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CONTEMPORARY ENDOCRINOLOGY ™

Selective Estrogen Receptor Modulators Research and Clinical Applications Edited by

Andrea Manni, MD Michael F. Verderame, PhD

HUMANA PRESS

SELECTIVE ESTROGEN RECEPTOR MODULATORS

CONTEMPORARY ENDOCRINOLOGY P. Michael Conn, SERIES EDITOR Developmental Endocrinology: From Research to Clinical Practice, edited by ERICA A. EUGSTER AND ORA HIRSCH PESCOVITZ, 2002 Challenging Cases in Endocrinology, edited by MARK E. MOLITCH, 2002 Selective Estrogen Receptor Modulators: Research and Clinical Applications, edited by ANDREA MANNI AND MICHAEL F. VERDERAME, 2002 Transgenics in Endocrinology, edited by MARTIN MATZUK, CHESTER W. BROWN, AND T. RAJENDRA KUMAR, 2001 Assisted Fertilization and Nuclear Transfer in Mammals, edited by DON P. WOLF AND MARY ZELINSKI-WOOTEN, 2001 Adrenal Disorders, edited by ANDREW N. MARGIORIS AND GEORGE P. CHROUSOS, 2001 Endocrine Oncology, edited by STEPHEN P. ETHIER, 2000 Endocrinology of the Lung: Development and Surfactant Synthesis, edited by CAROLE R. MENDELSON, 2000 Sports Endocrinology, edited by MICHELLE P. WARREN AND NAAMA W. CONSTANTINI, 2000 Gene Engineering in Endocrinology, edited by MARGARET A. SHUPNIK, 2000 Endocrinology of Aging, edited by JOHN E. MORLEY AND LUCRETIA VAN DEN BERG, 2000 Human Growth Hormone: Research and Clinical Practice, edited by ROY G. SMITH AND MICHAEL O. THORNER, 2000 Hormones and the Heart in Health and Disease, edited by LEONARD SHARE, 1999 Menopause: Endocrinology and Management, edited by DAVID B. SEIFER AND ELIZABETH A. KENNARD, 1999 The IGF System: Molecular Biology, Physiology, and Clinical Applications, edited by RON G. ROSENFELD AND CHARLES T. ROBERTS, JR., 1999

Neurosteroids: A New Regulatory Function in the Nervous System, edited by ETIENNEEMILE BAULIEU, MICHAEL SCHUMACHER, AND PAUL ROBEL, 1999 Autoimmune Endocrinopathies, edited by ROBERT VOLPÉ, 1999 Hormone Resistance Syndromes, edited by J. LARRY JAMESON, 1999 Hormone Replacement Therapy, edited by A. WAYNE MEIKLE, 1999 Insulin Resistance: The Metabolic Syndrome X, edited by GERALD M. REAVEN AND AMI LAWS, 1999 Endocrinology of Breast Cancer, edited by ANDREA MANNI, 1999 Molecular and Cellular Pediatric Endocrinology, edited by STUART HANDWERGER, 1999 Gastrointestinal Endocrinology, edited by GEORGE H. GREELEY, JR., 1999 The Endocrinology of Pregnancy, edited by FULLER W. BAZER, 1998 Clinical Management of Diabetic Neuropathy, edited by ARISTIDIS VEVES, 1998 G Proteins, Receptors, and Disease, edited by ALLEN M. SPIEGEL, 1998 Natriuretic Peptides in Health and Disease, edited by WILLIS K. SAMSON AND ELLIS R. LEVIN, 1997 Endocrinology of Critical Disease, edited by K. PATRICK OBER, 1997 Diseases of the Pituitary: Diagnosis and Treatment, edited by MARGARET E. WIERMAN, 1997 Diseases of the Thyroid, edited by LEWIS E. BRAVERMAN, 1997 Endocrinology of the Vasculature, edited by JAMES R. SOWERS, 1996

SELECTIVE ESTROGEN RECEPTOR MODULATORS Research and Clinical Applications Edited by

ANDREA MANNI, MD MICHAEL F. VERDERAME, PhD Division of Endocrinology, Diabetes, and Metabolism Pennsylvania State University College of Medicine Milton S. Hershey Medical Center Hershey, PA

HUMANA PRESS TOTOWA, NEW JERSEY

© 2002 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com

All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences arising from the information or opinions presented in this book and make no warranty, express or implied, with respect to its contents. Due diligence has been taken by the publishers, editors, and authors of this book to assure the accuracy of the information published and to describe generally accepted practices. The contributors herein have carefully checked to ensure that the drug selections and dosages set forth in this text are accurate and in accord with the standards accepted at the time of publication. Notwithstanding, since new research, changes in government regulations, and knowledge from clinical experience relating to drug therapy and drug reactions constantly occur, the reader is advised to check the product information provided by the manufacturer of each drug for any change in dosages or for additional warnings and contraindications. This is of utmost importance when the recommended drug herein is a new or infrequently used drug. It is the responsibility of the treating physician to determine dosages and treatment strategies for individual patients. Further, it is the responsibility of the health care provider to ascertain the Food and Drug Administration status of each drug or device used in their clinical practice. The publishers, editors, and authors are not responsible for errors or omissions or for any consequences from the application of the information presented in this book and make no warranty, express or implied, with respect to the contents in this publication. This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American National Standards Institute) Permanence of Paper for Printed Library Materials. Cover design by Patricia F. Cleary.

For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected] or visit our website at http://humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-912-9/02 $10.00 + $00.25]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging-in-Publication Data Selective estrogen receptor modulators : research and clinical applications / edited by Andrea Manni, Michael F. Verderame. p. ; cm. -- (Contemporary endocrinology) Includes bibliographical references and index. ISBN 0-89603-912-9 (alk. paper) 1. Selective estrogen receptor modulators. 2. Menopause--Hormone therapy. I. Manni, Andrea. II. Verderame, Michael, 1957- III. Contemporary endocrinology (Totowa, N.J.) [DNLM: 1. Selective Estrogen Receptor Modulators. 2. Estrogen Replacement Therapy--methods. 3. Postmenopause--drug effects. 4. Receptors, Estrogen--physiology. WP 522 S464 2002] RG186.S44 2002 618.1'75--dc21 2001026442

PREFACE The sharp decline in ovarian steroidogenesis occurring at the time of the menopause results in immediate adverse events that impair quality of life. These can include vasomotor instability and urogenital atrophy, as well as more long-term sequelae such as increased morbidity and mortality from cardiovascular disease and osteoporotic fractures. Hormone replacement therapy (HRT) with estrogen and progesterone (in the presence of an intact uterus) is clearly effective in alleviating symptoms of hot flashes and urogenital atrophy. Case control and cohort studies have also indicated that HRT reduces the risks of cardiovascular disease and osteoporotic fractures. However, results from the Heart and Estrogen/Progestin Replacement Study (HERS), which failed to demonstrate a benefit in cardiovascular mortality in women with established heart disease, emphasize the difficulty in drawing conclusions from epidemiological data. Despite its proven or implied benefits, HRT is associated with a variety of significant drawbacks that include increased risks of breast cancer, uterine cancer, deep vein thrombosis, gallbladder disease, and breast enlargement/tenderness. Because of these disadvantages, HRT is restricted to a relatively small fraction of postmenopausal women, and long-term compliance with treatment is estimated to be only 15–40%. Agents that retain the benefits of estrogens but at the same time avoid the risks are urgently needed to provide postmenopausal women with an optimal form of HRT. Selective Estrogen Receptor Modulators (SERMs) are a class of drugs with mixed estrogen agonistic/antagonistic activity that holds promise in fulfilling this need. Tamoxifen, the first and most studied of these compounds, has been in clinical practice for over 20 years in the treatment of women with hormone-responsive breast cancer. As a result of its antiestrogenic action in the breast, tamoxifen may, indeed, be effective as a chemopreventive agent for hormone-responsive breast cancer, while its partial estrogen agonistic effects on the skeletal system and on serum lipoproteins may offer protection from osteoporosis and cardiovascular disease. Although demonstration of these clinical benefits is still preliminary or lacking (e.g., reduction in heart disease risk), such mixed agonistic/antagonistic properties of tamoxifen provide proof of principle for the feasibility of developing new SERMs with an improved pharmacologic and therapeutic activity profile. A possible improvement in this regard may have been the introduction of raloxifene, which, in contrast to tamoxifen, has minimal estrogen-like activity in the uterus. As a result, its use has not been associated with an increased risk of endometrial cancer. Over the last several years, our knowledge of the basic cellular mechanisms governing estrogen action has grown exponentially. The simple model of estradiol binding to its cognate receptors (ER) followed by binding of the complexed receptor to estrogenresponsive elements of target genes has significantly expanded to include multiple additional interactive components. Several chapters in the Basic Studies section address in detail the cellular mechanisms of action of estrogens and SERMS, focusing on important aspects such as distinct ligand-dependent conformational changes in the ER that play a v

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Preface

critical role in the recruitment of coactivators and corepressors and the bidirectional crosstalk between estrogen receptor and growth-factor signaling. Differences in tissue distribution and function of ER-α and -β are also reviewed and discussed. Understanding of these basic mechanisms is critical for the design of new SERMs with improved tissue-specific estrogen agonistic/antagonistic activity resulting in maximal health benefits and minimal risks. The chapters in the Basic Studies section will provide a comprehensive updated review of the preclinical studies with currently available SERMs focusing on their effects on critical target organs such as the cardiovascular system and the brain. The Clinical Studies section will compare and contrast the influence of estrogens and currently available SERMs (primarily tamoxifen and raloxifene) on the major clinical endpoints, such as incidences of breast cancer, cardiovascular disease, osteoporosis, and cognitive impairment. Based on our current state of knowledge, a tentative approach to menopause-related health issues will be provided both for normal women as well as for women with a previous diagnosis of localized breast cancer. We believe that Selective Estrogen Receptor Modulators: Research and Clinical Applications will be of interest to basic scientists in endocrinology, tumor biology, and pharmacology, as well as a wide range of clinicians, including endocrinologists, medical oncologists, gynecologists, and family practitioners. We wish to thank the many contributors, who are distinguished leading experts in their fields and without whose major efforts this book would not have been possible. Andrea Manni, MD Michael F. Verderame, PhD

CONTENTS Preface ................................................................................................... v Contributors ......................................................................................... ix

BASIC STUDIES I

Molecular Mechanisms of Estrogen Receptor Function

1

Structure and Function of the Estrogen Receptor Stefan Nilsson, PHD and Jan-Åke Gustafsson, MD, PHD ....................... 3

2

Ligand-Induced Conformational Changes in Estrogen Receptors-α and -β Elizabeth A. Allegretto, PHD ................................................................ 19

3

Expression and Function of Estrogen Receptors-α and -β Jonathan Lindzey, PHD ........................................................................ 29

4

SERM Modulation of Gene Expression: Role of Coactivators and Corepressors Paul Webb, PHD ................................................................................... 57

5

Crosstalk Between Estrogen Receptors and Growth Factor Signaling Douglas Yee, MD and Carol A. Lange, PHD ........................................ 77

II

Tissue-Specific Effects of Estrogens and SERMs

6

Direct Estrogen Effects on the Cardiovascular System Munish K. Goyal, MD and Suzanne Oparil, MD ................................ 99

7

Estrogens and the Brain: Implications for the Treatment of Postmenopausal Women Bruce S. McEwen, PHD, Phyllis M. Wise, PHD, and Stanley Birge, MD ................................................................. 121

III Preclinical Studies 8

Insights into the Molecular Mechanism of SERMs Through New Laboratory Models Csaba Gajdos, MD, James Zapf, PHD, and V. Craig Jordan, PHD, DSc .................................................................................... 147

9

Third- and Fourth-Generation SERMs Fernand Labrie, MD, PHD, Claude Labrie, MD, PHD, Alain Bélanger, PHD, and Jacques Simard, PHD .......................... 167 vii

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Contents

CLINICAL STUDIES IV

Organ Specific Effects of Estrogens and SERMs

10

SERMs’ Effect on the Neuroendocrine System and the Reproductive Organs Nanette F. Santoro, MD, and Peter Kovacs, MD .............................. 191

11

Epidemiology of Cardiovascular Disease in Women: Role of Estrogens Jacques E. Rossouw, MD .................................................................. 207

12

SERMs Effects on Cardiovascular Risk Factors and Disease Richard R. Love, MD ......................................................................... 223

13

Estrogen and the Skeleton Michael Kleerekoper, MD, FACE and Ashish Verma, MD ................. 237

14

Effects of SERMs on Bone in Clinical Studies Aurelie Fontana, MD, and Pierre D. Delmas, MD, PHD .................... 245

15

Estrogens and SERMs: Clinical Aspects of Cognition with Aging and Neurodegenerative Disorders Alan J. Lerner, MD ............................................................................ 255

V SERMs and Endocrine Dependent Tumors 16

SERMs and Breast Cancer Prevention Jenny Chang, MD and C. Kent Osborne, MD ................................... 267

17

SERMs in Postmenopausal Women’s Health Jan L. Shifren, MD, and Leo Plouffe, Jr., MD, CM ........................... 279

VI

Roles of Estrogens and SERMs in Postmenopausal Hormone Replacement Therapy

18

Menopause Therapy: An Individualized Approach Nananda F. Col, MD, FACP, MPP, MPH, Michele G. Cyr, MD, FACP and Anne W. Moulton, MD, FACP ................................................. 299

19

Alternatives to Estrogen for Treatment of Menopause Richard J. Santen, MD and JoAnn V. Pinkerton, MD ..................... 313

20

Phytoestrogens in the Context of SERMs Susan R. Davis, MB, BS, PHD, FRACP ................................................... 345 Index .................................................................................................. 365

CONTRIBUTORS ELIZABETH A. ALLEGRETTO, PHD • Experimental Station, Dupont Pharmaceuticals Company, Wilmington, DE ALAIN BÉLANGER, PHD • Oncology and Molecular Endocrinology Research Center, Quebec City, Quebec, Canada STANLEY BIRGE, MD • Department of Geriatrics, Washington University School of Medicine, St. Louis, MO JENNY CHANG, MD • Breast Center, Baylor College of Medicine, Houston, TX NANANDA F. COL, MD, FACP, MPP, MPH • Department of Medicine, Harvard University Medical School and Brigham and Women’s Hospital, Boston, MA MICHELE G. CYR, MD, FACP • Department of Medicine, Brown University School of Medicine, Providence, RI SUSAN R. DAVIS, MB, BS, PHD, FRACP • The Jean Hailes Research Unit, Victoria, Australia PIERRE D. DELMAS, MD, PHD • Claude Bernard University of Lyon, Hospital Herriot, Lyon, France AURELIE FONTANA, MD • Claude Bernard University of Lyon, Hospital Herriot, Lyon, France CSABA GAJDOS, MD • Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, IL MUNISH K. GOYAL, PHD • University of Alabama at Birmingham, Birmingham, AL JAN-ÅKE GUSTAFSSON, MD, PHD • Department of Medical Nutrition, Karolinska Institute; Novum-Huddinge University Hospital, Huddinge, Sweden V. CRAIG JORDAN, PHD, DSC • Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, IL MICHAEL KLEEREKOPER, MD, FACE • Division of Endocrinology, Wayne State University, Detroit, MI PETER KOVACS, MD • Division of Reproductive Endocrinology, Department of Obstetrics/Gynecology and Women’s Health, Albert Einstein College of Medicine, Bronx, NY CLAUDE LABRIE, MD, PHD • Oncology and Molecular Endocrinology Research Center, Quebec City, Quebec, Canada FERNAND LABRIE, MD, PHD • Oncology and Molecular Endocrinology Research Center, Quebec City, Quebec, Canada CAROL A. LANGE, PHD • University of Minnesota Cancer Center, Minneapolis, MN ALAN J. LERNER, MD • Department of Neurology, Case Western Reserve University; Alzheimer Center, Cleveland, OH JONATHAN LINDZEY, PHD • Department of Biology, University of South Florida, Tampa, FL RICHARD R. LOVE, MD • Department of Medicine, University of Wisconsin, Madison, WI

ix

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Contributors

BRUCE S. MCEWEN, PHD • Laboratory of Neuroendocrinology, Rockefeller University, New York, NY ANNE W. MOULTON, MD, FACP • Department of Medicine, Brown University School of Medicine, Providence, RI STEFAN NILSSON, PHD • KaroBio AB, Novum, Huddinge, Sweden SUZANNE OPARIL, MD • University of Alabama at Birmingham, Birmingham, AL C. KENT OSBORNE, MD • Breast Center, Baylor College of Medicine, Houston, TX JOANN PINKERTON, MD • Midlife Health Center, University of Virginia Health Science Center, Charlottesville, VA LEO PLOUFFE, JR., MD, CM • Lilly Research Laboratory, Eli Lilly Company, Indianapolis, IN JACQUES E. ROSSOUW, MD • Women’s Health Initiative, National Heart, Lung, and Blood Institute, Bethesda, MD RICHARD J. SANTEN, MD • Division of Endocrinology, University of Virginia Health Science Center, Charlottesville, VA NANETTE F. SANTORO, MD • Director, Division of Reproductive Endocrinology, Department of Obstetrics/Gynecology and Women’s Health, Albert Einstein College of Medicine, Bronx, NY JAN L. SHIFREN, MD • Vincent OB/GYN Service, Massachusetts General Hospital; Harvard University Medical School, Boston, MA JACQUES SIMARD, PHD • Oncology and Molecular Endocrinology Research Center, Quebec City, Quebec, Canada ASHISH VERMA, MD • Division of Endocrinology, Wayne State University, Detroit, MI PAUL WEBB, PHD • Metabolic Research Unit, University of California at San Francisco, San Francisco, CA PHYLLIS M. WISE, PHD • Department of Physiology, University of Kentucky School of Medicine, Lexington, KY DOUGLAS YEE, MD • University of Minnesota Cancer Center, Minneapolis, MN JAMES ZAPF, PHD • Maxie Pharmaceutical, San Diego, CA

I

MOLECULAR MECHANISMS OF ESTROGEN RECEPTOR FUNCTION

1

Structure and Function of the Estrogen Receptor Stefan Nilsson, PHD and Jan-A˚ke Gustafsson, MD,

PHD

Contents Introduction Historical Perspective The N-Terminal A/B Domain DBD, The DNA Binding Domain The Ligand-Binding E/F Domain Mode of Target Gene Transcription Regulation Future Directions References

INTRODUCTION The two estrogen receptors (ER), α and β, belong to the steroid/thyroid hormone receptor family of ligand-dependent transcription factors. Through interaction between distinct domains of the ERs with natural and synthetic ligands, with various proteins and with DNA, they can modulate the activity of target genes and a number of other processes within the cell. We review in this chapter the domains of ERα and ERβ and their functional differences and role in ER biology, as well as what we know today about the different mechanisms of ER-dependent target gene regulation.

HISTORICAL PERSPECTIVE The estrogen receptors α (ERα) and β (ERβ) have a modular architecture similar to the other 50–60 members of the steroid/thyroid hormone receptor family (1–3) in that they are composed of independent but interacting functional domains (Fig. 1). The N-terminal A/B domain encodes the constitutive, ligand-independent but cell-type and gene-speciﬁc activation function 1 (AF1) (4–8). The C- or DNA-binding domain (DBD) contains a two-zinc ﬁnger motif important for DNA sequence-speciﬁc receptor binding and receptor dimerization (2,3,9,10). The carboxy-terminal E/F- or ligand-binding domain (LBD) mediates ligand binding, receptor dimerization, nuclear translocation, From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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Nilsson and Gustafsson

Fig. 1. Modular architecture of the estrogen receptors and other members of the nuclear receptor family. The N-terminal A/B domain harbors the autonomous, ligand-independent transcription activation function 1 (AF1) including sites for phosphorylation by the MAPK pathway. The highly conserved C-domain encodes Zn-binding motifs important for DNA binding. The hinge or D-domain is variable in length and primary sequence between members of the family and very ﬂexible to allow the Cand E/F domains to move freely. The multifunctional C-terminal ligand binding domain is responsible for binding of ligand, nuclear translocation, and homo- and heterodimerization. Also encoded within this domain is the second, ligand-dependent activation function 2 (AF2) through which coactivators interact with nuclear receptors.

Fig. 2. Schematic representation of the various isolated isoforms of ERβ. The ﬁrst reported ERβ, cloned from a rat prostate cDNA library, was 485 residues long, starting from M3. Later a longer isoform of ERβ with a 45 amino acids N-terminal extension was identiﬁed (M2). An isoform with a further 19 amino acids extension in the N-terminus (M1) was reported for both mouse and rat. In addition to the N-terminal isoforms also an 18 amino acids inframe insertion in the exon 5/6 splice junction in the LBD was isolated. As a consequence of this insertion this isoform had a much lower afﬁnity for E2 compared to ERα or any of the other N-terminally extended isoforms of ERβ. Transcripts encoding additional ERβ isoforms with variations at the extreme C-terminus were cloned from testis cDNA libraries. One such example is ERβcx, which has the last eighth exon replaced by unique exon sequences. Because of this change of C-terminal sequences ERβcx showed no ligand binding activity or capacity for transcriptional activity.

and transactivation of target gene expression via the second, ligand-dependent, activation function 2 (AF2) (2,3,5,6,11–14). In 1986 the cloning of the ﬁrst ER was published (15,16), today named ERα. Ten years later the existence of a second ER, ERβ, was reported (17). That ERα and ERβ are true ER subtypes rather than isoforms emanating from one and the same gene through differential splicing was conﬁrmed by their unique chromosomal localization, the human ERα gene localized to the long arm of chromosome 6 and the human ERβ gene to chromosome 14 (18). Since 1996 ERβ from various species (18–20) or differently sized ERβ isoforms have been reported (Fig. 2). N-terminally extended ERβ isoforms, an inframe ligand-

Chapter 1 / Estrogen Receptor Structure and Function

5

binding domain exon insertion, and splice variants at the extreme C-terminus with an exchange of the last exon for previously unknown 3′ exons have been identiﬁed (21–28). Various alternatively spliced forms have also been described for ERα (29–32). Whether all isoforms or differentially spliced versions of ERα and ERβ, respectively, exist as proteins or whether they have any signiﬁcant biological and physiological role warrants further investigation.

THE N-TERMINAL A/B DOMAIN The amino-terminal domains of ERα and ERβ respectively show variability both in terms of length (3,23) and amino acid sequence (18). The N-termini of both receptors are targets for phosphorylation by the mitogen-activated protein kinase (MAPK) signalling cascade (33–38) required for full AF1 activity and for promoting interaction with coactivator proteins and receptor-mediated transcriptional activity in the absence of cognate natural or synthetic ligands. Doubts about the presence of an AF1 function in ERβ similar in function to the AF1 in ERα have, however, been raised. In one study the transcriptional contribution of the A/B domain of ERα and ERβ was compared using N-terminal ERα/ERβ receptor chimeras (39). The study showed that replacement of the A/B domain of ERβ with the corresponding domain of ERα signiﬁcantly improved the transcriptional response of ERβ to 17β-estradiol (E2). Conversely, fusion of the ERβ A/B domain with ERα, replacing the A/B domain of ERα, caused a corresponding decrease in the response of ERα to E2. Tamoxifen-induced transcriptional agonism observed with ERα is absent in ERβ or in the ERβ N-terminus/ERα chimera but restored in the ERα/ERβ receptor chimera containing the ERα A/B domain. These data suggested an important functional difference in the N-terminal domains of ERα and ERβ (39). Furthermore, the transcriptional activity of ERα and ERβ, and mutant versions thereof, on a variety of estrogen response element (ERE) reporter-gene constructs in different cell lines has been analyzed (8,13,40). The conclusion from these studies was that the amino-terminal A/B domain of ERβ has no (or very weak) autonomous or ligand-independent transcriptional activity in comparison to ERα. In contrast, and opposite to the effect on ERα activity, sequential deletion of the ERβ N-terminus resulted in increased transcriptional activity of the receptor, suggesting that the ERβ A/B domain is rather a repressor domain and a putative site for interaction with corepressors than an activation domain (8,40). Nonetheless, although barely detectable, the weak transcriptional activity of the putative ERβ AF1 was mapped to amino acid residues 1–30 (human ERβ-485 (8) encompassing a six amino acid residue motif conserved among all identiﬁed mammalian ER amino-terminal domains. By progressive deletion of the ERα N-terminal region it was found that estrogens and antiestrogens required different segments of ERα AF1 for transcriptional activation (Fig. 3) (41). There was an absolute requirement for amino-acid residues 41–64 for antiestrogens like tamoxifen to activate transcription while a more dramatic drop in agonism in the presence of E2 was not observed until deletion progressed to amino acid residue 109. A segment comprised of amino acid residues 41–109 restores most of the partial agonism of tamoxifen and almost completely the activity of E2 when in the context of a full-length receptor (41). A prerequisite for transcriptional activity by ERs is interaction with coactivators (12). Both ERα and ERβ N-terminal domains interact with the coactivator glucocorticoid

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Fig. 3. Different functions localized to discrete regions of ERα N-terminal domain. Amino acids 1–37 are suggested to have an inhibitory effect on AF1 activity by direct or indirect interaction with the C-terminal ligand binding domain in the absence of ligand. An α-helical structure, encoded by amino acids 35–47, has been shown to have autonomous transactivation activity and sequences delimited by amino acids 41–64 are required for tamoxifen agonism. Region 87–109 is required for synergism between AF1 and AF2 and is required for full activity of E2. The GRIP-1 binding area covers sequences from amino acid 41–116 but sequences within the 41–64 region are of particular importance.

receptor-interacting protein (GRIP-1) but the interaction between GRIP-1 and the putative ERβ AF1 is much weaker than with AF1 of ERα (42). This may at least in part explain the difference in the autonomous activity of ERα and ERβ AF1 domains and their difference in response to tamoxifen. GRIP-1 markedly potentiated the tamoxifen agonism via ERα by interaction with the sequences of ERα AF1 shown to be required for tamoxifen agonism (Fig. 3) (41,42) while GRIP-1 only weakly, if at all, potentiated the transcriptional activity of tamoxifen-liganded ERβ (42). In a more recent article a stretch of 11 amino acid residues of ERα overlapping with the beginning of the 41–64 stretch required for tamoxifen agonism (Fig. 3) was shown to have autonomous transcriptional activity that could be compromised by the introduction of point mutations (43). This stretch of amino acids, proposed to adopt an α-helical structure, appeared to be more important for the ligand-independent activity of AF1 than its activity in the presence of E2. Furthermore, it was speculated that this α-helical core of ERα AF1 could be a surface for interaction with coactivators (such as GRIP-1) and for interaction with components of the basal transcription machinery (43). Interaction between the N-terminal AF1 and the C-terminal AF2 of ERα is required for full ERα transcriptional activity (40,44). Coexpression of ERα as two separate polypeptides, an N-terminal A/B-C-D and a C-terminal E/F polypeptide, in the presence of E2 or tamoxifen, reconstituted ERα on DNA as a transcriptionally competent complex containing both the AF1 and AF2 functions (44). In an extension of this study it was shown that the coactivator steroid receptor coactivator 1 (SRC-1) signiﬁcantly enhanced the association of the ERα AF1 and AF2 domains in the presence of both E2 and tamoxifen (45). A possible explanation for this enhancing effect of SRC-1 on AF1 and AF2 association is that AF1 and AF2 can interact with separate surfaces of the same coactivator as shown for GRIP-1 and SRC-1 (44,46), an important synergizing characteristic of these transactivation functions. Another type of N- and C-terminal interaction that instead results in inhibition of transcriptional activity has been described (43). That study showed that removal of either the C-terminal E/F domain or the most N-terminal

Chapter 1 / Estrogen Receptor Structure and Function

7

Fig. 4. Sequence of ERα DBD and estrogen response element. (A) Classical representation of DBD Zn-binding motifs. Amino acids in helical structures are boxed. The recognition helix (P-box) is positioned in the DNA major groove and residues that make direct or indirect (via water molecules) contacts with base pairs are shown in bold. Underlined residues at the beginning of the second Znbinding motif (D-box) participate in dimer formation. Asterisks indicate amino acid differences between ERα and ERβ. (B) Nucleotide sequence of a consensus estrogen response element with three nucleotide spacing between the half-sites (underlined).

37 amino acid residues increased the ligand-independent transcriptional activity of the receptor, suggesting a model where amino acid residues 1–37 function as a negative regulatory domain for AF1 activity (Fig. 3) in the absence of ligand by a direct interaction with the E/F domain. This hypothetical model, however, could not exclude that the Nand C-terminal interaction is indirect, involving other proteins such as corepressors (43).

DBD, THE DNA BINDING DOMAIN The 70–80 amino acid residues long DBD forms two zinc-binding motifs, which play an important role in receptor DNA sequence-speciﬁc recognition and binding and in receptor dimerization, respectively (9,10,47). Crystal structure of dimers of ERα DBD bound to a consensus ERE revealed that the side chains of four amino acids on the surface of the recognition helix (also termed the P-box) (Fig. 4) of each monomer make direct and indirect hydrogen bonds, via water molecules, with nucleotides in the ERE (10). Additional amino acids in both zinc-binding motifs make phosphate backbone contacts via ordered water molecules, greatly increasing the number of interactions between each receptor monomer and the DNA. Furthermore, direct and indirect contacts between amino acids of each ERα DBD dimerization interface (also termed the Dbox) (Fig. 4), located at the beginning of the second zinc-binding motif, contributes to the cooperative binding of two ER monomers to the ERE (10). The DBDs of ERα and ERβ show 97% homology (18) with identical P- and D-box

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sequences, critical for target DNA recognition, speciﬁcity and dimerization (9). Thus, ERα and ERβ are likely to bind with similar speciﬁcity and afﬁnity to different EREs. In addition, ERα and ERβ were found to bind to an ERE as either homo- or heterodimers (40,48–51).

THE LIGAND-BINDING E/F DOMAIN The ligand-binding domain (LBD) is multifunctional in that it mediates ligand binding, receptor dimerization, interaction with chaperone proteins, nuclear translocation, and transcription activation functions (2,3). The homology between ERα and ERβ E-domains is 59% and between their F-domains only 18% (18). Amino acid residues that line the ligand-binding cavity or interact with bound ligand span from helix 3 (H3) to H12 in both ERα and ERβ (52) and are almost identical between the two ER subtypes except for two residues. It is therefore not surprising that ERα and ERβ exhibit very similar although not identical ligand-binding characteristics (53) or show similar potencies in regulation of ERE-mediated reporter-gene expression in the presence of estrogen agonists or antagonists (54). The afﬁnity of a ligand for the ERs and the ligand-induced receptor conformation depend in part on the type of substituents, threedimensional (3D) structure, volume, and hydrophobic/hydrophilic character of the ligand and in part on the volume, shape, and plasticity of the ligand-binding cavity and the type of amino-acid residues lining the cavity. Complementarity of these features of charge, shape, and size between the ligand and the receptor ligand-binding cavity is crucial for receptor afﬁnity and selectivity of a ligand, ligand-induced receptor conformational change, and receptor/cofactor interactions, and subsequently the consequences on the biological effect of a ligand, either agonism or antagonism (52,55–60). The two amino acid differences within the ligand-binding cavity between ERα and ˙ 3 and ERβ ERβ have a direct impact on the overall volume of the cavity, ERα 490 A 3 ˙ (52), and may explain, to some extent, the distinct ligand-binding preferences 390 A or transcriptional consequences of ligands reported for ERβ (52–54,59). AF2 in the LBD constitutes the ligand-dependent transcription-activation function of nuclear receptors (6,11–14). In the crystal structure of ERα LBD in complex with E2 or diethylstilbestrol (DES) (56,57), it was shown that agonist-induced positioning of H12 over the ligand-binding pocket is crucial for the formation of the liganddependent AF2 coactivator recruitment and interaction surface (Fig. 5). Together with amino acid residues in H3, H4, and H5, H12 forms a shallow hydrophobic groove that can accommodate an LXXLL motif (NR-box) (Fig. 5), which is an essential component of coactivators and is needed for mediating coactivator-binding to agonist-bound receptors (14,57). In contrast, in the ERα- and ERβ-LBD raloxifene complexes (52,56), and the ERα-LBD 4-OH-tamoxifen complex (57), H12 was displaced from its agonist position over the ligand-binding cavity and instead occupied the hydrophobic groove formed by H3, H4, and H5, foiling the AF2 coactivator interaction surface. In fact, the position of H12 in the ER raloxifene and 4-OH-tamoxifen structures, mimicked the interactions formed by a coactivator NR-box peptide with agonist-bound LBD (52,57). Amino acid residues in H12 important for the AF2 activity of ERα are conserved in ERβ (52). Full length SRC-1 or a nuclear receptor (NR)-box containing a fragment thereof was shown to interact with AF2 of both ERα and ERβ with similar efﬁcacy (13). Mutation of amino acids in H12 previously shown to severely affect E2-dependent

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Fig. 5. AF2/LXXLL binding cleft: View of coactivator LXXLL binding site of human ERα-LBD. LEFT: Electrostatic surface representation of NR-box binding face of LBD. The inset at the lower right of the panel illustrates the approximate view of the LBD. Dashed box delineates the receptor AF2 site that is shown in close up. RIGHT: Close-up of coactivator binding groove in the complex between liganded ER and a 12-mer peptide corresponding to the NR-BoxII region of TIF2 (52). The LXXLL peptide binds as a helix so that all three consensus leucines (+1/+4/+5) are in contact with the LBD. The positions of the conserved lysine and glutamic acid residues that cap the LXXLL helix are marked.

transcriptional activation by ERα (6,11) also had a dramatic effect on ERβ-dependent transcriptional activity (13). Interestingly, however, while mutation of the conserved glutamic acid residue in H12 had only a minor effect on ERα transcriptional activity, it had a severe effect on ERβ AF2 activity. In another study (58) a mutated version of the second SRC-1 NR-box LXXLL motif showed ER subtype-selective afﬁnity, measured as the effect on the dissociation rate of ligands bound to the receptor. The data from these two studies suggest that although the core amino-acid sequence of AF2 and of H12 in ERα and ERβ is conserved, the surface required to interact with coactivators is not identical, possibly a result of subtle ER subtype-speciﬁc conformational differences caused by amino acid sequence differences between ERα and ERβ in other parts of their LBDs. The data also suggest that ERα and ERβ may have different coactivator requirements or at least a requirement for different NR-box sequences for optimal interaction with coactivator proteins. In addition, sequences ﬂanking the LXXLL motif of coactivator NR-boxes have been shown also to inﬂuence the afﬁnity and receptor selectivity of a coactivator for the agonist-induced AF2 surface (61,62). Based on information of the 3D structure of ERα (56) the major ER dimerization interface involves H11 of each monomer and H8 of one monomer interacting with the loop between H9 and H10 and with H10 in the neighboring monomer (Fig. 6). The dimerization interface is dominated by a stretch of hydrophobic residues at the beginning of H11 that intertwines to form a rigid backbone (Fig. 6). Mutational analysis has conﬁrmed the importance of the hydrophobic amino acids at the N-terminal end of H11 as the major contributor in ERα and ERβ homodimerization (63). Replacement of the L504, L508, and L511 (amino acid numbering of human ERα) with glutamic acid residues completely abolished ERE DNA binding and rendered the receptor incapable of transcriptional activation.

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Fig. 6. Schematic representation of ER-α LBD dimer (56). (A) Dimer viewed down twofold axis. Helixes that contribute to the dimer interface are labeled. (B) Close-up of central H11 helices of ER-α homodimer. Helixes are viewed looking down the dimer axis in an identical orientation to that in A. Hydrophobic residues that contribute to the core of the interface are labeled. (C) H11 helixes viewed perpendicular to dimer axis. Dotted box delineates central hydrophobic core of dimer formed by the N-terminal region of H11.

The function and relevance of the F-domain is largely unknown. Its amino acid sequence is not well conserved among species and the homology between ERα and ERβ is less than 18% (18). There is no obvious structure of the F-domain and deletion of the entire domain does not affect receptor protein stability, ligand binding, receptor dimerization or DNA binding. However, available data suggest that the ERα F-domain has a cell-type-speciﬁc modulatory function that affects the agonist/antagonist effectiveness of estrogens and antiestrogens (64,65).

MODE OF TARGET GENE TRANSCRIPTION REGULATION We long believed that there was only one ER that mediated the effect of natural and synthetic ligands and that estrogen sensitive genes were transcriptionally regulated by homodimers of ER binding to an ERE positioned either upstream of, or in the vicinity of, target gene promoters. Recently we have learned that there are at least two ERs, ERα and ERβ, transferring the biological and transcriptional effect of estrogens and antiestrogens and, furthermore, that they not only affect target gene expression as homodimers but perhaps more likely as heterodimers when expressed in the same cell (40,49–51). In a recent report by Tremblay and collaborators (51) they describe the approach of using ERα and ERβ with modiﬁed DNA recognition speciﬁcity to study the effect of ERα/ERβ heterodimers on transcription activation from a composite GRE 1/2-site–ERE 1/2-site palindromic response element (E/GRE). In their system,

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heterodimers of ERα and ERβ were able to stimulate target gene expression approx 60–70% of the level of ERα homodimers in the presence of 4-OH-tamoxifen while ERβ homodimers were inactive, which is in agreement with previous studies (20,39,54). In two parallel studies of transcriptional activity of ERα/ERβ heterodimers, however, the partial agonism of 4-OH-tamoxifen in the presence of ERα alone was completely suppressed by the coexpression of ERβ (40) and moreover, the presence of ERβ potentiated the antagonism of tamoxifen (66). The discrepancy in the results may depend on the different cells and promoters used and the relative levels of ERα and ERβ expressed in the cells, but also on the use of different DNA response element 1/ 2-site sequences and conﬁgurations. Tremblay et al. (51) also showed that MAPK pathway-dependent phosphorylation of both ERα and ERβ AF1 domains in ERα/ERβ heterodimers resulted in an enhanced transcriptional activity in the presence of E2 but that a single responsive MAPK phosphorylation site within the heterodimer is sufﬁcient for this effect. Furthermore, they showed that within the context of an ERα/ERβ heterodimer it is sufﬁcient for one ER monomer to bind E2 to get transcriptional activation but that the core of the AF2 must be intact in both monomers of the heterodimer to activate transcription (51). This may sound somewhat contradictory but could be explained by allosteric interactions between ERα and ERβ in the heterodimer and the stoichiometry of coactivator protein and ER heterodimer of 1:1 being sufﬁcient for transcriptional activity (51). Binding of E2 to one of the monomers may induce an ordered structure of its partner such that two NR-box motifs of the same coactivator can interact with each of the partners in the heterodimer, but if AF2 of one of the partners in the heterodimer is not intact the coactivator can only make one interaction via one of its LXXLL motifs with the heterodimer, which is too weak in strength to result in transcriptional activation by the liganded receptors. The scenario requiring one coactivator to make two contacts with the ER heterodimer, one with ERα and one with ERβ, to result in transcriptional activity is supported by the transcriptional suppression of ERα/ERβ heterodimers by an ERβ-speciﬁc LXXLL peptide (62). This ERβ-speciﬁc NR-box peptide was shown to inhibit E2-dependent transcriptional activity by ERβ homodimers and by ERα/ERβ heterodimers but not by ERα homodimers. Thus, disruption of one of two contact points made by a coactivator with the heterodimer may be sufﬁcient to inhibit the activity of the heterodimer (62) in analogy with the interpretation of the results by Tremblay et al. (51). In other studies of ERα/ERβ heterodimer activity (40,66) it was found that gradually increased expression levels of ERβ suppressed ERα transcriptional efﬁcacy. However, this effect was only seen at ERβ suboptimal hormone levels. At high E2 levels, sufﬁcient to saturate ERβ, the transcriptional activity of ERα was unaffected. In addition, the potency of E2-dependent ERα activity was shifted to the right in the presence of ERβ. These data support the conclusion that one role of ERβ is to modulate the transcriptional activity of ERα in cells where ERα and ERβ is coexpressed. As one possible mechanistic explanation for ERβ’s ERα modulatory activity it was shown that ERβ can bind constitutively, in the absence of hormone, to an ERE within the target gene promoter thereby decreasing the accessibility for ERα to DNA until levels of hormone are high enough to activate ERβ also (40,62). Different criteria have been used to classify ligands to ER as agonists or antagonists (67). During the last 10–15 years characterization of the transcriptional effect of ligands

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Fig. 7. When an agonist becomes an antagonist and an antagonist becomes an agonist. (A) By the ERE-dependent mechanism only E2 behaves as an agonist, stimulating gene expression. The SERMs raloxifene and tamoxifen show partial agonism when bound to ERα but no transcription stimulation by ERβ. The pure antagonist ICI 164,384 is unable to stimulate gene expression through either ER. (B) At an AP1 site not only E2 but also tamoxifen and ICI 164,384 act as efﬁcacious agonists in the presence of ERα. In contrast, E2 shows no agonism on an AP1 site when ERβ is present but both SERMs, and in particular raloxifene, and the pure antagonist ICI 164,384 have strong agonist activity. Thus, depending on the mechanism of gene regulation a ligand may appear as agonist or antagonist.

on ERE-containing reporter gene constructs have conveniently been used for identiﬁcation and classiﬁcation of natural and synthetic ER ligands as agonists or antagonists (54,68). Over the more recent years, however, we have learned that ER target-gene transcription regulation via binding to an ERE in the promoter region of estrogen sensitive genes is only part of the story. Today ER is known to regulate gene expression also by more indirect mechanisms, and therefore the terms agonist and antagonist should perhaps be used with more thoughtfulness (Fig. 7). By blocking the ability of the transcription factor NFκB [nuclear factor κ-B, a heterodimeric complex of the proteins p50 and p65 (RelA, c-rel, and other members containing the so-called Rel homology domain)] to bind to its response element on DNA (69–71) E2 can inhibit, for example, IL-6 cytokine gene expression. It was reported that it is the interaction between the ERα and the c-rel subunit of the NFκB complex that prevents NFκB from stimulating IL-6 expression through binding to its site in the IL-6 promoter (71). In a more recent study it was shown that both ERα

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and ERβ are able to transrepress RelA-dependent activation of ICAM-tk-luciferase expression in response to E2 (63). Mutational analysis of the importance of the ERα LBD indicated that distinct surfaces within the LBD are involved in transrepression of RelA activity. In particular, mutation of the hydrophobic residues at the N-terminal end of H11, which is part of the receptor dimerization interface (Fig. 6), rendered ERα incapable of down-regulating RelA transcriptional activity, suggesting an important role for the ER dimerization interface both in transactivation and transrepression. Furthermore, some of the residues within helixes that form part of the AF2 coactivator interaction surface are implicated in transrepression of RelA (63). However, the mechanism did not seem to involve competition for coactivator binding. Another mechanism has been described for target gene regulation by indirect binding of the ERs to DNA via physical interaction with another transcription factor (72–74). Using gel mobility shift assays it was shown that full-length ER or ER with intact Nor C-terminal domains could enhance the binding of the transcription factor Sp1 to its response element in an E2-independent fashion. In cells, however, Sp1-mediated reporter-gene expression depended on not only ER but also E2. That the ER and the Sp1 transcription factor interact physically was conﬁrmed by in vitro immunoprecipitation and pull-down experiments (73). Both ERα and ERβ have been shown to activate retinoic acid receptor α1 (RARα1) gene expression, presumably by the formation of an ER/Sp1 complex on GC-rich Sp1 sites in the RARα1 promoter (75,76). Interestingly, in the study of Zou et al., ERβ activated RARα1 promoter reporter constructs in the presence of estrogen antagonists such as 4-OH-tamoxifen, raloxifene and ICI 164,384, but not in the presence of E2, which instead blocked the effect of the antagonists (76). In MCF-7 human breast cancer cells, antiestrogens, but not E2, were shown to activate the transcription of the quinone reductase gene to increase nicotinamide adenine dinucleotide phosphate (NADPH)/quinoneoxidoreductase enzyme activity (77). The transcriptional effect of the ERs on the quinoneoxidoreductase gene was reported to be mediated via an electrophilic/antioxidant response element (EpRE/ARE). Furthermore, it was shown that ERβ was more efﬁcacious than ERα in stimulating gene expression from EpRE/ARE-containing reporter gene constructs (78) and that E2 antagonized the transcription-stimulatory effect of antiestrogens. The LIM/Homeodomain protein Islet-1 (member of a subset of homeodomaincontaining transcription factors deﬁned on basis of a common LIM domain; the acronym LIM is derived from the ﬁrst identiﬁed member of this family) is coexpressed with and physically interacts with the ER in vivo in the rat central nervous system (79). In vitro Islet-1 (ISL1) inhibits homodimerization of the ER, decreases ER/DNA interaction, and interferes with E2-dependent transactivation from an ERE-containing reporter gene, probably as a consequence of ER dimerization interference by ISL1. Interestingly, however, on reporter-gene constructs containing only binding sites for ISL1, coexpression of ER resulted in E2-dependent activation of reporter gene expression. As suggested by the authors the ER most likely functioned as a coactivator, ISL1 tethering an ER monomer to the DNA bound ISL1 protein. In addition, on E2 binding to the ER accompanied by repositioning of H12 into its agonist position, ER now may serve as a platform for recruitment of coactivators and potentiation of transcription activation (79). Based on data presented in the article the authors depicted three possible scenarios of crosstalk between ISL1 and ER that may explain their results mechanistically.

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Both ERα and ERβ can trigger AP1 (fos/jun)-dependent transcription with, however, requirement for different types of ligands (80,81). Whereas E2, DES, and the partial agonist/antagonist tamoxifen functioned as equally efﬁcacious agonists in the AP1 pathway in the presence of ERα, raloxifene showed only partial agonist activity. In contrast, in the presence of ERβ the antiestrogens tamoxifen and raloxifene were strong activators of AP1-dependent transcription whereas estradiol acted as an antagonist, inhibiting the activity of both tamoxifen and raloxifene (81). Mechanistic analysis to explain the ER subtype-speciﬁc and ligand-type-speciﬁc activity at AP1 sites resulted in two models, the AF1/AF2-dependent pathway (ERα in the presence of, e.g., E2, DES, and tamoxifen) and the AF1/AF2-independent but DBD-dependent pathway (ERα or ERβ in the presence of raloxifene and ICI 182,780) (82,83). In the AF1/AF2dependent pathway the authors do not foresee a direct interaction between ERα and the fos/jun heterodimer but rather an indirect interaction with the CBP/p300/p160 coactivator complex recruited by the fos/jun dimer. The authors propose that binding of ERα, via its AF1 and AF2 surfaces, to one of the p160 members (e.g. GRIP-1) of the fos/jun multiprotein complex triggers the coactivators to a higher state of transactivation potential (83). In the other, DBD-dependent pathway, it was proposed that ER activates AP1-dependent transcription by a remote mechanism without any participation in the fos/jun cofactor complex. Instead ER in complex with antagonists (e.g. ICI 182,780) or SERMs (e.g. raloxifene) attracts complexes of corepressors and histone deacetylases, pulling them away from the AP1 complex, thus allowing histone acetylases to act without opposition; that is, this pathway triggers AP1-dependent transcription by release of suppression (82,83).

FUTURE DIRECTIONS Although we know a great deal about ER domain structure and function and different modes by which the ERs regulate target-gene expression there are still many knowledge gaps to be ﬁlled. What does the overall 3D structure of full length homo- and heterodimers of ERα or ERβ look like?; what does it look like when bound to DNA; in complex with different agonists, antagonists, and SERMs; in complex with coactivators (not only NR-box motifs), corepressors, and other transcription factors with which the ERs interact? Perhaps more important, though, how should we be able to monitor and predict surface changes of the receptor and subsequently the cellular response to different agonists and antagonists? Technology to indirectly study surface changes induced by different ligands is available (84). This technology relies on the fact that peptides with different primary amino acid sequence can interact, more or less well, with agonist or antagonist bound ER depending on surfaces formed (or destroyed) as a result of type of ligand bound (agonist or antagonist) and the ligand-speciﬁc receptor conformational change induced (52,56,57). These peptides are likely to represent different proteins (e.g. transcription factors or coactivators) within the cell with which liganded ER interacts in order to transmit the ligand-speciﬁc signal. Solving 3D structures of various ER/ligand/peptide complexes may increase our understanding of how speciﬁc surface changes of the ERs are interpreted by different cell types, what impact these surface changes may have on the behavior of the cell, why certain receptor surface changes result in transactivation, and why certain receptor surface changes result in transrepression. After all, it is the surface of the receptor, modeled by the bound ligand, and not

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the afﬁnity of the ligand for the receptor per se, that the cellular machinery interprets before responding.

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49. Pace P, Taylor J, Suntharalingam S, Coombes RC, Ali S. Human estrogen receptor β binds DNA in a manner similar to and dimerizes with estrogen receptor α. J Biol Chem 1997;272:25832–25838. ˙ . Mouse estrogen receptor β forms estrogen 50. Pettersson K, Grandien K, Kuiper GGJM, Gustafsson J-A response element-binding heterodimers with estrogen receptor α. Mol Endocrinol 1997;11:1486–1496. 51. Tremblay GB, Tremblay A, Labrie F, Giguere V. Dominant activity of activation function 1 (AF-1) and differential stoichiometric requirements for AF-1 and -2 in the estrogen receptor alpha-beta heterodimeric complex. Mol Cell Biol 1999;19:1919–1927. 52. Pike ACW, Brzozowski AM, Hubbard RE, et al. Structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO J 1999;18:4608–4618. ˙ . Comparison 53. Kuiper GGJM, Carlsson B, Grandien K, Enmark E, Ha¨ggblad J, Nilsson S, Gustafsson J-A of the ligand binding speciﬁcity and transcript tissue distribution of estrogen receptors α and β. Endocrinol 1997;138:863–870. ˙ , Nilsson S. Differential response of 54. Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson J-A estrogen receptor α and estrogen receptor β to partial estrogen agonists/antagonists. Mol Pharmacol 1998;54:105–112. 55. McDonnell DP, Clemm DL, Hermann T, Goldman ME, Pike JW. Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol Endocrinol 1995;9:659–669. 56. Brzozowski AM, Pike ACW, Dauter Z, et al. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 1997;389:753–758. 57. Shiau AK, Barstad D, Loria PM, et al. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 1998;95:927–937. 58. Gee AC, Carlson KE, Martini PGV, Katzenellenbogen BS, Katzenellenbogen JA. Coactivator peptides have a differential stabilizing effect on the binding of estrogens and antiestrogens with the estrogen receptor. Mol Endocrinol 1999;13:1912–1923. 59. Meyers MJ, Sun J, Carlson KE, Katzenellenbogen BS, Katzenellenbogen JA. Estrogen receptor subtype-selective ligands: asymmetric synthesis and biological evaluation of cis- and trans-5,11dialkyl-5,6,11, 12-tetrahydrochrysenes. J Med Chem 1999;42:2456–2468. 60. Wijayaratne AL, Nagel SC, Paige LA, et al. Comparative analyses of mechanistic differences among antiestrogens. Endocrinology 1999;140:5828–5840. 61. Chang C-Y, Norris JD, Grøn H, et al. Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors α and β. Mol Cell Biol 1999;19:8226–8239. 62. Hall J, Chang C-Y, McDonnell DP. Development of peptide antagonists that target estrogen receptor β-coactivator interactions. Mol Endocrinol 2000;14:2010–2023. 63. Valentine JE, Kalkhoven E, White R, Hoare S, Parker MG. Mutations in the estrogen receptor ligand binding domain discriminate between hormone-dependent transactivation and transrepression. J Biol Chem 2000;275:25322–25329. 64. Montano MM, Ekena K, Krueger KD, Keller AL, Katzenellenbogen BS. Human estrogen receptor ligand activity inversion mutants: receptors that interpret antiestrogens as estrogens and estrogens as antiestrogens and discriminate among different antiestrogens. Mol Endocrinol 1996;10:230–242. 65. Montano MM, Moller V, Trobaugh A, Katzenellenbogen BS. The carboxy-terminal F domain of the human estrogen receptor: role of the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists. Mol Endocrinol 1995;9:814–825. ˙ . Estrogen receptor β acts as a dominant regulator of estrogen 66. Pettersson K, Delaunay F, Gustafsson J-A signalling. Oncogene 2000;19:4970–4978. 67. Jordan VC. Biochemical pharmacology of antiestrogen action. Pharm Rev 1984;36:245–276. 68. McDonnell DP, Vegeto E, Gleeson MAG. Nuclear hormone receptors as targets for new drug discovery. Bio/Technology 1993;11:1256–1261. 69. Ray A, Prefontaine KE, Ray P. Down-modulation of interleukin-6 gene expression by 17β-estradiol in the absence of high afﬁnity DNA binding by the estrogen receptor. J Biol Chem 1994;269:12940– 12946. 70. Stein B, Yang MX. Repression of the interleukin-6 promoter by estrogen receptor is mediated by NFκB and C/EBPβ. Mol Cell Biol 1995;15:4971–4979. 71. Galien R, Garcia T. Estrogen receptor impairs interleukin-6 expression by preventing protein binding on the NF-kappaB site. Nucleic Acids Res 1997;25:2424–2429. 72. Batistuzzo de Medeiros SR, Krey G, Hihi AK, Wahli W. Functional interaction between the estrogen

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Nilsson and Gustafsson receptor and the transcription activator Sp1 regulates the estrogen-dependent transcriptional activity of the vitellogenin A1 io promoter. J Biol Chem 1997;272:18,250–18,260. Porter W, Saville B, Hoivik D, Safe S. Functional synergy between the transcription factor Sp1 and the estrogen receptor. Mol Endocrinol 1997;11:1569–1580. Qin C, Singh P, Safe S. Transcriptional activation of insulin-like growth factor-binding protein-4 by 17β-estradiol in MCF-7 cells: Role of estrogen receptor-SP1 complexes. Endocrinology 1999;140: 2501–2508. Sun G, Porter W, Safe S. Estrogen-induced retinoic acid receptor α1 gene expression: role of estrogen receptor Sp1 complex. Mol Endocrinol 1998;12:882–890. Zou A, Marschke KB, Arnold KE, et al. Estrogen receptor β activates the human retinoic acid receptor α-1 promoter in response to tamoxifen and other estrogen receptor antagonists, but not in response to estrogen. Mol Endocrinol 1999;13:418–430. Montano MM, Katzenellenbogen BS. The quinone reductase gene: a unique estrogen receptor-regulated gene that is activated by antiestrogens. Proc Natl Acad Sci 1997;94:2581–2586. Montano MM, Jaiswal AK, Katzenellenbogen BS. Transcriptional regulation of the human quinone reductase gene by antiestrogen-liganded estrogen receptor-α and estrogen receptor β. J Biol Chem 1998;273:25443–25449. Gay F, Anglade I, Gong Z, Salbert G. The LIM/Homeodomain protein Islet-1 modulates estrogen receptor functions. Mol Endocrinol 2000;14:1627–1648. Webb P, Lopez GN, Uht RM, Kushner PJ. Tamoxifen activation of the estrogen receptor/AP1 pathway: potential origin for the cell-speciﬁc estrogen-like effects of antiestrogens. Mol Endocrinol 1995; 9:443–456. ˙ , Kushner PJ, Scanlan TS. Differential Paech K, Webb P, Kuiper GGJM, Nilsson S, Gustafsson J-A ligand activation of estrogen receptors ERα and ERβ at AP1 sites. Science 1997;277:1508–1510. Webb P, Nguyen P, Valentine C, et al. The estrogen receptor enhances AP-1 activity by two distinct mechanisms with different requirements for receptor transactivation functions. Mol Endocrinol 1999;13:1672–1685. Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P. Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 2000;74:311–317. Paige LA, Christensen DJ, Gron H, et al. Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta. Proc Natl Acad Sci 1999;96:3999–4004.

2

Ligand-Induced Conformational Changes in Estrogen Receptors-␣ and -␤ Elizabeth A. Allegretto,

PHD

Contents Abstract Introduction Protease Digestion Experiments ER Crystal Structures Peptide Phage Display Technology References

ABSTRACT Since the cloning of the second known estrogen receptor (ER), termed ERβ, there have been efforts to reevaluate estrogen signaling. ERα and ERβ are generated from separate genes and have marked nucleotide- and protein-sequence differences. Human ERα and ERβ share approx 96% amino-acid sequence identity in the DNA-binding domain (DBD), approx 53% sequence identity in the ligand-binding domain (LBD), and only about 30% identity in the amino terminal region. While both receptors bind to 17β-estradiol with equal afﬁnity (Kd ~ 0.5 nM) there are compounds that bind with varying afﬁnities to the two receptors. The biology of ERα and ERβ are likely to be quite different based on their tissue distribution. Additionally, transgenic mice that do not express either ERα or ERβ display distinct phenotypes. Because ERα and ERβ bind to endogenous estrogens with apparent equal afﬁnity, their ability to activate genes differently based on promoter context and/or cell-type context might be mediated by their ability to assume different conformations upon binding to the same and/or different ligands, thereby attracting different cofactor proteins and resulting in distinct biological activities. Partial proteolytic enzyme digestion has been used to detect differences in agonist-bound versus antagonist-bound receptor conformations. Additionally, the X-ray crystal structures of ligand-occupied ERα and ERβ LBDs show that clear changes occur in the receptors on binding to different classes of compounds. To date, however, the most sensitive technique for garnering

From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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Allegretto

information about subtle conformational changes induced by ligands in ERα and ERβ has been peptide phage display.

INTRODUCTION ERα and ERβ are similar to each other in that they both bind to endogenous estrogens with approximately equal afﬁnity and they both stimulate transcription from an estrogenresponsive element (ERE) in the presence of estradiol in cotransfection experiments (1–3). Differences between the two have emerged as efforts to study the receptors have increased. ERα and ERβ have been found to regulate transcription from activator protein 1 (AP-1) elements in a different manner (4). While estradiol acted as an agonist through ERα to stimulate transcription from a synthetic AP-1 element, it was inactive through ERβ, while ER antagonists such as 4-hydroxy-tamoxifen (4-OH-Tam), raloxifene, and ICI-164,384 acted as agonists through ERβ from an AP-1 site (4). Since then, several other groups have shown that ERα and ERβ exhibit different characteristics on various promoters in cotransfection assays (5–8). Cell and tissue distribution of ERα and ERβ are also quite different, with ERα being highly expressed in classical estrogen target tissues (mammary gland, uterus) and ERβ showing high levels of expression in ovary, prostate, thymus, and testis (2,9). Both receptors are also expressed in other cells such as those from brain, bone, and breast cancer (10–15). The differential promoter activity and cell-type expression of ERα and ERβ imply that various ligands may induce distinct conformational changes in ERα and ERβ that then allow binding of different coactivators or corepressors (see ref. 16 for review), ultimately accounting for their unique pharmacology. Different classes of ligands are known to bind to ERα with similar afﬁnity, but exert different activities depending on the promoter or cell context. For example, the known ER antagonists exhibit various proﬁles of activities. Some are classiﬁed as pure ERα antagonists, such as ICI-164,384 and ICI-182,180, which seem to block the actions of estradiol in all tissues tested (17). Other ER antagonists such as raloxifene and tamoxifen are classiﬁed as selective estrogen receptor modulators (SERMs) since they act as antagonists in the breast, but agonists in bone (18–22). Additionally, tamoxifen is a partial agonist in uterus (23) while raloxifene is not (19,21). The hypothesis that different ligand-induced receptor conformations correlate with diverse biology has been tested by various methods. Protease digestion was the ﬁrst method utilized to study this prior to cloning of the ERs (24). With the cloning of ERα in 1986 (25) and ERβ in 1996 (1,2) protein overexpression and crystal structure determination was made possible and yielded information on the overall structures of the ER LBDs with agonists or antagonists bound (26–28a). More recently, the use of peptide phage display technology (29) has enabled the mapping of minute changes in receptor conformation induced by different ligands (30–36).

PROTEASE DIGESTION EXPERIMENTS Workers ﬁrst started to probe intracellular receptor structure/function relationships by use of limited proteolytic enzyme digestion in the late 1970s. Glucocorticoid receptor (37), progesterone receptor (38), ER (39), and vitamin-D receptor (40) were subjected to partial digestion and the resulting fragments were analyzed in the effort to gather information on functional domain alignment and modularity prior to the cloning of the

Chapter 2 / Conformational Changes in ERα and β

21

receptors. The ﬁrst study in which this method was used to probe structural aspects of ERα in the presence of different ligands was performed in 1986 by Attardi and Happe (24). Rat uterine ER was radiolabeled in vivo with either the ER agonists [3H]-estradiol or [3H]-DES, or the ER antagonist [3H]-4-OH-Tam. Protein extracts were prepared and submitted to partial digestion with chymotrypsin and the resultant fragments from each labeled receptor were then separated by sucrose gradient sedimentation. Attardi and Happe found that the DES- and estradiol-labeled ER sedimentation patterns were similar to each other, but different from that of the 4-OH-Tam-ER complex. They concluded that because the agonist- and antagonist-bound ERs were differentially sensitive to limited proteolysis, the two classes of ligands were inducing different conformations in the receptor protein. They proposed that these different conformations might inﬂuence the interaction of receptor with DNA or chromatin, and hence their biological activity. Beekman et al., and McDonnell et al. extended these early ﬁndings using in vitro translated, [35S]-labeled recombinant ERα (41,42). ERα was incubated with estradiol or with ER antagonists of varying biological properties, including 4-OH-Tam, ICI164,384, and raloxifene, and then subjected to limited protease digestion and SDSPAGE (41,42). These three ER antagonists showed different activities in cotransfection assays in a promoter- and cell-dependent manner and therefore were hypothesized to induce different conformations in ERα (42,43). Discrete differences were observed in the digestion patterns of ER bound to estradiol versus ER complexed with any of the ER antagonists. However, the technique was unable to discriminate between any of the three antagonists based on the protease digestion patterns of their complexes with receptor (41,42). With the cloning of ERβ, workers compared ERα and ERβ using protease digestion techniques. Two groups showed that ligand-bound ERβ was more resistant to proteolytic cleavage than holo ERα (44,45). There was not a clear difference between the proteolytic digestion patterns of ERα and ERβ bound to any of the three ER antagonists tested (tamoxifen, ICI-164,384, and ICI-182,780) (44). With the discovery of ligands that interact differentially with each of the ER subtypes, work was done to compare these ligands using tryptic mapping. One study tested compounds with selectivity for ERα versus ERβ and vice versa using [35S]-labeled ERα and ERβ (45). These compounds included a pair of tetrahydrochrysenes, S,S-THC and R,R-THC, the S,S being an agonist on both ERα and ERβ and the R,R version an agonist through ERα, but an antagonist on ERβ. Also tested was propyl pyrazole triol (PPT) which is a potent and efﬁcacious ERα agonist and a weak ERβ antagonist. The three compounds were indistinguishable from estradiol in a tryptic digest of ERα. The R,R-THC and PPT compounds yielded similar patterns to ICI-182,780 when bound to ERβ whereas the S,S-THC-ERβ pattern was similar to estradiol bound to ERβ. Therefore, these experiments were able to differentiate antagonists from agonists bound to each of the receptors, but there were no discernable differences observed between the three agonists bound to ERα or between the two antagonists bound to ERβ. Although there were observed differences in the ability of these compounds to recruit coactivators to the receptors, any potential correlative conformational changes in the receptors were not detectable using protease digestion experiments (45). In summary, protease digestion experiments with ERα and ERβ bound to various ligands enabled the observation of crude conformational changes induced in the receptors by agonists versus antagonists. However, this method has not been useful to date in

22

Allegretto

discerning potential subtle differences in receptor conformation that would be hypothesized to take place based on the different biological activities of the various ligands within each compound class.

ER CRYSTAL STRUCTURES The crystal structures of the ERα LBD bound to estradiol or raloxifene were solved in 1997 (26). In 1998 ERα LBD was cocrystallized with a peptide from the receptor coactivator, glucocorticoid receptor interacting protein (GRIP-1), in the presence of the ER agonist DES, and the ERα LBD structure with 4-OH-Tam was solved (27). In 1999 the ERβ LBD structures with genistein and raloxifene were solved (28), and in 2001 the structure of the ICI-164,384-ERβ LBD complex was determined (28a). These structural studies demonstrated directly that there are dramatic differences between ER bound to agonist and ER bound to antagonist. The agonists and antagonists bind in the same pocket of the protein core, but result in distinct conformations. ERα LBD bound to estradiol or DES resulted in a structure with helix 12 of the ERα LBD ﬁtting tightly over the binding domain cavity where the ligand is situated (26,27). In the ERα LBD antagonist-bound structures (4-OH-Tam and raloxifene) the binding of ligand prevented the alignment of helix 12 over the core and it is repositioned (26,27). The agonist-induced position of helix 12 is thought to be necessary for formation of a competent activation-function-2 domain which allows interaction of receptors with coactivators (16,27). The DES-bound ERα LBD cocrystallized with an ER-interacting peptide sequence from GRIP-1 showed that the peptide bound to a hydrophobic groove formed in part by helix 12 on the surface of the DES-liganded ERα LBD (27). In the ER-4-OH-Tam structure, however, helix 12 of the LBD blocked this coactivator recognition groove by mimicking the interaction of the peptide with the receptor (27). The ERβ-raloxifene structure (28) is very similar to the ERα-raloxifene structure in that helix 12 is in the typical antagonist position not allowing coactivator to interact with receptor (26,27). In contrast to raloxifene, the binding of the “pure” antagonist, ICI-164,384, to ERβ prevented helix 12 from interaction with the ERβ LBD, hence completely destabilizing helix 12, which may help to explain its full antagonist proﬁle (28a). It will be of interest to compare this structure with that of ERα bound to pure antagonist, once it is determined. Genistein binds with higher afﬁnity to ERβ than ERα (9,46) and has been shown to be an ERβ-selective agonist in transfection assays (47). Genistein also exhibits estrogenic activities in vivo, causing increased uterine weight and decreased serum LDL (48), protection of smooth muscle vasculature (49) and protection against bone loss (50). The genistein-ERβ structure shows that helix 12 lies in a position more similar to antagonist-bound receptor than agonist-bound ERα (28). It is not clear why this would be the case, although it has been proposed that since genistein has shown less than 100% efﬁcacy in certain assays it may be a partial agonist (28). Additional crystal structures of ERβ bound to other agonists such as estradiol and ERα bound to genistein will be informative in this regard. The crystal structures of the ERs have afforded a molecular picture of how ligands interact with the receptor LBDs and have shown that clear conformational changes take place on binding of receptors to agonists versus antagonists. Although these structures have been instrumental in our understanding of receptor structure/function relationships, they don’t explain why raloxifene and 4-OH-Tam have different activities

Chapter 2 / Conformational Changes in ERα and β

23

in certain tissues or why estradiol can exert different activities through ERα and ERβ (see Introduction). One explanation would be that different receptor conformations are indeed induced by these ligands, but are not detectable by the protease digestion or crystal structure methods performed to date. To test this hypothesis, peptide phage display methodology was utilized.

PEPTIDE PHAGE DISPLAY TECHNOLOGY Peptide phage display methodology (29) has been used as a way to study distinct receptor-conformational changes induced in ERα and ERβ by a variety of ligands (30–36). It has been exquisitely sensitive to detecting subtle changes in receptor conformation induced with different ligands. The technique involves screening of libraries of M13 phage-displayed peptides using puriﬁed preparations of ERα or ERβ that have been immobilized on streptavidin-coated plates through a biotinylated ERE in the presence and absence of various ligands (30). Phage that were afﬁnity selected in this manner were then tested for interaction with each ER in the presence or absence of ligands by the use of anti-M13 antibodies in a phage ELISA. Time-resolved ﬂuorescence (TRF) assays were employed to demonstrate that the peptide portion of the phage was binding to the ERs by use of europium-labeled peptides (30). TRF was also used to perform dose-response studies of ligands in recruitment of peptides to ERα and ERβ and to quantitate the extent to which the various peptide-interacting surfaces are exposed in the ligand-ER complexes. The afﬁnity selection of phage by estradiol-bound ERα resulted in the identiﬁcation of several peptides that contained LXXLL motifs, the motif that is present in various receptor coactivator proteins that have been shown to interact with agonist-bound ER (16). Various other peptides that did not contain LXXLL motifs were also identiﬁed that preferred binding to unoccupied (apo) receptors or to 4-OH-Tam-bound ERs (30). Additionally, peptides that bound preferentially to either ERα or ERβ were detected. These peptides were then tested for binding to ERα and ERβ in the presence of several ER ligands. Each ligand tested altered the binding pattern of the peptides, yielding a distinct ﬁngerprint which was indicative of the different conformations induced by each of these ligands upon binding to the receptors (30,31). Additionally, peptides were identiﬁed that interacted differentially with ERα or ERβ in the presence of 4-OH-Tam or raloxifene (30). Several peptides showed preference for 4-OH-Tam-bound ERα over raloxifene-bound ERα, indicating for the ﬁrst time that there are conformational differences in the receptor structures induced by these two SERMs (see Table 1). Several of the SERMs are structurally similar triphenylethylene derivatives (4-OH-Tam, clomiphene, idoxifene, GW5638, GW7604), but induce different conformations in ERα as assessed by their ability to interact with various unique peptides, suggesting that even modest changes in ligand structure can affect receptor conformation (30–31a). GW5638 elicits distinct biology in comparison with these other compounds, and in fact it is in development for tamoxifen-resistant breast cancer (31a). This methodology has been extended to test the ability of ER-interacting peptides to affect receptor biology (32–35). In one study, peptides that interacted with ERα or ERβ in the presence of estradiol or 4-OH-Tam were tested for their ability to modulate ER-dependent transcriptional activity. Peptide-GAL4-DBD fusions were constructed and tested for their ability to inhibit ER transactivation from luciferase reporters driven

Table 1 Biological Activity and ER-Peptide Interaction Induction of Various ER Ligands Breast Uterus Bone Brain Peptide α/β I (30,31)

24

ERα ERβ Peptide α/β III (30,31) ERα ERβ Peptide α/β V (30,31) ERα ERβ Peptide α II (30,31) ERα ERβ EBIP-49 (34) ERβ EBIP-53 (34) EBIP-92 (34) ERβ

17β-Estradiol

Genistein

4-OH-Tam

Raloxifene

ICI-182,780

Agonist Agonist Agonist Agonist ++

Agonist (52) Agonist (48) Agonist (50) Agonist (52) ND

Antagonist (18) Partial Agonist (23) Agonist (20) Antagonist (55) 0

Antagonist (19) Antagonist (19,21,54) Agonist (22) Antagonist (56) 0

Antagonist (17,53) Antagonist (17,53) ? (53a) Not activea (53) 0

−

ND

++

+

−

0

ND

++

0

0

++ ++ ++ +

ND ++ + ++

+ 0 0 0

+ 0 0 0

+ 0 0 0

a ICI-182,780 is thought to be inactive in the brain because of its inability to cross the blood-brain barrier (57). Plus and minus signs indicate approximate relative efﬁcacy in changing interaction of peptide with receptor by the designated ligand versus vehicle control (++: higher relative ﬂuorescence units (RFU) induced, +: lower efﬁcacy of induction, 0: no change in interaction, −: decrease in RFU, ND: not determined).

Chapter 2 / Conformational Changes in ERα and β

25

by the C3 promoter or the collagenase promoter in transfected Hep G2 cells. 4-OHTam and estradiol each act as agonists from these promoter constructs in this assay (32). Peptides that had been shown to interact with 4-OH-Tam-bound ERα in the phage ELISA or TRF assay inhibited 4-OH-Tam-induced luciferase activity, whereas those that interacted with estradiol-bound ERα in vitro had no effect on 4-OH-Tam-induced luciferase activity from either of the promoters (32). Likewise, the peptides that interacted with estradiol-bound ERα inhibited the estradiol-induced luciferase activity from these promoters, but not that stimulated by 4-OH-Tam. Additionally, a peptide that interacted with ERβ, but not ERα, was found to block estradiol-induced luciferase activity through ERβ from an ERE, but not through ERα (33). These experiments showed that the peptides that were identiﬁed by afﬁnity selection of phage libraries to interact in vitro with ERα or ERβ also interacted with the receptors in cells and were able to inhibit their transcriptional activity. Therefore, these peptides acted as ER antagonists by blocking the receptor/cofactor interaction in cells, opening up the possibility that deliverable peptides such as these might be useful as drugs for breast cancer or other conditions (32–34). The conformational changes observed in the ERs on binding to different ligands is thought to result in the recruitment of speciﬁc cofactors. Whereas a number of these cofactors have been shown to interact with multiple members of the intracellular receptor (IR) family (16), studies have shown that speciﬁcity for individual receptors can be conferred by the ﬂanking regions of the coactivator LXXLL motifs (51). While these types of mutational studies are informative, they are limited by the number of permutations that can be generated. The use of combinatorial phage display has been useful in circumventing this problem. One study involved screening a phage display library with more than 108 variations of peptides containing LXXLL motifs (33). Three classes of LXXLL-containing peptides were selected by ERα in the presence of estradiol. These peptides were tested against ERβ, and several other IRs. Each class of peptide showed preferences for different receptors, indicating that the ﬂanking regions of the LXXLL core sequence are important for speciﬁcity of IR/cofactor interactions. Although peptides had been identiﬁed that interacted with ERβ without binding to ERα (33) or to TRβ (36), peptides that were speciﬁc to ERβ without interacting with several other IRs were identiﬁed by screening the LXXLL-containing peptidecombinatorial phage library with ERβ protein (34). These peptides disrupted the action of ERβ in cells, but did not affect the activity of any of the other IRs, including ERα. These reagents may prove to be instrumental in deciphering the action of ERβ versus ERα in cells or animals and may be useful in searching for novel ERβ-speciﬁc coactivators. Differences were observed in the ability of genistein and estradiol to interact with some of the ERβ-speciﬁc peptides, suggesting that there may be differences in the receptor conformations induced by genistein and estradiol (34, see Table 1). This is intriguing in light of the unique crystal structure of genistein-ERβ (28) and the interesting biological properties of genistein (28,48–50,52). Therefore, unique conformations are induced in ERα and ERβ with various compounds, resulting in the exposure of different receptor surfaces, some of which may be bona ﬁde interaction regions for speciﬁc coactivator proteins. This work supports the hypothesis that the diverse biological activities of various ER ligands may be caused in part by different receptor conformations induced by those compounds. The three techniques described have contributed to the concept that different ligands

26

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can induce distinct conformational changes in the ERs, allowing differential interaction with transcriptional accessory proteins, which may ultimately help determine the pharmacology of those ligands. The use of peptide phage display methodology could be extended in the future for screening libraries of compounds against a battery of identiﬁed ER-interacting peptides to ﬁnd unique ﬁngerprints. The ideal SERM is still an elusive entity, and several clinically useful SERM molecules might be designed with varying biological proﬁles, depending on the disease being targeted. New compounds with unique receptor conformational ﬁngerprints may help lead the way to discover novel SERMs with desirable proﬁles of activity.

REFERENCES 1. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 1996;93:5925–5930. 2. Mosselman S, Polman J, Dijkema R. ER beta: identiﬁcation and characterization of a novel human estrogen receptor. FEBS Lett 1996;392:49–53. 3. Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V. Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. Mol Endocrinol 1997;11:353–365. 4. Paech K, Webb P, Kuiper GG, et al. Differential ligand activation of estrogen receptors ER alpha and ER beta at AP1 sites. Science 1997;277:1508–1510. 5. Montano MM, Jaiswal AK, Katzenellenbogen BS. Transcriptional regulation of the human quinone reductase gene by antiestrogen-liganded estrogen receptor-alpha and estrogen receptor-beta. J Biol Chem 1998;273:25,443–25,449. 6. Zou A, Marschke KB, Arnold KE, et al. Estrogen receptor beta activates the human retinoic acid receptor alpha-1 promoter in response to tamoxifen and other estrogen receptor antagonists, but not in response to estrogen. Mol Endocrinol 1999;13:418–430. 7. Saville B, Wormke M, Wang F, et al. Ligand-, cell-, and estrogen receptor subtype (alpha/beta)dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 2000;275:5379–5387. 8. van den Wijngaard A, Mulder WR, Dijkema R, et al. Antiestrogens speciﬁcally up-regulate bone morphogenetic protein-4 promoter activity in human osteoblastic cells. Mol Endocrinol 2000; 14:623–633. 9. Kuiper GG, Carlsson B, Grandien K, et al. Comparison of the ligand binding speciﬁcity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 1997;138:863–870. 10. Shughrue PJ, Komm B, Merchenthaler I. The distribution of estrogen receptor-beta mRNA in the rat hypothalamus. Steroids 1996;61:678–681. 11. Onoe Y, Miyaura C, Ohta H, Nozawa S, Suda T. Expression of estrogen receptor beta in rat bone. Endocrinology 1997;138:4509–4512. 12. Windahl SH, Norgard M, Kuiper GG, Gustafsson JA, Andersson G. Cellular distribution of estrogen receptor beta in neonatal rat bone. Bone 2000;26:117–121. 13. Osterlund MK, Gustafsson JA, Keller E, Hurd YL. Estrogen receptor beta (ER beta) messenger ribonucleic acid (mRNA) expression within the human forebrain: distinct distribution pattern to ER alpha mRNA. J Clin Endocrinol Metab 2000;85:3840–3846. 14. Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS. Tissue distribution and quantitative analysis of estrogen receptor-alpha (ER alpha) and estrogen receptor-beta (ER beta) messenger ribonucleic acid in the wild-type and ER alpha-knockout mouse. Endocrinology 1997;138:4613–4621. 15. Jarvinen TA, Pelto-Huikko M, Holli K, Isola J. Estrogen receptor beta is coexpressed with ER alpha and PR and associated with nodal status, grade, and proliferation rate in breast cancer. Am J Pathol 2000;156:29–35. 16. Shibata H, Spencer TE, On˜ate SA, et al. Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action. Recent Prog Horm Res 1997;52:141–165. 17. Wakeling AE, Bowler J. Novel antioestrogens without partial agonist activity. J Steroid Biochem 1988;31:645–653. 18. Jordan VC. The strategic use of antiestrogens to control the development and growth of breast cancer. Cancer 1992;70:977–982.

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19. Cummings SR, Eckert S, Krueger KA, et al. The effect of raloxifene on risk of breast cancer in postmenopausal women: results from the MORE randomized trial. Multiple Outcomes of Raloxifene Evaluation. JAMA 1999;281:2189–97. 20. Love RR, Mazess RB, Barden HS, et al. Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer. N Engl J Med 1992;326:852–856. 21. Black LJ, Sato M, Rowley ER, et al. Raloxifene (LY139481 HCl) prevents bone loss and reduces serum cholesterol without causing uterine hypertrophy in ovariectomized rats. J Clin Invest 1994;93:63–69. 22. Johnston CC, Bjarnason NH, Cohen FJ, et al. Long-term effects of raloxifene on bone mineral density, bone turnover, and serum lipid levels in early postmenopausal women: three-year data from 2 doubleblind, randomized, placebo-controlled trials. Arch Intern Med 2000;160:3444–3450. 23. Kedar RP, Bourne TH, Powles TJ, et al. Effects of tamoxifen on uterus and ovaries of postmenopausal women in a randomized breast cancer prevention trial. Lancet 1994;343:1318–1321. 24. Attardi B, Happe HK. Comparison of the physiochemical properties of uterine nuclear estrogen receptors bound to estradiol or 4-hydroxytamoxifen. Endocrinology 1986;119:904–915. 25. Green S, Walter P, Greene G, et al. Cloning of the human oestrogen receptor cDNA. J Steroid Biochem 1986;24:77–83. 26. Brzozowski AM, Pike AC, Dauter Z, et al. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 1997;389:753–758. 27. Shiau AK, Barstad D, Loria PM, et al. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 1998;95:927–937. 28. Pike AC, Brzozowski AM, Hubbard RE, et al. Structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO J 1999;18:4608–4618. 28a.Pike AC, Brzozowski AM, Walton J, et al. Structural insights into the mode of action of a pure antiestrogen. Structure 2001;9:145–153. 29. Sparks AB, Adey NB, Cwirla S, Kay BK. In eds. Kay BK, Winter J, McCafferty J. Phage display of peptides and proteins, A Laboratory Manual. Academic, San Diego 1996; pp. 227–253. 30. Paige LA, Christensen DJ, Gron H, et al. Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta. Proc Natl Acad Sci USA 1999;96:3999–4004. 31. Wijayaratne AL, Nagel SC, Paige LA, et al. Comparative analyses of mechanistic differences among antiestrogens. Endocrinology 1999;140:5828–5840. 31a.Connor CE, Norris JD, Broadwater G, et al. Circumventing tamoxifen resistance in breast cancers using antiestrogens that induce unique conformational changes in the estrogen receptor. Cancer Res 2001;61:2917–2922. 32. Norris JD, Paige LA, Christensen DJ, et al. Peptide antagonists of the human estrogen receptor. Science 1999;285:744–746. 33. Chang Cy, Norris JD, Gron H, et al. Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta. Mol Cell Biol 1999;19:8226–8239. 34. Hall JM, Chang CY, McDonnell DP. Development of peptide antagonists that target estrogen receptor beta-coactivator interactions. Mol Endocrinol 2000;14:2010–2023. 35. Schaufele F, Chang CY, Liu W, et al. Temporally distinct and ligand-speciﬁc recruitment of nuclear receptor-interacting peptides and cofactors to subnuclear domains containing the estrogen receptor. Mol Endocrinol 2000;14:2024–2039. 36. Northrop JP, Nguyen D, Piplani S, et al. Selection of estrogen receptor beta- and thyroid hormone receptor beta-speciﬁc coactivator-mimetic peptides using recombinant peptide libraries. Mol Endocrinol 2000;14:605–622. 37. Wrange O, Gustafsson JA. Separation of the hormone- and DNA-binding sites of the hepatic glucocorticoid receptor by means of proteolysis. J Biol Chem 1978;253:856–865. 38. Schrader WT, Birnbaumer ME, Hughes MR, Weigel NL, Grody WW, O’Malley BW. Studies on the structure and function of the chicken progesterone receptor. Recent Prog Horm Res 1981;37:583–633. 39. Greene GL, Sobel NB, King WJ, Jensen EV. Immunochemical studies of estrogen receptors. J Steroid Biochem 1984;20:51–56. 40. Allegretto EA, Pike JW. Trypsin cleavage of chick 1,25-dihydroxyvitamin D3 receptors. Generation of discrete polypeptides which retain hormone but are unreactive to DNA and monoclonal antibody. J Biol Chem 1985;260:101,139–10,145. 41. Beekman JM, Allan GF, Tsai SY, Tsai MJ, O’Malley BW. Transcriptional activation by the estrogen

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receptor requires a conformational change in the ligand binding domain. Mol Endocrinol 1993;7:1266– 1274. 42. McDonnell DP, Clemm DL, Hermann T, Goldman ME, Pike JW. Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol Endocrinol 1995;9:659–669. 43. Tzukerman MT, Esty A, Santiso-Mere D, et al. Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 1994;8:21–30. 44. Van Den Bernd GJ, Kuiper GG, Pols HA, Van Leeuwen JP. Distinct effects on the conformation of estrogen receptor alpha and beta by both the antiestrogens ICI 164,384 and ICI 182,780 leading to opposite effects on receptor stability. Biochem Biophys Res Commun 1999;261:1–5. 45. Kraichely DM, Sun J, Katzenellenbogen JA, Katzenellenbogen BS. Conformational changes and coactivator recruitment by novel ligands for estrogen receptor-alpha and estrogen receptor-beta: correlations with biological character and distinct differences among SRC coactivator family members. Endocrinology 2000;141:3534–3445. 46. Witkowska HE, Carlquist M, Engstrom O, et al. Characterization of bacterially expressed rat estrogen receptor beta ligand binding domain by mass spectrometry: structural comparison with estrogen receptor alpha. Steroids 1997;62:621–631. 47. Kuiper GG, Lemmen JG, Carlsson B, et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 1998;139:4252–4263. 48. Dodge JA, Glasebrook AL, Magee DE, et al. Environmental estrogens: effects on cholesterol lowering and bone in the ovariectomized rat. J Steroid Biochem Mol Biol 1996;59:155–161. 49. Makela S, Savolainen H, Aavik E, et al. Differentiation between vasculoprotective and uterotrophic effects of ligands with different binding afﬁnities to estrogen receptors alpha and beta. Proc Natl Acad Sci USA 1999;96:7077–7082. 50. Ishimi Y, Miyaura C, Ohmura M, et al. Selective effects of genistein, a soybean isoﬂavone, on Blymphopoiesis and bone loss caused by estrogen deﬁciency. Endocrinology 1999;140:1893–1900. 51. McInerney EM, Rose DW, Flynn SE, et al. Determinants of coactivator LXXLL motif speciﬁcity in nuclear receptor transcriptional activation. Genes Dev 1998;12:3357–3368. 52. Santell RC, Chang YC, Nair MG, Helferich WG. Dietary genistein exerts estrogenic effects upon the uterus, mammary gland and the hypothalamic/pituitary axis in rats. J Nutr 1997;127:263–269. 53. Howell A, DeFriend DJ, Robertson JF, et al. Pharmacokinetics, pharmacological and anti-tumour effects of the speciﬁc anti-oestrogen ICI 182780 in women with advanced breast cancer. Br J Cancer 1996;74:300–308. 53a.Howell A, Osborne CK, Morris C, Wakeling AE. ICI 182,780 (Faslodex), development of a novel, “pure” antiestrogen. Cancer 2000;89:817–825. 54. Goldstein SR, Scheele WH, Rajagopalan SK, Wilkie JL, Walsh BW, Parsons AK. A 12-month comparative study of raloxifene, estrogen, and placebo on the postmenopausal endometrium. Obstet Gynecol 2000;95:95–103. 55. Cummings FJ, Gray R, Davis TE, et al. Tamoxifen versus placebo: double-blind adjuvant trial in elderly women with stage II breast cancer. NCI Monogr 1986;1:119–123. 56. Davies GC, Huster WJ, Lu Y, Plouffe L, Lakshmanan M. Adverse events reported by postmenopausal women in controlled trials with raloxifene. Obstet Gynecol 1999;93:558–565. 57. Wade GN, Blaustein JD, Gray JM, Meredith JM. ICI 182,780: a pure antiestrogen that affects behaviors and energy balance in rats without acting in the brain. Am J Physiol 1993;265:R1392–1398.

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Expression and Function of Estrogen Receptors-␣ and -␤ Jonathan Lindzey,

PH D

Contents Introduction Reproductive Tissues Nonreproductive Tissues Concluding Remarks Acknowledgments References

INTRODUCTION The crucial roles of estrogens in reproduction have been recognized and extensively studied for many years. Recent studies have also demonstrated that estrogens exert important effects on nonreproductive targets such as bone, the cardiovascular system, and neural sites involved in cognition. Historically, both reproductive and nonreproductive actions of 17β-estradiol (E2) were thought to be mediated by a single nuclear estrogen receptor (ERα) and possibly by membrane-bound ER. The recent description of a second nuclear ER (ERβ) (1,2) has greatly complicated our attempts to understand cellular mechanisms underlying physiological effects of estrogens. As described elsewhere in this volume, ERα and β exhibit substantial structural and functional homology. The ligand-binding domains have similar binding afﬁnities for E2 (3). Both receptors also have transactivating functions in the NH2 and COOH ends of the proteins, exhibit substantial homology in the DNA-binding domain and bind to identical estrogen response element sequences. In addition, ERα and β are able to form heterodimers (4). Despite similarities in structure and function of ERα and β, they exhibit different responses to certain ligands and can interact differently with transcription factors. For instance, ERβ binds to phytoestrogens with a higher afﬁnity than ERα (3). Also, E2/ERα complexes activate transcription at an AP1 site whereas E2-ERβ complexes inhibit transcription (5). Coupled with the potential for cellular colocalization and heterodimer formation, these context-dependent actions of ERα and β may provide for exceptionally complicated tissue-speciﬁc and gene-speciﬁc responses to estrogenic compounds. From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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A crucial ﬁrst step in distinguishing biological roles of ERα and β expression is to determine tissue- and cell-speciﬁc expression patterns for the two receptors. This review focuses on (1) comparisons of ERα and β expression patterns in reproductive and selected nonreproductive tissues, and (2) possible functions of these two receptors as inferred from patterns of expression and phenotypes of the recently developed estrogen receptor-α knockout (αERKO)(6) and estrogen receptor-β knockout (βERKO)(7) mouse models. Rather than providing an exhaustive review of the many studies examining ERα expression, this review focuses on more recent studies that provide comparisons of ERα and β expression patterns.

REPRODUCTIVE TISSUES Hypothalamus Estrogen action on the hypothalamus plays a critical role in the organization and activation of reproductive behaviors and physiology in both males and females. In many female vertebrates, E2 acts on the ventromedial hypothalamus to activate receptive behaviors such as the lordosis response in rats (8). In rodents, E2 also exerts negative and positive feedback on gonadotropin hormone-releasing hormone (GnRH) secretion by acting on the arcuate nucleus-median eminence and anterior hypothalamus-preopticperiventricular regions, respectively (9,10). Similarly, in male vertebrates, many of the behavioral and neuroendocrine effects of testosterone occur through aromatization into E2 and activation of ER in the anterior hypothalamus/medial preoptic area (MPO) (11). All of these crucial hypothalamic sites express ERα, β or both. Expression of ER␣ and ␤ in the Hypothalamus In ovariectomized wild-type and αERKO mice, in situ hybridization (ISH) detected ERβ mRNA in the MPO, suprachiasmatic nuclei (SCN), paraventricular nucleus (PVN), dorsomedial nucleus (DMN), medial tuberal nucleus, premammilary body, olfactory bulb, bed nucleus of the striate terminalis (BNST), and the amygdala (AMY) (12). Conversely, the ventromedial hypothalamus (VMH) and arcuate nucleus (AN) exhibited very low ISH signal. Autoradiography detected intense E2 labeling of cells in the preoptic area (POA), PVN, VMN, AN, and BST of WT mice (13,14). Although the total number of E2-labeled cells was reduced in αERKO females, labeled cells were detected in the POA, BNST, and PVN whereas very few cells in the VMH or AN were labeled. These data suggest that ERβ messenger RNA (mRNA) in the POA, BNST, and PVN is translated into functional receptor whereas the VMH and arcuate (ARC) express very little ERβ receptor in the mouse (14). In the female rat brain, ISH detected ERα mRNA in the BST, MPO, VMN, AN and medial amygdala (Fig. 1)(14–16). Signiﬁcant overlap in ERα and β mRNA expression was found in the MPO, BST, and medial amygdala (MeA) whereas very little ERβ was expressed in the VMN and AN (Fig. 1). Interestingly, the PVN and supraoptic nucleus (SON) expressed predominantly ERβ mRNA. Immunohistochemistry (IHC) determined that the major sites of ERβ immunoreactivity (IR) were in the lateral septum, BNST, PVN, SON and anterior medial amygdala (17). Some ERβ-IR cells were also found in the anterior hypothalamus, periventricular nucleus, MPOA and AN of both male and female rats. Thus, the distribution of ERβ-IR in female Sprague-Dawley rats is more circumscribed than ERβ mRNA expression. This discrepancy between mRNA

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FPO Print repla durin pre-p

Fig. 1. Autoradiograms of ERα (left) and ERβ (right) mRNA-positive cells in the rat brain by in situ hybridization. ERα mRNA is heavily expressed in the arcuate nucleus (AN) and ventromedial hypothalamic nucleus (VMN), whereas ERβ mRNA is largely absent from these nuclei. The paraventricular nucleus (PVN) and supraoptic nucleus (SO) express large amounts of ERβ mRNA whereas ERα mRNA is absent. The preoptic area (POA), bed nucleus of the striaterminalis (BST), and medial amygdala (MeA) express both ERα and β mRNAs. Other abbreviations: AN, anterior commissure; AHi, amygdalohippocampal area; CoA, cortical amygdala; DG, dentate gyrus; HIP, hippocampus; MTu, medial tuberal nucleus; ox, optic chiasm; 3V, third ventricle; ZI, zona incerta. (Reproduced with permission from Kuiper GG, Shughrue PJ, Merchenthaler I, Gustafsson JA. The estrogen receptor β subtype: a novel mediator of estrogen action in neuroendocrine systems. Front Neuroendocrinol 1998;19(4):253–286.)

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and protein expression may be caused by differences in transcription versus translation within hypothalamic regions, presence of splice variants not detected by available antibodies or, perhaps, technical issues such as antibody or probe quality. Using ISH in macaque monkeys, Gundlah et al. (18) reported high expression levels of ERβ mRNA in the POA, PVN and VMN with lower levels in the BNST, SON and mammillary bodies and very weak signal in the SCN and AN. Recent studies with macaque monkeys detected ERα/IR cells in the POA, PVN, VMN, ARC, ME (19,20), DMN, and periventricular nucleus of rhesus monkeys (20). The major differences between these patterns and the rodent are the higher density of ERβ mRNA in the VMN and the absence of ERβ mRNA signal in the anterior hypothalamus (AH), DMN, or medial tuberomammillary nucleus of the macaque. In addition, the macaque PVN expresses signiﬁcant amounts of ERα/IR whereas ERβ appear to be dominant in the PVN of rodents. In close agreement with rodent studies, ERβ mRNA was absent or at low levels in the human VMN and AN (21). However, in stark contrast to reports in other species, very low levels of ERβ (21) but high levels of ERα mRNA (22) were detected in the PVN and SON of humans. In addition, high levels of ERα mRNA were found in the AN and periventricular nucleus, with lower levels in the POA, AH, and VMH (22). Despite interspeciﬁc variability and some intraspeciﬁc inconsistencies between mRNA and protein expression, several useful patterns emerge from comparisons of the rodent and primate studies. First, the VMH and AN tend to express higher amounts of ERα compared to ERβ. Second, the PVN and SON in rodents almost exclusively express ERβ whereas primates express relatively greater amounts of ERα. Third, the POA tends to express both ERα and β in rodents and primates. Overlapping expression of ERα and β in the preoptic area, BNST and medial amygdala of rats raises the question of whether both receptors are coexpressed within the same cells (16,23). Indeed, in rats a high percentage of ERα/IR neurons within the periventricular preoptic nucleus, BNST, medial preoptic nucleus and medial amygdala express ERβ mRNA (24). This is particularly intriguing because of the presence of GnRH neurons in this region and the importance of the MPO for male sex behaviors. Although several studies failed to detect estrogen binding (25), ERα/IR (25), or ERβ mRNA (26) in GnRH neurons, an immortalized GnRH neuronal cell line (GT1-7) was recently reported to express mRNAs for both ERα and β. Furthermore, E2 downregulated GnRH mRNA levels in these cells (27). Recent in vivo work also demonstrated that a small population of rat GnRH neurons expresses ERα/IR (28) or ERα mRNA (29) whereas a larger percentage of GnRH neurons (~70%) in the MPO and MPN express ERβ mRNA (29). This same study used autoradiography to detect speciﬁc E2 binding within a small subset (~9%) of ERβ-positive GnRH neurons. Thus, contrary to earlier reports, some GnRH neurons may express ERα and/or β and, thereby, provide a mechanism for direct estrogen action on GnRH secretion. The expression of ERβ in rat SON and PVN is particularly interesting because oxytocin plays a critical role in lactation and parturition, and estrogens modulate oxytocin synthesis and secretion (30). It is now clear that arginine vasopressin (AVP) and oxytocin (OT) cells in rats express ERβ mRNA (31) and protein (32), with ERβOT and ERβ-AVP colocalization occurring primarily in the PVN and SON, respectively. This suggests a role for ERβ in regulating OT secretion in rodents. Nonetheless, βERKO mice exhibit no obvious defects in parturition or nursing that might be attributed to

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problems with the OT system (7). In addition, based on expression studies in primates, ERα may play a greater role in regulating the PVN and SON of primates. Functions of ER␣ and ␤ in the Hypothalamus The dominant expression of ERα in the AN of rodents and primates suggests that negative feedback effects of E2 on GnRH and LH secretion are mediated by ERα. This is supported by observations that serum LH levels [unpublished data, J. Lindzey; (33,34)] and pituitary luteinizing hormone (LH)β and follicle stimulating hormone (FSH)β mRNA levels (35) are dramatically elevated in female αERKO mice. Ovariectomy and E2 replacement studies further demonstrate that estrogen can suppress LHβ and FSHβ mRNA as well as serum LH and FSH in WT but not αERKO females (unpublished data, J. Lindzey). Furthermore, αERKO females are also acyclic and fail to exhibit LH surges (unpublished data, J. Lindzey). In contrast to αERKO females, preliminary reports ﬁnd that βERKO females exhibit normal cycling and normal basal LH and FSH levels (7). Thus, ERα is clearly responsible for E2 suppression of gonadotropins in female mice and, given the conserved pattern of high ERα expression in the AN, it is likely that this holds true for other species. ERα may also play a critical role in positive feedback effects of E2 involved in generating a preovulatory surge. Despite these data, several lines of evidence suggest that ERβ may play a role in regulating GnRH synthesis and secretion and hence serum LH levels. First, ERβ is coexpressed with ERα in MPO and periventricular regions involved in synthesizing and regulating secretion of GnRH (32). Second, ERβ is expressed in a subset of GnRH neurons and may therefore exert direct effects on GnRH neurons (29). Third, E2 treatments of αERKO females have been reported to increase serum LH levels (34). Although this suggests that ERβ might mediate some positive feedback effects of E2 on GnRH and LH in rodents, βERKO females exhibit spontaneous ovulation and can therefore be assumed to exhibit relatively normal patterns of gonadotropin synthesis and secretion (7). It is possible, however, that βERKO females may exhibit subtle changes in amplitude or timing of LH surges. In male vertebrates, the ability of testicular androgens to feedback and suppress gonadotropins is thought to rely partly on aromatization and activation of ER. Intact, male αERKO mice exhibit only 2-fold higher serum LH levels compared with 5- to 6-fold elevations following castration. Serum gonadotropins in αERKO males are also completely insensitive to exogenous E2 treatments (36). This suggests that ERβ does not mediate negative feedback effects on LH synthesis and secretion in male mice. Indeed, relatively normal serum-LH levels are maintained because androgen receptor signaling mechanisms effectively regulate hypothalamic GnRH and serum LH (34,36). This ﬁnding is consistent with observations that testicular feminized male (Tfm) mice have elevated serum LH (37). In humans, a male patient with an ERα mutation (38) and some patients with androgen insensitivity syndrome (AIS) (39) exhibit elevated serum LH. Thus, it appears that males rely both on ERα and androgen receptor (AR) for negative regulation of gonadotropins whereas ERβ plays no obvious role. Substantial ERα expression in the VMH of rodents and primates indicates that ERα probably plays a dominant role in controlling female sex behaviors. This is supported by an absence of receptive, proceptive, and maternal behaviors in intact αERKO females (40) or ovariectomized and estrogen-treated αERKO females (41,42). In addition, progesterone (P) appears to have little effect on receptive or proceptive behaviors in

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αERKO mice (41). By contrast, sex behaviors in intact βERKO females are normal (43) and maternal behaviors in βERKOs are sufﬁciently normal to rear young. Thus, ERα mediates the effects of E2 on receptivity, proceptivity, and parental behaviors in female mice. Because ERα is expressed at relatively high levels in the VMH of both rats and primates, it seems likely that ERα also regulates female reproductive behaviors in these species. The presence of both ERα and β in the AH region of rodents, suggests that both receptors may mediate some aspects of male sex behaviors. However, comparisons of αERKO and βERKO male behaviors indicate that ERα and androgen receptors mediate the actions of testosterone (T) on male sex behaviors. For instance, Ogawa et al. (44,45) observed mild deﬁcits in mounting behaviors but a complete absence of intromission when males were tested in their home cages. Wersinger et al. (46) found that both mounting and copulatory behaviors were severely compromised when males were tested in a neutral arena. These discrepancies in motivational aspects of mating (mounting) may be related to differences in the behavior-testing protocols employed. Nonetheless, these data support the hypothesis that ERα action is required for the sexual differentiation and/or activation of neural components mediating intromissive behaviors. In support of this conclusion, βERKO males exhibit normal mounting/intromissive behaviors and function as breeders within βERKO colonies (43). The lack of overt behavioral or endocrine phenotypes in the βERKO mice suggests that hypothalamic ERβ does not play a critical role in reproductive neuroendocrinology or behavior of mice. Two laboratories however demonstrated that E2 treatments of ovariectomized αERKOs increased levels of progesterone receptor (PR) mRNA and protein in the MPO (13,47) and PR/IR in the AN and caudal VMH (47). Given expression of ERβ in the MPO, induction of PR in the MPO may represent an ERβ effect. Although the very low levels of ERβ in the VMH and ARC complicate interpretations of the latter study, the highest density of ERβ mRNA appears in the caudal regions of the VMH. Therefore, it is possible that ERβ might play a role in regulating PR and hence progesterone effects on behavior or GnRH secretion. Still, it is quite evident that normal expression of ERα is required for normal reproductive behaviors and gonadotropin secretion.

Pituitary Two of the best-studied examples of estrogen action in the pituitary are E2 stimulation of lactotrope growth and prolactin (PRL) secretion (48,49) and regulation of gonadotropin synthesis and secretion (50). Although data presently point toward a dominant role of ERα in the adult pituitary, expression patterns suggest ERβ may play a greater role during development of the pituitary. Expression of ER␣ and ␤ in the Pituitary Ribonuclease protection assays (RPA) and reverse transcriptase polymerase chain reaction (RT/PCR) analyses of total RNA from mouse pituitaries detected signiﬁcant levels of ERα mRNA but very low levels of ERβ mRNA (51). On the other hand, rat studies support the existence of 1) species differences in pituitary expression of ERα and β, 2) lobe- and cell-speciﬁc expression, 3) sex differences in expression, and 4) developmental shifts in ERα versus ERβ expression. Several studies report an absence of ERα or β mRNA or protein expression in the

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Fig. 2. Autoradiograms of ERα (A,B) and ERβ (C–E) mRNA in the rat pituitary. Low magniﬁcation darkﬁeld photomicrographs show distribution patterns of ERα (B) and ERβ (C) mRNA in the three pituitary lobes. ERα mRNA signal is strong in the anterior lobe (AL) but absent in the intermediate (IML) and neural lobes. ERβ mRNA expression is strongest in the IML, with some scattered cells present in the AL and no labeled cells in the NL. Brightﬁeld photomicrographs illustrate the density of hybridization signal for ERα mRNA (A) or ERβ mRNA (D,E) over cells of the AL (large arrowheads). Arrows denote gonadotropelike cells without a signal for ERβ mRNA. (Reproduced from Shughrue et al., Comparative distribution of estrogen receptor-α (ERα) and β (ERβ) mRNA in the rat pituitary, gonad, and reproductive tract. Steroids 63, 1998:498–504, with permission from Elsevier Press.)

posterior lobe of rats (Fig. 2) (52,53) and cynomolgous monkeys (54) whereas ERαand β/IR were found in the posterior lobe of humans (55). It is unclear why such species differences might exist or whether this represents artifactual detection of receptors. Reports vary on the expression of ERα and β in the intermediate lobe of the pituitary. In Sprague-Dawley rats, ISH detected no ERα mRNA whereas the majority of ERβ signal was conﬁned to the intermediate lobe of adult rats (Fig. 2) (52). Other studies have either failed to detect (56) or found low levels of ERβ/IR in the intermediate lobes of adult rats (57). However, a recent colocalization study detected ERβ/IR in approx 25% of melanotropes of rats (58), and ERβ mRNA has also been detected in the intermediate lobe of cynomolgous monkeys (54). The anterior lobe presents the most interesting comparisons between ERα and β expression. In adult rats, ISH and IHC detected ERα mRNA (52) and protein (57) in a large number of anterior pituitary cells whereas ERβ expression was much lower (52,57) or absent (56). A colocalization study reported that 20–25% of lactotropes, gonadotropes, and corticotropes express ERβ mRNA (58). On the other hand, this same study found that a higher percentage of all cell types express ERα mRNA and that only 6–10% of anterior pituitary cells coexpress ERα and β mRNA. Additional colocalization studies found the majority of ERβ/IR positive cells colocalize with LHβ/IR (57). Similarly, in prepubertal rats, ERβ mRNA is coexpressed with FSHβ but not PRL (53). Although the adult rat pituitary uniformly expresses less ERβ than ERα, RT/PCR studies detected higher ERβ levels in pituitaries of prepubertal female rats followed by a developmental transition to higher ERα mRNA levels in pituitaries of postpubertal females (53). This same study demonstrated a reduction in number of ERβ mRNA-

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expressing anterior pituitary cells following puberty in females and lower ERβ mRNA expression per cell in adult males. IHC in rats also detected ERβ in the anterior pituitary as early as G12 whereas ERα was not detected until G17 (57). Furthermore, in both sexes, there were a greater number of ERα/IR positive cells in the adult pituitary compared to ERβ/IR positive cells (57). These preliminary studies suggest ERβ may play a role in mediating trophic effects of E2 on pituitary cell growth and development but that ERα actions may dominate in the adult pituitary. The pituitary of adult females rats also express alternatively spliced forms of ERα called truncated estrogen receptor proteins (TERP1 and 2). Both forms contain the steroid-binding domain and some unique sequences on the amino (NH2) terminus (59). These TERPs are upregulated by estrogen, are speciﬁc to female pituitaries, and can bind to both ERα and β and suppress transcriptional activity of full length ER (60,61). RT/PCR and ribonuclease protection assay (RPA) analyses of the rat pituitary have also found high levels of ERβ2, a splice variant of ERβ with very low E2 afﬁnity (62) and the ability to act as a dominant negative regulator of ERα and β (63). The physiological function of these splice variants is presently unknown. Function of ER␣ and ␤ in the Pituitary Estradiol has been reported to stimulate lactotrope cell numbers and growth during ontogeny and stimulates the synthesis and secretion of PRL in adults (49). As most studies failed to detect ERβ in lactotropes of adult rats however it appears that ERα mediates stimulatory effects of E2 on lactotrope function. This is supported by observations that both PRL mRNA (35) and serum PRL levels (64) are extremely low in adult female αERKO mice. Although PRL expression has not yet been determined in female βERKOs, there do not appear to be deﬁciencies in lactation or nursing that could be attributed to reduced PRL. As both ERα and β are expressed in rat gonadotropes, it is possible that both receptors play a role in E2 regulation of LH and FSH synthesis and secretion. By using dispersed pituitary cell cultures however my laboratory has shown that E2 pretreatment enhances GnRH-induced LH secretion by WT gonadotropes but has no effect on basal or GnRHinduced LH secretion by αERKO gonadotropes (unpublished data, J. Lindzey). As gonadotropin secretion seems normal in βERKO mice (7), these data indicate that ERα is sufﬁcient and necessary for E2 regulation of gonadotrope function in mice. Furthermore, these data suggest that the chronically high serum LH in female αERKO mice stems from enhanced GnRH secretion rather than E2 insensitivity at the level of the gonadotrope.

Ovary Ovarian Expression of ER␣ and ␤ Unlike the brain and pituitary, there are remarkably consistent patterns of ERα and β expression between different species. In mice, rats (1,3,65–68), and cows (69), ERβ mRNA and/or immunoreactivity are limited to granulosa cells of growing follicles. RNA analyses have also detected the ERβ2 splice variant in rat ovaries (62). Expression of ERα/IR is generally lower and present in interstitial, thecal, and occasionally the germinal epithelium and granulosa cell types of rodents (65,66,68). Similar distributions were found in primates where ERβ/IR was reported in the granulosa cells at all stages of follicular development in humans and marmosets (70).

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This same study reported fairly low and inconsistent ERα/IR in thecal, stromal, and granulosa cells of antral follicles. Human ovarian cell lines also express ERα and β with granulosa-derived cells expressing higher ERβ and epithelial-derived cells expressing higher ERα levels (71). Thus, there is a conserved pattern of expression between rodent models and primates. Interestingly, it appears that LH or hCG downregulates ERβ mRNA levels in rats (65) and humans (72). A recent report also describes a decreased level of ERβ mRNA in new corpora lutea (73). The consequences of ERβ downregulation in granulosa cells and the role of luteal ERβ remain to be determined. Function of Ovarian ER␣ and ␤ Delineating the role of ERα and β in the ovary is complicated because interstitial and granulosa cells undergo complex paracrine interactions during folliculogenesis (74). Fortunately, the apparent species conservation of cell-speciﬁc ERα and β expression in the ovary provides greater conﬁdence in extrapolating conclusions from αERKO and βERKO studies to other species. The data discussed below suggest that both receptors play a role in cell determination, follicular development, and ovulation. All tissue types are present in both αERKO and βERKO ovaries, and overt ovarian dysfunction becomes grossly evident only following elevations in gonadotropin secretion during pubertal and postpubertal periods (7,75). However, crossing of the two ERKO models produced double knockouts (DERKO) in which the maturing ovaries undergo a transdifferentiation characterized by the development of seminiferous tubulelike structures, Sertoli-like cells, and expression of MIS (76). As this phenotype was absent in prepubertal DERKOs or the single knockouts, it appears that a degree of ERα and β redundancy exists and that at least one ER is required for normal determination and maintenance of speciﬁc cell types in the adult ovary (76). The adult αERKO female is acyclic and infertile and exhibits severe ovarian deﬁcits. The causes of the ovarian deﬁcits may stem both from chronic LH hypersecretion and the effects of ERα ablation on E2 signaling within the ovary. Although ERα is not required for progression of follicles through the early antral stage or for expression of LH receptors on granulosa cells, follicles do not reach a preovulatory stage and ovulation does not occur (75). Instead, αERKO ovaries develop an increasing number of cystic, hemorrhagic follicles as a result of chronic exposure to very high LH levels (77) which in turn stem from an absence of negative feedback on the hypothalamus and pituitary. Signiﬁcantly, prior to development of this adult phenotype, peripubertal female αERKOs can be successfully superovulated (77). Although fewer oocytes are produced, the oocytes can be fertilized through in vitro fertilization assays. This suggests that the αERKO is minimally competent to respond to a normal pattern of gonadotropin stimulation and that αERKO oocytes are viable. In addition, αERKO follicles are competent to undergo luteinization when exposed to either a superovulatory regimen of pregnant mare serum gonadotropin (PMSG) and hCG (77) or high serum PRL levels (64). In contrast to the αERKO females, βERKO females spontaneously ovulate but exhibit severely reduced fecundity (7). Steroid levels and basal gonadotropin levels appear relatively normal although no studies have examined the size or timing of preovulatory gonadotropin surges in the βERKO model. Superovulation studies indicate that the reduced fecundity stems from reduced sensitivity of the Graaﬁan follicles to ovulatory surges of LH or hCG (7). Some follicles rupture and undergo luteinization

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but a signiﬁcant number of follicles fail to rupture and contain trapped oocytes. In addition, those oocytes that do rupture exhibit a reduced number of granulosa cells in the surrounding cumulus mass. This suggests that ERβ is responsible for induction of some factor or factors required for follicular rupture and granulosa cell integrity. Unlike folliculogenesis and ovulatory functions, steroidogenic capacity seems relatively normal in αERKO females, as there are no dramatic deﬁciencies in steroid production. For instance, ovarian P450scc RNA levels and serum P levels of αERKO females are comparable to those found in WT littermates (77). In addition, follicles and corpora lutea evidently produce large amounts of P in response to superovulation or elevated PRL (64). Finally, the chronically high serum LH levels in αERKO females drive high-circulating levels of E2 and T, an indication that 17α-hydroxylase and aromatase enzymes are expressed in the absence of ERα (77). Given that βERKO females are fertile and undergo normal uterine cycles, an absence of ERβ does not appear to have dramatically altered aromatase levels in the granulosa cells.

Uterus During normal menstrual or estrous cycles, estrogens drive the proliferative phase of the uterus in which luminal and glandular epithelial cells undergo hyperplasia, hypertrophy, and increased transcription and translation of E2-dependent genes (78). Estradiol is also implicated in decidual reactions and implantation of the blastocyst, and in preparing the myometrium for parturition (79). Consistent with these effects, all tissue components of the uterus express at least one of the ER. Expression of ER␣ and ␤ in the Uterus In the mouse, high levels of ERα mRNA are detected by RPA analyses of uteri from neonatal and adult mice (Fig. 3) (51,68). In addition, luminal and glandular epithelium, stromal cells, and myometrial cells all express ERα protein (68,80). By contrast, RPA analysis indicates that ERβ mRNA levels are vanishingly small in both neonatal and adult female mice (Fig. 3) (51,68) and that uterine ERβ/IR is absent in fetal, neonatal, and peripubertal mice (68). This is in contrast to a single report of ERβ/IR in the epithelium and stroma of mouse uteri (81). In rat uteri, ERα also appears to be the dominant receptor. ISH detected high levels of ERα mRNA in both epithelial and stromal cells whereas ERβ mRNA levels were very low (52,82). Similarly, IHC studies demonstrated that ERα protein is abundant in both glandular and luminal epithelial cells of rats, whereas ERβ/IR was limited to glandular epithelial cells (67). Other studies detected ERα/IR in epithelial, stromal, and myometrial cells but failed to detect ERβ/IR in rat uteri (56,66). In humans, ERα and ERβ mRNA were both detected in epithelial, stromal, and smooth muscle cells of normal uterine tissues although ERα expression was higher in all cell types (83). Although the overall level of ERβ expression was lower, the highest levels of ERβ mRNA were found in glandular epithelium compared to levels in luminal epithelial and stromal cells (83). In humans, both ERα and β protein were also detected in luminal epithelial and stromal cells whereas very weak ERβ/IR was detected in the glandular epithelium (55). Myometrial smooth muscle cells in rhesus monkeys and humans contain both ERα- and β mRNA (84,85) and it appears that myometrial ERα levels decline as ERβ protein levels increase near term (85).

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Fig. 3. Ribonuclease protection assays of total RNA (10 µg per lane) harvested from ovaries and uteri at neonatal days 1, 12, 19, and 26. Shown are protected bands for ERα, ERβ, and cyclophilin (for normalization purposes). Note the developmental increase in ovarian ERβ mRNA levels and the high levels of uterine ERα mRNA coupled with an almost complete absence of uterine ERβ mRNA at all ages leading up to puberty. (Reproduced with permission from Jefferson WN, Couse JF, Banks EP, Korach KS, Newbold RR. Expression of estrogen receptor β is developmentally regulated in reproductive tissues of male and female mice. Biol Reprod 2000;62(2):310–317.)

Function of Uterine ER␣ and ␤ Overall, it appears that uterine cells express higher levels of ERα than ERβ. This is particularly true for the mouse whereas there are more consistent reports of low levels of ERβ expression in uteri of rats and primates. These reports of ERβ in rats and humans suggests a role in uterine function but studies of αERKO and βERKO mice illustrate that ERα sufﬁces to regulate a vast number of classical estrogen responses. By examining the αERKO model we can conclude that differentiation of myometrial, stromal, and epithelial cell types does not rely on functional ERα. However, despite extremely high endogenous E2, the uterus of an intact αERKO is about 50% smaller and fails to exhibit hyperplasia, hypertrophy, water imbibition, hyperemia, or induction of estrogen responsive genes (progesterone receptor, c-fos, lactoferrin) following three days of estrogen treatment (86). Recent tissue recombination studies have further demonstrated that WT uterine stroma can drive E2-induced proliferation (87) of αERKO uterine epithelium. These data indicate that uterine ERα is the primary mediator of estrogen action in the mouse uterus and that stromal ERα is capable of inducing estrogenic effects in epithelial cells via paracrine mechanisms. This assessment is strengthened by the very low uterine levels of ERβ mRNA and protein and the ability of βERKO females to exhibit proliferative uterine responses and carry fetuses to term (7). Caution should be urged in extrapolating these data and conclusions to other species, however, as rats and primates appear to express both ERα and β in the uterus. Indeed, given that E2 is critical for preparing the myometrium for parturition, it is particularly

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intriguing that ERβ expression increases in the myometrium of humans near term (85). It will also be interesting to determine if expression of ERβ in the glandular epithelium of the rat and primate may play a role in priming these cells for the secretory phase of the uterine cycle. Obviously, ERα ablation eliminates many classical E2 responses, including estrogen induction of progesterone receptor (PR) (86). Nonetheless, some PR is constitutively expressed and is sufﬁcient to drive P regulation of calcitonin and amphiregulin in mouse uteri (88). This level of PR is also sufﬁcient to allow progesterone-dependent development of a decidual response in the αERKO uterus (88,89). In addition, 4hydroxy E2 and methoxycyhlor, an environmental estrogen, can activate DNA synthesis and increase expression of lactoferrin in αERKO uteri (90). Since these effects were not blocked by antiestrogens, these data suggest a novel uterine-E2-signaling pathway (non-ERβ).

Mammary Prior to puberty the mammary gland consists of a rudimentary ductal system projecting inward from the nipple (91). During pubertal development, this rudimentary ductal system grows out from the nipple, undergoes extensive branching and proliferation, and invades the adipose tissue of the mammary gland. Estrogen is required for ductal development, formation of terminal end-buds and proliferation of ductal epithelium (91). In addition, estrogen plays a role in further ductal growth and lobulo-alveolar growth during reproductive cycles and pregnancy (91). Expression of ER␣ and ␤ in the Mammary Gland Using both RPA and RT/PCR analyses, mouse mammary glands were found to express moderate amounts of ERα mRNA but very low levels of ERβ mRNA (51). IHC localized ERα/IR to both epithelial cells and stromal cells of neonatal mice with increasing amounts of stromal ERα in peripubertal females (92). Indeed, most proliferating cells in the terminal end-buds and ducts do not express ERα and this, therefore, suggests stromal ER stimulates epithelial proliferation via paracrine actions (93). A recent study found that epithelial cells in terminal end-buds of pubertal mice and ductal epithelial cells of proestrus females were weakly ERβ/IR and only scattered stromal cells were positive for ERβ/IR (94). As in the mouse, both epithelial and stromal cells of rat mammary glands contain ERα/IR. In addition, the numbers of ERα/IR positive epithelial cells decrease during pregnancy but increase dramatically during lactation (95). In contrast to the mouse, the rat has been reported to express ERβ protein in 60–70% of mammary epithelial cells (95). Interestingly, although approx 60% of the epithelial cells coexpressed ERα and β during pregnancy, very few proliferating cells expressed either receptor (95). In normal human mammary glands, a variable but low percentage (5–15%) of epithelial cells express ERα/IR and, as in rodents, proliferating cells do not express ERα (96,97). These studies failed to detect stromal ERα. RT/PCR analyses suggest however that ERβ mRNA levels exceed ERα mRNA and that ERβ/IR positive cells are found in both the ductal epithelial cells and the stroma (98). An ISH study in cynomolgus monkeys also detected ERβ mRNA in both epithelial and stromal cells (54). Thus, the available data suggest that rat and primate mammary glands express higher amounts of ERβ than the mouse glands.

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Function of ER␣ and ␤ The mammary gland of an adult αERKO female resembles that of a neonatal mouse (64,99). It possesses only a rudimentary ductal structure and lacks any lobulo-alveolar development. Conversely, adult female βERKO mice exhibit apparently normal mammary gland development and are able to successfully lactate, nurse, and rear pups (7). Although these studies indicate that mammary gland ERα is critical for normal development and function of the gland, some of the underdevelopment of the αERKO mammary gland may stem from chronically low PRL. When αERKO females were subjected to elevated PRL levels by pituitary grafts or subcutaneous pellets, ovarian follicles underwent luteinization and the combination of elevated E2 and P resulted in extensive ductal development and branching and lubulo-alveolar development (64,99). This raises the possibility that an ERα-independent signaling pathway may exist in the mammary glands of mice. Clearly, ERα is responsible for most of the ductal growth and branching that occurs in mouse mammary glands. However, as both rat and primate mammary glands appear to express substantially more ERβ than mouse mammary glands, both ERα and β may regulate mammary gland growth and function in rats and primates. There is however a consistent absence of ERα or β expression in proliferating epithelial cells. This suggests that, as in the uterus, stromal ER mediate mitogenic effects of estrogens on epithelial cells via paracrine factors.

Testis and Ductule Structures Estrogens were believed to regulate normal testis function primarily through regulation of gonadotropin synthesis and secretion. Recent studies demonstrated however that estrogens can stimulate spermatogenesis in hypogonadal mice (100) and promote human germ cell survival in an in-vitro assay (101). In addition, aromatase knockout mice exhibit arrested spermatogenesis (102) whereas aromatase deﬁcient humans exhibit varying degrees of compromised spermatogenesis and sperm function (103). These ﬁndings have renewed interest in the potential effects of estrogens directly on the testis. Expression of ER␣ and ␤ in the Testis In the mouse, RPA of total RNA from whole testis demonstrated that ERβ mRNA is more abundant than ERα at neonatal day 1 (Fig. 4) (68). ERβ mRNA levels then decline rapidly and ERα mRNA is expressed at higher levels than ERβ in the adult mouse testis (51). In agreement with this developmental shift in ERβ mRNA expression, ERβ/IR was detected in spermatogonia at neonatal days 5 and 12 but disappeared by day 26 (68). Both peritubular cells (104) and interstitial/Leydig cells (68) were ERα/ IR in the mouse whereas only one study reported ERβ-IR in Leydig cells of mice (105). Similar distributions were found in the rat, with ERβ restricted to Sertoli (56,106,107) and germ cells (107) and absent from interstitial cells of rat testes. Interstitial/Leydig cells (56,108) and round spermatocytes and spermatids (56) of rats were also reported to express ERα protein. As in rodents, ERβ-IR was detected in the seminiferous tubules of macaques (54) and Sertoli cells of humans (55). Both ERα- and β-IR were detected in early meiotic spermatocytes and elongating spermatids, and E2 was protective against germ cell apoptosis in culture (101). Leydig cells of fetal, neonatal, and adult marmosets expressed ERα protein (108) whereas no ERα-IR was found in peritubular or germ cells. Thus,

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Fig. 4. Ribonuclease protection assays of total RNA harvested from testes at postnatal days 1, 5, 12, 19, and 26. Shown are ERα, β, and cyclophilin (for normalization purposes). Note the decline in expression of ERα mRNA and very rapid decline in ERβ mRNA levels in the testis. (Modiﬁed and reproduced with permission from Jefferson WN, Couse JF, Banks EP, Korach KS, Newbold RR. Expression of estrogen receptor β is developmentally regulated in reproductive tissues of male and female mice. Biol Reprod 2000;62(2):310–317.)

despite some intra- and interspeciﬁc variability in the reported expression patterns, ERα is consistently found in Leydig or interstitial cells, and ERβ, when present, is expressed in Sertoli or germ cells. Expression of ER␣ and ␤ in Ductal Structures In the mouse, intense ERα-ir was found in cells of the efferent ductules whereas lower levels were found in the epididymis and none in the vas deferens (104). ERβir was also detected in the efferent ductules of mice (105). In adult male rats, ERα has also been detected in the epithelia and stroma of the efferent ducts (108,109). ERα was also found in the epithelia of the initial segment of the rat epididymis whereas weaker signals were detected in the rete testis and the caput, corpus, and caudal portions of the epididymis (109). As in the rat, macaques and marmosets expressed signiﬁcant amounts of ERα-ir in the nonciliated, absorptive cells of the efferent ductules (108,110). Indeed, it appears as though high levels of ERα are expressed in the efferent ductules of males of many species (111,112). Function of ER␣ and ␤ in the Testis and Ductules Comparisons of aromatase knockout (AKKO), αERKO, and βERKO mice demonstrate that estrogens and ERα are important for normal spermatogenesis and sperm maturation and delivery. Similar to aromatase deﬁcient humans, the ARKO mouse exhibits reduced spermatogenesis and increased apoptosis between 4.5 and 12 months of age (113). These changes appear to be independent of any changes in gonadotropins or androgens and therefore support a direct effect of estrogen on spermatogenesis or sperm maturation and survival. Indeed, this is consistent with observations that estrogen can promote initiation of spermatogenesis in mice (100) and prevent apoptosis of human germ cells (101). Given the expression of ERβ in Sertoli and germ cells, it seems possible that such effects might be partly mediated by ERβ. As male βERKO mice are fertile however ERβ is clearly not critical for successful spermatogenesis in mice.

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In contrast to male βERKO mice, male αERKO mice are completely infertile and exhibit a progressive degeneration of the testis and seminiferous tubules. Initially, young males (10 days) exhibit relatively normal testicular morphology. By day 20 there is a dilation of the lumen of the tubule although young adult males continue to exhibit normal spermatogenesis and normal epididymal sperm counts. With increasing age, however, there is a progressive decline in spermatogenesis, coupled with a dilation of the seminiferous tubules and decreased height of the seminiferous epithelium (114,115). Sperm harvested from αERKOs prior to onset of testicular dysmorphogenesis were unable to fertilize eggs during in vitro fertilization assays (114) thereby suggesting that αERKO sperm might be incompetent. However, a recent study demonstrated that when αERKO germ cells are transplanted into the testes of aspermic, sterilized WT males, the WT males are able to successfully sire offspring (116). Thus, infertility in αERKO males stems not from an inherent problem with the germ cells but from deﬁcits in the somatic tissues that assist with maturation and maintenance of the germ cells. Indeed, work by Hess et al. (117) demonstrated that the testicular dysmorphogenesis and decline in spermatogenesis stems from an inability of the efferent ducts to reabsorb ﬂuids, causing an increase in intratesticular ﬂuids. Although this may account for the dysmorphogenesis of the αERKO testes and declining spermatogenesis, it does not provide a direct explanation of the compromised sperm function. The deﬁcits in sperm number and motility found in aromatase deﬁcient humans indicate that estrogen also has important effects on human sperm function (103). Furthermore, reduced sperm motility in a patient with an ERα mutation also supports a role for ERα in human sperm function (38). Thus, ERα appears to play an important role in sperm function in both rodents and primates.

Prostate To date no physiological role has been found for estrogen action in the prostate. However, as with many male accessory sex structures, neonatal estrogen exposure in rodents results in abnormalities in the prostate. These abnormalities include enlarged prostate (118), squamous metaplasia (119), and altered expression of estrogen receptors (120,121), androgen receptors (122), and c-fos (123). Such changes could be caused by indirect effects on the hypothalamic/pituitary/gonadal axis; however, expression of ERα and β in the prostate suggest that some of these effects may be attributed to direct estrogen effects on the prostate. Expression of ER␣ and ␤ in the Prostate Periductal stromal cells of rats have been reported to express both ERα protein and mRNA whereas epithelial cells do not express ERα (120,124). By contrast, ERβ mRNA and protein have been found in the epithelial cells of rats (56,125) and primates (54,55,126). In the rat, there appear to be lower levels of ERβ at birth followed by an increase with development of luminal epithelial cells. RT/PCR and RPA analyses of the prostate have also found high levels of ERβ2, a splice variant of ERβ with very low E2 afﬁnity (62). Function of ER␣ and ␤ in the Prostate Localization of ERα and β to the stromal and epithelial cells, respectively, appears to be well conserved. Thus, phenotypes observed in knockout mice might also be found

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in other mammals. All reports on the prostate of αERKO mice indicate however that the prostate develops normally in the absence of ERα (114). Although the initial characterizations of βERKO mice reported some prostatic hyperplasia in older βERKO males, it appears that the development and function of the prostate is relatively normal in the absence of functional ERβ (7). The compartmentalization of ERα in stromal and ERβ in epithelial cells certainly suggests that these two receptors may alter different aspects of prostate development and function but this will require further work with knockout models and, more importantly, ERα and β-speciﬁc ligands.

NONREPRODUCTIVE TISSUES Hippocampus A number of extrahypothalamic brain regions express ER including the cortex, basal forebrain, and hippocampal regions. These sites are of particular clinical interest because of their role in cognition and recent studies indicating that estrogen inﬂuences some aspects of cognition. Not surprisingly, studies have detected E2-labeled cells in the basal forebrain, CA1-4, and dentate gyrus, and found that E2 altered synaptic density and excitability of hippocampal pyramidal cells (reviewed in 127). As discussed later, the distributions of ERα and β suggest that both receptors may play a role in cognitive functions. Hippocampal Expression of ER␣ and ␤ ISH detected low levels of ERβ mRNA in the mouse hippocampus (12) and recent RT-PCR studies conﬁrmed low levels of both ERα and β mRNA in the mouse hippocampus (128). In the ovariectomized rat, low levels of both ERα and β mRNA were detected in the cortex, dentate gyrus, and hippocampus (Fig. 1) (16,129). A recent autoradiographic study demonstrated nuclear estrogen binding sites in the pyramidal cells of CA1-3 (130). This distribution correlates well with ERβ/IR detected in the dentate gyrus, pyramidal cells of CA1-2 of the hippocampus (17,131), and glial cells of the dentate gyrus and CA1-3 (131). In addition, ERα/IR was also localized to pyramidal cells of CA1-3 (131). Recent work also suggests that cholinergic neurons in the basal forebrain of rats express primarily ERα mRNA (129,130). These data conﬁrm that ERα and β mRNA are translated into receptor protein in hippocampal regions involved in cognition and support the hypothesis that both receptors may mediate estrogen effects on cognition. Using RT/PCR, both ERα and β mRNA have been detected in the hippocampus of cynomolgous monkeys (132) whereas ISH localized dense ERβ signals in the dentate gyrus and CA2-3 and CA-3 of ovariectomized macaques (18). A single study in humans detected ERα/IR but, surprisingly, no ERβ/IR in the hippocampus (55). It is unclear whether this represents a true species difference in ERβ expression or simply technical difﬁculties stemming from the use of archival tissues or inadequate antigen retrieval. Although both ERα and β mRNAs have been localized to hippocampal areas of several species, expression levels and speciﬁc cell types that express ERα and β proteins are still debatable. In part this may stem from species differences or differences in ﬁxation, antigen retrieval, and antibody characteristics. Nonetheless, based on overlapping expression of ERα and β in areas such as the basal forebrain and hippocampus, it is possible that both ERα and β play a role in memory and learning.

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Function of Hippocampal ER␣ and ␤ Overlapping expression of ERα and β in the hippocampus suggests that both receptors mediate estrogen effects on memory and learning. Current studies utilizing αERKO and βERKO mice however only provide evidence that ERα and perhaps a membrane receptor play a role in cognition. In a recent study utilizing αERKO mice, it was found that E2 treatments impaired spatial discrimination in WT female mice but not in WT males or αERKO females (133). These data suggest that ERα mediates inhibitory actions of acute E2 treatments on some aspects of cognition. A more recent study examined estradiol-enhanced performance of αERKO and βERKO mice in an active avoidance task (134). In this study, ovariectomized or estrogen-treated βERKO mice exhibited no deﬁcits whereas ovariectomized αERKO mice exhibited deﬁcits compared to WT littermates. Surprisingly, E2 treatments restored performance in ovariectomized αERKOs to levels found in WT littermates. These results are puzzling but the authors suggest that ERα plays a role in organization of memory and that ERα- and βindependent signaling mechanisms may play a role in cognitive effects of E2 in the adult. Indeed, rapid nongenomic estrogen signaling has been proposed for a number of years and ﬁnds support in observations that E2 rapidly potentiates kainate-induced currents in CA-1 neurons of both αERKO and WT mice (135,136). As these effects were rapid and not suppressed by antiestrogens, these data support the idea that nonnuclear ER may play a role in estrogen effects on hippocampal function. The relatively subtle cognitive deﬁcits observed in αERKO mice may stem from the limited set of testing regimens employed or species differences in the role of estrogen, or they may indicate that nongenomic mechanisms mediate estrogens effects on cognitive centers. It is also important to remember however that estrogen’s effects on cognition are primarily manifested in aging menopausal woman. Thus, estrogen may simply help ameliorate effects of aging on neurons. For this reason, a more valid test of the cognitive role of ERα versus β should involve testing of aged knockout mice.

Cardiovascular System Recent studies have documented that E2 exerts a protective effect on the cardiovascular system in women (137,138). Although some of these effects may be a result of systemic changes, E2 may also have direct effects on the vasculature. This hypothesis is supported by the presence of both ERα and β in cardiovascular tissues, although it is presently unclear whether the protective effects of E2 involve ERα, β or nongenomic mechanisms. Cardiovascular Expression of ER␣ and ␤ Binding assays demonstrated that mouse aorta contain speciﬁc ER binding sites and that these sites are more abundant in males (139). In RPA analyses, ERβ was undetectable whereas low levels of ERα mRNA were detected in the aorta of mice (51). RT/ PCR analyses have however detected ERβ mRNA in the mouse (140) and rat aorta (62). This indicates that very low levels of ERβ mRNA are present and that the majority of ER binding in the mouse aorta may be caused by ERα. In primates, RT/PCR also detected ERβ in monkey aortic smooth muscle cells, coronary arteries (141), and aorta and cardiac muscle (142). IHC detected both ERα and β in major vessels such as the aorta, coronary artery, carotid artery, and inferior vena cava (55). Further studies in humans found that vascular smooth muscle contains

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greater amounts of ERβ than ERα and that women express higher amounts than men (143). A recent report of membrane estrogen receptors on human vascular endothelial cells added to this complexity (144). This membrane receptor is recognized by ERα antibodies and indicates that the ERα gene may code for a membrane receptor. Function of Cardiovascular ER␣ and ␤ As with other estrogen target tissues, it is difﬁcult to delineate speciﬁc roles for ERα and β because of the overlap of α and β in cardiovascular tissues. Several studies however support a role for ERα in cardiovascular function. For instance, one study found an age-associated methylation of the ERα gene promoter that is correlated with increased coronary atherosclerotic plaques in humans (145). This agrees with earlier ﬁndings that levels of atherosclerosis in mice may be correlated with the levels of ER binding (146). In addition, measurements of basal levels of nitric oxide (NO), a potent vasodilator, in the aorta of mice indicated that NO is higher in WT males compared to WT females and that αERKO males had reduced levels of NO (139). This suggests that the higher number of ERα receptors in the male mouse aorta may account for high NO levels. The physiological signiﬁcance of elevated basal NO is unclear however, as vasodilation responses of WT and αERKO males were similar when aortic rings were challenged with acetylcholine (139). Vascular injury models have demonstrated that endothelial and smooth muscle cells can proliferate in response to physical injury and that this thickening response is inhibited by E2; a potential mechanism for protection against vascular disease. Indeed, a role for ERβ was suggested by the observation that endothelial and smooth muscle cells increase expression of ERβ mRNA following aortic injury (147). On the other hand, the Mendehlson laboratory demonstrated that E2 inhibits the vascular injury responses of the carotid artery (increased mitotic activity and endothelial thickening) in both αERKO and βERKO mice (148). As suggested by the authors, these data may indicate that ERα and β form redundant pathways or that nongenomic pathways may play a role in vascular responses. Support for a nongenomic pathway is found in a study in which E2 treated human endothelial cells exhibited rapid increases in NO, activation of gaunyl cyclase, and activation of MAP kinase (144). This same study demonstrated that these effects were elicited by membrane-impermeant forms of E2 that bound with membrane proteins antigenically similar to ERα. This suggests an important alternative to traditional nuclear ER pathways. Estrogens may also affect myocardial tissue by alterations in growth following myocardial infarcts and alterations in excitability or contractility of the tissues (149,150). In rats, estrogens decrease numbers of Ca++ channels and, hence, contractility of the tissue (151). Indeed, αERKO males express higher numbers of L-type CA++ channels with resulting changes in duration of cardiac action potentials (151).

Bone The importance of estrogen action on human bone tissue is exempliﬁed by the link between menopause, declining estrogen levels, and osteoporosis and the efﬁcacy of estrogen replacement therapy in alleviating symptoms of osteoporosis (137). Estrogens are also involved in linear bone growth and epiphyseal closure as demonstrated by phenotypes of humans that possess mutations in the aromatase gene. Male patients

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with mutations in the aromatase gene exhibited tall stature, decreased bone density, continued linear growth, and lack of epiphyseal closure (38,152). In female patients with aromatase mutations, bone age was delayed, bone density was decreased, and no pubertal growth spurt was observed (153,154). Studies described in the next sections support the hypothesis that these estrogen effects may be mediated by both ERα and β. Expression of ER␣ and ␤ in Bone Recent studies have provided evidence of ERα and β expression in three critical cell types: chondrocytes, osteoblasts, and osteoclasts. Chondrocytes in growth plates of rabbits and humans express ERα mRNA (155,156) and, in pubertal female humans, also exhibit ERβ/IR (157). Human osteoblasts express ERα mRNA (155) and ERα/ IR (158). Furthermore, ERα mRNA appears to increase during osteoblast differentiation in culture (159,160). Osteoblasts have also been found to express ERβ mRNA in rats (161) and humans (162,163) and ERβ/IR in humans (163). Human osteoclasts were also found to express ERα and β mRNA and ERβ/IR with levels of ERα mRNA declining during osteoclast differentiation (164,165). Thus, ERα mRNA expression increases during maturation of osteoblasts but decreases during maturation of osteoclasts. Function of ER␣ and ␤ in Bone As chondrocytes, osteoblasts, and osteoclasts all express ERα and β to varying degrees, both receptors may play a role in estrogen effects on bone. Indeed, αERKO males exhibited varying degrees of decreased femur length (166,167) whereas female αERKO mice appeared to exhibit a more dramatic decrease in femur length (166). Neither sex of βERKO mice exhibited changes in femur length (168) whereas DERKO male mice had decreased femur length (168). These data suggest that ERα stimulates bone growth in mice. Bone mineral content (BMC) or density (BMD) were reduced in male αERKOs whereas female αERKOs were normal compared to age- and sex-matched controls (166). The lack of an effect in female αERKOs may stem from chronically high levels of serum T and, hence, compensatory action through androgen receptors. In contrast, βERKO females exhibited increased BMC whereas males were normal compared to age- and sex-matched WT littermates (168). Thus, it is possible that ERα increases osteoblast activity and hence BMD, whereas ERβ may play a role in suppressing osteoblast activity. The apparent opposition of ERα and β in regulating BMD of mice suggests that ERα- and β-speciﬁc ligands might target different aspects of bone physiology in humans. A male patient with a mutated ERα gene exhibited decreased BMD and tall stature stemming from continued linear growth and lack of epiphyseal closure (38). This agrees closely with phenotypes of aromatase deﬁcient humans and suggests that ERα mediates many of the obvious effects of E2 on bone growth, maturation, and density in humans.

CONCLUDING REMARKS Despite intensive research over the past few years, characterization of the physiological functions of ERα vs ERβ is still in its infancy. The obvious functional similarities and overlap in expression of these two receptors will make this characterization a difﬁcult task. There are three mechanisms by which we can begin to distinguish ERα from β effects: 1) tissue-, cell- and species-speciﬁc expression patterns, 2) phenotypes

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of knockout mice or naturally occurring mutations, and 3) effects of ERα- or β-speciﬁc agonists or antagonists. Tissues such as the testis, ovary, and prostate appear to exhibit well-conserved patterns of ERα and β expression with the two receptors localized to different cell types. However, tissues such as the hypothalamus, uterus, and mammary gland appear to exhibit species variation in ERβ expression. In addition, there are inconsistencies between reported patterns of mRNA and protein expression in several target tissues. Since ERβ biology is relatively young, reagents and techniques are still being ﬁne tuned. Therefore, reported species variation and inconsistencies in patterns of ERβ mRNA versus protein expression may represent true differences or may simply stem from quality of antibodies, choice of riboprobes, or conditions employed during ISH or IHC studies. Another potentially important explanation of these inconsistencies is the occurrence of splice variants for ERβ that might be detected by RNA analyses but missed during IHC studies. One of these variants, ERβ2, has an 18-amino acid insertion in the steroid-binding domain (63) and requires extremely high estrogen concentrations for receptor activation (169). This receptor is found in relatively high levels in the ovary, prostate, and pituitary of rats (62) and may function as a dominant negative regulator of ERα and β (63). Another ERβ splice variant, ERβcx, has a truncated COOH terminus and an insertion of an additional 26 amino acids. It is expressed in human testis, ovary, and prostate and can inhibit ERα transcription during transfection assays (170). Multiple ERα splice variants have also been documented over the years. These include deletions of exons 2–7, singly or in combinations, which have been detected in varying amounts within normal and malignant tissues (reviewed in 171). The presence of such ERα and β variants might result in false negatives and hence contribute to apparent inter- and intraspeciﬁc variation in expression patterns. Although it will be quite difﬁcult to localize such variants to speciﬁc cell types, future studies will need to take into account the coexpression of these variants with wild-type ERα and β in normal and pathological states. Reasonably consistent patterns of ERα and β expression are found in a number of hypothalamic nuclei and the ovary, testis, and prostate of both rodents and primates. In these instances, phenotypes of ER knockout mice may allow accurate predictions of ERα and β functions in these targets in rats and primates. Indeed, it seems fairly clear that ERα plays a dominant role in many aspects of reproduction. This is supported by the consistent patterns of ERα and β expression in the MPO, BST, VMN, AN, ovary, and testis of rodents and primates coupled with the many reproductive deﬁcits observed in αERKO mice and the relatively normal reproduction of βERKO mice. The one ﬁrm reproductive role for ERβ appears to be in ovulation, although the expression of ERβ in rat and primate uterus and mammary gland warrants further investigation into the role of ERβ in normal physiology and carcinogenesis in these tissues. Although the αERKO and βERKO models are presently our best tool for delineating the roles of ERα and β, there are obvious limitations to the use of αERKO and βERKO models. These limitations are most apparent in target tissues where there are signiﬁcant species differences in expression of ERα and β and in target tissues or cells where ERα and β are coexpressed. In these instances, ERα- and β-speciﬁc agonists and antagonists will be needed to delineate the roles of ERα and β. The recent development of an ERβ speciﬁc antagonist will assist with this process (172). The development and

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characterization of additional ERα- and β-speciﬁc agonists and antagonists will be crucial for further delineating the roles of ERα versus ERβ and for use in clinical settings.

ACKNOWLEDGMENTS I wish to thank Dr. Paul Shughrue and Retha Newbold for permission to reproduce portions of their work and for providing artwork.

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114. Eddy EM, Washburn TF, Bunch DO, et al. Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility. Endocrinology 1996;137(11):4796–4805. 115. Lindzey J, Korach K. Developmental and physiological effects of estrogen receptor gene disruption in mice. TEN 1997;8(4):137–145. 116. Mahato D, Goulding EH, Korach KS, Eddy EM. Spermatogenic cells do not require estrogen receptorα for development or function [see comments]. Endocrinology 2000;141(3):1273–1276. 117. Hess RA, Bunick D, Lee KH, et al. A role for oestrogens in the male reproductive system [see comments]. Nature 1997;390(6659):509–512. 118. vom Saal FS, Timms BG, Montano MM, et al. Prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses. Proc Natl Acad Sci USA 1997;94(5):2056–2061. 119. Singh J, Handelsman DJ. Morphometric studies of neonatal estrogen imprinting in the mature mouse prostate. J Endocrinol 1999;162(1):39–48. 120. Prins GS, Birch L. Neonatal estrogen exposure up-regulates estrogen receptor expression in the developing and adult rat prostate lobes. Endocrinology 1997;138(5):1801–1809. 121. Prins GS, Marmer M, Woodham C, Chang W, et al. Estrogen receptor-β messenger ribonucleic acid ontogeny in the prostate of normal and neonatally estrogenized rats. Endocrinology 1998;139(3): 874–883. 122. Prins GS, Birch L. The developmental pattern of androgen receptor expression in rat prostate lobes is altered after neonatal exposure to estrogen. Endocrinology 1995;136(3):1303–1314. 123. Salo LK, Makela SI, Stancel GM, Santti RS. Neonatal exposure to diethylstilbestrol permanently alters the basal and 17 β-estradiol induced expression of c-fos proto-oncogene in mouse urethroprostatic complex. Mol Cell Endocrinol 1997;126(2):133–141. 124. Chang WY, Wilson MJ, Birch L, Prins GS. Neonatal estrogen stimulates proliferation of periductal ﬁbroblasts and alters the extracellular matrix composition in the rat prostate. Endocrinology 1999;140(1):405–415. 125. Chang WY, Prins GS. Estrogen receptor-β: implications for the prostate gland. Prostate 1999; 40(2):115–124. 126. Enmark E, Pelto-Huikko M, Grandien K, et al. Human estrogen receptor β-gene structure, chromosomal localization and expression pattern. J Clin Endocrinol Metab 1997;82(12):4258–4265. 127. McEwen BS, Alves SE. Estrogen actions in the central nervous system. Endocr Rev 1999;20(3): 279–307. 128. Ivanova T, Beyer C. Ontogenetic expression and sex differences of aromatase and estrogen receptorα/β mRNA in the mouse hippocampus. Cell Tissue Res 2000;300(2):231–237. 129. Shughrue PJ, Scrimo PJ, Merchenthaler I. Estrogen binding and estrogen receptor characterization (ERα and ERβ) in the cholinergic neurons of the rat basal forebrain. Neuroscience 2000;96(1):41–49. 130. Shughrue PJ, Merchenthaler I. Evidence for novel estrogen binding sites in the rat hippocampus. Neuroscience 2000;99(4):605–612. 131. Azcoitia I, Sierra A, Garcia-Segura LM. Localization of estrogen receptor β-immunoreactivity in astrocytes of the adult rat brain. Glia 1999;26(3):260–267. 132. Register TC, Shively CA, Lewis CE. Expression of estrogen receptor α and β transcripts in female monkey hippocampus and hypothalamus. Brain Res 1998;788(1–2):320–322. 133. Fugger HN, Cunningham SG, Rissman EF, Foster TC. Sex differences in the activational effect of ERα on spatial learning. Horm Behav 1998;34(2):163–170. 134. Fugger HN, Foster TC, Gustafsson J, Rissman EF. Novel effects of estradiol and estrogen receptor α and β on cognitive function(1). Brain Res 2000;883(2):258–264. 135. Moss R, Gu Q. Estrogen: Mechanisms for a rapid action in CA1 hippocampal neurons. Steroids 1999;64(1–2):14–21. 136. Gu Q, Korach K, Moss R. Rapid Action of 17β-estradiol on kainante-induced currents in hippocampal neurons lacking intracellular estrogen receptors. Endocrinol 1999;140(2):660–666. 137. Barrett-Connor E, Grady D. Hormone replacement therapy, heart disease, and other considerations. Ann Rev Public Health 1998;19:55–72. 138. Nathan L, Chaudhuri G. Estrogens and atherosclerosis. Ann Rev Pharmacol Toxicol 1997;37:477–515. 139. Rubanyi GM, Freay AD, Kauser K, et al. Vascular estrogen receptors and endothelium-derived nitric oxide production in the mouse aorta: gender difference and effect of estrogen receptor gene disruption. J Clin Invest 1997;99(10):2429–2437.

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140. Iafrati MD, Karas RH, Aronovitz M, et al. Estrogen inhibits the vascular injury response in estrogen receptor α-deﬁcient mice. Nat Med 1997;3(5):545–548. 141. Register TC, Adams MR. Coronary artery and cultured aortic smooth muscle cells express mRNA for both the classical estrogen receptor and the newly described estrogen receptor β. J Steroid Biochem Mol Biol 1998;64(3,4):187–191. 142. Pau CY, Pau KY, Spies HG. Putative estrogen receptor β and α mRNA expression in male and female rhesus macaques. Mol Cell Endocrinol 1998;146(1,2):59–68. 143. Hodges YK, Tung L, Yan XD, Graham JD, Horwitz KB, Horwitz LD. Estrogen receptors α and β: prevalence of estrogen receptor β mRNA in human vascular smooth muscle and transcriptional effects. Circulation 2000;101(15):1792–1798. 144. Russell KS, Haynes MP, Sinha D, Clerisme E, Bender JR. Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling. Proc Natl Acad Sci USA 2000;97(11):5930–5935. 145. Post WS, Goldschmidt-Clermont PJ, Wilhide CC, et al. Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system. Cardiovasc Res 1999; 43(4):985–991. 146. Paigen B, Holmes PA, Mitchell D, Albee D. Comparison of atherosclerotic lesions and HDL-lipid levels in male, female, and testosterone-treated female mice from strains C57BL/6, BALB/c, and C3H. Atherosclerosis 1987;64(2,3):215–221. 147. Lindner V, Kim SK, Karas RH, Kuiper GG, Gustafsson JA, Mendelsohn ME. Increased expression of estrogen receptor-β mRNA in male blood vessels after vascular injury. Circ Res 1998;83(2):224–229. 148. Karas RH, Hodgin JB, Kwoun M, et al. Estrogen inhibits the vascular injury response in estrogen receptor β-deﬁcient female mice. Proc Natl Acad Sci USA 1999;96(26):15,133–15,136. 149. Mendelsohn ME, Karas RH. Estrogen and the blood vessel wall. Curr Opin Cardiol 1994;9(5): 619–626. 150. Grohe C, Kahlert S, Lobbert K, et al. Modulation of hypertensive heart disease by estrogen. Steroids 1996;61(4):201–204. 151. Johnson BD, Zheng W, Korach KS, Scheuer T, Catterall WA, Rubanyi GM. Increased expression of the cardiac L-type calcium channel in estrogen receptor-deﬁcient mice. J Gen Physiol 1997;110(2): 135–140. 152. Carani C, Qin K, Faustini-Fustini M, et al. Effect of testosterone and estradiol in a man with aromatase deﬁciency. N Engl J Med 1997;337(2):91–95. 153. Conte FA, Grumbach MM, Ito Y, Fisher CR, Simpson ER. A syndrome of female pseudohermaphrodism, hypergonadotropic hypogonadism, and multicystic ovaries associated with missense mutations in the gene encoding aromatase (P450arom) J Clin Endocrinol Metab 1994;78(6):1287–1292. 154. Mullis P, Yoshimura N, Kuhlman B, Lippuner K, Jaeger P, Harada H. Aromatase deﬁciency in a female who is compound heterozygote for two new point mutations in the P450arom gene: impact of estrogens on hypergonadotropic hypogonadism, multicystic ovaries, and bone densitometry. J Clin Endocrinol Metab 1997;82:1739–1745. 155. Kusec V, Virdi AS, Prince R, Trifﬁtt JT. Localization of estrogen receptor-α in human and rabbit skeletal tissues. J Clin Endocrinol Metab 1998;83(7):2421–2428. 156. Kennedy J, Baris C, Hoyland JA, Selby PL, Freemont AJ, Braidman IP. Immunoﬂuorescent localization of estrogen receptor-α in growth plates of rabbits, but not in rats, at sexual maturity. Bone 1999;24(1):9–16. 157. Nilsson LO, Boman A, Savendahl L, et al. Demonstration of estrogen receptor-β immunoreactivity in human growth plate cartilage. J Clin Endocrinol Metab 1999;84(1):370–373. 158. Braidman I, Baris C, Wood L, et al. Preliminary evidence for impaired estrogen receptor-α protein expression in osteoblasts and osteocytes from men with idiopathic osteoporosis. Bone 2000;26(5): 423–427. 159. Oreffo RO, Kusec V, Romberg S, Trifﬁtt JT. Human bone marrow osteoprogenitors express estrogen receptor-α and bone morphogenetic proteins 2 and 4 mRNA during osteoblastic differentiation. J Cell Biochem 1999;75(3):382–392. 160. Onoe Y, Miyaura C, Ohta H, Nozawa S, Suda T. Expression of estrogen receptor β in rat bone. Endocrinology 1997;138(10):4509–4572. 161. Windahl SH, Norgard M, Kuiper GG, Gustafsson JA, Andersson G. Cellular distribution of estrogen receptor β in neonatal rat bone. Bone 2000;26(2):117–121.

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162. Arts J, Kuiper GG, Janssen JM, et al. Differential expression of estrogen receptors α and β mRNA during differentiation of human osteoblast SV-HFO cells. Endocrinology 1997;138(11):5067–5070. 163. Vidal O, Kindblom LG, Ohlsson C. Expression and localization of estrogen receptor-β in murine and human bone. J Bone Miner Res 1999;14(6):923–929. 164. Oreffo RO, Kusec V, Virdi AS, et al. Expression of estrogen receptor-α in cells of the osteoclastic lineage. Histochem Cell Biol 1999;111(2):125–133. 165. Hoyland JA, Baris C, Wood L, et al. Effect of ovarian steroid deﬁciency on oestrogen receptor α expression in bone. J Pathol 1999;188(3):294–303. 166. Korach K, Taki M, Kimbro K. The effects of estrogen receptor gene disruption on bone. In: Paoletti R, editor. Women’s Health and Menopause. Amsterdam: Kluwer Academic Publishers and Fondazioni Giovanni Lorenzini; 1997. pp. 69–73. 167. Vidal O, Lindberg MK, Hollberg K, et al. Estrogen receptor speciﬁcity in the regulation of skeletal growth and maturation in male mice. Proc Natl Acad Sci USA 2000;97(10):5474–5479. 168. Windahl SH, Vidal O, Andersson G, Gustafsson JA, Ohlsson C. Increased cortical bone mineral content but unchanged trabecular bone mineral density in female ERβ(−/−) mice. J Clin Invest 1999;104(7):895–901. 169. Hanstein B, Liu H, Yancisin MC, Brown M. Functional analysis of a novel estrogen receptor-β isoform. Mol Endocrinol 1999;13(1):129–137. 170. Ogawa S, Inoue S, Watanabe T, et al. Molecular cloning and characterization of human estrogen receptor βcx: a potential inhibitor of estrogen action in human. Nucleic Acids Res 1998;26(15):3505– 3512. 171. Murphy L, Dotzlaw H, Leygue E, Douglas D, Coutts A, Watson P. Estrogen Receptor Variants and Mutations. J Steroid Biochem Mol Bio 1997;62(5–6):363–372. 172. Meyers MJ, Sun J, Carlson KE, Katzenellenbogen BS, Katzenellenbogen JA. Estrogen receptor subtype-selective ligands: asymmetric synthesis and biological evaluation of cis- and trans-5,11dialkyl-5,6,11, 12-tetrahydrochrysenes. J Med Chem 1999;42(13):2456–2468.

4

SERM Modulation of Gene Expression Role of Coactivators and Corepressors

Paul Webb,

PHD

Contents Introduction The Mechanism of Estrogen Action ER Action at Classical EREs ER Action at Alternate EREs Negative Estrogen Regulation Future Directions References

INTRODUCTION Estrogens stimulate breast cancer growth and lifetime estrogen exposure is a risk factor for development of breast cancer (reviewed in (1)). Thus, it is desirable to block estrogen action in cancer treatment and prevention. Estrogens also exert positive effects upon overall health, including prevention of osteoporosis and reduced cardiovascular disease. Thus, it is also desirable to either supply estrogens, or mimic estrogen action, in hormone replacement therapy. The selective estrogen receptor modulators (SERMs) are synthetic compounds that block the growth of estrogen-dependent breast cancers and are showing promise as breast cancer preventatives (2). Remarkably, the SERMs also exhibit unique proﬁles of estrogenlike effects in other tissues, such as the uterotropic activity of tamoxifen and the ability of tamoxifen and raloxifene to arrest bone loss (reviewed in (2–4)). The SERMs can even exhibit novel activities that are not shared with estrogens (5–10). It may be possible to harness these mixed agonist/antagonist behaviors to develop new hormone replacement therapies that would simultaneously function as cancer drugs or cancer preventatives. Understanding the mechanisms that underlie these diverse SERM behaviors will be an important step towards this goal. Estrogen signal transduction is mediated by estrogen receptor proteins (ERs), which are ligand-dependent transcription factors (11). Like other eukaryotic transcription factors, the ERs modulate transcription by binding to coactivator and corepressor

From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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proteins (12,13). This chapter reviews the way that the SERMs affect ER dependent gene expression by inﬂuencing ER interactions with coactivators and corepressors.

THE MECHANISM OF ESTROGEN ACTION There are two ERs (ERα and ERβ), which both belong to the nuclear receptor family of conditional transcription factors (14). Estrogens work by promoting ERα release from an inhibitory heat shock protein/chaperonin complex, and ERα then dimerizes and binds DNA and chromatin. Estrogens also promote conformational changes within the ERα molecule itself that allow the receptor to modulate gene expression and thereby alter the protein content of the cell. ERβ appears to bind to DNA constitutively, but still modulates gene expression in response to estrogens (15). Broadly speaking, the ERs inﬂuence the expression of two types of gene. In the bestknown pathway of estrogen action (classical response) the ERs stimulate transcription by binding as dimers to speciﬁc estrogen-response elements (EREs), which are found within the promoters of estrogen-regulated genes including the vitellogenins, ovalbumin, pS2, prolactin (reviewed in (16)) and C3 complement (17). The ERs also utilize undeﬁned protein/protein interactions to modulate gene expression through so-called alternate EREs, which bind heterologous transcription factors, but not the ERs (18). ER action at alternate EREs occur in positive estrogen regulation of the collagenase and ovalbumin proximal promoters and insulin-like growth factor 1 (IGF-1) gene (through activator protein 1 (AP-1) sites that bind jun/fos) (5,7,19–21), the cyclin D1 gene (through a cyclic adenosine monophosphate (AMP) response element that binds jun/ATF-2) (22), the E2F gene (through an SP-1 nuclear factor Y (NF-Y) complex) (23), Cathepsin D (through an upstream stimulatory factor (USF)-binding site (24), and the genes for retinoic acid receptor α-1, transforming growth factor (TGF)-β3, quinone reductase, brain creatine kinase, and estrogen-responsive B-box protein (EBBP) (through unknown transcription factors) (8–10,25–28). The ERs also repress transcription of the tumor necrosis factor (TNF) gene (through a TNF response element that binds a jun/activating transcription factor-2 (ATF-2)/ets/nF-κB complex) (29), the IL6 gene (through an nF-κB binding site) (30), and various red blood cell speciﬁc genes (through unknown transcription factors) (31). The following sections review how the ERs act at different types of response element and how the SERMs inﬂuence these processes by modulating ER interactions with coactivators and corepressors.

ER ACTION AT CLASSICAL ERES The ER structure/function requirements for classical estrogen responses are well understood (11,14). Like other nuclear receptors, the ERs are composed of three discrete domains, an N-terminal A/B domain (NTD), a central DNA-binding zinc ﬁnger domain (DBD) and a C-terminal ligand-binding domain (LBD) (Fig. 1). The ER DBD mediates speciﬁc ERE recognition. The LBD and the NTD contain separate activation functions (AFs) that mediate transcriptional enhancement. The LBD activation function (AF-2) is relatively strong and absolutely dependent on the presence of hormone for its activity. The NTD activation function (AF-1) is constitutive, usually weaker, and serves only to synergize with AF-2. ERα AF-1, however, does show strong independent activity in some cell types, at speciﬁc promoters and in response to mitogen activated protein (MAP) kinase phosphorylation (32).

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Fig. 1. Structural organization of the estrogen receptors (ERs). The ERs consist of three separable domains, the N-terminal domain (NTD), the DNA-binding domains (DBD, shown in black) and the ligand-binding domain (LBD). The LBD binds hormone (E) and contains a hormone-dependent activation function (AF-2) that is depicted as a patch on the LBD surface. The NTD contains a constitutive activation function (AF-1) that is regulated by MAP kinases.

ER activation functions work by binding to coactivators (Fig. 2A) (11,18). The existence of coactivators was ﬁrst inferred from the demonstration that nuclear receptor activation functions repress, or squelch, the activity of other activation functions, presumably by competing for endogenous limiting factors (33,34). ER AF-2 targets were directly detected using labeled ER/ligand binding domain (LBD)s as probes in FarWestern blots of cellular extracts (35,36). Each of the AF-2 interacting proteins was of relatively high molecular weight (140–220kDa) and many have now been cloned. Their identity as coactivators has been established by several independent criteria (reviewed in (12)). First, the coactivators bind ER activation functions. Second, the coactivators potentiate ER transcriptional activity, but not basal transcription. Third, overexpressed coactivators relieve squelching between nuclear receptors. Fourth, ER mutations and coactivator mutations that block ER/coactivator interactions also block transcriptional activation. Fifth, overexpression of coactivators masks the phenotype of partial ER mutants. Sixth, mutant versions of the coactivators interfere with ER action. Seventh, in accordance with the notion that coactivators bind to the ER/DNA complex and activate transcription, the coactivators enhance gene transcription in their own right when artiﬁcially tethered to DNA. Recently, it has also been shown that estrogen regulation is disrupted in mice bearing germ-line knockouts of genes that code for ER coactivators (37,38). The best understood ER AF-2 coactivators are the p160s (Fig. 2B), including glucocorticoid receptor interacting protein-1 (GRIP-1) [TIF-2/NcoA−2 {transcriptional mediatior/intermediary factor−2/nuclear receptor coactivator−2}], steroid receptor coactivator 1 (SRC-1)[NCoA−1], and activator of thyroid and retinoic acid receptors (ACTR) [pCIP, RAC3, TRAM-1 and AIB1]) (13). AF-2 binds to an amphipathic αhelix with the consensus sequence leucine-X-X-leucine-leucine (LXXLL), or nuclear receptor box (NR box), that is reiterated three or more times in each p160 (see 13), and references therein). The p160s work by remodeling chromatin. Each p160 contains two discrete activation domains, activation domain 1 (AD1) and activation domain 2 (AD2) (39–45). AD1 binds to CREB binding protein (CBP)/p300, which is a histone acetyl-transferase (HAT) (46,47), and both the p160s and CBP contact pCAF, another HAT (48). Some reports also suggest that the p160s themselves possess HAT activity (41,49). AD2 binds to CARM1, which is an arginine methyl-transferase (50). Histone acetylation and methylation are thought to open up chromatin and thereby improve the

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Fig. 2. (A) The estrogen/ER complex activates transcription by utilizing its DBD to bind to speciﬁc EREs and its activation functions (Afs) to recruit coactivators. AF-2 contacts NR boxes (LXXLL) within the p160 molecule and AF-1 binds the p160 C-terminus and CBP/p300. The coactivator complex then enhances transcription by remodeling chromatin. (B) p160 structure. The Pas/HLH contains strong homologies to other proteins (per, arnt, and sim) and is implicated in dimerization and DNA binding, but has not yet been shown to possess a similar function in the context of p160 proteins. The central region contains three distinct NR boxes (LXXLL) that bind to nuclear receptor AF-2 functions. AD1 is a strong activation function that colocalizes with the region of CBP/p300 binding. The C-terminus is complex and contains a glutamine (Q) rich region and second activation function. This region binds ERα AF-1 and also contains HAT activity, a pCAF site and a CARM1 binding site.

access of RNA polymerase and its associated proteins to the promoter (13). In accordance with this notion, the p160 ACTR and p300 have been shown to associate with the chromatin of estrogen-regulated genes and this event coincides with increased levels of local histone acetylation and enhanced gene transcription (51). Moreover, ERαdependent estrogen responses can only be reconstituted in vitro with templates that have been preassembled into chromatin (52,53). The ER AF-2 functions also bind other coactivators that contain LXXLL motifs (54,55). ERβ AF-2 binds strongly to TRAP220 (54), which is a component of a large multisubunit complex thyroid receptor associated protein (TRAP)s, also known as vitamin D receptor interacting protein (DRIP)/SRB- and MED-containing cofactor complex (SMCC)/activator-recruited cofactor (ARC) that was originally puriﬁed as a result of its interaction with liganded thyroid receptors (TRs) (56). The TRAPs enhance TR dependent transcription from naked DNA templates in vitro, but are also required for synergy between different classes of transcription factor at templates that have been preassembled into chromatin in vitro (56–58). This suggests that the TRAP complex

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Fig. 3. (A) Schematic of the AF-2 surface. The LBD is represented as a globular structure with ligand (striped) buried. Helixes 3, 4, 5, and 12 come together on the surface to form a hydrophobic cleft (gray shading) that acts as a binding site for the NR LXXLL motif. (B) Schematic of the way that SERMS block AF-2 activity. The SERM (striped) is partially buried, but protrudes through the LBD surface near the base of helix 12. Helix 12 is then forced back into the remainder of the hydrophobic cleft, thereby removing part of the NR box binding site (helix 12) and occluding the rest.

works by interacting with the basal transcription machinery and by remodeling chromatin. The estrogen receptor activation function 2 (ER AF-2) functions also bind to LXXLL motifs in a host of other potential coactivators including ERAPs (36), PPAR gamma coactivator-1 (PGC-1) (59,60), alteration/deﬁciency in activation (ADA)3 (61), the human homologues of the yeast SWI/SNF complex (62), and E6-associated protein (E6-AP) (63) and to proteins that do not substantially enhance ER transcriptional activity, including receptor interacting protein–140 Kd (RIP-140) (35,64), suppressor for gal1 (SUG1) and transcriptional mediator/intermediary factor-1 (TIF1) (65), although the physiological signiﬁcance of many of these interactions is not yet clear. AF-1 binds some of the same coactivators. ERα AF-1 binds to the GRIP-1 Cterminus (66,67), and to other p160s (66,68,69), and both ERα and ERβ AF-1 bind CBP/p300 (70). Interestingly, ERα AF-1 activity is potentiated by steroid receptor RNA activator (SRA) (71), a noncoding RNA transcript that associates with SRC-1, suggesting that the coactivators may be part of a ribonucleoprotein complex. Phosphorylated ERα AF-1 binds p68 RNA helicase (72), and phosphorylated ERβ AF-1 binds the p160s (73). Thus, AF-1 and AF-2 contact different surfaces of the same coactivator complex, and this ability may account for synergy between AF-1 and AF-2 (66,67).

SERMs Block Estrogen Response by Blocking AF-2 The SERMs inhibit ER action at EREs by blocking AF-2 activity (16,32). The molecular basis of this effect is now understood at the atomic level. A series of recent nuclear receptor crystal structures revealed that the LBDs form a compact globular αhelical structure that completely encloses the ligand (reviewed in (74)). The receptor crystal structures also allowed visualization of the organization of ER amino acids that were crucial for AF-2 activity (75–77), and revealed that each mapped to a hydrophobic cleft comprised of helixes 3,4,5, and 12 that is common to all nuclear receptors (Fig. 3A). A large-scale effort was then undertaken to identify all of the residues that

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comprised AF-2 in the TR (78). Each TR surface amino acid was mutated, and the resulting mutant TRs were examined for their ability to activate transcription, bind p160 coactivators, bind ligand and dimerize. The only residues that speciﬁcally affected TR transcriptional activity and p160 binding were located in the vicinity of the hydrophobic cleft. Moreover, mutations within the cleft diminished TR transcriptional activity and p160 binding in parallel. Similar results were also obtained with ERα (78,79). Thus, AF-2 is comprised of a large hydrophobic cleft that acts as a docking site for the p160 LXXLL motif. Subsequent cocrystals of the ERα, TR, and peroxisome proliferator activated receptor (PPAR) complexed with different p160 peptides revealed that there are two components of AF-2/p160 NR box recognition (80–82). First, charged lysine and glutamic acid residues in helix 3 and helix 12 form a charge clamp that binds to the carbamyl backbone of the LXXLL peptide and stabilizes the α-helix. Second, hydrophobic residues within the cleft make direct contacts with the side chains of the LXXLL leucines. The way that the SERMs block AF-2 activity was then clariﬁed by a series of SERM/ ER complex crystal structures (80,83,84). Generally, ER agonists like estradiol and ˚ , whereas antagonists, like tamoxifen and diethylstibestrol (DES) occupy approx 300 A raloxifene, are larger (85). The extra volume is contained within a side chain extension that is built upon a chemical backbone that partially resembles estrogen and interacts with the ER estrogen-binding pocket. In tamoxifen- and raloxifene/ERα complexes (80,83), and a raloxifene/ERβ complex (84), the SERM extension protrudes through the ER surface near the base of helix 12 (Fig. 3B). This results in a displacement of helix 12, which rotates 110° and folds back into the remainder of the hydrophobic cleft. Interestingly, both ERα and ERβ helix 12 contain a sequence (LLLEML) resembling the NR-box LXXLL sequence, and the interactions of helix 12 with the ER hydrophobic cleft strongly resemble those of the p160 NR box with the ER hydrophobic cleft (80,83). Thus, the SERMs inhibit AF-2 activity by altering helix 12 position so that half of the charge clamp (the helix 12 glutamic acid) that stabilizes the p160 NR-box carbamyl backbone is displaced and the remainder of the hydrophobic cleft, which binds the p160 LXXLL-motif leucine side chains, is occluded.

Estrogenlike Effects of SERMs Stem from ER␣ AF-1 ERα shows some activity in the presence of tamoxifen (86), and to a lesser extent, raloxifene and GW5638 (17,87,88). These effects can be quite strong in some cell types, in certain promoter contexts, and on mitogen stimulation and subsequent MAP kinase activation (32,89). Several lines of evidence indicate that tamoxifen agonist effects stem from AF-1. Truncation of the amino-terminal domain (NTD), which contains AF-1, abolishes all agonist effects at classical EREs. Moreover, the isolated NTD/ DBD region, which only contains AF-1, activates transcription with the same cell type and promoter speciﬁcity as tamoxifen-liganded ERα (86), and shows enhanced activity in response to mitogen stimulation (90–92). Finally, a natural ERα splice variant that lacks the NTD is unable to enhance gene transcription in the presence of tamoxifen and acts as a dominant negative for tamoxifen activation by full-length ERα (93). Thus, SERMs behave like estrogens at classical EREs by allowing ERα to bind to DNA and utilize AF-1 to recruit coactivators (Fig. 4A). The SERMs do not behave as agonists in the presence of ERβ (6,66,94). This is probably because ERβ AF-1 is relatively weak in coactivator binding (6,73). ERβ also

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Fig. 4. (A) Model to explain SERM agonist effects at classical EREs. ERα binds to EREs, but tamoxifen (T, shaded) alters helix 12 positioning, thereby preventing AF-2 from binding to coactivators. AF-1 still makes contacts with the p160/CBP complex, and these contacts are sufﬁcient to enhance transcription. (B) ERα NTD structure. The residues that are needed for AF-1 activity are shaded. The black area corresponds to box 1, which is responsible for tamoxifen activation in some breast cells. The gray area corresponds to the remainder of AF-1, which is required for synergy with AF-2 and tamoxifen response in other cell types. The positions of phosphorylated serine residues and the consensus MAP kinase phosphorylation site (PXXSP) are marked. Regions of AF-1 that contact known coactivators are marked above.

inhibits ERα-dependent estrogen and tamoxifen responses in heterodimers (15,95), however. The mechanism of this inhibitory effect is unknown, but it might also contribute to the inability of ERβ homodimers to allow SERM agonist effects. The fact that SERM agonist effects stem from ERα AF-1 indicates that it is important to understand how AF-1 binds coactivators. ERα AF-1 generally lies between amino acids 41 and 129 (Fig. 4B) (66,75,96–98). All of these residues are required for maximal tamoxifen response and for maximal synergy with AF-2 in most cell types. This region can be subdivided into distinct subdomains. The proximal region of the NTD, approximately spanning amino acids 41–100, shows constitutive activity and mediates GRIP-1 and CBP/p300 binding (66,70). A region spanning 100–130 is the MAP kinase target region and contains three serine residues (S104, S106 and S118) that are phosphorylated by MAP kinases (90–92,99,100) and other kinases (101). Indeed, S118 lies within a perfect consensus MAP kinase phosphorylation site (PXXSP), and accounts for more than 90% of ERα phosphorylation in vivo. Phosphorylation of these residues enhances ERα interactions with p68 RNA helicase (72). It is likely that AF-1 structure will prove to be even more complex. Progressive truncations of the proximal region of the NTD result in gradual reductions of transcriptional activity, rather than abrupt losses (see, for example, (96)). This suggests that

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even this region of AF-1 may be composed of multiple discrete activation domains. Indeed, tamoxifen responses exhibit somewhat different ERα/NTD sequence requirements in breast cells (98). Here, amino acids 41–64 (Box 1) are speciﬁcally required for tamoxifen response, but not synergy with AF-2, suggesting that Box 1 may be a discrete breast-cellspeciﬁc activation function. Finally, other ERα AF-1 coactivators may yet be identiﬁed. ERα AF-1 enhances transcription in yeast, even though yeast lacks p160s and CBP (86,97). Thus, AF-1 must contact unknown yeast coactivators and it will be interesting to ask whether homologues of these factors regulate AF-1 activity in humans.

SERMs Promote Corepressor Recruitment It is now appropriate to introduce another class of molecule, the corepressors (13). The TRs, retinoic acid receptor (RAR)s, and retinoid X receptor (RXR)s, as well as several orphan receptors, bind to their response elements in the absence of hormone and actively inhibit transcription. This repression is mediated by recruitment of corepressor proteins, including nuclear receptor corepressor (N-CoR) and silencing mediator for retinoid and thyroid receptor (SMRT), which bind to the unliganded receptor and are released on hormone binding. The TRs and RARs bind to speciﬁc ID sequences with the consensus I/LXXI/VI, which resemble the coactivator NR box (LXXLL) and are found reiterated several times within the corepressor C-terminus (102–105). The corepressor N-terminus contains distinct repression domains that bind other components of a large corepressor complex. In essence, this corepressor complex is thought to repress transcription because it contains histone deacetylases (HDACs), which bind either directly or indirectly through other corepressor proteins such as mSin3A, SMRT, and N-CoR (106–110). Thus, unliganded TRs and RARs repress transcription by binding a large complex that contains HDACs and works by reversing the effects of HAT in the coactivator complex. It was originally thought that the corepressors were not involved in steroid receptor action. This picture changed when N-CoR was shown to speciﬁcally associate with the tamoxifen-liganded ERα in a yeast two-hybrid screen (111), and coprecipitate with tamoxifen-liganded ERα in cell extracts (69). A large body of evidence now indicates that ERα AF-1 is under negative regulation by corepressors. First, transfected corepressors inhibit the activity of tamoxifen-liganded ERα (112). Second, microinjection of antibodies that recognize the corepressor complex leads to enhancement of tamoxifen activation, presumably by disrupting the corepressor complex (69,110). Third, tamoxifenliganded ERα enhances the activity of progesterone receptor AF-1, presumably by sequestration of active repressor complexes (113), and transfection of N-CoR reverses this effect. Thus, SERMs inhibit ER action both by blocking coactivator recruitment (Figs. 3 and 4), and by promoting corepressor recruitment (Fig. 5B). How do the nuclear receptors and ERs bind the corepressor complex? The TR interaction site for N-CoR consists of residues that overlap the coactivator binding site (102–104), and residues that lie beneath helix 12 (Fig. 5C). Indeed, removal of helix 12 promotes corepressor binding to the TRs, RARs, and RXR. The physical basis of ERα/N-CoR interactions then presents an obvious puzzle. The presumptive N-CoR target region of the cleft is occluded by helix 12 in the context of the SERM/ER complex (80,83,84). This suggests that either the existing ERα/SERM crystal structures are not representative of the corepressor binding conﬁguration, or that ERα recognizes corepressors in a completely distinct manner.

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Fig. 5. (A) Schematic of N-CoR Structure. Nuclear receptors bind to ID motifs with the consensus I/LXXI/VI that are reiterated three times in N-CoR and twice in SMRT. The N-CoR N-terminus contains separable silencing domains that bind to other components of the corepressor complex. (B) Model to explain the silencing role of N-CoR in ERα action. N-CoR binds to the tamoxifenliganded ERα and inhibits AF-1 activity by an unknown mechanism. (C) The paradox of ER/NCoR interactions. Residues that contribute to N-CoR and SMRT binding map to the hydrophobic cleft of the TR and extend under the usual position of helix 12 in the liganded TR structure. This suggests that helix 12 is displaced in the TR/N-CoR complex. Helix 12 covers the same residues in the context of the SERM/ER complex crystal structure. Thus, either the ERα crystal structures do not represent the true corepressor binding conﬁguration or ERα must bind to N-CoR in a fashion that is distinct from the TR.

The ERs also bind to an unrelated ER-speciﬁc repressor protein, REA (114). This 37 kDa protein binds to the ER/LBD, inhibits ER action, and reverses the action of p160 coactivators, but has little or no effect upon other nuclear receptors. Overexpression of REA strongly enhances SERM antiestrogenicity. This suggests that REA may play an important role in determining the sensitivity of estrogen target cells to SERM antiestrogenic effects. It will be very important to understand how the ERs bind REA and how the SERMs inﬂuence this process.

Different SERMs Repress AF-1 Activity to Different Degrees Curiously, the transcriptional activity of the DNA-bound ERα varies with different SERMs (86,88,115,116). Tamoxifen can allow quite signiﬁcant ERα transcriptional

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activity, but raloxifene and GW5638 allow less and ICI 182,780 allows little or none at all. The overall level of ERα-dependent tamoxifen activation equals the transcriptional activity of isolated AF-1 (86). Thus, because other SERMs exhibit less transcriptional activity than tamoxifen they must actively inhibit AF-1. While the molecular basis of this repressive effect is not yet clear, and may be related to the ability of some SERMs to downregulate ER levels (4), it is possible that at least part of this differential activity is related to differences in corepressor recruitment. Antiprogestins enhance progesterone receptor (PR) interactions with SMRT in vivo and the strength of the antiprogestin correlates with the strength of PR/SMRT interactions (117). The ability of some SERMs to inhibit AF-1 activity may correlate with the overall strength of ERα/corepressor interactions.

ER ACTION AT ALTERNATE ERES The ERs modulate transcription of genes that contain alternate EREs, which bind a variety of heterologous transcription factors but not the ERs. There are some indications that this process may be just as important as ER action at classical EREs (18,118). Tamoxifen enhances AP-1 activity in uterine cells, but not breast cells (5). This effect parallels the effects of tamoxifen upon cell division in these cell lines. Moreover, estrogens enhance AP-1 activity in some breast cell lines and repress AP-1 activity in others, depending on the levels of the AP-1 protein FRA-1 (19,119). Once again, these effects parallel estrogen effects on cell growth. Thus, ER action at AP-1 sites may resemble ER action at genes that play a role in cell division. Indeed, ERα does enhance the transcription of the genes that are clearly involved in growth response, such as cyclin D1, c-myc, IGF-1 and TGF-β3, and all of these responses are mediated by alternate EREs (reviewed in (18)). Two key facts are known about ER action at alternate EREs. First, the ERs work through poorly deﬁned protein/protein interactions. Second, SERMs often strongly enhance the transcription of this type of gene. Thus, any model to explain ER action at alternate EREs must explain the nature of the protein/protein interactions that are involved and why the SERMs show such strong activities. Our explorations of ER action at AP-1 responsive reporters have revealed that the ERs enhance AP-1 activity using two distinct mechanisms with different requirements for ER activation functions (5–7). ERα enhances AP-1 activity in the presence of estradiol and tamoxifen and, more weakly, in the presence of other SERMs. Estradiol action is independent of the ERα DBD, but does require both AF-1 and AF-2. By contrast, ERβ enhances AP-1 activity in the presence of SERMs, and SERMs with high antiestrogenicity at classical EREs, such as ICI 182,780 and raloxifene, show the most potent stimulatory effects at AP-1 sites. These effects are independent of ERβ activation functions. Thus, ERα and ERβ enhance AP-1 activity by AF-mediated and AF-independent pathways with distinct ligand preferences (Fig. 6). What are the mechanisms of these two pathways? For convenience, each pathway is discussed separately.

The AF-Mediated Pathway ERα enhances AP-1 activity in a manner that depends on both ERα activation functions (5–7). Moreover, GRIP-1 overexpression enhances estrogen response at AP-1 sites, and this effect requires the GRIP-1 C-terminus, which binds AF-1, and the GRIP-

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Fig. 6. ER enhances AP-1 activity by two distinct sets of protein/protein interactions. The ER activation functions play an important role in the AF mediated pathway at left, but the DBD is dispensable (indicated with gray shading). The ER activation functions are not required for the AF independent pathway (indicating with gray shading) but the DBD is required.

1 NR boxes, which bind AF-2 (6). Thus, the ERα activation functions seem to be working at AP-1 sites by contacting p160s, just as they do at EREs. In accordance with the notion that ERα action at AP-1 sites resembles ERα action at EREs, speciﬁc peptide inhibitors have been shown to block estrogen and tamoxifen response at both types of response elements (55). Nonetheless, whereas mammalian one-hybrid assays suggested that the estrogen-liganded ERα directly participates in the AP-1 complex in vivo, direct contacts between ERα and the AP-1 proteins proved to be dispensable for estrogen action at AP-1 sites (5,118). How then does ERα get to the AP-1 complex? Because jun-fos works by binding to CBP/p300 (120–122), which binds p160s, we have suggested that ERα might enhance AP-1 activity by binding to the AP-1 coactivator complex, rather than the AP-1 proteins themselves (Fig. 7A). This model has some surprising implications. The ERs work at classical EREs by recruiting coactivators. In the context of the AP-1-regulated promoter, jun-fos would provide the coactivator recruitment function. Thus, for ERα to enhance AP-1-responsive transcription it would have to work at a distinct step in transactivation. PPARγ is known to trigger the activity of its own coactivator, PGC-1, by favoring PGC-1 interactions with a downstream SRC-1/CBP complex (123). Our unpublished results indicate that free ERα strongly enhances the activity of coactivators that have been artiﬁcially tethered to DNA, suggesting that ERα does enhance, or trigger, the activity of its own coactivator complex. Moreover, AF-1, which is usually weak in coactivator recruitment, shows strong activity in triggering. We have therefore suggested that ERα enhances AP-1 activity by triggering the activity of the AP-1 associated CBP-p160 complex (6,18). This ﬁnding might help explain why tamoxifen shows strong activity at AP-1 sites and alternate EREs. We also speculate that AF-1-dependent triggering could explain the long-standing observation that ERα AF-1 is quite weak on simple ERE regulated promoters, but much stronger at complex promoters that contain both EREs and binding sites for other transcription factors. If the non-ER transcription factors were to prerecruit CBP-p160 complexes, and thereby relieve ERα of the job, then AF-1 dependent triggering would then come into play (Fig. 7B).

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Fig. 7. (A) Model for ERα action of AP-1 sites. The jun/fos complex enhances transcription by recruiting a CBP/p160 complex. ERα enhances AP-1 activity by binding to p160s that are present in this complex. Because jun/fos provides the recruitment function in this model, ERα must act at another step in transcriptional regulation. We have suggested that ERα triggers the AP-1 coactivator complex into full activity. (B) Model for AF-1 promoter speciﬁcity. Tamoxifen-liganded ERα binds to a promoter that also contains a binding site for heterologous factors (here AP-1) that recruit a CBP/p160 complex. The requirement for coactivator recruitment by ERα is bypassed and the role of AF-1 in triggering is revealed.

The AF Independent Pathway ERβ enhances AP-1 activity in the presence of SERMs, but not estrogens, and ERβ activation functions are not required for these SERM effects (6,7). Surprisingly, we also found that ERα mutants lacking AF-1 and AF-2 show reduced estrogen activation at AP-1 sites, but also display an ERβ-like phenotype in which the SERMs enhance AP-1 activity (6). Thus, both ERs have the potential to enhance AP-1 activity via an AF-independent pathway that is regulated by SERMs and inhibited by the presence of ER activation functions. There are several examples of endogenous genes that respond more strongly to SERMs than estrogens, including quinone reductase (8,9), TNFα (29), TGF-β3 (26), and RAR α-1 (10), suggesting that this pathway may be common to a range of genes with alternate EREs. How do SERMs activate AP-1 responsive transcription when estrogens do not? One

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Fig. 8. Model for SERM effects at AP-1 sites. The AP-1 responsive promoter is under negative regulation by HDACs. The SERM-liganded ER complex binds corepressors at a separate location and sequesters HDACs from the AP-1 site, thereby allowing the full activity of the AP-1 coactivator complex.

clue emerged from our early studies of ER action at AP-1 sites (5). The SERMliganded ER appeared to activate AP-1-responsive transcription without even directly participating in the AP-1 complex. We therefore suggested that the SERM/ER complex enhances AP-1 activity by binding unspeciﬁed corepressors at some location away from the AP-1 responsive promoter, and sequestering HDACs from the AP-1 complex (Fig. 8). A similar model has been proposed to explain the ability of unliganded TR to enhance AP-1 activity (124). Repressor sequestration would explain why: 1. SERM effects at AP-1 sites are independent of ER transactivation functions: They would require ER/corepressor binding surfaces. 2. SERM/ER complexes enhance AP-1 activity without participating in the AP-1 transcription complex: They would work by sequestration of repressors and by deﬁnition, must be away from the complex to function. 3. There is an correlation between the strength of SERM action at AP-1 sites and SERM antiestrogenicity at EREs: Both processes would reﬂect ER interactions with corepressors.

In summary, we suggest that ER action at AP-1 sites involves interactions with the same coactivators and corepressors that play a role at classical EREs. Here, however, both types of target protein enhance AP-1 activity. Similar mechanisms, or composites of the two, may be at play in ER action at a variety of alternate EREs.

NEGATIVE ESTROGEN REGULATION ERs inhibit the expression of genes that contain nF-κB binding sites. A recent study has revealed that the estrogens repress the TNF-alpha promoter, and that SERMs reverse this effect (29). Estrogen repression was found to be stronger in the presence of ERβ, independent of the ER/DBDs, but dependent on AF-2/p160 contacts. Thus, it is not surprising that the SERMs should block estrogen repression; SERMs are well known to block AF-2/p160 contacts.

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Fig. 9. Two possible models to explain the role of ER AF-2/coactivator contacts in transcriptional repression. The nF-κB (p50/p65) complex enhances transcription by binding to a CBP/p160 complex. The ER could sequester (squelch) p160s from the nF-κB coactivator complex or bind to p160s in the coactivator complex and recruit additional repressors (designated by an R).

The reasons ER AF-2/p160 contacts play a role in gene repression are less clear. Perhaps p160s act as corepressors in this context. Some other possibilities are outlined in Fig. 9. The ERs could sequester p160s from the nF-κB coactivator complex. Alternatively, the ERs could use p160s in the nF-κB coactivator complex as a platform to recruit repressor proteins.

FUTURE DIRECTIONS Ultimately, understanding SERM action will be as complicated as understanding how estrogens work. Estrogen action in different tissues probably represents the sum total of ER effects on many different types of genes and the subsequent interplay of gene products. Nonetheless, overall estrogen response probably involves relatively few core mechanisms of ER action. It may therefore be possible to understand SERM effects in terms of their overall ligand preference, and the known effects of SERMs on ER interactions with coactivators and corepressors. For example, SERMs block estrogen action in the normal breast and in breast cancer (1–3,125). Based on our knowledge of SERM action at simple reporter genes, estrogen action in the breast is probably dominated by AF-2/p160 interactions. Likewise, tamoxifen, but not raloxifene, shows estrogenlike uterotropic activity (2,125). Because tamoxifen often allows more ERα AF-1 activity than raloxifene, estrogen action in the uterus is probably dominated by ERα AF-1. Thus, ideal SERMs should prevent AF-2/coactivator interactions to block estrogen action in breast cancer, and either prevent AF-1/coactivator interactions or promote ERα/corepressor interactions to block AF-1 activity in uterus and prevent uterotropic effects. Understanding the ligand preference of SERM effects in other tissues will help us to understand these effects in terms of ER interactions with coactivators and corepressors. This, in turn, should help in the identiﬁcation of new SERMs with even more desirable properties.

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There is much more to learn about the mechanism of SERM action. While it is clear that ERα AF-1 activity plays an important role in tamoxifen response, it is unclear why ERα AF-1 activity varies in different contexts. It is also perplexing that ERα should only bind corepressors in the presence of SERMs. Could nature really have evolved a mechanism of ER regulation that is only important for synthetic drugs or do SERMs mimic a physiological effector of ER action? The near future promises to be an exciting time for SERM research. New technologies, such as the chromatin immunoprecipitation assay (51) and isolation of promoter-associated chromatin complexes (126) will soon allow us to detect coactivators and corepressors at individual promoters. It has already been possible to conﬁrm that estrogens promote p160 recruitment to estrogen-regulated promoters in breast cells, and that tamoxifen blocks this process (51). It will be informative to apply these new techniques to key genes that are stimulated or inhibited by SERMs in different tissues.

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5

Crosstalk Between Estrogen Receptors and Growth Factor Signaling Douglas Yee,

MD

and Carol A. Lange,

PHD

Contents Mechanisms of Steroid Hormone and Growth Factor Action Potential for Crosstalk and Implications for SERMs Clinical Evidence for SERM and Growth-Factor Receptor Interactions Summary References

MECHANISMS OF STEROID HORMONE AND GROWTH FACTOR ACTION Introduction The relevance of estrogen receptor-α (ERα) to breast-cancer biology is undisputed. Interruption of ER function by the selective estrogen receptor modulator (SERM) tamoxifen has been shown to reduce the risk of developing breast cancer, prolong overall survival in the adjuvant setting, and effectively palliate advanced disease. Although SERM therapy has fallen under the bland rubric of hormonal therapy, it must be recognized that in many ways SERMs represent the ideal therapy for cancer. As a therapy they are targeted and relatively free of side effects, and by measuring tumor ERα expression, their clinical utility can be predicted for any individual patient. It is therefore critical to understand how ERα functions. If ERα function were completely understood, then additional therapies (beyond simply interfering with estradiol binding to the receptor) could be developed for breast cancer. In addition, SERMs could be speciﬁcally selected for individual patients based on tumor characteristics. It is clear that ERα is a hormonally regulated transcription factor and its ability to bind estradiol is the basis for SERM action and effectiveness. It is now also established however that other factors affect ERα function and breast cancer biology. Other steroid hormone receptors, such as the progesterone receptor (PR) and peptide growth factor receptors clearly play a role in breast cancer biology. In this section, we will review the ability of growth-factor receptor-signaling pathways to interact with ERα. From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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Estrogen Receptor Function Beatson ﬁrst demonstrated that breast cancer growth was regulated by ovarian produced hormones (1). It took nearly 100 additional years to clone the principal effector of estradiol action. ERα is a member of a large superfamily of steroid hormone receptors (2) containing conserved domains compatible with their function as ligand-induced transcription factors. ERα contains a hormone-binding domain, a DNA-binding domain, and two transcriptional activation domains (AF-1 and AF-2). Estradiol binding to ERα results in dimerization and subsequent binding of the hormone-receptor complex to speciﬁc DNA palindromic sequences (estrogen response elements [ERE]) to initiate gene transcription. In this way, estradiol can induce the expression of growth-promoting genes within speciﬁc ERα-containing tissues. For example, gene expression of the PR is regulated by ERα. ER Isoforms This relatively simple model for ER action has recently been made more complex by the discovery of additional proteins that may partner with ERα to regulate estradiol function. Estrogen receptor-β (ERβ) has been identiﬁed in many tissues including breast cancer (3–6). ERβ shares structural homology with ERα and has the capacity to bind the same ligands as ERα, but ligand afﬁnity between the two species is different. In vitro, ERα and ERβ can also form heterodimers. Thus, it is likely that ERβ could inﬂuence estradiol or SERM action (7), but the precise relationships are incompletely understood (8,9). Other proteins, the estrogen-related receptors, may inﬂuence estrogen action presumably by interacting with the same EREs involved in ERα’s interaction with DNA (10–12). Thus, the requirement for ERα to function as a dimer allows other related proteins to partner with it, potentially inﬂuencing estradiol and SERM action. Coregulatory Proteins In addition to these estrogen receptor dimerization partners, there are additional protein/protein interactions that affect ERα function. The classic model of steroid receptor action predicted that hormonal signals activate their cognate nuclear receptors, which bind to speciﬁc DNA sequences and regulate transcription. This simpliﬁed view has now reached a new level of complexity with the discovery of diverse coregulators, which physically interact with and can either positively or negatively regulate the activities of their associated steroid receptors (reviewed in 13,14–17). For example, the cAMP response element binding protein (CREB) CBP/p300 (265 kDa CBP and a highly related protein, p300) coactivator of CREB, interacts directly with the ligandbinding domain of several steroid hormone transcription factors including estrogen, retinoic acid, glucocorticoid, and thyroid hormone receptors in a ligand-dependent manner (18). CBP/p300 also interacts with a large number of signaling molecules, and both sequence-speciﬁc and basal transcription factors including CREB, E1A, TFIIB, and AP-1 (c-Fos/Jun); CBP is required for activation of both CREB and AP-1 (19,20). Coregulators do not bind DNA directly, but act to bridge sequence-speciﬁc factors with components of the basal transcription machinery and can exhibit intrinsic enzymatic activities in order to modify protein or DNA (13–15). For example, CBP/p300 proteins are histone acetylating enzymes; acetylation of histones causes nucleosome spreading and elongation of DNA, making it more accessible to gene activation (21,22). Negative regulation of transcriptional complexes containing CBP occurs through association of

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activated Rsk90 (a downstream effector of mitogen-activated protein kinase (MAPK) with CBP following growth-factor stimulation (23). Several nuclear receptor corepressors have also been characterized, many of these molecules are associated with histone deacetylase activities (24). A 270 kDa protein termed N-CoR (nuclear receptor corepressor) interacts with a variety of nuclear steroid hormone receptors and silences basal transcription of unliganded receptors; N-CoR dissociates on ligand binding (25). In addition, N-CoR is recruited to antagonist-occupied PR and ERα, and thereby may actively repress transcription (reviewed in 26,27). Since SERM binding induces different conformational states of ERα (28), it is likely that coactivator and corepressor interactions are signiﬁcantly inﬂuenced by different ligands bound to ER. For example, the tamoxifen/receptor complex does not initiate transcription from the ERE, presumably because the conformation of the receptor in the tamoxifen-bound state is unable to interact with required additional transcriptional activators (29). ERα function may also be induced directly by posttranslational modiﬁcations. Particularly, ERα can be phosphorylated on serine and tyrosine residues (30–33). These phosphorylation events likely inﬂuence the ability of ER to interact with other proteins and could have a role in regulating responses to ligand. Thus, it is clear that ERα functions as a ligand-induced transcription factor. Although some evidence for direct estrogen-mediated activation of growth promoting pathways exists (34) and nonnuclear functions of ERα have been documented (35–36), it is felt that many of these stimulatory effects are caused by the transcriptional activation of ERα by estradiol. In breast cancer, inhibition of ERα function is an effective and powerful therapeutic tool. The discovery of additional ways ERα can be activated should yield new therapies directed at key growth-regulatory molecules. Thus, there is now substantial evidence that factors other than estradiol result in ERα activation.

Growth Factor Action It has long been known that breast cancer cells are stimulated to proliferate by peptide growth factors. Ligands of the insulin and epidermal growth-factor families have effects on breast cancer cells (37–40). When these initial observations were made, little was known about growth-factor signal transduction. As pathways utilized by growth factors have become better understood, it has become evident that the same molecules activated by growth factors can signal to ERα and inﬂuence its function. Growth-Factor Signaling Unlike steroid hormones which engage their receptors in the nucleus, peptide growth factors interact with speciﬁc cell-surface receptors. For a growth factor to inﬂuence the cell, the signaling event must be transmitted from the cell surface to appropriate compartments within the cell. In the case of proliferation, growth factors must engage the cell-cycle machinery and trigger the appropriate nuclear events to prepare the cell for division. Although it is outside the scope of this review to discuss all of these complex signal transduction pathways activated by growth factors, several generalizations can be made. Growth-Factor Receptors Most growth-factor receptors are enzymes. Once the extracellular domain of the receptor is engaged by ligand, a conformational change is induced that triggers biochemi-

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cal activation. Many growth-factor receptors are tyrosine kinases, and in many cases the intracellular domain of the receptor is both the kinase and the substrate. For example, insulinlike growth factor (IGF) binding to its receptor triggers autophosphorylation on several tyrosine residues. This autophosphorylation is required for subsequent signaling events (41). Some growth factor receptors, such as the receptors of the epidermal growth factor (EGF) family, are synthesized as a single polypeptide chain and contain an extracellular binding domain, a transmembrane region, and the intracellular kinase domain. Other receptors, such as the insulin and IGF receptor family members, are composed of two separate chains linked by cysteine bonds. In the case of the IGF-I receptor, the α subunit is entirely extracellular and is covalently linked to the β subunit. The β subunit contains a short extracellular portion, the transmembrane domain, and the intracellular kinase domain. The EGF and IGF receptors require dimerization or oligomerization to function. The four EGF-receptor family members (HER1 through HER4) can function as homo- or heterodimers. The IGF receptor is normally synthesized as a dimeric unit. In both cases, the dimeric structure is important for subsequent signal transduction. Certain classes of receptors are composed of dissimilar units which then are assembled into multimers after ligand binding. Speciﬁcally, all of the signaling components for transforming growth factor-β (TGFβ) are present in the cell membrane, but only after ligand binding do the appropriate subunits assemble to form the active signaling complex (42). The cytokines and interleukins also signal through multisubunit receptors, and even share subunits common to more than one receptor (43). Interestingly, most cytokine receptors do not contain intrinsic enzymatic activity; instead they recruit cytoplasmic tyrosine kinases to the complex in order to trigger subsequent events. Growth-factor receptors, although different in the details of their structure, share a common theme of activation. Once ligand binding occurs, a conformational change in the receptor results in activation of a biochemical event, usually enhanced kinase activity, which further changes the receptor complex. Postreceptor Phosphorylation Events Because growth-factor receptors generally autophosphorylate their intracellular domains, these newly phosphorylated amino acid residues provide docking sites for other proteins. Many of the proteins that bind the receptor have no enzymatic function. Instead, they serve as adaptors for other downstream-signaling molecules. Speciﬁc domains contained on the adaptor proteins, such as the src homology-2 (SH2) and the protein/tyrosine-binding (PTB) domains, recognize these phosphorylated tyrosine residues and allow the adaptor to bind to the activated receptor. Other domains contained in some adaptor proteins, such as the SH3 and plekstrin-homology domains, are also involved in binding receptors. Once the adaptor is bound to the phosphorylated receptor, the adaptor itself becomes a substrate for the receptor-tyrosine kinase. For example, after the IGF-I receptor is activated by ligand, it was shown that a 185 kDa protein rapidly became phosphorylated (44). A family of substrates was cloned and called insulin-receptor substrate (IRS) proteins (45). The IRS proteins contain more than 20 tyrosine phosphorylation sites and multiple serine/threonine residues which can also become phosphorylated. Once phosphorylated, additional proteins can be recruited and a signaling cascade is initiated.

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Fig. 1. Potential mechanisms of crosstalk between growth factor and steroid hormone receptors. This simpliﬁed diagram shows how EGF receptor could inﬂuence steroid hormone action. EGFR activation results in autophosphorylation of tyrosine residues in its intracellular domain (circled P). Docking molecules (such as grb2, p85 P13K, Shc) bind receptor and initiate a variety of downstream signaling cascades that could affect estrogen-receptor function. As noted in the text, phosphorylation of ERα or coregulatory molecules can inﬂuence transcriptional activation.

Signaling Pathways Engaged by Growth-Factor Receptors These initial receptor/substrate interactions allow for the recruitment of many different downstream signaling pathways. For example, many growth-factor receptors engage adaptor proteins that result in activation of the well-characterized Ras GTPase family members (Fig. 1 and reviewed in 46,47). EGF-receptor stimulation by either EGF or by TGFα leads to the initial phosphorylation of the Grb2 adaptor protein and subsequent activation of Ras (reviewed in 48,49). Insulin and IGF receptor can also activate Ras via the IRS and Shc adaptor proteins. Ras simultaneously activates cytoplasmic serine/ threonine protein kinases from both the Raf and MAPK kinases (MEK) kinase (MEKK) families (50). MEKKs and Rafs can independently phosphorylate and activate MEKs, leading to activation of a growing family of MAPKs or extracellular-signal regulated protein kinases (ERKs) (51–54). Thus, growth-factor receptor phosphorylation causes components of multiple protein kinase cascades to assemble. This series of three kinases, referred to as a MAP kinase module, are organized on intracellular scaffolds (55). More than a dozen mammalian MAPK family members have been discovered, and include the well-studied ERKs as well as several stress-activated enzymes (56). For example, expression of activated MEKK1 (a MAPKKK) leads to selective activation of speciﬁc MEK (MAPKK) and MAPK family members, including Jun kinase (JNK) (57) or p38 MAPK (58) in a cell-type and context dependent manner. JNK, also known as stress-activated protein kinase or SAPK, is activated by proinﬂammatory cytokines and stress stimuli such as UV-irradiation and heat shock (59); p38 MAPKs

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are similarly activated by cellular stresses and cytokines. JNK and p38 MAPK activities are associated with apoptotic stimuli, immune function, and differentiation (for review of MAPKs, see 56,60). Like MEKK, Raf, and MAPK, however, JNK and p38 MAPK family members are also activated in response to growth factors. A novel MAPK module consisting of three successive kinases (Fig. 1, [56]) has recently been characterized and may be an important mediator of growth factor action. ERK5/BMK1 (Big MAP Kinase) was identiﬁed as a new MAPK family member (53,54). ERK5 is potentially activated by EGF (61), via a mechanism involving the upstream kinases, MEKK3 and MEK5 (62). Although ERK5 activation is Ras dependent, it appears to involve a novel uncharacterized Ras effector and participation of Raf1 protein by direct protein-protein interaction. Interestingly, ERK5 activation does not correlate with Raf1 catalytic activity, yet ERK5 is required for Raf1-mediated cell transformation (63). Additional growth-factor receptor tyrosine kinase effectors that are activated independent of MAPKs have been well characterized; these include the P13K/AKT pathway, p70S6K, and STATs. One endpoint of activation of intracellular kinase cascades is modulation of the phosphorylation state, and thus the activity of additional downstream kinases, such as Rsk90 and MsK family members (64), and a large number of nuclear transcription factors, including STATs, Ets factor family members, serum response factor, c-jun, and c-myc (reviewed in 65). They in turn regulate the genes necessary for cell division, differentiation, or apoptosis. Thus, what appears to be a relatively simple event—binding of a ligand to a cell surface receptor—can lead to a complex network of signals that regulate cellular function. We are only beginning to understand how these pathways interact to affect cell biology. In addition to oneway activation, it is also evident that these downstream molecules can feed back on the receptor and its signaling components. Furthermore, downstream signaling molecules receive multiple inputs from overlapping and interacting pathways. Thus, growth-factor signal transduction can be viewed as a network of interactions. The ultimate effect of any growth factor on cell behavior is likely a result of the interaction between pathways that serve to either reinforce or diminish the individual network components. Clearly, steroid hormone receptors are likely to interact with multiple nodes on the growth-factor signaling network.

POTENTIAL FOR CROSSTALK AND IMPLICATIONS FOR SERMS Introduction In theory, there are several ways that growth-factor receptor signaling could affect function of steroid hormone receptors and inﬂuence SERM action. Transcriptional and posttranscriptional regulation of the steroid hormone receptor itself could be inﬂuenced by growth-factor signal transduction pathways. Since steroid hormone receptors require other coregulatory proteins, growth factors could also inﬂuence these accessory proteins. Additionally, steroid receptor transcriptional activity may be inﬂuenced by direct modiﬁcation (i.e. phosphorylation) of steroid hormone receptors. There is experimental evidence to show that all of these mechanisms exist.

Gene Regulation Normal human mammary epithelial cells express low basal levels of ER and PR (66,67). Only 7% of these cells stain positively for ERα and PR (66) and protein levels

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ﬂuctuate with cyclical changes in estrogen and progesterone levels during the menstrual cycle (68). It is clear from gene knockout studies that ERα and PR play essential roles in mammary gland development (69,70) and contribute signiﬁcantly to the development, progression, treatment, and outcome of human breast cancer (72). Roughly two-thirds of breast cancers express ERα; half of ERα-positive tumors also express PR (reviewed in 72). These tumors are often responsive to hormonal-based therapies, whereas ERαpositive/PR-negative have a lower response rate and ER-negative/PR-negative tumors are completely unresponsive. Interestingly, PR-positive tumors (even in the absence of detectable expression of ERα) are highly responsive to tamoxifen (73). What factors may contribute to the loss of ERα and/or PR expression? Several growth factors suppress ERα gene transcription and protein expression, including EGF (74), IGF-I (75), TGF-β (76), and c-erbB ligand gp30 (77), and heregulin (78). Stoica et al. (74) showed that inhibition of ERα gene transcription by EGF is blocked by tryphostins and wortmannin, indicating a speciﬁc role for tyrosine kinase receptor activation and PI3K. EGF however had no effect on ERα gene expression when cells were placed in serum-free medium, yet the addition of serum restored EGF regulation of ERα, implicating the requirement for additional factor(s). IGF-I-mediated down regulation of ERα gene expression is blocked by inhibitors of both PKA and PI3K (75). Interestingly, these growth factors all cause marked decreases in ERα messenger RNA (mRNA) and protein levels, apparently by suppression of ERα promoter activity. However, both EGF and IGF-I increase ERα transcriptional activity at endogenous ER-regulated genes and/or ERE-driven reporter genes. In contrast, gp30 blocks ERα activity by inhibiting ERα binding to its response element, as demonstrated by gel shift assays, while TGF-β1 has no effect on ERα activity (76). Longterm treatment of MCF-7 breast cancer cells with the phorbol ester TPA also decreased ERα mRNA and protein levels; ERα levels returned to control values following TPA removal (79). However, ERα from TPA pretreated cells failed to bind estrogen, and were nonfunctional in transcriptional or gel-mobility-shift assays. Mixing experiments indicated the presence of a TPA/PKC-inducible factor that interacts with and blocks ERα action, but has no effect on glucocorticoid receptor function; PKC inhibitors reversed the effects of TPA on ERα. PR gene expression is primarily positively regulated by functional ERα. Thus, SERM action is likely mediated in part via effects on ERα regulation of PR expression. PR gene expression is controlled by two promoter regions giving rise to transcripts encoding PR-A and -B isoforms. Although the PR gene lacks an identiﬁable ERE (80), the PRA promoter contains an ERE-half-site upstream of two Sp1 binding sites; estrogen enhances Sp1 binding and increases transcription. Because of its regulation by functional ERα, PR expression is an indicator of likely responsiveness to endocrine agents/SERMs. Thus, loss of PR expression is believed to be a result of lack of ERα or the presence of nonfunctional ERα. Estrogenic agents can have differential effects on PR-A versus PR-B promoter activities, thereby altering the cellular PR A/B ratio (81,82); breast cancers often exhibit elevated A/B ratios (83,84). Because PR-A and PR-B function differently, and PR-A can act as a repressor of steroid hormone transcriptional activity including that of PR-B and ERα (85,86), alterations in the relative PR A/B ratio result in altered gene regulation, and reverse progestin (and potentially SERM) action (82). Giangrande et al. (87) isolated peptides that differentially modulated PR-A and PR-B transcriptional activities and found that PR-A and PR-B interact with different sets of

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regulatory cofactors. PR-A bound to the corepressor SMRT with higher afﬁnity than PR-B; on agonist binding, PR-B but not PR-A was able to recruit the coactivators glucocorticoid receptor interacting protein 1 (GRIP-1) and steroid receptor coactivator 1 (SRC-1). Progestins also modulate PR mRNA transcripts (88) and protein levels (89). HOX5A, a member of a large family of potent transactivators required during development, activated the PR gene promoter and increased PR-B protein levels (90). Elevated nitric oxide synthase expression correlated with PR expression in human breast cancer cell lines and tumors (91). These factors suggest a mechanism for estrogenindependent PR gene regulation in ER−/PR+ breast cancers. Methylation of CpG islands in both ERα and PR gene promoter and/or 5-coding regions has been documented (92) and may silence ERα and/or PR gene expression in a fraction of breast cancers; this process could be indirectly inﬂuenced by growth-factor-mediated pathways that can regulate DNA methyltransferase expression (93,94).

Regulation of Coregulatory Proteins In addition to modiﬁcation of ERα and/or PR gene regulation, SERMs are predicted to effect interactions of a complex array of coregulatory proteins with ERα and/or PR. How does coregulator function relate to SERM action? Few studies have addressed the physiological signiﬁcance of coregulators of steroid hormones with regard to cell biology. Although most coregulators appear to interact with many nuclear receptors in vitro, the question of which coregulators speciﬁcally regulate the activities of which nuclear steroid receptors in vivo remains unclear. It is also clear that coregulators may act as integrators of multiple signal transduction pathways (reviewed in 2,26,95), and are often phosphoproteins, as phosphorylation-dependent interactions have been described (96). For example, AIB1 is a ligand-dependent ERα coactivator whose gene is ampliﬁed in ER-positive breast cancers. Phosphorylation of AIB1 by MAPK increases ERα transcriptional activity and stimulates the recruitment of p300 to ER/AIB1-containing transcriptional complexes (97). Shim et al. (98) found that ERα and SRC-1 were segregated in distinct subsets of cells in estrogen-responsive rat mammary epithelium, whereas they were coexpressed in stroma; expression patterns did not correlate with the ability of ERα to induce PR expression (98). Further studies are needed to deﬁne the full range of ER- and PRspeciﬁc coregulators and their tissue speciﬁcity. For example, observations that the SERM tamoxifen acts as an antagonist in the breast, but behaves as an agonist in the uterus, may be explained in part by the presence of excess ERα-co-repressors in the breast, but excess ERα co-activators in the uterus (26). Alternatively, ERα may be sequestered from a subset of its coactivators in the breast, but may colocalize with the same molecule(s) in the uterus. Similarly, breast tumors may anomalously underor overexpress ER/PR-speciﬁc coregulators during tumor progression, and thereby convert from tamoxifen-sensitive to tamoxifen-resistant phenotypes (26). These speculations remain to be proven. It is clear however that steroid receptors form multiprotein complexes by binding a wide variety of regulatory molecules, including coregulators. These proteins often act as distal effectors of growth-factor-mediated signaling pathways initiated at the cell surface. Because ligand binding to steroid receptors can alter or stabilize these interactions (99), there is likely to be great potential for disruption or alteration of steroid hormone-co-regulator interaction and function by SERMs. Such

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alterations will become more evident as the hormone dependence of speciﬁc interactions is further deﬁned in intact cells and in breast cancer models.

Direct Modiﬁcation Analogous to other members of the steroid receptor superfamily, ERα and PR are highly phosphorylated, and therefore sensitive to growth-factor-initiated signaling pathways. Although the role of direct phosphorylation of steroid hormone receptors and the exact kinases/signaling pathways involved remain largely undeﬁned, phosphorylation is inﬂuenced by ligand-binding and can affect receptor-ligand/receptor-SERM interactions. A variety of agents can activate ERα in the apparent absence of steroid-ligand, including cAMP, okadaic acid, dopamine, cyclin D1 (100), and EGF (101). Ligandinduced ERα transcriptional activity is further enhanced by activated Ras and/or growth factors that feed into activation of the MAPK pathway, including EGF and IGF (102,103). The activity of growth factors, Ras, and MAPKs on ERα is largely mediated by phosphorylation effects on the constitutive ligand-independent N-terminal activation function (AF-1); the activity of AF-2 is ligand-dependent. MAPK activation by growth factors results in phosphorylation of Ser118, located in AF-1 and enhances the transcriptional activity of ERα elicited by either estrogen or tamoxifen (103). The transcriptional activity of estrogen-occupied mutant ERα with alanine in place of Ser118 is not further enhanced by MAPK activation. Thus, in the presence of estrogen, ERα appears to undergo a state of hyperactivation following growth-factor stimulation of the MAPK pathway. Whereas EGF activation of ERα mapped to AF-1, phosphorylation of Ser118 was not sufﬁcient for estrogen-independent EGF signaling through ERα (101), indicating that additional phosphorylation sites, domains, and/or proteins are involved in the regulation of AF-1 function by growth factors. Ragatsky et al. (104) extended these results by showing that enhanced ERα transcriptional activity in response to activation of cyclin A-CDK2 complexes was mediated by phosphorylation of Ser104 and Ser106, but not Ser118. Enhanced ERα activity occurred in the presence and absence of estradiol as well as in the presence of tamoxifen, independent of AF-2 function (104). Other studies demonstrate however that AF-1 and AF-2 clearly interact to regulate N-terminal phosphorylation events. Chen et al. (32) recently found that efﬁcient phosphorylation of ERα Ser118 required the ligand-dependent recruitment of the TFIIH cyclin-dependent kinase (CDK7) to the ligand-binding domain/AF-2. Similar to ERα regulation, phosphorylation of ERβ on two N-terminal AF-1 MAPK consensus serine residues enhance ligand-independent transcription (105). The mechanism, however, involves recruitment of the transcriptional coactivator SRC-1 to phosphorylated receptors. Activation of human PR (hPR) appears to be almost entirely ligand dependent; there are few examples of ligand-independent activation of full-length native hPR (106). Similar to ERα, however, growth factors greatly inﬂuence PR signaling in the presence of progestins (107–111). Activation of cAMP-dependent protein kinase (PKA) by 8Br-cAMP produces synergy with PR agonists on PRE-regulated promoters, and converts the PR antagonists RU486 and ZK112993 to transcriptional agonists of PR-B, but not PR-A receptors in T47D human breast cancer cells (112); cAMP also alters the agonist/ antagonist balance of some ERα antagonists (reviewed in 95). Although the mechanism(s) of cAMP effects on ER/PR are unknown, regulation via phosphorylation of a

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common coactivator such as CBP/p300 or p160 family coactivators may be involved (113). Transcriptional synergy between progestins and EGF occurs at several promoters, including those regulating the mouse mammary tumor virus (114), p21WAF1 and c-fos genes (110). EGF and progestins upregulate cyclin D1, cyclin E, and p21WAF1 protein levels in a MAPK-dependent manner in T47D human breast cancer cells (109). Little is known about the functional role of PR phosphorylation, although several endogenously regulated phospho sites have been well characterized (reviewed in 115,116). For example, phosphorylation of PR Ser400 is both basally regulated and ligand-induced, and mediated by cyclin-dependent protein kinase 2 (CDK2) in vitro (117), suggesting a possible function in PR regulation of the cell cycle (108). In common with ERα, PR have N-terminal consensus MAPK phosphorylation sites. Ser294 and Ser345 are predominantly and latently phosphorylated after treatment of cells with progestins (118). In contrast to ERα, however, these residues reside within an inhibitory functional domain (IF) of PR, rather than an activation function (AF); the contribution of either of these sites to IF regulation is unknown. Ser294 however plays an essential role in PR protein turnover (119). In the presence of ligand, phosphorylation of PR on Ser294 by MAPK leads to rapid PR protein degradation by the ubiquitin/proteasome pathway (119). Inhibition of the 26S proteasome, inhibition of MAPKs by MEK inhibitors, or mutation of Ser294 to alanine all stabilized PR in the presence of ligand. Interestingly, phosphorylation of Ser294 also appears to be coupled to PR transcriptional activation in the presence of agents that activate MAPKs. Overexpression of constitutively active MEKK1 activated p42/p44 MAPKs in breast cancer cells and resulted in remarkable transcriptional synergy in the presence of the synthetic progestin R5020; this effect was both PR- and PRE-dependent (58). Stabilization of PR by ubiquitin pathway inhibitors, MEK inhibitors, or mutation of Ser294 to alanine blocked MEKKinduced transcriptional activation. MEKK1 expression resulted in direct phosphorylation of Ser294 as measured by phospho-speciﬁc PR antisera, and this was blocked by MEK inhibitors. Thus, phosphorylation of PR in response to MAPK activation may induce transcriptional synergy with progestins at growth-regulatory genes by driving increased PR degradation (see next paragraph). ER are also substrates for the ubiquitin/proteasome pathway, although the role of phosphorylation in this process, if any, remains undeﬁned (120). Yudt (121) however found that mutation of human ERα Tyr537 to phenylalanine resulted in decreased receptor stability. Similar to PR, stabilization of ERα by 26S-proteasome inhibitors blocks ERα transcriptional activity (122). Thus, the transcriptional activities/hormone responsiveness of both ERα and PR appear to be tightly linked to receptor stability/ turnover. These results favor a model whereby in the presence of ligand, one or more coactivators are recruited to steroid receptor complexes; this same factor either functions directly in the ubiquitin pathway, or associates with enzymes required for receptor ubiquitination (58,122). These interactions are likely to be affected by phosphorylation of receptors as is the case for PR (119), as well as associated coregulatory molecules. The same phosphorylation sites on ERα and/or PR can be regulated in response to steroid hormone binding as well as growth-factor treatment of cells. Which pathway is dominant? What are the points of interaction between pathways? How can we target either pathway for more effective or selective SERM action? Hormones and antihormones are predicted to induce differential phosphorylation of steroid hormone

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receptors (123); these heterogeneously phosphorylated receptors may regulate gene activity differently. Additionally, both ERα and PR are highly sensitive to growthfactor-initiated signaling pathways. Thus, changes in cellular phosphorylation state are likely to be important in determining the biologic activity of ER/PR and the effectiveness of SERMS. For example, the inhibitory action of tamoxifen is limited to the AF-2 function of ERα, whereas agonist activity is mediated primarily by interaction with AF-1. Thus, growth factors that lead to the activation of MAPK and/or CDK2 activities are predicted to profoundly affect the ability of tamoxifen and other SERMs to inﬂuence ERα transactivation. Since ERα is the primary modulator of PR gene expression and PR can suppress estradiol-stimulated ERα activity, it is also important to understand how SERMs will affect ER/PR interrelationships in the control of cellular responses to steroid hormones.

CLINICAL EVIDENCE FOR SERM AND GROWTH-FACTOR RECEPTOR INTERACTIONS The preceding sections suggest multiple ways by which growth-factor signaling pathways could interact with steroid hormone receptors and inﬂuence SERM action. Is there clinical evidence to support these hypothetical interactions? The epidermal growth-factor receptor (EGFR) system is perhaps the best-studied family of growth-factor receptors in breast cancer. Besides the prototypical EGFR, there are three related family members known as HER2, -3, and -4. HER2 was discovered to be ampliﬁed in primary human breast cancer. Moreover, the degree of gene ampliﬁcation was associated with prognostic signiﬁcance (124). The EGFR family members function as homo- or heterodimers and, except for HER3, are tyrosine kinases (125). Because ampliﬁcation and overexpression of these family members is common in breast cancer, the relationship between ERα and EGFR family members has been examined in several studies.

Expression of ER, PR, and EGFR Family Members In general, there is an inverse relationship between EGFR expression and ERα (126–128). The inverse relationship between HER2 and ERα is not as strong as between EGFR and ERα, and a substantial number of patients show both ERα and HER2 expression (129–132). Because HER2 expression could affect response to tamoxifen therapy, several studies have examined potential interactions between these two growthregulatory pathways.

HER2 Expression and Response to Hormonal Therapy There are now a number of reported studies that have examined the relationship between response to tamoxifen and HER2 status. Elledge et al. retrospectively studied patients enrolled on a cooperative group study treated with tamoxifen for metastatic disease. They showed that HER2 expression had no inﬂuence on tamoxifen response rate, time to treatment failure, or survival (131). In contrast, other studies have suggested that HER2 expression identiﬁes patients with poor response to tamoxifen. Newby et al. showed that patients who failed to respond to tamoxifen were more likely to express either EGFR or HER2. Of these nonresponding patients, more than half had expression of EGFR, HER2, or both in

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the pretreatment specimens, suggesting that signaling via these receptor pathways is associated with de novo tamoxifen resistance (132). This study also found that patients who initially responded to tamoxifen rarely had expression of EGFR or HER2. Another study examined patients with metastatic disease and supported the observation that HER2 expression is associated with a relatively poor response to tamoxifen (130). Both response rates and time to treatment failure were signiﬁcantly shorter in the HER2 positive patients. HER2 can also be found in the circulation of breast cancer patients. In ERα positive patients, 19.3% also had detectable serum levels of HER2. The response rate to patients with high serum HER2 levels was inferior (20.7% vs 40.9%) compared to patients with low serum levels. The median duration of treatment response was also inferior in patients with circulating HER2 (133). In a recently reported trial, levels of circulating HER2 were found to predict for inferior outcome in patients treated with second-line hormonal therapy (134). Similar studies have been performed in patients receiving tamoxifen as adjuvant therapy. In the Naples/Grupo Universitario Napoletano 1 (GUN-1) trial, operable breast cancer patients were randomized to receive tamoxifen or no further therapy after primary treatment of the tumor. Elevated levels of HER2 measured by immunohistochemistry were associated with a relative lack of efﬁcacy of adjuvant tamoxifen (135). Postmenopausal breast cancer patients randomized to 2 versus 5 years of tamoxifen also showed that HER2 positive patients did not appear to beneﬁt from prolonged tamoxifen treatment (136). A recently reported update studied lymph-node positive postmenopausal patients who were randomized to no further treatment versus tamoxifen (137). All premenopausal patients received cyclophosphamide, methotrexate, and ﬂuorouracil (CMF) adjuvant chemotherapy for nine cycles and were randomized to tamoxifen or no additional treatment. With a 15-year followup, the hazard ratio (HR) of death in the HER2negative group was 0.59 demonstrating a beneﬁt from tamoxifen treatment. In contrast, the HER2-positive patients had a hazard ratio of 1.09, which demonstrated no beneﬁt for tamoxifen in this group. Furthermore, there was a strong statistical interaction between HER2 and tamoxifen where none existed for CMF-treated patients. In fact, in HER2-positive patients treated with tamoxifen alone, there was a suggestion of a detrimental effect (HR = 2.23, conﬁdence interval: 0.95–5.23) although this was a subgroup analysis. Additional conﬂicting studies have been performed in patients receiving adjuvant therapy with or without additional chemotherapy. In Cancer and Leukemia Group B (CALGB) 8541, patients were randomized to receive varying doses of chemotherapy. All ERα-positive patients also received tamoxifen. This study was the ﬁrst to show beneﬁts from higher doses of doxorubicin for HER2 overexpressing patients (137). When this trial was reanalyzed however for patients receiving additional hormonal therapy, there was no evidence of an adverse effect of HER2 expression in the patients who received tamoxifen (138). Finally, there have been several reports presented at a meeting convened by the National Cancer Institute suggesting that there was no evidence of adverse effects of HER2 on response to tamoxifen in several cooperative group studies (139). As can be seen, there are an abundance of data concerning potential interactions between tamoxifen and HER2. Unfortunately, there is very little consensus or insight into how HER2 could affect ERα function. There may be several reasons for this confusion. First, many of the studies that suggest that HER2 expression is associated

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with decreased tamoxifen response have been presented only in abstract form. A more careful scrutiny of a published report may yield the reasons for the reported differences. Second, measurement of HER2 expression is not well standardized. Although the Food and Drug Administration has approved assays for measuring HER2, many of the studies were performed using different antibodies or techniques. Thus, it is possible that assay differences could account for the varying results. One study however has suggested that the results were independent of the HER2 assay (139). Third, many of the studies also included analysis of patients who had received both tamoxifen and chemotherapy. Because it is apparent that HER2 expression inﬂuences response to chemotherapy, these combined treatments add confounding factors. Last, simple overexpression is used as a surrogate indicator for activation of post-HER2-signaling pathways. As noted above, the signaling pathways engaged after growth-factor receptor activation are complex. Overexpression alone cannot substitute for a more detailed analysis of signal transduction.

SUMMARY It is clear that SERM therapy of breast cancer has been an extraordinarily useful treatment modality. At present, SERMs are directed at interruption of ERα binding to its cognate ligand, estradiol. In addition to ligand regulation of ERα, crosstalk between steroid hormone and growth-factor-receptor signaling pathways is known to occur at multiple levels; complex bidirectional interactions are well documented. Growth factors may effect estrogen and/or progesterone receptor gene expression, and receptor function in the breast. These interactions are necessary for the growth and development of normal breast tissue and most likely contribute to uncontrolled growth of the transformed breast epithelial cell. Thus, it is important to understand how growth factor to ER/PR crosstalk might inﬂuence the function of SERMs. The clinical evidence suggesting that HER2 inﬂuences the ability of tamoxifen to function in breast cancer is conﬂicting, but there is suggestive evidence that HER2 overexpression, and perhaps activation of its downstream signaling pathways, result in relative tamoxifen resistance. At present, we are far from understanding the mechanisms that account for these clinical observations. The pace of discovery is accelerating and the next few years should yield new insights and therapies based on an understanding of these key pathways involved in breast cancer biology.

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106. Bamberger AM, Bamberger CM, Gellersen B, Schulte HM. Modulation of AP-1 activity by the human progesterone receptor in endometrial adenocarcinoma cells. Proc Natl Acad Sci USA 1996; 93:6169–6174. 107. Beck CA, Weigel NL, Edwards DP. Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors. Mol Endocrinol 1992;6:607–620. 108. Groshong SD, Owen GI, Grimison B, et al. Biphasic regulation of breast cancer cell growth by progesterone: role of the cyclin-dependent kinase inhibitors, p21 and p27(Kip1). Mol Endocrinol 1997;11:1593– 1607. 109. Lange CA, Richer JK, Shen T, Horwitz KB. Convergence of progesterone and epidermal growth factor signaling in breast cancer. Potentiation of mitogen-activated protein kinase pathways. J Biol Chem 1998;273:31308–31316. 110. Richer JK, Lange CA, Manning NG, Owen G, Powell R, Horwitz KB. Convergence of progesterone with growth factor and cytokine signaling in breast cancer. Progesterone receptors regulate signal transducers and activators of transcription expression and activity. J Biol Chem 1998;273:31317– 31326. 111. Lange CA, Richer JK, Horwitz KB. Hypothesis: Progesterone primes breast cancer cells for crosstalk with proliferative or antiproliferative signals. Mol Endocrinol 1999;13:829–836. 112. Sartorius CA, Groshong SD, Miller LA, et al. New T47D breast cancer cell lines for the independnet study of progesterone B- and A-receptors: only antiprogestin-occupied B-receptors are switched to transcriptional agonists by cAMP. Cancer Res 1994;54:3868–3877. 113. Wada T, Qian XL, Greene MI. Intermolecular association of the p185neu protein and EGF receptor modulates EGF receptor function. Cell 1990;61:1339–1347. 114. Haraguchi S, Good RA, Engelman RW, Greene S, Day NK. Prolactin, epidermal growth factor or transforming growth factor-alpha activate a mammary cell-speciﬁc enhancer in mouse mammary tumor virus-long terminal repeat. Mol Cell Endocrinol 1997;129:145–155. 115. Takimoto G, Horwitz K. Progesterone receptor phosphorylation—Complexities in deﬁning a functional role. Trends Endocrinol Metab 1993;4:1–7. 116. Weigel NL. Steroid hormone receptors and their regulation by phosphorylation. Biochem J 1996; 319:657–667. 117. Zhang Y, Beck CA, Poletti A, et al. Phosphorylation of human progesterone receptor by cyclindependent kinase 2 on three sites that are authentic basal phosphorylation sites in vivo. Mol Endocrinol 1997;11:823–832. 118. Zhang Y, Beck CA, Poletti A, Edwards DP, Weigel NL. Identiﬁcation of a group of Ser-Pro motif hormone-inducible phosphorylation sites in the human progesterone receptor. Mol Endocrinol 1995;9:1029–1040. 119. Lange CA, Shen T, Horwitz KB. Phosphorylation of human progesterone receptors at serine-294 by mitogen-activated protein kinase signals their degradation by the 26S proteasome. Proc Natl Acad Sci USA 2000;97:1032–1037. 120. Nawaz Z, Lonard DM, Dennis AP, Smith CL, O’Malley BW. Proteasome-dependent degradation of the human estrogen receptor. Proc Natl Acad Sci USA 1999;96:1858–1862. 121. Yudt MR, Vorojeikina D, Zhong L, et al. Function of estrogen receptor tyrosine 537 in hormone binding, DNA binding, and transactivation. Biochemistry 1999;38:14146–14156. 122. Lonard DM, Nawaz Z, Smith CL, O’Malley BW. The 26S proteasome is required for estrogen receptor-alpha and coactivator turnover and for efﬁcient estrogen receptor-alpha transactivation. Mol Cell 2000;5:939–948. 123. Bagchi MK, Tsai SY, Tsai MJ, O’Malley BW. Ligand and DNA-dependent phosphorylation of human progesterone receptor in vitro. Proc Natl Acad Sci USA 1992;89:2664–2668. 124. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with ampliﬁcation of the HER-2/neu oncogene. Science 1987; 235:177–182. 125. Zwick E, Hackel PO, Prenzel N, Ullrich A. The EGF receptor as central transducer of heterologous signalling systems. Trends Pharmacol Sci 1999;20:408–412. 126. Klijn JG, Berns PM, Schmitz PI, Foekens JA. The clinical signiﬁcance of epidermal growth factor receptor (EGF-R) in human breast cancer: a review on 5232 patients. Endocr Rev 1992;13:3–17. 127. deFazio A, Chiew YE, Sini RL, Janes PW, Sutherland RL. Expression of c-erbB receptors, heregulin and oestrogen receptor in human breast cell lines. Int J Cancer 2000;87:487–498.

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TISSUE-SPECIFIC EFFECTS OF ESTROGENS AND SERMS

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Direct Estrogen Effects on the Cardiovascular System Munish K. Goyal, MD and Suzanne Oparil, MD Contents Estrogen and Coronary Artery Disease in Women Animal Models of Vascular Injury Effects of Estrogen on the Vascular Injury Response Mechanisms of Estrogen-Mediated Vasoprotection Inﬂammatory Markers Conclusions References

ESTROGEN AND CORONARY ARTERY DISEASE IN WOMEN Coronary artery disease (CAD) is the leading cause of death among women (1). The risk of CAD is low in premenopausal women and increases dramatically after menopause. Data from the Framingham Heart Study assessed sex-speciﬁc patterns of CAD and demonstrated that although men were at greater risk of heart disease than women at all ages, the difference in risk diminished as the participants got older, mainly because of a surge in the number of coronary events in women after age 45 (2). Whether this increased risk in women is a result of menopause with its associated loss of hormonal protection versus confounding factors such as aging has been debated. Observational studies have shown major reductions (approx 50%) in risk for CAD in postmenopausal women who take replacement estrogen or combined estrogen/progestin preparations (3). The largest of these, the Nurses Health Study, was established in 1976, when 121,700 female nurses between 30 and 55 years of age completed a questionnaire documenting their medical history and lifestyle. Every two years, followup questionnaires were sent out to update risk factors and new disease. Using data collected from this study, Grodstein et al. reported that hormone replacement therapy (estrogen or estrogen/progestin) decreased the risk of cardiovascular disease in postmenopausal women by approx 50% (4).

From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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Observational studies are limited, however, by selection bias because women on hormone replacement therapy tend to be better educated, have improved health care access, and are more attentive to lifestyle modiﬁcations for disease prevention (5,6). This deﬁciency can be overcome by randomized controlled trials, which eliminate treatment bias. The ﬁrst randomized controlled trial to examine the effects of estrogen with and without concomitant progestin on cardiovascular risk factors was the Postmenopausal Estrogen/Progestin Intervention (PEPI) Trial (7). PEPI compared the effects of conjugated equine estrogen (CEE) alone and estrogen plus three different progestin preparations to placebo on heart disease risk factors (HDL cholesterol, systolic blood pressure, serum insulin, ﬁbrinogen) in otherwise healthy postmenopausal women. The main ﬁndings of PEPI were signiﬁcantly increased HDL cholesterol levels (estrogen alone groups > estrogen + progestin groups) in all active treatment groups compared to placebo. In addition, LDL cholesterol and ﬁbrinogen levels were reduced in all active treatment groups compared to placebo. The possible long-term beneﬁts of this risk modiﬁcation were not appreciated because the PEPI trial was not powered to evaluate the effects of hormone replacement therapy on cardiovascular morbidity and mortality. The ﬁrst randomized controlled study to examine the effects of hormone replacement therapy on cardiovascular disease outcomes was the Heart and Estrogen/Progestin Replacement Study (HERS) (8). HERS was designed to test whether estrogen plus progestin therapy altered the risk for CAD events in postmenopausal women with known CAD. The study group included 2763 women randomized to 0.625 mg CEE plus 2.5 mg medroxyprogesterone acetate (MPA) versus placebo, who were followed for an average of four years. Surprisingly, the results indicated that women receiving combined hormone therapy did not experience an overall reduction in risk of fatal/ nonfatal MI. There was, however, a statistically signiﬁcant time trend, with increased cardiovascular events in the treatment group in the ﬁrst year, and a reduction in events after three years (Fig. 1). While PEPI showed a favorable effect on CAD risk factors in women apparently free of disease who were treated with hormone replacement therapy, HERS demonstrated no overall beneﬁt on CAD outcomes. Further, the Estrogen Replacement and Atherosclerosis (ERA) trial showed no change in the rate of progression of angiographically demonstrated coronary lesions in women with established CAD who were treated with hormones compared to a placebo group (9). Mechanisms that have been proposed to account for the discrepancy between the apparent beneﬁcial effects of estrogen in the primary prevention of CAD, based on observational studies and the PEPI trial, and the lack of beneﬁt (and possibility of harm) in secondary prevention trials (HERS, ERA) include procoagulant, proinﬂammatory, and atherosclerotic plaque destabilizing effects of estrogen, as well as altered estrogen-receptor (ER) expression in atherosclerotic arteries. Elevated levels of C-reactive protein, a marker of inﬂammation and an independent risk factor for the development of CAD morbidity and mortality (Fig. 2), have been reported in women on hormone replacement therapy (10). For example, an 85% increase in levels of C-reactive protein was noted in women in all four active treatment groups in the PEPI trial compared to the placebo group (11). Evidence for a possible procoagulant effect of hormone replacement therapy comes from a placebo-controlled trial that demonstrated higher levels of prothrombin fragments and soluble ﬁbrin in the hormone

101 Fig. 1. Kaplan-Meier estimates of the cumulative incidence of primary coronary heart disease (CHD) events (left) and to its constituents: nonfatal myocardial infarction (MI) (center) and CHD death (right). The number of women observed at each year of followup and still free of an event are provided in parentheses, and the curves become fainter when this number drops below half the cohort. Log rank P values are .91 for primary CHD events, .46 for nonfatal MI and .23 for CHD death. (Reproduced with permission from Hulley S, Grady D, Bush T, et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA 1998;280:605–613.)

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Fig. 2. Cumulative probability of death from cardiac causes in relation to C-reactive protein levels at enrollment. The number of patients in each group at the beginning of the study is given in parentheses. (Reproduced with permission from Lindahl B, Toss H, Siegbahn A, Venge P, Wallentin L. Markers of myocardial damage and inﬂammation in relation to long-term mortality in unstable coronary artery disease. N Engl J Med 2000;343:1139–1147.)

treatment group compared to placebo (12). Similar results were reported by Scarabin et al., who examined the effects of oral and transdermal estradiol/progesterone preparations in 45 healthy postmenopausal women (13). In contrast, in the PEPI trial, women randomized to placebo had greater increases in plasma ﬁbrinogen than women in the four active treatment arms (7). In addition, Koh et al. reported decreased levels of plasminogen-activator inhibitor type 1 (PAI-1), an inhibitor of ﬁbrinolysis, in postmenopausal women started on hormone replacement therapy (14). These conﬂicting ﬁndings illustrate the need for further studies to address on a molecular basis how estrogen modulates the inﬂammatory process. Another putative mechanism by which estrogen may precipitate CAD events is atherosclerotic plaque destabilization. The stability of a plaque depends largely on maintaining the integrity of its ﬁbrous cap, which is composed of extracellular proteins secreted by intimal smooth muscle cells (SMCs). Matrix metalloproteinases (MMPs), enzymes primarily secreted by macrophages, can degrade the collagenous cap, resulting in plaque rupture (15). Wingrove et al. demonstrated that estradiol induces dosedependent increases in MMP-2 expression in human vascular SMCs (VSMCs) in vitro (16). Similar ﬁndings were seen by Cannon et al., who reported increased levels of

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MMP-9 in postmenopausal women with CAD who were taking CEE (17). These ﬁndings, though provocative, provide no direct evidence that estrogen destabilizes plaques in vivo in either human or animal models. Further studies using advanced techniques of assessing coronary artery metabolism are needed to answer these critical questions. Since many or most of the vascular effects of estrogen are mediated through selective ERs, decreased expression of ERs in atherosclerotic vessels could contribute to the failure of estrogen to protect against the progression of CAD lesions and the occurrence of CAD events in women with established disease (18). Losordo et al. noted that the relationship between CAD and ER expression was statistically signiﬁcant in premenopausal women, where 10 of 12 women with normal arteries were ER positive, but only 1 of 6 atherosclerotic arteries demonstrated expression of ERs. Thus, we are left with the apparent paradox of hormone (estrogen)-related beneﬁt in reduction of cardiovascular disease risk factors and in primary prevention of CAD versus no beneﬁt, delayed beneﬁt or early harm in women with established CAD. The results of HERS suggest not starting hormone replacement in postmenopausal women with known CAD. HERS however did not assess the risks and beneﬁts of unopposed estrogen or of other estrogen and progesterone preparations (e.g., oral, transcutaneous estradiol, or native progesterone) in either primary or secondary prevention or of CEE plus MPA in healthy postmenopausal women, nor did it address continuing treatment in women with CAD who are already taking hormone replacement therapy. The Womens Health Initiative Randomized Trial (WHI), due to be completed in 2005, will clarify some of these issues (19). Interestingly, a small excess of coronary events was seen in the ﬁrst two years of hormone (CEE plus MPA) therapy in women participating in the randomized treatment portion of WHI, perhaps conﬁrming the results of HERS in a subset of women with occult CAD (20).

ANIMAL MODELS OF VASCULAR INJURY Although much has been learned about mechanisms of estrogen-induced vasoprotection in women, animal models are indispensable in advancing our understanding of the effects of sex hormones on the vasculature. Mechanical injury of arteries has been used widely as a rapid and convenient means of assessing cellular and molecular mechanisms of vascular disease and their modulation by estrogen. Balloon injury of the rat carotid artery is the most commonly used animal model of vascular injury (21). This is an experimental model of localized and highly controllable vascular damage in which the response to injury can be studied in vivo (22). With compression injury of the media and endothelial denudation, VSMCs undergo proliferation and migration, which then leads to the development of neointima (23). Within 48 hours of the initial insult, medial SMC proliferation occurs and a subset of these VSMCs migrate toward the luminal surface. A number of mitogenic and chemotactic factors, such as ﬁbroblast growth factor (FGF), transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), angiotensin II and osteopontin play a role in the proliferation of medial VSMCs and their migration into neointima (23,24). These VSMCs serve as progenitors for the neointimal cells that colonize the luminal surface of the injured vessel and continue to replicate and to secrete extracellular matrix (ECM) proteins. Once VSMC proliferation ceases, continued ECM formation

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Fig. 3. Mechanisms of response to endoluminal vascular injury.

contributes to expansion of the neointima (25), which continues until the injured vessel wall is reendothelialized. Clowes et al., using the Evans blue technique, found continued VSMC proliferation as late as three months after vascular injury in areas without an endothelium, in contrast to control arteries and injured segments covered by a new endothelium (26). A novel concept that has attracted increasing attention in recent years is the role of the adventitia in the response to endoluminal vascular injury (Fig. 3). Participation of the adventitia in the response to vascular injury has been suggested by pathologic ﬁndings of adventitial activation (inﬂammation and ﬁbrosis) in coronary arteries of victims of fatal CAD (27,28). In some individuals who died suddenly at an early age, adventitial inﬂammation appeared to antedate intimal disease. Further, neointima formation and/or atherosclerotic lesions have been observed in response to adventitial injury in various animal models, raising the possibility of alternative pathways of the vascular injury response and alternative routes of administering therapeutic agents (29,30). Studies demonstrate that endoluminal injury of the porcine coronary artery results in signiﬁcant remodeling of the adventitia, characterized by proliferation and differentiation of adventitial ﬁbroblasts to myoﬁbroblasts, which acquire α-smooth muscle actin (31,32). These adventitial responses were associated with increased neointima formation. The proliferating adventitial cells appeared to migrate into the neointima and transform into a myoﬁbroblast phenotype. Further, Li et al. introduced syngenic ﬁbroblasts that had been transduced with retroviral particles expressing β-galactosidase (LacZ) into the adventitia of right (injured) and left (uninjured) carotid arteries of ovariectomized rats immediately following balloon injury (33). LacZ expression appeared in the media within ﬁve days postinjury and in neointima at seven days, coincident with the onset of neointima formation. Expression of LacZ increased over

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time in the neointima and decreased in the adventitia and media. In contrast, LacZ expression was restricted to the adventitia in noninjured vessels seeded with transduced ﬁbroblasts. This is the ﬁrst direct demonstration that adventitial ﬁbroblasts are activated and migrate into neointima following endoluminal vascular injury, thus contributing to the injury response. The signaling pathway that mediates early activation of adventitial ﬁbroblasts following endoluminal vascular injury involves diffusion of chemoattractant/mitogenic factors to the adventitia from the damaged medial SMCs. Cell migration is regulated in large part by integrin receptors, a large conserved family of cell-surface polypeptides that mediate cell/matrix and cell/cell adhesion (34,35). Integrins play a role in a number of biologic processes, including proliferation, metastasis, differentiation, and wound repair. Vascular cells in vitro contain integrin receptors, and studies utilizing blocking antibodies selective for either β1 or β3 integrins revealed that β3 but not β1 inhibition prevented SMC migration in vitro as well as neointima formation in a rat carotid injury model in vivo (36). Of particular interest is αvβ3, which coordinates cellular binding to osteopontin (OPN), an extracellular matrix protein. Several lines of evidence suggest that OPN and αvβ3 integrin play a regulatory role in mediating the vascular injury response. Increased expression of both OPN and αvβ3 has been observed in injured blood vessels, and both neutralizing antibodies against OPN and a selective peptide antagonist of αvβ3 have been shown to suppress neointima formation in animal models (37,38). Further, VSMCs in vitro express OPN messenger RNA (mRNA) and protein, and the OPN produced is capable of directing the migration/adhesion of a number of cell types, including VSMC, monocytes, and adventitial ﬁbroblasts (24,39).

EFFECTS OF ESTROGEN ON THE VASCULAR INJURY RESPONSE The balloon-injured rat carotid-artery model has been used extensively to examine the effects of estrogen and other sex hormones on the vascular injury response. Initial observations indicated that there is sexual dimorphism in the neointimal response to vascular injury and that this dimorphism is estrogen dependent (40). Neointima formation was shown to be signiﬁcantly greater in intact male rats versus age-matched intact female rats, and neither gonadectomy nor gonadectomy plus testosterone replacement altered the neointimal response in male rats. In contrast, gonadectomy in female rats results in an increased neointimal response, comparable to that seen in intact males (Note: exogenous testosterone did not enhance this response). Interestingly, estradiol treatment markedly reduced (60%) neointima formation in gonadectomized rats of both sexes, but did not alter the response in intact males when compared to vehicle (Fig. 4). Subsequent in vitro studies showed that VSMCs from aortas of male and female rats manifest similar hypoproliferative responses to estrogen, suggesting that the lack of beneﬁt seen in intact males may be related to differences in hormonal milieu as opposed to phenotypic differences in the VSMCs (41). Further investigation is needed to elucidate the cellular and molecular basis for resistance to the vascular effects of estrogen in intact male subjects. Other studies using rabbit and mouse models have demonstrated similar inhibitory effects of estrogen on the vascular injury response (Fig. 5). (42,43) Foegh et al. investigated the effects of estradiol on arterial remodeling induced by balloon injury

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Fig. 4. Effects of gonadectomy, 17β-estradiol (E2) and testosterone on neointima formation of ballooninjured right common carotid artery of male and female Sprague-Dawley rats at 14 days after injury. Crosssectional areas of neointima are presented as mean ± SEM. *P < 0.5 compared with their respective intact control group. #P < 0.5 compared with their respective male group. (Reproduced with permission from Chen S, Li H, Durand J, Oparil S, Chen Y. Estrogen reduces myointimal proliferation after balloon injury of rat carotid artery. Circulation 1996;93:577–584.)

of rabbit arteries (44). They reported decreased myointimal thickening and 3H-thymidine incorporation during estrogen treatment, suggesting that the effect of estrogen on the injury response is in part caused by inhibition of cellular proliferation. Similar reductions in medial hyperplasia were reported by the Mendelsohn laboratory in the mouse carotid artery following endoluminal injury caused by passage of a wire (42,43). Studies in humans and animals have suggested that the administration of a progestin, although needed to prevent the hyperplastic/neoplastic effects of unopposed estrogen, may reduce the beneﬁts of estrogen replacement therapy in lowering the risk of CAD (7,45). For example, in the PEPI trial, women randomized to CEE and continuous or cyclical MPA had smaller increases from baseline in HDL cholesterol levels than those seen in women on unopposed estrogen. A study in ovariectomized monkeys demonstrated that animals treated with estradiol plus MPA manifested vasospasm in response to serotonin/thromboxane, whereas those treated with estradiol plus native progesterone did not (46). This suggests that certain progestins may have adverse effects on the cardiovascular system and others may afford some vasoprotection. Whether these adverse effects of progestins are clinically signiﬁcant is uncertain, especially in light of the work by Grodstein et al., who reported no difference in risk of CAD in women on estradiol plus progestin versus women on unopposed estrogen (4). Similar ﬁndings were reported by Psaty et al., who concluded that combined estrogen/progestin treatment did not increase the incidence of myocardial infarction when compared to unopposed estrogen (47).

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Fig. 5. Thin sections of carotid arteries of male and female wild-type BL/6-129 mice. Control and injured arteries were perfusion ﬁxed 2 wk postligation injury and stained with hematoxylin and eosin.

MECHANISMS OF ESTROGEN-MEDIATED VASOPROTECTION Estrogen Receptors Estrogen exerts many of its actions through two types of estrogen receptors: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). The ERs belong to the steroid receptor family, and many of the biological effects of estrogen involve ER-dependent modulation of gene expression (48,49). Once bound by estrogens, the ERs act as ligandactivated transcription factors and bind speciﬁc regions on DNA termed estrogen response elements (ERE). In the absence of a functional ERE, other transcriptional factors (e.g., AP-1) act as targets for the activated nuclear ER. The ER subtypes differ in their patterns of distribution, with ERα predominantly found in the uterus, testis, pituitary, kidney, and breast and ERβ localized to prostate, ovary, bladder, and brain. The heterogeneity of estrogen action may be partly explained by this differential tissue distribution (50).

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Structural differences between the two subtypes of ER may also contribute to the heterogeneity of ER function. ERs consist of a central domain responsible for speciﬁc DNA binding, a C-terminal domain involved in ligand binding and transactivation function, and an N-terminal domain that also contributes to activation. The ERα and ERβ share >90% homology in the DNA-binding domain (DBD) and a 55% homology in the ligand-binding domain (LBD); ERα and ERβ however are hypervariable in the N-terminal domain, suggesting differences in transactivation function. Paech et al. investigated the transactivation properties of the two wild-type ERs with different ligands at an AP-1 and an ERE site (51). Their experiments revealed that at an AP-1 site, estradiol activated transcription with ERα and inhibited transcription with ERβ. The ﬁnding that the ER subtypes respond differently to the same ligand suggests that estrogen may have tissue-selective effects on gene regulation, depending on the subtype of ER that is found in highest density in the tissue. Other studies have reported on the effects of ligands with different binding afﬁnities to ERα and ERβ (52). Using 17β-estradiol and genistein, a phytoestrogen with a 20fold higher afﬁnity to ERβ than to ERα, Makela et al. blunted the vascular injury response in ovariectomized rats; however, only 17β-estradiol (equal afﬁnity for both ERα and ERβ) treatment was associated with a dose-dependent uterotrophic effect. These results suggest the possibility that preferential targeting of ERβ may produce the desired vasoprotective effects without harmful uterotrophic effects. Evidence also suggests that, in addition to wild-type ERα and ERβ, variant ERs are present and are capable of anomalous transcriptional activities (53). Using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique, Hodges et al. isolated several variant ERs from human VSMCs. Some of these variant receptors appear to inhibit or enhance the effects of wild-type receptors, whereas others are constitutively expressed and do not require ligand activation. Their presence may help to explain tissue selectivity of estrogen action. Studies with monoclonal ER antibodies have detected ER protein in human VSMCs in vitro (54). In this study, immunostaining techniques further localized the ERs principally to the nucleus, although some cytosolic ERs were also detected. Using RT-PCR, ERα and ERβ mRNA have been detected in coronary arteries and cultured aortic SMCs of cynomolgus monkeys (55) and in human VSMCs (56). ERβ was found to be the predominant isoform expressed in VSMCs isolated from women, whereas VSMCs isolated from men expressed ERα and ERβ equally (56). The ﬁnding that ERβ is the prevalent isoform in VSMCs in women suggests the intriguing possibility that this isoform may be primarily responsible for the vasoprotective effects of estrogen, which are less evident in men and male laboratory animals than in females. Studies demonstrating selective increases in ERβ mRNA expression in vascular endothelial and SMCs after vascular injury lend further support to this hypothesis (57). Mice with homozygous deletion of either ERα (58) or ERβ (59) have been used to test in a rigorous fashion the function of the two ER isoforms in modulating the vascular injury response. Using the mouse model of carotid artery injury induced by passing a wire, Iafrati et al. compared the effects of estrogen on wild-type and ERα null mice. They noted that estrogen produced similar attenuation of vascular remodeling (medial hyperplasia) in wild-type and ERα null mice, suggesting that the vasoprotective effects of estrogen can be mediated by an ERα-independent pathway. Later studies by Karas

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et al. reported similar results when comparing the effects of estrogen on the carotid injury response in wild-type and ERβ null mice. Taken together, these studies suggest that in animal models, expression of either ER type is sufﬁcient to mediate the vasoprotective effects of estrogen. To test whether this beneﬁcial effect of estrogen is ER mediated versus a nonreceptor-mediated mechanism, Bakir et al. administered ICI 182,780, a nonselective ER antagonist, to intact female rats and, alone or in combination with estrogen, to ovariectomized rats (60). They demonstrated that ICI blocked the inhibitory actions of both endogenous and exogenous estrogen on neointima formation in female rats, indicating that the vasoprotective effects of estrogen are ER mediated. Estrogen has short, rapid vasodilator effects that are mediated by a nongenomic pathway (48). Two distinct mechanisms—effects on Ca2+ channels and effects on NO— have been implicated in these processes. Pharmacologic doses of estrogen have been noted to cause rapid vasodilation, accompanied by reduced calcium uptake, in contracted endothelium-denuded aortic rings from male rats (61). The vasorelaxation was not suppressed by a protein-synthesis inhibitor, a gene-transcription inhibitor, or an ER antagonist. Other studies have demonstrated that estradiol stimulates rapid release of NO from internal thoracic artery segments (62). These effects were inhibited by tamoxifen, indicating that the process is mediated by an ER. In addition, E2-BSA (estrogen bound to albumin) also stimulated NO release, demonstrating that the speciﬁc receptor in question was found on the cell surface, as E2-BSA is unable to penetrate the cell membrane. Although the study did not identify the speciﬁc cell-membrane receptor, work by Chen et al. documented that ERα could function in a nongenomic manner to activate nitric oxide synthase (NOS III) and cause rapid vasodilation (63). Further studies utilizing ERα-null mice are needed to determine whether ERβ or a yet undeﬁned ER is also involved in the nongenomic pathway of estrogen mediated vasodilation.

Early Response Genes Early response proto-oncogenes, such as c-myb and c-myc, clearly play an important role in regulating proliferation and differentiation of cell types known to participate in the vascular injury response. For example, the c-myb proto-oncogene is overexpressed in proliferating VSMCs in vitro, and suppression of c-myb mRNA by application of antisense oligonucleotides inhibits VSMC proliferation (64,65). Similar ﬁndings were reported by Gunn et al., who tested the effects of antisense oligonucleotides directed toward c-myb on VSMC proliferation in vitro and on neointima hyperplasia in vivo when delivered locally by porous balloon at the site of porcine coronary angioplasty (66). They observed both decreased c-myb expression and reduced neointima formation in the balloon-injured porcine coronary arteries treated with the antisense oligonucleotides. In the balloon injured rat carotid artery model, expression of c-myc was noted to rise rapidly (within 1 hour) and dramatically (×10) following balloon injury (67). Administration of mithramycin, a selective inhibitor of c-myc transcription, reduced both expression of c-myc and neointima formation in these vessels, suggesting that c-myc plays a role in VSMC proliferation and migration in the setting of vascular injury. Interestingly, there was a sexual dimorphism in expression of c-myc in response to vascular injury, with marked increases occurring in the male and delayed, attenuated responses in the female. Subsequent studies have indicated that estrogen is likely responsible for this effect (40).

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Growth and Chemoattractant Factors In response to endoluminal vascular injury, SMCs in the arterial wall produce a number of growth and chemoattractant factors that stimulate neointima formation. Experiments by Li et al. observed VSMC-dependent adventitial ﬁbroblast migration, determined by counting the number of blue-LacZ-expressing cells attached to Boyden type chambers (68). Compared to growth medium alone, chambers preconditioned with VSMC medium demonstrated a 2× increase in ﬁbroblast migration, suggesting that VSMCs release chemoattractant factors that promote ﬁbroblast migration. Addition of 17β-estradiol inhibited this migration in a dose-dependent fashion, and pretreatment of VSMCs with the selective estrogen inhibitor ICI-182,780 completely blocked the effects of estradiol. OPN is a key component of the extracellular matrix and has been hypothesized to help direct VSMC/adventitial ﬁbroblast migration in injured blood vessels in vivo. In order to test this hypothesis in vitro, Li et al. examined the expression of OPN in activated VSMCs and its responsiveness to estradiol (24). They noted a signiﬁcant dosedependent reduction in OPN mRNA expression in VSMCs pretreated with estrogen, as well as a reduction in adventitial ﬁbroblast migration in response to media conditioned by estrogen-treated VSMCs. Treatment with either a neutralizing antibody to OPN, an anti-β-integrin antibody, or a linear or cyclic Arg-Gly-Asp (RGD) peptide (inhibitor of cellular integrin/ανβ3 interactions with OPN) inhibited adventitial ﬁbroblast migration directed by exogenous OPN or VSMC-conditioned medium in a dose-dependent manner. These results indicate that estrogen attenuates adventitial ﬁbroblast migration in part by downregulating OPN expression in activated VSMCs in vitro. Whether or not these mechanisms play a role in estrogen-mediated vasoprotection in vivo is a topic for future study.

The Endothelium The endothelium plays a key role in maintaining vascular integrity and protecting against pathologic remodeling following vascular injury. The functions of the normal endothelium range from mediating the immune response to regulating vascular tone to inhibiting VSMC growth (69). Endothelial dysfunction, a sequel to endothelial injury, is characterized by a paradoxical vasoconstriction in response to compounds normally classiﬁed as endothelium-dependent vasodilators (e.g., acetylcholine, serotonin, histamine). Endothelial injury can be caused by a variety of factors, including low shear stress, hypertension, oxidized LDL, and smoking. A component of the vasoprotective action of estrogen relates to its favorable effects on endothelial structure and function. Estrogen enhances functional and anatomic reendothelialization in injured blood vessels, promotes vasodilation, upregulates constitutive NOS III expression, and enhances endothelial degradation of LDL cholesterol (48). Atherosclerotic arteries have impaired endothelial function, resulting in paradoxical vasomotor responses to acetylcholine. Both long-term and short-term estrogen treatment can improve endothelial function in atherosclerotic arteries. Williams et al. observed that short-term (20 minutes) estrogen therapy reversed acetylcholine-induced coronary artery vasoconstriction in surgically postmenopausal monkeys that had been fed an atherogenic diet and developed extensive coronary lesions (70). Similarly, Collins et al. observed that estradiol treatment enhanced the endothelium-dependent relaxation

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in the coronary arteries of postmenopausal women (71). Other studies have conﬁrmed these ﬁndings (72,73). Interestingly, estrogen failed to promote acetylcholine-induced vasodilation in the coronary arteries of men, suggesting that men are resistant to the vasoprotective effects of estrogen (71). Estrogen promotes reendothelialization and restores endothelial function in ballooninjured arteries, and this enhanced reendothelialization is thought to limit the extent of the response to injury (i.e., neointima formation) (26). Functional reendothelialization, assessed by measuring endothelium-dependent relaxation in balloon-injured carotid ring segments, and anatomical reendothelialization, assessed by the Evans blue technique, develops more rapidly in female than male rats, and adding supplemental estradiol to intact females further enhances reendothelialization (74). Similar results were reported by Krasinski et al., who observed a dose-dependent increase in reendothelialization as well as inhibition of neointima formation in balloon-injured carotid arteries of ovariectomized rats receiving exogenous estrogen (75). The mechanism by which estrogen exerts its effects on the endothelium is thought to be in part through expression of vascular endothelial growth factor (VEGF), an angiogenic growth factor with binding sites on endothelial cells. Krasinski et al. demonstrated increased VEGF mRNA expression in VSMCs exposed to estradiol (75), and Asahara et al. treated the intimal surface of balloon-injured rat carotid arteries with VEGF and observed the effects on reendothelialization and neointima formation (76). Vessels treated with VEGF achieved nearly complete reendothelialization after four weeks with a marked decrease in neointimal formation. These experiments establish a functional relationship between estrogen, increased VEGF expression, and reendothelialization in injured blood vessels that may explain estrogen-mediated vasoprotection in a variety of clinical and experimental settings.

Nitric Oxide Synthesis and Oxidant Injury NO, derived from the metabolism of L-arginine by NOS, is a mediator of diverse physiologic functions, including prevention of intimal hyperplasia (77) and maintenance of normal blood pressure and vascular architecture (78), as well as pathophysiologic processes such as atherosclerosis (79,80). In vitro NO is directly toxic to most cells; whereas in vivo, induction of NO production may be vasoprotective, as in prevention of cardiac allograft rejection (81). This paradoxical effect of NO, in which the same molecule is both protective and toxic, may be linked to local levels of NO generated by differential expression of NOS isoforms (82,83). In general, the isoforms are either CA2+-dependent and constitutively expressed (cNOS, types Ib and III) or Ca2+-independent and cytokine-inducible (iNOS, type II). NOS II is found in a variety of cell types, including cytokine-activated VSMCs and macrophages. Importantly, NOS II is not found in normal blood vessels, but is expressed in injured arteries within one day of the insult (84). Once activated, NOS II can produce more than 1000-fold more NO for a longer duration than NOS III (83). At these concentrations, NO produces the powerful biological oxidant peroxynitrite ONOO−, through a diffusion limited reaction with O2− (85). ONOO− directly attacks many biological targets and reacts with metals to form a nitroniumlike species (NO2+) that results in the formation of nitrotyrosine, which has been detected in human atherosclerotic lesions (86) and in the medial and neointimal compartments of damaged blood vessels.

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Altered expression of NOS II and NOS III, in particular an increase in NOS II and a reduction in NOS III, has been observed in balloon-injured arteries and is thought to play a functional role in the repair process. In both rat and pig carotid arteries, NOS II is undetectable prior to injury but appears in the medial compartment within 24 hours, and in the neointima at 1–2 weeks postinjury (84,87). NOS II transcriptional activity has been reported to be more robust and more resistant to regulation in explanted neointimal cells than medial SMCs (84). The functional consequences of altered NOS II and III activity in the setting of tissue damage have been the topic of considerable interest and controversy. NOS II-/-mice have been shown to have exacerbated transplant arteriosclerosis (88) and impaired closure of skin wounds (89). The impairment in wound healing was corrected by adenoviral-mediated transfer of human NOS II cDNA into the wound site. Similarly, transfer of the same NOS II cDNA to injured rat carotid and pig iliac arteries substantially reduced neointima formation (90). Further, NOS III-/-mice have been shown to have impaired remodeling in response to carotid-artery ligation (91). These ﬁndings appear to be consistent with the observations that administration of exogenous NO or NO donors reduces neointima formation in experimental models of vascular injury (92–96), suggesting that NO is vasoprotective in this setting. In contrast, neointima formation was greatly attenuated in carotid arteries of male NOS II-/-mice compared to wild-type controls following perivascular injury caused by placement of a periadventitial collar (97). Further, injury-induced proliferation of medial cells was attenuated in NOS II-/- mice compared to wild-type controls and mechanicalinjury-induced NOS II expression in wild-type VSMCs in vitro. These ﬁndings suggest that NOS II expression in injured blood vessels may promote neointima formation. This apparent paradox, i.e., that damaged vessels are protected from neointima formation by constitutive expression of endogenous NOS III or administration of exogenous NO or NO donors but further damaged by the consequence of enhanced expression of endogenous NOS II, may be a result of differences in quantity and compartmentalization of NO generated. Estrogen has directionally opposite effects on the two isoforms of NOS that are expressed in blood vessel walls: It enhances expression of NOS III in endothelial cells in vitro and blood vessel walls in vivo but inhibits NOS II mRNA as well as protein content and activity in rat and human VSMCs in vitro (63,84,85,98–100) and attenuates endotoxin-induced NO production in ovariectomized rats in vivo (100). Estradiol in concentrations as low as 10−11M has been shown to inhibit NOS II in cytokine-stimulated rat aortic SMCs in culture (98). This effect was ER-dependent (blocked by ICI-182,780). Studies in progress are examining the effects of estrogen treatment on NOS II and NOS III expression and function in injured blood vessels and relating these ﬁndings to vascular remodeling (i.e., neointimal formation and medial hypertrophy).

Antioxidant Properties Estrogen is an antioxidant, and this property may be useful in protecting the endothelium. The effects of estrogen on endothelium-mediated relaxation to bradykinin and substance P, as well as its effects on ex vivo oxidation of LDL, were studied in a swine model (101). Estrogen-treated swine maintained intact endothelial function, and LDL from estrogen-treated animals was more resistant to oxidation than LDL isolated from the ovariectomized control group. Similarly, Riﬁci et al. demonstrated that estradiol

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inhibited both copper-catalyzed and cell-mediated oxidation of LDL (102). The antioxidant properties of estrogen may in part account for its anti-atherosclerotic effects via a variety of mechanisms, including prevention of LDL oxidation (103) and rescue of NO from entering the ONOO− synthetic pathway (85).

INFLAMMATORY MARKERS Inﬂammatory mediators play an important role in the pathogenesis of atherosclerosis (104). Once the endothelium has been injured by agents such as ox-LDL, alterations in blood ﬂow, and free radicals, the inﬂammatory process is initiated, with release of a number of cytokines and growth factors, e.g. IL-1, TNF-β, and MCP-1 (monocyte chemotactic protein) and increased expression of cell-adhesion molecules (ICAM-1, VCAM-1) and selectins (E-, L-, P-selectin) that promote adherence of the inﬂammatory cells to the injured area (104). Increased circulating levels of inﬂammatory mediators such as C-reactive protein and of adhesion molecules such as ICAM-1 and E-selectin have been demonstrated in patients with cardiovascular disease. These molecules are now regarded as biomarkers for atherosclerotic disease (105). In vitro studies have demonstrated that estradiol can inhibit cytokine-induced activation of endothelial cells and subsequent transcription of cell-adhesion molecules such as E-selectin and VCAM-1 (106). This inhibition was negated by the estradiol antagonist, ICI 164,384, indicating an ER-mediated effect. Similar work by Koh et al. investigated the effects of hormone replacement therapy on soluble markers of inﬂammation (107). They reported that hormone treatment (transdermal estradiol or oral estradiol plus MPA) signiﬁcantly lowered ICAM-1 with mild reductions in VCAM-1 and E-selectin. Numerous clinical studies have described the effects of hormone therapy on inﬂammatory molecules (C-reactive protein, selectin, cell adhesion molecules) in women and have used these ﬁndings as surrogates for effects on cardiovascular disease risk. The results of these studies have yielded conﬂicting ﬁndings. C-reactive protein (CRP), an inﬂammatory marker that increases during acute phase reactions (108) and has been accepted as an independent risk factor for future cardiovascular events, has been shown to be elevated in women on hormone replacement therapy (109,110). In a cross-sectional study of 493 healthy postmenopausal women, median CRP levels were two times higher in women using any formulation of hormone replacement than in non-users (111). Similar ﬁndings were observed in a subpopulation of the PEPI trial, where women taking estrogen alone or in combination with progestins had signiﬁcantly higher levels of CRP than placebo (Fig. 6) (11). Neither study was designed or powered to address cardiovascular disease outcomes. Nevertheless, based on data from the Women’s Health Study and the Physician’s Health Study, which documented that men and women with higher baseline CRP levels experienced an increase in cardiovascular disease, it has been hypothesized that the adverse effects of hormone replacement therapy on CRP levels may provide a mechanism for the excess early cardiovascular events seen in the treatment arm of the HERS trial and in WHI (11,20). Although the effects of estrogen on CRP indicate a proinﬂammatory function, estrogen has also been shown to have an anti-inﬂammatory effect via decreased expression of adhesion molecules. For example, lower levels of soluble E-selectin were reported in hormone-treated women in the PEPI trial (Fig. 6) (11). Also, Caulin-Glaser et al.

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Fig. 6. Estimated mean level of E-selectin and C-reactive protein over time. ●, Indicates placebo; , CEE; ▲, CEE + MPA cyc; ■, CEE + MPA con; ◆, CEE + MPA. For C-reactive protein and E-selectin, the signiﬁcant differences were between the placebo and each active treatment arm. (Reproduced with permission from Cushman M, Legault C, Barrett-Connor E, et al. Effect of postmenopausal hormones on inﬂammation-sensitive proteins: The Postmenopausal Estrogen/ Progestin Interventions (PEPI) Study. Circulation 1999;100:717–722.)

observed a reduction in circulating adhesion molecules in postmenopausal women with coronary artery disease who were taking CEE versus a control group not on hormone replacement (112).

CONCLUSIONS Clariﬁcation of the effects of estrogen on inﬂammatory markers and cardiovascular disease outcomes, as well as fundamental investigation of the effects of hormone replacement on inﬂammatory processes, is urgently needed. These studies may elucidate

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the pathophysiology of the apparent adverse effects of hormone replacement therapy in postmenopausal women with established CAD. Together with studies of the cellular and molecular effects of estrogen and other postmenopausal hormones in animal models of acute vascular injury and atherosclerosis, these studies will provide a body of evidence on which to establish the role of hormone replacement therapy in the prevention and treatment of cardiovascular disease.

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7

Estrogens and the Brain Implications for the Treatment of Postmenopausal Women

Bruce S. McEwen, PHD, Phyllis M. Wise, PHD, Stanley Birge,

MD

Contents Abstract Introduction Biologic Basis of Estrogen Effects on Neural Function Estrogen Actions on Cognitive Function and Memory Processes Neuroprotective Actions of Estrogens Clinical Studies of Estrogen Deprivation on the CNS Summary and Conclusions References

ABSTRACT The actions of estrogens in the brain go well beyond the regulation of reproduction and include effects upon mood, cognitive function, motor coordination, pain, and protection of the brain from certain forms of damage. Multiple receptor mechanisms are believed to be involved, including at least two types of intracellular estrogen receptors (ERα and ERβ) and nonnuclear, nongenomic actions of estrogens that may involve a form of the intracellular receptors or other as-yet-unidentiﬁed receptor types. Selective estrogen response modulators (SERMs) interact with these many mechanisms of estrogen action in different ways, acting primarily as agonists or antagonists or having no effects in some cases. For this reason, it is difﬁcult to imagine that SERMs or any other substitute for estradiol itself will mimic all of the brain effects of 17-β estradiol itself. Rather, the challenge is to develop therapeutic strategies that emphasize particular beneﬁcial effects of estrogens on the brain, such as neuroprotection, while minimizing the possible antagonism of other estrogen actions that are beneﬁcial.

From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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INTRODUCTION With increasing life expectancy, women are likely to live a substantial part of their lives in a state of estrogen deﬁciency. Hot ﬂashes are for many women the most dramatic and noticeable consequence of loss of ovarian hormones and are treatable with estrogens. The loss of bone calcium and consequent osteoporosis, which develops much more gradually, is another consequence that has helped to establish the value of estrogen-replacement therapy (ERT) at the time of the menopause. Likewise, the loss of protection of the coronary arteries, leading to increased risk of cardiovascular disease in postmenopausal women, is another result of estrogen deﬁciency that has reinforced the value of ERT. Only quite recently has medical science recognized that the brain also suffers as a result of the loss of this circulating hormone. Ovarian failure appears to result in reversible changes in mental function, affect, and behavior. Epidemiological investigations indicate that the long-term consequences of estrogen deﬁciency in postmenopausal women may result in irreversible acceleration of brain aging. The clinical expression of these age-associated changes is a progressive loss of memory and deterioration in balance leading to increased risk of injurious falls and hip fracture. The estrogen-deﬁcient state may also increase vulnerability of postmenopausal women to ischemic brain injury and the earlier expression of Alzheimer’s disease. These studies have provided a new impetus to investigate estrogen effects on the brain. Studies over more than 30 years have indicated that estrogens target the brain of experimental animals (for summary, see (1)). Most of the earlier studies using animal models focused on estrogen actions on the hypothalamus that regulate ovulation and reproductive behavior. Only recently has it become apparent that estrogens exert many actions outside of reproductive function including actions on brain areas that are important for learning and memory and for emotions and affective state as well as motor coordination and pain sensitivity (see (2)). These effects reﬂect the actions of estrogens on a large number of brain areas outside of the hypothalamus. The problem in these brain regions has been recognizing the receptors and mechanisms by which estrogens produce their effects. This chapter focuses on ﬁve areas: ﬁrst, the cellular and molecular mechanisms by which estrogens produce their diverse effects on the brain; second, the brain regions and cell types in which estrogens produce their effects, emphasizing new knowledge regarding estrogen actions outside of the hypothalamus and pituitary gland; third, effects of estrogens on cognitive function; fourth, putative neuroprotective effects of estrogens; and ﬁfth, the potential clinical applications of this information, particularly in relation to cognitive function and dementia and especially with regard to actions of SERMs and other substitutes for estradiol itself as therapeutic agents.

BIOLOGIC BASIS OF ESTROGEN EFFECTS ON NEURAL FUNCTION Historical Perspective In the early 1960s, radioactive estrogens were used to identify estrogen receptors that bind to DNA and regulate gene expression (3). Using radioactive estrogens, the identiﬁcation and mapping of cells that contain estrogen receptors was extended from the uterus and mammary glands to the brain and pituitary gland (see (1)). At ﬁrst, only estrogen receptors in the hypothalamus and pituitary gland were studied because they

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are abundantly expressed in these regions of the brain/pituitary axis and estrogen receptors clearly mediate estrogen feedback actions on reproduction through receptors in these regions. From the beginning, however, nerve cells containing estrogen receptors were recognized in brain regions such as the hippocampus, cerebral cortex, midbrain, and brainstem; and yet only recently has attention been directed to their role in brain function. Recently, two types of estrogen receptors have been identiﬁed (4,5) and these are now referred to as ERα and ERβ. Estradiol binds with similar afﬁnity to both forms and traditional methods for cytosol binding and steroid autoradiography fail to discriminate between these two types of estrogen receptor. Both receptor types have a unique distribution within the brain, with some degree of overlap in which the possibility exists that colocalized receptors may form heterodimers. At the same time that progress with intracellular estrogen receptors has accelerated, other investigations of the functional effects of estrogens on nerve cell activity and protection of nerve cells from damage (neuroprotection) have uncovered rapid actions of these hormones that cannot involve activation or repression of gene expression, either because of their extreme rapidity or their structure/activity proﬁle in relation to the speciﬁcity of known intracellular estrogen receptors. These nongenomic actions of estrogens operate on the cell surface and affect the excitability of nerve cells and smooth muscle cells and the movement of the sodium, potassium, and calcium ions that create nerve impulses. We know very little about the molecular characteristics and the mechanism of action of these putative membrane receptors; yet they deserve consideration because of their potential importance in many estrogen actions.

Estrogen Receptors and Actions in the Central Nervous System We shall now examine the action of estrogens at the cellular and molecular level, discussing ﬁrst the intracellular receptors and then the nontraditional actions of estrogens (Fig. 1). ERα shows a characteristic distribution with high levels in the anterior pituitary, hypothalamus, hypothalamic preoptic area, and amygdala and much lower levels, with a more scattered distribution, in other brain regions. The discovery and cloning of ERβ (5–7) radically changed our view of estrogen action. Isoforms of ERβ have been identiﬁed recently, which have different afﬁnities for estrogens (8,9). These new forms of the estrogen receptor provide a basis for understanding how the absence or knock out of ERα (αERKO) (10,11) resulted in viable but sterile organisms and continued actions of estrogens on some tissues. A recent study (12) describes the knock out of ERβ: These βERKO mice appear quite normal and are able to reproduce, although they show some reduction in litter size. This contrasts to the αERKO mice, which are sterile and show altered sexual and other behaviors (see (13)). Measurements of messenger RNA (mRNA) for ERα and ERβ reveal distributions in the body which differ quite markedly from each other, with moderate to high expression of ERα in pituitary, kidney, epididymus, and adrenal, moderate to high expression of ERβ in prostate, lung, bladder, and brain, and overlapping high expression in ovary, testis, and uterus (14). In the brain, the distribution of ERα is fairly well established, but there is less certainty and more controversy surrounding the localization of ERβ. The autoradiographic maps of 3H-estradiol uptake and retention in the brain (15,16) are presumed to reﬂect binding to all forms of the estrogen receptor, particularly the ERα and the ERβ1 isoform, which have similar afﬁnities for 17β estradiol (14). Recent studies using 125I estradiol have

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Fig. 1. Schematic diagram of intracellular estrogen action via ERα and ERβ, as well as possible cell-surface effects of putative membrane estrogen receptors that produce neuroprotection (top) or affect intracellular signaling (bottom) via the cyclic AMP and MAP kinase pathways. Top Panel: Estradiol exerts its effects intracellularly via two principal receptor types, ERα and ERβ, and these are characterized by a distinct speciﬁcity for 17β-estradiol over 17α-estradiol. Estrogens also exert neuroprotective effects in part via a mechanism in which 17α-estradiol has equal or greater potency compared to 17β estradiol. Bottom panel: Estradiol acts either via cell-surface receptors or an intracellular estrogen receptor to activate two different second messenger pathways, one involving the MAP kinase cascade and the other involving cyclic AMP. Both pathways result in activation of gene transcription via at least three possible response elements: CRE, SRE, and AP-1. Note that in the case of intracellular second messengers there is some uncertainty concerning the involvement of ERα and ERβ in the signalling process versus the role of other, as yet uncharacterized, receptors (see text). AC, adenylate cyclase; cAMP, cyclic-3′,5′-AMP; CREB-P, phosphorylated form of CREB; ras, ras oncogene; MAPK, mitogen activated protein kinase; MAPKK, mitogen activated protein kinase kinase; fos-jun, fos-jun heterodimer. This ﬁgure republished from McEwen BS, Alves SH. Estrogen Actions in the Central Nervous System. Endocrine Rev 1999;20:279–307. Please see the source for details.

revealed the presence of cellular and cell nuclear labeling in brain areas not previously known to accumulate 3H estradiol, suggesting that the 125I estradiol is a more sensitive technique for revealing low levels of estrogen receptor binding activity in brain (17). In situ hybridization data suggest widespread distribution of ERβ mRNA throughout much of the brain including olfactory bulbs, cerebellum and cerebral cortex (18,19). Antisera that are currently available for ERβ and its multiple isoforms are not optimal

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to measure protein expression in brain and do not always provide speciﬁc signals in brain areas in which mRNA has been detected. ERα and ERβ are similar not only in afﬁnity for a number of estrogens and estrogen antagonists (14), but they also are similar in their ability to regulate genes in which the estrogen-response element (ERE) is the primary site of interaction (20). The major differences between ERα and ERβ concern their ability to regulate transcription via the AP-1 response element. ERα and ERβ can form heterodimers when expressed in the same cells, thus giving rise to additional possible variants as far as gene regulation (7). Thus far, endogenous colocalization of ERα and ERβ has been demonstrated in the hypothalamic preoptic area, the bed nucleus of the stria terminalis, and medial amygdaloid nucleus (21) (19) and probably exists in other brain regions. Actions of SERMs on the Nervous System The mixed agonist/antagonist effects of SERMs, operating via both ERα and ERβ, are reminiscent of earlier studies in which estrogen antagonists produced estrogenlike effects on some neurochemical endpoints and antagonistic effects on others. The antagonistic effects for Cl-628, a tamoxifenlike estrogen antagonist, were seen in terms of progestin receptor induction and lordosis behavior (22,23), whereas the agonistlike effects of CI-628 were seen for induction of choline acetyltransferase in the basal forebrain and repression of monoamine oxidase A in the amygdala (24). Recently, CI628 was shown to block estrogen-induced synapse formation in the hippocampus without having any agonistlike effects (25). One important implication of this ﬁnding is that nonsteroidal antiestrogens like CI-628 and possibly also SERMs such as tamoxifen and raloxifene will not have uniformly agonistic or antagonistic effects on the diversity of actions that estrogens normally produce in the brain. This has implications for the therapeutic applications of such agents and demonstrates that distinct studies of the actions of these agents on each endpoint of estrogen action will be required. Novel Estrogen Actions The variety of estrogen effects has been expanded to include rapid actions on excitability of neuronal and pituitary cells, the activation of cyclic AMP and mitogenactivated protein kinase (MAPk) pathways, effects on calcium channels and calcium ion entry, and protection of neurons from damage by excitotoxins and free radicals Table 1, Fig. 1). These estrogen actions occur through the two types of intracellular receptors (ERα and ERβ), or through other mechanisms for which receptors may not be required. Indeed, for several processes there are conﬂicting reports, based on structure/ activity studies and the actions of estrogen antagonists, as to whether intracellular receptors are involved. Thus, for estrogen actions on some aspects of calcium homeostasis, certain aspects of second messenger systems, and some features of neuroprotection novel mechanisms are implicated. In some of these actions stereospeciﬁcity for 17β over 17α estradiol is replaced by a broader speciﬁcity for the 3 hydroxyl group on the A ring. Membrane estrogen receptors have been reported on pituitary, uterine, ovarian granulosa cell, endothelial, and liver cell membranes, but these have been only partially characterized in binding studies and not yet shown to be linked to signal transduction mechanisms (2). At the same time, as summarized in Table 1 and Fig. 1, there are reported effects of estrogens on neuronal excitability and second messenger systems that have been difﬁcult to link to either novel receptor mechanisms or to genomic

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Table 1 Actions of Estrogens Related to Excitability and Cell Membrane Events Membrane binding sites Identiﬁed but not well-characterized in pituitary, liver, and endometrium, but not in brain. Some membrane sites may be related to intracellular ER (120,121). Genomic effects on membrane events E.g., induction of the MINK potassium channel in pituitary via genomic mechanism (122). Calcium channel expression in pituitary and hippocampus (123). Apparent nongenomic actions E.g., rapid excitation of electrical activity in cerebellum, hippocampus, striatum and cerebral cortex. Effects occur within seconds and are unlikely to involve a transcriptional activation: e.g., (53). Second messenger activation CREB phosphorylation: genomic vs. non-genomic mechanism unclear (124,125) MAP kinase activation: possible novel receptor pathway or involvement of classical ER in a novel signalling pathway (126) Calcium homeostasis Rapid actions: 17βE is more potent, but tamoxifen is an agonist on Ca++ currents (32). Rapid actions: 17αE is as potent as 17βE on calcium entry (127). Possible genomic actions: delayed and sustained increase in Ca channel activity (123). Neuroprotection Rapid actions: 17α Estradiol is as potent as 17β estradiol vs. oxidative damage (128). Genomic actions: 17β estradiol is more potent; antiestrogen blockade (129). Examples are provided for each topic. For detailed summary, see ref. (2). Note that these estrogen actions are not mutually exclusive but may represent different endpoints of interacting intracellular signalling cascades. Reprinted by permission from McEwen BS, Alves SH. Estrogen Actions in the Central Nervous System. Endocrine Rev. 1999;20: 279–307.

receptors. One reason for these difﬁculties is a lack, in many cases, of structure/activity studies that would rule in or rule out the participation of intracellular ER. The actions of estrogen may involve crosstalk between the traditional estrogen receptors and second messenger signaling pathways including the MAPk, adenylyl cyclase, and/or cAMP responsive element binding protein (CREB) pathway. These novel pathways may explain estrogen’s ability to interact with growth factors and neurotransmitters. These processes, as summarized in Table 1 and Fig. 1, are often interrelated at the level of intracellular signaling, and thus studies of these individual estrogen effects may some day converge when more is known about each of the mechanisms. Also important, but equally puzzling because of the lack of mechanistic details, are the novel ways in which estrogenic compounds protect nerve cells from damage by excitotoxins and free radicals (see discussion in the next section and (2) for review). In this realm, there are neuroprotective effects that are mediated via classical genomic receptors and which can be blocked by estrogen antagonists, but there are also other actions that are not and which appear to involve a novel mechanism in which 17α estradiol is as potent as the 17β estradiol. These actions of estrogens appear to reduce the production of or actions of free radicals in causing cell damage and promoting cell death through apoptosis.

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Estrogen Actions Throughout the Central Nervous System We now know that ovarian steroids have numerous effects on the brain throughout the lifespan, beginning during gestation and continuing on into senescence. Estrogens participate in the sexual differentiation of the brain during early embryonic or neonatal life, and these effects undoubtedly involve the intracellular estrogen receptors ERα and ERβ (26). The process of sexual differentiation involves the secretion of testosterone in fetal or early neonatal life and the actions of testosterone either through androgen receptors or via aromatization to estrogen in the defeminization and masculinization of brain structures and function (see (26,27)). Although initially believed to be conﬁned to the hypothalamus, structural and functional sex differences have been found in higher cognitive centers and in sensory and autonomic ganglia as well as structures of the limbic system of the brain and the midbrain, brainstem, and basal forebrain structures. Brain Regions and Neurochemical Systems Affected by Estradiol The following is a brief summary of some of the brain regions and neurochemical systems affected by estradiol, based on a recent review (2): Basal Forebrain Cholinergic System. Estradiol treatment upregulates cholinergic markers and NGF receptors, promoting neuronal survival; sex differences programmed during early development. Estrogen replacement therapy was reported to enhance the efﬁcacy of tacrine in treatment of cognitive function in dementia (28). Midbrain Serotonergic System. Estrogen treatment regulates tryptophan hydroxylase, serotonin transporters, and certain 5HT receptor subtypes, and sex differences in progestin-receptor expression and in 5HT turnover (29). Estrogen-replacement therapy was reported to have antidepressant effects and act as a facilitator of antidepressant actions of selective serotonin reuptake inhibitors (SSRIs) (30,31). Midbrain and Hypothalamic Dopamine System and Projections. Incertohypothalamic dopamine neurons show developmentally programmed sex differences in neuron number and function. Prolactin and estradiol, via intracellular ER, regulate the turnover of dopamine in hypothalamic nuclei. In contrast, for the nigrostriatal and mesolimbic dopamine systems, there are no known intracellular ER; yet, estrogen facilitates amphetamine- or apomorphine-stimulated dopamine release and locomotor activity in rats. In the corpus striatum, with males being less responsive than females to estradiol, there are four types of evidence for estrogen actions not involving the intracellular ER: ﬁrst, there is the lack of intracellular ER in striatum; second, estradiol acts rapidly, within seconds to minutes, to produce its effects; third, there is the pharmacological proﬁle of estrogen actions, particularly the ineffectiveness of diethylstilbestrol; fourth, estradiol conjugated to bovine serum albumin is able to mimic many of the effects of free estradiol even though the conjugated form cannot enter the cells. One possible explanation is the action of estradiol to reduce L-type calcium channel activity in striatal neurons via a G-protein coupled receptor (32). Brainstem Catecholaminergic Systems. Estradiol regulates tyrosine hydroxylase gene and immediate early gene expression, and does so apparently via intracellular ER. Estrogen treatment increases galanin mRNA in some neurons and this may affect noradrenergic tone by reducing noradrenaline release (33). Hippocampus. Estrogen treatment induces de novo synapse formation on pyramidal neurons, involving the participation of N-methyl-D-aspartate (NMDA) receptors. Estrogen treatment transiently downregulates γ-aminobutyric acid (GABA) and brain-derived

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neurotrophic factor (BDNF) activity in interneurons by a mechanism that is blocked by antiestrogens in a manner that is consistent with a key role for intracellular ERα in inhibitory interneurons (34–37). Spinal Cord. There are sex differences and estrogen modulation of nociception in humans (38). Sex differences in analgesia have been reported in mice along with sexspeciﬁc effects of estrogen on a form of nonopiod analgesia (39). Quantitative trait locus (QTL) mapping led to the identiﬁcation of a female-speciﬁc QTL on chromosome 8 (40). This female-speciﬁc mechanism, which is sensitive to estrogen modulation, is consistent with a gene that is turned off by testosterone exposure during sexual differentiation (41). Glial Cells. Estradiol regulates speciﬁc genes such as glial ﬁbrillary acidic protein and apolipoprotein estrogen within astrocytes and microglia via intracellular ER. Estrogen treatment regulates the morphology of astrocytes in hypothalamus and hippocampus, and these changes may reﬂect a role of glial cells in normal synaptic plasticity as well as lesion-induced plasticity. Cerebral Vasculature. Some intracellular ER are expressed in central nervous system (CNS) endothelia (42), and estrogen treatment regulates glucose utilization, possibly by inducing glucose transporter 1 in the endothelial cells of the blood-brain barrier (43).

Systems Involvement in Estrogen Actions These systems are involved in a variety of estrogen actions on mood, locomotor activity, pain sensitivity, vulnerability to epilepsy and attentional mechanisms and cognition, as summarized in (44) and (2) and brieﬂy summarized below. Affective State and Moods Estrogens affect the serotonergic, noradrenergic, dopaminergic, and cholinergic systems, all of which play a role in affective state and mood. Two disorders are particularly noteworthy, premenstrual syndrome (PMS) and depressive illness. For PMS, suppression of ovarian cyclicity reduces mood swings, although speciﬁc hormonal mechanisms are not known (30,45). High doses of estrogens have antidepressant effects in human subjects (46), and estrogen treatment inﬂuences the response to antidepressant drugs in animal models (47). Finally, as noted previously in the discussion of serotonin, estrogen treatment has been reported to enhance the efﬁcacy of Prozac treatment for depression (31). Motor Coordination and Movement Disorders Estrogens modulate activity of the cerebellum and the nigrostriatal and mesolimbic dopaminergic systems and have effects on normal and abnormal locomotor activity. High levels of estrogens antagonize the dopamine system and are recognized to exacerbate symptoms of Parkinson’s disease, whereas low estrogen levels facilitate dopaminergic function. See previous dopamine discussion. Excitability and Epilepsy Catamenial epilepsy varies according to the menstrual cycle, with the peak frequency of occurrence corresponding to the lowest ratio of progesterone to estradiol during the cycle. There are at least three potential mechanisms: First, estrogen induction of excitatory synapses in hippocampus, leading to decreased seizure thresholds (48); second,

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progesterone acts via the steroid metabolites, which act on the GABA(A) receptor to decrease excitability (49); third, gonadal hormones act on the liver to alter clearance rates of antiseizure medications (50,51). Pain Recent studies in mice indicate that males and females use functionally distinct pain pathways, and that gonadal steroids, particularly estrogens, play a major role in regulating these pathways (39). This topic is also summarized in the discussion of estrogen actions in the spinal cord. Cognitive Function Estrogens inﬂuence short-term verbal memory as well as performance on tests of ﬁne motor skills and spatial ability, as will be discussed in “Estrogen and Alzheimer’s Disease” later. There are sex differences in humans and in animals for strategies used in solving spatial navigation problems (52). Dementia See discussion in “Estrogen and Alzheimer’s Disease” later in this Chapter.

ER␤ and Nonreproductive Areas of the Brain In spite of the paucity of cell nuclear-localized ERα outside of the hypothalamus, hypothalamic preoptic region, and amygdala, estrogens have effects on many other brain regions and neurochemical systems involved in a host of nonreproductive brain functions. As noted above, the expression of ERβ and mRNA in many of these brain regions has raised the possibility of functional ER in these brain areas. At the same time, the presence of a few ERα containing nerve cells in inhibitory interneurons has led to the discovery, for example in the hippocampus, that these few nerve cells can have powerful trans-synaptic effects on neighboring neurons (36,37). In addition, the rapidity and structure/activity proﬁle of some of these effects has raised questions about the possible “nontraditional” and even nongenomic actions of estrogens in some brain regions, as exempliﬁed by electrophysiological studies in the hippocampus (53) and corpus striatum (32).

ESTROGEN ACTIONS ON COGNITIVE FUNCTION AND MEMORY PROCESSES Among the most novel and fascinating effects of estrogens are those on cognitive function, and there are at least four aspects of estrogen and of progesterone action in brain that are especially relevant to memory processes and their alterations during aging and neurodegenerative diseases. For details, see (2). First, as noted above, studies on female rats show that the estrogens and progestins regulate synaptogenesis in the hippocampus, a brain region important in spatial and declarative learning and memory. Formation of new excitatory synapses is induced by estradiol and involves the participation of a neurotransmitter, glutamic acid, acting through NMDA receptors (54,55). Inhibitory interneurons that innervate thousands of adjacent pyramidal neurons express ERα and are the most likely transsynaptic regulators of synapse formation on the pyramidal neurons (35). The downregulation of these synapses involves the action of progesterone, and synapse formation and destruction

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is a cyclic process during the estrous cycle of the female rat, with downregulation occurring after the proestrous surge of progesterone (56). Second, there are developmentally programmed sex differences in hippocampal structure that may help to explain differences in the strategies male and female rats use to solve spatial navigation problems. A similar sex difference in spatial problem solving is reported in humans (52). During the period of development when testosterone is elevated in the male, aromatase activity and estrogen receptors are transiently expressed in hippocampus, and recent data on behavior and synapse induction strongly suggest that this pathway is involved in the masculinization or defeminization of hippocampal structure and function. Third, as summarized earlier in “Estrogen Actions Throughout the Central Nervous System,” ovarian steroids have widespread effects throughout the brain, including brainstem and midbrain catecholaminergic neurons, midbrain serotonergic pathways, midbrain dopaminergic activity, and the basal forebrain cholinergic system. Fourth, estrogen effects on memory have been reported in animal models and in studies on humans (e.g., see (57)). The memories affected are ones in which the hippocampus plays a role along with the basal forebrain cholinergic system. Yet, there is some contradiction in terms of time course of the effects and types of memory affected between the reported estrogen actions and the known cellular processes, such as estrogen-induced synaptogenesis, and much more research is needed to reconcile morphological and neurochemical changes with the behavioral data (see (2) for summary). It cannot be overemphasized that, rather than one estrogen-regulated process, many types of estrogen action on a number of neurochemical and neuroanatomical substrates, combined with a number of molecular mechanisms, are likely to underlie the actions of estrogens on cognition and other aspects of behavior such as mood, pain perception, and nociception.

NEUROPROTECTIVE ACTIONS OF ESTROGENS Estradiol is an important growth and protective factor in the adult brain. It appears to attenuate the decline in neural function associated with normal aging, and protect the brain against neurodegenerative diseases and brain injury. Its important role in these functions leads to the realization that postmenopausal women, who are chronically hypoestrogenic, may suffer increasingly from cognitive dysfunction as they age and may be more vulnerable to neurological diseases and injury. During the past century our average life expectancy has increased by more than 30 to 40 years, but the age of the menopause has remained virtually unchanged. Thus, today a larger number of women are living a large portion of their lives in the postmenopausal state than ever before. In fact, until recently, most women continued to secrete estrogen until death. Therefore the chronic lack of estrogen that characterizes the postmenopausal state presents medical and societal challenges that our species has not faced before. It is critical that we deepen our understanding of the importance of estradiol in maintaining normal neural function so that we can better treat women during the postmenopausal years. During the past 10–20 years we have begun to understand that estrogens modulate far more diverse functions and inﬂuence far more tissues than merely those conﬁned to the traditional reproductive axis. Studies performed in animal models in the 1970s

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suggested that estradiol is a trophic hormone in the brain during fetal development. Although there were early indications that estradiol may be neurotrophic and protective in the adult as well, it has only been during the last several years that investigators have tested this possibility more thoroughly and accepted the concept that the adult brain remains highly plastic and hormone modulated. The ability of estradiol to inﬂuence neuronal structure and biochemistry has functional repercussions since it appears to improve some aspects of learning and memory. We have only begun to test the possible mechanisms of action through which estradiol exerts these effects. In adults, estradiol modulates neurite outgrowth, dendritic boutons, and synaptogenesis. A variety of studies have documented dramatic plasticity of synaptogenesis in the hippocampus (see discussion earlier in “Estrogen Actions Throughout the Central Nervous System” and (2) and arcuate nucleus (58), respectively. In the hippocampus, the number of spines on the dendrites ﬂuctuates with the reproductive cycle, attaining the highest density when estradiol is the highest (59). Parallel changes can be evoked by administering estradiol to ovariectomized rats. As summarized-elsewhere (2), these structural changes may underlie estrogen’s ability to inﬂuence long-term potentiation and neuronal excitability, improve cognitive behavior, and alleviate learning impairments that have been induced by a variety of injurious stimuli. In the arcuate nucleus, similar ﬂuctuations are apparent and may inﬂuence the ability of females to maintain regular estrous cyclicity. Estradiol also exerts profound protective effects against injury in adults (for review see (60)). Female rats exhibit less brain damage and cell death than male rats in numerous injury paradigms. Ovariectomy abolishes the sex-related differences and estrogen treatment decreases the extent of injury in both sexes. Many different models have been developed to explore estrogen’s ability to protect against ischemic injury, cerebral contusion, hypoxia, and drug-induced toxicity. The model that has been used most frequently is one in which investigators have attempted to mimic stroke-induced cerebral ischemia. Using this model, investigators have shown that males are more vulnerable than females to cerebral artery occlusion. Furthermore, castration reduces the extent of injury and estrogen treatment of males leads to protection against injury. Ovariectomized females are protected when they are treated with both physiological and pharmacological levels of estrogen replacement. It appears that physiological levels of estrogen exert protective actions through estrogen-receptor mediated actions whereas pharmacological levels of hormone may involve antioxidant (61), vasodilatory, and other nonreceptor-mediated mechanisms. Many in vitro models have been developed to study mechanisms of neuronal development, growth, repair, survival, plasticity, and death. Primary neuronal, tumor-derived neuronal cell lines, mixed neuron/astrocyte cell cultures, and organotypic explant cultures have been invaluable tools to study the underlying molecular mechanisms that neurons utilize to function properly under different conditions. A variety of neurotoxic insults have been used to mimic Alzheimer’s disease, stroke, and physical contusion. Thus, studies have inhibited mitochondrial function, suppressed glucose metabolism, altered nitric oxide production, or administered a variety of neurotoxic substances such as beta-amyloid peptide, excitatory amino acids, and free radicals. Numerous studies have shown that estrogen is trophic in in vitro cultures and can protect against injury induced by a variety of toxic stimuli. Some of these studies observed trophic effects within minutes of adding estradiol or estrogenlike compounds to cultures whereas

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others appear to require a longer exposure to steroids. Interestingly, many estrogenlike compounds that shared structural similarity in the presence of an aromatic A ring achieved equivalent neuroprotection, suggesting a mechanism of protection independent of binding to the classical estrogen receptor and transcriptional activation. Use of in vitro methods has allowed us to explore thoroughly the multiple mechanisms of action of estrogen and have emphasized that estrogen’s trophic and protective effects may be mediated via multiple cellular and molecular mechanisms. Some may be achieved by acting via binding to estrogen receptors (ERα or ERβ) that activate transcription of a variety of genes including those that encode for the neurotrophins and their receptors, cell-survival-factor proteins, and/or structural proteins that allow maintenance of synapses and neurite outgrowth. It has become increasingly clear however that estrogen may also protect neurons through multiple other molecular and cellular mechanisms that do not involve the classic estrogen receptors or that may involve the receptor, but act through novel mechanisms that include crosstalk with other second-messenger systems. Pharmacological levels of estrogens suppress lipid peroxidation, alter nitric oxide synthesis and release, and attenuate glutamate-induced elevations in intracellular calcium. Finally, as noted earlier in “Estrogen Receptors and Actions in the Central Nervous System” and in Fig. 1 and Table 1, estrogen-receptor interactions with multiple, different second-messenger pathways, including cAMP or MAP kinases may be involved. Estradiol appears to stimulate phosphorylation, and thus activation of CREB, extracellular signal-related kinase (ERK), and P13 kinase (Akt). These signaling molecules may act independently or in concert with estrogen receptors to mediate the protective actions of this steroid. Much more work must be done to better understand the multiple pathways through which estrogens may exert protective or trophic actions.

CLINICAL STUDIES OF ESTROGEN DEPRIVATION ON THE CNS Although neurobiologists have long recognized the role of ovarian hormones as modulators of neural function within the central nervous system, the clinical consequences of estrogen deﬁciency on brain function has not been appreciated by clinicians and their patients. There is now increasing evidence that estrogen deﬁciency of the postmenopausal woman may result in an irreversible acceleration of brain aging. One expression of these age-related changes is a progressive deterioration in balance leading to an increased risk of injurious falls and hip fracture. The estrogen deﬁciency state may also increase the risk of the postmenopausal woman to the earlier expression of Alzheimer’s disease. Today, women are living an ever-increasing portion of their lives in the postmenopausal state as a result of the dramatic increase in life expectancy enjoyed in this past century. This greater exposure to the estrogen deﬁciency state creates new challenges in the treatment of the postmenopausal woman and makes it imperative that the affect of estrogen and “designer estrogens” on brain function be included in the dialog with the patient considering hormone replacement therapy.

Short-Term Consequences of Estrogen Deprivation on the CNS For most women, the decline in ovarian function associated with the menopause is not accompanied by signiﬁcant changes in affect or behavior. In some, however, the changes are dramatic and incapacitating. Clinical trials in postmenopausal women have

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demonstrated improved quality of life and well being, and reduced symptoms of anxiety and depression. These effects of estrogen on mood have been attributed to the hormone’s ability to increase brain neurotransmitters and in particular, serotonin, through the inhibition of monoamine oxidase, the enzyme that catabolizes serotonin. This effect of estrogen may also account for the impressive additive effects of estrogen in the treatment of depression with SSRI (31). By itself, estrogen is not effective in the treatment of major depression that meets formal diagnostic criteria. However, approx 20% of older adults have signiﬁcant depressive symptomatology (62) that does not meet criteria for major depression but which has a signiﬁcant impact on quality of life and daily functioning (63). Carlson and coworkers (64) have demonstrated that in both elderly men and women, higher estradiol levels were associated with lower scores on the Geriatric Depression Scale (less mood symptomatology). A decrease in aspects of neurotransmission can also be expected to result in impairment of cognitive function. Following ovariectomy, short-term verbal memory declines (65). This decline is prevented with hormone replacement. The crossover design of these studies indicates that these consequences of acute estrogen deprivation are reversible. This group has also demonstrated (65) that the deterioration in memory performance was associated in part with the frequency of hot ﬂashes but could not be accounted for by estrogen’s effect on mood or insomnia. Other investigators have demonstrated that the hot ﬂash is associated with an abrupt decrease in regional cerebral blood ﬂow to the hippocampus (66) and may be the result of an sudden drop in the estrogendependent glucose transport across the blood-brain barrier (67). The hippocampus, that region of the brain that subserves the function of verbal memory, is uniquely sensitive to glucose deprivation as evidenced by autopsy studies of insulin-dependent diabetics. Thus hot ﬂashes may be associated with damage or loss of neurons within the hippocampus. This hypothesis would predict that women with a surgical menopause, and more likely to experience hot ﬂashes, would be more likely to manifest irreversible deﬁcits in verbal memory than women with a natural menopause. Indeed, several investigators have demonstrated this association (68). Women with a history of surgical menopause were twice as likely to experience cognitive impairment or dementia of the Alzheimer’s type than women undergoing a natural menopause. This increased risk of cognitive impairment and dementia of the Alzheimer’s type was evident after adjusting for duration of estrogen deﬁciency and other major surgeries. Although this presumed neuronal damage may be subclinical at the time of the menopause, it is postulated that this decrease in neuronal reserve may predispose the individual to the earlier expression of brain aging and Alzheimer’s disease. This concept could explain the observations in elderly postmenopausal women (69,70) that past users of estrogens, even those with less than one year of use (71), presumably at the time of the menopause for hot ﬂashes, perform as well as current users and better than nonusers on measures of cognitive function.

Long-term Consequences of Estrogen Deﬁciency on the Brain Estrogen and cognitive function Efforts to demonstrate effects of estrogen on cognitive function have been characterized by inconsistent results with some studies demonstrating improvement in cognitive function in women on estrogen replacement (70,72–75) and others failing to demonstrate signiﬁcant improvement (69,76,77). These differences can be attributed in part to small

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sample sizes, differing domains of cognitive function tested, differing durations of observation, inclusion of women receiving a progestin, and selection biases. In epidemiologic studies, nonparticipants and dropouts are several times more likely to have cognitive impairment than participants (78). This selective loss of the cognitively impaired mitigates against demonstrating a neuroprotective effect of the estrogen. Some of the problems inherent in observational studies of hormone-replacement users are circumvented by examining associations of endogenous estrogen levels or biological markers of endogenous estrogen levels with cognitive function. An example of a biologic marker of endogenous estrogen deﬁciency is the role of postmenopausal bone loss (79,80). The Study of Osteoporotic Fractures (SOF) (81), found a positive association with the rate of bone loss over six years with the magnitude of loss of cognitive function over this same time interval. Those women in the lowest quintile of bone density were also at greater risk of dementia. Another line of evidence for estrogen’s role in CNS aging is derived from brain imaging. In healthy older women, there is a decline in brain volume affecting primarily the hippocampus and parietal lobe that begins in the ﬁfth and sixth decade. A similar decline in brain volume is not seen in men until at least a decade later. The more rapid rate of decline in brain volume in women is associated with a more rapid decline in glucose metabolism after correction for atrophy in these same regions. It is these regions of the brain that are involved in memory and cognition and most sensitive to the neurodegenerative changes of Alzheimer’s disease. This sex difference can be attributed to the higher levels of estradiol in men than in women at any age after the menopause (82).

Estrogen and Alzheimer’s Disease There are several lines of evidence linking the estrogen deﬁciency state with the earlier expression of Alzheimer’s disease. A number of conditions that are associated with relative endogenous estrogen deﬁciency are associated also with an increased risk of Alzheimer’s disease. Women who take estrogen after the menopause reduce their risk of Alzheimer’s disease by about 50%. The prospective study of Tang and colleagues (83) illustrates that the reduced risk of this neurodegenerative disease can be attributed to a delay in the progression of this disease. They demonstrated that half of women who have never used estrogen will have developed the clinical changes of the disorder by the age of 85 whereas only 5% of women who have used estrogen after the menopause will have developed changes consistent with the diagnosis. An interesting observation of these authors is that women who took estrogens for less than one year, on average for only four months, had about a 50% reduction in their risk of expressing Alzheimer’s disease. We can assume that for most of these women, this limited exposure was for the treatment of hot ﬂashes at the time of the menopause. So how does this brief exposure to estrogen modify the expression of a disease some 20–30 years later? One explanation is that by treating hot ﬂashes, the neuronal damage associated with hot ﬂashes is prevented, thereby preserving a critical neuronal reserve which would serve to delay the expression of Alzheimer’s disease later in life. These observations provide one explanation for why there may be a narrow window of opportunity to signiﬁcantly modify the expression of this dreaded disease with estrogen. In addition, numerous observational studies in healthy nondemented women reveal that past users of estrogen are protected from the age-related decline in memory as well as current users (69,70,75). Again, as with Alzheimer’s disease, past use of less than one year had a signiﬁcant

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impact on cognitive decline (71). Matthews and coworkers (84) have made the signal observation that past users of estrogen but not current users of estrogen demonstrate a signiﬁcantly slower rate of decline over a three-year period of observation. The signiﬁcance of this study is that these women had a mean age of 72 at the time of the study. Thus, exposure of the brain at a much younger age results in an effect on the neurodegenerative process of aging that persists long after the hormone is discontinued. As noted in preceding sections, estrogen has a variety of actions on the CNS which could result in the delayed expression of aging and Alzheimer’s disease. These include the preservation of cerebral blood ﬂow and glucose transport, the protection of neurons from oxidative stress, amyloid toxicity, and the stimulation of neurotrophic factors that would facilitate the repair of damaged neurons. Because of these multiple effects of estrogens, one would anticipate that estrogen may delay the progression of Alzheimer’s disease as well and therefore be effective in the treatment of Alzheimer’s disease. Although there have been several small clinical intervention trials that would support that hypothesis, a recent (and the largest) placebo control trial (85) failed to ﬁnd any delay in the disease progression with up to one year duration of treatment with either 0.625 mg or 1.25 mg of conjugated equine estrogen. The latter results underscore the premise that the opportunity to affect the neurodegenerative disease progression may be limited to preclinical stages of the disease pathogenesis. There are several explanations that can be offered for the discrepancy between the dramatic ability of estrogen to delay the expression of this disease and estrogen’s inability to slow the disease progression. One explanation is that estrogen receptors are concentrated in those regions of the brian (the limbic system) that are the target of early stages of the disease. Once the disease has advanced beyond these regions, it is no longer responsive to estrogen. Mulnard and colleagues (85) suggest that “in the intact healthy brain, estrogen could play a key neuroprotective role delaying the initiation phase of the neurodegenerative disease onset.” This hypothesis is consistent with the delay in onset of Alzheimer’s disease observed with estrogen administration around the time of the menopause. It has also been suggested that there are two phases to the neurodegenerative process, an initiation phase and a propagation phase (86). This concept is exempliﬁed by the apolipoprotein E4 (ApoE 4) allele that is associated with the earlier age of onset Alzheimer’s disease but does not appear to alter the rate of progression of the disease (86–87). ApoE. ApoE is an injury-response protein that stimulates the regeneration of damaged neurons within the central nervous system (88,89) as well as peripheral nerves (90). The ability of ApoE containing low-density lipoproteins to stimulate neurite outgrowth is isoform speciﬁc. The ApoE4 isoform is without effect (91). In young transgenic mice expressing human amyloid precursor protein, ApoE expression enhances the clearance of amyloid-beta peptides (92) providing another potential mechanism for the protection of the brain from AD. Expression of ApoE in the brain is stimulated by estrogen (93). The ability of estrogen to restore synaptic density in a entorhinal-cortex-lesioned animal is dependent on the expression of ApoE (94). Thus, the increased risk of late onset AD in women can be attributed to a deﬁciency in the expression of the neuroprotective ApoE relative to women who have higher circulating levels of estradiol. As noted earlier in this section, clinical studies fail to support a protective role of estrogen or ApoE in the progression of the clinical manifestations of AD. In fact, it

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can be argued that after the ﬁrst two months of treatment, estrogen may accelerate the progression of AD as assessed by the Clinical Dementia Rating scale (85). This paradoxical affect of estrogen may be explained by the recent observation that in older transgenic mice, human ApoE expression enhances amyloid beta deposition and maturation of diffuse plaques to ﬁbrillar, neuritic plaques. The latter are believed to be more neurotoxic. Interestingly, ApoE4-expressing mice demonstrated a threefold greater neuritic-plaque burden than ApoE. These observations suggest that the response of the central nervous system to ApoE and therefore estrogen may be very age dependent, again suggesting that perimenopausal estrogen use may have a greater neuroprotective effect on brain aging and AD than later use for the treatment of the clinical manifestations of the neurodegeneration.

Estrogen and the Response to Stress Stress-induced increases in glucocorticoids can cause neuronal injury and loss, particularly in the hippocampus (95). These stress-induced effects of glucocorticoids on hippocampal neurons result in deﬁcits in learning and memory, and impairment of the inhibitory role of the hypothalamic pituitary axis (HPA) regulation (96). This impairment results in further increases in glucocorticoids in response to stress, increasing the vulnerability of the hippocampal neurons to further damage and cognitive impairment. Human studies provide an increasing body of evidence that stress-induced cortisol excess is associated with an accelerated decline in cognitive function as well as an increased risk of functional disability, cardiovascular disease, and Alzheimer’s disease (97). Estrogen may attenuate the HPA response to stress and preserve HPA regulation of cortisol release. Postmenopausal women exhibit larger age-related increases in cortisol secretion, higher 24-hour cortisol excretion, and a greater response to corticotropinreleasing hormone (CRH) stimulation than men of the same age (98). In response to the stress of a driving-simulation challenge, postmenopausal women exhibited a greater HPA response than men (99). Short-term estrogen replacement in postmenopausal women attenuates the glucocorticoid response to a psychological stress paradigm (100) and physical stress (101). Thus, the effects of estrogen on the HPA response to stress may be another pathway through which estrogen replacement may protect the brain from age-related neurodegeneration. A common condition associated with sustained elevations of cortisol is depression, a condition that has been identiﬁed as a risk factor for subsequent dementia in the elderly (102,103). Although readily amenable to treatment, depression is often unrecognized. Systematic screening for depression is imperative if we are to successfully identify those with this disabling disorder and to reduce their risk of Alzheimer’s disease.

Estrogen and Postural Stability An important consequence of brain aging is a deterioration in the speed whereby the brain processes complex sensory input and generates an appropriate response. Deterioration in central processing speed ultimately affects postural stability. The postural response to the brain’s perception of a change in the center of gravity is delayed to a point where that response is no longer able to avert a fall. Thus, we ﬁnd that age, female sex, and central processing speed, as assessed by the Trail Making task, are the best predictors of injurious falls in older adults (104). Further, results show a failure

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to generate any postural response to the loss of balance before hitting the ground (105). An unfortunate consequence of falls in the elderly is hip fractures. The incidence of fall-related wrist and hip fractures increases after the menopause. After age 70, the incidence of wrist fractures plateaus and declines, whereas hip fractures increase exponentially (105) in parallel with the exponential deterioration in central processing speed (106) (Fig. 4). It is postulated that because of the delay in central processing speed, the individual is unable to extend the forearm in time to break the fall so that the full energy of the fall is more likely to be directed to the hip. That energy is 2 to 3 times that necessary to fracture the proximal femur (107). Thus, after age 70, the age-related change in bone density fails to account for the observed incidence in hip fractures (108). These observations would predict that antiresorptive agents would have relatively little impact on fall-related (non-vertebral) osteoporotic fractures. Indeed, the FIT (alendronate) (109) and MORE (raloxifene) (110) trials were unable to demonstrate a signiﬁcant reduction in nonvertebral fractures while demonstrating signiﬁcant (43–48%) reductions in nonfall-related vertebral fractures. A role of estrogen in the deterioration of central processing speed, postural stability, and injurious falls is suggested by multiple lines of evidence. First, injurious falls are about three times more frequent at any age after the menopause in women than in men, consistent with the observation that men have approximately three times the level of circulating estradiol than women after age 60 (82). The deterioration in central processing speed and postural stability begins after the menopause (106,111). ERT prevents not only the deterioration in postural stability but also falls. Naessen and coworkers (111) found no difference in postural stability between premenopausal women, average age 25, and postmenopausal women, average age 68, who were on estrogen replacement therapy since menopause. In contrast, postmenopausal women, average age 68, who had not taken estrogen after the menopause, demonstrated a signiﬁcant deterioration in their balance. It is therefore not surprising that Honkanen and coworkers (112) found that postmenopausal women on estrogen replacement therapy enjoyed a 60% reduction in their risk of falling (excluding falls on ice). In summary, it appears that the estrogen deﬁciency state results in a deterioration in brain function and postural stability as a result of the loss of the neuroprotective effects of estrogen on factors contributing to brain aging.

Designer Estrogens (SERMs) and Brain Function The past several years have seen the emergence onto the health care scene of socalled designer estrogens (SERMs), drugs which act as antiestrogens on the breast and uterus but as estrogens on other target tissues such as the cardiovascular system and skeleton. Examples include tamoxifen and raloxifene (Evista). In vitro studies are conﬂicting as to the effects of these agents on neural tissue. Unfortunately, preclinical information (summarized earlier in “Estrogen Receptors and Actions in the Central Nervous System”) and clinical studies suggest these agents may be acting as antiestrogens on at least some aspects of brain function. Both drugs cause hot ﬂashes consistent with an inhibition of endogenous estrogen on the CNS. This inhibition of endogenous levels of estrogens in the postmenopausal woman may be sufﬁcient to determine a woman’s risk of AD. Conditions associated with relatively low levels of endogenous estrogens, myocardial infarction (113), osteoporosis

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(18), and being less than average body weight (114), are associated with an increased risk of AD. Inhibition of endogenous estrogen’s action on the brain may not only increase a woman’s risk of expressing AD but may also accelerate brain aging. In the major clinical trial designed to evaluate raloxifene’s effects on osteoporotic fractures, more than 7000 women were followed for three years on either raloxifene or placebo. Reduction in vertebral fractures was identical to the reduction in vertebral fractures observed with alendronate (Fosamax) in a comparable clinical trial. In contrast to alendronate, which reduced nonvertebral fractures by 14–38% and hip fractures by 20–50% (109,115), raloxifene reduced nonvertebral fractures by only 3% and increased hip fractures by 14% (110). One interpretation of this paradoxical response is that raloxifene is accelerating brain aging resulting in the slowing of the speed of processing sensory information and a deterioration in balance. Thus, the expected decrease in fallrelated osteoporotic fractures is offset by an increase in falls and nonvertebral fractures related to those falls. Additional evidence for an antiestrogenic effect of these agents on the brain is the observation that tamoxifen may increase the risk of depression (116) and memory problems (117).

Implications for the Management of AD and Brain Aging in the Postmenopausal Woman Because of the compelling evidence that estrogen deﬁciency accelerates brain aging and the expression of Alzheimer’s disease, the discussion of the risk/beneﬁt equation with our patients should now reﬂect this knowledge. That discussion will be enhanced if it includes an assessment of the patient’s risk of Alzheimer’s disease. An assessment of these factors with relatively high attributable risk could include: 1) a ﬁrst-degree relative with Alzheimer’s disease, 2) a loss of height of greater than 2 inches; 3) a positive screen for depression, and 4) less than a college education (Table 1). Given the high prevalence of the disease (30–40% over age 80) and the increasing life expectancy of the population, a 65-year-old woman has a life-time risk of developing Alzheimer’s disease approaching one in three. Given this information, women reluctant to consider ERT may be willing to endorse its use. This risk assessment may also prove to be relevant to the postmenopausal woman considering an antiestrogen or SERM for the prevention of breast cancer. Unfortunately, the beneﬁt of long-term breast antiestrogen use (greater than 5 years) in reducing breast cancer mortality in women with a recent diagnosis or at increased risk of breast cancer is unknown (118). Thus, the unknown beneﬁt from the long-term use of these agents in the prevention of breast cancer in otherwise healthy women must be weighed against the potential for accelerating brain aging and the expression of Alzheimer’s disease. Regrettably, the study of tamoxifen and raloxifene (STAR trial) in the prevention of breast cancer will not address this important concern because there is no control arm. Our limited knowledge of estrogen’s affect on the central nervous system raises three important questions: 1) when should ERT be initiated?, 2) for what duration?, and 3) in what form and dose? Although there is a suggestion that longer duration of ERT use is associated with a greater reduction in Alzheimer’s disease risk (118), this effect can also be attributed to the earlier onset of ERT, particularly at the time of the menopause. Those individuals with the longest duration of ERT are more likely to include individuals who began ERT at the time of the menopause for menopausal symptoms. This interpretation is consistent with the observation that past users, as well

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as current users of ERT, performed better on measures of cognitive function (84,119). After the disease becomes manifested clinically, there should be limited expectations for a signiﬁcant affect of estrogen on its course as evidenced by the recent estrogen intervention trial (85).Nonetheless, ERT remains an important adjunct with the exception of its use as an adjunct to cholinesterase inhibitors (28) and SSRIs (70) in the treatment of the symptoms of the disease. Thus, in contrast to estrogen’s effect on bone density where the increase in bone density is directly proportional to the number of years since the menopause, estrogen’s effect on the brain may have its greatest impact at the time of the menopause. The form and dose of estrogen is again a question that cannot be answered by the data available. Because endogenous estrogens appear to affect the expression of the disease, we can speculate that relatively low doses of estrogen, for example 0.3 to 0.625 mg of conjugated equine estrogens may be effective. However, one study suggested that higher doses, 1.25 mg, may be more effective in reducing Alzheimer’s disease risk (118). The role of progestins, whether cyclic or continuous, in attenuating the effect is also unknown. Clearly, these are issues that need to be addressed in future clinical trials such as the Women’s Health Initiative. Until those data are available, it may be argued that limiting a woman’s exposure to progestins, when feasible, may be prudent.

SUMMARY AND CONCLUSIONS The brain is an important target organ for the actions of estrogens. The multiple sites and diversity of actions of estrogens via different receptors and cellular mechanisms makes the study of estrogen action in the brain a daunting task. Very few generalizations can be made about the mechanisms of estrogen action in the brain until each estrogen effect is studied with respect to the receptor type and effector process that is involved, ie., whether it involves a classic or novel receptor mechanism and, in the case of classical receptors, whether it involves a direct binding of receptors to a DNA site, protein/protein interactions with other DNA-binding proteins, or the activation of a second messenger system. Until that time, attempts to design pharmaceutical agents other than estradiol itself for treatment of postmenopausal decline in neurological function run the risk of affecting only some of the estrogen-dependent mechanisms and/or acting as antagonists on others. This appears to be the case for the designer estrogens such as the SERM’s, although much more study is required before we are sure where they may be useful. Nevertheless, the continued effort to understand estrogen actions in the brain is warranted, because there is now an increasing body of evidence suggesting that the estrogen deﬁciency state of the postmenopausal woman accelerates the aging of the brain and the expression of Alzheimer’s disease. Brain aging is characterized by a subtle loss of primarily verbal memory and a slowing of the speed of processing sensory information. The clinical consequences of the latter are deterioration in balance and an increase in falls and fall-related fractures, particularly hip fractures. These observations provide an exciting opportunity to signiﬁcantly modify the progression and expression of these neurodegenerative processes that are the major cause of disability in older women. Recent studies suggests that the window of opportunity to signiﬁcantly affect this neurodegenerative disease may be limited to a preclinical stage and that period immediately following the menopause. This knowledge of estrogen’s role in brain aging

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and Alzheimer’s disease changes the risk/beneﬁt equation for estrogens and designer estrogens in the management of these problems in the postmenopausal woman.

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106. Ball LJ, Bisher GB, Birge SJ. A simple test of central processing speed: an extension of the Short Blessed Test. J Am Geriat Soc 1999;47:1359–1363. 107. Robinovitch SN, Hayes WC, McMahon TA. Prediction of femoral impact forces in falls on the hip. J Biomech Eng 1991;113:366–374. 108. Melton LJ, Kan SH, Wahner HW, Rigg K. Lifetime fracture risk: an approach to hip fracture risk assessment based on bone mineral density and age. J Clin Epidemiol 1988;41:985–994. 109. Cummings SR, Black DM, Thompson DE, Applegate WB, Barrett-Connor E, Musliner TA. Effect of alendronate on risk of fracture in women with low bone density but without vertebral fractures. JAMA 1998;280:2077–2082. 110. Ettinger B, Black DM, Mitlak BH, et al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene. Results from a 3-year randomized clinical trial. JAMA 1999;282:637–689. 111. Naessen T, Lindmark B, Larsen HC. Better postural balance in elderly women receiving estrogens. Am J Obstet Gynecol 1997;177:412–416. 112. Honkanen R, Komulainon M, Honkanen K. Hormone replacement therapy prevents falls in early postmenopausal women. European Congress on Osteoporosis. 1998;286:Abstract. 113. Aronson MK, Ooi WL, Morgenstern H, et al. Women, myocardial infarction, and dementia in the very old. Neurol 1990;40:1102–1106. 114. Berlinger WG, Potter JE. Low body mass index in demented outpatients. J Am Geriat Soc 1991;39:973–978. 115. Black DM, Cummings SR, Karpf DB, et al. Randomized trial of effect of alendronate on risk of fracture in women with existing vertebral fractures. Lancet 1996;348:1535–1541. 116. Cathcart CK, Jones SE, Pumroy CS, Peters GW, Knox SMCJH. Clinical recognition and management of depression in node negative breast cancer patients treated with tamoxifen. Breast Can Res Treat 1993;27:277–281. 117. Perez CA, Graham ML, Taylor ME, et al. Management of locally advanced carcinoma of the breast. Noninﬂammatory Cancer 1994;74:453–465. 118. Paganini-Hill A, Henderson VW. Estrogen replacement therapy and risk of Alzheimer’s disease. Arch Int Med 1996;156:2213–2217. 119. Schmidt R, Fazekas F, Reinhart B, et al. Estrogen replacement therapy in older women: a neuropsychological and brain MRI study. J Am Geriat Soc 1996;44:1307–1313. 120. Watson CS, Pappas TC, Gametchu B. The other estrogen receptor in the plasma membrane: Implications for the actions of environmental estrogens. Environmental Health Perspectives 1995;103:41–50. 121. Razandi M, Pedram A, Greene GL, Levin ER. Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: Studies of ERα and ERβ expressed in Chinese hamster ovary cells. Mol Endocrinol 1999;13:307–319. 122. Boyle M, MacLusky N, Naftolin F, Kaczmarek L. Hormonal regulation of K± channel messenger RNA in rat myometrium during oestrus cycle and in pregnancy. Nature 1987;330:373–375. 123. Joels M, Karst H. Effects of Estradiol and Progesterone on Voltage-Gated calcium and Potassium Conductances in Rat CA1 Hippocampal Neurons. J Neurosci 1995;15:4289–4297. 124. Zhou Y, Watters JJ, Dorsa DM. Estrogen rapidly induces the phosphorylation of the cAMP response element binding protein in rat brain. Endocrinology 1997;137:2163–2166. 125. Gu G, Rojo AA, Zee MC, Yu J, Simerly RD. Hormonal Regulation of CREB Phosphorylation in the Anteroventral Periventricular Nucleus. J Neurosci 1996;16:3035–3044. 126. Singh M, Setalo GJr, Guan X, Warren M, Toran-Allerand CD. Estrogen-induced activation of mitogen-activated protein kinase in cerebral cortical explants: Convergence of estrogen and neurotrophin signaling pathways. J Neurosci 1999;19:1179–1188. 127. Morley P, Whitﬁeld JF, Vanderhyden BC, Tsang BK, Schwartz J-L. A new nongenomic estrogen action: The rapid release of intracellular calcium. Endocrinology 1992;131:1305–1312. 128. Green PS, Gridley KE, Simpkins JW. Estradiol protects against β-amyloid (25–35)-induced toxicity in SK-N-SH human neuroblastoma cells. Neurosci Lett 1996;218:165–168. 129. Singer CA, Rogers KL, Stickland TM, Dorsa DM. Estrogen protects primary cortical neurons from glutamate toxicity. Neurosci Lett 1996;212:13–16.

III

PRECLINICAL STUDIES

8

Insights into the Molecular Mechanism of SERMs Through New Laboratory Models Csaba Gajdos, MD, James Zapf, and V. Craig Jordan, PHD, DSC

PHD,

Contents Introduction Tamoxifen to Treat and Prevent Breast Cancer Raloxifene and Its Analogs Drug Resistance to Tamoxifen Tamoxifen-Stimulated Tumor Growth A Molecular Mechanism to Explain Differences Between Tamoxifen and Raloxifene New Models to Investigate SERMs An Understanding of the Effectiveness of Five Years of Adjuvant Tamoxifen Conclusions References

INTRODUCTION It is estimated that in the US during 2000, breast cancer will account for 30% of all new cancer cases in women followed by lung and bronchus cancer combined (12%) and colon and rectum cancer combined (11%). Lung and bronchus cancer together are predicted to be the leading cause of cancer death (25%) followed by breast (15%) and colon and rectum cancer (11%) (1). More than 180,000 women were diagnosed with breast cancer in the United States in 1999 (2). Both osteoporosis and coronary heart disease, however, are important causes of death for women after menopause. During the past 20 years, two preventive strategies have emerged from the laboratory that have a profound impact on women’s health. Tamoxifen is the endocrine treatment of choice for all stages of breast cancer (3) and is the ﬁrst drug to be used to reduce the incidence of breast cancer in high-risk women (4). The recognition of selective estrogen receptor

From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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modulation in the 1980s (5) raised the possibility that compounds could be developed to prevent osteoporosis and prevent breast cancer as a beneﬁcial side effect (6). Raloxifene is the result. This chapter describes the laboratory principles that have provided the basis and foundation for the development of selective estrogen receptor modulators (SERMs). The mechanism of action of SERMs is unknown, but we believe by a study of drug resistance in breast cancer evidenced by tamoxifen-stimulated growth, we can gain insights into the molecular mechanism of SERM action that will provide clues for further study. Finally, we describe a new concept that might explain why the beneﬁts of tamoxifen as an antitumor agent last for years after tamoxifen therapy is stopped. We propose that tamoxifen-stimulated tumors can become supersensitive to physiologic estrogen so that tumor cells are destroyed by a woman’s own hormones after ﬁve years of tamoxifen is stopped (7).

TAMOXIFEN TO TREAT AND PREVENT BREAST CANCER The link between some breast cancers and an ovarian factor was established more than 100 years ago. George Beatson removed the ovaries from some premenopausal patients with metastatic breast cancer and found that the disease regressed and prognosis improved in some cases (8). Nearly forty years later Dr Antoine Lacassagne suggested that if breast cancer was caused by a special hereditary sensitivity to estrogen, then it could be prevented by a therapeutic antagonist to estrogen action in the breast (9). Unfortunately, there were no therapeutic antagonists of estrogen at that time, nor was there a target to design drug molecules. It would take a further 30 years to establish a mechanistic link between estrogen action and target-tissue growth (10–15). Jensen and associates (11) subsequently proposed the clinical estrogen receptor (ER) assay to predict hormone responsiveness in breast cancer and developed monoclonal antibodies to ER derived from MCF-7 cells to detect ER in tissue (16). This discovery was pivotal for the cloning and sequencing of the ER (17,18). The availability of the cDNA for the ER has made studies of the structure function relationships of SERM complexes possible and the crystallization of the ligand-binding domain with estrogens and antiestrogens a reality (19,20). Nonsteroidal antiestrogens were discovered in the 1950s (21). Possible considerations for their future development were as contraceptives, to treat habitual abortion, menstrual disturbances, endometriosis, menopause, and cancer. The ﬁrst clinically useful anticancer agent, tamoxifen, (Fig. 1) blocks the binding of estradiol to the ER derived from rat uterus (22–25) as well as human tumors (26,27). Daily administration of tamoxifen also inhibits the initiation and growth of DMBA-induced rat mammary carcinoma (28,29). The ﬁrst studies of tamoxifen as an antitumor agent in humans was undertaken at the Christie Hospital in Manchester, England (30). Tamoxifen the drug was given at 10 or 20 mg doses daily to late-stage breast cancer patients. The antitumor activity of tamoxifen was equivalent to the standard endocrine treatments, but with fewer side effects. Nearly 30 years ago, tamoxifen (ICI 46,474 or Novaldex) became the ﬁrst nonsteroidal antiestrogen to be approved for the treatment of advanced breast cancer. Continued effort in the laboratory to explore and understand the antitumor effects of tamoxifen (31–34) led to the hypothesis that long-term adjuvant therapy would be

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Fig. 1. Selective estrogen receptor modulators described in the text.

Table 1 Timelines for SERM Development Application

Laboratory Observation

Clinical Observation

Tamoxifen as a long-term adjuvant Tamoxifen as a chemopreventive SERM action

(33,34) (28,29,37) (5,44,58,70)

(3,35) (4) (60,61,64)

more likely to beneﬁt ER-positive patients. It is only after 20 years of testing adjuvant tamoxifen in randomized clinical trials, however, that it can be stated that survival and disease-free survival are related to the duration of adjuvant therapy and the ER status. Five years of adjuvant tamoxifen is superior to two years in ER-positive disease (35). One year of tamoxifen is without beneﬁt and patients who have an ER-negative tumor do not receive beneﬁt from tamoxifen irrespective of the duration of tamoxifen (3). The extensive experience with the safety of tamoxifen as a treatment of breast cancer naturally made this the drug of choice to test as a chemopreventive (36). There was a strong scientiﬁc rationale. Tamoxifen had been shown to prevent rat mammary carcinogenesis induced by different agents (28,29), and long-term treatment also prevented spontaneous carcinogenesis in mice infected with mouse mammary tumor virus

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(37). In the adjuvant trials tamoxifen had been shown to reduce the incidence of contralateral breast cancers (38–40). This observation made the drug the primary agent to test in high-risk women as a preventive. The National Surgical Adjuvant Breast and Bowel Project (NSABP) National Cancer Institute (NCI) P-1 (4) prospective clinical trial was opened in 1992. The speciﬁc aim of the trial was to test tamoxifen as a preventive for breast carcinoma in high-risk women determined by using a modiﬁed Gail model (41). Participants were randomized to tamoxifen 20 mg daily or to placebo for 5 years. Tamoxifen was found to reduce the risk of invasive breast cancer by 49% (2P < 0.001), with cumulative incidence through 69 months of followup totaling 43.4 versus 22 per 1000 women in the placebo and tamoxifen groups respectively. Similarly, tamoxifen produced a 50% decrease in the incidence of ductal carcinoma in situ. It is well known that with the long-term administration of tamoxifen a variety of endometrial changes occur in unselected women (42). Laboratory data suggest that tamoxifen has the potential to encourage the growth of preexisting disease harbored in the uterus (43,44). Animals bitransplanted with an MCF-7 breast tumor and the endometrial carcinoma EnCa 101 demonstrate target-site-speciﬁc effects with tamoxifen (44,45). Estradiol-stimulated growth of the breast tumor is controlled by tamoxifen whereas the endometrial tumor grows. Tamoxifen is stimulating rather than blocking growth. In the Oxford Overview Analysis (3) the incidence of endometrial carcinoma was approximately doubled in trials of one or two years and approximately quadrupled in trials of ﬁve years of tamoxifen. Even in trials of about ﬁve years of tamoxifen, however, the absolute increase in endometrial cancer was only about half as big as the absolute decrease in contralateral breast cancer. In the NSABP/NCI P-1 study the rate of endometrial cancer was also increased in the tamoxifen group, predominantly in women aged 50 years or older. In a recent case-control study (46) on the endometrial cancer risk in tamoxifen patients, the risk of endometrial cancer increased with the duration of tamoxifen. This study also emphasizes the signiﬁcance of known risk factors for endometrial cancer like obesity and prior estrogen use, since there was no increased risk of endometrial carcinoma in the absence of these factors in women taking tamoxifen. Tamoxifen also increases the risk of stroke, deep vein thrombosis, and pulmonary emboli as well as causing menopausal symptoms and potentially ocular toxicity. Some early animal studies in rats have shown an increased risk of hepatic tumors (47–49). These animal data were not, however, conﬁrmed in clinical trials because no liver tumors were observed in women taking tamoxifen (3,4). Rectal, ovarian, or other tumors did not increase in patients receiving tamoxifen (3,4). Nevertheless, the concerns about the safety of tamoxifen encourage the consideration of new agents and, most important, the development of a new paradigm for the prevention of breast cancer (6). The success of tamoxifen acted as a catalyst for the exploitation of the SERM concept.

RALOXIFENE AND ITS ANALOGS The nonsteroidal antiestrogen LY 117018 (Fig. 1) was ﬁrst reported in 1980 (50). The compound is less uterotropic in the mouse and rat than tamoxifen (51) and blocks the uterotropic actions of tamoxifen (52). Unfortunately, high doses are required because there is rapid phase II metabolism and rapid excretion. An analog LY 156358 or keoxifene, (Fig. 1) also has an excellent proﬁle as an antiestrogen (53) and despite the observation

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that there was no superiority over tamoxifen as an antitumor agent in laboratory models (54,55) was initially proposed for development as an anti breast-cancer agent. This was abandoned in the late 1980s following the observation that keoxifene was unable to show any responses in phase II trials in heavily pretreated patients (56). The breakthrough that altered the goals of drug development occurred with the ﬁnding that both tamoxifen and raloxifene maintained bone density in ovariectomized rats (57) but prevented the development of mammary cancer in rats given the carcinogen N-nitrosometylurea (55). The observation that tamoxifen was a SERM in bone was also reported and extensively studied by Turner (58,59). Because these data translated to the clinic with maintenance of bone density in postmenopausal women treated with tamoxifen (60,61), they had a profound impact on further drug development. First, tamoxifen could be tested in well women to prevent breast cancer because it might prevent rather than promote bone loss. Second, and perhaps most important, new agents could be considered for clinical testing but from another perspective. In 1990, it was proposed that SERMs could be developed to prevent osteoporosis in the general postmenopausal populations and would reduce the incidence of breast cancer in women who only had age as a risk factor (6). Although keoxifene had been abandoned as a treatment for breast cancer the compound already had shown that it maintained bone density in rats (57), had a reduced uterotropic activity in laboratory animals (53), had a reduced ability to stimulate the growth of endometrial cancer in the laboratory compared with tamoxifen (62), and prevented mammary carcinogenesis (55). The Eli Lilly Company made the decision to reevaluate and conﬁrm the site-speciﬁc estrogenlike actions of the compound (63) and subsequently initiated clinical trials to determine whether keoxifene, now called raloxifene, would prevent osteoporosis but with breast safety. This would provide an advantage over hormone replacement therapy (HRT) for the prevention of osteoporosis. The prospective clinical trials of raloxifene were successful in demonstrating an increase in bone density (64) and a signiﬁcant decrease in fractures of the spine (65). Raloxifene is now being tested as a preventive for breast cancer in the study of tamoxifen and raloxifene (STAR) trial because an evaluation of the osteoporosis trials provide the hypothesis, stated in 1990 (6), that SERMs would reduce the incidence of breast cancer (66). Since raloxifene decreases circulating cholesterol (67), it is also being tested as a preventive for coronary heart disease in high-risk women. If all those clinical trials show beneﬁt for high-risk women then raloxifene may be the ﬁrst multifunctional medicine (68). Drug development in this drug class has not stopped here however. Although raloxifene should not be used for the treatment of breast cancer because of its poor bioavailability, an analog, arzoxifene (LY353,381) (Fig. 1), with improved bioavailability (69), is in clinical trial for the treatment of advanced breast cancer. The discovery of agents with improved toxicology over tamoxifen has now consolidated a new group of pharmacological agents called SERMs with a potential for multiple applications. The current goal is to improve antitumor action and discover the molecular mechanism of SERM actions so that a whole range of medicines can be targeted at different diseases (70). The question is how to consolidate the therapeutic gains and advanced knowledge of SERM actions so novel drugs can be developed with greater speciﬁcity and targeted actions that can be predicted. We will describe our approach to the problem.

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DRUG RESISTANCE TO TAMOXIFEN Tamoxifen can be used with some success to treat breast-cancer patients (71). There is clinical evidence however that patients with advanced disease who initially respond to tamoxifen treatment from several weeks to years in duration will eventually experience disease progression. Similarly, many patients with node-positive disease and a signiﬁcant number of patients with node-negative disease treated with tamoxifen eventually experience disease recurrence during or after therapy (3). Indeed, if adjuvant tamoxifen is used for more than ﬁve years there is less beneﬁt than if patients stop treatment at ﬁve years (72). Although tamoxifen is classiﬁed as an antiestrogen it is not a pure antiestrogen and the drug has some well-documented estrogenlike actions (6). One possible explanation for drug resistance to tamoxifen is the development of tamoxifen-stimulated tumor growth. There is some clinical evidence for this with the documentation of a withdrawal response after progression of advanced disease being treated with tamoxifen (73,74). Patients who fail tamoxifen therapy often respond to second-line endocrine treatment such as an aromatase inhibitor. The reason for this is that tamoxifen resistant tumors retain the ER (75) but exploit circulating estrogen as the growth stimulus when tamoxifen treatment stops. An aromatase inhibitor, therefore, decreases the biosynthesis of estrogens and reduces the tumor-growth stimulus. Progress in understanding drug resistance to tamoxifen requires reproducible laboratory models that can mimic clinical experience. The models may not only prove to be useful for testing new agents for breast cancer treatments but also may provide some clues to the molecular mechanism of actions of SERMs. Thus, models of drug resistance could be unique systems to study the shift from a SERM being an antiestrogen to becoming an estrogen. Clearly, the cloning of cells that are supersensitive to the estrogenlike qualities of tamoxifen could provide a basis for the discovery of novel coactivator molecules or loss of corepressors that may subsequently be found to be responsible for SERM action in normal tissues.

TAMOXIFEN-STIMULATED TUMOR GROWTH Tamoxifen prevents cell replication so it can be argued that applying selection pressure to cell populations and waiting should be the best way to obtain a tamoxifen– stimulated breast cancer cell line. The MCF-7 cell line has been used extensively to develop resistant clones in vitro (76–79) and these cell lines have proved useful in identifying novel survival pathways or comparing and contrasting gene activation. The process of cell selection to develop a tamoxifen stimulated tumor however requires the evolution of angiogenic mechanism that will aid tumor growth and survival. By deﬁnition, cell-culture systems are constantly supplied with nutrients so there is no need to develop an angiogenic mechanism. Unfortunately, there is no literature about the validation of systems in vitro by transplantation into athymic mice and the subsequent demonstrations of SERM-stimulated growth. Without validation cell systems remain suspect as appropriate laboratory models of drug resistance. In contrast, MCF-7 cells have been used successfully to develop tamoxifen-stimulated tumors in vivo (80) that can be serially transplanted (81–83) and that will be prevented from growing by a pure antiestrogen (84,85). An early hypothesis for the mechanism of tamoxifen-stimulated growth implicated the local metabolism of tamoxifen to estrogens

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(86–88). Nonetheless, the subsequent demonstration that metabolically resistant analogs of tamoxifen still increased the growth of tamoxifen-stimulated tumors (89,90) resulted in the conclusion that the promiscuous estrogenlike effect of the tamoxifen molecule itself was responsible for enhancing cell growth through the recruitment of coactivators. Alternatively, the cell cycle was reactivated because the tamoxifen ER complex was no longer able to recruit corepressors (91).

A MOLECULAR MECHANISM TO EXPLAIN DIFFERENCES BETWEEN TAMOXIFEN AND RALOXIFENE Over the past ﬁve years, new models to describe the interaction of raloxifene and tamoxifen have been developed by several groups through a combination of X-ray crystallography and structure/function relationships of the ER. It is not possible to crystallize the whole ER for technical reasons, but a shortened ligand-binding domain has been crystallized with estradiol, DES, raloxifene, and 4-hydroxytamoxifen (19,20). The ﬁndings advance earlier structure/function studies that proposed that estrogens are locked within the ligand-binding domain to cause activation of the ER complex but the side chain of antiestrogens wedges the ligand-binding domain open so the ER is not fully activated (92,93). The X-ray crystallography demonstrated that estrogens are bounded within the hydrophobic ligand-binding domain where helix 12 folds across the top of the pocket sealing the estrogen inside. The correct positioning of helix 12 now permits coactivator binding so that estrogen-responsive genes can be transcribed (19,20) (Fig. 2). In contrast, raloxifene does not permit the locking of the ligand within the hydrophobic pocket and helix 12 is repositioned in the site on the surface of the ER normally occupied by a coactivator (Fig. 2). 4-Hydroxytamoxifen produces a similar repositioning of helix 12 but there are subtle differences in the structures of the ER complex (20) that can explain the differences in the estrogenlike properties of tamoxifen when compared to raloxifene. The X-ray crystallography revealed an intimate connection between the antiestrogenic side chain of raloxifene and amino acid 351 aspartate in the ligand-binding domain of ER (19), whereas the side chain of 4-hydroxytamoxifen is not as close to aspartate 351 (20) (Fig. 3). We propose that the remaining negative charge that surrounds the surface amino acid aspartate in the 4-hydroxytamoxifen ER complex is the key to the estrogenlike actions of the complex. This, we reason, could form the basis for a novel binding site for coactivators and explain the promiscuous estrogenlike effects of 4-hydroxytamoxifen compared to raloxifene. It had previously been suggested however that the estrogenlike actions of 4-hydroxytamoxifen occurred because activating functions (AF-1) at the far end of the ER from the ligand-binding domain was constitutive and unaffected by ligands (94). We have addressed these issues through a study of structure/function relationships of mutated ERs at target genes based on an initial observation of a naturally mutated ER discovered in a tamoxifen stimulated tumor (95). A single-point mutation in the cDNA for ER was detected in a MCF-7 tamoxifenstimulated tumor line using single-stranded conformational polymorphism (83). This resulted in a D351Y change in the amino acid sequence. The mutation was outside the traditional AF-2 region of the ligand-binding domain however, and studies using traditional techniques of transient transfection and artiﬁcial reporter genes showed very few changes in intrinsic activity for tamoxifen and raloxifene ER complexes when

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Fig. 2. (A) The locking of estradiol in the ligand binding domain of ERα. Helix 12 seals the steroid into a hydrophobic pocket and exposes sites in the complex that can bind coactivators. (B) The wedging of raloxifene in the ligand-binding domain of ERα. Helix 12 cannot seal the selective estrogen receptor (ER) modulator (SERM) into the hydrophobic pocket because the antiestrogenic side chain interacts with Asp 351, which acts as a pivot for the helix. Helix 12 now blocks coactivator binding.

compared with estradiol (96). In contrast, we chose to evaluate the structure/function relationship of wild type and mutant ERs using stable transfection of cDNAs in the ER-negative breast cancer cell line MDA-MB-231 (97). The rationale for the use of MDA-MB-231 cells was that they would be replete with transcription factors relevant to breast cancer. Additionally, we found that the complex promoter for transforming growth factor α (TGF-α) could be activated by ER (98) so this was selected as our gene target in situ.

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Fig. 3. Asp 351 in raloxifene- and tamoxifen-bound ERα structures. The distance between the alkylamine of tamoxifen and the carboxylate of Asp 351 is longer than expected for a hydrogen bond. We therefore hypothesize that Asp 351 is not shielded by the tamoxifen side chain. As a result residual charge is available for coactivator binding.

We found that there was a profound difference between tamoxifen and raloxifene ER complexes in the stable transfectants with wild type ER. Tamoxifen was a complete estrogen at the TFG-α gene (99) whereas raloxifene was an antiestrogen (100). Most important, the D351Y-transfected cells converted raloxifene from an antiestrogen to

Fig. 6. Left. Molecular modeling of the wild-type ligand-binding domain ER dimer showing the surface aa aspartate at position 351 and the tertiary amine of the antiestrogenic side chain of tamoxifen. Helix 12 is reported to occupy the site normally occupied by GRIP-1 that is needed to activate AF-2. Right. The carboxylic acid side chain of GW7604 is calculated to repel aspartate 351 thereby disrupting the surface charge. We suggest that the change in the positioning of the charge caused by GW7604 is critical to prevent coactivator binding in the region around aa351, which results in the loss of estrogenlike properties for the ER complex at the TGFα gene.

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Fig. 4. Estradiol (E2) or tamoxifen (TAM) ER complexes activate estrogen responsive genes by recruiting coactivators to different sites on the surface of the ER complex. The proposal (98) identiﬁed two distinct sites on the activated complexes through the use of phage display. The coactivator binding site for the E2/ER complex is almost certainly the well-recognized AF-2 site in the ligand binding domain but the TAM-ER binding site has recently been identiﬁed as a complex triple point interaction that recruits AF-1, a repositioned helix 12, and aspartate 351.

an estrogen (100). We conclude that aspartate at amino acid 351 was extremely important for the expression of antiestrogenic and estrogenic actions of raloxifene because a mutation to tyrosine changes the relationship of the ligand side chain and the protein (101). The interactions of the piperidine ring that shields the charge at the small aspartate is no longer possible when tyrosine is substituted so coactivators can now activate the D351Y/ER/raloxifene complex to transcribe TGF-α. The hypothesis was consolidated with further information about the 4-hydroxytamoxifen ER complex. Norris and coworkers (102) have used a phage-display assay to identify different coactivator binding sites on the estradiol and 4-hydroxytamoxifen ER complex. (Fig. 4) We reasoned that the 4-hydroxytamoxifen ER complex could have a binding site for coactivators that included aspartate 351. If the binding of coactivators at the novel site, (which we have named AF-2b), depends on a correctly positioned negative charge for LXXLX binding, then removal of the charge or dramatic displacement of the charge should result in loss of estrogenlike properties for the 4-hydroxytamoxifen ER complex. To test the ﬁrst point we prepared a D351 G cDNA stable transfectant in MDA-MB-231 and found that 4hydroxytamoxifen loses estrogenlike properties but retains antiestrogenic properties (103). Most important, these data demonstrate that it is possible to silence the constitutive activity of AF-1 in the 4-hydroxytamoxifen ER complex by an allosteric interaction at the ligand-binding domain. For the second point, we examined a novel derivate of tamoxifen called GW 7604 (Fig. 5). Unlike all other SERMs, GW 7604 and the pro drug GW 5638 have an antiestrogenic side chain that contains a carboxylic acid rather than a tertiary nitrogen atom. The pro drug has virtually no uterotropic activity in the rat but is estrogenlike in the bone (104,105) and is classiﬁed as a novel SERM with actions on the ER like a pure antiestrogen (106). We have shown by computer-assisted ligand docking (107) that GW 7604, the presumed active metabolite of GW 5638, dramatically repels amino acid 351 aspartate and this is correlated with a loss of

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Fig. 5. The structures of new analogs of tamoxifen with a novel acidic side chain

estrogenlike properties at the TGF-α gene (Fig. 6, see page 155) In broad terms, we reclassiﬁed GW 7604 as a raloxifenelike drug because D351Y ER weakly reactivates the TGF-α gene (107). In summary, we have built on previous models of estrogen and antiestrogen action (19,92,93,108) to describe a complex interaction between putative coactivators for the ER complex that could control SERM actions. The site for coactivator binding on the E2ER complex (AF-2) is distinct from the site on the 4-hydroxy tamoxifen ER complex (AF-2b) (103,107). Norris and coworkers originally proposed a new coactivator site on the ER, which they call AF-2a (109). We believe the site is more complex than originally thought however as it must involve AF-1, helix 12 and a correctly positioned and charged amino acid at the surface site 351. This new SERM site AF-2b could be a target for further drug discovery but only if there is selective speciﬁcity at the target site that would avoid general toxicity.

NEW MODELS TO INVESTIGATE SERMS The molecular classiﬁcation of SERMs and antiestrogens (70,108) has proved to be invaluable when integrated with the emerging knowledge of X-ray crystallography. A case in point is the new putative pure antiestrogen EM 800 (110,111) and its metabolite EM 652 (112) that is being evaluated as a preventive in breast cancer by the Schering Plough company. Although the structure of EM 652 is routinely represented as being similar to the pure antiestrogen ICI 182,780 the molecule has a strong structural similarity to raloxifene (Fig. 7). Indeed, we tested this hypothesis and were able to reclassify EM 651 as a raloxifenelike drug because the ligand reactivates D351Y to transcribe the TGF-α gene whereas a pure antiestrogen does not (113). Clearly, there would be an advantage to developing a SERM rather than a pure antiestrogen for the prevention of breast cancer. Additionally, there would be potential with EM 652 for further development as a preventive for osteoporosis and coronary heart disease.

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Fig. 7. Diagram of the structural similarities of raloxifene and EM-800 and its active metabolite, EM-652. The pure antiestrogen ICI 182,780 is a steroidal compound that has no estrogenic effects at any target. Raloxifene, which is a selective ER modulator, is currently used to prevent osteoporosis and could potentially prevent breast cancer. The structures of the nonsteroidal EM-800 and EM-652 have been compared with ICI 182,780, and the side chains seem to be in the same position. However, when the usual depiction of raloxifene (top right) is reoriented to sit in the LBD of the ER (center right), there is striking similarity to the structure of EM-652 (bottom right).

It would be naı¨ve to believe that the pharmacology of SERMs could be deciphered through an understanding of the molecular events in single cells alone. There are still many gaps in our knowledge and new translational models are needed to explore the clinical issue of drug resistance to SERMs in patients. To this end, we have made a commitment to develop new models of SERM resistance to breast and endometrial cancer in vivo so that cross resistance to tamoxifen can be evaluated for any new agent before valuable clinical resources are committed to national trials. All current investigators of tamoxifen-stimulated breast cancer growth are focused on the MCF-7 breast cancer cell line (114) but there is a requirement for diversity so that the multiplicity of possible drug-resistance pathways can be studied. About a quarter of breast cancers are mutated in the p 53 gene so it would be reasonable to create a laboratory model to study the observation that patients with p 53 mutations fail tamoxifen therapy more rapidly (115). Interestingly enough, MCF-7 cells contain wild type p 53 and it is generally found that estrogen withdrawal produces a variety of cell lines that are estrogen independent for growth but retain ER and are still responsive to antiestrogens (116–118). In contrast, the ER-positive T47D breast cancer cell line (119) is mutated for p 53 and loses ER under conditions of estrogen withdrawal (120). We chose to determine whether we could establish a new model of drug resistance to tamoxifen with the T47D cell line transplanted into athymic mice. Earlier studies

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had suggested that the cells were not only estrogen responsive for growth but also required a pituitary factor for optimal growth (121). Additionally, we hypothesized that tamoxifen would cause a rapid slip from ER positive to ER negative (120). We were incorrect, as transplantable estrogen-responsive tumors were rapidly converted to tamoxifen-stimulated tumors that retained the ER during high-dose (1.5 mg tamoxifen orally per day) tamoxifen treatment. The decrease in cell cycle control through mutated p 53 could potentially be the reason for the rapid failure of tamoxifen treatment both clinically and in the T47D model (122). We are addressing this issue further with MCF-7 cells engineered to be p 53 deﬁcient. For the future, we believe that a range of SERM- and antiestrogen-resistant models in vivo will prove to be invaluable to understand the process of drug resistance as a prelude to deciphering SERM action in normal cells.

AN UNDERSTANDING OF THE EFFECTIVENESS OF FIVE YEARS OF ADJUVANT TAMOXIFEN The recent Overview Analysis (3) clearly demonstrated the beneﬁcial effects of ﬁve years of adjuvant tamoxifen treatment both for node-negative and node-positive patients. There is a signiﬁcantly improved disease-free and overall survival rate in women treated with tamoxifen, and the beneﬁcial effects are observed for up to 10 years of followup. Based on these data the question could be raised, that if ﬁve years of treatment is superior to shorter treatment periods, why stop tamoxifen at ﬁve years, and why not go for longer duration of treatment? The NSABP (72) addressed this question by studying the effects of ﬁve versus more than ﬁve years of tamoxifen treatment for breast cancer in ER-positive lymph-node-negative patients. Using the data from the B-14 study as well as recruiting other patients with the same criteria, the following important observations were made: a) signiﬁcantly better disease-free, distant diseasefree and overall survival at ten years was found in patients treated with tamoxifen for ﬁve years compared to ﬁve years of placebo, b) tamoxifen therapy was associated with a 37% reduction in the incidence of contralateral breast cancer, c) advantages in diseasefree and distant-disease free survival were found in patients who discontinued tamoxifen therapy at ﬁve years compared to patients taking tamoxifen for ten years. It is possible that tamoxifen-stimulated drug resistance occurs with more than ﬁve years of adjuvant tamoxifen treatment but the question could be asked “Why does ﬁve years of tamoxifen confer a long-term survival advantage despite stopping tamoxifen?” Residual estrogen would be expected to reactivate any residual ER-positive breast cancer cells. Until recently there was no model of long-term tamoxifen therapy. The MCF-7 models (80,81) are representative of the development of drug resistance during the treatment of advanced breast cancer with tamoxifen. On average, tamoxifen therapy is effective for approximately one year. To address this deﬁciency we serially transplanted MCF-7 tamoxifen-stimulated tumors into tamoxifen-treated mice for up to ﬁve years (123). Based on extensive laboratory studies on the actions of E2 and tamoxifen on tumor growth regulation we propose the following sequential stages of hormone sensitivity in breast cancer that appear to follow a ﬁve-year cycle (Fig. 8): 1) tamoxifen acts as an antiestrogen by blocking tumor growth; 2) tamoxifen-stimulated tumors occur. These

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Fig. 8. Cyclical model of tumor sensitivity to tamoxifen over a 5-yr period. MCF-7 breast tumors are ER-positive and respond to E2, but tamoxifen blocks this E2-stimulated growth. After 1 yr of continuous tamoxifen treatment, however, the tumors are exclusively tamoxifen dependent, and 2 wk of E2 treatment results in complete tumor regression. After 6–8 wk of E2 treatment, some tumors will regrow, and these revert back to the original MCF-7 phenotype, with E2 stimulating growth and tamoxifen blocking E2-stimulated growth (MCF-7TAME) (124).

tumors can grow with either tamoxifen or E2; 3) Eventually only tamoxifen stimulates tumor growth, but E2 causes a dramatic regression of tumor size; 4) E2 subsequently stimulates the regrowth of some tumors, but tamoxifen blocks E2-stimulated growth. In our study, only physiological doses of estrogen were used to prevent tumor growth or induce tumor regression. We suggest that the repeated transplantation of these tumors in tamoxifen-treated animals has resulted in the selection of a MCF-7 tumor that is now supersensitive to the cytotoxic effects of estrogen. Overall, this new model system of changing hormonal sensitivity in breast cancer can be applied to provide an insight into the long-term control of breast cancer relapse following ﬁve years of adjuvant tamoxifen. We suggest that if the micrometastases around a woman’s body become supersensitized to the actions of estrogen by tamoxifen, then a women’s own estrogen may provoke an antitumor effect after ﬁve years of tamoxifen treatment (123). The conclusions would also imply that patients could beneﬁt from tamoxifen rechallenge following recurrence several years after their adjuvant therapy. Clearly, these conclusions could be tested in clinical trials.

CONCLUSIONS Overall, the progress made in the past decade has shown that there are exciting possibilities for using SERMs to understand physiology and to develop a menu of medicines to prevent a spectrum of diseases associated with the menopause. The development of tamoxifen (124) has opened the door to a new era of therapeutics where molecular biology is providing valuable mechanistic solutions that will aid new drug design in the future.

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54. Clemens JA, Bennett DR, Black LJ, Jones CD. Effects of a new antiestrogen, keoxifene (LY156758), on growth of carcinogen-induced mammary tumors and on LH and prolactin levels. Life Sci 1983; 32(25):2869–75. 55. Gottardis MM, Jordan VC. Antitumor actions of keoxifene and tamoxifen in the N-nitrosomethylureainduced rat mammary carcinoma model. Cancer Res 1987;47(15):4020–4. 56. Buzdar AU, Marcus C, Holmes F, et al. Phase II evaluation of Ly156758 in metastatic breast cancer. Oncology 1988;45(5):344–5. 57. Jordan VC, Phelps E, Lindgren JU. Effects of anti-estrogens on bone in castrated and intact female rats. Breast Cancer Res Treat 1987;10(1):31–5. 58. Turner RT, Wakley GK, Hannon KS, Bell NH. Tamoxifen prevents the skeletal effects of ovarian hormone deﬁciency in rats. J Bone Miner Res 1987;2(5):449–56. 59. Turner RT, Wakley GK, Hannon KS, Bell NH. Tamoxifen inhibits osteoclast-mediated resorption of trabecular bone in ovarian hormone-deﬁcient rats. Endocrinology 1988;122(3):1146–50. 60. Turken S, Siris E, Seldin D, et al. Effects of tamoxifen on spinal bone density in women with breast cancer. J Natl Cancer Inst 1989;81(14):1086–8. 61. Love RR, Mazess RB, Barden HS, et al. Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer [see comments]. N Engl J Med 1992;326(13):852–6. 62. Gottardis MM, Ricchio ME, Satyaswaroop PG, Jordan VC. Effect of steroidal and nonsteroidal antiestrogens on the growth of a tamoxifen-stimulated human endometrial carcinoma (EnCa101) in athymic mice. Cancer Res 1990;50(11):3189–92. 63. Black LJ, Sato M, Rowley ER, et al. Raloxifene (LY139481 HCI) prevents bone loss and reduces serum cholesterol without causing uterine hypertrophy in ovariectomized rats. J Clin Invest 1994;93(1):63–9. 64. Delmas PD, Bjarnason NH, Mitlak BH, et al. Effects of raloxifene on bone mineral density, serum cholesterol concentrations, and uterine endometrium in postmenopausal women [see comments]. N Engl J Med 1997;337(23):1641–7. 65. Ettinger B, Black DM, Mitlak BH, et al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3-year randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators [see comments]. JAMA 1999; 282(7):637–45. 66. Cummings SR, Eckert S, Krueger KA, et al. The effect of raloxifene on risk of breast cancer in postmenopausal women: results from the MORE randomized trial. Multiple Outcomes of Raloxifene Evaluation. JAMA 1999;281(23):2189–97. 67. Walsh BW, Kuller LH, Wild RA, et al. Effects of raloxifene on serum lipids and coagulation factors in healthy postmenopausal women. JAMA 1998;279(18):1445–51. 68. Jordan VC, Morrow M. Raloxifene as a multifunctional medicine? BMJ 1999;319(7206):331–2. 69. Sato M, Turner CH, Wang T, et al. LY353381.HC1: a novel raloxifene analog with improved SERM potency and efﬁcacy in vivo. J Pharmacol Exp Ther 1998;287(1):1–7. 70. Levenson AS, Jordan VC. Selective Oestrogen Receptor Modulation: Molecular pharmacology for the millennium. Eur J Cancer 1999;35:1628–1639. 71. Osborne CK. Tamoxifen in the treatment of breast cancer. N Engl J Med 1998;339(22):1609–18. 72. Fisher B, Dignam J, Bryant J, et al. Five versus more than ﬁve years of tamoxifen therapy for breast cancer patients with negative lymph nodes and estrogen receptor-positive tumors [see comments]. J Natl Cancer Inst 1996;88(21):1529–42. 73. Canney PA, Grifﬁths T, Latief TN, Priestman TJ. Clinical signiﬁcance of tamoxifen withdrawal response [letter]. Lancet 1987;1(8523):36. 74. Howell A, Dodwell DJ, Anderson H, Redford J. Response after withdrawal of tamoxifen and progestogens in advanced breast cancer [see comments]. Ann Oncol 1992;3(8):611–7. 75. Robertson JF. Oestrogen receptor: a stable phenotype in breast cancer. Br J Cancer 1996;73(1):5–12. 76. Bronzert DA, Greene GL, Lippman ME. Selection and characterization of a breast cancer cell line resistant to the antiestrogen LY 117018. Endocrinology 1985;117(4):1409–17. 77. Mullick A, Chambon P. Characterization of the estrogen receptor in two antiestrogen-resistant cell lines, LY2 and T47D. Cancer Res 1990;50(2):333–8. 78. Nawata H, Bronzert D, Lippman ME. Isolation and characterization of a tamoxifen-resistant cell line derived from MCF-7 human breast cancer cells. J Biol Chem 1981;256(10):5016–21. 79. Westley B, May FE, Brown AM, et al. Effects of antiestrogens on the estrogen-regulated pS2 RNA

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Gajdos, Zapf, and Jordan and the 52- and 160-kilodalton proteins in MCF7 cells and two tamoxifen-resistant sublines. J Biol Chem 1984;259(16):10030-5. Osborne CK, Coronado EB, Robinson JP. Human breast cancer in the athymic nude mouse: cytostatic effects of long-term antiestrogen therapy. Eur J Cancer Clin Oncol 1987;23(8):1189–96. Gottardis MM, Jordan VC. Development of tamoxifen-stimulated growth of MCF-7 tumors in athymic mice after long-term antiestrogen administration. Cancer Res 1988;48(18):5183–7. Gottardis MM, Wagner RJ, Borden EC, Jordan VC. Differential ability of antiestrogens to stimulate breast cancer cell (MCF-7) growth in vivo and in vitro. Cancer Res 1989;49(17):4765–9. Wolf DM, Jordan VC. The estrogen receptor from a tamoxifen stimulated MCF-7 tumor variant contains a point mutation in the ligand binding domain. Breast Cancer Res Treat 1994;31(1):129–38. Gottardis MM, Jiang SY, Jeng MH, Jordan VC. Inhibition of tamoxifen-stimulated growth of an MCF7 tumor variant in athymic mice by novel steroidal antiestrogens. Cancer Res 1989;49(15):4090–3. Osborne CK, Coronado-Heinsohn EB, Hilsenbeck SG, et al. Comparison of the effects of a pure steroidal antiestrogen with those of tamoxifen in a model of human breast cancer. J. Natl Cancer Inst 1995;87(10):746–50. Osborne CK, Coronado E, Allred DC, et al. Acquired tamoxifen resistance: correlation with reduced breast tumor levels of tamoxifen and isomerization of trans-4-hydroxytamoxifen. J Natl Cancer Inst 1991;83(20):1477–82. Osborne CK, Wiebe VJ, McGuire WL, et al. Tamoxifen and the isomers of 4-hydroxytamoxifen in tamoxifen-resistant tumors from breast cancer patients. J Clin Oncol 1992;10(2):304–10. Wiebe VJ, Osborne CK, McGuire WL, DeGregorio MW. Identiﬁcation of estrogenic tamoxifen metabolite(s) in tamoxifen-resistant human breast tumors. J Clin Oncol 1992;10(6):990–4. Wolf DM, Langan-Fahey SM, Parker CJ, et al. Investigation of the mechanism of tamoxifen– stimulated breast tumor growth with nonisomerizable analogues of tamoxifen and metabolites. J Natl Cancer Inst 1993;85(10):806–12. Osborne CK, Jarman M, McCague R, et al. The importance of tamoxifen metabolism in tamoxifenstimulated beast tumor growth. Cancer Chemother Pharmacol 1994;34(2):89–95. Lavinsky RM, Jepsen K, Heinzel T, et al. Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes. Proc Natl Acad Sci USA 1998;95(6):2920–5. Lieberman ME, Gorski J, Jordan VC. An estrogen receptor model to describe the regulation of prolactin synthesis by antiestrogens in vitro. J Biol Chem 1983;258(8):4741–5. Tate AC, Greene GL, DeSombre ER, et al. Differences between estrogen- and antiestrogen-estrogen receptor complexes from human breast tumors identiﬁed with an antibody raised against the estrogen receptor. Cancer Res 1984;44(3):1012–8. Berry M, Metzger D, Chambon P. Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. Embo J 1990;9(9):2811–8. Wolf DM, Jordan VC. Characterization of tamoxifen stimulated MCF-7 tumor variants grown in athymic mice. Breast Cancer Res Treat 1994;31(1):117–27. Anghel SI, Perly V, Melancon G, et al. Aspartate 351 of estrogen receptor alpha is not crucial for the antagonist activity of antiestrogens. J Biol Chem 2000;275(27):20867–20872. Jiang SY, Jordan VC. Growth regulation of estrogen receptor-negative breast cancer cells transfected with complementary DNAs for estrogen receptor [see comments]. J Natl Cancer Inst 1992;84(8): 580–91. Jeng MH, Jiang SY, Jordan VC. Paradoxical regulation of estrogen-dependent growth factor gene expression in estrogen receptor (ER)-negative human breast cancer cells stably expressing ER. Cancer Lett 1994;82(2):123–8. Levenson AS, Tonetti DA, Jordan VC. The oestrogen-like effect of 4-hydroxytamoxifen on induction of transforming growth factor alpha mRNA in MDA-MB-231 breast cancer cells stably expressing the oestrogen receptor. Br J Cancer 1998;77(11):1812–9. Levenson AS, Catherino WH, Jordan VC. Estrogenic activity is increased for an antiestrogen by a natural mutation of the estrogen receptor. J Steroid Biochem Mol Biol 1997;60(5–6):261–8. Levenson AS, Jordan VC. The key to the antiestrogenic mechanism of raloxifene is amino acid 351 (aspartate) in the estrogen receptor. Cancer Res 1998;58(9):1872–5. Norris JD, Paige LA, Christensen DJ, et al. Peptide antagonists of the human estrogen receptor. Science 1999;285(5428):744–6. MacGregor-Shafer JI, Liu H, Bentrem D, et al. Allosteric silencing of activating function 1 in the

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4-hydroxytamoxifen estrogen receptor complex by substituting glycine for aspartate at amino acid 351. Cancer Res 2000;60:5097–5105. Willson TM, Henke BR, Momtahen TM, et al. 3-[4-(1,2-Diphenylbut-1-enyl)phenyl]acrylic acid: a non-steroidal estrogen with functional selectivity for bone over uterus in rats. J Med Chem 1994; 37(11):1550–2. Willson TM, Norris JD, Wagner BL, et al. Discussion of the molecular mechanism of action of GW5638, a novel estrogen receptor ligand, provides insights into the role of estrogen receptor in bone. Endocrinology 1997;138(9):3901–11. Wijayaratne AL, Nagel SC, Paige LA, et al. Comparative analyses of mechanistic differences among antiestrogens. Endocrinology 1999;140(12):5828–40. Bentrem DJ, Dardes RC, Liu H, et al. Molecular mechanism of action at estrogen receptor alpha of a new clinically relevant antiestrogen (GW7604) related to tamoxifen. Endocrinology 2001;142:838– 846. McDonnell DP, Clemm DL, Hermann T, et al. Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol Endocrinol 1995;9(6):659–69. Norris JD, Fan D, Kerner SA, McDonnell DP. Identiﬁcation of a third autonomous activation domain within the human estrogen receptor. Mol Endocrinol 1997;11(6):747–54. Martel C, Labrie C, Belanger A, et al. Comparison of the effects of the new orally active antiestrogen EM-800 with ICI 182 780 and toremifene on estrogen-sensitive parameters in the ovariectomized mouse. Endocrinology 1998;139(5):2486–92. Tremblay A, Tremblay GB, Labrie C, et al. EM-800, a novel antiestrogen, acts as a pure antagonist of the transcriptional functions of estrogen receptors alpha and beta. Endocrinology 1998;139(1):111–8. Labrie F, Labrie C, Belanger A, et al. EM-652 (SCH 57068), a third generation SERM acting as pure antiestrogen in the mammary gland and endometrium. J Steroid Biochem Mol Biol 1999; 69(1–6):51–84. Schafer JI, Liu H, Tonetti DA, Jordan VC. The interaction of raloxifene and the active metabolite of the antiestrogen EM-800 (SC 5705) with the human estrogen receptor. Cancer Res 1999; 59(17):4308–13. Levenson AS, Jordan VC. MCF-7: the ﬁrst hormone-responsive breast cancer cell line. Cancer Res 1997;57(15):3071–8. Berns EM, Foekens JA, Vossen R, et al. Complete sequencing of TP53 predicts poor response to systemic therapy of advanced breast cancer. Cancer Res 2000;60(8):2155–62. Welshons WV, Jordan VC. Adaptation of estrogen-dependent MCF-7 cells to low estrogen (phenol red-free) culture. Eur J Cancer Clin Oncol 1987;23(12):1935–9. Katzenellenbogen BS, Kendra KL, Norman MJ, Berthois Y. Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breast cancer cells grown in the short-term and longterm absence of estrogens. Cancer Res 1987;47(16):4355–60. Pink JJ, Jiang SY, Fritsch M, Jordan VC. An estrogen-independent MCF-7 breast cancer cell line which contains a novel 80-kilodalton estrogen receptor-related protein. Cancer Res 1995;55(12):2583–90. Keydar I, Chen L, Karby S, et al. Establishment and characterization of a cell line of human breast carcinoma origin. Eur J Cancer 1979;15(5):659–70. Pink JJ, Bilimoria MM, Assikis J, Jordan VC. Irreversible loss of the oestrogen receptor in T47D breast cancer cells following prolonged oestrogen deprivation [published erratum appears in Br J Cancer 1997;75(10):1557]. Br J Cancer 1996;74(8):1227–36. Dembinski TC, Leung CK, Shiu RP. Evidence for a novel pituitary factor that potentiates the mitogenic effect of estrogen in human breast cancer cells. Cancer Res 1985;45(7):3083–9. Macgregor-Shafer JI, Lee ES, O’Regan RM, Yao K, Jordan VC. Rapid development of tamoxifen stimulated mutant p53 breast tumors (T47D) in athymic mice. Clin Cancer Res 2000;6:4373–4380. Yao K, Lee ES, Bentrem DJ, et al. Antitumor action of physiological estradiol on tamoxifen-stimulated breast tumors grown in athymic mice [In Process Citation]. Clin Cancer Res 2000;6(5):2028–36. Jordan VC. Tamoxifen: a personal retrospective. Lancet Oncology 2000;1:43–49.

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Third- and FourthGeneration SERMs Fernand Labrie, MD, PHD, Claude Labrie, MD, PHD, Alain Be´langer, PHD, and Jacques Simard, PHD Contents Introduction Breast Cancer as First Objective Menopause as Second Objective Need of a Pure Antiestrogen in Breast and Uterus AF-1 and AF-2 Functions of ER First-Generation SERMs Second- and Third-Generation SERMs Fourth-Generation SERM: (SCH 57068, EM 652) Summary References

INTRODUCTION The new antiestrogens called selective estrogen receptor modulators (SERMs) could well become the best example of the success achievable by biomedical and pharmaceutical research. In fact, the new antiestrogens induce three-dimensional structural changes of the estrogen receptor (ER) that lead to a multitude of different activities of the ER/ antiestrogen complex that are speciﬁc for each molecule and for each target cell type. Such ligand-induced modiﬁcations of the three-dimensional structure of ER that are unique to each antiestrogen can lead, at one extreme, to a complete blockade of the normal action of estrogens in some tissues whereas in others the same ER complex completely mimics or even surpasses the natural action of estrogens. Knowing that such an absolute selective action of antiestrogens is possible, the objective of pharmaceutical research is to design compounds that will act in a beneﬁcial way in all the tissues of special interest for women’s health. Since breast and uterine cancer were estimated to represent 35.5% of all new cancer cases and 18.3% of all cancer deaths in women in the United States in 1999 (1) and osteoporosis and cardiovasFrom: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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Fig. 1. Molecular structures of antiestrogens.

cular disease are the main causes of morbidity and mortality at postmenopause, the ideal compound would be the one having preventive as well as curative effects on all these diseases that most frequently affect women’s health. This ideal compound should also have an excellent safety proﬁle in order to insure compliance over 20 to 40 years of women’s life. What could only be a dream a few years ago has become a reality: Recent discoveries of pharmaceutical research offer women the hope to achieve a marked reduction in the incidence of breast and uterine cancer while protecting against bone loss and fracture as well as reducing the risk of cardiovascular diseases. Although estrogen replacement therapy at menopause can prevent bone loss and cardiovascular disease, there is evidence that estrogens are associated with an increased risk of breast cancer as well as endometrial cancer (2), thus seriously limiting the use of estrogen replacement therapy. We should therefore aim toward a compound having pure and potent antiestrogenic activity in the mammary gland and uterus while maximizing the beneﬁcial effects of estrogens in other tissues. Such characteristics seem to have been obtained with recently discovered SERMs. We thus brieﬂy compare the characteristics of the different series of SERMs with SCH 57068 (EM 652), a fourthgeneration SERM. Data obtained with tamoxifen (ﬁrst-generation SERM), raloxifene and LY353381 (second-generation SERM), and lasofoxifene (third-generation SERM) are presented (Fig. 1).

BREAST CANCER AS FIRST OBJECTIVE Breast cancer is the most frequent cancer in women, with 182,000 new cases and 40,800 deaths predicted in the United States in 2000 (3). Among all risk factors, estrogens are well recognized to play the predominant role in breast cancer development

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and growth (4–7). Existing surgical or medical ablative procedures however do not result in complete elimination of estrogens in women (8), because of the contribution of the adrenal glands, which secrete high levels of dehydroepiandrosterone (DHEA) and DHEA-sulfate which are converted into estrogens and androgens in peripheral target tissues (9–12). Considerable attention has thus focused on the development of blockers of estrogen biosynthesis and action (13–19). Since the ﬁrst step in the action of estrogens in target tissues is to bind to the estrogen receptor (20,21), a logical approach for the treatment of estrogen-sensitive breast cancer is the use of antiestrogens, or compounds that block the interaction of estrogens with their speciﬁc receptor. Until very recently, however, no agent with pure antiestrogenic activity under in vivo conditions has been available.

MENOPAUSE AS SECOND OBJECTIVE Despite its well-recognized beneﬁts on bone and the cardiovascular system (22–24) and its possible advantages on the risk and time of onset of Alzheimer’s disease (25–27), hormone replacement therapy (HRT) is practically limited to women with disabling vasomotor symptoms (28,29). A compound that could efﬁciently prevent breast cancer while, at the same time, preventing bone loss and cardiovascular disease would be a major breakthrough for the beneﬁt of women’s health. In fact, most of the other factors leading to a higher risk of breast cancer are practically impossible or very difﬁcult to control; these factors pertain to age at puberty, age at ﬁrst full-term childbirth, age at menopause, obesity, diet, and family history of breast cancer (BRCA1 and BRCA2 and yet other susceptibility genes to be deﬁned) (30–34).

NEED OF A PURE ANTIESTROGEN IN BREAST AND UTERUS Since clinical data suggest that long-term (ﬁve-year) tamoxifen adjuvant therapy is preferable to the short-term (two-year) use of the antiestrogen (35,36) and studies have shown the beneﬁts of long-term use of tamoxifen as a chemopreventive for breast cancer (37), it has become important to develop a pure antiestrogen in order to avoid the negative effects of the partial estrogenic activity of tamoxifen and thus make available a compound having activities limited to the desired therapeutic actions. The ﬁrst class of pure antiestrogens obtained were 7 α-substituted estradiol derivatives (13,15,17,18,38–40), especially ICI 164,384, EM 139, and ICI 182,780 (Fig. 1).

AF-1 AND AF-2 FUNCTIONS OF ER Classically, E2 binds to ER, leading to conformational changes that result in dimerization of E2/ER complexes with subsequent binding to speciﬁc estrogen-receptor element (ERE) located within the promoter area of genes responsive to estrogen (Fig. 2). The classical ERE element is an inverted hexanucleotide repeat separated by three nucleotides (41). Such binding of the dimerized ER/E2 complex to ERE results in activation of transcription. In analogy with many other nuclear receptors, ER plays a double life since it can be activated by kinase cascades independently from estrogens or from activation of the estrogen-binding site by an estrogenic ligand (Fig. 2). Polypeptide growth factors

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Fig. 2. Schematic representation of the dual activation mechanisms of ER by the AF-1 and AF-2 sites.

have been proposed as autocrine or paracrine mediators of classically estrogen-regulated mitogenesis (42,43). For example, exogenous administration of epidermal growth factor (EGF) mimics the effect of estrogens on the differentiation and proliferation of the reproductive tract in mice (44). Much evidence has accumulated to indicate that such action of EGF is mediated by ER (43–45). We will summarize later in “SCH 57068 Blocks Both AF-1 and AF-2 Functions of ERα and ERβ” the mechanisms of activation of AF-1 by Ras and EGF and, most interestingly, the complete inhibition of such action by the pure antiestrogen EM 652 (SCH 57068) (Fig. 1). In fact, AF-1 is thought to be responsible for the partial estrogenic activity of tamoxifen in cells that express ERα (46). It should be mentioned moreover that tamoxifen, contrary to EM 652, is unable to interfere with the activation of genes by factors acting through AF-1 (47), whereas such activation can be blocked by pure antiestrogens such as EM 652 and ICI 182,780.

FIRST-GENERATION SERMs Tamoxifen, the antiestrogen most widely used for the treatment of women with breast cancer, has shown clear clinical beneﬁts in advanced breast cancer, its efﬁcacy being comparable to that achieved with ablative and additive therapies (48). Because of its favorable proﬁle and clinical efﬁcacy, comparable to other endocrine therapies including oophorectomy and estrogens, tamoxifen has become the treatment of choice for patients with advanced or metastatic breast cancer (49–51). Tamoxifen, however, is known to possess mixed estrogenic and antiestrogenic activities (18,48,52) that are species-, tissue-, cell-, and even gene-speciﬁc (53,54). In support

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of the clinical evidence for the estrogenic activity of tamoxifen on human breast cancer growth (55,56), tamoxifen and its active metabolite 4-OH-tamoxifen have been found to stimulate the growth of human breast cancer cells in vitro and in vivo (53,57–65). Tamoxifen probably acts as an estrogen agonist more frequently than generally thought and this may explain some of the apparent paradoxes of endocrine treatments such as response to second endocrine therapy and withdrawal responses (51). All the analogs of tamoxifen, including toremifene, droloxifene, and idoxifene, also possess estrogenic effects analogous to those of tamoxifen (66,67). The most widely recognized adverse effects of tamoxifen are those related to the stimulatory effects on the endometrium including proliferation of the endometrium (68–70), formation of endometrial polyps (68,69,71,72), hyperplasia (68,73), and cancer (74–77). The ﬁnding that tamoxifen given as adjuvant to surgery improved survival (78) and decreased bone loss (79,80) has been a major stimulus to ﬁnd other and possibly more potent and speciﬁc compounds that would act as antiestrogens in the breast and uterus while having estrogenlike effects in the bones and cardiovascular system. In a breast cancer prevention study in high-risk women, treatment with the daily dose of 20 mg of tamoxifen for a median duration of 55 months decreased the risk of invasive breast cancer by 49% and the risk of ER-positive breast cancer by 69% (37). In the same trial including 13,175 women, however, the relative risk of endometrial cancer was increased by 2.53-fold (RR, 2.53; 95% conﬁdence interval: 1.35–4.97), thus conﬁrming previous data on the stimulatory effect of tamoxifen on the endometrium. The data showing that continuation of tamoxifen for four additional years gives results inferior to those obtained after only ﬁve years of treatment seriously limit the applicability of compounds of the class of tamoxifen for prevention of breast cancer. In fact, for an efﬁcient prevention of breast cancer, treatment should last as long as the risk of breast cancer does exist. If such treatment starts at menopause, or perimenopause, it should ideally continue for the remaining years of life. The clinical ﬁndings that led to discontinuation of trial B-14 and to the suggestion to limit tamoxifen use to ﬁve years likely result from the well-known resistance or loss of efﬁcacy which usually develops under chronic administration of tamoxifen (55,56,81,82). Following observations going back to Beatson (83), tamoxifen has clearly provided the proof of principle of the beneﬁcial effect of estrogen blockade in breast cancer at all stages of the disease. The optimal success of estrogen blockade, however, is likely to be achieved with the new antiestrogens showing pure antiestrogenic activity in the mammary gland and uterus. Analogs of tamoxifen have thus been synthesized and developed to various stages including clinical trials and commercialization. These compounds include toremifene, droloxifene, idoxifene, TAT-59, and GW5638. As mentioned earlier in this section, however, these compounds exert a stimulatory effect on the uterus which is comparable to that of tamoxifen.

SECOND- AND THIRD-GENERATION SERMs Clinical studies in postmenopausal women have conﬁrmed the protective effect of raloxifene on bone (84–87). Although the protective effects of raloxifene on bone mineral density are generally inferior to those achieved by estrogens in both animal

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Fig. 3. Breast cancer prevention: Cumulative incidence of all conﬁrmed breast cancers in the placebo and raloxifene groups (88).

and human studies, at least at the doses used, the risk of reduction of vertebral fractures observed at three years (87) is most encouraging. In fact, whereas 10.1% of placebo patients had at least one new vertebral fracture, this percentage decreased to 6.6% in the 60-mg group and to 5.4% in the 120 mg group of patients who took raloxifene. Such data correspond to a 50% reduction in vertebral-fracture risk in the group of women with low bone mass and a 30% reduction among women with previous vertebral fractures. A most important observation made with raloxifene pertains to the data showing a marked reduction in the risk of breast cancer. In the Multiple Outcomes of Raloxifene Evaluation (MORE) trial, treatment for three years with raloxifene decreased the risk of invasive breast cancer by 76% (88) (Fig. 3). These results, obtained after a relatively short-term administration of raloxifene, certainly raise the hope that the use of a compound having pure antiestrogenic activity in the breast and uterus could dramatically reduce breast cancer incidence and mortality in women. A signiﬁcant difference between the benzothiophenes (raloxifene and analogs) and the triphenylethylenes is the effect on the uterus: Whereas tamoxifen and its analogs exert relatively potent stimulatory effects on the uterus, raloxifene and its analogs have weak albeit statistically signiﬁcant stimulatory effects (89–91). As discussed in more detail later in “Effects on the Growth of Human Breast Cancer Cells in Vitro,” raloxifene exerts some weak but signiﬁcant stimulatory effects on the proliferation of human breast cancer cells in vitro (92). Moreover, an increase in uterine weight and uterine epithelial thickness has been reported previously in ovariectomized rats (90,91). LY 353381, an analog of raloxifene that shows improved in vivo potency (93), does possess comparable stimulatory effect on alkaline phosphatase in human uterine Ishikawa carcinoma cells as previously reported for raloxifene (65). LY 353381 (named “third generation SERM”) is thus unlikely to show superiority

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over raloxifene in terms of speciﬁcity of the effects or ratio of antiestrogenic/ estrogenic activity in the mammary gland and uterus. This stimulatory effect of LY 353381, in analogy with that of raloxifene, can be completely prevented by simultaneous addition of EM 652 (SCH 57068), thus supporting the intrinsic estrogenic activity of the compounds.

FOURTH-GENERATION SERM: SCH 57068, EM 652 Stimulated by the need of an improved therapy for breast cancer, considerable efforts have been devoted to the synthesis of compounds that would exert pure antiestrogenic activity in the mammary gland and uterus. As mentioned in the previous section, although tamoxifen has beneﬁcial effects on breast cancer, it clearly acts as an estrogen agonist in the endometrium with an increased rate of endometrial carcinoma in women taking tamoxifen under chronic conditions. Moreover, it is most likely that a pure antiestrogen will have beneﬁcial effects superior to those of tamoxifen on breast cancer prevention and treatment. In order to meet the objective of a completely tissue-speciﬁc antiestrogen, a long series of benzopyran derivatives were synthesized with the objective of developing an orally active compound having pure antiestrogenic activity in the mammary gland and uterus. EM 652 (SCH 57068) was thus the compound selected for clinical development (Fig. 1). In order to facilitate large-scale puriﬁcation, EM 800 (SCH 57050), the bipivalate derivative of EM 652 was synthesized. EM 800 is rapidly transformed into EM 652 in intact cells and following in vivo administration. The derivative currently used in our studies is EM 652.HCl (SCH 57068.HCl). The active compound EM 652 derived from EM 800 or EM 652.HCl behaves as a highly potent and pure antiestrogen in human breast and uterine cancer cells in vitro as well as in vivo in nude mice (65,66,94–96).

SCH 57068 Blocks Both AF-1 and AF-2 Functions of ER␣ and ER␤ Potential phosphorylation of serine 118 in human ERα (97–99) and serine 60 in mouse ERβ (100) through activation of the Ras-MAPK pathway has been shown to further maximize the E2 response of both estrogen receptors. To investigate if EM 652 could efﬁciently block this effect, we used the wild-type H-Ras and its dominant active form H-RasV12 in our transfection experiments (101). The inductions by both Ras forms were completely abolished with the addition of EM 652 in the medium, as with ICI 182,780, suggesting that EM 652 is effective in blocking the AF-1 activity of ERα. The same experiment was also conducted on ERβ where H-Ras and H-RasV12 augmented the E2 response in a similar manner. Again, EM 652 and ICI 182,780 abolished the Ras effect on ERβ in the presence of E2. We observed a similar pattern with SRC-1. SRC-1 is well known as a general coactivator for steroid receptors and has been shown to upregulate ERα-stimulated transcription (46,102). More recently, we demonstrated that SRC-1 interacts with ERβ and stimulates its transcriptional activity (100). This interaction occurred with the ligand-binding domain (LBD) of both ERs (46,100). Again, EM-652 was very potent in fully abolishing the E2 response of ERα and ERβ enhanced by SRC-1. These effects were not cell- or promoter-speciﬁc as demonstrated with the pS2 promoter in HeLa cells. Hence, EM 652 can be regarded as a pure antagonist that acts on both activation

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Fig. 4. Effect of increasing concentrations of EM 652 (SCH 57068), LY 353381, raloxifene, OHtamoxifene, droloxifene, toremifene or idoxifene on basal and E2-induced cell proliferation in MCF7 human breast cancer cells. Three days after plating, cells were exposed for seven (A, C) or eight (B) days to the indicated concentrations of compounds in the presence or absence of 0.1 nM E2. Media were changed at two- or three-day intervals. Data are expressed as the means ± SEM of triplicate dishes. When the SEM overlaps with the symbol used, only the symbol is illustrated.

domains of the ERs. Previous work (100) has shown that the SRC-1-induced ligandindependent activation of ERβ was not blocked by 4-hydroxy tamoxifen (OHT), which exerts an inhibition of ER limited to AF-2 (103), suggesting that SRC-1 might interact with other regions of the receptor.

Effects on the Growth of Human Breast Cancer Cells in Vitro It can be seen in Fig. 4A that the marked increase in MCF-7 cell proliferation induced by a seven-day incubation with 0.1 nM E2 was competitively blocked by a simultaneous exposure to EM 652, LY353381 or raloxifene at respective IC50 values of 0.23 ± 0.02, 0.77 ± 0.09, and 1.07 ± 0.12 mM, whereas as illustrated in Fig. 4C, the antagonistic activity of EM 652 or idoxifene was exerted at respective IC50 values and 0.49 ± 0.08 and .58 ± 12.8 mM. It can also be seen in Fig. 4A and 4C that the basal proliferation of MCF-7 cells was not affected by EM 652, whereas incubation with 0.02 nM LY 353381, 0.05 nM raloxifene or 2 nM idoxifene increased signiﬁcantly this estrogensensitive parameter by 120%, 63%, and 70%, respectively (all p < 0.01). To characterize with greater precision the dose-dependent stimulatory effect on MCF7 cell proliferation in the absence of E2 caused by all the compounds tested, we have

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next investigated the effect of a nine-day exposure to increasing concentrations of these mixed agonist/antagonist compounds in the presence or absence of 10 nM EM 652.HCl. As illustrated in Fig. 5, the dose-dependent maximal 60% increase in basal MCF-7 cell proliferation caused by raloxifene, LY353381, OH-tamoxifen, OH-toremifene, droloxifene, and idoxifene was completely blocked by a simultaneous incubation with the pure antiestrogen EM 652 (SCH 57068). These data strongly support the suggestion that the stimulatory effect of these mixed agonist/antagonist compounds on breast cancer cell proliferation is mediated through the estrogen receptors.

Effects on Alkaline Phosphatase in Human Endometrial Carcinoma Cells in Vitro Because data suggest that continuous long-term tamoxifen therapy is preferable to its short-term use (104) and studies are already in progress on the effect of long-term administration of tamoxifen or raloxifene to prevent breast cancer (37,105), it becomes important to make available a pure antiestrogen which, because of its lack of estrogenic activity, should theoretically be more efﬁcient than tamoxifen to treat breast cancer while simultaneously eliminating the excess risk of developing uterine carcinoma during its long-term use. We have therefore compared the effect of EM 800 or its active metabolite EM 652 with those of OH-tamoxifen, OH-toremifene, droloxifene, idoxifene, ICI-182,780, raloxifene, and the raloxifene analog LY 353381 on estrogen-sensitive alkaline phosphatase (AP) activity in human endometrial carcinoma Ishikawa cells. AP activity is well known to be stimulated by estrogens, whereas the other steroids— androgens, progestins, mineralocorticoids, or glucocorticoids—have no effect on this parameter (106). As illustrated in Fig. 6A, exposure to 0.05 up to 1000 nM LY 353381 caused a maximal 3.3-fold increase of AP activity, whereas the E2-induced AP activity was competitively, but again not completely, reversed by LY 353381 at an IC50 value of 1.8 ± 0.3 nM. On the other hand, this parameter was completely blocked by a simultaneous exposure to EM 652 at an IC50 value of 0.94 ± 0.13 nM. We have also observed that exposure to droloxifene or idoxifene increased by 7.3- and 6.9-fold AP activity at respective EC50 values of 4.0 ± 0.39 and 17.2 ± 1.5 mM (Fig. 6B,C). Direct comparison of the estrogenlike activity of these mixed agonist/antagonist compounds can best be seen in Fig. 7. Incubation with the indicated concentrations of LY 353381, raloxifene, OH-tamoxifen, OH-toremifene, droloxifene, or idoxifene increased AP activity by 3.1-, 2.1-, 4.3-, 4.8-, 4.0- and 4.6-fold, respectively. The blockade of the stimulatory effect of all these compounds on AP activity by simultaneous exposure to EM 652 well supports the suggestion that their stimulatory effect on this estrogen-sensitive parameter in human endometrial carcinoma is mediated through the estrogen receptor as previously reported (67).

Effects on Rat Endometrium in Vivo As mentioned earlier, raloxifene has been found to exert some stimulatory effect on uterine weight and endometrial epithelial height in ovariectomized rats (90). Direct comparison of the effect of raloxifene and EM 800 (precursor of EM 652) on endometrial epithelial height is illustrated in Fig. 8, which shows that the height of the epithelial cells lining the uterus in the ovariectomized rat is increased following raloxifene

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Fig. 5. Blockade by EM 652 (SCH 57068) of the stimulatory effect of raloxifenes, LY 353381, tamoxifen, droloxifene, toremifene or idoxifene on ZR-75-1 human breast cancer cell in the absence of E2. Three days after plating, cells were exposed for nine days to the indicated concentrations of compounds in the presence or absence of 10 nM EM 652. Media were changed at two- or threeday intervals. Data are expressed as the means ± SEM of triplicate dishes. When the SEM overlaps with the symbol used, only the symbol is illustrated.

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Fig. 6. Effect of increasing concentrations of EM 652 (SCH 57068), EM-800 (SCH 57050), LY 353381, droloxifene, and idoxifene on alkaline phosphatase activity in human Ishikawa cells. Alkaline phosphatase activity was measured after a ﬁve-day exposure to increasing concentrations of the indicated compounds in the presence or absence of 1.0 nM E2. The data are expressed as the means ± SEM of four wells. When SEM overlaps with the symbol used, only the symbol is illustrated.

treatment at the dose of 1 mg/kg for 37 weeks (Fig. 8E) whereas EM 800 has no effect (Fig. 8D). This complete absence of a stimulatory effect of EM 800 on endometrial epithelial height is in agreement with the absence of stimulatory effect of EM 800 on alkaline phosphatase activity in human Ishikawa uterine carcinoma cells (67). That the stimulatory effect of raloxifene on endometrial epithelial height is an estrogenic effect was illustrated by the ﬁndings that EM 652.HCl, although showing no effect by itself, almost completely reversed the stimulatory effect of raloxifene. Similar results were obtained with LY 353381, a close analog of raloxifene and lasofoxifene (data not shown).

Inhibition of Human Breast Cancer Xenografts in Nude Mice Estrone alone (OVX+E1) caused a 707% increase in ZR-75-1 tumor size during the 23 week treatment period (Fig. 9). Administration of the pure antiestrogen EM 652 at the daily oral dose of 50 µg to estrone-stimulated mice completely prevented tumor growth. In fact, not only was tumor growth prevented, but after 23 weeks of treatment, tumor size was 26% lower than the initial value at start of treatment (p < 0.04). This value obtained after treatment with EM 652 was not statistically different from that observed after ovariectomy alone (OVX) where tumor size decreased by 61% below initial tumor size. At the same dose (50 µg) and treatment period, the six other antiestrogens did not decrease initial average tumor size. Tumors in these groups were all signiﬁcantly higher than the OVX control group and the EM 652–treated group (p < 0.01). In fact, compared to pretreatment values, 23 weeks of treatment with droloxi-

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Fig. 7. Blockade by EM 652 (SCH 57068) of the stimulatory effect of LY 353381, raloxifene, OHtamoxifen, OH-toremifene, idoxifene and droloxifene on alkaline phosphatase activity in human Ishikawa carcinoma cells. Alkaline phosphatase activity was measured after a ﬁve-day exposure to the indicated concentrations of the speciﬁed compounds in the presence or absence of 100 nM EM 652. The data are expressed as the means ± SEM of four wells with the exception of the control groups where data are obtained from eight wells.

fene, toremifene, GW 5638, raloxifene, tamoxifen and idoxifene led to average tumor sizes 478%, 230%, 227%, 191%, 87% and 86% above pretreatment values, respectively (Fig. 9). After 161 days of treatment with a daily dose of 200 µg of tamoxifen, in the absence of estrone supplementation, the average tumor size increased to 196% over baseline (p < 0.01 vs OVX) (Fig. 10). The addition of 200 µg of EM 652 to 200 µg of tamoxifen daily completely inhibited the proliferation observed with tamoxifen alone (Fig. 10). The present study has the advantage of comparing seven antiestrogens under the same experimental conditions. Tamoxifen, idoxifene, raloxifene, toremifene, and GW 5638 show comparable effects as inhibitors of estrone-stimulated human breast cancer growth in nude mice by inhibiting estrone stimulation by 60–80%. In this study, droloxifene inhibited estrone stimulation by only 30%. On the same parameter, EM 652 has produced the greatest inhibition of tumor size or a 95% inhibition of estrone stimulation.

Inhibitory Effects on Cholesterol As can be seen in Fig. 11A, a 36% reduction of serum cholesterol was already observed with the lowest dose of EM 800 used, the serum cholesterol concentration being already deceased from 2.9 ± 0.18 mmol/L to 1.8 ± 0.09 mmol/L at the daily 25-

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Fig. 8. Hematoxylin and eosin-stained sections of rat uteri illustrating epithelial lining cells obtained from intact control (A), OVX control (B), OVX animals bearing an implant of 17β-estradiol (C) and OVX rats treated for 37 weeks with 1 mg/kg of EM 800 (D) or raloxifene (E). Note the absence of stimulatory effect of EM 800 on the uterine epithelial cells compared to the OVX control group whereas, at the same dose (1 mg/kg), a signiﬁcant hypertrophic effect of raloxifene was observed (original magniﬁcation ×220).

µg dose of EM 800 (p < 0.01). The daily 75-µg dose of EM 800 further decreased serum cholesterol to 1.6 ± 0.12 mmol/L (p < 0.01) and the 250-µg dose of EM 800 caused a maximal inhibition of 52% to a value of 1.4 ± 0.06 mmol/L (p < 0.01). A similar inhibitory effect of EM 800 was observed on serum triglyceride levels (Fig. 11B).

SUMMARY Fig. 12 summarizes the activities characteristic of 17β-estradiol and of the three classes of antiestrogens so far available, namely tamoxifen (ﬁrst-generation SERM), raloxifene and LY 353381 (second- and -third generation SERMs) and SCH 57068 (pure SERM) on the best-known parameters of women’s health. As can be seen in this schematic ﬁgure, tamoxifen, although exerting beneﬁcial effects on serum cholesterol, bone, and breast cancer, possesses relatively strong estrogenic activity in the endometrium as well as some stimulatory effect on breast cancer proliferation. Raloxifene, on the other hand, represents an important improvement in terms of decrease of the

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Fig. 9. Effect of treatment with seven antiestrogens for 161 days, on estrone-induced growth of human ZR-75-1 breast tumors in ovariectomized nude mice. Tumor size is expressed as the percentage of initial tumor area (Day 1 = 100%). Data are expressed as means ± SEM (n = 18–30 tumors/ group); ## p < 0.01 vs EM 652.HCl; ** p < 0.01 vs OVX. Antiestrogens were administered orally once daily at the dose of 50 µg/mouse under estrone stimulation obtained with subcutaneous 0.5cm silastic implants containing a 1:25 ratio of estrone and cholesterol.

stimulatory activity in the endometrium although some small estrogenic activity persists in the endometrium as well as on breast cancer cell proliferation. EM 652 (SCH 57068), on the other hand, is the only compound having pure antiestrogenic activity in the breast and endometrium while decreasing serum cholesterol and triglycerides, and protecting against bone loss. To our knowledge, on the parameters measured, secondand third-generation SERMs exert activities undistinguishable from those of raloxifene. The ideal compound for prevention of breast and uterine cancer should theoretically

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Fig. 10. Effect of treatment with tamoxifen, EM652, or tamoxifen plus EM652 for 161 days, on the growth of human ZR-75-1 breast tumors in ovariectomized nude mice. Tumor size is expressed as the percentage of initial tumor area (Day 1 = 100%). Data are expressed as means ± SEM (n = 18–30 tumors/group); ## p < 0.01 vs EM 652.HCl; ** p < 0.01 vs OVX. Antiestrogens were administered orally once daily at the dose of 200 µg/mouse in the absence of estrogen stimulation.

be the compound having the highest antagonistic effect and no agonistic or estrogenlike action in breast and uterine tissue while also being able to protect against bone loss and cardiovascular disease. The active metabolite EM 652 has been shown to be the antiestrogen closest to the deﬁnition of the perfect antiestrogen (107,108). In fact, EM 652 (SCH 57068) is a pure antagonist on breast tumor development and growth, and its effect is the most potent of all the antiestrogens tested (109,110); EM 652 has no stimulatory effect on the uterus and offers more potent protection against bone loss than does raloxifene (111).

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Fig. 11. Effect of daily oral administration of 25 µg, 75 µg, or 250 µg EM 800 for 9 mo on serum cholesterol (A) and triglyceride (B) levels in the rat. The number of animals per group was 9, 14, 16, and 20, respectively. Data are expressed as the means ±SEM. **, P<0.01 vs control (112).

Fig. 12. Schematic representation of the estrogenic and/or antiestrogenic action of estradiol, tamoxifen, raloxifene and EM 652 (SCH 57068) on the main parameters important for women’s health, namely the breast, endometrium, bone, and serum lipids.

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IV

ORGAN SPECIFIC EFFECTS OF ESTROGENS AND SERMS

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SERMs’ Effect on the Neuroendocrine System and the Reproductive Organs Nanette F. Santoro, MD, and Peter Kovacs, MD Contents Introduction SERMs and the Neuroendocrine System SERMs: Effects on the Ovary Tamoxiphen and Ovarian Cancer Risk Clomiphine and Ovarian Cancer Risk Endometrial Effects of SERMs Leiomyomas and SERMs Genitourinary Effects of SERMs Summary References

INTRODUCTION Estrogen-receptor modulation can be deﬁned as the role of any compound that interacts with the estrogen receptor and causes a biological effect. Selective estrogenreceptor modulators (SERMs) are compounds that interact with the estrogen receptor to cause desired effects. SERMs can be selectively agonistic, antagonistic or have both effects. It is helpful to conceptualize these agents as mixed agonists; that is, once bound to the receptor, either agonist or antagonist properties may be manifested. The exact actions of a given SERM are a function of its steric binding to receptor and its ability to interact with DNA or activating factors to effect a biological response. Clomiphene, tamoxifen and, most recently raloxifene have enjoyed widespread clinical use and have many beneﬁcial effects. All of these SERMs, however, have a set of side effects that limit their usage in certain clinical situations or lead to a need for increased surveillance and monitoring of treated women. The purpose of this chapter is to review the clinical data on these three compounds with respect to their effects on the hypothalamic/pituitary/ovarian axis, endometrium, myometrium, and genitourinary system.

From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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SERMS AND THE NEUROENDOCRINE SYSTEM The reproductive endocrine effects of tamoxifen have been a focus of intense investigation. Many studies have evaluated tamoxifen’s ability to increase endogenous estrogen production (1–13). Because tamoxifen’s effect on the reproductive axis is different in pre- and postmenopausal women, we will discuss them separately. Few studies have observed the effect of tamoxifen on follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol in postmenopausal women. Willis et al. investigated FSH, LH, and estradiol levels in 45 postmenopausal breast cancer patients, and compared levels before and during tamoxifen treatment. They observed a signiﬁcant decrease in FSH and LH levels in response to tamoxifen. Women whose breast cancer responded to tamoxifen therapy had no signiﬁcant change in their estradiol levels, whereas women whose disease progressed on tamoxifen had signiﬁcantly elevated circulating estradiol (3). Lahti et al. also reported lower FSH levels in response to tamoxifen therapy in postmenopausal breast cancer patients, but they failed to show any signiﬁcant change in estradiol levels when compared to controls (2). Both studies may be limited by difﬁculties in determining free and total estradiol in postmenopausal women. Cohen, in his review, reported lowered FSH and LH levels among postmenopausal women on tamoxifen. He found no consistent changes in estradiol levels (4). Tamoxifen has been used in premenopausal women as an ovulation induction agent. Premenopausal women with breast cancer have also been investigated (1,5–13). Sherman et al. followed ﬁve healthy premenopausal women who were taking tamoxifen and showed unchanged FSH and LH levels, but twofold increased estrogen levels. All participants remained ovulatory while on tamoxifen (5). Manni et al. followed 11 premenopausal breast cancer patients who were placed on tamoxifen. The dose of tamoxifen was increased until patients stopped menstruating, up to 100 mg a day. They found markedly elevated estrogen levels and unchanged gonadotropins. When patients stopped having regular menstrual function, their gonadotropins rose to the postmenopausal range, despite continued elevation of estradiol, well over 1000 pg/ml. Two patients out of ﬁve whose disease progressed on tamoxifen responded to oophorectomy. Their estradiol level decreased dramatically after oophorectomy (6). Rose et al. compared the endocrine effects of chemotherapy (CMF), chemotherapy plus prednisone and chemotherapy plus tamoxifen in premenopausal breast cancer patients. After several months of treatment almost all of the participants developed amenorrhea. Patients who also took tamoxifen had a signiﬁcant increase in circulating estradiol while they were having apparently normal menstrual cycles, but these levels decreased with the onset of amenorrhoea. Gonadotropin levels increased with the onset of amenorrhoea (7). Sawka et al. studied two groups of patients. In the ﬁrst group there were 65 premenopausal women with metastatic breast cancer. A second group of breast cancer patients participated in a randomized crossover trial comparing tamoxifen with ovarian ablation (oophorectomy or irradiation). Women who were assigned to tamoxifen and had disease progression received ovarian ablation. Data from 74 participants were analyzed. Thirtyeight women had stable or improved disease on tamoxifen. Women who became oligoor amenorrhoeic on tamoxifen had a better clinical response than those who continued to menstruate. Tamoxifen and ovarian ablation yielded comparable outcomes in premenopausal breast cancer patients. FSH levels remained unchanged while on tamoxifen, but estrogen levels increased, similar to the ﬁndings of Manni et al. (8). Jordan et al.

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looked at endocrine changes in response to chemotherapy, chemotherapy plus tamoxifen and chemotherapy plus long-term tamoxifen in breast cancer patients with node positive disease. Therapeutic regimens with tamoxifen resulted in higher estrogen levels. Gonadotropins were decreased in postmenopausal women, whereas they were increased in premenopausal women with the onset of ovarian insufﬁciency (9). Radvin et al. compared estradiol, progesterone, and gonadotropin levels in premenopausal breast cancer patients on tamoxifen and healthy, premenopausal women. They also reported increased estrogen and progesterone levels in women on tamoxifen. In contrast with some of the other studies, elevated FSH appeared to occur after tamoxifen exposure. Women who had no menses while on tamoxifen had low estrogen levels (11). Taken together, tamoxifen treatment of postmenopausal women consistently causes decreased gonadotropins. On the other hand, premenopausal women respond to tamoxifen with increased estrogen and unchanged or slightly increased gonadotropins. It appears that tamoxifen has a weak estrogenlike effect on the pituitary. This results in a negative feedback response in a low-estrogen environment (postmenopausal women) and decreased, though still postmenopausal gonadotropin levels. In women with normal premenopausal estrogen levels, however, tamoxifen competes with estrogen at the level of the hypothalamus or pituitary and interrupts the negative feedback loop. Elevated estrogen, in response to tamoxifen therapy, can adversely affect the treatment of breast cancer. GnRH agonist therapy or ovarian ablation have been considered as clinical maneuvers to maintain low estrogen levels. Increasing the tamoxifen dose to compete with estrogen at the level of the breast is also an alternative, but results in supraphysiologic circulating estradiol. It appears that patients with low estrogen levels have a better overall response to tamoxifen, though Sawka et al. failed to demonstrate a different response with tamoxifen or ovarian abalation. Clomiphene was the ﬁrst SERM that was used in clinical practice for ovulation induction. Clomiphene-induced neuroendocrine changes were studied by numerous authors (14–19). Pildes treated 36 anovulatory women with clomiphene and followed their urinary FSH and estrogen output. He found an increase of FSH output in 50% of women. He also reported an increase in urinary estrogen output (16). Vaitukaitis et al. treated healthy, regularly menstruating women with either estrogen or estrogen plus clomiphene early in their cycle and followed plasma levels of gonadotropins. They compared changes to those observed in spontaneous menstrual cycles. Estradiol treatment suppressed the early follicular rise of FSH, whereas the addition of clomiphene raised FSH levels to control values. Estradiol suppressed the normal follicular rise of LH, while clomiphene exaggerated it; similar levels were reached however by both groups of women in the late follicular phase. Progesterone levels were not affected by clomiphene (17). Ravid et al. compared gonadotropin and estrogen levels in oophorectomized women treated with estrogen or clomiphene and compared them to premenopausal women treated with clomiphene and postmenopausal women not on any hormone therapy. Basal estrogen was lower in postmenopausal and oophorectomized patients, with the lowest levels among castrated. In response to clomiphene, FSH and LH levels rose in the normally cycling women. In contrast, FSH and LH were signiﬁcantly suppressed in the ovariectomized group. Estrogen similarly suppressed gonadotropins in postmenopausal women (18). Adashi reviewed the site and mechanism of action of clomiphene. In premenopausal women in response to clomiphene an initial rise in gonadotropin levels can be observed. Following the rise in gonadotropins, a rise in

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estradiol level can be seen. By its competitive binding to estradiol receptors clomiphene displaces estrogen from the binding sites on the hypothalamus and prevents its negative feedback effect. This results in an increased frequency of pulsatile GnRH release. Clomiphene also increases the sensitivity of the pituitary to GnRH, similarly to estrogen, and results in increased gonadotropin release. The net result of these effects is a greater estradiol production and occasional multiple folliculogenesis. Gonadotropins are not necessarily suppressed by the higher circulating estradiol, because clomiphene prevents estrogen negative feedback. The complex neuroendocrine actions of clomiphene result in elevated levels of gonadotropins in the follicular phase of the cycle, followed by increased estrogen levels. Clomiphene can induce ovulation in close to 80% of anovulatory women. In postmenopausal women, with low circulating estrogen levels, clomiphene acts as an estrogen agonist and decreases gonadotropin levels but, because the ovary is no longer responsive, there is no effect on peripheral estrogen levels (19). Baker et al. evaluated raloxifene’s effect on the reproductive neuroendocrine system. Four hundred mg of raloxifene was given for ﬁve days to three groups of healthy, regularly cycling women, who took raloxifene at three different times during the menstrual cycle (follicular, periovulatory, luteal). Gonadotropin and estrogen levels were measured and were compared with pretreatment normal cycles. The general pattern of gonadotropin secretion was not altered by raloxifene. All women had spontaneous LH surges. Hormone results were reported as area under the curve (AUC). LH AUC for the whole cycle was not affected by raloxifene. When raloxifene was administered in the periovulatory period it did decrease LH AUC during the treatment interval. FSH AUC for the whole cycle was increased by raloxifene. Raloxifene also increased FSH AUC in any treatment phase. When raloxifene was administered during the follicular phase, it increased the estradiol AUC for the whole cycle and for the follicular phase as well. Raloxifene administered later in the cycle was not associated with increased estradiol levels. When raloxifene was given continuously for four weeks it increased FSH and estradiol levels in the ﬁrst half of the cycle. Raloxifene seems to have hypothalamic/pituitary properties similar to tamoxifen and clomiphene. The elevated gonadotropins are maintained despite increased estrogen levels (20).

SERMS: EFFECTS ON THE OVARY Tamoxifen’s effects on the ovary have also been evaluated. There are several case reports of ovarian cysts among tamoxifen-treated women. Schwartz et al. followed 17 postmenopausal women on tamoxifen and evaluated them with serial ultrasound examinations for uterine and ovarian changes. They had three patients with simple ovarian cysts at the initial examination. Two of these cysts remained unchanged while on tamoxifen and the third regressed. Two of the 17 women developed new simple ovarian cysts. All patients remained asymptomatic (21). Hochner-Celnikier reported newly developed ovarian cysts in ﬁve premenopausal women on tamoxifen. Those women with larger ovarian cysts had signiﬁcantly higher estradiol levels as well. Some cysts appeared as complex adnexal masses at the time of ultrasound examination. Two patients with larger ovarian cysts had surgical removal of the adnexa. The pathologic examination revealed corpus luteum cysts. This is consistent with the stimulatory effect of tamoxifen on the hypothalamic/pituitary/ovarian axis (22). Mourits et al. followed 142 breast cancer patients with serial ultrasounds. The study group included pre- and

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postmenopausal women. Some women were treated with chemotherapy followed by tamoxifen, some only received tamoxifen. Ovarian cysts were found in 25 women. Twenty-four of these women were taking tamoxifen. All cysts had benign characteristics on ultrasound. One-third of these cysts increased in size, one-third decreased and onethird did not change during followup. No patients had symptoms associated with these cysts. An interesting ﬁnding of the study is that all cysts appeared in women who had their last menstrual period within a year. No cysts developed in postmenopausal women with more than one year past their last menstrual period. Patients who were diagnosed with cysts had higher serum estradiol levels. These ﬁndings suggest that pre- and perimenopausal women undergoing tamoxifen therapy are more likely to develop functional ovarian cysts (23). Shushan et al. also followed women with breast cancer who were undergoing tamoxifen therapy and evaluated the incidence of abnormal ovarian ﬁndings on ultrasound examinations. Out of the 95 participants, 79 were postmenopausal, 16 premenopausal. They detected 11 ovarian cysts, ﬁve in postmenopausal women (6.3%) and six in premenopausal (37.5%). Nine of the 11 cysts had a benign appearance on ultrasound. Eight of the 11 cysts disappeared once tamoxifen was discontinued. Two patients had surgical removal of the adnexa secondary to persistent ovarian cysts. This study, unlike Mourits, reported cystic changes in postmenopausal ovaries as well. This presents a diagnostic dilemma for the physician, since sonographic ﬁndings might lead to an inappropriate intervention. On the other hand, metastatic disease from breast or GI cancer or even primary ovarian cancer are included in the differential diagnosis of the ultrasonographic ﬁndings reported herein (24).

TAMOXIPHEN AND OVARIAN CANCER RISK Several studies have tried to evaluate whether tamoxifen therapy puts patients at increased risk for ovarian cancer (25–27). Cohen et al. followed 175 postmenopausal breast cancer patients on tamoxifen and evaluated the ovarian pathology of the 16 women who required surgical removal of their adnexa for various indications. Ten of 16 women had ovarian pathology (one adenocarcinoma, three cases of breast cancer metastasis, six benign ﬁndings) (25). Cook et al. followed a cohort of breast cancer patients and evaluated them for the occurrence of second primary cancers. Thirtynine women developed primary ovarian cancer during the followup period, but no signiﬁcantly increased risk was shown with tamoxifen use (OR: 0.6 [95%CI: 0.2–1.8]) (26). McGonigle et al. evaluated pathologic specimens from 152 breast cancer patients who underwent surgical removal of their ovaries. At the time of surgery 99 women were taking tamoxifen. More than two-thirds of these women were postmenopausal at the time of oophorectomy. They found no difference in the occurrence of functional ovarian cysts or endometriomas. No tamoxifen-treated woman had ovarian cancer (27). It appears that women undergoing tamoxifen therapy are at a somewhat increased risk of developing ovarian pathology. Most of these ﬁndings are simple, functional ovarian cysts found incidentally. Cysts were more frequent among premenopausal women. Based on the above mentioned reports and the reports of the large tamoxifen trials (28,29), there is no evidence for an increased incidence of ovarian cancer among tamoxifen-treated women. Sonographic ﬁndings, however, can pose a diagnostic dilemma and the possibility of a malignancy (breast cancer metastasis or primary ovarian cancer) should not be dismissed without careful evaluation and followup.

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CLOMIPHENE AND OVARIAN CANCER RISK Studies looking at an association between clomiphene exposure and ovarian cancer lead to considerably greater controversy. In 1992, Whittemore evaluated reproductive factors and the risk of ovarian cancer. This metanalysis was based on 12 case-control studies, all of which used hospital or population controls. An increased risk of ovarian cancer was observed among infertile women who had used fertility drugs, when compared to fertile women (OR: 2.8 [95%CI: 1.3–6.1]). Nulligravidae who reported use of fertility drugs had an odds ratio of 27 (95%CI: 2.3–315.6) compared to nulligravid women who had no history of infertility and had not taken fertility drugs. The speciﬁc fertility medications could not be identiﬁed. The comparison of fertile women with infertile women may not be scientiﬁcally justiﬁed. Women who have no difﬁculty getting pregnant enjoy the protective beneﬁts of pregnancy and breast-feeding. They are more likely to be exposed to oral contraceptives, which will further decrease their ovarian cancer risk. Prolonged infertility and unprotected intercourse have long been found to be associated with increased ovarian cancer risk. The meaning of these ﬁndings remains unclear with respect to the counseling of infertile women who are contemplating the elective use of fertility drugs (30,31). Rossing et al. evaluated the risk of ovarian cancer in a cohort of infertile women exposed to clomiphene. They compared results to the general population of Washington State and compared subgroups within the cohort as well. An increased risk of ovarian tumors was observed among infertile women, regardless of drug exposure (RR: 2.5[95%CI: 1.3–4.5]). This ﬁnding is consistent with previous literature. Women with ovulatory problems appeared to be at exceptional risk. Among infertile women, those who used clomiphene had an increased risk of ovarian tumors. Clomiphene use for more than 12 cycles was associated with a relative risk of 11.1 (95%CI: 1.5–82.3). Women exposed to clomiphene were more likely to have infertility caused by ovulatory abnormalities, which in itself is a risk factor for ovarian cancer. The limited number of cases (n = 11) resulted in wide conﬁdence intervals (32). These reports and others raise but do not answer the issue of causality between clomiphene use and ovarian cancer. To date there is inadequate epidemiological and scientiﬁc support to establish causality. This relationship is particularly difﬁcult to study since ovarian cancer is a relatively rare disease and is very rare among women of reproductive age. Patient counseling is also particularly difﬁcult when discussing this issue. If fertility medications were to increase the risk of ovarian cancer, the possibility of pregnancy mitigating this risk is a motivating factor to many patients. Women beneﬁt from the cancer-reducing effects of pregnancy and breast feeding whether or not they have had antecedent infertility. It is advisable to discuss this possible association between clomiphene use and ovarian cancer with patients prior to prescribing it.

ENDOMETRIAL EFFECTS OF SERMS Estrogen has a proliferative effect on the endometrium. Excessive endogenous or exogenous estrogen can result in pathologic endometrial changes such as hyperplasia, polyps, or cancer. Several risk factors for endometrial cancer have been identiﬁed. Women with estrogen-secreting tumors (granulosa cell tumor), or liver disease (increased bioavailability of estrogens) are at increased risk. Obesity, hypertension,

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and diabetes are also associated with increased risk. Women with chronic anovulation and acyclic estrogen production, usually caused by polycystic ovary syndrome (PCOS) are also at risk for endometrial hyperplasia and carcinoma (33). Annually there are about 40,000 new endometrial cancer cases and 3,000 associated deaths (34). Endometrial cancer incidence was found to be increased in the 60s and early 70s. By the mid 70s the ﬁrst reports linking this increased incidence to unopposed estrogen use were seen in the medical literature. Since then, several case-control and cohort studies have addressed the issue of estrogen use and endometrial cancer (35–37). A four-to-sevenfold increase in risk of endometrial cancer has been reported with unopposed estrogen use, in a series of case-control studies (35–37). This increased risk was higher for early stage disease than for more advanced cancer (37), was increased with duration of use (35), and appeared to persist after cessation of estrogen (36). Progesterone is known to have antimitotic effects on the endometrium and has been added to hormone replacement therapy to counteract estrogen’s proliferative effect. Numerous studies have investigated the effect of combined hormone replacement therapy on the incidence of endometrial cancer (38–41). Persson et al. followed a large cohort of women on estrogen or on estrogen plus progestin and compared their risk of developing endometrial cancer with the expected population background risk. They showed that any hormone use was associated with an increased relative risk of 1.3 (95%CI: 1.0–1.7). Women who only took estrogen had a higher risk of 1.4 (95%CI: 1.1–1.9) and this risk increased with duration of use. Women on combined therapy were not at increased risk (RR: 0.9 [95%CI: 0.4–2.0]) (38). Voigt et al. also showed in their case-control study that women who took progesterone with estrogen were not at increased risk for developing endometrial cancer (RR: 1.3 [95%CI: 0.6–2.8]). More than 10 days of progesterone use was more protective than shorter progesterone exposure (RR: 0.9 [95%CI: 0.3–2.4] vs. 2.0 [95%CI: 0.7–5.3]) (39), a ﬁnding that has been conﬁrmed by others (40,41). A 1995 metanalysis of observational studies revealed an increased relative risk of 2.3 (95%CI: 2.1–2.5) with unopposed estrogen. This risk increased to 9.5 (95%CI: 7.4–12.3) with more than 10 years of use. Women were still at increased risk ﬁve years after discontinuation of estrogen use. The risk was greater for early-stage disease than for advanced disease. The risk among women who took estrogen with progestin was 0.8 (95%CI: 0.6–1.2) (42). The FDA approved tamoxifen in 1978 for the adjuvant treatment of breast cancer. The ﬁrst reports showing an increased risk of various endometrial pathologies were published in the mid 80s. These early reports were followed by several case-control studies evaluating the endometrial effects of tamoxifen. The large tamoxifen trials also reported their ﬁndings (28,29,43–46). Leeuwen et al. in a case-control study, observed a 1.3 (95%CI: 0.7–2.4) times increased relative risk for endometrial cancer among tamoxifen users; their ﬁndings however did not reach statistical signiﬁcance. The risk seemed to increase with duration of use (43). Bernstein et al. reported similar ﬁndings (OR: 1.52 [95%CI: 1.07–2.17]) (44). The Stockholm Breast Cancer Study Group found a sixfold increased risk for endometrial cancer with tamoxifen use. When the Scandinavian studies were analyzed together, an endometrial cancer relative risk of 4.1 (95%CI: 1.9–8.9) was observed among tamoxifen users (29). The Early Breast Cancer Trialists’ Collaborative Group in 1998 reported a signiﬁcantly increased risk (ratio of incidence rates: 2.58) of endometrial cancer among tamoxifen users based on 55 trials. They observed an excess of 33 endometrial cancers during ﬁve years of

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treatment in these trials (28). The National Surgical Adjunct Breast and Bowel Project [NSABP] B-14 trial revealed a 7.5-fold increased risk of endometrial cancer among tamoxifen users (0.2/1000 in the placebo group vs. 1.6/1000 in the tamoxifen group). Twenty-one of 24 cancers were FIGO stage I (46). In 1996, Daniel reported an increased risk of endometrial cancer, hyperplasia, and polyps among women on tamoxifen for breast cancer based on ﬁndings in case-control studies. The range of increased relative risk for endometrial cancer was 1.3–7.5 (47). In 1995, after reviewing the literature, Jordan et al. found 209 cases of endometrial cancer associated with tamoxifen use. They reported an overall modest increased incidence of endometrial cancer among tamoxifen users (2/1000 women/year vs 1/1000 women/year). They found a similar risk for endometrial cancer with short- and long-term tamoxifen treatment. Most (80%) of these cancers had a favorable histologic grade (grade 1 or 2) (48). It appears that tamoxifen has proliferative effects on the endometrium. Elkas et al. investigated tamoxifen’s effect on the expression of steroid receptors and several growth factors in the endometrium and compared it to that of estrogen’s. They showed that among breast cancer patients receiving tamoxifen, tamoxifen increased the expression of estrogen and progesterone receptors, and several growth factors to levels comparable to those seen in the proliferative endometrium in a normal menstrual cycle. Despite the increase in these growth factors and steroid receptors, tamoxifen did not lead to the same proliferative changes. They speculate that the proliferative process might be uncoupled from the presence of the necessary growth factors (49). Other endometrial pathologies are also found more frequently among women on tamoxifen. Simple, complex, atypical endometrial hyperplasia and endometrial polyps have been reported more frequently. Polyps were found in up to 30% of women treated with tamoxifen (50–53). Several endometrial monitoring techniques (ultrasound, endometrial biopsy, hysteroscopy) have been evaluated and compared. On ultrasound, the endometrium was found to be thicker and the uterus had a larger volume among tamoxifen users (54,55). Most studies used 5 mm as their cutoff point between thin and thick endometrium, although some used 6 mm. When women with thick endometria underwent endometrial sampling only about half of them had endometrial pathologies and very few were diagnosed with cancer. Several ultrasound studies demonstrate an unacceptably high false positive rate with the use of thickened endometrium as a screening tool (55,54). Briley et al. found that when women with postmenopausal bleeding and a thin endometrium on ultrasound were evaluated, 84/87 had no pathologies (56). This gives a 95% negative predictive value for a thin endometrium in a woman with postmenopausal bleeding, similar to what has been reported in women using HRT (57). Unfortunately, thick endometrium had a very poor positive predictive value (56). Cohen et al. evaluated symptomatic and asymptomatic breast cancer patients on tamoxifen. They found that women with vaginal bleeding had thicker endometria and had higher incidence of pathology but they did not report positive or negative predictive values (52). Timmerman et al. compared ultrasound to hysteroscopic monitoring of asymptomatic postmenopausal women treated with tamoxifen. They found ultrasound superior to hysteroscopy in identifying pathology when an endometrial thickness of 4 mm was used as a cutoff. Ultrasound however had a higher false positive rate (58). The poor positive predictive value of these screening techniques has led to a lack of consensus on routine screening of asymptomatic women on tamoxifen. Since the

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most common presenting symptom of endometrial cancer is vaginal bleeding (90% present with bleeding) and most cancers developing on tamoxifen have a favorable prognosis, women who present with bleeding or discharge should always be aggressively evaluated for endometrial pathology (59,60). In recent years another SERM, raloxifene, has been in the focus of investigation. Raloxifene has been shown to have favorable effects on the lipid proﬁle and, similarly to tamoxifen, it reduces breast cancer incidence. Several trials evaluated raloxifene’s effect on the endometrium. Boss et al. randomly assigned healthy postmenopausal women to placebo, 200 or 600 mg raloxifene, or 0.625 mg unopposed conjugated estrogen. Endometrial biopsies were obtained at baseline and at the end of the eightweek trial. Biopsies were evaluated with a scoring system designed to detect estrogen’s proliferative effect on the endometrium. At the end of the eight weeks, 77% of estrogentreated women demonstrated estrogenic effects on their endometria, compared to 15% of placebo treated and none in the raloxifene groups (61). Delmas et al. studied the effects of 30, 60, or 150 mg of raloxifene vs placebo in 601 postmenopausal women. They found no difference between the proportions of patients reporting vaginal bleeding. Women assigned to raloxifene, who reported vaginal bleeding, all had endometrial thickness less than 5 mm (62). Davies et al. reported results from six trials. Four trials involved randomization to raloxifene and placebo, one randomized to raloxifene and cyclic HRT, and one randomized to raloxifene and conjugated estrogen. These trials included 1157 postmenopausal patients. All trials assessed endometrial thickness and the incidence of vaginal bleeding. In the placebo-controlled trials, there was no difference in the percentage of women reporting vaginal bleeding, whereas in the HRT- and estrogen-controlled trials signiﬁcantly more estrogen- or HRT-treated women reported vaginal bleeding. Raloxifene did not change the endometrial thickness signiﬁcantly when compared to placebo. Estrogen and HRT signiﬁcantly increased endometrial thickness when compared to raloxifene (63). Cohen et al. reported results from two similarly designed trials. In both trials, postmenopausal patients were randomized to different doses of raloxifene or placebo. Both trials evaluated endometrial thickness and the incidence of vaginal bleeding. Neither endometrial thickness nor the incidence of vaginal bleeding was different between the different treatment groups (64). Goldstein et al. randomly assigned postmenopausal women to 60 or 150 mg raloxifene, conjugated equine estrogen, or placebo. All participants had baseline endometrial biopsies. During the one-year study period, women were evaluated with vaginal ultrasound. Those with thickened endometrium (>5mm) or vaginal bleeding were further evaluated with saline ultrasound and endometrial biopsies. Women assigned to estrogen had a signiﬁcantly higher incidence of endometrial hyperplasia. No hyperplasia was found among women on raloxifene. Signiﬁcantly more endometrial biopsies showed proliferative changes among estrogentreated women than among raloxifene-treated. Endometrial thickness was not changed by raloxifene. Estrogen use was associated with a greater incidence of vaginal bleeding (65). Clomiphene has been available for the longest time among SERMs in clinical practice and has been widely used for ovulation induction since the 1960s. Several studies have evaluated clomiphene’s effect on the endometrium in pre- and postmenopausal women (14,66–71). Van Campenhout et al. looked at clomiphene’s effect on the endometrium in oophorectomized women. They showed that when these women were treated with

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both estrogen and clomiphene, clomiphene prevented the estrogen-induced endometrial proliferative changes. When clomiphene was given alone, the endometrium remained atrophic (66). Rogers et al. evaluated endometrial thickness and histology among patients undergoing in vitro fertilization cycles with gonadotropins or gonadotropins with clomiphene. They found a signiﬁcantly reduced endometrial thickness among patients who took clomiphene. Histologic evaluation revealed a signiﬁcantly reduced glandular volume in patients exposed to clomiphene (70). Eden et al. evaluated endometrial thickness and uterine volume in their study of women taking clomiphene for ovulation induction. They reported signiﬁcantly reduced endometrial thickness when compared to nonclomiphene cycles. Clomiphene did not increase uterine volume (14). Dickey et al. also reported thinner endometrium on ultrasound examination in women taking clomiphene as part of their infertility medications (68). Lunan et al. reviewed clomiphene’s endometrial effects in women with pathologic conditions of the endometrium. They report data from studies that showed reversal of hyperplastic endometrium to atrophic endometrium (71). Kokko et al. studied clomiphene’s effect on endometrial cytosolic estrogen and progesterone receptors in postmenopausal women after estrogen plus clomiphene adminstration. The addition of clomiphene to estrogen resulted in signiﬁcantly more cases of endometrial atrophy. Clomiphene also lowered the cytosolic estrogen and progesterone content (69). Clomiphene appears to exert its antiestrogenic effect on the endometrium by decreasing the number of available estrogen receptors and by competing with estrogen for the remaining ones. Estrogen and tamoxifen increase the risk of endometrial pathologies including hyperplasia, polyp, and cancer. Progestins, if given for at least 10 days per month, reduce estrogen’s effect on the endometrium. Whether progestin has the same salutary effect on a woman treated with tamoxifen is unclear. Raloxifene and clomiphene seem to have no stimulatory effect on the endometrium. There is no reliable screening approach to detect clinically silent hyperplasia or carcinoma in women on HRT or tamoxifen. Patients on these treatments need to be encouraged to report any vaginal bleeding and unusual discharge, and if these occur, prompt evaluation with ultrasound, with or without saline enhancement, and/or tissue sampling should be performed.

LEIOMYOMAS AND SERMS Much less attention has been given to uterine leiomyomas in association with tamoxifen or other SERM therapy in the medical literature. Leiomyomas (ﬁbroids) are common uterine tumors, occurring in up to 50% of women (72). Fibroids are known to grow in an estrogenic environment and tend to shrink when estrogen levels are low (GnRH agonist treatment or menopause). Since tamoxifen appears to exert estrogen agonist properties on the endometrium, it might be expected to also function as a mixed agonist in the myometrium. Fibroid growth has been observed in both premenopausal and postmenopausal women while on tamoxifen, but results are not entirely consistent. Some patients have even required exploratory laparotomy to establish the diagnosis of rapidly growing pelvic masses (73–75). Two studies followed patients prospectively. Lumsden et al. followed six healthy premenopausal women who had ﬁbroids. These women were placed on tamoxifen and ﬁbroid size was assessed before treatment and after three months of tamoxifen use. They did not show any signiﬁcant change in ﬁbroid volume with tamoxifen therapy (1). On the other hand, Schwartz et al. followed

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17 postmenopausal patients who were treated with tamoxifen for breast cancer. Thirteen of the 17 women had at least one ﬁbroid at the initial sonographic evaluation. Followup ultrasound examinations showed a signiﬁcant increase in leiomyoma volume. The overall mean increase of leiomyoma volume was 68.3 ± 23%. Six women developed new leiomyomas (21). The limited number of reported cases to date (twenty-seven) and lack of prospective studies with large numbers of participants preclude a deﬁnitive statement about tamoxifen use and growth of leiomyomas. There is even less information available regarding clomiphene’s and raloxifene’s effect on uterine leiomyomas. Since clomiphene use in premenopausal women had been associated with a relative thinning of the endometrial lining, it might be predicted to be neutral or inhibitory to leiomyoma growth. In premenopausal women given clomiphene, an increased uterine volume was not observed (14). A case report of a premenopausal woman who had growth of a uterine ﬁbroid in conjunction with clomiphene therapy indicates a possible stimulatory effect on the myometrium, but is confounded by the probable elevated endogenous estradiol that would be expected after clomiphene administration (77). No human studies have evaluted raloxifene’s effect on ﬁbroid growth. Based on animal data, its properties as an endometrial antagonist of estradiol appear to be consistent with its myometrial effects. Porter et al. treated ovariectomized guinea pigs with estrogen to induce myoma growth. After discontinuation of therapy these ﬁbroids shrank. When estrogen was given again, the myomas grew back. If estrogen was given with raloxifene together these leiomyomas regressed rapidly (78). Fuchs-Young treated rat leiomyoma cell lines with estrogen and estrogen plus raloxifene. The estrogeninduced proliferative changes were reversed by raloxifene, consistent with an antiestrogen effect (79).

GENITOURINARY EFFECTS OF SERMS Vaginal cytology is a sensitive, speciﬁc, and inexpensive method for assessing estrogen action at the tissue level. There are several indices of hormone effect on the vaginal epithelium. The two most commonly used indices are the karyopyknotic index and the maturation index. The karyopyknotic index expresses the percentile relationship between superﬁcial squamous cells to all mature squamous cells. In periovulatory women, this may reach 50 to 85%. The index decreases in low-estrogen states. The maturation index expresses the percentile relationship between parabasal, intermediate, and superﬁcial cells. In a menstruating woman around the time of ovulation it is usually 0:35:65, meaning that 65% of cells are superﬁcial cells. In estrogen-deﬁciency states, the percent of parabasal cells increases and can account for more than 90% of cells (80). Based on its estrogen-agonist properties in the endo- and myometrium, tamoxifen would be expected to prevent genitourinary atrophy. The changes of the karyopyknotic index and maturation index were evaluated in postmenopausal women undergoing tamoxifen therapy (2,81–85). Friedrich et al. compared the maturation index in postmenopausal women with breast cancer who were undergoing tamoxifen therapy to postmenopausal women with breast cancer not on tamoxifen and to healthy postmenopausal women not receiving any hormone therapy. They showed that the maturation index increased in women taking tamoxifen and was signiﬁcantly higher than in the control groups (81). In another similarly designed study, Friedrich et al. reported

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similar changes with tamoxifen therapy (84). Bertolissi et al. prospectively studied the karyopyknotic and maturation index in 64 postmenopausal breast cancer patients. They showed a 13.5-fold increase in the karyopyknotic index within the ﬁrst month of tamoxifen treatment. Twenty-three percent of women had no changes in their karyopyknotic indexes. Tamoxifen treatment signiﬁcantly increased the percent of superﬁcial epithelial cells whereas the percent of parabasal cells decreased (83). Boccardo et al. compared the karyopyknotic indices of postmenopausal breast cancer patients on tamoxifen to similar patients not receiving tamoxifen. The karyopyknotic index in women on tamoxifen was higher than in women not exposed to tamoxifen (8% vs 2%). When women who previously had not been on tamoxifen were placed on it the karyopycnotic index rose signiﬁcantly (82). Lahti et al. compared the maturation index in postmenopausal breast cancer patients receiving tamoxifen to similar women who were not exposed to tamoxifen. They showed a signiﬁcantly lower percentage of parabasal cells in women receiving tamoxifen (8.1% vs 39.1%) and increased percentage of intermediate and superﬁcial cells (82.4% vs 55.3% and 14.4% vs 4.6%). All differences were statistically signiﬁcant (2). These reports show a remarkably consistent estrogen-agonist effect of tamoxifen on the vaginal epithelium. Mortimer et al., evaluated how tamoxifen induced changes of the vaginal mucosa affect the sexual functioning of women with breast cancer undergoing tamoxifen therapy. Forty-one of the 57 participants were sexually active. They were evaluated with vaginal smears to assess the maturation index and were asked to complete a questionnaire about sexual functioning. Somewhat surprisingly, tamoxifen-treated women reported a relatively high incidence of dyspareunia (54%) (85). Clomiphene’s effect on vaginal epithelium was assessed by several studies (15,66,86). Van Campenhout et al. studied the effects of estrogen and estrogen plus clomiphene in four women after oophorectomy. They showed a signiﬁcant increase in superﬁcial cells after estrogen treatment (50%). This increase was counteracted by clomiphene, decreasing the percentage of superﬁcial cells to less than 15%. When clomiphene was given alone it had no effect on the vaginal mucosa (66). Ruiz-Velasco et al. studied changes in vaginal cytology in 149 clomiphene-treatment cycles in premenopausal women undergoing ovulation induction. Clomiphene decreased the number of superﬁcial and intermediate cells and increased the percentage of parabasal cells, expressed by a decrease of the maturation value (86). Similar antiestrogenic properties of clomiphene were shown by Thompson et al., who showed the inhibition of proliferative changes induced by diethylstilbestrol on the vaginal epithelium in a woman with Turner’s syndrome. They showed no changes in the vaginal mucosa by clomiphene in postmenopausal women not on estrogen (15). Raloxifene treatment resulted in a decreased maturation index in healthy premenopausal women, consistent with its local antiestrogenic effect (20).

SUMMARY Selective estrogen-receptor modulators have been used for decades. Millions of women have used them as fertility medications or cancer treatment. Understanding their endocrine effect on all possible target organs permits the clinician to ﬁt the SERM to the patient, minimizing her side effects and maximizing the therapeutic beneﬁts. Reproductive-aged women taking tamoxifen have to understand that continued ovula-

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tory cycles are likely and pregnancy is theoretically possible while on this hormonal form of chemotherapy. The importance of the evaluation of any abnormal vaginal bleeding in women on tamoxifen should also be emphasized, as it appears to stimulate endometrial growth. Clomiphene and raloxifene, on the other hand, seem to have no stimulatory effect on the endometrium, although both stimulate the hypothalamic/ pituitary/ovarian axis in premenopausal women. To date there are not enough data to settle the debate over the causal relationship between clomiphene and ovarian cancer, but patients need to be made aware of the possible risk. Women taking SERMs who develop abnormal ovarian ﬁndings should be evaluated like any woman with sonographically abnormal ovarian ﬁndings. Patients need to be made aware that raloxifene and clomiphene cause atrophic changes in the vaginal mucosa and this could adversely affect their sexual life. Taking these effects into account, many women have beneﬁted from SERMs. There is every indication that more will continue to beneﬁt in the future, and that newer SERMs under development may present a superior risk/beneﬁt proﬁle to those already in use. Limiting use of SERMs to women only may soon be a notion of the past, as their applications in selectively treating dyslipidemia and osteoporosis may well extend beyond gender.

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65. Goldstein SR, Scheele WH, Rajagopalan SK, Wilkie JL, Walsh BW, Parsons AK. A 12-month comperative study of raloxifene, estrogen, and placebo on the postmenopausal endometrium. Obstet Gynecol 2000;95:95–103. 66. Van Campenhout J, Simard R, Leduc B. Antiestrogenic effect of clomiphene in the human being. Fertility and Sterility 1968;19:700–706. 67. Birkenfeld A, Beier HM, Schenker JG. The effect of clomiphene citrate on early embryonic development, endometrium and implantation. Human Reproduction 1986;1:387–395. 68. Dickey RP, Olar TT, Taylor SN, Curole DN, Matulich EM. Relationship of endometrial thickness and pattern to fecundity in ovulation induction cycles: Effect of clomiphene citrate alone and with human menopausal gonadotropin. Fertility and Sterility 1993;59:756–760. 69. Kokko E, Janne O, Kauppila A, Vihko R. Cyclic clomiphene citrate treatment lowers cystol estrogen and progestin receptor concentrations in the endometrium of postmenopausal women on estrogen replacement therapy. J Clin Endocrinol Metab 1981;52:345–349. 70. Rogers PAW, Polson D, Murphy CR, Hosie M, Susil B, Leoni M. Correlation of endometrial histology, morphometry, and ultrasound appearance after different stimulation protocols for in vitro fertilization. Fertility and Sterility 1991;55:583. 71. Lunan CB, Klopper A. Antiestrogens: a review. Clin Endocrinol 1975;4:551–572. 72. Mishell DR, Stenchever MA, Droegemueller W, Herbst AL. Comprehensive Gynecology 3rd Edition p. 491. 73. Ugwumadu AH, Harding K. Uterine leimyomata and endometrial proliferation in postmenopausal women treated with the anti-oestrogen tamoxifen. Eur J Obstet Gynecol Reprod Biol 1994 Apr;54(2): 153–6. 74. Leo L, Lanza A, Re A, et al. Leiomyomas in patients receiving Tamoxifen. Clin Exp Obstet Gynecol 1994;21(2):94–98. 75. Cohen I, Rosen DJD, Altaras M, Beyth Y. Tamoxifen treatment in postmenopausal breast cancer patients may be associated with ovarian overstimulation, cystic formation and ﬁbroid overgrowth. Br J Cancer 1994;69:620–621. 76. Dilts PV Jr, Hopkins MP, Chang AE, Cody RL. Rapid growth of leiomyoma in patient receiving tamoxifen. Am J Obstet Gynecol 1992;166:167–68. 77. Felmingham JE, Corcoran R. Correspondence. Br J Obstet Gynaecol 1975;82:431–432. 78. Porter KB, Tsibris JC, Porter GW, et al. Effects of raloxifene in guinea pig model for leiomyomas. Am J Obstet Gynecol 1998;179(5):1283. 79. Fuchs-Young R, Howe S, Hale L, Miles R, Walker C. Inhibition of estrogen-stimulated growth of uterine leiomyomas by selective estrogen receptor modulators. Mol Carcinog 1996;17(3):151–159. 80. Koss LG. Diagnostic cytology, 4th Edition JB Lippincott p. 300. 81. Friedrich M, Mink D, Villena-Heinsen C, Woll-Hermann A, Schmidt W. Tamoxifen and proliferation of vaginal and cervical epithelium in postmenopausal women with breast cancer. Eur J Obstet Gynecol Reprod Biol 1998;80(2):221–5. 82. Boccardo F, Bruzzi P, Rubagotti A, Nicolo G, Rosso R. Estrogen-like action of tamoxifen on vaginal epithelium in breast cancer patients. Oncology 1981;38:281–85. 83. Bertolissi A, Cartei G, Turrin D, Cioshi B, Rizzi V. Behaviour of vaginal epithelial maturation and sex hormone binding globulin in post-menopausal breast cancer patients during the ﬁrst year of tamoxifen therapy. Cytopathology 1998;9(4):263–70. 84. Friedrich M, Mink D, Villena-Heinsen C, Woll-Herman A, Wagner S, Schmidt W. The inﬂuence of tamoxifen on the maturation index of vaginal epithelium. Clin Exp Obstet Gynecol 1998;25(4):121–4. 85. Mortimer JE, Boucher L, Baty J, Knapp DL, Ryan E, Rowland JH. Effect of tamoxifen on sexual functioning in patients with breast cancer. J Clin Oncol 1999;17:1488–1492. 86. Ruiz-Velasco V, Bailon Uriza R, Conde BI, Salas E. Changes during clomiphene citrate therapy. Fertility and Sterility 1969;20:829–839.

11

Epidemiology of Cardiovascular Disease in Women Role of Estrogens

Jacques E. Rossouw,

MD

Contents Introduction Estrogen and Coronary Heart Disease Estrogen and Stroke Venous Thromboembolism Conclusions References

INTRODUCTION Cardiovascular disease is the major cause of mortality in adult women, accounting for a half-million deaths and 43% of all deaths in American women in 1997 (1). In the United States, more women than men now die from cardiovascular disease. Almost as many women as men die from coronary heart disease (CHD), more women than men die from stroke, and more die from pulmonary embolism. The average onset of CHD is later in women, and the incidence rate by age never catches up with that of men (Fig. 1). Because older women outnumber men, however, the overall mortality number for CHD is similar for the two sexes, and after age 75 numbers of deaths in women exceed those in men. Overall incidence and mortality rates and overall numbers of deaths from stroke are markedly higher in women. The incidence increases steeply with age, with no appreciable gender gap in age of onset. The incidence and mortality from venous thromboembolism (VTE) shows a similarly strong age effect. VTE is less common than either CHD or stroke, and in the postmenopausal age range may affect about 1 to 2 out of every 10,000 women annually, with much higher rates in high-risk women (2,3). Risk factors for CHD in women largely mirror those for men, but with some shifts in emphasis. Nonmodiﬁable risk factors include aging, the presence of arterial disease, and a family history of arterial disease. For every 10-year increase in age, the risk for CHD increases threefold. The presence of existing arterial disease (previous myocardial

From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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Fig. 1. Rates of mortality from coronary heart disease, stroke, and pulmonary embolism by age in women, United States, 1997 (inset: rates of mortality from coronary heart disease in men and women, by age) (1).

infarction, angina, stroke, TIA, peripheral arterial disease, or evidence of coronary or carotid artery disease by diagnostic procedure) is also a powerful risk factor, increasing the risk for CHD about ﬁvefold, while a family history of premature CHD increases the risk about twofold. Modiﬁable lifestyle factors such as smoking, inactivity, diet, body weight, and waist circumference individually are associated with a two- to ﬁvefold risk increase, and jointly these factors may increase the risk four to ﬁve times compared to women in the lowest risk categories (4). A recent report from the Nurses’ Health Study suggested that some two-thirds of the 31% decline in CHD incidence in that cohort over 16 years was a result of lifestyle factors: reductions in smoking, improved diet, and increasing use of postmenopausal hormone therapy, with some countervailing increase in risk because of an increase in the prevalence of obesity (5). Clustering of metabolically linked risk factors (body mass index, blood pressure, triglycerides, glucose, blood cholesterol, and HDL cholesterol) occurred frequently in the Framingham Offspring Study. Body mass index was an important determinant of risk factor clustering in women, and both high body mass index at baseline and increase in body mass over 16 years were associated with an increase in clustering. In women, the clustering of three or more of these factors may account for 48% of all CHD events, compared to 20% of events in men (6). Among the medically treatable risk factors, the presence of diabetes is associated with a ﬁvefold increase in risk, i.e. diabetes in women is as powerful a risk factor as the presence of existing heart disease (7). It is not clear that treatment of diabetes lowers the risk for macrovascular disease, but treatment of associated high blood cholesterol in diabetes does lower their risk for CHD (8). High blood pressure and high blood cholesterol (or LDL cholesterol) are

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Fig. 2. Excess risk for coronary heart disease attributable to lipids and lipoproteins, in men and women (A) age less than 65 years, and (B) age 65 or older (12).

each associated with a twofold increase in risk, and in clinical trials treatment lowers risk (9,10). Low HDL cholesterol is associated with a fourfold increase in risk and high triglycerides with an increase in one-third (11). In women under age 65, the relative risks associated with these lipids are very similar to those for men (Fig. 2). In older women, as in older men, the relative risks associated with lipids diminish, and the relative risk for LDL cholesterol is no longer signiﬁcant (12). This diminution of risk in older persons is almost certainly caused by confounding by comorbidity, and does not diminish the importance of lifelong lipid levels as the essential cause of atherosclerosis. Emerging risk factors include high levels of Lp(a), homocysteine, and C-reactive protein. Many of the risk factors for stroke (especially atherothrombotic stroke) are similar to those for CHD, with some additional factors and change in emphasis. The most consistent risk factors are age, presence of coronary disease or cerebrovascular disease (including TIAs), atrial ﬁbrillation, high blood pressure, smoking, and diabetes (13). Lipid disorders including high blood cholesterol are not particularly strongly associated with stroke, though interestingly, treatment with statin drugs does lower stroke risk (14). Venous thromboembolism is commonly secondary to a previous VTE, recent surgical procedures, immobilizaiton, fracture of a lower extremity, and cancer. Primary VTE is associated with age, inherited coagulation disorders, use of oral contraceptives or postmenopausal hormone therapy, smoking, obesity, and hypertension (13,15).

ESTROGEN AND CORONARY HEART DISEASE Until fairly recently, the conventional wisdom held that postmenopausal hormone therapy would reduce the risk for CHD (16–18). The conventional wisdom has been

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Table 1 Interpretation of Evidence that Postmenopausal Hormone Therapy is Cardioprotective Type of evidence Gender difference in age of onset of CHD Increase in CHD rates after menopause Observational studies suggest hormone users are at 35–50% lower risk Biological mechanisms: estrogen improves lipids, coagulation, vessel wall function

Usual interpretation

Alternative interpretation

Delayed onset in women caused by higher estrogen levels Caused by loss of cardioprotection from estrogen Because of estrogen effect

Earlier onset in men because of high androgen levels

Mechanisms mediate lower risk

The increase is caused by aging, not menopause Result of systematic bias in observational studies Estrogen effects are diverse, not possible to predict clinical outcome

shaken by the results of clinical trials, which have failed to show beneﬁt or have even produced evidence of increased risk in the ﬁrst years of treatment (19,20). HERS was a 4.1-year secondary prevention trial of conjugated equine estrogens plus medroxyprogesterone in 2763 women with an intact uterus, and its ﬁnding of increased risk in the ﬁrst year with possible subsequent beneﬁt is consistent with a cohort study of women with prior heart disease from the Nurses’ Health Study (19,21). The Estrogen Replacement and Atherosclerosis (ERA) trial was an angiographic trial of 309 women randomized to conjugated equine estrogens, conjugated equine estrogens plus medroxyprogesterone, or placebo (20). At the end of three years there was no difference in angiographic progression between the study groups. Nonetheless, the substantial epidemiologic evidence suggesting beneﬁt should not be dismissed lightly, and it remains possible that longer-term treatment will reduce risk of CHD. Of course, the apparent excess early risk needs to be explained, and methods found to avoid it, if postmenopausal hormone therapy is to regain its position as a recommended prevention treatment for CHD. The most convincing strands of evidence in humans that estrogen may prevent CHD comes from observational epidemiology and from studies of mechanism. Each type of evidence has strengths and weaknesses, and is subject to varying interpretations (Table 1).

Observational Epidemiology As noted above, women develop CHD somewhat later than men. The delay may be caused by higher endogenous estrogen levels in premenopausal women than in men, thus protecting women from premature CHD. Possibly tied into this observation is the fact that adult women have higher HDL cholesterol levels than men, which could provide an important underlying biological mechanism. The HDL cholesterol difference between women and men is not however really an estrogen effect, rather it is an androgen effect. Up to puberty, young men and women have similar HDL cholesterol

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Fig. 3. Log-linear plot of rates of mortality from coronary heart disease and cancer, showing that the increase in coronary heart disease is a function of age. There is a deﬂection downwards for the year-on-year increase in breast cancer mortality rates at about the age of menopause (1).

levels. At puberty, concurrent with the rise in endogenous testosterone levels, the HDL cholesterol levels in young men decline to the lower adult level (22). The 20% gender difference is maintained throughout adult life (23). There are two important corollaries to this observation. First, a 20% difference in HDL cholesterol levels predicts at least a 20% difference in CHD rates in the short term, and may predict an even bigger difference over a lifetime (24). Thus, the entire gender difference in CHD risk may be a result of the lifelong difference in HDL cholesterol levels. Second, the difference is caused by an androgen effect, which begs the question of whether most research to date has overemphasized estrogen and neglected androgen as possible candidates for explaining the gender difference in CHD incidence. It is often stated that CHD rates in women rise steeply after the menopausal age, and that this is again because of the relatively lower levels of estrogen after cessation of ovarian function. There is actually no evidence however, for an increase in the yearon-year incidence of CHD. This can be seen clearly when the rates are plotted on a logarithmic scale against age on a linear scale. The linear relationship seen on a semilogarithmic plot indicates that there is a constant proportional increase in CHD incidence with age, with no inﬂection upward at the average age of menopause (Fig. 3). This is strong evidence for an age effect, and evidence against an effect of menopause. In contrast to this ﬁnding for CHD, a similar plot for breast cancer does show an inﬂection downwards at about the age of menopause, consistent with the decreasing levels of the postulated estrogen risk factor. The Nurses’ Health Study investigators have reported that, after controlling for age and smoking status, the natural menopause is not associated with an increased risk for CHD (25). In addition to the lack of upward inﬂection for CHD rates, recent studies following women through the menopausal

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Fig. 4. Relative risk (and 95% conﬁdence intervals) for CHD incidence comparing current estrogen users with never-users, by dose and duration of estrogen use, in the Nurses’ Health Study (28).

transition have not indicated any marked effects on potential lipid mediators of CHD (e.g., LDL cholesterol, HDL cholesterol) (28). By far the most persuasive evidence in favor of a protective effect for estrogen comes from the large number of cohort studies comparing CHD risk in postmenopausal women currently using estrogen to never-users (27). These studies have shown consistently that CHD risk is 35–50% lower in estrogen users, after adjusting for other risk factors. There is a dose/response relationship, in that the beneﬁt is apparent at doses of 0.3 mg and 0.625 mg conjugated equine estrogens, but not at higher doses (Fig. 4) (28). The lower risk has been found in studies of estrogen alone, as well as in studies of estrogen used in combination with a progestin. For healthy women, the lower risk is found in those who have recently started estrogen as well as in long-term users. In women who have had a previous myocardial infarction, however, the Nurses’ Health Study investigators have reported a higher risk in the ﬁrst year of use, with lower risks after the second year (21). These ﬁndings are consistent with clinical trial ﬁndings from the secondary prevention Heart and Estrogen/progestin Replacement Study (HERS) (19). Other observational studies have suggested even greater risk reductions (36–80%) for hormone users with existing heart disease than for healthy women using hormones, but have not examined these temporal trends (reviewed in 29). For primary prevention, the observational epidemiology suggesting beneﬁt provides the rationale for clinical trials testing whether and to what degree current use of postmenopausal hormone therapy prevents a ﬁrst heart attack. Though strong and consistent, the observational epidemiology is not sufﬁcient to prove the case, because even the best studies may be subject to a variety of systematic biases that could lead to an overestimation of beneﬁt and an underestimation of harm from hormone therapy;

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hence the need for an unbiased estimate from clinical trials. These biases in observational studies have been reviewed elsewhere (27,30,31). Some, but not all, studies have shown that women who elect to use hormone therapy are healthier than those who elect not to. Health differences may be present before they commence therapy, and thus, the more favorable health outcome in hormone users may be a result of the characteristics of the women who take hormones, rather than the intrinsic effects of hormones. The differences in risk factors may be large, and in themselves could explain a large part of the lower risk in hormone users. For example, subsequent hormone users (sampled before they became menopausal) were more likely to be white, highly educated, lean, physically active, drink more alcohol, and have signiﬁcantly higher HDL cholesterol levels, as well as lower apoprotein B, blood pressure, and fasting insulin levels (32). The better observational studies adjusted for some of these variables, but could not adjust for variables that either were not measured or were not measured prior to the commencement of the hormone therapy (27,30,31). Women who use hormone therapy for a number of years are a highly select group with good compliance. The majority of women with a prescription for postmenopausal hormone therapy are no longer on that therapy by the end of the ﬁrst year, therefore those who remain on are rather special. On-treatment analyses of clinical trials using other prevention treatments (e.g., beta blockers) have demonstrated that patients who are good adherers to the study treatment (irrespective of whether they are in the active or placebo arms) have up to 60% reduced risk for death compared to poor adherers (33). This remarkable ﬁnding is a result of the characteristics of good adherers, who are likely to be more aware of their health than poor adherers, and therefore will be taking a number of health-promoting steps (either consciously or unconsciously) resulting in a lower mortality. Compliance bias is very powerful, and impossible to correct for in an observational study. The very deﬁnition of the index group of hormone users in an observational study, especially in the case of long-term current users, ensures that a very special group is selected for study. Since hormones are prescription drugs, women who use them are more likely to be in contact with the medical care system than other women. This leads to a greater likelihood that risk factors and early disease will be identiﬁed and treated, thus lowering mortality from CHD. Reports from the Nurses’ Health Study illustrate this surveillance bias phenomenon. For each of CHD and stroke, current hormone users have greater apparent risk reductions for mortality than for incidence. The risk reduction for CHD mortality was 53% compared to 40% for incidence, and for stroke mortality a risk reduction of 32% was reported, with no effect or a small increase (3%) for incidence (28,34). For breast cancer the apparent excess risk is greater for incidence than for mortality. The Nurses’ Health Study has reported a risk reduction of 24% overall for breast cancer mortality (note, however, that with prolonged treatment there was an increase in mortality risk), whereas for incidence there was an increased risk of 32–41% (34,35). The breast cancer ﬁndings may have biologic basis in that estrogen use may promote the growth of more benign types of cancer, but it remains possible that early identiﬁcation and treatment may contribute to a lower mortality. Given the substantial evidence that estrogen is a mediator in the pathogenesis of breast cancer, a lower breast cancer mortality risk in estrogen users is more likely to be caused by surveillance bias than to be a real effect. It is difﬁcult to make the case that the observed discrepancy

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between cardiovascular incidence and mortality relative risks has a biologic basis. From clinical trials of blood-pressure lowering and cholesterol lowering we know that management of these risk factors leads to parallel reductions in incidence and mortality. Therefore it seems unlikely that, if hormones reduce CHD or stroke risk, they would favor mortality disproportionately. A ﬁnal kind of bias that will give current hormone users a lower mortality than nonusers is the fact that many women who stop hormones do so because of intercurrent illness. Women who have recently stopped taking hormones have a markedly higher total mortality, as well as cause-speciﬁc mortality from cardiovascular disease and cancer (36). Thus, the women who remain on hormones are survivors with a lower mortality than women in the general population. Like compliance bias, it is very difﬁcult to correct for survivor bias in an observational study. There is no general agreement about the existence or magnitude of these biases in observational studies (37,38). On balance however it is likely that large biases of one sort or another are common, if not universal, among the observational studies of hormone users. In combination, these biases will lead to an overestimation of beneﬁt, and an underestimation of risk. Similar biases are to be expected in observational studies comparing persons who have selected a health behavior such as hormone use, vitamin supplement use, recreational physical activity, diet, or others with persons who have not made such a health decision. Any self-selected and self-maintained health behavior is likely to be accompanied by other behaviors or characteristics that will contribute to a more healthful outcome. Adjusting for baseline differences in risk factors will mitigate only one kind of bias (healthy user selection bias) but will not affect compliance, surveillance, or survivor bias. Even in the case of healthy user selection bias, there will always be uncertainty whether all factors that contribute to the health outcome have been accommodated, and it is likely that some cannot be accommodated because they were not measured or are unmeasurable. One of the more telling pieces of evidence favoring a role for estrogens would be a ﬁnding that, in contrast with a natural menopause, bilateral oophorectomy is associated with an increased risk for CHD in women who had never taken estrogen after menopause. This ﬁnding was reported from the Nurses’ Health Study; however it was based on very few cases and the increased risk was no longer signiﬁcant in the multivariate analysis (25). In the same study, the use of estrogens appeared to eliminate this increased risk but again the number of cases was too small for this ﬁnding to be conclusive. It is possible that women who have a hysterectomy with bilateral oophorectomy are at higher risk because of a higher prevalence of metabolic risk factors such as central obesity, high blood pressure, lipid disorders, and glucose intolerance in women requiring hysterectomy with bilateral oophorectomy. These factors were not adequately examined in the Nurses’ Health Study, which relies on self-reported data. The ﬁnding of an apparent lower risk in women who subsequently ever used estrogen would be subject to the same biases already discussed. In a more recent analysis, the Nurses’ Health Study investigators reported that a younger age at natural menopause (possibly indicating a longer period of estrogen deprivation) was associated with increased risk in smokers, but not in nonsmokers, and concluded that the association of CHD with age at menopause might be caused by residual confounding by smoking (39). The size of the residual bias in studies comparing current hormone users with

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Fig. 5. Comparison of relative risk (hormone users versus nonusers) for total cancers with relative risk for cardiovascular disease across 11 studies (*plus mortality data from Nurses’ Health Study) (34,40).

nonusers may be estimated by using the cancer relative risk as a measure of internal validity. There is no biologic reason why hormone users should have a lower risk of cancer mortality than nonusers. In fact, hormone users can be expected to have a higher risk of at least two cancers; breast cancer and endometrial cancer. Therefore, if an observational study ﬁnds a lower risk for all cancer mortality, this relative risk can be used as an indicator of the degree of residual bias. For example, in the Nurses’ Health Study the relative risk for all cancer death was 0.71 in hormone users compared to never-users. The risk reduction of 29% is unlikely to be real, and is better used as a minimum estimate of residual bias. Similarly, at least part of the apparent risk reduction of 53% for CHD mortality is unlikely to be real, and if we assume that the residual bias is similar to cancer and CHD, the CHD estimate will need to be lowered to 53−29 = 24% to yield a more realistic estimate of risk reduction. Even this may be an overestimate, because the healthy user and compliance biases are likely to be greater for CHD than for the less preventable cancer diagnoses. Across observational studies, the relative risks estimates for cancer and cardiovascular disease are correlated, so that those studies with the lowest relative risks for cancer also have the lowest relative risks for cardiovascular disease (Fig. 5) (40). On average, the risk reduction for cancer was approx 20% and that for cardiovascular disease was approx 40%, so that after allowing for the residual bias the risk reduction for cardiovascular disease may be 20% or less. The real risk reduction may be even less, since to date the clinical trials have failed to show overall beneﬁt for CHD (though longer-term trials are ongoing to ascertain the long-term effects particularly in women without prevalent CHD (41,42).

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Table 2 Potentially Protective/Adverse Effects of Hormone Therapy on the Cardiovascular System Potentially protective effects

Potentially adverse effects

Low-density lipoprotein cholesterol levels decrease High-density lipoprotein cholesterol levels increasea Lipoprotein(a) levels decrease Fibrinogen levels decreasea Plasminogen activator inhibitor-1 levels decrease Plasminogen levels increase Fibrin d-dimer fragment levels increase E-selectin levels decrease Endothelial function improves a

Triglyceride levels increasea Small, dense lipoprotein particles increase Factor VII levels increasea Prothrombin fragment 1 + 2 levels increase Tissue-type plasminogen activator levels decrease Activated protein C resistance increases Protein C, S levels decrease Antithrombin III levels decreasea C-reactive protein levels increase

Estrogen effect partially antagonized by progestins

Studies of Mechanisms The possibility that estrogen may reduce CHD risk has stimulated a wide variety of studies that attempt to explain the presumed beneﬁt (43). Because it was unexpected, fewer studies have been done to explain the apparent excess risk early in the course of treatment. Multiple mechanisms that may contribute to beneﬁt have been described, and a few of the more consistent mechanisms are shown in Table 2. These include: lipid effects such as lowering LDL cholesterol and Lp(a) and increasing HDL cholesterol levels, reduced ﬁbrinogen levels and enhanced ﬁbrinolysis (reduced PAI-1 and increased D-dimer levels), reduced homocysteine levels, antioxidant properties, and improved endothelial function (reduced E-selectin levels and improved ﬂow-mediated dilatation). On the other hand, several mechanisms that might increase risk have also been found: triglycerides increase, some coagulation markers increase (e.g., Factor VII, prothrombin fragments 1+2, activated protein C resistance), and the inﬂammatory marker C-reactive protein increases. Particularly in respect of coagulation and inﬂammation, it really is not possible to say whether the predominant effect will be favorable or unfavorable. Some randomized trials have concluded that the predominant effect on coagulation is proﬁbrinolytic, while others have concluded that on balance the effect is pro-coagulant (44,45). Compounding this difﬁculty is the fact that progestins may counteract some of the estrogen effects. An attractive hypothesis is that the early excess risk for arterial disease observed in HERS is caused by a procoagulant or inﬂammatory effect on susceptible plaques, while the favorable effects in the survivors may be a result of the later assertion of the generally favorable lipid effects. In HERS, lowering of Lp(a) in the active hormone therapy group appeared to be beneﬁcial. In a randomized subgroup analysis, the active hormone group with the highest Lp(a) levels at baseline was less likely to suffer an early event related to active hormones compared to placebo, and more likely to show beneﬁt in later years (46). The group with lower Lp(a) levels at baseline stood to gain less from lowering of Lp(a), and was more likely to have an early event on

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active hormone therapy compared to placebo. The role of direct vascular effects is unclear, because impaired endothelial function has not yet been established as a risk factor for CHD. Interestingly, current estrogen users do not appear to have a lower risk for angina, as one would expect if direct vascular effects were important (28). In a study of women in the ﬁrst year after acute myocardial infarction (MI), new hormone users had a higher incidence of unstable angina and a reduced incidence of death or MI (47). Overall, the studies of mechanisms have not resolved the core issue of whether estrogens protect against CHD. It is important to realize that almost all the studies of mechanism were done using oral estrogen preparations, and thus may have little relevance in explaining the gender difference in CHD. Ovarian estrogen directly enters the systemic circulation, unlike oral estrogens which undergo ﬁrst-pass hepatic circulation, and which need to be given at ten times the dose of nonoral estrogen in order to achieve similar blood levels. These doses of estrogens cause changes in the hepatic metabolism of a variety of proteins, including lipid apoproteins, coagulation proteins, and (probably) C-reactive protein (43,48–51). The large effects on lipids and coagulation proteins described for oral estrogens are greatly attenuated, absent, or in the opposite direction with transdermal estrogens (52–57). Transdermal estrogens have very modest effects on lowering LDL cholesterol and Lp(a), lower rather than raise triglycerides, have no effect on HDL cholesterol, and have a modest or no effect on coagulation protein levels. Transdermal estrogens retain the ability to improve endothelial function (58). Additional mechanistic studies as well as epidemiologic studies and clinical trials that focus on the role of nonoral estrogen preparations are needed.

ESTROGEN AND STROKE Unlike CHD, the epidemiologic studies have not shown a consistent relationship of current estrogen use with stroke incidence. Stroke mortality tends to be lower in current users, and the lower risk was signiﬁcant in a metanalysis, but there was no such effect for stroke incidence (59). Few of the studies have examined stroke subtypes in relation to estrogen use. In the Nurses’ Health Study, atherothrombotic stroke incidence was signiﬁcantly higher in current users than in never-users, and for all strokes the incidence tended to increase with increasing dose of estrogen (28). There appeared to be no effect on intracerebral hemorrhagic stroke or subarachnoid hemorrhage (Fig. 6). The caveats in regard to epidemiologic studies of CHD and estrogen apply equally to the studies of stroke. Because lipids are relatively less prominent as risk factors for stroke, a beneﬁt for stroke would seem less likely. Indeed, if suspicions that coagulation factors are relatively important in stroke are found to be correct, it seems quite possible that estrogen may increase the risk for at least atherothrombotic stroke.

VENOUS THROMBOEMBOLISM Oral contraceptives increase the risk for VTE, and the relative risk is particularly high in women with inherited thrombophilia, e.g., with the Leiden V mutation (60). Oral contraceptives also increase the risk for CHD and stroke in middle-aged smokers. However, until recently it was thought that the lower doses of estrogens used in postmenopausal hormone replacement therapy did not increase the risk for VTE. In 1996 three observational studies were published, each of which demonstrated a two-

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Fig. 6. Relative risk (and 95% conﬁdence interval) for incidence of all stroke, ischemic stroke, and subarachnoid hemorrhage, comparing current hormone users with never-users in the Nurses’ Health Study (28).

to-fourfold increased risk for idiopathic VTE in current hormone users but not in past users (61–63). The following year, these observational studies were supported by a letter from the HERS investigators conﬁrming similar relative risk for VTE in a randomized controlled clinical trial (64). Since that time, other studies have conﬁrmed the risk for VTE, including the detailed analysis of the HERS results (3). A metanalysis of six observational studies published prior to 1998 indicated that the risk for VTE associated with current hormone use is highest in the ﬁrst few years of use but does not disappear with continued use, is present in users of estrogen alone or with progestin, oral, and transdermal preparations, and in two studies there was a dose/ response relationship with higher risk at higher doses (2). The pooled results indicate a relative risk of 2.5 (1.3–4.8) for estrogen alone, 3.1 (1.6–5.8) for estrogen plus progestin, 2.8 (1.6–4.8) for oral hormones, and 2.1 (1.0–4.7) for transdermal estrogen. The absolute excess risks in these studies was 1 to 2 per 10,000 women per year. Other studies however indicate that the excess risk may be as high as 1 per 250 women per year in higher risk women, such as those with coronary heart disease or Alzheimer’s disease (3,65). For the most part, these were studies of women taking oral conjugated equine estrogens. There is relatively little information about other forms of estrogen. Two studies from Europe where estradiol is more commonly used found an excess risk only in the ﬁrst year of administration (2,66). The data on transdermal estrogen are sparse, and the relative risk is only marginally signiﬁcant even in the metanalysis (2). As in the case of oral contraceptives, evidence is starting to emerge that the relative risk associated with hormone therapy is particularly high in women with a phenotypic

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predisposition in the form of the activated protein C resistance, low levels of antithrombin, elevated levels of Factor IX, or a genotypic predisposition in the form of one of the prothrombotic mutations (67,68). The risk associated with hormone therapy however is increased also in women without these markers, and in the majority of cases a predictive marker has not been identiﬁed. It appears that hormone therapy is generally prothrombotic, precipitating VTE events in susceptible women, some of whom have a deﬁned marker and some of whom do not. Interestingly, premenopausal women have higher rates of VTE than men of a similar age, suggesting that endogenous estrogen may also be prothrombotic (69). At this point it is unclear whether the prothrombotic state following hormone therapy is conﬁned to conjugated equine estrogens, or extends to oral estradiol after the ﬁrst year, and in particular whether it extends to nonoral preparations as well. The absence of any marked change in blood coagulation proteins after the administration of nonoral estrogens would argue against implicating them, but more information is needed.

CONCLUSIONS The major risk factors for heart disease and stroke are the same in women as in men, and tested treatment leads to the same beneﬁt. Multiple metabolic risk factors often coexist in women, and diabetes confers a particularly high risk for arterial disease. The gender difference in the onset of CHD is unexplained. Questions are being raised about earlier assumptions that high levels of premenopausal estrogens protect women, and that use of postmenopausal hormone therapy will prevent heart disease. The epidemiologic studies of hormone users are prone to large and systematic biases, which may account for most if not all of the apparent beneﬁt. Trials of oral estrogens in women with prior arterial disease have failed thus far to show beneﬁt, and there are some suggestions of early harm. These ﬁndings may be related to the new knowledge that oral estrogens are prothrombotic, as shown by the increase in VTE particularly in the ﬁrst year or two of exposure to hormone therapy. Future research needs to focus on ﬁnding explanations for the early cardiovascular events, both arterial and venous, in order that women at higher risk can be appropriately counseled. It is still not known whether longer-term administration of oral estrogens confers beneﬁt, and what the overall risk/beneﬁt proﬁle is. It is not known whether the failure to show beneﬁt in the short term is because of the particular estrogen and estrogen/progestin combination used in these trials, or will apply also to other dosages and forms of oral estrogen and progestin, and in particular whether it will apply to nonoral routes of administration. Oral estrogens cause large perturbations of lipid, coagulation, and inﬂammatory markers, some of which are not in a beneﬁcial direction. The nonoral route of delivery is analogous to that of endogenous estrogen, and if the gender difference in onset of CHD is caused by estrogen, this route of delivery is more likely to show beneﬁt. Preferred strategies for the prevention of CHD in women include smoking cessation, healthy dietary habits, weight management, exercise, control of high blood pressure and high blood cholesterol (70). For prevention of recurrent CHD there are several proven effective and safe strategies, including low-dose aspirin, beta-blockers, intensive treatment of high blood cholesterol, and ACE inhibitors. Postmenopausal hormone use should not be considered for the prevention or treatment of CHD until such time as

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(a) strategies to screen out women at high risk for early events have been devised, and (b) the long-term clinical trials have shown beneﬁt for CHD. Hormones have multiple effects on several organ systems, and the overall risk and beneﬁt depend heavily on whether a reduction in the risk for CHD is realized. Therefore, it is critically important that this issue be settled before recommending the use of postmenopausal hormones for this indication.

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46. Shlipak MG, Simon JA, Vittinghof E, et al. Estrogen and progestin, lipoprotein(a) and the risk of recurrent coronary events after the menopause. JAMA 2000;283:1845–52. 47. Alexander KP, Newby KL, Hellkamp AS, et al. Initiation of hormone replacement therapy after acute myocardial infarction is associated with more cardiac events during follow-up. J Am Coll Cardiol 2001;38:1–7. 48. Campos H, Sacks FM, Walsh BW, et al. Differential effects of estrogen on low density lipoprotein subclasses in healthy postmenopausal women. Metabolism 1993;42:1153–8. 49. Walsh BW, Li H, Sachs FM. Effects of postmenopausal hormone replacement therapy with oral and transdermal estrogen on high density lipoprotein metabolism. J Lipid Res 1994;35:2083–93. 50. Su W, Campos H, Judge H, et al. Metabolism of apo(a) in apo B100 of lipoprotein(a) in women: effect of postmenoopausal estrogen replacement. J Clin Endocrinol Metab 1998;83:3267–76. 51. Cushman M, Legault C, Barrett-Connor E, et al. Effect of postmenopausal hormones on inﬂammationsensitive proteins: the Postmenopausal Estrogen/Progestin Interventions (PEPI) study. Circulation 1999;100:717–22. 52. Crook D, Cust MP, Gangar KF, et al. Comparison of transdermal and oral estrogen-progestin replacement: effects on serum lipids and lipoproteins. Am J Obstet Gynecol 1992;166:960–5. 53. Crook D. The metabolic consequences of treating post-menopausal women with non-oral hormone replacement therapy. Br J Obstet Gynecol 1997;104(suppl 16):4–13. 54. Meschia M, Bruschi F, Soma M, et al. Effects of oral and transdermal hormone replacement therapy on lipoprotein(a) and lipids: a randomized controlled trial. Menopause 1998;5:157–62. 55. Kroon UB, Silfverstolpe G, Tengborn L. The effects of transdermal estradiol and oral conjugated estrogens on hemostasis variables. Thromb Haemost 1994;71:420–3. 56. Scarabin P-Y, Alhenc-Gelas M, Plu-Bureau G, Taisne P, Agher R, Aiach M. Effects of oral and transdermal estrogen/progesterone concentrations on blood coagulation and ﬁbrinolysis in postmenopausal women. A randomized controlled trial. Arterioscler Thromb Vasc Biol 1997;17:3071–8. 57. Koh KK, Mincemoyer R, Bui MN, et al. Effects of hormone replacement therapy on ﬁbrinolysis in postmenopausal women. N Engl J Med 1997;336:683–90. 58. Gerhard M, Walsh BW, Tawakol A, et al. Estradiol therapy combined with progesterone and endothelium-dependent vasodilation in postmenopausal women. Circulation 1998;98:1158–63. 59. Paganini-Hill A. Estrogen replacement therapy and stroke. Prog Cardiovasc Dis 1995;38:223–242. 60. Waselenko JK, Nace MC, Alving B. Women with thrombophilia: assessing the risks for thrombosis with oral contraceptives or hormone replacement therapy. Seminars Thromb Hemost 1998;24(suppl 1):33–39. 61. Daly E, Vessey MP, Hawkins MM, Carson JL, Gough P, Marsh S. Risk of venous thromboembolism in users of hormone replacement therapy. Lancet 1997;348:977–980. 62. Jick H, Derby LE, Myers MW, Vasilakis C, Newton KM. Risk of hospital admission for idiopathic venous thromboembolism among users of postmenopausal estrogens. Lancet 1997;348:981–983. 63. Grodstein F, Stampfer MJ, Goldhaber SZ, et al. Prospective study of exogenous hormones and risk of pulmonary embolism in women. Lancet 1996;348:983–987. 64. Grady D, Hulley SB, Furberg C. Venous thromboembolic events associated with hormone replacement therapy (Letter). JAMA 1997;278:477. 65. Mulnard RA, Cotman CW, Kawas C, et al. Estrogen replacement therapy for treatment of mild to moderate Alzheimer disease. JAMA 2000;283:1007–15. 66. Hoibraaten E, Abdelnoor M, Sandset PM. Hormone replacement therapy with estradiol and risk of venous thromboembolism—a population-based case-control study. Thromb Hemost 1999;82:1218–21. 67. Lowe G, Woodward M, Vessey M, Rumley A, Gough P, Daly E. Thrombotic variables and risk of idiopathic venous thromboembolism in women aged 45–64 years. Relationship to hormone replacement therapy. Thromb Hemost 2000;83:530–5. 68. Rosendaal F, personal communication. 69. Rosendaal FR. Thrombosis in the young: epidemiology and risk factors. Thromb Haemost 1996;78:1–6. 70. Mosca L, Collins P, Herrington DM, et al. Hormone replacement therapy and cardiovascular disease. A statement for healthcare professionals from the American Heart Association. Circulation 2001; 104:499–503.

12

SERMs Effects on Cardiovascular Risk Factors and Disease Richard R. Love,

MD

Contents Introduction Interpretation Caveats and Available Information on Cardiovascular Risk Factor and Endpoint Changes Animal Models for Cardiovascular Effects of SERMs Cardiovascular Risk Factor (Intermediate Endpoint) Effects of Three SERMs Predictors of the Collective Impact of SERM Changes on Lipid and Hemostatic Cardiovascular Risk Factors Cardiovascular Events and Tamoxifen Treatment Deﬁnitive Studies of Cardiovascular Events and SERMs Conclusions References

INTRODUCTION If SERMs are going to be used extensively in clinical medicine, their effects on rates of cardiovascular disease, particularly in postmenopausal women, need to be favorable. This is because cardiovascular disease is the major cause of death in older women in Western countries (1). Until recently, the effects of hormones on cardiovascular risk factors (intermediate endpoints)—on lipids, lipoprotein, and coagulation factors were considered to be good predictors of impact on cardiovascular diseases themselves— fatal and nonfatal myocardial infarction, sudden death, atherosclerotic disease, and stroke. The use of hormone replacement therapy, with associated lipid-lowering effects, has been widely believed to be associated with reductions in cardiovascular disease of 30 to 40%. The results of the Heart and Estrogen/progestin Replacement Study (HERS) has however suggested that the relationships of presumed risk factors to disease endpoints is more complex (2). In this study, contrary to the predictions from epidemiologic studies, the hormonal therapy did not confer protection against coronary heart disease

From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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(2). These trial results emphasize for SERMs the need to focus on cardiovascular diseases also. SERMs of two types have been widely evaluated in clinical trials and are approved for use in the United States. The prototype SERM is tamoxifen, a triphenylethylene for which there are the most extensive and long-term data. A second triphenylethylene is chlorotamoxifen or toremifene. The third more-recently approved SERM is a benzothiophene: raloxifene. The cardiovascular risk factor and disease effects of these three compounds are the focus of this chapter. This presentation considers ﬁrst some critical issues in interpreting the available clinical data; next, a brief review of some animal data on tamoxifen and raloxifene; next, the extensive data on risk-factor effects of each of the three approved SERMs; and ﬁnally, the data on cardiovascular disease from trials of tamoxifen.

INTERPRETATION CAVEATS AND AVAILABLE INFORMATION ON CARDIOVASCULAR RISK FACTOR AND ENDPOINT CHANGES The discussion in this chapter concerns target-organ toxicities of three SERMs: tamoxifen, raloxifene, and toremifene. Two areas deserve attention to permit rational understanding and interpretation of current data. First, based on the belief that SERMs acted primarily through combination with cellular estrogen receptor protein, whose presence was more frequently detectable in the tumors of postmenopausal women, initial clinical studies and indeed the preponderance of mature studies have been accomplished in postmenopausal hormone-receptor-positive tumor-bearing populations. These include both therapeutic and toxicologic investigations. The side effects database in premenopausal women is remarkably small. One SERM, tamoxifen, causes hormonal perturbations with large estrogen increases in premenopausal women, and its tissue effects appear clearly, in at least two tissues, bone and vagina, to be modulated by the estrogen milieu (3–5). Thus, the tissue effects to be discussed in this chapter are primarily those observed from studies in postmenopausal women, and these may be very different from those eventually described in premenopausal subjects. A second critical perspective necessary here concerns the levels of reliability of clinical data from different sources. We can conceive a hierarchy of data sources according to reliability (6). At the optimal end of such a hierarchy might be data from overviews or metanalyses of all randomized trials speciﬁcally designed to address particular questions. Less reliable data successively might be from groups of trials, single trials, prospective and retrospective series studies, and then clinical observations, and ﬁnally speculations based on some biologic facts. The data from each of these kinds of studies may be biased in critical ways which should prevent their generalization to larger populations. Three speciﬁc types of bias are of signiﬁcant concern in interpreting the published SERM data and those under development. The ﬁrst is subject selection bias. Clinical studies have, of ethical necessity, investigated volunteers. Such populations are different from the universe of individuals to whom we may wish ultimately to apply SERM treatment. Essentially all adjuvant studies of tamoxifen and other SERMs in breast cancer have been carried out in Caucasian populations whose dietary exposures and hormonal proﬁles might modulate the SERMs’ therapeutic and biologic effects. Two tamoxifen prevention trials have recruited volunteers demonstrated to be more healthy than their general population

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Table 1 Animal Model Intermediate Endpoint Effects of Tamoxifen, Toremifene, Raloxifene Endpoint

SERM

Effect

Total cholesterol

Tamoxifen Raloxifene

Lowering by 40–60% Lowering

Antioxidant

Tamoxifen Raloxifene Toremifene

Inhibition of oxidation of LDL Inhibition of oxidation of LDL Inhibition of oxidation of LDL

Lesion effects

Tamoxifen

Inhibition of spontaneous and established arterial fatty lesions in mice (11) Suggested decreased progression of coronary artery lesions in monkeys (12,13)

Tamoxifen Raloxifene

counterparts (7,8). One explanation for the ﬁndings regarding long-term cardiovascular endpoint effects of tamoxifen in metanalyses is that there is a powerful healthy-volunteer effect (9). Several reported studies involve subsets of patients in such clinical trials. An additional issue is that the longer-term followup data on cardiovascular disease (in Early Breast Cancer Trialist Collaborative Group [EBCTCG] metanalyses, e.g.) are mostly for patients given limited-duration tamoxifen therapy. A second bias of concern in interpreting data about cardiovascular risk factor or other endpoints is surveillance bias. The issue is that the process of screening or intensive surveillance, particularly using laboratory or radiologic technologies, leads to detection of effects which might not otherwise be recognized or develop to a level of clinical signiﬁcance. A third source of bias concerns the methods used to detect particular effects or endpoints and when they are applied to subjects studied. This type of bias is very important in interpreting the available data about cardiovascular toxicities and effects from SERMs. To begin with, the adjuvant studies that have been the major source of such data were designed to ascertain major breast-cancer-associated endpoints. The obtaining of data on cardiovascular effects of SERMs was often almost incidental, and the methods for this were imprecise. This is absolutely critical to appreciate in interpreting other and newer data about cardiovascular effects. The adjuvant studies were not designed to look at cardiovascular endpoints, and thus the use of those, particularly in the EBCTCG metanalyses, is hazardous. A further aspect of the interpretation of available data about SERM cardiovascular effects concerns important other numerical and statistical issues. The statistical strategies appropriate to analysis of the occurrence of certain risk factors and endpoints, not deﬁned a priori as being of concern, must be carefully considered.

ANIMAL MODELS FOR CARDIOVASCULAR EFFECTS OF SERMS In vitro and in vivo animal models of the effects of SERMs on cardiovascular risk factors provide some supporting background data for observations in humans. Data on three endpoints are summarized in Table 1. Like estrogen, SERMs in animals appear to lower cholesterol; a possibly greater effect is seen with tamoxifen, but the direction of the effect is consistent (10). Available data suggest this change is mediated by estrogen-receptor binding.

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Antioxidant effects of agents are important in atherogenesis. The oxidation of lowdensity lipoprotein (LDL) cholesterol is a critical event, and this is observed to be regularly inhibited by each of the three approved SERMs; additionally, these effects of SERMs are markedly greater than those of estrogen (10). While other intermediate endpoint effects have been observed with SERMs, lesion effects are probably the most interesting, and the results of investigations perhaps foretell the uncertainty about disease effects in humans. What is seen is that different effects characterize investigations in different animal species models. In a mouse model, arterial fatty-lesion inhibition has been clearly observed with tamoxifen (11). In partial contrast, however, only suggested decreases in coronary-artery lesions were observed in a monkey model, in studies of both tamoxifen and raloxifene (12,13).

CARDIOVASCULAR RISK FACTOR (INTERMEDIATE ENDPOINT) EFFECTS OF THREE SERMS Although the database for risk factor effects of the three currently approved SERMs is large—for tamoxifen particularly so—much of the published data are from uncontrolled studies. Because longitudinal changes in these cardiovascular risk factors can be seen consequent to study participation and aging effects, particularly for high-density lipoprotein (HDL) cholesterol, interpretation of the true effects is best attempted from randomized controlled trial data. Even in these studies, subset data are reported and must be interpreted with caution. For purposes of this review, randomized studies from eight groups are analyzed (Table 2). These studies have been in both disease-afﬂicted (but in some, disease-free) and healthy postmenopausal populations. There has been little suggestion that results for the analyses of interest are inﬂuenced by disease status. This overview can provide a reasonable picture of the likely direction of SERM effects on cardiovascular risk factors but less certain estimates of magnitude of effects.

Lipids and Lipoproteins In the early 1980s, Lerner summarized data from animal and clinical studies of drugs similar to tamoxifen showing cholesterol-lowering effects (27). In 1984 Rossner and Wallgren ﬁrst reported reductions in HDL and LDL cholesterol in women treated with 40 mg of tamoxifen daily, double the usual current dose (28). Later in the 1980s, other uncontrolled and confounded data were published (summarized in (14)); and then in 1990, this author published data from a two-year placebo-controlled trial (14). These and subsequent data (Table 3) have given a very consistent picture of the magnitude of lipid/cholesterol-lowering effects of tamoxifen. The overall range of percentage total cholesterol lowering has been 8–13%, and this author developed data exploring why this range might be expected (15). Speciﬁcally, the percentage of decrease increases with increasing total cholesterol levels at baseline; at lower levels, approx 5 mmol/L or 200 mg/dL, the average decrease found was 8%, while at levels above 6.7 mmol/L or 250 mg/dL, the percentage decrease was double this level (15). The onset of this lipidlowering effect is rapid—it occurs over 2 weeks—and similarly, a loss of effect occurs on cessation of medication, although more slowly. In smaller studies of toremiﬁne (Table 2), similar percentage decreases to those seen

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Table 2 Tamoxifen CV Intermediate Endpoint Studies Descriptive Characteristics Author (Ref) Tamoxifen

Love (14–18)

Design a

RCT placebo

Grey (19)

RCT placebo tamoxifen Decensi (20,21) RCT placebo, tamoxifen (subsets of patients took b HRT)

Toremifene Gylling (22)

Saarto (23) Raloxifene

Delmas (24) Walsh (25) Valk-de Roo (26)

a

Subset from RCT of tamoxifen, toremifene RCT tamoxifen, toremifene RCT placebo, 3 doses of raloxifene RCT placebo, HRT, 2 doses raloxifene RCT placebo, raloxifene, HRT

Total N

Population

140 Breast cancer disease-free (subsets in postmenopausal several reports) 57 Healthy postmenopausal 3479 Healthy hysterectomized (2925 in women pre- and analysis postmenopausal here) 1117 in tam. group 24

Metastatic breast cancer postmenopausal

49

Breast cancer disease-free postmenopausal

601

Healthy postmenopausal

390

Healthy postmenopausal

56

Healthy postmenopausal

RCT, randomized controlled trial; bHRT, hormone replacement therapy

with tamoxifen are observed (Table 3, (22,23)). Data from raloxifene studies indicate a possibly lower cholesterol-lowering effect with a 60 mg dose (Table 3, 24,26)). The principal change that leads to a reduction in total cholesterol levels with SERMs is a major reduction in LDL cholesterol (Table 3). The greatest percentage reductions are seen with tamoxifen, in the range of 20% (Figure 1), with modestly lesser effects seen with toremifene and raloxifene. Gylling, in a detailed metabolic study, demonstrated that tamoxifen and toremifene inhibit a conversion step in the cholesterol synthetic pathway (22). Data on cholesterol effects of the three approved SERMs on HDL are mixed and do not show a clear direction of effect (Table 3). In the largest study, by Decensi (20), there was no clear change in almost 1000 patients treated with tamoxifen to which they were randomly assigned. Although the Saarto study showed an increase of 14% in HDL cholesterol with toremifene, this conclusion was based on observations in only 23 cases (23), and there was no placebo control group in this study. Gylling observed no HDL change at all in 14 subjects with toremifene treatment (22). Triglyceride levels increase with tamoxifen treatment 18–28% (14,15). Although other authors have not noted this effect and Veronesi and Decensi have not published

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Table 3 Cardiovascular Lipid and Lipoprotein (Intermediate Endpoint) Effects of Tamoxifen, Chlorotamoxifen (Toremifene), and Raloxifene in Postmenopausal Women Endpoint

SERM

Effecta

References

Total cholesterol

Tamoxifen Toremifene Raloxifene

↓8–13% ↓11–12% ↓6–10%

(14,15,19,20) (22,23) (24,26)

LDL cholesterol

Tamoxifen Toremifene Raloxifene

↓14–20% ↓15–20% ↓10–14%

(14,19,20) (22,23) (24–26)

HDL cholesterol

Tamoxifen Toremifene Raloxifene

No change No change No change

(14,20) (22,23) (24–26)

Triglycerides

Tamoxifen Toremifene Raloxifene

↑18–28% ? no change ↑7%

(7,14) (22,23) (24–26)

Apolipoprotein A

Tamoxifen Toremifene Raloxifene

↑7% ↑13% ↑3%

(15) (23) (25)

Apolipoprotein B

Tamoxifen Toremifene Raloxifene

↓7–12% ↓10% ↓9%

(15,23) (23) (25)

Lipoprotein(a)

Tamoxifen Toremifene Raloxifene

↓40% ↓40% ↓7%

(15,23) (23) (25)

a

% change vs. placebo

speciﬁc data on this subject (7,20), Veronesi speciﬁcally invokes “the unexpected ﬁnding with hypertriglyceridemia” as a matter of concern in their prevention trial, which led to cessation of recruitment (7). Clinically, individuals with glucose intolerance and a possible proclivity to hypertriglyceridemia seem most likely to develop marked increases in this lipid. Increased hepatic synthesis of very-low-density lipoprotein may be the mechanism for this hypertriglyceridemia. Triglyceride changes with toremifene or raloxifene have not been seen (Table 3). A consistent picture is seen with effects of SERMs on apolipoprotein A; modest increases characterize available data for each of the three approved SERMs (Table 3). This author found a 7% increase with tamoxifen (15), Saarto found a 13% increase with toremifene (23), and Walsh found a 3% increase with raloxifene (25). Increased synthesis of this lipoprotein is suggested as the explanation. Similarly, a decrease in apolipoprotein B is seen consistently with all three SERMs: 12% with tamoxifen (15), 10% with toremifene (23), and 9% with raloxifene (25). Increased apolipoproten B receptor (which lowers LDL cholesterol) may be the change responsible for this apolipoprotein B reduction.

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Fig. 1. Mean fasting levels of LDL cholesterol over 2 years in the Wisconsin Tamoxifen Study (14). LDL cholesterol levels are lower at all time points and decreased signiﬁcantly from baseline and compared to the placebo group with tamoxifen treatment (p < .001).

Finally, data from studies are consistent in suggesting that levels of lipoprotein (a) are reduced by each of the three SERMs (Table 3). The magnitudes of the reductions vary considerably among studies but may be great and signiﬁcant.

Hemostatic Effects As might be expected, because of the effects of SERMs on lipoproteins and lipids synthesized by the liver, these hormones also have effects on other proteins synthesized in that organ, proteins important in the atherosclerotic thrombosis process and coagulation. There are fewer data on many of the hemostatic proteins, and as will be discussed, interpreting likely net impact of their changes is difﬁcult. Data on tamoxifen and raloxifene support a conclusion that ﬁbrinogen levels decline 10–20% with each of these SERMs (Table 4) (Fig. 2). More recently, there has been more interest in blood levels of homocysteine as a risk factor for vascular disease and venous thrombosis (29), and SERM studies have investigated this factor. Homocysteine is an amino acid derived from metabolic conversion of methionine. Both tamoxifen and raloxifene appear to lower blood levels of homocysteine (Table 4). In the most rigorous study, by Cattaneo (21), the higher the baseline level of homocysteine, the greater the reduction following tamoxifen therapy. Although changes are suggested in plasminogen-activator inhibitor levels with tamoxifen and raloxifene, these seem inconsistent with other changes or absence of other hemostatic changes, and thus are probably of limited consequence. Platelet counts are clearly and consistently 7% lower with tamoxifen therapy; no patients (or at least only rare and possibly thrombocytopenic patients before therapy) develop clinically worrisome thrombocytopenia with this SERM.

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Table 4 Cardiovascular Hemostatic Effects of Tamoxifen and Raloxifene SERM

Effecta

References

Fibrinogen

Tamoxifen Raloxifene

↓14–18 ↓10–22%

(16,19,30) (25,26)

Homocysteine

Tamoxifen Raloxifene

↓4–20% ↓5%

(18,21) (31)

Plasminogen activator inhibitor

Tamoxifen Raloxifene

↓15% ↑6%

(32) (25)

Platelet count Antithrombin III

Tamoxifen Tamoxifen

↓7% ↓8–16%

(16,20) (16,30)

a

% change from baseline or vs. placebo

Fig. 2. Mean ﬁbrinogen levels at baseline and 6 months in patients receiving placebo (●) or tamoxifen (■). The difference in the change in the two graphs is signiﬁcant at p = 0.0003 (16).

Similarly, antithrombin III levels are consistently lower in studies with tamoxifen, but individual patients do not develop such low levels as to be at known clinical risk for thrombosis on this basis (16). Other hemostatic protein changes have been investigated with inconclusive results. Proteins C and S do not appear to change signiﬁcantly with tamoxifen treatment (17). Fibrinopeptide A or prothrombin fragments 1 and 2 do not appear to change in consistent directions with either tamoxifen or raloxifene (10).

SERMs and Nonlipid Nonhemostatic Cardiovascular Risk Factors There are other clinically very signiﬁcant risk factors for cardiovascular disease that might be inﬂuenced by SERM treatment. About some of these there are mostly reassuring

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data demonstrating an absence of major effects. In women, diabetes is a signiﬁcant cause of small-vessel heart disease, and thus the impact of SERMs, increasingly being given long term, or glucose metabolism is potentially important. Both we (33) and Grey (19) found no changes in fasting glucose levels with tamoxifen therapy, and Valk-de Roo found no effects of raloxifene on either glucose or insulin levels (26). Although power for small but clinically important changes in blood pressure was low in both published studies addressing this question for tamoxifen and raloxifene, no effects were found (26,33). This author found no association of exercise levels with tamoxifen therapy (33), and both this author and Grey (19) showed no signiﬁcant changes in weight with tamoxifen therapy. As might be expected, a gradual increase in weight was observed in patients over time with or without tamoxifen treatment; this natural history has sometimes led to the erroneous conclusion that tamoxifen does cause weight gain. Finally, Grey investigated abdominal fat distribution with tamoxifen treatment and could ﬁnd no change (19). This observation is consistent with the other previously discussed ﬁndings on absence of glucose and insulin effects of SERMs.

PREDICTORS OF THE COLLECTIVE IMPACT OF SERM CHANGES ON LIPID AND HEMOSTATIC CARDIOVASCULAR RISK FACTORS There is an extensive literature covering the relationships of lipids and hemostatic risk factors and cardiovascular disease in women. Bush (34), Law (35), Kannel (36), and Anderson (37) provided guidelines for the relationships of lipids and lipoproteins in particular to the development of coronary heart disease. While the Framingham model (37) was based on longitudinal data from that town’s cohort, it has been uncertain whether it is comprehensive in its consideration of all potentially important risk factors. In pulling together our lipid data in 1994, my colleagues and I used Anderson’s model to estimate the potential effects of tamoxifen on risk for cardiovascular disease (33). This model did not include lipoprotein (a) or ﬁbrinogen. In a low-risk cohort, we estimated a 19% reduction in coronary heart disease over ﬁve years, whereas an 8% reduction might be observed in a high-risk cohort. The healthy-volunteer effect referred to earlier suggests that in tamoxifen trials the cohort may be low risk, but the low event rates altogether may be making observation of beneﬁt difﬁcult. Several risk factors for cardiovascular disease have not been included in the Anderson model (37). Triglyceride is an independent risk factor and, as reviewed earlier (in “Lipids and Lipoproteins”), generally changes with SERMs are to increase risk (38). Lipoprotein (a) is considered a powerful risk factor, for which changes are favorable (39). Fibrinogen is important also; indeed, Framingham data suggested this as particularly important in women (40), and recent reviews have continued to emphasize its importance (41,42). Finally, homocysteine has received increasing attention as a potent risk factor for cardiovascular disease (29). The multiplicity of effects of SERMs on risk factors, the suggestions that some effects are favorable and others are not, and the uncertainties regarding the magnitudes of effects and modiﬁers of these in individual patients all support the position that focusing on cardiovascular events is critical for understanding important clinical effects of SERMs.

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Table 5 Cardiovascular Events and Tamoxifen Treatment Author (Reference)

Total N

McDonald (43)

1070

Rutqvist (44) Tamoxifen 40 mg vs. placebo

2365

McDonald (45)

1312

Costantino (8)

2885

Fisher (46)

13,388

EBCTCG (9) Metanalysis

Event Fatal myocardial infarction ↓RR.4 (p = 0.009) Hospital admission cardiac disease RR .68 for control group. Marginal MI beneﬁt. Myocardial infarction reduced with tamoxifen; level of effect varied with duration and presence of concurrent use. Hospitalization rates (for MI) lower with tamoxifen. CHD deaths RR .66 (NS) Myocardial infarction RR 1.11 (NS) New Q wave RR 1.36 (NS) Non breast-cancer death

CARDIOVASCULAR EVENTS AND TAMOXIFEN TREATMENT The available data on cardiovascular events and SERMs are for tamoxifen within the contexts of adjuvant therapy trials with this SERM (9). Critical data are summarized in Table 5. With publication of the author’s lipid data in 1990, interest in the impact of tamoxifen on cardiovascular events increased signiﬁcantly, and the McDonald report from the Scottish trial a year later (43) was seen as conﬁrmatory of the suggested riskfactor beneﬁts. Although the numbers of myocardial infarct events in that trial were small, 10 vs. 25, the randomized trial context of the evaluation and the methods used have encouraged investigators that the effect observed was real. The next report, by Rutqvist using different methods but again within the context of a clinical trial, supports the conclusion of cardiac beneﬁt, but despite larger numbers of cases, was not able to increasingly prove beneﬁt statistically (44). A more extensive study by McDonald in 1995 of the same cohort he had originally studied, adding the premenopausal women in the adjuvant Scottish trial, demonstrated beneﬁt again, but as noted the level varied with duration and continuation of use of tamoxifen (45). Costantino and colleagues evaluated coronary heart disease (CHD) mortality in women participating in a large adjuvant study of tamoxifen; enrollees had to have a hormone receptor-positive tumor, which may indicate that their risk factors (for breast cancer and otherwise) were different from the population at large or other populations (8). Although CHD deaths were fewer with tamoxifen treatment, the demonstrated difference was not statistically signiﬁcant (8). Finally, in evaluating tamoxifen in adjuvant trials, the Early Breast Cancer Trialists’ Collaborative Group (1998) did metanalyses of these trials and was not able to demonstrate beneﬁt in reduction of non breast-cancer deaths (9). To date, data from only one trial of tamoxifen in healthy women have been published (46,47). These data showed nonsigniﬁcant increases rather than decreases in cardiovascular events with tamoxifen treatment. Unfortunately, there are no ongoing efforts to

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Table 6 Pulmonary (PE or DVT) and Cerebrovascular Events and Tamoxifen Treatment Author (Reference)

Design

Total N

Event

RR

McDonald (45)

RCT tamoxifen, placebo 1312

PE or DVT Cardiovascular

2.1 (ever used) 1.6 (ever used)

Rutqvist (44)

RCT tamoxifen, placebo 2365

Thromboembolism 1.06

Fisher (46)

13,388

PE DVT Stroke TIA

3.01 1.60 1.59 .76

Veronesi (7)

5408

DVT Stroke

2.0 1.8

PE, pulmonary embolism; DVT, deep vein thrombosis; TIA, transient ischemic attack; RR, relative risk.

follow this trial cohort for cardiovascular or other events. Two ongoing tamoxifen prevention trials in Europe involve much smaller populations and are continuing longterm followup (7,48). Some of these same trials (McDonald Scottish (43), Rutqvist Swedish (44); Fisher NSABP P1 (46), Veronesi, Italian prevention (7)) have provided the best available data on pulmonary embolism (PE), deep venous thrombosis (DVT), and cardiovascular events with tamoxifen treatment (Table 6). The results have been both consistent and worrisome. Deep venous thrombosis and associated pulmonary embolism appear to be clearly increased 1.5–3-fold with tamoxifen treatment. This conclusion is consistent with clinical reports, although as indicated earlier, the factors that mediate this process are unclear. Completed stroke has been found to be more frequent in the Scottish adjuvant trial and the American and Italian prevention trials (7,45,46). This result has been unexpected, and the possible mediators of such adverse events (thromboses or embolizations) are unclear. In particular, the ﬁbrinogen-reducing effect of tamoxifen might be predicted from the Framingham data to be protective against stroke (40). In the American prevention study, transient ischemic attacks were less frequent with tamoxifen (46), which suggests either a spurious chance result for stroke or that different mechanisms are important in the two processes. There seem to be several possible explanations for why these and other trials (those in Table 5) have shown marginal cardiovascular effects despite data demonstrating consistent effects on powerful cardiovascular risk factors. First, in fact, the balance of SERM/tamoxifen effects may be marginal in inﬂuencing rates of these events. As suggested above, perhaps we have overemphasized some factors when others are equally or more important—ones inﬂuenced adversely by tamoxifen. Triglycerides are an example. It would seem, however, to this author that beneﬁt from SERMs on cardiovascular events is likely, and thus focusing on the nature of the evidence so far seems more appropriate. There are several reasons why, despite a developing beneﬁcial effect of SERMs/tamoxifen on cardiovascular events, these favorable occurrences have not been convincingly observed. There may be effects of stopping the tamoxifen, which has been standard practice (e.g., the Costantino study (8)), that limit the beneﬁts to

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date. In the EBCTCG data, this effect and the fact that many of the trials involved short-term therapy may account for an absence of demonstrable beneﬁt. Additional explanations may lie in the methods and maturity of these trials. The adjuvant trials were not designed as cardiovascular endpoint trials, and in these circumstances the data on these endpoints may be less rigorous than needed. As Costantino has emphasized, for some trials the followup to date is short and inadequate to demonstrate beneﬁt (8). Probably the most likely explanation for absence of cardiovascular beneﬁts, if beneﬁts there are to be, is that participants in the adjuvant and prevention trials are healthy volunteers. Veronesi (7) and Gail (47) have called attention to the lower-than-expected rates of cardiovascular events in their prevention studies. As noted in an introductory section of this chapter, healthy-volunteer effects are a selection bias for trials, and these can exert powerful effects on ability to demonstrate endpoints. It seems likely that, in both adjuvant tamoxifen and tamoxifen prevention trials, participants at baseline and perhaps consequent to the dynamics of trial membership are already at lower risk than average of coronary heart disease and other cardiovascular events. These circumstances lead both to lower event rates and to lesser beneﬁts from the SERM treatment. The observation that with greater levels of cholesterol there are greater absolute and percentage reductions in this lipid with tamoxifen treatment (33) illustrates how such lower risks might reduce or mask beneﬁts.

DEFINITIVE STUDIES OF CARDIOVASCULAR EVENTS AND SERMS The preceding discussion suggests that the maturing of ongoing studies, both of adjuvant and preventive therapies with SERMs, may yet provide deﬁnitive answers to the questions about cardiovascular events and these therapies. As noted, although the American prevention trial with tamoxifen had objectives related to heart-disease mortality, no further followup is in process to more rigorously address this area (46). The European prevention studies, although smaller, may provide data on cardiovascular endpoints as they further mature (7,48). The only trial in progress speciﬁcally designed to investigate the impact of a SERM on cardiovascular endpoints involves raloxifene— the Raloxifene Use for the Heart (RUTH) trial (10). This trial is planned to involve 10,000 postmenopausal subjects, and given its speciﬁc objectives, will doubtless address the issues that have made difﬁcult demonstrating deﬁnitive beneﬁt in the trials to date.

CONCLUSIONS SERMs—tamoxifen, raloxifene, and toremifene—have some powerful effects and some less certain effects on risk factors for cardiovascular disease. Studies addressing the impact of tamoxifen on cardiovascular endpoints suggest beneﬁt but are limited in many ways. Maturing of data from current trials and data from a new trial of raloxifene will hopefully clarify the issues in the coming several years.

REFERENCES 1. Wenger NK. Coronary heart disease: an older woman’s major health risk. BMJ 1997;315(7115):1085– 1090. 2. Hulley S, Gray D, Bush T, et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA 1998;280(7):605–613. 3. Love RR, Mazess RB, Barden HS, et al. Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer. N Engl J Med 1992;326(13):852–856.

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4. Powles TJ, Hickish T, Kanis JA, Tidy A, Ashley S. Effect of tamoxifen on bone mineral density measured by dual-energy x-ray absorptiometry in healthy premenopausal and postmenopausal women. J Clin Oncol 1996;14(1):78–84. 5. Love RR, Kurtycz DF, Dumesic DA, Laube DW, Yang FY. The effects of tamoxifen on the vaginal epithelium in postmenopausal women. J Womens Health Gend Based Med 2000;9(5):559–563. 6. Coates A. Clinical trials. In: Love RR, editor. Manual of Clinical Oncology. 6th ed, p. 224–232, New York: Springer-Verlag, 1994. 7. Veronesi U, Maisonneuve P, Costa A, et al. Prevention of breast cancer with tamoxifen: preliminary ﬁndings from the Italian randomised trial among hysterectomised women. Lancet 1998;352:93–97. 8. Costantino JP, Kuller LH, Ives DG, Fisher B, Dignam J. Coronary heart disease mortality and adjuvant tamoxifen therapy. J Natl Cancer Inst 1997;89(11):776–782. 9. Anonymous. Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet 1998;351(9114):1451–1467. 10. Barrett-Connor E, Cox DA, Anderson PW. The potential of SERMs for reducing the risk of coronary heart disease. TEM 1999;10(8):320–325. 11. Grainger DJ, Witchell CM, Metcalfe JC. Tamoxifen elevates transforming growth factor-beta and suppresses diet-induced formation of lipid lesions in mouse aorta. Nat Med 1995;1(10):1067–1073. 12. Williams JK, Wagner JD, Li Z, Golden DL, Adams MR. Tamoxifen inhibits arterial accumulation of LDL degradation products and progression of coronary artery atherosclerosis in monkeys. Arterioscler Thromb Vasc Biol 1997;17(2):403–408. 13. Clarkson TB, Anthony MS, Jerome CP. Lack of effect of raloxifene on coronary artery atherosclerosis of postmenopausal monkeys. J Clin Endocrinol Metab 1998;83(3):721–726. 14. Love RR, Newcomb PA, Wiebe DA, et al. Effects of tamoxifen therapy on lipid and lipoprotein levels in postmenopausal patients with node-negative breast cancer [see comments]. J Natl Cancer Inst 1990;82(16):1327–1332. 15. Love RR, Wiebe DA, Newcomb PA, et al. Effects of tamoxifen on cardiovascular risk factors in postmenopausal women. Ann Intern Med 1991;115(11):860–864. 16. Love RR, Surawicz TS, Williams EC. Antithrombin III level, ﬁbrinogen level, and platelet count changes with adjuvant tamoxifen therapy. Arch Intern Med 1992;152(2):317–20. 17. Mamby CC, Love RR, Feyzi JM. Protein S and protein C level changes with adjuvant tamoxifen therapy in postmenopausal women. Breast Cancer Res Treat 1994;30(3):311–4. 18. Love RR, Anker G, Yang Y, et al. Serum homocysteine levels in postmenopausal breast cancer patients treated with tamoxifen. Cancer Lett 1999;145(1–2):73–77. 19. Grey AB, Stapleton JP, Evans MC, Reid IR. The effect of the anti-estrogen tamoxifen on cardiovascular risk factors in normal postmenopausal women. J Clin Endocrinol Metab 1995;80(11):3191–3195. 20. Decensi A, Robertson C, Rotmensz N, et al. Effect of tamoxifen and transdermal hormone replacement therapy on cardiovascular risk factors in a prevention trial. Br J Cancer 1998;78(5):572–578. 21. Cattaneo M, Baglietto L, Zighetti ML, et al. Tamoxifen reduces plasma homocysteine levels in healthy women. Br J Cancer 1998;77(12):2264–2266. 22. Gylling H, Pyrhonen S, Mantyla E, Maenpaa H, Kangas L, Miettinen TA. Tamoxifen and toremifene lower serum cholesterol by inhibition of ∆8-cholesterol conversion to lathosterol in women with breast cancer. J Clin Oncol 1995;13(12):2900–2905. 23. Saarto T, Blomqvist C, Ehnholm C, Taskinen M-R, Elomaa I. Antiatherogenic effects of adjuvant antiestrogens: a randomized trial comparing the effects of tamoxifen and toremifene on plasma lipid levels in postmenopausal women with node-positive breast cancer. J Clin Oncol 1996;14(2):429–433. 24. Delmas PD, Bjarnason NH, Mitlak BH, et al. Effects of raloxifene on bone mineral density, serum cholesterol concentrations, and uterine endometrium in postmenopausal women. N Engl J Med 1997; 337:1641–1647. 25. Walsh BW, Kuller LH, Wild RA, et al. Effects of raloxifene on serum lipids and coagulation factors in healthy postmenopausal women. JAMA 1998;279(18):1445–1451. 26. de Valk-de Roo GW, Stehouwer CDA, Meijer P, et al. Both raloxifene and estrogen reduce major cardiovascular risk factors in healthy postmenopausal women. A 2-year, placebo-controlled trial. Arterioscler Thromb Vasc Biol 1999;19(12):2993–3000. 27. Lerner LJ. The ﬁrst nonsteroidal antiestrogen—MER 25. In: Sutherland RL, Jordan VC, (eds). Nonsteroidal Antiestrogens, Molecular Pharmacology and Antitumor Activity, p. 1–16. Sydney: Academic Press, 1981.

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28. Rossner S, Wallgren A. Serum lipoproteins and proteins after breast cancer surgery and effects of tamoxifen. Atherosclerosis 1984;52(3):339–346. 29. Refsum H, Ueland PM, Nygard O, Vollset SE. Homocysteine and cardiovascular disease. Annu Rev Med 1998;49:31–62. 30. Chang J, Powles TJ, Ashley SE, et al. The effect of tamoxifen and hormone replacement therapy on serum cholesterol, bone mineral density and coagulation factors in healthy postmenopausal women participating in a randomised, controlled tamoxifen prevention study. Ann Oncol 1996;7(7):671–675. 31. Walsh BW, Paul S, Wild RA, et al. The effects of hormone replacement therapy and raloxifene on C-reactive protein and homocysteine in healthy postmenopausal women: a randomized, controlled trial. J Clin Encodrinol Metab 2000;85(1):214–218. 32. Mannucci PM, Bettega D, Chantarangkul V, Tripodi A, Sacchini V, Veronesi U. Effect of tamoxifen on measurements of hemostasis in healthy women. Arch Intern Med 1996;156(16):1806–1810. 33. Love RR, Wiebe DA, Feyzi JM, Newcomb PA, Chappell RJ. Effects of tamoxifen on cardiovascular risk factors in postmenopausal women after 5 years of treatment. J Natl Cancer Inst 1994;86(20):1534–1539. 34. Bush TL, Fried LP, Barrett-Connor E. Cholesterol, lipoproteins, and coronary heart disease in women. Clin Chem 1988;34(8B):B60–B70. 35. Law MR, Wald NJ, Thompson SG. By how much and how quickly does reduction in serum cholesterol concentration lower risk of ischaemic heart disease? BMJ 1994;308(6925):367–372. 36. Kannel WB. Metabolic risk factors for coronary heart disease in women: perspective from the Framingham Study. Am Heart J 1987;114(2):413–419. 37. Anderson KM, Wilson PW, Odell PM, Kannel WB. An updated coronary risk proﬁle. A statement for health professionals. Circulation 1991;83(1):356–362. 38. Austin MA. Plasma triglyceride as a risk factor for coronary heart disease. The epidemiologic evidence and beyond. Am J Epidemiol 1989;129(2):245–259. 39. MBewu AD, Durrington PN. Lipoprotein (a): structure, properties and possible involvement in thrombogenesis and atherogenesis. Atherosclerosis 1990;85(1):1–14. 40. Kannel WB, Wolf PA, Castelli WP, D’Agostino RB. Fibrinogen and risk of cardiovascular disease. The Framingham Study. JAMA 1987;258(9):1183–1186. 41. Woodward M, Lowe GD, Rumley A, Tunstall-Pedoe H. Fibrinogen as a risk factor for coronary heart disease and mortality in middle-aged men and women. The Scottish Heart Health Study. Eur Heart J 1998;19(1):55–62. 42. Ernst E, Resch KL. Fibrinogen as a cardiovascular risk factor: a meta-analysis and review of the literature. Ann Intern Med 1993;118(12):956–963. 43. McDonald CC, Stewart HJ. Fatal myocardial infarction in the Scottish adjuvant tamoxifen trial. The Scottish Breast Cancer Committee. Br Med J 1991;303(6800):435–7. 44. Rutqvist LE, Mattsson A. Cardiac and thromboembolic morbidity among postmenopausal women with early-stage breast cancer in a randomized trial of adjuvant tamoxifen. The Stockholm Breast Cancer Study Group [see comments]. J Natl Cancer Inst 1993;85(17):1398–406. 45. McDonald CC, Alexander FE, Whyte BW, Forrest AP, Stewart HJ. Cardiac and vascular morbidity in women receiving adjuvant tamoxifen for breast cancer in a randomised trial. BMJ 1995;331:977–980. 46. Fisher B, Costantino JP, Wickerham DL, et al. Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst 1998;90(18): 1371–1388. 47. Gail MH, Costantino JP, Bryant J, et al. Weighing the risks and beneﬁts of tamoxifen treatment for preventing breast cancer. J Natl Cancer Inst 1999;91(21):1829–1846. 48. Powles T, Eeles R, Ashley S, et al. Interim analysis of the incidence of breast cancer in the Royal Marsden Hospital tamoxifen randomised chemoprevention trial. Lancet 1998;352(9122):98–101.

13

Estrogen and the Skeleton Michael Kleerekoper, MD, FACE and Ashish Verma, MD Contents Introduction Estrogen and Skeletal Growth Estrogen Deﬁciency and Bone Loss Estrogen Replacement and the Prevention of Bone Loss Estrogen and the Prevention of Osteoporotic Fractures References

INTRODUCTION The crucial role of estrogen in the normal growth and development of the skeleton in girls has been known for decades. Recent case reports in men with estrogen-receptor deﬁciency (1) or deﬁciency of aromatase enzyme (2) have underscored an equally important role for estrogen in skeletal development in boys. The role of declining ovarian estrogen production in bone loss in women has been well documented since the initial hypothesis of Albright in the 1940s (3). Several recent studies have now pointed out a potential important role for estrogen in the age-related bone loss in men (4,5). Important studies in the last several years have provided insight into possible mechanisms underlying these clinical observations, particularly the role of estrogen in modulating local cytokine production in skeletal tissue (6). The ﬁnding of estrogenreceptor beta (ERβ) as the dominant estrogen receptor in bone (7) answers many questions regarding the previously documented low estrogen-receptor density in this clearly estrogen-dependent tissue. Increasingly sophisticated assays for measurement of circulating estrogen levels in very low concentrations has shed new light on these issues (8).

ESTROGEN AND SKELETAL GROWTH Hormonal inﬂuences on the growth and development of the skeleton in children and adolescents has recently been reviewed in detail (9,10). There are no signiﬁcant From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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differences in bone mineral density (BMD) between girls and boys at ages 8 through 11 (11) even though there are already signiﬁcant BMD differences between nonHispanic whites and African-American children at this age. This ethnic difference continues throughout life accelerating at puberty (12,13) and the gender difference begins to appear at puberty (14). The earlier puberty in girls is accompanied by an earlier growth spurt than in boys including an earlier increase in BMD. Clear relationships between stages of puberty and BMD increases have been well documented in girls (15). Girls with delayed or absent puberty do not have this normal pubertal increase in BMD (16). A recent report of a 28-year-old male with estrogen-receptor deﬁciency (1) highlights the importance of estrogen on skeletal growth and development in males. This patient had delayed epiphyseal closure with marked skeletal undermineralization in the presence of normal circulating testosterone levels and elevated estrogen levels. Further evidence of the role of estrogen in the accumulation of skeletal mass in males is provided by the report of estrogen therapy resulting in a signiﬁcant increase in bone mass in males with aromatase deﬁciency (2,17).

ESTROGEN DEFICIENCY AND BONE LOSS In all experimental animal models, bilateral oophorectomy is associated with a decrease in bone mass, a disruption of the microarchitecture of the skeleton, and a decrease in bone strength (18). Estrogen deﬁciency induced in this manner stimulates osteoclast-mediated bone resorption. Several lines of evidence point to an increase in local cytokine production, particularly IL-1 and IL-6, as the stimulus for increased osteoclastic activity (19,20). There is both an increase in osteoclast number and increase in activity of each osteoclast. Antibodies to IL-6 block the increased bone resorption induced by bilateral oophorectomy. Bilateral oophorectomy followed by estrogen administration prevents the increase of IL-1 and IL-6 and prevents the increase in osteoclastic activity. Repression of the IL-6 promoter by estrogen appears to be mediated via (NF-κB) and C/EBP β (21). Surgical menopause (bilateral oophorectomy) (22) and natural menopause (23) in women have also been shown to be associated with acceleration of bone loss because of stimulation of osteoclast-mediated bone resorption. Studies implicate increased IL-1 and IL-6 production in this phenomenon in women, as it does in the experimental animal. Diseases and therapies associated with a signiﬁcant reduction in ovarian estrogen production are also associated with increased bone resorption and increased bone loss. This includes premature ovarian failure and declining ovarian function caused by disease of the hypothalamus (athletic amenorrhea, anorexia, and bulimia), or pituitary (prolactinoma). Intermittent therapy with gonadotropin-releasing hormone (GnRH) agonists lowers pituitary gonadotropin synthesis and release with a resultant decrease in ovarian estrogen production. This too is associated with bone loss that can be prevented by concomitant administration of estrogen. Osteoclast-mediated bone resorption occurs on skeletal surfaces, cancellous, endocortical, and to a lesser extent periosteal. Per unit of bone mass, or bone volume, there is substantially greater cancellous bone surface than endocortical or periosteal surface. At menopause, only 20% of total skeletal mass is cancellous bone, but 80% of total skeletal surface is cancellous bone. As a direct result of this, the major effects of

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estrogen-deﬁciency-induced increases in bone resorption are seen in cancellous bone, with cortical thinning because of endocortical bone resorption occurring later (24). In the bone remodeling cycle, osteoclast-mediated bone resorption is tightly coupled to osteoblast-mediated bone formation (25). The increase in bone formation resulting from estrogen deﬁciency, however, is not sufﬁcient to overcome the estrogen-deﬁciencyinduced increase in bone resorption. Nonetheless, this coupling of resorption and formation, to a great extent, controls the rate of bone loss. This is in contrast to other circumstances, such as the effects of glucocorticosteroids on the skeleton, which increase bone resorption, but also inhibit bone formation. Glucocorticosteroid-induced bone loss is substantially more rapid than the bone loss associated with estrogen deﬁciency. Although low levels of estrogen persist throughout life following bilateral oophorectomy or the naturally occurring menopause, the rate of bone loss appears to slow down within 10 years after menopause. The mechanism for the slowing down of postmenopausal bone loss is unclear. One possibility is that this is simply a reﬂection of the reduced amount of skeletal surface, particularly cancellous bone surface, induced by the more rapid early postmenopausal bone loss. Throughout the postmenopausal period, the rate of bone loss remains higher than in the estrogen replete premenopausal woman. Several recent studies indicate just how precisely estrogen levels modulate bone metabolism. In women in their 70s and older, where measurable estrogen levels in the circulation are quite low, there is nonetheless a relationship between the variability even in these low estrogen levels and bone density (26), as well as hip-fracture rates (27). The comments made about estrogen deﬁciency and bone loss in women apply equally well to testosterone deﬁciency and bone loss in men. Bilateral orchiectomy in men is associated with accelerated bone loss (28) just as is bilateral oophorectomy in women, and both primary and secondary diseases of the testes, with the concomittant declining testosterone production, are associated with accelerated bone loss. Surprisingly, in the older male, crosssectional studies have shown a closer relationship between circulating estrogen levels and bone mass than between circulating testosterone levels and bone mass (28,29). Prospective studies have not yet been done relating estrogen levels to rates of bone loss or fractures in men. To further complicate the respective roles of estrogens and androgens in bone loss in aging men and women, a potential role for androgen deﬁciency in the pathogenesis of osteoporosis in women has recently been reported (30). This work conﬁrmed that serum estradiol was an independent determinant of BMD at the lumbar spine in women as was bioavailable testosterone. Only sex-hormone-binding globulin (SHBG) but neither estradiol nor testosterone correlated with biochemical markers of bone remodeling.

ESTROGEN REPLACEMENT AND THE PREVENTION OF BONE LOSS As previously mentioned, in experimental animals, bilateral oophorectomy induces accelerated bone loss and this can be prevented by administration of estrogen. The same is true following a surgical menopause in women (22). The situation is a little different following natural menopause in women because the decline in ovarian estrogen production is more gradual and begins up to four years prior to the last menstrual period. There is probably a threshold level of estrogen, or a threshold for the rate of decline in estrogen that triggers the increase in osteoclastic bone resorption (31). These theoretical thresholds are almost certainly reached, at least in some women, prior to

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the last menstrual period. This means that many women at the time of their last menstrual period have a substantial increase in the surface extent of resorption cavities. When estrogen is given, the birth rate of new resorption cavities is substantially curtailed, but the coupled bone formation process continues to ﬁll in existing resorption cavities. This phenomenon is known as closure of the remodeling space. This will be seen as a transient increase in bone mass reaching a plateau as the remodeling space is completely closed. This is amply demonstrated in the NIH-sponsored postmenopausal estrogen/ progestin intervention studies (PEPI) (32). Importantly, a recent further analysis of the PEPI trial documented that only a small fraction (1–2%) of women adherent to hormone replacement therapy experienced any reduction in BMD during three years of followup (33). Equally important, this study demonstrates that not all women experience bone loss in the early postmenopausal years even if not provided with estrogen replacement. Presumably this reﬂects the effects of residual endogenous estrogen and possibly endogenous androgen as has been postulated. There is substantial variability in the size of the remodeling space at the time of the last menstrual period. There is substantial matching variability in the measured apparent increase in bone mass when estrogen is replaced. Some of the bone loss induced by estrogen deﬁciency is irreversible. The longer the estrogen deﬁciency is allowed to proceed uncorrected, the greater the amount of irreversible bone loss (25). Estrogen deﬁciency however is associated with ongoing bone loss throughout life. Whenever estrogen is provided to an estrogen-deﬁcient woman, no matter how long she has been estrogen deﬁcient, there will be inhibition of bone resorption and a dramatic slowdown in the rate of loss. Again, if there is a large remodeling space at the time estrogen is given, there will be a seeming increase in bone mass; if there is a very small remodeling space, because the rate of bone loss is very small, there will be little measurable increase in bone mass (34). These observations, however, do suggest that it is possible and pertinent to consider estrogen replacement at any time in the life of a postmenopausal estrogen-deﬁcient woman. The earlier the estrogen is administered, the less irreversible bone loss will occur. If estrogen replacement is discontinued or interrupted, then rates of bone loss increase seemingly to the rate that was operative at the time estrogen was administered. The effects of estrogen deﬁciency on bone resorption are most evident at the time of bilateral oophorectomy, with the most rapid decline in ovarian estrogen production. If adequate estrogen is given at the time of bilateral cophorectomy, bone loss will be prevented—and will be prevented as long as estrogen is administered. As soon as estrogen is discontinued, however, the rate of loss will approach that which would have operated at the time of the bilateral oophorectomy; this is similar for rates of loss following natural menopause (23). The role of the selective estrogen-receptor modulators (SERMs) tamoxifen (35) and raloxifene (36) in the preservation of bone mass in postmenopausal women is discussed in detail elsewhere in this textbook and will not be elaborated on here. The positive effects of SERMs on the skeleeton however underscores the importance of estrogen or estrogen agonists in preserving skeletal health in women with estrogen deﬁciency.

ESTROGEN AND THE PREVENTION OF OSTEOPOROTIC FRACTURES The major determinants of risk of a fragility fracture complicating osteoporosis are bone mass, bone microarchitecture, and propensity to fall. From the preceding discus-

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sion, it is apparent that estrogen deﬁciency affects both bone mass and bone microarchitecture in such a way that osteoporotic fragility fractures are increasingly prevalent in older postmenopausal women (37). It is equally apparent that estrogen replacement will preserve skeletal mass and microarchitecture that is present at the time estrogen replacement is begun. Finally, the beneﬁcial effects of estrogen will be apparent only as long as estrogen is continued. Crosssectional epidemiologic studies have demonstrated that estrogen use for a minimum of 10 years postmenopause is associated with a signiﬁcant reduction in the occurrence of osteoporotic hip fracture (38). Some studies have suggested that ﬁve years of estrogen therapy may be sufﬁcient, but not as effective as 10 years. By age 75, a time when most women who have begun postmenopausal estrogen therapy have long since discontinued that therapy, a history of ever-use estrogen is no more protective against hip fracture than a history of never-use of estrogen (39). Importantly, current use of estrogen even at age 75, remains protective against hip fracture. Regrettably, prospective studies of the effect of estrogen on fracture are very limited (40,41) and none have included hip fracture as an outcome variable of interest. Controlled clinical trials with raloxifene (42) have however clearly demonstrated the effectiveness of this SERM in reducing the rate of osteoporotic vertebral fractures (the studies were not designed to study the effect on hip fracture). It is enticing to extrapolate from this to conclude that controlled trials with estrogen in which fracture is the primary outcome variable of interest would be equally positive. Such an extrapolation would be premature. Nonetheless the most logical conclusion from all of this is that maximum beneﬁt from estrogen replacement therapy for the prevention of osteoporotic fragility fractures will be derived in the woman who starts estrogen as early as possible after the menopause and continues with that estrogen therapy for as long as possible, preferably throughout the remainder of her life.

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11. Nelson DA, Simpson PM, Johnson CC, Barondess DA, Kleerekoper M. The accumulation of whole body skeletal mass in third- and fourth-grade children: effects of age, gender, ethnicity, and body composition. Bone 1997;20:73–78. 12. Gilsanz V, Roe TF, Mora S, Costin G, Goodman WG. Changes in vertebral bone density in black girls and white girls during childhood and puberty. N Engl J Med 1991;325:1597–1600. 13. Gilsanz V, Skaggs DL, Kovanlikaya A, Sayre J, Lore ML, Kaufman F, Korenman SG. Differential effects of race on the axial and appendicular skeletons of children. J Clin Endocrinol Metab 1998; 83:1420–1427. 14. Takahashi Y, Minamitani K, Kobayashi Y, Minagawa M, Tasuda T, Niimi H. Spinal and femoral bone mass accumulation during normal adolescence: comparison with female patients with sexual precocity and with hypogonadism. J Clin Endocrinol Metab 1996;81:1248–1253. 15. Theintz G, Buchs B, Rizzoli R, Slosman D, Clavien H, Sizonenko PC, Bonjour JP. Longitudinal monitoring of bone mass accumulation in healthy adolescents: evidence for a marked reduction after 16 years of age at the levels of lumber spine and femoral neck in female subjects. J Clin Endocrinol Metab 1992;79:1060–1065. 16. Holmes SJ, Shalet SM. Role of growth hormone and sex steroids in achieving and maintaining normal bone mass. Horm Res 1996;45:86–93. 17. Rochira V, Faustini-Fustini M, Balestrieri A, Carani C. Estrogen replacement therapy in a man with congenital aromatase deﬁciency: effects of different doses of transdermal estradiol on bone mineral density and hormonal parameters. J Clin Endocrinol Metab 2000;85:1841–1845. 18. Schot LP, Schuurs AH. Pathophysiology of bone loss in castrated animals. J Steroid Biochem Mol Biol 1990;37:461–465. 19. Ershler WB, Harman SM, Keller ET. Immunologic aspects of osteoporosis. Dev Comp Immunol 1997;21:487–499. 20. Stein B, Yang MX. Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-kappa B and C/EBP beta. Mol Cell Biol 1995;15:4971–4979. 21. Lindsay R, Hart DM, Forrest C, Baird C. Prevention of spinal osteoporosis in oophorectomized women. Lancet 1980;2:1151–1154. 22. Slemenda C, Longcope C, Peacock M, Hui S, Johnston CC. Sex steroids, bone mass, and bone loss: A prospective study of pre-, peri-, and postmenopausal women. J Clin Invest 1996;97:14–21. 23. Riggs BL, Melton LJ. Medical process series: Involutional osteoporosis. N Engl J Med 1986;314:1676– 1686. 24. Han ZH, Palnitkar S, Rao DS, Nelson D, Parﬁtt AM. Effects of ethnicity and age or menopause on the remodeling and turnover of iliac bone: implications for mechanisms of bone loss. J Bone Miner Res 1997;12:498–508. 25. Cummings SR, Browner WS, Bauer D, Stone K, Ensrud K, Jamal S, Ettinger B. Endogenous hormones and the risk of hip and vertebral fractures among older women. N Engl J Med 1998;339:733–738. 26. Chapurlat RD, Garnero P, Breart G, Meunier PJ, Delmas PD. Serum estradiol and sex hormonebinding globulin and the risk of hip fracture in elderly women: the EPIDOS study. J Bone Miner Res 2000;15:1835–1841. 27. Stepan JJ, Lachman M, Zverina J, Packovsky V. Castrated men exhibit bone loss: effect of calcitonin treatment on biochemical indices of bone remodeling. J Clin Endocrinol Metab 1989;69:523–527. 28. Riggs BL, Khosla H, Melton LJ. A unitary model for involutional osteoporosis: Estrogen deﬁciency causes both type I and type II osteoporosis in postmenopausal women and contributes to bone loss in aging men. J Bone Miner Res 1998;13:763–773. 29. Carlsen CG, Soerensen TH, Eriksen EF. Prevalence of low serum estradiol levels in male osteoporosis. Osteoporosis Int 2000;11:697–701. 30. Zofkova I, Bahbouh R, Hill M. The pathophysiological implications of circulating androgens on bone mineral density in a normal female population. Steroids 2000;65:857–61. 31. Falch JA, Oftebro H, Haug E. Early postmenopausal bone loss is not associated with a decrease in circulating levels of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D or vitamin D binding protein. J Clin Endocrinol Metab 1987;64:836–841. 32. Anonymous. Effects of hormone therapy on bone mineral density: results of the postmenopausal estrogen/progestin interventions (PEPI) trial. The writing group for PEPI. JAMA 1996;276:1389–1396. 33. Greendale GA, Wells B, Marcus R, Barrett-Connor E. For the Postmenopausal Estrogen/Progestin Interventions Trial Investigators. How many women lose bone mineral density while taking hormone

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39. 40. 41. 42.

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replacement therapy? Results from the postmenopausal estrogen/progestin interventions trial. Arch Intern Med 2000;160:3065–3071. Rosen CJ, Chesnut CH, Mallinak NJ. The predictive value of biochemical markers of bone turnover for bone mineral density in early postmenopausal women treated with hormone replacement or calcium supplementation. J Clin Endocrinol Metab 1997;82:1904–1910. Fritsch M, Jordan VC. Long-term Tamoxifen Therapy for the Treatment of Breast Cancer. Cancer Control 1994;1:356–366. Prestwood KM, Gunness M, Muchmore DB, Lu Y, Wong M, Raisz LG. A comparison of the effects of raloxifene and estrogen on bone in postmenopausal women. J Clin Endocrinol Metab 2000; 85:2197–2202. Looker AC, Orwoll ES, Johnston CC Jr, Lindsay RL, Walner HW, Dunn WL, et al. Prevalence of low femoral bone density in older U.S. adults from NHANES III. J Bone Miner Res 1997;12:1761–1768. Michaelsson K. Baron JA, Farahmand BY, Johnell O, Magnusson C, Persson PG, et al. Hormone replacement therapy and risk of hip fracture: population based case-control study. The Swedish hip fracture study group. BMJ 1998;316:1858–1863. Felson DT, Zhang Y, Hannan MT, Kiel DP, Wilson PW, Anderson JJ. The effect of postmenopausal estrogen therapy on bone density in elderly women. N Engl J Med 1993;329:1141–1145. Nachtigall LE, Nachtigall RH, Nachtigall RD, Beckman EM. Estrogen replacement therapy I: a 10year prospective study in the relationship to osteoporosis. Obstet Gynecol 1979;53:277–81. Lufkin EG, Riggs BL. Three-year follow-up on effects of transdermal estrogen. Ann Intern Med 1996;125:77. Ettinger B, Black DM, Mitlak BH, Knickerbocker RK, Nickelsen T, Genant HK, et al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3-year randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators. JAMA 1999;282:637–45.

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Effects of SERMs on Bone in Clinical Studies Aurelie Fontana, MD and Pierre D. Delmas, MD, PHD Contents The Effects of Tamoxifen on Bone Metabolism The Effects of Raloxifene on Bone Metabolism Hypotheses on the Mechanism of Action to Decrease Skeletal Fragility Conclusion References

Osteoporosis is a disease characterized by low bone mass and microarchitectural deterioration of bone tissue resulting in increased bone fragility and in an increase in fracture risk (1). The menopause induces an accelerated bone loss within ﬁve years, followed by a linear rate of bone loss that may accelerate after the age of 75 years. Hormone-replacement therapy (HRT) prevents postmenopausal bone loss but its longterm use is probably necessary to reduce the risk of fragilities fractures, as most of them occur after the age of 60 years (2). Long-term compliance to HRT however is limited by side effects such as uterine bleeding and breast tenderness and by the fear of breast cancer, the risk of which appears to increase after prolonged treatment (3). The clinical interest in SERMs in the management of osteoporosis is related to these limitations of HRT. The concept of SERMs is derived from the observation that tamoxifen, used in breast cancer for its antiestrogen effects on breast tissue, has estrogenlike effects on the skeleton and lipoproteins. Although tamoxifen has an excellent beneﬁt/risk ratio as an adjuvant treatment in breast cancer, its use in healthy postmenopausal women is questionable because of its increased risk of endometrial cancer. Several synthetic compounds have been developed and some of them appear to be devoid of the undesirable estrogen effects on breast and endometrial tissue. Among these compounds, raloxifene has undergone extensive clinical investigation that will be reviewed. Preclinical studies on ovariectomized rats have shown that raloxifene preserves bone mass (4,5) and increases bone strength in ovariectomized rats. There was no signiﬁcant difference in bone strength of the femoral neck, femur, and lumbar spine after estrogen or raloxifene treatment (6). From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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THE EFFECTS OF TAMOXIFEN ON BONE METABOLISM Effects on Bone Turnover Data on the effects of tamoxifen on bone turnover in healthy women are limited. A small study on 10 healthy elderly women (70–85 years of age) receiving tamoxifen 20 mg/d for 10 weeks, has shown a decrease in the urinary excretion of pyridinoline (Pyr) and deoxypyridinoline (DPyr) by 23% and 25% respectively. These bone resorption markers returned to baseline values after therapy was discontinued (7). In a recent study, 30 postmenopausal women with breast cancer operated on six to eight weeks earlier were treated either with toremifene (40 mg/d) or with tamoxifen (20 mg/d) (8). At six months and 12 months, there was a signiﬁcant decrease of the urinary Pyr of about 20% and 30% respectively in the tamoxifen-treated group. In the toremifene group the decrease of urinary Pyr was similar at six months but signiﬁcantly less at 12 months (10%) when compared with the tamoxifen group (30%). The decrease of urinary Dpyr was about 20% at 6 and 12 months during tamoxifen use and respectively 15% and 5% during toremifene therapy with no signiﬁcant difference between the two treatments. The same authors have studied other markers of bone turnover in a similar population (9). At 12 months of treatment, the decrease was about 40%, 25%, and 22% for urinary crosslinked aminoterminal telopeptide of type I collagen (NTX), osteocalcin and serum aminoterminal propeptide of type I collagen (PINP) in the tamoxifen group. The decrease in bone turnover markers was signiﬁcantly less in the toremifene treated group and neither tamoxifen nor toremifene induced signiﬁcant changes in bone alkaline phosphatase (ALP), and serum crosslinked carboxyterminal telopeptide of type I collagen (ICTP). In summary, these data suggest that tamoxifen acts as a partial estrogen agonist on bone and reduces bone turnover in postmenopausal women.

Effects on BMD In Premenopausal Women A large trial in healthy pre- and postmenopausal women has evaluated the toxicity and feasibility of tamoxifen for chemoprevention of breast cancer. Women were randomized to receive either tamoxifen (20 mg/d) or placebo for eight years. Total hip and lumbar spine BMD was available from 179 women (10,11). In women who remained premenopausal during the trial (n = 125), lumbar spine BMD signiﬁcantly decreased from baseline in the tamoxifen-treated group (about 3.3% ± 1.6 at three years) and was signiﬁcantly lower in women on tamoxifen compared with women on placebo at 1, 2, and 3 years of treatment. Total hip BMD was signiﬁcantly decreased from baseline at three years of treatment only (1.6% ± 1.4) in women treated with tamoxifen contrasting with a signiﬁcant increase in the placebo group (2.7% ± 2.3 at three years). Compared with the placebo, total hip BMD was signiﬁcantly lower in the tamoxifen group at two and three years of treatment. Thus, in premenopausal women tamoxifen acts as an antiestrogen on bone tissue resulting in bone loss. In Postmenopausal Women Several studies have shown a prevention of bone loss in postmenopausal patients with breast cancer treated by tamoxifen compared with healthy postmenopausal controls suggesting an estrogenlike effect on bone despite antiestrogenic activity on the breast

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(12,13). In a large prospective, double masked, randomized, controlled study Love et al. (14) have shown an increased BMD at the spine (1.2% at 2 years) in breast cancer patients treated with tamoxifen (20 mg/day) compared with a decreased BMD in the placebo group (2% at two years). The rate of bone loss at the radius did not differ between the two groups. These results were maintained at ﬁve years of treatment (15). A small study of about 30 postmenopausal patients with a nonmetastatic breast cancer treated either by tamoxifene (20 mg/d) or by toremifene (40 mg/d) has shown after 12 months of treatment a mean increase in BMD of 2% at the lumbar spine and 1% at the femoral neck (9). Tamoxifen has also been shown to reduce bone loss at the spine and hip by about 50% in patients with a recent artiﬁcial menopause caused by adjuvant chemotherapy of breast cancer, whereas the use of cyclic treatment with risedronate, a new bisphosphonate, resulted in an increased BMD in these women (16). In healthy postmenopausal women, tamoxifen has induced an increase in lumbar spine BMD of 1.5% compared to placebo (17,18). In the tamoxifen chemoprevention of breast cancer trial (11), BMD assessment was available in 59 postmenopausal women treated either with tamoxifen 20 mg/d or placebo. BMD increased by 1.2% and 1.7% per year at the lumbar spine and total hip respectively, contrasting with a nonsigniﬁcant decrease in placebo-treated women. In summary, tamoxifen induces an increase in lumbar spine and total hip BMD of 1% to 2% in postmenopausal women.

Effects of Fractures A large placebo-controlled study (19) using tamoxifen (20 mg/day) in the prevention of breast cancer in more than 13,000 patients at high risk showed a nonsigniﬁcant reduction of about 20% in the overall incident of hip, Colle’s, and spine fracture. Because the population was not selected on the basis of a low BMD, the number of fracture events was quite low and therefore the study was underpowered to show a reduction of osteoporotic fractures. Love et al. (15) have reported no signiﬁcant difference in the rate of fracture between tamoxifen (20 mg/d) and placebo after 5 years of treatment in postmenopausal women with breast cancer but, again, the number of events was low (respectively 7 vs 10 fractures). In conclusion, tamoxifen has an estrogenlike effect on the skeleton in postmenopausal women, reducing bone turnover markers and preventing bone loss, with an overall effect that appears to be less pronounced than with estrogen-replacement therapy. The effect of tamoxifen on the incidence of fragility fractures has not been adequately studied.

THE EFFECTS OF RALOXIFENE ON BONE METABOLISM Effects on Bone Turnover Short-term effects of raloxifene on bone turnover have been reported in 250 healthy postmenopausal women receiving either placebo, raloxifene 200 mg or 600 mg, or conjugated equine estrogens 0.625 mg once daily (20). After eight weeks of treatment, a signiﬁcant decrease was observed in serum alkaline phosphatase, serum osteocalcin, urinary pyridinoline, and urinary calcium with the two doses of raloxifene. Changes in the bone turnover markers were not signiﬁcantly different between the raloxifeneand estrogen-treatment groups. A recent randomized double blind study compared markers of bone turnover in postmenopausal patients treated with raloxifene (60 mg/d) or conjugated equine estrogen (0.625 mg/d) (21). After 24 weeks of treatment, all

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Fig. 1. Median percent change (± standard error) in serum osteocalcin and urinary type I collagen C-telopeptide to creatinine in postmenopausal women treated with raloxifene or placebo for two years. From ref. (22).

markers of resorption and formation decreased signiﬁcantly from baseline in the two groups except for deoxypyridinoline. Changes were signiﬁcantly different between the two groups with a marked decreased in urinary type I C-telopeptide (CTX) and NTX in the estrogen-treated women (respectively 53% and 43% from baseline) compared with a decrease of 23% and 22% in CTX and NTX in raloxifene-treated women. For bone formation markers (osteocalcin, bone alkaline phosphatase, and C-terminal type I procollagen peptide), the mean changes were also signiﬁcantly greater in the estrogentreated group. Bone turnover was assessed in a study including 600 postmenopausal women without osteoporosis randomly assigned to receive 30, 60, or 150 mg of raloxifene or placebo daily for 24 months. Each of the three groups of raloxifene had a signiﬁcant decrease of serum osteocalcin, bone alkaline phosphatase (BAP) and CTX compared with the placebo (22). Bone turnover decreased to premenopausal levels with a reduction of serum osteocalcin, BAP and CTX of 23%, 15% and 34% respectively with the 60-mg dose (Fig. 1). Similar effects on bone turnover have been found in osteoporotic patients treated with raloxifene (23,24). The Multiple Outcomes of Raloxifene Evaluation (MORE) study has assessed BMD and risk fractures in 7705 postmenopausal women deﬁned

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Fig. 2. Percentage change of bone mineral density in postmenopausal osteoporotic women treated for three years by raloxifene 120 mg/d (■), 60 mg/d (▲) or placebo (❍). Adapted from ref. 23.

as low BMD or vertebral fractures (24). Women were randomized to receive placebo or 60 or 120 mg of raloxifene daily. After 36 months of treatment, serum osteocalcin decreased by a median of 8.6%, 26.3% and 31.1% and the urinary CTX decreased by 8.1%, 34%, and 31.5% respectively in the placebo, 60 mg and 120 mg raloxifene groups. The decrease was signiﬁcant in both raloxifene groups compared with the placebo.

Effects on BMD In the same multicentric European study including 600 early postmenopausal women (55 years old) without osteoporosis treated by 30, 60, or 150 mg of raloxifene or placebo during 24 months, raloxifene prevented bone loss at all skeletal sites at the three doses with a 2.4% increase in BMD at the lumbar spine and total hip when compared to placebo in the 60-mg raloxifene group (22). In 129 postmenopausal osteoporotic women (50–75 years of age) treated for two years with 60 or 120 mg raloxifene or placebo, BMD increased by 3.2% at the lumbar spine and 2.1% and 1.6% respectively in the femoral neck and total hip with the 60mg dose (25). In the MORE study (24) including 7705 postmenopausal women with osteoporosis, BMD increased signiﬁcantly compared with the placebo by 2.1% and 2.6% at the femoral neck and spine in the 60-mg raloxifene group and respectively by 2.4% and 2.7% in the 120-mg raloxifene group after 36 months of treatment (Fig. 2). In a recent study that compared the skeletal effects of raloxifene (60 mg/d) with conjugated equine estrogen (0.625 mg/d) over 24 weeks, the increase in lumbar-spine and total-body BMD was twice as high for conjugated estrogen than for raloxifene. At the proximal femur, the increase in BMD was not signiﬁcantly different between both groups (21).

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Fig. 3. Reduction in new vertebral fractures in 6828 postmenopausal osteoporotic women treated by raloxifene or placebo over three years. RR = relative risk, CI = conﬁdence interval. From Lufkin EG, Whitaker MD, Nickelson T, et al. Treatment of established postmenopausal osteoporosis with raloxifene: a randomized trial. J Bone Miner Res 1998;13:1747–1754.

Effects on Fractures The MORE study has assessed risk fractures in 7705 postmenopausal women deﬁned as low BMD or vertebral fractures (24). Women were randomized to receive placebo or 60 or 120 mg of raloxifene daily. All patients received daily supplements of 500 mg of calcium and 400 to 600 IU of cholecalciferol. Women were divided into two groups. Group 1 included women with a T score below −2.5 at the femoral neck or lumbar spine, without prevalent vertebral fractures. Group 2 included women with low BMD and one vertebral fracture, or 2 or more vertebral fractures regardless of the BMD level. All women had vertebral X rays at baseline, 24 and 36 months. X rays were also performed in case of a painful syndrome suggestive of a recent vertebral fracture. Baseline and follow-up X-rays were available in 6828 women (88% at endpoints). After 36 months of treatment, the risk of new vertebral fracture deﬁned by vertebral morphometry was reduced in both groups receiving raloxifene with a relative risk of 0.7 (95%CI: 0.5–0.8) with 60 mg and 0.5 (95%CI: 0.4–0.7) with 120 mg. Although the absolute risk was about fourfold higher in women with prevalent vertebral fractures, the reduction with raloxifene was signiﬁcant both in women with and without prevalent vertebral fractures (Fig. 3). With the recommended daily dose of 60 mg, the relative risks were 0.7 (95%CI; 0.6–0.9) and 0.5 (95%CI: 0.4–0.8) in women with and without prevalent vertebral fractures respectively. Overall there was no difference in the incidence of fractures in the 120-mg raloxifene group and in the 60-mg group (5.4% and 6.6% respectively). There was, however, a lower incidence of vertebral fractures in

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the 120-mg raloxifene group in women with prevalent vertebral fractures but not in women without such fractures. The overall occurrence of nonvertebral fractures was low, with a three-year incidence of 9.3% in the placebo group and 8.5% in the raloxifene-treated women. There was a 10% reduction with raloxifene that did not reach the level of signiﬁcance (RR, 0.9; 95%CI: 0.8–1.1). The cumulative incidence curve of nonvertebral fractures is lower with raloxifene than with placebo after 18 months of treatment, a trend that needs to be assessed in the followup of this study.

HYPOTHESES ON THE MECHANISM OF ACTION TO DECREASE SKELETAL FRAGILITY The strong relationship between bone mass and the risk of fracture led to a deﬁnition of osteoporosis that does not require fractures and to the World Health Organization threshold (T score < −2.5) for the diagnosis of osteoporosis (26,27). Nonetheless, the relationship between the magnitude of the increase in BMD and the change in fracture risk during therapy is a matter of controversy, because of unexpected ﬁndings in clinical trials. First, despite a marked increase of BMD at the lumbar spine, ﬂuoride does not appear to decrease signiﬁcantly the incidence of vertebral fractures at either high (28) or low doses (29). Second, the bisphosphonate alendronate was found to induce a marked 50% reduction of vertebral and nonvertebral fractures despite a relatively small increase in BMD ranging from 2% to 8% depending on the skeletal site (30). Third, raloxifene was found to decrease incidence of vertebral fracture by 30–50%, despite an even smaller 2.6% increase of spinal BMD (24). These results suggest that the increase in BMD may not fully explain fracture reduction and that other mechanisms may be involved, as recently reviewed (31). Prevalent fractures, especially of the spine, and the rate of bone turnover assessed by markers of bone resorption, predict fracture risk independent of the level of BMD in untreated women (32,33) and may inﬂuence the ability of antiresorptive therapy, especially raloxifene, to decrease vertebral fracture incidence in osteoporosis. Although the percentage of vertebral-fracture reduction from placebo is of comparable magnitude in patients with or without prevalent vertebral fractures, the actual incidence of vertebral fractures in patients treated with either alendronate or raloxifene is ﬁve times higher in those with prevalent vertebral fractures, despite comparable increases in BMD for a given drug (24,30). Patients with fractures probably have marked abnormalities of bone architecture, especially in the trabecular envelope, which are not captured by BMD measurement, as suggested by bone histomorphometry (34). The role of baseline bone turnover and of changes in bone turnover in predicting the antifracture efﬁcacy of bone resorption inhibitors has not been adequately studied. Antiresorptive drugs induce a rapid decrease of resorption markers reaching a plateau after three months, and a slower decrease of formation markers that reach a plateau after 6 to 12 months. The most relevant question, however, is to know if the reduction of bone turnover predicts the risk of fracture under therapy, independent of changes in BMD. The rapid effect of antiresorptive therapy on fracture rate is probably related to the inhibition of trabecular plate perforation and of loss of connectivity caused by decreased osteoclastic activity, which may in turn be reﬂected by the magnitude of the decrease of resorption markers. In the MORE study, patients treated with raloxifene

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who showed the largest decrease of serum osteocalcin and bone alkaline phosphatase at six months had the lowest incidence of vertebral fractures over three years (35). In contrast, the change of BMD is poorly predictive as shown recently (36). These data suggesting that the short-term decrease of bone turnover can predict the long-term antifracture efﬁcacy of raloxifene need to be conﬁrmed. The mechanisms by which antiresorptive therapy, in particular SERMs, reduce skeletal fragility is explained only partially by increases in BMD. Prevalent fractures remains an important determinant of fracture risk under therapy and the decrease of bone turnover may be an independent predictor of the reduction of fracture risk with raloxifene. Other mechanisms may be important and should be explored in preclinical models.

CONCLUSION Raloxifene, the ﬁrst of the second-generation SERMs to be widely available, represents a signiﬁcant improvement over tamoxifen in its effects on bone. It prevents postmenopausal bone loss and reduces the incidence of vertebral fractures. The extension of the MORE study at ﬁve years will tell whether or not raloxifene also decreases the incidence of nonvertebral fractures. SERMs represent a new and promising class of agents for the management of postmenopausal women’s health. Data will be necessary in patients with high risk of breast cancer in whom SERMs could be an alternative for the prevention and treatment of osteoporosis. Finally, the recent observation that estrogens may play a role in bone metabolism of men (37) and that SERMs prevent bone loss and induce a decrease in total serum cholesterol without affecting the prostate in orchidectomized male rats (38) raises the possibility that they also may be of interest for the treatment of elderly men.

REFERENCES 1. Kanis JA, Melton LJ III, Christiansen C, Johnston CC, Khaltaev N. The diagnosis of osteoporosis. J Bone Miner Res 1994;9:1137–41. 2. Felson DT, Zhang Y, Hannan MT, et al. The effect of postmenopausal estrogen therapy on bone density in elderly women. N Engl J Med 1993;329:1141–1146. 3. Collaborative Group on Hormonal therapy in breast cancer. Breast cancer and hormone replacement therapy: collaborative reanalysis of data from 51 epidemiological studies of 52,705 women with breast cancer and 108,411 women without breast cancer. Lancet 1997;350:1047–59. 4. Sato M, McClintock G, Kim J, Turner CH, et al. Dual-energy x-ray absorptiometry of raloxifene effects on the lumbar vertebrae and femora of ovariectomized rats. J Bone Miner Res 1994;9:715–24. 5. Black LJ, Sato M, Rowley ER, et al. Raloxifene (LY139481 HCL) prevents bone loss and reduces serum cholesterol without causing uterine hypertrophy in ovariectomized rats. J Clin Invest 1994;93:63–69. 6. Turner CH, Sato M, Bryant HU. Raloxifene preserves bone strength and bone mass in ovariectomized rats. Endocrinol 1994;135:2001–2005. 7. Kenny AM, Prestwood KM, Pilbeam CC, Raisz LG. The short-term effects of tamoxifen on bone turnover in older women. J Clin Endocrinol Metab 1995;80:3287–3291. 8. Marttunen MB, Hietanen P, Titinen A, et al. Effects of tamoxifen and toremifene on urinary excretion of pyridinoline and deoxypyridinoline and bone density in postmenopausal patients with breast cancer. Calcif Tissue Int 1999;65:365–368. 9. Marttunen MB, Hietanen P, Titinen A, Ylikorkala O. Comparison of effects of tamoxifen and toremifene on bone biochemistry and bone mineral density in postmenopausal breast cancer patients. J Clin Endocrinol Metab 1998;83:1158–1162. 10. Powles TJ, Hardy JR, Ashley SE, et al. A pilot trial to evaluate the acute toxicity and feasibility of tamoxifen for prevention of breast cancer. Br J Cancer 1989;60:126–33.

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11. Powles TJ, Hickish T, Kanis JA, et al. Effect of tamoxifen on bone mineral density measured by dual energy X-Ray absorptiometry in healthy premenopausal and postmenopausal women. J Clin Oncol 1996;14:78–84. 12. Turken S, Siris E, Seldin D, et al. Effects of tamoxifen on spinal bone density in women with breast cancer. J Natl Cancer Inst 1989;81:1086–88. 13. Kristensen B, Ejlertsen B, Dalgaard P, et al. Tamoxifen and bone metabolism in postmenopausal lowrisk breast cancer patients: a randomized study. J Clin Oncol 1994;12:992–997. 14. Love RR, Mazess RB, Barden HS et al. Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer. N Engl J Med 1992;326:852–856. 15. Love RR, Barden HS, Mazess RB, Epstein S, Chappell RJ. Effect of tamoxifen on lumbar spine bone mineral density in postmenopausal women after 5 years. Arch Intern Med 1994;154:2585–2588. 16. Delmas PD, Balena R, Confavreux E, et al. Bisphosphonate Risedronate prevents bone loss in women with artiﬁcial menopause due to chemotherapy of breast cancer: a double blind, placebo-controlled study. J Clin Oncol 1997;15:955–962. 17. Chang J, Powles TJ, Ashley SE, et al. The effect of tamoxifen and hormone replacement therapy on serum cholesterol bone mineral density and coagulation factors in healthy postmenopausal women participating in a randomised, controlled tamoxifen prevention study. Annals Oncol 1996;671–675. 18. Grey AB, Stapleton JP, Evans MC, et al. The effect of antiestrogen tamoxifen on bone mineral density in normal late postmenopausal women. Am J Med 1995;99:636–41. 19. Fisher B, Costantino JP, Wickerham DL, et al. Tamoxifen for prevention of breast cancer: report of the national surgical adjuvant breast and bowel project P-1 study. J Natl Cancer Inst 1998;90:1371–1388. 20. Draper MW, Flowers DE, Huster WJ, Neild JA, Harper KD, Arnaud C. A controlled trial of raloxifene (LY139481) HCl: Impact on bone turnover and serum lipid proﬁle in healthy postmenopausal women. J Bone Miner Res 1996;11:835–842. 21. Prestwood KM, Gunness M, Muchmore DB, Lu Y, Wong M, Raisz LG. A comparison of the effects of raloxifene and estrogen on bone in postmenopausal women. J Clin Endocrinol Metab 2000; 85:2197–2202. 22. Delmas PD, Bjarnasson NH, Mitlak BH, et al. Effects of raloxifene on bone mineral density, serum cholesterolconcentrations, and uterine endometrium in postmenopausal women. N Engl J Med 1997; 337:1641–47. 23. Lufkin EG, Whitaker MD, Nickelson T, et al. Treatment of established postmenopausal osteoporosis with raloxifene: a randomized trial. J Bone Miner Res 1998;13:1747–1754. 24. Ettinger B, Black DM, Mitlak BH, et al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene. JAMA 1999;282:637–645. 25. Meunier PJ, Vignot E, Garnero P, et al. Treatment of postmenopausal women with osteoporosis or low bone density with raloxifene. Osteoporisis Int 1999;10:330–336. 26. Anonymous. Consensus development conference: Diagnosis, prophylaxis and treatment of osteoporosis. Am J Med 1993;94:646–650. 27. World Health Organization. Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. WHO technical report series 843. Geneva: WHO; 1994. 28. Riggs BI, Hodgson SF, O’Fallon WM, et al. Effects of ﬂuoride treatment on the fracture rate in postmenopausal women with osteoporosis. N Engl J Med 1990;322:802–809. 29. Meunier PJ, Sebert JL, Reginster JY, et al. Fluoride salts are no better at preventing new vertebral fractures than calcium-vitamin D in postmenopausal osteoporosis: The FAVOS study. Osteoporosis Int 1998;8:4–12. 30. Black DM, Cummings SR, Karpf DB, et al. Randomised trial of effects of alendronate on risk of fracture in women with existing vertebral fractures. Lancet 1996;348:1535–1541. 31. Delmas PD. How does antiresorptive therapy decrease the risk of fracture in women with osteoporosis? Bone 2000;27:1–3. 32. Garnero P, Hausherr E, Chappuis MC, et al. Markers of bone resorption predict hip fracture in elderly women: The EPIDOS prospective study. J Bone Miner Res 1996;11:1531–1538. 33. Ross PD, Davis JW, Epstein RS, Wasnich RD. Pre-existing fractures and bone mass predict vertebral fracture incidence in women. Ann Intern Med 1999;114:919–923. 34. Parﬁtt AM, Mathews CHE, Villanueva AR, et al. Relationship between surface, volume and thickness of iliac trabecular bone in aging and in osteoporosis: Implications for the microanatomic and cellular mechanism of bone. J Clin Invest 1983;72:1396–1409. 35. Bjarnasson NH, Christiansen C, Sarkar S, et al. Six months change in biochemical markers predict

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3-year response in vertebral fracture rate in postmenopausal osteoporotic women: Results from the MORE study. J Bone Miner Res 1999;14 (Suppl 1):S157. 36. Bjarnasson NH, Christiansen C, Duong T, Delmas PD. Pretreatment BMD and vertebral fracture status as well as change in osteocalein are all predictors for the risk of incident vertebral fracture during raloxifene therapy. Osteoporosis Int 2000;11 (Suppl 2):S173. 37. Riggs B, Khosla S, Melton LJ. A unitary model for involuntional osteoporosis: Estrogen deﬁciency causes both type I and type II osteoporosis in postmenopausal women and contributes to bone loss in aging men. J Bone Miner Res 1998;13:763–773. 38. Ke QZ, Qi H, Crawford DT, et al. Lasofoxifene (CP-336,156), a selective estrogen receptor modulator, prevents bone loss induced by aging and orchidectomy in the adult rat. Endocrinology 2000;141:1338– 1344.

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Estrogens and SERMs Clinical Aspects of Cognition with Aging and Neurodegenerative Disorders

Alan J. Lerner,

MD

Contents Introduction Estrogens, Aging, and Cognition Alzheimer’s Disease Parkinson’s Disease Other Neurodegenerative Disorders References

INTRODUCTION The effects of estrogens in the central nervous system (CNS) are pleiotropic, with both direct and indirect effects, and their inﬂuence is felt in diseases that range from developmental chromosomal abnormalities to conditions that manifest in the later decades of life. This chapter reviews the clinical aspects of these interactions in neurodegenerative diseases, although estrogen effects have been described in many diseases of the CNS.

ESTROGENS, AGING, AND COGNITION Hormone-replacement therapy (HRT) is one of the most common medical therapies employed in the United States. It has gained popularity in part from recognition that many disorders associated with aging including osteoporosis, coronary artery disease, and aging of the endocrine system may be alleviated by HRT. Rates of usage of HRT vary by region, degree of education, and prior hysterectomy. These usage factors however, may not always correlate with usage in “at-risk” individuals (1). Interest in the cognitive effects of estrogen-replacement therapy (ERT) has been investigated in a number of human models since the 1950s. In surgically or naturally menopausal women, ERT has been associated with enhanced short-term and longterm memory, and increased capacity for learning paired associations. Visual memory, however, does not improve with ERT (2). In women aged 32 to 36 years old with

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uterine myomas given a gonadotropin-releasing hormone agonist (GnRH-a) for 12 weeks, verbal memory scores declined signiﬁcantly. This was followed by GnRH-a with estrogen or placebo, with the former group showing improvements to baseline verbal scores, but continued poorer performance in the GnRH-a and placebo cohort. These results suggest that estrogen deprivation effects are reversible with replacement, at least in the short term (3). Serum levels of endogenous estrogens are not consistently correlated with cognitive performance in older women (4). Animal studies using paradigms such as ovariectomized animals and standardized behavioral tests have shown a wide variety of interactions of estrogens with cholinergic, dopaminergic, and serotoninergic neurotransmitter systems important for modulating memory, mood, and affect (5–9). Estrogens also facilitate hippocampal neuronal long-term potentiation, a model for memory formation (10). Estrogens also protect against memory-impairing compounds such as anticholinergics and benzodiazepines (11–13), and reduce neuronal responses to oxidative stress (14). Human studies have shown conﬂicting results in longitudinal and crosssectional studies, with most studies showing only a modest effect on selected neuropsychological variables such as explicit memory or visual/spatial skills, but not immediate memory (15–19). It is likely that the memory-promoting effects of acute estrogen replacement derive from its pleiotropic action in the central nervous system, including interactions with estrogen receptors with changes in gene expression, as well as upregulation of other neurotransmitter systems, as indicated by extensive data from animal models. Estrogen may also affect cerebral blood ﬂow. Using Positron Emission Tomography (PET), postmenopausal women receiving estrogen were shown to have increased activation in the inferior parietal lobule while storing verbal material and decreased activation of that area during storage of nonverbal material. This was not reﬂected clinically however, in terms of enhanced performance. PET scans performed in subjects in the Baltimore Longitudinal Study on Aging showed increased right-hemisphere activation in women on ERT, who also performed better on neuropsychological tests of ﬁgural and verbal memory (19–21). By contrast, in Alzheimer’s disease patients, cerebral blood ﬂow as measured by single photon emission computed tomography scan showed no differences after 12 weeks of conjugated estrogens (22). Consistent with the variability of the studies of direct hormone replacement on cognition are results from crosssectional and longitudinal epidemiological studies. In the Rancho Bernardo studies (23), the age-related decrement in cognitive function, adjusted for age and education, showed no effect of estrogen treatment. A later study (24) showed no gender differences in cognitive decline by age between men and women never on HRT, providing no support for the concept that HRT may be trophic in terms of preserving cognition. Indeed, in a retrospective analysis of this population looking at serum hormone levels, endogenous estrogen levels did not show any difference on cognitive testing, whereas higher levels of testosterone predicted better performance on the Mini-Mental Status examination, especially the attentional task (25,26). Similarly, a nested multicenter study for osteoporosis found no relation of serum estradiol to cognitive score, but high estrone levels were actually associated with lower digit symbol scores (4). Studies in more homogeneous populations have shown somewhat different results. In a study of women in Utah (90% of whom were Mormon, who do not use tobacco

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or ethanol, and have low rates of cardiovascular disease and cancer), Steffens et al. (27) found that non-use of HRT was an independent risk factor for lower cognitive test scores. Japanese-American women who were using unopposed estrogens had a modest beneﬁt in age-related cognitive change, especially compared to women using estrogen/progestin combinations (28).

ALZHEIMER’S DISEASE Alzheimer’s disease (AD) is the most common neurodegenerative disease associated with aging. It can also be classiﬁed as a complex polygenetic disease, such as hypertension, coronary artery disease, or schizophrenia as there are both genetic and environmental factors that result in the phenotypic expression of what is clinically called AD. Because of the demographic shifts and remarkable medical advances of the last century, aging populations are now the fastest-growing portions of the population, and women constitute a majority of this population owing to their increased life expectancy relative to men.

Steroids and Alzheimer’s Disease Evidence for steroid effects in AD include the role of glucocorticoids in mediating damage to hippocampal neurons and the multiple roles implicated for estrogens, from maintaining synapses to enhancing cerebral blood ﬂow. Androgens may have antiglucorticoid effects and may be aromatized to estrogens in the brain. There are no clinical methods for determining human levels of estrogens at the cellular level, but the aromatase effect may spare males from relative estrogen deﬁciency with aging (29). Aromatase distribution is more concentrated in the temporal lobe than the frontal lobe, the former being a brain region important for encoding new memories (30). Astrocytes also express aromatase messenger RNA (mRNA), suggesting another way in which they may support neuronal viability across the lifespan (31). In one autopsy study, however, the levels of testosterone were equal in male and female brains, and AD brains did not differ from controls in estradiol concentration (32).

Structure/Activity Relationships and Neuroprotection Structure and function may play a role in steroids’ ability to act as neuroprotective agents. In a model of neuroprotection involving in vitro cell viability after serum deprivation, only steroids with a phenolic A ring were found to be protective. These compounds include 17β-estradiol, diethylstilbestrol, and compounds such as 17α-estradiol that binds only weakly to estrogen receptors. Testosterone, dihydrotesterone, progesterone, prednisolone, aldosterone, and cholesterol, all lacking a phenolic A ring, had no protective effect in that assay (33). Steroidal structure also affects its antioxidant properties. Estriol and 17β-estradiol have relatively strong antioxidant properties, whereas cortisone and corticosterone are mildly antioxidant in an assay involving generation of peroxy radicals. Testosterone, progesterone, androstenedione, dehydroepiandrosterone (DHEA), and estrone had no antioxidant effect, however (34–36). RU486 has strong antioxidant properties, preventing peroxide accumulation caused by beta-amyloid, hydrogen peroxide, and glutamate in mouse and rat hippocampal neurons (37). Other antioxidants such as alpha-tocopherol and selegiline may slow progression of AD to deﬁned endpoints such as loss of activities

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of daily living (38), but it is unknown whether treatment with any steroids would have the same effect. Using an informatics approach linking estrogens to AD, Smalheiser and Swanson (39) identiﬁed that estrogenic antioxidant activity needed further study. They identiﬁed processes such as estrogen regulation of calbindin D28k, induction of cathepsin D and other proteases, inhibition of Apo E levels, and enhanced neuronal response to glutamate as possible mechanisms related to estrogen effect at the molecular level, at which the link to AD is largely unexplored.

Estrogen Replacement Therapy and AD Risk Besides aging, a large number of environmental risk factors have been investigated in AD. Particular interest has focused on whether certain exposures may be protective, such as the relationship of education to the concept of functional neuronal reserve, expressed as the commonly held idea of “Use it or lose it.” The results of epidemiological studies are inconsistent, but protective risk factors may include postmenopausal ERT, smoking, more education, and chronic exposure to nonsteroidal antiinﬂammatory medications. Positive risk factors include history of depression, head injury (particularly in individuals with the Apo E e4 allele), and possibly thyroid disease. In contrast to the modest ﬁndings in enhancing cognition, ERT does appear to be somewhat protective for developing AD in most studies, and appears to be a doserelated (daily dose and duration of treatment) phenomenon (40–42). Exposure of only one year postmenopausal appears to provide some risk reduction, both in terms of disease development and age of onset, even many years later. The crude odds ratio for ERT exposure in several studies has been about 0.4. The relative risk reduction from ERT may interact with other risk factors such as Apo E genotype, smoking, history of AD in ﬁrst-degree relatives, and age of onset (43). The public health aspects of this have begun to be investigated, and more study is needed to determine whether agents such as SERMs will produce similar effects. Judging from the experience with ERT use in cardiovascular disease and osteoporosis, it will take many years and long-term prospective studies of primary prevention to understand the full implications on a risk/ beneﬁt basis. The impact of AD however is often so catastrophic that further study is necessary. The ﬁndings in the at-risk population differ from the negative studies in established AD (see “Estrogens as Treatment for Alzheimer’s Disease” later). This may reﬂect the long preclinical tail of AD, so that even mild dementia represents widespread failure of neural networks, impervious to the trophic effects of estrogens (44). The biology of how ERT can modify AD development is complex, with several components likely to be interacting. In addition to providing increased blood ﬂow and antioxidant effects, estrogen plays a role in normal brain development, and estrogen receptors are present in brain areas important to memory systems (e.g., hippocampus). Estrogen also induces changes in Apo E mRNA expression, and protects against betaamyloid mediated neurotoxicity and apoptosis (45–47).

Clinical Features of AD The clinical expression of AD is quite variable, with diagnostic criteria requiring a mixture of memory deﬁcits in addition to deﬁcits in other aspects of cognition such as language, praxis, agnosia, or executive function disturbance (e.g., planning, abstraction,

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judgment). Behavioral manifestations are common and include depression, agitation, delusions and hallucinations, or anxiety syndromes. Sleep disorders and purposeless behaviors such as pacing may also occur in individual patients. Few studies have addressed the gender speciﬁcity of clinical expression. Studies of gender difference, and whether estrogens play any role in modifying behavior in AD need to be interpreted cautiously in light of underlying gender differences in lateralization, visuo-spatial and language abilities occurring in normal development. In a longitudinal study of AD patients compared to normal elderly, female AD patients performed worse than male AD patients on tests of naming, vocabulary, and word recognition skills, but not on syntactic measures (48). These deﬁcits persisted during longitudinal followup, and contrast with no gender differences on these measures in normal elderly. Henderson and Buckwalter (49,50) also found gender differences on the Mini-Mental status examination score related to language dysfunction. No gender differences were found using the Blessed test (a cognitive screening instrument), and the rate of deterioration was similar in both sexes (51). The biological basis for this difference in language abilities in AD may be a result of several factors. AD women have a much stronger correlation of disease severity with postdexamethasone cortisol levels than do men (52). Regional cerebral blood ﬂows measured by PET in AD have been described, with women having higher mean nonweighted metabolic rates than men (53). Noncognitive symptoms may also show gender differences. Males tend to have more problems with verbal aggressiveness, preoccupation with bodily function, and apathy. In multiple regression-model analysis of the Dementia Behavior Disturbance Scale, there were no gender differences in behavior severity. Males scored higher however on scales of apathy and vegetative signs, whereas females showed more reclusive behavior and emotional lability with behaviors such as hoarding, refusing help, and inappropriate laughter and crying (54). Differences in baseline behavior may contribute to these ﬁndings and differences in levels of androgens and estrogens may play a role in behavioral expression.

Estrogens as Treatment for Alzheimer’s Disease The major basis for current pharmacological treatment of AD is based on the cholinergic hypothesis. The cholinergic system radiates from basal forebrain nuclei, and degenerates early in AD, and cholinergic markers correlate with disease state. Enhancement of cholinergic function by acetylcholinesterase inhibitors has been demonstrated to improve cognitive functioning compared to placebo, and may have effects on noncognitive symptoms and vegetative symptoms as well (55,56). A retrospective review of data from the 30-week tacrine trial showed the most robust improvements in neuropsychological testing occurred in women with concurrent ERT or recent treatment (within three months of trial enrollment (57). In patients with established cognitive impairment, early open-label studies, and more recent randomized studies using current AD diagnostic criteria showed trends toward improvements in several neuropsychological and behavioral ratings. Many of these studies however have been quite small, and of relatively short duration (less than six months) (58–60). Three larger, longer, multicenter, randomized, placebo-controlled studies in established AD women failed to show any positive beneﬁts of ERT, diminishing hope that ERT may emerge as an adjunctive treatment in AD (22,61,62). One pilot

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study of transdermal estrogen showed positive results after eight weeks, particularly in attentional tasks. The reason for this disparity is unclear, but may relate to higher estrogen bioavailability with transdermal delivery (44,63).

Endocrine Modulation of Behavior in AD A number of case reports have reported use of estrogenic compounds, medroxyprogesterone or leuprolide in demented males. Men are more likely to show physical aggression as well as apathy in the course of their AD (54). In two men treated brieﬂy with diethylstilbestrol, agitation was reported to occur less often (64). Medroxyprogesterone use has been reported in a male with vascular dementia and unwanted sexual behavior. Although positive cognitive effects were not observed, the patient responded successfully after other medications had failed (65). Three demented patients with aggression and agitation as well as pacing restlessness improved greatly with medroxyprogesterone and leuprolide (66). A single case of dementia and features of the KluverBucy syndrome with verbal, physical, and sexual aggression responded to leuprolide (67). Four additional patients with disruptive sexual behavior responded favorably to medroxyprogesterone and were treated for one year without side effects (68). In the largest study to date, 15 patients (13 women and 2 men) with advanced AD and severe behavioral disturbance were randomized to placebo versus 0.625 mg conjugated estrogens, which increased up to 2.5 mg/d over three weeks (69). The estrogen-treated group showed less physical and verbal aggression over the study period. No differences in cognition or mood were found, and side effects were minimal. These reports suggest a possible role for estrogen therapy as an augmentative agent in severe behavioral disturbances in AD.

PARKINSON’S DISEASE In 1817, James Parkinson described a new disorder he referred to as paralysis agitans, now referred to as idiopathic Parkinson’s disease (PD). The cardinal neurological features include tremor, muscle rigidity, bradykinesia, and postural instability. The major pathologic hallmark of PD is less of dopaminergic projections from the substantia nigra, and the formation of Lewy bodies, intracellular aggregates composed of alphasynuclein (4q21-q23). PD is generally not an inherited disease, but mutations in alphasynuclein have been associated with familial PD.

Epidemiology PD affects perhaps one million individuals in North America and shows dramatic age-related increases in incidence and prevalence. It is more common in men than women. The prevalence of PD is approximately 150 per 100,000, increasing after age 65 to nearly 1100 per 100,000 (70). In addition to age, some studies (71) have reported a protective effect of smoking, although sampling artifacts may explain this association. The prevalence of PD is higher in industrialized societies, and epidemiological associations have been made with rural living and consumption of well water. These associations may reﬂect exposure to environmental toxins such as pesticides with homologies to the known toxin MPTP (72,73). Estrogen-replacement therapy may protect against PDassociated dementia without protecting against PD development itself (74).

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Estrogen Effects on Dopaminergic Systems In vitro experiments show that 17 β-estradiol possesses antiapoptotic properties on nigral cells and diminishes nigral cell death induced by oxidative stress (14,45,75,76). This protective effect may occur more with female cells than male cells, and is not blocked by either estrogen-receptor antagonists or protein-synthesis inhibitors (14,76). Possible mechanisms of action include an antioxidant effect, as similar neuroprotective effects are seen with 17 α-estradiol that lacks active hormonal effects. Estrogens also have prodopaminergic activity through inhibition of catechol-Omethyl transferase (COMT, EC 2.1.1.6), one of two major enzymes involved in dopamine breakdown. COMT activity varies throughout the menstrual cycle, and 17 βestradiol reduces COMT mRNA levels in MCF-7 cells in a time- and dose-dependent manner, probably by binding to the COMT promoter region (77).

Clinical Studies Clinical studies in PD have been hopeful but far from deﬁnitive, usually because of small sample size and limited periods of followup. Parkinsonian women receiving conjugated estrogens in a double-blind study showed greater “on” time, decreased “off” time, and improved motor scores on the Uniﬁed Parkinson’s disease rating scale (UPDRS) (78). In a crossover study utilizing high-dose transdermal 17 β-estradiol, there was a signiﬁcant reduction in the threshold for antiparkinsonian effects of IV levodopa, indicating a prodopaminergic effect of the 17 β-estradiol (79). A retrospective study of early PD patients showed a relationship of estrogen use and lower symptom severity in women with early PD not taking L-dopa. The authors conclude that estrogens should not be avoided and may be beneﬁcial in early PD (80).

OTHER NEURODEGENERATIVE DISORDERS There are very few clinical data to support the use of estrogens or SERMS in the other forms of neurodegenerative disorders including Huntington’s disease (HD), chromosome 17 linked fronto-temporal dementia (in its various forms including Progressive Supranuclear Palsy and Corticobasal degeneration and Pick’s disease) or Diffuse Lewy body disease. In a crossover study of four men and six women with HD, only two showed improvements on a motor rating scale after one month of treatment with conjugated estrogen (81).

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SERMS AND ENDOCRINE DEPENDENT TUMORS

16

SERMs and Breast Cancer Prevention Jenny Chang,

MD

and C. Kent Osborne,

MD

Contents Introduction Estrogen Receptor Function Activity of SERMs SERMs for Chemoprevention of Breast Cancer Newer SERMs for Therapeutic Evaluation Future Studies—NSABP P2 (Study of Tamoxifen and Raloxifene) Conclusions and Recommendations References

INTRODUCTION In Western Europe and the United States, mortality from breast cancer has decreased steadily since 1987 (1). This decline has been attributed to improvements in the treatment of women with early breast cancer, especially adjuvant therapy with tamoxifen. However, breast cancer still represents the leading cause of cancer and the second leading cause of cancer deaths for women in the United States (2). Further reductions in breast cancer incidence may come from breast cancer prevention. Measures such as lifestyle changes including diet and exercise, and chemoprevention offer the potential to reduce the incidence of this disease. Estrogen is crucial for the development and the progression of breast cancer, and endocrine therapies designed to antagonize this pathway are the oldest treatments for breast cancer (3). With the recognition that endocrine therapy in patients with breast cancer reduces contralateral breast cancer incidence, chemoprevention trials with antiestrogens (more properly called selective estrogen receptor modulators or SERMs, because of their dual, tissue-speciﬁc estrogen agonist/antagonist activities) in women at risk of breast cancer were initiated to determine if blocking estrogen’s proliferative effects in breast epithelium would lower the risk of breast cancer. The results of one of the studies has led the United States Food and Drug Administration (FDA) to approve the use of tamoxifen to reduce

From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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the incidence of breast cancer in women at high risk for this disease. This review focuses on the use of tamoxifen and other SERMs in the chemoprevention of breast cancer.

ESTROGEN RECEPTOR FUNCTION There are several potential molecular targets for chemoprevention. The estrogen receptor (ER), retinoic-acid receptors, growth factors, their receptors and their signaling molecules, other nuclear transcription factors such as AP-1 that mediate the effects of growth factors, and proteins regulating apoptosis are among the many possible targets. Of all these targets, ER has been the most extensively studied. Estrogen is critical for the development of breast cancer. Premalignant lesions, especially atypical ductal hyperplasia (ADH), are associated with a four- to six-fold increase in risk of breast cancer, and they nearly always, in contrast to normal ductal epithelial cells, express high levels of ER. Moreover, patients who have high plasma estrogens, or women who do have high levels of ER in their normal duct epithelium seem to be at higher risk of breast cancer (4,5). A mutant ER that is exquisitely sensitive to low levels of estrogen has been identiﬁed in a signiﬁcant proportion of premalignant lesions (6). ER is a transcription factor normally located in the nucleus of estrogen-responsive cells. ER contains several functional domains (7). These include a DNA-binding domain that recognizes speciﬁc elements in the promoter region of target genes called estrogenresponsive elements (EREs). The C-terminal region contains the hormone-binding domain, as well as the hormonally regulated transactivation domain AF-2, which is important for estrogen-mediated transcriptional activity. A hormone-independent amino terminal activation domain, AF-1, has also been identiﬁed. A third activation domain, AF-2A, has also been found in the beginning of the hormone-binding domain. When estrogen or other ligands like tamoxifen bind to the receptor, dimerization of the receptors facilitates the binding of the dimer to the ERE in the promoter region of target genes (8). This binding of the dimer to the ERE induces gene transcription with the accumulation of speciﬁc messenger RNA (mRNA). Increased levels of proteins encoded by these genes then alter the cellular phenotype. One of the many estrogenic effects in breast cells is enhanced cell proliferation and cell survival. ER does not act alone. Other nuclear proteins can interact with the ER dimer to modify the expression of certain genes. Some of these receptor-interacting proteins function as coactivators to amplify transcriptional activity of ER, whereas others function as corepressors (8–10). Molecules that have been identiﬁed as ER coactivators include SRC-1 and SRC-2, CBP/p300, TIF-2, TRIP-1, and AIB-1 (SRC-3, RAC-3). Conversely, N-CoR and SMRT function as corepressors and have been reported to bind to ER in the presence of tamoxifen (11–13). Recent studies suggest that coactivators contain either intrinsic histone acetyl transferase activity or recruit other molecules containing such activity, whereas corepressors form complexes with histone deacetylase activity. These acetylators or deacetylators modify chromatin structure in such a way as to either facilitate or block gene transcription (14). The presence of corepressors may be critical for the antiestrogen activity of drugs like tamoxifen, and the relative abundance of various coactivators and corepressors could theoretically contribute to the agonistic or antagonistic effects of tamoxifen seen in different tissues or on different gene promoters (7). Thus, ligands like tamoxifen

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Fig. 1. Potential determinants of the agonist/antagonist activity of ER.

can have dual functions; they may be antiestrogenic in some species, in some tissues, or on certain genes, and estrogenic in other species, in other tissues, or on other gene promoters. The precise molecular explanation for these dual properties is not clear, but it is an area of intense investigation. In addition, two different receptors, ER-α and ER-β, products of different genes, have been identiﬁed (7). These receptors are distributed somewhat differently in different tissues, and preliminary data suggest that they may have different functions. Thus, SERMs like tamoxifen act selectively at different sites. In the breast, they generally function as antiestrogens at least on genes regulating proliferation, whereas in the bone and liver they are more estrogenic. The presence of the two types of ER (ER-α, ER-β) could also contribute to the mechanism of target-site speciﬁcity. ER-β has no intrinsic AF-1 activity and could act primarily as an inhibitor. There may also be differences between the ER forms in their ability to interact with other pathways. For instance, tamoxifen-bound ER-β can bind to AP-1 sites and activate genes involved in cell proliferation that are also activated by polypeptide growth factors. Thus, ligandbound ER may regulate gene expression through both classical ER pathways as well as other nonclassical mechanisms. It is therefore reasonable to speculate that the particular ensemble of ligands, receptors, interacting proteins, other transcription factors, and the promoter of a particular gene will together determine whether a ligand like tamoxifen, or other SERMs, will have predominant antagonist or agonist activity, and it could explain the mixture of estrogen agonist/antagonist activities of ER in different cell types (Fig. 1). Theoretically, SERMs could be developed with various tissue activities based on their ER-binding properties. Speciﬁc SERMs could selectively alter ligand-induced receptor conformation in a way that facilitates interaction with speciﬁc coactivator or corepressor proteins resulting in speciﬁc activity proﬁles. It is possible to develop speciﬁc SERMs with more estrogenic or more antiestrogenic qualities that would be optimal for different clinical indications.

ACTIVITY OF SERMS In terms of breast cancer prevention, patients and physicians are most concerned about several key organs and tissues including the breast, the vagina, the central nervous system (hot ﬂashes, dementia), bone, liver (synthesis of cholesterol), and endometrium

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Table 1 Antiestrogenic and Estrogenic Activities of Raloxifene and Tamoxifen Tissue

Raloxifene

Tamoxifen

Breast Vagina CNS Bone Liver Endometrium

AE AE AE E E Weak E

AE AE AE E E E

AE, antiestrogenic activity predominates E, estrogenic activity predominates

(Table 1). Tamoxifen and raloxifene, a newer SERM, have similar effects on most of these tissues (15). Importantly for breast cancer prevention, these two agents are predominantly antiestrogenic in the breast. Unfortunately, they are not estrogenic in the vagina or in the central nervous system, so they do not improve symptoms of atrophic vaginitis or hot ﬂashes. An unknown concern is the effect of these drugs on the processes leading to dementia. Epidemiologic data suggest that estrogen might help to prevent dementia. Both of these drugs are estrogenic in the bone, at least in postmenopausal women, and can help preserve bone density and reduce fractures. Both are also estrogenic in the liver and reduce blood cholesterol levels of low density lipoprotein (LDL). The major difference between these two drugs is on the endometrium where raloxifene seems to have less estrogenic activity than tamoxifen. The estrogenic activity of tamoxifen probably accounts for the slight increase in endometrial cancer seen with prolonged tamoxifen therapy (16). The perfect SERM for use in chemoprevention of breast cancer would be antiestrogenic in the breast and endometrium, and estrogenic in the vagina, the central nervous system, bone, and liver. This perfect SERM has not been developed, but theoretically, it is possible to design such a drug given the new knowledge of the molecular biology of ER.

SERMS FOR CHEMOPREVENTION OF BREAST CANCER NSABP P1 (BCPT) Study: Tamoxifen There is considerable clinical experience with tamoxifen in breast cancer treatment as it was approved by the FDA for use in advanced disease in 1978. The most recent worldwide overview of the Early Breast Cancer Trialist Cooperative Group (EBCTG) conﬁrmed the efﬁcacy of tamoxifen as adjuvant treatment in women with ER-positive cancers (17). This analysis demonstrated that the use of adjuvant tamoxifen for 5 yr reduced cancer recurrence by 47%, with a corresponding reduction in overall mortality of 26% (p < 0.00001). Tamoxifen was associated with a signiﬁcant reduction (47%) in the incidence of contralateral breast cancer. The side-effect proﬁle of tamoxifen was also acceptable with menopausal symptoms being the most common toxicity. The most serious adverse effect of prolonged tamoxifen is an increased incidence of endometrial cancer, a side effect that is uncommon relative to the favorable effects of the drug on

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Table 2 Characteristics of the Raloxifene and NSABP Tamoxifen Prevention Trials Study design

Raloxifene, randomized, placebo controlled

Tamoxifen, randomized, placebo controlled

Number of women Mean age (yr) % < 50 years Breast cancer risk Followup time Primary endpoint

7704 67 0 low to normal 3 yr fractures

13388 55 38% high 4 yr breast cancer

Table 3 Results of the Raloxifene and Tamoxifen Prevention Trials Breast cancer Fractures CV disease DVT, pulmonary emboli CVA Endometrial cancer

Raloxifene

Tamoxifen

↓ (RR = 0.26) ↓ ND ↑ NR ND

↓ (RR = 0.51) ↓ ND ↑ ↑ (not statistically signiﬁcant) ↑

RR, relative risk ND, no difference NR, not reported ↑, increased incidence ↓, decreased incidence

breast cancer mortality. Based on these results, large-scale chemoprevention studies with tamoxifen were undertaken. The Breast Cancer Prevention Trial (BCPT, P1), initiated by the National Surgical Adjuvant Breast and Bowel Project (NSABP) in 1992, was a large-scale, phase III chemoprevention trial (Table 2). This trial randomized 13,388 women, 35 years of age or older, to tamoxifen 20 mg/d or placebo for 5 yr (16). Eligibility was based on a breast cancer risk that was at least equivalent to that of an average 60-year-old woman with a 5-yr predicted risk for breast cancer of 1.66%, according to the Gail model (based on family history, age, age at ﬁrst birth, age of menarche, previous breast biopsies, or a history of lobular carcinoma in situ or ADH, or being at least 60 years of age) (18). The early results of this study showed that tamoxifen signiﬁcantly reduced the risk of invasive breast cancer, primarily ER-positive tumors, by 49% with an accumulative incidence of 175 versus 89 patients developing invasive breast cancer, in placebo versus tamoxifen groups, respectively (Table 3). Tamoxifen also signiﬁcantly reduced the risk of noninvasive breast cancer by 50%. Not only did the beneﬁt from tamoxifen begin soon after it was started, but the breast cancer incidence curves continue to separate long after therapy is stopped, suggesting an ongoing risk reduction with each additional year of followup. Although not statistically signiﬁcant because of a small number of

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cases, the greatest beneﬁt was seen in individuals with a history of lobular carcinoma in situ (LCIS) (56% reduction in risk; 18 events with placebo, 8 with tamoxifen) and those with a history of atypical hyperplasia (AH) (87% reduction in risk; 23 events with placebo, 3 with tamoxifen). Tamoxifen also reduced fractures of the hip, radius, and spine (19% reduction), but it had no effect on the rate of ischemic heart disease in this initial analysis (16). Tamoxifen was associated with an increased rate of endometrial cancer (risk ratio 2.53, 36 cases in the tamoxifen arm versus 15 in the placebo arm), as well as vascular events such as thrombophlebitis (110 in the tamoxifen arm versus 77 in the placebo arm). The only death from endometrial cancer reported to date was a patient on the placebo arm. Concern that the tumor-promoting effect of tamoxifen might result in more aggressive and therefore higher-stage endometrial cancers was allayed by the observation that only International Federation of Gynecology and Obstetrics (FIGO) stage I endometrial cancers were seen in women who received tamoxifen. The risk of endometrial cancer caused by tamoxifen is similar to that seen with estrogen-replacement therapy. Furthermore, the increased risk was predominantly in women ≥ 50 years old. An increase in the need for surgery in women with preexisting cataracts was also noted. Tumors that did develop in patients on the tamoxifen arm were about the same size when compared to placebo (29% versus 22% > 2 cm in diameter), and were slightly more likely to have four or more positive lymph nodes (12% versus 6%). The major impact of tamoxifen was to reduce the incidence of ER-positive tumors. There was no evidence of a signiﬁcant difference in the rate of ER-negative tumors. These data suggest that the major effect of tamoxifen at this short followup may be the inhibition of progression of subclinical ER-positive tumors to clinically evident cancers. If tamoxifen has a true prevention effect to inhibit the evolution of premalignant disease to cancer, then one might expect a reduction in both ER-negative and ER-positive cancers because nearly all precursor lesions are ER-positive and loss of ER is a late event that occurs at the stage of carcinoma in situ disease. The striking risk reductions observed in the subset of study participants with a prior diagnosis of AH lends support to this idea. Alternatively, ER-negative tumors could evolve through a different pathway. The central ﬁnding of a 49% reduction in the incidence of breast cancer in the targeted high-risk population served as the basis for approval by the FDA. The deﬁnition of the term prevention as used in the BCPT, as well as concerns over the general public’s understanding of the term, led the FDA to approve reduction in breast cancer incidence in high-risk women rather than prevention of breast cancer as the indication for the use of tamoxifen in this patient population.

European Tamoxifen Prevention Trials Two smaller European studies evaluating tamoxifen versus placebo have not yielded statistically signiﬁcant results regarding the efﬁcacy of tamoxifen in breast cancer risk reduction, in apparent contradiction to the results of BCPT. Among 2,471 women followed for a median of 17 mo in the British trial (19), a relative risk (RR) of 1.06 was observed for tamoxifen versus placebo groups. In the Italian trial 5,408 women were followed for a median of 46 mo and a signiﬁcant reduction in breast cancer incidence was not found (20). Among the potential causes for the failure of these studies to conﬁrm the results of BCPT are differences in trial design and implementation.

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Most important is a substantially lower statistical power of the two European studies, which together had fewer than half the number of events in the BCPT (111 cancers in the British and Italian studies versus 265 in BCPT). Hormone-replacement therapy (HRT) was permitted in both the Italian and British studies presenting a possible confounding factor. In the British trial, however, HRT appeared to have no impact on the effect of tamoxifen on breast cancer occurrence, and the Italian data actually showed a preventive effect from tamoxifen among this subset of women who were taking HRT. In the Italian study, the small sample size, the lower risk for breast cancer in study participants, and a 26% dropout rate, further decreased the statistical power. Interestingly, among the patients who took their assigned agent for at least one year, there was a trend in favor of women assigned to tamoxifen. In the British trial, eligibility was based primarily on family history and, as a result, more women with genetic susceptibility were included. A possible explanation for the lack of beneﬁt in this trial is that these women might have differences in breast biology that renders them less sensitive to tamoxifen. The role of tamoxifen in women with genetic predisposition to breast cancer, such as that conferred by mutations in the breast cancer susceptibility genes BRCA1 and BRCA2, remains to be clariﬁed. An argument that tamoxifen may be less effective in such women is supported by the ﬁnding of a greater proportion of ER-negative tumors in women with BRCA1 mutations, although it is difﬁcult to extrapolate this observation to the prevention setting. The efﬁcacy of tamoxifen in reducing breast cancer risk in mutation-positive women is now being evaluated in a subset of women in the BCPT.

MORE Trial: Raloxifene The effect of raloxifene, another SERM similar to tamoxifen but with less uterotropic activity, on the risk of breast cancer in postmenopausal women was evaluated in the multiple outcomes of raloxifene evaluation (MORE) trial (Table 2). This study was a multicenter, randomized, double-blind trial in which postmenopausal women with osteoporosis on raloxifene or placebo were followed at 180 centers in the United States and Europe (21). The primary endpoint of this study was to determine whether three years of raloxifene could reduce the risk of fractures. Participants were also monitored for the occurrence of breast cancer, a secondary endpoint of the trial. In this study, the mean age was 67 years and 80% of the patients were above age 60. The breast cancer risk in patients in the raloxifene trial was lower than that in the BCPT (18). The median duration of followup in the raloxifene study was 40 mo. Signiﬁcantly fewer cases of breast cancer were observed in women assigned to raloxifene (13 out of 5,129 women versus 27 out of 2,576 women on placebo, relative risk 0.24). Raloxifene reduced the risk of ER-positive cancer by 90% but had no effect on ER-negative breast cancer, similar to tamoxifen in the BCPT. Raloxifene, like tamoxifen, increased the risk of venous thrombotic disease, but it did not increase the risk of endometrial cancer. Hence, in this study of postmenopausal women with osteoporosis, the risk of invasive breast cancer was decreased by 76%. Raloxifene has been approved by the FDA for prevention of osteoporosis, but it was not approved for breast cancer risk reduction because this was not a primary study endpoint. Although the BCPT study and MORE trial reported exciting early results, neither drug is without side effects and newer alternatives that are less toxic or more effective are needed.

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NEWER SERMS FOR THERAPEUTIC EVALUATION SERMs can be classiﬁed into three major categories: triphenylethylene derivatives like tamoxifen and toremifene, other nonsteroidal compounds including raloxifene, and steroidal compounds which have little or no estrogenic activity. Some of these compounds are in later stages of clinical testing for women with breast cancer.

Triphenylethylenes Apart from tamoxifen, three other triphenylethylene compounds have had extensive testing and they include toremifene, droloxifene, and idoxifene. Toremifene (Fareston: Schering Corp., Kenilworth, NJ) differs from tamoxifen only by the presence of one chlorine atom at position four, and its preclinical and clinical activities are very similar to those of tamoxifen. Unlike tamoxifen however toremifene does not produce DNA adducts and is not a hepatocellular carcinogen in rats. This potential advantage is of doubtful clinical signiﬁcance. Toremifene is approved for the treatment of stage IV breast cancer in postmenopausal women. Toremifene at 60 mg/d has comparable efﬁcacy to tamoxifen 20 mg in phase III clinical trials of advanced disease. The side-effect proﬁle is similar to that of tamoxifen. Toremifene has estrogenic effects in the uterus similar to tamoxifen. Its estrogenic effects in bone may be somewhat less than tamoxifen (22). Studies in the adjuvant setting are in progress. Droloxifene (3-OH-tamoxifen) has a spectrum of activity similar to that of both tamoxifen and toremifene. Like toremifene, droloxifene does not cause DNA adduct formation and is not a hepatocellular carcinogen in rats (23). Because it was less effective than tamoxifen in a Phase III trial, it is no longer in development. Idoxifene is a metabolically stable analog of tamoxifen with a substitution of an iodine atom at the fourth position and a pyrrolidine side chain. Idoxifene produces fewer DNA adducts than tamoxifen. Like tamoxifen, it has estrogen agonist effects in bone and liver, and less uterotropic activity. Unfortunately, further development of this compound has been abandoned because of an unexpected and unexplained increase incidence of uterine prolapse and polyps, which precluded its study in breast cancer prevention and dampens enthusiasm for its further evaluation in breast cancer (24). TAT-59. This phosphorylated derivative of 4-hydroxy-tamoxifen is being developed for the treatment of breast cancer. It has high binding afﬁnity for ER compared to tamoxifen, and is a more potent antagonist in rat-mammary carcinoma, as well as human breast cancer xenografts growing in athymic mice (25). GW5638. This carboxylic derivative of tamoxifen was developed with the primary goal of achieving more complete antiestrogenic activity in breasts and the uterus while maintaining estrogenic activity in bone and in the cardiovascular system (26). Like other SERMs, this drug possesses antitumor activity in human breast cancers transplanted into athymic mice. The carboxylic acid moiety predicts that GW5638 may have low penetration into the CNS, and as such, may be active systemically while avoiding induction of postmenopausal symptoms such as hot ﬂashes.

Other Nonsteroidal Compounds EM800 is an orally active prodrug of the active compound EM652 (benzopyrene). The latter shows higher afﬁnity for ER than estradiol, 4 hydroxy tamoxifen, or the steroidal antiestrogen ICI 182,780, giving it the highest afﬁnity for ER relative to other

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SERMs (27). The drug is a potent inhibitor of ER-positive breast cancer cells in vitro and of breast cancer xenografts in athymic mice. In addition to its potent inhibition of breast cancer growth in preclinical models, EM800 has estrogen agonist activity in bone and liver, while demonstrating more antiestrogenic effects in the uterus. Whether these potential advantages translate to improved clinical management of patients with breast cancer awaits the results of ongoing studies. LY353381 (SERM 3) and LY357489 are newer, more potent raloxifene analogs. They are more potent inhibitors of in vitro breast cancer growth than raloxifene or tamoxifen, and are devoid of intrinsic estrogen agonist effects on cultured breast cancer cells. SERM3 is a more potent agonist than raloxifene in bone and liver, and it completely inhibits estrogen-induced uterine weight gain in mature rats (28). SERM3 is targeted for breast cancer prevention and treatment given its promising preclinical proﬁle.

Steroidal Antiestrogens ICI 182,780 (Faslodex, AstraZeneca Pharmaceuticals) is a derivative of estradiol with a long hydrophobic side chain at the seven alpha position. ICI 182,780 demonstrates a pure antiestrogenic proﬁle. The mechanism of action of this steroidal antiestrogen differs signiﬁcantly from other SERMs with mixed agonist/antagonist properties. In contrast to other SERMs, ICI 182,780 blocks ER transcription coming from both AF-1 and AF-2 domains. This drug may also impair ER dimerization but most importantly, it induces ER degradation with a marked reduction in cellular concentration of ER (29). ICI 182,780 demonstrates exciting antitumor activity in preclinical models. Serial biopsies from tumors in patients treated with either tamoxifen or ICI 182,780 show that the latter is a more potent inducer of apoptosis (30). ICI 182,780 may not cross the blood/brain barrier and may therefore not cause hot ﬂashes, an important clinical problem with other SERMs. This cumulative data suggest that although ICI 182,780 may be a superior antitumor agent, it may not be the most desirable SERM for breast cancer prevention in normal women because of its predominant antiestrogenic proﬁle. Results of randomized studies comparing ICI 182,780 with other hormonal agents like tamoxifen in advanced breast cancer should be available in the near future.

FUTURE STUDIES—NSABP P2 (STUDY OF TAMOXIFEN AND RALOXIFENE) The side effects of tamoxifen have motivated continuing search for a better chemopreventive agent for breast cancer with a more desirable risk to beneﬁt ratio. As a result, in July 1999, the NSABP began its second breast cancer prevention trial, NSABP P2: a Study of Tamoxifen and Raloxifene, or STAR. Based on the toxicity proﬁle of raloxifene and with its preliminary breast cancer prevention data, STAR was designed as an equivalency trial. Secondary endpoints include the incidence of endometrial cancer, ischemic heart disease, and fractures, as well as toxicity of each agent and the participants’ quality of life. Unlike the BCPT, the target population of STAR is to include only postmenopausal women because it is only in this population that raloxifene studies have been conducted. The eligibility criteria for the STAR trial resembles that for the BCPT except for the postmenopausal restriction. Risk-eligible postmenopausal volunteers are currently being randomized in a double-blinded fashion to receive either tamoxifen 20 mg/d or raloxifene 60 mg/d for a duration of 5 yr. Participants will be

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stratiﬁed according to age, breast cancer risk, race, and history of LCIS, as well as by hysterectomy status. To answer the question of equivalency, posed as a primary endpoint of this study, the target enrollment was set at 22,000 eligible women.

CONCLUSIONS AND RECOMMENDATIONS Results from the major trials of chemoprevention with SERMs indicate that these agents reduce the incidence of breast cancer in women at risk of the disease. Nonetheless, unresolved issues concerning long-term side effects, duration of therapy, and use in women with BRCA1 and BRCA2 mutations, are important questions that will only be answered with longer followup of patients in these clinical trials. Other issues remain to be resolved. First, there was no reduction in cardiovascular mortality in the BCPT, although one could emerge with additional years of followup. Possible late side effects such as impaired cognitive function need evaluation. Is tamoxifen estrogenic in the CNS and will it reduce the incidence of Alzheimer disease, or will an increased incidence result from its antiestrogenic activity? The optimal age to start treatment is not clear but patients were above 35 years on the BCPT and postmenopausal on the MORE trial. The optimal duration of treatment is still not known. Duration of therapy in these trials was about 5 yr. Are the effects durable, or are we simply suppressing rather than truly preventing cancers? What about women with hereditary breast cancer susceptibility; is the reduction in breast cancer incidence similar to that in sporadic cases? Can we extrapolate the results of these studies to the general population of women? Is survival then improved by preventive treatment, and what is the magnitude of beneﬁt over time with additional years of followup. A possible clue for the true prevention effect of tamoxifen comes from examining the 6% of women on tamoxifen in the BCPT study who have previous biopsies showing atypical ductal hyperplasia and who may, therefore, be still in premalignant stage. In this subset of women there was a dramatic 87% reduction in breast cancer incidence with tamoxifen. Only three patients to date have developed breast cancer on tamoxifen who had atypical hyperplasia compared to 23 on the placebo arm. Although these data are derived from the subset analysis, they do suggest that tamoxifen may prevent breast cancer and that this effect may be greater than its beneﬁcial effects on subclinical cancers. The decision regarding who should be considered for preventive tamoxifen must be based on the risks associated with the drug and the beneﬁts that might be derived from it. As a starting point, women with risk factors that were used as eligibility criteria for the BCPT (Gail model risk > 1.66% over 5 yr or a history of LCIS or AH) may be considered. Other high-risk women such as those with BRCA1 or BRCA2 mutation might also be considered, although these groups were not speciﬁcally tested in the BCPT. To synthesize the multiple effects of tamoxifen into a uniﬁed risk/beneﬁt model, Gail et al. subjected the risks (endometrial cancer, stroke, pulmonary embolism, deep venous thrombosis) and the beneﬁts (breast cancer and fracture reduction) of tamoxifen as observed in the BCPT into a quantitative analysis (31). The groups that beneﬁt most from preventive tamoxifen include younger women at a higher risk, and women over the age of 50 years who are not at risk of uterine cancer. In contrast, the risk to beneﬁt ratio is far less clear for women who are 50 years or older, postmenopausal, have not had a hysterectomy, and with no history of LCIS. A woman’s ﬁnal decision regarding

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whether or not to implement preventive tamoxifen must involve discussion with her physician and other knowledgeable health professionals. A major challenge at this point is to design future studies with agents that have a preventive effect on both ER-positive and ER-negative tumors. Smaller, shorter phase II studies, which rely on surrogate biomarker endpoints, and which require fewer resources to allow for evaluation of more candidate agents in at-risk populations, should be conducted. Only the most promising agents to evolve from phase II trials with a suitable toxicity proﬁle will go on to be tested for their impact on the clinical endpoints of breast cancer incidence and mortality in large, expensive phase III trials. The use of newer SERMs and other compounds to reduce breast cancer incidence with favorable ancillary beneﬁts should be tested with this model before large-scale phase III clinical studies. In so doing, further reduction in breast cancer incidence and mortality may be observed in the future.

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20. Veronesi U, Maisonneuve P, Costa A, Sacchini V, Maltoni C, Robertson C, et al. Prevention of breast cancer with tamoxifen: preliminary ﬁndings from the Italian randomised trial among hysterectomised women. Italian Tamoxifen Prevention Study. Lancet 1998;352:93–97. 21. Ettinger B, Black DM, Mitlak BH, et al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: Results from a 3-year randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators. JAMA 1999;282:637–645. 22. Vogel CL, Shemano I, Schoenfelder J, Gams RA, Green MR. Multicenter phase II efﬁcacy trial of toremifene in tamoxifen-refractory patients with advanced breast cancer. J Clin Oncol 1993;11: 345–350. 23. Rausching W, Pritchard KI. Droloxifene in new antiestrogen: its role in metastatic breast cancer. Breast Cancer Res Treat 1994;31:83–94. 24. Coombes RC, Haynes BP, Dowsett M, et al. Idoxifene: report of a phase I study in patients with metastatic breast cancer. Cancer Res 1995;55:1070–1074. 25. Toko T, Sugimoto Y, Matsuo K, et al. TAT-59, a new triphenylethylene derivative with antitumor activity against hormone-dependent tumors. Eur J Cancer 1990;26:397–404. 26. Willson TM, Henke BR, Momtahen TM, Lubahn DB, et al. 3-[4-(1,2-Diphenylbut-1-enyl)phenyl] acrylic acid: a non-steroidal estrogen with functional selectivity for bone over uterus in rats. J Med Chem 1994;37:1550–1552. 27. Martel C, Provencher L, Li X, et al. Binding characteristics of novel nonsteroidal antiestrogens to the rat uterine estrogen receptors. J Steroid Biochem Mol Biol 1998;64:199–205. 28. Sato M, Turner CH, Wang T, Adrian MD, Rowley E, Bryant HU. LY353381.HCl: a novel raloxifene analog with improved SERM potency and efﬁcacy in vivo. J Pharmacol Exp Ther 1998;287:1–7. 29. Dauvois S, White R, and Parker MG. The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling. J Cell Sci 1993;106:1377–1388. 30. Ellis PA, Saccani-Jotti G, Clarke R, et al. Induction of apoptosis by tamoxifen and ICI 182780 in primary breast cancer. Int J Cancer 1997;72:608–613. 31. Gail MH, Costantino JP, Bryant J, et al. Weighing the risk and beneﬁts of tamoxifen treatment for preventing breast cancer. J Natl Cancer Inst 1999;91:1829–1846.

17

SERMs in Postmenopausal Women’s Health Jan L. Shifren, MD and Leo Plouffe, Jr., MD, CM Contents Introduction Structural Chemistry and Pharmacology of SERMs Tissue-Speciﬁc Effects of SERMs Conclusions References

INTRODUCTION Selective estrogen receptor modulators (SERMs) are a group of structurally diverse compounds that bind to estrogen receptors and act as either estrogen agonists or antagonists, depending on the target cell and hormonal milieu (1). Four SERMs are currently available for clinical use: clomiphene citrate, tamoxifen citrate, toremifene citrate, and raloxifene hydrochloride (Table 1). With the exception of clomiphene citrate, these compounds are used primarily or exclusively in postmenopausal women. This chapter brieﬂy reviews the structural chemistry and pharmacology of the four clinically available SERMs. A discussion of the tissue-speciﬁc effects of different SERMs in postmenopausal women follows, with emphasis on current and potential future clinical uses of tamoxifen, raloxifene, and toremifene.

STRUCTURAL CHEMISTRY AND PHARMACOLOGY OF SERMS The clinically available SERMs belong to one of two distinct families based on chemical structure: triphenylethylenes (clomiphene, tamoxifen, and toremifene) or benzothiophenes (raloxifene) (Fig. 1). Differences in molecular structure are believed to account for some of the contrasting effects of SERMs in various body tissues (1). The current clinical indications and key pharmacologic properties of clomiphene, tamoxifen, toremifene, and raloxifene are summarized in Tables 1 and 2.

TISSUE-SPECIFIC EFFECTS OF SERMS SERMs are among the best-studied medications for use in postmenopausal women. The effects of SERMs on the breast, the skeleton, and the cardiovascular system as From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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Table 1 SERMs Approved For Use in the United States (in Chronological Order of Introduction to Clinical Practice) Compound

Trade Name (U.S.A.)

Clomiphene citrate (2,3)

Clomid Serophene

Tamoxifen citrate (4)

Nolvadex

Toremifene citrate (5)

Fareston

Raloxifene hydrochloride (6)

Evista

Approved Use(s) Treatment of ovulatory dysfunction in the management of infertility Primary or adjuvant treatment of breast cancer Reduction in the risk of breast cancer in women at high risk for breast cancer Treatment of metastatic breast cancer in postmenopausal women with estrogen receptorpositive or receptor statusunknown tumors Treatment and prevention of osteoporosis in postmenopausal women

Recommended Dosage 50–150 mg/d for 5 d each cycle 20–40 mg/d for up to 5 yr 20 mg/d for 5 yr

60 mg/d

60 mg/d

Fig. 1. Chemical structures of SERMs currently in clinical use.

well as on gynecologic and other tissues have been or are currently being studied in several large, randomized, controlled clinical trials (Table 3). The tissue-speciﬁc effects of SERMs demonstrated in key trials are summarized in the following sections.

Breast Effects Tamoxifen The estrogen-antagonist effects of tamoxifen in human breast tissue were ﬁrst demonstrated in the early 1970s. In two small clinical trials of patients with advanced, widely

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Table 2 Pharmacologic Properties of Approved SERMs Peak plasma concentration Plasma half-life Primary route of elimination Metabolized by cytochrome P450 pathway

Clomiphene

Tamoxifen

Toremifene

Raloxifene

6h 5d Fecal Yes

5h 5–7 d Fecal Yes

3h 5d Fecal Yes

Variesa 28 h Fecal No

a Functions of systemic interconversion and enterohepatic cycling of raloxifene and its glucuronide metabolites.

Table 3 Summary of Selected Clinical Trials Involving SERMs (Completed or Ongoing) Trial Tamoxifen Breast Cancer Adjuvant Therapy Metanalysis of 55 randomized trials (7) NSABP B-14 (8) Breast Cancer Prevention NSABP Breast Cancer Prevention Trial (9) Italian breast cancer prevention trial (10) United Kingdom breast cancer prevention trial (11) Study of Tamoxifen and Raloxifene (STAR) Toremifene Metastatic Breast Cancer United States trial (12) Nordic trial (13) Eastern European trial (14) Breast Cancer Adjuvant Therapy Finnish trial (15) International Breast Cancer Study Group—Trial 12 (15) International Breast Cancer Study Group—Trial 14 (15) Raloxifene Osteoporosis Prevention Osteoporosis prevention trial (16) Osteoporosis Treatment Multiple Outcomes of Raloxifene (MORE) Trial (17) Breast Cancer Prevention Study of Tamoxifen and Raloxifene (STAR) Cardiovascular Disease Prevention Raloxifene Use for the Heart (RUTH)

No. of Participants

37,427 4,063 13,388 5,048 2,471 22,000 (target)

648 415 463 1,460 (target) 1,140 (target) 840 (target)

601 7,705 22,000 (target) 10,000 (target)

disseminated breast cancer, tamoxifen reduced tumor size in approximately one third of the women (18). Since then, tamoxifen has become one of the most widely used therapeutic agents in clinical oncology. Breast Cancer Treatment. Tamoxifen was initially approved for the treatment of metastatic breast cancer in postmenopausal women. It was subsequently approved for

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Table 4 Gail Model for the Assessment of Breast Cancer Risk (27) Individual risk for breast cancer is calculated using Age Age at menarche Age at ﬁrst live birth Number of previous breast biopsies Number of ﬁrst-degree relatives with breast cancer

treatment of early and advanced breast cancer in women of all ages and in men, and as adjuvant therapy for early breast cancer. Tamoxifen is thought to exert its effects on breast tissue by competitively binding to estrogen receptors and inhibiting the expression of estrogen-regulated genes, including those for the secretion of growth and angiogenic factors by tumors, thus slowing cell proliferation (19,20). Tamoxifen may directly induce apoptosis in human breast-cancer cells (20,21). Numerous breast-cancer trials have demonstrated beneﬁts with tamoxifen therapy in dosages ranging from 20 to 40 mg/d in postmenopausal women (22). A recent update of a metanalysis conducted by the Early Breast Cancer Trialists’ Collaborative Group (which included followup of 37,000 women with operable breast cancer who had received up to 5 yr of adjuvant tamoxifen therapy in 55 randomized, clinical trials that began prior to 1990) found that tamoxifen signiﬁcantly reduced breast cancer recurrence and extended 10-yr survival (7,9). Tamoxifen therapy primarily beneﬁted women with estrogen receptor (ER)-positive tumors, which is the predominant tumor type among postmenopausal women (23). Among women with ER-positive tumors who received tamoxifen for 5 yr, the annual recurrence and death rates were decreased by 50% and 28%, respectively (7). Treatment beyond 5 yr has not been associated with additional disease control beneﬁts (8,24,25). Breast Cancer Prevention. Clinical trial data showing that 5 yr of adjuvant tamoxifen therapy signiﬁcantly reduced the risk for contralateral breast cancer by 47% in women with invasive disease provided the rationale for studying tamoxifen for primary prevention (7,26). In 1992, the National Surgical Adjuvant Breast and Bowel Project (NSABP) launched the Breast Cancer Prevention Trial (BCPT) to evaluate the efﬁcacy of tamoxifen (20 mg/d) for breast-cancer prevention in 13,388 U.S. and Canadian women aged 35 years and older who were considered at high risk for breast cancer (9), as determined by age over 60 and the Gail model for breast-cancer risk demonstrating an increased risk or a history of lobular carcinoma in situ (LCIS) (27). Premenopausal as well as naturally and surgically postmenopausal women were included (Table 4). The trial results were released earlier than expected, in April 1998, after evidence of tamoxifen’s efﬁcacy reached a predetermined efﬁcacy threshold for completion. Overall, the incidence of breast cancer in the tamoxifen group was 3.4 cases per 1,000, compared with 6.8 cases per 1,000 in the placebo group (Fig. 2) (9). After 5 yr of treatment, tamoxifen reduced the incidence of invasive breast cancer by an average of 49% (RR: 0.51; 95% CI: 0.39–0.66). The prophylactic treatment beneﬁts were evident in all age groups studied (Fig. 3). The risk for breast cancer was reduced by 44% in women aged 35 to 49 years, by 51% in women aged 50 to 59 years, and by

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Fig. 2. NSABP tamoxifen breast cancer prevention trial results: annual breast cancer cases. (Adapted from ref. 9.)

Fig. 3. NSABP tamoxifen breast cancer prevention trial results: invasive breast cancer cases in all age groups. (Adapted from ref. 9.)

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Fig. 4. NSABP tamoxifen breast cancer prevention trial results: cumulative rate of noninvasive breast cancer cases in all age groups. (Adapted from ref. 9.)

55% in women aged 60 years and older. Tamoxifen also reduced the incidence of noninvasive breast cancer (ductal carcinoma in situ [DCIS]) by 50% (RR: 0.50; 95% CI: 0.33–0.77) in all age groups and in women at all levels of breast-cancer risk (Fig. 4) (9). In addition, tamoxifen decreased breast-cancer risk in women with a history of lobular carcinoma in situ or atypical hyperplasia (by 56% and 86%, respectively) as well as in women in all baseline risk categories. For both invasive and noninvasive breast cancers, the risk reduction associated with tamoxifen therapy was attributed to a 69% decrease in the occurrence of ER-positive tumors; tamoxifen had no signiﬁcant impact on the incidence of ER-negative tumors (9). Preliminary analyses of two European tamoxifen breast-cancer-prevention trials failed to conﬁrm the ﬁndings of the BCPT (10,11). The study designs of these trials however differed in several ways. The European trials focused on different population subsets than the BCPT, enrolled fewer patients, allowed concomitant estrogen use, and had higher dropout rates (28) (Table 5). Although both the BCPT and Royal Marsden Hospital (UK) trial enrolled women at high risk for breast cancer, eligibility for BCPT was based on a combination of family history as well as other risk factors. The Royal Marsden Hospital trial speciﬁcally focused on women with a strong family history of breast cancer—a population known to have a higher incidence of ER-negative tumors, making them less likely to derive beneﬁt from prophylactic SERM therapy (11). The Italian trial enrolled relatively young postmenopausal women recruited from the general population who had undergone prior hysterectomy, many with oophorectomy, who were at comparatively low risk for breast cancer (the rate of breast cancer among women in the placebo group was 2.3 cases per 1,000 woman-years) (10). In addition, the dropout rate in the Italian trial was very high (26%). For these reasons, the BCPT is considered to be the most deﬁnitive breast-cancer-prevention study to date (29). The American Society of Clinical Oncology recently published a report cautiously afﬁrming the use of tamoxifen for primary breast cancer prophylaxis in high-risk women. The group emphasized the need for a thorough evaluation of both personal and family medical histories as well as individualized assessment of the beneﬁts and

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Table 5 Comparison of the BCPT with European Tamoxifen Prevention Trials Comparison

BCPT (9)

Sample size 13,388 Age <50 39.3% First degree relatives with breast cancer None 23.8% 1 relative 56.8% 2 relatives 19.4% HRT users 0% Follow-up, woman-years 52,401 Breast cancer events 368 Cancer incidence/1000 Placebo 6.8 Tamoxifen 3.4

Royal Marsden Hospital (11)

Italian (10)

2,471 62%

5,048 38%

0% 82.6% 17.4% 41% 12,355 70

79% 18.2% 2.8% 14% 20,731 41

5.0 4.7

2.3 2.1

risks of treatment (29). Additional studies are currently being conducted to further assess the reduction in the incidence of breast cancer with tamoxifen therapy (18). Toremifene Toremifene (60 mg/d) is generally used for the treatment of metastatic breast cancer in postmenopausal women with ER-positive tumors or tumors of unknown receptor status (12–14). Like tamoxifen, the antitumor effects of toremifene in the breast are primarily mediated through competitive binding to the estrogen receptor (30). Toremifene was directly compared with tamoxifen in three trials of previously untreated postmenopausal women with advanced breast cancer (Table 3). Overall, the efﬁcacy of both SERMs was comparable (12–14). Studies evaluating toremifene as second-line therapy, after tamoxifen, for the treatment of advanced disease suggest that these compounds are clinically crossresistant, with little or no response to toremifene occurring after failure with tamoxifen (31,32). Several trials comparing toremifene and tamoxifen as adjuvant therapy for women with node-positive breast cancer are currently being conducted, but the ﬁnal results are not yet available (15) (Table 3). Toremifene is being studied for its ability to prevent breast cancer in high-risk women (33). Raloxifene Cummings and associates reported data on the incidence of newly diagnosed breast cancer in a cohort of 7705 postmenopausal women with osteoporosis enrolled in the Multiple Outcomes of Raloxifene Evaluation (MORE) study, an osteoporosis-treatment trial in postmenopausal women with no prior or active history of breast cancer (Fig. 5) (34). After a median 40 mo of therapy, the incidence of newly diagnosed invasive breast cancer was reduced by 76% (RR: 0.24; 95% CI: 0.13–0.44) among patients treated with raloxifene compared with placebo. As with tamoxifen, this reduction was mainly caused by a 90% reduction in the occurrence of ER-positive tumors (RR: 0.10; 95% CI: 0.04–0.24); raloxifene had no signiﬁcant effect on the frequency of ER-

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Fig. 5. MORE study results: effect of raloxifene on cumulative breast cancer incidence (30). From Cummings SR, Eckert S, Krueger KA, et al. The effect of raloxifene on risk of breast cancer in postmenopausal women: results from the MORE randomized trial. JAMA 1999;281:2189–2197. Copyrighted 1999, American Medical Association.

negative tumors (RR: 0.88; 95% CI: 0.25–3.0). The reduction in risk with raloxifene was comparable for both the 60 and 120 mg/d dosages. A large number of women from the MORE study who completed 48 mo of therapy will be followed up for an additional 48 mo in the Continued Outcomes Relevant to Evista (CORE) study. Additional evaluation of the effects of raloxifene on breast cancer incidence will be performed in the Raloxifene Use for The Heart (RUTH) trial, a study of the effects of raloxifene on cardiovascular disease (CVD) outcomes being conducted in 10,000 postmenopausal women at high risk for CVD. Use of raloxifene speciﬁcally for breast-cancer prevention in postmenopausal women at high risk for the disease is currently under investigation in the Study of Tamoxifen and Raloxifene (STAR) trial, which was launched in 1999 (18). Sponsored by the NSABP, this double-blind, randomized comparison of tamoxifen (20 mg/d) and raloxifene (60 mg/d) is the largest breast-cancer-prevention study ever conducted, involving more than 300 institutions throughout the United States, Canada, and Puerto Rico. Approximately 22,000 women aged 35 to 59 years who are at increased risk for breast cancer or aged 60 years and older will receive either tamoxifen or raloxifene for at least 5 yr. Enrollment is expected to continue until 2005, after which time some of the initial results will become available.

Skeletal Effects Raloxifene Studies of raloxifene have focused primarily on its skeletal effects. To date, raloxifene is the only SERM proven to be effective for both prevention and treatment of postmenopausal osteoporosis (16,17). Like estrogen, raloxifene acts as an antiresorptive agent on bone, increasing bone mineral density (BMD) in the spine and hip by 1% to 3%

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compared to placebo within 12 mo, and reducing the levels of biochemical markers of bone turnover as early as 3 mo (16,17,35). The 36-mo results from the MORE trial showed that raloxifene (60 or 120 mg/d) decreased vertebral fracture risk by approx 50% (17). At the indicated clinical dosage of 60 mg/d, raloxifene speciﬁcally reduced the incidence of new vertebral fractures by 55% among women with osteoporosis (BMD T-score at or below −2.5) and with no preexisting vertebral fracture (6). Among women who did have preexisting vertebral fractures, the risk reduction was 30%. These results are comparable to the vertebral fracture risk reductions reported in double-blind clinical trials with bisphosphonates such as alendronate (36,37) and risedronate (38). The risk for nonvertebral fractures did not differ signiﬁcantly between the placebo group and the raloxifene groups (RR: 0.9; 95% CI: 0.8–1.1 for both raloxifene groups combined). The MORE study however was not designed or adequately powered to assess efﬁcacy at individual sites other than the spine, including hip, wrist, or ankle (17). Tamoxifen The skeletal effects of tamoxifen were initially studied in women with breast cancer to determine whether this SERM had a negative impact on the skeleton (39). The ﬁndings, derived from studies of women with breast cancer, showed that tamoxifen treatment accelerated BMD loss in premenopausal women but exerted a small protective effect on BMD in postmenopausal women (40–43). Fracture data from subjects enrolled in the BCPT showed a statistically nonsigniﬁcant overall reduction of 19% in clinical fractures (9). Toremifene Data on the skeletal effects of toremifene are limited. They have been obtained primarily from breast-cancer trials. These studies suggest that toremifene is a weak antiresorptive agent, as evidenced by slight reductions in the biochemical markers of bone turnover (42). Over 12 mo of treatment, however, toremifene was associated with a small decline in BMD. No fracture data have been reported for toremifene, and further studies are needed to fully assess its skeletal effects.

Effects on Lipids and Other Markers of Cardiovascular Disease Risk Raloxifene The effects of raloxifene on lipids and other intermediate markers of CVD risk have been evaluated in several trials. Compared with placebo, raloxifene reduced total serum cholesterol (T-Chol) and low-density lipoprotein cholesterol (LDL-C) by approx 10%. Although raloxifene did not signiﬁcantly affect high-density lipoprotein cholesterol (HDL-C) levels, it did increase levels of the HDL2-C subfraction. Unlike estrogen, raloxifene did not increase triglyceride levels (16,44,45). Raloxifene has been shown to increase apolipoprotein A-1 and decrease ﬁbrinogen, apolipoprotein B, lipoprotein(a) (Lp[a]), and homocysteine levels (44–46). Raloxifene appears to differ from estrogen in its effects on certain markers of vascular inﬂammation (47). For example, in head-to-head comparisons of raloxifene and combination estrogen/ progestin replacement therapy (HRT), HRT increased circulating levels of C-reactive protein, but raloxifene did not (45,46). The positive effects of raloxifene on serum lipids and on other markers of CVD

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risk are in many ways similar to those observed with estrogen therapy. Based on studies showing that decreases in LDL-C and ﬁbrinogen levels are associated with reductions in CVD events, the positive effects of raloxifene could potentially decrease CVD risk by up to 30% (44). Whether the beneﬁcial effects of raloxifene on lipid levels and other markers of CVD risk will translate into reduced risk for cardiac outcomes is currently being studied in the RUTH trial. Launched in 26 countries in 1998, the RUTH trial will enroll approximately 10,000 postmenopausal women aged 55 years and older who either have, or are at high risk for, coronary heart disease (CHD). Results are expected by 2006 (48). Tamoxifen Data on the effects of tamoxifen on lipids and other intermediate markers of CVD risk have come primarily from large breast-cancer treatment trials. These trials found that tamoxifen reduced T-Chol by 7% to 17% and LDL-C by 6% to 29% but had no signiﬁcant effects on HDL-C or triglycerides. Tamoxifen therapy resulted in decreased levels of ﬁbrinogen, apolipoprotein B, Lp(a), and homocysteine (49–53). The effects of tamoxifen on clinical cardiovascular events have been evaluated. Three randomized breast-cancer-treatment trials conducted by the NSABP, the Scottish Cancer Trials Breast Group, and the Stockholm Breast Cancer Study Group found that adjuvant tamoxifen therapy signiﬁcantly reduced the incidence of fatal and nonfatal myocardial infarction (54–57). A recent metanalysis however, in which the authors reanalyzed clinical outcomes data, failed to show a signiﬁcant reduction in cardiovascular mortality associated with tamoxifen therapy (7). Similarly, the BCPT found that tamoxifen had no effect on the average annual rate of CHD, with few CHD events being recorded (9). Toremifene The effects of toremifene on lipids and other intermediate markers of CVD risk have been studied in small breast-cancer trials. In these studies, toremifene was comparable to tamoxifen in lowering T-Chol, LDL-C, apolipoprotein B, Lp(a), ﬁbrinogen, and homocysteine levels (49,58). In one study, toremifene produced an increase in HDL-C, whereas tamoxifen did not (49).

Endometrial and Other Gynecologic Effects Tamoxifen Almost all data concerning the gynecologic effects of tamoxifen come from breastcancer-treatment or prevention trials. The most compelling ﬁndings concern the association between tamoxifen therapy and the subsequent development of endometrial cancer. The ﬁrst cases of endometrial cancer in women taking tamoxifen were reported in the early 1980s (59–61). Most tamoxifen studies however were poorly controlled and reported relatively few cancer cases (62–65). Large-scale clinical trials, including the NSABP B-14 trial and the BCPT, did not assess endometrial status at the initiation of tamoxifen therapy but monitored the incidence of endometrial cancer during the course of each study (7,9,66,67). Overall, these major studies showed that tamoxifen increased endometrial cancer risk by 2- to 7.5-fold. This elevated risk was seen within the ﬁrst 2 yr of treatment, might increase over time, and could adversely affect overall mortality (7,9,66).

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The current consensus is that tamoxifen therapy increases endometrial cancer risk (66,68,69). Contrary to earlier reports however, it appears that the stage and grade of endometrial cancers observed in women taking tamoxifen are similar to those in women in the placebo group (9). In addition to increasing the risk for endometrial cancer, tamoxifen therapy also has been linked to an endometrial condition called subendothelial cystic atrophy (63,65). This condition is characterized by cystic proliferation of the basalis layer of the endometrium, which increases ultrasonographically determined endometrial thickness by several millimeters. It occurs in approx 50% or more of all postmenopausal women treated with tamoxifen (64,70–73). These changes, which are best visualized with transvaginal ultrasonography, can be documented within the ﬁrst 6 to 12 mo of treatment (71,73). Whether or not the heightened risk of endometrial cancer associated with tamoxifen correlates with increased endometrial thickness remains unclear (74,75). A number of other endometrial effects have been attributed to tamoxifen, particularly in postmenopausal women. These include endometrial bleeding and thickening, polyps, growth of leiomyomas, stimulation of endometriosis, endometrial hyperplasia, and rare endometrial or myometrial malignancies (63,71,76,77). Tamoxifen has been linked to estrogenic changes of the vaginal mucosa and an increased incidence of vaginal discharge (9,78). The increased vaginal discharge may account for increased reporting of vaginal symptoms by women taking tamoxifen, including interference with sexual activity. In the absence of appropriately controlled trials, all of these putative associations should be regarded cautiously, as they may be the result of reporting biases. For women with breast cancer, the beneﬁts of tamoxifen therapy—increasing diseasefree survival and decreasing the incidence of recurrent and contralateral tumors— usually far outweigh the risks, including the increased risk for endometrial cancer (66,79). The risk/beneﬁt proﬁle of tamoxifen for breast cancer prevention in healthy postmenopausal women with intact uteri is less certain and requires individualized patient assessment (29). Toremifene Clinical data concerning the gynecologic effects of toremifene are very limited. Available information suggests that the effects of toremifene on the endometrium are similar to those of tamoxifen, but additional clinical data are needed before any deﬁnitive conclusions can be drawn (14,22,71,80). Raloxifene Studies focusing on the endometrial effects of raloxifene were undertaken early in drug development (81). Preliminary observations that raloxifene did not adversely impact the endometrium were subsequently conﬁrmed in separate clinical trials involving from several hundred to more than 5000 healthy postmenopausal women (16,34,82– 85). These studies found that raloxifene did not cause endometrial cystic atrophy, as evidenced by the lack of change in endometrial thickness over observation periods of up to 3 yr. An assessment of 969 healthy postmenopausal women monitored for 36 mo showed no statistically signiﬁcant difference in the incidence of endometrial carcinoma between raloxifene- and placebo-treated women (85). Baseline endometrial screening was per-

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Table 6 Adverse Events Most Commonly Reported With Approved SERMs Adverse Event Common Hot ﬂashes Endometrial stimulation Vaginal bleeding/discharge Leg cramps Nausea Rare Venous thromboembolic events Endometrial cancer (increased risk) Ocular problems Corneal opaciﬁcation a

Tamoxifen

Toremifene

Raloxifene

Yes Yes Yes No No

Yes Yes Yes No Yes

Yes No No Yes No

Yes Yes

Probable Probable

Yes Not seena

Yes

Yes

Not seena

By >40 months follow-up

formed through transvaginal sonography; the incidence of vaginal bleeding and rates of endometrial sampling during the study were comparable among the treatment and placebo groups.85 Similar ﬁndings that raloxifene did not have an adverse impact on the endometrium were reached in the MORE trial. After a median 40 mo of followup in 6000 postmenopausal women with osteoporosis, the risk of endometrial cancer in the raloxifene group did not differ from that in the placebo group (RR: 0.8; 95% CI: 0.2–2.7) (34). Although endometrial ﬂuid accumulation occurred slightly more frequently among older postmenopausal women treated with raloxifene than among those receiving placebo, it was not associated with an increased risk for either benign endometrial changes or carcinoma and is consistent with endometrial ﬂuid increases associated with aging and subsequent endometrial atrophy (86). Histologic studies of endometrial tissue have found no differences between raloxifene and placebo over 12 mo of treatment or between raloxifene and continuous HRT over 24 mo of treatment (84,87). Raloxifene appears to have a neutral effect on the vaginal epithelium, with patientreported symptoms of vaginal dryness or discharge or other complaints being comparable to those reported for placebo (82). Similarly, raloxifene therapy did not increase the incidence of urogenital events (82). Additional studies focusing on the urogynecologic effects of raloxifene are in progress.

Common Side Effects Tamoxifen Aside from the gynecologic symptoms described in the previous section, the only other commonly occurring side effect associated with tamoxifen therapy is an increased incidence of hot ﬂashes (9) (Table 6). The frequency of hot ﬂashes varies greatly and appears to be highly dependent on the study population (9). Toremifene The most common side effects associated with toremifene therapy are hot ﬂashes, vaginal discharge, and nausea (34,80). (Table 6). The incidence of hot ﬂashes and vaginal discharge with toremifene is comparable to that observed with tamoxifen.

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Raloxifene Hot ﬂashes also are reported with raloxifene therapy (Table 6). Their frequency and severity vary with dosage and the time since menopause (82,85). Another side effect reported with raloxifene therapy is leg cramps. In clinical trials, leg cramps occurred in approx 6% of raloxifene-treated women and 2% of placebo-treated women (82). These cramps were generally reported as mild, were not associated with other adverse events, and appeared to be idiopathic.

Severe Adverse Events Although a rare event, tamoxifen and raloxifene have been linked to an increased risk for venous thromboembolic events (VTEs), including deep vein thrombosis and pulmonary embolism (Table 6). This increased risk is comparable to the risk found with estrogen therapy (9,34,88,89). The incidence of VTEs in women taking toremifene is probably comparable to that in women taking tamoxifen or raloxifene, but the limited sizes of toremifene clinical trials to date do not allow a conclusive determination (13,14,90). Tamoxifen and toremifene have been linked to rare ocular changes, such as corneal opaciﬁcation (13,14,91,92). Tamoxifen slightly increases the risk for cataracts (RR: 1.14; 95% CI: 1.01–1.29) (9). There have been no reports of adverse ocular events associated with raloxifene.

CONCLUSIONS SERMs—compounds that behave like estrogen in some tissues but block the action of estrogen in others—provide a growing array of clinical applications that have impressive potential for improving postmenopausal health in the next century. Based on the results of large, randomized, placebo-controlled clinical trials, SERMs have received worldwide approval for a variety of indications. Tamoxifen and toremifene have been approved for the treatment of breast cancer; tamoxifen has been approved for the prevention of breast cancer in women at high risk for the disease; and raloxifene has been approved for the prevention and treatment of postmenopausal osteoporosis (9,16,17,20). SERMs continue to be the focus of intense research activity. Ongoing areas of research with SERMs include prevention and treatment of osteoporosis, cancer, and cardiovascular disease. The urogynecologic and cognitive effects of SERMs are being investigated (93). Many of these research efforts are exploring the possibility that SERMs may offer some of the beneﬁts of estrogen-replacement therapy but carry a lower risk for adverse effects. Ultimately, studies such as the STAR and RUTH trials will provide the long-term clinical outcomes data needed to assess the broadening range of indications for SERMs in the maintenance of postmenopausal health.

REFERENCES ˙ , Kushner PJ, et al. Differential ligand 1. Paech K, Webb P, Kuiper GGJM, Nilsson S, Gustafsson J-A activation of estrogen receptors ER-α and ER-β at AP1 sites. Science 1997;277:1508–1510. 2. Clomid package insert. Hoechst Marion Roussel, Kansas City, Mo, 1996. 3. Serophene package insert. Serono Laboratories, Inc, Norwell, Mass, 1994. 4. Nolvadex package insert. Zeneca Pharmaceuticals, Wilmington, Del, 1998. 5. Fareston package insert. Schering Corporation, Kenilworth, NJ, 1997. 6. Evista package insert. Eli Lilly and Company, Indianapolis, 1999.

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7. Early Breast Cancer Trialists’ Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet 1998;351:1451–1467. 8. Fisher B, Dignam J, Bryant J, et al. Five versus more than ﬁve years of tamoxifen therapy for breast cancer patients with negative lymph nodes and estrogen receptor-positive tumors. J Natl Cancer Inst 1996;88:1529–1542. 9. Fisher B, Costantino JP, Wickerham DL, et al. Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst 1998;90:1371–1388. 10. Veronesi U, Maisonneuve P, Costa A, et al. Prevention of breast cancer with tamoxifen: preliminary ﬁndings from the Italian randomised trial among hysterectomised women. Italian Tamoxifen Prevention Study. Lancet 1998;352:93–97. 11. Powles T, Eeles R, Ashley S, et al. Interim analysis of the incidence of breast cancer in the Royal Marsden Hospital tamoxifen randomised chemoprevention trial. Lancet 1998;352:90–101. 12. Hayes DF, Van Zyl JA, Hacking A, et al. Randomized comparison of tamoxifen and two separate doses of toremifene in postmenopausal patients with metastatic breast cancer. J Clin Oncol 1995;13: 2556–2566. 13. Pyrho¨nen S, Valavaara R, Modig H, et al. Comparison of toremifene and tamoxifen in post-menopausal patients with advanced breast cancer: a randomized double-blind, the ‘nordic’ phase III study. Br J Cancer 1997;76:270–277. 14. Gershanovich M, Garin A, Baltina D, Kurvet A, Kangas L, Ellme´n J. A phase III comparison of two toremifene doses to tamoxifen in postmenopausal women with advanced breast cancer. Eastern European Study Group. Breast Cancer Res Treat 1997;45:251–262. 15. Holli K. Adjuvant trials of toremifene vs tamoxifen: the European experience. Oncology 1998; 12(suppl 5):S23–S27. 16. Delmas PD, Bjarnason NH, Mitlak BH, et al. Effects of raloxifene on bone mineral density, serum cholesterol concentrations, and uterine endometrium in postmenopausal women. N Engl J Med 1997; 337:1641–1647. 17. Ettinger B, Black DM, Mitlak BH, et al. Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3-year randomized clinical trial. JAMA 1999; 282:637–645. 18. Jordan VC, Glusman JE, Eckert S, et al. Raloxifene reduces incident primary breast cancers: integrated data from multicenter, double-blind, placebo-controlled, randomized trials in postmenopausal women. Breast Cancer Res Treat 1998;50:227. 19. Arteaga CL, Osborne CK. Growth factors as mediators of estrogen/antiestrogen action in human breast cancer cells. In: Lippman M, Dickson R, eds. Regulatory Mechanisms in Breast Cancer pp. 289–304. Kluwer Boston: Kluwer 1991. 20. Osborne CK. Tamoxifen in the treatment of breast cancer. N Engl J Med 1998;339:1609–1618. 21. Ellis PA, Saccani-Jotti G, Clarke R, et al. Induction of apoptosis by tamoxifen and ICI 182780 in primary breast cancer. Int J Cancer 1997;72:608–613. 22. Ragaz J, Coldman A. Survival impact of adjuvant tamoxifen on competing causes of mortality in breast cancer survivors, with analysis of mortality from contralateral breast cancer, cardiovascular events, endometrial cancer, and thromboembolic episodes. J Clin Oncol 1998;16:2018–2024. 23. Yasui Y, Potter JD. The shape of age-incidence curves of female breast cancer by hormone-receptor status. Cancer Causes Control 1999;10:431–437. 24. Tormey DC, Gray R, Falkson HC. Postchemotherapy adjuvant tamoxifen therapy beyond ﬁve years in patients with lymph node-positive breast cancer. Eastern Cooperative Oncology Group. J Natl Cancer Inst 1996;88:1828–1833. 25. Stewart HJ, Forrest AP, Everington D, et al. Randomised comparison of 5 years of adjuvant tamoxifen with continuous therapy for operable breast cancer. The Scottish Cancer Trials Breast Group. Br J Cancer 1996;74:297–299. 26. Rutqvist LE, Cedermark B, Glas U, et al. Contralateral primary tumors in breast cancer patients in a randomized trial of adjuvant tamoxifen therapy. J Natl Cancer Inst 1991;83:1299–1306. 27. Gail MH, Brinton LA, Byar DP, et al. Projecting individualized probabilities of developing breast cancer for white females who are being examined annually. J Natl Cancer Inst 1989;81:1879–1886. 28. Pritchard KI. Is tamoxifen effective in prevention of breast cancer? Lancet 1998;352:80–81. Comment. 29. Chlebowski RT, Collyar DE, Somerﬁeld MR, Pﬁster DG. American society of clinical oncology

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51. Grey AB, Stapleton JP, Evans MC, Reid IR. The effect of the anti-estrogen tamoxifen on cardiovascular risk factors in normal postmenopausal women. J Clin Endocrinol Metab 1995;80:3191–3195. 52. Guetta V, Lush RM, Figg MD, Waclawiw MA, Cannon RO III. Effects of the antiestrogen tamoxifen on low-density lipoprotein concentrations and oxidation in postmenopausal women. Am J Cardiol 1995;76:1072–1073. 53. Cattaneo M, Baglietto L, Zighetti ML, et al. Tamoxifen reduces plasma homocysteine levels in healthy women. Br J Cancer 1998;77:2264–2266. 54. Costantino JP, Kuller LH, Ives DG, Fisher B, Dignam J. Coronary heart disease mortality and adjuvant tamoxifen therapy. J Natl Cancer Inst 1997;89:776–782. 55. McDonald CC, Alexander FE, Whyte BW, Forrest AP, Stewart HJ. Cardiac and vascular morbidity in women receiving adjuvant tamoxifen for breast cancer in a randomised trial. The Scottish Cancer Trials Breast Group. BMJ 1995;311:977–980. 56. McDonald CC, Stewart HJ. Fatal myocardial infarction in the Scottish adjuvant tamoxifen trial. The Scottish Breast Cancer Committee. BMJ 1991;303:435–437. 57. Rutqvist LE, Mattsson A. Cardiac and thromboembolic morbidity among postmenopausal women with early-stage breast cancer in a randomized trial of adjuvant tamoxifen. J Natl Cancer Inst 1993; 85:1398–1406. 58. Gylling H, Pyrho¨nen S, Ma¨ntyla¨ E, Ma¨enpa¨a¨ H, Kangas L, Miettinen TA. Tamoxifen and toremifene lower serum cholesterol by inhibition of delta 8-cholesterol conversion to lathosterol in women with breast cancer. J Clin Oncol 1995;13:2900–2905. 59. Jordan VC. Tamoxifen and endometrial cancer. Lancet 1989;1:733–734. Letter. 60. Neven P, De Muylder X, Van Belle Y. Tamoxifen and the uterus and endometrium. Lancet 1989; 1:375. Letter. 61. Mathew A, Chabon AB, Kabakow B, Drucker M, Hirschman RJ. Endometrial carcinoma in ﬁve patients with breast cancer on tamoxifen therapy. NY State J Med 1990;90:207–208. 62. Barakat RR. Tamoxifen and endometrial neoplasia. Clin Obstet Gynecol 1996;39:629–640. 63. Barakat RR. Tamoxifen and the endometrium. In: Foon KA, Muss HR, eds. Biological and Hormonal Therapies of Cancer. Kluwer, Boston, 1998, pp. 195–207. 64. Schwartz LB, Snyder J, Horan C, Porges RF, Nachtigall LE, Goldstein SR. The use of transvaginal ultrasound and saline infusion sonohysterography for the evaluation of asymptomatic postmenopausal breast cancer patients on tamoxifen. Ultrasound Obstet Gynecol 1998;11:48–53. 65. Goldstein SR. Unusual ultrasonographic appearance of the uterus in patients receiving tamoxifen. Am J Obstet Gynecol 1994;170:447–451. 66. Fisher B, Costantino JP, Redmond CK, Fisher ER, Wickerham DL, Cronin WM. Endometrial cancer in tamoxifen-treated breast cancer patients: ﬁndings from the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14. J Natl Cancer Inst 1994;86:527–537. 67. Fornander T, Rutqvist LE, Cedermark B, et al. Adjuvant tamoxifen in early breast cancer: occurrence of new primary cancers. Lancet 1989;1:117–120. 68. ACOG committee opinion. Tamoxifen and endometrial cancer. Int J Gynecol Obstet 1996;53:197–199. 69. Jennings TS, Creasman MT. Effects on the reproductive tract: clinical aspects. In: Lindsay R, Dempster DW, Jordan VC, eds. Estrogens and Antiestrogens, 1st ed. Lippincott-Raven, Philadelphia, 1997, pp. 223–242. 70. Lahti E, Blanco G, Kauppila A, Apaja-Sarkkinen M, Taskinen PJ, Laatikainen T. Endometrial changes in postmenopausal breast cancer patients receiving tamoxifen. Obstet Gynecol 1993;81:660–664. 71. Toma´s E, Kauppila A, Blanco G, Apaja-Sarkkinen M, Laatikainen T. Comparison between the effects of tamoxifen and toremifene on the uterus in postmenopausal breast cancer patients. Gynecol Oncol 1995;59:261–266. 72. Hulka CA, Hall DA. Endometrial abnormalities associated with tamoxifen therapy for breast cancer: sonographic and pathologic correlation. Am J Roentgenol 1993;160:809–812. 73. Uziely B, Lewin A, Brufman G, Dorembus D, Mor-Yosef S. The effect of tamoxifen on the endometrium. Breast Cancer Res Treat 1993;26:101–105. 74. Tesoro MR, Borgida AF, Maclaurin NA, Asuncion CM. Transvaginal endometrial sonography in postmenopausal women taking tamoxifen. Obstet Gynecol 1999;93:363–366. 75. Bertelli G, Venturini M, Del Mastro L, et al. Tamoxifen and the endometrium: ﬁndings of pelvic ultrasound examination and endometrial biopsy in asymptomatic breast cancer patients. Breast Cancer Res Treat 1998;47:41–46. 76. McGonigle KF, Shaw SL, Vasilev SA, Odom-Maryon T, Roy S, Simpson JF. Abnormalities detected

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VI

ROLES OF ESTROGENS AND SERMS IN POSTMENOPAUSAL HORMONE REPLACEMENT THERAPY

18

Menopause Therapy An Individualized Approach

Nananda F. Col, MD, FACP, MPP, MPH, Michele G. Cyr, MD, FACP, and Anne W. Moulton, MD, FACP Contents Introduction Overview of the Evidence A Framework for Choosing Treatment on an Individual Basis Findings from the Decision Model: Comparing Raloxifene, HRT, and Alendronate Summary References

INTRODUCTION The declining estrogen levels that occur at menopause increase women’s risk for developing certain chronic conditions, and can result in both short- and long-term symptoms. Risks for osteoporosis and coronary heart disease (CHD) rise substantially after menopause. Osteoporosis results in more than 1.3 million fractures annually (1), and CHD claims approximately 230,000 women’s lives each year (2). Vasomotor symptoms occur in approx 70% of perimenopausal women, and include hot ﬂashes, night sweats, and sleep disturbance. Although these typically subside within several years, they may have signiﬁcant impact on a woman’s quality of life if left untreated. Genitourinary symptoms usually develop within 2 to 5 yr after menopause and include vaginal dryness, dysuria, urinary urgency, frequency, and stress or urge incontinence. Many women choose to use estrogen for symptom relief during the menopause transition as it is 95% effective in relieving vasomotor symptoms and is helpful in preventing many genitourinary symptoms. Although short-term treatment (2–5 yr) is often sufﬁcient for symptom relief, women who are interested in preventing chronic conditions such as osteoporosis and heart disease need to consider longer use of estrogen or other

From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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treatments. Long-term use of estrogen, however, has been associated with increased risks for breast cancer (3–6), venous thromboembolism (VTE) (7–10), gallbladder disease (11), and endometrial cancer (if used without a progestin in women with an intact uterus) (12). These increased risks have led many menopausal women to consider treatments other than hormone replacement therapy (HRT), such as alendronate and the selective estrogen receptor modulators (SERMS) raloxifene and tamoxifen. Each of these treatments has a unique risk/beneﬁt proﬁle. Deciding which treatment, if any, is appropriate for an individual patient requires a careful assessment of the risks and beneﬁts of each treatment, along with the individual’s risk factor proﬁle and goals for treatment.

OVERVIEW OF THE EVIDENCE Alendronate. Alendronate, a bisphosphonate, is a nonhormonal therapeutic agent that has been shown to be effective in both the prevention and treatment of osteoporosis. Alendronate inhibits bone resorption, increasing bone mineral density by 3 to 7% over a 4-yr period (13,14) and reducing the risks for spinal and hip fractures among women with established osteoporosis or with previous spinal fractures by 30 to 50% (15,16). The Fracture Intervention Trial (17) reported a 36% reduction in the risk for any type of clinical fracture (RR=0.64) after 4 yr of treatment among osteoporotic women (whose T-score at femoral neck was −2.5 or less). No apparent beneﬁt, however, was found among osteopenic women (whose T-score at the femoral neck fell between −1.16 and −2.5)—the RR was 1.03−1.14) (17). The beneﬁcial effects of alendronate on bone mineral density (BMD) are only apparent with ongoing treatment. After discontinuation of treatment, the rate of bone loss parallels that observed in women not receiving treatment (15). Alendronate has no known carcinogenic effects and no known impact on CHD, venous thrombosis, or menopausal symptoms. Its clinical use is limited by its limited bioavailability, tendency to cause esophagitis, and cost. This daily medication must be taken on an empty stomach with eight ounces of water, and the patient must remain in an upright position for 30 minutes after ingestion to avoid potentially fatal esophageal ulcers. FDA approval is pending for once-weekly dosing, which appears to be as effective and safe as the daily regimen, and is more convenient. Raloxifene. Raloxifene is a nonsteroidal compound with mixed estrogenic and antiestrogenic properties (18–21). It has been shown to decrease the risk of vertebral fractures and to increase BMD in the hip and spine (22). It is FDA approved for the prevention of osteoporosis in the United States and was recently approved for the treatment of osteoporosis in Europe. A recent randomized study comparing the effects of raloxifene and HRT on lumbar spine and total body BMD found that HRT (conjugated equine estrogen [CEE]) had an approx twofold greater effect (23). Raloxifene has been shown to lower serum total cholesterol and HDL levels, and to have a mixed effect on clotting factors that may affect CHD risk (24,25). It does not appear to increase the risk of endometrial cancer (26). Randomized trials following 10,385 postmenopausal women reported a signiﬁcantly decreased risk of breast cancer among raloxifene users after 3 yr of treatment compared to placebo (RR 0.38, 95% CI:0.22–0.68) (27). A smaller subset of 7704 women (mean age 67) randomized to raloxifene were found to have a RR of breast cancer of 0.26 (95% CI:0.13–0.52), but median followup was only 29 mo, and only 32 cases of breast cancer occurred (28). Because of the relatively

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Table 1 Impact of Tamoxifen on Clinical Events, Based on Findings from the NSABP P-1 Trial Outcome (age 50–59) Invasive breast cancer Endometrial cancer Deep venous thrombosis Pulmonary embolism Hip fracture

Rate per 1000 women Placebo

Rate per 1000 women Tamoxifen

Relative Risk

95% CI

6.28 0.76 0.88 0.31 0.84

3.10 3.05 1.51 1.00 0.46

0.49 4.01 1.71 3.19 0.55

0.29–0.81 1.70–10.90 0.85–3.58 1.12–11.15 0.25–1.15

short duration of followup, raloxifene’s long-term impact on breast cancer cannot be established with any certainty at this time. Raloxifene was found to signiﬁcantly increase the risk of VTE: the RR was 3.1 (95% CI:1.5–6.2) (28). Raloxifene has no beneﬁcial effect on menopausal symptoms and induces hot ﬂashes in approx 25% who begin therapy (compared to 18% receiving placebo). The severity of hot ﬂashes does not seem to be affected by raloxifene. The risk of hot ﬂashes caused by raloxifene occurred only within the ﬁrst six months after initiating treatment. Raloxifene does not appear to be associated with insomnia, night sweats, or vaginal atrophy. Tamoxifen. Tamoxifen is a nonsteroidal agent with antiestrogenic effects that suppresses the proliferation of neoplastic breast epithelial cells (29) and decreases the risk of recurrence and contralateral disease among women with breast cancer (30). Tamoxifen was recently approved for the primary prevention of breast cancer among women at high risk for breast cancer. While acting as an estrogen antagonist in the breast, tamoxifen also has estrogen agonist effects on bone metabolism in postmenopausal women (31,32), lipid proﬁles (33), coagulation factors (34), and the endometrium (35). Tamoxifen’s effectiveness in preventing breast cancer was demonstrated in the randomized, controlled National Surgical Adjuvant Breast and Bowel Project (NSABP) P-1 Trial (36) conducted among 13,388 U.S. women at high risk for breast cancer followed for an average of 48 mo. In this trial, tamoxifen halved the risk of invasive breast cancer, but quadrupled the risk of endometrial cancer among women over age 50 (Table 1). Tamoxifen also increased the risk of pulmonary embolism (RR 3.19, 95% CI:1.12–11.15), deep venous thrombosis (RR 1.71, 95% CI:0.85–3.58), cataracts (RR 1.14, 95% CI:1.01–1.29) and menopausal symptoms, and decreased the risk of hip fracture, though not signiﬁcantly so (RR 0.55, 95% CI:0.25–1.15). Tamoxifen has been shown to induce or worsen menopausal symptoms, including hot ﬂashes and vaginal discharge, in approx 20% of users. Although tamoxifen’s protective effect on breast cancer was shown to be statistically signiﬁcant in the NSABP-P-1 Trial (36), two smaller randomized, controlled trials conducted in Europe found tamoxifen to have no effect (37,38). This discrepancy in ﬁndings has been attributed to differences in the cohort baseline risk, inclusion of women concurrently taking HRT in the European trials, variation in the duration of treatment, and lack of statistical power (39). It is unlikely that tamoxifen confers signiﬁcant protection against CHD, although considerable controversy remains. Three randomized trials suggested that tamoxifen

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Table 2 Impact of Selected Treatments on Bone Mineral Density and Serum Lipid Proﬁle

Treatment Estrogen norethisterone Estrogen + MPA Alendronate Raloxifene

Bone Mineral Density Femoral necka

Serum Total Cholesterolb

HDLb

Chol/HDL Ratiob

+3.7% +1.8% +1.4% +0.97%

NA −6.2 —c −6.4

NA +2.0 — −3.7

NA +0.92 — +0.97

a

% change. % absolute change from baseline. c There are no data showing that alendronate has any impact on serum cholesterol levels. b

might reduce the risk of CHD among postmenopausal women previously diagnosed with breast cancer (40–44). Only one of these trials however found a statistically signiﬁcant reduction in CHD mortality. Furthermore, these studies were confounded by the presence of cancer and therapies administered for cancer recurrence (45). Tamoxifen had no impact on the development of future fatal and nonfatal CHD events in the much larger P-1 trial (the risk ratio for ischemic heart disease was 1.15, 95% CI:0.81– 1.64). The metanalysis by the Early Breast Cancer Trialists’ Collaboration Group, including more than 36,000 patients, yielded a null effect on noncancer mortality (rate ratio, 0.99). It is important to note that none of these trials were designed to monitor CHD as a primary study endpoint. Little data are available concerning concurrent HRT and tamoxifen use. The Royal Marsden Hospital Trial included 523 women using both tamoxifen and HRT. Subgroup analyses found no interaction between HRT use and tamoxifen’s effect on breast cancer. Hormone-Replacement Therapy. HRT increases BMD initially and reduces the risk of hip fracture by 68% (46) after ﬁve years of use, though protection wanes after discontinuation. No randomized, controlled studies are available at this time linking HRT use to clinical fractures, though numerous observational studies have suggested a substantial protective effect in both the prevention and treatment osteoporosis (47). A recent randomized, controlled trial compared the effects of four years of treatment with different formulations of HRT to alendronate (5 mg/d) on bone mineral density (15). The HRT preparations that contained progestins with androgen activity (norethisterone, the progestin commonly used in Europe) had a more pronounced effect on BMD than the HRT formulations using progestins without androgen activity (medroxyprogesterone acetate [MPA], the progestin most commonly used in the United States) or alendronate (see Table 2) (15). The impact of HRT on CHD has been the subject of considerable controversy (48). Randomized trials have found HRT to have a beneﬁcial effect on serum total cholesterol and HDL levels and to have a favorable effect on PAI-1 and other coagulation factors that may affect CHD risk (49). The Nurses Health Study, which followed approx 60,000 menopausal women for 16 years, found that HRT use was associated with a signiﬁcant and substantial 40% reduction in CHD risk (RR 0.60, 95% CI:0.47–0.76) (50). This study, however, was not randomized and some of the observed beneﬁt of HRT could

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have been attributed to the “healthy user” effect—women who used HRT were healthier than women who did not use HRT. Although this well-designed study attempted to control for known risk factors for CHD using statistical techniques, it is impossible to disentangle cause and effect in the absence of randomization. A recent randomized trial examining the effect of combination HRT (estrogen with MPA) on women with established CHD (the HERS study) found HRT to have no beneﬁcial effect on secondary prevention of CHD (51). This study found an increased risk of CHD events during the ﬁrst year of treatment, followed by a protective trend after 3 yr of treatment. The generalizability of these results to women taking estrogen alone or to women without CHD has been questioned. A subgroup analysis of the HERS cohort suggested that women who had elevated levels of Lipoprotein A (Lp(a)), an independent risk factor for CHD, who received HRT had signiﬁcant reductions in Lp(a) after 1 yr of treatment and a 15–22% decreased risk of CHD events. Among women with Lp(a) levels above the median, the risk of CHD events was lowered by HRT during years two through ﬁve of therapy (RR 0.68, 95% CI:0.48–0.95) (52). The most recent estimates from the Nurses Health Study (53) concerning the impact of HRT on established CHD (secondary prevention) concurred with the HERS trial results, ﬁnding an increase in CHD risk during the ﬁrst year of use (RR=2.1), a decrease in risk after two years of treatment (RR=0.56), and no residual beneﬁt after discontinuation of treatment. The impact of HRT on breast cancer risk has also been the subject of considerable controversy. Numerous studies have found a small, but signiﬁcantly increased, risk of breast cancer associated with long-term HRT use (greater than 5 yr). A pooled reanalysis of 51 epidemiological studies revealed an overall RR of 1.35, (95% CI:1.21–1.49) after ﬁve years of use for current users (54). Most of the older studies addressed the effect of unopposed estrogen. Recent studies, however, have questioned the impact of adding a progestin on breast cancer risk. The Nurses Health Study reported no signiﬁcant difference between the impact of unopposed estrogen (RR 1.32, 95% CI:1.14–1.54) and combination HRT (RR 1.41, 95% CI:1.15–1.74) respectively (3), though the number of women using combination therapy was relatively small. Two recent case-control studies have suggested that progestin does increase the risk of breast cancer (55). The smaller of these studies (55) reported a RR of 1.4 (95% CI:1.1–1.8) after 4 yr of combination treatment. This estimate, however, was based on a very small sample size, with only 39 cases of breast cancer in women using combination HRT for more than four yr. A considerably larger case-control trial reported that combination HRT increased the risk of breast cancer (RR=1.51) after 10 yr of use, compared to estrogen alone (RR=1.24 after 15 yr of use) (56). Estrogen has no impact on endometrial cancer if progestin is used concomitantly (47). When estrogen is used alone, the risk for endometrial cancer increases eight-to-tenfold (12). There has been ongoing interest in estrogen’s role in the prevention and treatment of Alzheimer’s disease. A randomized, controlled trial of estrogen therapy for mild to moderate Alzheimer’s disease showed no effect of 1 yr of therapy on disease progression, or on global, cognitive, or functional outcomes (57). A metanalysis of observational studies addressing estrogen’s role in cognition and dementia revealed an observed risk of dementia in estrogen users of 0.71 (95% CI:0.53– 0.96) compared to nonusers (58). The studies contributing to the metanalysis, however, suffer signiﬁcant methodological problems and a deﬁnitive conclusion cannot be reached

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in the absence of a randomized, controlled trial. The Women’s Health Initiative Memory Study will address the question of HRT’s effect on cognitive function and risk of Alzheimer’s disease. Data from the Nurse’s Health Study were analyzed to assess the effect of estrogen on colorectal carcinoma and adenoma (59). Current estrogen use was associated with a decreased risk of colorectal cancer (RR=0.65, 95% CI:0.50–0.83). This effect disappeared ﬁve yr after discontinuing the treatment. An earlier metanalysis found no clear relationship between the use of HRT and risk for colorectal cancer (60). Because these represent observational data only, randomized, controlled trials are needed to substantiate these ﬁndings.

A FRAMEWORK FOR CHOOSING TREATMENT ON AN INDIVIDUAL BASIS To help choose therapy, we developed a decision analytic Markov model that compared various treatment options (HRT, alendronate, raloxifene, and tamoxifen) to no treatment among postmenopausal women. The model simulates the lifetime incidence of breast cancer, endometrial cancer, hip fracture, VTE, and CHD, using published regression models that link individual risk factors to future disease incidence. All women began the simulation without evidence of any of these diseases. With each subsequent simulated year, cohort members could develop breast cancer, endometrial cancer, VTE, or CHD, sustain a hip fracture, or die from other causes as occur in the general population at a rate determined by the woman’s attained age. Breast cancer risk was based on a composite risk score that included information about a woman’s family history, age at menarche, age at ﬁrst live birth, and number of previous benign breast biopsies, using the Gail model (61). The baseline age-adjusted incidence rates for breast cancer were derived from recent Surveillance, Epidemiology, and End Results (SEER) incidence data (62). The odds of developing CHD (63,64) were calculated according to individual risk proﬁles, using coefﬁcients from logistic regression equations that link speciﬁc coronary risk (diabetes, blood pressure, age, tobacco use, and left ventricular hypertrophy) to disease incidence. Because there are presently no data linking the use of raloxifene to CHD endpoints, raloxifene’s impact on total cholesterol and HDL levels was used to simulate its effect on CHD. Endometrial cancer risk was based on age- and race-adjusted incidence rates drawn from recent SEER data. Risk for endometrial cancer was based on the presence of speciﬁc risk factors including obesity, nulliparity, late age at menopause, and use of unopposed exogenous estrogen, using a relative risk scale to describe varying levels of risk according to the presence or absence of speciﬁc risk factors. High risk was deﬁned in these analyses as having a risk factor that corresponds to twice the average risk (RR=2) and highest risk as threefold. Data describing the impact of raloxifene, tamoxifen, HRT, and alendronate that were used in the decision model are presented in Table 1. Data on the impact of tamoxifen were based on ﬁndings from the P-1 trial for women 50 years or older. Even though tamoxifen’s protection against hip fractures was of borderline statistical signiﬁcance, we applied the point estimate for risk reduction found in the P-1 trial because tamoxifen has consistently been found to increase bone mineral density in postmenopausal women.

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We assumed that tamoxifen was taken for only ﬁve years, whereas we assumed ongoing treatment with the other choices.

FINDINGS FROM THE DECISION MODEL: COMPARING RALOXIFENE, HRT, AND ALENDRONATE No single treatment option was consistently better or worse than the other options for all women. If gains in life expectancy are used to compare the expected beneﬁts of long-term treatment, then raloxifene was preferred over both HRT and alendronate for women at high risk for breast cancer, with gains in life expectancy greater than six months compared to no treatment (Fig. 1). Women at high risk for CHD had larger gains in life expectancy from HRT than they would from either raloxifene, alendronate, or no treatment, if HRT confers long-term protection against CHD. Women at lowest risk for hip fracture, breast cancer, and CHD would not beneﬁt substantially from any of these treatments. Gains in life expectancy from alendronate were less than three months for most women. Although alendronate was never associated with higher gains in life expectancy than either HRT or raloxifene, the use of life expectancy to compare treatments may underestimate the clinical beneﬁts of alendronate because osteoporotic fractures tend to occur late in life. Also, life expectancy gains do not reﬂect the substantial morbidity associated with osteoporosis (vertebral fractures and long-term disability from hip fractures). The gains in life expectancy from each of the therapeutic choices examined are relatively high when compared to other preventive strategies. Gains in life expectancy from mammography are approximately 1 month and gains from annual PAP smears are approximately three months (65). Examining the impact of treatment choice on certain clinical events (Table 3), HRT, alendronate, and raloxifene should have somewhat similar efﬁcacies in preventing hip fracture (the simulated relative risks were 0.57, 0.54, and 0.58, respectively). The number needed to treat (NNT) for 10 yr to prevent a single hip fracture was approximately 1000 for women at low risk for hip fracture, 600 for women at average risk, and 200 for women at high risk, with relatively small differences between raloxifen, HRT, and alendronate (Table 3). The predictions from the decision model are very sensitive to the assumptions contained in the decision model. Raloxifen and alendronate have only been studied in clinical trials for a relatively few years; their long-term impact on osteoporosis and breast cancer is not known at this time. Although we have much longer clinical experience with HRT, we have no randomized data concerning the primary prevention of CHD or osteoporotic fractures at this time. The model extrapolates short-term ﬁndings over a longer period of time, which may or may not be appropriate until long-term data are available. While we await randomized trials to report, this approach can be used to approximate the long-term implications of treatment. Three clinical examples are discussed below to facilitate this process. Clinical Examples Example 1: A 68-year-old woman with a history of deep venous thrombosis in her 40s presents for followup of test results. She is now 20 years postmenopausal and her

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Fig. 1. Identiﬁcation of optimum treatment, based on gains in life expectancy, according to a woman’s risk factors for breast cancer and CHD. Referring to Table 3, each risk factor can be linked to its risk weight and then all risk weights summed to determine the aggregate risk score for CHD and breast cancer, as plotted here. Using Clinical Example 2 to illustrate this process, based on having menarche at age 12 (add 0.10 points), 1 previous breast biopsy (add 0.24 points), and one ﬁrstdegree relative with breast cancer who is nulliparous (add 1.01 points), her total breast cancer risk score is 0.10 + 0.24 + 1.01 = 1.35. To determine her risk score for CHD, more information about her CHD risk factors (systolic blood pressure, cholesterol/HDL ratio) would be necessary. For women interested in estimating the impact of HRT on their personal risks for developing CHD, breast cancer, and hip fracture, as well as on life expectancy, these risk scores can also be applied to ﬁgures published elsewhere (66). From Col. NF, Pauker SG, Goldberg RJ, Eckman MH, Orr RK, Ross EM, Wong JB. Individualizing therapy to prevent long-term consequences of estrogen deﬁciency in postmenopausal women. Arch Int Med 1999;159:1458–1466.

bone mineral densitometry reveals osteoporosis. She has no menopausal symptoms except some vaginal dryness that she treats with over-the-counter preparations, and has no other past medical history. Her family history is negative for cardiovascular disease and breast cancer. She has a remote smoking history, normal cholesterol, and normal blood pressure. Comment: Because of her previous DVT, HRT, raloxifene, and tamoxifen would all be contraindicated as each of these treatments increases her risk of recurrent DVT or PE. Because she has osteoporosis, treatment with either alendronate or other therapies (such as calacitonin nasal spray, Miacalcin) should be discussed. Alendronate is more effective, but is more inconvenient pending the approval of weekly dosing. Treatment should also include sufﬁcient calcium, vitamin D, and appropriate exercise.

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Table 3 Expected Effect of Treatment Choice and Level of Risk on the Number Needed to Treat (Compared to Conservative Care) to Prevent One Case of Selected Clinical Events 10-Yr NNTa Risk Levelb Low

Average

High

Treatment

Hip Fracture

CHD

Breast Cancer

HRT Alendronate Raloxifene Tamoxifenc HRT Alendronate Raloxifene Tamoxifen HRT Alendronate Raloxifene Tamoxifen

909 909 1000 — 588 588 625 >700 213 217 227 >700

130 — 1000 — 46 — 435 — 19 — 244 —

−208 — 222 — −149 — 161 85 −49 — 52 51

a NNT is the number needed to treat for 10 years to prevent one case; a negative sign indicates the number needed to induce one case of breast cancer. b Low risk for hip fracture is deﬁned as the absence of any risk factors among women whose weight has increased >20% since age 25; average risk mirrors the distribution of risk factors in the US population for 50-year-old women; high risk corresponds to the presence of two risk factors for hip fracture. Risk factors are listed in Table 4. Low risk for CHD is deﬁned as the absence of any risk factors and having a total serum cholesterol/ HDL ratio of <2.8 and systolic blood pressure <125. Low risk for breast cancer is deﬁned as having no ﬁrst degree relatives with breast cancer, no previous breast biopsies, and being less than 25 years of age at ﬁrst live birth. Average risk mirrors the distribution of risk factors in the US population for 50-year-old women. High risk corresponds to having two or more ﬁrst degree relatives with breast cancer. c Assumes that tamoxifen is taken for only 5 yr but that the protective effect on breast cancer continues for an additional 5 yr.

Example 2: A 48-year-old woman with FSH >40 ng presents to discuss menopause treatment. She has occasional—not bothersome—hot ﬂashes. Her past medical history is signiﬁcant for hypertension controlled on a diuretic, and hypercholesterolemia normalized with a statin. She has had a breast biopsy that revealed ﬁbrocystic breast disease. She is nulliparous and had menarche at age 12. Her sister was diagnosed with breast cancer in her early 50s and she wants to avoid any increase in her risk for breast cancer. Comment: This patient is at high risk for developing breast cancer and heart disease and is taking appropriate measures to reduce her CHD risk. Although HRT is not absolutely contraindicated in this patient, it would not be appropriate given her stated desire to avoid increasing her risk for breast cancer. While HRT would likely improve her hot ﬂashes, her symptoms are so mild at this time as to not justify treatment. Raloxifene or tamoxifen would be considerations as they might help to reduce her risk of breast cancer, although either of these treatments might worsen her menopausal symptoms and tamoxifen would increase her risk for endometrial cancer. The choice between raloxifene and conservative care depends on her preferences. A brief trial of raloxifene to determine its effect on symptoms could be offered to the patient to help her make a more informed decision.

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Table 4 Risk Factors Predicting the Lifetime Risk of Selected Diseases Risk Weighta

Risk Factor CORONARY HEART DISEASE Systolic blood pressure (mmHg) 100 110 120 130a 140 150 160 170 180 Total cholesterol/HDL ratio 2 3 4a 5 6 7 8 9 10 Left ventricular hypertrophy on ECG History of diabetes Cigarette smoking BREAST CANCER Age at menarche (years) <12 12,13a >13 Number of previous benign breast biopsies 0a 1 ≥2 Age at ﬁrst live birth (yr) <20

20–24 25–29 or nulliparousa ≥30

0.0 0.09 0.17 0.24a 0.31 0.37 0.43 0.48 0.54 0.0 0.29 0.49a 0.66 0.79 0.90 0.99 1.08 1.15 0.59 0.38 0.28

0.20 0.10a 0.01 0.0a 0.24 0.48 Number of ﬁrst degree relatives with breast cancer 0 1 ≥2 0 1 ≥2 0a 1 ≥2 0 1 ≥2

0 0.96 1.92 0.22 0.99 1.75 0.44a 1.01 1.59 0.66 1.04 1.43 continued

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Table 4 (continued) Risk Weighta

Risk Factor HIP FRACTURE History of maternal hip fracture Previous hyperthyroidism Current use of long-acting benzodiazepines On feet ≤4 hr/d Inability to rise from chair Resting pulse rate >80 beats/min Height >165 cm at age 25 Self-rated health: fair poor Decrease in weight since age 25 20–40% 41–60% Decreased calcaneal bone densityc

1 1 1 1 1 1 1 per 6 cmb 1 2 1 2 1

a

Indicates population-based average values for perimenopausal women. For every 6 cm beyond this height, risk weight increases by 1. c For every 0.10 g/cm2 (one standard deviation) below the mean of 0.41 g/cm2 for calcaneal bone density measures, risk weight increases by one. Bone density measures taken at other sites, such as the trochanter or femur, may be used as a proxy by applying one risk weight for each standard deviation below the mean. b

Example 3: 51-year-old has not had a menstrual period for over a year. She is having fairly frequent hot ﬂashes and signiﬁcant sleep disruption from night sweats. She has no signiﬁcant past medical history or family history. She does not smoke, has normal blood pressure and cholesterol. Comment: A short course of HRT could be offered to provide symptomatic relief. Hot ﬂashes usually last 18–36 months, therefore the patient could be treated for 12–18 months and then HRT could be slowly tapered, assessing symptoms.

SUMMARY We have critically reviewed the effects of the major treatment options available to menopausal women, including hormone-replacement therapy (HRT), alendronate, and the selective estrogen-receptor modulators (SERMS) raloxifene and tamoxifen. The impact of these medications on osteoporosis, heart disease, breast cancer, venous thromboembolism (VTE), and menopausal symptoms is discussed and a simple method for weighing the risks and beneﬁts of these treatments on an individual basis is presented. Limitations of the available evidence for these treatments are discussed as well. Clinical examples illustrate the most appropriate choice of therapy based on a woman’s riskfactor proﬁle. Tamoxifen is most appropriate for women who have a substantially elevated risk for breast cancer, but not deep venous thrombosis or endometrial cancer. Raloxifene is the best choice for women at high risk for osteoporosis and breast cancer but low risk for VTE. Short-term hormone replacement therapy (HRT) is the optimum treatment for women who have severe menopausal symptoms; long-term HRT is appropriate for women at high risk for osteoporosis and low risk for breast cancer and VTE,

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recognizing that the impact of HRT on coronary heart disease (CHD) has not been clearly deﬁned at this time and that existing models use data from observational studies. The magnitude of the gain or loss in life expectancy from treatment choice depends on an individual’s risk factors for hip fracture, breast cancer, endometrial cancer, CHD, and VTE. Individual preferences to treat menopausal symptoms or to avoid speciﬁc outcomes such as breast cancer need to be considered in the context of any gains or losses in survival from treatment.

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Alternatives to Estrogen for Treatment of Menopause Richard J. Santen, MD and JoAnn V. Pinkerton,

MD

Contents Introduction Evidence Linking Breast Cancer to Hormone Replacement Therapy Goals for Use of Alternatives to HRT Urogenital Atrophy Vasomotor Instability Alternative/Herbal Therapy Neurocognitive Dysfunction Safety and Side Effects of Alternatives to Estrogen Practical Approach to Use of Alternatives to Estrogen Conclusions Acknowledgements References

INTRODUCTION Women frequently choose alternatives to hormone-replacement therapy (HRT) for treatment of menopause even though medical indications for estrogens are present (1–5). Among all menopausal women in the United States, only 20–40% ever take HRT (5). Among those given prescriptions, only 65% continue use of HRT after the ﬁrst prescription and only 25% for more than three years (6). Many of these women feel that menopause is a natural transition in life and that menopause-associated problems do not require therapy. Potential complications such as deep venous thrombosis, pulmonary emboli, and stroke inﬂuence the decision process of others. For the estimated 2.5 million survivors of breast cancer in the United States, concern about recurrence is the determining factor in seeking alternatives to use of HRT. For a large number of additional women, fear of developing breast cancer as result of taking HRT is a major

From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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consideration. Before discussing alternatives to HRT, we consider it necessary to ﬁrst examine the evidence linking HRT to breast cancer risk since this issue is the most common reason for women to seek alternatives.

EVIDENCE LINKING BREAST CANCER TO HORMONE REPLACEMENT THERAPY More than 50 case-control and prospective-cohort studies have examined the risk of breast cancer attributable to HRT but the results are conﬂicting (7). No prospective, randomized, placebo-controlled trials have been completed to date. Critical analyses note the potential biases inherent in retrospective studies. For example, women who choose to take HRT could be at higher risk of developing breast cancer for reasons poorly understood. This confounding effect could explain an apparent but not real increase in breast cancer risk in users of HRT. Because of these factors, experts disagree whether existing evidence supports the conclusion that HRT causes breast cancer. The authors of this chapter believe that it is prudent to advise patients that an increased risk of breast cancer is likely, although not yet proven. The strategy is to ask patients to make decisions based upon the “worst case” analysis that the data linking HRT to breast cancer are in fact true. Our thinking on this issue is inﬂuenced by a wide range of biologic, experimental and clinical data which involve animal experiments and studies of oophorectomy, antiestrogens, and HRT in women. Administration of estrogens to a variety of animal species results in breast cancer (8). Oophorectomy before the age of 35 reduces the lifetime risk of breast cancer by 75% in women (9). Antiestrogens cause a 50% reduction in diagnosed breast cancer when taken over a 4 to 5 year period (10). Epidemiologic data from prospective-cohort studies suggest that the risk of breast cancer increases with use of estrogens as hormonereplacement therapy. Taken together, these data provide strong but indirect evidence that HRT increases the risk of breast cancer. Recent reports suggest that progestins add to the risk of breast cancer imparted by estrogens alone (11,12). In interpreting these data, it is important to know whether or not progestins exert mitotic or antimitotic effects on the breast. Progestins oppose the proliferative effects of estrogens on the human endometrium and prevent endometrial cancer. Investigators have hypothesized that progestins might abrogate the carcinogenic effects of estrogen on the breast through a similar antiproliferative action (13) whereas others argue that progestins exert proliferative and thus procarcinogenic effects on the breast (14). Depending upon their structure and the tissues in which they are studied, the various progestins can exert either androgenic, synandrogenic, antiandrogenic, estrogenic, glucocorticoid-like, or progestational effects (15–28). These disparate actions of progestins on human breast cells in culture have confounded interpretation regarding effects on proliferation. Critical evaluation of these data do not establish whether the predominant effect of progestins is to stimulate or inhibit breast cell proliferation. Convincing evidence of the proliferative effect of progestins on breast derives from quantitative studies of mammographic density in women receiving HRT (29–33). Glandular tissue enhances the density of mammograms and adipose tissue reduces it. Thus, breast density can serve as a surrogate marker for long-term glandular cell proliferation. Mammographic density analysis of the Postmenopausal Estrogen/Progestin Interventions (PEPI) trial (29) of 307 eligible candidates out of 875 women was performed.

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Eligibility criteria included available baseline mammogram and a followup mammogram at 12, 24, or 36 months, 80% compliance with assigned medication and no use of estrogen for ﬁve years prior to baseline. At 12 months, the percentage of women with density grade increases was 0% (95% CI:0.0–4.6%) in the placebo group; 3.5% (95% CI:1.0–12.0%) in the conjugated estrogen alone group; 23.5% (95% CI:11.9–35.1%) in the conjugated estrogen plus cyclic MPA group; 19.4% (95% CI:9.9–28.9%) in the conjugated estrogen plus daily MPA group; and 16.4% (95% CI:2.4–73.3%) with the conjugated estrogen plus cyclic micronized progesterone group. A recent study (33) suggests that mammographic density may be a marker for increased risk for breast cancer. Women with extremely dense breasts had a relative risk of breast cancer of 6.14 compared to women with predominantly fatty replaced breasts. These results are consistent with the studies (34) of others and provide compelling evidence of the proliferative as opposed to the antiproliferative effects of the progestins on human breast tissue in vivo. If there is a risk of breast cancer with estrogen, the effect is small and therefore large numbers of women are needed to identify an effect. The recent Collaborative Group metanalysis involving more than 50,000 women with breast cancer provided four cogent reasons why prior studies of HRT and breast cancer risk were conﬂicting (7). First, the increased risk of breast cancer with HRT dissipates within 3 to 5 yr after stopping this medication. Thus, studies comparing “ever users” with “never users” would be unlikely to demonstrate a risk from HRT unless most of the “ever users” were still taking estrogens. Second, the risk of breast cancer from estrogens is linear over time with a 2.3% increase in relative risk per year of use. Most earlier studies included women taking HRT for a relatively short period of time and thus only at a minimally increased risk. Third, the risk of breast cancer from HRT seems to be limited to thin women with a body mass index of less than 25kg/m2. Studies that included a high proportion of obese women would not be expected to demonstrate an increased risk from HRT. Fourth, the risk of breast cancer falls for a four-year period after the menopause because of declining levels of estrogen. Consequently, it is necessary to compare groups of women who have been menopausal for a similar duration to detect an increased risk of breast cancer. The recent Collaborative Group metanalysis took each of the four factors into account when examining their large database (7). Notably, they found a 2.3% increased relative risk of breast cancer with each year of HRT use. Data from this study, while retrospective, provide reasons for the conﬂicting nature of prior studies and strong evidence for a small but signiﬁcant effect of HRT to increase the risk of breast cancer. For a long period of time, the medical community believed that progestins might have an antimitogenic effect on breast that paralleled that on the uterus. With this rationale, it was believed that progestins might protect the breast from the carcinogenic properties of estrogen. A recent study by Schairer et al. (11) examined this issue in a large observational study. These investigators reported that estrogen alone increased the relative risk of breast cancer by 1% per year and estrogen plus a progestin by 8% per year. The Nurses Health Study (35) published only in abstract form, appeared to conﬁrm the ﬁndings of Schairer et al. Women followed for 860,000 patient years exhibited a 2% increase in relative risk per year with estrogens alone and a 9% increase per year with estrogen plus a progestin. Additional studies by Ross et al. (12) and by Magnussen et al. (36) also support the conclusion that progestins add to the risk of

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breast cancer imparted by estrogens. Other published studies do not support these ﬁndings. When stringent criteria are utilized to deﬁne which of these studies are valid, however, the weight of evidence provided by valid studies supports the conclusion that progestins do add to breast cancer risk (37). Several recent reports concluded that the risk of breast cancer with HRT increases linearly with time. This concept is highly relevant for women taking estrogen plus a progestin long term to prevent osteoporosis or heart disease. Based upon the Nurses Health Study, 10 years of use of estrogen plus a progestin would increase the relative risk of breast cancer by 90% (35). In contrast, 1 yr of use to prevent hot ﬂashes would be associated with only a 9% increased risk. Media reports about breast cancer and HRT focus only upon relative risk and do not consider absolute and attributable risk. A 90% increase in relative risk does not mean that 90% of women will develop breast cancer. One must calculate the incidence of breast cancer in the population and multiply by relative risk to determine how many women will actually develop breast cancer. One must then calculate attributable risk, by subtracting the number of women developing breast cancer in the user and nonuser groups. When this calculation is carried out, one ﬁnds that only 1 in 9925 50-year-old women will develop a breast cancer attributable to estrogen alone when use is limited to 2 yr. Only 1 in 1241 will develop breast cancer attributable to estrogen plus a progestin over this time period. When using HRT for 10 years, these statistics will be 1 in 397 with estrogen alone and 1 in 50 for estrogen plus progestin (37). Careful analysis of these data lead one to conclude that deﬁnitive evidence is not in hand to link HRT with increased breast cancer risk. The evidence, however, is compatible with biologic data regarding carcinogenesis and is supported by the fact that antiestrogens prevent breast cancer over at least short intervals of up to ﬁve years. The authors consider it prudent to advise patients that estrogens may cause breast cancer and that progestins may add to that risk. The Women’s Health Initiative data which should become available in approximately the year 2005 may help clarify this issue. Whether use of HRT in survivors of breast cancer is advisable has been debated for several years. Observational data are now available from more than 1000 women given HRT after an initial diagnosis of breast cancer (38). No increase in expected rate of recurrence has been observed. At the present time, however, there have been no randomized, controlled, prospective trials to examine this issue. One trial designed to examine the safety of HRT under these circumstances found that most women are reluctant to take HRT once they have been diagnosed as having breast cancer (38). A large consensus conference concluded that until evidence of safety is available, the use of alternatives to HRT in this setting would be preferable (38).

GOALS FOR USE OF ALTERNATIVES TO HRT The considerations regarding estrogens and breast cancer discussed in the previous section emphasize the need for alternative therapies for menopause-associated problems. Major therapeutic goals to be achieved by estrogen alternatives include the amelioration of symptoms from urogenital atrophy, vasomotor instability, and neurocognitive dysfunction, and the prevention of heart disease and osteoporosis (38). Additional

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desired effects include a reduction in incidence of Alzheimer’s disease, macular degeneration, colon cancer, and mandibular bone loss (38). This review examines data regarding the relative effectiveness of HRT comapred to nonestrogen alternatives such as HMGCo-A reductase inhibitors or statins for prevention of heart disease; bisphosphonates, calcitonin, raloxifene, tamoxifen, and statins for prevention of osteoporosis; low-dose vaginal estrogen for urogenital atrophy; clonidine, megastrol acetate, and selective serotonin reuptake inhibitors (SSRIs) for treatment of vasomotor instability; and SSRIs for treatment of depression and mood changes. In providing a practical approach to making recommendations we compare the risks and beneﬁts of systemic hormone replacement therapy with those of its alternatives. We believe that use of an evidencebased medicine framework provides a logical means to approach decision making and patient education.

Prevention of Cardiovascular Disease Heart disease represents the most frequent cause of death for women over the age of 60. Based on epidemiological and observational evidence, HRT is believed to be beneﬁcial for prevention of cardiovascular disease in menopausal women but this is being questioned based on recent data. The HMG-CoA reductase inhibitors, or “statins,” have also been used for the same purpose. We will review here the relative efﬁcacy of these two approaches for primary and secondary prevention of heart disease. Estrogens and Primary Prevention of Cardiovascular Disease Treatment Strategies. Two therapeutic goals, primary and secondary prevention, have been evaluated with respect to estrogens and cardiovascular disease. The primary prevention strategy attempts to reduce the incidence of initial cardiovascular events. Secondary prevention seeks to diminish the rate of new events in subjects with known cardiovascular disease. The precise efﬁcacy of estrogens for primary prevention of heart disease can only be estimated from observational studies as no randomized, placebo-controlled, double-blind trials have been completed. These observational data provide substantial support for the cardioprotective effects of estrogen and the magnitude of this effect. Evidence from more than 30 epidemiological studies suggests a 35–50% decrease in the incidence of initial coronary events with estrogen-replacement therapy (39–45). Mechanism of Effect. Estrogen acts on the cardiovascular system by at least two different mechanisms: positive effects on the serum lipid proﬁle and direct actions on blood vessels. About 25% of the cardioprotective effect is estimated to be a result of lipid changes including increased HDL, decreased LDL, decreased Lp(a), decreased LDL oxidation, and decreased vascular LDL update (46). The fact that estrogens improve lipid proﬁles has been used as evidence that estrogens should prevent heart disease. In the absence of prospective randomized trials, lipid levels are used as “indirect endpoints (surrogate endpoints)” to support the possibility that estrogens are cardioprotective. In the randomized, placebo-controlled, double-blind PEPI trial (47), 875 women received various estrogen/progestin regimens. Estrogen alone decreased LDL cholesterol levels and increased HDL cholesterol. Concomitant use of medroxyprogesterone acetate with estrogen blunted the increase in HDL cholesterol but did not affect the

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reduction in LDL cholesterol. Triglyceride levels increased with either therapy when compared with placebo. Micronized progesterone exerted less of a negative effect on HDL than medroxyprogesterone acetate (47). The remaining 65–75% of the cardioprotective effect of estrogen is believed to result from lipid-independent effects on the heart and blood vessels and on plaque formation. Possible mechanisms are increased nitric oxide, decreased endothelin, increased PG12, and decreased thromboxane A2 (48,49). A recent review discussed the various means whereby estradiol exerts direct effects on the vasculature (50). When delivered transdermally, estrogen is believed to exert its effects predominantly through nonlipid effects. This means of administration abrogates ﬁrst-pass effects on the liver with resultant lipid alterations but allows direct blood vessel and vascular actions (51). Evidence for Efﬁcacy. Data from the Nurse’s Health Study provide the strongest support for a cardioprotective effect of estrogen (52,53). In this prospective observational study, 59,337 women were followed for a total of 662,891 patient-years. The authors reported that the relative risk of developing heart disease in current hormone-replacement therapy users was 0.60 for estrogen alone and 0.39 for estrogen plus a progestin (52). They also calculated the absolute numbers of patients beneﬁted. After adjustment for multiple risk factors, it was estimated that 33 new cardiac events would be prevented per year per 100,000 women initiating estrogen usage at age 50 (52). For those starting at age 60, the number prevented would be 66. We believe that the data are more meaningful to patients if extrapolated to 10 yr of estrogen use. This requires the assumption of linearity of effects over time. Making such calculations, we estimate that by taking estrogen continuously for 10 yr, 330 new events per 100,000 would be prevented in 50-year-old women and 660 events in 60-year-old women (54). Dr. Barrett-Connor and others advise caution in interpreting these observational data because of several confounding biases (55–58). Women who take estrogen in the United States are self-selected for several factors that reduce the risk of cardiovascular disease— the “healthy woman” bias. There may be a “prevention” bias for women who spend a signiﬁcant amount of time in doctor’s ofﬁces and become better informed about health issues, or a “compliance” bias since these self-selected women continue to take estrogen long term. Compliance with taking a placebo has been shown to be associated with a decrease in incidence of heart disease. Data from the Coronary Drug Project showed that subjects who took 80% of their placebo had a relative risk (RR) of cardiovascular disease of .53, or a 50% reduction in heart disease (38). Deﬁnitive data to prove a beneﬁcial cardioprotective effect of estrogen awaits completion of the large, randomized, placebo-controlled trial called the Women’s Health Initiative. Data from this study are expected to be available in about the year 2005. Comparison of Statins with Estrogens for Primary Prevention of Cardiovascular Disease Lipid Levels. The ability of various agents to alter lipid levels is considered to be an indrect endpoint to predict efﬁcacy in preventing heart disease. With this as a rationale, two randomized studies have compared the effects of estrogen and the HMG Co-A reductase inhibitors (statins) on lipid levels (59,60) (Fig. 1). Australian Study. This trial compared the effects of postmenopausal hormonereplacement therapy and simvastatin on lipids in 58 women with hypercholesterolemia (59). Continuous combined high-dose estrogen plus progestin (Premarin 1.25 mg,

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Fig. 1. Comparative effects of estrogens, selective estrogen receptor modulators, and statins on lipid levels. E, estrogen preparation; ST, statin preparation; TAM, tamoxifen; RAL, raloxifene. When the bars are together without a space, this indicates a direct “head-to-head” comparison of one therapy with another. When the bars are separate, this indicates a trial that studied a single agent. The dark shaded E vs. ST trial represents that of Darling (59). The lightly shaded E vs. ST trial is that of Davidson et al. (60). The TAM trial is that of Love et al. (40) and the RAL trial is that of Delmas et al. (68). LDL-C, LDL cholesterol; HDL-C, HDL cholesterol; TG, triglyceride; Lp(a), lipoprotein(a). This ﬁgure compares available data from multiple studies and does not exclusively represent direct comparisons in the same study. Interpretation of the data must take into consideration the limited nature of available information and the need for direct comparisons of all available therapeutic modalities.

Medroxyprogesterone 2.5 mg) was compared with 40 mg of simvastatin daily. Both hormone therapy and simvastatin caused signiﬁcant reductions in LDL cholesterol (24% and 36%, respectively), but simvastatin was more effective than hormone therapy (p < 0.001). Both treatments caused a signiﬁcant increase (7%) in HDL cholesterol. The two treatments differed signiﬁcantly in their effect on triglyceride levels (p < 0.001) with simvastatin reducing these levels by 14% and hormone therapy increasing them by 29%. Also of note was the statistically signiﬁcant reduction in Lp(a) lipoprotein levels with hormone therapy (a decrease of 11 mg/dL or 27%), whereas simvastatin had no signiﬁcant effects. The clinical signiﬁcance of these differences is as yet unknown. North American Study. Davidson et al. (60) reported the effects of conjugated estrogens alone, pravastatin alone, and the combination for management of hypercholesterolemia in postmenopausal women. Participants (n=76) were randomly assigned to receive conjugated estrogens, 0.625 mg/d; pravastatin sodium, 20 mg/d; conjugated estrogens plus pravastatin; or a placebo for 16 w. For the purposes of this chapter, we present only the comparative data between pravastatin and estrogen. Among participants treated with conjugated estrogens, levels of calculated low-density lipoprotein choles-

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terol (LDL-C) decreased by 13.5%, whereas increases in levels of HDL-C (22.5%) and triglycerides (4.2%) were seen (Fig. 1). Participants in the pravastatin group achieved reductions of 25.4% in calculated LDL-C levels. Levels of HDL-C increased slightly (3.7%) and triglycerides decreased by 12.1%. The pravastatin group had a more signiﬁcant reduction in LDL-C. Estrogen improved HDL-C signiﬁcantly (22.5% vs. 3.7%) but also increased triglycerides. Higher Doses of Statins. Dose escalation may further improve lipid levels as reported by Stein et al. (61). This study evaluated the lipid-altering efﬁcacy and safety of simvastatin 80 mg/d, a dose twice the maximum currently recommended. LDL decreased signiﬁcantly (p < .001) from baseline at weeks 18 and 24, with mean reduction of 38% and 46% for the 40- and 80-mg groups. Simvastatin 80 mg/d provided substantial reductions in LDL cholesterol, allowing most patients to reach target levels; it also had an excellent safety and tolerability proﬁle. Effects of Statins on New Coronary Events and Survival Primary Prevention of Heart Disease. The Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPs/TexCPS) examined the effect of 20 mg/d of lovastatin (or 40 mg/d if LDL cholesterol remained above 110 mg/dl) versus placebo as primary prevention of acute coronary events (62). The study involved 5608 men and 997 women without a prior history of cardiovascular disease and with average LDL cholesterol and below average HDL cholesterol levels. Overall, the relative risk of ﬁrst coronary event was 0.63 after an average followup of 5.2 years in patients receiving lovastatin. This group also had a decreased relative risk for myocardial infarction, unstable angina, coronary revascularization procedures, coronary events, and cardiovascular events. The effect of treatment with lovastatin on the rate of ﬁrst acute major coronary events was relatively greater in women than in men (46% vs. 37% reduction); however the actual number of women who had a primary endpoint was small (20 of 997) and there were no statistically signiﬁcant differences in treatment effects between sexes. Other Agents with Potential for Prevention of Cardiovascular Disease Tamoxifen. The SERMS exert both estrogen-agonist and estrogen-antagonistic effects, depending on the tissue examined. Tamoxifen, used for prevention and treatment of breast cancer, is one of these which exerts estrogenic effects on the liver and thus lowers LDL cholesterol levels. Lipid data are available from the studies of Love et al. (63), which compared basal lipid levels with those obtained after 5 yr of receiving either placebo or tamoxifen in 62 women (Fig. 1). After ﬁve years of use, tamoxifen decreased LDL cholesterol concentrations by 30%. HDL cholesterol also fell by 6.4% (7% for placebo) and triglycerides increased 45.6% (23.8% for placebo). Lipoprotein (a) was decreased with tamoxifen by 3.5% compared to a 1% decrease with placebo. Data from the Royal Marsden Hospital tamoxifen chemoprevention trial demonstrated similar effects on lipid levels (38,64). Thus, tamoxifen appears to decrease LDL cholesterol, to reduce lipoprotein (a) and to increase triglycerides (63–65). The tamoxifen prevention trial conducted by the NSABP (10) was originally powered to detect a cardiovascular protective effect. Only one-third of accrued patients however were postmenopausal rather than the two-thirds projected. For this reason, the number of cardiovascular events was insufﬁcient for adequate analysis. Nonetheless, the trend

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toward a similar number of events in tamoxifen- as in placebo-treated patients suggests that tamoxifen may not be cardioprotective (10). Raloxifene. This SERM is approved by the FDA for the prevention and treatment of osteoporosis. In preclinical studies in cholesterol-fed rabbits, raloxifene reduced aortic accumulation of cholesterol (67), suggesting the potential of inhibiting atherosclerosis. In randomized studies on lipid effects in humans (68,69), actions similar to those of estrogens on lipids but of somewhat smaller magnitude were observed (Fig. 1). Reductions in total cholesterol of 6.6% and LDL by 10.9% were seen with no change in HDL or triglycerides. Both lipoprotein (a) and ﬁbrinogen were reduced. Although not directly compared, this corresponds to an increase of 10.6% in HDL and an increase of 10% in triglycerides with HRT. Raloxifene exerts direct effects on the cardiovascular system while also inducing lipid alterations. In ovariectomized placebo-treated cynomolgous monkeys however (70), neither vasoactive effects nor protective effects against atherosclerosis were seen at either low or high doses of raloxifene. In comparison, (according to blood levels), very-high-dose Premarin provided nearly complete protection. In a recent double-blind controlled 6 mo trial of 390 postmenopausal women (71), raloxifene at 60 mg and 120 mg/d signiﬁcantly lowered serum homocysteine by 8% (P = 0.014) and 6% (p + 0.024) comparable to the 7% (P = 0.014) with 0.625 mg CEE/2.5 mg MPA. Of interest, however, in the same study, raloxifene did not increase C-reactive protein (−6% and −4%, P > 0.2), which is felt to be beneﬁcial whereas HRT increased C-reactive protein levels by 84% (P < 0.001). A prospective study is in progress (RUTH) to study cardiovascular endpoints during raloxifene treatment of postmenopausal women at risk for cardiovascular events that may help understand the effect of raloxifene on protection against heart disease. Phytoestrogens. Phytoestrogens are plant-derived compounds that are isoﬂavones, bind to estrogen receptors, and have both estrogen agonist and antagonist properties (72). A recent metanalysis (73) of the effect of soy on cholesterol in humans revealed that 47 g/d of soy was associated with a 12.9% decrease in LDL cholesterol, 9.3% decrease in total cholesterol, and no change in HDL cholesterol. Interestingly, the greatest effect was seen in those with the highest pretreatment cholesterol. The amounts of total soy isoﬂavones that exert clinical effects approximate 40–80 mg/d (74–76). A recent study in forty oophorectomized cynomolgus monkeys (76) found that dietary intake of soy protein was associated with reductions in plasma cholesterol with lower total, VLDL, and LDL cholesterol and signiﬁcantly higher HDLs compared to groups that received casein-lactalbumin intact soy protein, an isoﬂavone-rich semipuriﬁed soy extract, or casein soy protein plus CEE. Cholesterol absorption was signiﬁcantly lower in the soy protein group. In this study, surprisingly, soy protein but not the semipuriﬁed soy extract, rich in isoﬂavones, had the lipid-lowering effect. No clinical data are available that report the incidence of new cardiovascular events in women receiving soy products. Secondary Prevention of Cardiovascular Disease Estrogen. Nonrandomized secondary prevention studies (77–80) suggest that HRT use in women with established CVD reduces risk of death and future cardiovascular events. Surprisingly, the randomized, prospective HERS trial (81,82) could not conﬁrm

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this beneﬁcial effect. The HERS study involved 2763 women, mean age 66.7 with severe coronary heart disease who used continuous combined estrogen (CEE .625 mg) and progesterone (MPA 2.5 mg) or placebo, with 4.1 yr of followup. The HRT group showed an increase in CHD and mortality at year one when compared to those using placebo. Interestingly, a followup report found that the increase in new cardiovascular events was limited to those patients with increased levels of lipoprotein (a) (83). With continued use, it appeared that a beneﬁcial effect of HRT developed over time, with fewer deaths in years four and ﬁve. At the end of ﬁve years on study however, the number of deaths, heart attacks, and CHD rates did not differ between the two groups. Increases in thromboembolism of 4.1/1000 woman years (RR 2.89), and gallstones (RR 1.38) were seen. Estrogen effects on lipid were found with an 11% decrease in LDL cholesterol and a 10% increase in HDL cholesterol. Many believe that the early death rate represented a prothrombotic effect of estrogen and that the lack of differences between groups was caused by a detrimental effect of MPA, the progestin used in the hormone group (81). Clarkson’s studies in monkeys (84), demonstrated that MPA could completely abrogate the ameliorative effects of estrogens on the coronary arteries. The HERS study has called into question the efﬁcacy of estrogens for secondary prevention. Additional studies will be required to dissect out the prothrombotic from cardioprotective effects of estrogen in women with known cardiovascular disease (82). Even though this study contradicts the results of nonrandomized trials, the data raise strong doubts about the efﬁcacy of estrogens for secondary prevention and highlight the fact that the statins have been shown to be efﬁcacious in this setting. A second randomized trial for secondary prevention, the estrogens and atherosclerosis (ERA) trial compared the effects of placebo, Premarin alone, and Premarin plus MPA in randomized groups of women followed prospectively at multiple medical centers (84a). The primary endpoint of the study was the degree of coronary artery narrowing as assessed by coronary arteriography after three years of treatment. Neither Premarin alone nor Premarin plus MPA resulted in a reduction of coronary narrowing when compared to the placebo group. Taken together, the HERs and ERA studies call into serious question the rationale for using HRT for secondary prevention of heart disease. Statins. Randomized, prospective, controlled trials demonstrate that the statins reduce new cardiovascular events by approximately 30% and have favorable side-effect proﬁles. Unfortunately, the statin studies have entered a much larger proportion of men than women. Nevertheless, the statins appear to reduce coronary events in women to about the same extent as in men. It also improved survival in patients aged 60 or more. When results from men and women were combined (85,86), it was found that simvastatin produced highly signiﬁcant reductions in the risk of death and morbidity in patients with CHD followed for a median of 5.4 yr, relative to patients receiving standard care. Over the median followup period, one or more major coronary events occurred in 622 (28%) of the 2223 patients in the placebo group and 431 (19%) of the 2221 patients in the simvastatin group for a highly signiﬁcant 34% risk reduction with P < .00001. The results indicate that the addition of simvastatin 20–40 mg/d to the treatment regimens of CHD patients, with characteristics similar to those of postmenopausal women, should be beneﬁcial. Conclusions from these non “head-to-head” comparisons of the statins with HRT for primary and secondary prevention can only be tentative. Only the statins have been demonstrated by proper randomized trials to prevent major coronary events. Based on

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our analysis, it is reasonable to consider the statins appropriate estrogen alternatives for the primary or secondary prevention of heart disease. It should be noted that the American College of Cardiology now recommends the statins in preference to HRT for primary and secondary prevention of cardiac disease, and the American Heart Association does not recommend starting estrogens solely for prevention of heart disease.

Prevention and Treatment of Osteoporosis Estrogen therapy reduces bone resorption and has been used both to prevent and to treat osteoporosis. The PEPI trial (87) revealed that HRT improved bone mineral density (BMD) by up to 4% in the lumbar spine and 2% in the hip at three years. To have the most protective effect on bone density, estrogen therapy is best initiated at menopause. Nonetheless, Ettinger (88) suggests that estrogen given to older osteoporotic women can also increase bone density 5–10% and reduce fracture risk. Based on epidemiologic studies (89–91), HRT users have a decreased risk of fracture (RR of .45). Bone loss may accelerate after discontinuation of estrogen. Ten years after discontinuation, the bone mineral density and fracture risks were similar in women who had used estrogen compared to never users (92). Data regarding fracture risk reduction with estrogens derives almost exclusively from observational studies (93–95). One clinical trial of transdermal estrogen therapy in 75 postmenopausal women with osteoporosis observed for one year revealed a relative risk of vertebral fractures of 0.39 compared to placebo (96). A series of 20 studies (95) revealed a risk reduction ranging from 80% to no effect in some studies. The reduction appears to average approximately 50%. Several studies estimated a lifetime reduction of vertebral and hip fractures and increased overall survival from prevention of these events (44,89,97). A prospective-cohort study (The Study of Osteoporotic Fractures) (98) among 9704 women 65 and older found the relative risk for nonspinal fractures for women on estrogen to be 0.66. Current users experienced a reduced hip fracture relative risk of 0.60. For women who started estrogen within 5 yr of menopause, the RR was 0.29 for hip fracture and 0.50 for all nonspinal fractures. More precise comparisons must await further studies. Antiresorptive Agents for Prevention of Osteoporosis Antiresorptive agents such as the bisphosphonates, calcitonin, tamoxifen, and raloxifene are useful for the prevention or treatment of osteoporosis. Bisphosphonates are stable analogs of pyrophosphate and exert effects primarily on the osteoclast. Alendronate and risedronate are approved by the FDA for prevention and treatment of osteoporosis. Comparison of Alendronate and Estrogen. Data regarding the relative efﬁcacy of the bisphosphonates versus estrogen are available for effects on bone density but not for fracture prevention. The most commonly used bisphosphonate, alendronate, appears to exert an antiresorptive potency similar to that of estrogen when used at low dose (5 mg) (Fig. 2). The Early Postmenopausal Intervention Cohort (EPIC) (99,100) study of recently menopausal women included placebo, alendronate (2.5 or 5 mg/d) and openlabel estrogen/progesterone. Patients receiving placebo plus calcium lost bone. Those receiving either 2.5 or 5 mg/d of alendronate increased bone mass between 1 and 2% over baseline but not to the same extent as the estrogen/progesterone group at 2% (Fig. 2).

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Fig. 2. Comparative effects of alendronate (A), tamoxifen (TAM), estrogen (E), placebo (P), raloxifene (R) and nasal calcitonin (NT) on percent change in bone mineral density as assessed by DEXA scan. When the bars are together without a space, this indicates a direct “head-to-head” comparison of one therapy with another. When the bars are separate, this indicates a trial that studied a single agent. A-10 and A-5 represent alendronate at 10 and 5 mg. E vs A-5 represents estrogen vs. 5 mg of alendronate. This ﬁgure compares available data from multiple studies and does not exclusively represent direct comparisons in the same study. Interpretation of the data must take into consideration the limited nature of available information and the need for direct comparisons of all available therapeutic modalities.

With measurement of total body bone density at two years, estrogen/progesterone improved bone density almost 2% whereas 5 mg of alendronate increased this parameter 1% and 2.5 mg of alendronate maintained baseline levels compared with a 2% loss with placebo. The four-year followup conﬁrms the continued efﬁcacy of alendronate over this time period. The bone mineral density in patients receiving placebo decreased by 1–6% at four years (101). Alendronate therapy at 5 mg/d increased bone mineral density at the spine 3.8 ± 0.3%, hip 2.9 ± 0.2%, and total body 0.9 ± 0.2%. Continued increments in bone mineral density were seen between years two and four in women receiving alendronate. Thus for prevention of bone loss, both estrogen/progesterone and 5 mg of alendronate are effective with slightly better response with estrogen/progesterone. Dose-response studies indicate that 10 mg of alendronate may be more efﬁcacious than 5 mg/d (99–102). At the end of three years, BMD was higher in patients treated with 10 mg/d alendronate than in patients receiving placebo by (mean ± SE) 8.8 ± 0.4% at the lumbar spine, by 5.9 ± 0.5% at the femoral neck, and by 7.8 ± 0.6% at the trochanter (102). The 5-mg dose exerted lesser effects. Although no head-to-head comparison studies have been conducted, it would appear that 10 mg/d of alendronate would result in a similar or greater increase in bone mass than that seen with estrogen therapy. Fracture and BMD Data with Alendronate. No direct comparative data to evaluate the reduction of fracture risk with estrogen versus bisphosphonates are yet available. Consequently, it is necessary to compare data from other randomized or observational studies to draw tentative conclusions. Three multicenter, double-blind, placebo-controlled studies provide compelling evidence of the vertebral antifracture efﬁcacy of

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alendronate. The ﬁrst (102) involved 994 postmenopausal women (mean age, 64 yr) with osteoporosis, who received either placebo for three years, alendronate 5 mg/d for 3 yr, alendronate 10 mg/d for 3 yr or alendronate 20 mg/d for 2 yr and then 5 mg/d for the third year. All patients received 500 mg elemental calcium. BMD increased in patients receiving all alendronate dosage regimens and decreased in patients receiving placebo. Vertebral fractures occurred in 6.2% of patients receiving placebo and 3.2% of patients receiving alendronate; this represented a 48% reduction in numbers of women sustaining fractures (P < 0.04). Two or more new vertebral fractures occurred in 4.2% of patients receiving placebo (15 of 355) and 0.6% of patients receiving alendronate (3 of 526), a risk reduction of 87%. Patients in the placebo group who sustained new fractures lost 23.3 mm in height. Alendronate-treated patients who sustained one or more fractures lost only 5.9 mm in height, consistent with less-severe fractures. Nonvertebral fractures occurred in 60 of 590 women receiving placebo and 73 of 1012 receiving alendronate. The cumulative incidences (placebo vs. alendronate) were 12.6% and 9%, a 29% reduction in risk compared with placebo (P < 0.05). In the Fracture Intervention Trial (FIT) (103), 2027 women (mean, 71 years) with one or more vertebral fractures at baseline and reduced BMD were randomized at 11 centers in the United States to receive either placebo (n = 1005) or alendronate (n = 1022, 5 mg/d for two years, 10 mg/d in year three). Calcium and vitamin D were supplemented if the diet contained <1000 mg/d calcium. After three years, BMD increased signiﬁcantly by 6.2% above placebo at the spine and by 4.7% above placebo at the total hip region. The rate of new clinically apparent vertebral fractures decreased by 47% compared to placebo. Similar decreases were seen in the frequency of hip and wrist fractures, but not for other types of fractures. A followup to this study examined women without preexisting vertebral fractures. Women received 5 mg/d of alendronate for two years followed by 10 mg/d thereafter (104). Only those with osteoporosis (i.e., bone density at the femoral neck of >2.5 SD below the normal adult mean) responded with a signiﬁcant reduction of clinically evident fractures. The radiologically detected fractures were reduced by 44% in the total group of women taking alendronate (relative risk 0.56; 95% CI:0.39–0.80). Subgroup analysis demonstrated a signiﬁcant reduction only in those with a baseline bone density T score of > 2.5 SD and close to a signiﬁcant reduction in those with scores of −2.5–2.0 (relative risk 0.54 (95% CI:0.28–1.04). Too few episodes occurred in the remainder to be meaningful (i.e., 10 vs. 8 events). These well-designed trials suggest that alendronate is effective in preventing vertebral and nonvertebral fractures when compared to placebo, and 10 mg appears to be the most effective dose. Although hip fractures were reduced, the number of actual fractures was small. Once a week dosing with alendronate appears to be as effective as daily dosing and may improve compliance and decrease potential for GI side effects.

Bone Density and Fracture Data for Risedronate Risedronate has been shown to improve bone density and decrease vertebral fracture rates. In a prospective multicenter trial (105), 450 women assigned to the placebo arm and 489 on 5 mg of risedronate completed all three years of the trial. Treatment with 5 mg/d of risedronate, compared with placebo, decreased the cumulative incidence of new vertebral fractures by 41% (95% CI:18%–58%) over 3 yr (11.3% vs 16.3%; P = .003). A fracture reduction of 65% (95% CI:38%–81%) was observed after the ﬁrst

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year (2.4% vs 6.4%; P < .001). The cumulative incidence of nonvertebral fractures over 3 yr was reduced by 39% (95% CI:6%–61%) (5.2% vs 8.4%; P = .02). Bone mineral density increased signiﬁcantly compared with placebo at the lumbar spine (5.4% vs 1.1%), femoral neck (1.6% vs −1.2%), femoral trochanter (3.3% vs −0.7%), and midshaft of the radius (0.2% vs −1.4%). Bone formed during risedronate treatment was histologically normal. The overall safety proﬁle of risedronate, including gastrointestinal safety, was similar to that of placebo. Miller et al. presented preliminary results of the Hip Intervention Program with 9497 women at least 70 years of age enrolled into either the low bone density group (with T-score <−4 or <−3 with a clinical risk factor for fracture) or the clinical risk factors group (80 and over with one risk factor, no BMD requirement, or a femoral neck score <−4) (106). A total of 216 hip fractures were observed. In the low-bone-density group the risk of hip fractures with low femoral neck BMD decreased by 39% (p = 0.02) and the risk of hip fractures in patients with low femoral neck BMD and one or more prevalent vertebral fractures at baseline decreased by 58% (p = 0.004). The risk of hip fractures was not decreased in the group enrolled because of fall-related, nonskeletal risk factors. The overall risk of hip fracture decreased by 24% (p = 0.05%, NS).

Nasal Calcitonin for Osteoporosis Nasal calcitonin is approved for treatment of osteoporosis in women who are four or more years postmenopausal. It is not effective in preventing bone loss in early postmenopausal women (107,108). A randomized double-blind trial of approx 230 postmenopausal women with osteoporosis in each group received either placebo or three doses of nasal calcitonin (100, 200, and 400 IU/d) for three years. An increase of 1.0–1.5% over baseline in lumbar spine but not other skeletal sites was seen at the end of year one (signiﬁcant) with no dose response apparent. Statistical signiﬁcance over placebo was lost in years two and three. Overgaard et al. (109) studied the effects of intranasal salmon calcitonin in a 2-year, double-blind, placebo-controlled trial of women aged 68–72 years randomized to receive 50, 100, or 200 IU calcitonin or placebo daily plus 500 mg calcium supplement. Among 162 women completing the study, spinal BMD increased by 1% in the placebo group and by 3% in the group receiving 200 IU calcitonin. The numbers of fractures varied according to the method of assessment of vertebral morphometry. By one method, fractures occurred in seven patients receiving placebo and ﬁve receiving calcitonin; by the second method, fractures occurred in six patients receiving placebo and four receiving calcitonin. Peripheral fractures occurred in two placebo- and two calcitonin-treated patients. Although a decreased number of fractures was seen, pooling of all doses was not preplanned and the small numbers of fractures leave uncertainty. Four-year interim results (110) from the ﬁve-year multicenter study Prevent Recurrence of Osteoporotic Fractures (PROOF) were presented in abstract form at the 1998 European Congress on Osteoporosis. 1255 postmenopausal women with established osteoporosis were randomized to receive either placebo, or Salmon Calcitonin nasal spray (100, 200, or 400 IU/d) plus 1000 mg of calcium and 400 IU of Vitamin D. At four years, there was a 36% reduction in relative risk of new fracture with 200 IU compared to placebo (p = .020). Reductions of 18% and 23% in new fractures were seen among those treated with 100 IU or 400 IU compared to placebo. The mean increment in BMD over baseline for placebo, 100 IU, 200 IU and 400 IU was 0.7,

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1.2, 1.2 and 1.6% respectively. The increases in lumbar spine BMD were statistically signiﬁcantly increased in all treatment groups compared to placebo over baseline and at two years compared to placebo, and up to three years for the 400 IU. Despite modest increases in BMD, the abstract analysis suggests nasal calcitonin spray reduces the risk of new vertebral fractures in postmenopausal women with established osteoporosis. The data suggest a protective effect on hip fractures but the study is not adequately powered to detect differences in hip fracture rates. At four years, a 51% reduction in hip/femur fractures was noted in the 200 IU dose but the numbers are quite small, 1.3% (4/315) of patients in 200 IU group versus 2.6% (8/305) of patients in the placebo group.

SERMs for Prevention of Osteoporosis Studies in postmenopausal women using tamoxifen for prevention of breast cancer have shown average increases in BMD of 1–2% per year (111). Premenopausal women experience a transient bone loss for the ﬁrst 2 yr with tamoxifen, felt to be caused by the antiestrogenic effect of tamoxifen. In the Tamoxifen Prevention Trial (10), a reduction in fractures of hip, spine, and other areas was observed which approached but did not reach statistical signiﬁcance. Three large, randomized, placebo-controlled osteoporosis-prevention trials (68,69,112–114) with raloxifene demonstrated signiﬁcant increaases in bone mineral density of 2% over placebo in hip, spine, and total body. Raloxifene acts as an antiresorptive agent, as does estrogen. Compared to placebo, there is between a 2 and 2.5% increase in spine, total hip, and total body (P < 0.001 for all comparisons) BMD. In the North American trial, there were 12% who lost 1% per year in the spine and hip. Raloxifene increased total-body bone mineral density from 1.8% to 2.5% over placebo after 24 months. The effect was similar to that seen with conjugated equine estrogens and medroxyprogesterone acetate or 5 mg of alendronate per day. In the total hip, the raloxifene group improved to a greater extent than those given placebo (1.8% to 2.3%). The effect was similar in magnitude to that observed in women receiving conjugated equine estrogens and medroxyprogesterone acetate or 5 mg of alendronate. The effects were greater than effected by 200 IU of nasal calcitonin or tamoxifen. The MORE trial is a multicenter, double-blind, controlled study of 7705 recently postmenopausal (avg. 4.5–5 yr) women with osteoporosis (mean age 66.5) treated with raloxifene (60 mg or 120 mg) or placebo followed for 36 months. All received 500 mg of calcium and 400–600 IU Vitamin D. Primary endpoints were radiographic vertebral fractures and bone density (112). Raloxifene at 60 mg showed signiﬁcant reduction in vertebral fractures with 30% reduction (RR 0.70, 95% CI:0.56–0.86) in women with prevalent fractures and 52% reduction (RR (0.48, 95% CI:0.29–0.71) in women without prior fractures.

Comparison Among Therapeutic Agents for Treatment of Osteoporosis For women with documented osteoporosis, there are no direct comparisons of effectiveness of different agents (Fig. 2). Ten mg of alendronate has been shown to increase density in the lumbar spine by 5 to 7% and femoral neck by 4 to 6%. Estrogen alone or with progesterone increased bone density by 2 to 5% in the lumbar spine and by 1 to 6% in the hip in the PEPI trial. Nasal calcitonin at 200 IU improved bone density 2–3 percent at two years in lumbar spine. Raloxifene increased BMD 2–3% per year,

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tamoxifen 1–2%. Although we have long-term epidemiologic data about the effect of estrogen on prevention of osteoporosis, we are lacking prospective controlled data for estrogen and long-term data about alendronate, nasal calcitonin, and raloxifene. Head-to-head comparisons of various agents in preventing fracture are nonexistent. Efﬁcacy is estimated by comparing results from different trials. Epidemiologic studies suggest a relative reduction of fracture rate of 55% with estrogen use, even for women over 70. For alendronate, the decrease in lumbar spine and hip fractures is 50% (115); for risedronate 41% (105); for nasal calcitonin, the decrease in lumbar fractures presented in abstract is 36% (110) and for raloxifene 38–52% (112). Based on these data, one can conclude that alendronate, risedronate, nasal calcitonin, and raloxifene could serve as alternatives for estrogen for prevention and treatment of osteoporosis. In women with osteopenia or osteoporosis, raloxifene reduced incidence of newly diagnosed breast cancer by up to 71% at ﬁve years, as shown by the MORE trial (116,117). A recent metanalysis of all raloxifene trials conﬁrmed a similar reduction (116). The practical signiﬁcance of this ﬁnding is that raloxifene might be chosen over alendronate or calcitonin in women with an increased risk of breast cancer. A surprising new ﬁnding is that the statins may stimulate bone formation and reduce fracture incidence. Both statins and nitrogen-containing bisphosphonates (alendronate and risedronate) work by inhibiting different steps in the pathways that branch from the synthesis of cholesterol from acetyl CoA (118). Mundy and collaborators (118) utilized a high-throughput screening assay to ﬁnd agents capable of stimulating bone formation. Of the approx 30,000 compounds screened, the statins were the most effective in stimulating the enzyme used to screen for bone stimulatory activity. Since publication of this ﬁnding, there have been ﬁve retrospective case-control analyses of fracture reduction with the statins (119–121). Each found an approx 50% reduction. An additional study reported increases in bone density in Type II diabetic patients receiving statins but not in those receiving other types of lipid-lowering agents. Prospective, randomized trials will be required to determine whether currently approved or new statins improve bone density and reduce the risk of fractures and to provide the rationale for approval of the statins for prevention and treatment of osteoporosis. If these trials are positive, an agent will be available to take the place of estrogen both for the prevention of cardiovascular disease and also for osteoporosis.

UROGENITAL ATROPHY Symptoms of lack of estrogen in the urogenital tract include frequent urination, urinary urgency or leakage, vaginal dryness, itching or burning in the vagina or vulva, and dyspareunia. Oral estrogens relieve all of these symptoms and reduce the frequency of urinary tract infections. Vaginal moisturizers and lubricants can be helpful in patients not willing or able to take estrogens but do not completely relieve symptoms in the majority of patients. One moisturizer, Replens (122,123) used three times weekly, was compared to conjugated estrogen cream at a dose of 1.25 mg/d in a randomized 12 wk trial with 30 patients. This study showed that Replens reduced vaginal pH to 4.8 compared to estrogen at 4.4, diminished atrophy in 60% of patients compared to 100% with estrogen, and relieved symptoms but not to the same extent as estrogen. Another approach is to use sufﬁciently low doses of vaginal estrogen to achieve local effects without systemic estrogen absorption. Standard doses of vaginal estrogen

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increase plasma estrogen levels and have many of the problems associated with oral estrogen use. Very low doses of vaginal estrogen however do not increase plasma levels substantially and yet can be effective. In one study examining this possibility (124), plasma estrone and estradiol levels were measured in women receiving vaginal Premarin with gradual increases in doses from 0.3 to 2.5 g/d. At the highest dose used, the investigators found signiﬁcant increases in systemic levels of estradiol up to 60 pg/mL, a level comparable to that seen with oral Premarin. However, the lowest dose (0.3 g/d) produced complete maturation of vaginal mucosa with only minimal increases in plasma estrone and estradiol. Hands et al. recently used this very-low-dose regimen and showed no increase in plasma estrone over baseline after six months of use (125). Disadvantages of the vaginal method involve irregular application intervals, bolus absorption and low absorption capacity of a fat-based vehicle, necessitating use of emollient (which results in stickiness, messiness, compliance issues). Newer methods of delivering estrogen locally into the vagina without signiﬁcant systemic absorption include a vaginal estrogen ring device (Estring) (126,127), low dose vaginal creams currently being tested and developed and new vaginal estradiol tablets. The vaginal ring provides nearly complete relief of symptoms. Use of this device is associated with a decreased frequency of urinary tract infections in elderly women prone to this problem (128). In open-label studies (but with blinded review of vaginal cytology), similar efﬁcacy was observed with the vaginal-ring device as with conjugated systemic estrogens with respect to vaginal secretions, color, tissue integrity, urethral meatus integrity, and patient acceptance. Data reviewed indicate minimal systemic absorption from this device (38). Only 4% of women using the ring had endometrial thickness greater than 5 mm measured by ultrasound compared with 10% using the vaginal ring. Three percent experienced vaginal withdrawal bleeding from the ring compared to 21% using vaginal cream. A note of caution, however, is that the estring was associated with an increase in bone density in postmenopausal women, a ﬁnding suggesting some degree of systemic absorption (129). Our preliminary data support the possibility that very low doses of estradiol given vaginally can achieve local vaginal effects without systemic absorption of estradiol (130). Currently available radioimmunoassays for estradiol may not be sufﬁciently sensitive to detect minor increments in plasma estradiol with use of Estring or vaginal creams (130). Further studies are required to determine the safety of vaginal estradiol in women surviving breast cancer. This group of patients could potentially be highly sensitive to even minimal amounts of circulating estradiol and further safety studies are required in them. Taken together, these data suggest that low-dose vaginal estrogen can provide a means of bypassing the need for systemic estrogen use in postmenopausal women. This route has not been associated with an increase in thromboembolic events nor in development of breast cancer.

VASOMOTOR INSTABILITY Symptoms related to this problem include hot ﬂashes, night sweats, sweating, insomnia or early morning awakening, mood disturbances, muscle aching, and fatigue. The use of a placebo consistently reduces the number and severity of hot ﬂashes by about 25% (131). A recent study demonstrated that a soy preparation was no more effective

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Fig. 3. Mean hot-ﬂash scores for women during the baseline week, the 2-wk crossover period, and the 4-wk period of open-label megestrol acetate (reprinted with permission from Loprinzi C, Michalak JC, Quella SK et al. Megestrol acetate for the prevention of hot ﬂashes. N Engl J Med 1994;331:347– 352. Copyright  1994 Massachusetts Medical Society. All rights reserved.

than placebo (132). It should be noted however that placebo in nearly all studies reduces hot ﬂashes by approx 30% (133,134). With vitamin E there was a statistically signiﬁcant 10% improvement over placebo (131). Clonidine is slightly more effective (133,134). Side effects include drowsiness, fatigue, and symptoms of low blood pressure in some patients. Anecdotal observations by the authors of this chapter however suggest that only a subset of women respond to this medication and others are completely unresponsive. No means are available to identify those who will be responders and an empirical trial is necessary to determine efﬁcacy. Megestrol acetate (135), at a dosage of 40 mg/d appears to be as effective as estrogen, with an 80% level of control of hot ﬂashes (Fig. 3). A major side effect is weight gain. Long-term safety of Megestrol acetate, particularly in patients surviving breast cancer, is not known. The Selective Serotonin Reuptake Inhibitor (SSRI) class of drugs decrease hot ﬂashes more effectively than does placebo (136). Use of these agents may for the ﬁrst time provide an effective alternative to estradiol for treatment of hot ﬂashes. Two recent studies, one a multicenter trial, utilized randomized, placebo-controlled, prospective designs to evaluate the efﬁcacy of SSRIs for vasomotor instability in a large number of patients. Both reported (in abstract form only) greater than 50% reduction in hot ﬂash scores with these agents versus 20% with placebo (137,138). These, in conjunction with several observational studies, provide substantial evidence that these agents are effective for hot ﬂashes and could be used as ﬁrst-line therapy. The popular literature suggests that soy products and phytoestrogens may be effective for treating hot ﬂashes. Studies (74,139) however that have used phytoestrogens for relief of hot ﬂashes show conﬂicting results, varying from little effect, to a signiﬁcant reduction, to a reduction in the severity but not frequency of hot ﬂashes. One randomized controlled study of 104 postmenopausal women (132) compared 60 g of isolated soy protein versus 60 g of placebo casein daily. Soy protein signiﬁcantly reduced the mean number of hot ﬂashes per 24 hours after 4, 8 and 12 weeks of treatment with a 45%

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reduction in hot ﬂushes versus a 30% reduction with placebo (p < .01) with comparable adverse events, primarily GI side effects in both groups. Note the soy protein substantially reduced the frequency of hot ﬂushes compared to placebo but did not eliminate them or reduce them to the same degree seen with HRT.

ALTERNATIVE/HERBAL THERAPY Many women use herbs for treatment of menopausal symptoms without physician knowledge. Quality control of herbal products is problematic. Safety, effectiveness, and toxicity data are lacking. No studies have demonstrated the efﬁcacy of soy products in treatment of menopause. Common other menopausal remedies used by women include Dong Quai, Ginseng, Black Cohosh, Vitex/Chasteberry, DHEAS, Melatonin, and St. John’s Wort. Dong Quai was recently shown to be no more effective than placebo in a randomized double-blind trial (140).

NEUROCOGNITIVE DYSFUNCTION For symptoms of sleep disturbance, trazodone or benzodazepines and hypnotics have been used. Depression should be identiﬁed and treated with antidepressants and mood stabilizers. The incidence of depression increases at the time of menopause, presumably in response to estrogen deprivation. The SSRI class of drugs should be as effective in women with menopause-associated depression as in endogenous depression not associated with estrogen deﬁciency. This issue however has not been studied with carefully controlled studies. More research is needed to identify the frequency and severity of these symptoms and the use of nonestrogenic medications for their treatment. St. John’s Wort, an herb, is currently being studied for the treatment of mild depression.

SAFETY AND SIDE EFFECTS OF ALTERNATIVES TO ESTROGEN In choosing between estrogen and its alternatives, the wide range of beneﬁts and risks of each need be considered. The additional beneﬁcial effects of estrogen include a potential reduction in incidence or severity of Alzheimer’s disease (141–143), macular degeneration (144), colon cancer (145), mandibular bone loss (146) and prevalence of osteoarthritis (147). On the other hand, one must also consider negative side effects such as uterine bleeding and the risk of uterine cancer requiring progesterone to protect the endometrium. Data regarding increased risk of thromboembolic events with HRT were initially observational (148). The HERs study, however, compares HRT use to placebo and clearly demonstrated an increased risk of DVT and pulmonary emboli with HRT (81,82). The risk of gallstones is also increased by HRT (149). The risk of breast cancer from HRT has already been discussed. These risks of HRT can be compared with those of surrogates for estrogen. Regarding the statins, creatinine phosphokinase (CPK) levels and renal and hepatic function have been altered and rhabdomyolysis induced (150). Increments in CPK may occur in patients who have no symptoms of myopathy. Combinations of lipid-lowering agents, particularly nicotinic acid and the statins, increase the risk of rhabdomyolysis. These effects occur uncommonly and use of these agents has now become widespread. For women with breast cancer, bisphosphonates may be particularly attractive because

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of reduced risk of fractures from skeletal metastasis and potential for slowing of progression of metastasis (151). By slowing remodeling, bone apparently becomes less hospitable for metastasis to grow. Reports of gastrointestinal symptoms including erosive esophagitis (152) have been reported with alendronate although no signiﬁcant differences have been found in controlled studies compared to placebo. Ettinger (153) recently reported a retrospective database analysis and telephone interview survey of 812 women with mean age 68.7. He found that one out of three women receiving alendronate for osteoporosis had upper GI complaints in a large Kaiser HMO with one of eight women requiring medical treatment of Acid-Related Disorders (ARD). Excluding women with prior history of ARD, the RR was 1.6 for alendronate users over nonusers. A signiﬁcant trend was seen with increasing incidence as women age and with concurrent use of nonsteroidal antiinﬂammatory drugs. This study underscores the need to minimize the risk of esophagitis and to improve drug absorption by recommending that alendronate be taken ﬁrst thing in the morning, on an empty stomach, with 8 oz water, and to remain upright for 30 min. As a part of the three-year FIT prospective study, women receiving alendronate who developed new clinically recognized vertebral fractures reported an average of 3.2 fewer days of bed rest (p < .001) and 11.4 fewer days of limited activity caused by back pain than women receiving placebo (154). The nasal form of calcitonin has few side effects—rhinitis, nasal discomfort or ulceration, nausea, facial ﬂushing and diarrhea, and, like subcutaneous calcitonin, may have an analgesic effect on bone pain (155). With regard to raloxifene’s effect on breast, studies suggest a 50–70% decrease in risk of breast cancer at three years. Although of interest, prevention of breast cancer was not a primary endpoint of the study. A deﬁnitive trial comparing tamoxifen with raloxifene for prevention of breast cancer (the STAR trial) is ongoing. From animal studies, raloxifene was not thought to have estrogenic effects on the endometrium. This appears to be born out by clinical trials. In the MORE trial, minimal increase in uterine thickness occurred and no increase in incidence of endometrial cancer (117). Uterine thickness was unchanged from placebo with no stimulation of atrophic endometrium. Side effects included leg cramps and an increased relative risk of DVT of 2.5. Raloxifene does not appear to be an effective agent to relieve the symptoms of vasomotor insufﬁciency and in fact, 9.7% of women noted this problem on raloxifene versus 6.4% on placebo. No effect was seen on vaginitis, migraine, headache, anxiety, or emotional lability. Very few data are available with respect to cognitive function, mood, or memory (68–69). Tamoxifen has been used primarily for prevention of recurrent breast cancer but is now approved for prevention of breast cancer in women at high risk for this disease. The Tamoxifen Prevention Trial (10), a prospective, randomized, blinded, controlled study of approx 13,000 high-risk women found a 45% decrease in development of breast cancer after 4.5 years of treatment. Risks include increased incidence of uterine cancer, cataracts and blood clots (10). The absolute excess of deaths from endometrial cancer during the whole decade after randomization was, in each of the three tamoxifen duration categories, about 1 or 2 per 1000 (corresponding to an annual excess of about 0.2 per 1000). There was a nonsigniﬁcant tendency for the excess of endometrial cancer deaths to be greater in the trials of longer duration of tamoxifen. Although this trend may well be real, the absolute excess was not large. Among 3673 women allocated to

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5 years of tamoxifen in trials that provided cause-of-death information, there were seven endometrial cancer deaths during 26,400 woman-years of followup before any recurrence of breast cancer, and the cumulative risk during the whole of the ﬁrst decade was about two deaths from endometrial cancer per 1000.

PRACTICAL APPROACH TO USE OF ALTERNATIVES TO ESTROGEN In menopausal women not willing to take HRT, ﬁve speciﬁc problems should be considered for potential treatment: vasomotor instability, urogenital atrophy, neurocognitive disturbances, treatment or prevention of osteoporosis, and prevention of cardiovascular disease. A careful history and physical examination can identify and evaluate the severity of hot ﬂashes, symptoms of urogenital atrophy and depression, mood disorders or memory problems. To assess the risk of cardiovascular disease, the history focuses on familial factors, diet, smoking history, prior hypertension, prior MI, angina, weight, and presence of diabetes. The physical exam evaluates blood pressure and presence of heart disease. Fasting glucose and lipid levels are obtained. Regarding osteopenia or osteoporosis, a dual X-ray absorptiometry (DEXA) scan is obtained to determine the degree of bone loss. This evaluation provides the data needed to decide whether to treat one or more of the ﬁve potential problems. Usually, women require therapy for only one menopausal problem and frequently two, but rarely more than two. Even though HRT manages all ﬁve problems, a focused, patient-tailored approach usually does not require a large number of medications and is practical for the patient. The authors of this chapter have utilized a sequential approach to treatment of each of these problems (Fig. 4). For vasomotor instability this includes avoidance of precipitating events initially followed by use of 800 IU of vitamin E daily and then clonidine, an SSRI, and megestrol acetate. More recently we have been using the SSRIs as the initial therapy. Treatment is only utilized if the severity of symptoms warrant. Clonidine is started as one transdermal patch (TTS #1) for one week, followed by two patches for the second week followed by three patches the third week. Patients should not increase the number of patches if side effects occur. During the fourth week, use of the TTS #1, #2, or #3 replaces the one, two or three patches used during the induction phase. If hot ﬂashes persist at night, supplementary tablets of clonidine from 0.1 to 0.4 mg can be given at nighttime. Only a percentage of patients respond to this regimen and the others do not. For those not responding, the next step is use of the SSRIs. Paroxetine 20–40 mg/d or venlafaxine 75 mg/d for example may be effective. As noted above, more recently we have been using the SSRIs as the initial therapy. This change is based on the results of the large multicenter trial from the North Central Clinical Oncology Group whose ﬁndings were presented at the annual meeting of ASCO in the spring of 2000. Finally, megestrol acetate 40 mg/d has been shown to be effective and can be utilized. Some women have an exacerbation of hot ﬂashes for one week before diminution of this symptom (135) and weight gain is a common complaint. For urogenital atrophy, the ﬁrst approach is Replens or vagisel for vaginal moisturization, and astroglide, a water soluble lubricant, for symptoms of vaginal dryness and dyspareunia. The next step is either use of the Estring or 0.5 g of Premarin cream or

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Fig. 4. Algorithm for treatment of the ﬁve major problems associated with estrogen deﬁciency with methods not using systemic estrogen therapy.

100 µm Estrace vaginal cream daily for three weeks followed by twice weekly thereafter. We do not use a progestin in combination with low-dose vaginal estrogen. For depression or anxiety associated with depression, we utilize standard SSRI therapy in conventional doses. Anxiety can be treated with anxiolytic medication provided it is not a part of a syndrome with a predominance of depression. Memory changes are not amenable to alternatives to estrogen. For women with osteopenia or osteoporosis, initial measures include a minimal calcium intake of 1500 mg/d, and vitamin D 800 IU/d if urinary calcium levels on this regimen are below 100 mg/24 h (156–160). We routinely measure 24-h urinary calcium to ensure adequacy of calcium intake and absorption. Exercise to include at least 30 min of weight-bearing exercise at least three times per week is recommended. Bone formation is stimulated by a combination of gravitational and weight-bearing forces such as the low repetitive muscle activity combined with weight load found in walking,

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jogging or low-impact aerobics (157–158). If the DEXA T score is ≥ 1.5 (with one risk factor), we recommend bisphosphonates, raloxifene, or tamoxifen. The choice between raloxifene, tamoxifen, and bisphosphonates such as alendronate or risendronate depends upon several factors. If the patient has an increased risk of developing breast cancer based upon the Gail model (i.e., >1.67% at ﬁve years), we recommend tamoxifen for patients without a uterus and raloxifene for those with a uterus. The rationale for this is that raloxifene, while not proven to prevent breast cancer, is indicated for treatment of osteopenia or osteoporosis and may reduce the risk of breast cancer. In those without a uterus, tamoxifen is effective in enhancing bone mass and probably in preventing fracture, and is effective in reducing the risk of breast cancer. The absence of a uterus eliminates the risk of tamoxifen in causing endometrial cancer. For those at high risk of breast cancer (i.e., >4% chance of developing breast cancer within ﬁve years) and with a uterus, tamoxifen would be recommended. Under these circumstances, the beneﬁts of tamoxifen in preventing breast cancer would outweigh the risks of developing endometrial carcinoma. Bisphosphonates are ﬁrst line for prevention or treatment of bone loss alone. Studies are underway with dosing of alendronate once a week to improve compliance and tolerability. Risedronate is now available and is an alternative for patients with bone loss who can’t tolerate alendronate or as an alternative to alendronate. Nasal calcitonin is usually reserved for patients with contraindications or side effects from bisphosphonates and raloxifene. For patients with substantial risk of heart disease, we recommend a prudent lowcholesterol, low-fat diet, exercise, cessation of smoking, and control of blood pressure. If the LDL and HDL levels meet criteria for treatment according to standard guidelines (161), we recommend use of a statin in sufﬁcient dosage to lower LDL cholesterol levels to less than 130 mg/dL. In those with preexisting heart disease, the targets are to lower LDL cholesterol to less than 100 mg/dL.

CONCLUSIONS Women with an absolute contraindication to use of estrogen such as breast cancer survivors or patients who are fearful of taking HRT are candidates for these alternatives to estrogen. A recent consensus conference made recommendations regarding use of estrogen surrogates in breast cancer survivors. The consensus statement (38) is quoted because it serves as the basis for recommendations in this review, both for breast cancer survivors and for those unable or unwilling to take estrogen. “Effective means are now available to treat or improve problems associated with the menopause without using estrogen replacement therapy. A tailored treatment strategy, which identiﬁes the needs of each individual patient, is recommended. The physician and patient can then make informed choices to address speciﬁc problems and to treat each patient individually. Treatments such as “statins” now exist for prevention of heart disease and new therapies are available for both prevention and treatment of osteoporosis. These can be used in place of estrogen. Bone speciﬁc agents such as bisphosphonates and nasal calcitonin are available for prevention or treatment of osteoporosis. Provision of low dose estrogen to the vagina locally, either via vaginal ring or cream, provides relief of symptoms of urogenital atrophy without increasing plasma estrogen levels substantially, although further studies of plasma estrogen concentrations with highly sensitive assay methods are necessary to determine if increments in systemic estrogen levels occur with these

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local delivery methods. Treatment of the symptoms of vasomotor instability is highly effective with Megestrol acetate, less so with clonidine, and marginal with vitamin E. Symptoms related to the effects of estrogen deﬁciency on the CNS may respond to CNS active agents such as anti-depressants but this area requires further study. The SERMs should be tested long term in clinical trials in survivors of breast cancer for prevention of osteoporosis and heart disease but are not effective for relief of short term menopausal symptoms.”

ACKNOWLEDGMENTS A major portion of the material in this chapter is reproduced from Endocrine Reviews (Pinkerton, JV and Santen, RJ Endocrine Review, June 1999) with the permission of The Endocrine Society; from Santen, R.J. and Pinkerton, J.V. Risk of breast cancer with progestins in combination with estrogen as hormone replacement therapy. J Clin Endocrinol and Metab 2001;86:16–23, and from Santen R.J. and Pinkerton, J.V. The treatment of estrogen deﬁciency in women at risk for breast cancer and survivors of the disease. In: Sex Hormone Therapy. Henry Burger (ed), Kluwer Academic Publishers, Norwell, Massachusetts, 2000, pp. 43–81.

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134. Laufer LR, Erlik Y, Meldrum DR, Judd HL. Effect of clonidine on hot ﬂashes in postmenopausal women. Obstet Gynecol 1982;60:583–586. 135. Loprinzi CL, Michalak JC, Quella SK, et al. Megestrol acetate for the prevention of hot ﬂashes. N Engl J Med 1994;331:347–352. 136. Loprinzi C, Pisansky T, Fonseca R, et al. Pilot evaluation of vanlafaxine hydrochloride for the therapy of hot ﬂashes in cancer survivors. J Clin Oncol 1998;16:2377–812. 137. Loprinzi CL, Kugler JW, Sloan J, et al. Venlafaxine alleviates hot ﬂashes: an NCCTG trial. Proceedings of ASCO. J Clin Oncol 19: abstract number 4, 2000. 138. Loprinzi CL, Quella SK, Sloan JA, et al. Preliminary evaluation of ﬂuoxetine (Prozac) for treating hot ﬂashes in breast cancer survivors. Proceedings of the 22nd Annual San Antonio breast cancer Symposium. Breast Cancer Res Treat 1999;58:34, Abstract 37. 139. Lien E. Hormone therapy and phytoestrogens. J Clin Pharm Ther 1996;2:101–111. 140. Hirata JD, Swiersz LM, Zell B, Small R, Ettinger B. Does dong quai have estrogenic effects in postmenopausal women? A double-blind, placebo-controlled trial. Fertility and Sterility 1997;68: 981–6. 141. Kowall NW. Alzheimer disease 1999: a status report. Alzheimer Disease and Associated Disorders 1999;13 Suppl 1:S11–16. 142. Birge SJ. The role of estrogen in the treatment of Alzheimer’s disease. Neurology 1997;48(5 suppl 7):S36–S41. 143. Yaffe K, Sawaya G, Lieberburg I, Grady D. Estrogen therapy in postmenopausal women: effects on cognitive function and dementia. JAMA 1998;279:688–695. 144. Smith W, Mitchell P, Wang JJ. Gender, oestrogen, hormone replacement and age-related macular degeneration: results from the Blue Mountains Eye Study. Aus NZ J Ophthalmol 1997;25(suppl 1): S13–15. 145. Grodstein F, Martinez ME, Platz EA, et al. Postmenopausal hormone use and risk for colorectal cancer and adenoma. Ann Int Med 1998;128:705–712. 146. Paganini-Hill A. The beneﬁts of estrogen replacement therapy on oral health. Arch Int Med 1995;155:2325–2329. 147. Felson DT, Nevitt MC. The effects of estrogen and osteoarthritis. Curr Opin Rheumatol 1998;10:269– 272. 148. Daly E, Vessey MP, Hawkins MM, Carson JL, Gough P, Marsh S. Risk of venous thromboembolism in users of hormone replacement therapy. Lancet 1996;348:977–980. 149. Petitti DB, Sidney S, Perlman JA. Increased risk of cholecystectomy in users of supplemental estrogen. Gastroenterol 1988;94:91–5. 150. Nakahara K, Kuriyama M, Sonoda Y, et al. Myopathy induced by HMG-CoA reductase inhibitors in rabbits: a pathological, electrophysical and biochemical study. Toxicol Appl Pharmacol 1998;152:99–106. 151. Boissier S, Magnetto S, Frappart L, Cuzin B, Ebetino FH, Delmas PD, Clezardin P. Bisphosphonates inhibit prostate and breast carcinoma cell adhesion to unmineralized and mineralized bone extracellular matrices. Cancer Res 1997;57:3890–4. 152. De Groen PC, Lubbe DF, Hirsch LJ, et al. Esophagitis associated with the use of Alendronate. N Engl J Med 1996;335:1016–1021. 153. Ettinger B, Pressman A, Schein J. Clinic visits and hospital admissions for care of acid-related upper gastrointestinal disorders in women using alendronate for osteoporosis. Am J Man Care 1998; 4:1377–1382. 154. Nevitt MC, Thompson DE, Black DM, Rubin SR, Ensrud KE, Yates AJ, Cummings SR. Effect of alendronate on limited activity days and bed disability days caused by back pain in postmenopausal women with existing vertebral fractures. Arch Int Med 2000;160:77–85. 155. Gennari C, Agnusdei D, Camporeale A. Use of calcitonin in the treatment of bone pain associated with osteoporosis. Calcif Tissue Int 1991;49:Suppl 2:S9–S13. 156. Dawson-Hughes B, Harris S, Krall E, et al. Effect of calcium and vitamin D supplementation on bone density in men and women 65 years of age or older. N Engl J Med 1997;337:670–676. 157. Friedlander A, Genant H, Sadowsky S, et al. A two-year program of acrobics and weight training enhances bone mineral density of young women. J Bone Min Res 1995;10:574–585. 158. Kerr D, Morton A, Dick I, Prince R. Exercise effects on bone mass in postmenopausal women are site-speciﬁc and load dependent. J Bone Miner Res 1996;11:218–225.

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159. Blumsohn A, Herrington K, Hannon RA, Shao P, Eyre DR, Eastell R. The effect of calcium supplementation on the circadian rhythm of bone resorption. J Clin Endocrinol Metab 1994;79:730–5. 160. Eastell R. Drug therapy: treatment of postmenopausal osteoporosis. N Engl J Med 1998;338:736–746. 161. Knopp RH. Drug treatment of lipid disorders. N Engl J Med 1999;341:498–511.

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Phytoestrogens in the Context of SERMs Susan R. Davis,

MB, BS, PHD, FRACP

Contents Introduction Phytoestrogen Classiﬁcation and Metabolism Measurement of Phytoestrogens Food Sources of Phytoestrogens Phytoestrogens as Estrogen Agonists/Antagonists Phytoestrogen Action in the Breast and Uterus Other Potential Biological Effects Clinical Effects of Phytoestrogens in Women Premenopausal Women Postmenopausal Women Conclusion References

INTRODUCTION Selective estrogen receptor modulators (SERMs) are characterized by their ability to act as either estrogen agonists or antagonists in different tissues, with different genes, and in different hormonal milieux. The effects of the most-studied SERMs are partially understood by their interactions with the ligand-binding pocket of the estrogen receptors (ER) α and β, as described elsewhere in this text. Phytoestrogens are plant constituents with a phenol structure similar to estrogen. They are found in a wide variety of edible plants and have acquired the misnomer of “nature’s SERMs.” Although these compounds may exhibit both estrogenic and antiestrogenic effects, the extent to which antiestrogenic effects are caused by ER antagonism is not known. It appears most feasible that they may act as weak estrogens under some circumstances, with apparent antiestrogenic effects resulting from either non ER-mediated mechanisms or their ability to bind ERα and β in the cell and exert weak agonism in the presence of estradiol. Hence the ultimate actions of these compounds in speciﬁc cells is determined by many factors including the presence or absence of estradiol, the concentration of the phytoestrogen being studied, the presence or absence of other phytoestrogens, the From: Contemporary Endocrinology: Selective Estrogen Receptor Modulators: Research and Clinical Applications Edited by: A. Manni and M. F. Verderame  Humana Press, Totowa, NJ

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relative levels of ERα and ERβ, and the diverse mix of coactivators and corepressors present. Effects vary according to the phytoestrogen studied, cell line, tissue, species, and response being evaluated. Furthermore, in attempting to translate in vitro ﬁndings to humans, the interindividual diversity and complexity of dietary phytoestrogen absorption and metabolism make the bioactivity of these compounds unpredictable. Observational epidemiological studies, primarily comparing Asian and Western populations, suggest that consumption of a phytoestrogen-rich diet ameliorates estrogen deﬁciency symptoms in postmenopausal women, and may protect against breast cancer, bone loss, and cardiovascular disease. Consequently there has been an overwhelming trend toward increasing consumption of phytoestrogen rich foods, and various formulations of concentrated isoﬂavone extracts are being heavily promoted. More recent intervention studies question the validity of the proposed beneﬁts of phytoestrogen supplementation, with little data in postmenopausal women to support a role for phytoestrogens as an alternative for conventional hormone replacement therapy (HRT). In addition, it remains unclear the extent to which the reported beneﬁcial health effects, most consistently as favorable effects on lipids, are a result of phytoestrogens alone, or other components of whole food. Overall exposure to these compounds may not necessarily be good, and perhaps inappropriate or excessive exposure may be detrimental. A greater understanding of the speciﬁc intracellular effects of the various phytoestrogens in different tissues, the relationships between timing and duration of exposure and disease, and randomized, controlled clinical trials are needed before deﬁnitive recommendations regarding their therapeutic use can be made.

PHYTOESTROGEN CLASSIFICATION AND METABOLISM Phytoestrogens have as their common denominator a phenolic group similar to that of estrogenic steroids. The ability of various phytoestrogens to occupy the ligandbinding pocket of either ER and exhibit estrogenic effects appears to reside in this phenolic group. Phytoestrogens are ubiquitous throughout the plant world and have been categorized according to their chemical structures as isoﬂavones, lignans, and coumestans (Fig. 1). Isoﬂavones and coumestans (also known as isoﬂavanoids) are sometimes described as intrinsic estrogen components, as they are synthesized within the plant itself. Resorcylic acid lactones are often referred to as phytoestrogens, however these compounds are produced by molds that contaminate cereal crops and are therefore more accurately called mycoestrogens (1). Isoﬂavones and isoﬂavone glycosides are among the most common phytoestrogens, with more than 70 isoﬂavones and 40 isoﬂavone glycosides identiﬁed (2). They are heterocyclic phenols closely related in structure to estrogenic steroids (3). They occur mainly in the plant species of the family Leguminosae, namely peas, beans, and lentils, and also in species of the genera Graminae, Rosaceae (Prunus spp), Iridaceae (Iris supp), and Solanaceae (Nicotiniara tabac) (4). Few of these compounds have been found to be estrogenic. A few are administered as herbal therapies, for example oatstraw and red clover which are rich in isoﬂavones, and squaw vine and false unicorn root which are said to have estrogenic properties but have been little researched. Although possibly not the most estrogenically active, genistein and daidzein are the

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Fig. 1. Classiﬁcation of principal dietary phytoestrogens.

most studied isoﬂavones, occurring in plants as the inactive glycosides genistin and daidzin and their respective methyl ethers, biochanin A and formononetin (1). These precursors are hydrolysed to genistein and daidzein within the human gastrointestinal tract. Genistein may be further metabolished to p-ethyl phenol and daidzein to Odesmethylangiolensin (ODMA) and equol (5,6). This metabolism in humans involves complex enzymatic interconversions inﬂuenced by multiple factors that have been fully described elsewhere (7–9). Of signiﬁcance, not all humans metabolize daidzein to equol such that individuals may be classiﬁed as equol excretors or nonexcretors (10,11). Coumestans are structurally similar to isoﬂavones, and coumestrol and 4-methoxy coumestrol are the most commonly occurring coumestans that exhibit estrogenic activity (2). Alfalfa is rich in coumestrol, and is widely available as a herbal tea or tablet. Little is known about the metabolism of the coumestans in humans, however the metabolism in a variety of animals has been more extensively investigated (1). Lignans occur in vascular plants in the form of glycosides (12). The principal lignans in plants are matairesinol, secoisolariciresinol, lariciresinol, and isolariciresinol. These are the precursors of enterolactone and enterodiol, which exhibit estrogen activity in various systems (13). As for isoﬂavones, enterolactone and enterodiol are formed from their plant precursors within the gastrointestinal tract. Enterodiol may also be oxidized to enterolactone (5). Following absorption from the gut the metabolically active phytoestrogens undergo

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enterohepatic circulation and may be excreted in the bile (14) or deconjugated by the intestinal microﬂora, reabsorbed, reconjugated by the liver and excreted in the urine (15,16). Hence measurement of urinary phytoestrogens is a reliable estimate of absorption and systemic exposure. Only trace amounts of daidzein and genistein are detected in the feces (9). Resorcylic acid lactones are metabolites of fungai, principally Fusarium, which are ﬁeld organisms that thrive in poorly stored grains, oil seeds, and hay. They are concentrated in the seed hull of grains which is usually removed during food processing (1). The mycoestrogens identiﬁed as having estrogenic activity are zearalenone and the isomers α- and β-zearalanol (1). Isoﬂavones and lignans have been detected in human urine, plasma, feces, semen, bile, saliva, and breast milk (17–19). After two weeks of soy supplementation premenopausal women were found to have higher levels of isoﬂavones in nipple aspirates than in the circulation (20). The concentrations of different phytoestrogen metabolites vary widely between individuals even when a controlled amount of an isoﬂavone or lignan supplement is administered. This may be caused by differences in gut microﬂora, inﬂuence of gender, antibiotic use, and the presence of bowel disease (10,19,21). Concomitant food intake particularly ﬁber, vegetables, fruit, protein, and fat, as well as the duration of exposure to phytoestrogens in the diet also inﬂuence phytoestrogen metabolism (9,22,23).

MEASUREMENT OF PHYTOESTROGENS Phytoestrogens may be measured by high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GCMS) and more recently liquid chromatography mass-spectrometry (LCMS). All three analytical techniques have advantages and disadvantages. HPLC analysis is the simplest method. Following extraction, chromatography retention times are compared to retention times of known standards, and the mass is calculated from the area under the curve obtained from standard curves. GCMS is slightly more complicated. Once the compounds are extracted they need to be derivatized in order to be run through the gas chromatograph, a process that can be tedious and complicated. The advantage of GCMS lies in the ability of MS being to conﬁrm the identity of the peak by its molecular weight. LCMS is a newer technique, which combines the advantages of HPLC and GCMS, but at greater expense. All three techniques have been used for measurement of phytoestrogens in human blood and urine, however international standards still need to be established. The principal analytes measured include the isoﬂavones daidzein and genistein, the isoﬂavan equol, and the lignans enterodiol and enterolactone. Measurement of plasma or urine levels however tell us nothing of the tissue levels of genistein and other phytoestrogens that may be achieved in different sites in the body.

FOOD SOURCES OF PHYTOESTROGENS The isoﬂavones most likely to be estrogenically active occur almost exclusively in legumes and beans, particularly the soybean (1,24,25). Miso, soy bean paste, and tempeh are prepared by fermenting soy beans such that most of their isoﬂavone content is as aglucones (26). Tofu and soy milk are produced by hot water extraction and superheated water treatment respectively and contain only the β-glucosides genistin and daidzin.

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Soy protein concentrate produced by aqueous alcohol extraction contains <5% isoﬂavones (26), and second-generation soy foods, for example soy yogurt and soy noodles, have even less isoﬂavone content, whereas soy sauce contains almost none (27–29). Other consumable sources include whole-grain cereals, black cohosh, and red clover. The latter extracts are now sold over the counter as an isoﬂavone concentrate in tablet form. Lignans are the main phytoestrogens found in the diets of vegetarians, occurring widely in cereals, fruits, and vegetables. Flaxseed, also known as linseed, is the richest source of lignans (6,25,30). Coumestans occur predominantly in association with germination, and thus occur in the highest concentrations in the soybean sprout, alfalfa, and fodder crops (1). The phytoestrogen content of speciﬁc foods varies considerably according to the genetic differences in plants, location, season, infection with fungal disease, and differences in food-processing techniques (31).

PHYTOESTROGENS AS ESTROGEN AGONISTS/ANTAGONISTS The estrogenic effects of phytoestrogens were ﬁrst observed in Western Australia in 1946 as reproductive dysfunction in sheep, later known as Clover Disease (32). Subsequently, similar effects were reported in humans in association with the consumption of tulip bulbs in Holland, which were not uncommonly substituted for onions toward the end of the Second World War. Menstrual disturbances, including dysfunctional uterine bleeding, were reported in women who consumed the bulbs, which are rich in estrogenically active compounds (33). Isoﬂavones stimulate uterine growth in laboratory animals and in animals grazing on clover-rich pasture (32,34). Long-term grazing of ewes on subterranean clover results in obvious estrogenic effects such as cystic endometrial hyperplasia (32) and transformation of the cervix to a more uterine appearance (35). Paradoxically, masculinization of the external genitalia in the form of partial fusion of the labia of the vulva and clitoral hypertrophy may also occur. These apparently androgenic effects are similar to, but less severe than those seen in ewe lambs exposed to testosterone in late gestation (36). The role of various phytoestrogens in humans as estrogens however remains controversial as the biological actions of these compounds are extremely complex.

Phytoestrogens Exert Their Estrogenisity Primarily Via Binding the ERs␣ and ␤ The ligand-binding pockets of ERα and β are very capacious and this results in considerable promiscuity of these receptors. Ligands for ERα and β can act as pure agonists or antagonists, or they can have partial or selective agonist/antagonist activity. Whether an ER ligand acts as an agonist or antagonist is determined by several factors including the topographical manner in which the molecule interacts with the ligandbinding domain of the receptor (37), the diverse expression and interactions and activity of coregulators (coactivators and corepressors) in different tissues, and the nature of the response elements with which the receptors interact on the estrogen-regulated genes (38). Because of the complexities of ligand/ER interactions and the insufﬁcient number of studies addressing the diverse circumstances in humans in which they may have clinical effects, our understanding of the biological potencies of various phytoestrogens

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and how or under what circumstances they can act as antiestrogens or estrogen agonists remains relatively limited. The weight of available data however indicates that phytoestrogens for the most part do not act as SERMs. Rather they may act as weak estrogens under some circumstances, with apparent antiestrogenic effects caused by either nonER-mediated mechanisms or their ability to bind ERα and β and exert minimal agonism in the presence of estradiol. Knowledge of the consequences of the interactions between various ligands with ERα and β has been enhanced by the crystal structures of the ERα and β ligandbinding domains (LBD) complexed with several ligands. Most recently genistein has been found to ﬁt well into the ERβ LBD (39). Although agonists and antagonists bind at the same site within the core of the LBD, each induces speciﬁc conformations in the transactivation domain, known as AF-2. The effect of each ligand on the positioning of helix 12 provides a structural mechanism by which a ligand may act as an agonist or antagonist. Whereas compounds such as tamoxifen and raloxifene distinctly place helix 12 in an antagonist position, the positioning of helix 12 with genistein bound is more like that of estradiol. Thus genistein appears more likely to activate both ERs as an agonist (39). This is consistent with the ﬁndings of Kuiper and others who were unable to demonstrate any antiestrogenic activity of the most common phytoestrogens as described in next paragraph. The reported estrogenic potencies of the phytoestrogens studied to date vary considerably. Most of these compounds appear to be signiﬁcantly less potent in bioassays than 17β-estradiol but results from in vivo bioassays vary according to species, route of administration, and endpoints determined (1). Importantly, ER-binding studies published prior to the knowledge of two ERs did not differentiate between ERα and ERβ, particularly studies using uterine cytosol. The relative estrogenic potencies of the most common phytoestrogens have been evaluated in terms of estrogen-speciﬁc enhancement of alkaline phosphatase activity in human endometrial adenocarcinoma cells of the Ishikawa-Var I line. With 17βestradiol arbitrarily assigned a value of 100, the relative estrogenic potencies of various isoﬂavanoids were: coumestrol 0.202, genistein 0.084, equol 0.061, daidzein 0.013, biochanin A <0.006, and formononetin <0.006 (40). Kuiper and others have more recently reported the relative binding afﬁnities (RBA) of several phytoestrogens to ERα and ERβ (41). The RBA of each competitor was calculated as a ratio of concentrations of estradiol and competitor required to reduce the speciﬁc radioligand binding by 50%, with the RBA value for estradiol arbitrarily set at 100. In most instances the RBAs were at least 1000-fold lower than estradiol. Coumestrol, genistein, apigenin, naringenin, and kaempferol showed stronger competitive binding with estradiol for ERβ than with ERα. Kuiper et al. also demonstrated the ability of several phytoestrogens to stimulate the transcriptional activity of both ER subtypes at concentrations of 1 to 10 nM (41), with coumestrol showing the greatest estrogenic potency for ERα, and genistein and coumestrol being equivalent but most potent with respect to ERβ. These observations are consistent with human cell-culture bioassays in which coumestrol ahs been found to be the most potent phytoestrogen. No antiestrogenic activity could be detected for the many phytoestrogens studied, whereas zearalenone, a mycoestrogen, acted as a full agonist for ERα and a mixed agonist/antagonist for ERβ. Hall and McDonnell have demonstrated that ERα and ERβ can form heterodimers

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in target cells (42) and that at subsaturating hormone levels, ERβ functions as a transdominant inhibitor of ERα. This may be a major mechanism by which speciﬁc phytoestrogens appear as antagonists in an estrogenic milieu, whereas they are in fact acting as subliminal agonists. Interestingly, ERβ compared with ERα messenger RNA (mRNA) is highly expressed in vascular endothelium and smooth muscle cells, the ovary, and the prostate (40). Binding afﬁnity however does not automatically translate into biological action, which may be more profoundly determined by transcriptional activation and translational efﬁcacy, both of which are subject to multiple inﬂuences. Thus effects of genistein and other phytoestrogens in these tissues are yet to be determined.

PHYTOESTROGEN ACTION IN THE BREAST AND UTERUS There has been much interest in potentially anticarcinogenic actions of isoﬂavones. Laboratory rats fed soy-enriched diets have less breast tumor development following stimulation with the directly- and indirectly-acting tumour-inducers N-methyl=N-nitrosourea and dimethylbenz (a) anthracene respectively (3). Speciﬁc phytoestrogens exert proliferative effects in human breast cancer cell lines at low concentrations but at higher concentrations inhibit the proliferation of ERpositive and ER-negative breast cancer cells as follows (43,44). In the human ER-positive MCF-7 breast cancer cell line the effects are biphasic. At 0.1 to 10 µM concentrations, coumestrol, genistein, biochanin A, apigenin, luteolin, kaempferol, and enterolactone induce DNA synthesis by 150 to 235%, with continuous stimulation of DNA synthesis by MCF-7 cells at low concentrations of genistein and coumestrol over 10 d (3,45). Inhibition of DNA synthesis by 50% requires concentrations of the various phytoestrogens listed of 20 to 90 µM (45). For human breast cancer MCF-7, MDA-468, and T47D cells, the IC50 for serum-stimulated growth was reported by Peterson and Barnes as 22 to 37 µM (46). Wang and others also reported biphasic effects of genistein in MCF-7 cells, which are ER-positive (45). Genistein stimulated estrogen-dependent pS2 mRNA expression and cell proliferation at low concentrations but suppressed both at high concentrations. Genistein failed to stimulate proliferation of ER-negative MDA-MB-231 cells, but suppressed their proliferation at high concentrations (43). When MCF-7 cells were coincubated with estradiol and genistein (0.1–10 µM) genistein acted as an antiestrogen, suppressing pS2 mRNA expression (43), and Peterson and Barnes have reported similar results using equivalent concentrations (46). Circulating levels of isoﬂavones in humans consuming a high soy diet are reported by others to be in the range of 0.5 to 4 µM (18,47), and Dalais et al. have detected peak circulating levels of genistein of 0.15 to 0.37 µg/mL (0.56 to 1.4 µmol) following 45g isoﬂavone supplementation per day (48). Thus published data indicate induction of DNA synthesis in tumor cell lines at concentrations close to probable plasma levels in humans, even with high isoﬂavone intakes, and inhibition only at very high concentrations. However when ER-positive cells are exposed to a combination of estradiol and genistein, the latter appears to act as an antiestrogen. Whether this is because of genistein acting as an antagonist at the level of the ER by putting the LBD of the ER into an antagonist conformation, or by genistein ameliorating the effects of estradiol by competitive but agonistic binding is

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not known. The antiproliferative effects of genistein and other compounds observed in ER-negative breast cancer cell lines support the likelihood of an ER-independent effect (43,45). Although initially reported as stimulating uterine growth (32,34), the net effect of phytoestrogens on the endometrium may be antagonistic. In ovariectomized (OVX) rats a soybean extract diet alone was not estrogenic. When administered together with conjugated equine estrogen (CEE), a low-soy diet in OVX rats has additive estrogenic actions on markers of endometrial proliferation. In contrast, a high soy diet in OVX rats antagonises the effects of CEE with reductions in the uterine luminal epithelial height and lactoferrin response (36). Thus data from cell-culture and animal studies remain incomplete, as is our understanding of the cellular effects of these compounds. Their effects on breast cancer cells varies with ER status and dose. Isoﬂavones may have little or no effect on endometrial cell growth, but when administered concomitantly with estradiol may be antiestrogenic at moderate to high concentration.

OTHER POTENTIAL BIOLOGICAL EFFECTS Genistein has been found to reversibly arrest cell-cycle progression of human gastric carcinoma cells (HGC-27) at the G2/M boundary of the cell cycle of concentration of 25 µM and above (49). A proposed mechanism by which genistein inhibits cancer cell growth is inhibition of tyrosine kinase activity of activated growth-factor receptors and cytoplasmic tyrosine kinases, essential for the transduction of mitogenic signals. Genistein may inhibit epidermal growth-factor receptor (EGF-R) tyrosine autophosphorylation (50). Peterson and Barnes (46) however were unable to demonstrate this effect, and concluded that isoﬂavones exert their inhibitory effects either downstream from EGF-R tyrosine autophosphorylation or via some other mechanism. Genistein has also been reported to inhibit topoisomerase II, an essential nuclear enzyme involved in DNA replication (51). This is a potential mechanism by which genistein causes cell-cycle arrest at G2. Other studies imply that by inhibiting topoisomerase II, genistein and coumestrol may have deleterious effects on mammalian cells (52). Potential anticarcinogenic effects of isoﬂavones include inhibition of angiogenesis (53) and cell-cycle progression (54), suppressive effects on endogenous estrogen production as a result of aromatase enzyme inhibition (55,56) and inhibition of 5-alpha reductase (57). Isoﬂavones may also stimulate the synthesis of sex-hormone-binding globulin (SHBG) (58,59), as well as possess antioxidant properties (60) whereas lignans exhibit digitalislike activity (61). In the estrogen-deprived aromatase knockout (ArKO) mouse model a diet rich in isoﬂavones does not induce estrogenization. Female ArKO mice exposed to isoﬂavones in utero and maintained on an average intake of 40 µg/g bodyweight per day exhibit atrophied reproductive tracts and failure of arborization of prepubertal mammary ducts as do their unsupplemented counterparts (62). It may be that high-dose phytoestrogens do not exert estrogenic activity in otherwise estrogen-deﬁcient animals or that the isoﬂavones act as antiestrogens on reproductive tissues in this model. Both male and female ArKO mice are characterized by excessive central fat deposition. Male ArKO mice randomized to soy chow diet develop less central adiposity than ArKO mice fed a wheat chow diet (mean ± sd central fat pad 0.36 ± 0.15 g/animal

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versus 0.71 ± 0.39 g/animal p < 0.05 for soy and wheat diets respectively, (unpublished data kindly provided by Arnold, B, Fisher, CR, and Simpson, ER). These ﬁndings suggest components in soy, possibly isoﬂavones, may protect against central fat deposition.

CLINICAL EFFECTS OF PHYTOESTROGENS IN WOMEN Observational epidemiological studies suggested that Asian women who consume a soy-rich diet and who have high urinary phytoestrogen excretions experienced fewer menopausal symptoms than Western women. This statement has since been refuted (63). Subsequent randomized, controlled clinical trials of phytoestrogens in the management of menopausal symptoms have demonstrated no consistent beneﬁt from consumption of a phytoestrogen-rich diet on menopausal hot ﬂushes above that of placebo therapy. The trials have included phytoestrogens in the form of soy ﬂour, soy-enriched breads, and a mixed diet including tofu and soy subfractions in the form of isoﬂavone supplements. The confounders in these studies are varied potency and variety of soy products used, the individual variation in dietary metabolism of phytoestrogens, the background intake and inadvertent ingestion of phytoestrogens, and the natural resolution of hot ﬂashes over time. Claims are made that a high-soy diet or tablet phytoestrogen supplements are cardioprotective and may also protect against osteoporosis and breast cancer. Whereas there are some data indicative of a cardioprotective effect of soy, there is little to support protection against bone loss, and that phytoestrogens prevent breast cancer cannot be substantiated at this time. Nonetheless, the beliefs that the consumption of foods high in phytoestrogens results in reduced risks for hormonedependent cancer and cardiovascular disease have resulted in phytoestrogen food fadism in some countries. Dietary habits and lifestyle characteristics may explain differing disease risk patterns between various populations. Studies of disease prevalence in migrant populations support the hypothesis that environment conditions, particularly exposure in childhood, inﬂuence the development of breast cancer (64,65). An association however does not automatically translate into cause and effect. Consumption of a plant food (phytoestrogen) rich diet may merely be an excellent marker for a healthy lifestyle package that includes ingestion of other favorable nutrients in vegetables, cereals, and beans, lower energy consumption, greater energy expenditure, and lower body weight with resultant health beneﬁts. The so-called health beneﬁts may as much be a result of exclusion of deleterious factors such as caffeine, alcohol, and saturated fats as to the inclusion of phytoestrogens. In fact very few population studies linking high phytoestrogen intake with reduced risks for cancer and heart disease have included and adjusted for other beneﬁcial dietary components or known major risk factors. The major dietary studies of phytoestrogens have mostly been limited to the effects of soy. Other phytoestrogen sources and the interactions between various foods and their constituents also warrant investigation.

PREMENOPAUSAL WOMEN A cyclical pattern of lignan excretion has been observed during the menstrual cycle in humans and Vervet monkeys (6). It is probable that changing hormone levels during the menstrual cycle inﬂuence the metabolism and absorption of the dietary lignan precursors (15). High intakes of phytoestrogens may modify menstrual cycle length.

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Soy protein (60g/d) has been reported to increase follicular phase length, with suppressed midcycle peaks of follicle stimulating hormone (FSH) and luteinizing hormone (LH) (55). Ingestion of 36 oz of soymilk each day was associated with decreases in 17βestradiol and luteal phase progesterone in young ovulating women (22). In contrast daily ﬂaxseed supplementation (10g/d) has been reported to increase luteal phase duration but to have no effect on follicular phase length (66). An increase in SHBG did not occur in these studies in contrast to other reports (17,67). Martin et al. (68) demonstrated different dose-dependent inhibitory effects of lignans and isoﬂavones on steroid binding by SHBG, and postulated that phytoestrogens may modulate SHBG activity and so inﬂuence the delivery of estradiol to target tissues, and hence inﬂuence menstrual cycle length. It is often said that phytoestrogens protect women from hormone-dependent cancers by increasing menstrual cycle length and reducing overall lifetime estrogen exposure. Although appealing, this hypothesis does not take into account the tissue-speciﬁc effects of these exogenous estrogenic compounds nor the timing of exposure. Daily soy supplementation for two weeks in one study increased the proliferation rate of breast epithelium of premenopausal women (69), and in another was associated with lowered levels of nipple aspirate lipoprotein D and raised pS2 levels consistent with an estrogenic effect (20). No antiestrogenic effects of soy were detected in the latter. These outcomes were not predictable if extrapolating from the effects of isoﬂavones on circulating levels of 17β-estradiol alone (22).

POSTMENOPAUSAL WOMEN Menopausal Symptoms It is commonly said that Japanese women have a lower frequency of hot ﬂashes compared with postmenopausal western women and this has been partly attributed to their high phytoestrogen consumption (70). The validity of this has been challenged (63), such that the apparent low frequency of hot ﬂashes in Japanese women may be primarily a result of underreporting of symptoms rather than a lower prevalence of symptoms. The ﬁrst study to demonstrate that certain dietary phytoestrogens can exert mild estrogenic effects in postmenopausal women was published in 1990, and revealed an increase in vaginal-cell maturation index, an indicator of estrogen activity (71). Dalais et al. also reported an increase in vaginal cytology maturation index in women treated with soy supplements (72). These effects on vaginal cytology do not correlate with urinary isoﬂavone excretion (73). The reports of effects of phytoestrogen supplementation on postmenopausal vasomotor symptoms are not consistent. There are considerable differences between studies with no clear correlation between estrogenic changes in vaginal cytology and effects on vasomotor symptoms. When the effects of soy versus wheat ﬂour supplementation on hot ﬂashes and vaginal cytology were evaluated in a subset of women, there was no beneﬁt with soy in this population after 12 weeks (74). The effects of soy- versus linseed- versus wheat-supplemented diets on postmenopausal hot ﬂashes have also been reported. The reduction in hot ﬂash rate after 12 weeks was greatest in the wheat-diet phase, which was associated with very low urine isoﬂavone levels. Following 12 weeks of soy grits (45g/d) vaginal cytology index increased 103% despite no signiﬁcant reduction in hot ﬂashes with this supplement (72). In contrast Albertazzi et al. have

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reported a small reduction in hot ﬂashes in postmenopausal women supplemented with soy versus placebo, however the effects were not dramatic (75). Knight et al. (76) and Baber et al. (77) have reported the effects of an over-thecounter tablet preparation of isoﬂavones extracted from red clover (40mg/tablet) versus placebo in postmenopausal women. Doses of both 40mg/d and 160mg/d had no beneﬁt over placebo on reduction in hot ﬂashes. Furthermore isoﬂavone supplementation at low and high dose did not alter vaginal pH, SHBG, FSH or total cholesterol (76,77), although Knight et al. reported an increase in HDL cholesterol with therapy. There are certainly difﬁculties in objectively assessing vasomotor symptoms in studies because of the natural resolution of this symptom over time and the high placebo response rate. Conventional estrogen therapy however has been shown to effectively reduce hot ﬂashes in comparison to placebo, and for phytoestrogens to be a viable alternative to HRT the same standard should consistently apply. Phytoestrogens do not appear to improve other symptoms that characterize the menopausal transition such as anxiety, arthralgia, myalgia, and headaches.

Cardioprotection The ingestion of vegetable protein, particularly soy and soybean based foods, has been linked with a lower prevalence of coronary heart disease and improvements in a number of risk factors for atherosclerosis (67,78). To distinguish the relative contributions of the protein and nonprotein (isoﬂavone and saponin) components of soy, several researchers have used intact soy protein (soy+) and alcohol-extracted soy isolate (soy−), a process that removes a substantial portion of isoﬂavones and saponins (78). Feeding male and female Rhesus monkeys soy+ results in LDL cholesterol and VLDL cholesterol values 30–40% lower than controls (79). Honore et al. have examined the effects of soy protein on coronary vascular reactivity in atherosclerotic male and female rhesus monkeys randomized to soy+ and soy− diets. Acetylcholine induced coronary artery constriction in the female monkeys fed soy− whereas the arteries in the females fed soy+ dilated. Acute intravenous genistein administration resulted in vasodilation in previously constricted arteries of soy-fed females. In the males, acetylcholine induced vasoconstriction that was not reversed with the soy+ diet (80). There is some debate whether the beneﬁcial effects of soy observed are a result of the isoﬂavone moiety or other components of soy such as saponins. Greaves and others randomized ovariectomized female cynomolgus monkeys to casein protein, casein plus puriﬁed genistein and daidzein, or intact soy protein (81). The soy+ group had signiﬁcantly lower total cholesterol, LDL cholesterol and VLDL cholesterol, and signiﬁcantly higher HDL cholesterol concentrations than the other groups. Puriﬁed isoﬂavones selectively increased HDL cholesterol but had no effect on LDL and VLDL cholesterol. This implies that other nonprotein components of soy, possibly saponins or phytic acids, are responsible for the changes in lipids and lipoproteins reported (81). Cassidy et al. reported a 9% reduction in total cholesterol in a small study of normolipemic premenopausal women treated with a 60g soy protein supplement (55). Similarly, consumption of 25g soy protein-enriched bread results in a decreased total serum cholesterol and increased HDL cholesterol in hypercholesterolemic men (82). A soybean protein diet in subjects with Type II hyperlipoproteinemia may lower cholesterol on average by 20% (83). A metanalysis of thirty-eight published, controlled, clinical trials of soy protein consumption averaged at 47g/d and serum lipid and lipoprotein

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concentrations found that consumption of soy protein is signiﬁcantly associated with mean reductions in total cholesterol (9.3% decrease, 95% CI:0.35–0.85 mmol/L), LDL cholesterol (12.9% decrease, 95% CI:0.30–0.82 mmol/L), and triglycerides (10.5% decrease, 95% CI:0.003–0.29 mmol/L) (78). The hypocholesterolemic effect appears to be signiﬁcantly related to pretreatment plasma-cholesterol levels (78). A recent study of normolipemic postmenopausal women supplemented with a 40mg phytoestrogen pill demonstrated a 22% increase in HDL cholesterol and no signiﬁcant change in other parameters (84). Teede et al. (85) investigated the cardiovascular effects of dietary intact soy in 213 healthy men and postmenopausal women. They reported signiﬁcant reductions in mean, systolic, and diastolic blood pressures with soy in this normotensive population. Soy had no effect over placebo on vascular function (systemic arterial compliance, pulse wave velocity) and endothelial function (ﬂow mediated vasodilation) in women. Soy induced greater reductions in the LDL/HDL ratio and triglycerides, but Lp(a) lipoprotein increased compared to placebo. No beneﬁcial effects were seen for soy for total and LDL cholesterol or HDL cholesterol. Simons et al. in a much smaller study of postmenopausal women have reported similar ﬁndings (86). Dietary soy may inhibit platelet aggregation and have antioxidant effects (71,87). Tikkanen et al. have evaluated the effects of soy supplementation in healthy men on LDL oxidation in vitro (88). Compared with control diet, the lag phases of the LDL oxidation curves were prolonged by a mean of 20 min following soy intake, implying a reduced susceptibility to oxidation. Only small amounts of genistein and daidzein were detected in puriﬁed LDL indicating the modifying effects of the isoﬂavones may have occurred in vivo. In summary, the consumption of soy foods is associated with favourable effects on lipids and lipoproteins in some studies with variable effects on vascular function in primates and humans. Whether the observed effects result from the isoﬂavone component of soy or other moieties is still not clear. It is not known how much soy needs to be ingested and how often to achieve any cardioprotective effects. Although a healthy alternative to animal protein, soy protein cannot be regarded as a valid alternative to estrogen to reduce cardiovascular disease risk in postmenopausal women until based on evaluable data.

Phytoestrogens and Bone The genesis of osteoporosis is multifactorial with major inﬂuences being genetics, aging, hormone deﬁciency, and diet. The incidence of osteoporosis has been reported to vary geographically with some studies indicating lower rates in Asian women compared with Western women (89,90) and again phytoestrogens are implicated as a protective factor. The effects of phytoestrogens on bone have been thoroughly reviewed by Anderson and Garner (91). They suggest phytoestrogens may have effects on osteoblasts and osteoclasts, probably mediated via the ERs for the former, whereas effects on osteoclasts appear to be non ER mediated (91). Dietary soybean supplementation has been shown to signiﬁcantly reduce bone loss in ovariectomized rats (p < 0.001) (92). A synthetic ﬂavonoid, ipriﬂavone (7 isopropoxyisoﬂavone) inhibits osteoblast recruitment and function, and treatment with 600 mg/d has prevented short-term bone loss at the distal radius in osteoporotic postmenopausal women (93). In a study of postmenopausal women receiving casein, soy protein, or

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soy protein fortiﬁed with isoﬂavones for six months, the group treated with the fortiﬁed soy manifested an increase in bone mineral content and density compared with controls (94). Similarly, it has been reported that postmenopausal women consuming soyenriched bread (45g) had an increase in bone mineral content in contrast to women consuming wheat kibble bread (72). This study was not controlled for ﬁber content of the different breads, which may affect calcium absorption, although it was probably too short term for the latter to have inﬂuenced the outcome. Gambacciani et al. (95) have reported reduced urinary hydroxyproline excretion with ipriﬂavone. Although these studies are encouraging, they in the most part have included few participants, have not been controlled for confounding factors such as exercise, and the intervention has been relatively short term. Thus there is insufﬁcient published data to support a speciﬁc role for phytoestrogens in the prevention of osteoporosis. Meanwhile, it is recommended that women choosing soy milk, or other soy products as dairy alternatives select calcium fortiﬁed products to maximize osteoporosis prevention.

Cancer There is much interest in the role of phytoestrogens in protecting against some types of cancer with data accumulating from animal, in vitro, and human epidemiological cancer studies. The incidence of hormone-dependent tumors is lower in Asia and Eastern Europe than in Western countries (67,96) and among vegetarians (97,98). Breast, ovarian, and colon cancer show a negative correlation with cereal and phytoestrogen intake when comparing cancer mortality rates and food availability data between countries (96).

Clinical Studies of Phytoestrogens and Breast Cancer Breast cancer is a common cause of morbidity and mortality among women in Western countries. The incidence of breast cancer is lower in Asia and Eastern Europe than in Western countries and among vegetarians. Although the higher intake of phytoestrogens by Asian populations is associated with reduced breast cancer rates, this has not been consistent in all studies and this has stimulated research into the possible role of diet and breast cancer occurrence. The evidence that diet has a role in the development of breast cancer initially came from population and migration studies. Japanese immigrants to North America have a higher incidence of breast cancer than their counterparts in Japan (64). Japanese women in their homeland with breast cancer have a high number of in situ tumours with fewer nodal metastases, and those with metastases have less nodal spread than women with breast cancer in the United States or Britain (25). Furthermore, in Japanese women there is a signiﬁcant graded inverse association between risk of breast cancer and the consumption of miso (soybean paste soup) (99). Lee et al. (100) did not observe a beneﬁcial relationship between soy intake and breast cancer in postmenopausal Singaporean women. Similarly Yuan et al. found no association between soy and breast cancer in a study in China, but rather an inverse correlation between ﬁber intake and other nutrients and breast cancer rates (101). Most recently lower levels of ERα gene expression have been demonstrated in normal breast tissue in Japanese women compared with Caucasian women and this may account in part for the ethnic differences in breast cancer rates (102). Baghurst and Rowan have investigated the relationship between dietary ﬁber and beast cancer in a case-controlled study, and reported a progressive

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reduction in breast cancer relative risk with each quintile of increasing ﬁber intake for women (97). The women in the highest quintile of nonstarch polysaccharide intake had a relative risk of 0.46. Possible mechanisms include an interruption of the enterohepatic circulation of both endogenous estrogens and phytoestrogens, and hence an overall reduction in total circulating estrogens secondary to the high-ﬁber diet, the high phytoestrogen content of the dietary ﬁber, the exclusion of other carcinogenic foods, or the concurrent ingestion of other inﬂuencing macronutrients. In early reports breast cancer patients have been observed to have low urinary lignan excretion reﬂecting a low ﬁber intake (67). Ingram et al. measured urinary isoﬂavone and lignan excretion in women newly diagnosed with breast cancer and community controls (103). After adjustment for age at menarche, parity, alcohol intake, and total fat intake, high excretion of equol and enterolactone but not genistein and daidzein were associated with a substantial reduction in breast cancer risk, with signiﬁcant trends through the quartiles. the measurement of high enterolactone excretion again reﬂects an increased vegetable and/or ﬁber diet in the women who did not develop breast cancer, however the high excretion of equol is most closely linked to a high legume intake. Murkies et al. studied urinary phytoestrogen in Australian women immediately following diagnosis of breast cancer and reported lower isoﬂavone excretion in cases compared with controls despite similar dietary patterns (104). Protective effects of isoﬂavones have been seen in animal models of experimentally induced breast cancer measured by tumor number, incidence, metastases, and latency (105–107). Prepubertal genistein-treated rats develop fewer mammary gland terminal end buds, with signiﬁcantly fewer cells in the S-phase of the cell cycle, and more lobules than control at 50 days old (108). This is consistent with the ﬁndings of immigration studies which suggest exposure to a phytoestrogen-rich diet in childhood may be protective (64). Prepubertal phytoestrogens may cause precocious maturation of breast terminal end buds to more differentiated lobules, and result in subsequent breast cancer protection. In contrast, postpubertal exposure, without breast maturation from signiﬁcant phytoestrogen ingestion in childhood, or another stimulus for breast maturation such as full term pregnancy, could potentially increase breast cancer risks via an agonistic estrogen action. As described earlier, breast cell lines saturated with predominantly genistein exhibit receptor stimulation at low concentrations (44,50,109) and dose-dependent inhibition at increased concentrations (34,105,109). Genistein also appears to act as an estrogen antagonist in an estrogenic milieu. It is premature to make ﬁrm recommendations regarding phytoestrogen intake and breast cancer prevention or management. Asian women may be genetically less susceptible to breast cancer, and a high phytoestrogen intake may be a positive marker for an overall high-ﬁber diet and exclusion of other carcinogenic foods rather than being protective in itself. The inclusion of one serving of soy per day in an otherwise diverse diet that has a high content of fruit, vegetables and grains, would appear to have multiple positive health beneﬁts for younger women. In contrast, increasing circulating isoﬂavone levels in postmenopausal women, who are estrogen deﬁcient, to levels known to stimulate breast cancer cells in vitro, may be potentially dangerous. Alternatively, isoﬂavones may be protective against breast cancer for postmenopausal women using estrogen replacement, by acting as antiestrogens in the estrogenic milieu. There is no evidence that phytoestrogen supplementation in tablet form is protective against breast cancer,

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or even safe. Concurrent use of high-dose phytoestrogen supplements and tamoxifen in women with breast cancer should also at this time be discouraged until further information is available because of the potential for isoﬂavones to antagonize the desired antiestrogen effects of tamoxifen (110). Women with breast cancer are best advised to optimize their nutrient intake and nonsaturated fat protein sources such as ﬁsh with small amounts of soy as food.

CONCLUSION The discovery that pharmacological compounds can be estrogenic in some tissues and antiestrogenic in others fueled interest in the possibility that phytoestrogens may be nature’s SERMs. Available data however do not support this concept. Elegant crystallography has demonstrated that phytoestrogens such as genistein can occupy the LBD of both ERs, and competitive binding studies conﬁrm various phytoestrogens compete more strongly for ERβ, than for ERα. Both transactivation studies and studies of cancer cell lines however consistently demonstrate agonism of isoﬂavones and lignans at physiological concentrations. The weight of the data is that the purported antiestrogenic effects of phytoestrogens in cancer cell lines are non ER mediated. In other tissues the recent ﬁnding that ERβ functions as a transdominant inhibitor of ERα may mean that phytoestrogens that preferentially bind ERβ dampen the transactivation of ERα by estradiol but are not ER antagonists as such. Furthermore the favorable effects of soy on lipoprotein lipids may primarily be a result of other important chemical constituents. The relationships between our health and the food we eat cannot be adequately explained by nutrient composition alone. The focus of nutrition research on compounds such as phytoestrogens strongly indicates that these phytochemicals may have physiological effects in humans, but their therapeutic role, particularly in postmenopausal women, remains unclear. There are no speciﬁc examples of phytoestrogen toxicity in human beings in countries where phytoestrogens have been consumed for centuries. It is difﬁcult for humans to consume, from natural soy foods, the amounts of isoﬂavones necessary to reach the toxological levels found to induce pathological effects in animals (111). The trend toward the consumption of concentrated isoﬂavone supplements, particularly in pill form, will facilitate high intakes and the potential for dangerous effects from megadosing is of concern.

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62. Fisher CR, Greaves K, Parlow AF, Simpson ER. Characterization of mice deﬁcient in aromatase “ArKO” because of targeted disruption of the cyp 19 gene. Proc Natl Acad Sci USA 1998;95:6965–70. 63. Albery LN. Plenary Lecture. The politics of Menopause. Eighth Congress of the International Menopause Society 3-7 November 1996;A15:4. 64. Shimizu H, Ross RK, Bernstein L, Yatani R, Henderson BE, Nack TM. Cancer of the prostate and breast among Japanese and white immigrants in Los Angeles County. Br J Cancer 1991;63:963–6. 65. Serra-Majem L, La Vecchia C, Ribas-Barba L, et al. Changes in diet and mortality from selected cancers in south Mediterranean countries, 1960–1989. Eur J Clin Nutr 1993;47(Supplement 1): S25–S34. 66. Phipps WR, Martini MC, Lampe JW. Effect of ﬂaxseed ingestion on the menstrual cycle. J Clin Endocrinol Metab 1993;77:1215–9. 67. Adlercreutz H. Western diet and western diseases: some hormonal and biochemical mechanisms and associations. Scand J Clin Lab Invest 1990;50(Suppl 210):3–23. 68. Martin ME, Haourigui M, Pelissero C, Benassayag C, Nunez EA. Interactions between phytoestrogens and human sex steroid binding protein. Life Sci 1996;58(5):429–36. 69. McMichael-Phillips DF, Harding C, Morton MS, et al. Effects of soy-protein on epithelial proliferation in the histologically normal human breast. Am J Clin Nutr 1998;68:1431s–1435s. 70. Lock M. Ambiguities of aging: Japanese experience and perceptions of menopause. Culture, Medicine & Psychiatry 1986;10:23–46. 71. Wilcox G, Walqvist ML, Burger H. Oestrogenic effects of plant foods of post menopausal women. BMJ 1990;301:905–6. 72. Dalais FS, Rice GE, Wahlqvist ML, et al. Effects of dietary phytoestrogens in postmenopausal women. Climacteric 1998;1:124–9. 73. Baird DD, Umbach DM, Lansdell L, et al. Dietary intervention study to assess estrogenicity of dietary soy among postmenopausal women. J Clin Endocrinol Metab 1995;80(5):1685–90. 74. Murkies AL, Lombard C, Strauss BJG. Dietary ﬂour supplementation decreases postmenopausal hot ﬂushes: effect of soy and wheat. Maturitas 1995;21:189–95. 75. Albertazzi P, Pansini F, Bonaccorsi G, Zanotti L, Forini E, De Aloysio D. The effects of dietary soy supplementation on hot ﬂushes. Obstet Gynecol 1998;91:6–11. 76. Knight D, Howes JB, Eden JA. The effect of Promensil, an isoﬂavone extract, on menopausal symptoms. Climacteric 1999;2:79–84. 77. Baber RJ, Templeman C, Morton T, Kelly GE, West L. Randomized placebo-controlled trial of an isoﬂavone supplement and menopausal symptoms in women. Climacteric 1999;2:85–92. 78. Anderson JW, Johnstone BM, Cook-Newell ME. Meta-analysis of the effects of soy protein intake on serum lipids. N Eng J Med 1995;333:276–82. 79. Anthony MS, Clarkson TB, Weddle DL. Effect of soy protein phytoestrogens on cardiovascular risk factors in rhesus monkeys. J Nutr 1995;125(3 Suppl):803–4. 80. Honore EK, Williams JK, Anthony MS. Soy isoﬂavones enhance coronary vascular reactivity in athersclerotic female macaques. Fertility and Sterility 1997;67(1):148–54. 81. Greaves K, Parks J, Williams KD, Wagner JD. Intact dietary soy protein, but not adding an isoﬂavone rich protein to casein, improves plasma lipids in ovariectomized cynomolgus monkeys. J Nutr 1999;129:1585–1592. 82. Bakhit RM, Klein BP, Essex-Sorlie D. Intake of 25g of soybean protein with or without soybean ﬁbre alters plasma lipids in men with elevated cholesterol concentrations. J Nutr 1994;124:213–22. 83. Sirtori CR, Agradi E, Conti F. Soybean-protein diet in the treatment of typeII hyperlipoproteinaemia. Lancet 1977;1:275–7. 84. Knight DC, Howes JB, Eden JA. The effect of Promensil, an isoﬂavone extract, on menopausal symptoms. Climacteric 1999;2:79–84. 85. Teede HJ, Dalais FS, Kotsopoulos D, et al. Phytoestrogen dietary supplementation improves lipid proﬁles and blood pressure: a double blind, randomized, placebo-controlled studying men and postmenopausal women. [Abstract] Climacteric 2000;2(Suppl 1):127. 86. Simons LA, von Konigsmark K, Simons J, Celemajer DS. Phytoestrogens do not inﬂuence lipoprotein levels or endothelial function in healthy, postmenopausal women. Am J Cardiol 2000;85(11):1297– 301. 87. Burger HG, Dudley EC, Groom NP, et al. Prospectively measured changes in FSH, oestradiol, testosterone and the dimeric inhibins in relation to ﬁnal menses. J Clin Endocrinol Metab 1999; 84(11):4025–30.

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INDEX Affect, estrogen and, 128, 132–133 Air Force/Texas Coronary Atherosclerosis Prevention Study (FCAPs/TexCPS), 320 Alendronate, 138, 251, 304–307, 332, 335 BMD and, 324–325 estrogen cf., 323–324 fractures and, 325 in HRT, 302 life expectancy and, 305–307 menopause and, 300 osteoporosis and, 138, 323–324 Alkaline phosphatase (AP), 175, 177 figure αERKO mice, 30, 47, 48, 123 hypothalamus in, 33–34 mammary glands of, 41 ovaries in, 37–38 pituitary in, 36 prostate in, 44 testis and ductules of, 42–43 uterus of, 39–40 Alzheimer’s disease (AD), 132, 133, 134–136, 137–138, 138–139 clinical features, 258–259 endocrine modulation of behavior in, 260 environmental risk factors, 258 ERT and, 258 estrogen and, 259–260, 303 gender differences, 259 steroids and, 257–258 American Society of Clinical Oncology, 284–285 Animal models, 225–226. See also Mice Antiestrogens. See also Selective estrogen receptor modulators (SERMs) nonsteroidal, 148 steroidal, 275 Antithrombin III levels, 230 Apolipoproteins, 135–136, 228 Aromatase knockout (ArKO) mice, 42, 352–353 Arzoxifene, 151 Atherosclerosis, 110–111, 355 Atherosclerotic plaque destabilization, 102–103 Atypical ductal hyperplasia (ADH), 268 Autophosphorylation, 80

Baltimore Longitudinal Study on Aging, 256 Benzopyrene. See EM 652 Benzothiophenes, 172 βERKO mice, 30, 47, 48, 123 hypothalamus in, 33–34 mammary glands of, 41 ovaries in, 37–38 pituitary in, 36 prostate in, 44 testis and ductules of, 42–43 uterus of, 39–40 Biases, in studies, 213–215, 224–225, 318 Bisphosphonates, 331–332, 335 Blood pressure, 208 Body mass index, 208 Bone estrogen and, 46–47, 238–239 phytoestrogens and, 356–357 raloxifene and, 171–172 Bone loss ERT and, 239–240 menopause and, 238 orchiectomy and, 239 prevention of, 239–240 Bone mass oophorectomy and, 238 Bone mineral density (BMD), 47, 151, 251, 300, 323 alendronate and, 324–325 ethnic differences in, 238 HRT and, 302 raloxifene and, 249, 327 risedronate and, 325–326 statins and, 328 tamoxifen and, 246–247, 327 Bone resorption, 238–239 Bone turnover, 251–252 raloxifene and, 247–249 tamoxifen and, 246 Brain aging of, 132, 135, 136, 138, 139 decline in volume, 134 estrogens and, 121, 122, 127–128, 130–131

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366 Brain-derived neutrophic factor (BDNF), 127–128 Breast cancer, 147 alternatives to estrogen and, 335 bisphosphonates and, 331–332 diet and, 357 EM 652 and, 174–175, 176 figure ERα and, 77 ERs and, 13 estrogen and, 148, 267, 268, 315 HRT and, 168, 303, 314–316 isoflavones and, 358–359 mammographic density and, 314–315 mortality, 267 nude mice and, 177–178 oophorectomy and, 314 ovarian factors, 148 ovariectomy and, 177 phytoestrogens and, 351–352, 357–359 progestins and, 314, 315–316 raloxifene and, 151, 172, 273, 285–286, 332 SEER incidence data, 304 SERMs and, 70, 77, 89, 138, 168–169, 269– 270, 276–277 tamoxifen and, 148–150, 158–159, 170–171, 192–193, 270–273, 280–285, 301, 332 toremifene and, 285 Breast Cancer Prevention Trial (BCPT), 271–272, 273, 282–284, 288 P-1, 150, 270–272, 301 Calcitonin, nasal, 326–327, 332, 335 Cancer and Leukemia Group B (CALGB) 8541, 88 Cardiovascular disease (CVD), 207 alternatives to estrogen and, 333 estrogens and, 317–318, 321–322 HRT and, 99–103 phytoestrogens and, 321 prevention, 317–323 raloxifene and, 287–288, 321 risk factors, 226–231 SERMs and, 223–234, 231–234 statins and, 318–320, 322–323 tamoxifen and, 288, 320–321 toremifene and, 288 Cardiovascular system estrogen action on, 45–46 HRT and, 216 table phytoestrogens and, 355–356 Castration, 131. See also Orchiectomy

Index CBP/p300, 78–79 Central nervous system (CNS) ERs and, 123–126 ERT and, 138–139 estrogen and, 127–128 estrogen deprivation and, 132–139 SERMs and, 125 Children, skeletal growth in, 237–238 Chlorotamoxifen, 224 Cholesterol. See also Lipoproteins EM 800 and, 178–179, 182 figure raloxifene and, 151, 227 SERMs and, 225 tamoxifen and, 225, 226, 227 CI-628, 125 Clinical Dementia Rating Scale, 136 Clomiphene endometrium and, 199–200 genitourinary effects, 202 leiomyomas and, 201 ovarian cancer and, 196 ovulation and, 193–194 Clonidine, 330, 333 Cognitive function. See also Alzheimer’s disease (AD); Brain estrogen and, 129–130, 133–134. Colon cancer, 147 Continued Outcomes Relevant to Evista (CORE) study, 286 Corepressors, 64–65, 69, 268–269 Coronary Drug Project, 318 Coronary heart disease (CHD)/Coronary artery disease (CAD), 99, 207. See also Coronary artery disease deaths from, 207 estrogen and, 209–217 gender differences, 207, 210–211 HRT and, 302–303 HRT vs. SERMs, 305 menopause and, 211–212, 304 oophorectomy and, 214 oral contraceptives and, 217 prevention of, 219–220 progestin and, 106 raloxifene and, 151, 300, 304 risk factors, 207–209 tamoxifen and, 301–302 Coumestans, 346, 347, 349 Coumestrol, 352 C-reactive protein, 113, 287 HRT and, 100, 102 Creatinine phosphokinase (CPK) levels, 331

Index Cyclic AMP (cAMP) pathway, 125 Cyclic AMP (cAMP) response element binding protein (CREB), 59, 78, 126 Cytokines, 80

367

Estrogen affect and, 128, 132–133 alendronate cf., 323–324 Alzheimer’s disease and, 134–136, 138–139, 259–260, 303 Daidzein, 346–347, 348, 356 antioxidant properties, 112–113 Deep vein thrombosis, 150, 233. See also Venous benefits of, 331 thromboembolism (VTE) BMD and, 323 Dehydroepiandrosterone (DHEA), 169 bone loss and, 238–239 Dementia. See Alzheimer’s disease brain and, 127–128 Depression, 128, 133, 331, 334 breast cancer and, 148, 267, 268, 315 Diabetes CHD and, 209–217 CHD and, 208 CNS and, 127–128 SERMs and, 231 cognitive function and, 129–130, 133–134 Double knockout mice (DERKO), 37 dementia and, 303 Droloxifene, 171, 175, 274 depressive illness and, 128, 133 Dual X-ray absorptiometry (DEXA) scan, 333 endometrium and, 196–197 Ductules, estrogen action in, 41–43 endothelium and, 110–111 epilepsy and, 128–129 Early Breast Cancer Trialists’ Collaborative Group fractures and, 240–241, 323 (EBCTCG), 197, 232, 234, 270, 282, 302, 315 hippocampus and, 127–128, 133 Early response genes, VSMCs and, 109 inflammatory markers and, 113–114 EM 652, 157–158, 173–174, 175, 177, 274–275 memory and, 130, 133, 134 EM 800, 157–158, 175, 177, 178–179, 179 figure, motor coordination and, 128 182 figure, 274–275 neuroprotective actions, 130–132 Endometrial cancer, 150, 198–199 NO and, 109, 112 alternatives to estrogen and, 335 osteoporotic fractures and, 240–241 ERT and, 168 pain and, 129 estrogen and, 196–197 PMS and, 128 menopause and, 304 postural stability and, 136–137 raloxifene and, 270, 273, 289–290, 332 skeletal growth and, 237–238 tamoxifen and, 270–271, 272, 288–289, 332– stress and, 136 333 stroke and, 217 Endometrium urogenital atrophy and, 328–329 clomiphene and, 199–200 vascular injury and, 105–107 EM 800 and, 175, 177, 179 figure Estrogen receptor alpha (ERα), 3–5, 19–20, 107 monitoring techniques, 198 A/B domain, 5 progesterone and, 197 activation of, 85 raloxifene and, 175, 177, 179 fig., 199, 289–290 AF-1 in, 5, 6 SERMs and, 196–200 AF-2 in, 6, 8 tamoxifen and, 197–198, 270 AF-2/p160, 70–71 Endothelium, estrogen and, 110–111 AP-1 and, 66–67 Epidermal growth factor (EGF), 80, 87, 170 bone and, 46–47 Epilepsy, 128–129 breast cancer and, 77 ERKO mice. See αERKO mice; βERKO mice cardiovascular system and, 45–46 Esophagitis, 332 coregulatory proteins, 78–79 Estradiol, 11, 20, 23, 25, 351 C-terminal, 6 binding to ERβ, 78 ductules and, 41–43 binding to ERs, 123, 124 figure ERβ cf., 20 brain and, 130–131 estradiol binding to, 78 inflammatory markers, 113 function, 78–79 VTE and, 218 gene expression, 83

368 Estrogen receptor alpha (ERα) (cont.) gene transcription, 83 hippocampus and, 44–45 hypothalamus and, 30–34 mammary and, 40–41 N-terminal region, 5–6 ovary and, 36–38 phytoestrogens and, 349–351 pituitary and, 34–36 prostate and, 43–44 testis and, 41–43 uterus and, 38–40 Estrogen receptor beta (ERβ), 3–5, 19–20, 107 A/B domain, 5 AF-1 in, 5 AF-2 in, 9 AP-1 and, 68 bone and, 46–47 cardiovascular system and, 45–46 ductules and, 41–43 ERα cf., 20 hippocampus and, 44–45 history, 3–5 hypothalamus and, 30–34 isoforms, 4–5, 123 mammary and, 40–41 N-terminal region, 5–6 ovary and, 36–38 phytoestrogens and, 349–351 pituitary and, 34–36 prostate and, 43–44 SERMS and, 62–63 testis and, 41–43 uterus and, 38–40 Estrogen receptors (ERs), 122–123 A/B domain, 3, 5–7, 58 activation functions (AFs), 58–66, 169–170, 173–174 AF-1, 58, 61, 62–64, 65–66, 268 AF-2, 58, 59–62, 268 AF-2/p160 contacts, 69–71 AF-independent pathway, 68–69 AF-mediated pathway, 66–68 amino-acid sequences, 19 amino-terminal domains, 5, 19, 62 AP-1 and, 20, 68 breast cancer and, 13, 268–269 carboxy-terminal domain, 3–4 C domain, 3–4 cell and tissue distribution, 20 CNS and, 123–126 coactivators, 5–6, 59–61, 67–68, 71, 268 corepressors, 64–65, 69, 71, 268–269

Index Estrogen receptors (ERs) (cont.) crosstalk with signaling pathways, 126 crystal structures, 22–23 C-terminal region, 268 D domain, 4 figure DNA-binding domain (DBD), 3, 7–8, 19, 58, 108, 268 domains, 58 E-domains, 8 E/F domain, 3–4 F-domains, 8, 10 function, 78–79, 268–269 gene expression by, 58 gene transcription regulation, 10–14 isoforms, 78 knockout mice. See ERKO mice ligand-binding domain (LBD), 3–4, 8–10, 19, 58, 108 ligand-induced conformations, 19–26 membrane, 125–126 modular architecture, 3–5 N-terminal domain (NTD), 3, 5–7, 19, 58, 62 transcriptional activity, 5–7 Estrogen Replacement and Atherosclerosis (ERA) trial, 100, 210, 322 Estrogen-replacement therapy (ERT) Alzheimer’s disease and, 258 bone loss and, 239–240 breast cancer and, 168 CNS and, 138–139 cognitive effects, 255–256 endometrial cancer and, 168 Estrogen-response elements (EREs), 7, 20, 58, 66, 78, 107, 268 Estrogens brain and, 122 CVD and, 317–318, 321–322 hypothalamus and, 122 mechanism of action, 58 statins cf., 318–320 Estrone, 177 Evista. See Raloxifene Extracellular-signal regulated protein kinases (ERKs). See Mitogen-activated protein kinase (MAPk) Falls, injurious. See Postural stability Fibrinogen, 231, 288 Fibroids. See Leiomyomas Fluoride, 251 Follicle-stimulating hormone (FSH), 192–194 Fosamax. See Alendronate Fos/jun complex. See Jun/fos complex

Index Fracture Intervention Trial (FIT), 137, 300, 325 Fractures, 251 alendronate and, 325 estrogen and, 240–241, 323 hip, 132, 137 HRT and, 302 life expectancy and, 305–307 raloxifene and, 241, 250–251, 273, 287 risedronate and, 325–326 SERMs and, 328 tamoxifen and, 247 Framingham Heart Study, 99, 231 Framingham Offspring Study, 208 Gail model, 282 table Gender differences in Alzheimer’s disease, 259 in BMD, 47, 238 in brain injury, 131 in CHD, 207, 210–211 in HDL cholesterol, 210–211 in reendothelialization, 111 Genistein, 22, 25, 346–347, 348, 350, 351, 352, 356, 358 Glucocorticoid receptor-interacting protein (GRIP-1), 5–6, 59, 61, 66–67 Gonadotropin-releasing hormone agonists (GnRH-a), 256 GW 5638, 66, 171, 178, 274 HDL. See High-density lipoproteins (HDLs) Heart and Estrogen/Progestin Replacement Study (HERS), 100, 210, 212, 216–217, 218, 223–224, 303, 321–322 Helix 12, 8–9, 22, 350 HER2, tamoxifen and, 87–89 Herbal therapy, 331 High-density lipoproteins (HDLs), 209, 287, 317–318 androgen effect, 210–211 phytoestrogens and, 355–356 SERMs and, 227 Hip fractures, 132, 137 Hip Intervention Program, 326 Hippocampus, estrogen and, 44–45, 127–128, 133 Histone deacetylases (HDACs), 64 Homocysteine, 229, 231 Hormone-replacement therapy (HRT), 151, 169, 255 biases in studying, 213–215 BMD and, 302 breast cancer and, 303, 314–316 cardiovascular disease and, 99–103, 223

369 Hormone-replacement therapy (HRT) (cont.) cardiovascular system and, 216 table CHD and, 209–210, 212–213, 302–303 compliance bias, 213 in European trials, 273 fractures and, 302 life expectancy and, 305 menopause and, 302–304 phytoestrogens cf., 346 surveillance bias, 213–214 users vs. nonusers, 213–215 VTE and, 217–219 Hot flashes, 133, 134, 291, 301, 330–331, 333, 354–355 4-Hydroxy-Tamoxifen (4-OH-Tam), 20, 21, 23, 24 table, 25. See also Tamoxifen Hypothalamic pituitary axis (HPA) regulation, 136 Hypothalamus, estrogens and, 30–34, 122 ICI-164,384, 20 ICI-182,180, 20, 24 table ICI-182,780, 14, 66, 157, 175, 275 Idoxifene, 171, 175, 178, 274 Insulinlike growth factor (IGF), 80 Insulin-receptor substrate (IRS) proteins, 80 Interleukins, 80 Isoflavones, 346, 348, 349, 352, 358–359 Jun-fos complex, 13–14, 67–68 Jun-N-terminal kinase (JNK), 81–82 Karyopyknotic index, 201–202 Keoxifene, 150–151 Lasofoxifene, 168 Leiden V mutation, 217 Leiomyomas, 200–201 Lignans, 346, 347, 348, 349 LIM/Homeodomain protein Islet-1, 13 Lipid levels Australian study, 318–319 North American study, 319–320 Liver tumors, 150 Low-density lipoproteins (LDLs), 208–209, 226, 229, 231, 287, 288, 317–318, 355–356. See also High-density lipoproteins (HDLs); Very low density lipoproteins (VLDLs) Lung cancer, 147 Luteinizing hormone (LH), 192–194 LXXLL motifs, 8, 11, 23, 25, 60–61 LY 117018, 150 LY 156358, 150–151 LY 353381, 168, 172–173, 175, 177 figure, 275 LY 357489, 275

370 Mammary gland, estrogen action in, 40–41 Mammography, 306 Markov model, 304 Maturation index, 201, 202 MCF-7 cells, 148, 150, 152–153, 158, 159–160, 174–175, 176 figure Megestrol acetate, 330, 333 MEK kinase (MEKK), 81–82 Memory, estrogen and, 130, 134 Menopause alendronate and, 300 bone loss and, 238 CAD/CHD and, 99, 211–212, 304 endometrial cancer and, 304 individualized treatment for, 304 life expectancy and, 122, 130 phytoestrogens and, 353, 354–355 raloxifene and, 300–301 SERMs and, 169, 300–302 symptoms of, 299–300 tamoxifen and, 301–302 Mice knockout. See ERKO mice nude. See Nude mice Mitogen-activated protein kinase (MAPk), 81–82 pathway, 125, 126 phosphorylation, 5, 11, 58, 63 Motor coordination estrogen and, 128 Multiple Outcomes of Raloxifene Evaluation (MORE) trial, 137, 172, 248–249, 250, 251–252, 273, 285–286, 287, 290, 327 Mycoestrogens, 346, 348 Naples/Gruppo Universitario Napoletano 1 (GUN1) trial, 88 National Surgical Adjuvant Breast and Bowel Project (NSABP), 159, 197–198, 282– 284, 288, 320. See also Breast Cancer Prevention Trial (BCPT); Study of Tamoxifen and Raloxifene (STAR) B-14, 171 P-1. See under Breast Cancer Prevention Trial P-2. See Study of Tamoxifen and Raloxifene (STAR) Neuroendocrine system, SERMs and, 192–194 Nitric oxide (NO) estrogen and, 109, 112 N-methyl-D-aspartate (NMDA) receptors, 127 North Central Clinical Oncology Group, 333 Nuclear receptor corepressor (N-CoR), 64–65 Nude mice, breast cancer and, 177–178 Nurses’ Health Study, 99, 208, 210, 211, 212, 213, 214, 217, 302, 303–304, 315, 316, 318

Index Oophorectomy. See Ovariectomy Oral contraceptives, 217 Orchiectomy. See also Castration bone loss and, 239 Osteopenia, 333, 334 Osteoporosis, 245 alendronate and, 300, 323–324 alternatives to estrogen and, 333, 334–335 antiresorptive agents, 323–325 definition, 251 estrogens and, 137 life expectancy and, 306–307 nasal calcitonin for, 326–327 phytoestrogens and, 356–357 raloxifene and, 138, 245, 273, 327 SERMs and, 327–328 Ovarian cancer clomiphene and, 196 tamoxifen and, 195 Ovariectomy, 192 bone mass and, 238 brain injury and, 131 breast cancer and, 177, 314 CHD and, 214 Ovaries estrogen action in, 36–38 tamoxifen and, 194–195 Ovulation clomiphene and, 193–194 tamoxifen and, 192 Oxford Overview Analysis, 150 Pain, estrogen and, 129 Parkinson’s disease (PD), 128, 260–261 Peptide phage display, 23–26 Physicians’ Health Study, 113 Phytoestrogens, 330, 345 anticarcinogenic actions, 351 benefits, 353 bone and, 356–357 breast and, 351–352 breast cancer and, 357–359 cardiovascular system and, 355–356 classification, 346–347 CVD and, 321 diet and, 346 epidemiological studies, 346, 353 estrogenic effects, 349–351 food sources, 348–349 HDL cholesterol and, 355–356 helix 12 and, 350 LDL cholesterol and, 355–356 measurement of, 348 menopause and, 353, 354–355

Index Phytoestrogens (cont.) metabolism, 347–348 osteoporosis and, 356–357 plant origins, 346–348 in premenopausal women, 353–354 SERMs cf., 345, 350 uterus and, 352 vasomotor symptoms and, 354 VLDL cholesterol and, 355 Pituitary, estrogen action in, 34–36 Postmenopausal Estrogen/Progestin Interventions (PEPI) trial, 100, 102, 113, 240, 314–315, 317–318, 323 Postural stability, 132, 136–137 Pravastatin, 319–320 Premarin, 322 Premenstrual syndrome (PMS), estrogen and, 128 Prevent Recurrence of Osteoporotic Fractures (PROOF), 326 Progesterone, endometrium and, 197 Progestin receptor (PR), 83–87 Progestins, 302 breast cancer and, 314, 315–316 CAD and, 106 Propyl pyrazole triol (PPT), 21 Prostate, estrogen action in, 43–44 Protein/tyrosine-binding (PTB) domains, 80 p160s, 59–61, 67, 69–71 Pulmonary emboli, 150, 207, 233. See also Venous thromboembolism (VTE)

371 Raloxifene (cont.) ocular changes with, 291 osteoporosis and, 138, 241, 273, 327 side effects, 291 skeletal effects, 286–287 tamoxifen cf., 153–157 uterus and, 172–173 vaginal epithelium and, 290 VTE and, 273, 300 X-ray crystallography of, 153 Raloxifene Use for the Heart (RUTH) trial, 234, 286, 288 Rancho Bernardo studies, 256 Ras GTPase family, 81 Rectum cancer, 147 Resorcylic acid lactones, 346, 348 Retinoic acid receptors (RARs), 13, 64 Retinoid X receptors (RXRs), 64 Rhabdomyolysis, 331 Risedronate, 325–326, 335 Royal Marsden Hospital trial, 284, 302, 320 R,R-THC, 21

Saponins, 355 SCH 57068. See EM 652 Scottish Cancer Trials Breast Group, 288 Selective estrogen receptor modulators (SERMs), 20, 148, 151, 167–168 AF-1 and, 62–64, 65–66 AF-2 and, 61–62 alternate EREs and, 66 Quinone reductase gene, 13 animal models, 225–226 Raf kinase family, 81–82 AP-1 and, 14, 68–69 Raloxifene, 20, 22, 24 table, 137, 148, 168, 224, apolipoproteins and, 228 306–307 biases in data, 224–225 analogs, 150–151, 172, 175 breast cancer and, 70, 77, 89, 138, 168–169, bone and, 171–172 269–270, 276–277 bone density and, 151 cardiovascular disease and, 223–234, 231–234 bone turnover and, 247–249 cholesterol and, 225 brain function and, 137–138 clinical trials, 281 table breast cancer and, 151, 172, 273, 285–286, 332 CNS and, 125 CHD/CVD and, 151, 287–288, 300, 304, 321 coregulatory proteins and, 84–85 cholesterol and, 151, 227 corepressors and, 64–65, 71 EM 652 and, 157–158 diabetes and, 231 EM 800 and, 158 figure endometrium and, 196–200 endometrial cancer and, 273, 332 ERβ and, 62–63 endometrium and, 199, 270, 289–290 estrogen repression and, 69 fractures and, 273, 287 first-generation, 170–171 genitourinary effects, 202 fourth-generation, 173–179 leg cramps, 291 fractures and, 328 leiomyomas and, 201 generations of, 168 life expectancy and, 304–307 genitourinary effects, 201–202 menopause and, 300–301 growth-factor receptor signaling and, 82–89 neuroendocrine system and, 194 HDL and, 227

372

Index

Selective estrogen receptor modulators (cont.) Tamoxifen, 20, 77, 147, 168, 170–171, 224. hemostatic effects, 229–230, 231 See also 4-Hydroxy-Tamoxifen (4-OH-Tam) ideal, 26 adjuvant, 148–149, 159–160 leiomyomas and, 200–201 analogs, 171 lesion effects, 226 antithrombin III levels, 230 lipoproteins and, 229, 231 BMD and, 246–247, 327 menopause and, 169, 300–302 bone density and, 151 neuroendocrine system and, 192–194 bone turnover and, 246 osteoporosis and, 327–328 brain function and, 137–138 oxidation of LDL and, 226 breast cancer and, 148–150, 158–159, pharmacologic properties, 279, 281 table 170–171, 192–193, 270–273, 280–285, phytoestrogens cf., 345, 350 301, 332 second- and third-generation, 171–173 cardiovascular events and, 232–234 selective action of, 269 CHD and, 301–302 triglyceride and, 231 cholesterol and, 225, 226, 227 Selective Serotonin Reuptake Inhibitor (SSRI), CVD and, 288, 320–321 133, 330, 331, 333, 334 deep vein thrombosis and, 150 Serotonin, 128, 133 drug resistance to, 152 Silencing mediator for retinoid and thyroid receptor endocrine treatments and, 171 (SMRT), 64–65 endometrial cancer and, 150, 270–271, 272, Simvastatin, 318–319 288–289, 332–333 Skeleton endometrium and, 171, 197–198, 270 estrogen and, 237–241 ERα AF-1 activity and, 70 raloxifene and, 286–287 ER-positive tumors and, 272 tamoxifen and, 287 estradiol and, 192 toremifene and, 287 European trials, 272–273, 284 Soybeans, 348–349 fractures and, 272 Soy foods, 330–331, 353, 355–356. See also FSH and, 192 Isoflavones genitourinary atrophy and, 201–202 Spatial problem solving, 130 gynecologic effects, 288 S,S-THC, 21 hepatic tumors and, 150 St. John’s Wort, 331 HER2 and, 87–89 Statins homocysteine and, 229 CVD and, 318–320, 322–323 ischemic heart disease, 272 estrogens cf., 318–320 Italian trial, 284 risks of, 331 karyopyknotic index and, 201–202 Steroidal antiestrogens, 275 leiomyomas and, 200–201 Steroid receptor coactivator 1 (SRC-1), 6, 59, 173–174 length of treatment, 171 Steroids, Alzheimer’s disease and, 257–258 LSH and, 192 Stockholm Breast Cancer Study Group, 197, 288 menopause and, 301–302 Stress, estrogen and, 136 mixed estrogenic and antiestrogenic activity, Stress-activated protein kinase (SAPK), 81 170–171 Stroke, 150, 233 multiple effects, 276–277 deaths from, 207 ocular changes with, 291 estrogen and, 217 ovarian ablation and, 192–193 oral contraceptives and, 217 ovarian cancer and, 195 risk factors for, 209 ovary and, 194–195 Study of Osteoporotic Fractures (SOF), 134, 323 ovulation and, 192 Study of Tamoxifen and Raloxifene (STAR), 138, pulmonary emboli and, 150 151, 275–276, 286 raloxifene cf., 153–157 Subendothelial cystic atrophy, 289 side effects, 290 Surveillance, Epidemiology, and End Results skeletal effects, 287 (SEER), 304 stroke and, 150 Synaptogenesis, 131 thrombocytopenia and, 229

Index Tamoxifen (cont.), triglyceride levels and, 227–228 tumor growth and, 152–153 vaginal mucosa and, 289 vascular events and, 272 X-ray crystallography of, 153 TAT-59, 171, 274 T47D cells, 158–159 Testis, estrogen action in, 41–43 Thrombocytopenia, 229. See also Venous thromboembolism (VTE) Thrombophilia, 217 Thyroid receptor associated proteins (TRAPs), 60–61 Thyroid receptors (TRs), 60, 62, 64–65 Topoisomerase II, 352 Toremifene, 171, 175, 178, 224, 274 breast cancer and, 285 cholesterol and, 226–227 CVD and, 288 gynecologic effects, 289 ocular changes with, 291 side effects, 290 skeletal effects, 287 Transforming growth factor-β (TGFβ), 80 Transient ischemic attacks (TIAs), 233 Triglycerides SERMs and, 231 tamoxifen and, 227–228 Triphenylethylenes, 172, 274 Truncated estrogen receptor proteins (TERP1 and 2), 36

373 Urogenital system, 201–202, 328–329, 333–334 Uterus estrogen action in, 38–40 phytoestrogens and, 352 raloxifene and, 172–173 tumors. See Leiomyomas Vaginal epithelium, raloxifene and, 290 Vaginal mucosa, tamoxifen and, 289 Vascular endothelial growth factor (VEGF), 111 Vascular injury animal models of, 103–107 estrogen and, 105–107 Vascular smooth muscle cells (VSMCs), 103–105, 108, 109 Vasomotor instability, 329–331, 333, 354 Vasoprotection, estrogen-mediated, 107–113 Venous thromboembolism (VTE), 207, 209, 291 See also Deep vein thrombosis; Pulmonary embolism estradiol and, 218 HRT and, 217–219 oral contraceptives and, 217 raloxifene and, 300 Venous thrombotic disease, raloxifene and, 273 Very low density lipoproteins (VLDLs) phytoestrogens and, 355 Vitamin E, 330 Women’s Health Initiative (WHI), 103, 113, 303, 316, 318

CONTEMPORARY ENDOCRINOLOGY ™ P. Michael Conn, Series Editor

Selective Estrogen Receptor Modulators Research and Clinical Applications Edited by

Andrea Manni,

MD and

Michael F. Verderame,

Ph D

Division of Endocrinology, Diabetes, and Metabolism Pennsylvania State University College of Medicine Milton S. Hershey Medical Center, Hershey, PA

Hormone replacement therapy with selective estrogen receptor modulators (SERMs) promises the highly desirable benefits of estrogen and progesterone replacement therapy, while avoiding their associated risks of breast and uterine cancer, gallbladder disease, and weight gain. In Selective Estrogen Receptor Modulators: Research and Clinical Applications, leading experimental and clinical researchers from a wide range of disciplines present a wealth of fresh scientific information on the biochemistry, molecular biology, pharmacology, and clinical activity of SERMs. The basic science chapters of the book focus—with an eye to the development of the ideal SERM—on the complex mechanisms of estrogen action, including ligand-dependent conformational changes in alpha and beta, and the recruitment of coactivators and corepressors which modulate the estrogen receptor transcriptional activity and contribute to its crosstalk with growth factor signaling. The clinical presentations review the data accumulated on currently available SERMs, primarily tamoxifen and raloxifene, in cancer treatment and prevention, as well as their effects on the reproductive, vascular, skeletal, and central nervous systems. A novel approach to menopause-related health issues is also provided for women with and without a previous diagnosis of localized breast cancer. Multidisciplinary and state-of-the-art, Selective Estrogen Receptor Modulators: Research and Clinical Applications provides for experimentalists and clinicians alike a comprehensive overview of the basic mechanisms of estrogen and SERMs action, as well as evidence-based guidelines for their clinical use in postmenopausal women. FEATURES

䊏 Integrates a large body of diverse and 䊏 Addresses the questions most frequently frequently conflicting scientific and clinical data asked by women at the time of menopause 䊏 Provides evidence-based guidelines on how 䊏 Reviews the effects of estrogen and SERMs to individualize HRT on brain function CONTENTS Basic Studies. I Molecular Mechanisms of Estrogen Receptor Function. Structure and Function of the Estrogen Receptor. Ligand-Induced Conformational Changes in Estrogen Receptors-α and -β. Expression and Function of Estrogen Receptors-α and -β. SERM Modulation of Gene Expression: Role of Coactivators and Corepressors. Crosstalk Between Estrogen Receptors and Growth Factor Signaling. II TissueSpecific Effects of Estrogens and SERMs. Direct Estrogen Effects on the Cardiovascular System. Estrogens and the Brain: Implications for the Treatment of Postmenopausal Women. III Preclinical Studies. Insights into the Molecular Mechanism of SERMs Through New Laboratory Models. Third- and FourthGeneration SERMs. Clinical Studies. IV Organ Specific Effects of Estrogens and SERMs. SERMs’ Effect on the Neuroendocrine System and the Reproductive Organs. Epidemiology

of Cardiovascular Disease in Women: Role of Estrogens. SERMs Effects on Cardiovascular Risk Factors and Disease. Estrogen and the Skeleton. Effects of SERMs on Bone in Clinical Studies. Estrogens and SERMs: Clinical Aspects of Cognition with Aging and Neurodegenerative Disorders. V SERMs and Endocrine Dependent Tumors. SERMs and Breast Cancer Prevention. SERMs in Postmenopausal Women’s Health. VI Roles of Estrogens and SERMs in Postmenopausal Hormone Replacement Therapy. Menopause Therapy: An Individualized Approach. Alternatives to Estrogen for Treatment of Menopause. Phytoestrogens in the Context of SERMs. Index.

90000 Contemporary Endocrinology™ SELECTIVE ESTROGEN RECEPTOR MODULATORS RESEARCH AND CLINICAL APPLICATIONS ISBN: 0-89603-912-9 humanapress.com

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