2-KO) showed reduced expression of NPY and AgRP, but no differences in POMC expression, suggesting a direct effect for CaMKK2 only on the orexigenic NPY neurons. The selective CaMKK2 inhibitor STO-609 has been shown to inhibit NPY expression and reduce food intake and body weight in wild-type (WT) animals. Ghrelin has been shown to induce Ca2þ signalling in NPY neurones and in the ARC (Kohno et al., 2003, 2008) and this has led to the postulation that the rise in the intracellular Ca2þ leads to the activation of CaMKK2 which then mediates the stimulatory effect of ghrelin on hypothalamic AMPK. Hypothalamic AMPK activity is reduced in the CaMKK2-KO mice (Anderson et al., 2008) and CaMKK2-KO mice were shown to have no increase in food intake following systemic ghrelin injection (Anderson et al., 2008). This confirms CaMKK2 as the AMPK upstream kinase that mediates the effects of ghrelin on hypothalamic AMPK activity.

The downstream effectors of ghrelin–AMPK in appetite regulation The downstream effect of the ghrelin–GHS-R– CaMKK2–AMPK axis in the stimulation of

appetite involves an AMPK-dependent inhibition of the de novo fatty acid synthesis pathway in the VMN (Lopez et al., 2008). AMPK is an important regulator of the fatty acid biosynthetic pathway as it can phosphorylate and inhibit acetyl-coenzyme A carboxylase (ACC), thereby preventing the production of malonyl-CoA. The reduction in malonyl-CoA leads to the stimulation of CPT1 and the transport of fatty acids into the mitochon­ dria, ultimately facilitating mitochondrial fatty acid oxidation. Ghrelin was shown to induce a decrease in the FAS mRNA levels in the VMN as well as the hypothalamic FAS activity and protein levels (Lopez et al., 2008). These effects were not seen in rats treated with compound C or with the stereotactic delivery of a-1/a-2 dominant negative AMPK adenoviruses, clearly showing that the ghrelin effect on FAS mRNA expression is AMPK dependent (Lopez et al., 2008). Ghrelin also increases the phosphorylation of ACC with consequent decreased levels of malonyl-CoA and increased levels of CPT1 in the hypothalamus at 2 h (He et al., 2006; Obici et al., 2003; Pocai et al., 2006). This ultimately leads to an increase in food intake. This mechanism of action is further supported by the decrease in the orexigenic effect of ghrelin at 2 h after icv administration of etomoxir, an inhibitor of CPT1 (He et al., 2006; Obici et al., 2003; Pocai et al., 2006). Recently, UCP2 was also shown to mediate the effects of ghrelin on NPY/AgRP neuronal activity (Andrews et al., 2008). UCPs are inner-membrane mitochondrial proteins which modulate energy expenditure via ‘uncoupling’ ATP generation, thereby dissipating the mitochondrial proton gradient necessary for ATP synthesis and generating heat (Wu et al., 1999). Ghrelin has previously been shown to enhance UCP2 mRNA expression in the pancreas (Sun et al., 2006), liver (Barazzoni et al., 2005) and white adipose tissue (Tsubone et al., 2005). Ghrelin was shown to increase hypothalamic UCP2 mRNA expression, mitochondrial respira­ tion and mitochondrial proliferation in WT mice (Andrews et al., 2008). These effects were lost in UCP2-KO mice. UCP2 was also shown to mediate the effects of ghrelin in inducing NPY/AgRP

195

mRNA expression and activating NPY/AgRP neurons. Ghrelin had no direct effect on POMC neuronal action potential frequency or on POMC mRNA expression, and possibly indirectly hyperpolarizes POMC neurons by activating inhibitory NPY/AgRP inputs (Cowley et al., 2003). Consistent with this, ghrelin treatment increased inhibitory synapses and significantly reduced the number of excitatory synapses on POMC neurons in WT but not in UCP2-KO. The UCP-dependent effects on neuronal plasticity and activity translate into ghrelin’s orexigenic effects, as UCP2-KO mice do not show an increase in food intake in response to either ip or intra-mediobasal hypothalamic ghrelin treatment. AMPK has been shown to require UCP2 for its effect on feeding as the effect of the AMPK activator AICAR on food intake was lost in UCP2-KO mice (Andrews et al., 2008). The effect of ghrelin on AMPK activity was still present in UCP2-KO mice, thereby suggesting that AMPK is situated upstream of UCP2 (Andrews et al., 2008). In addition, the ghrelin-induced increase in UCP2 mRNA levels was diminished by concomitant icv administration of compound C or etomoxir, placing the AMPK–CPT1 pathway upstream of UCP2. UCP2, by scavenging ROS production triggered by ghrelin, is involved in mediating the effects of ghrelin on NPY/AgRP mRNA levels, on mitochondrial uncoupled respiration, on NPY neuronal firing and on food intake, and functions as a downstream effector of the ghrelin–AMPK– CPT1 pathway (Andrews et al., 2008).

Ghrelin and the cannabinoid system The cannabinoid system plays an important role in the hypothalamic neuronal regulation of appetite and body weight (Pagotto et al., 2006). Cannabi­ noids are known to induce food intake via the CB1 (Kirkham and Tucci, 2006). We have shown that cannabinoids stimulate hypothalamic AMPK activity, suggesting that AMPK mediates the orexigenic effects of cannabinoids (Kola et al., 2005). Cannabinoids are also known to play a role in mediating the anorexigenic effects of leptin (Di Marzo et al., 2001).

Recently, we have shown that an intact cannabinoid-signalling pathway is required for the effects of ghrelin on appetite and AMPK (Kola et al., 2008). The effects of ghrelin on hypothalamic AMPK activity, on PVN neuronal activity and ultimately on appetite were abolished in the absence of CB1 or in the presence of the CB1 antagonist rimonabant (Kola et al., 2008). Ghrelin also stimulated intrahypothalamic 2-arachydol glycerol and anandamide levels (Kola et al., 2008). Interestingly, a bilateral ghrelin–cannabinoid interaction is also suggested, as ip injection of cannabinoids in rats resulted in increased plasma ghrelin levels (Zbucki et al., 2008) and in agree­ ment, rimonabant injection reduces ghrelin levels, suggesting that the orexigenic effects of cannabi­ noids may also be connected to an increase in ghrelin secretion from the gastric X/A-like cells.

Ghrelin in obesity, anorexia, type 2 diabetes and determination of stature Ghrelin stimulates food intake and promotes positive energy balance in the body. Circulating ghrelin levels negatively correlate with the body mass index (BMI) in general, as seen in the obese patients with reduced serum ghrelin levels (Krsek et al., 2003). Obese subjects also have blunted nocturnal plasma ghrelin levels. Relationships between polymorphisms in the ghrelin or GHS-R gene and obesity have been reported (Kojima et al., 2004; Liu et al., 2007). For example, patients with the Leu72Met polymorphism in the ghrelin genome are pheno­ typically obese at an earlier age compared to homozygotic Leu72 allele patients. The Arg51Gln ghrelin genome variant is seen in 6.3% of obese subjects (Ukkola et al., 2001). This polymorphism changes the C-terminal pro­ cessing site of ghrelin and consequently results in reduced production of ghrelin. Four naturally occurring GHS-R mutations, 1134T, V160M, A204E and F279L, have been reported to date, and they all affect the constitutive activity of GHS-R (Liu et al., 2007). In contrast, we have found that common polymorphisms in ghrelin

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and its receptor genes are not major contributors to the development of polygenic obesity, although common variants may alter body weight and eating behavior and contribute to insulin resistance, in particular in the context of early-onset obesity (Gueorguiev et al., 2009b). In contrast, Prader–Willi syndrome (PWS) patients have high ghrelin levels despite their high BMI (Cummings et al., 2002). PWS is a complex genetic disorder caused by a loss of one or more paternal genes in the region 15q11–15q13 and is characterized by mild men­ tal retardation, short stature, muscular hypoto­ nia and obesity secondary to hyperphagia (Nicholls and Knepper, 2001). The high ghrelin levels might in part contribute to their hyper­ phagia and will normalize following recovery to ideal body weight. PWS patients also have a 3–4 fold increase in mean plasma GH concen­ tration than the reference population (Kojima et al., 2004). Short-term octreotide treatment has been shown to decrease the fasting levels of ghrelin in children with PWS (Haqq et al., 2003). Patients suffering from anorexia nervosa have high levels of plasma ghrelin and GH (Kojima et al., 2004). Their levels return to control levels following weight gain. Ghrelin and its receptor play an important role in glucose metabolism and energy homeostasis, and their genes have been studied for susceptibil­ ity alleles for type 2 diabetes but neither the ghrelin gene nor the GHS-R gene variants were found to be associated with type 2 diabetes (Garcia et al., 2009). In Danish adults, Larsen et al. (2005) failed to identify association of Arg51Gln, Leu72Met and Gln90Leu with type 2 diabetes (as assessed by HOMA insulin resistance index and fasting serum glucose and insulin). The negative association for the Leu72Met poly­ morphism with type 2 diabetes was reinforced by work from other groups, in a separate Danish cohort (Bing et al., 2005), on two separate Japanese cohorts (Ando et al., 2007; Choi et al., 2006; Kuzuya et al., 2006), on two separate Kor­ ean cohorts (Choi et al., 2006; Kim et al., 2006) and for all three polymorphisms in Old Order Amish members (Steinle et al., 2005). However,

an increase in insulin sensitivity and a reduced risk for type 2 diabetes were observed in Caucasians with Leu72Met polymorphism (Berthold et al., 2009; Zavarella et al., 2008). These contradicting results may be explained by the differences in the study designs and the population studied, such as the race and gender of the patients. Ghrelin gene has also been studied in relation to height, weight or body mass index and no significant genotype or haplotype associations were found with adult or childhood height, weight or body mass index (Garcia et al., 2008; Gueorguiev et al., 2007, 2009a, 2009b).

Ghrelin influences anterior pituitary function We demonstrated that the ghrelin mRNA is expressed in normal human pituitaries (Korbo­ nits et al., 2001), and studies in the rat showed that ghrelin is specifically expressed in the somato­ trophs, lactotrophs and thyrotrophs (Caminos et al., 2003). The local production of ghrelin might have direct paracrine and/or autocrine effects on the pituitary function. Pituitary ghrelin mRNA levels are age- and gender-dependent (Caminos et al., 2003; Leontiou et al., 2007). It has been shown that the genetic expression of ghrelin in the pituitary is higher during the early ontogeny stage of rats. In female rats, the level of ghrelin expression gradually decreased during development. By contrast, the variation of ghre­ lin expression level seen in male rats during development is insignificant (Caminos et al., 2003). GHS and ghrelin have been shown to stimulate the release of GH in a dose-related pattern, which is more marked in humans than in animals (Kojima et al., 1999; Muccioli et al., 2002; Smith et al., 1997). Intravenous (i.v.), ip, subcutaneous (s.c.) and icv injection of ghrelin in rats resulted in dose-related increased secretion of GH, suggest­ ing that ghrelin probably acts both directly and indirectly on the somatotrophic pituitary cells to stimulate the release of GH (Asakawa et al., 2001b; Date et al., 2000; Kojima et al., 1999; Wren et al., 2000).

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The GH-releasing activity of ghrelin is mediated by the activation of the GHS-R1a. Ghrelin binds to the GHS-R1a in somatotrophic pituitary cells and this complex activates phospholipase C (PLC) through the Gq11-protein, thereby activating the inositol phosphate cascade. The resultant genera­ tion of inositol 1,4,5-triphosphate (IP3) and dia­ cylglycerol (DAG) causes the release of Ca2þ from intracellular stores and activation of protein kinase C, respectively (Eglen et al., 2007; Luttrell, 2008). Activation of protein kinase C leads to inhibition of potassium channels, thereby depolar­ izing the somatotroph. Following depolarization, the initial Ca2þ influx induced by the action of ghrelin on somatotrophs is also followed by a sustained Ca2þ influx through activated L-type voltage-gated Ca2þ channels and Naþ influx through Naþ channels (Yamakage and Namiki, 2002). The increase in intracellular calcium results in GH secretion. GHS and ghrelin have also been shown to sti­ mulate the release of PRL and ACTH, respec­ tively, from the lactotroph and the corticotroph cells in the anterior pituitary (Arvat et al., 2000; Ghigo et al., 2001). The stimulatory effect of GHS on PRL secretion in human is independent of both gender and age, whereas the stimulation of ACTH secretion is independent of gender but shows peculiar age-related variations (Arvat et al., 2000; Ghigo et al., 2001; Muccioli et al., 2002).

Ghrelin and pituitary adenomas Ghrelin and GHS-R mRNAs are expressed in a variety of pituitary adenomas (Leontiou et al., 2007). We have shown that ghrelin mRNA

expression was relatively low in corticotroph ade­ nomas and relatively high in somatotroph adeno­ mas compared to normal pituitaries (Korbonits et al., 2001). Ghrelin expression in the pituitary shows a cell-specific pattern similar to the expres­ sion of Pit-1 gene (Caminos et al., 2003). Ghrelin increases the transcription of Pit-1 gene in neona­ tal rat anterior pituitary cells (Garcia et al., 2001) but not in adult pituitary cells (Soto et al., 1995). GH-producing adenomas have been shown to express ghrelin and GHS-R at both mRNA and protein level (Kim et al., 2001; Korbonits et al., 1998, 2001). The levels of ghrelin mRNA expres­ sion were negatively correlated with the size of the adenoma (Kim et al., 2001). GH-producing adeno­ mas can lead to two clinical conditions, depending on the age when it occurs: gigantism in childhood and acromegaly in adulthood. Both these condi­ tions are characterized by persistently elevated blood GH levels. Acromegalic patients had lower ghrelin levels (Table 2) compared to that measured in normal subjects (Cappiello et al., 2002). This might be because the possibility that the high GH and insulin-like growth factor 1 (IGF-1) levels seen in acromegaly exert a negative feedback effect on ghrelin production. Alternatively, it might be sec­ ondary to the insulin-resistant state, and thus it is the hyperinsulinaemia seen in the acromegalic patients that leads to lower levels of ghrelin, since insulin levels are known to be negatively correlated to ghrelin levels. The normalization of GH and IGF-1 levels after successful surgical treatment of acromegaly were shown to be followed by a rise in ghrelin levels (Freda et al., 2003; Katergari et al., 2008; Kozakowski et al., 2005; Wasko et al., 2006) whereas in acromegalic patients receiving

Table 2. Ghrelin levels in patients with acromegaly, Cushing’s disease and hyperprolactinaemia Endocrine disorders

Types of adenomas

Basal ghrelin levels compared to controls

Acromegaly

GH-releasing adenoma

# (Cappiello et al., 2002)

Cushing’s disease

ACTH-releasing adenoma

# (Otto et al., 2004)

Hyperprolactinaemia

Prolactinoma

! (Ciccarelli et al., 1996)

=(Libe et al., 2005) !

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medical therapy with octreotide ghrelin levels are further suppressed, despite the suppression of the elevated levels of GH and IGF-1 (Freda et al., 2003; Kozakowski et al., 2005; Wasko et al., 2006). This could be caused by the direct effect of octreotide on ghrelin-producing gastric cells (Freda et al., 2003). Prolactinomas are the most common type of pituitary tumor and secrete PRL. Lactotroph ade­ nomas have been shown to express both GHS-R and ghrelin mRNA (Korbonits et al., 2001). Cul­ tured PRL-secreting pituitary adenomas have been shown to secrete PRL in vitro following the administration of ghrelin (Rubinfeld et al., 2004). However, this increase in PRL secretion was not seen in patients with prolactinomas receiving GHSs (Table 2; Ciccarelli et al., 1996). Cushing’s disease is mainly caused by an ACTH-secreting corticotroph pituitary adenoma. Both ghrelin and GHS-R are expressed in cortico­ troph pituitary adenomas. It remains controversial as to whether ghrelin levels are altered in this condition as some studies have reported a decreased level of circulating ghrelin in hypercor­ tisolism (Otto et al., 2004), whereas others showed no variation in ghrelin levels in patients with Cushing’s disease (Table 2; Libe et al., 2005). In corticotroph pituitary adenomas, ghrelin was shown to accumulate within the secretory granules of the ACTH-producing tumor cells and

to stimulate an increase in intracellular Ca2þ (Leontiou et al., 2007). From these observations, it is postulated that the expression of ghrelin and GHS-R in corticotroph pituitary adenoma forms a local autocrine loop that leads to secretion of ACTH, although this has not been clearly demon­ strated (Martinez-Fuentes et al., 2006). The expression of ghrelin and the GHS-R in pituitary adenomas may have a role in the mechanism underlying the development and the tumorigenesis of the adenoma cells through auto­ crine and/or paracrine pathways (Kim et al., 2001). We have shown that ghrelin stimulated proliferation of a GH-secreting rat pituitary cell line, GH3, via activation of the mitogen-activated protein kinase (MAPK) pathway (Leontiou et al., 2007). The involvement of GHS-R1a in this con­ text remains unclear as des-acyl ghrelin, which does not bind to the GHS-R1a, was shown to have a similar proliferative effect on the cell line.

Ghrelin and neuroendocrine tumors of the gastro­ entero-pancreatic tract Ghrelin and its receptors are expressed widely in the gastro-entero-pancreatic (GEP) tract. They exert many different functions, as shown in Fig. 2 (El-Salhy, 2009; Leontiou et al., 2007). Neuroen­ docrine tumors are a heterogeneous group of rare

Stimulates gastric acid secretion

Stimulates ileal peristalsis

Stimulates gastric motility

Role of Ghrelin in GEP Tract

Inhibits pancreatic insulin secretion

Inhibits cholecystokinin-induced pancreatic protein secretion

GEP tumorigenesis Fig. 2. Different functions of ghrelin in the gastro-entero-pancreatic (GEP) tract.

199

neoplasms and consist of cells that are a cross between hormone-producing cells and nerve cells. The main primary sites of neuroendocrine tumors are the GEP tract and the lung. Ghrelin is known to stimulate proliferation of neoplastic cells (Nanzer et al., 2004). Ghrelin immunoreactivity was shown in a majority of gas­ tric, pancreatic, intestinal and lung endocrine tumors studied (Papotti et al., 2001; Rindi et al., 2002; Volante et al., 2002). These tumors included gastric carcinoids, insulinomas, gastrinomas, glu­ cagonomas and VIPomas. We have previously demonstrated the expression of ghrelin and GHS-R in a pancreatic insulinoma and gastrinoma (Korbonits et al., 1998). The co-expression of GHS-R in the tumor cells suggests a potential autocrine/paracrine loop in neoplastic conditions. However, the plasma levels of ghrelin in patients with GEP tumors were similar to that observed in the controls, except in one patient who had a pancreatic ghrelinoma (Corbetta et al., 2003). This indicates that hypersecretion of bioactive peptides such as gastrin, insulin, glucagons and VIP seen in neuroendocrine tumors was not asso­ ciated with changes in ghrelin levels. Patients with ghrelinoma have hyperghrelinaemia (Corbetta et al., 2003; Tsolakis et al., 2004). Ghrelin expression has also been observed in some, but not all, GEP tumors occurring in some patients with familial multiple endocrine neoplasia 1 (MEN-1) syndrome (Iwakura et al., 2002; Raffel et al., 2005).

Abbreviations ACC ACTH AgRP AMPK ARC CaMKK CB1 CPT1 FAS GH GHS GHS-R GOAT icv ip IGF-1 malonyl-CoA MBOAT4 NPY POMC PRL PVN UCP2 VMN

acetyl-coenzyme A carboxylase adrenocorticotrophin agouti-related peptide AMP-activated protein kinase arcuate nucleus calmodulin kinase kinase cannabinoid type 1 receptor carnitine palmitoyl transferase 1 fatty acid synthase growth hormone growth hormone secretagogue growth hormone secretagogue receptor ghrelin O-acyltransferase intracerebroventricular intraperitoneal insulin-like growth factor 1 malonyl coenzyme A membrane-bound O-acyltransferase 4 neuropeptide Y pro-opiomelanocortin prolactin paraventricular nuclei uncoupling protein 2 ventromedial nucleus

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ISSN: 0079-6123

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2010 Elsevier B.V. All rights reserved.

CHAPTER 9

Pathogenesis of pituitary tumors Run Yu and Shlomo Melmed
Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA

Abstract: Pituitary tumors are common and mostly benign neoplasia which cause excess or deficiency of pituitary hormones and compressive damage to adjacent organs. Oncogene activation [e.g. PTTG (pituitary tumor-transforming gene) and HMGA2], tumor suppressor gene inactivation (e.g. MEN1 and PRKAR1A), epigenetic changes (e.g. methylation) and humoral factors (e.g. ectopic production of stimulating hormones) are all possible pituitary tumor initiators; the micro-environment of pituitary tumors including steroid milieu, angiogenesis and abnormal cell adhesion further promote tumor growth. Senescence, a cellular defence mechanism against malignant transformation, may explain the benign nature of at least some pituitary tumors. We suggest that future research on pituitary tumor pathogenesis should incorporate systems approaches, and address regulatory mechanisms for pituitary cell proliferation, development of new animal models of pituitary tumor and isolation of functional human pituitary tumor cell lines. Keywords: pituitary adenoma; cell cycle; senescence; pathogenesis

is about 80/100 000 based on a detailed study in England (Fernandez et al., 2009). Clinically and histochemically, pituitary adenomas are classified based on the cell type from which the adenoma originates and the hormones which are secreted by the adenoma (Melmed, 1999). The anterior pituitary comprises several cell types that each secrete specific hormones. Tumors derived from lactotrophs (prolactin-secreting) are termed prolactinoma and cause hypogonadism and galactorrhoea. Somatotropinomas or GHomas are tumors derived from somatotrophs (GH-secreting) and cause gigantism and acromegaly. Corticotropinomas or ACTHomas are tumors derived from corticotrophs (ACTH-secreting) which result in Cushing’s disease. Thyrotropinomas or TSHomas are derived from thyrotrophs (TSH-producing) and cause hyperthyroidism. Gonadotrophs also form tumors but only

Introduction to pituitary adenomas The pituitary gland is composed of two anatomically, functionally and phylogenetically distinct parts: anterior pituitary (adenohypophysis) and posterior pituitary (neurohypophysis). The anterior pituitary is the central regulator of the endocrine system, coordinating signals from the hypothalamus centrally and endocrine organs peripherally. The pituitary gland effectively regulates hormonal secretion of most classical endocrine organs. Tumors frequently arise in the pituitary gland and they constitute up to 15% of intracranial neoplasms (Melmed, 2003). The prevalence of pituitary tumors


Corresponding author. Tel.: (310) 423-4691; Fax: (310) 423-0119; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82009-6

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few of them produce active LH or FSH that results in hypergonadism; the majority of gonadotropinomas do not secrete clinically important hormones and are thus non-functioning. Other non-functioning pitui­ tary tumors do not produce known hormones or hormone sub-units and are thus termed null cell adenoma. Clinically, the most common pituitary tumors are non-functioning and prolactinoma. Acromegaly and especially Cushing’s disease are relatively rare. Thyrotropinoma and functioning gonadotropinoma are extremely rare. Most pituitary tumors are sporadic but some are components of familial tumor syndromes (Brandi et al., 2001; Marx et al., 1999). Except in a few iso­ lated cases, both sporadic and familial pituitary adenomas are benign and slow-growing. The patho­ physiological consequences of a pituitary adenoma are related to over-production of particular pituitary hormones or due to tumor compression and damage to the normal pituitary and vital organs around the pituitary. Pituitary adenomas are the most common cause of both hyperpituitarism and hypopituitarism in adults (Melmed, 1998, 2003). Understanding the pathogenesis of pituitary tumors is critical for pre­ vention, diagnosis, treatment and surveillance of these tumors.

Physiological pituitary cell proliferation Mechanisms underlying pituitary cell proliferation are best illustrated by four models: embryonic development, pregnancy, the estrous cycle and in deficiencies of peripheral hormones induced by the pituitary. Lineage-specific regulation of pituitary development and proliferation is regulated by homeobox genes during embryonic development but is beyond the scope of this section (Drouin 2006; Keegan and Camper, 2003; Scully and Rosen­ feld, 2002). During embryonic development, the nuclear transcription repressor Six6 inhibits in a tissue-specific manner transcription of pituitary p27, a cyclin-dependent kinase inhibitor (CDKI), allowing expansion of an early pituitary progenitor cell population and pituitary development (Li et al., 2002). Mice and humans deficient in Six6 exhibit pituitary hypoplasia (Gallardo et al., 1999; Li et al., 2002). Isolated deletion of key cell cycle regulator

cyclin-dependent kinase 4 or securin [pituitary tumor-transforming gene (PTTG)] does not affect embryonic pituitary development but both result in postnatal pituitary hypoplasia (Chesnokova et al., 2007). Hormones such as estrogen and melatonin stimulate embryonic pituitary cell proliferation in animal models (Danilova et al., 2004; Wu et al., 2009). Lactotroph hyperplasia occurs during estrous and during pregnancy, which is mostly due to physiologically elevated estrogen levels (BenJonathan and Liu, 1992). Relatively little is known on the mechanisms of estrogen-dependent pituitary hyperplasia; PTTG and pro-angiogenic factors basic fibroblast growth factor (bFGF) and vascu­ lar-epithelial growth factor (VEGF) are up-regu­ lated by estrogen, which may explain the estrogeninduced lactotroph proliferation (Heaney et al., 1999). Finally, primary deficiency of hormones secreted by peripheral endocrine organs (thyroid, gonad and adrenal) results in hyperplasia of the cognate trophic pituitary cells due to removal of tonic inhibition by these hormones directly on pitui­ tary and indirectly on the hypothalamus (Al-Gah­ tany et al., 2003). For example, in primary hypothyrodism, abnormally low thyroxine levels release direct inhibition by thyroxine on thyro­ trophs and also increase TRH, both stimulating thyrotroph proliferation and resulting in pituitary thyrotroph hyperplasia (Alkhani et al., 1999). Hypothalamic trophic hormones and inhibitory hormones maintain an appropriate pituitary cell mass under most physiological conditions. Regulation of physiological pituitary cell prolif­ eration illustrates that intrinsic pituitary factors and humoral factors are both important for pitui­ tary cell proliferation. Under physiological condi­ tions, humoral factors are more important regulators of pituitary cell proliferation, but intrin­ sic pituitary factors are required to enable humoral factors to exert their functions.

General principles of pituitary tumor pathogenesis The pathogenesis of pituitary tumors appears to be similar to that of other benign tumors. Tumorigenesis generally encompasses two steps (which can be

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Oncogene activation TSG inactivation Aneuploidy

Normal pituitary

Additional mutations Angiogenesis Cell adhesion

Microscopic adenoma

Additional mutations Unknown changes

Gross adenoma Senescence

Aggressive/ malignant Senescence

Hyperplasia Humoral factors

Fig. 1. Overview of pituitary tumor pathogenesis. Four stages of pituitary tumor pathogenesis are shown. Microscopic adenoma refers to small adenoma that can only be identified by histological examination. Gross adenoma refers to pituitary tumor that is visible on MRI. Oncogene activation, tumor suppression gene (TSG) inactivation and aneuploidy probably contribute to transformation from normal pituitary to microscopic adenoma. Humoral factors can cause pituitary hyperplasia but it is not clear if they also cause transformation from hyperplasia to microscopic adenoma (dashed line). Additional mutations and tumor micro-environment allow the progression to gross adenoma while further mutations may facilitate malignant transformation, which requires escape from cell senescence. See text for details.

somewhat overlapping), initiation and promotion (Nery, 1976; Potter, 1980). At the initiation step, a cell acquires genetic features that endow it with a higher proliferative potential. At the promotion step, additional genetic abnormalities, growth fac­ tors, pro-angiogenesis factors, and tumor micro­ environment together promote the further growth. Tumor initiation may be more specific for a given type of tumor while tumor promotion is rather non­ specific and common to most tumors. Etiologically, pathogenesis of pituitary adenomas can be either due to intrinsic anterior pituitary cellular defects (in most pituitary tumors) or due to humoral factors originat­ ing outside the pituitary (in a small number of pituitary tumors) (Fig. 1). The importance of pituitary tumor micro-environment is also being appreciated.

Monoclonality of pituitary tumors Pituitary tumors are true tumors rather than hyperplastic pituitary cells. The monoclonal origin of pituitary tumors is supported by X-chromo­ some inactivation pattern in tumors of female patients (Alexander et al., 1990; Herman et al., 1990). Further experiments helped refine the monoclonal theory by demonstrating that some

pituitary tumors may contain several tumor clones arising independently from expansion of indivi­ dual cells (Clayton et al., 2000). The monoclonal nature of pituitary tumors strongly suggests that they are derived from single pituitary cells that possess unique and unchecked proliferative advantage caused by intrinsic defects. Although the nature of most of the intrinsic defects is cur­ rently not known, they could be in the forms of oncogene activation or tumor suppressor gene inactivation caused possibly by external forces such as radiation (Hassounah et al., 2005), while in most cases, these defects are presumably inher­ ited or spontaneous.

Oncogene activation Oncogene activation probably is responsible for the pathogenesis of most sporadic pituitary tumors. Proto-oncogenes usually encode proteins in these classes: protein tyrosine kinases (e.g. src), protein serine/threonine kinases (e.g. raf), tran­ scriptional factors (e.g. c-myc), small G proteins (e.g. ras), cell cycle regulators (e.g. cyclin D and PTTG) and others. Unlike the few classical viral oncogenes, most oncogenes reported or suggested in the literature so far have not fulfilled the gold

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standard for an oncogene, that is, inducing specific tumors in a certain cell type. Activating mutations of oncogenes can be either inherited or somatic and they increase the activity of mutated oncopro­ teins. Over-expression of wild-type oncogenes usually results from chromosomal re-arrangement or gene duplication and abnor­ mally enhances oncoprotein functionality by mass effects. Classical oncogenes are not commonly found in pituitary tumors and are not considered to contri­ bute significantly to pathogenesis of most pituitary tumors. Activating ras oncogene mutations are found in a very invasive prolactinoma and in meta­ static pituitary carcinomas, but not in most pituitary tumors (Boggild et al., 1994; Herman et al., 1993; Karga et al., 1992; Pei et al., 1994). Expression of c­ myc is increased in some pituitary tumors but c-myc expression levels do not correlate with tumor cell proliferation rates, suggesting that c-myc expression alone is not responsible for unchecked cell prolif­ eration (Boggild et al., 1994; Woloschak et al., 1994). c-fos expression levels are abnormally high in a small number of sporadic pituitary tumors (Hoi Sang et al., 1988; Woloschak et al., 1994). How exactly these classical oncogenes are activated in pituitary adenomas is largely unanswered. Activa­ tion of oncogenes CCND1 (over-expressed cyclin D1), ptd-FGFR4 (truncated isoform of fibroblast growth factor) and PTTG (over-expressed securin) correlate with pathogenesis of most types of pitui­ tary adenomas. Oncogene gsp (mutant G protein a sub-unit) is mostly associated specifically with soma­ totropinoma pathogenesis and will be discussed later below. CCND1 encodes cyclin D1 and is a putative oncogene for pituitary tumors. Cyclin D isoforms (including D1, D2 and D3) promote cell cycle transition from G0 to S phase and they are the first group of cyclins to be up-regulated when quiescent cells (G0) start to proliferate (Hunter and Pines, 1994; Motokura and Arnold, 1993; Sherr, 1995). Cyclin Ds stimulate cyclin D-depen­ dent kinases CDK4 and CDK6 which phosphor­ ylate the Rb tumor suppressor gene product. Hyperphosphorylated Rb no longer binds to S phase transcription factor E2F and releases it to allow cell cycle progression into S phase

(Harbour and Dean, 2000; Nevins et al., 1997). CCND1 is located on chromosome 11q13 which is often re-arranged in pituitary tumors (Metzger et al., 1999; Sherr, 1995). A common CCND1 polymorphism was examined in sporadic human pituitary tumors (Hibberts et al., 1999). Of 60 tumors studied, 15 harboured allelic imbalance at the CCND1 locus, suggesting CCND1 gene duplication. As expected for an oncogene, the allelic imbalances were more commonly found in invasive or non-functioning tumors than in non-invasive tumors or somatostatinomas. CCND1 allelic imbalance or chromosome 11q13 re-arrangements and cyclin D1 over-expression do not necessarily co-exist in the same tumors. Cyclin D1 over-expression therefore is unlikely caused by gene rearrangement and CCND1 may not play a primary role in pituitary tumorigenesis (Metzger et al., 1999). Furthermore, cyclin D1 staining and tumor grade did not correlate in the study of 60 pituitary tumors. ptd-FGFR4 [Pituitary tumor-derived, N-termin­ ally truncated isoform of fibroblast growth factor (FGF) receptor-4] is the result of abnormal transcrip­ tion initiation of the FGFR4 gene (Ezzat et al., 2002). The novel FGFR4 isoform is expressed in 40% of pituitary tumors examined but not in normal pitui­ tary tissue. ptd-FGFR4 expression in pituitary tumors correlates well with tumor invasiveness and ptd­ FGFR4 expression transforms NIH 3T3 cells in vitro thus may function as an oncogene. Further evidence is derived from transgenic mice expressing the FGFR4 isoform under the prolactin promoter with development of lactotroph pituitary tumors. Mechanisms for ptd-FGFR4-induced pituitary tumorigenesis appear to be disruption of cell adhe­ sion (Ezzat et al., 2004). Expression of N-cadherin and b-catenin, two important cell adhesion mole­ cules, is diminished in cells expressing ptd-FGFR4. The importance of cell adhesion in pituitary tumor pathogenesis id further discussed below.

Pituitary tumor-transforming gene PTTG was originally isolated by mRNA differen­ tial PCR display from rat pituitary tumor GH3 cells, using normal rat pituitary cells as controls

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(Pei and Melmed, 1997). Subsequently, human and mouse homologues were cloned as well (Wang and Melmed, 2000; Zhang et al., 1999a). The rat, mouse and human PTTGs all transform NIH 3T3 cells as shown by soft agar assay in vitro and tumor formation in nude mice in vivo (Pei and Melmed, 1997; Wang and Melmed, 2000; Zhang et al., 1999a). Human PTTG is expressed in pituitary tumors but not in the normal pituitary. PTTG therefore meets the criteria of a pituitary oncogene. Studies on PTTG function illustrate several important features of pituitary tumorigenesis.

PTTG and the protein it encodes PTTG is located on chromosome 5q33-35 and contains five exons. Thus far, mutations in PTTG have not been found either in the coding region or in the promoter (Kanakis et al., 2003; Zhang et al., 1999b). PTTG expression is cell cycle-dependent and peaks at G2/M in tumor cells (Yu et al., 2000a). PTTG protein (also called securin) is composed of 202 amino acid residues and has a predicted molecular weight of 22 kDa. Serine 165 within the SH3-binding domain is a consensus site for kinases cdc2 (Ramos-Morales et al., 2000) and MAPK (Pei, 2000) but the function of serine 165 is controver­ sial (Boelaert et al., 2004). It is not required for the securin function of PTTG demonstrated by live-cell imaging or for FGF-2 transactivation but may suppress transformation capacity. PTTG has diverse physiological functions in cell cycle reg­ ulation, DNA damage repair and gene transcrip­ tion (Vlotides et al., 2007).

PTTG expression in pituitary tumors Multiple studies have demonstrated that PTTG is expressed at high levels in all pituitary tumors but not in the normal pituitary gland (Hunter et al., 2003; McCabe et al., 2002; McCabe et al., 2003; Saez et al., 1999; Zhang et al., 1999b). In one early study (Zhang et al., 1999b), PTTG was over-expressed in most pituitary tumors (23/30 of

non-functioning, 13/13 growth hormone-secreting, 9/10 prolactinoma and 1/1 ACTH-secreting), and PTTG expression levels correlate closely with invasive tumor behavior.

Pituitary tumorigenesis by PTTG over-expression The role of PTTG in pituitary tumorigenesis has been directly demonstrated in transgenic mice that express human PTTG targeted to the mouse pitui­ tary with the promoter of a sub-unit of glycopro­ tein hormones (Abbud et al., 2005). Pituitary size increases in male transgenic animals and serum LH, testosterone, GH and/or IGF-I levels are ele­ vated. Gonadotroph and thyrotroph, as well as somatotroph focal hyperplasia and adenoma are observed.

Mechanisms for PTTG tumorigenesis The precise mechanisms for PTTG tumorigen­ esis are not clear but several possible PTTGregulated pathways have been implicated. Most human solid tumors are aneuploid (Sandberg et al., 1988; Yunis, 1983) and pituitary tumors are no exceptions (Daniely et al., 1998; Harada et al., 1989; Hui et al., 1999; Joensuu and Klemi 1988; Ludecke et al., 1985). PTTG is a mamma­ lian securin which regulates metaphase to ana­ phase transition during mitosis by inhibiting an enzyme (separase) from cleaving the bonding between sister chromatids (Zou et al., 1999). PTTG over-expression inhibits sister chromatid separation and directly causes aneuploidy as observed by live cell imaging (Yu et al., 2003). PTTG binds to Ku-70 of the Ku-70/Ku-80 com­ plex as part of a regulatory sub-unit of DNAdependent protein kinase (DNA-PK); the cata­ lytic sub-unit DNA-PKcs participates in doublestrand DNA break repair (Bernal et al., 2002). PTTG may disrupt DNA repair by binding to Ku-70. PTTG also has a complex relationship with the tumor suppressor p53. On the one hand, PTTG binds to p53 and inhibits DNA binding, transactivation and apoptosis mediated by p53 (Bernal et al., 2002). On the other hand,

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PTTG also induces p53-dependent and p53­ independent apoptosis (Yu et al., 2000b). Thus the exact effect of PTTG on p53 may be context-dependent. Other possible mechanisms for PTTG tumuorigenesis stem from the PTTG transactivating function. PTTG up-regulates expression levels of bFGF, an angiogenic factor, in the pituitary and in NIH 3T3 cells (Heaney et al., 1999; Zhang et al., 1999b). PTTG also transactivates c-myc, Cyclin D3, matrix metallo­ proteinase 2 gene (MMP2), and inhibits p21 expression in CHO cells (Chesnokova et al., 2007; Malik and Kakar, 2006; Pei, 2001; Tong et al., 2007).

Low PTTG levels prevent tumorigenesis PTTG appears to be required for pituitary tumuorigenesis (Fig. 2). In mice, PTTG deletion results in pituitary hypoplasia with low cell prolif­ eration rates, and protection against pituitary tumor development induced by heterozygous Rb deletion (Chesnokova et al., 2007). In the

PTTG/ pituitary, the ARF/p53/p21-dependent senescence pathway is activated, as also evidenced by increased activity of senescence-associated b-galactosidase (SA-b-gal) activity. The PTTG role in pituitary tumorigenesis suggests that PTTG may be a potential therapeutic target for pituitary tumors.

Senescence in pituitary tumor pathogenesis Senescence refers to premature irreversible cell proliferation arrest (Arzt et al., 2009; Zhang, 2007). Senescent cells maintain cellular function and viability, but are devoid of proliferative potential. Premature senescence is not related to telomere shortening; rather, it is a response to cellular stresses such as oncogene overexpression, oxidative stress, DNA damage and withdrawal of key nutrients or growth factors. Premature senescence is a protective mechanism against tumorigenesis but decreases normal cel­ lular functions by virtue of leaving a smaller mass of residual cells with differentiated

Pituitary trophic status

Tumor potential

+Hyperplasia

αGSU.PTTG Rb+/– Hyperplasia

αGSU.PTTG Normal

WT Hypoplasia

Pttg–/– Rb+/– +Hypoplasia

Pttg–/–

Fig. 2. Pituitary PTTG content correlates with gland plasticity and tumor formation potential. On the left side of the inverted triangle are listed mouse models with descending pituitary PTTG content, with or without the combination with tumorigenic Rbþ/. Horizontal bars represent observed effects of the different genotypes on pituitary trophic status, which correlates with pituitary tumorigenic potential (arrow). (From Fig. 10 in Vlotides et al. (2007), with permission.)

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functions. Mechanisms leading to senescence mainly converge to the p53/p21 and p16/pRb pathways. p53 is activated by oncogene overexpression, DNA damage or oxidative stress, and increases p21 expression, thus conferring cell cycle arrest. p16 levels are also up-regulated by similar stresses and p16 inhibits cyclin-depen­ dent kinases and prevents pRB phosphorylation. Senescence may thus function as a natural surveillance protecting against tumorigenesis. Furthermore, in benign tumors such as pituitary tumors, senescence may prevent malignant transformation. Cellular responses to PTTG over-expression exemplify an oncogene-induced senescence. Tran­ sient PTTG over-expression results in p53-depen­ dent and p53-independent apoptosis in all cell types examined (Yu et al., 2000a, 2000b). Prior to apoptosis, cell cycle arrest develops in cultured cells over-expressing PTTG, suggestive of senes­ cence (Yu et al., 2000b). Senescence has recently been directly demonstrated in somatotropinomas (Chesnokova et al., 2008) (Fig. 3). p21, a CDKI, is an important mediator of cell senescence. Unlike in CHO cells, pituitary PTTG expression

stimulates p21 expression and PTTG and p21 levels correlate. In 38 such tumors, 29 exhibited strong and 9 weak p21 immunoreactivity. p21 over-expression is limited to benign somatotropi­ nomas and p21 was not detected in four GH-pro­ ducing pituitary carcinomas, 21 non-secreting pituitary oncocytomas, 7 null cell adenomas, 5/8 ACTH-producing tumors or 3/5 gonadotropinproducing tumors. b-galactosidase activity and protein levels, markers of senescence, are stronger in benign somatotropinomas than in normal pitui­ tary tissue. SA-b-gal activity and p21 levels also correlate strongly in these tumors. Thus at least in somatotropinomas, PTTG mediates senescence, which may explain the benign nature of most of these tumors. It is interesting that both PTTG under-expression and over-expression are asso­ ciated with senescence in pituitary cells which pre­ vents tumorigenesis in the former case, and inhibits malignant transformation in the latter. Mechanisms for these observations are not clear but may be due to dysregulated cell cycle control and DNA damage caused by disequilibrium of intracellular PTTG levels (Chesnokova et al., 2007, 2008).

p21 A

SA-β-Gal

B

T

T

C

D

T

T

E

F

T

Normal

Normal

Tumor T

T

Carcinoma

Fig. 3. Senescence in human somatotropinomas. (Left) (A–E) Immunohistochemistry of sections derived form 5 somatotropinomas stained for p21. Normal pituitary and a breast carcinoma are also shown for comparison. Note the clear p21 staining in somatotropinomas but not in normal pituitary or in breast carcinoma. (Right) Immunohistochemistry of the normal pituitary and somatotropinoma sections stained for p21 and SA-b-gal activity. Note the correlation between p21 staining and SA-b-gal activity. (From Figs. 5 and 6 in Chesnokova et al. (2008), with permission.)

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Tumor suppression gene inactivation Familial pituitary tumor syndromes and transgenic pituitary tumor models both indicate that tumor suppression gene inactivation is responsible for specific tumor formation in the pituitary. Tumor suppressor genes are defined as the genes whose homozygous deletion or inactivation causes tumorigenesis (Alexander, 2001). The classical ‘two-hit’ theory of tumorigenesis points out that an individual may inherit a heterozygous inactivat­ ing mutation of a tumor suppressor gene; while the normal allele of the tumor suppressor gene still functions to suppress tumorigenesis, tumori­ genesis will occur later in life after loss of the normal allele (Alexander, 2001). Familial tumor syndromes manifesting several pituitary tumor types are discussed here and those which present with specific pituitary tumor types are discussed under ‘Pathogenesis of specific types of pituitary tumors’. Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant genetic disease, encom­ passing tumors mainly in three organs: para­ thyoid adenoma, pancreatic endocrine tumors and pituitary adenomas, and other tumors that do not produce classical hormones (Brandi et al., 2001; Marx et al., 1998). By age 40, pituitary tumors develop in 40% of patients with MEN1; approximately half of these tumors are prolacti­ nomas, the remainder being somatotropinomas, mixed prolactinoma–somatotropinomas, nonfunctioning tumors, corticotropinomas and thyr­ otropinomas. Tumors encountered in other organs include foregut carcinoids, adrenal corti­ cal adenomas and meningiomas, and they exhibit various penetrance rates. MEN1 results from inactivating mutations of the tumor suppressor gene MEN1 (Agarwal et al., 2005; Marx et al., 1999). MEN1 is localized on chromosome 11q13 with 10 exons and encodes the protein menin. Mostly a nuclear protein, menin interacts with nuclear and cytoplasmic partners, including pro­ teins that regulate gene transcription, DNA pro­ cessing and repair, cytoskeletal organization, GTPase activity and protein degradation. Hun­ dreds of inactivating MEN1 mutations have been identified throughout the gene but there does

not seem to be a clear genotype–phenotype cor­ relation. Although MEN1 mutations are cer­ tainly responsible for most patients with MEN1, it is not clear why MEN1 mutations result in tumors, and it is even more an enigma why tumors only arise in selected organs while menin is universally expressed in all tissues. Homozygous Men1 deletion in mice is embryo­ nically lethal while heterozygous Men1 deletion causes tumors in endocrine pancreas, parathyr­ oid, thyroid, adrenal cortex and pituitary (Crab­ tree et al., 2001). About 1/5 of patients with clinical diagnosis of MEN1 (any two tumors arising from parathyroid, endocrine pancreas and pituitary) do not have detectable MEN1 mutations. Mutations of other genes thus may also cause MEN1. In rare cases, the gene for CDKI p27, CDKN1B/p27Kip1, is a tumor suppressor gene in patients with clinical MEN1 but without MEN1 mutations (Georgitsi et al., 2007; Pellegata et al., 2006). Homozygous deletion of Cdkn1b (the gene for p27 in rats) is associated with a novel MEN-like syndrome MENX, an autosomal recessive syndrome with overlapping features of human MEN1 and MEN2. These animals develop pheochromocyto­ mas or paragangliomas, parathyroid adenomas, pituitary tumors and thyroid neoplasia. Homozy­ gous deletion of the p27 gene in mice results in intermediate lobe pituitary tumors without other stigmata of MEN1. p27 deletions have not been found in sporadic human pituitary tumors, except for those in the two families described above (Yu et al., 2006). Other transgenic mouse models also demon­ strate that tumor suppressor gene inactivation causes pituitary adenoma. Heterozygous deletion of the classical tumor suppressor gene Rb results in intermediate- and anterior-lobe pituitary tumors (Chesnokova et al., 2008; Jacks et al., 1992). In agreement with the ‘two-hit’ theory, these tumors exhibit no Rb expression, suggesting post-zygotic inactivation of the normal allele before tumorigen­ esis. In human pituitary tumors, Rb deletion or mutations are not common (Cryns et al., 1993; Pei et al., 1995; Simpson et al., 1999), and Rb protein is undetectable in only <5% of non-functioning pituitary tumors. In contrast, in 6–20% of

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somatotropinomas, Rb protein is undetectable (Donangelo et al., 2005; Simpson et al., 1999), sug­ gesting possible epigenetic inactivation of Rb in some somatotropinomas. Mouse models also sug­ gest that CKIs prevent pituitary tumorigenesis. Homozygous deletion of CKI p18 causes inter­ mediate lobe pituitary tumors (Franklin et al., 1998); and p21 deletion enhances tumor growth in Rb-deficient mice (Chesnokova et al., 2008). In some human pituitary tumors, p18 expression is lower than in normal pituitary (Morris et al., 2005); mutations in the p21 or p18 genes, however, have not been identified (Yu et al., 2006).

Epigenetic pathogenesis of pituitary tumors Epigenetic modification of genes involved in reg­ ulating cell proliferation is important in pituitary tumorigenesis. CDKN2A encodes CDKI p16 whose function is to inhibit cyclin D1-dependent kinase CDK4 from interacting with cyclin D1 and phosphorylating Rb. Rb hyperphosphorylation would lead to cell cycle progression from G1 to S. Although mutations or loss of CDKN2A have not been identified in pituitary tumors, hypermethylation of CDKN2A exon 1 is found in nonfunctioning pituitary tumors (Simpson et al., 1999). In these tumors, p16 expression levels assessed by immunocytochemistry are low and p16 is localized abnormally to the cytoplasm rather than the nucleus. Using cDNA-representational difference analy­ sis, GADD45� (growth arrest and DNA damageinducible gene 45g) and MEG3 (maternal imprinting gene 3) were identified as potential tumor suppressor genes protecting the normal pituitary from developing tumors. The two genes are expressed in the normal pituitary but not in non-functioning pituitary tumors (Zhang et al., 2002, Zhang et al., 2003). GADD45� is a p53 target gene and encodes a protein that regulates cell proliferation and apoptosis. It is also expressed at low levels in functioning tumors such as in somatotropinomas and prolactinomas. Forced GADD45� expression in rat pituitary tumor cell lines significantly decreases cell proliferation rates. MEG3 encodes an mRNA

that is not translated, and is expressed in gonado­ trophs but not in non-functioning pituitary tumors which are mostly derived from gonadotrophs. Interestingly, suppressed expression of both GADD45� and MEG3 appear to be mediated by DNA hypermethylation (Gejman et al., 2008; Ying et al., 2005). Epigenetic loss of expression of the three genes is thus demonstrated by DNA hypermethylation in pituitary tumors. These genes do not fulfill the criteria of tumor suppressor genes as patients har­ bour both normal alleles in normal tissues, while both alleles are inactivated by hypermethylation in pituitary tumors. Whether or not hypermethylation is caused by environmental car­ cinogens or radiation exposure or by a sponta­ neous loss of an unidentified classical tumor suppressor gene, or by activation of an unknown oncogene, remains to be elucidated.

Humoral factors Rarely, excess trophic hypothalamic hormones elaborated by neuroendocrine tumors or hypotha­ lamic hamartomas lead to hyperplasia and hormo­ nal hypersecretion of a particular pituitary cell type. Unambiguous pituitary tumors are not com­ mon in this situation. GH-releasing hormone (GHRH), CRH and GnRH excess by these tumors produce acromegaly, Cushing’s syndrome and sexual precocity or hypergonadism, respec­ tively (Tasdemiroglu and Kaya, 2004). Absence of peripheral hormone negative feedback inhibi­ tion due to primary endocrine organ insufficiency or ablation for therapeutic purposes may lead to hyperplasia and adenoma of the particular pitui­ tary cell type. Thyrotroph and corticotroph hyper­ plasia or adenoma (or the growth of an existing corticotroph adenoma) is well known in patients with severe hypothyroidism and bilateral adrena­ lectomy, respectively (Al-Gahtany et al., 2003). In some animal models, excess exogenous estrogen results in prolactinoma (Heaney et al., 1999). Once the humoral factor excess is corrected, pitui­ tary hyperplasia and adenoma are often reversed, strongly supporting that the humoral factor excess is causative for the hyperplasia and adenoma.

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Tumor micro-environment Tumor angiogenesis, tumor cell interaction and tumor–stroma interaction are important for understanding pituitary tumor pathogenesis. These tumor micro-environment parameters probably are not critical in tumor initiation but are certainly important for tumor promotion and tumor invasiveness. The role of angiogenesis in pituitary tumor pathogenesis is complex. Pituitary tumors require angiogenesis for nutrient supply. Most pituitary tumors, however, are less vascularized than the normal pituitary, which may explain their benign nature. This microvascular density as a measure­ ment of angiogenesis is nonetheless positively cor­ related with tumor size and aggressive behaviors (Turner et al., 2000b; Vidal et al., 2001). PTTG appears to promote angiogenesis, FGF-2 and VEGF-A expression in an estrogen-induced pitui­ tary tumor model (Heaney et al., 1999), and con­ ditioned media from PTTG-over-expressing NIH3T3 cells stimulate proliferation, migration and tube formation of human umbilical vein endothelial cells (Ishikawa et al., 2001). Angio­ genesis may be a therapeutic target for pituitary tumors. In a mouse model of pituitary tumor with a heterozygous deletion of the Men1, treatment with an anti-VEGF-A monoclonal antibody inhi­ bits pituitary adenoma growth, lowers prolactin levels and increases mean tumor doubling-free survival (Korsisaari et al., 2008). Cell–cell adhesion is an important regulator of tumor growth. Expression of cell adhesion mole­ cules H-cadherin and E-cadherin is reduced in all major types of pituitary tumors, compared to nor­ mal pituitary (Qian et al., 2007). Furthermore, expression levels of E-cadherin are much lower in high-grade tumors than in low-grade ones, sug­ gesting that loss of adhesion molecules is asso­ ciated with tumor aggressiveness. Lower expression of these two cadherin molecules is caused by tumor-specific methylation of their gene promoters. Interestingly, loss of membra­ nous E-cadherin is associated with nuclear redis­ tribution in pituitary tumors, suggesting that cleavage of the extracellular domain of E-caherin and nuclear translocation may underlie pituitary

tumor local invasion (Elston et al., 2009). b-cate­ nin, a cadherin-associated protein, regulates cell differentiation and proliferation. b-catenin expres­ sion is reduced in prolactinoma and abnormally accumulated in pituitary tumor nuclei (Qian et al., 2002; Semba et al., 2001). Mechanisms for which reduced plasma membrane expression and simul­ taneous nuclear accumulation of adhesion mole­ cules are associated with tumor aggressive behavior remain unclear. The extracellular matrix is composed of poly­ saccharides and fibrous proteins and acts as a barrier of tumor invasion and angiogenesis. MMPs are endopeptidases that degrade the extra­ cellular matrix. Expression of collagenase MMP-9 is up-regulated in invasive pituitary tumors, carci­ nomas and macroprolactinomas, and is positively correlated with vascular density (Gong et al., 2008; Turner et al., 2000a). Components of the extracellular matrix may also regulate pituitary tumor hormone secretion and cell proliferation. For example, laminin inhibits growth of cortico­ tropinoma and prolactinoma cells in vitro (Kuchenbauer et al., 2001; Kuchenbauer et al., 2003).

Pathogenesis of specific types of pituitary tumors In this section of the chapter, we apply the princi­ ples to the pathogenesis of all the major types of pituitary tumors and describe their specific patho­ genetic mechanisms.

Pathogenesis of somatotropinoma Somatotropinoma causes gigantism or acrome­ galy, a disease of excessive levels of GH (Melmed, 2006). GH is normally produced and secreted in the pituitary somatotrophs. During embryonic growth, transcription factors regulate the somato­ trophs to develop and differentiate into a pheno­ type of synthesizing and secreting GH (Melmed, 2003; Melmed 2006). The transcription factor PROP1 (Prophet of Pit-1) is the major homeobox protein for somatotroph development. PROP1 regulates Pit-1 which then binds to the growth

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hormone gene promoter to directly modulate somatotroph differentiation and proliferation and GH gene transcription. After birth, GH produc­ tion and secretion are under nuanced hypothala­ mic control through stimulatory GH-releasing hormone (GHRH) and ghrelin, and inhibitory somatostatin. Migrating through the hypophyseal portal system to the anterior pituitary and bind their respective somatotroph cell surface recep­ tors, GHRH and ghrelin stimulate GH production and secretion while somatostatin inhibits GH secretion. Potential derangement in any of the above mechanisms would favour excess GH secretion and somatotropinoma formation. Spontaneous somatotropinomas are responsible for acromegaly in most patients. Most somatotropinomas are benign and composed of various types of GHsecreting cells. Densely granulated and sparsely granulated somatotropinomas demonstrate unique morphology but the latter does not exhibit aggressive clinical behavior. Some somatotropino­ mas comprise both somatotroph and lactotroph tumor cells; other somatotropinomas are com­ posed of only one cell type that secrete both GH and prolactin. Other cell types secreting GH and other hormones include acidophil stem cells that secrete both GH and prolactin, and plurihormonal cells that produce GH, prolactin, TSH and a sub­ unit. Sometimes, cells in a somatotropinoma produce GH but do not secrete sufficient GH to induce clinical acromegaly, resulting in clinically silent somatotropinoma. Rarely, invasive pituitary carcinomas secrete GH as well.

Molecular pathogenesis of somatotropinomas in familial tumor syndromes Somatotropinoma is a feature of MEN1 as dis­ cussed earlier. It is more specifically associated with the rare familial disorder Carney complex which is also an autosomal dominant genetic dis­ ease (Boikos and Stratakis, 2007). Clinical presen­ tations of Carney complex include hormonal hypersecretion, lentigines (hyperpigmentation of the skin) and myxomas of the heart. Most (up to 75%) of patients with Carney complex exhibit

elevated levels of GH, IGF-1 or prolactin, but only 10% of patients actually present with clinical acromegaly. Patients with Carney complex initi­ ally develop somatotroph hyperplasia, followed by somatotropinoma formation. Pituitary abnormal­ ities are associated with inactivating mutations of PRKAR1A, the regulatory sub-unit isoform 1A of protein kinase A (PKA) (Kirschner et al., 2000). PRKAR1A is localized on chromosomal 17q22-24, has 11 exons, and encodes a 48 kDa protein comprised of 381 amino acid residues. The most abundant PKA regulatory sub-unit isoform, PRKAR1A is highly expressed in the brain, endo­ crine organs including the pituitary, fat and bone. Without cAMP presence, two PRKAR1A mole­ cules form a heterotetramer with two catalytic sub-units and inhibit the intrinsic activity of the catalytic sub-units. With increased intracellular cAMP levels, cAMP binds to each of two PRKAR1A molecules and causes conformational changes on PRKAR1A, releasing the catalytic sub-units to phosphorylate protein targets. Inacti­ vating PRKAR1A mutations interupt efficient binding to the catalytic sub-units, causing consti­ tutive activation of the catalytic sub-unit and resulting in modulation of the functions of numer­ ous protein targets of this kinase. Heterozygous deletion of functional PRKAR1A does not cause somatotropinomas in mice but a pituitary-specific homozygous deletion of PRKAR1A results in ele­ vated levels of GH, somatotroph hyperplasia and somatotropinomas (Kirschner et al., 2005; Yin et al., 2008). Murine phenotypes of global hetero­ zygous deletion of PRKAR1A are consistent with low penetrance of somatotropinomas in patients with Carney complex. Occasionally, in patients and in animal models, the wild-type PRKAR1A allele is retained in tumor tissue so that decreased expression rather than complete absence of pro­ tein kinase A regulatory sub-unit is sufficient to cause tumorigenesis, a phenomenon termed ‘hap­ loinsufficiency’ (Burton et al., 2006). Furthermore, another chromosomal locus 2p16 is also impli­ cated in a few patients with Carney complex (Kirschner et al., 2000). Isolated familial acromegaly (caused by somato­ tropinomas) is a very rare familial disorder; only about 40 affected families have been described

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(De Menis and Prezant 2002). It appears to be an autosomal dominant genetic disease with various degree of penetrance. Pituitary tumors occur more frequently in younger patients, and about 25% of patients present with gigantism. The genetic causes of isolated familial acromegaly are not clear but loss of heterogeneity on chromosome 11q13 on a locus different from MEN1 has been identified (Gadelha et al., 2000). Presumably, an unidentified tumor suppressor gene at that locus may cause this disorder. Familial pituitary adenoma is another rare but loosely defined familial pituitary syndrome; only a small number of family members over several generations develop somatotropinoma or prolac­ tinoma so that penetrance is rather low (Vieri­ maa et al., 2006). The identification of a putative tumor suppressor gene in patients with this syn­ drome illustrates a systemic approach to unravel the genetic basis of familial syndromes. Wholegenome single-nucleotide polymorphism geno­ typing was carried on family members with or without somatotropinoma or prolactinoma (Vierimaa et al., 2006). Linkage analysis demon­ strated a linkage on chromosome 11q12 to 11q13 which includes the locus for isolated familial acromegaly and MEN1. MEN1 mutations were not found in family members with pituitary tumors, and linkage was further narrowed to a 7 Mb region which harbours 295 genes. Compar­ ing expression patterns of individual genes in affected and unaffected family members revealed that the most significant difference in expression occurred in the AIP gene. AIP, aryl hydrocarbon receptor interacting protein, is also named ARA9, XAP2, or FKBP37 (Melmed, 2007). Heterozygous nonsense germline muta­ tion Q14X was discovered only in affected family members but not in unaffected family members or in the general population. Thus AIP is a puta­ tive tumor suppressor gene. Another germline AIP mutation (R304X) was identified in an Ita­ lian family with acromegaly but AIP mutations were not evident in a German and a Turkish family with familial acromegaly. AIP encodes 6 exons and the AIP protein is composed of 330 amino acid residues. AIP struc­ ture is homologous to that of immunophilin

FKBP52 and contains an N-terminal FKBP-homo­ logous domain and 3 C terminal tetratricopeptide repeats which interact with other proteins (Melmed, 2007; Vahteristo and Karhu, 2007). AIP was identified as a binding partner for the hepatitis B virus X protein, and is one of the three proteins forming an intracellular complex with the aryl hydrocarbon receptor (AHR). AIP functions to maintain stabilization, to facilitate nuclear– cytoplasmic trafficking, and to regulate transacti­ vation of genes by xenobiotics. A mutant AHR that does not bind AIP apparently functions nor­ mally, suggesting that AIP interaction is not required for AHR signalling. Mechanisms for AIP tumor suppressor function are not clear. As ligand-dependent AHR signalling inhibits cell proliferation likely by Rb interaction and p27 induction, it is possible is that AIP loss-of-function mutations may interfere with AHR inhibition of cell proliferation which may be related to tumorigenesis. In 73 families with familial isolated pituitary adenomas (FIPA, defined as  2 members in one family harbouring anterior pituitary tumors but without clinical evidence of MEN1 or Carney complex), 11 (15.1%) harbour various germline mutations in AIP but penetrance of AIP muta­ tions could not be ascertained (Daly et al., 2007). In another study, 4 of 17 family members of a Brazilian family with familial somatotropinoma harbour AIP mutations and 2 of the 4 members exhibit somatotropinomas (Toledo et al., 2007). Cumulatively, a total of 170 patients were included in the 3 studies of familial pituitary ade­ nomas. AIP mutations in germline DNA or in tumor DNA have been found in 32 of 72 patients harbouring somatotropinomas (44%) but only in 4 of 98 patients with other types of familial pituitary adenomas (4%). AIP mutations in familial non­ somatotropinoma cases are about 10-fold as pre­ valent as those observed in sporadic pituitary tumors (see below), suggesting that AIP muta­ tions are more closely associated with familial pituitary adenomas, especially somatotropinomas. In patients with sporadic pituitary tumors, AIP mutations in either germline DNA or in tumor DNA are much rarer and are found in only ~2% and most of these patients harbour

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somatotropinoma (Melmed, 2007; Vahteristo and Karhu, 2007). AIP mutations are extremely rare (only one case, 0.3%) among other pituitary ade­ nomas. The prevalence of AIP mutations is even lower after the Finnish Q14X founder mutation is excluded. The low frequency of AIP mutations indicates that most sporadic pituitary tumors are not related to AIP mutations. The overwhelming evidence from these studies demonstrates that AIP mutations are predominantly associated with familial somatotropinomas. Furthermore, as AIP mutations are associated with the rare famil­ ial somatotropinoma syndrome and obtaining accurate family histories for somatotropinomas is often challenging, it is plausible that those asso­ ciated with AIP mutations may actually be famil­ ial. Thus based on the extremely low rates of AIP mutations in non-somatotropinoma sporadic pitui­ tary adenomas, AIP mutations do not appear to be important genetic factors for most pituitary tumors encountered clinically.

Molecular pathogenesis of somatotropinomas in non-familial somatotropinomas Most non-familial somatotropinomas are truly sporadic but can be also syndromic as in McCune–Albright Syndrome (MCS). Non-famil­ ial somatotropinomas are presumably caused by oncogene activation. MCS is a clinical syndrome affecting the bone, skin and endocrine organs. Classical MCS features include polyostotic fibrous dysplasia, café-au-lait macules and pre­ cocious puberty. Nearly 20% of patients with MCS exhibit biochemical GH excess but only a few patients actually harbor clear somatotropi­ nomas on MRI (Akintoye et al., 2002; Galland et al., 2006). Prolactinoma and corticotropinoma are also present in MCS. MCS is not an inher­ ited disease but is caused by de novo post-zygo­ tic mutations of GNAS, resulting in mosaicism (Diaz et al., 2007). GNAS locates on chromo­ some 20q13 and encodes Gsa, the stimulatory sub-unit of the G protein. Gsa activates adenylyl cyclase which converts ATP to cAMP; cAMP being a classical second messenger that binds regulatory sub-units of PKA to release the

catalytic sub-unit to phosphorylate a series of target proteins. Activating mutations of GNAS results in a overactive Gsa, leading to abnor­ mally increased PKA activity. As discussed ear­ lier, inactivating mutations of the regulatory sub-unit 1A causes Carney complex also by pro­ moting unchecked PKA activity. Thus overac­ tive PKA is common to both Carney complex and MCS. GNAS is ubiquitously expressed in all cells of the body but MCS is only manifest in a few organs; the specificity of MCS organ invol­ vement is probably due to the mosaic nature of GNAS mutations. Gsa activating mutations are also present in sporadic somatotropinomas. GNAS mutations are relatively specific in somatotropiomoas: about 40% of Caucasian patients with this condi­ tion harbor GNAS mutations (Landis et al., 1989; Lyons et al., 1990). GNAS is imprinted in normal pituitary so that only the maternal allele is expressed (Hayward et al., 2001); as a result, acti­ vating mutations of GNAS mostly occur in the maternal allele in somatotropinomas. In a small number of somatotropinomas, the paternal GNAS allele is also expressed and mutations of the pater­ nal allele can also be found. The presence of GNAS mutations is not correlated with clinical features of patients with somatotropinomas (Diaz et al., 2007). Other elements of the cAMP–PKA pathway may also be involved in somaotropinoma pathogenesis. cAMP response element (CRE)­ binding protein (CREB), a transcription factor, is a direct target of PKA. Phosphorylated CREB binds to CRE in the promoter region of genes encoding multiple proteins regulating cell prolif­ eration. Phosphorylated CREB is detected in all 15 somatotropinomas studied, regardless of GNAS mutations (Bertherat et al., 1995), suggest­ ing CREB activation as a final common pathway in somatotropinoma pathogenesis. Although abnormal PKA activation is a common mechanism for pathogenesis of somatotropinomas in Carney complex, MCS and sporadic acromegaly, it is not clear as to how overactive PKA leads to somatotropinoma pathogenesis (Fig. 4). PKA reg­ ulates multiple cellular processes both directly and indirectly (Spaulding, 1993). One possibility is that up-regulated cAMP–PKA–CREB pathways cause

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Ectopic GHRH

*

GHRH RIα

PRKAR1A Carney

RII

GHRH-R

α

Protein kinase A

*

C

RIα

RII

C

C

β

α cAMP

Isolated

C p-CREB

γ

*

GNAS

Adenylyl cyclase

*

Cell growth

GH

Fig. 4. GHRH signalling and GHoma tumorigenesis. The pituitary somatotroph cells harbour cell surface GHRH receptors coupled to stimulatory G protein (Gs) with a and bg sub-units. Upon GHRH binding, Gsa dissociates from bg and stimulates adenylyl cyclase which catalyses ATP to form cAMP. cAMP binds the regulatory (R) sub-units of protein kinase A, releasing the catalytic (C) sub­ unit, which phosphorylates cAMP response element (CRE)-binding protein (CREB). Phosphorylated CREB stimulates transcription of genes that may lead to cell proliferation. Not shown is the activation of ERKs by the PKA pathway. Ectopic GHRH, activating mutations of Gsa, inactivating mutations of PKA regulatory sub-unit and abnormal CREB phosphorylation all activate the GHRH signalling pathway. See text for details. (From Fig. 3 in Yu, R. & Melmed, R. (2009) Current concept in molecular pathogenesis of acromegaly. In J. A. H. Wass (Ed.), Acromegaly (pp. 19–40). Bristol, UK: BioScientifica, with permission.)

uncontrolled cell proliferation. Another PKA tar­ get is Rap1, a member of the Ras small G proteins (Wang et al., 2006). Rap1 activation leads to activa­ tion of extracellular signal regulated kinases (ERKs), intracellular mediators of cell proliferation for numerous growth factors. PKA-independent Rap1 activation directly by cAMP potentially could be associated with tumorigenesis in MCS and sporadic acromegaly but Rap1 activation alone may not be essential for tumorigenesis in Carney complex in which PKA is indeed activated due to inactive regulatory sub-unit, bypassing cAMP. Presently it is not possible to test these hypotheses in human somatotrophs as there are no functional human pituitary cell lines. In the GC rat somatotrophinoma cell lines, mutant Gsa or over-expressed wild-type Gsa increases PKAphosphorylated CREB levels (Bertherat et al., 1995), but it is not clear whether or how CREB phosphorylation by PKA promotes cell prolifera­ tion or increases GH secretion in somatotrophs.

On the other hand, dominant negative CREB decreases proliferation of lactotrophs (Ishida et al., 2007). In mouse NIH 3T3 fibroblasts, a mutant Gsa increases basal adenylyl cyclase activity and cAMP levels but does not modulate cell proliferation or transformation (Zachary et al., 1990). Rather, mutant Gsa augments pro­ liferative effects of forskolin (an activator of adenylyl cyclase) and a phosphodiesterase inhibi­ tor (which inhibits cAMP degradation). Thus GNAS mutation in itself may not be sufficient to initiate somatotropinoma tumorigenesis but can potentiate proliferative effects caused by other factors. It is intriguing that inappropriate PKA activation is specifically related to somatotropi­ noma tumorigenesis but not other pituitary tumors and this phenomenon has not been clearly explained. PKA overactivity may resemble an exaggerated signalling pathway of the GHRH receptor (coupled to Gs) that physiologically induces somatotroph proliferation.

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Humoral factors Ectopic GHRH secreted by neuroendocrine tumors lead to GH hypersecretion and somatotro­ pinoma (Melmed et al., 1988). Somatotropinomas are not encountered in patients with GH resis­ tance (Kornreich et al., 2003).

Tumor micro-environment Pituitary GH secretion is also regulated by cell adhesion molecules N-cadherin and neural cell adhesion molecule (N-CAM) (Rubinek et al., 2003). N-cadherin mRNA is selectively expressed in somatotropinomas. In vitro, CAM stimulation increased GH secretion from cultured GH ade­ noma cells.

Somatotropinoma senescence Oncogene-induced cell senescence is identified in somatotropinomas and may explain why these tumors are invariably benign. Aneuploidy and DNA damage due to PTTG over-expression likely activate senescence pathways by stimulating p21 (Chesnokova et al., 2008).

prolactinoma pathogenesis as in non-functioning pituitary tumors, it is also over-expressed without significant HMGA2 locus duplication (Pierantoni et al., 2005). HMGA2 tumorigenesis mechanisms are not clear but may include regulation of cyclin B (De Martino et al., 2009). The most important humoral factor for prolactinoma pathogenesis is estrogen, which plays a permissive role. Dopa­ mine agonists are the most important therapeutic agents for prolactinoma, mimicking endogenous inhibition of lactotrophs by dopamine. Epidermal growth factor (EGF) stimulates prolactin tran­ scription and prolactinoma cell proliferation and antagonists to EGF receptor inhibit prolactin release and prolactinoma growth (Vlotides et al., 2008). Other sub-types of the ErbB family (one sub-type is the EGF receptor), p185her2/neu and ErbB3, are expressed in prolactinomas, especially aggressive ones. Heregulin, the ErbB3 ligand, specifically induces prolactin expression and tyr­ osine phosphorylation and ErbB3 and p185c-neu heterodimerization (Vlotides et al., 2009). The tyrosine kinase inhibitor gefitinib inhibits heregu­ lin signalling, suggesting p185her2/neu and ErbB3 as targets for treating aggressive prolactinoma. Cell adhesion molecules E-cadherin and b-cate­ nin expression is inversely correlated to aggres­ sive behaviors such as invasiveness and high tumor cell proliferation rates (Qian et al., 2002)

Pathogenesis of prolactinoma Lactotrophs are under tonic inhibition of hypothalamic dopamine and are stimulated by estrogen. Prolactinoma is the second common pituitary tumor but specific pathogenesis of pro­ lactinoma is not well characterized. Pituitary oncogene activation (CCND1, PTTG, etc.) and tumor suppressor gene inactivation (MEN1) as discussed above are both present in prolactinoma. General over-expression of a putative oncogene HMGA2 (high mobility group A2), a non-histone chromatin-associated protein, results in prolactinand GH-secreting pituitary tumors in transgenic mice (Fedele et al., 2002). The HMGA2 locus on chromosome 12q14-15 is amplified and HMGA2 over-expressed in human prolactinomas (Finelli et al., 2002). HMGA2 is not specific for

Pathogenesis of corticotrophinoma (ACTHoma) Corticotrophs are stimulated by hypothalamic CRH and inhibited by cortisol. As these tumors are rare, very little is known of their specific pathogenesis. It is assumed that the principles of pituitary tumorigenesis also apply for corticotro­ pinoma. Peroxisome proliferator-activated recep­ tor-g is expressed exclusively in corticotrophs and may be important for ACTH secretion and corti­ cotroph survival (Heaney et al., 2002). Ectopic secretion of CRH by neuroendocrine tumors occa­ sionally causes corticotropinoma (Ilias et al., 2005). Bilateral adrenalectomy without sufficient cortisol replacement may induce microscopic corticotropinoma progressing into gross tumors.

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Pathogenesis of thyrotropinoma (TSHoma) Thyrotropinomas are extremely rare and tend to large and invasive. Besides TSH, these tumors may also secrete GH or prolactin. True, spontaneous thyrotropinomas may be associated with multiple endocrine neoplasia syndrome type 1 or more commonly, are spora­ dic (Beck-Peccoz et al., 1996; Sanno et al., 2001). Over-expression of the pituitary-specific transcription factor Pit-1 may play a role in thyrotropinoma pathogenesis (Teramoto et al., 2004). Gsp and other oncogenes are not found in these tumors. Polyclonal thyrotroph hyper­ plasia in primary hypothyroidism occasionally mimics thyrotropinoma and is reversible upon thyroxine replacement. Pathogenesis of gonadotropinoma Although gonadophinoma is the most common pituitary tumor, few studies address its unique pathogenesis directly but many studies on pitui­ tary tumorigenesis include it as a type among several studied. MEG3, a possible tumor suppres­ sor gene, is expressed only in gonadotrophs but not in gonadotropinomas thus loss of MEG3 may result in gonadotropinoma growth (Ying et al., 2005). Prospects of the study on pituitary tumor pathogenesis Pituitary tumor pathogenesis is challenging to study due to unique tumor biology and behavior, difficulty in accessing the pituitary gland, and lack of human pituitary cell lines and few faithful ani­ mal models for human pituitary tumors. Much remains to be learned to understand the patho­ genesis of pituitary tumors. Oncogene activation, tumor suppressor gene inactivation and tumor micro-environment are all required for pituitary cell transformation. We suggest that future research on pituitary tumor pathogenesis should incorporate systems approaches, address regula­ tory mechanisms for pituitary cell proliferation

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 10

Molecular genetics of the AIP gene in familial pituitary tumorigenesis Asil Tahir, Harvinder S. Chahal and Márta Korbonits
Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK

Abstract: Pituitary adenomas usually occur as sporadic tumors, but familial cases are now increasingly identified. As opposed to multiple endocrine neoplasia type 1 and Carney complex, in familial isolated pituitary adenoma (FIPA) syndrome no other disease is associated with the familial occurrence of pituitary adenomas. It is an autosomal dominant disease with incomplete variable penetrance. Approximately 20% of patients with FIPA harbour germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene located on 11q13. Patients with AIP mutations have an overwhelming predominance of somatotroph and lactotroph adenomas, which often present in childhood or young adulthood. AIP, originally identified as a molecular co-chaperone of several nuclear receptors, is thought to act as a tumor suppressor gene; overexpression of wild-type, but not mutant AIP, reduces cell proliferation while knockdown of AIP stimulates it. AIP is shown to bind various proteins, including the aryl hydrocarbon receptor, Hsp90, phosphodiesterases, survivin, RET and the glucocorticoid receptor, but currently it is not clear which interaction has the leading role in pituitary tumorigenesis. This chapter summarizes the available clinical and molecular data regarding the role of AIP in the pituitary gland. Keywords: pituitary tumorigenesis; AIP gene; germline; somatotrophinomas; lactotroph adenomas

population studies in earlier series reported 19– 28 cases per 100 000 inhabitants (Clayton, 1999), while in a more recent active case-finding study 72–94 cases were detected per 100 000, probably reflecting increased vigilance and better imaging modalities (Daly et al., 2006b). Similar data showed an almost four-fold increased prevalence of pituitary adenomas (76 cases per 100 000 inhabitants) compared to older epidemiological studies (Fernandez et al., 2009). A recent screening study concentrating on acromegaly used insulin-like growth factor-1 (IGF-1) levels as a screening mod­ ality in a small population cohort (Schneider et al., 2008). It found a very high prevalence of acromegaly

Overview of pituitary tumorigenesis Pituitary tumors are common neoplasms accounting for approximately 15% of all primary intracranial lesions (Jagannathan et al., 2005). A meta-analysis by Ezzat et al. (2004) on the prevalence of pituitary tumors shows that they are found in 14.4–22.5% of autopsy and radiological studies. A minority of these will present with clinical and biochemical symptoms:


Corresponding author. Tel.: +44 20 7882 6238; Fax: +44 20 7882 6197;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82010-2

229

230

(103.4 cases of acromegaly per 100 000). This is in sharp contrast with the two recent epidemiological studies of 12.5 (Daly et al., 2006b) and 8.6 (Fernan­ dez et al., 2009) acromegaly cases per 100 000 inha­ bitants as well as the earlier data of 5.3–6 cases per 100 000 (Holdaway and Rajasoorya, 1999; Monson, 2000); confirmation of these data in the future will be adamant for our understanding of disease frequency. The pathogenesis of sporadic pituitary adeno­ mas remains unclear. Pituitary tumors are widely considered to be of monoclonal origin, since somatic cell mutations appear to precede clonal expansion in cells of corticotroph, lactotroph and non-functioning tumors (Herman et al., 1990). The most common pituitary tumor types are pro­ lactinomas (39–50%), non-functioning pituitary adenomas (NFPAs) (23–27%), growth hormone (GH)-secreting adenomas (16–21%), adrenocor­ ticotropic hormone (ACTH)-secreting adenomas (4.7–16%), thyroid-stimulating hormone (TSH)­ secreting adenomas (0.4%) and luteinizing hor­ mone (LH)/follicle-stimulating hormone (FSH)­ secreting adenomas (0.9%) (Yamada, 2001). Recent epidemiological data show similar fre­ quencies for prolactinomas (57%), NFPAs (28%), GH-secreting adenomas (11%) but lower prevalence in ACTH-secreting adenomas (2%) (Fernandez et al., 2009). The majority of these tumors is histologically benign, but can result in serious clinical syndromes due to hormonal excess and/or mass effect. Pituitary adenoma formation has been asso­ ciated with a variety of oncogene and tumor sup­ pressor gene abnormalities (Table 1). The first and probably only commonly occurring oncogene reported to have a role in sporadic pituitary tumorigenesis is the gsp oncogene. This is a mutated variant of the GSa subunit gene GNAS, which transmits the effects of the hypothalamic growth hormone-releasing hormone (GHRH) to the pituitary. Somatic mutations of this imprinted gene occur on the maternal allele in up to 40% of somatotrophinomas (Hayward et al., 2001). Other oncogenes and tumor suppressor genes implicated in pituitary tumorigenesis are listed in Table 1. The majority of pituitary adenomas arise spor­ adically. However, there is growing evidence to

suggest that 5% or more of adenomas are familial in origin, linked to certain genetic conditions with familial inheritance patterns (Beckers and Daly, 2007; Leontiou et al., 2008a). Multiple endocrine neoplasia type-1 (MEN1) syndrome is an autosomal dominant disease characterized by several endocrine tumors, in particular, pituitary, parathyroid and pancreatic neoplasms. Up to one third of patients with MEN1 have the occurrence of pituitary adeno­ mas, which consist of prolactinomas in 60% of patients, somatotrophinomas in 20% and cortico­ trophinomas or NFPA in less than 15% (Marini et al., 2006; Scheithauer et al., 1987; Thakker, 1998). Around 70–80% of patients harbour a mutation or gene deletion in the tumor suppres­ sor MEN1 gene located on 11q13. However 20–30% of cases that are phenotypical of MEN1 show no MEN1 mutation. These patients are described to have an “MEN1 phenocopy” (Burgess et al., 2000; Hai et al., 2000). Identifica­ tion of a homozygous frameshift mutation in CDKN1B, encoding the cyclin-dependent kinase inhibitor p27(Kip1) in a naturally occurring rat strain produces a dramatic reduction in p27 pro­ tein and a phenotype resembling the human MEN1 and MEN2 syndromes (Pellegata et al., 2006). Germline nonsense mutations of the human CDKN1B gene in MEN1 mutation-nega­ tive patients have been discovered in five families with MEN1 syndrome (Agarwal et al., 2009; Georgitsi et al., 2007b; Pellegata et al., 2006), but in general are very rare (Igreja et al., 2009; Owens et al., 2009; Ozawa et al., 2007). Carney complex (CNC) is characterized by several tumors including endocrine tumors (Bertherat, 2001; Carney et al., 1985; Pack et al., 2000). Over 60% of patients have a muta­ tion in the tumor suppressor protein kinase A type I-a regulatory subunit (PRKAR1A) gene, located on chromosome 17q24 (Horvath and Stratakis, 2008; Kirschner et al., 2000). Another locus associated with this disease was identified on chromosome 2p16 (Stratakis et al., 1996). The prominent features of CNC are increased produc­ tion of GH and prolactin due to hyperplasia or adenoma of somatotrophs and mammotrophs. Other endocrine abnormalities include primary

Table 1. Genes affected in pituitary tumorigenesis. (Modified from Tichomirowa et al., (2009).) Gene Tumor suppressor genes AIP


BMP-4 p27Kip1(CDKN1B)


p16INK4A (CDKN2A) p18INK4C (CDKN2C) GADD45 gamma MEG3a MEN1
p53 PKA (PRKAR1A)
Retinoblastoma WIF 1

ZAC1

Defect

Reference

Germline mutations in some FIPA families, particularly in families with somatotrophinomas, somatomammotrophinomas or a mixture of prolactinomas and somatotrophinomas. No somatic mutations Reduced expression in prolactinomas and an inhibitory role in corticotrophinomas Germline heterozygous nonsense mutation in MEN4 (a rare MEN1-like syndrome). Reduced protein expression in sporadic adenomas, especially ACTH-secreting ones, but no somatic mutations identified Hypermethylation of promoter region in pituitary adenoma development Hypermethylation of promoter region in pituitary adenoma development Growth suppressor controlling pituitary cell proliferation. Promoter methylation in non-functioning adenomas, prolactinomas and somatotrophinomas Hypermethylation of promoter region results in loss of expression found in nonfunctioning adenomas and gonadotrophinomas Inactivating germline mutations in all pituitary adenoma types Somatic inactivating mutations (very rare) or overexpression in a subset of pituitary carcinomas Truncating mutations in Carney complex leading to somatomammotroph hyperplasia and adenomas Hypermethylation of promoter region in pituitary adenomas and rare cases of pituitary carcinomas Hypermethylation of promoter region in pituitary adenomas, especially in nonfunctioning adenomas Inhibitors of the Wnt pathway (WIF1, SFRP2, frizzled B = SFRP3 (FZDB), SFRP4) were all downregulated in pituitary tumors compared to normal pituitaries Hypermethylation of promoter region in pituitary adenomas, especially in nonfunctioning adenomas

Daly et al. (2007); Leontiou et al. (2008a)

Giacomini et al. (2009) Dahia et al. (1998); Georgitsi et al. (2007b); Lidhar

et al. (1999)

Ruebel et al., (2001)

Kirsch et al., (2009) Zhang et al. (2002) Zhao et al., (2005); Zhang et al., (2003) Scheithauer et al. (1987); Zhuang et al. (1997) Tanizaki et al. (2007); Thapar et al., (1996) Veugelers et al. (2004)

Bates et al. (1997); Simpson et al., (2000)

Elston et al. (2008)

Theodoropoulou et al. (2006)

(Continued)

Table 1. (Continued ) Gene

Defect

Reference

DAP1 PTAG

Loss of DAP kinase expression in invasive adenomas Pituitary tumor apoptosis by CpG island methylation and loss of transcription

Simpson et al., (2002) Bahar et al. (2004)

Biallelically expressed in tumors. Somatic activating mutations in up to 40% of somatotrophinomas and mosaicism in McCune–Albright syndrome Important in regulation of cell progression through the G1 phase of the cell. Overexpression in non-functioning adenomas and somatotrophinomas Increased expression in invasive pituitary tumors Somatic activating mutations in pituitary carcinomas Point mutations in invasive pituitary adenomas Alternative transcription initiation, associated with more invasive tumors in patients with somatotrophinomas

Hayward et al. (2001); Landis et al. (1989)

Oncogenes Gsp Cyclin D1 PTTG RAS PKC Ptd-FGFR4


Genes associated with familial pituitary adenomas are marked with an asterisk.

Wang et al., (2001) Zhang et al. (1999) Pei et al., (1994) Alvaro et al. (1993) Ezzat et al., (2002); Morita et al. (2008)

233

pigmented nodular adrenocortical disease (PPNAD), testicular tumors, thyroid tumors and nodules (Stergiopoulos and Stratakis, 2003). Clinical acromegaly and pituitary adenoma formation occurs in 10% of patients with CNC (Horvath and Stratakis, 2008; Stergiopoulos and Stratakis, 2003). The McCune–Albright syndrome is an autosomal dominant disease characterized by a mosaic mutation in the gene encoding GSa gene (GNAS). This condition is characterized by multiple endocrine disorders, such as multinodular goitres, multinodular adrenal hyperplasia, preco­ cious puberty and pituitary adenomas. Increased GH and prolactin secretion is common in these patients due to nodular and diffuse hyperplasia of somatomammotroph cells and due to GH-produ­ cing adenomas (Horvath and Stratakis, 2008). No familial cases have been described to date. Familial pituitary tumors have also been described in several families with no other asso­ ciated endocrine diseases or tumors. These adeno­ mas are inherited in an autosomal dominant pattern with incomplete penetrance. This condition is com­ monly classified as familial isolated pituitary ade­ noma (FIPA), an umbrella term to include all cases of familial isolated pituitary tumors not associated with MEN1 and CNC (Valdes-Socin et al., 2000). It includes the subgroup of isolated familial somatotro­ phinoma (IFS) (Gadelha et al., 1999; Yamada et al., 1997) and the aryl hydrocarbon receptor-interacting

protein (AIP) mutation-positive group termed pitui­ tary adenoma predisposition (PAP) (Vierimaa et al., 2006). Linkage studies have shown that the locus for one of the genes causing FIPA is located on chromo­ some 11q13.3, but it is independent of the MEN1 gene (Benlian et al., 1995; Gadelha et al., 2000; Luccio-Camelo et al., 2004; Soares et al., 2005; Yamada et al., 1997). In 2006 a Finnish group reported heterozygous mutations in the AIP gene located in the 11q13 area 2.7 Mb downstream of MEN1 (Vierimaa et al., 2006). Since then mutations have been found in more than 40 families and numer­ ous seemingly sporadic, usually early-onset, pituitary adenoma patients (Table 2) (Cazabat et al., 2007; Chahal et al., 2010, Daly et al., 2007; Leontiou et al., 2008a; Vierimaa et al., 2006).

History of familial pituitary adenomas Reports of familial pituitary adenomas date back to more than a century ago, though information is sparse. The term acromegaly was first used by Pierre Marie in 1886, but patients with acromegaly have been described much earlier. It was only at the beginning of the 20th century with postmortem examinations of patients with acromegaly that it became clear that the cause of acromegaly was associated with pituitary adenomas and that gigant­ ism had a similar cause with onset of the disease

Table 2. AIP mutations reported to date Number of patients showing clinical features

Mutation type

AIP mutation

References

Base pair substitution resulting in nonsense mutation

c.40C>T p.Q14X

10 acromegaly 5 gigantism 4 prolactinomas 1 somatomammotroph

Georgitsi et al. (2007a); Raitila et al. (2007); Vierimaa et al. (2006)

c.64C>T p.R22X

1 acromegaly Sporadic

Barlier et al. (2007)

c.70G>T p.E24X

1 acromegaly 6 gigantism

Leontiou et al. (2008a)

c.241C>T p.R81X

4 acromegaly

Leontiou et al. (2008a); Toledo et al. (2008)

c.424C>T p.Q142X

2 acromegaly 1 gigantism 1 prolactinoma

Daly et al. (2007)

(Continued)

234 Table 2. (Continued )

Mutation type

Base pair substitutions resulting in missense mutation

AIP mutation

Number of patients showing clinical features

c.490C>T p.Q164X

1 acromegaly 1 gigantism

Igreja et al. (2010)

c.601A>T p.K201X

2 acromegaly Sporadic (both from same country)

Cazabat et al. (2007)

c.649C>T p.Q217X

2 acromegaly

Daly et al. (2007)

c.715C>T p.Q239X

2 gigantism

Daly et al. (2007)

c.721A>T p.K241X

1 childhood macroprolactinoma (de novo germline mutation)

Beckers et al. (2008)

c.804A>C

2 acromegaly 11 acromegaly

Toledo et al. (2007)

p.Y268X c.910C>T p.R304X

5 gigantism 3 prolactinoma

Cazabat et al. (2007); Daly et al. (2007); Igreja et al. (2010); Leontiou et al. (2008a); Vierimaa et al. (2006)

c.47G>A p.R16H

Several normal subjects as well as several sporadic acromegaly, 1 familial acromegaly No loss of heterozygosity No loss of PDE binding

Cazabat et al. (2007); Daly et al. (2007)

c.145G>A p.V49M

1 gigantism Sporadic, no loss of heterozygosity No loss of PDE binding

Iwata et al. (2007)

c.308A>G p.K103R

1 Cushing’s disease Sporadic childhood onset Loss of PDE binding

Beckers et al. (2008)

c.713G>A p.C238Y

3 acromegaly

Leontiou et al. (2008a)

c.721A>G p.K241E

1 prolactinoma 1 NFPA

Daly et al. (2007)

c.769A>G

1 thyrotrophin-releasing tumor Sporadic No loss of PDE binding

Montanana et al. (2009)

c.811C>T p.R271W

5 acromegaly 2 prolactinoma

Daly et al. (2007); Jennings et al. (2009)

c.896C>T p.A299V

1 sporadic acromegaly Also found in an asymptomatic carrier of R304X on different alleles No loss of binding

Georgitsi et al. (2007a) Igreja et al. (2010)

p.I257V

References

(Continued )

235 Table 2. (Continued )

Mutation type

Frameshift mutation

AIP mutation

acromegaly somatomammotroph prolactinoma NFPA (prolactin positive) (unknown diagnosis) Cushing’s disease

References

c.911G>A p.R304Q

2 1 2 1 1 1

c.74_81delins7 p.L25PfsX130

1 gigantism 2 acromegaly 1 somatomammotrph adenoma 1 prolactinoma 3 gigantism

Igreja et al. (2010)

Frameshift mutation in exon 2 Details not available

1 gigantism

Yaneva et al. (2008)

Not available p.P114fs

1 acromegaly

Beckers et al. (2008)

c.404delA p.H135LfsX20

1 gigantism Sporadic

Cazabat et al. (2007)

c.5oodelC p.P167HfsX3

3 acromegaly, 3 acromegalaid features

Khoo et al. (2009)

c.517-521delGAAGA p.E174fsX47

3 acromegaly

Daly et al. (2007); Naves et al. (2007)

c.542delT p.L181fsX13

1 acromegaly

Georgitsi et al. (2007a)

c.622dupC p.C208LfsX15

2 acromegaly

Igreja et al. (2010)

c.824-825insA p.H275QfsX12

1 gigantism Sporadic

Georgitsi et al. (2007a)

c.854-857delAGGC p.Q285fsX16

1 acromegaly 1 gigantism

Daly et al. (2007)

c.919insC p.Q307RfsX103

1 somatomammotroph 1 prolactinoma

Beckers et al. (2008)

IVS2-1G>C (c.100­ 1G>C) Predicted to result in exon 2 skipping

1 acromegaly

Georgitsi et al. (2007a)

c.249G>T p.G83AfsX15

1 gigantism, 1 acromegaly 1 prolactinoma

Igreja et al. (2010)

IVS3þ15C>T (c.468þ15C>T) May disrupt splicing enhancer site at beginning of intron 3

1 acromegaly

Montanana et al. (2008)

c.286-287delGT p.V96PfsX32

Splice-site base pair substitution

Number of patients showing clinical features

Cazabat et al. (2007); Georgitsi et al. (2007a); Igreja et al. (2010); Leontiou et al. (2008a); Vargiolu et al. (2009)

Iwata et al. (2007)

Sporadic

Sporadic

(Continued)

236 Table 2. (Continued ) Number of patients showing clinical features

References

IVS3-1G>A (c.469-1G>A) Predicted to result in exon 3 skipping

1 acromegaly

Vierimaa et al. (2006)

IVS3-2A>G (c.469-2A>G) Predicted to result in exon 3 skipping

1 acromegaly

c.807C>T p.F269= disrupts exon 6 splicing

2 acromegaly

Leontiou et al. (2008a)

Promoter

c.-270-269CG>AA and c.-220G>A

2 gigantism

Igreja et al. (2010); Leontiou et al. (2008a)

In-frame deletions

c.66-71delAGGAGA p.G23_E24del

1 acromegaly

Georgitsi et al. (2007a)

c.138-161del24 p.G47_R54del

2 acromegaly

Daly et al. (2007)

c.742_744delTAC p.Y248del

1 gigantism sporadic

Georgitsi et al. (2008)

c.878-879AG>GT, p. E293G and c.880-891del CTGGACCCAGCC p.L294_A297del

1 acromegaly Not known if sporadic of familial

Georgitsi et al. (2007a)

In-frame insertion

c.805_825dup p.F269_H275dup


2 gigantism 1 acromegaly

Leontiou et al. (2008a)

Large gene deletions

c.100-1025_279þ357 p.A34_K93 Ex2del

1 NFPA (GH/PRL pos) 1 acromegaly 1 somatomammotroph adenoma 1 pituitary adenoma (no surgery)

Georgitsi et al. (2008); Igreja et al. (2010)

c.1104-109_279þ578 Ex1_Ex2del

2 acromegaly

Georgitsi et al. (2008)

Whole gene deletion

2 gigantism

Igreja et al. (2010)

c.1-?_993þ?delWhole gene deletion

2 gigantism 1 acromegaly

Igreja et al. (2010)

Mutation type

AIP mutation

Sporadic Cazabat et al. (2007)

Sporadic

Mutations only identified in sporadic patient are marked with “sporadic”. Functional assay data is from Igreja et al. (2010).
This mutation has previously been annotated c.794_823dup, p.A274_H275ins10 (EF643650) (Leontiou et al., 2008a) while the current appropriate annotation is c.805_825dup, p.F269_H275dup (accession number NM_003977.1).

during childhood (de Herder, 2008). The first recorded patients with isolated familial acromegaly, as far as we understand, are Baptiste and Antoine

Hugo in the early 20th century. Their heights were 2.30 m and 2.25 m and they had three brothers and two sisters of normal height (Fig. 1). The report

237

Fig. 1. The Hugo brothers. The first documented case of possible isolated familial acromegaly. Baptiste and Antoine Hugo tower in height above the rest of their immediate family. Squares represent males, circles females and filled symbols affected subjects.

238

describes typical features of acromegaly including hypogonadism, osteoporosis, frontal bossing, prog­ nathism and enlarged internal organs. Postmortem examination of Antonio Hugo revealed a huge pituitary adenoma (50× 25 × 23 mm) with suprasel­ lar, retrosellar and left parasellar expansion (de Herder, 2008). Erdheim (1903) was the first to describe familial pituitary tumors to occur in the setting of MEN in 1903. Wermer later described a family with four sisters affected with pituitary adenomas, hypercal­ caemia and adenomatosis of the pancreas and gut. He suggested that the condition was inherited in an autosomal dominant pattern (Wermer, 1954). Isolated pituitary adenomas were not formally described until 1967, when a prolactinoma family was reported (Linquette et al., 1967). This was followed by a description of two acromegaly families (Himuro et al., 1976; Levin et al., 1974) and later a corticotroph adenoma family (Salti and Mufarrij, 1981) and slowly isolated familial pitui­ tary adenomas became appreciated as a new clin­ ical disease (McCarthy et al., 1990; Pestell et al., 1989). Currently over 200 families have been described with FIPAs; however, this number will increase as this condition is being more recognized. In 1995, a family with three patients affected by gigantism or acromegaly was described with no association to the immediate vicinity of the MEN1 gene locus (Benlian et al., 1995). In 1997, loss of heterozygosity (LOH) on chromosome 11q13 was reported in pituitary adenomas of two siblings with familial acromegaly. This locus comprised the MEN1 gene, so it was suggested that familial acromegaly was either an alternative form of the MEN1 syndrome or there was an independent gene at the locus nearby that was involved in the pathogenesis (Yamada et al., 1997). A study on two additional families con­ firmed the involvement of the 11q13 locus (Gadelha et al., 2000). The hypothesis of an inde­ pendent gene was later confirmed by a study on eight novel families (Soares et al., 2005). Subse­ quently the original four families with association to the 11q13 area (Benlian et al., 1995; Gadelha et al., 2000, Yamada et al., 1997) as well as four of the eight subsequent families with LOH at

11q13 were shown to harbour an AIP mutation (Fig. 2) (Iwata et al., 2007; Leontiou et al., 2008a) following the identification of the AIP gene (Vierimaa et al., 2006).

AIP protein AIP is also known as hepatitis B virus (HBV) X-associated protein (XAP2) or aryl hydrocarbon receptor (AhR)-associated protein (ARA9) (Carver and Bradfield, 1997; Kuzhandaivelu et al., 1996). It is a molecular co-chaperone pro­ tein and known to interact with the nuclear recep­ tor AhR (aryl hydrocarbon receptor) as well as other proteins (see below). The AIP gene is located at 11q13 and consists of six exons coding for a 330-amino-acid protein. The AIP protein is widely expressed in the body, including the pituitary. It has a similar structure to the glucocor­ ticoid receptor (GR)-associated immunophilin FKBP52 (FK506-binding protein 52) and the immunophilin FKBP12 (Petrulis et al., 2000). Despite the PPI (peptidyl-prolyl cis-trans iso­ merases)-like domain on the N-terminal segment of the protein being similar to the immunophilin FKBP52, AIP does not bind the immunosuppres­ sant macrolide FK506 and does not act like an immunophilin (Kazlauskas et al., 2002). At the C-terminal part of the protein there are three tetratricopeptide repeats (TPRs) and a final a-helix (Fig. 3). The three TPR domains are degenerate sequences of 34 amino acids compris­ ing two a helices and they have a crucial role in mediating the protein–protein interactions of AIP (Kazlauskas et al., 2002).

AIP and AhR interaction AhR, a ligand-activated transcription factor and two molecules of the chaperone Hsp90 together with co-chaperones AIP and p23 form a stable complex (Carver et al., 1998) (Fig. 4). The ligand-bound AhR is translocated to the nucleus, dissociates from Hsp90 and AIP to heterodimerize with aryl hydrocarbon receptor nuclear transloca­ tor (ARNT) (Petrulis et al., 2000) to bind to the

239

manufacturing processes. The AhR–dioxin inter­ action can result in hepatocellular damage, thymic involution, epithelial hyperplasia, teratogenesis, and carcinogenesis (Bunger et al., 2003; Tomita et al., 2003; Walisser et al., 2004). AhR can also bind polycyclic aromatic hydrocarbons that are present in car and diesel exhaust, cigarette smoke and charbroiled or smoked food, and were shown to have mutagenic and carcinogenic effects (Park et al., 2009). AhR was shown to transport activated polycyclic aromatic hydrocar­ bons into the nucleus where they cause oxidative damage resulting in DNA strand breaks and mutations. Interestingly, this effect was shown to

xenobiotic/dioxin-inducible response element (XRE/DRE). This interaction results in the regu­ lation of multiple genes, including drug-metaboliz­ ing enzymes such as the cytochrome CYP1 family (Carver et al., 1998; Hillegass et al., 2006; Meyer et al., 1998). AhR is an orphan nuclear receptor with a role in regulating proliferation and differentiation in hepatocytes, immune system homeostasis and tumorigenesis (Barouki et al., 2007). AhR has been shown to control the effects of environmen­ tal toxins such as dioxin. Polychlorinated dibenzo­ dioxins can be formed through volcanic activity, forest fires, combustion, chlorine bleaching and

Family D

A.

1

Acromegaly

2

64y Carrier

B.

3

4

32y

32y

7500 6000

6

C.

LOH 150

5

200

210

220

200

210

220

7

8

36y

34y

9

CAG – arginine

D11s1917 Leukocyte DNA Subject D3

4500 3000 1500 0 150 2000 1600

D11s1917 Tumor DNA Subject D3

1200 800 400 0

Fig. 2. (Continued)

TGA – stop codon

240

D.

Stop

1

2

3

4

5

6

α helix PPI-like region 1

12

TPR1 90

182

TPR2

215 234

TPR3

267

301 330

Fig. 2. (A) Family tree [Family D (Soares et al., 2005)] showing two affected subjects and three carrier family members. (B) Loss of heterozygosity (LOH) study of affected patient D3: leukocyte-derived DNA shows the two peaks of a heterozygous marker (D11s1917) with a smaller fragment inherited from the mother and a larger fragment inherited from the father (Soares et al., 2005). In the pituitary adenoma-derived DNA the smaller marker (inherited from the non-carrier mother) is lost while the larger marker, lying on the same (paternal) allele where the AIP mutation is located, is retained. Therefore the germline heterozygosity of the D11s1917 marker is lost in the tumor tissue: there is LOH in this tumor tissue. The loss of the normal maternal allele resulted in no functional AIP present in the tumor cell leading to tumorigenesis. (C and D) Following the identification of AIP mutations in familial isolated pituitary adenoma families, sequencing of the AIP gene revealed a heterozygote mutation at c.241C>T resulting in a stop codon (p.R81X) in the second exon of the gene (Leontiou et al., 2008a). The schematic drawing of the AIP gene and protein is shown marking the various protein domains and amino acids.

Human AIP

TPR1

TPR2 α-helix TPR3

Fig. 3. The structure of AIP. Hypothetical structure of AIP based on the structure of FKBP51 showing the three TPR domains and the final a-helix. (Courtesy of David Barford, London, UK.)

be independent of AhR–ARNT binding to XREs suggesting that AhR plays a role as a nuclear transporter for these polycyclic aromatic hydrocarbon-type genotoxins (Park et al., 2009). AhR ligand dioxin can disrupt the regulation of the gonadotroph axis. Studies suggest that smoking during pregnancy causes decline in fertility of chil­ dren and can result in an earlier menopause for women (Matikainen et al., 2001). This direct effect of the endocrine pathway has been linked to AhR activation. The use of the AhR antagonist resvera­ trol helps maintain ovarian cell function, suggesting that AhR could play a role in the destruction of ovarian cells via apoptosis (Jurisicova et al., 2007). In response to dioxin stimulation AhR can form an AhR–ARNT–ERa (estrogen receptor a) com­ plex, which can activate ERa-responsive genes even in the absence of estrogen. On the other hand, ERa is less responsive to estrogen stimula­ tion when it is in the AhR–ARNT–ERa complex.

241 AhR - Hsp90 - AIP complex

Hsp 90

AhR

P23

AIP

Fig. 4. AhR–Hsp90–AIP–p23 complex. Unliganded AhR in the cytoplasm forms a multiprotein complex with the chaperone Hsp90, the co-chaperone AIP and the co-chaperone p23.

AhR can also increase the degradation of ERa or the androgen receptor (Ohtake et al., 2007). AhR is part of the E3 ligase system that can attach ubiquitin molecules to proteins, which then are marked to degradation by the proteosome. Although AIP does not seem to play a role in the ERa ubiquitination (Ohtake et al., 2007), this interaction of AhR might be relevant for the mechanism of precocious puberty reported in a 2-year-old infant with an AIP mutation (Naves et al., 2007). There is conflicting data about the role of AIP to stimulate or inhibit the activity of AhR. The majority of these studies have been carried out in vitro in transient expression systems. Some studies have shown that AIP increases AhR levels in the cytoplasm, as well as increasing the level of AhR-mediated gene induction (LaPres et al., 2000; Meyer and Perdew, 1999). AIP can affect the subcellular localization of AhR via influencing its dynamic nucleocytoplas­ mic shuttling (Kazlauskas et al., 2000; Petrulis

et al., 2000, 2003). However, this may not be a typical mode of action, as newer studies have reported that AIP expression has minimum affect on the subcellular localization or level of expression of the AhR in transient expression assays (Ramadoss et al., 2004). Other studies have shown that AIP has an inhibitory effect on AhR transcription (Hollingshead et al., 2004; Pollenz and Dougherty, 2005). It was sug­ gested that AIP increases the stability of the AhR protein due to decreased ubiquitination of the receptor (Kazlauskas et al., 2000). Some studies report that AIP has a negligible effect in maintaining AhR protein levels (Hollingshead et al., 2004; Pollenz and Dougherty, 2005). In sporadic pituitary adenomas AIP and AhR expression was shown to be positively correlated (Jaffrain-Rea et al., 2009). Another study showed that AIP may regulate ARNT levels as ARNT was less frequently expressed in pituitary adenomas with AIP mutations. Silencing AIP in a rodent pituitary adenoma cell line GH3, resulted in a

242

reduction of ARNT (Heliovaara et al., 2009) and a significant increase in cell proliferation (Leontiou et al., 2008b). The downregulation of ARNT could be due to an imbalance in AhR–ARNT complex formation caused by aberrant cAMP signalling (Heliovaara et al., 2009).

Other interacting partners of AIP In addition to Hsp90, AIP was shown to bind to another heat shock protein, Hsc70 from the Hsp70 family. It seems that AIP preferentially forms a com­ plex with Hsc70 rather than with Hsp90 in the absence of AhR (Meyer et al., 1998; Yano et al., 2003). AIP has been reported to interact with cyclic nucleotide phosphodiesterases (PDEs), which cata­ lyze the degradation of cAMP and cGMP. PDEs are important in physiological processes and have a wide range of action by controlling intracellular cyclic nucleotides. There are 11 different PDE families in humans, which can encode for >50 dif­ ferent isoenzymes with varying properties and spe­ cificities for substrates (Soderling and Beavo, 2000). PDE4 isoforms have unique N-terminal sequences divided into upstream conserved regions 1 and 2 (UCR1 and UCR2) (Bolger et al., 1993; Houslay et al., 1998). The second TPR domain of AIP has been found to interact with two regions on the UCR2 of the N-terminal part of a PDE4A5/4 (rat/human) subtype (Bolger et al., 2003). PDE4A5 is a cAMP-specific phosphodiesterase isoform expressed in many different tissues. The PDE4A5– AIP interaction was originally identified in a yeast two-hybrid assay, but was also shown in mammalian cells, where AIP co-immunoprecipitated with PDE4A5 in transfected COS7 cells and in rat brain. This resulted in inhibition of PDE4A5 activ­ ity, which did not occur if there was a truncation of the unique N-terminal region of PDE4A5 or a mutation in the UCR2 region. The interaction was specific to PDE4A5 and did not occur with any other PDE4 isoforms (Bolger et al., 2003). AIP also acts to restrict the phosphorylation of PDE4A5 by protein kinase A (PKA), a process that increases the activity of PDE4A5, and it also causes an increase of affinity for PDE inhibitor rolipram (Bolger et al., 2003). These data suggest

that AIP reduces the activity of PDE4A5 via three different mechanisms. These effects do not support the hypothesis that the tumor suppressor effect of AIP in somatotroph cells would involve the interac­ tion with PDE4A5, although cell-type-specific dif­ ferences cannot be ruled out. Nevertheless, it has been shown that mutations identified in FIPA patients disrupt the binding of AIP and PDE4A5 in an in vitro assay, but it is not known if this has direct relevance to pituitary tumorigenesis (Igreja et al., 2010; Leontiou et al., 2008a). AIP has also been reported to interact with another PDE isoform called PDE2A (de Oliveira et al., 2007). PDE2A binds to the TPR domain in the C-terminal region of AIP and acts to degrade cAMP and cGMP. The structure of PDE2A consists of two central conserved cyclic GMP, Adenylyl cyclase, FhlA binding segments (GAF-A) and GAF-B domains, a catalytic C-terminal and an Nterminal segment with unknown function. The GAF-B domain is essential for effective binding to AIP. This interaction has been shown in a yeast twohybrid assay and by co-immunoprecipitation in COS-1 cells and rat brain lysates (de Oliveira et al., 2007). There was no change of enzyme activity of PDE2A following AIP binding. However, AIP– PDE2A interaction was shown to inhibit the trans­ location of AhR from the cytoplasm of the nucleus, indicating that PDE2A could have a role in AhR complex regulation. It was suggested that cAMP could be an endogenous ligand of AhR (OeschBartlomowicz et al., 2005). If AIP binds and brings PDE2A in close vicinity of AhR and reduces local cAMP concentration, then this mechanism could lead to reduced AhR translocation and transcrip­ tional activity (de Oliveira et al., 2007). Proteomics screening has identified another interacting partner to AIP called survivin (Kang and Altieri, 2006). This is an inhibitor of the apop­ tosis pathway and has a specific function to maintain cell viability and cell division. Survivin was shown to directly interact with the TPR domains in the C-terminal of AIP. AIP binds and stabilizes survivin and elevates cellular anti­ apoptotic threshold. Clearly this mechanism is not easily compatible with the suggested tumor suppressor role of AIP. Knockdown of AIP with siRNA or competition of the survivin–AIP

243

complex by peptidyl mimicry destabilizes survivin levels in cells and reduces cell viability in MCF-7 breast cancer cells (Kang and Altieri, 2006). This is in contrast to data in pituitary cell line GH3 where siRNA for AIP was shown to increase cell viability (Heliovaara et al., 2009; Leontiou et al., 2008b). AIP was shown to be interacting with the long and short isoforms of rearranged during transfec­ tion gene (RET) (Vargiolu et al., 2009). RET is a known oncogene and activating mutations lead to the syndrome of MEN2. On the other hand, in the absence of RET ligand GDNF (glial cell linederived neurotrophic factor) RET stimulates apoptosis (Canibano et al., 2007). AIP binding to RET disrupts the binding of AIP to survivin. As AIP protects survivin from degradation, it is sug­ gested that RET–AIP binding could lead to survi­ vin degradation and increased apoptosis. However, pathogenic mutations in AIP (K241E, R271W and R304Q) and RET (E768D, V778I, L790F, V804M, Y806C, S891A, M918T, and S922F) do not prevent the binding of the two molecules in vitro, therefore further studies are needed to clarify the relevance of the RET–AIP interaction in pituitary tumorigenesis. A study by Yano et al. used two-hybrid screen­ ing to identify an interaction of AIP with Tom20 (translocase of the outer membrane of mitochon­ dria 20). The C-terminal of Tom20 was required to bind the TPR domain of AIP. This preprotein receptor forms translocator complexes and imports mitochondrial preproteins into the mito­ chondria. The formation of a ternary complex of Tom20, AIP, and preprotein preornithine trans­ carbamylase was observed. Overexpression of AIP in cell culture was shown to enhance preor­ nithine transcarbamylase import. siRNA knock­ down of AIP impaired this import. An in vitro binding assay demonstrated that AIP specifically binds to mitochondrial preproteins that directly target Tom20. AIP binds to Tom20 more strongly than preproteins and can transfer preprotein to Tom20. It was suggested that AIP acts as a pro­ tein chaperone by preventing substrate proteins from aggregation and maintaining preprotein import into mitochondria (Yano et al., 2003). In addition to AhR, three other nuclear recep­ tors have been shown to bind AIP: b thyroid

hormone receptor 1 (TRb1), peroxisome prolif­ erator-activated receptor a (PPARa) and GR. Froidevaux et al. conducted a yeast two-hybrid screening study on mouse hypothalamus, which showed the TPR domain of AIP to interact with the TRb1. This receptor is a central feedback regulator of the hypothalamo–hypophyseal–thyr­ oid axis and hence thyroid hormone homeostasis. In situ hybridization of mice hypothalamus demonstrated the expression of AIP and TRH transcripts in the same region of the paraventricu­ lar nucleus. Further immunocytochemistry experi­ ments showed that AIP and TRb1 were also expressed in the same mice neurons. siRNA knockdown of AIP in vitro affected the stability of TRb1. In vivo, siRNA abrogated specifically the TRb1-mediated (but not TRb2) activation of hypothalamic TRH transcription (Froidevaux et al., 2006). Although we have observed a few AIP carrier subjects with primary hypothyroidism (not necessarily related to their genetic condition; Korbonits, unpublished observation), no consis­ tent specific thyroid axis abnormality has been described in patients with AIP mutations. PPARa has also been shown to interact with AIP. This nuclear receptor acts as a ligandinducible transcription factor. PPARa has an important role in lipid metabolism and homeosta­ sis. It also mediates the carcinogenic effects of peroxisome proliferators on liver cells in mice (but not in humans) (Yang et al., 2008). Experi­ ments have shown that PPARa co-immunopreci­ pitates with AIP in mouse liver cytosol (Sumanasekera et al., 2003). In vitro binding assays show that cells co-expressing PPARa and AIP have a reduced peroxisome proliferator response. This suggests that the PPARa–Hsp90– AIP complex has a repressor function (Sumanase­ kera et al., 2003). In yeast studies AIP was not shown to be part of the GR–Hsp90 complex (Carver et al., 1998). However, a recent study in human cells found that AIP can bind to the Hsp90–GR complex and inhibit the transcriptional activity of the GR and delay its nuclear entry (Laenger et al., 2009). These features are similar to the effects of FKBP51, which has no effect in yeast on GR function while inhibiting GR activity in

244

mammalian cells. No abnormalities have been described of the hypothalamo–pituitary–adrenal axis in patients with AIP mutations. AIP was shown to bind to two viral proteins: HBV X antigen (Meyer et al., 1998) and Epstein–Barr virus (EBV)-encoded nuclear antigen-3 (EBNA-3) (Kashuba et al., 2000). The X protein of HBV plays an important role in viral replication in animals. Data suggest that the X gene product may contribute to HBVinduced tumorigenesis. The X protein is capable of inducing transformation of NIH3T3 cells and mouse hepatocytes (Kuzhandaivelu et al., 1996). EBNA-3 plays an important role in the effect of EBV on cell transformation possibly via disrup­ tion of the G2/M checkpoint (Krauer et al., 2004). EBNA-3 was also shown to bind specifi­ cally to the TPR domain of AIP and to interact with AhR in the cell cytoplasm and resulted in nuclear translocation of AhR (Kashuba et al., 2000). The binding of AIP to the transforming proteins of two evolutionarily very distant viruses may indicate the involvement of AIP and possibly aryl hydrocarbon receptor signal transduction pathway in virus-induced cell transformation.

G proteins transfer extracellular signals of G-protein-coupled seven transmembrane recep­ tors to intracellular second messengers. G proteins have three subunits, a, b and g. The a subunit has four subtypes, stimulatory Gs, inhibitory Gi, Gq and G12. Members of the G12 family G12 and G13 appear to be expressed ubiquitously and have been shown to interact with various partners (Worzfeld et al., 2008) including a TPR-containing protein, phosphatase 5 (Laenger et al., 2009; Yamaguchi et al., 2002). Recently, AIP was also found to bind to G12 and this AIP–G12 interaction inhibits AIP binding to AhR, leading to reduced AhR signalling (Nakata et al., 2009).

Tumor suppressor role of AIP AIP is thought to act as a tumor suppressor gene. LOH was shown in the tumors in FIPA families (Gadelha et al., 2000, Soares et al., 2005; Yamada et al., 1997). According to the Knudson two-hit hypothesis (Knudson, 1971) the first hit is due to an inherited germline of one allele and the second hit is a somatic deletion of another allele (Fig. 5). Leontiou et al. showed that overexpressing the

TUMOR

First ‘hit’ due to a germline mutation. Red line represents germline AIP mutation, blue line represents normal AIP allele

Second ‘hit’ due to a deletion in the normal AIP allele. Tumor tissue shows loss of heterozygocity around the AIP area

Gene and protein expression of the mutant allele can be detected in the tumor tissue depending on the size and the detection methoidd. Second ‘hit’ due to a deletion in the normal AIP allele. Tumor tissue shows loss of heterozygocity around the AIP area

Fig. 5. Knudson two-hit hypothesis (Knudson, 1971).

245 P < 0.001 P < 0.001

620000

P < 0.001

Number of cells at 72hr

600000 580000 560000 540000 520000 500000 480000 460000

Empty vector

Wildtype

R304X

Insertion

C238Y

Fig. 6. MTS assay results in HEK-293 cells transiently transfected with wild-type and mutant AIP plasmids showing reduced proliferation of wild-type AIP-transfected cells compared to empty vector control and mutated AIP-transfected cells (Leontiou et al., 2008a).

AIP gene decreases the rate of cell proliferation in three different cell types. Cells were transiently transfected with wild-type AIP, empty vector and AIP mutants. Overexpression of wild-type AIP considerably reduced cell proliferation on a cell viability assay compared to empty vector and this effect was lost in cells transfected with AIP mutant plasmids (Fig. 6) (Leontiou et al., 2008a). siRNA knockdown of AIP in GH3 cells further suggests a tumor suppressor role for AIP (Heliovaara et al., 2009; Leontiou et al., 2008b). In sporadic pituitary adenomas AIP mRNA and protein expression is paradoxically increased, suggesting that lack of AIP expression is unlikely to play a role in the pathogenesis of sporadic adenomas. It is not clear, however, which interacting partner of AIP is involved in the tumor suppressor effect.

AIP mutations Along the whole length of the AIP gene 49 different mutations have been discovered. These mutations include deletions, insertions, segmental

duplications, nonsense, missense mutations and deletions of whole exons or the whole gene (Table 2). Most of the pathogenic missense mutations directly affect the TPR domains or the C-terminal a-helix. This supports the earlier evidence that the third TPR and the last five C-terminal amino acids are important for the activ­ ity of AIP (Petrulis and Perdew, 2002). A common genetic “hotspot” for mutations of AIP at the 304 residue (R304X and R304Q) has been shown to be affected in several families with FIPA (Cazabat et al., 2007; Daly et al., 2007; Georgitsi et al., 2007a; Igreja et al., 2010; Leontiou et al., 2008a; Vargiolu et al., 2009; Vierimaa et al., 2006). Other potential hotspots are at the 241 locus found in three FIPA patients (K241E and K241X) (Daly et al., 2007), the 271 locus (R271W, Daly et al. (2007); Jennings et al. (2009)) and at the 81 locus (R81X) found in two FIPA families and in a sporadic giant (Igreja et al., 2010; Leontiou et al., 2008a, Toledo et al., 2008). There have been numerous sporadic and a few familial patients described with the Q14X mutation, but all of these were from Finland suggesting that these cases are

246

due to a possible founder mutation in northern Finland. It is important to distinguish between rare polymorphisms and real pathogenic muta­ tions that affect pituitary tumorigenesis; for example the R16H change that has been dis­ covered in two patients with sporadic acrome­ galy and was also found in 3 out 360 controls (Cazabat et al., 2007; Daly et al., 2007; Geor­ gitsi et al., 2007a), whereas an in vitro func­ tional study did not show a change in function (Igreja et al., 2010). While these data do not rule out the pathogenic role of this change, caution needs to be taken when predicting dis­ ease in asymptomatic carriers of this change.

Clinical features of FIPA FIPA is an autosomal dominant disease with incomplete penetrance showing a heterogeneous genetic background (Fig. 7). Approximately 20% of all types of FIPA families and 40% of somato­ troph adenoma families harbour a mutation in the AIP gene, while the rest of the families probably have a mutation in a currently unknown gene (or genes). FIPA families can be homogeneous, display­ ing pituitary tumors of the same type, or Gonadotropinoma, 1.74%

heterogeneous, displaying different pituitary tumor types (Beckers and Daly, 2007; Daly et al., 2007; Georgitsi et al., 2008; Leontiou et al., 2008a; Vierimaa et al., 2006). The most commonly occurring phenotype is somatotroph and lactotroph adenomas, especially in the AIP mutation-positive families. All the families har­ bouring an AIP mutation have either pure somatotrophinomas or mixed somatotrophino­ mas and somatomammotrophinomas clinically or at least on immunostaining. It was also shown that the same mutation can result in different clinical phenotypes in different families. Currently no AIP mutations have been reported in FIPA families homogeneous for prolactinomas. Age – FIPA patients in general are diagnosed younger than those with sporadic pituitary adeno­ mas. The average age of onset tends to be later in heterogeneous FIPA families when compared to homogenous families. Within the FIPA cohorts AIP mutation-positive families have an earlier onset of disease than patients in AIP mutation-negative families (Daly et al., 2007; Leontiou et al., 2008a). In a cohort of 20 families with AIP mutations 16 had at least one member with gigantism and/or disease onset <18 years, while only 3 of the 44 AIP-negative FIPA Thyrotropinoma, 0.25%

Mixed, 0.50%

Corticotrophinomas 2.99% NFPA 9.94%

Pure prolactinoma, 24.38% Somatotroph & somatomammotroph adenomas 60.2%

Fig. 7. Pie chart showing the distribution of different tumor types in 402 identified patients with FIPA.

247

families had a member with gigantism and/or disease onset <18 years. The youngest patient with a known AIP mutation was diagnosed at the age of 6 years with a large pituitary adenoma, while the eldest AIP mutant patient was diagnosed at the age of 66 years with a large prolactinoma (Igreja et al., 2010). Sex distribution – There is no major difference in the sex distribution of the disease. The slightly higher prevalence (62%) in women is possibly related to the higher incidence of prolactinomas, which are mainly microadenomas. There is a higher rate of maternal transmission in homoge­ nous kindreds when compared to heterogeneous kindreds. This could be due to the higher level of mother–daughter homogenous prolactinomas. Paternal transmission is more common in families with heterogeneous somatotrophinomas (Beckers & Daly, 2007, Daly et al., 2006a). Penetrance – It was clear from the early descrip­ tion of the disease that the penetrance is low (Benlian et al., 1995, Pestell et al., 1989). Vierimaa et al. described a penetrance of 15% in their ori­ ginal large family with AIP mutation (Vierimaa et al., 2006), while further studies on large families with AIP mutations show a penetrance of pituitary tumors of 30% (three affected subjects out of nine AIP mutation carriers) (Naves et al., 2007). The vast majority of AIP-negative FIPA families consist of two known affected family members while AIP-positive families usually consist of 2–5 members having pituitary adenomas. These observations suggest that penetrance of the dis­ ease is higher in AIP-positive families than in AIP-negative families. Igreja et al. (2010) showed that the mean number of affected members in AIP-positive families is 3.2 (SD 1.8) and 2.2 (SD 0.5, p < 0.001) in families without the mutation. The factors influencing penetrance are difficult to determine and possibilities of a second locus have been investigated (Khoo et al., 2009). While some families occasionally can indeed show very high percentage of penetrance, the earlier reported higher levels of penetrance is probably due to the lack of availability of all family members at risk (Daly et al., 2007; Leontiou et al., 2008a).

AIP mutations have been described in some apparently sporadic patients, commonly in child­ hood-onset acromegaly (Georgitsi et al., 2008), although in our cohort two of the 16 childhoodonset or studied giants carried an AIP mutation (Igreja et al., 2010; Leontiou et al., 2008a). The mutations usually have also been found in one of the parents as well but in one case a new mutation has been described in a childhood-onset corticotroph adenoma (Beckers et al., 2008). Tumor behavior – Adenomas in patients with FIPA are usually macroadenomas [88.5% in AIP mutation-positive families and 71.2% in AIP mutation-negative families, statistically not differ­ ent (Daly et al., 2007)] with AIP mutation families showing more aggressive and larger adenomas than AIP mutation-negative familial or sporadic tumors (Daly et al., 2007; Leontiou et al., 2008a). Patients with FIPA typically do not respond well to somatostatin analogue therapy (Leontiou et al., 2008a). Clinical management – Clinical management of patients with FIPA is challenging and clinical experience of long-term follow-up of patients and asymptomatic carriers is lacking. AIP muta­ tion screening including testing for single basepair changes as well as large deletions is now available in an accredited National Health Ser­ vice laboratory in the UK. If an AIP mutation is identified in a FIPA family, possible carriers should be tested for the mutation. Carriers should undergo regular clinical assessment and biochemical tests long term with a baseline mag­ netic resonance imaging (MRI) and occasional imaging follow-up. It needs to be considered that tumors sometimes are heterogeneous within a family. It is also important to emphasize that pituitary adenomas are commonly discovered on MRI imaging in the general population. Given the very young age of onset of pituitary disease in some families, predictive genetic testing of children in AIP mutation-positive families may be considered under the age of 4. In AIP muta­ tion-negative families all family members with 50% chance to inherit the gene should be clini­ cally followed up.

248

Summary The syndrome of FIPA has only recently been recognized as a clinical entity. Our knowledge of its heterogeneous clinical and genetic features has grown immensely in the last 3 years, but further novel clinical characteristics, genes and pathways need to be discovered to gain a full picture of this rare syndrome.

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 11

Somatostatin and somatostatin receptors: from basic concepts to clinical applications Maria Cristina De Martino
, Leo J. Hofland and Steven W. J. Lamberts Department of Internal Medicine, Division of Endocrinology, Erasmus Medical Center, Rotterdam, The Netherlands

Abstract: Somatostatin (SS) and SS receptors (ssts) are broadly expressed in the human body where they exert many physiological actions. Moreover, they can be expressed in many pathological tissues. Particularly, a high density of ssts has been described in human neuroendocrine tumors (NETs). SS and ssts have a therapeutic and diagnostic value in several clinical conditions. For this reason stable SS-analogues have been developed. Among SS-analogues, octreotide, octreotide long-acting-release (LAR), lanreotide-sustained-release (SR) and lanreotide autogel (ATG) are approved for clinical use and pasireotide is in a late phase of clinical development. Presently, the SS-analogues are the standard treatment option for acromegalic patients and play a prominent role in the symptomatic control of patients with gastroenteropancreatic-neuroendocrine tumors (GEP-NETs). SS-analogues are able to control hormonal hypersecretion and reduce tumoral growth in the majority of cases. However, some patients are resistant to SS-analogue treatment and other patients (often GEP-NETs), after a variable period of treatment, develop tachyphylaxis to these compounds. The mechanisms behind this treatment resistance or tachyphylaxis are presently under investigation. The understanding of these mechanisms might help to develop new treatment modalities for patients not responding to the currently available SS-analogues. The high tumoral expression level of ssts, characteristic of many NETs, has been the rational to develop radiolabelled SS-analogues to visualize sst-expressing tumors and to treat unresectable tumors. Indeed, SS-analogues coupled with 111In are used to perform sst-scintigraphy, which is a very useful first-line imaging technique in the diagnosis and follow-up of GEP-NETs. Moreover, SS-analogues conjugated to 111 In or to other radioisotopes, such as 177Lu or 90Y, have promising effects in the treatment of advanced NETs. ssts are expressed in some non-neuroendocrine tumors as well and in some non-tumoral diseases, suggesting that SS-analogues might have a role in the diagnosis and treatment of these pathological conditions as well. The development of novel SS-analogues with new pharmacokinetic and pharmacodynamic characteristics may further improve the clinical applications of such compounds. Keywords: somatostatin receptor; somatostatin; somatostatin analogue; neuroendocrine tumor; acromegaly


Corresponding author. Tel.: þ31-10-7034394; Fax: þ31-10-7035430; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82011-4

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pharmacodynamic properties of these compounds, their effects in different cell types and tissues and finally their clinical applications (Moller et al., 2003; Patel, 1999; Weckbecker et al., 2003). Presently, many SS-analogues are used for in vitro and in vivo studies and some of them have an important clinical use, particularly in the treat­ ment and diagnosis of neuroendocrine tumors (NETs). The name NETs generally refers to benign or malignant neoplasms originating from neuroendo­ crine cells, which belong to the diffuse endocrine system (pituitary, parathyroids, pancreatic islets, para-follicular areas of the thyroid, adrenal medulla, sympathetic ganglia, digestive and the

Introduction Since its discovery in 1973, many functional roles have been attributed to somatostatin (SS) (Lamberts et al., 1991). In the late 20th century, the genes of five somatostatin receptor (sst) sub­ types have been cloned and characterized (Patel, 1999). These discoveries initiated novel studies evaluating the expression of ssts subtypes in SS target tissues, as well as evaluating the intracellu­ lar mediators activated by SS binding to these receptors. Moreover, several SS-analogues (Fig. 1), with sst subtype binding profiles different from the natural SS, were developed and further studies were dedicated to characterize the

Gly

Ala

Cys

Lys

Ans

Phe Phe

S

Trp Lys

S Thr Cys

Ser

Thr

Phe

SS-14

D-Phe

D-βNal

Cys

Cys

Tyr

Phe S

S

D-Trp Lys

S

In-DTPA

D-Phe

Cys

Thr

Cys

Octreotide (SMS201-995)

111

2-aminoethyl-carbamoyl

177

Cys

Lu-DOTA

D-Phe

D-Trp

S

Phe

[111In-DTPA] Octreotide (Octreoscan)

90

S

Thr Cys

Tyr(Bzl)

Pasireotide (SOM230)

Tyr

S

Lys

S

Cys

Lys

HyPro

Val

Lanreotide (BIM 23014)

Phe

Thr-ol

Lys

S

Thr Thr-ol

D-Trp Phg

D-Trp

Y-DOTA

D-Phe

Cys Tyr

D-Trp

S

Lys

S

Cys

[177Lu-DOTA,Tyr3] Octreotate

Lys Thr

Thr Thr-OH

D-Trp

Thr-ol

Cys

[90Y-DOTA,Tyr3] Octreotide

Fig. 1. Structures of natural SS and the most clinically developed SS-analogues. Please see online version of this article for full color figure.

257

A1

B1

A2

B2

Fig. 2. Immunohistochemical detection of sst2A receptors in a human gastrinoma. (A) sst2A immunoreactivity, (B) preabsorption with the corresponding immunizing peptide demonstrating specificity of staining. A1 and B1: magnification 200×; A2 and B2: magnification 400×. Please see online version of this article for full color figure.

respiratory tracts). NETs originate from normal SS target tissues and generally display a high den­ sity of sst expression (Fig. 2). Therefore, the effects of SS-analogues in several kinds of NETs have been investigated, leading to the clinical use of these drugs to control symptoms of hormonal hypersecretion, and in some cases, to inhibit tumor growth, in patients with growth hormone (GH)- or thyroid-stimulating hormone (TSH)-secreting pituitary adenomas, gastroenteropancreatic­ neuroendocrine tumors (GEP-NETs) and more recently adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas and some other kinds of NETs. In the management of patients with NETs, the ultimate goal of the treatment is the complete removal or destruction of the tumor with, as much as possible, safe procedures. This approach would allow the cure of the patient, but unfortunately, this is often not possible. When cure cannot be achieved, a very important objective of the treatment is the control of clinical symptoms, the control of tumor growth, as well as the improvement of patient’s sur­ vival and quality of life. The management of patients with NETs may require a multidisciplinary approach and different kinds of treatment as surgery, radio­ therapy (mainly for pituitary tumors), biotherapy (SS-analogues, dopamine agonists, interferon-a),

peptide receptor radionuclide therapy (PRRT) using radiolabelled SS-analogues (mainly for GEPNETs), chemotherapy and chemoembolization. Unfortunately, in patients with the most aggressive tumors, many of these treatments only have a palliative effect and for them new treatment options are still required (Plockinger et al., 2009). The frequent homogeneous and high expres­ sion of ssts in NETs, particularly sst2, has been the rationale to develop radiolabelled SS-analo­ gues (Fig. 1) with high affinity for the sst2, which are used to visualize sst2-positive tumors (Lamberts et al., 2002a). SS-analogues conjugated with 111In or 177Lu are used to visualize sst-expressing tumors with sst-scintigraphy. Moreover, the con­ jugation of SS-analogues with 111In or other radio­ nuclides, such as 177Lu or 90Y, is a promising treatment option for patients with inoperable or metastatic sst-positive NETs (particularly GEP-NETs) (Forrer et al., 2007; Kwekkeboom et al., 2009a). Future developments in the field will probably follow several directions. These include the devel­ opment, and the further progression in clinical trials, of new compounds, acting as SS-agonist, with new pharmacokinetic and pharmacodynamic properties, the development and the clinical experimentation using SS-dopamine (DA) chimaeric compounds, acting via combined targeting of ssts and dopamine receptors (DRs). Hopefully, radiolabelled SS-ana­ logues will be used in all reference centres for the treatment of patients with NETs, leading to the routine use of sst-scintigraphy in the diagnosis of these tumors and to a better standardized and more widespread use of PRRT. The development of SS-analogues linked to chemotherapeutic com­ pounds may lead to sst-based targeted chemother­ apy. Combining SS-analogues with other kinds of treatment, such as other biotherapies [dopamine agonist, mammalian target of rapamycin (mTOR) inhibitors], is currently under investigation. The availability of new compounds or combination treatment strategies, together with novel advances in understanding the sst expression and their function in NETs, in some non-neuroendocrine tumors and in some non-tumoral diseases may lead to new diagnostic and/or treatment indications for SS-analogues or their conjugated compounds.

258

Somatostatin, somatostatin receptors, signalling and molecular interactions SS is a cyclic peptide that in mammals exists in two biologically active isoforms, consisting of 14 (SS-14) and 28 (SS-28) amino acids. In humans, both these isoforms are derived from the cleavage of a common 116-amino-acid precursor, encoded by a single gene located on chromosome 3q28 (Moller et al., 2003; Patel and Galanopoulou, 1995). In SS, a particular tetra-amino-acidic sequence (Phe8–Trp8–Lys9–Thr10) was found to be essential for the binding to the specific membrane receptors (Fig. 1) (Veber et al., 1979) that mediate the physiological actions of SS (Lamberts et al., 1996). Up to date, five human sst subtypes have been characterized and cloned. The subtypes have a 40–60% identical amino acid sequence (Lamberts et al., 1996; Moller et al., 2003). In humans, there are five different genes each encoding for one specific subtype of sst. The human transcript of the sst2 gene can result in two splice variants that differ only for the length of the cytoplasmatic portion (sst2A and sst2B). All ssts bind SS with a similar high affinity (Table 1) (Hofland and Lamberts, 2003; Patel, 1999). Recently, the SS/sst system has become more complex by the discovery of a third actor, that is, cortistatin (CST) (de Lecea et al., 1996). In humans, CST exists in two biologically active isoforms, consisting of 17 (CST-17) and 29 (CST­ 29) amino acids. Both isoforms are derived from

the cleavage of a common precursor and, similar to SS, they bind with high affinity to all the five ssts (Dalm et al., 2008; Fukusumi et al., 1997; Patel, 1999). Novel putative CST-specific receptors have been proposed, but none of them have been definitely considered very convincing (Dalm et al., 2008; Siehler et al., 2008; Volante et al., 2008). ssts belong to the seven-transmembrane segment receptor superfamily and functionally couple to G-proteins (Hofland and Lamberts, 2003; Lamberts et al., 1996; Moller et al., 2003). ssts share common signal transduction pathways, such as the inhibition of adenylate cyclase and calcium channels, as well as the stimulation of potassium channels and phosphotyrosine phosphatase (PTP) (Hofland and Lam­ berts, 2003; Schonbrunn, 2008). PTPs are proposed to be involved in the inactivation of several growth factor receptors [platelet-derived growth factor receptor (PDGF-R), vascular endothelial growth factor receptor type 2 (VEGF-R2), insulin receptor (insulin-R), epi­ dermal growth factor receptor (EGF-R)] and their mediator (ERK1/2), as well as in the activation of proteins involved in cell–cell and cell–matrix adhesion. PTPs are considered as inhibitors of growth factor-regulated cell prolif­ eration and invasiveness, and are probably the major mediator of SS-induced anti-neoplastic effects (Florio, 2008). Among the ssts, sst3 appears to be the subtype mostly related to the pro-apoptic and

Table 1. SS-analogues binding affinity (IC50 nmol/l) to the different ssts Compound

SST1

SST2

SST3

SST4

SST5

DR2

SS-14 Octreotide Lanreotide Pasireotide BIM-23244 BIM-23A760

0.93–2.26a–d 280–1140a–e 180–2330a–e 9.3c >100a,d,e 662–853a,d

0.15–0.26a–d 0.38–0.6a–e 0.54–0.75a–e 1c 0.29–0.3a,d,e 0.03a,d

0.56–1.43a–d 7.1–34.5a–e 14–107a–e 1.5c >100a,d,e 52–160a,d

1.5–1.77a–d >1000a–e 230–2100a–e >100c >1000a,d,e >1000a,d

0.29–1.4a–d 6.3–7a–d 5.2–17a–e 0.16c 0.67–0.7a,d,e 3.1–42a,d

ND ND ND ND ND 15a,d

ND: not determined. a Saveanu et al. (2006). b Shimon et al. (1997). c Bruns et al., (2002). d Jaquet et al. (2005). e Ren et al. (2003).

259

anti-proliferative effects of SS and SS-analogues (Hofland and Lamberts, 2003). The selectivity of SS actions in different tissues is probably related to the differential expression of sst subtypes and their intracellular transduction pathways in the different normal and pathological tissues (Lamberts et al., 1996). Recently, it has been suggested that different SS-analogues, in the same cell type, may elicit differential effects, by the activation of different subsets of intracellular mediators. This phenom­ enon seems to depend on the typical agonist– receptor interactions (Schonbrunn, 2008). Similar to many other G-protein-coupled receptors, ssts undergo agonist-induced endocy­ tosis following the binding of SS to the recep­ tors. Subsequentially, the internalized sst is directed to different intracellular compartments, leading to either recycling or degradation of ssts (Hofland and Lamberts, 2003; Tulipano and Schulz, 2007). This phenomenon seems to be different among the different sst subtypes, being more evident for the sst2, sst3 and sst5 than for others. Moreover, while the recycling seems to be the most common process following the internalization of sst2 and sst5, degradation seems to be the most common for sst3 (Jacobs and Schulz, 2008). This intracellular traf­ ficking of ssts seems to be more pronounced in some tissues than in others (i.e. different in differ­ ent kinds of tumors). The co-expression of different sst subtypes or the presence of different intracellu­ lar components involved in this trafficking could form the basis for these differences (Hofland and Lamberts, 2003; Jacobs and Schulz, 2008; Tulipano and Schulz, 2007). The internalization of ssts is a mechanism potentially useful to treat sst-expressing tumors because it can help to bring radioisotopes or chemotherapeutic compounds into tumor cells, providing the basis for targeted radiotherapy or chemotherapy. On the other hand, the degrada­ tion of ssts may induce downregulation of the receptors, one of the process possibly involved in the tachyphylaxis to SS-analogue treatment (Hofland and Lamberts, 2003). Other mechan­ isms, probably involved in tachyphylaxis, are receptor uncoupling from second messenger

activation, the overgrowth of ssts-negative clones or mutations in ssts genes (Hofland and Lam­ berts, 2003; Tulipano and Schulz, 2007). Cells expressing ssts often express other kinds of seven-transmembrane segment receptors, which can reciprocally influence their activity. ssts and DR have been reported to physically interact by forming hetero-dimers with enhanced functional activity (Rocheville et al., 2000). These hetero­ dimers can also modulate the agonist-induced desensitization of the individual receptors (Hofland and Lamberts, 2003; Tulipano and Schulz, 2007). The above mechanisms, together with the het­ erogeneous expression of ssts in pathological tis­ sues, cause the variable spectrum of clinical responses observed when SS-analogues are used to treat human diseases. Moreover, the thorough understanding of these processes is crucial to develop novel SS-analogues that could improve the clinical utility of this class of compounds.

SS-analogues SS has functions that can have a therapeutic (and diagnostic) value in several clinical conditions. However, the use of the natural SS isoforms, due to their short half-life (less than 3 minutes) would require continuous intravenous administration (Lamberts et al., 1996). SS-analogues are com­ pounds that bind ssts, but differ from SS with respect to pharmacokinetics and pharmacody­ namics. Moreover, they can be conjugated with radioisotopes or chemotherapeutics for receptortargeted therapies (Weckbecker et al., 2003). Presently, the current clinically available octa­ peptide SS-analogues are a standard treatment option for acromegalic patients and symptomatic patients with GEP-NETs. Octreotide (SMS201-955) acetate (Fig. 1) has been the first stable molecule available for clinical use. It is a cyclic octapeptide, similar to SS, but with a considerable longer half-life (about 2 hours), and different binding properties to ssts. Octreotide binds with high affinity to sst2 and a relative high affinity to sst5, but with moderate affinity to sst3 and low affinity to sst1 and sst4 (Table 1) (Hofland and Lamberts, 2003; Lamberts

260

et al., 1996). It can be administered intravenously (i.v.) or subcutaneously (s.c.). The standard dosage for the treatment of NETs varies between 100–1500 mg/day s.c., divided in 2–4 administra­ tions (Ben-Shlomo and Melmed, 2008a; Oberg et al., 2009). Octreotide long-acting-release (LAR) is a depot formulation consisting of octreo­ tide acetate encapsulated in microspheres that, administrated intramuscularly (i.m.), gradually releases the octreotide. The standard dosage is 10–40 mg/month (Ben-Shlomo and Melmed, 2008a; Oberg et al., 2009). Lanreotide-sustained-release (SR) is the sus­ tained-release formulation of the SS-analogue lan­ reotide (BIM-23014) (Fig. 1), which has high binding affinity for sst2 and a relative high binding affinity for sst5 (Table 1) (Hofland and Lamberts, 2003; Lamberts et al., 1996, 2002a; Oberg, 2009). The standard dosage is 30–60 mg i.m. every 7–14 days. Recently, lanreotide autogel (ATG), the depot formulation of lanreotide (deep s.c. injection, every 30–60 days), is replacing the lanreotide-SR (Feelders et al., 2009; Oberg, 2009; Oberg et al., 2009). The availability of long-acting depot formula­ tions, compared to the daily administration of octreotide s.c., has considerably improved the acceptance and the compliance of patients to the treatment (Lamberts et al., 2002a). The above-indicated SS-analogues are generally safe and well tolerated. Side effects include abdom­ inal discomfort, bloating and sometimes steator­ rhoea, but these usually subside within a few weeks. In some patients treated for a long time, it is possible to observe the development of gallstones and persistent steatorrhoea (de Herder, 2005). Octreotide and lanreotide have a significantly higher affinity for sst2 than for sst5 (Table 1). Sev­ eral SS-analogues with different binding affinity have been developed and used for in vitro and in vivo studies (Weckbecker et al., 2003). Among these, an interesting compound is the BIM­ 23244, a SS-analogue with comparable high bind­ ing affinity for sst2 and sst5. In vitro studies using this compound have demonstrated that a co-acti­ vation of sst2 and sst5 suppresses GH production in octreotide-resistant GH-secreting adenomas,

suggesting that this kind of compounds could improve the clinical utility of SS-analogues (Saveanu et al., 2001). Pasireotide (SOM-230) is a novel SS-analogue that binds with high affinity to sst2, sst3 and has a very high binding affinity to sst5 (Fig. 1; Table 1). In vitro data show that pasireotide has a higher affinity and functional activity for sst5 compared to octreotide. Studies in different animal species sug­ gest that pasireotide produces a stronger and more long-lasting suppression of insulin like growth factor type I (IGF-I) production than octreotide (Schmid, 2008). In animal models, pasireotide induced tumor shrinkage, while octreotide only stabilized tumor size, suggesting that pasireotide is stronger than octreotide in inhi­ biting tumor growth (Schmid, 2008). Preclinical and clinical data support the potential clinical use of pasireotide for the treatment of patients with acromegaly, Cushing’s disease (CD) and of patients with GEP-NETs not fully responding to octreotide (Schmid, 2008). The hetero-dimerization of ssts with DRs has been the rationale to develop new compounds that can bind and activate at the same time one or more subtype of ssts and DR type 2 (D2) and also their dimers (Ferone et al., 2007). Among these compounds, BIM-23A760 is a preferential agonist for sst2 and D2. In vitro studies demon­ strated that this compound is more potent than octreotide in suppressing GH secretion in GH-secreting adenomas (Jaquet et al., 2005). The above-listed SS-analogues in the clinical setting can be defined “cold” analogues to differ­ entiate them from SS-analogue coupled to radio­ active molecules that have been developed and are presently used for the diagnosis and treatment of NETs (see below).

Physiological role and distribution of SS and ssts in normal tissues SS and ssts are widely distributed in the human (Table 2) and rodent body and a differential expression of the two native SS and of the five ssts have been found in different cell types and tissues (Moller et al., 2003; Patel, 1999). Among

261

Table 2. Distribution of ssts in the human body SST1

SST2

Caput and neck

Cerebrum1,2 Hypothalamus2 Pituitary3,4 Retina5 Parathyroid6 Thymus7

Cerebrum1,2 ,8

Cerebellum2

Pituitary2 ,3,4 Retina5 Thyroid6 Thymus7

Thorax Lung1 Bronchial gland6 Heart11

Lung12 Bronchial gland6 Heart11

Abdomen and pelvis Stomach6,8,14 Small intestine6,8

Colon and rectum6 ,15 Colon smooth cells16

Kidney6,17 6

Liver

Pancreas1,6 ,18,19 20

Adrenal Placenta21

SST3







SST5







Cerebrum1,2 ,9

Cerebellum2

Pituitary2 ,4 Retina5 Thyroid6 Parathyroid6 Thymus7

Cerebrum1,2

Cerebellum2 Retina5 Thyroid6 Parathyroid6

Cerebellum2 ,10

Pituitary2 ,3,4,10 Retina5 Thyroid6 Parathyroid6

Bronchial gland6

Lung1,13 Bronchial gland6 Heart11

Bronchial gland6 Heart10 Skeletal muscle10

Stomach6,12 ,14,22 Small intestine6,12 Colon and rectum6,15,23 Colon smooth cells16



Kidney6 ,8,17 ,24 6

Liver



Pancreas6 ,18,19 25 Spleen

Adrenal20 26,27 Prostate Endometrium28

Stomach6,14

Small intestine6

Colon and rectum6 ,15 Colon smooth cells16

Kidney17 1,6

,9,18,19

Pancreas

Adrenal20

Stomach6,14

Small intestine6 Colon and rectum6,15



Kidney6 ,17 6

,18,19

Pancreas

Adrenal20 26 Prostate Placenta21

Stomach6,14 Small intestine6,10

Colon and rectum6 ,15 6

,17

Kidney

Liver6



Pancreas6 ,18,19 10,20

Adrenal Prostate26 Placenta10

Bone marrow29 PBMC
30 T- and B-cell lines31 Monocytes/ macrophages/ dendritic cells30,32

PBMC
30 T-lymphocytes30 B-lymphocytes30 T- and B-cell lines31









Immune system




SST4

PBMC: peripheral blood mononuclear cell;

the expression of different ssts in these organs have been reported to be different in different cell types or different areas of the same organ. 1 21 Rohrer et al. (1993). Caron et al. (1997).

2 Thoss et al. (1996). 22 Zaki et al. (1996).

3 Miller et al. (1995). 23 4 Casini Raggi et al. (2002).

Panetta and Patel (1995). 5 24 Klisovic et al. (2001). Reubi et al., (1993).

6 Taniyama et al. (2005). 25 Reubi et al., (2001).

7 Ferone et al. (1999). 26 8 Sinisi et al. (1997).

Yamada et al. (1992a). 9 27 Yamada et al. (1992b). Reubi et al., (1995).

10 O’Carroll et al., (1994). 28 Green et al. (2002).

11 Smith et al. (2005). 29 12 Oomen et al., (2000).

Gugger et al., (2004). 13 30 Varecza et al. (2009). Lichtenauer-Kaligis et al. (2004).

14 Le Romancer et al. (1996). 31 van Hagen et al. (1999).

15 Laws et al., (1997). 32 16 Dalm et al. (2003).

Corleto et al. (2006). 17 Bhandari et al. (2008). 18 Kumar et al. (1999). 19 Strowski and Blake (2008).

262

the physiological actions of SS, the most well known are the inhibitions of GH, insulin, glucagon and gastrin secretion. However, SS also inhibits the secretion of other hormones (particularly other gastrointestinal and pituitary hormones) and a number of exocrine secretions. SS acts as a neurotransmitter in the nervous system, it modu­ lates gastrointestinal motility and immune cell activity and it displays anti-proliferative effects in some tumoral cell types (Siehler et al., 2008). CST is widely expressed throughout the human body and seems to play a role in the nervous and immune systems (Dalm et al., 2008). In the human central and peripheral nervous system, SS is produced in many regions, but it is particularly abundant in the deeper layers of the cortex, in limbic areas, in the striatum, in the periaqueductal central grey, in the areas of major sensory pathways and in the hypothalamus (80% in the anterior periventricular nucleus with axonal projections to the median eminence) (Patel, 1999). In the rat central nervous system, ssts are abun­ dantly expressed in the deeper layers of the cere­ bral cortex and in large areas of the limbic system (Reubi and Maurer, 1985). The subtype expres­ sion at the mRNA and protein level displays high distinct patterns of distribution (Dournaud et al., 1998; Patel, 1999). Thoss et al. demonstrated in the human brain the presence of a sst distribution pattern overall similar to that previously reported in the rat brain (Thoss et al., 1996). However, in human brain, different from the rat, sst5 mRNA expression appeared to be very low and restricted to the cerebellum and pituitary (Thoss et al., 1996). SS modulates cognitive (i.e. the transition between the sleep phases and the consolidation of long- and short-term memory), locomotor, sen­ sory and autonomic activity. However, some of these functions have been supposed to be more specifically related to the effect of CST (Dalm et al., 2008; Siehler et al., 2008). SS inhibits the secretion of noradrenalin, thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH) and SS itself, in the hypothalamus, and of dopamine from middle brain (Patel, 1999). The mRNA of all five receptors has been found in adult rat and foetal human pituitary (Panetta and Patel, 1995; Patel, 1999), while the mRNA of

sst4 has not been found in the human adult pitui­ tary gland (Panetta and Patel, 1995). The inhibi­ tion of GH is the most important role of SS in the pituitary and seems to be predominantly mediated by sst2 and sst5 (Moller et al., 2003; Shimon et al., 1997). In the normal pituitary gland, SS also inhi­ bits basal and stimulated TSH secretion (Patel, 1999) and it has been suggested that SS has reg­ ulatory effects on ACTH release under physiolo­ gical conditions, and that sst (in particular sst2) expression is under negative regulatory control by glucocorticoids (Hofland, 2008). In the human endocrine pancreas there is a dis­ crete population of cells that produce SS (D-cells), which is involved in the regulation of glucagon and insulin secretion (Dubois, 1975; Patel, 1999). Studies performed with immunohistochemistry have demonstrated the presence of all five sst sub­ types in the human pancreas. However, the patterns of specific subtype expression vary in the different studies (Strowski and Blake, 2008). Strowski and Blake recently reviewed the function and expres­ sion of ssts in the endocrine pancreas and concluded that sst2 seems to be the most expressed subtype in human pancreatic A- and B-cells, suggesting a major role of this receptor as insulin and glucagon secretion inhibitor. However, the data regarding the cell-specific expression of other ssts and the poten­ tial role of these receptors in endocrine pancreatic functions are still controversial (Strowski and Blake, 2008). In the gastrointestinal tract (GIT) SS is produced by specialized neuroendocrine cells of the mucosa and by neurons of the submucosal and mysenteric plexuses. The expression of ssts has been found in several cells and tissues of the GIT, where SS inhibits several endocrine and exocrine secretions (gastric acid, pepsin, bile, colonic fluid) and generally suppresses the smooth muscular moti­ lity (Patel, 1999; Reubi, 1992). In many other rat and human tissues, the expres­ sions of SS, CST and ssts have been found. Particu­ larly during the past decade, several studies have characterized the ssts subtype expression in the human immune system. SS seems to inhibit immu­ noglobulin synthesis and lymphocyte proliferation in lymphoid tissues. However, the role of SS and CST in these tissues and cells still needs to be addressed (Dalm et al., 2008; Moller et al., 2003).

263

Distribution of ssts in pathological tissues sst expression has been described in several patho­ logical tissues, particularly in tumors and inflam­ matory tissues (Dalm et al., 2008; Reubi, 2003; Reubi et al., 1997). High expression levels of sst have been found in NETs, which often originate from SS target tissues, such as pituitary adenomas, GEP-NETs (Fig. 2), paragangliomas, pheochromocytomas, medullary thyroid carcinomas (MTCs) and small cell lung cancers (SCLCs) (Grozinsky-Glasberg et al., 2008; Kvols et al., 1992; Lamberts et al., 1992; Reubi, 1997; Reubi et al., 1992a, 1992c; Schaer et al., 1997). Moreover, ssts have also been found to be expressed in other kinds of tumors, such as breast and prostate cancers, malig­ nant lymphomas, meningiomas, astrocytomas, etc. (Lamberts et al., 2002a; Reubi et al., 1992b, 1992d; Vikic-Topic et al., 1995; Volante et al., 2008). In the majority of human sst-expressing tumors, sst2 is the most abundantly expressed subtype and sst4 the least expressed. Moreover, a wide heterogeneity of sst subtype expression has been reported in different tumors and within the same tumor (Grozinsky-Glasberg et al., 2008; Hofland and Lamberts, 2003; Volante et al., 2008). The expression of ssts has also been found in several non-tumoral pathologies, particularly in inflammatory and autoimmune disease, such as in the lesions of granulomatose disease and rheu­ matoid arthritis (RA) (Dalm et al., 2008; van Hagen et al., 2008).

Clinical use of sst expression for the diagnosis of disease The role of ssts in the diagnosis of tumors Currently, 111In-pentereotide (OctreoScan), a [111In-diethylenetriamine-pentaacetic acid (DTPA)] conjugate of octreotide (Fig. 1), is the first-choice radiolabelled SS-analogue to perform sst-scintigra­ phy (de Herder et al., 2006a; Kwekkeboom et al., 2009b). This compound (tracer) is injected in

patients and is selectively accumulated in tissues with high sst2, after which planar and emission computed tomography images are made with a g-camera. This technique allows the in vivo visuali­ zation of sst2 expressing pathological tissues (de Herder et al., 2006a; Lamberts et al., 2002a). Alternatively, 99mTc-EDDA-hydrazinonicotinyl (HYNIC)-Tyr3-octreotide (Tc-TOC), or 99mTc EDDA/HYNIC-octreotate can be used (Gabriel et al., 2005a, 2005b; Hubalewska-Dydejczyk et al., 2005). The clinical use of this technique in the initial analysis of patients with NETs, mainly to image primary and metastatic tumors, is important to determine the optimal treatment (Lamberts et al., 2002a). Particularly, OctreoScan is the first option imaging technique in the diagnosis of GEPNETs, with the exception of insulinomas (de Her­ der et al., 2006a, 2006b). Guidelines for the standard use of 111In-pente­ treotide scintigraphy in the management of patients with NET have been recently published from European Neuroendocrine Tumor Society (ENETS) (Kwekkeboom et al., 2009b). The com­ mon indications of 111In-pentetreotide scintigraphy have been reported in the above-mentioned guide­ lines and they include (a) the detection and localiza­ tion of a variety of suspected NETs and some nonneuroendocrine tumors and their metastases, (b) the definition of the staging in patients with NETs, (c) the determination of sst status (patients with sst­ positive tumors may be more likely to respond to octreotide therapy), (d) the follow-up of patients with known disease to evaluate potential recur­ rence, (e) the selection of patients with metastatic tumors for PRRT and (f) the prediction of the effect of PRRT (Balon et al., 2001; Kwekkeboom et al., 2009b). 111 In-pentetreotide scintigraphy can visualize sst2-positive tumors with high sensitivity, but rela­ tively low specificity, because several nontumoral, pathological conditions, such as immune diseases, surgical scar tissues, respiratory infec­ tions, and normal healthy organs, such as the pituitary, thyroid, spleen, liver, and renal parench­ yma, also show uptake of the tracer and can thus be visualized. Moreover, the gallbladder, bowel, renal collecting systems, ureters and urinary

264

bladder are visualized with 111In-pentetreotide scintigraphy as well because they are involved in the clearance of the tracer (Balon et al., 2001). The sensitivity of 111In-pentetreotide scintigra­ phy is different in different kinds of tumors (Balon et al., 2001; Kwekkeboom et al., 2009b). Indeed, in the diagnosis of pituitary adenomas, GEP-NETs (except insulinomas), paragangliomas, SCLC and meningiomas the reported detection rate with sst­ scintigraphy is higher than 75%, indicating a very high sensitivity of this imaging technique in detect­ ing these tumors. An intermediate sensitivity (detection rate 40–75%) has been reported in the diagnosis of insulinomas, MTC, pheochromocy­ toma, breast cancer and lymphomas (Balon et al., 2001; Kwekkeboom et al., 2009b). The lower sensitivity of 111In-pentetreotide scintigraphy in detecting insulinomas, particularly the benign subtype, compared to others pancreatic NETs could be related to the lower expression of the sst2 subtype in these tumors (de Herder et al., 2006b). In addition, the limitations of the techni­ que in detecting pheochromocytomas seems to be mainly due to the kidney uptake of the tracer that can cover the uptake of the adrenal mass (Balon et al., 2001). In patients with pituitary tumors, the use of sst­ scintigraphy has been proposed in order to select patients for SS-analogue treatment or to differ­ entiate scar tissue from tumor recurrence after pituitary surgery. However, sst-scintigraphy seems not to be cost-effective for this indication and presently it does not play a relevant role in clinical diagnosis and follow-up of patients with these tumors (de Herder et al., 2006a). In the diagnosis of pheochromocytomas, 111In­ pentetreotide scintigraphy has to be considered as a secondary option after 123I-MIBG scintigraphy (a technique with a higher sensitivity for the diag­ nosis of these tumors). However, it can have an important role in the staging of malignant pheo­ chromocytomas and in the detection of extraadrenal pheochromocytomas and paragangliomas (de Herder et al., 2006a; Gabriel et al., 2005b; Tenenbaum et al., 1995; van der Harst et al., 2001). In patients with MTC, sst-scintigraphy has been used as complementary imaging technique to

computerized tomography (CT) or 18FDG-positron emission tomography (PET). Presently, the treatment with SS-analogues and radiolabelled SS-analogues is not generally accepted in these patients. Therefore, sst-scintigraphy does not play a crucial role to determine the optimal treatment and it is not routinely used in the management of these patients (de Herder et al., 2006a). 111 In-pentetreotide scintigraphy shows high sensitivity for the detection of the extension of disease in patients with lymphomas, particu­ larly in Hodgking’s disease and for the detec­ tion of supradiaphragmatic localizations (Dalm et al., 2008).

The role of ssts in the diagnosis of non-tumoral diseases New potential fields in which sst-scintigraphy may have a role in the diagnosis are immune-mediated diseases. However, only few studies have been performed and although they seem to be promis­ ing, further evaluations are still necessary (Dalm et al., 2008). An increased uptake of 111In-pentetreotide, and more recently of (99m)Tc-HYNIC-octreotide, has been found in the orbital tissues of patients with active thyroid-associated ophthalmopathy. There­ fore, it has been proposed that sst-scintigraphy could have a role in the follow-up of patients with these diseases (Krassas, 2002; Sun et al., 2007). The use of 111In-pentetreotide scintigraphy in patients with granulomatous diseases has been evaluated in a study considering 13 patients with sarcoidosis, 4 patients with tuberculosis and 3 patients with Wegener’s disease. In this study all patients were positive at sst-scintigraphy, suggest­ ing a high sensitivity of this technique in detecting granulomatous diseases (Dalm et al., 2008; van Hagen et al., 1994a). The use of [111In-DTPA]-octreotide scintigraphy has been evaluated in a cohort of 14 patients with RA and 4 patients with severe osteoarthritis (OA) (van Hagen et al., 1994b). In this study, the majority of painful or swelling joints of patients with RA were visualized with the scintigraphy and the degree of pain and swelling was well correlated

265

with the positive scintigraphy findings in the joints. Moreover, in patients with OA, the uptake of tracer in the affected joints was significantly lower than that in patients with RA (van Hagen et al., 1994b). These results suggest a higher uptake of [111In-DTPA]-octreotide in active RA lesions, but the potential value of sst-scintigraphy in the clinical evaluation of patients with active RA has not been addressed yet (Dalm et al., 2008).

Clinical significance of sst expression in the treatment of disease The role of ssts in the treatment of NETs GH-secreting pituitary tumors GH production from GH-secreting adenomas causes acromegaly, a chronic, systemic and invali­ dating disease. Human GH-secreting pituitary tumors express multiple sst subtypes, but sst2 and sst5 are generally the most abundantly expressed (van der Hoek et al., 2007). SS-analogues, parti­ cularly the long-acting formulations, represent a cornerstone in the medical treatment of acrome­ galic patients. First-line surgery is indicated in patients with small-enclosed tumors, without systemic compli­ cations, or in those requiring immediate tumor debulking because of acute chiasmatic syndrome or presenting other emergency conditions. Primary medical therapy with SS-analogues is indicated in patients who have low chances to be cured by neurosurgery or with serious

co-morbidity. Moreover, it may be a reasonable choice in elderly patients (Feelders et al., 2009; Melmed et al., 2009). SS-analogues play an important role as adjuvant treatment, in patients not cured by neurosurgery, in which tumor debulking could increase the response to these compounds (Feelders et al., 2009). In patients to be treated with radiotherapy, medical treatment is useful to control, at least in part, the disease during the waiting time to obtain the maximal effects from radiotherapy (Landolt et al., 2000). Moreover, it has been suggested that SS-analogues before surgery might amelio­ rate the patient’s general conditions and positively influence the outcome of surgery (Feelders et al., 2009; Melmed et al., 2009). Long-term therapy can suppress GH secretion by GH-secreting adenomas, thereby normalizing IGF-I levels in about 40–80% of patients with acromegaly (Table 3) (Ben-Shlomo and Melmed, 2008b; Colao et al., 2009a; Feelders et al., 2009; Freda et al., 2005; Lamberts et al., 2002b; Melmed et al., 2009). In these patients the biochemical con­ trol is often associated with improvement of the clinical syndrome, but the biochemical control does not necessary fully correspond to the co-mor­ bidities and vice versa (Colao et al., 2004; Colao et al., 2009a; Feelders et al., 2009; Lamberts et al., 1996; Melmed et al., 2009). SS-analogues can induce a significant tumor shrinkage (>20%) in about 75% of treated patients (mean 50% reduction in tumor volume) (Fig. 3) (Bevan, 2005; Melmed et al., 2005, 2009). The higher percentage of patients who show

Table 3. Effects of SS-analogues on hormonal production and on tumor shrinkage in acromegalic patients Percentage of patients meeting efficacy criteriaa

Percentage of patients meeting tumor shrinkage criteriab

Therapy

GH

IGF-I

Primary therapy

Secondary therapy

All

Octreotide-SC Octreotide-LAR Lanreotide-SR All patients

53 57 48 52

54 67 47 55

51 80 31 52

27 28 9 21

45 57 24 42

a b

Freda et al. (2005). Bevan (2005).

266 A.

B.

Fig. 3. MRI images showing shrinkage of a GH-secreting tumor in an acromegalic patient before (A) and after (B) 12-months treatment with octreotide LAR 30 mg/28 days. (Acromegalic patient from the series of Professor Annamaria Colao, Department of Endocrinology, Federico II University of Naples, Italy.)

tumor shrinkage compared to the biochemical control suggest that these two effects are mediated, at least in part, by different molecular mechanisms (Table 3) (Bevan, 2005). The rate of shrinkage has been found to be higher in de novo treated patients. Several studies have tried to identify predictors of tumor response such as the tumor size, the biochemical response or the dosage of the drugs but until now they have produced equivocal results (Bevan, 2005). Table 3 shows the data regarding the effects of SS-analogues on hormonal production [as reported by Freda et al. (2005) in a recent meta-analysis] and on tumor shrinkage [as reported by Bevan et al. (2005)]. Ben-Shlomo and Melmed (2008b) recently reviewed this thopic as well. In their review the authors included data on the effects of lanreotide-ATG and they divided the group of patients treated with

lanreotide-SR into two groups, according to the dosage of lanreotide-SR used. They showed that lanreotide-ATG is able to control the hormonal production in about 50% of treated acromegalic patients. Moreover, they reported stronger effects of lanreotide-SR on hormonal production than the effects reported by Freda et al., parti­ cularly at high dosage. However, all these data were derived from different studies, with often variability in the sample selection, in time of therapy, dosages of drugs used and the evalua­ tion of the study outcomes. Therefore, the exact percentage of patient responders or non-respon­ ders to the different SS-analogues is still matter of discussion. Acromegalic patients sensitive to SS-analogs treated with these drugs generally do not present tachyphylaxis and can be long-term controlled (Hofland and Lamberts, 2003).

267

Recent studies suggest that in acromegalic patients, primary therapy with SS-analogues is an effective alternative to surgery, and treatment may be safely continued in patients with acromegaly, according to the individual indications and prefer­ ences of the patient (Colao et al., 2009a, 2009b). Octreotide and lanreotide seem to be equally effective; however, randomized clinical trials that directly compare the effects of the two compounds are still lacking (Feelders et al., 2009). A recent prospective randomized controlled clinical trial suggests that high-dose treatment with octreotide-LAR (up to 60 mg/28 days) could be a safe and effective option for acromegalic patients not completely controlled by standard dose of SS-analogues (Giustina et al., 2009). Dopamine agonists can enhance the effects of SS-analogues (Flogstad et al., 1994). SS-analogues can be used in combination with other dopamine agonists or the GH receptor antagonist pegvisomant in acromegalic patients who require medical treatment and whom are not controlled with SS-analogues on monother­ apy. In particular cases, the co-administration of SS-analogues can be considered in patients responding to pegvisomant monotherapy because it can reduce the required pegvisomant dose, resulting in better cost-effectiveness (Feelders et al., 2009; Melmed et al., 2009). Preclinical data suggest that pasireotide can be useful in the treatment of acromegalic patients, particularly the subset resistant to other SS-analo­ gues. Indeed, in vitro studies demonstrated that pasireotide is effective in lowering the GH production in primary cultures of human GHsecreting pituitary tumors (eight of nine were responders) including one octreotide-resistant adenoma (Hofland et al., 2004). Early phase clin­ ical trials have shown that pasireotide is more powerful than octreotide in lowering GH levels in acromegalic patients (van der Hoek et al., 2004a, 2005). Phase II clinical trials have recently been completed. Preliminary data show that shortterm treatment with pasireotide efficiently con­ trols GH and IGF-I and reduces tumor volume in patients with acromegaly (Schmid, 2008). Promising in vitro results suggest that the clin­ ical development of chimaeric compounds,

targeting sst2/D2 or sst2/sst5/D2 receptors may offer a further opportunity for the treatment of patients resistant to conventional treatments (Feelders et al., 2009).

Other pituitary tumors ACTH-secreting pituitary adenomas cause CD, the most common cause of endogenous hypercor­ tisolism, a systemic clinical condition associated with severe morbidities and an increased mortality (Biller et al., 2008; Pivonello, et al., 2008). The first-line treatment of CD is surgery, but remission rates range between 60 and 80% and disease recurrence is frequent. Therefore, medical treatment is often required (Biller et al., 2008; Pivonello et al., 2008). The majority of ACTH-secreting adenomas express the mRNA of sst1, sst2 and sst5, but the currently available sst2-preferring analogues are not able to reduce in vivo ACTH and cortisol secretion (Hofland, 2008). It has been suggested that the low expression of sst2 proteins on the cell surface due to a hypercortisolism-induced downregulation of sst2 on the tumor could be responsible for this lack of inhibition (de Bruin, et al., 2009; Hofland, 2008; Schmid, 2008; van der Hoek et al., 2004b). The sst5 is the predominantly expressed sst in ACTH-secreting adenomas and it appears to be more resistant to the hypercotisolism-induced downregulation (Hofland, 2008). In human ACTH-secreting adenoma cell cul­ tures, pasireotide was more potent than octreotide in suppressing the ACTH release and was able to suppress cell proliferation (Batista et al., 2006; Hofland et al., 2005). Therefore, sst5 seems a promising target for medical treatment in CD (Hofland, 2008; Hofland et al., 2005; Schmid, 2008). A phase II clinical trial, in patients with CD, testing short-term effectiveness of pasireo­ tide, has recently demonstrated that 15 days pasir­ eotide treatment lowers the 24-hour urinary cortisol secretion in the majority of patients (76%) (Boscaro et al., 2009). However, only 20% of patients normalized with respect to the 24-hour urinary cortisol secretion. Anyway, this

268

study evaluated the effects of short-term treat­ ment, while the results of longer clinical trials are still necessary to better evaluate the long-term effectiveness of pasirotide in the treatment of patients with CD. The majority of ACTH-secreting adenomas express D2 (Pivonello et al., 2004) and treatment with the dopamine agonist cabergoline has been recently proposed as an optional treatment in patients with CD unsuccessfully treated by surgery (Pivonello et al., 2009). Therefore, therapy co-targeting sst and D2, using a combination of the available compounds or by using the new compounds, such as the SS-dopamine chimaeras, could be future treatment options for patients with CD. TSH-secreting pituitary adenomas, a very rare cause of hyperthyroidism, often express ssts (Lamberts et al., 1996). Although surgery is the first-line treatment for TSH-secreting adenomas, it is often not radical, presenting a success rate lower than 50%, probably because these tumors tend to be large and highly fibrotic (Beck-Peccoz and Persani, 2002). SS-analogues can play an important role, as adjuvant treatment, in patients not cured by sur­ gery or in the waiting time for the complete effects of radiotherapy. Moreover, they can play a role as primary treatment in the waiting time for surgery or as primary treatment in patients not candidate for surgery (Beck-Peccoz and Persani, 2002; van der Hoek et al., 2007). SS-analogues are able to control TSH secretion and the subsequent free triiodothyronine (FT3) and free tetraiodothyronine (FT4) level in the majority of patients having a TSH-secreting adenoma and they can induce tumor shrinkage in about 50% of them. Escape from these effects is observed in about 10% of patients (Beck-Peccoz, et al., 1996; Beck-Peccoz and Persani, 2002). However, the rarity of these tumors makes it difficult to determine whether primary therapy with SS-analogues can be con­ sidered as the optional primary treatment in all patients. Presently, in the management of prolactin (PRL)-secreting pituitary adenomas, medical treatment with dopamine agonists is the first-

line treatment and the currently available SSanalogues do not play a role (van der Hoek et al., 2007). A very heterogeneous group of pituitary tumors are the non-functioning adenomas (NFAs). A con­ sistent proportion of them (up to 90%) secrete low amounts of intact follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and/or their a- and b-subunits either in vitro or in vivo (Colao et al., 2009c) Despite favourable anti-proliferative effects of octreotide treatment on NFA cells in vitro, few clinical trials have been reported in NFAs, and they have observed a tumor reduction only in 11–13% of cases (Colao et al., 2009c; van der Hoek et al., 2007).

GEP-NET In patients with GEP-NETs, the clinical presen­ tation can differ largely and can include signs and symptoms of tumor growth and/or tumor hypersecretion (secreting-tumors). The secriting GEP-NETs produce biologically active agents (peptide hormones and amines) that can cause a variety of invalidating symptoms including diarrhoea, flushing, hypokalaemia, achlorhydria, hypoglycaemia and the so-called “carcinoid” syndrome. SS-analogues play a primary role in the treat­ ment of hypersecretion-related symptoms in patients with GEP-NETs who are no candidate for surgery, for patients who are candidate for surgery during the pre-surgical period, or for patients having residual disease after surgery (adjuvant treatment) (Akerstrom et al., 2009; Oberg et al., 2009). SS-analogues can reduce the serum concentra­ tion of tumor markers and are able to control tumor-related symptoms in about 60–80% of patients (Lamberts et al., 2002a; Modlin et al., 2008). In the treatment of patient with GEP-NETs, SSanalogues have been reported to inhibit tumor growth in about 50% of cases, by inducing stabiliza­ tion of the tumor volume (Arnold et al., 1996; Panzuto et al., 2006; Saltz et al., 1993l; Shojamanesh et al., 2002). Patients who after 6 months of

269

treatment present a stable disease, generally live longer than patients who are unresponsive (Arnold et al., 2005; Panzuto et al., 2006). In a recent study, evaluating the effects SSanalogues on tumor growth in 31 patients with progressive metastatic GEP-NETs, stabilization of tumor growth has been observed more fre­ quently in patients with gastrointestinal tumors than in patients with pancreatic tumors and less frequently in patients with distant extra-hepatic metastases (Panzuto et al., 2006). Only in a very small percentage of patients, SS-analogues have also been reported to decrease tumor size (Granberg et al., 2008; Shojamanesh et al., 2002). High-dose SS-analogues have been reported to control clinical symptoms and tumoral growth in some patients non-responder to the standard doses (Anthony et al., 1993; Eriksson, et al., 1997; Faiss et al., 1999; Welin et al., 2004), but prospective clinical trials should be performed to confirm these preliminary data. The inhibition of tumor growth has been sup­ posed to be responsible for the prolonged survival in patients with metastasized GEP-NETs treated with SS-analogues, but, at least in part, the improvement in the quality of life and the reduc­ tion of possible complications, derived by the con­ trol of clinical syndrome, plays a crucial role as well (Lamberts et al., 2002a). However, SS-analogues cannot be considered as a curative treatment, because tumor progres­ sion, sooner or later, occurs in nearly all patients with unresectable GEP-NETs. In addition, patients with GEP-NETs treated with SS-analo­ gues, generally after a variable time of remission (median 8–12 months), develop insensitivity to the treatment, despite the increase of the adminis­ tered dose (de Herder, 2005). The mechanisms responsible for this tachyphylaxis, and the duration of the responses to SS-analogues, have not been fully characterized yet (Hofland and Lamberts, 2003). Tachyphylaxis with SS-analo­ gues is rarely observed in acromegalic patients during treatment, and it is one of the major limitations of SS-analogues treatment in patients with GEP-NETs (Hofland and Lamberts, 2003; Lamberts et al., 2002a)

The role of SS-analogues in the treatment of patients with non-secreting GEP-NETs is still con­ troversial (Oberg et al., 2009). Considering that clin­ ical data with respect to the anti-proliferative effects of SS-analogues are not convincing enough yet, presently, there is not a clear indication for the use of SS-analogues in these patients (de Herder, 2005). However, the interim results of a placebocontrolled, prospective, randomized clinical trial, evaluating the effects of octreotide-LAR in a group of 85 patients with well-differentiated meta­ static NETs have been very recently published, showing that octreotide-LAR is able to inhibit tumor progression in patients with both secreting or non-secreting tumors (Rinke et al., 2009). These results support the use of SS-analogue treatment in patients with secreting as well as non-secreting NETs. However, further investigations are still required to clarify the impact of SS-analogue treat­ ment on time to tumor progression and overall survival. Preclinical studies in animal models reported that pasireotide can reduce the systemic IGF-I levels and tumor shrinkage. The inhibition of GH and IGF-I induced by pasireotide, different from octreotide, did show only partial tachyphy­ laxis (Schmid, 2008). These results demonstrated that pasireotide may have a (indirect) potential effect on tumor growth and could elicit biologi­ cal effects without induction of tachyphylaxis, suggesting potential important clinical applica­ tion for the treatment of NETs. Preliminary data of a phase II clinical trial show that 30% of patients with metastatic carcinoid tumors, not well controlled by standard dose of octreotideLAR, present a significant symptomatic improvement when treated with pasireotide (Oberg, 2009). Another important application of sst-targeting therapy in the treatment of GEP-NETs is the use radiolabelled SS-analogues to perform PRRT. Several radiolabelled SS-analogues have been used for the treatment of unresectable sst-positive GEP-NETs with promising, but variable results. The studies evaluating the effects of PRRT with the most frequently used radioligands (Fig. 1), [90Y-DOTA,Tyr3]octreotide (DOTATOC) and [177Lu-DOTA,Tyr3]octreotate (177Lu-octreotate),

270

in patients with GEP-NETs, have reported 20–30% of partial remissions (Bodei et al., 2003; Kwekkeboom et al., 2005; Waldherr et al., 2002). Higher remission rates have been correlated with high tumoral uptake during the pre-treatment sst­ scintigraphy (Kwekkeboom et al., 2009a). A ben­ efit of 3.5–6 years has been estimated in patients treated with 177Lu-octreotate, compared with similar patients involved in other intervention or observational studies (Kwekkeboom et al., 2009a). Generally, PRRT has been found to be rela­ tively safe, particularly when compared with other treatment options for unresectable GEPNETs, such as chemotherapy. However it can cause minor adverse effects and rarely severe toxi­ city mainly for kidney, bone marrow and liver (Forrer et al., 2007; Kwekkeboom et al., 2009a). Nevertheless, randomized clinical trials are still required to better establish the optimal treatment schedule and specific indications for the use of different radioligands (or combination of radi­ oligands) in different patients. Therefore, up to date, we still lack the specific recommendations for the use of the different radiolabelled SSanalogues and often the choice is made on the basis of their availability in the local clinical setting (Kwekkeboom et al., 2009a).

Other NETs Like GEP-NETs, medical treatment plays an important role in managing patients with unresect­ able tumors, also in other kinds of NETs. In this context there is some evidence that SS-analogues may improve symptoms related to active agents in patients with metastatic MTCs and bronchial car­ cinoid tumors. MTCs could be a potential target for SS-analo­ gues treatment, since the majority of them (75%) express octreotide-sensitive receptors (sst2 and to a lesser extent sst5) (Papotti et al., 2001). Octreo­ tide has been reported to improve symptoms in some cases (diarrhoea, weight loss, flushing), but a reduction in tumor mass or an improvement in patient survival rate has not been clearly demon­ strated yet (Lupoli et al., 1996; Mahler, Verhelst

et al., 1990; Modigliani et al., 1992; Vitale et al., 2000, 2001). Only few patients have been treated with radi­ olabelled SS-analogues with encouraging results (Virgolini et al., 2002), but clinical trials are still missing. Bronchial carcinoids can have a broad range of clinical behavior and patients can be asympto­ matic (13–51%) or have complex clinical manifes­ tations, such as carcinoid syndrome (typical or atypical) or ectopic Cushing’s syndrome (2–6%) (Soga and Yakuwa, 1999). In patients with meta­ static pulmonary carcinoids octreotide has been reported to be effective in controlling the clinical syndrome (Filosso et al., 2002; Granberg et al., 2001; Hearn, et al., 1988; van Hoek et al., 2009), but contradictory data have been reported regard­ ing the effects on tumor volume (Filosso et al., 2002; Granberg et al., 2001; Hearn et al., 1988). A recent paper comparing the in vitro effects of pasireotide with octreotide in primary cell cultures of a human bronchial carcinoid tissue suggests that pasireotide might also be useful in the in vivo treatment of these tumors (van Hoek et al., 2009). Although in some cases a clinical beneficial effect has been reported, to date, there are no convincing clinical data to support the use of SS-analogues in the treatment of patients with pheochromocyto­ mas (Invitti et al., 1993; Kopf et al., 1997; Koriyama et al., 2000; Lamarre-Cliche et al., 2002). However, PRRT has been reported to induce stabilization of disease in some cases of malignant pheochromo­ cytomas (Valkema et al., 2002). Thymic and ovarian carcinoids are very rare tumors in which the experience with the use of SSanalogues is limited to some case reports (Boix et al., 2002; Vergani et al., 1998; Watson et al., 1990). The use of new SS-analogues, such as pasireo­ tide, chimaeric compounds, or the combination of SS-analogues with other biological compounds, could have a potential role in the treatment of these tumors, as discussed above for GEP-NETs.

The role of ssts in the treatment of other cancer The role of SS-analogues in the treatment of non-neuroendocrine cancer has been supported

271

from many preclinical studies suggesting that SSanalogues can directly inhibit cell proliferation, interfere with growth factor effects and inhibit tumor angiogenesis (Weckbecker et al., 2003). Moreover, several kinds of tumors express sst receptors and can be visualized by sst-scintigraphy. However, the clinical experience in this regard has produced disappointing results. Clinical trials suggest that the combination of octreotide with tamoxifen in the treatment of patients with breast cancer does not improve the results obtained with tamoxifen alone (Bajetta et al., 2002; Ingle et al., 1999). The results of a randomized phase II study indicated that the combination of luteinizing hormone-releasing hormone analogue, an SSanalogue, and dexamethasone may be equally effective as salvage chemotherapy in patients with hormone-refractory prostate cancer, in terms of the clinical and PSA response, overall survival, and time to progression (Dimopoulos et al., 2004). In patients with advanced hepatocellular carci­ noma SS-analogues have been reported to improve symptoms (Dimitroulopoulos et al., 2002, 2007; Kouroumalis et al., 1998) and in a subset of patients with high tumoral sst expression SS-analogue treatment has been reported to improve the survival and the quality of life (Becker et al., 2007; Dimitroulopoulos et al., 2002, 2007; Kouroumalis et al., 1998). Preclinical studies suggest that pasireotide has stronger effects than octreotide on tumor growth inhibition (Schmid, 2008). Moreover, this com­ pound has high binding affinity for four of the five sst subtypes that might overcome the problem of a heterogeneous expression of ssts observed in tumors. Therefore, pasireotide could have stron­ ger anti-tumor effects also in non-neuroendocrine tumors, although there is no clear evidence for that at the moment.

The role of ssts in non-tumoral diseases SS-analogues may suppress the growth and inflammatory factors determining Graves’s ophthalmopathy (Weckbecker et al., 2003).

Indeed, clinical studies have suggested that treat­ ment with SS-analogues improves the clinical ocular signs of inflammation in patients with this disease (Stiebel-Kalish et al., 2009). The pathological proliferation of blood vessels characteristic for diabetic retinopathy can be inhibited by SS-analogues. Early clinical investiga­ tions suggest that octreotide is useful in patients with diabetic retinopathy, in which conventional photocoagulation therapy has failed (Krassas et al., 2007). The anti-secretory effect of SS-analogues on the pancreas has given the rationale for the clinical use of octreotide in the days following pancreatic surgery and in the treatment (and prevention after endoscopic retrograde cholan­ giopancreatography) of acute pancreatitis. However, the efficacy of these treatments is still controversial (Bang et al., 2008; Bruns et al., 2009). Many other clinical applications have been pro­ posed, but until now the clinical use of SS-analo­ gues is only for the previous reported indications in the treatment of NETs.

Future applications and prospective The pharmaceutical industry is proceeding to develop novel SS-analogues with different phar­ macodynamic characteristics (such as pasireotide) that may be useful to increase the efficacy of SSanalogue treatment and to extend the indication of treatment to other diseases (i.e. CD). Lanreotide-ATG has been very recently approved for clinical use and a new slow release formulation of pasireotide is progressing in clinical trials. The availability of these new formulations will probably further improve the compliance to treatment and the quality of life of patients. Regarding the currently available analogues, further studies are still required to clarify the efficacy and the safety of the high-dose treatment in patients with NETs, to compare the efficacy of different compounds, to clarify the role of SS-analogues in particular cases where there is no clear indication for treat­ ment (such as non-secreting GEP-NETs or

272

non-neuroendocrine tumors), as well as to better address the clinical role of primary medical treatment with SS-analogues. Future progress is needed with regard to the clinical use of radiolabelled SS-analogues. The description of a high density of sst2 receptors in other pathological conditions might increase the indication of sst-scintigraphy or PRRT. More­ over, it is conceivable that the availability of SS-analogues targeting other ssts will be fol­ lowed by the development of new radioligands targeting different ssts that might be useful to detect or treat pathological conditions with a relatively lower expression of sst2 (i.e. insulino­ mas). In this respect, more knowledge on the cellular uptake of such radioligands need to be obtained. Octreotide has been conjugated with gallium, 68 Ga-DOTA-D-phe1tyr3-octreotide forming 68 ( Ga-DOTATOC) and 66Ga-DOTA-D-phe1­ tyr3-octreotide (66Ga-DOTATOC). These radi­ oligands have been used in clinical investigations in patients with NETs to perform PET (de Herder et al., 2006a). This imaging technique might be a future option as well. The use of SS-analogues in combination with mTOR inhibitors seems to be promising in the treatment of NETs. A recent clinical trial has evaluated the effects of the octreotide in combi­ nation with everolimus (mTOR inhibitor) in patients with advanced low-/intermediate-grade neuroendocrine tumors, reporting tumor shrink­ age (partial response) in 22% and disease stabi­ lization in 70% of a series of 60 treated patients (Yao et al., 2008). Several clinical trials are pre­ sently ongoing to better address the efficacy and safety of this combination treatment. Considering the effects on tumor growth, this treatment could potentially be indicated in non-secreting NETs as well. SS-analogues can be used as vectors to specifi­ cally accumulate cytotoxic compounds in sst­ expressing tumors (Hofland and Lamberts, 2003). This approach, named sst-targeted che­ motherapy, may reduce the systemic adverse effects and increase the efficacy of chemotherapy in the tumors. Preclinical studies have demon­ strated promising effects in several

neuroendocrine and non-neuroendocrine tumor models (Engel et al., 2005, 2007; Keller et al., 2005, 2006; Seitz et al., 2009; Treszl et al., 2009; Ziegler et al., 2009). In the “era of biotherapy” we expect that the studies in the field of SS and ssts will lead to the clinical development of novel SS-analogues, of chimaeric compounds, of SS-targeted chemother­ apy and new indications for the use of “cold” SS-analogues and radiolabelled SS-analogues. Moreover, new combinations of treatments will be introduced into clinical practice. This progress, together with a further understanding of the molecular pathogenetic alterations of disease, will probably lead to a tailored patient-based treatment of disease in the coming decades, in which the study of predictors of response to treat­ ment (i.e. a positive sst-scintigraphy in a patient with a GEP-NET) will help to decide the best therapeutical approach for that individual patient.

Acknowledgements The authors thank Professor Annamaria Colao, Department of Endocrinology, Federico II Uni­ versity of Naples, Italy for providing the MRI image of an acromegalic patient from her series.

Abbreviations ACTH CD CRH CST CT D2 DA DR EGF-R ENETS FSH FT3 FT4

adrenocorticotropic hormone Cushing’s disease corticotropin-releasing hormone cortistatin computerized tomography DR type 2 dopamine dopamine receptor epidermal growth factor receptor European Neuroendocrine Tumor Society follicle-stimulating hormone free triiodothyronine free tetraiodothyronine

273

GEP-NET GH GIT IGF-I insulin-R lanreotide-ATG lanreotide-SR LH MTC mTOR NET NFA OA octreotide-LAR PDGF-R PET PRL PRRT PTP RA SCLC SS-analogue sst TRH TSH VEGF-R2

gastroenteropancreatic­ neuroendocrine tumor growth hormone gastrointestinal tract insulin like growth factor type I insulin receptor lanreotide autogel lanreotide-sustained-release luteinizing hormone medullary thyroid carcinoma mammalian target of rapamycin neuroendocrine tumor non-functioning adenoma osteoarthritis octreotide long-acting-release platelet-derived growth factor receptor positron emission tomography prolactin peptide receptor radionuclide therapy phosphotyrosine phosphatase rheumatoid arthritis small cell lung cancer somatostatin analogue somatostatin receptor thyrotropin-releasing hormone thyroid-stimulating hormone vascular endothelial growth factor receptor type 2

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 12

Somatostatin analogues: treatment of pituitary and neuroendocrine tumors Annamaria Colao
, Antongiulio Faggiano and Rosario Pivonello Department of Molecular & Clinical Endocrinology and Oncology, “Federico II” University of Naples, Naples, Italy

Abstract: This chapter summarizes the most recent data on the use of the somatostatin analogues (SSAs), octreotide (OCT) and lanreotide for the treatment of patients with pituitary and neuroendocrine tumors (NETs). These two analogues have a high affinity for somatostatin receptor (SSR) sub-types 2 and 5. The major indications of these compounds are GH- and TSH-secreting pituitary adenomas, secreting NETs and non-functioning NETs in progression. Pasireotide is a new analogue, with a receptor pattern different from previous analogues since it binds with high affinity to SSR types 1, 2, 3 and 5. This analogue will be available to treat patients with ACTH-secreting adenomas in a short time. A recent study has also demonstrated a beneficial effect of OCT long-acting release in patients with non-functioning NETs independently from their progression status. These data open the treatment with SSAs in all NET patients. Keywords: somatostatin; pituitary tumors; somatostatin analogues; neuroendocrine tumors (NET); octreotride

as GH, TSH and all known gastrointestinal hor­ mones (Reichlin, 1987). The very short half-life of natural SS (about 3 min in blood) has prompted the development of synthetic analo­ gues with longer half-life to use for treatment purposes: short-acting compounds such as octreo­ tide (OCT), which needs to be administered sub-cutaneously (s.c.) several times per day, or long-acting compounds such as octreotide longacting release (LAR) and lanreotide slow-release (SR) or Autogel (ATG), with a monthly administration, intra-muscular or s.c., are currently available for treatment of pituitary and neuroen­ docrine tumors (NETs). More recently, a new analogue, pasireotide, in under investigation for treatment of acromegaly, Cushing’s diseases and NETs. This analogue differs from LAR and ATG

Introduction Somatostatin (SS), which was initially discovered as a growth hormone (GH) release inhibiting hormone, is a polypeptide hormone present in two natural forms of 14 and 28 amino acids. It is widely distributed throughout the body and binds with high affinity to five different subtypes of specific SS receptors (SSRs) on the cell surface, which belong to the G-protein-coupled receptor family (Table 1), as previously reported. SS acts as a controller of endocrine function by inhibiting the secretion of various hormones, such


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E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82012-6

281

282 Table 1. Properties of SSRs and binding affinity for individual receptor sub-types of native SS and SSAs

Properties Affinity Somatostatin-14 Somatostatin-28 Octreotide Lanreotide Pasireotide

SSRt1

SSRt2

SSRt3

SSRt4

SSRt5

# angiogenesis Stops cell cycle

# hormone secretion Stops cell cycle

" apoptosis

Stops cell cycle

# hormone secretion Stops cell cycle

2.26 1.85 1140 2330 9.3

0.23 0.31 0.56 0.75 1

1.43 1.3 34 107 1.5

1.77 ND 7030 2100 >100

0.88 0.4 7 5.2 0.16

in its somatostatin receptors (SSRs) binding affi­ nity (see below) and is foreseen to be available for treatment in 2010–2011. Pituitary adenomas and NETs are rare tumors, responsible of some specific clinical syndromes such as acromegaly, Cushing’s disease, secondary hyperthyroidism, gastrointestinal syndromes and carcinoid syndrome, all due to the excess secretion of the hormones produced by the tumor. There are also pituitary tumors and NETs lack­ ing a specific hormone excessive secretion and causing intra-cranial or gastrointestinal compres­ sive symptoms and/or metastasis (in the liver mainly): these tumors are called non-secreting or non-functioning and represent 30–60% of all tumors of this type. The characteristics of these tumors is to express, on their membrane surface, SSRs as well as many others including dopamine receptors and other growth factor receptors. This chapter presents an update of available data in the setting of treatment with somatostatin analogues (SSAs) in patients with pituitary tumors and NETs.

Pharmacological characteristics of SSA The currently available SSA for treatment of pituitary tumors and NETs are s.c. OCT, intra­ muscular LAR and s.c. ATG. OCT, the first SSA used in clinical practice, was introduced in the early 1980s to overcome the 2 min half-life of native SS (Lamberts et al., 1996).

OCT has pleiotropic effects: it inhibits GH, and consequently IGF-I, and TSH secretion from the pituitary gland, insulin, glucagon, pan­ creatic bicarbonate and polypeptide, cholecysto­ kinin, gastrin, motilin, secretin, vasoactive intestinal peptide, neurotensin and gastric acid from the endocrine gastrointestinal system; decreases blood flow to the gut, intestinal moti­ lity, and carbohydrate, water and electrolyte absorption (Lamberts et al., 1996). OCT displays some effects on other human malignan­ cies as a result of inhibition of the secretion of growth hormone, insulin and gastrointestinal hormones; direct or indirect (via GH) inhibition of IGF-I production or of its binding proteins; direct inhibition of angiogenesis; or direct antiproliferative effects on tumor cells, mediated by SSRs. LAR is enclosed in microspheres of a slowly biodegrading polymer that allows prolonged drug release (Astruc et al., 2005); after the injection LAR levels rise briefly because of release from the surface of the microspheres, then they fall and begin to rise again about 7–14 days after the injec­ tion and remain elevated for an average of 34 days. Steady-state conditions are usually achieved after two to three injections. The usual starting dose of LAR is 20 mg with titration down to 10 mg or up to 30, 40 or 60 mg, based on the response of excess hormone levels. ATG consists of a solution of lanreotide in water with no additional excipients (Antonijoan et al., 2004). ATG was found to have linear pharmaco­ kinetics for the 60–120 mg doses and provided a prolonged dosing interval and good tolerability.

283

The dosage range is 60–120 mg every 28–56 days. Pasireotide (SOM230), the new analogue under strict investigation to treat patients with acromegaly, Cushing’s disease and NETs and will possibly be available in 2010–2011. This is a cyclohexapeptide with a high affinity for SSR types 1, 2, 3 and 5 (Schmid, 2008). SOM230, exhibits an affinity binding profile for human SSRs more similar to native SS than to either OCT or lanreotide (Ben-Shlomo et al., 2009). Details of binding affinity of different analogues on SSRs and the properties of these receptors are shown in Table 1.

Effects of somatostatin analogues in patients with pituitary tumors GH-secreting adenomas Acromegaly is a rare and slow developing disease with an estimated annual incidence of three to four cases/million population and a current esti­ mated prevalence of 40 cases/million population, caused by a benign pituitary adenoma in more than 95% of cases (Colao et al., 2004). Progres­ sive somatic disfigurement characterizes the disease that is accompanied by a wide range of systemic manifestations: coarsened facial features, exaggerated growth of hands and feet, soft tissue hypertrophy, hyperhydrosis, goitre, osteoarthritis, carpal tunnel syndrome, fatigue, visual abnormal­ ities, increased number of skin tags, colon polyps, sleep apnoea and daytime somnolence, reproduc­ tive disorders and cardiovascular disease (Colao et al., 2004). Besides the negative effects of high circulating GH and IGF-I levels, the tumor mass itself may induce compression of the optic nerve tract or chiasm, cranial nerve palsies, headache, hydrocephalus, as well as various degrees of pitui­ tary function insufficiency (Colao et al., 2004). Approximately 60% of acromegalic patients die from cardiovascular disease, 25% from respira­ tory disease and 15% from malignancies (Melmed, 2009). SSAs are the most widely used drugs to control acromegaly (Melmed et al., 2002). An analysis of

466 patients treated with OCT showed sup­ pressed GH levels below 2.5 mg/l in 29.2%, nor­ malizes IGF-I levels in 39.9% and reduces tumor size (>20% of reduction in maximal diameter) in 38.6% of patients (Colao and Lombardi, 1998). Most clinical signs and symptoms of acromegaly, such as sweating, soft tissue swelling, fatigue, and headache, are generally relieved after the admin­ istration of the first doses of OCT. The s.c. for­ mulation is not currently used as it has been replaced by the slow-release ones such as LAR or ATG. However, some investigators still use the OCT at the beginning of treatment, for 1 or 2 weeks, to investigate the tolerability of the drug. The efficacy of LAR has been investigated in many studies showing a considerable variability among the data probably due to wide range in number of patients studied and in duration of treatment. A critical analysis of studies report­ ing on the efficacy of SSRs in acromegaly showed GH suppression in 54% and GF-I nor­ malization in 63% of unselected subjects (Freda et al., 2005). These data were obtained in patients treated after unsuccessful surgery while recent evidence suggests that LAR and lanreotide-ATG might also be used as first-line treatment in selected patients with acromegaly (Colao et al., 2006a; Murray and Melmed, 2008). In particular, several studies reported reduction in tumor size after treatment with LAR or ATG given first-line (Bevan, 2005): overall, 103 of 180 patients (57%) had a decrease in tumor size. For first-line therapy patients (defined as patients without prior sur­ gery or radiation), 81 of 101 patients (80%) had tumor shrinkage (Bevan, 2005). For adjunctive therapy patients, 22 of 79 patients (28%) experi­ enced tumor shrinkage (Bevan, 2005). An increase in tumor size during SSA therapy is very rare, being reported in less than 2% of patients in the trials analysed in a meta-analysis from Freda et al. (2005) or by Colao et al. (2006b) in their own series. In the latter study (Colao et al., 2006b), the two patients with increase in tumor size during treatment were both males not responding to the treatment. No difference in the amount of tumor shrinkage was found according

284

Tumor shrinkage (%)

50 25 0 –25 –50 –75

as

as

om

om

en

en

ad

ad

ve

ro

In

va

si

ac rm lla se tra Ex

En

cl

os

ed

m

M

ac

ic

ro

ro

-a d

-a d

en

en

om

om

as

as

–100

Fig. 1. Tumor shrinkage, expressed as per cent decrease of tumor volume compared to baseline, in 99 newly diagnosed patients treated with LAR or lanreotide for 12 months. The patients were grouped according with initial tumor volume. (Data have been re­ drafted from Colao et al. (2006a).)

with the initial tumor size, though micro-adeno­ mas have the highest degree of no shrinkage (Fig. 1): of 13 patients with micro-adenomas five did not have any shrinkage (38.5%) as compared to 19 of 89 patients (21.3%) with macro-adenomas. Tumor shrinkage after treat­ ment with SSAs has then been confirmed by several other authors (Cozzi et al., 2006; Jallad et al., 2005; Mercado et al., 2007) but most studies have reported data on tumor volume using LAR only for 12–24 months. Limited data using lanreotide-ATG are, however, in line with those obtained with LAR (Colao et al., 2009a; Mazziotti and Giustina, 2009). Moreover, a recent study performed in a selected series of patients who received SSA therapy only for 5 years, achieved a progressive shrinkage of tumor size, suggesting that some minor decrease of tumor mass might occur in responsive patients beyond the first year of treatment (Colao et al., 2009b) (Fig. 2). Of relevance, control of GH and IGF-I levels during SSA treatment is followed by improve­ ment of cardiac, respiratory and articular com­ plications (Colao et al. 2004). In particular, 12

months of treatment with either LAR or lanreo­ tide-SR induced decreased left ventricular mass and improvement of cardiac performance in a significantly greater extent than surgery, while patients cured with surgery had better glucose control than those controlled with SSAs (Colao et al., 2008a). However, no deterioration of glu­ cose tolerance was found in a 12 months pro­ spective study in de novo patients treated with SSA (Colao et al., 2009c). In fact, deterioration of glucose tolerance was found to be associated with active acromegaly and development of obe­ sity more than with the use of SSA in a conse­ cutive series of newly diagnosed patients with acromegaly treated first-line with SSA for 12 months (Colao et al., 2009c). Disease control is essential to improve car­ diac performance or at least to stop the dete­ rioration of the acromegalic cardiomyopathy: patients with uncontrolled acromegaly for 5 years experienced a worsening of left ventri­ cular performance on effort as compared to those achieving disease control after surgery alone or surgery associated with OCT (Colao et al., 2001).

285 A.

Lamberts, 2004), SSA have been used to com­ plete the effects of surgery with high success. TSH levels have been reported to normalize in ~80% of patients and tumor shrinkage occurs in 50% of cases during treatment with OCT (Chanson et al., 1993; Comi et al., 1987; Fukuda et al., 1998). Lanreotide treatment was similarly shown to suppress plasma TSH levels and to normalize fT4 and fT3 levels, suggesting its use in the long-term medical management of these adenomas (Kuhn et al., 2000). Similarly, LAR was successful in reducing TSH, fT3 and fT4 levels without causing significant side effects (Caron et al., 2001). In our experience, in five patients with TSH-secreting pituitary adenomas LAR and lanreotide were efficient in normal­ izing thyroid hormone profile after 3–6 months of treatment at low dose without any significant side effect (Colao et al., 2003). Data on treat­ ment with SSA in this tumor histotype are still limited because of the rarity of the disease. However, it has been noticed that the dosage of SSA required to normalize TSH levels is lower than that required to normalize GH levels in acromegaly.

350 300 250 200 150 100 50

Tumor volume (mm3)

B.

C.

0 1800 1600 1400 1200 1000 800 600 400 200 0 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 Basal

1

2

3

4

5

Fig. 2. Tumor shrinkage in patients with micro-adenomas (A), macro-adenomas extrasellar or enclosed (B) and adenomas invasive (C) treated with SSAs for 5 years as monotherapy. (Data are re-drafted from Colao et al. (2009b).)

TSH-secreting pituitary adenomas TSH-secreting adenomas are rare, frequently are macroadenomas at diagnosis and thus pre­ sent with mass effect symptoms such as head­ ache, visual disturbance, together with variable symptoms and signs of hyperthyroidism (BeckPeccoz et al., 1996). Based on these premises, trans-sphenoidal surgery often fails to normalize TSH and thyroid hormone levels: the preva­ lence of persistent hyperthyroidism after surgery is reported to average 50% of cases (Beck-Peccoz et al., 1996). Since these adeno­ mas express high amount of SSRs (Hofland and

Clinically non-functioning pituitary adenomas A few clinical trials have been conducted to eval­ uate potential effects of s.c. OCT in patients with clinically non-functioning pituitary adenomas: results have been recently reviewed (Colao et al., 2008b). As summarized in Table 2, tumor shrink­ age was reported only in 12% of patients with the vast majority of patients having stable remnant tumors. Importantly, only in a small minority of the patients (5%), increase of tumor size was documented during treatment with s.c. OCT. However, generally the patients follow-up has been too short (6 months in average) to draw final conclusions on a potential effect of SSAs in preventing tumor re-growth. A still unexplained finding was reported by Warnet et al. (1997), who showed that OCT treatment was followed by a rapid improvement in headache and visual disturbances, without any change in tumor volume. This effect was probably not linked to a

Table 2. Summary results of OCT s.c. treatment in patients with clinically non-functioning pituitary adenomas


Visual field

First author

Year of publication

Warnet A Turpin G De Bruin TWA Katznelson L Gasperi M Merola B Plockinger U Liuzzi A Warnet A Borson-Chazot F Colao A

1889 1991 1992 1992 1993 1993 1994 1991 1997 1997 1999

Summary

No. pts

Tumor volume Improved (%)

Unchanged (%)

Worsened (%)

5

60.0

40.0

0.0

4 3 5 9

75.0 33.3 20.0 11.1

25.0 66.6 60.0 88.9

0.0 0.0 20.0 0.0

20 22 16

5.0 40.9 50.0

95.0 40.9 37.5

0.0 18.2 12.5

84

32.1

59.6

8.3

No. pts

Increased (%)

Octreotide Unchanged (%)

Decreased (%)

2 1 4

0.0 0.0 0.0

100.0 100.0 100.0

0.0 0.0 0.0

8 19 14 20 7 16 9

25.0 0.0 14.3 15.0 42.8 0.0 22.2

75.0 94.3 85.7 70.0 42.8 100.0 77.8

0.0 5.7 0.0 15.0 14.3 0.0 0.0

100

12.0

83.0

5.0

Dose (mg/day)

Duration (months)

100–300 300–450 1200 200–750 300 150–300 300–1500 300–500 300–600 300 300–600

1–12 6 3–6 2 3–6 1–12 3 3–12 2 1 12

Modified from Colao et al. (2008b).
PubMed search using as key words ‘octreotide, lanreotide and clinically non-functioning adenomas’ or ‘octreotide, lanreotide and functionless adenomas’. Last search December 19, 2009.

287

direct effect on tumor shrinkage but likely to a direct effect on the retina and the optic nerve (Lamberts et al., 1995). Approximately one third of patients receiving s.c. OCT experienced an improvement in visual field defects after 1 week to 12 months treatment (Table 2).

ACTH-secreting pituitary adenomas Currently, there are several medications used in patients with active or recurrent Cushing’s dis­ ease, i.e. adrenal inhibitors such as ketoconazole, metyrapone, etomidate, mitotane, aminoglutethi­ mide, trilostane or neuromodulators such as cabergoline or bromocriptine (Nieman and Ilias, 2005; Pivonello et al., 2008) but no medical ther­ apy is currently approved for ACTH-secreting adenomas. The current available SSA LAR and lanreotide-ATG are not effective in this disease, as these are preferential ligands of SSR type 2 which is down-regulated by excess cortisol, while corticotroph adenomas express SSR type 5 (Hof­ land et al., 2005). Therefore the new analogue pasireotide (SOM230) might represent a potential agent to effectively treat Cushing’s disease. A recent phase II study demonstrated that pasireo­ tide (SOM230) given at the dose of 600 mg twice daily for 15 days reduced urinary free cortisol in 76% of patients with de novo, persistent or recur­ rent Cushing’s disease (Boscaro et al., 2009). In 17% of these patients, urinary free cortisol was normalized (Boscaro et al., 2009). These data, obtained in a very short period of time, sugest that the multi-receptor ligand SSA pasireotide (SOM230) might be a promising pituitary-targeted medical therapy for Cushing’s disease. A 12­ month treatment study is ongoing to verify the efficacy of pasireotide in ACTH-secreting pitui­ tary tumors.

Effects of SSA in patients with NETs NETs are rare, heterogeneous, ubiquitous tumors with variable biological behavior and an estimated incidence of one to five cases/100,000/ year (Yao et al., 2008). More recent estimates

show a steady increase in the incidence of NETs and a sharp increase in their prevalence and survival, highly likely because of their better diagnosis and management. The biological beha­ vior and the variable clinical presentations, with the potential occurrence of different functioning endocrine syndromes, make these tumors a very heterogeneous pathological entity (Faggiano et al., 2008). As a consequence, the therapeutic strategy should be diversified and tailored to the clinical–biological profile of NETs. The treatment of NETs is multi-modal, with the intent to control symptoms, to reduce levels of circulating hormones, to prevent further tumor growth and, if possible, to obtain a decrease of tumor size, thus improving the sur­ vival and quality of life of patients. The current therapeutic procedures include surgery, emboli­ zation, chemoembolization, radiofrequency or brachytherapy of liver metastases, biotherapy, radiometabolic treatment and chemotherapy (Arnold et al., 2009; Kaltsas et al., 2004; Modlin et al., 2008; Oberg, 2009). Surgery remains the treatment of choice and the only approach that can achieve a cure in patients with NETs, while medical treatment should be considered as an adjuvant to surgery unless either the general condition of the patient or other contraindications preclude surgery (Kaltsas et al., 2004). The last WHO classification of NETs classifies these tumors on the basis of the grade of differ­ entiation and proliferative activity, regardless of the site of origin and secretory activity (Solcia et al., 2000). This classification identifies a sub­ group with poor differentiation and high prolifera­ tive activity that needs more aggressive treat­ ments. However, most of the NETs of any site have a more or less indolent clinical trend and can benefit from peculiar therapeutic approaches with biological agents with anti-secre­ tive and anti-proliferative activity. The use of these agents requires knowledge of the biological mechanisms underlying neuroendocrine tumori­ genesis. Neuroendocrine cells produce several hormones, peptides, amines and other molecules with different biological activity and, among these, chromogranins have a particular relevance for the

288

biochemical and immunohistochemical diagnosis and are used as markers of treatment efficacy. Other proteins, such as SSR types 1–5, are useful not only for the diagnosis, because they are the biological prerequisite for the use of selective scintigraphy for these receptors, but also for prognostic definition and therapeutic decisions (Ferolla et al., 2008). The expression of SSRs is present in more than 90% of NETs and thus SSAs are widely used in patients with these tumors. SSAs have direct and indirect effects on NETs: the former include the arrest of tumor growth and stimulation of apoptosis, while indir­ ect anti-proliferative effects occur through antiangiogenic mechanisms, immunomodulatory effects and through the inhibition of factors stimulating tumor growth. Clinically these biolo­ gical activities can result in reduction or stabiliza­ tion of tumor mass (Kwekkeboom et al., 2009). SSAs are effective in controlling symptoms derived by the carcinoid syndrome and in sup­ pressing hormone secretion in secreting NETs. The prevalence of objective responses that are obtained with the use of slow-release analogues in NETs is similar to that observed with short half-life analogues. At standard dosages, SSAs improve the symptoms related to functioning NETs in 64% of patients, with a biochemical response in 66% of cases (Grozinsky-Glasberg et al., 2008). Only in a small percentage of cases (~10%) there is a reduction of more than 50% of tumor mass, while a stabilization of the same occurs in 35–50% of patients (GrozinskyGlasberg et al., 2008). However, the indication for using SSAs in patients with NETs is limited to patients with secreting tumors or with non-func­ tioning tumors in a stage of recurrent disease. Recently, a placebo-controlled, double-blind, phase IIIB study in patients with well-differen­ tiated metastatic midgut NETs has been per­ formed to investigate the effect of SSAs in the absence of a clinical syndrome (Rinke et al., 2009). Importantly, the median time to tumor progression in the LAR and placebo groups was significantly different being 14.3 months in the former and 6 months in the latter (Rinke et al., 2009). The most favourable effect was observed in patients with low hepatic tumor load and

resected primary tumor (Rinke et al., 2009). These results have opened the perspective of treatment with SSA in patients with non-func­ tioning NETs before any recurrence of the dis­ ease is documented. As already mentioned in the section above, an important advance in SSA treatment has been represented by development of slow-release formu­ lations that do not require multiple daily injections and which, in patients with NETs, also reduce the phenomenon of resistance or escape. In fact, a remarkable improvement of diarrhoea, frequently associated with excess of hormones and peptides secretion, has been observed; also improvement gastrointestinal disorders, quality of life and overall well-being has been reported (Oberg, 2009). In patients with multiple endocrine neoplasia type 1 (MEN1) treated with SSA for duodeno-pan­ creatic NETs, Faggiano et al. (2008) reported nor­ malization of hypercalcaemia and hypercalciuria secondary to primary hyperparathyroidism from adenomas/hyperplasia of the parathyroid (Fig. 3). This effect was hypothesized to be mediated by SSRs expressed on parathyroid adenomas.

Safety and tolerability of somatostatin analogues LAR, lanreotide-SR and ATG are generally well tolerated by most patients. Common adverse effects of treatment are similar to those of shortacting OCT and include gastrointestinal distur­ bances and injection site reactions. Up to 80% of patients receiving SSAs experience nausea, abdominal discomfort, diarrhoea, malabsorption of fat and flatulence especially after the first injec­ tion (Table 3). Symptoms are, however, generally mild and less than 10% of the patients require treatment withdrawal because of side effects. These symptoms are usually transient and mild to moderate in severity. Symptoms start within hours after the first injection of the drug, their severity is dose dependent, and they usually sub­ side spontaneously in 10–14 days, despite contin­ ued treatment. The occurrence of these adverse effects can be readily understood from the phy­ siologic actions of SS on the gastrointestinal tract

289 p < 0.05

Serum PTH levels (pmol/l)

20 15 10 5 0 –3

0

3

6

p < 0.01 p < 0.05

p < 0.01 Serum phosphorus levels (mmol/l)

Serum calcium levels (mmol/l)

3

2

1

0 –3

0

3

1.2

p < 0.05

0.8

0.4

0.0

6

–3

Renal phosphate threshold (mmol/l GF)

24-h urine Ca/Cr mg ratio

p < 0.01 p < 0.05

0.6 0.4 0.2 0.0 –3

0

3

6

p < 0.01

p < 0.01 0.8

3

0

6

Months

p < 0.05

1.25 1.00 0.75 0.50 0.25 0.00 –3

0

3

6

Months

Fig. 3. Variations of serum concentrations of PTH, calcium and phosphorus, 24-h urine calcium: creatinine (Ca/Cr) mg ratio, renal phosphate threshold (expressed as mean + SEM) before (Time: –3 and 0 month) and after (Time: 3 and 6 months) therapy with LAR in eight patients with MEN1-dependent primary hyperparathyroidism. (Data from Faggiano et al. (2008).)

and exocrine pancreas, in particular, the prolongation of the gastrointestinal transit time, the decrease of endogenous fluid secretion in the jejunum and of the intestinal absorption of water and electrolytes. The spontaneous resolution of these symptoms supports the concept of a rapid adaptation to the effects

of SS of the function of the gastrointestinal tract and pancreas. Pain at the injection site has been reported to occur in about 9% of patients and has been shown to be dose dependent. Withdrawal of medication as a result of gastro­ intestinal or injection site events is rare (Freda,

290 Table 3. Side effects of depot SS analogue therapy Site Gastrointestinal tract

Biliary tract Glucose metabolism

Diarrhoea Abdominal discomfort Flatulence Constipation Nausea Vomiting Early Persistent All types New gallstones Hypoglycaemia Hyperglycaemia

Transient hair loss Hypothyroidism Sinus bradicardia Vitamin B12 deficiency Injection site pain

36.4% 29.1% 25.7% 18.8% 10.3% 6.5% 49% <10% 50% 15% 2% 7–15% 3–6% 2% <1% <1% 24%

Data modified from Freda (2002).

2002; Gillis et al., 1997; Murray and Melmed, 2008; Roelfsema et al., 2008).

Future directions The dosages of SSAs currently used are probably insufficient to determine control of hormone secre­ tion and tumor growth both in acromegaly and NETs. Two independent studies (Colao et al., 2007a; Giustina et al., 2009) have shown that patients with acromegaly inadequately controlled with the dose of LAR of 30 mg given intramuscu­ larly every 28 days might achieve lower IGF-I levels using 40 or 60 mg every 28 days. These results have been previously reported also in patients with NETs, even if the experience is lim­ ited (Eriksson et al., 1997). Moreover, pituitary tumors and NETs express not only SSRs but also dopamine receptors (Ferone et al., 2009; Pivonello et al., 2004; Srirajaskanthan et al., 2009); these receptors, at the level of the membrane, can form homo- or hetero-dimers (Rocheville et al., 2000). The treatment with SSAs combined with dopa­ mine agonists has already been demonstrated effi­ cacious in patients with acromegaly (Colao et al., 2007b). With the development of chimeric

molecules that can bind SSR sub-types 2 and 5 as well as dopamine receptors (dopastatins), there is expectation of promising clinical results as in vitro studies are showing positive results on cell prolif­ eration and hormone secretion (Ferone et al., 2009; Srirajaskanthan et al., 2009). Dopastatins resulted more effective in inhibiting cell growth in a non-small lung carcinoma cell line compared with either sub-type-specific SSAs or dopamine agonists, demonstrating in this cell system, constitu­ tively expression of both SS and dopamine receptors (Ferone et al., 2005). Indeed, a differential cytotoxi­ city of chimeric compounds was recently observed in bronchopulmonary and small intestinal NET cell lines (Kidd et al., 2008). Conversely, in gastric enter­ ochromaffin-like cell, the dopastatin BIM-27A760 did not display an additive effect on histamine secre­ tion and cell proliferation (Kidd et al., 2007). The responses of each individual cell line suggested that NETs from diverse locations and arising from differ­ ent neuroendocrine cells may require cell-specific anti-proliferative agents based on the unique recep­ tor profile of each individual lesions (Kidd et al., 2008). There are several ongoing pre-clinical and clinical trials, involving pasireotide, dopastatin and combitation treatments with everolimus (a mammalian target for rapamycin (m-TOR) inhibitor) aimed at increasing the percentage of objective response and/or to reducing the impact of the phenomenon of tachyphylaxis (Ferone et al., 2009). The activation of multiple SSRs by pasireotide significantly reduced cell prolifera­ tion in the NET cell line NCI-H727 (Ono et al., 2007), and inhibited cell growth and catechola­ mine secretion in cell cultures of pheochromocy­ toma, also inducing apoptosis (Pasquali et al., 2008).

Conclusions Pituitary tumors and NETs are rare diseases asso­ ciated with increased morbidity and mortality. SSRs are currently the most widely used drugs to control hormone excess in GH- and TSHsecreting pituitary adenomas and NETs, are well tolerated and with minimal side effects.

291

New drugs are under investigation to overcome the limitation of currently available analogues, namely, pasireotide and dopastatins.

Abbreviations ATG LAR MEN1 NET OCT SS SSR SSA SR

lanreotide Autogel octreotide long-acting release multiple endocrine neoplasia type 1 neuroendocrine tumor octreotide somatostatin somatostatin receptor somatostatin analogue lanreotide slow-release

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293 Kaltsas, G. A., Besser, G. M., & Grossman, A. B. (2004). The diagnosis and medical management of advanced neuroendo­ crine tumors. Endocrine Reviews, 25(3), 458–511. Katznelson, L., Oppenheim, D. S., Coughlin, J. F., Kliman, B., Schoenfeld, D. A., & Klibanski, A. (1992). Chronic somatos­ tatin analog administration in patients with a-subunit-secret­ ing pituitary tumors. Journal of Clinical Endocrinology and Metabolism, 75(5), 1318–1325. Kidd, M., Drozdov, I., Joseph, R., Pfragner, R., Culler, M., & Modlin, I. (2008). Differential cytotoxicity of novel somatos­ tatin and dopamine chimeric compounds on bronchopul­ monary and small intestinal neuroendocrine tumor cell lines. Cancer, 113(4), 690–700. Kidd, M., Modlin, I. M., Black, J. W., Boyce, M., & Culler, M. (2007). A comparison of the effects of gastrin, somatostatin and dopamine receptor ligands on rat gastric enterochromaf­ fin-like cell secretion and proliferation. Regulatory Peptides, 143(1–3), 109–117. Kuhn, J. M., Arlot, S., Lefebvre, H., Caron, P., Cortell-Rudelli, C., Archambaud, F., et al. (2000). Evaluation of the treatment of thyrothropin-secreting pituitary adenomas with a slow release formulation of the somatostatin analog lanreotide. Journal of Clinical Endocrinology and Metabolism, 85(4), 1487–149. Kwekkeboom, D., Kam, B., Van Essen, M., Teunissen, J., Van Eijck, C., Valkema, R., et al. (2009). Somatostatin receptor based imaging and therapy of gastroenteropancreatic neuroendocrine tumors. Endocrine-Related Cancer. Decem­ ber 8 [Epub ahead of print]. Lamberts, S. W.J., de Herder, W. W., van der Lely, A. J., & Hofland, L. J. (1995). Imaging and medical management of clinically nonfunctioning pituitary tumours. Endocrinologist, 5, 448–451. Lamberts, S. W., van der Lely, A. J., de Herder, W. W., & Hofland, L. J. (1996). Octreotide. The New England Journal of Medicine, 334(4), 246–254. Liuzzi, A., Dallabonzana, D., & Oppizzi, G. (1991). Is there a real medical treatment for the ‘non-secreting’ pituitary ade­ nomas? (abstract). Journal of Endocrinological Investigation, 14(Suppl. 1), 18. Mazziotti, G., & Giustina, A. (2009). Effects of lanreotide SR and Autogel on tumor mass in patients with acromegaly: A systematic review. Pituitary. February 3 [Epub ahead of print]. Melmed, S. (2009). Acromegaly pathogenesis and treatment. The Journal of Clinical Investigation, 119(11), 3189–3202. Melmed, S., Casanueva, F. F., Cavagnini, F., Chanson, P., Frohman, L., Grossman, A., et al. (2002). Guidelines for acromegaly management. Journal of Clinical Endocrinology and Metabolism, 87(9), 4054–4058. Mercado, M., Borges, F., Bouterfa, H., Chang, T. C., Chervin, A., Farrall, A. J., et al. (2007). A prospective, multicentre study to investigate the efficacy, safety and tolerability of octreotide LAR (long-acting repeatable octreotide) in the primary therapy of patients with acromegaly. Clinical Endocrinology (Oxford), 66(6), 859–868. Merola, B., Colao, A., Ferone, D., Selleri, A., Di Sarno, A., Marzullo, P., et al. (1993). Effects of a chronic treatment with

octreotide in patients with functionless pituitary adenomas. Hormone Research, 40(4), 149–155. Modlin, I. M., Oberg, K., Chung, D. C., Jensen, R. T., de Herder, W. W., Thakker, R. V., et al. (2008). Gastroenter­ opancreatic neuroendocrine tumours. The Lancet Oncology, 9(1), 61–72. Murray, R. D., & Melmed, S. (2008). A critical analysis of clinically available somatostatin analog formulations for therapy of acromegaly. Journal of Clinical Endocrinology and Metabolism, 93(8), 2957–2968. Nieman, L. K., & Ilias, I. (2005). Evaluation and treatment of Cushing’s syndrome. American Journal of Medicine, 118(12), 1340–1346. Oberg, K. (2009). Somatostatin analog octreotide LAR in gastro-entero-pancreatic tumors. Expert Reviews Anticancer Therapy, 9(5), 557–566. Ono, K., Suzuki, T., Miki, Y., Taniyama, Y., Nakamura, Y., Noda, Y., et al. (2007). Somatostatin receptor subtypes in human non-functioning neuroendocrine tumors and effects of somatostatin analogue SOM230 on cell prolif­ eration in cell line NCI-H727. Anticancer Research, 27(4B), 2231–2239. Pasquali, D., Rossi, V., Conzo, G., Pannone, G., Bufo, P., De Bellis, A., et al. (2008). Effects of somatostatin analog SOM230 on cell proliferation, apoptosis, and catecholamine levels in cultured pheochromocytoma cells. Journal of Mole­ cular Endocrinology, 40(6), 263–271. Pivonello, R., De Martino, M. C., De Leo, M., Lombardi, G., & Colao, A. (2008). Cushing’s syndrome. Endocrinology and Metabolism Clinics of North America, 37(1), 135–149. Pivonello, R., Matrone, C., Filippella, M., Cavallo, L. M., Di Somma, C., Cappabianca, P., et al. (2004). Dopamine recep­ tor expression and function in clinically nonfunctioning pitui­ tary tumors: Comparison with the effectiveness of cabergoline treatment. Journal of Clinical Endocrinology and Metabolism, 89(4), 1674–1683. Plo¨ ckinger, U., Reichel, M., Fett, U., Saeger, W., & Quabbe, H. J. (1994). Preoperative octreotide treatment of growth hormone-secreting and clinically nonfunctioning pituitary macroadenomas: Effect on tumor volume and lack of corre­ lation with immunohistochemistry and somatostatin receptor scintigraphy. Journal of Clinical Endocrinology and Metabo­ lism, 79(5), 1416–1423. Reichlin, S. (1987). Secretion of somatostatin and its physiolo­ gic function. Journal of Laboratory and Clinical Medicine, 109(3), 320–326. Rinke, A., Müller, H. H., Schade-Brittinger, C., Klose, K. J., Barth, P., Wied, M., et al. (2009). Placebo-controlled, dou­ ble-blind, prospective, randomized study on the effect of octreotide LAR in the control of tumor growth in patients with metastatic neuroendocrine midgut tumors: A report from the PROMID study group. Journal of Clinical Oncology, 27(28), 4635–4636. Rocheville, M., Lange, D. C., Kumar, U., Patel, S. C., Patel, R. C., & Patel, Y. C. (2000). Receptors for dopamine and somatostatin: Formation of hetero-oligomers with enhanced functional activity. Science, 288(5463), 154–157.

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 13

The MENX syndrome and p27: relationships with multiple endocrine neoplasia Sara Molatore and Natalia S. Pellegata
Institute of Pathology, Helmholtz Zentrum M€ unchen-German Research Center for Environmental Health, Neuherberg, Germany

Abstract: In the past 3 years new insight into the etiopathogenesis of hereditary endocrine tumors has emerged from studies conducted on MENX, a rat multiple endocrine neoplasia (MEN) syndrome. MENX spontaneously developed in a rat colony and was discovered by serendipity when these animals underwent complete necropsy, as they were found to consistently develop multiple endocrine tumors with a spectrum similar to both MEN type 1 (MEN1) and MEN2 human syndromes. Genetic studies identified a germline mutation in the Cdkn1b gene, encoding the p27 cell cycle inhibitor, as the causative mutation for the MENX syndrome. Capitalizing on these findings, we and others identified heterozygous germline mutations in the human homologue, CDKN1B, in patients with multiple endocrine tumors. As a consequence of these observations a novel human MEN syndrome, named MEN4, was recognized which is caused by mutations in p27. Altogether these studies identified Cdkn1b/CDKN1B as a novel tumor susceptibility gene for multiple endocrine tumors in both rats and humans. In this chapter we present the MENX syndrome and its phenotype, and we compare it to the human MEN syndromes; we discuss the current state of knowledge regarding the genes associated to inherited MEN, with a particular focus on CDKN1B; we present recent clinical and basic findings about the MEN4 syndrome and the functional characterization of the CDKN1B mutations identified. These findings are placed in the broader context of how p27 dysregulation might affect neuroendocrine cell function and trigger tumorigenesis. Keywords: multiple endocrine neoplasia; MENX; tumor susceptibility genes; p27; animal models

Erdheim described in an article the case of a patient having acromegaly, due to a pituitary adenoma, and three enlarged parathyroid glands (Erdheim, 1903). In the following decades several more cases having various combinations of neuroendocrine tumors were described and importantly in the 1950s these diseases were recognized as genetic disorders inher­ ited as dominant traits. Over the years, the clinical symptoms, the histo-pathological and biochemical features of these multi-tumor syndromes were defined, and finally a consensus about the

Introduction The term ‘multiple endocrine neoplasia’ (MEN) was introduced by Steiner et al. in 1968, but the first description of a patient presenting with multiple endocrine malignancies dates back to the beginning of the ninetieth century, precisely to 1903 when
Corresponding author. Tel.: þ49-89-3186-2633; Fax: þ49-89-3186-3360;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82013-8

295

296

phenotypic presentation associated to the different variants of these syndromes came in the 1960s through the work of Wermer, Williams, Pearse and Sipple among others (reviewed in Carney, 2005). MEN syndromes are characterized by tumors invol­ ving two or more endocrine glands, although tumors can also develop in organs other than these glands. They are autosomal-dominant diseases and have high penetrance and variable clinical expression. Two main syndromes were known until recently: MEN type 1 (MEN1) and type 2 (MEN2), which present with a different tumor spectrum and have different causative mutations. Then, in 2002 a rat strain was discovered where MENs develop consis­ tently. This multi-tumor syndrome, named MENX, has a MEN1-like phenotype and has a different genetic basis than either MEN1 or MEN2. Following studies on MENX, a novel human MEN syndrome (MEN4) was recognized, which associates with mutations in the same susceptibility gene as MENX: the gene encoding p27. As the pathophysiology and molecular genetics of MEN1 and MEN2 tumors syndromes have been reviewed in detail in other publications (Lemos and Thakker, 2008; Lodish and Stratakis, 2008; Marini et al., 2006; Marx, 2005), they will only briefly be discussed here. Emphasis will be on the MENX and the MEN4 syndromes and on the functions of the gene/protein causing them. To fully comprehend how the various MEN syndromes relate to one another, the possible interactions among the proteins associated with

these syndromes will be discussed. As often in cancer research, animal models will be presented that have helped us understand the function of the MEN-associated proteins in tumorigenesis, and specifically in neuroendocrine tumor development.

Multiple endocrine neoplasia type 1 MEN1 is characterized by tumors of the parathyr­ oid, endocrine pancreas and anterior pituitary and affects 1 in 30 000 people (Table 1). The most common clinical manifestation of MEN1 is increased activity of the parathyroid gland (pri­ mary hyperparathyroidism, 1HPT) which occurs in  90% of patients. Hyperparathyroidism alters the normal balance of calcium concentration in the blood, leading to kidney stones, bone thinning, high blood pressure (hypertension), weakness and fatigue. While the sporadic cases of 1HPT pre­ sent as single-gland adenomas, MEN1-associated cases often present as diffuse hyperplasia or multi­ ple adenomas. Pancreatic islet cell tumors occur in 60–70% of MEN1 patients and are usually multi-centric. The most common type is gastrinoma, which can lead to the development of peptic ulcers in the stomach and duodenum, followed by insulinoma, causing hypoglycaemia. Gastrinoma, causing the Zollin­ ger–Ellison syndrome, is the most common cause of morbidity in MEN1 patients.

Table 1. Tumor spectrum of the human and rat MEN syndromes Gene:

Cdkn1b gene

MEN1 gene

Syndrome:

MENX

MEN1

þ (100%)
þ þ þ (100%)
þ þ

þ þ

Tumor phenotype: Pituitary adenoma Parathyroid adenoma Medullary thyroid tumors Pheochromocytoma Paragangliomas Insulinoma Neuroma Others
†

Frequency of the tumor type. The other tumors are reported in Table 2.

þ

RET gene MEN2A

þ þ þ þ

MEN2B

þ þ þ

CDKN1B MEN4

þ þ

þ†

297

Anterior pituitary tumors occur in 15–42% of MEN1 patients and are in general larger, are often multi-focal and behave more aggressively than their sporadic counterpart. Between 25 and 90% of the MEN1-associated pituitary adenomas are prolactinomas. Approximately 25% of pituitary tumors secrete growth hormone (GH) or GH together with prolactin. Excess of GH secretion causes acromegaly while excess of pro­ lactin may cause spontaneous breast milk flow (galactorrhea). A small percentage of pituitary tumors (3%) secrete the adrenocorticotropic hormone (ACTH), causing Cushing’s disease. Most of the remainder are non-functional (Agarwal et al., 2004).

The MEN1 gene and the encoded Menin protein MEN1 is caused by inactivating mutations of the MEN1 gene, which maps to 11q13, consists of 10 exons and is ubiquitously expressed. The protein encoded by MEN1 is called Menin and does not show similarity to any other protein or protein domains in the database (SWISSPROT). The MEN1 locus was first mapped to chromo­ some 11 by linkage in 1988 (Larsson et al., 1988) and the gene was cloned 9 years later (Chandrasekharappa et al., 1997). MEN1 is mutated in the germline of MEN1 patients but it is also often found somatically mutated in sporadic MEN1-associated tumors, indicating that func­ tional Menin plays a critical role in neuroendo­ crine tissue homeostasis. Since the cloning of MEN1, more than 500 different germline and somatic mutations have been identified. Muta­ tions of MEN1 occur mostly in coding exons, but also in intronic sequences and comprise all types of changes, with a relatively high incidence of frameshift mutations (Lemos and Thakker, 2008). The mutations are scattered throughout the Menin protein sequence and there is no geno­ type–phenotype correlation, which could help stratifying the patients for tumor risk in view of a more effective clinical management. MEN1 is considered a tumor suppressor gene, meaning that it functions as a negative regulator of cell proliferation. The classical theory about how

tumor suppressors are involved in the tumorige­ netic process comes from the seminal work of Alfred Knudson and has been referred to as the ‘two-hit’ model of cancerogenesis (Knudson, 1971). In this model the first hit is a heterozygous germline mutation in the tumor suppressor gene (in this case MEN1), present in all cells at birth. The second hit is a somatic mutation in the gene, usually a deletion, which occurs in the predisposed cell (in this case an endocrine cell) and causes the loss of the remaining wild-type allele. Thus, this hypothesis postulates that tumor suppressor genes need to be biallelically inactivated in order to confer growth advantage. In agreement with this theory, the tumors of MEN1 patients show loss of the wild-type allele, suggesting that loss of func­ tion of Menin is necessary for tumor development and that the protein behaves as a ‘canonical’ tumor suppressor in neuroendocrine tissues. How­ ever, this is not always the case as in other tissues, such as leukaemia and prostate, Menin was found to promote tumorigenesis (Paris et al., 2009; Yokoyama et al., 2005). Approximately 30% of the clinically suspected MEN1 patients do not exhibit MEN1 mutations, suggesting that other predisposing genes may play a role in this phenotype, as indeed it was recently demonstrated and is discussed in the next sections. The physiological functions of Menin and its role in tumorigenesis are actively investigated. Menin has many interacting partners, including transcription factors member of the AP-1 family such as Jun and JunD, the nuclear factor (NF)-�B, the Smad family of transcription factors, cell cycle regulators and a variety of other transcription fac­ tors and cell structural elements (reviewed in Agarwal et al., 2005). Menin also interacts with proteins involved in DNA damage-dependent cell cycle arrest or in DNA repair after damage (Yang and Hua, 2007). Through these interactions Menin plays a role in transcriptional regulation, cell proliferation and DNA repair. An interesting issue that concerns Menin, but also other proteins predisposing to neuroendo­ crine tumors, is that it is ubiquitously expressed but the MEN1 syndrome is associated with tissueselective tumorigenesis. Recent studies have helped tackle this issue. It has been reported that

298

Menin associates with members of the trithorax group of transcription factors, such as the mixedlineage leukemia (MLL), MLL2 and Ash2 to form a nuclear complex that is able to methylate histone H3 at lysine 4 thereby activating transcription. Histone modification at specific DNA sites is a very important epigenetic mechanism for main­ taining gene transcription in terminally differen­ tiated cells. It was shown that Menin recruits this MLL-containing complex with histone methyltransferase (HMTase) activity to specific promo­ ter sequences including those of homeobox domain (HOX) genes (Hughes et al., 2004), which are developmental control genes, and of the cyclin-dependent kinase inhibitors (CDKIs) Cdkn1b (p27) and Cdkn2c (p18), which negatively regulate cell cycle progression (Karnik et al., 2005). Interestingly, Menin proteins bearing patient-derived mutations do not activate CDKI transcription, underpinning the importance of Menin-mediated regulation of CDKI transcription in neuroendocrine cells (Karnik et al., 2005). Based on these results, we can conceive a model in which the Menin–MLL complex with HMTase activity can promote transcription of different genes depending on the tissue context. These target genes may be differentially sensitive to Menin malfunction, thereby explaining the selective tumor spectrum of MEN1 (Gracanin et al., 2009).

Multiple endocrine neoplasia type 2 MEN2 is a syndrome of medullary thyroid carci­ noma (MTC) with or without pheochromocytoma and multi-gland parathyroid adenoma. The preva­ lence of MEN2 has been estimated at ~1 in 30 000 people. Several variants of MEN2 exist depending on the clinical presentation of the patients. MEN2 type A (MEN2A) is the most common variant, presenting with MTC and pheochromocytoma in about 20–50% of cases, and with MTC and pri­ mary hyperparathyroidism (parathyroid ade­ noma) in 5–20% of cases. MEN2 type B (MEN2B) is characterized by MTC, pheochromo­ cytoma (in 50% of cases), marphanoid habitus and mucosal and digestive ganglioneuromatosis.

MEN2B is the least common (about 5% of all MEN2 cases) but the most aggressive variant (Table 1). Other rare variants of MEN2 include familial medullary thyroid cancer (FMTC) with­ out other associated endocrinopathies and FMTC with Hirschprung’s disease (Marini et al., 2006). The disease presentation in hereditary MTC is usually bilateral and multi-centric, in contrast to the sporadic cases where it is commonly single and unilateral. The pheochromocytomas that develop within MEN2 are usually bilateral and hormonally active. The first clinical manifestation of MEN2 is usually MTC, indicating that the thyroid para-fol­ licular cells from which MTC derives are the most sensitive to the effect of the underlying genetic mutation. Since this tumor is quite unresponsive to radiation therapy and standard chemotherapy, and it is prone to metastatic spread, MTC repre­ sents the most common cause of death for affected patients. prophylactic thyroidectomy before the tumor spreads beyond the thyroid gland is there­ fore the recommended treatment for mutationpositive patients.

The RET proto-oncogene and the encoded protein MEN2 is caused by mutations in the RET (REar­ ranged in Transfection) proto-oncogene. RET maps to chromosome 10q11.2 and is composed of 21 exons. RET encodes a receptor tyrosine kinase that binds members of the glial cell linederived neurotrophic factor (GDNF) family. These factors play a crucial role in the develop­ ment, function and maintenance of the nervous system, regulating multiple aspects of neural development, such as cell proliferation and survi­ val, migration and axon growth. RET is physiolo­ gically expressed in neural-crest-derived cells, such as thyroid C cells and adreno-medullary cells, and is required for the proper development of the kidney and the enteric nervous system in mammals (Arighi et al., 2005). Alternative splicing of the RET gene results in the production of three different isoforms of the protein RET (Myers, Eng, Ponder, & Mulligan, 1995). Each protein is divided into three domains: an N-terminal extracellular domain with four

299

cadherin-like repeats and a cysteine-rich region close to the cell membrane; a single hydrophobic transmembrane domain and an intracellular por­ tion with two tyrosine kinase sub-domains, TK1 and TK2. Upon ligand binding to the extracellular domain, the receptor dimerizes owing to the cysteine-rich region and its dimerization leads to autophosphorylation of intracellular tyrosine residues, which in turn activates downstream signal transduction pathways (Runeberg-Roos and Saarma, 2007). RET transduces signals that promote cell growth, cell motility, survival and differentiation through different downstream pathways such as RAS/MEK/ERK, PI3K/AKT/ NF-�B and others (de Groot et al., 2006). Germline RET mutations associated with MEN2 are gain-of-function mutations and bring about the activation of the tyrosine kinase activity, independently from the presence of the physiolo­ gical ligands. This condition constitutively activates downstream pathways of signal transduction. It is worth mentioning that RET can also undergo germline loss-of-function mutations and this associates with Hirschsprung’s disease, a rare condition characterized by the absence of gang­ lion cells in the lower digestive tract (Lantieri et al., 2006). The lack of ganglion cells is due to impaired neural crest cell migration during embryonic development, process mediated by wild-type RET expression. Therefore, RET is the paradigm of a single gene that causes different types of cancer in man when targeted by different genetic alterations In MEN2, the position of the mutated base in RET is strongly associated with the disease phe­ notype with regard to age of onset and aggres­ siveness of MTC and the presence or absence of other endocrine tumors. For example, the major­ ity of cases of MEN2A is due to mutations in the extracellular cysteine-rich domain of RET (exons 10 and 11); the C634R missense mutation (exon 11) is strictly associated with pheochromo­ cytoma and primary hyperparathyroidism; most of MEN2B cases are associated with mutations in the intracellular tyrosine kinase receptor domains (exon 16), in particular at codon 918 (Marini et al., 2006; Raue and Frank-Raue,

2007 2009). The good genotype–phenotype cor­ relation of RET mutations in MEN2 provides a unique model for early prevention and cancer cure, as well as for stratification of mutation carriers in different risk groups. This remarkable feature has greatly improved the clinical man­ agement of the affected individuals and their families. Downstream of RET signalling there are a vari­ ety of transduction pathways. Which of these pathways mediate the oncogenic effect of RET in neuroendocrine cells is not completely under­ stood. The effects of RET activation have been extensively studied in MTC cell lines carrying MEN2-associated mutations in RET (such as the TT human thyroid cells) (Carlomagno et al., 1995) or in transgenic mouse strains expressing RET mutant proteins. It has been demonstrated that, as a consequence of gain-of-function muta­ tions in RET, the NF-�B family of transcription factors is constitutively active in MTC cells and their activation is required for RET-induced transformation of these cells (Ludwig et al., 2001). Recently, immunohistochemical analysis of MTCs from either transgenic mice expressing a MEN2B–RET mutation or from MEN2 patients demonstrated an increase in nuclear staining for b-catenin. Interestingly, b-catenin nuclear locali­ zation was more pronounced in metastases then in primary human MTCs. Augmented nuclear localization was associated, in vitro, with the acti­ vation of a b-catenin-dependent transcription pro­ gramme. Indeed, b-catenin is a multi-faceted protein that plays a role in cell adhesion, when it is localized at the plasma membrane, and in gene transcription, when it localizes to the nucleus. When b-catenin is in the cytosol, it is targeted for proteasomal degradation by the adenomatous polyposis coli/Axin/glycogen synthase kinase-3 complex (MacDonald et al., 2009). It was observed that activated RET phosphorylates b-catenin and promotes its detachment from the plasma membrane while, at the same time, pro­ tecting the protein from proteasomal degradation, so that b-catenin can accumulate in the nucleus. In non-endocrine cells over-expressing MEN2­ type mutant RET proteins, functional b-catenin is required for RET-induced cell proliferation and

300

tumor growth in nude mice. Therefore, activation of b-catenin signalling might mediate some of the oncogenic functions of RET in neuroendocrine cells and might explain the early metastatic poten­ tial of MTC (Gujral et al., 2008). In vitro studies on fibroblast cell lines have sug­ gested that activated RET regulates the expres­ sion of the CDKIs p18 and p27, thereby promoting cell proliferation. This was also con­ firmed in the pheochromocytoma samples from MEN2 patients: low immunohistochemical stain­ ing for both CDKIs was observed in tumors com­ pared to normal adrenal tissue (Joshi et al., 2006). Concomitant with reduction of CDKI expression, there was an increased expression of D-type cyclins, resulting in an enhanced proliferative sti­ mulus. Therefore, downstream of oncogenic RET signalling are multiple pathways that promote the proliferation and possibly the cell adhesion prop­ erties of neuroendocrine cells.

The MEN1-like syndromes The MENX syndrome The MENX multi-tumor syndrome was discov­ ered in a Sprague–Dawley rat colony in Neuher­ berg which spontaneously started to develop multiple neuroendocrine tumors, specifically: anterior pituitary adenoma, adrenal pheochromo­ cytoma as well as extra-adrenal pheochromocy­ toma (paraganglioma), thyroid C-cell hyperplasia, parathyroid hyperplasia and pancrea­ tic islet cells hyperplasia (Fritz et al., 2002) (Fig. 1 and Table 1). These malignancies show a clear progression from hyperplasia to tumor with time: affected rats develop with complete penetrance adrenal medullary hyperplasia at 3–4 months of age, which develops into pheochromocytoma by 6–8 months. They also present (100%) with ante­ rior pituitary hyperplasia at about 4 months of age, which progresses to adenoma by 8–12 months. The morphology and histo-pathology of the rat tumors is similar to that of their human counterpart, and especially to that of tumors developing within the context of inherited predis­ position, being the rat tumors bilateral (adrenal,

thyroid and parathyroid) and/or multiple (pitui­ tary, parathyroid). Affected rats have an average life span of 10 + 2 months whereas their wild-type littermates live ~24–30 months (Pellegata et al., 2006). Mutant rats are bigger in size than their wildtype littermates and show organomegaly, parti­ cularly of spleen and thymus. In addition to the tumor phenotype, affected animals develop macroscopically visible bilateral juvenile catar­ acts, a phenotype that precedes the appearance of neoplastic disease. In contrast to the human MEN syndromes, MENX is inherited as a reces­ sive trait, therefore affected rats are homozy­ gous for the underlying genetic mutation. The symptomatology of affected rats is compatible with high blood pressure being the leading cause of morbidity, although formal proof is yet to be provided. In order to identify the gene responsible for this disease, classical linkage studies were per­ formed and they initially mapped the MENX locus to an interval of ~22 cM on the distal part of rat chromosome 4 (Piotrowska et al., 2004). This excluded the homologues of genes that predispose human patients to neuroendo­ crine tumors (i.e. Men1, Vhl, Nf1, Sdhd and Sdhb) since they map to other rat chromosomal regions. However, on the distal part of rat chro­ mosome 4 maps the rat homologue of the RET gene (causing MEN2 in humans). Therefore, efforts were employed to determine whether this gene could cause MENX, but genotyping of additional animal crosses excluded Ret as being the candidate gene for the rat syndrome (Piotrowska et al., 2004). Therefore, these genetic studies indicated early on that the causative genetic event in MENX was the mutation of a gene never before associated to the predisposition to neuroendocrine tumors. The MENX locus was subsequently finemapped to a ~3 Mb interval, a size amenable to search for candidate genes. Considering both the tissue expression profile in silico and the putative function of the transcripts mapped to this region, a few genes were selected for mutation screening. Among them was Cdkn1b, encoding the CDKI p27. Several observations made Cdkn1b a possible

301 Wild-type

Mutant B.

C.

D.

E.

F.

G.

H.

Pancreas

Thyroid gland

Pituitary gland

Adrenal gland

A.

Fig. 1. Histology of adrenal (A, B), pituitary (C, D) and thyroid (E, F) glands and of pancreas (G, H) of wild-type and age-matched MENX-affected (Mutant) rats. Multi-focal hyperplastic lesions in the anterior pituitary are indicated by arrows (D). A nodule of thyroid C cell hyperplasia is also indicated (F). Pancreatic islet cells are indicated by arrows in panels G and H: since the pictures were taken with the same magnifications the increase in size of the islets in mutant rats can be readily appreciated. H&E stain original magnification: 20× (A, B, C, D); 25× (E, F); 40× (G, H).

candidate for the MENX syndrome: (1) p27 is ubiquitously expressed, so also in organs that develop tumors in affected rats; (2) p27 knockout mice develop intermediate lobe pituitary gland tumors, an organ that is also affected in the MENX syndrome and (3) p27 is a negative regu­ lator of the cell cycle and a putative tumor sup­ pressor, and this is consistent with the recessive mode of inheritance observed in MENX rats. In fact, based on the ‘two-hit’ model for tumor

suppressor gene cancerogenesis, we expect such a gene to require biallelical inactivation in order to give cells the survival advantage required for tumor development. Upon sequencing of the Cdkn1b gene in affected and unaffected littermates, a tandem duplication of eight nucleotides in exon 2 (c. 520–-528dupTTCAGAC) which causes a frame­ shift was identified in affected rats. At the pro­ tein level the mutated allele encodes a protein predicted to have a novel C-terminal sequence starting at codon 177. While the wild-type p27 protein is 198 amino acids long, the mutated protein, as a result of the frameshift, is predicted to be 221 amino acids long. This Cdkn1b muta­ tion segregated with the disease in all affected rats tested (>200) and was not observed in unaf­ fected littermates nor in control wild-type rats of seven commercially available inbred strains (Fig. 2A). Following the identification of the causative genetic mutation, the expression of the Cdkn1b gene in MENX rats was explored in more detail to assess its expression pattern in mutant ani­ mals. The analysis of various tissues of affected rats at young age (before they develop malig­ nancies) and wild-type littermates (controls) showed no significant difference in the level of Cdkn1b mRNA between the two groups. Furthermore, correct splicing of the mutant mRNA was demonstrated in affected rat tissues. When the expression of the encoded p27 protein was assessed by western blotting, a faint band corresponding in size to the predicted mutant p27 protein could be observed only in a few tissues (i.e. thymus and thyroid). Immunohisto­ chemical staining performed with an anti-p27­ specific antibody, showed extremely low (thyr­ oid, pituitary, thymus, parathyroid and brain) or lack of (adrenals, lung, kidney, liver and testis) p27 immunoreactivity in tissues of affected rats, while the same tissues presented with strong nuclear positivity for p27 in wild-type rats (Fig. 2B). This suggests that the MENX muta­ tion does not affect the transcription or proces­ sing of Cdkn1b mRNA, but affects the amount of mutant p27 protein by post-transcriptional or post-translational mechanisms.

302 A. = Unaffected rats (+/+, +/mut) +/mut

= Affected mut/mut rats

+/mut

M Mutant Wild-type

mut/mut

+/+

A TGTTTCAGAC TTT CAG ACGGT TCCCC A TGTT TCA GA CGGT TCCCC Ser Asp Gly Ser Ser Asp Phe Gln Thr

+/mut A TGTTTCA GA CGGTTCCCC

TTTCAGAC

B. Mutant

Thyroid

Adrenal

Wild-type

p27 Fig. 2. Identification of the Cdkn1b mutation in MENX and assessment of p27 expression. (A) Pedigree of a breeding group segregating the MENX phenotype. Genomic DNA of the animals was amplified with primers spanning the Cdkn1b mutation and the alleles (wt and mutated) were resolved on polyacrylamide gels. For each Cdkn1b genotype the corresponding sequence chromatogram is shown. The insertion in the mut/mut rat sequence is indicated by a rectangle. Open square/circle: unaffected male/female; filled square/circle: affected male/female. M, molecular size marker. (Modified with permission from Pellegata et al. (2006). Copyright © 2006 by the National Academy of Sciences, U.S.A.) (B) Immunohistochemical analysis demonstrates extremely reduced/loss of expression of p27 in vivo. (Top) p27 staining of the adrenal gland of 2-month-old wild-type and mutant littermates. The medulla of the mutant animal shows no positivity for p27 (Inset). (Bottom) p27 staining of the thyroid gland. The mutant rat shows very faint nuclear positivity for p27 in 10% of the cells (inset). Immunoperoxidase original magnification: (top) ×200; (bottom left) ×400; (bottom right) ×100; insets: ×640.

303

The identification of a new multiple endocrine neoplasia syndrome in man (MEN4) If mutations in RET virtually explain all the MEN2 cases, 30% of patients having a MEN1­ like phenotype do not carry MEN1 gene muta­ tions. While this could be due to the presence of mutations in non-coding or regulatory sequences that are not commonly analysed, it leaves open the possibility that some of these patients may bear mutations in additional, as yet unidentified sus­ ceptibility genes. Following the identification of Cdkn1b as the gene predisposing MENX rats to multiple endocrine tumors, it seemed meaningful to check whether mutations in the human homo­ logue (CDKN1B) could explain some of these MEN1-like cases. Therefore, individuals with a MEN1-like phenotype but lacking mutations in the MEN1 gene were screened for the presence of CDKN1B germline mutations. A female proband with GH-secreting pituitary adenoma (causing acromegaly; 30 years) and primary hyperparathyroidism (46 years) was found to carry a germline heterozygous TGG->TAG (c. G692A) nonsense mutation at codon 76 (p. W76X). Extended pedigree analysis confirmed that in the proband’s family this mutation segre­ gates with the predisposition to tumors belonging to the MEN1 tumor spectrum. Indeed, a mutationpositive sister of the proband showed a renal angiomyolipoma (55 years), a tumor associated with the MEN1 syndrome (Pellegata et al., 2006) (Table 2). Since in the proband’s family this change was inherited together with a MEN1-like tumor phenotype and because it had a dramatic effect on the protein sequence, this was consid­ ered a pathogenic mutation. Georgitsi et al. (2007) analysed the CDKN1B gene in a larger series of mainly European patients having a MEN1-like phenotype, and no mutations in MEN1, or presenting with familial or sporadic pituitary adenomas. They identified a heterozygous 19 bp duplication in CDKN1B exon 1 in a female patient who had developed three lesions compatible with MEN1: small-cell neu­ roendocrine cervical carcinoma (45 years), ACTH-secreting pituitary adenoma (Cushing’s disease) (46 years) and hyperparathyroidism (47

years) (Table 2). This duplication causes a frame­ shift (c. K25fs) and the variant mRNA is predicted to encode a protein having a different amino acid sequence, after codon 25, when compared to wildtype p27, and being 69 amino acids shorter than the normal protein. Similarly to the p27W76X variant, this genetic alteration causes a dramatic change in the p27 protein sequence, and therefore it was considered a pathogenic mutation. In a study of American patients with a family history of endocrine tumors and showing 1HPT as a common feature, three individuals were found to carry the following germline CDKN1B changes: ATG-7G->C; p27P95S; stop->Q (Agarwal, Ozawa, Mateo, & Marx, 2009). The patient having the nucleotide change in the 50 untranslated region of CDKN1B (ATG-7G->C) presented with pri­ mary hyperparathyroidism (61 years) and a nonfunctioning bilateral adrenal mass (63 years). The patient with the missense variant at codon 95 pre­ sented with 1HPT, Zollinger–Ellison syndrome and masses in the duodenum and pancreas (50 years). The individual with the Stop->Q mutation showed primary hyperparathyroidism (50 years). Since these three changes affect, at least in vitro, the amount of p27 protein or its binding to other proteins, they were classified as potentially pathogenic. The discovery of patients with germline CDKN1B mutations and a MEN1-like tumor phe­ notype prompted the OMIM database for Mende­ lian Disorders to recognize a novel MEN syndrome, named MEN4, which is caused by p27. Due to the limited number of cases so far reported, the precise tumor spectrum associated with the MEN4 syndrome is currently not defined (Tables 1 and 2). As new cases with germline CDKN1B mutations are identified, it will be inter­ esting to see whether tumor types outside the context of MEN1 are also a feature of MEN4. It is interesting to note that germline mutations in Cdkn1b/CDKN1B induce a different pheno­ type in rats and humans (Table 1). Indeed, while the first pathological manifestation of MENX is hyperplasia of adreno-medullary cells, which pro­ gresses to tumor with time, pheochromocytoma was not observed in the mutation-positive patients. This might be due to a species-dependent

Table 2. Clinical characteristics of the mutation carriers and molecular phenotype of the p27 variant proteins identified to date Molecular phenotype

p27 variant

Other affected in the family

Protein expression in vitro

Protein localization in vitro

Protein interaction

Protein expression in vivo

Protein localization in vivo Cytoplasm/ normal tissue

Pellegata et al. (2006) Georgitsi et al. (2007)

Syndrome

Clinical phenotype of proband

p27W76X

MEN1­ like

1HPT, GHpituitary adenoma

1

Wt

Cytoplasm

ND

NO/tumor tissue


p27K25fs

MEN1­ like

0

ND

ND

ND

NO/tumor tissue (LOH)

p27ATG7G->C

MEN1­ like

0

Reduced

ND

ND

ND

ND

Agarwal et al. (2009)

p27P95S

MEN1­ like

0

Wt

ND

GRB2/ Reduced

ND

ND

Agarwal et al. (2009)

p27stop>Q

Fam. 1HPT

1HPT, ACTHpituitary adenoma, carcinoid tumor of the uterine cervix 1HPT (1 PT tumors), bilateral adrenal mass NF, uterine fibroids 1HPT (2 PT tumors), ZES, mass in duodenum and tail of pancreas 1HPT (3 PT tumors)

3

Reduced

ND

CDK2/wt

ND

ND

Agarwal et al. (2009)

1HPT, primary hyperparathyroidism; PT, parathyroid tumor; NF, non-functioning; ZES, Zollinger–Ellison syndrome; LOH, loss of heterozygosity; Wt, wild-type = normal.
There is no expression in the tumor.

References

305

and tissue-specific sensitivity to impaired p27 function (i.e. rat adreno-medullary cells are more sensitive to it than their human counterpart). Alternatively, it might be due to distinct biological properties of the p27 mutations in MENX or in MEN4 patients. In this context it is worth bearing in mind that while MENX-affected rats are homo­ zygous for the causative Cdkn1b mutation, MEN4 patients carry heterozygous mutations in CDKN1B.

The CDKN1B gene and the encoded p27 protein The CDKN1B gene maps to 12p13, consists of two coding exons and encodes for the ubiquitously expressed, 198 amino acids long p27 protein. p27 is a well-characterized CDKI that controls the progression from G1 to the S phase of the cell cycle in response to either mitogenic and antimitogenic stimuli. p27 belongs to the KIP/CIP family of CDKI together with p21 and p57. The other CDKI family (named INK4) comprises p15, p16, p18 and p19. In the eukaryotic cell cycle the progression from one phase to the next depends on the activities of a series of cyclin/cyclin-dependent kinase (CDK)

complexes, consisting of a catalytic sub-unit (CDK) and a positive regulatory sub-unit (cyclin). The entry into the G1 phase is determined by assembly and activation of cyclinD/CDK4 or cyclinD/CDK6 complexes. The subsequent pro­ gression through G1/S transition and S phase is dependent on cyclinE/CDK2 and cyclinA/CDK2 complexes. These complexes inactivate, through phosphorylation, members of the retinoblastoma (Rb) family causing the release of the E2F family of transcription factors which in turn activate the transcription of genes responsible for G1 to S progression. p27’s main function is to bind and inhibit cyclinE/Cdk2 complexes (Fig. 3). However, p27 also binds cyclinD/CDK4–6 complexes and acts as an essential assembly factor for such com­ plexes. In contrast to cyclinE/CDK2, cyclinD/ CDK4–6 complexes remain catalytically active when bound to p27. Binding to cyclinD/CDK4–6 sequesters p27 away from cyclinE/CDK2 com­ plexes, thereby allowing for cell cycle progression (Slingerland & Pagano, 2000). p27 is mainly nuclear in growth-arrested cells (where it inhibits cyclinE/CDK2) but as cells progress along the cell cycle (i.e. upon mitogenic stimulation) it is trans­ located to the cytoplasm, thereby withdrawing its inhibition on cyclinE/CDK2 function.

ATP Cyclin E

CDK2

ADP

p

RB

p

RB E2F

E2F

Genes required for the S phase

Cyclin E

CDK2

RB E2F

p27

Fig. 3. Schematic representation of the regulation of Rb function by p27.

306

p27 in many cell types is a rate-limiting effec­ tor of cell cycle exit. Hence, the intracellular amount of p27 is tightly regulated, being high during quiescence and low during S phase, when the cells are committed to proliferation. This fine regulation of the levels of p27 is principally achieved through ubiquitin-dependent proteaso­ mal degradation. Two main pathways involved in p27 degradation have been identified. The first is mediated by the Skp2-dependent SCF (skp-cullin-f-box) E3 ligase and requires phos­ phorylation of p27 by cyclinE/CDK2 at a con­ served threonine (Thr187) (Montagnoli et al., 1999; Pagano et al., 1995). Skp2-mediated degra­ dation of p27 occurs in the nucleus in G1-S and G2 phases of the cell cycle. The second pathway is mediated by the KPC ubiquitin ligase and is responsible for the degradation of p27 in the cytoplasm at the G0–G1 transition (Kamura et al., 2004). The function of p27 is also modulated by altering its intracellular localization. Indeed, as mentioned, p27 is mainly a nuclear protein and in this compartment can bind and inhibit cyclin/

ATG-7G->C K25fs

P95S

25

l119T Q104X

W76X

Cyclin

CDK complexes, but it can be sequestered in the cytoplasm where it may have additional functions that are not completely understood. Noteworthy, a pro-oncogenic role for p27, linked to its cytoplasmic localization and inde­ pendent from its ability to bind to cyclin/CDK complexes, has been proposed based on studies of mice strains with specific mutations in p27 (Besson et al., 2007). This pro-oncogenic activity might be related to the ability of cytoplasmic p27 to associate with key proteins such as Rac and RhoA and promote cell migration (Besson et al., 2004; McAllister et al., 2003). In addition to cyclin/CDKs and Rac/RhoA, p27 interacts with signal transduction molecules such as Grb2, activator of Ras (Moeller et al., 2003), and adaptor proteins such as Jab1, which is involved in the shuttling of p27 between nucleus and cytoplasm (Tomoda et al., 1999). In Fig. 4 are indicated the main interacting part­ ners of p27 and the protein domains mediating those interactions. It is important to mention that p27 is also involved in cell differentiation. Indeed, p27

Stop->Q

Cdk

Lenght:198 aa 153–166 NLS

50

CycA 52

90

Cdk2 90 96

Grb2 96

151

Jab1 118

158

Scatter domain (Rac) 198

118

RhoA 170

198

Stathmin Fig. 4. Schematic structure of the p27 protein. Indicated are the regions mediating the binding to the interacting partners. Above the protein are reported the germline mutations and the somatic mutation (boxed) so far identified in patients. The W76X and I119T mutations were found both as somatic and as germline changes (underlined).

307

controls the development of the mouse cerebral cortex by regulating in a concerted manner cell cycle exit, differentiation of cortical progenitor cells into neurons and migration of neurons to the cortical plate (Nguyen et al., 2006). These functions are independent from the capacity of p27 to inhibit cyclin/CDK complexes. In conclu­ sion, p27 is a complex molecule whose multiple functions are regulated through a plethora of pro­ tein–protein interactions and post-translational modifications, alteration of intracellular localiza­ tion and efficiency of degradation. Several observations have classified p27 as a putative tumor suppressor: (1) it functions as a negative regulator of cell cycle; (2) its overexpres­ sion blocks cell growth in several cell types; (3) its absence predisposes mice to tumor formation. However, p27 is an atypical tumor suppressor since, in contrast to other well-known tumor sup­ pressors such as p53 and RB1, it is not somatically mutated in human cancers. Indeed, only a handful of potentially pathogenic somatic changes have been reported in the hundreds of human tumor samples analysed since the cloning of the CDKN1B gene: a nonsense mutation in an adult T-cell leukemia/lymphoma (p27W76X) (Moro­ setti et al., 1995); a nonsense mutation in a breast cancer sample (p27Q104X) (Spirin et al., 1996); a missense change in an unclassified myeloproli­ ferative disorder (p27I119T) (Pappa et al., 2005) (Fig. 4). The effect of these somatic mutations on the function of the encoded p27 protein was not determined by the authors. Noteworthy, one of these three somatic changes, specifically p27W76X, was later found to cause MEN4 in a family with MEN1-like phenotype. For a protein that is so rarely mutated, such as p27, it can hardly be a coincidence that the same mutation would arise independently in a leukaemia sample (somatic) and in the germline of a family that develops endocrine tumors. Therefore it is tempt­ ing to speculate that this truncated p27 protein might have a pro-oncogenic effect in different tumor tissues. While p27 is not mutated in human cancers, it is frequently down-regulated and its reduced expression is associated to a poorer prognosis in cancers of the colon, breast, prostate, liver,

lung, bladder, ovary, stomach and others (Chu et al., 2008). In colorectal carcinoma samples it was observed that loss or reduction of p27 expression was associated with accelerated proteasome-dependent degradation of the protein (Loda et al., 1997). In some tumor tissues, increased expression of the ubiquitin ligase Skp2, which is involved in p27 degradation, has been observed and this is inversely correlated with the level of p27 (Hershko, 2008). Although increased p27 degradation has not been demon­ strated in all tumors with decreased expression of p27, the observation that low protein levels often associate with no change in CDKN1B mRNA expression has been considered indirect evidence that enhanced proteolysis is responsible for p27 down-regulation in such tumors. What about the level of p27 expression in neuroendocrine tumors? Decreased expression of p27 has been reported in human sporadic corticotroph pituitary adenoma and carcinoma, where its level of expression inversely correlates with the aggressiveness of the tumor (Lidhar et al., 1999). No differences in CDKN1B mRNA expression were observed between pitui­ tary adenomas and normal pituitary tissue. Instead, there was an inverse correlation between p27 and Skp2 protein expression in pituitary tumors, suggesting that increased Skp2 might mediate, at least in part, the down-regula­ tion of p27 (Musat et al., 2004). Reduction/loss of p27 expression occurs fre­ quently also in sporadic adrenal pheochromocy­ toma (56%) and its reduction does not associate with loss of heterozygosity (LOH) at the p27 locus nor with mutations of the gene (Pellegata et al., 2007). So, again it might be due to post­ translational mechanisms, although in these tumors mRNA expression was not studied. Parathyroid adenomas have lower p27 immu­ noreactivity than normal parathyroid cells, while there appear to be no difference in the level of CDKN1B expression between tumors and control tissues (Erickson et al., 1999). In conclusion, several sporadic neuroendocrine tumors show low p27 expression underpinning the importance of an impaired protein function in their development.

308

Functional characterization of the CDKN1B mutations predisposing to neuroendocrine tumors Five germline CDKN1B mutations have been identified to date in MEN4 patients and are indicated in Fig. 4. We can observe that these mutations are point mutations or insertions creating a frameshift, and that they occur either in various domains of the p27 protein or in the 50 untranslated region of the gene. Although few, they offer us the possibility to better under­ stand which features of p27 are important in mediating tumor suppression. Therefore, let us consider what we know so far about the mole­ cular phenotype associated with these mutations. Proceeding from the 50 end of the gene, one patient was reported to carry a nucleotide change in the Kozak sequence at position –7 with respect to the translational initiation site (ATG-7G>C). The Kozac sequence is recognized by the ribo­ some as the translational start site and it is highly conserved among vertebrate mRNAs. Transfec­ tions were performed using the wild-type or the variant CDKN1B cDNA and showed that while the amount of mRNA produced by both alleles is similar, the amount of p27 protein produced by the variant-containing cDNA is less than that pro­ duced by the wild-type allele, suggesting deficient translation of p27 protein (Agarwal et al., 2009). The frameshift mutation at codon 25 (p27K25fs) was associated with LOH of the wildtype CDKN1B allele and was not further analysed in vitro. The p27W76X mutation encodes for a trun­ cated protein which, upon transfection into recipi­ ent rat fibroblast cells, is mislocalized to the cytoplasm (Pellegata et al., 2006). Therefore, this change prevents the mutant p27 protein from entering the nucleus, the cell compartment where p27 exerts its cell cycle regulatory function by binding to cyclin–CDK complexes. We previously pointed out that this mutation might be selected by the tumor cells as it was also identified as a somatic change in a leukemia sample (Morosetti et al., 1995). The cytoplasmic localization of the p27W76X mutant protein in vitro is consistent with this hypothesis, since it has been proposed

that p27 may actively contribute to tumorigenesis when confined to this compartment. p27 interacts, among others, with the Grb2 pro­ tein, which is an adaptor protein that binds to the guanine nucleotide exchange factor Sos and recruits it to the plasma membrane where it can activate the Ras/mitogen-activated protein kinase pathway. p27 binds to Grb2 via a proline-rich region between amino acids 91 and 95 (Fig. 4) and prevents the association of Grb2 with Sos. It was demonstrated that the p27P95S variant binds to Grb2 with reduced efficiency in vitro because the change affects the last residue in the prolinerich domain mediating the binding. The relation­ ship between p27 and activation of Ras pathway is still not clear, as no alterations in the activation of this pathway were identified in cells lacking p27 (p27–/– mouse embryonal fibroblasts) and accord­ ingly having excess of Grb2–Sos (Moeller et al., 2003). However, the change in the binding char­ acteristics of the p27P95S protein suggests a potential effect on the activation of the Ras signal transduction pathway. Expression analysis of the p27 stop->Q var­ iant protein showed a higher molecular weight than wild-type p27, as predicted from the exten­ sion of the open reading frame by 60 amino acids to the next in-frame stop codon. In vitro, the amount of the encoded mutant protein was lower than that of the wild-type one, although no differences in expression between the mutant and normal CDKN1B allele at the mRNA was evident (Agarwal et al., 2009). The proteasome inhibitor MG132 rescued the expression of the p27stop>Q protein bringing it to wild-type levels. Therefore, the stop->Q variation encodes a protein unstable in vitro and degraded by the proteasome. The in vitro characterization of the p27 mutant/ variant proteins performed to date has shown that they have a lower steady-state level than wild-type p27 (ATG-7G>C; stop->Q), are mislocalized to the cytoplasm (p27W76X) or show reduced protein-binding efficiency (p27P95S). It would be interesting to expand the character­ ization of the p27W76X mutant molecule to include its effect on cell migration, as this has been linked to the pro-oncogenic effect of p27.

309

A thorough molecular characterization of these few naturally occurring CDKN1B mutations may broaden our understanding of the relationship between p27 and neuroendocrine tumor predispo­ sition. In addition, knowing the properties of the mutant p27 proteins associated to hereditary tumors may facilitate the development of targeted therapeutic strategies for a more effective clinical management of the patients carrying those muta­ tions and of their families.

Contribution of animal models to our understanding of the role of p27 in neuroendocrine tumorigenesis Already before the identification of germline Cdkn1b/CDKN1B mutation in rats and patients predisposed to neuroendocrine tumors, the impor­ tance of p27 in the maintenance of endocrine organs had been illustrated by the phenotype of various mice models bearing a deletion of this gene. It all started in 1996, 2 years after the cloning of the Cdkn1b gene (Polyak et al., 1994), when, to determine the effect of its inactivation in mamma­ lian development, three independent research groups generated p27 knockout mice (Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996). The disruption of the murine gene caused a gene dose-dependent increase in animal size without altering the normal body proportions. Although the weight of most organs had increased in proportion to the whole body weight, others were disproportionately enlarged including thy­ mus, spleen, testis, ovary, pituitary gland, adrenal gland and pancreatic islets (Rachdi et al., 2006). In particular, in the pituitary was evident an adeno­ matous transformation of the cells in the pars intermedia. This was the sole tumor phenotype observed. None of the hormones that are usually produced by the pituitary gland was expressed in the cells of the adenomatous tumor tissue. Since increased animal size can be associated with endo­ crine abnormalities (as for transgenic mice expres­ sing GH or insulin growth factor-1), the possibility of an endocrine contribution to the increased size of p27-deficient mice was evaluated by measuring

hormone levels in serum that resulted though nor­ mal. Taking into account different phenotypic and biochemical parameters of the p27–/– mice, the resulting picture was incompatible with gigantism due to endocrine-related effects. Further analysis demonstrated that the augmented size of several organs was due to increased cell density. Another phenotype caused by disruption of p27 was female infertility, possibly due to an alteration of endo­ crine signalling that affects the cells involved in the hypothalamic–pituitary–ovarian axis. Taken together, these findings established p27 as an essential regulator of body growth in mice, with specific endocrine effects on particular cell types. The phenotype of p27 –/– mice seems to derive from a combination of alteration in cell prolifera­ tion, and perturbation of specific endocrine cell functions (Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996). Using as starting point the information gained with the p27-deficient mouse, many double knock­ out murine models were subsequently generated, in order to clarify the role of Cdkn1b as tumor suppressor gene and its involvement in organo­ genesis and tumor development. It was shown that loss of p27 collaborates with several onco­ genic stimuli by increasing tumor multiplicity or reducing tumor latency, or both. A prominent neuroendocrine tumor phenotype is observed in several of these animal models. In mice the loss of another CDKI gene, Cdkn2c (p18), belonging to the INK4 family, leads to enlargement of many organs, such as the spleen, thymus, heart, testis and adrenal gland (Franklin et al., 1998). Moreover, intermediate lobe pitui­ tary hyperplasia (in some animals even adenoma) was observed with nearly complete penetrance. Crossing p18 –/– mice into a p27 –/– background resulted in animals that spontaneously develop different types of hyperplastic tissues and/or tumors in the pituitary, adrenals, thyroid, para­ thyroid, testes, pancreas, duodenum and stomach. Interestingly, six of these hyperplastic tissues and tumors were in endocrine organs, so that the spec­ trum of affected tissues overlaps that commonly seen in patients with MEN syndromes. In addition, these double mutant mice invariably died from pituitary adenomas by 3 months of age, indicating

310

a shorter tumor latency than in either p18 –/– or p27 –/– single mutants that develop tumors by 10 months of age (Franklin et al., 1998; Franklin et al., 2000). Therefore, p18 and p27 collaborate to sup­ press pituitary tumorigenesis. Other genes involved in the regulation of the cell cycle were also inactivated together with p27. For example, breeding cyclinD1-deficient mice into a p27 –/– background essentially rescued the developmental defects of cyclinD1-null mice (mis­ aligned teeth, reduced body size and retinal abnormalities). In contrast, the tumor phenotype of p27-deficient mice was not corrected by loss of cyclinD1: the double mutants developed pituitary tumors with similar histo-pathology and latency as p27 –/– animals. This provided evidence that p27 acts downstream of cyclinD1 and it is an important mediator of cyclinD1-dependent developmental pathways (Geng et al., 2001). Mice with mutations that impair the function of the Rb pathway in cell cycle control (i.e. Rb þ/–, chimeric Rb –/– mice) develop intermediate lobe pituitary tumors with high penetrance (Jacks et al., 1992, Williams et al., 1994). For this reason, Park et al. (1999) examined the phenotype of double mutant Rb –/þ, p27 –/– mice: they developed pituitary adenocarcinoma with loss of the remaining wild-type Rb allele, and a high-grade thyroid C cell carcinoma (MTC). Both tumors were detected quite early in the double mutants and they appeared more aggressive than in the single Rb –/þ mutant mice, as they displayed at higher frequency severe features such as haemorrhage, necrosis and invasion. These results speak of a collabora­ tion between these two proteins in suppressing endocrine tumorigenesis. Being the role of p53 in tumor predisposition well known and being the p53-deficient mice deeply characterized, two groups investigated the functional collaboration between p53 and Cdkn1b in tumor suppression by generating p53–/–, p27 –/– double mutant mice. The tumor spectrum of the double mutant mice closely resembled that of the single p53 mutants, but, in addition to lymphomas and sarcomas (char­ acteristic of the p53 –/–mice), they developed a consistent number of other tumor types,

including adrenal medullary hyperplasias and pituitary gland enlargement, indicative of early stages of adrenal and pituitary tumor develop­ ment (Damo et al., 2005; Philipp-Staheli et al., 2004). This indicates strong synergy between the two proteins in tumor suppression. PTEN (Phosphatase and TENsin homologue) is a tumor suppressor gene frequently mutated in a variety of human tumors. In mice, monoallelic inactivation of this gene alone or in combination with p27 inactivation predisposes animals to prostatic intraepithelial neoplasia and bilateral hyperplasia of the adrenal medulla (pheochro­ mocytoma) (Di Cristofano et al., 2001). The double knockout mice showed increased tumor multiplicity and decreased tumor latency com­ pared to Pten þ/– animals, suggesting again collaboration between Pten and p27 in suppressing tumor growth in various neuroendo­ crine organs. Another model of endocrine tumorigenesis is represented by human growth hormone-releasing hormone (hGHRH) transgenic mice. These mice exhibit somatotrope hyperplasia and once crossbred with p27 –/– mice earlier appearance and increased penetrance of pituitary tumors was observed (Teixeira et al., 2000). In conclusion, ablation of p27 accelerates tumor formation in mice in co-operation with several oncogenic events and often confers a neuroendo­ crine-selective tumor phenotype.

Is p27 a dose-dependent tumor suppressor in neuroendocrine cells? There has been some debate as to whether Cdkn1b/CDKN1B is a bona fide tumor suppressor gene. The reduced expression in many human tumor types and the finding that lack of p27 in mice predisposes to tumor formation supports the hypothesis of a tumor suppressor role of p27 in tumorigenesis. However, since hemizygous loss of p27 in haematopoietic malignancies occurs in the absence of inactivation of the wild-type allele (Komuro et al., 1999; Sato et al., 1995), it was sus­ pected that p27 might not be a ‘canonical’ tumor suppressor, and that maybe it did not need to be

311

completely inactivated to confer a proliferative advantage to the cells. As it is often the case, animal models have helped tackle the issue of the importance of the dose of p27 in tumor formation. p27 –/– mice, in addition to being analysed for their spontaneous tumor phenotype, were also studied following exposure to chemical carcinogens or radiation to determine the effect of DNA damage on tumor development in the absence of p27. After treat­ ment with g-irradiation or the chemical carcino­ gen N-ethyl-N-nitrosourea (ENU), p27-null mice showed an increased mortality mainly due to the development of pituitary tumors and intestinal adenomas. In addition, also adrenal tumors, both medullary and cortical, occurred at higher frequency in p27 –/– mice (Fero et al., 1998; Payne and Kemp, 2003). When subjected to the same treatments, heterozygous p27þ/– mice were also significantly more susceptible than wild-type mice to tumorigenesis of the lung, intestine and pituitary, but at intermediate rates compared with wild-type and double knockout animals. Following the ‘two-hit’ model of cancerogenesis, increased tumor formation is often observed in mice heterozygous for a germline deletion of a tumor-suppressor gene, and in the tumors of these animals the remaining wild-type allele is usually inactivated. In contrast to this hypothesis, no mutations, deletions or silencing of the wildtype Cdkn1b allele were found in the tumors from p27 þ/– mice, allele which remained then active. Therefore, reduction to one dose of func­ tional p27 predisposes mice to tumor formation. It was later observed that p27 þ/– mice are more susceptible to tumor formation than p27-null animals in murine models of mammary and prostate cancer, implying that the remaning p27 actively contributes to tumor formation (Gao et al., 2004; Muraoka et al., 2002). Studies on mice with a mutation in p27 that abolish its translocation to the cytoplasms have shown that expressing a p27 protein that is con­ fined to the nucleus, where it can bind cyclin/CDK complexes, makes the animals in part resistant to tumorigenesis induced by urethane. This suggests that a cytoplasmic function of wild-type p27, lost by the nuclear mutant protein, might be mediating

the tumor-promoting effect of p27 (Besson et al., 2006). These and similar studies have lead to the hypothesis that complete loss of p27 is not selected by the tumor cells. To better understand this sce­ nario, we must not forget that p27 is required for the assembly of cyclinD/CDK4–6 complexes, which are essential promoting factors of cell cycle progression. Thus, in mice p27 behaves as a dose-dependent tumor suppressor whose residual function needs not to be abolished in order for tumorigenesis to take place. In human tumors often the expression level of p27 is reduced in hyperplasic and dysplastic lesions, and further reduced in carcinoma, as if the loss of the residual p27 function is selected during tumor progression (Chu et al., 2008). To clarify whether p27 acts as a dose-depen­ dent tumor suppressor in the context of neuroen­ docrine tumorigenesis in humans, it would be important to assess whether the tumor tissues of MEN4 patients (heterozygous for germline p27 mutations) show loss of p27 expression through inactivation of the wild-type allele. So far, unfortunately, only two tumor tissues of mutation-positive individuals were available for protein analysis. One was a neuroendocrine cer­ vical carcinoma that developed in a patient car­ rying the germline heterozygous 19 bp duplication in CDKN1B exon 1 (p27K25fs), which also showed LOH at 12p13 indicating the loss of the wild-type allele. Accordingly, this tissue showed no p27 immunoreactivity (Geor­ gitsi et al., 2007). The renal tumor tissue (angiomyolipoma) of a patient carrying the p27W76X mutation was sub­ jected to immunohistochemical staining and showed no expression of p27, although both CDKN1B alleles were present (DNA level) and also expressed (mRNA level) (Pellegata et al., 2006). One tumor carrying the ATG-7C->C change was studied for loss of the wild-type CDKN1B allele at the DNA level, which was not found, but it was not subjected to immunohistochemistry for p27 (Agarwal et al., 2009). So, whether this change associates with lower p27 levels in patients’ tissues, as it occurs upon transfection in vitro, is currently unknown.

312

Therefore, although in many human tumor types biallelic inactivation of CDKN1B is exceed­ ingly rare, in neuroendocrine tumors it may occur at increased frequency indicating that in these tissues p27 behaves as a ‘canonical’ tumor sup­ pressor. Of course this hypothesis is based only on two tumor tissues. The analysis of additional tumors from mutation-positive patients will clarify whether a selection for the complete inactivation of p27 function takes place in specific cell context, such as neuroendocrine cells. Of course p27 might also possess pro-oncogenic functions, mainly when it is confined to the cyto­ plasm (Besson et al., 2007). Therefore, a thorough characterization of the functional properties of the mutant p27 proteins is a prerequisite to correctly interpret their contribution to tumor development.

Interaction between Menin or RET and p27 In the previous sections, we have introduced the function of the proteins predisposing to the var­ ious MEN syndromes, with a special focus on their physiological and pathological function in neu­ roendocrine cells. Now let us explore in more detail how these molecules are functionally related to one another, when known, most nota­ bly in the context neuroendocrine cells where their abnormal function promotes tumorigenesis. Let us first consider Menin and p27. As men­ tioned earlier, in Menin-knockout mouse fibro­ blasts Menin binds to a MLL-containing complex with HMTase activity and recruits this complex to the Cdkn1b (p27) and Cdkn2c (p18) promoters thereby activating their transcription (Fig. 5A). Men1 þ/– mice develop multiple endocrine tumors with a spectrum that, for the most part, recapitulates that of the human MEN1 syndrome (Crabtree et al., 2001). These mice present with pancreatic b-cell hyperplasia and studying these cells Karnik et al. (2005) formally demonstrated that p27 is an essential downstream target of the Menin-mediated anti-proliferative pathway in these cells. Interestingly, it was shown that several MEN1-associated mutations when expressed in mouse Men1-deficient cells inhibit the ability of

Menin to activate Cdkn1b transcription, further strengthening the hypothesis that control of cell proliferation is an important physiological func­ tion of Menin in neuroendocrine cells. These find­ ings position p27 downstream of Menin in the pathway that ultimately leads to regulation of cell division. This model of functional interaction between the two proteins can also explain the phenotype of mice obtained by crossing Men1 þ/– animals into a p27 –/– background, which show as sole tumor phenotype intermediate lobe pituitary tumors. Interestingly, neither the frequency nor the latency of tumors arising in Men1 þ/–, p27 –/ – double mutants was significantly altered when compared with Men1 þ/– single knockout mice (Bai, Pei, Nishikawa, Smith, & Xiong, 2007). This indicates that in vivo Menin and p27 do not co-operate to suppress tumor growth in endocrine cells and support the model that both proteins impinge on the same pathway that ultimately leads to regulation of cell proliferation. If p27 and Menin function along the same pathway also in humans, then concominant alterations of both proteins should not be selected during the tumorige­ netic process, as they would be redundant. Determin­ ing the status of p27 in the tumors of MEN1 patients should help clarify whether this hypothesis is true. To the best of our knowledge, there has been so far only one case report of a MEN1 patient whose retroper­ itoneal carcinoid did not show p27 expression (Sakurai, Murakami, Sano, Uchino, & Fukushima, 2009). However, more cases are needed to provide conclusive evidence of whether alterations in Menin and p27 are mutually exclusive. Concerning possible interactions between RET and p27, we have briefly mentioned that adrenal tumors of MEN2 patients (bearing germline RET mutations) show decreased p27 expression (Joshi et al., 2006). To understand the molecular mechanisms mediating this effect, it is important to mention studies conducted on an in vitro model of neuronal differentiation, the NT2/D1 human teratocarcinoma cell line. GDNF induces differ­ entiation of NT2/D1 cells in neuronal-like cells through the activation of RET signalling. In these cells, RET activation causes the accumula­ tion of p27 which in turn binds to and inhibits CDK2 and blocks cell proliferation (Baldassarre

313 A. Nucleus MLL Menin Me H34K H34K

H34K H34K

H3K4

Pol II

B.

Me H34K

Me H34K

Me H3K4

Me H3K4

p27

GDNFs

RET

p27 Fig. 5. Model of p27 regulation by Menin (A) or RET (B). (A) Menin activates transcription of the Cdkn1b gene by promoting Histone 3 (H3) Lysine (K) 4 methylation (Me) of its promoter sequences. (B) Activation of RET by GDNF in NT2/D1 cells increases the amount of p27 protein through molecular pathways still to be determined.

et al., 2002) (Fig. 3). Indeed, if p27 expression is abolished in these cells by small interfering-(si) RNA-mediated knockdown of CDKN1B there is no cell cycle block in response to GDNF treatment, indicating that p27 is necessary for the cell growth inhibition promoted by this neurotrophic factor. Therefore, in this cell context, p27 is a downstream target of activated RET signalling (Fig. 5B). Whether this mechanism of regulation takes place also in neu­ roendocrine cells is presently not known.

The studies here summarized establish a functional relationship between Menin or RET and p27, and suggest that p27 is a common read­ out of both Menin and RET pathways at least in the specific tissue context analysed (Fig. 5). Further studies are warranted to provide conclu­ sive evidence as to whether the relationships among these proteins here outlined take place according to this model also in human neuroendo­ crine cells.

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Is MENX a good model of human neuroendocrine tumors? Tumors of the neuroendocrine system are rela­ tively rare. Their molecular pathobiology is poorly understood, in part because of the scar­ city of biological material for comprehensive bio­ logical studies, and clinical trials for such tumors are challenging to set-up and carry to comple­ tion. In this scenario, it would be extremely relevant to possess a faithful animal model to achieve a deeper understanding of the biology of neuroendocrine tumors and to develop and test novel targeted therapeutic approaches. Hence the interest on the MENX model. A prerequisite to further exploit this animal model for molecular studies or for the preclinical evaluation of anti-tumor drugs is to ascertain that MENX tumors are indeed a good model of human neuroendocrine tumors and the results obtained on the rat tumors can be extrapolated to their human counterpart. Preliminary molecular studies performed on pheochromocy­ toma arising in MENX-affected rats seem to support the hypothesis that MENX might be a good model of human neuroendocrine tumors. A genome-wide scan for allelic imbalance (AI) was performed on a series of rat pheo­ chromocytoma to identify recurrent regions of chromosomal loss. This experimental approach has been historically employed to identify loci where putative tumor suppressor genes involved in tumor progression are located. Recurrent AI at candidate regions on rat chromosomes 8 and 19 were observed in ~30% of MENX-associated pheochromocytoma (Shyla et al., 2009). Inter­ estingly, the regions often lost in rat tumors are syntenic to regions previously associated with human pheochromocytoma development, both in sporadic and hereditary cases (Table 3). Due to the high concordance of affected loci between rat and human tumors, studies of the MENX-associated pheochromocytomas should facilitate the identification of novel candidate genes implicated in their human counterpart. Interestingly, the rat tumors seem to share com­ mon genetic mechanisms also with human

sporadic neuroendocrine tumors, which repre­ sent the majority of the cases and whose genetic etiology is less understood than that of the her­ editary cases. Ongoing studies of the transcriptome and the proteome of MENX-associated tumors will pro­ vide conclusive evidence about how faithful a model of human neuroendocrine tumors this syn­ drome is.

Conclusions The rat MENX syndrome has provided us with a novel tumor susceptibility gene for neuroendo­ crine tumors in both rats and humans: Cdkn1b/ CDKN1B (p27). This finding underscores the important role of cell cycle regulation in neuroen­ docrine cells, which are highly sensitive to impaired function of CDKIs, as demonstrated by several mice models. We can speculate that in neuroendocrine cells defective p27 function(s) cannot be compensated by the other members of the KIP/CIP family of inhibitors as it occurs in tissues unaffected by p27 malfunction, thereby explaining the associated tissue-selective tumorigenesis. The proteins responsible for the ‘classical’ MEN syndromes, Menin and RET, feed into a p27­ dependent pathway regulating cell proliferation in a tissue-specific manner. Despite extensive information on the function of p27 as a regulator of important processes such as cell cycle, cell migration and neuronal differ­ entiation, the molecular mechanisms mediating its role in tumor susceptibility in humans still need to be deciphered, mainly because the germline mutations so far identified are few and their functional characterization is incom­ plete. What is also not clear is whether in the neuroendocrine cells of mutation-positive patients p27 works as a dose-dependent tumor suppressor, as it has been demonstrated in mice and postulated for other human tumors. The functional characterization of the naturally occurring mutations in CDKN1B and the identi­ fication of additional mutation-positive patients will help clarify these issues.

Table 3. Regions on rat chromosomes 8 and 19 showing AI in MENX pheochromocytoma cases and their syntenic regions on the human genome already shown to be involved in pheochromocytoma or paraganglioma (extra-adrenal pheochromocytoma) development Copy number changes frequency (%)

Chromosomal change

LOH/AI loci in rat MENX-PC

Syntenic region in human genome

LOH and/or CGH data available for human PC

Losses

8q11-q12

11q14.3-q22.3

Lui et al. (2002)

11q13-qter

8q22-q24

11q22.3-q24.1

Dannenberg et al. (2000)

11q

Edstrom et al. (2000)

11q13-qter, 11q13-q23, 11q14-qter 11q14-qter, 11q22-qter

Gains

PC 21/36 (58%) 29 (28%)


15q21.3-q24.3

Lui et al. (2002)

15q15-qter

Edstrom et al. (2000)

15q, 15q22-qter

8q31-q32

3q21.3-q24

sporadic PCs, extra-adrenal PCs sporadic PCs, 2/11 (18%) 1/11 (9%)

1/36 (3%) 2/17 (12%)

15cen-q21, 15q23-qter 3q

9/15 (60%)

Lui et al. (2002)

3q

1/5 (20%)

abdominal PGLs abdominal PGLs VHL-associated PCs sporadic PCs,

2/11 (18%)

Mulligan, Gardner, Smith, Mathew, and Ponder (1993)

Type of PC and/or PGL VHL-associated PCs

3/17 (18%)

11q 8q24

PGL

abdominal PGLs MEN2-associated PCs, sporadic PCs MEN2-associated PCs

(Continued)

Table 3. (Continued ) Copy number changes frequency (%)

Chromosomal change

LOH/AI loci in rat MENX-PC

Syntenic region in human genome

LOH and/or CGH data available for human PC

Losses

Dannenberg et al. (2000)

3q

Gains

PC 14/25 (56%)

3q Edstrom et al. (2000)

3q22-q25, 3q

19q11†

4q31.21-q31.23

3q

Lui et al. (2002)

4q

Edstrom et al. (2000)

4q, 4q21-qter

4q28-qter, 4q

Type of PC and/or PGL sporadic PCs,

1/4 (25%) 9/23 (39%)

3q Cascon et al. (2005)

PGL

5/11 (45%) 21/29 (72%) 1/5 (20%) 3/22 (14%)

extra-adrenal PCs MEN2A-associated PCs, NF1-associated PCs, sporadic PCs abdominal PGLs PCs MEN2-associated PCs

2/11 (18%)

MEN2A-associated PCs, sporadic PCs abdominal PGLs

PC, pheochromocytoma; PGL, paraganglioma; MEN2, multiple endocrine neoplasia type 2; VHL, von Hippel–Lindau disease; NF1, neurofibromatosis type 1; LOH, loss of heterozygosity;

AI, allelic imbalance; CGH, comparative genomic hybridization.

Modified with permission from: Shyla et al. (2009). Copyright © 2009 by John Wiley & Sons.


The per cent of copy number changes was identified in the group of adrenal and extra-adrenal pheochromocytomas. † The19q11 locus corresponds to the 26.3–34.4 Mb rat chromosomal region of chromosome 19.

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Acknowledgments The authors thank the members of the laboratory for stimulating discussions; the Mouse Pathology group (Institute of Pathology, Helmholtz Zentrum München) for help with the animal pathology screening. This work was in part supported by a grant from the Deutsche Krebshilfe (#107973) to N.S.P. and by a Fellowship from Centro per la Comunicazione e la Ricerca, Collegio Ghislieri, Pavia, Italy (S.M.).

Abbreviations 1°HPT ACTH AI CDK CDKI ENU FMTC GDNF GH H&E hGHRH HMTase HOX LOH MEN MLL MTC NF-kB

primary hyperparathyroidism adrenocorticotropic hormone allelic imbalance cyclin-dependent kinase cyclin-dependent kinase inhibitor N-ethyl-N-nitrosourea familial medullary thyroid cancer glial cell line-derived neurotrophic factor growth hormone haematoxylin and eosin human growth hormonereleasing hormone histone methyltransferase homeobox domain loss of heterozygosity multiple endocrine neoplasia mixed-lineage leukaemia medullary thyroid cancer nuclear factor-kB

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 14

Hormonal therapy of prostate cancer Fernand Labrie
Research Center in Molecular Endocrinology, Oncology and Human Genomics, Laval University and Laval University Hospital Research Center (CRCHUL), Qu�ebec, Canada

Abstract: Of all cancers, prostate cancer is the most sensitive to hormones: it is thus very important to take advantage of this unique property and to always use optimal androgen blockade when hormone therapy is the appropriate treatment. A fundamental observation is that the serum testosterone concentration only reflects the amount of testosterone of testicular origin which is released in the blood from which it reaches all tissues. Recent data show, however, that an approximately equal amount of testosterone is made from dehydroepiandrosterone (DHEA) directly in the peripheral tissues, including the prostate, and does not appear in the blood. Consequently, after castration, the 95–97% fall in serum testosterone does not reflect the 40–50% testosterone (testo) and dihydrotestosterone (DHT) made locally in the prostate from DHEA of adrenal origin. In fact, while elimination of testicular androgens by castration alone has never been shown to prolong life in metastatic prostate cancer, combination of castration (surgical or medical with a gonadotropin-releasing hormone (GnRH) agonist) with a pure antiandrogen has been the first treatment shown to prolong life. Most importantly, when applied at the localized stage, the same combined androgen blockade (CAB) can provide long-term control or cure of the disease in more than 90% of cases. Obviously, since prostate cancer usually grows and metastasizes without signs or symptoms, screening with prostate-specific antigen (PSA) is absolutely needed to diagnose prostate cancer at an ‘early’ stage before metastasis occurs and the cancer becomes noncurable. While the role of androgens was believed to have become non-significant in cancer progressing under any form of androgen blockade, recent data have shown increased expression of the androgen receptor (AR) in treatment-resistant disease with a benefit of further androgen blockade. Since the available anti-androgens have low affinity for AR and cannot block androgen action completely, especially in the presence of increased AR levels, it becomes important to discover more potent and purely antagonistic blockers of AR. The data obtained with compounds under development are promising. While waiting for this (these) new anti-androgen(s), combined treatment with castration and a pure anti-androgen (bicalutamide, flutamide or nilutamide) is the only available and the best scientifically based means of treating prostate cancer by hormone therapy at any stage of the disease with the optimal chance of success and even cure in localized disease. Keywords: intracrinology; anti-androgens; androgen blockade; early diagnosis; cure; long-term control


Corresponding author.

Tel.: (418) 652-0917; Fax: (418) 651-1856;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82014-X

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Introduction One in eight men will be diagnosed with prostate cancer during their lifetime with 192 280 new cases being predicted in the United States in 2009 (Jemal et al., 2009). Despite the 33% decrease in deaths from prostate cancer in the United States during the 15 years between 1992 and 2007 as estimated by the American Cancer Society (Jemal et al., 2007), prostate cancer remains the second cause of cancer deaths with 27 360 deaths predicted for 2009 in the United States alone (Jemal et al., 2009). Prostate cancer is thus a major medico-social problem comparable to that of breast cancer in women. The main objective of physicians managing patients with cancer is to permanently free them from the disease. It is thus a major progress to see that androgen blockade is now increas­ ingly recognized as curative, conditional to its use in localized (when it is curable) instead of advanced and metastatic (when it has become non-curable) disease. These news are particu­ larly timely since more than 95% of patients can now be diagnosed by simple PSA screening and can thus be treated at the localized and only potentially curable stage (Labrie et al., 1996b), thus providing an explanation for the important decrease in prostate cancer deaths observed since 1992 (Jemal et al., 2007). The extremely long delay in recognizing the curative potency of androgen blockade can be explained by two misinterpretations concerning androgen blockade which, unfortunately, are still at the basis of the official guidelines of some uro­ logical associations distributed to guide the clinical practice of their members. These two common misinterpretations are as follows: 1. Application to localized prostate cancer of observations made in advanced disease which are characteristics specific to metastatic disease and which do not apply to localized disease. As well indicated by Professors Akaza and Namiki (Akaza, 2008; Namiki et al., 2008), the erroneous belief of a temporary efficacy of androgen blockade due to the relatively rapid development of resistance to treatment is a

characteristic typical and limited to advanced and metastatic disease. There have never been valid reasons to apply to localized prostate cancer these observations of resistance to treatment which exclusively belong to advanced disease. In fact, contrary to the situation in metastatic prostate cancer, a continuous and very long-term positive response with the high probability of a cure is observed in localized disease (Akaza, 2008; Labrie et al., 2002; Namiki et al., 2008) when optimal or CAB is used. This possibility of cure is however conditional to the start of CAB sufficiently early at time of diagnosis (Labrie et al., 2002). The conclusion that androgen blockade can be curative and does not simply delay progression has been reached in many studies including a meta-analysis of the controlled clinical trials performed as adjuvant hormonal treatment in non-metastatic prostate cancer (Fleshner et al., 2007). The author of this meta­ analysis has concluded that androgen blockade given as adjuvant to surgery or radiotherapy should be classified as a treatment of curative intent for patients with poor prognosis non-metastatic prostate cancer. It should be mentioned that such positive results could even be observed using a non-optimal androgen blockade, namely monotherapy, while much better results are achieved with CAB without additional negative effects (Akaza, 2008; Akaza et al., 2007; Labrie, 2004; Labrie et al., 2002; Namiki et al., 2008). 2. A second extremely common error, not to say generalized, is the use of monotherapy as first treatment, a treatment much inferior to CAB even though a significant rate of cure (33%) can be obtained with monotherapy in localized prostate cancer (Peto and Dalesio, 2003). However, a major limitation of monotherapy (castration alone or an antiandrogen alone) is that 40% of active androgens are left in the prostate under monotherapy (Labrie, 2007; Labrie et al., 2009a; Labrie et al., 1985). These androgens made locally in the prostate continue to stimulate prostate cancer after any

323

treatment limited to castration or an antiandrogen alone, thus permitting continued stimulation of cancer proliferation and metastasis at distance where resistance to treatment always develops and cure becomes impossible (Huggins and Hodges, 1941).

prostate cancer is almost always possible with current androgen blockade …’. At the metastatic stage, on the other hand, while monotherapy first used by Huggins and Hodges (1941) has not been shown to prolong survival, a 20% prolongation of prostate cancer-specific survival can be obtained with CAB applied at start of treatment (Bennett et al., 1999; Caubet et al., 1997; Craw­ ford et al., 1989; Denis et al., 1998; Janknegt et al., 1993; Labrie et al., 1996a; Labrie et al., 1982; Labrie et al., 1985; Prostate Cancer Triallists’ Collaborative Group, 2000). It is important to indicate that since the anti-androgen was generally added at time of progression following castration alone, the above­ mentioned studies compare early versus late CAB and not placebo versus CAB as generally believed.

In localized disease, the simple addition to castration of a pure anti-androgen in order to block the action of the androgens made locally in the prostate increases the potential of cure from 33% observed with monotherapy (Fleshner et al., 2007; Peto and Dalesio, 2003; Prostate Cancer Triallists’ Collaborative Group, 2002) to more than 90% (Labrie, 2007; Labrie et al., 2002) (Fig. 1B). It is very important to read Professor Akaza (2008) saying: ‘cure of A. Metastatic prostate cancer 1. Monotherapy Non-statistically significant effect

0

20

40

60

80

100%

2. Combined androgen blockade

*

First and only statistically significant treatment

0

20

40

60

80

100%

B. Localized prostate cancer 1. Monotherapy

*

Statistically significant

0

20

40

60

80

100%

80

100%

2. Combined androgen blockade Cure in > 90% 0

20

40

60

Fig. 1. Comparison of the efficacy on prostate cancer-specific survival of monotherapy (castration alone or anti-androgen alone at high dose) (1) and combined androgen blockade (CAB) (castration þ pure anti-androgen) (2) administered in metastatic (A) and localized (B) prostate cancer. A1: No positive study available

A2: Bennett et al. (1999), Caubet et al. (1997), Crawford et al. (1989), Denis et al. (1998), Janknegt et al. (1993), Labrie et al.

(1985) and Prostate Cancer Triallists’ Collaborative Group (2000)

B1: Peto and Dalesio (2003) and Prostate Cancer Triallists’ Collaborative Group (2002)

B2: Labrie et al. (2002)

The asterisk (
) indicates that since the anti-androgen was added at time of progression following castration, these studies compare early versus late CAB and not placebo versus CAB.

324

In other words, a greater difference in survival, even at the advanced metastatic stage, should have been obtained if a true comparison between placebo and CAB had been studied. It is, in fact, well recognized that a significant number of positive responses are observed when a pure anti-androgen is added at time of progression in patients who had castration as first treatment (Labrie et al., 1988). These responses observed at time of addition of the anti-androgen decrease the difference between the castration alone and CAB groups. Discovery of the local formation of androgens from DHEA of adrenal origin by the action of the enzymes of intracrinology (Labrie, 1991; Labrie et al., 1989b) has indicated the need to develop CAB (Labrie et al., 1982; Labrie et al., 1985), a treatment which adds to castration (medical or surgical) a pure anti-androgen in order to block the action of the androgens made locally in the prostate from DHEA. In fact, all the enzymes required to make androgens from DHEA are expressed in the prostate (Luu-The et al., 2008; Pelletier, 2008). It is important to mention that recent data indi­ cate that androgen blockade is important, not only as first line therapy in both localized and meta­ static diseases, but could also play a role in pros­ tate cancer which has become resistant to first line androgen blockade (Chen et al., 2004; Scher and Sawyers, 2005; Taplin and Balk, 2004).

Two sources of androgens of approximately equal importance are present in men: castration removes only 60% of androgens in the prostate while bicalutamide (Casodex) alone, at the 150 mg daily dose, has an effect similar to castration Intracrinology An important advance in our understanding of the biology and endocrinology of prostate cancer is the observation that humans are unique among animal species in having adrenals that secrete large amounts of the inactive precursor steroids DHEA, and its sulfate DHEA-S, which are converted into active androgens in a large series of peripheral tissues, including the prostate (Fig. 2).

Intact – normal GnRH Testosterone

Pituitary gland LH

ACTH

Testosterone Testis

DHEA DHT

Prostate

Adrenal

Fig. 2. Schematic illustration of the two sources which provide approximately equal amounts of androgens to the normal prostate and prostate cancer. (1) The testicles secrete testosterone released in the blood stream while (2) the adrenals secrete dehydroepiandrosterone (DHEA) in the circulation, the precursor being converted into testosterone and then into dihydrotestosterone (DHT) in the prostate.

The local synthesis of active steroids in per­ ipheral target tissues has been named intracri­ nology (Labrie, 1991; Labrie et al., 2004; Labrie et al., 2003; Labrie et al., 1989a). The active androgens made locally exert their action by binding to the prostatic AR without being released in significant amounts in the extracel­ lular environment or general circulation. Most importantly, the active androgens made in per­ ipheral tissues are inactivated locally as glucur­ onides before their elimination through the circulation (Fig. 3). Contrary to the previous belief that the testes are responsible for 95% of total androgen production in men (as could be inferred from the 95–97% decrease in serum testosterone observed after castration) (Fig. 4), it is now well established that the prostate makes the androgens testo and DHT locally in relatively large amounts.

Very limited effect of castration on total androgen availability in the prostate While the serum levels of testo are reduced by 97.4% following castration in 69–80-year-old

325

GnRH CRH

LH

ACTH

Adrenal gland

Testo

Circulation

DHEA DHEA

Testo

4-Dione E1

ADT–G 3α–diol–G

Testis

Circulation

E2

DHT

E2

ADT–G E1S

Testo E 2

Circulation

A-Dione

3α–Diol–G

Peripheral target tissues Circulation

DHEA

Fig. 3. Schematic representation of the testicular and adrenal sources of sex steroids in men. The adrenal glands – as well as secreting cortisol that decreases CRH secretion, which otherwise stimulates ACTH levels – secrete large amounts of DHEA; this precursor is converted in specific target tissues into androgens and/or estrogens via the process of intracrinology. Only small amounts of these peripherally made sex steroids diffuse into the circulation. The androgens are metabolized into the metabolites ADT and 3a-diol which are then further transformed into the more water-soluble glucuronide derivatives and released into the blood where they can be measured as parameter of total androgenic activity. Approximately 60% of androgens are made in the testicles as testosterone which is distributed in the peripheral tissues by the circulation. ACTH, adrenocorticotropin; CRH, corticotrophin-releasing hormone; DHEA, dehydroepiandrosterone; 4-dione, androstenedione; A-dione, 5a-androstanedione; DHT, dihydrotestosterone; E1, estrone; E1S, estrone sulfate; E2, estradiol; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; testo, testosterone; ADT-G, androsterone glucuronide; 3a-diol-G, androstane-3a-diol-3 or 17-glucuronide.

men (Fig. 4A), the sum of the metabolites of androgens (ADT-G, 3a-diol-3G and 3a-diol­ 17G), the only accurate and valid parameter of

total androgenic activity measurable in the circu­ lation (Labrie et al., 2006), is only reduced by 58.9% (Fig. 4B), thus indicating that a very impor­ tant amount (41.1%) of androgens is still present in the prostate after complete elimination of testi­ cular androgens. Such data are in close agreement with the concentration of intraprostatic DHT that shows that, on average, 39% of DHT is left in the prostate after castration in various studies, namely 45% (Labrie et al., 1985), 51% (Bélanger et al., 1989), 25% (Nishiyama et al., 2004) and 35% (Mostaghel et al., 2007). In another study, it was observed that intraprostatic DHT levels remained at 50% of pre-treatment values after castration (Yoon et al., 2008). The observations based upon the best and vali­ dated parameters of androgenic activity, where all steroids are measured by the mass spectrometry technology show that ~40% of androgens are made in the prostate in 69–80-year-old men. Since serum DHEA decreases markedly with age starting in the thirties (Labrie et al., 2005), and testicular androgen secretion decreases only slightly, it is most likely that intraprostatic androgens of adrenal origin have an even greater relative and absolute importance at younger ages. The logical conclusion from these data is that castra­ tion is an insufficient treatment for prostate cancer since, on average, it eliminates only 60% of andro­ gens in the prostate.

Very limited blockade of total androgens achieved with an anti-androgen alone Since an anti-androgen used alone can, like castra­ tion, show easily detectable effects on serum PSA and also clinically, due to the particularly high sensitivity of prostate cancer to androgen depriva­ tion, it is important to remember that, at best, administration of an anti-androgen alone can only partially block androgens, thus leaving an important amount of DHT free to stimulate AR and prostate cancer growth and metastasis. In fact, the data obtained from phase III stu­ dies have shown that daily 150 mg bicalutamide monotherapy provides a survival outcome simi­ lar, and sometimes inferior, to that observed

326 A.

B.

C.

5

40

100

30

75

3 ng/ml

−97.4%

ng/ml

4

−58,9%

−61%

20

50

10

25

2

1

0

0

0 Intact

Castrated

Testosterone

Intact

Castrated

ADT–G + 3α–diol–3G+17G

Intact

Castrated

DHT in prostate

Fig. 4. Effect of castration on the concentration of serum testo (A), total androgen pool (sum of serum ADT-G, 3a-diol-3G and 3a-diol-17G) (B) and intraprostatic DHT, the predominant androgen in the prostate (C). Data in C are the average of values published in Bélanger et al. (1989), Labrie et al. (1985), Mostaghel et al. (2007) and Nishiyama et al. (2004). Data are presented as means + SEM (Labrie et al., 2009b).

with castration alone in patients with locally advanced (no metastases) disease (Iversen et al., 2001; Iversen et al., 2000; Tyrrell et al., 1998; Wirth et al., 2004). The 50 mg dose of bicalutamide, on the other hand, has been shown to be clearly inferior to castration (Bales and Chodak, 1996). Very unfortunately, the 50 mg daily dose of bicalutamide used alone is the approved dose in the United States, Canada and most other countries. In Japan, the approved dose is 80 mg. Because, as mentioned above, 40–50% of andro­ gens are left in the prostate following castration (Fig. 4C), two conclusions are obvious. First, when used in association with medical or surgical castration, bicalutamide should be used at the 150 mg daily dose to efficiently block the action of the 40–50% of androgens left in the prostate after castration. On the other hand, monotherapy with a daily dose of 150 mg bicalutamide leaves, like cas­ tration, about 40–50% of androgens free to con­ tinue to stimulate the AR, thus indicating the need to simultaneously block the secretion of testicular androgens by a GnRH agonist or orchiectomy.

Accordingly, although the use of a non-steroidal anti-androgen alone provides some advantages in terms of secondary effects, especially loss of libido and sexual dysfunction compared to castration, an anti-androgen alone is only a partial therapy of prostate cancer with the high risk of negative con­ sequences on survival. It is relevant to mention that no PSA progres­ sion has been observed for up to 7 years in patients with localized or locally advanced pros­ tate cancer who received CAB with a GnRH ago­ nist and 250 mg flutamide 3 times daily (Labrie, Cusan, Gomez, Belanger and Candas, 1999). Such results are quite different from the data obtained following monotherapy with bicalutamide at the dose of 150 mg daily (Wirth et al., 2004) where clinical progression already occurred in 8.5% of patients at 5.3 years of follow-up, whereas 14.0% had progressed in the placebo group (p < 0.0001). Although statistically significant, due to the large number of patients (8113 patients), a 5.5% differ­ ence in disease progression between monotherapy with bicalutamide and placebo is a small effect compared with the data obtained with CAB in

327

patients at a comparable stage of the disease where no (0%) PSA progression occurred before 7 years of treatment (Labrie et al., 2002; Labrie et al., 1999). Although not randomized, this last study per­ formed in a small number of patients is highly suggestive of the marked superiority of CAB in localized and locally advanced disease compared with monotherapy. In fact, an even greater dif­ ference was seen in the group of patients chosen for watchful waiting where 29.4% of patients with localized disease progressed with bicaluta­ mide whereas 44.2% of those with locally advanced disease progressed with bicalutamide (Wirth et al., 2004), thus clearly demonstrating the high risk of watchful waiting and the super­ iority of CAB where, as mentioned above, no progression occurred in a group of 26 men until 7 years of follow-up (Labrie et al., 2002; Labrie et al., 1999).

Clinical effects of monotherapy versus combined androgen blockade as first line therapy in localized disease Monotherapy (GnRH agonist alone, orchiectomy alone or anti-androgen alone) has a significant but far from optimal effect on localized prostate cancer

The availability of a safe and highly efficient method of medical castration has generated renewed interest in the treatment of prostate can­ cer and has stimulated an unprecedented number of clinical trials, which rapidly led to the world­ wide commercialization of a series of GnRH agonists having equivalent characteristics, mechanisms of action and efficacy. This marked the end of the requirement for surgical castration, a procedure that is psychologically difficult to accept by the majority of men. Most importantly, this was the end of the need to administer high doses of estrogens to achieve medical castration at the expense of serious cardiovascular effects (Peeling, 1989; Robinson and Thomas, 1971; VACURG, 1967). The importance of medical castration achieved with GnRH agonists was well recognized by Jacobi and Wenderoth (1982) who stated: ‘What medical developments have urologists witnessed since orchiectomy and estrogen treatment by Huggins 40 years ago? Gestagens, antiandrogens, adrenal inhibitors, antiprolactins, antiestrogens, cytotoxic agents? In principle, the gain in terms of efficacy and the loss as a result of toxicity have never been balanced to a degree which could establish one of the aforementioned drugs as the generally accepted standard treatment to replace estrogens. GnRH analogues may prove to be the first nontoxic medical castration measure applic­ able for general use in the future’.

Major progress achieved by the introduction of medical castration with GnRH agonists Clinical effects observed with monotherapy Medical castration with a GnRH agonist achieved in 1980 in the first prostate cancer patient who received this treatment (Labrie et al., 1980) has been a landmark in the field of prostate cancer. Soon after our observation that administration of the GnRH agonist buserelin led to an almost complete inhibition of serum testosterone and DHT levels within 2 weeks of administration by the intranasal route, a less than optimal route of administration, a detailed comparison of the effect of various doses of the same GnRH agonist was performed comparing the intranasal and subcutaneous routes (Faure et al., 1982).

Since prostate cancer is the most sensitive of all cancers to hormone therapy, a positive effect on serum PSA and on the clinical evolution of the cancer can be easily observed with such a sub­ optimal blockade of androgens. Physicians and their patients should not, however, be satisfied by a sub-optimal positive result obtained with monotherapy, since such an effect is only a fraction of what can be achieved by more complete androgen blockade. In fact, although significant positive results are observed with monotherapy, much bet­ ter results and even cure of the cancer can be achieved by CAB applied to localized disease.

328

Following the meta-analysis of androgen block­ ade with monotherapy in localized prostate cancer (Fig. 1B1), Peto stated that ‘prostate cancer is usually treated with surgery or radiation, but a few cancer cells may remain and cause an oftenfatal recurrence. Since the mid-80s, oncologists have increasingly followed up with either surgical removal of the testes, or with newer anti-hormone drugs’ (Peto and Dalesio, 2003). The meta-analy­ sis which looked at several studies involving 5000 men showed that 74% of patients who received early hormone monotherapy were still alive 10 years later, compared with 62% of those who did not (Arnst, 2003; Peto and Dalesio, 2003) (Fig. 1B1). The conclusion of this meta-analysis on the effect of monotherapy in localized and locally advanced prostate cancer was that the risk of dying from prostate cancer within 10 years decreased by one-third if hormonal treatment was given immediately rather than after the dis­ ease had progressed (Peto and Dalesio, 2003). Since patients originally on placebo were treated by monotherapy at time of progression, this onethird decrease in the risk of dying from prostate cancer was not the result of the comparison between castration and placebo (or no androgen blockade), but between early and late androgen blockade. It is also very important to consider that these results were obtained with only partial

blockade of androgen or monotherapy. These data led Peto to the conclusion that ‘Hormone treatment as a whole works much better than previously thought’. In fact, all clinical trials of androgen blockade have shown prolongation of life or a reduced death rate from prostate cancer in patients with localized or locally advanced disease (Table 1). During 3.7–9.3 years of follow-up, the first six studies have shown reductions in deaths from prostate cancer ranging from 37.5 to 81% (Bolla et al., 1997; Granfors et al., 1998; Hanks et al., 2000; Labrie et al., 1999; Messing et al., 1999; Pilepich et al., 1997). A seventh study provided no data on cancer-specific deaths, but a 45% decrease in overall deaths was reported (D’Amico et al., 2004).

Simple addition of a pure anti-androgen to castration (Fig. 1B2) can achieve cure in more than 90% of localized prostate cancers instead of the 33% decrease in deaths obtained by monotherapy (Fig. 1B1). Clinical data With today’s knowledge, monotherapy (castration alone or anti-androgen alone) can achieve only 50–60% of androgen blockade (Fig. 4). As

Table 1. Effect of the use of androgen blockade on prostate cancer death rates

Study

Advantage

Median follow-up (years)

p

EORTC 415 patients RTOG 85-31 276 patients Laval University Screening Trial 21 400 subjects Messing et al. (1999) 98 patients Granfors et al. (1998) 91 patients RTOG 92-02 1554 patients D’Amico et al. (2004) 201 patients

77% decrease in cancer-specific death

3.7

0.01

37.5% decrease in cancer-specific death for Gleason score 8–10 67% decrease in cancer-specific death

4.5

0.03

8

0.0002

81% decrease in cancer-specific death

7.1

0.001

39% decrease in cancer-specific death

9.3

0.06

59% decrease in cancer-specific death for Gleason score 8–10 45% decrease in overall death

5

0.007

4.5

0.04

329 Table 2. Androgen blockade in prostate cancer Combined androgen blockade

GnRH agonist alone GnRH antagonist alone Orchiectomy DES Proscar alone Proscar þ castration Cyproterone acetate alone Cyproterone acetate with castration Flutamide alone Ninutamide alone Bicalutamide alone Megace Medroxyprogesterone acetate

GnRH agonist, GnRH antagonist or Orchiectomy þ Flutamide or Nilutamide or Bicalutamide (150 mg or more daily)

mentioned above, although GnRH agonist ther­ apy in localized prostate cancer has shown impor­ tant benefits in terms of survival in localized prostate cancer, the knowledge that 40% of androgens (Fig. 4C) remain in the prostate after castration indicates that superior results should be expected from the use of CAB or the combination of an GnRH agonist with a pure anti-androgen (Table 2). Early data already indicated that the benefit of CAB versus monotherapy is greater for patients with minimal metastatic disease than for those with extensive metastatic disease (Crawford et al., 1989; Denis et al., 1998). The availability of GnRH agonists which are much more acceptable than surgical castration or high doses of estrogens (Labrie et al., 1980) has greatly facilitated or even permitted the development of androgen blockade at the localized stage. It has been of major importance to observe that CAB can achieve long-term control or cure of pros­ tate cancer in at least 90% of patients with localized or locally advanced disease (Fig. 5) provided that treatment is given continuously, uninterrupted, for at least 7 years (Labrie et al., 2002) (Table 2). The effect of CAB on long-term control or pos­ sible cure of prostate cancer was evaluated by the absence of biochemical failure or the absence of PSA rise for at least 5 years after cessation of con­ tinuous treatment (Fig. 5). A total of 57 patients with localized or locally advanced disease received

Long-term control or cure (%)

Monotherapy or combination

100

11/12 7/8

80 60 40

1/3

3/8

20 0/11

0 0

2

4 6 8 Continuous CAB (years)

10

12

Fig. 5. Effect of duration of treatment of localized prostate cancer with continuous combined androgen blockade (CAB) on the probability of long-term control or ‘cure of the disease’ illustrated by no recurrence of PSA rise for at least 5 years after cessation of CAB. The point at 4.75 years of treatment (33%) refers to the three patients treated with CAB for 3.5–5.0 years and followed for at least 5 years, while the point at 5.75 years refers to the eight patients treated continuously with CAB for 5.0–6.5 years before cessation of treatment. The point at 8.25 years, on the other hand, refers to the eight patients treated continuously for 6.5–9.0 years while the point at 11 years refers to the thirteen patients treated for 10.0–11.7 years with continuous CAB before stopping treatment. All patients were followed for at least 5 years after cessation of continuous CAB or until PSA rise. Only 1 patient has died from prostate cancer while 18 have died from other causes (Labrie et al., 2002).

CAB for periods ranging from 1 to 11 years. With a minimum of 5 years of follow-up after cessation of long-term CAB, only two PSA rises occurred among 20 patients with Stage T2–T3 cancer who stopped treatment after continuous CAB for more than 6.5 years, for a non-failure rate of 90% (Fig. 5). On the other hand, for the 11 patients who had received CAB for 3.5–6.5 years, the non-failure rate was only 36%. The serum PSA increased within 1 year in all 11 patients with stage B2/T2 treated with CAB for only 1 year, thus indicating that active cancer remained present after shortterm androgen blockade despite undetectable PSA levels. Most importantly, in all patients who had biochemical failure after stopping CAB, serum PSA rapidly decreased again to undetectable levels when CAB was restarted and PSA remained at such low levels afterward. Of these patients, only one patient had died of prostate cancer at last follow-up (Labrie et al., 2002).

330

With the knowledge of the above-described data, it seems reasonable to suggest that the minimal duration of continuous CAB in localized prostate cancer should be 6 years, thus providing an ~50% probability of long-term control or pos­ sible cure of the cancer. With longer duration of CAB, the probability increases to about 90% at 8–10 years of treatment. The present data indi­ cate that possible cure of the disease can be obtained in almost all patients with localized prostate cancer treated continuously with CAB for 7 years or more, thus raising hope for the successful treatment of patients who fail after surgery, radiotherapy or brachytherapy where no or minimally effective alternative therapeutic approach exists. A series of recent studies performed in Japan clearly illustrate the very high efficacy of CAB in localized disease (Akaza, 2008; Akaza et al., 2006; Egawa et al., 2004; Namiki et al., 2008; Ueno et al., 2006). In a prospective study performed in stages C and D prostate cancer patients (Akaza, 2006; Akaza et al., 2004) comparing GnRH agonist monotherapy and CAB (GnRH agonist þ bicalutamide 80 mg/ day), the effect of CAB was more pronounced in patients with C than with D disease. In fact, only 5.8% (3/52) of patients progressed under CAB com­ pared to 42.6% (20/47) with monotherapy, thus showing a marked superiority of CAB compared to monotherapy, especially in stage C or locally advanced disease. These data strongly support our results showing an even much greater advantage of CAB in stage B disease (Labrie et al., 2002). Based upon the above-summarized data, at least for older men, primary hormone therapy is a valid therapeutic option for localized or locally advanced prostate cancer (Akaza et al., 2006). A similar conclusion was reached in a retrospective study of 447 stage B prostate cancer patients who received androgen blockade alone or radical pros­ tatectomy combined with androgen blockade. No difference in disease-specific survival was found at 9.2 years, thus indicating the predominant effect of androgen blockade and the absence of effect of prostatectomy in men receiving androgen block­ ade (Egawa et al., 2004). Data on the current treatment of prostate can­ cer in Japan show that primary androgen blockade

is the treatment chosen for localized and locally advanced prostate cancer in a high proportion of cases. In the survey of the Japanese Urological Association published in 2005, androgen blockade alone was used as primary treatment in 40% of T1 patients and over 50% of T2 patients. Moreover, from the data collected in 2001–2003 by the Japa­ nese Prostate Cancer Surveillance Group (J-CaP) (Akaza et al., 2004), about 60% of patients who receive androgen blockade receive CAB. In addi­ tion, about 70% of patients who receive androgen blockade receive hormone therapy as first treat­ ment. A similar trend is seen in the United States from the Cancer of the Prostate Strategic Urologic Research Endeavour (CaPSURE) (Cooperberg et al., 2003).

No resistance or loss or lack of response to CAB exists for the treatment of localized disease Recognition of the absence of development of resistance to androgen blockade in localized pros­ tate cancer is extremely important. In fact, it is very frequently stated that androgen blockade should not be administered early because resistance to treatment will develop and one might as well wait to use androgen blockade at a later stage of the disease. In fact, deferring treatment is a very ser­ ious error since it implies that, very often, it will then be too late to achieve an otherwise possible cure. In fact, when the cancer has reached the bones, the resistance to treatment can no more be avoided and cure has become impossible. It should be realized that when prostate cancer is first detected, even by screening, the cancer is not small since its diameter is of the order of 1 cm or more. This is the only appropriate time to start treatment with the very strong hope of a cure.

Combined androgen blockade in advanced prostate cancer Although androgen blockade should move to the treatment of localized disease, metastatic disease remains frequent and will always be a therapeutic challenge. Again, it is important to indicate that

331

the observations made with metastatic disease, especially the relatively short duration of response to CAB and the appearance of resistance to treat­ ment should not be applied to localized disease. The results obtained in a large series of clinical trials in patients with advanced prostate cancer have demonstrated that CAB compared to castra­ tion alone has the following advantages: (1) more complete and partial responses, (2) improved control of metastatic pain, (3) longer disease-free survival and (4) longer survival. As already men­ tioned, the combination of a pure anti-androgen (e.g. flutamide, nilutamide or bicalutamide) with a GnRH agonist was the first treatment shown to prolong life in patients with advanced prostate cancer (Bennett et al., 1999; Caubet et al., 1997; Crawford et al., 1989; Denis et al., 1998; Labrie et al., 1982; Labrie et al., 1985; Prostate Cancer Triallists’ Collaborative Group, 2000). Analysis of all the studies performed with flu­ tamide and nilutamide associated with medical or surgical castration compared with castration plus placebo shows that overall survival is increased by an average of 3–6 months (Bennett et al., 1999; Caubet et al., 1997; Crawford et al., 1989; Denis et al., 1993; Denis et al., 1998; Dijkman et al., 1997; Janknegt et al., 1993; Prostate Cancer

Triallists’ Collaborative Group, 2000; Schmitt et al., 2001) (Fig. 6). It is essential not to include the data obtained with cyproterone acetate, a compound showing intrinsic androgenic activity in all in vitro and in vivo assays (Labrie et al., 1987; Luthy et al., 1988; Plante et al., 1988). These preclinical data have translated into a shortened survival when cyproterone acetate was added to castration and compared with cas­ tration alone in men with metastatic disease (Prostate Cancer Triallists’ Collaborative Group, 2000). Since about 50% of patients in that age group die from causes other than prostate cancer, this 3–6-month difference in overall survival trans­ lates into an average of 6–12 months of life gained when cancer-specific survival is ana­ lyzed. These additional months, or sometimes years, of life can be obtained by simply adding a pure anti-androgen (flutamide, nilutamide or bicalutamide at a proper dose) to castration. These data demonstrate the particularly high level of sensitivity of prostate cancer to andro­ gen deprivation, considering that such statisti­ cally significant benefits on survival are obtained, even at the very advanced stage of metastatic disease.

Favours CAB

Favours castration

PCTCG: nilutamide (n = 1751) PCTCG: flutamide (n = 4803) PCTCG: nilutamide + flutamide (n = 6554)

* **

Caubet: NSAA (n = 3732)

*

Caubet: NSAA (n = 1978)

**

Caubet: NSAA (n = 2357)

**

Klotz: NSAA (n = 5015)

*

Debruyne: nilutamide (n = 1191)

*

Bennett: flutamide (n = 4128)

*

0.5 *2p < 0.05; **2p < 0.01

1.0

2.0

Hazard ratio and 95% confidence limits

Fig. 6. Summary of meta-analyses comparing combined androgen blockade [combination of medical or surgical castration associated with a pure anti-androgen (NSAA), namely flutamide or nilutamide] versus medical or surgical castration alone. Adapted from Klotz et al., 2001.

332

With the clinical data summarized above, the controversy or uncertainty concerning CAB should be part of history and the addition of a pure anti-androgen at a proper dose should be recognized by all as providing an average advan­ tage of 3–6 months of life in metastatic disease at a time when no alternative treatment even exists. Further improvement of the hormonal therapy of metastatic disease is very difficult. By far the best and today’s only possibility of improvement for the prostate cancer patient is treatment of localized disease. In fact, in analogy with the treat­ ment of all other types of cancers, the beneficial effects are much greater when the same treatment is applied at an earlier stage of the disease. In the United States, where three million of all men currently alive are expected to die from pros­ tate cancer (Statbite, 2004), 6 additional months of life per individual correspond to 1.5 million years overall, whereas 12 additional months correspond to 3 million years.

Resistance to treatment in metastatic disease Contrary to the situation in localized disease where resistance to CAB practically does not exist, resistance to hormone therapy is the stan­ dard observation in metastatic prostate cancer, thus creating a major and unresolved therapeutic challenge. In fact, recent evidence indicates that AR functioning is required for the growth of prostate cancer at all stages, including castra­ tion-resistant disease (Taplin, 2007). These data show that endocrine therapy-resistant prostatic carcinomas generally display uniformly high AR expression (Ruizeveld de Winter et al., 1994; van der Kwast et al., 1991). In a recent study, a sig­ nificant increase in AR mRNA levels was observed in the cancerous prostatic cells com­ pared with the benign tissue biopsies (Levesque et al., 2009). Treatment with flutamide has already been shown to decrease AR expression in prostatic carcinoma tissue (van der Kwast et al., 1996). Even at time of progression in patients with metastatic prostate cancer treated by castration alone, the benefits of additional androgen

blockade are illustrated by the observation of a positive response in 30–60% of patients in pro­ gression by hypophysectomy, adrenalectomy or aminoglutethimide (Drago et al., 1984; Labrie et al., 1985; Maddy et al., 1971; Murray and Pitt, 1985). In a relatively large-scale study of 209 meta­ static prostate cancer patients showing disease progression after orchiectomy or treatment with high doses of estrogens or a GnRH agonist alone, the addition of flutamide permitted to achieve complete, partial and stable responses in 6.2, 9.6 and 18.7% of cases, respectively, for a total clinical benefit of 34.5%. The mean duration of response was 24 months (Labrie et al., 1988). Contrary to the generalized opinion that patients in relapse after castration have exclu­ sively ‘androgen-insensitive’ tumors, the abovementioned data suggest that ‘androgen-sensitive’ tumors are present at all stages of prostate can­ cer in all patients and that maximal androgen blockade should always be administered. Instead of being ‘androgen insensitive’, most of the tumors which continue to grow after castration are androgen sensitive and able to grow in the presence of the ‘low’ level of androgens of adre­ nal origin left after ‘castration’ (Labrie et al., 1988). ‘Control of their growth requires further androgen blockade’. This affirmation was well supported by already available clinical data and fundamental observations (Labrie and Veilleux, 1986). As evidence for the androgen sensitivity of prostate cancer progressing under androgen blockade, Fowler and Whitmore (1981) have observed a rapid and severe exacerbation of the disease in 33 out of 34 patients in relapse within the first 3 days of testosterone adminis­ tration, thus clearly showing that at least part of prostate cancer cells remain androgen sensitive even at the advanced stage of progression under androgen blockade (Fowler and Whitmore, 1981). Based upon the above-summarized data, it becomes important to develop more potent but always pure blockers of androgen formation or action, the blockade of AR being the most obvious and therapeutically well-supported target. Positive clinical data have recently been reported with a new anti-androgen (Tran et al., 2009),

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although the true role of this compound used in relapsing patients where the anti-androgen was apparently stopped at time of administration of the new agent remains to be assessed. Similar uncertainty applies to the 50% (or more) decrease in serum PSA in 28 (67%) of 42 patients observed following administration of abiraterone in patients relapsing under antiandrogen treatment (Attard et al., 2009). Con­ sidering the well-demonstrated clinical benefits and the important decrease in serum PSA which follow simple cessation of administration of the anti-androgen (Dupont et al., 1993; Kelly et al., 1997), the true clinical benefits of these compounds remain undefined. It is clear, how­ ever, that a more potent blocker of AR having pure antagonistic activity could play an impor­ tant role in prostate cancer therapy at all stages of the disease.

More potent anti-androgens, the key to more successful treatment of prostate cancer While androgen blockade has been known for a long time to be efficacious as first line therapy at all stages of prostate cancer (Akaza, 2008; Huggins and Hodges, 1941; Janknegt et al., 1993; Labrie et al., 2002; Labrie et al., 1985; Namiki et al., 2008), recent data indicate a continuous role of the AR in treatment-resistant metastatic disease (Chen et al., 2004). While the percentage of objective responses in patients with metastatic prostate cancer is higher and the duration of response is longer when CAB is used (Crawford et al., 1989; Jan­ knegt et al., 1993; Labrie et al., 1985), progres­ sion of the disease always occurs if the patients have metastatic disease at start of treatment. Somewhat surprisingly, however, it was found that a large percentage of patients show a posi­ tive clinical response upon discontinuation of the anti-androgen with a decrease in serum PSA by more than 90% in 47% of patients (Dupont et al., 1993). This paradoxical phenom­ enon became generally recognized (Collinson et al., 1993; Herrada et al., 1996; Kelly and Scher, 1993; Kelly et al., 1997). A possible

explanation of the paradoxical effect of antiandrogen withdrawal is the development of hypersensitivity to androgens (Labrie and Veil­ leux, 1986; Labrie et al., 1988), or intracellular changes making the anti-androgen act as a par­ tial androgen agonist. The traditional therapeutic approach for metastatic disease which has become resistant to a specific androgen blockade, usually partial androgen blockade achieved by medical or sur­ gical castration or an anti-androgen alone, deserves re-evaluation following the observa­ tions of higher AR levels and/or maintenance of responsiveness to androgens in treatmentresistant cancer (Chen et al., 2004; Harris et al., 2009; McPhaul, 2008; Mostaghel et al., 2009; Mulders and Schalken, 2009; Scher and Sawyers, 2005; Taplin and Balk, 2004). While addition of an anti-androgen was the usual approach in patients showing resistance to monotherapy by castration, the patients who had become resistant to CAB were not consid­ ered candidates for further hormonal manipula­ tions and were treated by non-hormonal approaches, specially chemotherapy with no clear success (Eisenberger et al., 1985). In fact, elevated AR expression has been found to lead to resistance to anti-androgen therapy in mouse xenograft prostate cancer models (Chen et al., 2004). In any case, such data indicate that AR blockade could well remain an important ther­ apeutic target even at the last stage of the dis­ ease when resistance to various forms of androgen blockade has developed. Although the available anti-androgens fluta­ mide, bicalutamide and nilutamide have pure AR antagonistic activity and have shown major benefits in prostate cancer therapy (Crawford et al., 1989; Janknegt et al., 1993; Labrie et al., 1985; Prostate Cancer Triallists’ Collaborative Group, 2000), the affinity of all these com­ pounds for AR is very low (Labrie et al., 1997; Labrie et al., 1999; Luo et al., 1996; Simard et al., 1997) and leaves an estimated 5–10% of DHT free to continue to stimulate AR and prostate cancer growth (Labrie et al., 1987). There is thus the need to discover and develop novel anti-androgens having higher

334

affinity for the human AR in order to take optimal advantage of the well-demonstrated sensitivity of prostate cancer to androgens. Using the structural information on the ligandbinding domain (LBD) of the human AR (Cantin et al., 2007; Pereira de Jesus-Tran et al., 2006), we have synthesized a long series of novel molecules having higher or much higher affinity for the human AR and/or higher anti-androgenic potency in intact cell models than flutamide, bicalutamide or nilutamide, the only presently pure AR antago­ nists available for therapeutic use in men suffering from prostate cancer. In addition to the increased AR levels, the local and autonomous synthesis of androgens may explain the observation that androgen deprivation in prostate cancer xenograft models demonstrated only transient cell cycle arrest, with little evidence of apoptosis, followed by rapid progression (Agus et al., 1999). Moreover, as mentioned above, cancerous prostate tissue was found to synthesize more DHT than the benign prostatic tissue (Nishiyama et al., 2007). With the above-summarized information, it is reasonable to believe that the drug most urgently needed for prostate cancer is a more potent blocker of AR. With the available weak anti-androgens, only partial androgen blockade can be achieved – true total androgen blockade needs more potent compounds. As mentioned above, the problem with current anti-androgens is their low potency which leaves 5–10% of DHT in the prostate cancer tissue (Fig. 7) free to continue to stimulate cancer growth and metastasis. Moreover, with the available pure but weak anti-androgens (Labrie et al., 1997; Labrie et al., 1999; Luo et al., 1996; Simard et al., 1997), CAB must be continued for many years (six or more), even in localized disease (Fig. 5). With a more potent anti-androgen, complete apoptosis and cell death should be achieved more rapidly, thus greatly facilitating cure of localized disease. The compounds which we have synthesized are designed to impede repositioning of the mobile carboxy-terminal helix 12, which blocks the ligand-dependent transactivation function (AF-2) located in the AR ligand-binding domain (ARLBD). Using

Intraprostatic DHT

% 100 75

Cancer growth

decreases but

continues −

resistance

develops

50 25 0 Intact

Monotherapy castration or anti-androgen alone

Combined androgen blockade castration + anti-androgen

Fig. 7. Comparison of the intraprostatic concentrations of DHT in intact men, in men castrated or receiving an antiandrogen alone (monotherapy) and in men receiving combined androgen blockade (castration þ a pure antiandrogen having relatively low potency, namely flutamide, bicalutamide or nilutamide).

crystal structures of the human ARLBD (hARLBD), we first found that H12 could be directly reached from the ligand-binding pocket (LBP) by a chain positioned on the C18 atom of an androgen steroid nucleus. A set of DHTderived molecules bearing various C18 chains were thus synthesized and tested for their capa­ city to bind to human androgen receptor (hAR) and act as antagonists. Although most of those having very high affinity for hAR were agonists, several very potent antagonists were obtained, confirming the structural importance of the C18 chain. To understand the role of the C18 chain in their agonistic/antagonistic properties, the structure of the hARLBD complexed with one of these agonists, EM-5744, was determined at a 1.65 Å resolution (Cantin et al., 2007). We have identified new interactions involving Gln738, Met742 and His874 that explain both the high affinity of this compound and the inability of its bulky chain to prevent the repositioning of H12. These structural information were helpful to refine the structure of the chains placed on the C18 atom in order to obtain efficient H12-direc­ ted steroidal anti-androgens. With the aim of designing such new antiandrogens, we decided to make use of earlier

335

structural findings on the human estrogen recep­ tor (hER), a receptor structurally related to the hAR. The hER is unable to interact with co­ activator partners when a ligand bearing a welloriented bulky chain is bound to its ligand-bind­ ing site (Brzozowski et al., 1997). Indeed, as revealed by comparison of the crystal structures of the hER ligand-binding domain (hERLBD) in complex with a natural estrogen [estradiol (E2)] and a potent SERM (selective estrogen receptor modulator, raloxifene), agonist and antagonist molecules bind at the same site within the LBD. However, they exhibit different binding modes, inducing a distinct conformation in the transactivation domain (AF-2) characterized by a different positioning of H12. More precisely, the size and structure of raloxifene prevent the molecule from being completely confined within the steroid-binding cavity. Consequently, its bulky side chain protrudes from the cavity and impedes H12 from adopting the position found in the E2–hERLBD complex structure, a confor­ mation essential for interaction with transcrip­ tional co-activators. Concerning hAR, the crystal structures of its LBD (hARLBD) in complex with the natural androgens DHT and testo (Pereira de Jesus-Tran et al., 2006; Sack et al., 2001) have shown that H12 occupies therein the same position as that observed in the E2–hERLBD structure. Such data suggest that this helix is essential for the function of the AF-2 of hAR and, like in the hER, participates in the interaction with co-acti­ vators. This has been confirmed by the structure of the liganded hARLBD in complex with a pep­ tide derived from physiological co-activators (Estebanez-Perpina et al., 2005; He et al., 2004; Hur et al., 2004). Using all the available structural information of the hAR, we then proceeded to molecular model­ ling studies to find the best position on an andro­ gen nucleus (here DHT) for introducing a bulky chain able to reach the site normally occupied by H12. Finally, the combined data from molecular modelling and structure/activity relationship stu­ dies served as a basis for the design and improve­ ment of the chain structure, with the aim of maximizing the affinity of these steroidal-based

compounds for hAR. This rational approach yielded several different DHT-based ligands able to bind hAR with high affinity (many folds over that of DHT). In our in vitro tests, a small sub­ group proved to be very efficient antagonists of DHT stimulation, thus indicating that the particu­ lar structure of the bulky chain is of paramount importance for its activity.

Hormone therapy is greatly underused in prostate cancer Despite the fact that it is well recognized since 1940s (Huggins and Hodges, 1941) that the stan­ dard and even the only efficient treatment of metastatic prostate cancer is androgen blockade, a recent survey of 9110 men aged 65 years or older who died from prostate cancer between 1991 and 2000 has surprisingly indicated that 38% of black and 25% of white men in the United States did not receive hormone therapy before dying from prostate cancer (Lu-Yao et al., 2006). If such a large proportion of patients with metastatic prostate cancer do not receive hor­ mone therapy, one can understand the difficulty to implement CAB instead of the partial and limited blockade obtained with monotherapy (Table 3). This deficient use of androgen block­ ade in prostate cancer can be contrasted with the respective 93.5 and 98% rates of use of beta blockers after myocardial infarction (National Committee for Quality Assurance, 2003, p. 60) and tamoxifen in estrogen-receptor-positive breast cancer (Buzdar and Macahilig, 2005). This deficiency in the field of prostate cancer may be due to the fact that doctors underesti­ mate the risks of death from prostate cancer compared to other causes of death. Table 3. Frequent errors related to androgen blockade 1. Monotherapy (GnRH agonist alone, orchiectomy alone or anti-androgen alone) instead of combined androgen blockade 2. Too short duration of treatment 3. Treatment started too late 4. Intermittent treatment

336

Early diagnosis is essential in order to be able to apply the available curative treatments of prostate cancer It is very important to realize that the use of the currently available approaches for the diagnosis and treatment of prostate cancer can virtually eliminate death from this disease. With the cur­ rent techniques, screening can detect prostate can­ cer at a clinically localized stage in 99% of cases (Labrie et al., 1996b). Radical prostatectomy, radiotherapy or brachytherapy can be instituted immediately with curative intent following such early diagnosis. Moreover, excellent results can be expected with CAB alone, particularly in older patients where the choice of CAB can be easier. In fact, CAB can also be used alone as primary therapy with excellent results, as shown in important recent studies (Akaza et al., 2006; Egawa et al., 2004; Homma et al., 2004; Labrie et al., 2002; Ueno et al., 2006). Most importantly, CAB must be used immedi­ ately in patients for whom radical prostatectomy, radiotherapy or brachytherapy fails. As men­ tioned above, it is often erroneously believed that resistance will develop to androgen blockade in localized disease and that this treatment should therefore be delayed until a later stage of the disease. This belief is incorrect. In fact, the use of CAB to treat localized prostate cancer does not lead to resistance to treatment as long as the can­ cer is limited to the prostate or to tissue near the prostate at time of starting treatment. However, as mentioned above, if the treatment is deferred, the possibility of cure is very often lost because the cancer has the opportunity to metastasize to the bones where cure is practically impossible. In metastatic disease, the response to androgen blockade is short and resistance to treatment can­ not be avoided. It should be appreciated that when prostate cancer is first detected, even by screening, the tumor is not small. Immediate treat­ ment is the only treatment that offers a strong hope of cure. In fact, CAB must be started imme­ diately at time of diagnosis. While showing the high efficacy of hormonal therapy in localized prostate cancer, the present data clearly indicate that long-term treatment with

the best available drugs, somewhat similar to the 5 years of tamoxifen in breast cancer, is required for optimal control of prostate cancer. Great caution should be taken, however, when using serum PSA as surrogate marker. In fact, serum PSA rapidly and easily decreases to undetectable levels under androgen blockade although the cancer remains present for much longer periods of time, usually for many years as demonstrated in our recent study (Labrie et al., 2002). For this reason, inter­ mittent therapy should not be recommended outside prospective and randomized clinical trials.

Conclusion With the present knowledge, it is clear that all available means should be taken to diagnose pros­ tate cancer early and to use efficient therapy immediately in order to prevent prostate cancer from migrating to the bones when cure or even long-term control of the disease is an exception. It is clear that the only means of preventing prostate cancer from migrating to the bones and becoming incurable is efficient treatment at the localized stage. In fact, since radical prostatectomy, radio­ therapy and brachytherapy (implantation of radioactive seeds in the prostate) can achieve cure in about 50% of cases, these approaches are all equally valid choices as first treatment of loca­ lized prostate cancer with a curative intent. Androgen blockade should also be considered as first line treatment of curative intent, especially for elderly men and those having other serious health problems. The most important, however, is to follow closely serum PSA after surgery, radiotherapy and brachytherapy and to start CAB as soon as signs of recurrence of the cancer appear. It is also clear from the data summarized above that CAB alone could well be the most efficient therapy of localized prostate cancer while it has already been recognized as the best therapy for metastatic disease. Clearly, the rational use of the presently avail­ able diagnostic and therapeutic approaches could decrease prostate cancer death by at least 60% (Labrie et al., 1996b; Labrie et al., 1999). As an example, between 1991 and 1999, the death rate

337

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L. Martini (Eds.)

Progress in Brain Research, Vol. 182

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 15

Pheochromocytoma and paraganglioma Vitaly Kantorovich1 and Karel Pacak2,� 1

Division of Endocrinology and Metabolism, Department of Internal Medicine, College of Medicine, University of

Arkansas for Medical Sciences, Arkansas Cancer Research Center, AR, USA

2 Section on Medical Neuroendocrinology, Reproductive and Adult Endocrinology Program, NICHD, NIH, Bethesda,

MD, USA

Abstract: Pheochromocytoma is a very special kind of tumor full of duplicity. On the one hand it represents its own microworld with unique clinical, biochemical and pathological features, while on the other it constitutes a tremendously significant part of whole body system, playing a vital role for practically every organ system. It has a very special character – sometimes like a child it can be sweet and predictable, while at times it can behave like a deadly wild beast, crashing and tearing everything on its path in a fierce rage. It also consists of the amazingly intelligent neuroendocrine cells that possess a magical ability to make miraculous substances of many kinds. But most of all, it is a system that is able to drive our curiosity and the itch of “Cogito, ergo sum” to limitless depths and year by year it still amazes us with new and unexpected discoveries that move our understanding of multiple pathways and metabolic events closer to the ultimate truth. Recent discoveries of succinate dehydrogenase (SHD) and prolyl hydroxylase (PHD) mutations, for example, propelled our understanding of neuroendocrine tumorigenesis as a whole, as well as physiology of mitochondrial respiratory chain and phenomenon of pseudohypoxia in particular. Good old discoveries make their way from dusty repositories to shine with new meaning, appropriate for the current level of knowledge. This acquired wisdom makes us better physicians – knowing the specific expression makeup of catecholamine transporters, GLUTs and SRIFs allows for better tailored imaging and therapeutic manipulations. There are still long ways to go, keeping in mind that pheochromocytoma is but so very special, and we are optimistic and expect many great things to come. Keywords: pheochromocytoma; paraganglioma; catecholamines; metanephrines; positron emission tomography

unflattering “treacherous murderer”. These apparently “mixed feelings” relate to the rarity of disease in a population of usual suspects – patients with poorly controlled and labile hypertension of one side and horrific devastation of acute pheo­ chromocytoma crisis that strikes unexpectedly and unsuspectedly, leaving traumatic memories of acute medical disaster that champions any intensive care story (Brouwers et al., 2003;

Past There are probably few names that have not been used to describe pheochromocytoma – from somewhat complimenting “great masquerader” to �

Corresponding author. Tel.: 1-301-402-4594; Fax: 1-301-402-0884;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)82015-1

343

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Manger and Gifford, 2002; Pacak et al., 2001e). For the sake of simplicity, we will use the term “pheochromocytoma” as an inclusive reference for both the adrenal tumor (true pheochromocy­ toma) and its extra-adrenal counterpart (para­ ganglioma), but diverge into more detailed terminology when necessary. Autopsy studies have shown that significant numbers of pheo­ chromocytomas remain undiagnosed until death, and that up to 50% of these unrecognized tumors may have contributed to patient mortality (Sutton et al., 1981). On the other hand, pheochromocyto­ mas represent an amazing pathophysiological phe­ nomenon, where a single tumor comprises part of the diffuse neuroendocrine system (DNES). The history of pheochromocytoma research is rich while its present is dynamic with continuous change in our view on the disease and its management based on rapidly changing knowledge and under­ standing of the underlying genetics and other mechanisms leading to it. The initial discovery of pheochromocytoma represents the pinnacle of descriptive medical science and closely associates with the develop­ ment of neuroendocrinology as a whole. First, adrenal pheochromocytoma was described by Frankel (1886), extra-adrenal pheochromocytoma by Alezais and Peyron (1908), while the name was coined by Pick (1912) for dusky (phaios) colour (chroma) features on staining with chromium salts. It is also important to recognize the visionary work of Alfred Kohn, who between 1898 and 1902 introduced the terms “chromaffin” and “paraganglion” and shaped a paradigm of systemic nature of chromaffin tissue and existence of dif­ fuse paraganglionic system. The association between paroxysmal hypertension and pheochro­ mocytoma was described by L’Abbe et al. (1922). Epinephrine was isolated from a pheochromocy­ toma by Kelly et al. (1936) and Holton (1949) demonstrated the presence of norepinephrine in a pheochromocytoma. Von Euler and Holtz inde­ pendently reported the occurrence of norepi­ nephrine in the human body (Holtz et al., 1947; von Euler, 1946a , 1946b). The first successful surgical removal of pheochromocytoma was per­ formed by Roux in 1926 (Manger and Gifford, 1996).

Present Epidemiology According to different reviews and statistics, pheochromocytomas account for ~0.05–0.1% of patients with any degree of sustained hyperten­ sion (Bravo and Tagle, 2003; Manger and Gifford, 1996, 2002). This probably accounts for only half of persons harbouring the tumor, since about half the patients with pheochromocytoma have only paroxysmal hypertension or are nor­ motensive. With the current prevalence of sus­ tained hypertension in the adult population of Western countries about up to 30% (Epstein and Eckhoff, 1967; McBride et al., 2003; Page, 1976), the prevalence of pheochromocytoma can be estimated at between 1:4500 and 1:1700, with an annual incidence of 3–8 cases per 1 million per year in the general population (Pacak et al., 2001a). Pheochromocytoma occurs at any age, but most often in the fourth and fifth decade, and it occurs equally in men and women. At least 24% are familial (Neumann et al., 2002) and those tumors are often multi-focal and bilat­ eral (Bravo and Tagle, 2003; Manger and Gif­ ford, 2002). Furthermore, malignant pheochromocytoma or paraganglioma may account from 1% to about 90% of cases, depend­ ing on the type of the tumor, its localization (adrenal tumors are very rarely metastatic) and genetic background (mainly SDHB-related tumors) (Brouwers et al., 2006; Timmers et al., 2007c). About 10% of patients with pheochro­ mocytoma present with metastatic disease at the time of their initial workup (Scholz et al., 2007). In children, pheochromocytomas are more fre­ quently familial (40%), extra-adrenal (8–43%), bilateral adrenal (7–53%), or multi-focal or bilat­ eral if located in the adrenal gland (Barontini et al., 2006; Caty et al., 1990; De Krijger et al., 2006; Ludwig et al., 2007; Ross, 2000). These tumors peak at 10–13 years, with a 2:1 male pre­ dominance before puberty (Caty et al., 1990; Hume, 1960; Ross, 2000). Previous studies sug­ gested that less than 10% of paediatric pheochro­ mocytomas are malignant (Caty et al., 1990; Kaufman et al., 1983; Reddy et al., 2000; Ross,

345

2000) with reported mean survival rates of 73% at 3 years and 40–50% at 5 years after diagnosis (Coutant et al., 1999; Hume, 1960; Loh et al., 1997). There are currently new data suggesting that pheochromocytomas in children show much higher genetic incidence (Neumann et al., 2002), are commonly associated with succinate dehydro­ genase (SDH) subunit B (mainly in those patients with no family history of pheochromocytoma or paraganglioma) and often lead to metastatic dis­ ease even in very young children (Pacak et al., unpublished observations).

Anatomy and physiology Chromaffin cell ganglia (also called paraganglia) – with adrenal medulla representing the largest of them – are neuroendocrine organs, diffusely scat­ tered throughout the human body and associated with autonomic nervous system explaining why pheochromocytomas may be found practically in any location. It seems that paraganglia may play a role of prenatal regional chemo- and/or oxygen sensor, relying on maternal pressor backup, while after the birth the body needs both functions equally, which is better provided by the adrenal. Abnormalities in postnatal oxygen sensing may play a significant role in the generation of hypoxia or “pseudohypoxia” phenomenon and play a pivo­ tal role in further tumorigenesis (Gottlieb and Tomlinson, 2005). Pheochromocytomas are derived from the adre­ nal gland; paragangliomas arise from parasympa­ thetic-associated tissues (most commonly along the cranial nerves and vagus (e.g. glomus tumors, chemodectoma and carotid body tumor) and from extra-adrenal sympathetic-associated chromaffin tissue (often designated extra-adrenal pheochro­ mocytomas). While paragangliomas arise mainly from chromaffin tissue adjacent to sympathetic ganglia in the abdomen and less commonly in the chest or pelvis, both adrenal and extra-adrenal tumors display similar histopathological characteristics. It is difficult, if not impossible, to distinguish malignant from benign pheochromocytomas based on histopathological features. Only the

tumor invasion of tissues and the presence of metastatic lesions (most commonly in the liver, lungs, lymphatic nodes and bones) are consistent with the diagnosis of malignant pheochromocy­ toma (Yu and Pacak, 2002). Chromaffin cells have the ability to synthesize and secrete various amines and peptides (i.e. adrenocorticotropic hor­ mone (ACTH), chromogranins, neuropeptide Y, calcitonin, angiotensin-converting enzyme, renin, vasoactive intestinal polypeptide, adrenomedullin and atrial natriuretic factor), as well as antigens (Trojanowski and Lee, 1985), protein gene product 9.5 (Thompson et al., 1983), galanin (Bauer et al., 1986), renin (Mizuno et al., 1985), angiotensinconverting enzyme (Gonzalez-Garcia and Keiser, 1990) and synaptophysin (Miettinen, 1987) or parathyroid hormone-related protein (Kimura et al., 1990).

Catecholamines Biochemical reactions resulting in the production of catecholamines take place in numerous cells throughout the body, including central and per­ ipheral nervous systems, DNES, gastrointestinal tract and kidneys (Eisenhofer and Goldstein, 2004). Pathophysiological relevance of this pro­ duction depends mostly on the ability of the cell to possess appropriate enzymatic machinery, as well as on the ability of significant volume production of catecholamines. It is also important to appreciate that intracellular catecholamine synthesis parallels simultaneous cellular metabo­ lism of produced catecholamines independently of their release (Eisenhofer et al., 2003a). This metabolic phenomenon serves as a cornerstone of current biochemical diagnostic strategy of pheochromocytomas. The L-stereoisomer of amino acid tyrosine from a dietary source or following hydroxylation of phenylalanine in the liver serves as a substrate for the initiation of catecholamine synthesis. It is converted to dihydroxyphenylalanine (dopa) by tyrosine hydroxylase (TH) (Fig. 1) and repre­ sents the rate-limiting step in catecholamine biosynthesis (Udenfriend, 1966). Tissue expres­ sion of this enzyme is largely confined to

346 COOH OH

CH2

CH NH2

Tyrosine

Tyrosine hydroxylase

COOH CH2

OH OH

DOPA DOP A

CH NH2 OH

CH2 OH

Dopamine

CH

PMNT

Norepinephrine CH2

OH

CH2

CH

NH2

OH

L-DOPA L -DOPA decarboxylase

OH

Dopamine β--hydroxylase hydroxylase

CH NH2

OH OH

OH

CH2

CH

CH NH

Epinephrine CH3

Fig. 1. Catecholamine biosynthesis.

dopaminergic and noradrenergic neurons of the central nervous system, sympathetic nerves of peripheral nervous system, chromaffin cells of the adrenal medulla and extramedullary para­ ganglia. It is also expressed in enterochromaffin cells of enteric carcinoids (Jakobsen et al., 2001). The following step represents the produc­ tion of dopamine by decarboxylation of dopa aromatic L-amino acid decarboxylase. The dopa­ mine formed in neurons and chromaffin cells is translocated from the cytoplasm into vesicular storage granules. Large amounts of dopamine are also produced as an end product of catecho­ lamine synthesis in peripheral non-neuronal cells of the gastrointestinal tract and kidneys (Eisenhofer and Goldstein, 2004). The dopa­ mine formed in noradrenergic neurons and chromaffin cells is converted to norepinephrine by dopamine ß-hydroxylase (DBH), an enzyme

that is found only in the vesicles of cells that synthesize norepinephrine and epinephrine. In adrenal medullary chromaffin cells, norepi­ nephrine is metabolized by the cytosolic enzyme phenylethanolamine N-methyltransferase (PNMT) to form epinephrine (Axelrod, 1966). Epinephrine is then translocated into chromaffin granules, where the amine is stored awaiting release (Johnson, 1988). Expression of PNMT in extra-adrenal pheochromocytomas is negli­ gent, which explains the preferential production of norepinephrine by these tumors, compared to both norepinephrine and epinephrine produc­ tion by adrenal pheochromocytomas (Brown et al., 1972). Translocation of catecholamines into vesicular granules for storage is facilitated by two vesicular monoamine transporters: VMAT1 and VMAT2 (Fig. 2) (Henry et al., 1998). Catecholamines

347 Secreted catecholamines

Catecholamine transporter-mediated cellular reuptake

Catecholamine exocytosis

VMAT-mediated vesicular reuptake

Secretory vesicles Stored catecholamines

Fig. 2. Catecholamine metabolism.

show higher affinity to VMAT2 and differential expression of these transporters throughout the sympatho-adrenal system may correlate with dif­ ferences in uptake of metaiodobenzylguanidine (MIBG; see below) (Jakobsen et al., 2001). Sto­ rage vesicles represent a complex functional unit that continuously maintains a highly dynamic equilibrium between passive outward leakage of catecholamines into the cytoplasm counterba­ lanced by VMATs-driven inward active transport (Eisenhofer et al., 2003b). Catecholamines share the acid environment of the storage granule matrix with adenosine triphosphate (ATP), pep­ tides and proteins, the most well known of which are the chromogranins (O’Connor et al., 1994). The chromogranins are ubiquitous components of secretory vesicles, and their widespread pre­ sence among endocrine tissues has led to their measurement in plasma as sensitive, albeit rela­ tively non-specific markers of neuroendocrine tumors, including pheochromocytomas or

paragangliomas. Two populations of chromaffin cells have been described with morphologically distinct vesicles that preferentially store either norepinephrine or epinephrine, and which release the two catecholamines differentially in response to different stimuli. Most adrenal pheochromocy­ tomas secrete both norepinephrine and epinephr­ ine; about a third exclusively produce norepinephrine, and a much smaller proportion exclusively produce and secrete epinephrine. For a better understanding of the pathophysiol­ ogy and clinical symptomatology of pheochromo­ cytoma and paraganglioma, one needs to be aware about the following physiological concepts. All the three main catecholamines (dopamine, epinephrine and norepinephrine) have tightly regulated metabolism and organ-specific secre­ tion. While dopamine is a major central neuro­ transmitter, its peripheral levels can be elevated in rare dopamine-secreting pheochromocytomas. Adrenal gland, on the other hand, normally

348

secretes both epinephrine (predominantly) and norepinephrine directly into the circulation, of which about 90% is rapidly removed by extraneuronal hepatic monoamine transport system. Paraganglia exclusively secretes norepinephrine, which is a major neurotransmitter within sympa­ thetic nervous system. For norepinephrine released by sympathetic nerves, about 90% is removed back into nerves by neuronal uptake, 5% is removed by extraneuronal uptake and 5% escapes these processes to enter the bloodstream. Overall, these processes result in circulatory halflife for catecholamines of less than 2 minutes (Eisenhofer, 2001). Catechol-O-methyltransferase (COMT) is responsible for a major pathway of catecholamine metabolism, catalyzing O-methylation of dopa­ mine to methoxytyramine, norepinephrine to nor­ metanephrine and epinephrine to metanephrine, as well as methylation of dihydroxyphenylglycol (DHPG) to 3-methoxy-4-hydroxyphenylglycol (MHPG). COMT is not present in catechola­ mine-producing neurons. Normetanephrine and metanephrine are produced in small amounts and only at extraneuronal locations, with the sin­ gle largest source representing adrenal chromaffin cells, which account for over 90% of circulating metanephrine and 24–40% of circulating norme­ tanephrine (Eisenhofer et al., 1995). About 90% of the vesicular monoamine (VMA) formed in the body is produced in the liver, mainly from hepatic uptake and metabolism of circulating DHPG and MHPG (Eisenhofer et al., 1996). In humans, VMA and the sulphate and glucuronide conju­ gates of MHPG represent the main end products of norepinephrine and epinephrine metabolism and are eliminated mainly by urinary excretion.

Clinical presentation Most clinical signs and symptoms associated with hypercatecholaminaemia are non-specific, which may in part explain underdiagnosis of phaeochro­ nocytoma (Tables 1 and 2) resulting in extensive differential diagnosis (Manger and Gifford, 1996, 1977; Young and Landsberg, 1998). Although paroxysmal or resistant hypertension is still a

Table 1. Symptoms and signs of pheochromocytoma Clinical setting

Symptoms

Multi-system crisis

Hypertension and/or hypotension, multiple organ failure, temperature = 40C, encephalopathy

Cardiovascular

Collapse Hypertensive crisis Shock or profound hypotension Acute heart failure Myocardial infarction Arrhythmia Cardiomyopathy Myocarditis Dissecting aortic aneurysm Limb ischaemia, digital necrosis or gangrene

Pulmonary

Acute pulmonary oedema Adult respiratory distress syndrome

Abdominal

Abdominal bleeding Paralytic ileus Acute intestinal obstruction Severe enterocolitis and peritonitis Bowel perforation with generalized peritonitis Bowel ischaemia Mesenteric vascular occlusion Acute pancreatitis Cholecystitis Megacolon

Neurological

Hemiplegia Limb weakness

Renal

Acute renal failure Acute pyelonephritis Severe haematuria

Metabolic

Diabetic ketoacidosis Lactic acidosis

major cause for diagnostic workup of pheochro­ mocytoma, there is a steadily increasing propor­ tion of cases found as part of investigation for adrenal incidentalomas or accidentally found extra-adrenal paragangliomas showing few, if any, symptoms. Incidentalomas seem to be asso­ ciated with lower levels of catecholamines (Kopetschke et al., 2009). At least about 20–30% of patients with pheochromocytoma or paragan­ glioma are asymptomatic or have only minor signs and symptoms; therefore, the diagnosis is easily missed, often with tragic consequences. There also seems to be a difference in clinical presentation

349 Table 2. Differential diagnosis for pheochromocytoma

• Neuroblastoma; ganglioneuroblastoma; ganglioneuroma • Adrenal medullary hyperplasia • Hyperadrenergic essential hypertension • Baroreflex failure • Thyrotoxicosis • Anxiety, panic attacks • Migraine or cluster headaches • Autonomic epilepsy • Abrupt clonidine withdrawal • Amphetamines • Cocaine • Alcoholism • Ingestion of tyramine-containing foods or proprietary cold preparations while taking monoamine oxidase inhibitors

• Hypoglycaemia, insulin reaction • Paroxysmal tachycardias including postural tachycardia syndrome

• Angina pectoris or myocardial infarction • Mitral valve prolapse • Abdominal catastrophe/aortic dissection • Cardiovascular deconditioning • Renal parenchymal or renal artery diseases • Intracranial lesions, cerebral vasculitis and haemorrhage • Menopausal syndrome • Lead poisoning • Toxemia of pregnancy • Unexplained shock • Acute intermittent porphyria

between adrenal and extra-adrenal tumors, driven predominantly by different secretory profile – nor­ epinephrine in extra-adrenal tumors, and epinephrine and norepinephrine in adrenal tumors. Sustained or paroxysmal hypertension (equally present) is the most common clinical sign (85–90%). Up to 13% of the patients typically present with persistently normal blood pressure (Bravo and Tagle, 2003), but this proportion can be much higher in patients with adrenal inciden­ talomas or in those who undergo periodic screen­ ing for familial pheochromocytoma (Neumann et al., 2002). Pheochromocytoma may also pre­ sent with hypotension, commonly seen in patients harbouring tumors that are secreting

epinephrine or compounds causing vasodilata­ tion, or after higher doses of anti-hypertensive therapy. Hypotension, postural or alternating, may occur secondary to hypovolaemia, abnormal autonomic reflexes, downregulation or differen­ tial stimulation of a- and b-adrenergic receptors, or the type of co-secreted neuropeptide (Bravo and Gifford, 1993; Bravo and Tagle, 2003; Bravo et al., 1981). Headache occurs in up to 90% of patients with pheochromocytoma (Manger and Gifford, 1996), being mild or severe, short or long in duration, and may last for up to several days. Excessive generalized sweating occurs in ~60–70% of patients presenting with pheochro­ mocytoma (Manger and Gifford, 1996). Other complaints are palpitations and dyspnea, weight loss despite normal appetite (caused by catecho­ lamine-induced glycogenolysis and lipolysis) or weight gain (constipation), and generalized weakness (Gifford et al., 1985). Some patients present with new and commonly more severe episodes of anxiety or panic attacks (Bravo and Gifford, 1993). Palpitations, anxiety and nervousness are more common in patients with pheochromocytomas that produce epinephrine (Bravo and Gifford, 1993). This set of clinical symptoms has to be differentiated from hyperadrenergic hypertension, anxiety or panic attacks, which is characterized by tachycardia, sweating, anxiety and an increased cardiac output (Esler et al., 1977). These patients often have increased levels of catecholamines in blood and urine and may be excluded by the use of the clonidine suppression test (Bravo et al., 1981). Pheochromocytoma is known to be associated with a wide range of cardiac complications, includ­ ing ischemic or hypertrophic cardiomyopathy, supra- and ventricular tachyarrhythmias and acute ischemic events. It is important to suspect pheochromocytoma in case of unexplained acute cardiac event. On the other hand, we have observed only few cardiac complications in patients with long-standing pheochromocytomas or paragangliomas and extensive hypercatechola­ minaemia, probably related to significant downregulation of adrenergic receptors (Pacak et al., unpublished observations).

350

Pheochromocytoma-induced metabolic or hae­ modynamic attacks may last from a few seconds to several hours, with intervals between attacks varying widely. A typical paroxysm is characterized by a sudden major increase in blood pressure; a severe, often pounding headache; profuse sweating over most of the body, especially the trunk; palpi­ tations; prominent anxiety or a sense of doom; skin pallor; nausea, with or without emesis; and pain in the abdomen, the chest or other locations where a tumor is usually located (Manger and Gifford, 1996; Plouin et al., 1981). After an episode, patients usually feel drained and exhausted, and some may urinate more frequently. Rarely, paroxysmal blood pressure elevations during diagnostic procedures such as endoscopy, anaesthesia (caused by a sud­ den fall in blood pressure or any activation of the sympathetic nervous system that occurs during the induction phase of general anaesthesia) or inges­ tion of food or beverages containing tyramine (e.g. certain cheeses, beers, wines, bananas and chocolate) should arouse immediate suspicion of pheochromocytoma. The use of certain drugs such as histamine, metoclopramide, glucagon, etc. may also precipitate a hypertensive episode (Barancik, 1989; Bittar, 1979; Cook and Katritsis, 1990; Jan et al., 1990; Manger and Gifford, 1996; Page et al., 1969; Schorr and Rogers, 1987; Steiner et al., 1968). Moreover, micturition or bladder distension in the case of a pheochromocytoma of the urinary blad­ der can cause clinical symptoms and signs of cate­ cholamine excess. These episodes should be differentiated from pseudopheochromocytoma, which refers to the large majority of individuals (often women) with severe paroxysmal hyperten­ sion, whether normotensive or hypertensive between episodes, (Kuchel, 1985), related to a short-term activation of the sympathetic nervous system. In contrast to pheochromocytoma, patients with pseudopheochromocytoma more often pre­ sent with panic attacks or anxiety, flushing, nausea and polyuria (Kuchel, 1985, 2004; White and Baker, 1986). Less common clinical manifestations include fever of unknown origin (hypermetabolic state) and constipation secondary to catecholamineinduced decrease in intestinal motility (Bouloux and Fakeeh, 1995). Observed flushing is rare, but

can follow the vasoconstrictive episode of pallor or Raynaud’s phenomenon, associated with the attack (Manger and Gifford, 1996). Patients may also present with tremor, seizures, hyperglycaemia, hypermetabolism, weight loss (usually only in patients with malignant pheochromocytoma), fever and even mental changes (Manger and Gifford, 1996). The hyperglycaemia is usually mild, occurs with the hypertensive episodes, is accompanied by a subnormal level of plasma insu­ lin (because of a-adrenergic inhibition of insulin release) (Colwell, 1969) and usually does not need treatment; however, it can be sustained and severe enough to require insulin and even to present as diabetic ketoacidosis (Edelman et al., 1992). Hypercalcaemia has been reported in some patients with pheochromocytoma perhaps due to parathyroid hormone-related peptide (Heath and Edis, 1979; Steiner et al., 1968; Stewart et al., 1985). In addition, pheochromocytoma has pre­ sented as Cushing’s syndrome with the tumor as the ectopic source of ACTH (Spark et al., 1979). Rarely, pheochromocytoma has produced vasoac­ tive intestinal peptide with resultant watery diar­ rhoea, hypokalaemia and achlorhydria (Verner– Morrison syndrome) (Viale et al., 1985). Lactic acidosis without shock or sepsis has been reported (Bornemann et al., 1986). We have recently dis­ covered a cohort of patients stating to have experi­ enced exercise-induced nausea and vomiting on the first presentation of disease. We propose that the act of exercising increases the amount of cir­ culating catecholamines in pheochromocytoma/ paraganglioma patients and may potentially lead to the activation of the a1- and a2-adrenergic receptors in the area postrema inducing nausea and vomiting (Pacak et al., unpublished observations). In contrast to adult patients, in whom sustained hypertension is found in only 50% of cases, more than 70–90% of children present with sustained hypertension (Caty et al., 1990; Reddy et al., 2000). Pheochromocytoma is the underlying cause in 1–2% of cases of paediatric hypertension and should be considered after exclusion of the more common causes such as renal diseases and renal artery stenosis (Ross, 2000). Sweating, visual problems, weight loss, and nausea and vomiting

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are more common in children than in adults (Fonseca and Bouloux, 1993), as are polyuria and polydypsia. In addition, children commonly present with palpitations, anxiety, hyperglycae­ mia, pallor and/or flushing, while others occasion­ ally present with a reddish blue mottling of the skin and a puffy red and cyanotic appearance of the hands (Manger and Gifford, 1996). Because neuroblastomas (the most common solid tumors in childhood), ganglioneuroblastomas and gang­ lioneuromas can synthesize and excrete catecho­ lamines and their metabolites, the differential diagnosis of a pheochromocytoma is somewhat difficult or, at times, impossible.

Genetics of pheochromocytoma Up to 24–28% of pheochromocytomas are inher­ ited (Neumann et al., 2002). Hereditary pheochro­ mocytoma is associated with multiple endocrine neoplasia type 2 (MEN2A or MEN2B), von Reck­ linghausen’s neurofibromatosis type 1 (NF-1), von Hippel–Lindau (VHL) syndrome and familial paraganglioma caused by germ-line mutations of genes encoding SDH subunits B, C and D (Table 3), as well as recently discovered SDH5 (see below). In general, the traits are inherited in an autosomal-dominant pattern. Tumorigenesis theories and related applications are discussed in the section below.

Multiple endocrine neoplasia type 2 syndrome MEN2 is an autosomal, dominantly inherited syndrome (Sipple’s syndrome) that consists of pheochromocytoma, medullary carcinoma of the thyroid and hyperparathyroidism (Pacak et al., 2005; Sarosi and Doe, 1968; Sipple, 1961). It affects about 1 in 40,000 individuals and is characterized by medullary thyroid carcinoma, pheochromocy­ toma and parathyroid hyperplasia/adenoma (Brandi et al., 2001). The syndrome is caused by mutation of RET proto-oncogene located on chro­ mosome 10 (10q11.2), which encodes a receptor tyrosine kinase from glial cell line-derived neuro­ trophic factor (GDNF) family and is specifically

expressed in neural crest-derived cells such as the calcitonin-producing C cells in the thyroid gland and the catecholamine-producing chromaffin cells in the adrenal gland (Mulligan et al., 1993). RET plays a role in normal gastrointestinal neuronal and kidney development (Lore et al., 2001). RET consists of 21 exons with 6 “hot spot exons” (exons 10, 11, 13, 14, 15 and 16) in which mutations are identified in 97% of patients with MEN2. Pheochromocytoma (at least 70% of which are bilateral) develops on a background of adrenome­ dullary hyperplasia and becomes manifest (e.g. biochemically or on imaging) in about 50% of patients. MEN2-associated pheochromocytomas are almost exclusively benign (with <5% reported to be malignant) and localized to the adrenals (Casanova et al., 1993; Neumann et al., 1993). The peak age is around 40 years, but children as young as 10 years can be affected (Jadoul et al., 1989). Patients with MEN2-related pheochromo­ cytoma often lack sustained hypertension or other symptoms (they occur only in about 50%). Because MEN-related pheochromocytomas secrete epinephrine, stimulation of ß-adrenergic receptors causes palpitations and tachycardia. Therefore, their detection is mainly based on ele­ vated plasma metanephrine and epinephrine levels. In patients with pheochromocytomas that produce exclusively normetanephrine, MEN2 is excluded. In addition, as with most epinephrinesecreting pheochromocytomas, the hypertension is more likely to be paroxysmal than sustained. For these reasons the diagnosis is easy to miss. MEN2B patients have pheochromocytoma; medullary carcinoma of the thyroid; ganglioneur­ omatosis; multiple mucosal neuromas of eyelids, lips and tongue; and some connective tissue dis­ orders that include marfanoid habitus, scoliosis, kyphosis, pectus excavatum, slipped femoral epi­ physis and pes cavus (Khairi et al., 1975). This syndrome also appears to be caused by germ-line mutations in the RET proto-oncogene on chromo­ some 10, but these mutations affect the tyrosine kinase catalytic site of the protein (Eng et al., 1994). In children with MEN2B-associated pheo­ chromocytomas, a higher risk of malignancy com­ pared with MEN2A or sporadic disease is found (Ross, 2000).

Table 3. Familial pheochromocytoma and paraganglioma syndromes Syndrome

Gene/protein/pathway

Phenotype

Pheo phenotype

Multiple endocrine neoplasia type 2

Chr 10 (10q11.2) – AD RET proto-oncogene Receptor tyrosine kinase Growth and differentiation

MEN2A – MTC Hyperparathyroidism MEN2B – MTC Mucosal neuromas Megacolon Marfanoid habitus Intestinal ganglioneuroma

Prevalence of 50% Adrenergic phenotype MEN2A – mostly benign Bilateral in 30% MEN2B – higher malignancy rate

Neurofibromatosis type 1 (von Recklinghausen’s disease)

Chr 17 (17q11.2) – AD NF1 tumor suppressor gene Neurofibromin Downregulation of p21-ras

Peripheral neurofibromas Café au lait spots and freckling Iris hamartomas Hyperparathyroidism MTC Hypothalamic tumors

Prevalence of 0.1–5.7% Adrenergic phenotype

Cerebelloretinal haemangioblastomatosis (von Hippel–Lindau syndrome, VHL)

Chr 3 (3p25-26) – AD

Retinal angiomas

Prevalence of 10–20%

VHL tumor suppressor gene HIFa Hypoxia-induced pathway

NS haemangioblastomas Renal cell cancer Cystadenomas

Noradrenergic phenotype

SDHB: Chr 1 (1p36) SDHC: Chr 1 (1q21) SDHD: Chr 11 (11q23) Subunits of MC II Pseudohypoxia-induced pathway Abnormal apoptosis Increased angiogenesis Complex inheritance

Parasympathetic paraganglioma Sympathetic paraganglioma Renal cell cancer

Mostly extra-adrenal SDHB – malignant PPT SDHC/D – HNPGL SDHD – multi-focal Mostly noradrenergic phenotype Sometimes dopaminergic phenotype

SDH syndromes

Abbreviations: MTC – medullary thyroid cancer; NS – nervous system (in this case – cerebellar and spinal haemangioblastomas); SDH – succinate dehydrogenase; AD – autosomal dominant mode of inheritance; MC II – mitochondrial complex II – component of electron transport chain involved in the Krebs cycle; HNPGL – head and neck paraganglioma.

353

von Hippel–Lindau syndrome

Neurofibromatosis type 1

Another neuroectodermal syndrome commonly associated with pheochromocytoma is VHL syn­ drome, which is caused by mutations in chro­ mosome 3 (3p25-26), which encodes the VHL tumor suppressor gene (Latif et al., 1993). Pheochromocytomas in VHL disease typically develop according to Knudson’s two-hit model, an inherited germ-line mutation of VHL and loss of function of the wild-type allele of the VHL gene. The disease has been divided into two types based on the significant geno­ type–phenotype correlations observed. Type 1 has mainly large deletions or mutations and expresses the full phenotype of vascular lesions of the retina, cysts or solid tumors in the brain or spinal cord, pancreatic cysts, renal cell carci­ noma, epididymal cystadenoma and endolym­ phatic sac tumors, but no pheochromocytoma. Type 2 has missense mutations, pheochromocy­ toma and the full phenotype (Chen et al., 1995). Patients are often asymptomatic when they pre­ sent with other aspects of this disease. This syndrome is quite variable in terms of the dif­ ferent organ systems involved and the extent of involvement from patient to patient and from family to family. Overall, less than 30% of patients with a VHL germ-line mutation develop a pheochromocytoma. Pheochromocy­ tomas as part of the VHL syndrome have an exclusively noradrenergic phenotype reflecting the production of norepinephrine only (Eisen­ hofer et al., 2001b). These tumors are mainly located intra-adrenally and are bilateral in about 50% of patients, with a less than 7% incidence of metastases. They arise on the background on adrenomedullary hyperplasia. These tumors are commonly found based on periodic annual screening, or during searches for other tumors that are part of this syndrome. Therefore, when detected, these tumors are commonly small and often fail to be detected by nuclear imaging methods. Furthermore, about 80% of pheochromocytomas found in VHL patients during screening are asymptomatic and not associated with hypertension.

von Recklinghausen’s NF is now divided into two types: NF-1 has neurofibromas of peripheral nerves, whereas NF-2 has central neurofibromas. NF-1 is inherited in an autosomal-dominant pat­ tern. The association with pheochromocytoma is one between a relatively common disease and a rare disease. Thus, although less than 1–2% of patients with NF have pheochromocytoma, about 5% of patients with pheochromocytoma have NF (Kalff et al., 1982). Pheochromocytoma associated with NF-1 is caused by germ-line mutations in chromosome 17 (17q11), where the NF-1 gene encodes for the protein neurofibromin. These mutations lead to inactivation of this tumor sup­ pressor gene and its protein (Colman and Wal­ lace, 1994). Similar mutations introduced into the NF-1 gene in mice lead to pheochromocytoma, which is otherwise rare in these animals (Jacks et al., 1994). Pheochromocytoma in patients with NF-1 is rarely seen in children, because it usually occurs at a later age (around 50 years). Only 12% of NF-1 patients are diagnosed with bilateral and multi-focal pheochromocytomas, and less than 6% of patients have metastatic pheochromocytoma (Walther et al., 1999). The incidence of pheochro­ mocytomas in NF-1 is relatively low (about 1%) compared with other hereditary syndromes, and routine screening of such patients is not generally recommended. However, if a patient with NF-1 has hypertension, then a pheochromocytoma should be considered and excluded (Kalff et al., 1982).

Succinate dehydrogenase gene family Recently, paraganglioma and pheochromocytoma susceptibility has been associated with germ-line mutations of the SDH gene family (Baysal et al., 2000). The SDH genes (SDHA, SDHB, SDHC and SDHD) encode the four subunits of complex II of the mitochondrial electron transport chain and are also part of the Krebs cycle (Astrom et al., 2003; Gottlieb and Tomlinson, 2005), both essential for the generation of ATP. SDHB and SDHD mutations can lead to complete

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loss of SDH enzymatic activity, a phenomenon that has been linked to tumorigenesis through upregulation of hypoxic–angiogenetic responsive genes (Gimenez-Roqueplo et al., 2002; van Nederveen et al., 2009). Except for the SDHA gene, mutations of SDHB, SDHC and SDHD genes are associated with the presence of familial paraganglioma or pheochromocytoma. Frameshift, missense and nonsense mutations were identified for the SDH gene family. In recent studies it has been found that about 4–12% of “sporadic” pheochromocytomas and up to 50% of familial pheochromocytomas express either SDHD or SDHB mutation (Astuti et al., 2001). The SDHB, SDHC and SDHD traits are inherited in an autosomal dominant fashion and give rise to familial paraganglioma (PGL) syndromes 4, 3 and 1, respectively (Table 3). The penetrance of these traits is incomplete, however. Furthermore, SDHD-related disease is characterized by maternal genomic imprinting. Due to silencing of the maternal allele by methylation, individuals who inherit a mutation from the mother remain free of paraganglioma, but may still pass on the mutation to their offspring. Although no muta­ tions in the SDHA have been associated with pheochromocytomas yet (most likely due to the fact that two genes encodes for SDHA), mutation of SDH5 was detected in patients with head and neck paragangliomas (Hao et al., 2009). SDHB mutations predispose to mainly extraadrenal pheochromocytomas with a high malig­ nant potential, and less frequently to benign parasympathetic head and neck paragangliomas (Amar et al., 2005a; Benn et al., 2006; Brouwers et al., 2006; Havekes et al., 2007; Neumann et al., 2004; Timmers et al., 2007c). SDHD mutations are typically associated with multi­ focal parasympathetic head and neck paragan­ gliomas and usually benign extra-adrenal and adrenal pheochromocytomas (Benn et al., 2006; Neumann et al., 2004). Metastastic pheochromo­ cytoma is rare in SDHD mutation carriers, but can occur (Havekes et al., 2007; Timmers et al., 2007d). SDHC mutations are rare, and are almost exclusively associated with parasympa­ thetic head and neck paraganglioma (Schiavi et al., 2005), although rare cases of SDHC-

associated extra-adrenal pheochromocytoma have been reported (Mannelli et al., 2007; Peczkowska et al., 2008). In a recent large pro­ spective study, Burnichon et al. (2009) sup­ ported previous findings of high frequency of SDHD and SDHC mutations in head and neck paragangliomas, as well as higher frequency of abdominal and pelvic disease and overall malig­ nancy in SDHB mutations. We have observed the same high frequency of malignancy in our paediatric population (Pacak et al., unpublished observation). The majority of SDHB-related paragangliomas secrete either norepinephrine or both norepinephrine and dopamine (Timmers et al., 2007c), a profile that is consis­ tent with mainly extra-adrenal tumors. Some SDHB-related PGLs exclusively overproduce dopamine, but not other catecholamines (Eisenhofer et al., 2005; Timmers et al., 2007c). Therefore, measurement of plasma levels of dopamine or its O-methylated metabolite methoxytyramine should be considered in SDHB-related pheochromocytoma. About 10% of SDHB-related sympathetic paragangliomas are “biochemically silent” (Timmers et al., 2007c; Timmers et al., 2009). Both mediastinal paragangliomas and paragangliomas of the large para-aortic paraganglion, described by Emil Zuckerkandl, usually associate with SDHx mutations (Ghayee et al., 2009; Van Nederveen et al., 2006).

Other pheochromocytomas MEN1 (Wermer’s syndrome) consists of hyper­ parathyroidism, pituitary adenomas and pancrea­ tic islet cell tumors. Pheochromocytoma is not usually part of this complex; however, the occur­ rence of pheochromocytoma and pancreatic islet cell tumors has been reported in some families (Carney et al., 1980). Various crossover syn­ dromes have been reported in which pheochro­ mocytoma has been associated with characteristics of MEN1, MEN2A, MEN2B, von Recklinghausen’s neurofibromatosis (NF), VHL and the Zollinger–Ellison syndrome (Cameron et al., 1978).

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Pheochromocytoma may also occur as part of Carney’s triad (i.e. gastric leiomyosarcoma, pul­ monary chondroma and extra-adrenal pheochro­ mocytoma) (Carney, 1983). The syndrome is very rare; less than 30 cases have been reported, and only 25% of patients manifest all three parts of the triad. It occurs sporadically and is clearly nonfamilial (Margulies and Sheps, 1988). Recently, a new syndrome called the Stratakis–Carney syn­ drome, or the “dyad of gastrointestinal stromal tumor and paraganglioma” associated with SDHB, SDHC and SDHD mutations were identi­ fied (Pasini et al., 2008).

new era of diagnosis and treatment for pheochromocytoma. Diagnostic algorithm had also become multi­ directional: in some patients it starts from clin­ ical symptomatology through biochemistry and imaging, while in others it begins by incidental discovery of adrenal mass and continues into “all-possible-adrenal-pathology-rule out” workup. Yet in another group it will be initiated by surgical discovery of adrenal or paraganglial tumor during an unrelated surgery. In such case, pathological examination will become the main diagnostic procedure, followed by metastatic workup.

Diagnosis of pheochromocytoma Biochemical diagnosis of pheochromocytoma The diagnostic algorithm changes continuously in case of pheochromocytoma. For right or wrong, current diagnostic approach is not driven by high clinical suspicion, but rather by the defensive “rule out” nature of modern healthcare. Massive number of patients with resistant hypertension, anxiety disorders and adrenal inci­ dentalomas will undergo questionably indicated workup for pheochromocytoma and very few will be found to have one, decreasing the like­ lihood of pheochromocytoma even in a patient with a positive result. This fact, on the other hand, elevates the need for certainty of right diagnosis to considerable importance, which results in repeated biochemical tests and use of at least two imaging modalities (Ilias and Pacak, 2004; Pacak et al., 2007). There had also been tremendous develop­ ments in diagnostic procedures. Biochemical testing is based not on the episodically secreted catecholamines, but rather on continuously produced metabolites, metanephrines, which significantly increased our ability to “catch” pheochromocytoma. Imaging studies include high-resolution anatomical techniques that enable discovery of smaller adrenal, extraadrenal and metastatic lesions, while functional studies based on physiology of chromaffin cells enable “metabolic” localization of tumors. Singlephoton emission computed tomography (SPECT) techniques together with image fusion opened a

Historically, biochemical testing of pheochromo­ cytoma faced high quality expectations. It had to have high sensitivity with reasonable false-test­ ing parameters, as well as be reliable in detecting relatively small increase in catecholamines. These expectations were driven by potential dreadfulness of misdiagnosis, as well as the fact that catecholamine secretion is mostly episodic and may not correlate with existence of hyper­ tension. Metanephrines, on the other hand, are produced continuously within pheochromocy­ toma tumor cells, and independently of catecho­ lamine release, thus obviating any need for collection of blood or urine samples during hypertensive episodes (de Jong et al., 2007; Eisenhofer et al., 1999a; Lenders et al., 1995; Lenders et al., 2002; Raber et al., 2000; Sawka et al., 2003; Unger et al., 2006; Vaclavik et al., 2007). Expert recommendations from the Inter­ national Symposium on Pheochromocytoma for initial biochemical testing include measurements of fractionated metanephrines in urine or plasma, or both, as available (Grossman et al., 2006; Pacak et al., 2007). There was no consen­ sus on whether plasma or urine measurements should be the preferred test. Both tests offer similarly high diagnostic sensitivity (provided appropriate reference ranges are used), so that a negative result for either test appears equally effective for excluding pheochromocytoma.

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However, because of differences in specificity, tests of plasma free metanephrines exclude pheochromocytoma in more patients without the tumor than do tests of urinary fractionated metanephrines. The conditions under which blood or urine samples are collected can be crucial to the relia­ bility and interpretation of test results. Blood for measurements of plasma free metanephrines or catecholamines should ideally be collected from patients supine for at least 20 minutes before sampling (Eisenhofer, 2003). The extent of the increase of a positive test result is also crucially important in judging the likelihood of a pheochromocytoma. Most patients with the tumor have increases in plasma or urinary metanephrines well in excess of those more commonly encountered as false-positive results in patients without the tumor. Increases in plasma concentrations of normetanephrine or of metanephrine four times above the upper reference limit are almost non-existing in patients without pheochromocytoma, but occur in about 70–80% of patients with the tumor (Lenders et al., 2002). Similarly, increases in urinary outputs of normetanephrine above 1500 mg/day (8.2 mmol/day) or of metanephrine above 600 mg/day (3.0 mmol/day) are rare in patients without pheochromocytoma, but occur in about 70% of patients with the tumor. Diet­ ary constituents or medications can either cause direct analytical interference in measurements of catecholamines and metabolite levels (tricyc­ lic anti-depressants) or may influence the phy­ siological processes that determine these levels (labetalol, buspirone, acetaminophen), as can do renal failure, etc. (Bouloux and Perrett, 1985; Eisenhofer et al., 2003c; Lenders et al., 1993; Roden et al., 2001). In rare dopaminesecreting pheochromocytomas, measurement of plasma methoxytyramine was shown to provide additional diagnostic value (Eisenhofer et al., 2005). The provacative glucagon test is inherently dangerous and rarely used, while clonidine sup­ pression test may be useful and bear lower risk (Bravo et al., 1979; Grossman et al., 1991;

Lawrence, 1967; Lenders et al., 2010; Sheps and Maher, 1966). Clonidine (Catapres) is now the drug used most often in suppression tests to identify a pheochromocytoma (Bravo and Gifford, 1984). It is a centrally acting a2-adrenergic agonist that suppresses central sympathetic nervous outflow, and this normally results in lower levels of plasma catecholamines. Blood is drawn for plasma catecholamines and metanephrines before and 3 hours after the oral administration of clonidine 0.3 mg/ 70 kg body weight (to maximum of 0.5 mg). Normal plasma norepinephrine or normeta­ nephrine levels, or their respective decrease by 50 or 40% exclude pheochromocytoma (Eisen­ hofer et al., 2003c). Pheochromocytomas differ considerably in the rates of catecholamine synthesis, turnover and release, and in the types of catecholamines and metabolites produced. Adrenal pheochromocy­ tomas may produce near exclusively norepinephrine, or both norepinephrine and epinephrine, which will show up in urine as metanephrine and normetanephrine. In contrast, extra-adrenal pheochromocytomas almost invari­ ably produce norepinephrine and urinary norme­ tanephrine only (Brown et al., 1972; Eisenhofer et al., 1999b; Eisenhofer et al., 2008). Because of the considerable variation in catecholamine release among patients with pheochromocytoma, plasma concentrations or urinary excretion of catecholamines are poorly correlated with tumor size (Eisenhofer et al., 1995). In contrast, because of the metabolism of catecholamines within tumors and the independence of this pro­ cess on catecholamine release, urinary excretion or plasma concentrations of metanephrines show strong positive correlations with tumor size and can be useful in judging the extent and progression of disease (Eisenhofer et al., 1999a; Stenstrom and Waldenstrom, 1985). Malignant pheochromocytoma usually shows norepinephric profile, as well as high tissue, plasma and urinary levels of dopa and dopamine, the immediate precursors of norepinephrine (Goldstein et al., 1986; John et al., 1999; van der Harst et al., 2002).

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Anatomical imaging In most institutions, computed tomography (CT) of the abdomen, either with or without contrast, pro­ vides the initial method of localizing both adrenal and extra-adrenal pheochromocytoma because this imaging technique is easy, widely available and rela­ tively inexpensive. CT can be used to localize adre­ nal tumors 1 cm or larger and extra-adrenal tumors 2 cm or larger (sensitivity is about 95%, but specificity is only about 70%) (Maurea et al., 1996). Pheochro­ mocytomas will usually exhibit a density of more than 10 HU and an inhomogeneous appearance. Radiographic contrast seems to be safe in patients with pheochromocytoma and does not precipitate exaggerated release of catecholamines into circula­ tion (Baid et al., 2009). Magnetic resonance imaging (MRI) with or without gadolinium enhancement is a very reliable method and may identify more than 95% of tumors; it is superior to CT in detecting extra-adre­ nal tumors (Schmedtje et al., 1987). On MRI T1 sequences, pheochromocytoma has a signal like that of the liver, kidney and muscle, and can be differentiated with ease from adipose tissue. Che­ mical shift MRI characterizes adrenal masses based on the presence of fat in benign adenomas and the absence of fat in pheochromocytoma, metastases, haemorrhagic pseudocysts or malig­ nant tumors. The hypervascularity of pheochro­ mocytoma makes them appear characteristically bright, with a high signal on T2 sequence and no signal loss on opposed-phase images. More parti­ cularly, almost all pheochromocytomas have a more intense signal than that of the liver or muscle and often more intense than fat on T2-weighted images. However, such intense signals can be eli­ cited by haemorrhage or haematomas, adenomas and carcinomas, so an overlap with pheochromo­ cytoma must be considered and specific additional imaging is needed to confirm that the tumor is pheochromocytoma (Prager et al., 2002). Among the advantages of MRI imaging of pheochromocy­ toma are its high sensitivity in detecting adrenal disease (93–100%) and the lack of exposure to ionizing radiation (Honigschnabl et al., 2002). However, its overall sensitivity for detection of

extra-adrenal, metastatic or recurrent pheochro­ mocytoma is lower compared with that of adrenal disease (90%). Overall, the specificity of MRI is about 70% (Maurea et al., 1996). Anatomical ima­ ging should initially focus on the abdomen and pelvis (Pacak et al., 2007). Functional imaging Functional imaging of pheochromocytomas made a significant leap in recent years (Havekes, 2009). These tumors “are made” to be functionally imaged through utilization of specific transporters, which enable cellular accumulation of isotope and thus, imaging of primary or metastatic pheochromocy­ toma cells. One can further subdivide these into specific, which are based on catecholamine trans­ porters and vesicular monoamine transporters (VMATs), and non-specific, showing increased tissue metabolic activity or expressing somatostatin receptors. The first include 123I­ or 131I-MIBG scintigraphy, 18F-fluorodopamine, 18F-dihydroxy­ phenylalanine (18F-dopa), 11C-hydroxyephedrine and 11C-epinephrine positron emission tomography (PET), enabled by the presence of norepinephrine transporter system on in the cellular membrane and VMATs (Hoegerle et al., 2002; Pacak et al., 2001b; Pacak et al., 2001d; Pacak et al., 2002; Shapiro et al., 2001; Shulkin et al., 1999). Others include 18 F-fluorodeoxyglucose (FDG)-PET scanning (based on membrane expression of glucose transporters) or somatostatin receptor scintigraphy (Epelbaum et al., 1995). Although CT and MRI have excellent sensitivity, these anatomic imaging approaches lack the specificity required to unequi­ vocally identify a mass as a pheochromocytoma. The higher specificity of functional imaging – the test of choice is currently 123I-MIBG scintigraphy – offers an approach to overcome the limitations of anatomic imaging (Pacak et al., 2007). Metaiodobenzylguanidine scintigraphy Whole-body scanning using MIBG labelled with radioiodine (123I or 131I) is a historic cornerstone of functional imaging for pheochromocytoma.

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Other tumors arising from neuroendocrine cells – chemodectomas, non-secreting paragangliomas, carcinoids and medullary carcinomas of the thyroid – may also take up 123I- or 131I-MIBG (von Moll et al., 1987). MIBG labelled with 131I provides negative results in over 50% of patients with proven pheochromocytomas (Havekes, 2009). Much better sensitivity is, however, available with MIBG labelled with 123I (Nakatani et al., 2002; Shapiro et al., 2001). Another advantage of 123I over 131I­ labelled MIBG is its additional utility for imaging by SPECT. The agent also has a shorter half-life compared with 131I-MIBG (13 hours vs. 8.2 days), so that higher doses can be used (Shapiro et al., 2001). The sensitivity of 123I-MIBG scintigraphy is 92–98% for non-metastatic pheochromocytoma (van der Horst-Schrivers et al., 2006), but only 57–79% for metastases (Timmers et al., 2007b; van der Harst et al., 2001; van der Horst-Schrivers et al., 2006). The accumulation of MIBG can be decreased by several types of drugs: (1) agents that deplete catecholamine stores, such as sym­ pathomimetics, reserpine and labetalol; (2) agents that inhibit cell catecholamine transporters, includ­ ing cocaine and tricyclic anti-depressants and (3) other drugs such as calcium channel blockers and certain a- and b-adrenergic receptor blockers (Solanki et al., 1992). It is suggested that most of these drugs be withheld for about 2 weeks before undergoing MIBG scintigraphy. Both 123I-MIBG and 131I-MIBG require saturated solution of potas­ sium iodine (SSKI, 100 mg twice a day for 4 or 7 days, respectively) to be used to block thyroid gland accumulation of free 123/131I. The study is relatively expensive, and the patient must usually be scanned at 24 hours and again at either 48 or 72 hours after injection of the radioisotope to determine whether images that appear on the early scan are physiolo­ gical and will fade, or are tumors and will persist or increase in intensity on the later scan.

Positron emission tomography PET imaging is done within minutes or hours after injection of short-lived positron-emitting agents. Low radiation exposure and superior spatial reso­ lution are among the advantages of PET, whereas

cost and limited availability of the radiopharma­ ceuticals and PET equipment (including cyclo­ tron) still prohibit more widespread use. Most PET radiopharmaceuticals used for the detection of pheochromocytoma enter the pheo­ chromocytoma cell using the cell membrane norepinephrine transporter. Dopamine is a better substrate for the norepinephrine transporter than most other amines, including norepinephrine. 18 F-fluorodopamine is a positron-emitting analo­ gue of dopamine and was found to be a good substrate for both the plasma membrane and intracellular vesicular transporters in catechola­ mine-synthesizing cells (Goldstein et al., 1993). It is superior to 123/131I-MIBG in patients with metastatic pheochromocytoma. 11C-hydroxyephe­ drine and 11C-epinephrine are other PET imaging agents that have been shown to have a limited diagnostic yield because of their less than perfect sensitivity and/or specificity (Shulkin et al., 1992), while 18F-fluorodopa has good sensitivity in detecting metastatic disease and perhaps the high­ est yield to detect head and neck paragangliomas (Hoegerle et al., 2002; Timmers et al., 2007a). Increased glucose metabolism characterizes various malignant tumors, and thus the uptake of glucose labelled with 18F-fluoride is useful in the imaging of these tumors. Malignant pheo­ chromocytomas accumulate 18F-FDG more avidly compared to benign pheochromocytomas; nevertheless, FDG cannot distinguish malignant from benign disease. This radiopharmaceutical is non-specific for this tumor; however, it can be useful in those patients in whom other imaging modalities are negative, and in rapidly growing metastatic pheochromocytoma that is becoming undifferentiated, losing the property to accumu­ late more specific agents (Ilias and Pacak, 2004). Moreover, 18F-FDG PET is the preferred techni­ que for the localization of SDHB-associated metastatic pheochromocytoma – the so-called “flip-flop phenomenon” (Timmers et al., 2007b). Impairment of mitochondrial function due to loss of SDHB function may cause tumor cells to shift from oxidative phosphorylation to “aerobic” glycolysis, a phenomenon known as the “Warburg effect” (Warburg, 1956). Higher glucose requirement because of a switch to less

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efficient pathways for cellular energy production may explain the increased 18F-FDG uptake by malignant SDHB-related pheochromocytoma.

Octreoscan Somatostatin receptor scintigraphy using octreo­ tide has also been used in patients with pheochro­ mocytoma (Kaltsas et al., 2001); however, the sensitivity of this imaging modality is low, espe­ cially in the detection of solitary tumors, and this modality is inferior to MIBG scintigraphy (Kaltsas et al., 2001). However, in patients with metastatic pheochromocytoma, Octreoscan can be useful, especially in those tumors that express somatosta­ tin receptors and are negative on MIBG scintigra­ phy and 18F-fluorodopamine-PET (Ilias and Pacak, 2004). Octreoscan can also prove to be a sensitive imaging modality in head and neck paragangliomas but larger studies are needed.

Treatment of pheochromocytoma The best therapeutic modus operandi for a pheo­ chromocytoma, as with most of other neuroendo­ crine tumors, is finding an experienced operator (surgeon) (Manger and Gifford, 1977, 1996 ). In the perfect world, a team consisting of an internist, an anaesthesiologist and a surgeon will medically prepare the patient for a safe surgery, maintain blood pressure within normal values during the surgery and safely withdraw from therapy after the surgery. In the same perfect world, the job of the endocrinologist will be to diagnose pheochro­ mocytoma, localize it, design and execute efficient preoperative therapy for at least 2 weeks prior to surgery and follow up the patient after successful operation. In reality, as usually is the case, the situation is complicated by the fact that many patients have insidious clinical course and will have end organ damage or metastatic disease by the time of diagnosis. In those patients, medical therapy will be chronic, which requires apprecia­ tion of possible adverse effects and compliance. On the other hand, some of the tumors will be found during unrelated surgeries and precipitated

by manipulation around or at the unsuspected tumor. These tumors usually present with severe intra-operative pheochromocytoma crisis and are extremely hard to manage because of tremendous amount of catecholamines released to the circula­ tion (Mannelli, 2006; Pacak, 2007).

Medical therapy and preparation for surgery a-Adrenoceptor blockers Phenoxybenzamine (Dibenzyline; irreversible non-competitive a-adrenoceptor blocker) is most commonly used for preoperative management. The initial dose of long-acting phenoxybenzamine is usually 10 mg twice a day (usual total daily dose is 1 mg/kg); this is increased until the clinical manifestations are controlled, patient becomes normotensive or side effects appear (postural hypertension). Some patients may require much larger doses, and the dosage may be increased in increments of 10–20 mg every 2–3 days. Other a-blocking agents also of use are prazosin (Minipress), terazosin (Hytrin) and doxazosin (Cardura) (Nicholson et al., 1983). All three are specific, competitive and therefore short-acting a1-adrenergic antagonists, and all three have the potential for severe postural hypotension immedi­ ately after the first dose. Labetalol (Normodyne or Trandate), a drug with both a- and b-antago­ nistic activities, may also be used in a dosage of 200–600 mg twice daily (Van Stratum et al., 1983). The advantages of labetalol are that an a-blocker and a b-blocker are given simultaneously, and both oral and intravenous (IV) formulations of the drug are readily available. However, with labetalol, one is forced to use a fixed ratio of ato b-antagonistic activity (i.e. 1:4 or 1:6), while the desired ratio is about 4:1 or more, which means that an efficient anti-hypertensive dose will almost certainly associate with bradycardia. In some patients it may also cause hypertension (perhaps by its greater effect on b-adrenoceptors than a-adrenoceptors) (Briggs et al., 1978). Pre- or postoperative postural hypotension can be treated with normal or high-salt diet to restore volume depletion.

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Others In patients with clinical manifestations caused by b-adrenoceptor stimulation (e.g. tachycardia or arrhythmias, angina, or nervousness), b-adrenergic receptor blockers such as propranolol, atenolol or metoprolol are indicated. A b-blocking agent should never be used in the absence of an efficient a-blockade because of loss of b-adrenoceptor­ mediated vasodilatation, exacerbation of epinephr­ ine-induced vasoconstriction and a resultant serious and life-threatening elevation of blood pressure – an unopposed b-blockade (Eisenhofer et al., 2007). We had previously also suggested that in rare cases a phenomenon of unopposed a-blockade can occur (Kantorovich and Pacak, 2005). Metyrosine (a-methyl-L-tyrosine) competitively inhibits TH, the rate-limiting step in catechola­ mine biosynthesis and significantly but not com­ pletely depletes catecholamine stores, making pre- and in-surgical blood pressure control easier (Sjoerdsma et al., 1965). Treatment is started at a dosage of 250 mg orally every 6–8 hours, and thereafter the dose is increased by 250–500 mg every 2–3 days or as necessary up to a total dose of 1.5–4.0 g/day. The drug readily crosses the blood–brain barrier and often causes sedation, depression, anxiety, galactorrhea and rarely causes extrapyramidal signs (e.g. parkinsonism) in older patients. Various calcium channel block­ ers have also been used to control blood pressure both before and during surgery (Colson and Ribstein, 1991). While both a- and b-adrenocep­ tor blockers may elevate plasma-free normeta­ nephrine levels (Eisenhofer et al., 2003c), calcium channel blockers do not affect plasma metanephrine levels.

Hypertensive crisis Hypertensive crises can manifest as severe head­ ache, visual disturbances, acute myocardial infarc­ tion, congestive heart failure or cerebrovascular accident. It is a true medical emergency and should be treated as such in preferable setting of the intensive care unit that allows continuous monitoring of the patient’s haemodynamic

parameters. Phentolamine (Regitine) is one of the choice medications, used as repeated (every 2 minutes) intravenous 5 mg boluses or as a con­ tinuous infusion (100 mg of phentolamine in 500 ml of 5% dextrose in water). Alternatively, nifedipine (10 mg orally or sublingually) can also be used to control hypertension, while labetalol should be used with caution (see above). Some drugs (e.g. tricyclic anti-depressants, metoclopra­ mide and naloxone) can cause hypertensive crisis in patients with pheochromocytoma.

Surgery For most abdominal pheochromocytomas smaller than 6 cm, laparoscopy has replaced laparotomy as the procedure of choice because of significant postoperative benefits (Vargas et al., 1997; Win­ field et al., 1998). The operative mortality at the Mayo Clinic from 1980 to 1986 was 1.3%, or 1 in 77 patients (Sheps et al., 1990). The long-term survival of patients after successful removal of a benign pheochromocytoma is essentially the same as that of age-adjusted normals and with improve­ ment in current technique and multi-disciplinary approach, the procedure seems to be relatively safe (Stenstrom et al., 1988).

Postoperative management Acute withdrawal of hypercatecholaminaemia results in postoperative hypotension, which can be significant especially if both metyrosine and phenoxybenzamine were used preoperatively, because of catecholamine synthesis inhibition by first and long-term blockade by second. Volume replacement is the treatment of choice and the volume of fluid required is often large (0.5–1.5 times the patient’s total blood volume) during the first 24–48 hours after removal of the tumor and lower volumes thereafter (i.e. 125 ml/hour). Postoperative hypertension may be related to pain, volume overload, autonomic instability, essential hypertension or residual tumor. Repeat measurements should be made several weeks after surgery and later follow up yearly, for a total of

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5 years, if the patient remains asymptomatic or at any time if symptoms reappear. At least 25% of patients remain hypertensive, but this is usually easily controlled with medication (Amar et al., 2005b; Young and Landsberg, 1998).

Malignant pheochromocytoma Malignant pheochromocytoma is established by the presence of metastases at the sites where chro­ maffin cells are normally absent (Linnoila et al., 1990). Pheochromocytoma metastasizes via haematogenous or lymphatic pathways, and the most common metastatic sites are lymph nodes, bone, lung and liver (Bravo, 1994; Glodny et al., 2001; Goldstein et al., 1999; Kopf et al., 2001; Schlumberger et al., 1992). Among all pheochro­ mocytomas, the frequency of malignant pheochromocytomas ranges from 1 to 90% (Amar et al., 2005a; Benn et al., 2006; Brouwers et al., 2006; Glodny et al., 2001; Timmers et al., 2007c; Timmers et al., 2009). It is recognized that one half of malignant tumors present are found at the initial presentation, whereas the second half develop at a median interval of 5.6 years (Mornex et al., 1992). The prevalence of underlying SDHB mutations among patients with malignant pheochromocytoma is 30%, and even higher (48%) if the primary tumor originates from an extra-adrenal abdominal location (Brouwers et al., 2006). Two groups of patients can be distinguished based on the location of metastatic lesions. The first group represents short-term survivors with the presence of metastatic lesions, especially in liver and lungs. Their survival is usually less than 2 years. The second group repre­ sents long-term survivors with the presence of bone metastatic lesions. Patients in this group can survive more than 20 years after the initial diagnosis. The overall 5-year survival rate varies between 34 and 60% (John et al., 1999; Mundschenk and Lehnert, 1998). The survival of patients with metastatic disease due to an underlying SDHB mutation is lower in nonSDHB patients (Amar et al., 2007). We have recently found an unexpected high frequency of SDHB mutation in paediatric malignant

pheochromocytomas and paragangliomas (Pacak et al., unpublished observation). Recent advances in biochemical testing and nuclear imaging techni­ ques, as discussed previously, have greatly improved our ability to diagnose and localize malignant pheochromocytoma at much earlier stages. Clinical manifestations of malignant pheochro­ mocytoma are similar to that of its benign counter­ part, and patients may have minimal symptoms or symptoms caused by local invasion despite marked hypercatecholaminaemia (Bravo and Gifford, 1984; Glodny et al., 2001; Goldstein et al., 1999; Kopf et al., 2001; Mornex et al., 1992; Schlumber­ ger et al., 1992; Timmers et al., 2007c). Similar to benign pheochromocytomas, malignant pheochro­ mocytomas predominantly secrete norepinephrine (Eisenhofer et al., 2001a; Schlumberger et al., 1992; Stumvoll et al., 1997). However, larger tumor burden associates with higher levels of plasma and urinary metanephrines (Goldstein et al., 1999; Mundschenk and Lehnert, 1998). Immaturity and dedifferentiation of malignant tissue associate with increased dopamine excretion because of an intraneuronal loss of dopamine-b­ hydroxylase (Eisenhofer et al., 2001a; John et al., 1999; Mornex et al., 1992; Schlumberger et al., 1992), while normal epinephrine concentrations, together with that of excessive norepinephrine levels result from the tumor’s inability to N-methylate (Stumvoll et al., 1997). No clinical or pathological features reliably predict malignancy, although young age, extra-adrenal tumor location, large tumor size, adrenal pheochromocytomas that fail to take up MIBG and persistent postoperative arterial hypertension have all been associated with an increased likelihood of malignancy (Elder et al., 2003; Glodny et al., 2001; Goldstein et al., 1999; John et al., 1999; Mornex et al., 1992; Rao et al., 2000; Schlumberger et al., 1992; Stumvoll et al., 1997; Yon et al., 2003). Successful management of malignant pheo­ chromocytoma requires a multi-disciplinary approach (Scholz et al., 2007). The treatment regimen should be individualized to meet the goal of controlling endocrine activity, decreas­ ing tumor burden, and alleviating local symp­ toms. Pharmacological treatment of malignant

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pheochromocytoma does not differ from benign disease. However, for patients with multiple metastatic lesions, radical surgical resection is often impossible and at present it is not clear whether it would prolong patients’ survival. However, debulking often results in smaller tumor burden that may respond much better to radio- and chemotherapy, and a significant decrease in catecholamine levels, reflecting improvement of many symptoms and signs. The first-line systemic treatment for malignant pheochromocytoma is targeted radiotherapy using 131 I-MIBG. 131I-MIBG therapy is used in MIBGpositive tumors, especially those that are unresect­ able as single or multiple doses. The procedure is well tolerated, with minimal toxicity that includes nausea; mild bone marrow suppression, especially thrombocytopenia; mildly elevated liver enzymes; and some renal toxicity. Overall, about one third of patients show partial response (less than 50% reduction of tumor mass) and improvement in symptoms and signs (Loh et al., 1997). Radiother­ apy using radiolabelled somatostatin analogues such as [111In]-pentetreotide appears to be largely ineffective in metastatic pheochromocytoma (Lamarre-Cliche et al., 2002), while the use of needs [90Y-DOTA]-D-Phe1-Tyr3-octreotide further investigation (Forster et al., 2001). In rapidly progressive metastatic pheochromo­ cytoma, chemotherapy rather than MIBG therapy is recommended. A combination of cyclophos­ phamide, vincristine and dacarbazine showed a 57% complete or partial tumor response, while 79% had either a complete or partial biochemical response (Averbuch et al., 1988). Patients with large tumor burdens may present with mas­ sive release of catecholamines – “catecholamine storm” – within the first few hours after adminis­ tration of the first course of chemotherapy (Quezado et al., 1992). External beam radiation is used for palliation of chronic pain and symptoms of local compres­ sion arising from these tumors (Siddiqui et al., 1988). However, no systemic effects on tumor burden or hormone levels were observed. Suc­ cessful infarction of pheochromocytoma by embolization has been demonstrated in indivi­ dual case reports (Takahashi et al., 1999).

Recently, radiofrequency ablation, cryotherapy, percutaneous microwave coagulation, emboliza­ tion, as well as use of anti-angiogenesis medica­ tions have been used for the treatment of malignant pheochromocytoma (Pacak et al., 2001c).

Thoughts about future In general The future of pheochromocytoma-related science lies in part in its past, while understand­ ing of its pathology changes with better under­ standing of the physiology of the sympatho­ adrenal system. It is usually seen as the “emergency response” – fight or flight system, but on the other side paraganglia is an organic part of peripheral nervous system, while norepi­ nephrine is a major central and peripheral neu­ rotransmitter. From this point of view, it seems quite artificial to see pheochromocytoma as just an isolated endocrine pathology. Just by itself, adrenal medulla together with paraganglia repre­ sents a truly diffuse neuroendocrine organ. One can suggest that direct secretion of catechola­ mines into circulation by medulla as opposed to local uptake-based neuronal transmission of paraganglia, together with rapid metabolism, to assure that there will be no significant spillover, subdivides them into functionally different and somewhat functionally unrelated organs. Differ­ ence in enzymatic milieu with noradrenergic secretory pattern of paraganglia versus adrener­ gic/noradrenergic profile of medulla could further support this notion. Development of paragangliomas in chronic hypoxic conditions supports the thought that in addition to periph­ eral neurotransmission paraganglia still has a “side job” of oxygen sensing. This is especially true for parasympathetic paraganglia, the carotid body, in particular. These paraganglia were thought to predominantly secrete acetylcholine and not catecholamines, but we know now that they do contain catecholamine secretory gran­ ules and are capable of secretion, as well as paraganglioma formation.

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On genetic testing

On being nervous

The Shakespearian question on whenever “to do or not to do” the genetic testing for patients with pheochromocytomas and paragangliomas hangs as Damocles’ sword above the provider’s head, changing shape and size with every paper on frequency and malignant potential of these tumors. The condition is not as clear as with MEN2, where there is a well-documented corre­ lation between genotype and phenotype, as well as age-related morbidity. In VHL, 98% of pheo­ chromocytomas associate with missense rather than other type of gene mutation, while muta­ tions at codons 634 (2A) and 918 (2B) are mostly “pheochromocytomagenic” in MEN2. Numerous views and opinions have been pub­ lished lately, but there is still no clear-cut agree­ ment on this issue (Cascon et al., 2009; GimenezRoqueplo et al., 2006; Gimenez-Roqueplo et al., 2008; Pigny et al., 2009). Clinical observations and history as well as biochemical phenotype can help guide the necessity of genetic test to some extent. VHL tumors have noradrenergic phenotype, while pheochromocytomas of MEN2 usually have an adrenergic biochemical profile. Thus, we strongly suggest that appropri­ ate genetic testing be done based on clinical presentation, localization of pheochromocytoma, as well as biochemical phenotype. In patients with metastatic pheochromocytoma with primary extra-adrenal tumor in abdomen physicians should think about SDHB gene testing first, while head and neck paraganglioma will be more suggestive of SDHD or SDHC mutations (Amar et al., 2007; Benn et al., 2006; Brouwers et al., 2006; Neumann et al., 2004). Paediatric cases are far more concerning. Recent descrip­ tion of malignant paragangliomas presenting at relatively early age suggest the need for more aggressive approach (Havekes et al., 2008; Pro­ danov et al., 2009). Our recent unpublished observations of high rate of malignancy in pae­ diatric paraganglioma cases further support this notion. Unless larger studies come up with wellstructured guidelines, all paediatric patients without family history of these tumors should be tested for SDHB/D gene mutations.

Let’s go back to the anxious personality. Although acute stress will not result in sustained elevation of catecholamine levels and end-organ damage but severe or prolonged stress might do so. We had recently seen a patient with adrenal incidentaloma and prominent noradrenergic hypercatecholami­ naemia (Kantorovich et al., presented at the Endo­ crine Society Meeting, 2009). This patient was later found to have malignant thymoma-associated paraneoplastic Morvan’s syndrome, which associ­ ates, among other features, with overactivity of sympathetic nervous system. Another phenom­ enon worth attention is catecholamine-induced cardiomyopathy. Traditionally, described as hypertrophic or dilated, it lately expanded to mys­ terious “broken heart” and Japanese octopus traptype (tako-tsubo) cardiomyopathies. With no addi­ tional pathogenic causative mechanisms other than previously suggested coronary vasospasm or abnormalities in myocardial microvasculature as a result of high catecholamine levels, these clinical entities are widely reported in association with and without pheochromocytomas as classic apical or inverted forms (Bielecka-Dabrowa et al., 2010; de Souza et al., 2008; Kim et al., 2007, Madhavan et al., 2009; Sanchez-Recalde et al., 2006). Of great interest in this context is a recent paper showing that pheochromocytoma-conditioned growth media causes significantly more cardio­ myocyte damage then norepinephrine only, sug­ gesting existence of additional factors, secreted by the tumor (Mobine et al., 2009). Our feeling is that in the condition of any severe hypercatecholami­ naemia – stress- or pheochromocytoma-related, there are several factors that will predict the exten­ sion of cardiac damage – the rapidity and level of catecholamine rise, the known ability of cardio­ myocyte adrenergic receptor desensitization and the individual distribution and amount of epicardial sympathetic innervation and endo-/ myocardial adrenergic receptors density. This feel­ ing is strongly supported by our recent observation of lack of cardiac abnormalities seen on high-reso­ lution cardiac MRI in series of patients with longstanding pheochromocytomas (Pacak et al., unpublished observations).

364

On tumorigenesis Familial pheochromocytoma–paraganglioma syn­ dromes widened our understanding of tumorigen­ esis significantly, although not without some additional confusion. Currently, two major and seemingly independent, but probably somewhat cross-talking pathways result in pheochromocy­ toma formation. The first takes place in the neural crest-derived neuroendocrine cells (medullary thyroid cells, parathyroid chief cells, adrenal chro­ maffin cells, etc.) and results in multiple tumors, related to this embryological system. It is repre­ sented by RET and NF1 gene mutations and displays abnormalities in cell cycle and differentia­ tion, still containing numerous spots of “terra incognita”. The second, functional rather than embryonic, shows an avalanche of newly discov­ ered data and relates to hypoxia-driven pathway of tumorigenesis. It started with VHL syndrome, but expanded over recent years into a solid system that significantly extended our understanding of cellular metabolism. Pseudohypoxic conditions of VHL-related mutation in HIF (hypoxia-inducible factor) support both the oxygen sensing function of sympatho-adrenal system and tumorigenic cap­ abilities of “hypoxia”. But it not just a scavenging VHL protein, which when mutated, precludes ubi­ quitination and further degradation of HIF-1a, which dimerizes with HIF-1b to become a potent hypoxia-driven transcription factor, resulting in pheochromocytoma or paraganglioma. It is even not just HIF-1a anymore. It can be any of oxygensensing a units – HIF-1a or HIF-2a – and the whole pathway seems to contain a growing num­ ber of spots, susceptible to mutations and asso­ ciated with similar functional and pathogenic outcome, as well as syndromatic presentation (Ladroue et al., 2008). Addition of SDH system to this pathway is both logical and confusing. Mutation of mitochondrial protein involved in electron transfer would clearly associate with abnormal oxygen sensing and would be expected to induce hypoxic drive, but SDH mutations are also associated with accumulation of succinate through the system’s involvement in Krebs cycle, which generates independent metabolic stress, capable of HIF induction. Another SDH-related

confusion is caused by heterogenicity of clinical pictures between different subunits mutations. Of these, SDHB stands alone as the most malig­ nant of all pheochromocytoma–paraganglioma­ related conditions. Malignant disease is found in a relatively young age. This brings us back to embryological phenomenon of paraganglia being the leading part of the system at antepartum with relative involution in postpartum period. It is not clear if this malignant phenotype is part of rever­ sal to embryological phenotype or just a dediffer­ entiation. The last would be more reasonable if one adds the fact that SDHB tumors are more glucose-avid on PET images compared to other pheochromocytomas/paragangliomas. One would also suspect that “Warburg effect” in these tumors is a clear advantageous phenomenon of aggressive malignancy rather than a compensatory manoeuvre of the diseased mitochondria. While one would expect SDH-complex abnormalities to generate potentially more intracellular hypoxia and probably induce higher Warburg effect, recent studies showed that VHL but not SDH-related mutations drive Warburg effect in inherited pheochromocytomas (Favier et al., 2009; Kaelin, 2009). Interesting are findings of association of pheochromocytoma/paraganglioma with abnormalities in p53, which has been previously reported to play a significant role in the genera­ tion of the “Warburg effect” (Ma et al., 2007).

On being a part of DNES There is a definite lack of clear characterization of the last being a part of the DNES. With the unfortunate departure of the amine precursor uptake and dercar­ boxylation (APUD) system (Andrew et al., 1998), there seems to be no effort to generate a replacement that would be accepted by the field. Neuroendocrine cell can take up any amine precursor and with appropriate the enzymatic machinery it can produce any neuroendocrine product – amine, peptide, etc. It is well known that carcinoids can produce catecho­ lamines. While this fact does not make carcinoid a pheochromocytoma, the functional usefulness of the APUD classification is hard not to miss. They both possess similar uptake, synthetic (amines,

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peptides, chromogranin) abilities, secretory granules, somatostatin receptors and catecholamine uptake transporters. They definitely differ embryologically with pheochromocytomas and paragangliomas being derivatives of the neural crest (ectoderm), while car­ cinoids coming from endoderm. Using the term of DNES with or without stressing their functional simi­ larities rather than embryonic differences may ease the pain of artificial division of the field, while it can definitely gain if intellectual forces would join together in the search for better imaging and treat­ ment, which does have some similarities in both cases.

In summary There are more things to expect from the field in the future. Our understanding of the basic mechanisms of cell function with regard to differ­ ential expression of catecholamine transporters on the cell surface, as well as different VMATs in the membrane of the vesicle will allow development of new imaging and treatment modalities in the future, and also the wide use of the latest molecu­ lar techniques – as in the case of proteomics approach in the discovery of SDH5 mutation. Bet­ ter understanding of molecular pathology of SDH mutations will widen our understanding of patho­ genesis and better screening and treatment of the disease.

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Subject Index

acetyl-coenzyme A carboxylase (ACC), 194

acromegaly, 197–198, 216, 229–230, 233, 236, 260,

265, 283, 290, 297, 338

activating function (AF)-1 transactivation

domain of GR, 3

activation-deficient GR mutant possessing, 8

acute lymphoblastic leukemia (ALL), 8

glucocorticoid therapy for

prednisone with vincristin, 13

adaptation syndrome, 149

adipocyte differentiation, p38 MARK role in, 180

adipocytokines, 168

See also metabolic syndrome (MS) in later life

adrenal and intracrine steroidogenic pathways, 110

adrenalectomy (ADX), 38

adreno corticototropic hormone (ACTH), 1, 150

Adverse Event Reporting System (AERS)

database, 132

aging

age-related sarcopenia, 121

and changes in brain volume, 35

cognitive decline and, 45

hippocampal plasticity

and cognitive ability, 47

and menopause, 48

related deficits, 49

spatial learning and memory, 48–49

AhR–dioxin interaction, 239

AhR–Hsp90–AIP–p23 complex, 241

AKT-mediated phospho-inhibition of

Fox03a/ FKHRL

Bim transcription, 19

AKT signaling with perifosine and glucocorticoid

induced apoptosis, 20

allostatic load, 45

See also chronic stress

Alzheimer’s disease

and aging, 33

behavioral symptoms, 79

Cache County cohort study, 86

cerebral atrophy, 79

cognitive decline in, 78

early menopause and, 82

risk study, 83

endogenous estrogens and risk, 82

episodic memory impairment, 82

estrogen containing hormone therapies, 51

factors contributing to net estrogen

effects on, 90

FDG–PET study, 79

hormone therapy in women with

randomized, double-blind, placebo-

controlled trials of, 84

and symptoms, 83

HPA axis dysregulation, 34

implications for, 82

metabolic derangements in, 79–81

neurofibrillary tangle formation during, 82

neuronal damage and DHEA administration, 126

neuronal loss, 34

oxidative stress, early event in, 81

pathological features, 79

sex steroid effects, 90

WHIMS findings, 89

women with Down’s syndrome, risk in, 82

a-melanocyte-stimulating hormone (a-MSH), 192

AMP-activated protein kinase (AMPK), 191,

193–194

b-amyloid

concentrations and treatment with estradiol, 81

formation and mitochondrial dysfunction, 81

oligomers, 79

androgen receptor (AR)

AR–leptin interactions in hypothalamus, 177

AR-transfected COS-7 cells

S42 treatment, 185

whole-cell binding studies, 183–184

co-expression of OBRB and, 178

375

376

androgen receptor (AR) (Continued)

endogenous AR expression in hypothalamus, 177

gene variations, 176

intracrine action mechanism, 179

structure and regulation, 182

activation function-1 (AF-1), 183

activation function-2 (AF-2), 183

conformational changes, 183

as target for drug development for MS, 182–186 androgens

action sites in women, 117

activity and brain, 176–178

androgen deprivation therapy (ADT), 180

androgen insufficiency syndrome, 182

and AR, 176

post-menopausal women synthesis, 111

androsterone glucuronide (ADT-G) serum levels, 116

Angelmann syndromes, 162–163

anthracycline, topoisomerase II inhibitor for

ALL, 13

anti-apoptotic Bcl-2 protein

aberrant expression, 18

apolipoprotein E, 156

apoptotic peptidase-activating factor 1 (Apaf-1), 10

arachidonic acid release, 7

arcuate (ARC) and PMV nuclei, 176

aromatase enzymes, transformation DHEA into

estrogens, 120

aryl hydrocarbon receptor interacting protein

(AIP), 218, 238

and AhR interaction, 238–242

AIP–G12 interaction, 244

binding to Hsp90–GR complex, 243

b thyroid hormone receptor 1 (TRb1) binding

to, 243

interaction with Hsc70, 242

interaction with Tom20, 243

mutations in, 233–236, 245

and PDEs interaction, 242

AIP–PDE2A interaction, 242

PDE4A5–AIP interaction, 242

and PPARa interaction, 243

RET–AIP interaction, 243

structure of, 240

and survivin interaction, 242

tumor suppressor role of, 244–245

Knudson two-hit hypothesis, 244

and viral proteins, 244 aryl hydrocarbon receptor nuclear translocator (ARNT), 238–239

asparaginase for ALL, 13

assisted reproduction technologies (ARTs),

161

blood pressure (BP) levels in children, 166

children born after

long-term health issues of, 162

neuroendocrine findings in, 164

DHEA-S elevation, 169

epigenetic changes, 163

genomic imprinting disorders, risk of, 163

infants born by, 162

neuroendocrine impact on offspring

hypothalamic–pituitary–thyroid axis of, 169–170

on postnatal growth, 163

on pubertal timing of, 168–169

offspring

and cardiometabolic risk, 165–168

imprinting disorders of, 162–163

overall health of, 170–171

postnatal growth, 165

women health during, 162

atherosclerosis as model for critical window

hypothesis, 85–86

auto-induction of GR transcription, 16

Baltimore Longitudinal Study of Aging, 83

Barker’s fetal origin of adult disease

hypothesis, 162

b-catenin, 210, 216, 221, 299–300

B-cell lymphoma (Bcl) anti-apoptotic proteins

Bcl-2 and Bcl-XL, 81

Beckwith–Wiedemann syndromes, 162–163

bicalutamide monotherapy, 325–327

body mass index standard deviation score

(BMI SDS), 165

bone mineral density (BMD) and DHEA, 120

brain and androgen activity, 176–178

breast atrophy and DHEA, 124

CA3 dendritic retraction, hippocampus role on, 39–43

calcium mobilization and glucocorticoids, 10

calmodulin

calmodulin kinase kinase 2 (CaMKK2), 193

377

and DNA fragmentation, 9–10

See also ghrelin

cAMP-driven PKA pathway

and glucocorticoid-induced apoptosis, 19

with forskolin, 20

cAMP phosphodiesterases (PDEs),

glucocorticoid-resistant CEM cells in, 20

cannabinoid receptor gene (CNR1), 156

carcinoids, 364–365

cardiometabolic risk of offspring, 165–168

cardiovascular disease (CVD) and DHEA, 122

Carney complex (CNC), 217, 230, 233

caspases

caspase-3 activation in lymphocytes, 10

caspase-9 deficiency and glucocorticoid-induced

apoptosis, 11

morphological and biochemical changes during

apoptosis, 10

catechol-O-methyltransferase (COMT), 348

CEM leukemic cell line

p38 protein and glucocorticoid-induced

apoptosis, 19

CEM T-ALL cell line, GR LBD (L753F)

mutation in, 17

Center for Food Safety and Applied Nutrition

(CFSAN) post-marketing database, 132

central nervous system (CNS)

androgen–AR system, 176

ceramide biosynthesis, apoptosis in

glucocorticoid-treated thymocytes, 10

choline acetyltransferase (ChAT) levels and

OVX, 54

chromogranins, 347

chronic lymphoblastic leukemia (CLL), 8

glucocorticoid therapy for, 13

high-dose methylprednisolone (HDMP),

clinical trials of, 14

rituximab, 14

chronic stress

brain aging and, 45

elevated glucocorticoids influencing spatial

ability, model for, 44

on hippocampal CA3 dendritic retraction, 40

model for, 43–45

hippocampal dependent spatial recognition

memory on Y-maze, 40

HPA axis on age-related cognitive decline,

influence of, 45–47

learning on spatial T-maze, 41

rats with, spatial recognition memory assessment

metyrapone injection effect on, 42

spatial learning and memory in young adults,

38–39

circadian salivary cortisol rhythm, 153

clonidine, 356

suppression test, 349

c-myc in human leukemic CEM cells, 9

cognitive performance representation, 37

combined androgen blockade (CAB), 330–332

conjugated equine estrogens (CEEs)

combination therapy with synthetic progestin and MPA, 53

containing therapy, 53

estrogenic effects of, 53

corticosteroid-binding globulin (CBG), 1

corticotropin-releasing hormone (CRH), 150

secretion, 1

cortisol serum level and aging, 127

cortistatin (CST), 258

critical window hypothesis

atherosclerosis as model, 85–86

and cognitive outcomes, 86

Cushing’s disease (CD), 3, 198, 207, 260, 267–268,

270, 287, 297

cvannabinoids, 195

cyclin-dependent kinase inhibitors (CDKIs), 298

cyclophosphamide, vincristine, doxorubicin and

prednisone (CHOP), 15

cyproterone acetate, 331

dehydroepiandrosterone (DHEA), 97, 324

Abbreviated Sex Function (ASF)

questionnaires, 119

aging problems, 99–100

anti-carcinogenic activity of, 128–129

anti-metabolic activity, 178–180

biological role in, 179

and bone

bone mineral density (BMD), 120

muscle and lean body mass, 121–122

and brain, 125–127

and breast, 124

CVD and, 124–125

DDSP with LC-PTP structure, comparison of, 179

Dietary Supplement and Health Education Act

for, 114

378

dehydroepiandrosterone (DHEA) (Continued)

endometrium, effect on, 119

formulations quality, 114–115

human T-lymphocytes and, 179

increased longevity/decreased mortality, 129–131

and insulin sensitivity, 122–123

intravaginal application, 118

change in serum estrogens/androgens, 119

sexual dysfunction and, 119

and lipids, 123

obesity and, 123–124

problem with, 98–99

residual intraprostatic, values of, 98

safety profile of, 131–132

secretion change with age, 108

self-administration and, 99–100

in serum variability of, 112

sex steroids from, 98

enzymatic mechanisms, 112–113 physiological and precursor, 132

sexual function, 127

side effects, 131

skin and, 128

studies in

men with, 106–107 women with, 101–105

tissue-specific effects of, 114

treatment of anaemia, 132

and vaginal atrophy, 116

women and men, treated with, 115–116

dehydroepiandrosterone sulphate (DHEA-S), 175

and atherosclerosis, 179

BMD and post-menopausal women, 179

dementia, 78

midlife women without, 87–88

older women without, 88–89

dendritic retraction

within CA3 region of hippocampus

antidepressant treatment for, 36

depression

and aging, 33

brain structure changes and, 35

des-acyl ghrelin, 191

dexamethasone (Dex), 3

dexamethasone-induced gene 2 (Dig2)

in murine lymphoma cell lines, 9

suppression test, 151

dexamethasone, etoposide, ifosfamide and

cisplatin (DVIP), 15

Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR), 33

diastolic blood pressure (DBP), 161

Dietary Supplement and Health Education Act

for DHEA, 114

diffuse neuroendocrine system (DNES), 344–345,

364–365

dihydroxyphenylglycol (DHPG), 348

dimethylbenzanthracene (DMBA)-induced

mammary carcinoma, 129

dopamine, 346

dopamine b-hydroxylase (DBH), 346

doxorubicin, bleomycin, vincristine,

cyclophosphamide, procarbazine, etoposide and prednisone (BEACOPP) regimens, 14–15 dystrophic nerve processes, 79

early menopause and cognitive risk, 82–83

Early Versus Late Intervention Trial with

Estradiol (ELITE)

estradiol and estrogens treatment, clinical trials

for, 87

trials, 77

E-caherin expression, in pituitary tumors, 216

electron transport chain and of ATP

synthetase, 80

endogenous glucocorticoids

emotion and nociception, 2

energy

demands of healthy brain, 79

generation in mitochondria, 79

Epidemiologic evidence of DHEA in the etiology of neoplasia, 132

epigenetic alterations, 156–157

epinephrine, 346

Epstein–Barr virus (EBV)-encoded nuclear

antigen-3 (EBNA-3), 244

erasure process, 163

estradiol, 79

amyloid precursor protein, up trafficking of, 81

Bcl-2 and Bcl-XL expression, 81

changes in serum levels, 118

delayed-match-to-position spatial T-maze,

effects on, 56

genomic and non-genomic effects, 85–86

379

induced spatial working memory, 55

injections, reference memory performance, 36

spinal fluid concentrations of, 81

sub-clinical atherosclerosis and, 86

for test cognitive effects of hormone therapy in

animal model, 53

treatment study, 55

estrogens

actions on basal forebrain cholinergic neurons, 82

activational effects of, 53

age-related changes in responsiveness to

treatment with, 55

antiinflammatory actions, 81

anti-oxidant effects, 81

brain, 79

containing hormone therapies, 53

and brain bioenergetics data studies, 81

effects on cognition, 56

estrogenemia, 85

estrogen–progestin trial of women, 83

estrogen replacement therapy (ERT), 112

healthy cell bias hypothesis of action, 85

and hippocampal morphology, 36

neurotransmitter systems and, 81–82

nuclear and mitochondrial genome, 80

and progestin replacement therapy, 112

receptors types

estrogen receptor alpha (ERa), 78

estrogen receptor beta (ERb), 78

regional blood flow, 80

euthyroid hyperthyrotropinaemia (EH), 169

extracellular signal related kinase (ERK) signaling

and TCR signaling, 11–12 familial isolated pituitary adenomas (FIPA), 218–219, 233

AIP mutations, 233–236

clinical features of, 246–247

history of, 233, 236–238

fasting glucose to insulin ratio (FGIR), 167

fatty acid synthase (FAS), 192

fetal thymic organ culture (FTOC)

in vitro evidence for mutual antagonism model, 12

flip-flop phenomenon, 358

follicle stimulating hormone (FSH)

and cognition, 58

Fox03a/FKHRL1 transcription factor, 8

g-aminobutyric acid (GABA) type A receptor, 126

inhibitory role in CNS, 155

gastroenterohepatic-neuroendocrine tumors

(GEP-NETs), 263, 268

gastro-entero-pancreatic (GEP) tract, ghrelin

functions in, 198–199

genomic imprinting, 163

gestational diabetes mellitus (GDM), 168

ghrelin, 189

anorexia nervosa and, 196

and appetite regulation, 191–192

UCP2 as downstream effector of ghrelin–AMPK–CPT1 pathway, 194–195 and cannabinoid-signalling pathway, 195

and determination of stature, 196

discovery of, 189–190

effects on pituitary function, 196–197

expression in GEP tumors, 198–199

functions of, 190

and GHS-R, 191

on hypothalamic AMPK activity by

CaMKK2, 194

obesity and, 195–196

orexigenic effect, and hypothalamic

intracellular signalling pathway, 192–193 AMPK, role of, 193–194

and pituitary adenomas, 197–198

structure and expression of

des-acyl ghrelin, 191

expression, 190

fatty acid modification, 191

ghrelin gene, 190

ghrelin–GOAT system, 191

obestatin, 191

proghrelin, 191

synthesis, 190

type 2 diabetes and, 196

ghrelin O-acyltransferase (GOAT), 191

glial cell line-derived neurotrophic factor

(GDNF), 298

glucocorticoid-induced leucine zipper (GILZ)

role in glucocorticoid-induced apoptosis, 9

glucocorticoid receptor (GR), 2

domain structure of, 4

expression by promoters, 6

GRa receptor, 4

GRb expression, 4

380

glucocorticoid receptor (GR) (Continued)

in T-ALL CEM cells, 16

homodimer and gene repression, 6

as multiprotein heterocomplex, 6

mutations in, 17–18

nuclear transcription

factors and, 6

and homodimerization, 7

protein isoforms, 3

signaling

cytoplasmic effects, 6–8

genomic effects, 6

translational isoforms, 5

glucocorticoids

calcium mobilization and, 10

cell cycle arrest, 2

chemotherapy

limitations of, 15

use in, 3

cognition, 47

CRH and ACTH synthesis, 1

Fox03a/FKHRL1 transcription factor, up

regulated by, 8

glucocorticoid action regulating genes

(FKBP5), 156

‘Glucocorticoid Cascade Hypothesis’, 46

glucocorticoid receptor (GR), 2

glucocorticoid response elements (GREs), 3

‘Glucocorticoid Vulnerability Hypothesis,’ 46

gluconogenesis and lipolysis, 2

induced alterations in GR expression, 16–17

induced apoptosis, 2

cytoplasmic signaling, 9–10

execution of, 10–11

glucocorticoids

genes differentially regulated during, 8

genomic signaling, 8–9

of haematological malignancies, 13

of healthy lymphocytes, 11–13

in osteosarcoma cells, 16

signaling cascade, 12

induced apoptosis of lymphoid cells and GR

expression, 3–6

structure, 3

induced CA3 dendritic retraction, 43

inhibition of inflammation, 2

intracellular ceramide concentrations, 10

kinome, interactions with, 19

mitochondrial production

ceramide and hydrogen peroxide, 8

lysosomal release of cathepsin B, 8

of reactive oxygen species, 8

physiological effects, 2

potassium efflux in thymocytes and CEM

T-ALL cells, 10

regulated gene expression, 7

resistance in haematomalignancies

altered expression of GR isoforms, 15–16

side effects, 3

targeting Bcl-2 family members, 20

TCR signaling and, 8

therapy of

acute lymphoblastic leukemia, 13

chronic lymphoblastic leukemia, 13–14

Hodgkin’s and non-Hodgkin’s lymphoma,

14–15

multiple myeloma, 14

toxicity, 43

treatment in murine lymphoma cell lines, 8

vulnerability hypothesis, 43

glucose tolerance test, 122

glutamate excitotoxicity, 81

glycolysis defined, 80

glycolytic hexokinase II enzyme, 9

gonadal hormones, spatial learning and memory,

49, 51

gonadectomy (GDX), 51

gonadotropinoma, 207–208, 222

gonadotropin-releasing hormone (GnRH)

agonists, 98

gonadotropins, 58–59

gossypol (AT-101) and dexamethasone-induced

cell death, 20

G-protein-coupled receptors, 78

granzyme A, glucocorticoid treatment in B-ALL

cells, 9

growth hormone (GH), 189, 297

growth hormone (GH)omas, 207

See also acromegaly; ghrelin

growth hormone secretagogue (GHS)

GHS receptor (GHS-R), 189, 191

haematological malignancies

chemotherapy phases, 13

and glucocorticoid-induced apoptosis

synthetic glucocorticoids for treatment, 13

381

haematomalignant cell lines

cell-type-specific expression of GR promoter

transcripts, 16

healthy cell bias hypothesis of action of estrogens,

85

hematuria, 131–132

hexokinase glycolytic enzymes, 80

high-demand time-delayed memory retention

tests, 52

high-density lipoprotein (HDL) and DHEA

administration, 123

high-dose methylprednisolone (HDMP), clinical

trials for CLL, 14

high sensitivity C-reactive protein (hsCRP), 167

Hirschsprung’s disease, 299

histone acetyltransferase (HAT) activity, 183

Hodgkin’s disease

glucocorticoid therapy for, 14–15 hormone therapy

and episodic memory, 87

follow-up analysis of participants in studies, 86

for osteoporosis, 86

and risk of Alzheimer’s disease, 83

human glucocorticoid receptor b (hGRb) expression, 4

human GOAT, 191

human estrogen receptor (hER), 335

hunger hormone. See ghrelin

hypertension, 3

hyperthyrotropinaemia, 170

hypertriglyceridaemia, 184

hypothalamic–pituitary–adrenal (HPA) axis, 33

and AD onset, 34

aging and

calcium conductance, 46

perforated synapses, 46

spine density, 46

synapse number, 46

hippocampal dependent learning and memory, influence on spatial learning and memory, 37–38

hippocampal synapse number and aging, 35

stress

and age-related cognitive decline, 45–47

and gonadal steroid influence, 36

hypothalamic–pituitary–gonadal (HPG) axis

and aging influence on hippocampal dependent

functions

clinical implications, 49–51

reproductive senescence, 48

spatial learning, 48–49

spatial memory, 47–48

spatial learning and memory

estrogens and progesterone, 51–56

gonadotrophins, 58–59

testosterone, 56–58

hypothalamus, role of ARC neurons in, 192

immunophilin chaperone proteins, 6

imprinting disorders of offspring, 162–163

See also assisted reproduction technologies

(ARTs)

insulin

insulin growth factor binding protein (IGFBP3),

164–165

insulin growth factor-1 expression, 80

insulin receptor substrates (IRSs), 185

resistance

and hyperinsulinemia, 184

and obesity, 168

sensitivity index and DHEA administration, 122

intracrinology, 97, 324

androgens from DHEA, 109–110

changes in DHEA secretion with age, 108

intracrine system, 108–109

intracytoplasmic sperm injection (ICSI) procedure, 161

intrauterine growth restriction (IUGR), 162

intravaginal estrogen formulations, 116–117

intrinsic mitochondrial apoptosis pathway, 9

in vitro fertilization (IVF), 161–162

JNK-mediated phosphoinhibition of Bim, 19–20 kinase suppressor of Ras 2 (KSR2), 193

Knudson two-hit hypothesis, of cancerogenesis,

244, 297, 353

Krebs tricarboxylic acid cycle, 80

mitochondrial enzymes within, 81

Kronos Early Estrogen Prevention Study

(KEEPS), 77

estradiol and estrogens treatment, clinical trials

for, 87

labetalol, 359–360

land radial-arm maze, spatial learning effect on, 39

382

lanreotide autogel (ATG), 260, 281

See also somatostatin analogues (SSAs)

lanreotide-sustained-release (SR), 260

See also somatostatin analogues (SSAs)

laser-capture micro-dissection of neurons, 80

leptin signalling

and androgen receptor in hypothalamus, 176–178

leptin receptor (OBRB), 176

STAT3 activation, 176

leucopenia, 3

leukocyte protein tyrosine phosphatase

(LC-PTP), 179

Leu72Met polymorphism in ghrelin, and obesity,

196

ligand-binding domain (LBD) of GR, 3

lipocortin-1 activation, 7

lipogenic gene expression in liver and adipocytes,

184–185

locus caeruleus/norepinephrine–sympathetic

nervous system (LC/NE–SNS), 150

low-density lipoprotein (LDL) and DHEA

administration, 123

low working memory load block trials, 50–51

lutenizing hormone (LH)

brain and spatial cognition, effects on, 58

lymphocytes

GRg protein expression in, 4

specific protein tyrosine kinases Lck and

Fyn, 8

magnocellular cholinergic neurons, 81–82

major depressive disorder (MDD), 152

major histocompatibility complex (MHC)

encoded molecules, 11

mammalian target of rapamycin (mTOR)

anti-apoptotic Mcl-1 protein expression and, 20

JNK signaling and, 19

signaling pathway, 19

maternal amnesia phenomenon, 56

McCune–Albright Syndrome (MCS), 219, 233

Mcl-1-specific inhibitor obatoclax and

glucocorticoid-induced apoptosis, 20

mechlorethamine, vincristine, procarbazine and

prednisone (MOPP)

combination chemotherapy, 14

medial prefrontal cortex (mPFC), 155

medroxyprogesterone acetate (MPA), 53

impaired spatial memory retention and, 56

membrane-bound GR (mGR), 8 Menin, role in tumorigenesis, 297–298 menopause on brain activities, 79

cognition and, 48, 51

DHEA and

circulating levels of, 111

source of sex steroids at, 111

early menopause, 82

and episodic memory, 86–87

estradiol and estrone, 79

forgetfulness and, 87

hormone deficiency and, 97

hormone-related menopausal symptoms, 116

hormone therapy, 85

and intracrine mechanisms, 108–109

Menopause-Specific Quality of Life

(MENQOL) and ASF questionnaires, 119

model from, 52

risk of Alzheimer’s disease and, 82

surgical menopause, 52–53

SWAN study, 87

systemic estrogens after, 111–112

VCD-induced transitional, 52

See also intracrinology

metabolic syndrome (MS) in later life, 162

adipocytokines, 168

AR as target for drug development for,

182–186

BP and triglyceride concentrations, 167

cardiovascular risk factors, 167

chronic inflammation markers, 167

criteria for, 167

health-care professionals for, 168

insulin resistance and, 168

occurrence of, 168

PCOS and, 168

roles of T and estradiol in men, 180–182

3-methoxy-4-hydroxyphenylglycol (MHPG), 348

metyrosine, 360

mitochondria

calcium sequestration within, 81

and dysfunction, 81

electron transport and remediation of oxidative

stress, 80

energy, generation in, 79

ERb in, 80

383

membrane disruption by proapoptotic Bcl-2

family members, 10

mitochondrial reactive oxygen species and

ceramide production, 10

reactions in cell cytoplasm and, 80

mitogen-activated protein kinase (MAPK), 179

ERK and JNK, glucocorticoid-induced

apoptosis inhibition, 19

pathway, 198

mixed lineage leukemia (MLL), 298

moderate working memory load block trials,

50–51

Morris water maze, spatial learning effect on, 39

Multi-Institutional Research in Alzheimer Genetic

Epidemiology (MIRAGE) study, 86

multiple endocrine neoplasia (MEN) syndromes,

295–296

MEN4, 303

CDKN1B gene and encoded p27 protein, 305

CDKN1B germline mutations in, 303,

308–309

functions of p27, 306–307

mutation in p27, 303–305

p27 and cyclin/CDK complexes activities, 305

pathways involved in p27 degradation, 306

pro-oncogenic role for p27, 306

MEN type 1 (MEN1), 296

clinical manifestation of, 296

loss of function of Menin, 297

Menin–MLL complex with HMTase activity, 298

mutation in MEN1, 297

tumor spectrum of, 296–297

MEN type 2 (MEN2), 351

activation of b-catenin signalling, 299–300

effects of RET activation in MTC cell lines, 299

familial medullary thyroid cancer (FMTC), 298

medullary thyroid carcinoma (MTC),

presentation in, 298

MEN2 type A (MEN2A), 298

MEN2 type B (MEN2B), 298

mutations in RET proto-oncogene, 298–299

proliferation of neuroendocrine cells, 300

RET protein domains, 298–299

MENX multi-tumor syndrome, 300

Cdkn1b gene, mutation in, 300–302

discovery of, 300

life span and phenotype of affected rats, 300

p27 expression, assessment of, 301–302

multiple endocrine neoplasia type 1 (MEN1)

syndrome, 199, 214, 230

See also multiple endocrine neoplasia (MEN)

syndromes

multiple myeloma (MM)

glucocorticoid therapy for

thalidomide/dexamethasone (THal/Dex), 14

murine GOAT, 191

mutual antagonism, 11

myalgia

and DHEA treatment, 132

N-cadherin, 210

neuroendocrine tumors (NETs), 256–257, 282, 287

developments in treatment of, 257

management of patients with, 257

MENX model and, 314–316

role of ssts in treatment of, 265

ACTH-secreting adenomas, 267–268

bronchial carcinoid tumors, 270

GEP-NET, 268–270

GH-secreting pituitary tumors, 265–267

metastatic MTCs, 270

TSH-secreting pituitary adenomas, 268

SS-analogues use in treatment of, 256,

287–288

sst expression in, 257

neurogenesis in dentate gyrus and spatial

memory, 46

neuronal loss with aging, 35

neurosteroid, 79

N-methyl-D-aspartate (NMDA) receptors, 126

non-functioning adenomas (NFAs), 268

Non-Hodgkin’s disease

glucocorticoid therapy for, 15

norepinephrine, 346

NR3C1 gene of GR, 3

N-terminal transactivation domain (NTD) of GR, 3

nuclear export signal (NES), 3

nuclear receptor, 182

Nurses’ Health Study of bilateral oophorectomy, 131

obesity and DHEA, 123–124

obestatin, 191

octreotide long-acting-release (LAR), 260, 282

See also somatostatin analogues (SSAs)

octreotide (SMS201-955) acetate, 259

oophorectomy and menopause, 82

384

osteoporosis, 3

ovariectomy (OVX), 51

b-amyloid in brains accumulation, 81

choline acetyltransferase (ChAT) levels

and, 54

cognitive decrements, 52

cognitive effects study, 60

disproportional impairments, 56

and hormone treatment, 54

induced memory changes, 52

LH levels and, 59

maze learning and memory, 52

related memory changes in middle-age, 52

spatial reference and working memory, 52

young OVX monkeys

learning and memory, studies in, 53

oxidative phosphorylation, 81

paraganglia, 345, 362

paraganglioma. See pheochromocytoma

pasireotide (SOM-230), 260, 267–268, 270–271,

283, 287

peptide receptor radionuclide therapy (PRRT),

257, 270

peroxisome proliferator-activated receptor a

(PPARa), 243

peroxisome proliferator-activated receptor

gamma (PPARg) and DHEA, 124

pheochromocytoma, 343

anatomical imaging of

computed tomography (CT), 357

magnetic resonance imaging (MRI), 357

anatomy and physiology, 345

biochemical testing of, 355–356

catecholamines

metabolism, 347–348

synthesis, 345–346

translocation, 346–347

clinical presentation

cardiac complications, 349

in children, 350–351

differential diagnosis, 349

fever and constipation, 350

headache and sweating, 349

hyperglycaemia, 350

hypertension, 349

metabolic/haemodynamic attacks, 350

nausea and vomiting on exercising, 350

signs and symptoms, 348

diagnosis of, 355

epidemiology

age related factor, 344

in children, 344–345

prevalence, 344

functional imaging

metaiodobenzylguanidine scintigraphy,

357–358

future research, considerations for, 362–365

genetics of, 352

MEN2 syndrome, 351

other syndromes, 354–355

SDH syndromes, 353–354

von Hippel–Lindau syndrome, 353

von Recklinghausen’s neurofibromatosis type

1 (NF-1), 353

history of, 344

malignant pheochromocytomas

clinical manifestations of, 361

location and survival in, 361

management of, 361–362

treatment for, 362

medical therapy and preparation for surgery

a-adrenoceptor blocker, use of, 359

b-adrenergic receptor blockers, use of, 360

calcium channel blockers, use of, 360

hypertensive crises and, 360

octreoscan, use of, 359

positron emission tomography for, 358–359

surgery for, 360

and postoperative management, 360–361

treatment of, 359

phenoxybenzamine, 359

phentolamine, 360

phenylethanolamine N-methyltransferase

(PNMT), 346

phosphodiesterases (PDEs), 242

phosphofructokinase glycolytic enzymes, 80

phosphotyrosine phosphatase (PTP), 258

PI3K–AKT activation signaling, glucocorticoid-

resistant MM cells in bone marrow, 20

pituitary adenoma predisposition (PAP), 233

pituitary tumor pathogenesis

corticotrophinoma (ACTHoma) pathogenesis,

221

future research on, 222

prolactinoma pathogenesis, 221

385

senescence in, 212–214 somatotropinoma, 216–217

Carney complex, 217

familial isolated pituitary adenomas (FIPA),

218–219

familial pituitary adenoma, 218

GHRH signalling and GHoma tumorigenesis,

220

humoral factors, 221

isolated familial acromegaly, 217–218

non-familial somatotropinomas, pathogenesis

in, 219–220

senescence, 221

tumor micro-environment, 221

thyrotropinoma (TSHoma), pathogenesis of, 222

tumor micro-environment parameters and

angiogenesis, role of, 216

cell–cell adhesion, 216

tumor–stroma interaction, 216

two-hit’ theory of tumorigenesis, 214–215 pituitary tumors, 207, 229–230, 282

classification of, 207–208

epigenetic pathogenesis of, 215

genes implicated in tumorigenesis, 231–232

humoral factor excess and, 215

monoclonal nature of, 209

oncogene activation and, 209–210

CCND1, 210

ptd-FGFR4, 210

PTTG, 210–212

pathogenesis of, 208–209

pituitary cell proliferation, mechanisms of, 208

tumor suppressor gene inactivation and,

214–215

See also pituitary tumor pathogenesis

pituitary tumor-transforming gene (PTTG), 210

expression in pituitary tumors, 211

mechanisms for tumuorigenesis, 211–212

PTTG protein (securin), 211

role in pituitary tumorigenesis, 211–212

plaques, 79

plasma interleukin-6 (IL-6) concentrations, 167

plasma membrane potassium channels inhibition

and glucocorticoid-induced apoptosis, 10

polycystic ovary syndrome (PCOS), 168

post-traumatic stress disorder (PTSD), 149

attention deficit hyperactivity disorder and, 156

and brain

neuroanatomical findings, 155

concepts and definition, 150

cortisol and norepinephrine responsible for,

154

diversity in neuroendocrinology

co-morbid disorders, 158

early life experiences, 157

epigenetic alterations, 156–157

genetic vulnerability, 156

trauma exposure and time, 157

type of stressor/trauma, 158

emotional stressful stimuli, 152

GABAA/benzodiazepine (BZ) receptor

complex, 155

HPA axis and, 150, 156

ACTH levels, 151

basal cerebrospinal fluid (CSF), 151

consecutive blood sampling in, 151

enhanced cortisol suppression, 151

glucocorticoid receptors (GRs), 151

longitudinal interactions between SNS and,

152–155 morning plasma NE and evening salivary cortisol concentrations, 154

plasma cortisol levels, 151

urinary cortisol levels, 151

maternal PTSD, 157

mPFC in, 155

NE hyperactivity in, 152

neurotransmitters and neuropeptides, 155

pathophysiology of symptom clustering in, 150

and post-traumatic symptomatology, 152

prevalence of, 156

p27 protein, 305

as canonica tumor suppressor in human tumor,

311–312

as dose-dependent tumor suppressor in mice,

310–311

expression in tumor tissues, 307

functional relationship between Menin/ RET

and, 312–313

functions of, 306–307

low level of expression in neuroendocrine

tumors, 307

role in neuroendocrine tumorigenesis, study on

animal models

breeding of cyclinD1-deficient mice into

p27–/–mice, 310

386

p27 protein (Continued)

crossing of p18–/–mice into p27–/–mice,

309–310

hGHRH transgenic mice and p27 double

mutant mice, 310

p27-deficient mice, 309

p53–/–, p27–/–double mutant mice, 310

Pten and p27 double mutant mice, 310

Rb–/þ, p27–/–mice, study on, 310

structure of, 306

as tumor suppressor, 307

p38 protein and glucocorticoid-induced apoptosis,

19

Prader–Willi syndrome (PWS), 196

prednisone, 3

for ALL, 13

pro-apoptotic BH3-only Bcl-2 family member

Bim

and activation of downstream apoptotic

mediators, 12

expression, 8

failure to induce expression, 18–19

transactivation, 11

up-regulation, glucocorticoid-induced

apoptosis, 9

progesterone

detrimental cognitive effects, 56

pseudopregnant estropause state, 56

programmed cell death, 81

prolactinomas, 198, 207

PROP1 (Prophet of Pit-1), 216

prostate cancer, hormonal therapy in

advanced, CAB in, 330–332

androgen blockade as treatment, 322

anti-androgen administration and, 325–327

bicalutamide monotherapy, 325–326

effect on prostate cancer death rates, 328

errors related to, 335

at localized stage, 322–323

at metastatic stage, 323–324

misinterpretations regarding, 322–323

anti-androgens with higher affinity for human

AR, development of, 333–335

clinical effect of monotherapy, 327–328

early detection and use of CAB, 336

localized, effect of CAB on long-term control,

328–330

minimal duration of continuous CAB, 329–330

no resistance to CAB on treatment, 330

studies in Japan, 330

medical castration with GnRH agonists,

importance of, 327

prevalence in United States, 322

resistance to hormone therapy in metastatic

cancer, 332–333

sources of androgen in men, 324

androgens in prostate after castration, 324–325

intracrinology, 324

prostatic cancer

ADT for, 180

and GnRH agonists, 98

prostate-specific antigen (PSA) expression

levels in, 183

prostatic-specific antigen (PSA), 132

pseudohypoxia, 345

p23 stabilizing protein, 6

psychological trauma, 149

psychosis, 3

pyruvate kinase glycolytic enzymes, 80

raloxifene. See selective estrogen receptor

modulator (SERM)

Reed–Sternberg cells, 14

reverse pharmacology, 190

rituximab, chimeric monoclonal CD20 antibody

for CLL, 14

RU-486, GR antagonist, 10

SCSAN ARMS database, 132

sebum production and DHEA, 131

selective androgen receptor modulator (SARM)

muscle strength and BMD, 183

two-step screening method, 183

UCP-1 expression in, 183

selective estrogen receptor modulator (SERM), 109

senescence, 212–213

serine/threonine kinase GSK3

mediator of glucocorticoid-induced Bim

up-regulation, 8–9

serotonin (5-hydroxytryptamine 5-HT), 155

sex steroids

comparable amounts of adrenal origin in men

and women, 110–111

precursor of, 132

in pre-menopausal women, ovarian and adrenal

sources, 99

387

sources for post-menopausal women, 100

testicular and adrenal sources in men, 99

sleep apnea syndrome, 182

small for gestational age (SGA), 162

somatostatin analogues (SSAs), 259–260, 282

effects of

ACTH-secreting pituitary adenomas, 287

clinically non-functioning pituitary

adenomas, 285–287

GH-secreting adenomas, 283–285

in patients with NETs, 287–288

TSH-secreting pituitary adenomas, 285

future research, directions for, 290

pharmacological characteristics of, 282

octreotide-LAR, 282–283

octreotide (OCT), 282

pasireotide (SOM230), 283

safety and tolerability of, 288–290 somatostatin (SS) and somatostatin receptor (sst), 256, 281

future applications of, 271–272

physiological role and distribution, 260

in gastrointestinal tract (GIT), 262

in pathological tissues, 263

sst2 expression in human pancreas, 262

ssts distribution in human body, 261

and SS-analogues, 259

BIM-23244, 260

lanreotide, 260

octapeptide, 259–260

pasireotide (SOM-230), 260, 267–268, 270–271

ssts, role of

in diagnosis of non-tumoral diseases, 264–265

in diagnosis of tumors, 263–264

in non-tumoral diseases, 271

in treatment of NETs, 265–270

in treatment of non-neuroendocrine cancer,

270–271 sst subtypes, 258–259

degradation of, 259

internalization of, 259

See also somatostatin analogues (SSAs)

SREBP-1c activation in liver, 184–185

Stanford V regimens, 14

steroid hormone receptors function, 78

steroidogenic acute regulatory protein (StAR), 120

Stratakis–Carney syndrome, 355

stress-induced CA3 dendritic retraction in females, 37

stressor, 149

stress-related hormone levels, 162

Study of Women’s Health Across Nation

(SWAN), 87

succinate dehydrogenase (SDH), 345

SDH gene family, mutations of, 353–354

supraphysiological plasma T levels, 181

surgical menopause, 82

sympathetic nervous system (SNS), 149, 151

synthetic glucocorticoids as drugs, 2

leukemia and lymphoblastomas, clinical

treatment of, 13

systemic lupus erythematosus (SLE)

and DHEA treatment, 131–132

systolic blood pressure (SBP), 161

T-ALL 6TG1.1 cell line, GR LBD (L753F) mutation in, 18

tangles, 79

T-cell death-associated gene (TDAG8)

thymocytes from, 9

T-cell receptor (TCR)

activation-induced apoptosis, 11

cell death and, 11

ERK signaling and, 11–12

TCR-linked mGR–multiprotein complex, 8

tertiary model for interactions between aging, spatial learning and memory, 47

Thal/Dex/melphalan regimen for MM, 14

thalidomide/dexamethasone (THal/Dex) therapy

for MM, 14

thymocyte

development, systemic glucocorticoids in

regulation role, 11–13

thyroid stimulating hormone (TSH) levels, 169

thyrotropinomas, 207, 222

T replacement therapy, 182

triglycerides

and DHEA administration, 123

SREBP-1c and CPT-1 pathway in liver and

visceral fat of ORX, 186

tumor necrosis factor (TNF) and caspase

cascade, 10

tumor suppressor genes, 214

type 2 diabetes and DHEA, 122

tyrosine hydroxylase (TH), 345

Tyr1138 phosphorylation, 176

388

UCPs, mitochondrial proteins, 194–195

uncoupling protein-1 (UCP-1), 183

vaginal atrophy

correction with intravaginal DHEA, 117

and DHEA levels, 97

intravaginal estrogen formulations, 116–117

vaginal dryness, 116

VAGINORMTM, 119

vasomotor symptoms, 85

vesicular monoamine (VMA), 348

transporters, 346–347 4-vinylcyclohexene diepoxide (VCD) induced follicular depletion

cognitive effects study, 60

and OVX effects on cognition, 58–59

induced transitional menopause, 52

visceral fat, 181

visceral obesity, 175–176

voltage-dependent anion channel (VDAC), 9

von Hippel–Lindau (VHL) syndrome, 353 Warburg effect, 358, 364

water maze, glucocorticoids removal via

adrenalectomy (ADX), 38

Women’s Health Initiative (WHI)

studies, hormone therapies on, 51

WHI Memory Study (WHIMS), 53

dementia outcomes in, 85

hormone therapy and Alzheimer risk, 83

WHI Study of Cognitive Aging

(WHISCA), 53

Yerkes–Dodson law, Hebbian version, 37

Y-maze, spatial learning effect on, 38

young adults, spatial learning and memory in

chronic stress and glucocorticoids, influence of, 38–39 zinc-finger DNAbinding domain (DBD) of GR, 3