Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy (Elsevier Insights)

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

G.V. Sherbet

School of Electrical, Electronic and Computer Engineering, University of Newcastle upon Tyne, UK The Institute for Molecular Medicine, Huntington Beach, California, USA

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO

Elsevier 32 Jamestown Road London NW1 7BY 225 Wyman Street, Waltham, MA 02451, USA First edition 2011 Copyright © 2011 Elsevier Inc. All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangement with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-387819-9 For information on all Elsevier publications visit our website at elsevierdirect.com This book has been manufactured using Print On Demand technology. Each copy is produced to order and is limited to black ink. The online version of this book will show color figures where appropriate.

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To Shri Sai Baba

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Preface

If the objective is worthy and significant Anticipate and overcome the hindrances Proceed with determination And reach the goal Thiru Valluvar (Tamil poet, second century, India)

Thirukkural (Chapter 68, verse 676) Growth factors have a long history, encompassing over six decades since their discovery. In that time, research has identified and characterized a multiplicity of other mediators of signalling in cell growth, differentiation, apoptosis and survival in normal and aberrant cell biology. There has been an abiding interest in the mode of function of these biological phenomena, which has resulted in an extensive and deeper understanding of their mechanisms. Many modes of function such as cell-cycle regulation, modulation of cell motility by remodelling of the extracellular matrix and induction of invasion by the initiation of epithelial–mesenchymal transition, induction of angiogenesis and vascular permeability have inevitably led to intense studies into their role in tumour growth and progression, where there are perceivable parallels between growthfactor-related molecular events and the clinical stage of cancer progression. These have not only pushed forward the boundaries of our knowledge but also led to the clinical deployment of growth factors, their receptors and downstream signalling elements in a significant approach to cancer management. Growth factors are a topic dear to my heart, which has formed part of my continuing study of cancer metastasis. I have all along been conscious of the complexities of the subject and the enormity of undertaking an overview. These I regarded as a challenge rather than an impediment. As the Thirukkural above has stated, I considered the effort of providing a coherent continuum of the function of growth factors as worthwhile and important, not only for those engaged in their research and peripheral disciplines but also for undergraduate and graduate students as a broad dissertation of the subject. I hope I have succeeded in this endeavour; time will tell.

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Preface

I received much support and help from Dr. M.S. Lakshmi, who read the manuscript and made suggestions. She also supplied the Thirukkural and translation quoted above. I thank her for her time and effort. My thanks also go to Professor Bayan Sharif and Professor Satnam Dlay, who provided me with an excellent environment for study and research. G.V. Sherbet

Abbreviations

ADAM A disintegrin and metalloproteins domain-containing protein ALL Acute lymphocytic leukaemia AMH Anti-Müllerian hormone (Müllerian inhibiting substance) APC Adenomatous polyposis coli APL Acute promyelocytic leukaemia AR Androgen receptor ARE Androgen response elements AREG Amphiregulin BMP Bone morphogenetic protein BTC β-Cellulin CAMKII Calmodulin kinase CCN1/Cyr61 CCN family of genes (the cysteine-rich 61/connective tissue growth factor) cdk Cyclin-dependent kinase CFC Cripto/FRL1/Cryptic CNS Central nervous system CRABP Cellular RA-binding proteins CTGF Connective tissue growth factor DAG Diacylglycerol Dhh Desert hedgehog DOS Daughter of Sevenless EBV Epstein-Barr virus ECM Extracellular matrix EDG Endothelial differentiation gene EGF Epidermal growth factor EMT Epithelial mesenchymal transition ER Oestrogen receptor EREG Epiregulin ERK Extracellular signal-related protein kinase ES Embryonic stem cells ESCC Oesophageal squamous cell carcinomas FAK Focal adhesion kinase FGF Fibroblast growth factor ERE Oestrogen response element FGF-BP FGF-binding proteins FGFR FGF receptor

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FLRT Fox FSH GDF GH GM-CSF GPCR GRE GSK HB-EGF HCG HER2 HESC HGF Hh HIF-1 HLH HMG HPV HRG IFITMS IFN IGF IGFBP IGFR Ihh IP IPT IR IRS JAK JNK LEF LH LIF LIM LTF MAD MAPK MEK MEKK mER MH MCP MMP

Abbreviations

Fibronectin-leucine-rich transmembrane protein Forkhead box transcription factors Follicle stimulating hormone Growth and differentiation protein Growth hormone (Somatotropin) Granulocyte–macrophage colony-stimulating factor G-protein coupled receptors Glucocorticoid receptor elements Glycogen synthase kinase Heparin binding epidermal growth factor Human chorionic gonadotropin Human epidermal growth factor receptor (erbB2) Human embryonic stem cell Hepatocyte growth factor Hedgehog proteins Hypoxia-inducible factor 1 transcription activator Helix-loop-helix transcription factors High Mobility Group transcription factors encoded by Sox genes Human papilloma virus Heregulin IFN-induced transmembrane protein Interferon Insulin-like growth factor IGF binding proteins IGF receptor Indian hedgehog Interferon induced protein Ig domain of HGF/MET receptor Insulin receptor Insulin receptor substrate proteins Janus kinase Jun N-terminal kinase Lymphoid enhancer factor Luteinising hormone Leukaemia inhibitory factor Homeodomain proteins with Cys-His motifs Lactoferrin MAX dimerisation protein Mitogen-activated protein kinase A threonine and tyrosine kinase that phosphorylates MAPK MAPK kinase kinase Membrane located oestrogen receptor MAD homology domain Monocyte chemoattractant protein Matrix metalloproteinase

Abbreviations

MPA mTOR NCAM NGF NO NOS NSCLC OPG PACE PDF PDGF PDGFR PI3K PIN PIP2 Pitx PKA PKC PLGF PPAR PR PSA PSI PTCH PTHRP RANK RANKL RAR Rb RCC RTK S1P Shh siRNA SMO SRC STAT Tbx TFF1 TGF TIMP TNBC TNF TNFR TRADD

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Medroxyprogesterone acetate, a synthetic progestin Mammalian target of rapamycin Neural cell adhesion molecule Nerve growth factor Nitric oxide Nitric oxide synthase Non-small cell lung carcinoma Osteoprotegerin Paired basic amino acid cleaving enzyme Prostate derived factor Platelet-derived growth factor Platelet derived growth factor receptor Phosphoinositide-3 kinase Prostatic intraepithelial neoplasia Phosphatidylinositol 4,5-bisphosphate Paired-like homeobox transcription factor Protein kinase A Protein kinase C Placental growth factor Peroxisome proliferator-activated receptor Progesterone receptor Prostate-specific antigen Plexin semophorin integrin domain of MET receptor Patched glycoprotein binding hedgehog proteins Parathyroid hormone-related protein Receptor/activator of NF-κB transcription factor The receptor/activator of NF-κB ligand Retinoic acid receptors Retinoblastoma protein Renal cell carcinomas Receptor tyrosine kinase Sphingosine 1-phosphate, a GPCR ligand Sonic hedgehog Small interference RNA Smoothened, a component of Hh signalling Steroid receptor co-activator Signal transducers and activators of transcription T-box gene family of transcription factors Trefoil protein Transforming growth factor Tissue inhibitor of metalloproteinase Triple-negative breast cancer Tumour necrosis factor TNF receptor TNFR associated death domain

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TRAIL TRAF TSH TSP TWEAK TWEAKR uPA uPAR VD3 VDR VDRE VEGF Vg-1 VHL VWC WISPs

Abbreviations

TNF associated apoptosis inducing ligand TNF associated factor Thyroid stimulating hormone Thrombospondin TNF family type II transmembrane ligand TWEAK receptor Urokinase-type Plasminogen Activator uPA receptor 1,25-dihydroxyvitamin D-3 cholecalciferol Vitamin D3 receptor Vitamin D3 response element Vascular endothelial growth factor TGF-β homologue from Xenopus laevis Von Hippel-Lindau Von Willebrand type C repeat module Wnt inducible signalling pathway protein

Introduction

Recent years have seen a considerable emphasis on the study of growth factors and their mode of function. The biological and phenotypic effects of growth factors were recognised decades ago. The elucidation of their mode of function and the recent recognition that growth factors as well as the downstream elements associated with their function might be potential targets in the therapeutic management of several human diseases has added great impetus to the study of growth factors and their attributes. At the fundamental level is the involvement of growth factors and their receptors in cell differentiation and morphogenesis, which intrinsically links the processes of differentiation with neoplasia. With differentiation seen as being antagonistic to neoplastic transformation, and to tumour initiation, development and invasion and metastasis, the wide spectrum of growth factor function can be visualised as a composite of factors that are positive or negative regulators of differentiation, cell proliferation and cancer. Consistent with this is the regulation of the modes of signal transduction that is seen in response to the stimuli received by the cell from the extracellular environment. The initiation of tumorigenesis, and the development and progression of the tumour, can be identified as distinct phases (Figure 1). It is generally recognised that deregulation of genetic programmes lies at the root of neoplastic transformation and determines the aberrant behaviour of cancer cells from initiation to metastatic deposition at distant sites. The metastatic cascade can be divided into distinct compartments. Also, the genetic profile that determines the phenotype identifiable with the given compartment and the mechanisms of acquisition of biological features that characterise the specific compartment are increasingly being unravelled (Sherbert, 2006). The association of events of metastasis with deregulated activation of genetic programme and growthfactor-driven signalling systems perceivably correlates in a temporal dimension with progression. Besides, the overlapping outcomes of some genetic alterations and unfettered activation of signalling pathways indicate the operation of a degree of causal interaction between aberrant genetic activity and inappropriate growth factor signalling. To summarise, growth factors do appear to be able to influence markedly every phase of tumorigenesis; they can not only positively or negatively influence cell proliferation but also induce cell motility and aid in cancer dissemination and deposition, and growth of metastatic lesions. This emphasises their relevance in normal and abnormal differentiation and neoplastic phenomena. The focus and format adopted here is on these biological events and the participation of growth factors in them (Table 1). Finally, it was inevitable that the progress made in recent years, the considerable emphasis placed on growth factors and the elucidation of their mode of function would

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Introduction

Neoplastic transformation Genetic damage, environmental and occupational factors, inherited cancer susceptibility, deregulation of differentiation, aberrancy of cellular phenotype Cellular hyperplasia Deregulation of cell proliferation and apoptosis, Growth factor signalling and function, and tumour growth Benign disease

In situ neoplasm

Local invasion Acquisition of invasive behaviour; alterations in cell membrane properties, extracellular matrix remodelling, alterations in intercellular adhesion, modulation of cytoskeletal dynamics, and cell locomotion Angiogenesis; invasion of the vascular/lymphatic system (Programming of the synthesis and function of angiogenic factors, invasion of vascular and lymphatic compartment, evasion of immunological surveillance) Extravasation at metastatic sites Transport of residual cells and dissemination to distant locations in the host organisms, extravasation into the parenchyma of metastatic site Metastatic tumour Establishment and expansion of metastatic tumour

Figure 1  The cascade of events in the development of metastasis and the associated genetic programme and phenotypic properties. There is no rigorous comparmentation in the temporal frame of the function and expression of the genetic programme leading to the appearance and expression of phenotypic behaviour related to the compartments that are grossly identifiable (from Sherbert, 2006).

be viewed in the clinical setting. Indeed, this has led to the recognition that growth factors, their receptors as well as downstream elements associated with their function might be potential targets in therapeutic management of human diseases. Humanised monoclonal antibodies raised against growth-factor receptors have proved to be valuable for targeted cancer treatment and in the management of patients.

Introduction

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Table 1  Genetic Changes and Growth Factor/Receptor Expression Influencing Events of Metastasis Biological Event

Genes and Growth Factors

Deregulation of differentiation, morphogenesis and pattern formation

Bone Morphogenetic Protein, Fibroblast Growth Factor, Vascular Endothelial Growth Factor and Transforming Growth Factor-β; hedgehog family proteins; microRNAs

Tumour growth

Cell-cycle regulation p53, Retinoblastoma protein (Rb), cyclins, Cyclin-dependent kinase (cdk) inhibitors BRCA1, BRCA2, Adenomatous polyposis coli (APC); suppressor genes; control of cell population expansion; Phosphoinositide-3 kinase (PI3K)/Akt/PTEN, promotion of apoptosis; anti-apoptosis genes; Transforming growth factor (TGF), insulin-like growth factor (IGF); microRNAs

Invasive ability

Extracellular matrix (ECM) remodelling associated proteolytic enzyme system/ remodelling, modulation of cell adhesion, cell shape, motility, cell membrane malleability, modulation of cytoskeletal dynamics; cadherin/ catenin complex cytoskeletal linkage; CD44/ cytoskeletal linkage; microRNAs

Tumour vascularisation, microvessel formation

Activation of angiogenic factors, endothelial cell proliferation Vascular endothelial growth factor (VEGF), Hypoxia-inducible factor-1 (HIF-1), Hepatocyte growth factor (HGF); Matrix metalloproteinase (MMP), Tissue inhibitor of metalloproteinase (TIMP)

Metastasis suppressors

nm23 and KISS, and promoters S100A4, MTA etc.

1 Convergence of Growth-Factor

Signalling Pathways in Developmental Systems and Pathogenesis

The dynamics of differentiation and growth and the initiation and progression of disease processes involves a harmonised mobilisation and activity of a wide variety of mediators and biological response modifiers; prominent among them are growth factors, steroid hormones and cytokines. They influence and markedly impinge upon the cellular processes, regulate the cell division cycle and integrate it with apoptosis and cell survival. They can modulate mechanical properties of cells namely cell membrane malleability, alter cytoskeletal dynamics and promote cell motility. These effects are highly relevant in cancer development and spread. Also relevant are factors involved in the induction of neovascularisation, which allows cancer cells to invade, spread and metastasise. Essentially, they impart specific signals to cells in a paracrine or autocrine fashion, which determine cell fate in terms of differentiation, patterning in development and cell motility both in normal and in pathogenic microenvironments. Although the phenotypic outcome can vary, one can see much overlap in their function and considerable cross-communication and interaction of signalling systems that channel the flow of information imparted to the cell. Most of the critical determinants can be grouped into definable families on the basis of criteria of their structural makeup. More often than not, members of a family of growth factors also share signal transduction systems yet they can generate distinct phenotypic features. It follows that successful analysis of the fundamentals of their function requires an examination of the individual determinants as well as the intricacies of signalling together with interactive modes of generation of phenotypes. This is the basic premise of looking at growth factor families, steroid hormones and cytokines, and any commonality or merger of signalling cascades in their function as signalling molecules.

Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00001-3 © 2011 Elsevier Inc. All rights reserved.

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2 Growth Factor Families

Many growth factors have been identified. There is no clearly established convention for their classification. Here, certain basic features are adopted for classification such as the structure and molecular organisation of the growth factors, the nature of receptors that they bind and the signalling cascades they activate. One can distinguish two families of ligands by these criteria: (1) membrane-receptor binding family; (2) intracellular-receptor binding family (Table 2.1).

The Membrane-Receptor Binding Family of Growth Factors Of the two major families, the membrane-receptor binding family has been subdivided here into the cystine-knot group of growth factors, and the EGF- and FGFfamily growth factors (Figure 2.1).

Cystine-Knot Group of Growth Factors Cystine-knot proteins are small proteins composed of approximately 30 amino acid residues, with a characteristic tertiary fold. In this, three intramolecular disulphide bonds are formed wherein cysteine 1 in the sequence is connected to cysteine 4, cysteine 2 to cysteine 5, and cysteine 3 to cysteine 6. A knot forms when the disulphide bond between cysteine 3 and cysteine 6 crosses the loop formed by the two other disulphides and the interconnecting backbone. The cystine knot is highly conducive to protein stability. It is one of three knot configurations. These are resistant to acid and alkali treatments, and thermal and proteolytic attacks. This stability been attributed to conformational rigidity endowed by disulphide linkage of the cystine knot (Kolmar, 2008; 2009). Apart from its structural integrity, the cystine knot probably has a role in dimerisation of proteins and may be involved in receptor binding of growth factors. Interleukin-6 (IL-6) adopts a cystine-knot-like fold apparently preparatory to receptor recognition (Hymowitz et al., 2001). Numerous cystine-knot proteins occur in nature and they display a phylogenetic relationship. This suggests that the cystine-knot tertiary structure might be Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00002-5 © 2011 Elsevier Inc. All rights reserved.

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

Table 2.1  Growth Factor Families Receptor Family

Signalling Cascade

Other Defining Features

Membrane-Receptor Binding Family of Ligands Cystine-Knot Group TGF-β family TGF-β, nodal, activin, inhibin

Type RII and RI [ALK4, 5, 7], Smad2/3 → Smad4; also RIII in some ligands

BMP, GDFs

RII, RI [ALK1, 2, 3, 6], Smad1/5 → Smad4

VEGF

VEGFR-1, -2, and -3

PDGF, IGFs, CTGF, HGF, thrombospondin Cytokines, interferons, interleukins, GM-CSF

EGF Family EGF, TGF-α

STAT/PI3K; Ras/MAPK/ ERK/PKC

Forkhead box

DAG lipase/Ca2-CAM kinase and PI3K; MEK/ MAPK

Forkhead box factors, bHLH, T-box

Neuregulin, amphiregulin, epiregulin, β-cellulin

FGF Family

Intracellular-Receptor Binding Family of Ligands (a) Cytoplasmicreceptor binding ligands: glucocorticoids

ER, PR, VD3R, RAR

(b) Nuclear-receptor binding growth factors: oestrogen, progesterone, VD3, retinoids Note: See also Table 4.

characteristic of signalling molecules of higher organisms (see Vitt et al., 2001). Several growth factors, for example TGF-β, NGF, PDGF and ECM proteins, are cystine-knot proteins (Vitt et al., 2001). Notable cystine-knot proteins include the following: mucins (ECM proteins), which are linked with Norrie disease (the X-linked syndrome of blindness, deafness and mental retardation, whose protein is Norrin); the slit-like proteins with the ECM slit domain containing leucine-rich

Growth Factor Families

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Figure 2.1  Classification of growth factors into families, representing a resume of the scheme adopted here for discussion.

and EGF-like repeats; von Willebrand factor; and the BMP antagonists such as chordin and noggin, which are associated with neural induction; and the BMP-regulated sclerostin. Here, cystine-knot growth factors are treated as a distinct division of the growth factor family. Included in this division are the TGF-β family proteins, e.g. TGF-β, Nodal, BMP, activin and GDF. These growth factors function by activating specific membrane receptors and then downstream activating the Smad signalling cascade. As stated earlier, VEGF, PDGF, NGF, IGF, TSP and CTGF are notable examples of growth factors possessing the characteristic cystine-knot tertiary structure. Another important cystine-knot group comprises cytokines and other immune modulators, which are membrane-receptor binding ligands. These function in an autocrine, paracrine or endocrine fashion. They are inducers of proliferation and differentiation of immune cells and haematopoiesis; prominent among them are the interleukins, interferons and granulocyte-macrophage colony-stimulating factors (GM-CSFs). Hormones such LH, FSH, HCG and TSH also possess the cystine-knot tertiary feature, despite a lack of sequence homology, but they are not included within the purview of this book.

The Intracellular-Receptor Binding Family of Ligands The second subfamily is represented by growth factors that bind to and function through intracellular receptors. Included in this subfamily are growth regulators such as glucocorticoids, which bind cytoplasmic receptors, and those that bind nuclear receptors, such as ER, PR, VD3R, and retinoids.

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3 Growth Factors in Differentiation and Morphogenesis

A wide spectrum of growth factors determines and regulates development, ­differentiation and morphogenetic pathways. Differentiation and morphogenesis are not the outcome of the function of a single dedicated factor. They can be a composite phenotypic manifestation of the function of a cohort of factors using interacting and cross-linking pathways of information flow and mutual inter-regulation of the pathways by this cross-talk. A prime example is the Hedgehog (Hh) signalling system, which interacts and co-ordinates flow of signalling activated by many growth factors (Warburton et al., 2003). These include the transforming growth factor-β (TGF-β) family constituents and retinoids, whose signals coalesce downstream of Hh receptor activation into generating the differentiated phenotypes, morphogenetic patterns and cell motility that are characteristics of developing systems as well as some pathogenetic conditions. Although DNA replication and repair are also functions subserved by Hh signalling, the TGF-β family appears to induce proliferation by an independent pathway. Overall, the spectrum of effects of stimulation of growth and differentiation, morphogenesis and cell motility is so broad and extensive that one wonders how the large number of ligands of the TGF-β family and other colluding ligands could achieve the apparent spatial and temporal specificity in the enduring and complex biological processes. Growth factor signalling is totally dependent upon the presence of the appropriate receptor for the ligand to bind. Members of the TGF-β family use the TGF-β family receptors and the canonical signalling pathway. It is needless to emphasise that these receptors would be expressed all the time and in all cells, merely waiting for the ligands to arrive. Obviously mechanisms exist that dictate and direct the interaction between different pathways of signalling, different modes of interaction, and self- and inter-regulation of the signal flow. The possibility has to be entertained that colluding factors could conceivably induce the expression of receptors that are required for a specified differentiation or proliferation pathway. Another level of complexity encountered is how some ligands can transduce their signals using the same pathways and yet can induce the emergence of differentiated phenotypes. Some of these thoughts are amply borne out by currently available evidence. These, and the means of possible and potential regulation of signalling and phenotype specification, are addressed here. Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00003-7 © 2011 Elsevier Inc. All rights reserved.

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

TGF-β Family Growth Factors in Differentiation and Morphogenesis TGF-β is a superfamily of several growth factors, including the prototype TGF-β isoforms TGF-β1, TGF-β2 and TGF-β3. Others of note are the bone morphogenetic proteins (BMPs), inhibins, activin, growth and differentiation factors (GDFs), Vg-1 and anti-Müllerian hormone (AMH). Members of the TGF-β family can be divided into many groups based on their sequence homology. The comparatively low sequence homology of Nodal with other members of the TGF-β family has prompted its designation as a peripheral member of the family. Here, however, I differentiate TGF-β ligands into two major groups: (1) TGF-beta/Activin/Nodal ligands; (2) the BMPs together with the GDFs (see Table 3.1). Generally, TGF-β family growth factors are transcribed as precursor proteins that are processed into mature functional proteins by the agency of furin and PACE4 Table 3.1  The TGF-β Family Signalling Cascade TGF-β Ligand

Receptor

Signalling Components

TGF-β isoforms

TGF-β receptors I and II

Smads→transcription factors

Activin

TGF-β receptor family; activin-like receptor kinase (ALK 1/2); Type III

Smads→transcription factors FOXH1→activation of activin response element

Activin receptors (Act R); Smad Inhibin

TGF-β type III, betaglycan; inhibin binding protein InhBP/p120

Nodal

Delta2/Notch; FGF/MEK/ERK

FoxH1 (Forkhead box H1)→Pitx gene

ALK7, ALK4, and either ActRIIA or ActRIIB; EGF–CFC

EGF–CFC one-eyed pinhead; Tbx spadetail→Pitx2c Sox32 with Pou5f1 activate Sox17; Sox17 repressed by BMP

Lefty (antagonist of Nodal)

ActRII (Lefty competes for receptor)

AMH

AMHR types I and II

Inhibition of gonadal tumours through AMHR II

BMPs

ALK 1/2; ActR; BMPR

Smads→helix loop helix

Type III

Hey1; Runx

VEGFR GDFs

ActR

Note: The relevant references are provided in the text.

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(Paired basic Amino-acid Cleaving Enzyme) (also known as SPC1 and SPC4, respectively) convertases belonging to the SPC (subtilisin-like proprotein convertase) family. Cleavage at the furin processing site with RXXR motif RX(R/K)R (Thomas, 2002) releases the mature peptide, a feature shared by TGF-β proteins. However, the amino-acid motifs can differ, for example TGF-β has RHHR or RHRR, Nodal has RQRR and so on. Viral proteins have RX(K/R)R or RRTR sequences. The astacin family are metalloproteinases, which includes BMP1, are capable of activating the growth factors such as GDFs. TGF-β, despite its nomenclature and appellation as a factor that transforms normal cells, has a wide range of functions. TGF-β ligands regulate cell proliferation, differentiation, migration, cell adhesion, cell survival and apoptosis besides participating in other normal cellular function and disease processes. Vg-1 has an important function in pattern formation in the development as pattern formation in Xenopus. Inhibin and activin are peptide growth factors involved in the control of the biosynthesis and secretion of follicle-stimulating hormone (FSH) from the anterior pituitary. Activin stimulates but inhibin blocks these processes. AMH is produced by the Sertoli cells of the foetal testis and in granulosa cells of growing follicles of the ovary. In the ovary, AMH regulates primordial follicle recruitment and the responsiveness of growing follicles to FHS. BMPs and GDFs fully participate in cell differentiation and pattern formation. In the context of this book, it should be noted that BMPs are also known to inhibit endothelial cell migration and neovascularisation.

TGF-β in Tumour Growth, Invasion and Metastases The systemic spread of cancer cells is followed by target-site-specific deposition, leading then to the development of overt metastasis. Here again growth factors and the activation of genes regulating the cell proliferation and growth come into play. TGF-β performs diverse and pleiotropic physiological functions participating in cell adhesion, migration and proliferation, as a part of normal growth and differentiation. Its effects are tissue specific, stimulatory of the growth of mesenchymal cells and inhibitory of epithelial, endothelial and lymphoid cell growth (Frater-Schroder et al., 1986; Kerhl et al., 1986a, 1986b; Shipley et al., 1985; Tucker et al., 1984). In the context of cancer it is essential to emphasise that TGF-β can reputedly suppress tumorigenesis in early stages of cell transformation and growth, yet it can promote progression of advanced cancer. Obviously there would exist comprehensible and comprehensive mechanisms that regulate TGF-β function. TGF-β occurs as a part of an inactive pro-protein (Lawrence et al., 1985; Pircher et al., 1986), which is processed to yield the mature active ligand. In fact, the mature ligand is a 25 kilodalton (kDa) fragment that is derived from the carboxy (C) terminus of the pro-protein. The amino (N)-terminal remnant of the pro-protein generates a 75 kDa homodimer (latent TGF-β binding protein, LTBP) (Derynck et al., 1985). These two proteins are linked by non-covalent bonds and when dissociated from LTBP, TGF-β becomes biologically active (Flaumenhaft et al., 1993). At a separate and discrete level of

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

function, differential signalling and the activation of a different genetic profile will probably be at the heart of this bivalent action, given that there are marked differences in the biological requirements of disease progression (Table 3.2).

The TGF-β Signalling Cascade Signalling by TGF-β ligands can be distinguished into a canonical or conventional pathway and a non-canonical mode. The canonical system involves a family of transmembrane receptors including types I, II and III with downstream components called the Smad proteins. The non-canonical course does not activate the Smad cascade, but it transduces the signal by engaging other systems of transduction totally or partly independently of the Smad cascade. The pleiotropic responses exerted by TGF-β ligands appear to be an outcome of the distinctive canonical and non-­canonical signalling systems. The suppression of cell proliferation, promotion of apoptosis and inhibition of tumour progression are an outcome of the Smad pathway, whereas the Table 3.2  TGF-β Signalling-Associated Genetic Profile in Cell Differentiation, Invasion and Metastasis Phenotypic Feature

Signalling Pathway

Cell differentiation, proliferation, tumour growth

P53-Rb/stathmin/p53 downstream effectors, for example, p21waf, p16, etc. P53/stathmin/microtubule dynamics/cell division; p53 and downstream target apoptosis family genes; caspases; calpain/Fas (?); inhibition of PTEN/ Akt-mediated cell survival; immunomodulation TNF-mediated immune suppression

Invasion, intercellular adhesion

Epithelial mesenchymal transition; modulation of cytoskeletal dynamics; cadherin/catenin complex cytoskeletal linkage; CD44/cytoskeletal linkage ECM-associated proteolytic enzyme system/ECM remodelling

Tumour vascularisation

Activation of MMP/TIMP Activation of angiogenic factors VEGF/endothelial cell proliferation MetAP2/p53-mediated inhibition of endothelial cell proliferation

Tumour dissemination, osteotropism and development of overt metastases

P53/Hdm2; osteotropism

Note: These phenotypic features are not intended as representing those exclusively linked with TGF-β signalling. Rather, they suggest how different genetic programming might result in the bivalent function of TGF-β (based on Sherbet (2006) and references cited in the text).

Growth Factors in Differentiation and Morphogenesis

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opposing phenotypic changes occur when Smad signalling is abrogated or the alternative non-Smad signalling system is activated. Furthermore, many outcomes are a result of cross-talk between various signalling pathways.

The TGF-β Receptor Group Receptor Types I and II in Signalling by TGF-β Ligands Members of the TGF-β family transduce their effects through a relatively uncomplicated system of two types of receptor, the type I and type II (RI and RII) receptors (see below for type III accessory or co-receptors). These receptors are transmembrane proteins consisting of a ligand-binding extracellular domain, a transmembrane domain and a cytoplasmic serine/threonine kinase domain. Seven type I receptors, ALK 1–7 and five type II receptors have been identified. Upon ligand binding, the type II receptor initially engages type I to form a heterotetramer receptor complex. Ligand binding activates the serine/threonine kinase of RII, which then phosphorylates RI on specific serine and threonine residues in the juxtamembrane GS (glycine and serine-rich) domain. TGF-β binds type II with high affinity. Downstream are the cytoplasmic Smad proteins, which carry the signal to the nucleus, and nuclear DNA-binding proteins that form complexes with Smad to form transcription factors (Derynck et al., 1998; Heldin et al., 1997; Massagué, 1998; Massagué et al., 2005; Wrana et al., 1992). Three Smad types are distinguishable: the receptor-regulated Smads (R-Smads, Smads 1, 2, 3, 5 and 9) (Wu et al., 2001), the common mediator co-Smad4 required in signalling by all members of the TGF-β family, and the inhibitory I-Smads. I-Smads inhibit the activation of R-Smads and the co-Smad. Smad6 and Smad7 inhibit signalling downstream of TGF-β RI receptors (Itoh et al., 2001). I-Smad6 specifically inhibits BMP type I receptor signalling. However, Smad7 is less specific, being able to inhibit signalling by several TGF-β RI-related receptors, for example BMP type I and activin receptors. The inhibitory function of Smad6 itself is regulated by its binding to the cytoplasmic protein called AMSH (associated molecule with the SH3 domain of STAM) (Itoh et al., 2001).

Receptor Type III Endoglin-Mediated TGF-β Signalling The canonical Smad signalling system involves regulatory components; among them are endoglin (CD105) and betaglycan, which are often described as accessory type III receptors of the TGF-β family ligands. Essentially they assist the binding of the ­ligands to the type I and II receptors. Type III receptor might indeed function in both liganddependent and -independent signalling. Endoglin functions upstream of ALK1/Smads 1, 2 and 3. Endoglin binds TGF-β receptors to recruit members of the TGF-β family to form active receptor complexes. TGF-β binds to endoglin in the presence of RII, but endoglin can bind RII when the ligand is absent. In endothelial cells, ALK5 receptor and the endothelial cell-specific ALK1 receptor promote endoglin activity in the presence of RII. The TGF-β family ligands activin-A and BMPs can bind endoglin in

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consort with other receptors (see Barbara et al., 1999; Koleva et al., 2006; Lebrin et al., 2005; Mercado-Pimentel et al., 2007; Scharpfenecker et al., 2007).

Receptor Type III Betaglycan in TGF-β Signalling Betaglycan is a transmembrane proteoglycan. It was recognised some time ago to function as a co-receptor for TGF-β to which it binds with high affinity. It has several binding sites for TGF-β and accentuates signalling by TGF-β and TGF-β family ligands. The external domain of betaglycan has two ligand negatives, one in the distal and a second one in proximal half in relation to the membrane, and the ectodomain displays a bi-lobular structure, with each lobule folding and binding TGF-β independently of the other (Mendoza et al., 2009). Functionally, betaglycan leads to the suppression of cell proliferation and invasion. It can mediate TGF-β ligand-­dependent or independent signalling through the canonical Smad system and the non-Smad pathways.

The Canonical Smad Pathway There are two important functional domains in Smads: the MH (Mad homology) domain MH2 that occurs at the C terminus of Smads and it is linked through a nonconserved region to MH1. Although MH2 interacts with other proteins, MH1 binds the DNA (Attisano et al., 2001; Kim et al., 1997). Hariharan and Pillai (2008) have attributed the differences in function between R-Smads and I-Smads to the loss of some structural elements from MH1 and MH2 of R-Smads and alterations in the structural flexibility of these domains. The processing of the signal seems to be regulated by R-Smads together with the I-Smad. This leads to the phosphorylation of type I, which associates with Smads, known as receptor-regulated Smads (R-Smads) (Heldin et al., 1997; Massagué, 1998) and phosphorylates the R-Smad complex. This complex now binds Smad4 (Lagna et al., 1996), a tumour-suppressor gene product (Hahn et al., 1996) and translocates to the nucleus. In the nucleus, the total complex of R-Smad/Smad4 activates the appropriate transcription factor, leading to the expression of the early response genes (Figure 3.1). TGF-β family members, including the TGF-β isoforms, activin, Nodal, inhibins, AMH, GDFs, BMPs, etc., all bind to the members of the same family of receptors and then share the same downstream signalling pathway. Given that the TGF-β family uses the TGF-β family receptors and the canonical signalling pathway involving Smads, questions arise about how such a multiplicity of phenotypic effects is generated by the large number of constituent members of the family and how the specificity of signal transduction is achieved in the face of the simplicity of the signalling cascade. Not only are there many members in this family, but they also control a wide diversity of cellular functions while sharing the receptor and signalling downstream elements. Some of these growth factors appear capable of generating the same phenotype of differentiation; others can generate more than one phenotype. One can envisage many possible ways by which these effects might be achieved: (1) ligand binding dictated by differential affinity and specificity to the receptors; (2) the adoption of specific means of targeting the ligands to the receptors;

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TGF-β family ligand ↓ -----------------------------------------------------------Type II receptor ↓ Type I/II-II/I [Heterotetrameric receptor complex] ↓ ---------------------------------------------------------------I – P* ↓ R-Smad – P* ↓ Smad4 ↓ ----------------------------------------------------------R-Smad – P*/Smad4 ↓ Activation of transcription factors ↓ Early response genes -----------------------------------------------------------

Figure 3.1  The TGF-β cascade of signalling shows the ligand binding to type II receptor leading to the recruitment of type I receptor and the formation of heterotetrameric complex which phosphorylates and activates type I (I–P*). The activated type I receptor in turn phosphorylates cytoplasmic R-Smads. These bind to Smad4. The R-Smad–P*/Smad4 complex now within the nucleus activates transcription factors that can induce the expression of early response genes. Generally, it would seem that BMP and GDF signal through Smad1/5 and the type I receptors ALK 1, ALK2, ALK3 and ALK6, whereas TGF-β, activin and Nodal activate Smad2/3 through ALK4, ALK5 and ALK7 (Shi and Massague, 2003).

(3) the recruitment of different R-Smads to constitute the downstream signalling chain; (4) specificity of activation of transcription factors; (5) inhibition of the signalling pathway; (6) regulation of molecular processing leading to ligand activation and consequent encroachment upon its function. These potential modes for achieving signalling specificity to activate responsive genes that dictate differentiation and phenotype are appropriate here during the discussion of the biological functions of the different members of the TGF-β family. In the present context of delineating the signalling cascade, it is of note that the interactions between the receptors and Smads, and between the Smads and the transcription factors, depend upon the structural features of MH2 domains, which confer the faculty of selective interactions between them (Chen et al., 1998).

The Effects of TGF-β on Cell Proliferation and Apoptosis Tumour growth results from a disequilibrium of cell proliferation and cell loss by apoptosis. In normal differentiation a stringent regulation of equilibrium between these opposing pathways is essential. When cell population expansion within the

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

tumour is counterbalanced by the loss of cells by apoptosis and necrosis due to inadequate vascularisation, tumour growth is limited. However, with vascularisation this balance is tipped towards increased cell proliferation and tumour growth. It has been postulated that TGF-β signals activate the Smad pathway and lead to apoptosis. Signalling independently of Smad also operates, as discussed in detail in a later section on Non-Smad (Non-Canonical) Signalling. This has been postulated as providing a means to generating diametrically opposite effects of induction of proliferation or promotion of cell survival and enhancement of migration. TGF-β has been found to activate NF-κB and inhibit PTEN (Chow et al., 2010). This would allow Akt-mediated signalling to cell survival. Another mode of signalling, which is independent of Smad but occurs through the Raf/ERK/MAPK pathway leading to apoptosis or to cell survival, has been proposed. In this, a common downstream effector, namely Prohibitin, is said to subserve a dual function as inducer of apoptosis or promoter of survival by its ability to regulate mitochondrial membrane permeability (Zhu et al., 2010). This is an attractive concept which would seemingly reconcile the opposing effects of TGF-β. Royce et al. (2010) reported loss of Smad protein expression more often in rectal tumours than in those arising in the colon. They argued that loss of Smad is an early event of colorectal carcinogenesis. It is possible that in this situation tumorigenesis might take the survival pathway through Raf/ERK/MAPK or Akt signalling (Figure 3.2). An alternative explanation is provided by Daly et al. (2010), who proposed that high levels of Smad tend to generate suppressor function, whereas at low levels Smads perform a tumour-promoting function. Ras is said to modulate Smad levels and might thus provide a means of switching from Smadmediated apoptosis to low Smad-mediated survival (Figure 3.2). As discussed below, Smad-dependent as well as -independent pathways also operate in the induction of cell migration by TGF-β. An additional parameter worthy of further study is the possibility raised recently that the Smads might differentially affect biological properties of migration, and possibly of metastatic ability of cancer cells. TGF-β was seen

Figure 3.2  Some postulates relating to the bivalent function of TGF-β signalling through Smad and non-Smad pathways.

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to transcribe Smad2 and Smad3 differentially, and this corresponded with VEGF expression (Petersen et al., 2010). Indeed, Smad4 might inhibit cancer progression, as shown by Zhang et al. (2010) using murine tumour models. MDM2 and its human homologue HDM2 regulate the function of p53. HDM2 is overexpressed frequently in the final stages of progression. It has been shown recently that TGF-β activated canonical Smad3/4 signalling, specifically associated with HDM2 promoter, enhanced the expression of HDM2 and led to consequent ubiquitination and destabilisation of p53 (Araki et al., 2010). Of further interest is these authors’ demonstration that the activation of Smad signalling and of HDM2 occurred mainly in late-stage carcinomas. This provides strong correlative evidence of TGF-β function in the late stages of progression. Other downstream targets of TGF-β/Smad signalling have been identified; epigenetic silencing is believed to take place as a consequence of aberrant Smad signalling. Among them are ADAM19 and the newly identified FBXO32. The silencing of these genes has been reported to occur in advanced stages of ovarian cancer and to correlate with poor prognosis (Chou et al., 2010). Despite the perceived correlation, it is conceptually a long way from a mechanistic explanation of how the silencing of these target genes affects prognosis, for ADAM proteins themselves target many proteins. They can transactivate growth factor receptors, release membrane bound growth factors, induce angiogenesis and have been associated with tumour development and progression.

Cell Proliferation, Invasion and Metastasis Mediated by Type III Receptors Given that type III receptors can function in either ligand-dependent or -independent fusion, it follows that their expression per se might influence cell proliferation, invasion and metastasis. Both endoglin and betaglycan do indeed influence these features of cell behaviour and cell phenotype. Endoglin is a transmembrane glycoprotein that displays significant tissue specificity of expression. It is expressed in the vascular endothelium and has been associated with endothelial proliferation and cell migration (Burrows et al., 1995; Fonsatti et al., 2000; Miller et al., 1999). The attribution of its specificity of expression in endothelial cells is further strengthened by the fact that its promoter displays strong activity in endothelial cells compared with promoters of other endothelial cell components and with epithelial cells and fibroblasts (Graulich et al., 1999). Furthermore, it has been shown that the transcription factor KLF6, which is induced in endothelial cells during vascular injury, transactivates TGF-β, TGF-β receptors and TGF-β-stimulated genes. Overexpression of KLF6 transactivates the endoglin promoter (Botella et al., 2002). The importance of endoglin for endothelial proliferation is clearly indicated by the reduced cell proliferation and migration, inhibition of capillary formation, diminished nitric oxide (NO) synthase activity and reduced VEGF secretion in mice heterozygous for endoglin (CD105/) attributable to reduced endoglin in the heterozygous state. Besides, both in vitro and in vivo, there was a marked reduction of vasculature (Jerkic et al., 2006a, 2006b). A  full or normal complement of endoglin is necessary for maximal angiogenesis.

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

With its association as a key element in angiogenesis associated with tumours and in tissue regeneration and inflammatory phenomena, endoglin has naturally been focused on as a potential therapeutic target. Of much interest that could boost clinical targeting of endoglin is the finding that serum levels of endoglin were higher in patients with metastatic disease than in those with no metastasis. The tumours investigated included colorectal and breast cancers (Takahashi et al., 2001). However, the number of patients studied was small and there was a large spread of endoglin values in both groups, which makes it difficult to draw firm conclusions about the possible significance of the findings. Nonetheless, further studies of this kind are warranted. The presence or absence of betaglycan expression has also been the focus of attention in relation to tumour progression. Loss of betaglycan is encountered frequently in breast cancer and there is a view that this might correlate with disease progression. betaglycan exerts significant inhibitory effects on cell migration, proliferation and angiogenesis and has indeed been regarded by some as a tumour suppressor. Bilandzic et al. (2009) found that the expression of betaglycan messenger RNA (mRNA) was markedly reduced in ovarian granulosa cell tumours. Also, two cell lines displayed reduced levels of betaglycan expression and were poorly responsive to TGF-β and inhibin A. The response to the ligands was restored by transfecting the cells with betaglycan, with increased adhesion and reduced cellular invasion in vitro. Iolascon et al. (2000) encountered loss of type III receptor in late stages of neuroblastomas, but added that the expression of types I and II seemed to be unaffected. The loss of betaglycan in ovarian cancer seems to correlate with tumour grade. Compatible with the findings of Bilandzic et al. (2009), in vitro betaglycan appeared to inhibit migration and invasive behaviour of ovarian cancer cells. Also, it seemed to potentiate the inhibitory effects on cell migration exerted by inhibin and the ability of the latter to inhibit metalloproteinases (Hempel et al., 2007a). This team of investigators has also described similar findings in prostate cancer, with loss of betaglycan correlating with tumour stage and PSA expression. They also showed that restoration of betaglycan expression led to inhibition of cell migration and invasion in vitro (Turley et al., 2007). Earlier, Copland et al. (2003) reported loss of type III receptor in samples of renal cell carcinoma. Of interest is their finding that loss of type II receptor after loss of type III seemed to lead to the acquisition of metastatic ability. This finding has not been followed up and tested as it should have been on account of the potential significance in the clinical context. For instance, it might be worthwhile checking the effects of restoring type II receptor expression using trichostatin A, which is able to activate the type II receptor gene promoter and induce the expression of type II mRNA (Kashiwagi et al., 2010). Earlier, the farnesyltransferase inhibitor L-744832 was shown to restore type II expression. Alcock et al. (2002) demonstrated that TGF-β signalling could be induced by the inhibitor, which was associated with the re-expression of type II receptor and decreased DNA methyltransferase 1. Indeed, the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine seems to lead to re-expression of type II transcript and protein; this is also accompanied by an increase in TGF-β promoter activity (Ammanamanchi et al., 1998). It is

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needless to stress that the phenotypic effect of TGF-β would need to be convincingly demonstrated. One ought to add a caveat, however, that there is room for debate here, with the demonstration that the expression of TGF-β1 and receptors I, II and III was twofold greater in high-grade than in low-grade lymphomas (Woszczyk et al., 2004). The study involved the investigation of a small group of patients, and the contraindications require further evidence. Besides, it should be remembered that it is possible that, as in the context of angiogenesis, TGF-β ligands are part of a network that determines the outcome in terms of cell migration and invasion.

Endoglin as a Chemotherapeutic Target The involvement of endoglin in the regulation of TGF-β signalling and its association with angiogenesis have evoked much interest in its potential as a therapeutic target. As previously noted, endoglin in its full complement is angiogenic. Plasma endoglin levels are higher in a proportion of patients with breast cancer, which has been correlated with reduced response to hormonal therapy and overall survival (Vo et al., 2010). In the clinical context it is of note that clinical trials are taking place of humanised antibodies raised against endoglin, testing the efficacy of antibody conjugates with cytotoxic drugs and antibody conjugates engineered to metabolise pro-drugs to generate active drugs. Anti-endoglin antibodies have successfully inhibited the formation of lung metastasis in experimental assays (Uneda et al., 2009). Results from phase 1 clinical trials of TRC105, TRC102 and TRC093 were presented at the American Society of Clinical Oncology meeting in June 2010. The success of these is yet to be evaluated. Otten et al. (2010) have listed several preclinical and clinical trials targeted at members of the TGF-β family.

Therapeutic Potential of Betaglycan The ability of betaglycan to inhibit cell migration and the association of its loss with tumour progression have not attracted much attention or prompted studies to view its therapeutic deployment. Recombinant betaglycan was reported to inhibit tumour growth and metastasis in vivo by inhibiting-tumour-associated angiogenesis (Bandyopadhyay et al., 2002a, 2002b). Very little further activity has taken place in this area. Corticosteroids have been shown to induce upregulation of betaglycan expression. There is also a degree of uncertainty engendered by the fact that dexamethasone, hydrocortisone and aldosterone exert different effects. Besides, this is an isolated study and confirmation is required of these initial findings. betaglycans are not alone in receiving scant attention in this way. CD44 is another example. A fundamental problem exists which might be intractable, an impediment common to many biological phenomena. Many proteoglycans exert complex effects by participating in and regulating signalling systems and physically participating in the regulation of adhesion, cell migration, morphogenesis and differentiation. This has inevitably led to the postulate that a balance between the signalling and structural roles might be an

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

important factor in the perceived opposing effects exerted on tumour development and progression (Mythreye and Blobe, 2009).

TGF-β and Organ-Specific Metastatic Spread Tumours often display specific patterns of metastasis. Organ specificity of metastatic deposition was recognised and related to the mode of dissemination and to homotypic and heterotypic interactions occurring between tumour and host cells. Cell membrane components participate prominently in determining the pattern of metastatic spread (Brunson et al., 1978; Brunson and Nicolson, 1979; Nicolson, 1962; Nicolson and Winkelhake, 1975; Sherbet, 1982, 1987). Osteotropic metastasis is a well-documented phenomenon; especially of note in the present context is the propensity of TGF-β to localise in the bone. TGF-β functions as a tumour suppressor in the early stages of cancer but promotes progression in the late stage of the disease. A key component involved in the promotion of progression is cadherin. A clear link has been established between induction of epithelial mesenchymal transition (EMT) by TGF-β and the association of E-cadherin in this process. TGF-β induces EMT and loss of E-cadherin. This aids invasive behaviour. Induction of EMT downregulates E-cadherin together with changes in cell morphology and polarity. In cells where TGF-β signalling is countermanded by the Nodal gene Lefty, a marked inhibition occurs of Smad activation and inhibition of EMT. Further downstream signalling seems to involve JNK, for inhibition of JNK signalling blocks induction of EMT by TGF-β (Mariasegaram et al., 2010). Snail1, a transcriptional repressor of E-cadherin, is not only an inducer of EMT but is known to interact with Smads. In breast epithelial cells, the Snail1/Smad3/4 complex then targets the promoters of genes encoding a tight junction protein and E-cadherin (Vincent et al., 2009). Although expression of E-cadherin is reduced, vimentin and N-cadherin show increased expression (Deckers et al., 2006; Zeisberg et al., 2003). Other TGF-β ligands such as BMPs often alter EMT in a different direction. BMP7 is known to inhibit EMT. In fact, it seems to promote the reverse process of mesenchymal epithelial transition. Cells induced into mesenchymal epithelial transition by BMP7 show reduced expression of the bHLH transcription factor TWIST. However, TWIST levels could be restored by ALK2 (BMP receptor) short interfering RNA (siRNA), which inhibits Smad 1, 5 and 8 signalling (Na et al., 2009). Nonetheless, the ligands signal through the Smad system. The specificity of function seems to derive from the component Smad proteins. Different TGF-β ligands could be engaging different Smads depending upon the nature of Smad-binding elements. Then downstream the Smad complexes could recognise and bind well-defined specific DNA motifs. Buijs et al. (2007) not only recognised the inhibitory effect of BMP7 on EMT, but also that BMP expression was inversely related to metastatic potential and correlated with E-cadherin/vimentin ratio. They also demonstrated that extraneous BMP7 inhibits Smad3/4, besides BMP signalling through Smad1/4/5 being enhanced by the presence of TGF-β in the bone. Experimental studies in vivo showed that BMP7 administration led to inhibition of metastatic growth in the bone. Cell suspensions inoculated into animals treated daily

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with BMP7 resulted in decreased metastatic deposition in the bone. Another suggested action is that BMP7 might, through Smad1, mediate activation of the inhibitory Smad7. The transcription factor NF-κB is a regulator of gene expression that is conducive to the generation of the invasive phenotype and to cell survival. It is, indeed, often quoted as being capable of suppressing the epithelial phenotype and promoting EMT. NF-κB function might directly or indirectly involve and require Smad. TGF-β ­activates its receptor and activates NK-κB. For this, Smad4 is needed, but NF-κB activation does not occur when the inhibitory Smad7 is present. Smad7 inhibits IκB-α phosphorylation (Grau et al., 2006). IκB-α binds to and sequesters NF-κB and inhibits the function of the latter. Phosphorylation of IκB-α leads to the degradation of IκB-α/NF-κB complex (DiDonato et al., 1995). The RelA subunit NF-κB is also required for the DNA binding of Smad complexes in TNF signalling (Bitzer et al., 2000; Kon et al., 1999). Again, of much relevance in this context is that TNF-α can activate NF-κB and in turn activate Smad 7 and thus inhibit the canonical Smad signalling. NF-κB is also involved with the transduction non-canonically of the TGF-β signal by the PTEN/Akt pathway. As noted earlier, Snail1 transcription repressor of E-cadherin is prominent in the induction of EMT. Interestingly, Akt has been reported to activate NF-κB, leading to the induction of Snail expression (Julien et al., 2007). The molecular mechanisms associated with NF-κB function in osteotropism are increasingly being elucidated. The cytokine RANKL (receptor/activator of NF-κB ligand) is a member of the TNF family. The interaction of RANKL with its receptor RANK (receptor/activator of NF-κB) induces osteotropic migration of breast cancer and melanoma cells (Jones et al., 2006). RANKL occurs in osteoblasts and stromal cells of the bone marrow. RANKL is involved with the differentiation of osteoclast progenitor cells into mature osteoclasts and it signals through membrane RANK expressed in precursor and mature osteoclasts. Osteoclast formation can occur independently of RANKL, for example by the mediation of TNF-α and interleukins. The effects of inhibiting RANKL function or RANK on osteolysis and the formation of osteolytic metastasis have been investigated in tumour models in vitro and in vivo. Osteoprotegerin, an antagonist of RANKL, effectively inhibits tumour growth and the formation of osteolytic lesions in SCID-immunodeficient mice injected with PC3 prostate cancer cells (Armstrong et al., 2008). Inhibition of IKK, a component of NF-κB, has been shown to inhibit osteoclast activity and prevent osteolytic metastasis of experimental tumours in vivo (Idris et al., 2009). IGFs and TGF-β occur in the osteolytic setting and might be involved in the osteolytic metastasis of breast cancer. TGF-β is able to upregulate the parathyroid-related protein (PTHRP) from tumours, which can enhance the expression of RANKL and can activate osteoclasts. Interleukins have been implicated in PTHRP-independent osteolysis (Kozlow and Guise, 2005). Rose and Siegel (2010) have suggested that TGF-β and RANKL are important in the development of metastatic breast cancer in the bone, which is a frequent metastatic target of breast cancer. Prostate cancers also show osteotropism and are deemed to produce osteolytic lesions. TGF-β family ligands, including TGF-β and BMPs, appear to upregulate transcription factors such as the bHLH family repressor Hey1 at early stages of stimulating

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bone-marrow stromal progenitor cells to differentiate (Sharff et al., 2009). Also downstream Runx2, an important regulator of osteoblast differentiation, drives multipotent mesenchymal cells to differentiate into osteoblast lineage while inhibiting them from differentiating into the adipocytic and chondrocytic lineages (Komori, 2006). Prostate cancer cells treated with TGF-β or 5α-dihydrotestosterone enhanced Runx2 transcription. This appeared to increase cell proliferation, and xenografts of treated cells resulted in the formation of larger tumours (van der Deen et al., 2010). Although this study provides an interesting prelude to the potential role of Runx2 in the formation of osteolytic lesions by prostate cancer, definitive experimental evidence has yet to come.

Non-Smad (Non-Canonical) Signalling TGF-β ligands play significant roles in embryonic development, cell proliferation and apoptosis, cell migration, the formation of the ECM edifice and its modulation, EMT, as well as the reverse processes of mesenchymal epithelial transition, regulation in adaptive immunity of CD4 cell differentiation into effector cells, the function of the regulatory Treg cells and in many disease processes. The pleiotropic responses of epithelial cells to, and the diametrically divergent effects exerted by, TGF-β ligands are an outcome of distinctive signalling systems that transduce the growth factor signals. The inhibitory effects of these ligands, namely suppression of cell proliferation, promotion of apoptosis and inhibition of tumour progression, are attributable to the canonical Smad signalling pathway, whereas the opposing effects of promotion of growth, invasion and metastasis emerge either as a consequence of the abrogation of Smad signalling or activation of alternative non-Smad signalling systems. As noted earlier, the induction of EMT by TGF-β is associated with the loss or downregulation of E-cadherin together with associated changes in cell morphology and polarity. Smad abrogation or inactivation blocks EMT. Furthermore, Snail1, a transcriptional repressor of E-cadherin, has been shown to form a complex with Smad; the Snail1/Smad complex then targets the promoters of genes encoding a tight junction protein and E-cadherin (Vincent et al., 2009). So there are clear indications of the functioning of the canonical signalling pathway in EMT induction. The reverse process of mesenchymal epithelial transaction also seems to be a Smad-driven process. It is needless to reiterate many phenotypic forms of outcome are determined by interaction between various signalling pathways, as in the acquisition of cell motility and invasive behaviour upon EMT transition, Ras signalling, Wnt/β-catenin/cadherin, Notch and hedgehog systems, among others. TGF-β has been known for some time to signal by non-Smad or non-canonical pathways. Many important non-Smad means of signalling have been identified. These include MAPK, Erk and JNK and its elements p38 (Adachi-Yamada et al., 1999; Atfi et al., 1997; Derynck and Zhang, 2003; Hartsough and Mulder, 1995; Hocevar et al., 1999; Zhou et al., 1999). PI3K/Akt, with associated and integrated operation of PTEN and mTOR pathways, and Rho GTPase signalling are also known to be activated by TGF-β (Assinder et al., 2009; Barrios-Rodiles et al., 2005; Lamouille and Derynck, 2007; Ozdamar et al., 2005). RhoB is a direct transcription target of TGF-β, and it is an active participant in the induction of cell migration by

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TGF-β. This is indicated by the inhibition of TGF-β-induced migration by silencing of RhoB gene expression using siRNA or a dominant negative form of RhoB (Vasilaki et al., 2010). It was recognised some time ago that TGF-β rapidly activates Ras and leads to the recruitment of Raf to the plasma membrane, which in turn activates MEK1/Erk in many cell types (see Mulder et al., 1992; Yan et al., 1994). Another important element in Erk activation is the phosphorylation of tyrosines of type I and II receptors by TGF-β, which recruit Grb2/Sos to activate Erk through Ras, Raf and MAPK and to the regulation of EMT. EMT involves E-cadherin, increased MMP activity and induction of actin stress fibres. Erk activation is one of the non-Smad pathways of TGF-β-mediated induction of EMT (Davies et al., 2005; Zavadil et al., 2001). P38 and JNK are activated by MKKs (mitogen-activated protein kinase kinases) MKK3/6, and TGF-β has been shown to activate JNK through MKK4 (Engel et al., 1999; Frey et al., 1997; Hocevar et al., 1999; Weston et al., 2007). Smad-independent signalling occurs in the development of Th17 and transcription factor Foxp3() regulatory T-cells (Treg cells), although loss of Smads 2 and 3 slightly reduces Treg induction. This might be because an enhancer element of Foxp3 has been shown to bind Smad3 and NFAT (Tone et al., 2007), which could partly contribute to Foxp3 regulation by TGF-β. Regulatory T-cell development appears to involve Erk and/or JNK pathways, whereas the p38 pathway seems to signal the development of Th17 effector cells (Lu et al., 2010). TGF-β has been shown to activate Akt independently of Smad and induce the acquisition of phenotypic effects of migration, apoptosis and vascularisation (Bakin et al., 2000; Lamouille and Derynck, 2007; Shin et al., 2001; Vinals et al., 2001; Wilkes et al., 2005). Akt activation may be linked with the suppression of PTEN. This seems to occur through NF-κB activation, for inhibition of its activation prevents the downregulation of PTEN and the enhanced cell motility (Chow et al., 2010). Cell proliferation and survival signalling can take the serine–threonine kinase mTOR (mammalian target of rapamycin) p70 S6K1 (the 40S ribosomal S6 kinase) pathway and is activated by many factors, growth factors and insulin among them. The pathway involves Akt kinase, an upstream regulator of mTOR (Asnaghi et al., 2004). The mTOR pathway involves two mTOR complexes, namely mTORC1 and mTORC2. The mTORC1 complex comprises raptor (the regulatory-associated protein of TOR) and GβL. The complex mTORC1 regulates phenotypic alterations by activating its effectors S6K and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) (Hara et al., 2002; Kim et al., 2002; Nojima et al., 2003). The activation of the latter results in enhanced ribosomal biosynthesis and the synthesis of proteins required for G1–S transition and cell proliferation. The complex mTORC2 comprises mTOR and the rictor (rapamycin-insensitive companion of TOR), and GβL can phosphorylate Akt and might be involved in cytoskeletal reorganisation (Sarbassov et al., 2004, 2005). It can form complexes with additional adaptor proteins (Guertin and Sabatini, 2007; Jacinto et al., 2006; Makino et al., 2006; Yang et al., 2006). S6K1 is overexpressed in breast cancer and is associated with poor prognosis. S6K1 functions downstream of Akt and mediates cell proliferation. TGF-β family members can also signal in a Smad-independent fashion, taking PI3K and S6K, which are

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activated by Akt and MAPK pathways (Figure 3.2). TGF-β inhibits cell proliferation through PP2A (protein phosphatase 2A). PP2A complexes with S6K, dephosphorylates and inactivates the kinase (Petritsch et al., 2000). The part played by small GTPases in the TGF-β signalling pathway has attracted much attention. Small GTPases are involved in the regulation of differentiation, cell division and cytoskeletal dynamics. They are monomeric guanine nucleotide binding proteins of small molecular size (21–30 kDa). The small GTPase superfamily includes the Ras, Rho, Rab, Arf and Ran subfamilies. Ras and Rho signal crucial events in the life of the cell. Rho GTPases are involved in the organisation of the actin cytoskeleton and determination of cell polarity. Albeit seen to be involved with non-Smad signalling, small GTPases do indeed indulge in cross-talk with Smadmediated TGF-β signalling. Rho and Rac may interact with E-cadherin signalling. Rho, Rac and Ras can interact with growth factor and PI3K signalling. Rho GTPase seems to be involved in TGF-β-induced promotion of EMT (Barrios-Rodiles et al., 2005; Lee et al., 2010; Ozdamar et al., 2005). Similarly, overexpression of HSP72 abrogates EMT induced by TGF-β by blocking Smad3 phosphorylation and nuclear translocation. The abrogation effect requires physical interaction between Smad3 and the peptide-binding domain of HSP72. As further confirmation, inactivation of endogenous HSP72 increases phosphorylation of Smad3 and promotes EMT. Another system that interacts with TGF-β-induced promotion of EMT is integrin signalling, which is an important element in EMT. Bianchi et al. (2010) encountered upregulation of ανβ-integrin, which occurred through activation by TGF-β of the Smad pathway (Bianchi et al., 2010). There is general recognition that TGF-β ligands interact with Wnt/β-catenin signalling (Labbe et al., 2007). TGF-β is known to induce production of Wnt (Zhou et al., 2004). Also it activates β-catenin, a component of the canonical Wnt pathway, by signalling through Smad3 (Labbe et al., 2000) and signalling downstream in the conventional format of LEF1/TCF to lead to the activation of specific target genes. Smad-mediated signalling has been adduced as evidence for the formation of the β-catenin/LEF1 complex, leading to the loss of E-cadherin and to the induction of EMT (Medici et al., 2006). Indeed, these various signalling pathways can interact and modulate Smad signalling. Such interaction could conceivably lead to the pleiotropic effects exerted by TGF-β as well as determining the pattern of effects displayed by them during the course of cancer progression. In this instance, one can visualise how the growth inhibitory effects and the ability to promote apoptosis wane, and PI3K- and Ras-mediated responses, for instance involving growth promotion, might be acquired with alterations in and restructuring of the ECM with the generation of phenotypes with invasive faculties. Thus, both canonical and noncanonical signalling systems seem to retain their participation in, and influence the induction of, EMT and invasion.

Non-Canonical TGF-β Signalling in Cancer Invasion and Angiogenesis The participation of TGF-β in cancer invasion is supported by its ability to promote EMT. The conversion of epithelial cells into mesenchymal cells or EMT is a

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developmental programme, which is characterised by changes in cell morphology, reduced intercellular adhesion and enhanced cell migration. These changes occur in consonance with reduced expression or loss of E-cadherin, enhanced expression of N-cadherin and vimentin, and β-catenin translocation, possibly in association with its signalling implication. Many transcription factors are upregulated in the implementation of the EMT programme. Epithelial tumours undergo this process and seem to acquire invasive capacity and the potential to metastasise (see Lee et al., 2006). EMT implementation does enhance cell migration of scirrhous gastric cancer cells, which was inhibited by blocking TGF-β receptor kinase activity (Shinto et al., 2010). Lenferink et al. (2010) regard the ­apoptosis-associated protein clusterin as being involved downstream in the EMT programme activated by TGF-β. Activation of EMT occurs not only with TGF-β, but also with EGFR and HER2 receptors, suggesting the involvement of non-Smad signalling in the process. EGF might induce EMT by activating Ras and Erk1, which in turn can inhibit the EMTpromoting route paved by Smads1/4/5. In a similar vein, activation of the inhibitory Smad7 is a route adopted by IFN through STAT1 and TNF-α through NK-κB.

MicroRNAs, EMT and TGF-β Function Short single-stranded non-coding RNA molecules called microRNAs (miRNAs) play an important part in gene regulation; they differ from siRNAs in that siRNAs are ­double-stranded, small RNA molecules. miRNAs regulate gene expression by binding to the 3 untranslated regions of target genes and inhibit translation of or destabilise target gene transcripts. Their expression in a tissue-specific manner has been implicated with differentiation (Mishra and Bertino, 2009). miRNAs have been closely linked with many aspects of cell behaviour related to tumour growth and progression. Some species of miRNA are often downregulated in expression in neoplasia and have been regarded as tumour suppressors (Calin and Croce, 2006; Spahn et al., 2010; Zhang et al., 2007), where induced re-expression has been advocated as a possible means of anticancer therapy. miRNAs are also key players in differentiation and pattern formation in early embryonic development, which also involve EMT. Some miRNAs appear to inhibit cell migration and invasion. miRNA-146a and miRNA-146b seem to inhibit invasion and in vivo migration by MDA-MB-231 breast cancer cells (Bhaumik et al., 2008). A variety of genes and modes of signalling are targeted by miRNAs in bringing about inhibition of cell invasion. miRNAs have been closely implicated in activating the EMT programme. Several miRNAs have been seen to mediate the induction of EMT by TGF-β. miRNA-21 is often upregulated in cancers and promotes TGF-β-mediated activation of EMT (Zavadil et  al., 2007). In contrast, the members of the miRNA-200 family and miRNA-205 are said to be downregulated in cells that have undergone EMT stimulated by TGF-β. miRNA-200 and miRNA-205 suppress EMT by inhibiting transcription factors ZEB1 and ZEB2, which are known to promote EMT (Burk et al., 2008; Gregory et al., 2008; Korpal et al., 2008). The repression of E-cadherin is one prominent feature associated with the induction of EMT (Park et al., 2008). The expression of miRNA-200 and the ZEB factors is inversely related in epithelial

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ovarian cancers, and the miRNA could be promoting mesenchymal to epithelial transformation, the reverse process of EMT (Bendoraite et al., 2010). PDGF overexpression in PC3 cells leads to EMT with associated alterations in the cell phenotype. This seems to result from the downregulation of miRNA-200s. A result of this is the upregulation of ZEB1, ZEB2 and Snail2, which repress E-cadherin transcription (Kong et al., 2008, 2009). Sánchez-Tilló et al. (2010) found that ZEB1 recruits the co-repressor BRG1. Blocking this interaction induced the expression of E-cadherin and inhibited EMT, confirming the importance of E-cadherin in the transition. Furthermore, from the disposition of the ZEB1/BRG1 complex and E-cadherin, they suggested the involvement of this mechanism in tumour invasion. However, as stated above, miRNAs can have a contrary function. miRNA-101 targets the histone methyltransferase EZH2 and inhibits invasive behaviour of PC2 cells (Cao et al., 2010). The trimeric catalytic subunit PRC2/EEDEZH2 complex methylates lysine residues of histones, thus altering chromatin structure and regulating the transcription of target genes. Several genes, for example those encoding cell-cycle regulators, morphogenetic determinants, motility modulators and transcription factors, are repressed by the PRC2/EED-EZH2 complex. Nonetheless, the redeeming feature of the work reported by Cao et al. (2010) is the demonstration that miRNA-101 is regulated by AR (androgen receptor). They appear to be suggesting that suppression of EZH2 by the miRNA might be mediated by AR. However, androgens/ARs have recently been shown to achieve this through Rb/p130 pathways. EZH2 expression is lower in androgen-sensitive than in refractory cells. Androgen treatment increased the expression of E-cadherin, a target of EZH2, and inhibited migratory behaviour (Bohrer et al., 2010). As noted earlier, the suppression of E-cadherin is the cause of suppression of EMT by miRNA-200. Gastric cancer cell migration is inhibited by overexpression of miRNA-375, which seems to happen by inhibition of JAK2. Also, an inverse relationship seems to exist between miRNA-375 and JAK2 protein levels in gastric tumours (Ding et al., 2010). Quite clearly, various miRNAs inhibit migratory behaviour, albeit taking recourse to different target genes. Alterations in cell motility and acquisition of invasive ability are not an exclusive feature of cancer. Indeed, they are also an important part of embryonic development, differentiation and the developmental programme. EMT is activated in the specification of tissue types, which also involves TGF-β family ligands. The differentiation programme often finds active participation of TGF-β family ligands, such as TGF-β, Nodal, BMP and GDF. BMPs, for example, participate in neural induction by the Spemann’s embryonic organiser, constituted by the anterior end of the primitive streak stage embryo. BMP activity has been noticed along the dorso-­ventral axis with the presumptive neural ectoderm, which is induced to differentiate into neural tissue. Nodal is another TGF-β ligand that is active in early embryonic development and patterning. The formation of the germ layers, namely ectoderm, endoderm and mesoderm, and pattern formation are dependent upon Nodal signalling, which is an essential requisite for endoderm and mesoderm induction (Kimelman, 2006; Schier, 2003; Schier and Talbot, 2005; Shen, 2007). Nodal and GDF function has been postulated in this patterning, with the proposed ability of GDF to activate Nodal signalling and inhibition of BMP by Nodal ligands

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(referenced under the appropriate section pp. 36, 50). EMT is a differentiationrelated programme that is activated by TGF-βs, and possibly by activin and Nodal. In contrast, GDFs and BMPs could support the reverse process of MET. A common mode of regulating EMT-activating ligands might involve miRNAs. miRNAs have been implicated in the differentiation process. According to Choi et al. (2007), the Nodal ligand Squint and the Nodal inhibitors Lefty1 and Lefty2 are targets of miRNA-430. Nodal signalling appears to be regulated by the miRNAs by a balance of their respective activities. Thus, when Squint and the Nodal inhibitors are protected using antisense oligonucleotide morpholinos complementary to binding sites in miRNA-430, the result is enhanced or reduced Nodal signalling respectively. Activity of Nodal is also regulated by ­miRNA-15 and miRNA-16 by targeting the Nodal type II receptor. Putative miRNA-15 and -16 binding sites have been identified in this receptor (Martello et al., 2007). The migration of mesoderm from the anterior primitive streak requires the downregulation of E-cadherin and induction of EMT. The T-box transcription factor eomesodermin (Eomes) appears to be involved in EMT through the regulation of E-cadherin. Arnold et al. (2008) showed that in Eomes-deficient embryos the induction of EMT is blocked. In other words, Eomes is required for the downregulation of E-cadherin. During gastrulation, patterning of the mesoderm and endoderm along the proximodistal axis of the PS is regulated by graded levels of Nodal activity (Ben-Haim et al., 2006; Vincent et al., 2003). Further, Nodal and Eomes interact genetically and might be co-ordinately regulated. Arnold et al. (2008) have shown that developmental abnormalities occur in Eomes/Nodal double heterozygotes, which supports the concept of interaction between Eomes and Nodal. They suggest that such interactions are compatible with the finding of an activin response element in the promoter of Xenopus Eomes genes (Ryan et al., 2000). However, the precise nature of interaction between Nodal and Eomes is uncertain. miRNAs may promote apoptosis and inhibit cell proliferation. miRNA-200c functions by inducing apoptosis, possibly involving CD95/Fas. Zaman et al. (2010) found miRNA-145 to be downregulated in prostate cancer, and reported that when overexpressed by transfection, prostate carcinoma cells PC-3 displayed increased apoptosis. Apart from promoting apoptosis, miRNAs have been reported to function as tumour suppressors by targeting c-myc, the cell-cycle regulator cyclin A1 and the homeobox protein Six1. Six1 is targeted by miRNA-185 (Imam et al., 2010). These authors emphasised their findings with the observation that this miRNA shows decreased expression in breast, ovarian and renal cancers. Equally, however, there are reports that certain miRNAs are overexpressed in cancers. So re-expression as well as their downregulation might exert tumour inhibitory effects (Garofalo et al., 2008; Hammond, 2006) and the effects might be totally dependent upon the nature of the endogenous miRNA. miRNA-21 is highly expressed in tumours, for example breast cancers (Si et al., 2007). It is anti-apoptotic (Carletti et al., 2010; Chan et al., 2005) as well as being able to induce cell proliferation (Asangani et al., 2008; Roldo et al., 2006; Si et al., 2007). miRNAs that promote cell proliferation and tumour growth appear to do so by different mechanisms. miRNA-21 inhibited the tumour suppressor PDCD4

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(Programmed Cell Death 4) protein (Asangani et al., 2008; Frankel et al., 2008). PDCD4 is often downregulated in cancers and its tumour suppressor function seems to be due at least in part to inhibition of MAPK signalling pathway (Yang et al., 2006). The regulation of anti-apoptosis gene bcl-2 might also be involved in some instances (Si et al., 2007). Suppression of PTEN (Bar and Dikstein, 2010; Zhang et al., 2010) and inhibition of apoptosis (Yu et al., 2010) is one of the mechanisms. Correlation has also emerged between expression of some miRNAs with that of p53 and unfavourable prognosis (Pu et al., 2010). Other cell-cycle regulatory proteins may also be involved in the control of cell-cycle progression by miRNAs. Davis et al. (2009) found that PDGF induced proliferation of vascular smooth muscle cells and the transcription of miRNA-221, and further downregulated the targets c-Kit and p27Kip1. The downregulation of p27kip1 would have promoted cell proliferation. The downregulation of c-kit is believed to downregulate the expression of myocardin. These effects of miRNA-221 are in accord with the results of Felicetti et al. (2008), who found that the promyelocytic leukaemia zinc finger transcription factor PLZF represses miRNA-221 and -222. When PLZF is blocked and the function of the miRNAs is restored, p27Kip1 and c-kit are inhibited, leading to enhanced proliferation of melanoma cells. Obviously in both these studies, c-kit seems to play a different role from its conventional role of promoting angiogenesis. Stem cell factor and c-kit serve as an important signalling pathway to angiogenesis, but in the experiments with miRNA-221 and -222, it appears possible that c-kit might be activated by some other ligand (also see below). In the context of cell proliferation, it would be reasonable to cite the seemingly significant association of miRNAs with differentiation. Induction of differentiated phenotype in cancer has long been perceived, at least on a theoretical basis, as a mode of assuaging the effects of cancer. miRNA-1 and miRNA-206 are believed to be downregulated in poorly differentiated rhabdomysarcoma, but they do occur in differentiated skeletal muscle, suggesting their association with the differentiated state (Tauli et al., 2009). However, one can only view this as a point of departure towards induction of differentiation by promoting the function of miRNAs. There are also suggestions that miRNAs might be responsible for drug resistance and radiation sensitivity of tumours (van Jaarsveld et al., 2010). The acquisition of drug resistance has in parallel shown upregulated expression of certain miRNAs and P-glycoprotein (Li et al., 2010). Therefore, they might be important in developing treatment strategies. miRNAs basically target different genes with varying outcomes. The expression or lack of expression of miRNAs has been attributed to epigenetic mechanisms, so silencing or activation of miRNA genes, as appropriate, might be a potential avenue of approach. It has been noticed that miRNA expression can be highly variable, even within one tissue type (Pereira et al., 2010). This lack of uniqueness of expression (Pereira et al., 2010) could be another obstacle, though not insurmountable, in applying miRNAs as tools in treatment or as an aid in assessing progression and prognosis. Besides, miRNAs seem to interact with and regulate signalling by ER and AR, and in turn are themselves regulated by steroid receptors (Tessel et al., 2010) and other growth factors such as the BMPs. IFN-β has been shown to modulate rapidly the expression of miRNAs that target hepatitis C viral genomic

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RNA (Pedersen et al., 2007). Also IFN-β may be subject to regulation by miRNAs in innate immune responses (Witwer et al., 2010). IFN-γ receptor α is a target of miRNA-155 in Th1 cell differentiation (Banerjee et al., 2010). miRNAs have been strongly implicated in TNF-α signalling in intercellular adhesion and can target cell proliferation pathways involving PTEN and Akt. They also participate in apoptosis signalling through death receptor by the Fas ligand, TNF-α, TRAIL and activation of caspases. These have been discussed in some detail in the section dealing with TNF-α (p. 118). EMT plays an essential role in cancer development and progression. Hence, as noted earlier, miRNAs can mediate EMT programming activated by TGF-β and PDGF. The promotion of progression through stimulation of invasion is actuated by the suppression by miRNAs of E-cadherin expression. PDGF overexpression can lead to EMT with associated activation of EMT. Changes in cell behaviour, repression of E-cadherin transcription and cell proliferation induced by PDGF are mediated by miRNA-221. miRNA-221 seems to be a pro-progression miRNA. It is upregulated in expression with miRNA-222, inducing in vitro expression of malignant features. Interestingly this seems to be reversed by inhibiting the expression of the miRNAs, which also inhibit invasive behaviour and cell growth (Zhang et al., 2010). In hepatocellular carcinoma, miRNA-221 targeted the anti-apoptosis protein Bmf and inhibited apoptosis. Besides, overexpression of the miRNA seemed to correlate with a more aggressive phenotype with earlier recurrence after resection (Gramantieri et al., 2009). However, one should recall that miRNA-221 is reported to be inhibitory of angiogenesis. Its expression is downregulated in prostate cancer, which seems to relate to aggressive behaviour of tumours and correlates with recurrence (Spahn et al., 2010). These authors also found that miRNA-221 expression was negatively related to the expression of c-kit receptor for stem-cell factor, which has been implicated in angiogenesis. Davis et al. (2009) and Felicetti et al. (2008), on the other hand, reported that PDGF induced transcription of miRNA-221 and proliferation of vascular smooth muscle cells. However, they found c-kit downregulated as well. It may be that the conventional c-kit signalling promoting angiogenesis might subsist in prostate carcinoma in which miRNA-221 downregulation has the effect of upstaging the effects of c-kit, and that in the presence of PDGF c-kit signalling might have altered. It is fairly obvious from the above discussion that many questions still remain to be resolved. Not too infrequently, the same species of miRNA or members of the same family can be overexpressed in some tumour types but downregulated in others. In other words, a consistent pattern of expression is not always encountered. With the numerical abundance of miRNAs, the spectrum of positive and negative regulator species and the variety of biological effects they exert, attribution to individual miRNAs of regulatory properties with any degree of specificity remains elusive.

TGF-β Operates in Other Signalling Systems of Cell Adhesion and Motility A significant role has been assigned to TGF-β and other chemokines in the promotion of cell migration. The chemokine CCL23 and its receptor, for instance, induce

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chemotaxis and migration of endothelial cells and angiogenesis (Hwang et al., 2005). An important requirement for successful invasion is cell adhesion. There is some indirect evidence that TGF-β might contribute to cellular adhesion in collaboration with certain integrins. Hypoxia increases TGF-β and α2-, α3- and α5-integrin expression with concomitant increase in adhesion ability of gastric cancer cells (Noda et al., 2010). The Smad pathway has also been invoked in the promotion of invasion by the interaction of the metastasis promoter S100A4 with Smad. S100A4 has been reported to bind to the N-terminal region of Smad3. In an MCF7 derivative cell line, S100A4 enhanced MMP-9 expression and invasion induced by TGF-β; by inference, this could be a result of S100A4–Smad interaction (Matsuura et al., 2010). This is a novel attribution to S100A4. S100A4 is known to interact and bind with many cellular proteins, not least p53 (Sherbet and Lakshmi, 2006). The binding and sequestration of p53 by S100A4 leads to the abrogation of the cell-cycle regulatory property of p53 and to unregulated tumour growth. The Smad-mediated effect of S100A4 further supplements the versatility of the protein to alter cell behaviour and promote tumour progression. Another possibility is suggested for Smad involvement in TGF-β-induced migration. Vasilaki et al. (2010) have found that TGF-β activates MEK/MAPK signalling, which leads to Smad3 binding to a promoter element of the small GTPase RhoB gene and inducing its transcription. Smad mediation is abolished when this promoter element is mutated. Vasilaki et al. (2010) also showed that inhibition of RhoB inhibited TGF-β-induced cell migration (Figure 3.2). Coupled with the ability to induce angiogenesis, TGF-β would be a major contributor to the process of cancer progression to the metastatic state. TGF-β seems to induce tumour-associated vascularisation by inducing VEGF expression with other angiogenic factors. Inhibition of TGF-β function reduces both vascularity and VEGF expression (Wilson et al., 2010). An additional criterion worthy of consideration is that Smads might differentially affect biological properties of migration, and possibly the metastatic ability of cancer cells. TGF-β differentially transcribed Smad2 and Smad3, which corresponded with VEGF expression (Petersen et al., 2010). Indeed, Smad4 has been shown to inhibit cancer progression, albeit in murine tumour models (Zhang et al., 2010). It is needless to emphasise that because other TGF-β family ligands transactivate the same receptors and use the canonical signalling system, it would be reasonable to suggest that other ligands of the family might function in a similar fashion. So Smadmediated and -independent effects on cancer invasion and metastasis can be due to several factors, including selective recruitment of Smads, and to switching of signalling pathways with interaction between the two modes (Figure 3.2).

Interaction between Smad and Non-Smad Signalling Interaction between, and mutual regulation of, Smad and non-Smad signalling is an obvious means of influencing the biological function of TGF-β. As already pointed out, loss or deficiency of Smad signalling does not necessarily lead to neoplasia, for growth and progression of cancer might take recourse to other signalling pathways

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(Yang and Yang, 2010). In other words, abrogation of the canonical pathway might lead to the determination of biological outcome by non-Smad or an alternative signalling pathway. Apart from conceptualisation of the context-dependent phenotypic effect of TGF-β, one might be able to glean therapeutically valuable information from a scrutiny of this form of switching of signalling.

Signalling Links between BMP, Oestrogen and ER An outstanding example of mutual regulation of Smad pathway with other signalling systems is provided by BMP function. Oestrogens are mitogenic steroids functioning through activation of ER-α and ER-β receptors. In an environment with the presence of TGF-β, the mitogenic influence of the steroids would be alleviated. Indications are that interactions might be occurring between TGF-β ligands and ER signalling at genomic and non-genomic levels. ER-α has been found to form complexes with Smad and the ubiquitin ligase Smurf, thus leading to the degradation of Smad by ubiquitination and to the inactivation of TGF-β signalling (Ito et al., 2010). On the other hand, a genomic route is probably suggested by other recent findings. Matsumoto et al. (2010) have shown that BMP2 enhances the expression of ER-α and ER-β in murine myoblastic C2C12 cells and that oestradiol enhanced BMP-induced Runx2 and osteocalcin expression, which is related to the mineralisation of the bone at late stages of differentiation. Intriguingly, however, oestradiol seemed to increase BMP2-induced phosphorylation and activation of Smad1/5/8 and Id-1 promoter activity. Id-1 encodes the helix–loop–helix protein that is associated with epithelial cell proliferation, invasion and differentiation. BMP, but not TGF-β, strongly activates the Id-1 promoter in a Smad-dependent manner (Korchynskyi and ten Dijke, 2002). Yang et al. (2007) found that ER MCF7 breast cancer cells expressed high levels of BMP6 and E-cadherin, but low levels of δEF1 transcript, whereas ER-negative MDA-MB-231 cells expressed BMP6 and E-cadherin mRNA at significantly reduced levels. This inverse relationship existed also in breast cancer tissues. BMP6 caused inhibition of δEF1 transcription, which correlated with upregulation of E-cadherin mRNA expression. As further confirmation, these authors showed that extraneous δEF1 inhibited E-cadherin expression. With E-cadherin expression known to be intimately linked with invasive behaviour, these findings suggest a clear genomic effect of ER. Otsuka et al. (2009) found that BMP2, -4, -6, -7 and activin suppressed oestradiol-induced cell proliferation; BMP6, -7 and activin were more effective than BMP2 and -4. Besides, they suggested that p38 MAPK signalling and the expression of steroid sulphatase, which catalyses the production of oestrogens, might play an important part in suppressing the mitogenic effects of oestrogen in breast cancer cells. One can summarise with the comment that TGF-β and BMP can signal phenotypic changes in cell proliferation, invasion and differentiation by interacting with ER signalling, either by non-genomic routes or by directly influencing ER-mediated transcription of target genes. Although these findings relate to specified states of differentiation and invasion, the participation of the oestrogen/ER, and the induction of ER by BMP, provides reasonable grounds for suggesting their potential significance in developing new strategies for breast cancer management. Ye et al. (2010) found

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reduced BMP10 expression in breast cancer, which correlated with disease progression and poor prognosis. MDA-231 cells transfected with a full-length BMP16 showed inhibited cell proliferation and motility in vitro. Davies et al. (2007) reported that BMP2 expression was lower in breast tumours than normal tissues. BMP2 transcript levels correlated with moderate to poor prognosis. In contrast, BMP2 was reported to be overexpressed by ovarian carcinoma cells in vitro. Treatment with recombinant BMP2 led to rapid phosphorylation of Smad1/5/8 and Erk/MAPKs, and increased expression of Id-1, Smad6 and Snail mRNAs. Furthermore, high BMP2 levels correlated with poor survival of patients (see Le Page et al., 2006, 2009). Helms et al. (2005) noticed high expression of BMP receptor RIB (BMPRIB) was associated with high tumour grade, enhanced tumour growth and poor prognosis in ER carcinomas, which they attributed to the activation of BMP/Smad signalling. Equally this could have resulted from ER-mediated inhibition of TGF-β signalling. Also, oestradiol has been shown to regulate the expression of BMPRII and BMPRIB, but here BMPRIB was expressed in ER-negative tumours as well. Although BMPs function mainly by binding to their own dedicated receptors BMPRI and II, they can also bind other TGF-β family receptors, albeit with perceived differences in the affinity of recognition by BMPs of their own receptors as well as other members of the receptor family. Furthermore, the effects are not identified with any specific BMPs. The pleiotropic nature of BMP-mediated phenotypic responses has been fully recognised. So the situation is somewhat complex and probably not amenable to a simple explanation. Overall, the inescapable conclusion is that it is premature to suggest strategies based on BMP expression in cancer management.

Functional Synergy and Regulation between Smad and MAPK Pathways Growth factors, GPCRs and several other biological response modifiers including stress and phorbol esters signal through a cascade of kinases of the MAPK system. MAPK and its downstream effector kinases MEK, MKK and MEKK are sequentially activated by phosphorylation. Further downstream they signal through components of the system, such as Erks, p38 and SAPK/JNK, by activating specific transcription factors and immediate early and early response genes leading to cell-cycle regulation, proliferation and apoptosis, and activation of developmental programmes. Non-Smad pathways have been shown to participate in TGF-β signalling. MAPK signalling has been linked with many processes mediated by TGF-β. As discussed earlier, TGF-β activates MEK/Erk as a means to the recruitment of Smad3 to a noncanonical system involving RhoB, which possesses a Smad binding element in its promoter (Vasilaki et al., 2010). An important biological effect of TGF-β is the promotion of EMT. Erythropoietin prevents this effect of TGF-β. Erythropoietin has been found to inhibit Smad3 phosphorylation and its nuclear translocation. MAPK signalling appears to be involved in this process, as indicated by the inhibition of both erythropoietin effects and activation of the Smad route by TGF-β by inhibition of MAPK signalling (Chen et al., 2010). A synergistic and collaborative function

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of the Smad pathway with non-Smad p38/MAPK has been described in the induction by TGF-β of vascular smooth muscle cell differentiation. This effect of TGF-β occurred in parallel with upregulation of KLF4 (Kruppel-like factor 4) and TGFBRII receptors. Furthermore, KLF4 activated both Smad2 and Smad3, and p38/MAPK (Li et al., 2010). A collaborative function of this kind has also been reported between Smad2 and MAPK (ERK1/2, JNK, p38) pathways in the induction of collagen IV synthesis in rat mesangial cells (Jiang et al., 2010).

Nodal Signalling in Development, Differentiation and Pattern Formation Nodal is a member of the TGF-β family of growth factors. However, it is regarded as a peripheral member rather than a core constituent. It bears only approximately 25–40% sequence homology to the core members of the family. Its nomenclature derives from its prominent expression in the Hensen’s node of the primitive-streak stage embryo. Vertebrates display three major developmental axes: anterior–posterior, dorsal– ventral, and left–right. The left–right axis is not invariably symmetrical, especially in relation to the visceral organs. Nodal is expressed asymmetrically after gastrulation in the left lateral plate mesoderm, determining the left–right asymmetry (Lohr et al., 1997; Rebagliati et al., 1998a). That it forms the key component in neural induction has been demonstrated by a mutation in the so-called cyclops locus, which encodes Nodal protein (Rebagliati et al., 1998b). Indeed, Nodal also needs to be controlled with precision in the formation of the endoderm, as well as neural differentiation. The asymmetric expression of Nodal corresponds with the direction of heart looping, torsion and embryonic turning (Collignon et al., 1996b). Alterations in Nodal expression seem to relate closely to the incidence of mirror-image-reverse (situs inversus) and inversion of embryonic turning (Lowe et al., 1996).

Regulation of Nodal Signalling As a member of the TGF-β family, the varied functional information of Nodal is channelled along the canonical pathways involving the Smad proteins that target specific transcription factors so that appropriate genetic information is downloaded.

Differential Activation of Contributory Pathways Early embryonic development involves Nodal, which is associated with left–right patterning of the embryonic axis. Many TGF-β members have been attributed with the ability to modulate Nodal signalling, namely to activate Nodal signalling or suppress it. Thus, depending upon the signalling by the modulating ligand, Nodal signalling could be altered or switched in either direction. This has the potential of activating transcription factors in a selective manner, as required for the specification

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of differentiation of the presumptive progenitor cells. This is achievable also by differential activation of related pathways, as demonstrated in the differentiation of the notochord and the neural plate in early embryogenesis. Indeed, Nodal seems to inhibit the differentiation of ectodermal cells into neural tissue while promoting and regulating mesoendodermal differentiation. Hudson and Yasuo (2006) have described a detailed study of Nodal signalling in the development of the notochord in the ascidian urochordate (sea squirt) Ciona intestinalis. Of the single row of 40 cells constituting the notochord, the anterior 32 ‘primary’ notochord cells arise from the A-line, anterior vegetal hemisphere of the eight-cell stage blastomere. The posterior eight notochord cells come from the B-line posterior vegetal hemisphere cells of the blastomere. The specification of the posterior eight notochord cells depends upon Nodal signalling received from lateral locations along the B-line cells as apparent from the downregulation of expression of the notochord specific gene Ci-Brachyury upon inhibition of Nodal signalling. On the other hand, Nodal signalling of the A-line cells functions through the Delta2/Notch pathway. On similar lines are the findings of Hudson et al. (2007) that three signalling pathways, namely Nodal, Delta2/Notch and FGF/MEK/ERK, are integrated to specify the differentiation of neural-plate precursor cells by the co-ordinated functioning of these three signalling pathways.

Selective and Differential Activation of Transcription Factors Nodal signalling functions in conjunction with other TGF-β family members but also requires other mediating functional elements. The Pitx (paired-like homeobox transcription factor) acts downstream of these genes and displays a marked left–right asymmetric expression. Pitx2 expression pattern corresponds with that of Nodal and Lefty in the left lateral plate mesoderm. In subsequent development the left handedness of the expression of Pitx2 appears in the heart and certain visceral organs (Logan et al., 1998; Piedra et al., 1998; Ryan et al., 1998; St Amand et al., 1998; Yoshioka et al., 1998). Furthermore, Pitx2 expression is regulated by Nodal by the mediation of FoxH1 (Forkhead box H1). The locus Schmalspur encodes FoxH1 transcription factor. Maternal Schmalspur transcripts are found in the animal pole during oogenesis and then in specific domains in the organiser, notochord and lateral plate mesoderm (Pogoda et al., 2000). FoxH1 is expressed in early developmental stages, binds Smad proteins and mediates signalling by TGF-β family ligands (Attisano et al., 2001; Chen et al., 1997). Yoshida and Saiga (2008) showed that Nodal requires a single FoxH1-binding site present in an intronic enhancer to impel Pitx expression in the left epidermis. Essner et al. (2000) have identified two isoforms of pitx2: pitx2a and pitx2c in the zebra fish. These not only show distinct patterns of expression but also appear to be regulated by distinct genetic pathways during mesoendoderm and asymmetric organ development. The endoderm is patterned along the anterior–posterior axis to evolve into distinct derivatives. The regulation of early mesoendoderm Pitx2c expression is dependent upon EGF–CFC (epidermal growth factor–Cripto-1/FRL1/cryptic)

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genes called one-eyed pinhead required for Nodal signalling (Gritsman et al., 1999) and spadetail of the T-box (Tbx) gene family of transcription factors, and can be induced by expression of the ectopic homeobox transcription factor Goosecoid. In zebra fish development, the gene Schmalspur seems essential in Nodal signalling because it appears to influence the expression of other genes involved in Nodal signalling. Schmalspur interacts with notail and spadetail T-box genes (Rojo et al., 2001). Thus Nodal, Schmalspur and associated genes might be essential genetic ­factors for maintaining expression of pitx2c in axial mesoendoderm. One should hasten to add that there are no human equivalents of these zebra fish Tbx transcription factors (Lardelli, 2003). It is needless to say that Nodal signalling would need to be regulated so that mesoderm and endoderm formation, the positioning of the anterior–posterior axis, neural patterning and left–right axis specification are accurately and stringently controlled. Deficiency or inhibition of Nodal signalling is known to inhibit differentiation of mesoderm and endoderm, and Nodal is regarded as a key inducer of mesoendoderm during gastrulation. Vallier et al. (2004) investigated the effect of recombinant Nodal protein in HESC (human embryonic stem cells) cultures and in HESC cultures carrying Nodal transgene. Control HESC displayed a marked upregulation of neuroectoderm markers, whereas in contrast cells exposed to recombinant Nodal protein showed a reduction of neuroectoderm markers. Embryoid bodies formed of HESC expressing Nodal showed no upregulation of neuroectoderm markers. The regulation of Nodal signalling can be due to certain inhibitors or be subject to the influence of co-receptor required for the signalling process. The Lefty and Cerberus families of proteins function as extracellular antagonists of Nodal signalling. Cerberus belonging to the cystine-knot superfamily is an inhibitor of Nodal and the TGF-β signalling pathway. Cerberus proteins are secreted during gastrulation in embryogenesis (Entrez Gene Data Base, 2009). Lefty proteins, of which two subtypes have been identified (Kosaki et al., 1999), belong to the TGF-β family, but they antagonise Nodal signalling and so are involved in the regulation of the left–right symmetry (Hamada et al., 2002). The implication and the consequences of the loss of such regulation is emphasised by Perea-Gomez et al. (2002). They demonstrated the development of abnormalities of primitive streak formation in Cerberus-like(/); Lefty1(/) mutants. These defects could be rectified by deleting a copy of the Nodal gene in Cerberus-like(/); Lefty1(/) double-mutant embryos. ALK4 and either ActRIIA or ActRIIB function as receptors for Nodal. Now the inhibition of Nodal can be rescued by an excess of ActRIIA or ActRIIB, which can be interpreted as indicating that Leftymediated inhibition of Nodal signalling might function by competitive binding to the common receptor ActRIIA or ActRIIB (Sakuma et al., 2002). Lefty interacts with EGF–CFC proteins and prevents them from being a part of the Nodal receptor complex. Cripto is an EGF–CFC that interacts with the type I receptor ALK4 through the conserved CFC motif in Cripto. This Cripto interaction with ALK4 is required in both Nodal binding to the ALK4/ActR-IIB receptor complex and Smad2 activation (Yeo and Whitman, 2001).

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

Nodal, miRNA and EMT TGF-β family ligands, such as TGF-β, Nodal, BMP and GDF, show active participation in developmental and differentiation programmes, such as formation of the germ layers, neural induction and differential patterning of development. Nodal is eminently active in early embryonic development and patterning. Nodal signalling is essential for the formation of the ectoderm, endoderm and mesoderm, and for endoderm and mesoderm induction. TGF-βs, and possibly also by activin and Nodal, activate the differentiation programme EMT. miRNAs are important modulators and regulators of gene expression. As discussed in detail in an earlier section, miRNAs mediate EMT activation by Nodal. According to Choi et al. (2007), the Nodal ligand Squint and the Nodal inhibitors Lefty1 and Lefty2 are the targets of miRNA430. Nodal signalling is regulated by the miRNA by a balance of their respective activities. Disruption of this balanced function markedly modulates Nodal signalling. Thus when Squint and the Nodal inhibitors are protected using antisense oligonucleotide morpholinos complementary to binding sites in miRNA-430, the result was enhanced or reduced Nodal signalling respectively. Activity of Nodal is also regulated by miRNA-15 and miRNA by targeting the Nodal type II receptor. They identified putative miRNA-15 and -16 binding sites in this receptor (Martello et al., 2007). The activation of EMT is associated with the downregulation of E-cadherin and induction of cell motility. Downregulation of E-cadherin occurs in the migration of mesoderm from the primitive streak between ectodermal and endodermal layers. Arnold et al. (2008) have shown that the T-box transcription factor eomesodermin (Eomes) is involved in EMT through the regulation of E-cadherin. Eomes-deficiency blocks EMT, implicating the requirement of Eomes for the downregulation of E-cadherin. During gastrulation, patterning of the mesoderm and endoderm along the proximodistal axis of the primitive streak is regulated by graded levels of Nodal activity (Ben-Haim et al., 2006; Vincent et al., 2003). Nodal and Eomes interact genetically and might be co-ordinately regulated. Developmental abnormalities have been encountered in Eomes/Nodal double heterozygotes, which strongly support the concept of interaction between Eomes and Nodal. It is significant in this context that an activin response element has been identified in the promoter of Xenopus Eomes genes (Ryan et al., 2000).

Requirement of Cofactors in Nodal Regulation Another mode of Nodal regulation that easily comes to mind is that exerted by the requirement of certain cofactors. Camus et al. (2006) encountered greatly augmented neuroectodermal differentiation in Nodal-negative mutants. Cripto (EGF–CFC protein) is a co-receptor for Nodal. Cripto has a single divergent EGF-like motif as well as a novel cysteine-rich domain termed the CFC (Cripto/FRL1/Cryptic) motif. Cripto is bound to the cell membrane by a glycosyl–phosphatidyl inositol link. The involvement of EGF–CFC proteins in determining the specification of the anterior–­ posterior and left–right body axes, as well as in formation of the primary germ layers during gastrulation in early embryonic development has been well established

Growth Factors in Differentiation and Morphogenesis

35

(Shen and Schier, 2000; Whitman, 2001). The loss of CFC function by mutation leads to left–right laterality defects (Bamford et al., 2000), obviously consequential upon abrogation of proper signalling by Nodal. Cripto engages Nodal to an activin receptor complex consisting of a type I ­serine– threonine receptor ActRIB dimer (ALK4) and a dimeric type II activin receptor, either ActRII or ActRIIB. The CFC motif mediates the interaction of Cripto with ActRIB, whereas the EGF motif mediates binding Cripto to Nodal. For this to happen the EGF motif is modified by the addition of an O-linked fucose monosaccharide. Effectively this glycosylation seems to regulate Nodal signalling (Schiffer et al., 2001; Yan et al., 2002). Nodal signalling cannot take place if EGF–CFC co-receptors are not available, but Activin signalling can do so by virtue of it being able to transduce its signal independently of a co-receptor (Kumar et al., 2001; Yan et al., 2002). ALK7 seems able to mimic Xenopus Nodal signalling and has been postulated as a more specific receptor for Nodal (Reissmann et al., 2001). The unavailability of Cripto (/) in murine embryonic stem cells seems to subvert Nodal signalling and make them prone to neuronal and neuroectodermal/­ epidermal type differentiation (Sonntag et al., 2005). Cripto (/) embryos show normal organisation of anterior prosencephalic and mesencephalic regions (Liguori et al., 2003). On similar lines is the finding that Lefty expression also drives human embryonic stem cells into ectodermal and neural differentiation (Dvash et al., 2007; Watanabe et al., 2005), attributable quite obviously to Nodal inhibition. In contrast, human embryonic stem cells exposed to recombinant Nodal protein showed a reduction in the induction of neuroectodermal markers (Vallier et al., 2004).

Functional Stability of Nodal The stability of proteins is viewed mainly in terms of molecular folding and unfolding, which is in turn influenced by hydrophobic interactions and hydrogen bonds. Thus proteins are destabilised when the polar groups in the molecule are sequestered. In contrast, the sequestration of hydrophobic bonds and initiation of disulphide linkages promote protein stability. Molecular modifications also impinge upon another aspect of stability, namely functional stability. As noted earlier, growth factors can function either in an autocrine fashion or as paracrine factors. In the TGF-β family, the processing of the precursor has a significant bearing on the mode of function. The prodomain inhibits the activity of many proteins and offers a target for protein stabilisation. This is because the presence of the prodomain confers a compact structure on the molecule and allows greater stability. The removal of the prodomain, for example in Nodal, reduces Nodal stability but appears to promote autocrine signalling. The stability of mature Nodal is enhanced by the insertion of an N-glycosylation site, and with this modification Nodal can assume a paracrine mode of signalling (Le Good et al., 2005). Le Good et al. (2005) have pointed out that the glycosylation site is present in several TGF-β family proteins. Possibly this alteration might be related to their ability to function as a paracrine growth factor. On similar lines is the finding that BMP4 is activated by furin-mediated cleavage at sites within the prodomain. Cleavage at the site adjacent to the mature protein allows cleavage

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

at the upstream site within the prodomain. BMP4 derived from precursor with an uncleaved upstream site is less active and functions at a shorter range. However, upon being cleaved correctly, BMP4 signals rapidly and over a greater range (Cui et  al., 2001). In other words, the mode of processing determines the mode of autocrine or paracrine mode of functioning. The processing of Nodal can occur virtually in situ, for example as in the extraembryonic ectodermal cells that provide a regulation and maintenance environment for trophoblast stem cells. These extra-embryonic ectodermal cells produce furin and PACE4 required for the activation of Nodal signalling. The activated Nodal seems then to induce the expression of FGF4, which regulates the differentiation of the trophoblast stem cells (Beck et al., 2002; Guzman-Ayala et al., 2004). Beck et  al. (2002) showed that Nodal might be inducing Cripto, which is a ligand and a coreceptor for Nodal as stated earlier. Explants from embryos carrying double-negative mutations of furin and PACE4 producing unprocessed Nodal do not induce Cripto, but recombinant mature Nodal can induce Cripto in these explants. Furthermore, Cripto is also induced by BMP4, hence Beck et al. (2002) have suggested that furin and PACE4 are both involved with the Nodal- and BMP4-mediated signalling pathway in this regulatory system. Indeed, the uncleaved Nodal maintains the production of furin and PACE4 in the extra-embryonic source and BMP4, so amplifying Nodal signalling (Ben-Haim et al., 2006).

Bone Morphogenetic Proteins Bone differentiation and morphogenesis involve many growth factors and cytokines. Bone morphogenetic proteins (BMPs) form a large family of secreted proteins and constitute an important subgroup of the TGF-β family. BMPs have been known as important regulators of early embryonic development and participate prominently in the differentiation of bone, cartilage and connective tissue. Indeed, they play a major regulatory role in differentiation, growth and apoptosis of a variety of cell types, for example epithelial, mesenchymal, haematopoietic and neuronal cells. They are also involved with the development of the nervous system, heart, kidneys, lungs, skin and gonads (Hogan, 1996). BMPs are highly conserved in evolution. They are formed from protein precursors by enzymatic cleavage by furin, a proprotein convertase. Cleavage at the furin site with an RXXR motif releases the mature peptide, a feature shared by TGF-β proteins. The C terminus of BMPs contains seven cysteine amino-acid residues. BMPs are regarded as subgroup of the TGF-β family by virtue of the structural homology between them and other members of the TGF-β family. The sequence homology extends to the conserved seven cysteine residues (Elima, 1993; Wozney, 1992). These are involved in intramolecular linkage, and one cyteine participates in homo- or heterodimerisation into active BMPs. Several BMPs have been identified, for example BMP2–7, BMP8a, BMP8b, BMP9 (also called GDF2), BMP10 and BMP15. The BMPs share the TGF-β family receptor system and signalling cascade with other members of the TGF-β family. They show a varied profile of expression in

Growth Factors in Differentiation and Morphogenesis

37

human tissues. The expression profiles may be related to tissue morphogenesis and function. With such a wide spectrum of participation in cellular function, it is hardly surprising that BMPs would be implicated in pathogenesis by the abnormal or aberrant expression of their own receptors. BMP1 is distinguishable from the rest of the BMPs and is considered to form a subgroup of secreted metalloproteinases with the astacin-like proteinases. Astacins are a family of zinc-endopeptidases of which Meprins are the mammalian homologues (Johnson and Bond, 1997). Astacins are involved in the formation of the extracellular matrix by the generation of mature ECM components. BMP1 is known to activate TGF-β1, GDF8 and GDF11 (also called BMP11) and BMP2 and BMP4 precursors into mature and functionally active growth factors. BMP1 activates BMP2 and BMP4 by cleaving the extracellular antagonist Chordin (Ge and Greenspan, 2006a, 2006b). Bone morphogenesis consists of several sequential events including chemotaxis, mitosis and differentiation (Reddi and Huggins, 1992). Bone development, remodelling and repair involve the recruitment of primary mesenchymal progenitor cells and their differentiation into osteoblast lineage. BMPs are known to stimulate chemotactic migration of these progenitor cells (Fiedler et al., 2002). The existence of a large number of BMPs has suggested a possible functional redundancy (Reddi, 1994). On the contrary, Cheng et al. (2003) have suggested that BMP-induced osteoblast lineage-specific differentiation of mesenchymal stem cells displays a hierarchical model of involvement and function of specific BMPs, and that BMPs9 and 10 bind to ALK1, the endothelial-specific receptor of the TGF-β receptor family. David et al. (2008) found that human serum is capable of inducing Smad1/5 phosphorylation, which was inhibited by anti-BMP9 antibodies, suggesting clearly that BMP9 is the growth factor that induced phosphorylation. Furthermore, it is intriguing that BMP inhibited the formation of endothelial sprouts and so inhibited both VEGF and bFGF-induced neovascularisation. This is an aspect of the capability of BMPs that would be of considerable significance in the context of secondary spread of cancer. BMPs 2, 4 and 9 appear to be involved in iron homeostasis. These BMPs and IL6 upregulate the expression of hepcidin, a peptide hormone that preserves iron homeostasis. The STAT site in the human hepcidin promoter is required together with some additional regulation of responsiveness to BMP9 (Truksa et al., 2007a, 2007b). BMPs seem to function with a co-receptor, the hemojuvelin (HJV) receptor, in regulating hepcidin expression and iron metabolism. HJV selectively recruits the BMP type II receptors ActRIIA and BMPRII for the BMPs (Xia et al., 2008). The hepcidin promoter has a BMP-responsive element, which mediates hepcidin mRNA expression (Falzacappa et al., 2008). Interestingly, GDF15 has been reported to suppress the expression of hepcidin and is found to be overexpressed in thalassaemia (Tanno et al., 2007). However, it is unclear at present if the BMPs and GDFs together regulate iron homeostasis by their opposing effects on hepcidin expression. The eponymous nomenclature of BMPs as major determinants of bone morphogenesis has undoubtedly focused attention on chondrogenesis. However, BMP function is not restricted to bone morphogenesis. Indeed, early embryogenetic programmes actively involve BMPs, for example in the differentiation of neural tissue,

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

the morphogenesis of the neural plate into forebrain and then into optic and otic placodes, and possibly also into the subsequent events of differentiation. BMP function can be detected very early in embryonic development in the perceived restriction of competence for neural differentiation in a temporal and spatial sequence. BMP12 is also frequently referred to as GDF7. The mature protein has 146 amino-acid residues, with the conserved seven cysteine residues. BMP12/GDF7 shares sequence homology with GDF5 and GDF6 and has been attributed with pleiotropic function of chondrogenesis, bone morphogenesis and neuronal differentiation multiple functions. Signalling by BMP12/GDF7 includes BMPRIB and BMPRII or Activin RII and Smads. A close involvement with neuroectodermal differentiation coupled with epidermal and neural crest differentiation amply testifies to the unquestioned significance of BMP participation in developmental processes. BMPs can induce the differentiation of the lateral plate mesoderm. Not only are they associated with these early stages of development and differentiation, but they also appear to take part in specialised processes as well as in terminal processes of differentiation such as induction of dendritic extensions and maturation of ovarian follicles. The expression of BMPs in the growth and maturation of ovarian follicles follows a seemingly stage-dependent and regulated pattern. They are also involved with the development of the heart, kidneys, lungs and skin. Their involvement in many forms of human disease has been documented (see references in Table 3.3).

Heterogeneity of BMP Signalling Cascade and Phenotypic Effects BMPs appear to be pleiotropic in function, being able to regulate cell proliferation, differentiation and morphogenesis. Often the phenotypic effects might be contradirectional. The complexities of BMP functioning have been obvious for some time. Although BMPs 2, 6, 7 and 9 are all capable of inducing osteogenesis, BMP3 seems to inhibit the process, but only osteogenesis induced by BMP9 is inhibited (Kang et  al., 2004). This quite clearly reflects a divergence in signalling to determine the same differentiated phenotype. Another dimension to the complexity is added by connective tissue growth factor (CTGF), which is a downstream mediator of TGF-β. TGF-β upregulates CTGF expression in chondrogenesis and osteogenesis (Oka et al., 2007), and as a fibrosis-promoting agent participates in wound healing and fibrosis. It initiates mesenchymal cell condensation which occurs preparatory to cartilage formation. Cells stimulated in vitro by TGF-β1 show a concomitant upregulation of CTGF (Song et al., 2007). Luo et al. (2004) demonstrated an upregulation of CTGF in the early stages of BMP9 and Wnt3A stimulations. Wnt was able to provide osteogenic stimulus independently of BMP9; however, when constitutively expressed, CTGF led to the inhibition of both BMP9 and Wnt3A. This suggests a close link-up between BMP and Wnt signalling pathways. Given that BMPs as members of the TGF-β family use the TGF-β family receptors and the canonical signalling pathway involving Smads, how is such multiplicity of phenotypic effects generated by the large number of BMPs identified so far? Besides, more than one BMP would appear to be capable of generating the same

BMP ID

Phenotype

BMP9

Chondrogenesis

Receptor/Signalling Cascade

Transcription Factors

References

STAT

Truksa et al. (2007a, 2007b)

BMPRIb/II; ActRI, Ib; II; ALK1; Smads 1–5/ Smad8→Smad4

BMP

BMP2

Lopez-Coviella et al. (2006); Brown et al. (2005) Helix–loop–helix Hey1; Runx

Sharff et al. (2009)

Sox genes

Chimal-Monroy et al. (2003)

Neural tissue differentiation

Sox, Fox; Helix–loop–helix Zic, Pou

Sasai et al. (2001)

Neural differentiation in Xenopus ventral ectoderm

Sox1

Nitta et al. (2006)

Embryonic stem cells

Sox, STAT [with FGF; Activin and Nodal]

Sun et al. (2008)

Chondrogenesis in fracture repair

Sox genes

Uusitalo et al. (2001)

Osteoblast differentiation

p53

Chandar et al. (2005)

Homeobox 7

Xu et al. (2008)

Chondrogenesis Formation of dendrites in retinal ganglion cells together with GDF11

Ihh, Wnt

Growth Factors in Differentiation and Morphogenesis

Table 3.3  Signalling Systems and Phenotypic Effects of BMPs

Hocking et al. (2008) (Continued) 39

40

Table 3.3  (Continued) Phenotype

BMP4

Lateral mesoderm differentiation

Receptor/Signalling Cascade

Neuroectodermal differentiation

BMP7

References Tonegawa et al. (1997)

Neural crest differentiation; Neural induction

Transcription Factors

Requires notch function

Endo et al. (2002)

Sox

Aberdam et al. (2007)

FRL-1 suppresses BMPresponsive genes, Xvent-1 and Xmsx-1, activates MAP kinase

Yabe et al. (2005)

Epidermal differentiation

Notch Delta Np63 pathway

Aberdam et al. (2007)

Chondrogenesis development of limb

Homeobox Barx2 and Sox

Meech et al. (2005)

Osteoblast differentiation

p53

Chandar et al. (2005)

Induction of otic placodes

Pax-2, Sox-3, and Notch expressed in parallel

Grove and Bronner-Fraser (2000)

Osteoblast differentiation

p53

Chandar et al. (2005)

Oocyte-specific; granulosa cell development and fertility

FOXL2 and FOXO3A

Lakhal et al. (2008)

Development of the kidney, eye, distal elements of limb

Dudley et al. (1995); Luo et al. (1995)

Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

BMP ID

Growth Factors in Differentiation and Morphogenesis

41

phenotype of differentiation. As stated before, one can envisage many possible ways by which these effects might be achieved: (1) ligand binding dictated by differential affinity and specificity to the receptors; (2) the recruitment of different R-Smads to constitute the downstream signalling chain; (3) specificity of activation of transcription factors; (4) inhibition of signalling pathway. BMPs function mainly by binding to their own dedicated receptors BMPRI and BMPRII. They can also bind other TGF-β family receptors. Differences are often seen in the affinity of recognition by BMPs of their own receptors as well as other members of the receptor family. These appear to be dictated by structural parameters. Furthermore, specific amino-acid residues could also play important roles in this process, which in turn would determine BMP cross-receptor affinity (Mace et al., 2006). Scharpfenecker et al. (2007) have reported that BMP9 of endothelial cells binds to ALK1 and endoglin, the cell-surface glycoprotein that constitutes a component of TGF-β receptor complex, with high affinity, but only weakly to ALK2 and to the BMP receptor BMPR-II (BMPRII) and ActR-II. Furthermore, binding affinity appears to change dependent upon the available receptors. Thus binding to ALK2 improves when BMPR-II or ActR-II is also expressed. Another interesting finding is that ALK1 and BMPR-II are the major binding targets of BMP9, but ALK2 and BMPR-II are the target receptors in myoblasts. Potentially this could be the mechanism involved in the inhibition of the growth of endothelial sprouts and the consequent inhibition of neo-angiogenesis from endothelial cells, and induction of differentiation in the myoblast system. Again in basal forebrain cholinergic neurons exposed to BMP9, several genes encoding proteins that control the cell cycle and growth, ECM proteins and adhesion-mediating proteins are upregulated. Also BMP9 induces the expression of the nerve growth factor receptor (Lopez-Coviella et al., 2005). Here one can visualise receptor affinity as well as tissue specificities and differential expression of genes coming into play in the generation of phenotypic features. Sharff et al. (2009) showed that BMP9 upregulated Hey1 of the hairy/enhancer of split-related protein of the basic helix–loop–helix family of transcription repressors early in the stimulation of bone-marrow stromal progenitor cells to differentiate. They have suggested that although Hey1 may be a direct target, downstream Runx2 could play an important regulatory role in the differentiation of the progenitor cells. Runx2 is an important regulator of osteoblast differentiation: it drives multipotent mesenchymal cells to differentiate into osteoblast lineage while inhibiting them from differentiating into the adipocytic and chondrocytic lineages (Komori, 2006). Gene families encoding other transcription factors also seem to be associated with the function of BMPs. Sox genes encode transcription factors of the HMG (High Mobility Group). They have been implicated in developmental as well as oncogenic processes. Sox proteins participate in chondrogenesis apparently by regulating the expression of cartilage proteins (Lefebvre et al., 1997, 1998). Sox3 is prominently expressed in the developing central nervous system and may have a role in neural tissue differentiation (Collignon et al., 1996a).

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

Amplification and overexpression of Sox genes has been seen in many tumour types (Dong et al., 2004). Uusitalo et al. (2001) showed that chondrogenic differentiation of mesenchymal progenitor cells in fracture repair involved L-Sox5 (the elongated form), Sox6 and Sox9, and that they mediated the promotion of chondrogenesis by BMP2. Gene transfer experiments demonstrated that BMP2 enhanced the upregulation of these Sox genes and type II collagen. BMPs also participate in other differentiating systems. Induction of neural differentiation by Spemann’s embryonic organiser constituting the anterior end of the primitive streak stage embryo is due to BMP activity along the dorso-ventral axis, with the presumptive neural ectoderm induced to differentiate into neural tissue. Yabe et al. (2003) investigated the role of BMP4 in neural induction. The overexpression of FRL-1, a member of the EGF–CFC gene family, in animal cell caps induced the expression of early markers of neural induction. When overexpressed FRL-1 gene suppressed the expression of the BMP-responsive genes Xvent-1 and Xmsx-1 in the animal pole cell caps. FRL-1 seems to activate MAP kinase to suppress BMP and induce neural differentiation. BMP function is inhibited and probably regulated by the binding by Cerberus, a known inhibitor of the TGF-β signalling pathway. Cerberus is secreted during gastrulation phase of embryogenesis (Entrez Gene Data Base, 2009). BMP4 is associated with neuroectodermal differentiation with activation of the transcription factor Sox1, but epidermal differentiation occurs through activation of notch Delta Np63 (Aberdam et al., 2007). Using a Xenopus early embryogenesis model, Nitta et al. (2006) have shown that overexpression of Sox1 induced neural markers in ventral ectoderm. Sox expression seems to be differentially and spatially regulated in relation to the differentiating primordial morphogenesis. There is an apparent link-up here with BMPs. BMP function in this differentiation pathway might also involve Sox, Zic family (zinc finger proteins), Pou domain factors, helix–loop–helix and Fox transcription factors (Sasai et al., 2001). BMP4 mediates the differentiation of the neuroectoderm into neural crest cells and indeed can inhibit non-neural epidermis from so differentiating. Here the Notch ligand Delta1 is needed for BMP4 expression and function (Endo et al., 2002), although the mechanism of this function of Notch is yet to be elucidated. Possibly this differential selection of differentiation pathway could be attributed to the co-functioning of Sox and Notch. Sox2 overexpression is said to upregulate Notch1 and inhibit neurogenesis unless the Notch pathway is inhibited (Bani-Yaghoub et al., 2006). One might also bear in mind here as an explanatory comment that the expression of Hey1 is induced by Notch. Besides, Notch expression has implications for developmental processes such as neurogenesis (Greenberg and Jin, 2006). Chondrogenesis during limb development seems to be regulated by BMP4, which activates several genes, for example coding for neural cell adhesion molecule (NCAM), ECM proteins and collagen II markers. Activated in parallel is the Homeobox Barx2 transcription factor, together with Sox (Meech et al., 2005). Chimal-Monroy et al. (2003), investigating limb chondrogenesis in some detail, have concluded that induction of Sox transcription factors Sox8 and Sox9 precedes activation of BMP signalling. Sox10 seems in some way to contribute to the acquisition of differentiation competence to limb mesoderm. The process of cartilage

Growth Factors in Differentiation and Morphogenesis

43

differentiation then begins in a sequence, with the induction of BMPRIb followed by the induction of Sox5, Sox6 and Sox9. Overall there is substantial cross regulation in Sox genes in their function. The early stages of in vitro chondrogenic differentiation of human mesenchymal stem cells display high expression of BMP2 and Sox together with Ihh (Indian hedgehog), Wnt genes and chondrogenic marker genes. Activin and Nodal were also identified as important signalling pathways in addition to BMP2 (Xu et al., 2008). Consistent with this is the report by Cameron et al. (2009) who noted that several genes are differentially expressed in the initiation of chondrogenesis. Notable among them were BMP8a and FGFR4, together with genes coding for the transcription factors Sox and Fox family members. The human FOX (Forkhead-box) genes constitute a large family of genes, with as many as 44 members that encode transcription factors (Kaestner et al., 2000). These are actively associated with normal development, proliferation, differentiation and ageing. Fox genes are also overexpressed in many human cancers and are associated with transcriptional regulation by the Shh pathway (Katoh and Katoh, 2004). It would seem therefore that divergence of pathways leading to different cellspecific differentiation might be a consequence of activation of specific transcription factors at the appropriate stage of acquisition of competence. Furthermore, this seems to be another provision at an additional level in the signalling sequence for determining the selection of differentiative pathways in the total picture of embryonic development and morphogenesis. This could be due to their functioning synergistically with other BMPs and in consort with other growth factors, such as TGF-β, FGF, IGF, CTGF and so on. Although proliferation and differentiation might be viewed as divergent pathways, there is evidence that reconciles these. Montesano et al. (2008) showed that although BMP per se is not mitogenic, when combined with FGFs 2, 7, 10, EGF and HGF, it can enhance cell proliferation induced by these growth factors. BMPmediated induction of osteoblast differentiation is modulated by TGF-β1, FGF-2 and PDGF-AB, which seem to upregulate the expression of one type of BMP receptor (Singhatanadgit et al., 2006). Li et al. (1998, 2001) reported that GH and IGF-I enhance the expression of BMP2 and BMP4 messenger RNAs four- to fivefold in human dental pulp fibroblasts in vitro. Also, the BMP receptor BMPRIA was expressed in response to GH (growth hormone). These authors recognised the possibility of the involvement of factors other than IGF with GH in this process and have an interesting suggestion that the combinations of different factors with GH might confer specificity of GH function. GH did not influence the expression of bone sialoprotein and E11 protein, which are regarded as markers of later stages of differentiation. VEGF and BMP4 have been reported to participate in bone formation from periosteal grafts. Fibroblasts in these grafts differentiated into chondrocytes to form cartilage. Subsequent vascular invasion into cartilage coincided with expression of VEGF in the graft and surrounding areas, followed by replacement of cartilage by bone (Ueno et al., 2003). Possibly, this effect of VEGF might be occurring independently of BMP4, because BMP9 has been found to inhibit neo-angiogenesis. So it could be that BMP9 might have been present in the system at assay. More recently, it has emerged that BMP9 can inhibit the proliferation and migration of bovine

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

aortic endothelial cells induced by bFGF (basic fibroblast growth factor) and neo-angiogenesis induced by VEGF (Scharpfenecker et al., 2007). In the context of this discussion on the effects of BMPs on cell migration, it would not be out of place to cite some work that exemplifies signalling interactions between BMP and TGF-β. TGF-β is known to be able to exert bimodal effects dependent upon the stage of tumour development. As noted before, it can inhibit tumour growth in the early stages of neoplasia but promote tumour invasion by activating the developmental programme EMT. BMP7 is known to inhibit EMT and indeed promote the reverse process of mesenchymal epithelial transition. Cells induced into MET by BMP7 show reduced expression of the bHLH transcription factor TWIST. However, TWIST levels could be restored by ALK2 (BMP receptor) siRNA, which inhibits Smad 1, 5 and 8 signalling (Na et al., 2009). Buijs et al. (2007) recognised the inhibitory effect of BMP7 on EMT, and found that BMP expression was inversely related to metastatic potential and correlated with the E-cadherin/vimentin ratio. Experimental studies in vivo showed that BMP7 administration led to inhibition of metastatic growth in the bone. Cell suspensions inoculated into animals treated daily with BMP7 resulted in decreased metastatic deposition in the bone. Another suggested action is of BMP7 mediating Smad1 activation of the inhibitory Smad7, which in turn would be expected to inhibit canonical Smad signalling mediating the EMT-inducing activity of TGF-β. Of considerable interest in terms of the interacting pathways of signalling is that between BMP and GDF in skeletal development. Retinoic acid (RA) synthesis involves the enzyme Aldh1a2. Both BMPs and GDFs appear to reduce the expression of Aldh1a2 and in this way inhibit RA signalling (Hoffman et al., 2006). Ovarian granulosa cells have been studied in relation to the control of hormonedependent cell differentiation. The process of granulosa cell differentiation requires FSH, which works in conjunction with oestradiol. This differentiation process is known to be accompanied by the expression of specific hormone receptors. The induction of differentiation of ovine granulosa cells by exposure to FSH and oestradiol has been shown to regulate the expression of BMPRII, BMPRIB and GDF receptor genes (Chen et al., 2009). GDF expression regulated by FSH influences the formation of primordial follicles (Wang and Roy, 2006). The TGF-β family of growth factors are secreted proteins and can function in an autocrine fashion or as paracrine effectors. In either case, their functions are greatly facilitated by targeting them to their receptors. BMPs and some GDFs seem to be targeted by the agency of fibrillin glycoprotein, which is 350 kDa in size. Fibrillins form the extracellular microfibril network. Mutations in fibrillin-1 and -2 are associated with Marfan syndrome, a heritable condition that affects the connective tissue, and is reflected in abnormalities of long bones, eyes, and cardiovascular and nervous systems. There is implicit recognition that microfibrils are concerned with the regulation of TGF-β/BMP signalling (Ramirez et al., 2007). Arteaga-Solis et al. (2001) suggested that a functional link-up exists between fibrillin-2 and BMP7 in limb patterning. In the context of this functional association, it follows that targeting of the growth factors is an important element in successfully relaying the signal to the cell. This seems to be achieved by the interaction between

Growth Factors in Differentiation and Morphogenesis

45

fibrillin and the prodomain of BMPs and GDFs. The N terminus of fibrillin-1 possesses high-affinity binding sites BMP2, -4, -7, -10 and GDF5, and formation of such complexes seems to provide tissue-specific targeting of the growth factors (Sengle et al., 2008).

Growth and Differentiation Factors Growth and differentiation factors (GDFs) are members of the TGF-β family. More than a dozen GDFs have been identified. They participate in development and differentiation, and the reconstruction and maintenance of many tissues and organs. GDFs show wide tissue distribution and might indeed possess tissue- and cell-type-specific functions. GDFs have also been implicated in disease processes, especially those highlighted here, such as the growth, invasive and metastatic behaviours of cancers. The wide spectrum of biological function subserved by GDFs has been amply investigated in the past few years. This has been achieved in conjunction with other members of the BMP/GDF subgroup of the TGF-β family. Many of the biological effects occur in participation with BMPs with shared pathways of signalling. As members of the TGF-β family, GDFs bind to and trigger the flow of information with the recruitment of receptors of other members of the TGF-β family; synergistic effects occur with activin, Nodal and others.

GDFs in Development and Differentiation In common with other members of the TGF-β family, GDFs participate in embryonic development, pattern formation and cell motility. The participation of GDF in embryonic development begins, as in the case of BMPs, with the primitive streak stage and the formation of the neural plate and its morphogenesis into the forebrain. Indeed, GDFs occur virtually ubiquitously in foetal tissues as well as in a variety of human cancers. GDFs might often function in conjunction with other members of the TGF-β family, for example Nodal and BMPs. GDFs are also associated with other developmental features, such as granulosa cell development, development of primordial follicles and oocyte maturation. These functions are controlled by the relevant hormones by affecting the expression of GDFs. Mutations in GDF genes are known to produce ovarian failure. The induction of dendrite extension in retinal ganglion cells has been ascribed to GDFs. From a physiological viewpoint, of interest is their association with the acutephase response and the production of acute-phase proteins. They are known to be involved in paracrine regulation of corticotroph function. Apart from these normal functional states, GDFs have been found in synovial tissue from rheumatic disease and determine its response to glucocorticoids. Of considerable interest in terms of their relevance to cancer is the potential regulation of ECM proteins, which might be conducive to cell motility. GDFs have been reported to function synergistically with other growth factors and to promote or suppress tumour progression. Also significant in this context is that different GDFs can exert totally opposite effects on tumour

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

progression. Finally, GDFs are expressed in the differentiation and development of the prostate and in prostatic intraepithelial neoplasia in murine models. The most extensively studied differentiation function of GDFs is chondrogenesis. The development of the skeletal system, with the formation of bone tissue, the development of long bones and fracture repair, involves the process called endochondral ossification, in which cartilage is replaced by bone tissue. The differentiation processes in the early stages of endochondral ossification require proliferation of mesenchymal cells, their aggregation and formation of condensations. These continue to grow and differentiate into chondrocytes, giving rise to cartilage anlagen, eventually upon maturation to bone formation. Chondrogenic growth and differentiation can be associated with the expression of a variety of markers such as glycosaminoglycans, the large aggregating chondroitin sulphate proteoglycan (commonly referred in an abbreviated form as aggrecan) and collagens with expression in parallel with C proteins, GDFs and BMPs. GDFs also occur quite prominently in the skeletal function, being an important element in the regulation of collagen component of ligaments and tendons. These contain collagen type I fibrils in a proteoglycan matrix. They also possess fibroblasts that are arranged in a parallel pattern. GDFs influence the composition of ligaments and tendons (see Table 3.4 for references).

Co-ordination of GDF Signalling with Other TGF-β Family Members GDFs can work synergistically with other major signalling partners or ligands of the TGF-β family, for example the BMPs, AMH and Nodal among them. Also retinoids have been cited as potential co-functionaries of GDF. The conjoint function of GDF with VEGF is of considerable significance from the viewpoint of cancer invasion and metastasis.

Synergistic Signalling of GDFs with BMPs BMPs do work in synergy but some GDFs are also known to inhibit the function of certain BMPs. So there are complex bi-modal and self-regulatory mechanisms and the switching of signalling pathways involved in the function of these ligands. GDF3 at very high doses activates Nodal signalling with overexpression of Nodal mRNA. However, it inhibits BMP when GDF3 mRNA is overexpressed (Levine et al., 2009). The switching of pathways leads to the activation of transcription factors that determine the specificity of the outcome of the differentiation process. Nodal signals with FGF and BMP to determine endoderm specification. It specifies endoderm differentiation through the Sox17 transciption factor function, and with the repression of FGF and BMP signalling. Endodermal differentiation involves transcription factor Sox17 together with Sox32 and Pou5f1. Nodal is believed to activate Sox32, and Pou5f1 synergistically activates Sox17, whereas BMP and FGF repress Sox17 in the ventral and dorsal endoderm respectively. In other words, both BMP and FGF are involved in the success of Nodal in determining endodermal specification. Chan et al. (2009) have identified three conserved modules, A, B and C. The Pou5f1-binding element on the B module and the Sox32-binding element on the C module work

GDF Type

Putative Pathway/Functional Focus

References

GDF1

Associated at the primitive streak stage with forebrain development; functions with Nodal using ALK4 and 7 (?) receptors

Andersson et al. (2006)

BMP inhibition and activation of Nodal signalling

Levine et al. (2009)

Expressed during cartilage formation with Wnt, FGF and BMP

Cameron et al. (2009)

Promotes osteogenic differentiation

Zeng et al. (2006)

Chondrogenesis in limb bud; uses Homeobox Barx2 and Sox9, functions in parallel with BMP4

Meech et al. (2005)

Induces VEGF expression in vitro; regulates expression of receptors of other VEGF family members

Zeng et al. (2007); Sena et al. (2007)

GDF2 [BMP9] GDF3 GDF4 GDF5

Growth Factors in Differentiation and Morphogenesis

Table 3.4  Growth and Differentiation Factors and Their Putative Functions and Signalling Pathways

Rheumatic synovial tissue; decreased by glucocorticoids Signalling mediated by BMPR-IB

Bramlage et al. (2008)

ECM proteins gene expression in mouse inter-vertebral disc cells

Sieber et al. (2006); Kotzsch et al. (2009)

Promotes neurite outgrowth, synergy with neural cell adhesion molecules

Cui et al. (2008)

Binds to N-terminal domain of fibrillin of the microfibril system

Niere et al. (2006); Sengle et al. (2008)

GDF6

Tendon collagen matrix

GDF7 [BMP12]

Shares sequence homology with GDF5 and GDF6. Pleiotropic function of chondrogenesis, bone morphogenesis, and neuronal differentiation. Signalling includes BMPRIB and BMPRII or Activin RII and Smads

Mikic et al. (2009)

47

(Continued)

48

Table 3.4  (Continued) Putative Pathway/Functional Focus

References

GDF8 [Myostatin]

Binds activin receptor type IIB; paracrine regulation of corticotroph function

Taketa et al. (2008)

Skeletal muscle development

Walsh and Celeste (2005)

Myogenic differentiation

Yang et al. (2008)

Different stages of larval embryonic development of Epinephelus coioides

Ko et al. (2007)

Inversely related to mammary epithelial cell differentiation

Manickam et al. (2008)

Sertoli cell functions, possibly through ALK (activin-like kinase)-5, and -6, BMPRII

Nicholls et al. (2009)

Development of primordial follicles; oocyte maturation; functioning through HOX cofactors

Lee et al. (2008); Mery et al. (2007); Ota et al. (2008); Sadeu and Smitz (2008); Wang and Roy (2006)

Increases inhibin expression in granulosa cells by stimulating Smad2

Roh et al. (2003)

Smad3-mediated signalling

Spicer et al. (2006)

Oocyte-specific; granulosa cell development and fertility; possible activation of Forkhead transcription factors (FOXL2 and FOXO3A)

Lakhal et al. (2008)

GDF9

GDF9, GDF9b [BMP15]

Mutations associated with ovarian failure

Dixit et al. (2005)

High expression possibly relates to good prognosis in breast cancer

Hanavadi et al. (2007)

Promotes FSH-induced follicular development; anti-apoptosis effect through PI3K/Akt

Orisaka et al. (2006)

Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

GDF Type

Knight and Glister (2006)

Promotes follicular development with VEGF

Shimizu (2006)

GDF10

Human GDF10 mRNA foetal cochlea, foetal lung, testis, retina, pineal gland, other neural tissues; head and neck tumours

Katoh and Katoh (2006a)

GDF11 [BMP11]

Formation of dendrites in retinal ganglion cells together with BMP2

Hocking et al. (2008)

High expression relates lymph node metastases and poor overall survival in colorectal cancer

Yokoe et al. (2007)

Iron homeostasis

Lakhal et al. (2009)

Overexpressed in thalassaemia; suppresses expression of hepcidin

Tanno et al. (2007)

Acute phase response; production of acute phase proteins

Gung et al. (2009)

Expressed in the differentiation and development of the prostate and in prostatic intraepithelial neoplasia in a murine model

Noorali et al. (2007)

GDF15

Growth Factors in Differentiation and Morphogenesis

Oocyte-specific expression at early stage, promotes follicle growth and maturation; early stage arrest of follicle development and infertility with inactivating mutations

Induced in liver injury High levels in metastatic gastric cancers

Zimmers et al. (2006)

Expression related to invasiveness

Lee et al. (2003); Baek et al. (2009)

Expression related to androgen sensitivity; weak staining in benign glandular than adenocarcinoma foci

Karan et al. (2003)

Prostatic tumours with high expression resistant to clocetaxel and mitoxantrone

Huang et al. (2007)

Promotes apoptosis induction by CD437 (retinoid like molecule) in H460 lung cancer cells

Kadara et al. (2006)

49

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

synergistically. So it would be interesting to ascertain if there are specific targets of BMPs and FGFs to achieve Sox17. This might be provided by the non-conserved R module, which seems to exert a repressive effect on both the ventral and dorsal sides. It is clear that the mutual regulation of Nodal and BMP/FGF signalling occurs as a consequence of selective activation or repression of some of the transcription factors occurring in differentiation.

GDF and Nodal Signalling in Embryonic Development Early embryonic development involves Nodal, which is associated with left–right patterning of the embryonic axis. Andersson et al. (2006) have advocated an interaction between Nodal and GDF1 in this patterning. This is based on the finding that severe abnormalities, such as the absence of notochord and prechordal plate, and malformation of the foregut and organising centres, implicated in the development of the anterior head and branchial arches, occurred in GDF1(/)/Nodal(/) mutants, but no abnormalities were encountered in GDF1(/) or Nodal(/) single mutants. Andersson et al. (2006) also found FoxA2 (Forkhead box transcription factor A2) and Goosecoid downregulated in the anterior primitive streak of double-mutant embryos. However, the Nodal element in Hensen’s node functioned independently of GDF1. The invagination of the epiblast cells at the anterior primitive streak does indeed differentiate into the axial mesoendoderm; epiblast cells invaginate from Hensen’s node anteriorly as a mesodermal component that is involved in the induction of the neural plate in the presumptive neuroectoderm. This would suggest then that GDF is not required with Nodal in the patterning of the neural plate. This could be because the neuroectodermal induction occurs by the agency of the underlying mesoendoderm, which expresses Goosecoid. Goosecoid expression then spreads gradually to the ectodermal layer (Thisse et al., 1994). So even though Nodal seemingly functions independently of GDF1, the transfer of the transcription factor could be taking place in the process of induction. On the other hand, it may be that other transcription factors might be involved. The Goosecoid (GSC homeobox 2) gene located on chromosome 22q11.21 encodes a transcription factor. This is known to participate actively in the processes of cell invagination from Hensen’s node, constituting the Spemann organiser and the regulation of neural induction by the organiser (Zhu et al., 1999). Using wild-type and Goosecoid-mutant mouse nodes, these authors demonstrated that the wild-type mouse nodes can induce the neural-specific transcription factors Sox2 and Sox3 but Goosecoid (/) nodes cannot do so. As discussed earlier, GDF3 has been attributed with the ability to activate Nodal signalling, but this happens only when it is overexpressed and inhibits BMP. In other words, the presence of GDF3 could be switching the signalling to the Nodal pathway seemingly by inhibiting BMP signalling (Levine et al., 2009). This has the potential of activating transcription factors in a selective manner, as required for the specification of differentiation of the presumptive progenitor cells. This is achievable also by differential activation of related pathways, as demonstrated in the differentiation of the notochord in early embryogenesis as discussed previously. Inhibin expression in the ovary is stimulated by FSH and other members of the TGF-β family,

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51

for example activin and BMP. GDF9 stimulates inhibin A and B in granulosa cells. FSH and GDF9 functioned synergistically. Treatment with both FSH and GDF9 also increased mRNA expression for inhibin-α and -β subunits. The signalling appears to be mediated by Smad2 as GDF9 treatment increased phosphorylation of Smad2, but not Smad1 (Roh et al., 2003). GDF9 participates with BMP15 in spermatogenesis. GDF9 is found mainly in round spermatids; it is co-expressed with ALK5 and BMP receptor type II in round spermatids and Sertolic cells. It upregulated monomeric as well as dimeric inhibin B production (Nicholls et al., 2009).

Collaborative Function of GDFs and Hormones Apart from other members of the TGF-β family, the function of BMPs and GDFs is certainly influenced by hormones according to physiological requirements. FSH stimulates germ-cell maturation, stimulating growth of ovarian follicles, in particular affecting granulosa cells which produce sex steroid hormones. Thus FSH appears to be able to regulate BMP receptor expression. Chen et al. (2009) found BMPRII, BMPRIB and ALK-5 expression to be higher in the granulosa cells of large follicles than of small follicles. FSH at 1–10 ng/ml downregulated the expression of BMPRIB, but not BMPRII and ALK-5. However, these last two receptors were downregulated at higher (10 ng/ml) FSH dosage. When combined with oestradiol (1 ng/ml), FSH (5 ng/ml) upregulated the expression of all three receptors. One can conclude from this that BMP and ALK are associated with FSH function, and that clearly the expression of these receptors is closely related to the stage of follicular development.

GDF Expression in Cancer With their extensive involvement in developmental regulation, it is hardly surprising that GDFs, like other members of the TGF-β family, are implicated in pathogenesis. Hanavadi et al. (2007) reported that highly aggressive breast cancer did not express GDF9, and forced expression of the growth factor in breast cancer cells inhibited invasive behaviour. High expression of GDF9 and GDF9B (BMP15) correlated with good prognosis in breast cancer; patients were disease-free for up to 10 years of follow-up. GDF15 (also often referred to as macrophage inhibitory cytokine-1 MIC-1), appears to take part in normal development and morphogenesis of the prostate. Its expression, both through mRNA and protein, modulates in the course of differentiation and development. GDF15 expression has been investigated in many forms of cancer. Its presence has been related to progression and invasiveness of tumours (Bauskin et al., 2006). It is upregulated in gastric cancer tissues and cell lines. Gastric cancer cell lines expressing high levels of GDF15 are highly invasive in vitro (Lee et al., 2003). GDF15 is secreted by gastric cancer cell lines and high levels of the growth factor are detected in the sera of cancer patients. GDF15 was found in metastatic gastric cancers at greatly enhanced levels (Baek et al., 2009), which suggests a possible relationship with progression of the disease. Yokoe et al. (2007)

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

found high expression of GDF11 in lymph node metastases of colorectal cancer, and high expression was associated with poorer prognosis. This is in sharp contrast to findings relating to GFD9. So these observations can at best be regarded as provisional. Contrary to expectation from the above studies, GDF15 has been implicated in the promotion of apoptosis. It has been shown to enhance apoptosis induced by the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437). There are numerous reports that CD437 is capable of inhibiting tumour growth (Langdon et al., 1998; Liang et al., 1999; Schadendorf et al., 1994, 1996; Sun et al., 1997). This could be a reflection of its ability to induce apoptosis (Adachi et al., 1998; Lu et al., 1997; Marchetti et al., 1999; Mologni et al., 1999). Strelau et al. (2008) found low levels of GDF15 expression in primary glioblastoma; possibly, therefore, it has no effect on growth. Besides, whether GDF15 itself can induce apoptosis is yet uncertain. IL-1, TNF-α and TGF-β induce the expression of GDF15 in macrophages, inhibiting macrophage activation and inflammation (Bootcov et al., 1997). The cell-cycle regulator and tumour suppressor p53 induces GDF15 expression (Tan et al., 2000). Indeed, it might exert anti- or pro-apoptotic effects in other cell types and organ systems (Ago and Sadoshima, 2006). Changes in GDF15 expression also seem to accompany the development of prostatic intraepithelial neoplasia (PIN). The differences between normal differentiation and development and PIN might be an apparent defect in the post-translational regulation of expression of the growth factor (Noorali et al., 2007). Karan et al. (2003) found high GDF15 expression in androgen-independent LNCaP-C81 cells and their metastatic variant LNCaP-Ln3 compared with androgen-sensitive LNCaP-C33 cells. Also, prostatic adenocarcinomas stain weakly in the benign prostatic glandular area compared with areas of adenocarcinoma. Patients with prostate cancers of Gleason grades 3/4, which overexpress GDF15, have been reported to be refractory to neoadjuvant chemotherapy with clocetaxel and mitoxantrone (Huang et al., 2007). The androgens testosterone and dihydrotestosterone regulate the growth and differentiation of the normal prostate and the growth of prostatic cancer. Several growth factors including interleukins, TNF, MCSF, TGF-β and androgens are known to regulate the expression of GDF15 (Bootcov et al., 1997; Karan et al., 2002; Zimmers et  al., 2006). Overall, it would seem that GDF15 and androgens not only influence each other’s expression but they also markedly affect the progression of prostate cancer.

Activin and Inhibin Signalling Activins and inhibins are heterodimeric glycoproteins related to the TGF-β family. As their names imply, they are mutually antagonistic in function. The activins are dimer proteins composed of β-subunits, βA or βB, and occur in three variations as activin A (βAβA), activin B (βBβB) or activin AB (βAβB). The inhibins are composed of one activin β-subunit and a unique α-subunit. Two isoforms, inhibin A (α/βA) and inhibin B (α/βB), have been identified and investigated. In common with

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53

other members of the family, they are formed as large precursor proteins that are processed into mature proteins 32–34 kDa in size related to the degeree of glycosylation (Cook et al., 2004). The processed protein is secreted into the extracellular compartment. The subunits dimerise through disulphide bonds in the endoplasmic reticulum and are transported to the Golgi apparatus. The dimers undergo furinmediated cleavage at the RXRR site, dislocating the mature peptide from the propeptide. The biological activity is dependent upon this process (Mason et al., 1996). The α-subunit possesses seven conserved cysteine residues characteristic of the TGF-β prototype (Thompson et al., 2004). The sites of production and target tissues of these proteins have been identified. The processed inhibin dimers and the α-subunit are exclusively produced by the granulosa cells of the ovary. The inhibin β-subunits and activins are produced by many tissues (Luisi et al., 2001). The pituitary gonadotrope and the ovary are the two major foci of inhibin function (Woodruff and Mather, 1995). Activins have multiple tissue targets (Luisi et al., 2001).

Signalling Antagonism between Activin and Inhibin Inhibin and activin are involved in the control of the biosynthesis and secretion of FSH from the anterior pituitary. Activin stimulates but inhibin blocks these processes (Carroll et al., 1989; Weiss et al., 1993). FSH and members of the TGF-β family, such as GDF9, TGF-β and activin, in turn appear to regulate inhibin expression to complete a loop of ovarian function (Drummond et al., 2000; Findlay et al., 2001, Kaivo-Oja et al., 2003, Lanuza et al., 1999, Roh et al., 2003). The signalling system adopted by inhibins and activins conforms to the established pattern adopted by the TGF-β family. Activins relay their signals by binding to type II ActRII or IIB serine/threonine kinase receptors and, in the canonical fashion, to phosphorylate type I ALK4 serine/threonine kinase receptor and activate the Smads, so initiating the expression of responsive genes. Inhibins also bind to ActRII, thus providing a means of regulation by competitive signalling; however, they do not bind to ALK4 as do activins, possibly providing another tier of regulation of information flow. Mutational analysis of the activin βA subunit has allowed the identification of the residues required for activin receptor binding, activity and inhibition of its function by inhibin through its own receptors (Cook et al., 2005). betaglycan (TGF-β RIII), the cell-surface chondroitin sulphate/heparan sulphate proteoglycan, is known to bind through different molecular domains to several members of the TGF-β and bFGF families (Andres et al., 1989, 1992). It seems to function as a high-affinity co-receptor for inhibin (Lewis et al., 2000). Betaglycan binding of inhibin greatly enhances the affinity of inhibin for activin receptors. From this intervention of betaglycan, inhibin can disrupt the interaction of activin with its own receptors and the interaction of activin receptors with other members of the TGF-β family (Cook et al., 2004). Harrison et al. (2003) have suggested that five hydrophobic amino-acid residues on the extracellular domain ALK4 are involved in the binding of activin.

54

Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

The signalling antagonism can be viewed from another angle. The intricate interactive regulation of FSH expression by the inhibin/activin system and the reciprocation of the regulatory process by inhibin and activin by the gonadotropin and certain members of the TGF-β family have been unravelled to some degree. A differential effect of TGF-β and FSH was recognised some time ago. According to Lanuza et al. (1999), FSH-dependent stimulation of inhibin A is twice as high as that of inhibin B. TGF-β, on the other hand, influenced inhibin B far more than inhibin A. Activin A also exerted a similar differential effect on inhibin B stimulation. Further, detailed investigations of the expression of inhibins A and B by ovarian cell cultures from 4- to 12-day-old rats in response to FSH and TGF-β have revealed that the production of inhibins A and B in response to these paracrine and autocrine stimuli follows a reasonably well-defined pattern (Drummond et al., 2000; Findlay et al., 2001). Therefore, one has to entertain the possibility that such a regulation of inhibins would influence activin signalling in the development and differentiation of ovarian follicles. One also has to complete this picture by including a factor called follistatin, which binds activin with high affinity. Follistatin is a glycoprotein that inhibits FSH production; this FSH suppression effect of follistatin has been attributed to its ability to bind and sequester activin (Besecke et al., 1997; de Winter et al., 1996; Hashimoto et al., 1997; Leal et al., 2002). The availability of activin is an important determinant of FSH secretion by the pituitary gland (Padmanabhan et al., 2002). Another factor that might intervene in the signalling accomplishment by activin is Cripto, which mediates Nodal signalling. Cripto can bind activin and ActRII/IIB. When Cripto is engaged with Nodal signalling, it is capable of inhibiting activin signalling (Gray et al., 2003); this could have potential consequences for inhibin function.

4 Vascular Endothelial Growth Factor Neovascularisation is an essential ingredient of embryonic development, wound healing and chronic inflammation. It is a prerequisite for the successful growth and metastasis of tumours. Several growth factors possess the ability to induce neovascularisation. The focus here is on VEGF (vascular endothelial growth factor) and HGF (hepatocyte growth factor). The angiogenic ability of FGFs and TGF-β has already been discussed in some detail. These growth factors appear to bind to heparan sulphate proteoglycans. In this way, they might be localised at the cell surface and thus allow these ligands to transduce their signals by binding to appropriate receptors. VEGF family has several members, for example VEGFs A–E, placental growth factor (PLGF) and the snake venom VEGF-F, representing structurally homologous growth factors. By convention, VEGFA is referred to plainly as VEGF. The VEGF gene is located on chromosome 6p21.1, VEGFB on 11q13 and VEGFC on4q34.3. The VEGFs are represented by many splice isoforms. The VEGF gene is spliced in exon 8 at proximal or distal sites. The distal splice isoforms are designated as ‘b’ isoforms. VEGF thus has VEGF121, VEGF121b; VEGF165, VEGF165b; VEGF145, VEGF 145b; VEGF189, VEGF189b; and VEGF206. VEGFB occurs as two splice isoforms. The VEGFs are cystine-knot growth factors that are specific for vascular endothelium, because they bind to receptors that are found only on endothelial cells. VEGFs function as major angiogenic factor in normal as well as pathological conditions; the ‘b’ isoforms are inhibitors of angiogenesis. The snake venom and viral VEGFs are said to have unique function (Yamazaki and Morita, 2006). VEGFs and VEGF receptor homologues also occur in invertebrates where they are involved with cell migration and neurogenesis.

VEGF Signalling VEGFs seemed to have emerged early in evolution as suggested by their presence together with their receptors in invertebrates, which do not possess a vascular system (Holmes and Zachary, 2005). VEGFs play an important role in the development of pathological conditions such as rheumatoid arthritis (Koch et al., 1994) and in neoplastic development and progression. The VEGFs activate specific VEGFR-1 (also called Flt-1, fms-like TRK), VEGFR-2 (Flk-1, foetal liver kinase) and VEGFR-3 tyrosine kinase receptors. VEGFA, VEGFB and PLGF bind and function through VEGFR-1 (de Vries et al., 1992; Olofsson et al., 1998; Park et al., 1994). VEGFR-2 binds VEGFA in a highly specific manner but it can also bind VEGFC and VEGFD receptors, VEGFC and D act through VEGFR-2 and -3 Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00004-9 © 2011 Elsevier Inc. All rights reserved.

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

(Otrock et al., 2007; Simiantonaki et al., 2007; Zachary and Gliki, 2001; Clauss, 2000). These receptors are ubiquitously expressed, but different signalling properties have been ascribed to them (Waltenberger et al., 1994; Shibuya et al., 1990). In common with the EGFR family, upon ligand binding VEGF receptors dimerise and autophosphorylate, which in turn leads to the activation of several signalling systems, namely the Ras/MAP-kinase, PI3K/PTEN/Akt, the Jak/Stat and the PLC-γ/PKC pathways. The phosphorylated tyrosine residues provide recognition and docking sites for SH2 (src homology) and GRB2 (growth factor receptor-bound protein 2). VEGFR-1 is able to phosphorylate PLC-γ (Ito et al., 1998; Sawano et al., 1997; Seetharam et al., 1995). It can interact with the regulatory subunit of PI3K (Cunningham et al., 1995). VEGF-2 is also known to activate PLC-γ, generating DAG and IP3 (Zachary and Gliki, 2001). Among the pathways that VEGF-2 activates is Ras/ Raf/MEK/ERK signalling. The promotion of cell proliferation occurs in addition by the activation Akt. VEGFR-3 also treads these same signalling pathways. The VEGF ligands can potentiate each other’s phenotypic effects. Also, more than one receptor is expressed in a cell type. In these cases, the receptors can form heterodimers and in this way contribute to superior signalling, as noted with VEGF-2 and VEGF-3 (Alam et al., 2004) (Figure 4.1).

Angiogenic Properties of VEGF Proximal and Distal Splice Variants The isoforms resulting from exon 8 splicing at the proximal splice site promote angiogenesis, whereas the distally spliced ‘b’ isoforms may bind the receptors but do not activate signalling and so are competitively anti-angiogenic. For instance, VEGF165b and VEGF165 bind VEGF receptor 2 with the same affinity, but the former does not activate the receptor or downstream signalling (Woolard et al.,

Figure 4.1  VEGF sigalling modes and interaction with other angiogenic agents, for example HGF, TGFβ and EGF. Also shown is the putative involvement of Notch signalling, linked with the hypoxia-mediated PI3K route to cell proliferation.

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2004). So the splicing of VEGF pre-RNA may be regarded as possessing a function that might regulate angiogenesis. Of the distally spliced ‘b’ variants, the VEGF165b variant has been the subject of much investigation. VEGF165b inhibits angiogenesis in retinopathy in murine models and age-related macular degeneration. It inhibits retinal neovascularisation in mice. It was suggested that experimental alteration in the balance of VEGF165 and 165b expression might be a viable way of regulating angiogenesis (Konopatskaya et al., 2006). Woolard et al. (2004) found that experimental tumours expressing the VEGF165b variant grew more slowly compared with those expressing VEGF165. VEGF165b expression is downregulated in prostate cancer (Woolard et al., 2004) and renal carcinoma (Bates et al., 2002). Downregulation has been encountered also in colorectal cancer, which was related to poor prognosis (Diaz et al., 2008). Modification in splicing by selecting the splicing site can switch the pattern of generation of variants from the anti-angiogenic to the pro-angiogenic form (Qui et al., 2009). IGF-1 seems to be able to promote VEGF165 formation than VEGF165b. Altering splice-site selection by altering the activity of RNA-binding splice factors, such as ASF/SF2, Nowak et al. (2010) have shown the occurrence of corresponding changes in retinal neovascularisation using a murine model. In fact, they suggest that such manipulated conversion of splicing from pro- to anti-angiogenic forms could be considered as a possible therapeutic strategy.

Synergy of VEGF with Other Angiogenic Signalling Systems Another situation emerges from the observations that suggest that VEGF signalling may be accentuated by contributions from other growth factors such as HGF, which is discussed in detail in the following pages. Both VEGF165 and HGF activate the same signalling pathways, but do not activate each other’s receptors. However, they do function synergistically, which suggests downstream differences in signalling (Sulpice et al., 2009). HGF is also angiogenic, but this study indicates that they could be contributing to different aspects of the processes of angiogenesis. In other words, although the signalling pathways might be shared, there could be switching mechanisms that target specific aspects of angiogenesis. On the other hand, it might be as Sulpice et al. (2009) have suggested: different kinetics and strength of activation might operate under the angiogenic environment. How these signals are balanced is unclear. It was suggested that activation of PI3K/Akt signalling by angiogenic factors can lead to VEGF regulation and in turn to angiogenesis. Furthermore, because Akt is itself regulated by PTEN, this form of regulation could subsist in a given angiogenic environment (Sherbet, 2003). Recently Ma et al. (2009) found that short interfering (siRNA)-mediated inhibition of PTEN expression led to an increase in VEGF secretion and to the promotion of proliferation, and migration of endothelial cells together with increased tubule formation in HUVEC cultures. The upregulation of VEGF by Akt appears to involve the recognition of Sp1 transcription factor binding sites in the VEGF promoter (Pore et al., 2004). The transcription factor HIF-1 (hypoxia-inducible factor 1) is an important

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mediator of the hypoxia signalling pathway. Hypoxia upregulates VEGF expression through PI3K/Akt signalling, with the expression of Akt being mediated by STAT3 signalling (Xu et al., 2005). So both of these transcription activators can be implicated in hypoxia-induced angiogenesis.

Notch Signalling in Angiogenesis Notch signalling is another interacting pathway that has been implicated in VEGF function and induction of angiogenesis. VEGF is able to induce expression of Notch1 and the Notch ligand Delta4 in human arterial endothelial cells. Particularly noteworthy is that Delta4, although detected in normal tissues, is highly expressed in vasculature associated with human xenografts. It is also found in blood vessels of the kidney and in breast cancer. The link with angiogenesis resides in the induction of Delta4 by hypoxia. HIF1-α has been shown to be upregulated in MCF7 breast cancer cell lines transfected constitutively with active Notch1 (see Chiaramonte et al., 2006). Another equally attractive view is that VEGF and Notch-Delta might subserve different aspects of angiogenesis (Thurston and Kitajewski, 2008). This perception is justified by the findings that VEGF can induce angiogenesis by HIF1-mediation, which can in turn upregulate Delta4 and stimulate signalling in determining the morphogenetic processes of angiogenesis. Equally, Notch could be functioning through PDGF, the downregulation of which can lead to Notch-1 inactivation together with the downregulation of VEGF and MMP (Wang et al., 2007).

Sphingosine 1-Phosphate (S1P) Cross-Talk in VEGF Signalling Sphingosine 1-phosphate (S1P) has been postulated to cross-communicate with the VEGF signalling system. S1P released by platelets induces migration, proliferation of endothelial cells and brings about cytoskeletal changes in these cells. S1P binds G-protein-coupled receptors (GPCRs) of the endothelial differentiation gene (EDG) family (Lee et al., 1998, 1999; Wang et al., 1999; Lee OH et al. 1999). S1P has been closely linked with angiogenesis. The signalling downstream involves PI3K and Akt. S1P can also function through the Ca2/calmodulin pathway. VEGF also activates eNOS through VEGFR-2, PI3K and Akt (Tanimoto et al., 2002). S1P is also able to transactivate VEGFR directly and activate the PI3K/Akt signalling system (Figure 4.1). The notion was mooted some time ago by Igarashi et al. (2003) that S1P might activate VEGF promoter function. S1P has been found to induce migration of human ML-1 thyroid follicular cancer cells and stimulate VEGF-A secretion; this is reduced by inhibiting S1P. Also, S1P-induced cell migration as well as signalling through Akt is inhibited when VEGFR-2 is inhibited, suggesting a close interaction between S1P and VEGFR-2 (Balthasar et al., 2008). However, a provisional opposite view was expressed wherein inhibition of VEGF-R2 had no tangible effects on S1P-induced ERK and Akt signalling (Ronco and Sanyal, 2005). Heo et al. (2009) have implicated the G(αi/o)-pathway-linked PLC to Ras signalling, Akt, ERK and MAPK signalling, in the induction of VEGF by S1P. So the pathways are shared between

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VEGF and S1P, which suggests strongly that the two might collude in promoting cell migration and angiogenesis.

The Interaction of ADAM Proteins with VEGF Signalling ADAM (a disintegrin and metalloprotease domain)-containing proteins are associated with several disease processes such as arthritis, inflammation, and tumour development and progression. ADAM proteins are capable of transactivating growth factor receptors, such as EGFR. They have been deeply implicated in cell signalling. ADAM-17, ADAMTS1 and MMPs can facilitate signalling by releasing growth factor ligands from their membrane-bound location. The presence of ADAMs in endothelial cells and their potential role in angiogenesis has been recognised and documented. They can induce endothelial cells to differentiate and induce the formation of capillary-like structures and neovascularisation. Of interest in the context of VEGF signalling is the demonstration that ADAM-17 induces the formation of capillary-like networks of HUVEC cells in Matrigel scaffolds and induces in vitro invasion. Of further interest is that ADAM-17 expression is linked with VEGF expression and MMP-2 activation (Gooz et al., 2009). In sharp contrast, ADAMTS1, which has three TSP-1 domains, possesses anti-angiogenic properties. It binds to and sequesters VEGF, preventing its phosphorylation, and inhibits endothelial cell proliferation (Luque et al., 2003). The effect seems to be specific and mediated by the carboxy (C)-terminal heparin-binding domain because ADAMTS1 did not bind VEGF121. So VEGF suppression could be a reason why the C-terminal region of ADAMTS-1 can inhibit both tumorigenicity and metastatic spread encountered in experimental metastasis assays (Kuno et al., 2004). VEGF can upregulate ADAMTS1 (Xu et al., 2006), which suggests the presence of a regulatory loop that might tightly control neovascularisation. In a similar fashion, ADAM15 and VEGF expression are inter-related in the ischemic retina. ADAM15 upregulates the expression of VEGF, VEGFR1 and VEGFR2. Downregulation of VEGF by experimental means also downregulates ADAM-15, thus describing a regulatory loop (Xie et al., 2008). Compatible with their role in VEGF-mediated promotion of angiogenesis is their association with tumour development. Expression of ADAMs has been correlated with invasion and proliferation of breast cancer cells in vitro. ADAM-17 is overexpressed in primary and metastatic colonic cancers; in breast cancer its expression correlated with tumour grade, and the levels of active protein increased progressively with progression to metastatic disease.

Genetic Polymorphism in VEGF in Disease States Single nucleotide polymorphisms (SNPs) in the VEGF gene have been reported in the context of many human diseases. Genetic alterations can influence the expression of growth factors. Their effects on cancer risk, susceptibility, progression and prognosis have been described. SNPs in EGF have been reported in several human tumours, for example melanomas, glioblastoma and gastric cancer (see pp. 174–176). In malignant melanoma EGF polymorphism reflected disease-free survival (Okamoto et al.,

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2006). It is encountered also in prostate cancer where it is believed to influence disease relapse interval (Teixeira et al., 2008). SNPs in the VEGF gene have shown some correlation with disease state of melanomas (Howell et al., 2002). Tahara et al. (2009) focused on the G1612A and C936T polymorphisms in 3-untranslated region (UTR) in a series of patients with pre-malignant intestinal metaplasia. The 1612GA genotype occurred more frequently in patients over 65 years old with metaplasia and Helicobacter pylori infection. The C936T polymorphism showed no such association. The lack of significance of 936T/C gene polymorphisms in colorectal cancer has been confirmed (Wu et al., 2009). But Wu et al. (2009) do draw attention to the fact that the distribution of EGF 61G/G homozygotes and the G-allele frequency were higher in the cancer group than in the control group. Hofmann et al. (2008) have also found no relationship between VEGF polymorphism and tumour features related to the progression of colorectal cancer. Ungerback et al. (2009) also found no link between SNPs in the 5 UTR and the clinical and pathological state of the disease. Also, there was no association between VEGF messenger RNA (mRNA) expression and the presence of SNPs, although VEGF expression was generally higher in colorectal cancer. This apparent divergence of perceived effects could indicate that different SNPs might affect the disease differently. It has emerged from recent findings that although some SNPs directly relate to disease state and its aggressiveness, others might be associated with reduced risk of disease and yet others possess protective effects. Such a possibility has been encountered in colorectal cancer (Maltese et al., 2009). Indeed, in non-Hodgkin’s lymphoma, the  936TT genotype and alleles, so also certain other polymorphisms, have been linked with decreased risk of invasion (Diao et al., 2009).

Is the Production of SNPs and VEGF Linked? SNPs of VEGF have been investigated for a possible correlation with levels of VEGF production. Renner et al. (2000) found three (702C/T, 936C/T, 1612G/A) SNPs. VEGF plasma levels were significantly lower in carriers of the 936T allele; the other two were not associated with any alterations of VEGF levels. Only 1 out of 13 SNPs in the 5 UTR of the gene was correlated with increased VEGF production in LPS-stimulated PBMCs (peripheral blood mononuclear cells) (Watson et al., 2000). So the situation is at best provisional in terms of the relationship of VEGF expression and the presence of SNPs. According to Cosin et al. (2009), the most widely studied 936C/T polymorphism may occur with the risk of endometriosis, but the increased VEGF levels found in these patients did not relate to the presence of the polymorphisms, which is consistent with the findings of Ungerback et al. (2009) for colorectal cancer.

Functional Synergy of SNPs of Angiogenic Agents Many angiogenic factors might be operating in the cellular and tumour environment. It is possible that SNPs present in them might display additive or synergistic outcome. SNPs of VEGF appear to produce such synergistic effects, with MMPs and TSP1 also displaying SNPs. Sfar et al. (2009) looked at the SNPs VEGF-1154G/A, VEGF634G/C, MMP9-1562C/T and TSP1-8831A/G in patients with prostate cancer.

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The higher the number of SNPs of VEGF and MMP9, the greater was the risk of developing prostate cancer. A similar situation existed for VEGF SNPs and TSP1 SNPs, but TSP1 SNPs were not associated with risk per se. The presence of three SNPs in the angiogenic factors was associated with a 20-fold increase risk of developing aggressive and high-grade tumours. VEGF SNP 634G    C itself has been suggested to reflect some possible susceptibility to developing gastric cancer (Guan et al., 2009). So although it is easy to appreciate that genetic changes of this kind might affect VEGF expression and in this way influence tumour progression, further work is required to establish firmly any such relationships. In some cell types VEGF itself may be regulated by other growth factors such as interleukin-1, selectins (Kvanta, 1995), FGF2, EGF and PDGF (Koochekpour et al., 1995). Sera and synovial fluids from patients with rheumatoid arthritis contain high levels of VEGF. Furthermore, growth factors such as interleukins enhance VEGF expression clearly related to transcription, as indicated by increased binding of the transcription factor AP-1 to the VEGF promoter (Cho et al., 2006). That there might be a shared mediation of PKC pathway in the regulation of VEGF by these growth factors is suggested by the experiments of Kvanta (1995), where enhanced VEGF expression was induced by phorbol esters which activate the kinase. In superficial bladder cancer, ketoconazole, which inhibits all-trans RA catabolysing cytochromes, inhibited VEGF and TGF-α expression and improved survival time of the patients (see Hameed and El-Metwally, 2008). It would have been of interest to know what proportion of these patients showed recurrent disease, which not infrequently tends to be invasive.

Neovascularisation and Tumour Progression Neovascularisation is an important requirement for several biological processes such as embryonic development, wound healing and chronic inflammation. To provide a historical perspective, VEGF has long been known to induce new vascular structure associated with tumours, which facilitates their growth and dissemination. Tumour growth in the avascular phase is self-limiting on account of constraints on the diffusion of nutrients and catabolic products imposed by the tumour surface area in relation to its volume, whereas in the vascularised phase these constraints are lifted. Tumours induce the proliferation of proximal capillaries. New capillaries arise mainly from small venules in response to the angiogenic stimulus imparted by the tumour. Initially, a local dissolution of the basement membrane occurs, possibly mediated by proteinases followed by a migration of endothelial cells towards the angiogenic source. The endothelial cells then align end to end and form a ‘sprout’, which subsequently develops a lumen. VEGF is a potent mitogen. Its expression correlates significantly with vascular density associated with tumours. As noted earlier, the isoforms arising from exon 8 proximal splicing promote angiogenesis, but the ‘b’ isoforms generated by splicing at distal sties bind but do not activate the receptors and are competitively antiangiogenic. The variant anti-angiogenic VEGF165b has received much attention in

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relation to its expression in cancer progression. It is found in many normal tissues but its expression is downregulated in prostate cancer, renal carcinomas and metastatic melanomas (Bates et al., 2002; Pritchard-Jones et al., 2007; Woolard et al. 2004). Compatible with this is the recent report that in a xenograft experimental model overexpression of VEGF165b inhibited the growth of tumour xenografts and inhibited tumour-associated endothelial cell proliferation and migration (Rennel et al., 2008). The basis for the difference in angiogenic ability of VEGF165 and VEGF165b has not been fully elucidated so far. Both of them bind VEGFR1 and VEGFR2 with equal affinity. However, a marked difference has been detected in the activation of VEGFR2 and ERK signalling. VEGF165 seemed able to generate a sustained activation of the receptor and the signalling pathway, whereas VEGF165b could achieve only a weak and transient activation (Suarez et al., 2006). This would be a reasonable explanation when both ligands are expressed, but for situations where VEGF165b expression is downregulated one has to look for alternative explanations such as possible changes relating to the generation of splice variants. It is possible that internal switching mechanisms exist that modulate the switching pattern to alter the valance of pro- versus anti-angiogenic variants. The choice of splice site is believed to depend upon splice factors such as ASF/SF2. Nowak et al. (2010) have shown that the expression of the splice variants can be altered by modulating the activity of RNA-binding splice factor ASF/SF2. IGF-1 promotes proximal splicing rather than distal splicing. Upon treatment of epithelial cells with IGF-1, splicing at proximal site seemed to increase, which resulted in an increased production of VEGF165 and less of VEGF165b. This switch in choice seems to be regulated by the phosphorylation state of splice factors such as ASF/SF2. Switch to the proximal site is inhibited by PKC inhibition and inhibition of SRPK1/2 (SR protein kinases). IGFinduced nuclear localisation of ASF/SF2 is blocked by the inhibition of SRPK1/2. Inhibition of SRPK1/2 indeed inhibits angiogenesis in vivo (Nowak et al., 2010). In contrast, TGF-β1 induced splicing at the distal site, which was prevented by inhibition of p38 MAPK and CLK/sty (CDC-like kinase, CLK1) which phosphorylate SR protein-splicing factors ASF/SF2, SRp40 and SRp55 (Nowak et al., 2008). Hypoxia has been found to function as a specific inducer of alternative splicing of Cyr61 (cysteine rich 61), an angiogenic matrix protein (Hirschfeld et al., 2009). Hypoxia also regulates the generation of VEGF165 rather than VEGF165b (Varey et al., 2008). Further investigation into VEGF165b generation is crucial because it seems to bind to and negate the effects of the anti-VEGFA monoclonal antibody commercially known as Avastin (bevacizumab) (Varey et al., 2008).

Targeting VEGF and VEGFR in Cancer Treatment Tumour-induced neovascularisation, angiogenesis and lymphangiogenesis provide tumour cells with a channel for dissemination to form distant metastases. Targeting VEGF and VEGFR to inhibit the induction of microvasculature by tumours and inhibit angiogenic signalling as a mode of cancer treatment has received

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considerable attention. One can identify many types of inhibitor, such as the humanised monclonal antibodies Avastin and ranibizumab, siRNAs, miRNAs (microRNAs), TRK inhibitors and VEGF-Trap decoys. Angiogenic signals of VEGF are transduced by binding specifically to VEGF receptors. So a strategy similar to that adopted with EGF family growth factor receptors has been evolved to target VEGFR, inhibit its function and inhibit the signalling by VEGF. Avastin, a humanised monoclonal antibody, is such an inhibitor. Avastin with paclitaxel chemotherapy (National Cancer Institute and Eastern Cooperative Oncology Group (ECOG), 2005) has been found to improve disease-free survival and improved response rate in patients with advanced breast cancer. However, because of serious side effects, there is much resistance to the use of Avastin (see Sherbet, 2009). Toxicities associated with Avastin are haemorrhage, hypertension, proteinuria, cardiac toxicity and difficulties related to wound healing. Avastin has been approved in Europe for first-line treatment of women with metastatic breast cancer, but has not been recommended for use in UK (National Institute for Clinical Excellence June 2008 guidelines). VEGF-Trap is a dimeric soluble receptor constituting sections of the extracellular domain of VEGFR1 and VEGFR2 (Huang et al., 2003). Therefore, unlike Avastin, VEGF-Trap is capable of binding VEGF as well as other ligands that can bind VEGFR1 and VEGFR2. Clinical trials to test its efficacy in the treatment of patients with metastatic breast cancer are under way (NCT00369655). Herceptin targeted at the HER2 receptor inhibits tumour growth and reduces VEGF expression, thus effectively inhibiting microvascular density and vascular permeability (Le et al., 2008). In experimental animals, Herceptin inhibited corneal neovascularisation and in parallel reduced VEGF expression in the epithelial and endothelial layers of the cornea (Guler et al., 2009). In breast cancer cell lines, MCF-7 and T-47D HER2 expression is low, but when expression is induced an increase is seen in VEGF and IL-8 expression. Herceptin, on the other hand, inhibits the vascularisation of xenograft tumours together with the downregulation of VEGF as well as IL-8. However, this is accompanied by the upregulation of the anti-angiogenic TSP-1 (Wen et al., 2006). In summary, there is adequate confirmation that Herceptin inhibits angiogenesis. So, saliently, Herceptin has the ability to compound its own effects arising from HER-2 inhibition by exerting an inhibitory effect on neovascularisation. A similar overexpression of HER2 with enhanced VEGF-C has been reported in epithelial ovarian cancer. Herceptin inhibits both (Hsieh et al., 2004). A combination of Avastin with platinum-based chemotherapy and taxols (paclitaxel or docetaxel) is currently being assessed. Attempts are also being made to target other angiogenic receptors. Among these are small molecule inhibitors, namely Sunitinib, Sorafenib and Pazopanib, which inhibit RTKs. They target VEGF and PDGF receptors together with other tyrosine kinases. Both Sunitinib and Sorafenib inhibit angiogenesis. Sunitinib has been found to reduce tumour size. This is probably a combined effect of inhibition of cell proliferation and its ability to upregulate pro-apoptosis genes and inhibit Akt-mediated signalling (Yang et al., 2010). Sorafenib also induces apoptosis, which can be potentiated by MEK inhibitors (Nguyen et al., 2010). According to Llobet et al. (2010), the apoptotic effect is enhanced by TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) ligands. Also, these authors do not regard

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Raf/MEK/ERK inhibition as being involved in Sorafenib-induced apoptosis or its sensitisation by TRAIL. A third option is available for targeting angiogenesis. This is by inhibiting the mTOR pathway. The signalling pathway called mTOR (mammalian target of rapamycin) involves the effector serine–threonine kinase S6K1 (40S ribosomal S6 kinase 1), which is activated by Akt (Asnaghi et al., 2004). Its importance in cancer has been highlighted by S6K1 overexpression in breast cancer and its association with poor prognosis. Because S6K1 functions downstream of Akt, obviously mTOR would mediate cell proliferation. Notably, it integrates ER, EGFR and IGFR signalling and is implicated in angiogenesis. The inhibition of mTOR using everolimus on its own or in combination with gefitinib or cetuximab to block EGFR function has been shown to be effective in cancer cell lines that are resistant to EGFR inhibitor. EGFR inhibitors were found at least partly to regain effectiveness when combined with everolimus. Furthermore, in a xenograft model of colon cancer, everolimus and gefitinib inhibited EGFR-related signalling of growth and VEGF production (Bianco et al., 2008). The flavanoid silibinin, which inhibits tumour growth, is known to downregulate iNOS. It was found to inhibit hypoxia-induced HIF-1α and VEGF production. Finally, the angiogenic and lymphangiogenic effects exerted by FGF-2 have been attributed to mTOR activation (Matsuo et al., 2007). These events correlated with the inactivation of mTOR signalling (Garcia-Maceira and Mateo, 2009). From these studies the conclusion is inescapable that mTOR inhibitors might be a viable approach for combination therapy. The small non-coding microRNAs (miRNAs), being regulators of gene expression, have been studied for their role in angiogenesis. miRNA-126 is an endothelialcell-specific miRNA that been closely implicated in the function of VEGF and bFGF. VEGF signalling leading to the induction of angiogenic sprouts is mediated by the induction of miRNA-126 by the zinc finger transcription factor klf2a (Nicoli et al., 2010). miRNA-20b is also able to regulate VEGF signalling. In MCF-7 breast cancer cells, VEGF function is mediated by miRNA-20b and HIF-1 and STAT3 (Cascio et al., 2010). miRNA-221, in contrast, seems to be inhibitory of angiogenesis. Its expression is downregulated in prostate cancer, is related to aggressive behaviour of tumours and correlated with recurrence (Spahn et al., 2010). These authors also found miRNA-221 expression was negatively related to the expression of c-kit receptor for stem cell factor, which has been implicated in angiogenesis. However, miRNA-221 requires further study, for equally it has been claimed that overexpression of miRNA-221 with that of miRNA-222 correlated with malignant behaviour of cells, and inhibition of their expression inhibited cell growth and invasive behaviour (Zhang et al., 2010). An interesting sideline in this study is the alteration in the malignant phenotype, which could be reversed by the induction of PTEN. However, one can enlist further support for the importance of miRNA in cancer in the finding that genetic polymorphism of miRNA196a2 might be predictive of risk of gastric cancer (Peng et al., 2010). Overall, it would seem that angiogenesis might be regulated by miRNAs. Apart from this, the link of miRNAs with cell proliferation has prompted the targeting of miRNAs as a means of controlling tumour growth and progression through inhibition of angiogenesis. Nonetheless, it should be noted in any attempts to devise anti- or pro-miRNA strategy that miRNAs target numerous genes, and collateral effects resulting from interference with genes might be counterproductive.

5 Hepatocyte Growth Factor

Hepatocyte growth factor (HGF) is among a group of factors possessing angiogenic ability, which have often been described as heparin-binding growth factors. HGF, together with VEGF and FGFs and TGF-β, bind to heparan sulphate proteoglycans, which affords a means of localising them on the cell surface and presenting them to their appropriate receptors in the most favourable conformation in order to facilitate the interaction of the growth factors with the receptors.

Molecular Structure of HGF HGF, initially known as the scatter factor or cytotoxic factor (Gherardi et al., 1989; Higashio et al., 1990; Rubin et al., 1991), is secreted by fibroblasts and is mitogenic for epithelial and endothelial cells as well as melanocytes, but does not affect fibroblasts (Rubin et al., 1991). It contains 728 amino acid residues. It is secreted as an inactive precursor which is processed into the active form. The active form of HGF is a heterodimer of disulphide-linked α and β chains (Nakamura et al., 1989; Hartmann et al., 1992; Kataoka et al., 2003). The α-chain is folded at the amino (N)-terminal domain, which is followed by four Kringle domains. Carboxy (C)-terminal fragments of the α-chain known as NK1, NK2 and NK4, generated by proteolytic cleavage, also bind the HGF receptor MET competitively but cannot activate the receptor and so inhibit signalling (Chan et al., 1991; Cioce et al., 1996; Date et al., 1997). As discussed below, NK4 has been deployed with some success to restrain tumour invasion and growth. The β-chain begins with valine 495; it is also proteolytically processed (Perona and Craik, 1995; Hedstrom, 2002).

HGF/MET Signalling The MET gene encodes the HGF tyrosine kinase receptor. The mature receptor is formed by disulphide linkage between the extracellular α-subunit and the transmembrane β-subunit derived by post-translational proteolytic cleavage of a single-chain Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00005-0 © 2011 Elsevier Inc. All rights reserved.

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precursor molecule. Four domains have been identified in the extracellular region: the Sema domain (semaphorin domain), which encompasses the α-subunit and the N-terminal part of β-subunit and comprises the PSI (plexin semophorin integrin) domain (cysteine-rich MET-related sequences), and three glycine–proline-rich repeats and four IPT (Ig) domains (Birchmeier et al., 2003; Trusolino and Comoglio, 2002). The Sema domain is required for ligand binding and receptor dimerisation (Kong-Beltran et al., 2004). The PSI domain is said to form a wedge between the ligand-binding Sema and the Ig domains and is believed to account for the correct positioning of the ligand-binding site of the receptor (Kozlov et al., 2004). The MET receptor is activated by dimerisation upon ligand binding (Prat et al., 1998). Ligand binding activates the kinase activity of the receptor, leading to the phosphorylation of residues Tyr 1234 and 1235. Several signal transducers are recruited to the tyrosines. Transducers such as GRB2, SHC, SRC and p85 of PI3K directly interact with the docking sites of MET or through a scaffolding protein (Pelicci et al., 1995; Maina et al., 1996; Weidner et al., 1996). HGF/MET activates several signalling pathways, namely Ras, PI3K, MAPK/STAT and β-catenin/Wnt, leading to the biological effects of cell scattering, proliferation, resistance to apoptosis, invasion and angiogenesis. These pathways are focused on here in the context of cancer cell invasion, angiogenesis and formation of metastases (Figures 5.1 and 5.2). HGF induces cell proliferation and invasion by activating PI3K/Akt and ERK signalling pathways, the latter a component of the MAPK system. It induces invasion of cholangiocarcinoma cells in this way. The biological effects of HGF are interfered with or complemented by several interacting signalling systems. The induction

Figure 5.1  The signalling pathways interacting with HGF. HGF activates MET and the MAPK/ERK pathway to induce cell proliferation. EGF can upregulate MET and in this way EGF  HGF can combine efforts towards induction of cell proliferation. HGF can enhance cell population by inducing resistance to apoptosis and induce invasion through activation of PAX/ Sox transcription factors. HGF/MET activation of the MAPK/ERK pathway can also lead to the downregulation of TSP, which in turn again seems to induce invasive behaviour. As shown in Figure 5.2, the downregulation of TSP is also conducive to angiogenesis. So Figure 5.1 should be read in conjunction with Figure 5.2 below. The related references are cited in the text.

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Figure 5.2  HGF interaction with other signalling systems to integrate the phenotypic effects of cell proliferation, invasion and angiogenesis, which form the essential ingredients of cancer cell dissemination and metastasis. References are provided and discussed in the text.

of invasion was reported to be accompanied by alteration of membrane-located E-cadherin expression. The ECM proteolytic system of MMPs or PA was unaffected. Inhibition of PI3K abolished the increased invasion. PI3K induced ERK1/2 phosphorylation, but inhibition of this route suppressed invasion only in one of the two cell lines tested (Menakongka and Suthiphongchai, 2010). These authors have suggested that different signalling systems might operate in different cell lines. On the other hand, it might possibly be due to the interaction of different effectors with HGF signalling. The cadherins are closely involved with catenin/Wnt signalling. HGF/MET does indeed activate the β-catenin/Wnt pathway (Previdi et al., 2010). PI3K and MAPK pathways might be activated either independently or in concerted function with other effectors such as EGF or thrombospondin (TSP). EGF has been found to induce MET expression, which might bind to and be activated by HGF and enhance proliferation by ERK1/2 activation. Inhibition of ERK signalling has led to the inhibition of induction of cell proliferation (Accornero et al., 2010). Rho et al. (2009) established PC-9 sublines resistant to EGFR inhibitors which displayed MET activation. They also found the T790M mutation in the EGFR gene, which has been linked with drug resistance compared with the wild-type gene. However, this mutation is also known to occur in disease progression quite independently of the receptor tyrosine kinase (RTK) inhibitors gefitinib or erlotinib (Kosaka et al., 2004). Furthermore, NSCLCs carrying activating mutations within the EGFR kinase domain may be more susceptible to the RTK inhibitor gefitinib. Mutant EGFRs have been found to activate Akt/STAT signalling and induce cell proliferation. In these cells, inhibition of mutant EGFR reversed the effect and induced

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apoptosis (Sordella et al., 2004). So the significance of the perceived link between MET and EGFR requires further investigation. Another interacting factor is TSP (thrombospondin). HGF seems to be able to induce invasive behaviour in certain ovarian carcinoma cells through downregulation of TSP. In this process HGF seems to signal through the MAPK pathway. When this route is blocked, HGF-mediated increase of invasive behaviour is also blocked (Wei et al., 2010). TSP-1 is an inhibitor of angiogenesis, so its downregulation by HGF is compatible with the pro-angiogenic effects of HGF. As noted in the preceding pages, VEGF165 and HGF activate the same signalling pathways and function synergistically. However, they do not activate each other’s receptors; this suggests differences in signalling downstream of the activation of their respective receptors (Sulpice et al., 2009). HGF is not only angiogenic, it also possesses the ability to promote cell proliferation, resist apoptosis and induce cell motility or invasion. In other words, there could be mechanisms that switch function specifically to target invasion. It was suggested some time ago that activation of PI3K/Akt signalling by angiogenic factors can lead to VEGF regulation and in turn to angiogenesis. The collusion between VEGF and HGF could augment cell proliferation, angiogenesis and invasion, with significant effects on cancer cell dissemination (Figures 5.1 and 5.2). It should be noted here that a putative link between MET, VEGF165 and NRP1 (neuropilin-1), a VEGF co-receptor, has emerged recently. VEGF165 has been found to promote interaction between MET and NRP1 and promote MET phosphorylation in prostate cancer cells. Activation of MET seems to occur in parallel with the upregulation by VEGF of the Bcl-2 family anti-apoptotic Mcl-1, possibly through activation of Src kinase and STAT3 (Zhang et al., 2010). Angiogenesis is regulated by NO (nitric oxide), iNOS (inducible NO synthase) and COX-2. NO is known to enhance and NOS antagonists to inhibit VEGF synthesis. NO also upregulates COX-2, so a strong link has been established between NO, VEGF and angiogenesis. In this context it might be noted that HGF induces COX-2 through MET activation (Scarpino et al., 2009). HGF is known to activate the zinc finger transcription factor egr-1 (early growth response-1) and induce the transcription of VEGF and PDGFA. In fact, egr-1 binding sites have been detected in the promoter regions of these genes. Here HGF seemed to activate MEK1/2 and PKC pathways to activate egr-1 (Worden et al., 2005). HGF upregulates both egr-1 and the transcription repressor Snail. Snail is said to repress the expression of E-cadherin and the tight junction protein claudin-3 of epithelial cells, which aids the processes of cell scattering and migration (Grotegut et al., 2006). The transcription factors PAX (paired box) and Sox are said directly to interact with MET, induce cell proliferation and resistance to apoptosis, and increase cell migration. PAX6(5a), a variant form of PAX6, is expressed in pancreatic carcinoma cell lines at higher levels than PAX6 protein. These proteins bind to an enhancer element of MET promoter. Inhibition of PAX leads to an inhibition of their biological effects (Mascarenhas et al., 2009, 2010). HGF is known to activate PAX in the induction of cell proliferation, providing a clear activation signal to PAX/MET, leading to proliferation and invasion. General indications are that this could occur

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independently of ERK1/2 (Yablonka-Reuveni et al., 2008). Plexin B1 seems to block the activation of MET in melanoma and possibly inhibit invasion in this way (Stevens et al., 2010). This could be why Plexin is regarded as a metastasis suppressor in melanomas.

MET as a Therapeutic Target MET is expressed in many forms of cancer. The gene is amplified or the protein is overexpressed with marked relationship to tumour grade, lymph node involvement, invasion and metastasis. The angiogenic ability of HGF was reported many years ago and expression levels correlated with breast tumour growth with attendant elevation of MET to correspond with tumour progression (Nagy et al., 1995, 1996). The involvement of HGF in angiogenesis was strengthened by the finding that MET expression was upregulated by bFGF and TGF-β, which are powerful inducers of angiogenesis (Hiscox and Jiang, 1996). Subsequent to these early works, much evidence has emerged about the role of HGF as an active participant in differentiation and morphogenesis. HGF is a potent mitogen and an inhibitor of apoptosis, a promoter of cell invasion and motility, and an inducer of angio/lymphangiogenesis. With these attributes much effort has been expended on the role of HGF/MET in cancer invasion, metastasis and in predicting prognosis. Consistent with research and development in other areas of growth factors, MET has been viewed as a potential anticancer therapeutic target. Several types of MET inhibitor have been designed, namely monoclonal antibodies against MET or its ligand that prevent MET activation, and inhibitors of MET activation. Small interference RNAs have also been considered as potential inhibitors. Among monoclonals is the humanized mAb IgG2, which is directed against HGF Rilotumumab (Amgen Inc.). Rilotumumab prevents interaction between HGF and MET, and inhibits MET phosphorylation and signalling. Phase I clinical trials are currently underway (see Giordano, 2009). Rilotumumab is said to be well tolerated by patients with solid tumours. Mild side effects have been encountered. Most patients showed favourable response to treatment and disease-free progress from 7 to 40 weeks (Gordon et al., 2010). Small-molecule MET kinase inhibitors such as SU11274, K252a, PHA665752 and PF2341066 have been designed. Davis et al. (2010) tested SU11274 and Rilotumumab on clear-cell sarcoma cell lines. Both inhibited cell growth in culture. The antibody effectively suppressed xenografted cells. Kenessey et al. (2010) also reported that SU11274 blocked MET-mediated signalling in melanomas, decreased cell proliferation and increased apoptosis. In vitro, SU11274 at subapoptosis concentrations inhibited cell migration. Furthermore, when xenografted into SCID mice, the tumours showed reduced growth. Qian et al. (2009) have described another kinase inhibitor, EXEL-2880 (XL880, GSK1363089), which targets a range of growth factor receptors, notably MET and VEGFR. They reported loss of anchorage-dependent proliferation in vitro and in vivo; there was reduced tumour growth and inhibition of lung metastasis in experimental assays. The latter is

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probably a reflection of alterations in adhesive faculty of the tumour cells or changes in endothelial permeability. Inhibition of angiogenesis by targeting HGF and MET has provided an attractive route to therapy of cancer progression. One approach has been to use NK4, the N-terminal hairpin domain and four kringle domains of HGF. NK4 competitively binds to MET, but it cannot activate the receptor and inhibits HGF/MET signalling. NK4 is said to function as an inhibitor of angiogenesis (Nakabayashi et al., 2003; Nakamura et al., 2010). Nakabayashi (2003) had shown earlier that NK4 inhibited VEGF-induced endothelial cell proliferation and migration, and effectively blocked angiogenesis in vivo. Besides, NK4 has been attributed with the ability to suppress HGF-induced cell proliferation and motility and to promote apoptosis (Yue et al., 2010). Serine proteinases have also been deployed as inhibitors. These would prevent mature HGF being formed from its inactive precursor and in this way effectively inhibit HGF signalling (Parr et al., 2010). However, the NK4 fragment is also generated by proteolytic cleavage of HGF by serine and other peptidases as well as cathepsins, so the serine proteinase inhibitors might function in an antagonistic fashion in NK4-mediated suppression of biological effects.

6 The Platelet-Derived Growth Factor Family

The platelet-derived growth factor (PDGF) is a family of growth factors, which also includes VEGFs. PDGFs are highly mitogenic to mesenchymal cells. They are coupled with migratory behaviour of mesenchymal cells, and with differentiation in developmental and adult organisms (Hoch and Soriano, 2003). Four PDGFs, namely PDGFA, PDGFB, PDGFC and PDGFD, have been identified (Fredriksson et al., 2004; Reigstad et al., 2005). The PDGFs function as homo- or heterodimers; PDGFs C and D form only homodimers, but PDGFA and PDGFB can also function as heterodimers (Hart et al., 1990; Heldin et al., 1986; Stroobant and Waterfield, 1984), or are represented in another way as five dimeric isoforms: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD. PDGFs possess the characteristic PDGF domain composed of eight conserved cysteines, which participate in the formation of inter- and intramolecular bonds. The amino (N)-terminal region of PDGFC bears homology with the CUB (acronym of complement proteins C1r/C1s, sea urchin protein uEGF with EGF-like domains and BMP) domain, which occurs in extracellular and plasma membrane-associated proteins. The carboxy (C)-terminus contains a growth factor domain (GFD), which has 25% sequence homology with VEGF and 23% homology with the PDGFA chain. A serum-sensitive cleavage site between the two domains allows release of the GFD from the CUB domain (Gilbertson et al., 2001). PDGFD also possesses the N-terminal CUB domain. Two membrane receptors, PDGFR-α and PDGFR-β, are involved in PDGF signalling. In common with other growth factor receptors, both PDGFR-α and PDGFR-β dimerise and autophosphorylate on several tyrosine residues. The tyrosine phosphorylated sites serve to bind various SH2 domain-containing proteins. PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC can bind to and activate PDGFR-α; PDGF-BB and PDGF-DD specifically bind to and activate PDGFR-β. Besides, PDGF-AB, PDGF-BB and PDGF-CC can also activate PDGFR-α/ PDGFR-β heterodimers (Fredriksson et al., 2004).

Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00006-2 © 2011 Elsevier Inc. All rights reserved.

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PDGF in Cell Transformation and Cancer PDGF signalling has received much attention since the demonstration that the product of the v-sis oncogene, which can induce cell transformation, bears considerable sequence homology with PDGFB and greater than 50% homology with PDGFA (Doolittle et al., 1983; Johnsson et al., 1984; Josephs et al., 1984; Waterfield et al., 1983). The oncogenic protein transforms cells that express the receptors (Deuel et al., 1983; Garret et al., 1984; Huang et al., 1984; Leal et al., 1985). PDGF exerts a marked influence on the biological behaviour of cells that is identifiable with migratory behaviour, and with differentiation in developmental and adult organisms, with cell transformation and with cancer development and progression. These phenotypic tasks are performed by well-defined systems of signal transduction coupled with interaction with signalling systems involved in the function of other growth factors.

Putative Collaborative Participation of PDGF in Angiogenic Signalling Neovascularisation is a pre-eminent requirement of cancer invasion and metastasis. Some recent work has suggested the collusion of PDGF with signalling pathways such as the HIF-1 transcription factor, FGF2 and HB-EGF. In pancreatic adenocarcinoma the levels of heparanase, itself an angiogenic agent, correlated with the expression of PDGFA and with HIF-1a, with the latter showing correlation with bFGF and HB-EGF. HIF-1 is that is induced by VEGF. Also, heparanase expression correlated independently with the presence of lymph node metastasis (Hoffmann et al., 2008). These data suggest a putative link between heparanase, PDGF and metastasis involving angiogenesis induction. Heparanases cleave the heparan sulphate glycosaminoglycans (HSPG) and regulate cell proliferation and angiogenesis associated with wound healing and inflammation. HSPGs interact with constituents of major ECM components, and remodel and alter ECM in a way that can lead to changes in intercellular adhesive function and related invasive ability (Bernfield et al., 1999; Iozzo, 1998). Furthermore, HSPGs appear to be capable of regulating several angiogenic agents, for example bFGF, VEGF and HGF, besides factors such as PDGF-BB and HB-EGF that promote proliferation and cell migration (Bechard et al., 2001; Catlow et al., 2008; Mulloy and Forster, 2000; Powers et al., 2000; Raman et al., 2003; Robinson et al., 2005). One should reiterate here the need for direct evidence for this cross-talk of PDGF with angiogenic signalling. An alternative route to angiogenesis is by modulating VEGF expression in collusion with Notch signalling. Wang et al. (2007) have shown that downregulation of PDGF expression led to inactivation of Notch-1 in parallel with the downregulation of VEGF and MMP.

PDGF and Epithelial–Mesenchymal Transition The conversion of epithelial cells into mesenchymal cells or epithelial–mesenchymal transition (EMT) is a developmental programme. EMT is characterised by changes in cell morphology and reduced intercellular adhesion leading to enhanced cell

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migration. Also associated are reduced expression or loss of E-cadherin, enhanced expression of N-cadherin and vimentin, and β-catenin translocation, possibly in association with its signalling implication. Many transcription factors are upregulated in the implementation of EMT. Epithelial tumours undergo this process, which results in the acquisition of invasive capacity and the potential to metastasise (see Lee et al., 2006). Activation of EMT may occur with TGF-β, and EGFR and HER2 receptors. With this backdrop, it is of considerable interest that PDGF has been linked with the induction of EMT in the promotion of cell proliferation, invasion and metastasis. EMT plays an essential role in cancer development and progression. So it is notable that microRNAs (miRNAs) have been implicated in the regulation of EMT. PDGF modifies cell motility by altering the adhesive property and machinery of cells. The activation of EMT in this process has inevitably attracted much attention. PC3 prostate cancer cell transfectants expressing high levels of PDGF-D display a marked change in cell morphology, loss of E-cadherin and enhanced expression of vimentin, an intermediate filament protein of mesenchyme origin, which participates in cell adhesion and motility (Kong et al., 2008). Jechlinger et al. (2006) described the presence of an autocrine loop involving PDGF/PDGFR and TGF-β. They suggest that PDGF activates PDGFR in an autocrine fashion, which in turn activates Ras/ PI3K signalling to sustain cell proliferation and survival during EMT. The activation of EMT is blocked by PDGFR antibodies and this induces apoptosis. As stated earlier in the context of EMT induction by TGF-β, miRNAs, which are short, single-stranded non-coding RNA molecules, play an important part in gene regulation. Their expression has been implicated with differentiation, and they have been closely linked with alterations in cell behaviour related to tumour growth and progression. miRNAs basically target different genes with varying outcomes, some functioning as tumour suppressors and others as promoters of tumour development and progression. The members of miRNA-200 family and miRNA-205 are said to be downregulated in cells that have undergone EMT stimulated by TGF-β. Experimentally induced expression of the miRNA-200s blocks TGF-β-mediated activation of EMT. The miRNA-200s and miRNA-205 block the repression of E-cadherin expression (Gregory et al., 2008; Korpal et al., 2008; Park et al., 2008). PDGF overexpression in PC3 cells leads to EMT with associated alterations in the cell phenotype. This seems to result from the downregulation of miRNA-200s. A result of this is the upregulation of ZEB1, ZEB2 and Snail2, which represses E-cadherin transcription (Kong et al., 2008, 2009). Sánchez-Tilló et al. (2010) found that ZEB1 recruits the corepressor BRG1. Blocking this interaction induced expression of E-cadherin and inhibited EMT confirming the importantance of E-cadherin in the transition. Furthermore, from the disposition of the ZEB1/BRG1 and E-cadherin, they have suggested the involvement of this mechanism in tumour invasion. However, as stated previously, miRNAs can have a contrary function. miRNA21 is often upregulated in cancers and promotes TGF-β-mediated activation of EMT (Zavadil et al., 2007). Davis et al. (2009) found that PDGF-induced proliferation of vascular smooth muscle cells, induced the transcription of miRNA-221 and further downregulated the targets c-kit and p27Kip1. Felicetti et al. (2008) found

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that the promyelocytic leukaemia zinc finger transcription factor PLZF represses miRNAs-221 and 222. When PLZF is blocked and the function of the miRNAs is restored, p27Kip1 and c-kit are inhibited, leading to enhanced cell proliferation of melanoma cells. One should recall here that the c-kit encoded TRK has been linked with the induction of cellular migration, cell proliferation and survival, albeit yet to be fully established, and p27Kip1 is a cyclin-dependent kinase inhibitor that negatively regulates G1 cell cycle progression.

PDGF Signalling in Cell Proliferation and Migration The mitogenic role played by PDGF in early development, and its relevance to cell differentiation and cell migration, has been fully documented (Hoch and Soriano, 2003). Therefore, its involvement in angiogenesis (as discussed earlier) and cancer invasion and progression has been investigated as a normal course of events. Some evidence has accumulated which suggests the activation of the PI3K pathway results in cell migration and proliferation (Fischer et al., 2007). Besides PI3K, the proliferative signal from PDGF might activate the ERK1/2 pathway (Holmström et al., 2008). These authors compared and contrasted PDGF signalling with that of EGF. They seem to suggest EGF and PDGF share the ERK1/2 pathway, but EGF does not activate PI3K or PKC signalling. However, EGF can and does induce cell migration. So the distinction apparent here is not a decisive signalling-pathway-related outcome towards cell migration or proliferation. A distinction between signal transduction in motility and cell proliferation is not evident from the data presented by Gentilini et al. (2007), who found both PI3K/Akt and ERK1/2 pathways need to be activated for the induction of motility. Human medulloblastoma cells respond to PDGF-BB by PDGFR-β activation and activation of Akt/ERK signalling together with the transactivation of EGFR, which correlate with enhanced migration, cell survival and proliferation. Inhibition of PDGFR tyrosine kinase with imatinib abrogated PDGFBB-stimulated effects, suggested to be mainly owing to the inhibition of PDGFR-β activation (Aoki et al., 2007) and enhanced expression of the Akt regulator PTEN (Abouantoun and MacDonald, 2009). These findings provide further support to the thesis that an integration of the PI3K/Akt and ERK1/2 pathways operates in cell motility and proliferation. Downstream, the transcription factor NK-κB (Rel) mediates the induction of proliferation (Smith et al., 2008). Hata et al. (2010) stably transfected U87 or LN229 glioma cells with PDGFBB. Transfectant clones that showed high and low PDGFBB secretion were selected. In vitro human mesenchymal stem cells derived from bone marrow tended to show greater migration towards high PDGFBB producing glioma cell clones than towards the low-level producers. Antibodies against PDGFBB inhibited this chemotactic migration. So it seems that PDGF can function as an inducer of cell migration as well as a chemoattractant. It would have been useful to know whether there were differences in the motility of high and low PDGFBB producers. PDGF can act as a paracrine factor as it often does in epithelial tumours, or in an autocrine fashion as in leukaemias and sarcomas. It would be prudent to say here that chemotaxis and induction of cell motility are two separate aspects of the same biological

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phenomenon. The two aspects can be uncoupled on the basis of the mode of PDGF function.

Co-operation with GPCR Pathway in Mitogenic Function G-protein-coupled receptors (GPCRs) constitute a major system of transduction of growth factor signals. Agonists of GPCRs are not remarkable as mitogenic agents, but their effects appear to be accentuated by interaction with growth factors that function by activating RTKs. GPCRs co-operate with EGF and PDGF in mitogenic function (Ediger and Toews, 2000; Gosens et al., 2003; Krymskaya et al., 2000).

Signalling Specificity Growth factors have been known to activate many early response genes to elicit specific cellular responses. The early response genes co-ordinate cell proliferation, apoptosis and differentiated phenotypic features. The participation of PDGF in a wide spectrum of function and the ability of this growth factor to activate several pathways, including PI3K, Ras, STAT, Src kinases and NF-κB, has been discussed previously. This then leads one to the quest of devices and mechanisms that might bring about specificity in PDGF function. It would be expected and it has been demonstrated that PDGF activates several early response genes such as egr-1, ets-1, fos and jun. Apart from transcription factors, early response genes respond to stimuli from membrane matrix proteins, cytokines and proteins that are involved in the cytoskeletal machinery. The induction of cell proliferation and migration by PDGF is an important aspect of cardiovascular biology, both normal and aberrant (see Raines, 2004). PDGF induces JE/MCP-1, the chemokine in vasuclar smooth muscle cells. PDGF contributes significantly to vascular remodelling and the formation of neointima in atherogenesis. PDGF can induce intimal hyperplasia. PDGFR-β is activated by PDGFBB and by PDGFAB when both PDGFR-β and PDGFR-α are expressed together (Davies et al., 2000; Giese et al., 1999; Sirois et al., 1997). This is an important feature of atherogenesis in the development of arteriosclerosis. PDGF, as alluded to already, is a major inducer of angiogenesis. So the focus here is to identify and establish perceived links between activation of specific transcription factors and the cardiovascular- and angiogenesis-related function of PDGF.

PDGF Activates ets-1 Transcription Factor Many growth factors activate the transcription of early response genes. It follows that to attain phenotypic specificity of function, growth factors might be activating specific immediate early response genes, which would in turn activate the expression of delayed early and late response genes. The pattern of activation in terms of negative and positive regulation of substantive delayed response and late response genes would inevitably depend upon the expression pattern of the immediate early response genes. Potentially a feedback regulation of early response gene activation can also be envisaged. These prospective modes add a further tier of growth

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factor-mediated regulation of phenotypic signalling by specific, immediate, early gene activation. PDGFBB activates ets-1 transcription factor with PKC mediation during the regulation of vascular smooth muscle cell migration and proliferation (Naito et al., 1998). Ets-1 can negatively regulate the expression of certain markers of smooth muscle cell differentiation (Dandré and Owens, 2004). PDGFBB induces thrombomodulin in vascular smooth muscle cells by activating the PI3K/Akt and mTOR (mammalian target of rapamycin) pathway and the ets-1 transcription factor. The induction of thrombomodulin is blocked by the inhibition of src kinase, PI3Kinase and mTOR (Lo et al., 2009). Angiotensins are believed to induce angiogenesis by the mediation of many angiogenic growth factors such as nitric oxide synthase and metalloproteinases (see Escobar et al., 2004). Angiotensin upregulates VEGF expression. In mesangial cells, angiotensin II has been found to increase ets-1 expression by the intervention of Cox-2 (Pearse et al., 2008); the increase in ets-1 in turn upregulates PDGFD (Liu et al., 2006). Ets-1 binds to a specific promoter site and activates PDGFA (Santiago and Khachigian, 2004). The effects of angiotensin on angiogenesis might occur through PDGF-induced upregulation of MCP-1. Angiotensin II enhances MCP-1 expression in pancreatic ductal adenocarcinoma cells by ERK1/2 signalling (Chehl et al., 2009).

Early Growth Response (egr) Zinc Finger Transcription Factors The zinc finger transcription factor egr-1 (early growth response-1) shows specific participation in angiogenesis, atherosclerosis and other cardiovascular conditions (see Khachigian, 2006). The angiogenic factor HGF activates egr-1; HGF also activates egr-1 to induce the transcription of PDGFA and VEGF but not IL-8. Consistent with this, egr-1 binding sites were found in the PDGFA and VEGF, but not IL-8, promoter. Here, HGF seemed to activate MEK1/2 and PKC pathways to activate egr-1 and induce the expression of PDGFA and VEGF (Worden et al., 2005). HGF is also known as the scatter factor. In this biological effect of cell scattering, HGF upregulates both egr-1 and the transcription repressor Snail. Snail is said to repress the expression of E-cadherin and the tight junction protein claudin-3 of epithelial cells, which aids the processes of cell scattering and migration (Grotegut et al., 2006). The activation of egr-1 in conjunction with angiogenesis is apparent in the experimental model wherein SDF-1 (the stromal cell derived factor-1) induces VEGF expression in HUVEC cells, which is mediated by the activation of ERK1/2 pathway and the induction of egr-1 (Neuhaus et al., 2003). Angiopoietin-1 (Ang-1) also upregulates egr-1 expression in HUVEC cells within a couple of hours of exposure to ERK1/2, PI3K/Akt signalling, with the biological manifestations of upregulated endothelial cell proliferation and migration (Abdel-Malak et al., 2009). This networking function can lead to the formation of negative regulatory loops as well. Egr-1 has also been found to be anti-angiogenic under specific conditions, such as continual expression (Huang et al., 2006; Lucerna et al., 2006). This is possibly a consequence of upregulation of transcription repressors that function in a negative regulatory fashion.

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Other growth factors might also enlist egr-1 in their function. EGF requires egr-1 to elicit proliferative responses, and the latter can be modulated by negative regulators of egr proteins (Mayer et al., 2009). EGF is known to generate other responses as well as proliferation. It binds to egr-1 promoter and regulates the expression of the Klotho gene. EGF activates the Ras/MEK/ERK signalling cascade in regulating Klotho expression (Choi et al., 2010). However, Klotho is a single-pass transmembrane protein associated with chronic kidney failure, osteoporosis and arteriosclerosis. Obviously, EGF is able to subserve a totally different phenotypic function than normally attributed to it, of activating the standard signalling pathways and transcription factor but also activating a different substantive or delayed response gene. Another postulate is the networking of early response genes and a feedback regulation of signalling. This is suggested by the finding that when these are mutated there is a back-propagation of their altered status, which affects the same cell types and influences the signalling pathways that PDGF activates (Schmahl et al., 2007).

MCP-1 in PDGF Effect MCPs (macrophage chemoattractant proteins) are C-C family chemokines that induce chemotaxis of monocytes, basophils and T-lymphocytes. The C-C chemokines are classified according to the arrangement of cysteine residues into CC chemokines with adjacent first two conserved cysteine residues, CXC chemokines that have one intervening amino acid residue, chemokines with three intervening residues, and chemokines that do not have the first and third of the four conserved cysteine residues. Among MCPs are MCP-1 (CCL2 chemokine C-C motif ligand- 2), MCP-3 (CCL7) and MCP-4 (CCL-13) proteins (see section on chemokines). MCP genes are immediate early genes encoding secreted cytokines characterised by the CXC motif of two cysteines separated by one amino acid. They bind to CCR2 and CCR4 receptors and are ligated to endothelial cells by glycosaminoglycan (see Entrez Gene summary). Over two decades ago, Rollins et al. (1988) cloned and characterised the JE murine/rat homologue of the human MCP-1 as a PDGF-induced gene. Poon et al. (1996) showed that aortic vascular smooth muscle cells exposed to PDGFBB displayed monocyte chemotactic activity mediated by MCP-1, and even earlier that serine–threonine kinases are involved in the signalling process (Kawahara et al,. 1994). The latter finding naturally linked MCP-1 with growth factor-mediated activation of RTKs. MCP-1 may play an important part in tumour development by virtue of its chemoattractant property to induce cell migration. Its expression in tumour and stromal cells is said to correlate with macrophage infiltration into the tumour. Also interesting is the apparent association of stromal but not tumour cell MCP-1, detected by immunohistochemical means, with patient survival (Fujimoto et al., 2009). MCP-1 is associated with the function of many growth factors and quite closely with angiogenesis. Although designated as an immediate early response gene, MCP-1 has been postulated to induce a transcription factor called MCPIP (MCP1-induced protein). The induction of MCPIP is required for the formation of capillary-like structures in HUVEC cell cultures. Inhibition of MCPIP suppressed

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MCP-1-mediated induction of VEGF, HIFα and tubule formation. These findings were also confirmed by transfection of HUVECs with an MCPIP expression vector. The transfectants showed tubule formation and expressed cadherin genes cdh12 and cdh19 (Niu et al., 2008). TGF-β is a powerful angiogenic factor. In addition it can enhance the expression of VEGF and MCP-1. Accentuation of TGF-β signalling by cathepsin G also resulted in the upregulation of VEGF and MCP-1 (Wilson et al., 2010). TGF-β induces MCP-1 by Smad3 receptor and PKC-δ signalling (Zhang et al., 2009). In glomerular podocytes in culture, TGF-β activated PI3K signalling and induced MCP-1, which then modulated the actin cytoskeleton (Lee et al., 2009). The activation of mononuclear phagocytes by MCP-1 does involve actin polymerisation and the upregulation of integrins and ICAMs (Gu et al., 1999; Ikeda et al., 1993; Rollins, 1997), thus promoting cell migration. Nonetheless, this mode of MCP-1 function, as well as signalling by TGF-β which occurs through the Smad receptor system, constitutes a novel idea.

Relationship of PDGF Expression to Cancer Invasion and Progression The abundantly obvious alterations imposed by PDGF on cell motility, cell proliferation and angiogenesis have led to the exploration of PDGF as a marker of clinical aggression of cancer, metastatic potential and as a prognostic aid. Therapy targeted against PDGFR is being evolved, and some TRK-specific inhibitors, for example Imatinib, are in clinical trials. PDGF is overexpressed in many human cancers. Breast cancers overexpress PDGFA and PDGFR-α, which has been found to correlate with presence of disseminated tumour in the lymph nodes (Carvalho et al., 2005). In colorectal cancer, vascular invasion was more pronounced in patients with tumours expressing high PDGFBB than in those with tumours expressing low PDGFBB. Also, high PDGFBB expression correlated with poor prognosis (Nakamura et al., 2008). Belizon et al. (2009) also found enhanced levels of PDGFBB in colorectal tumours compared with benign adenomas. However, they were unable to establish a definitive relationship between growth factor levels and tumour stage. In patients with NSCLC, PDGF-A expression correlated with metastasis to the lymph nodes, and the co-expression of PDGFB and VEGFR3 indicated poor prognosis (Donnem et al., 2010). A similar relationship subsists in oesophageal squamous cell carcinomas, where PDGFBB expression correlated with tumour size and depth of invasion, spread to the lymph nodes and poor prognosis (Matsumoto et al., 2007). Interactions between tumour cells and tumour stroma symbolise an important factor in the development and progression of cancers. Colon and prostate cancers express PDGFB receptor in the stromal component. In breast cancer, stromal expression of the receptor correlates with HER2 expression and tumour grade, and is associated with ER-negative cancers. Also, high stromal receptor levels indicated shorter recurrence-free survival (Paulsson et al., 2009).

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Genetic Changes in PDGFs and PDGFRs The overexpression of PDGFs and/or PDGFRs might be an outcome of gene amplification with or without any structural rearrangement of the genes. Activating mutations and rearrangements of PDGFR genes have been detected. It was reported some time ago that PDGFR gene overexpression in a glioma was accompanied by the amplification of the PDGFR gene carrying a deletion of a region that coded for 81 amino-acid residues corresponding to the immunoglobulin-like domain in the extracellular region of receptors (Kumabe et al., 1992). However, a recent detailed study revealed no mutations of PDGFA or PDGFR, but PDGFA was expressed in most gliomas investigated and one-fifth of samples showed gene amplification (Martinho et al., 2009). The histologically benign ameloblastoma of the odontogenic ectoderm consistently expresses PDGFAA and PDGFR-α, but only 1 out of 29 patients showed a mutation in exon 12 of the PDGFR-α gene (Sulzbacher et al., 2008). PDGFR-α is expressed in a large majority of anaplastic large-cell lymphomas (5 out of 7, 71.4%) and in nasal type NK/T-cell lymphoma (9 out of 12, 75%), but expression was not related to mutation of the PDGFR-α gene, of which exons 10, 12, 14 and 18 were analysed (Chen et al., 2008). Holtkamp et al. (2006) found PDGFR-α gene amplification and expression in a series of malignant peripheral nerve sheath tumours; two tumours carried somatic PDGFR-α mutations in exons coding for the extracellular domain. There were no point mutations or polymorphisms. In a similar vein are findings relating to a series of the rare form of bone cancer called chordoma, which have shown PDGFR-β overexpression and activation, and less prominently of PDGFR-α and c-kit, although there was evidence of activating mutations (Tamborini et al., 2006). So a somewhat limited review of PDGFA and PDGFR-α expression would lead one to suggest overexpression of the growth factor and its receptors, with amplification of corresponding genes possibly occurring frequently in cancers, although only infrequently accompanied by genetic alterations.

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7 Nerve Growth Factors

Nerve growth factor (NGF) was discovered and characterised in the early 1950s. NGF together with BDNF (brain-derived neurotrophic factor) and the neurotrophins (NTs: NT3, NT4, NT5 and NT7) constitute the neurotrophin family of growth factors. NTs play a wide variety of roles, for example in neural differentiation, survival of neuronal cells (Lessmann et al., 2003), and neuroprotection and repair (see Sofroniew et al., 20001). They have been associated with many disease processes. They achieve a wide diversity of functions by activating many signalling systems. NTs bind to two types of receptor: Trk and p75NGFR. The latter is a TNF family receptor. Both the precursor forms and proteolytically processed NTs bind p75NGFR, but only the mature processed NTs bind Trks with specificity. NTs are secreted proteins and function by binding to their receptors, so the signalling characteristics have come to the forefront. Although historically NGF was accorded the epithet of a neuroprotective and survival promoter, it participates also in apoptosis and is detected in many human cancers. The functional diversity and phenotypic outcome of NTs is engendered by specificities connected with receptors activated by the ligands. It is in this context that the discussions of the NTs are here oriented.

NT Signalling Upon receptor binding, NTs activate the conventional signalling pathways, namely Ras/ERK/MAPK and PI3K/Akt, leading to cell survival and differentiationrelated functions. Three Trk receptors have been identified, TrkA, TrkB and TrkC, which carry differential binding abilities for the neurotrophins. TrkA binds NGF, TrkB binds BDNF and NT4, and TrkC binds NT3. The last one also binds TrkA and TrkB (Huang and Reichardt, 2003). Ligand binding induces the dimerisation of the receptors. Heterodimers of TrkA and p75NGFR promote cell survival but homodimers of p75NGFR lead to apoptosis. As a consequence of the signalling downstream, the immediate early genes fos, egr1 and egr2 are activated. The promoters of these genes are targeted by the recruitment of the transcription factors Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00007-4 © 2011 Elsevier Inc. All rights reserved.

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C/EBP (CCAAT/enhancer binding protein) -α and -β together with NeuroD (Calella et al., 2007). C/EBP-α and the co-repressors CtBP1/2 (carboxyl-terminal binding proteins 1 and 2) regulate gene function. NGF, presumably mediated by TrkA, promotes the formation of a complex between the nuclear speckle protein acinus and CtBP2, which leads to the downregulation of cyclin D1 and the inhibition of cell proliferation in leukaemic cells (Chan et al., 2009). Terasawa et al. (2009) found NGF to be capable of activating ERK1/2 signalling and inducing the expression of microRNA (miRNA), miR221 and miR22, in PC12 cells. These short, singlestranded non-coding RNA molecules might regulate immediate early gene expression. TrkA might take recourse to p53 in regulating cell proliferation. It has been shown that TrkA phosphorylation is promoted by p53 by suppressing the phosphatase SHP-1. Activated in this way, TrkA seems to inhibit cell proliferation (Montano, 2009). Of note here is that TrkA activation occurs in the absence of NGF. In breast cancer cultures, in contrast, TrkA can be activated by NGF to promote cell proliferation, which could take place by conventional signalling pathways. One cannot exclude the possibility that p75NGFR might contribute through p53 to the inhibition of cell proliferation. In fact TrkA can upregulate p75NGFR. A PC12-variant cell line that had no TrkA constitutive expression of p75NGFR was greatly reduced (Rankin et al., 2005). Furthermore, although Trk mutations are probably somewhat rare events, constitutive activation by mutation cannot be excluded. The binding of mature NTs to Trks signals through Ras/MAPK or PI3/Akt, which leads to cell survival. However, ligand binding to p75NGFR can lead to neural cell death with JNK and p53 mediation. In contrast, ligand binding to p75NGFR is also known to activate the NF-κB pathway, which inhibits apoptosis and promotes cell survival (Figure 7.1a and b). Inhibitors of both p75NGFR and Trk signalling have the effect of inducing apoptosis, as shown in breast cancer cells (Naderi and Hughes-Davies, 2009). It was recognised some time ago that PLC-γ/PKC signalling is involved in neuronal development. Growth factors are regarded as being able to activate this pathway. A link of this pathway to NGF is the finding that NGF phosphorylated PLC-γ through the agency of Trk (Kim et al., 1991; Vetter et al., 1991) (Figure 7.1a and b).

Genetic Alterations in NT Receptors Genetic alterations appear infrequently in NTs or their receptors. Splice variants of Trks do occur and deregulation of receptor function by loss-of-function mutations has also been found. Single-nucleotide polymorphisms (SNPs) have been encountered in investigations of patients with neurological and non-neurological conditions. Whether these are functional in the disease process is debatable. A T  C transition at position 2198 from the transcription start site of the NGF promoter was reported by Alam et al. (2005), which is believed to affect VDR binding. Two SNPs of NGF have been identified in patients with multiple sclerosis and postulated to possess functional features in its development and progression (Akkad et al., 2008). Scaruffi et al. (1999) found SNPs (C  T) in the TrkA tyrosine kinase domain at 6773, 7232

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Figure 7.1  (a and b) Signalling pathways activated by NTs, which are discussed in the text. The ligands display differential binding to the receptors. The precursor forms and proteolytically processed NTs can both bind p75NGFR, but only the mature processed NTs bind Trks with specificity. Ligand binding induces the dimerisation of the receptors. Heterodimers of TrkA and p75NGFR promote cell survival but homodimers of p75NGFR lead to apoptosis. BDNF recruits C/EBP to activate early response genes; NGF, on the other hand, promotes complex formation of the nuclear speckle protein acinus with CtBP2, a co-repressor that functions with C/EBP, downregulates cyclin D1 expression and inhibits cell proliferation. Thus the diversity of phenotypic effects is created and often with opposite effects.

and 7301 nucleotides, in medulloblastomas. SNPs were detected in Trks in human medullary thyroid carcinomas. The medullary carcinoma arises from C cells (parafollicular cells). Trks are differentially expressed in C-cell hyperplasia but, in later stages of the carcinoma, there is no perceived perceptible link between the genetic variations and receptor expression levels (Gimm et al., 2001). Again, in prostate cancer the TrkA gene showed four polymorphisms in three exons. The incidence of these was similar to that found in the control group (George et al., 1998). There is one report concerning p75NGFR, which is related to melanomas where 2 out of 10 samples showed reduced p75NGFR levels. The two samples carried an identical point mutation in the transmembrane domain of the protein (Papandreou et al., 1996).

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Expression of NTs and Their Receptors in Cancer NTs are expressed in many forms of cancer. Inevitably much effort has been directed towards how they might influence the growth and spread of cancers. NGF is expressed in most (63 out of 109) oesophageal squamous cell carcinomas, and expression levels correlate with tumour stage, the presence of metastatic tumour and poor prognosis (Tsunoda et al., 2006). It is frequently expressed in the tumour component of breast cancers (Adriaenssens et al., 2008). TrkA is expressed in breast cancer cell lines. Overexpression of the receptor has led to enhanced growth, induction of angiogenesis and metastasis of breast cancer cells xenografted into immune compromised mice (see Lagadec et al., 2009). TrkA was associated with advanced ovarian cancer compared with borderline and FIGO (International Federation of Obstetricians and Gynaecologists) stage I carcinomas, but the expression of p75NGFR was comparable in the three groups (Odegaard et al., 2007). Mostly Trk expression is clearly linked with cell proliferation and inhibition of apoptosis, but Blasco-Gutierrez et al. (2007) found TrkC expression decreased with increasing tumour grade, possibly suggesting NT3 activity. This requires confirmation, for Bloom grading incorporates mitotic index and state of tumour differentiation, so it is difficult to visualise how TrkC activation might import good prognosis. It is not beyond the realms of possibility that TrkC might transactivate p75NGFR. Clearly there are areas for taking this forward. BDNF-induced activation of TrkB has been seen in ovarian carcinomas with consequent induction of cell migration and inhibition of apoptosis (Au et al., 2009). High BDNF and TrkB but not p75NGFR expression occur in both prostate cancer and BPH, whereas other NTs and receptors are detected in low levels (Bronzetti et al., 2008). TrkA and TrkB but not TrkC are detected in human lung adenocarcinomas (Perez-Pinera et al., 2007). There are clear indications, however, that the presence of p75NGFR might indicate good prognosis in B cell precursor-acute lymphoblastic leukaemia. The expression of p75NGFR was higher in the low-risk category of patients than in the high-risk group. Although this does not allow one to accept the reverse of the argument, a subgroup of patients who showed disease relapse had low p75NGFR compared with those who remained in remission (Troeger et al., 2007). The expression of p75NGFR messenger RNA (mRNA) was low or totally undetectable, and the protein was not detected in retinoblastoma or in normal retinal tissue (Dimaras et al., 2006). Direct correlations between p75NGFR invasive behaviour and longer disease-free and overall survival have been observed in breast cancer. Also, p75NGFR is inversely correlated with the presence of ER and PR (Reis et al., 2006). Moon et al. (2009) found NGF more frequently (38 out of 45) in the tumour component of hepatocellular carcinomas than in normal hepatocytes (29 out of 45). Of much interest was that no p75NGFRs were found in the tumour cells. Because ligand-activated p75NGFR can induce apoptosis, they suggest by inference that loss of p75NGFR could lead to carcinogenesis. NGF-expressing tumours had a higher PCNA index. This could indicate that NGF might activate Trk receptors, but there is no information about the state of their expression.

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NGF is capable of inducing cell migration. Breast cancer cell lines exposed to NGF show enhanced invasion in Matrigel assays (Dolle et al., 2005). Schneider et al. (2001) described the distribution of NT ligands and their receptors in pancreatic cancer. From their pattern of distribution these authors suggested that these might be involved with the neural invasion often encountered in pancreatic cancer. Pancreatic tumours that express high levels of NGF frequently show perineural invasion. Indeed, expression of NGF correlated with tumour spreads to the lymph nodes. TrkA expression relates significantly with perineural invasion. Both NGF and TrkA negatively correlate with the proliferation marker Ki67 (Ma et al., 2008). This confirms the earlier report of the link between TrkA expression and perineural invasion (Dang et al., 2006). Here, one has to entertain the possibility that the negative relationship of Trk with proliferation could be a result of intervention by p75NGFR activation. This is also compatible with its association with good prognosis. Recently, Wang et al. (2009) have reported that p75NGFR expression was weaker in pancreatic carcinoma cells than normal tissue. Furthermore, they found that expression correlated positively with perineural invasion. Upon transfection with p75NGFR, cancer cells displayed chemotaxis towards NGF, which has been postulated as a reason for perineural invasion of cancer cells. In oesophageal squamous cell carcinomas also, p75NGFR correlated inversely with tumour stage, progression and prognosis (Tsunoda et al., 2006). Dang et al. (2009) found that high expression of p75NGFR was prognostically a good indicator and corresponded with longer overall survival. Overall, the role of p75NGFR in cancer progression is rather provisional at present. Using prostate carcinoma cell lines, Festuccia et al. (2007) demonstrated that the presence of TrkA and TrkB correlated with invasive ability and that both NGF and BDNF were able to induce cell migration. BDNF has been attributed with the ability to induce angiogenesis. It is expressed in multiple myeloma cells, and of much interest is that decrease of BDNF expression corresponded with remission. Primary myeloma cells and the myeloma cell line RPMI18826 induced migration and formation of capillary-like structures in HUVEC cultures. Both cell lines expressed BDNF, so the effects could be partly inhibited by anti-BDNF antibodies (Hu et al., 2007). Here, one should take cognizance of the fact that the myeloma cells also expressed VEGF, which could account for the only partial inhibition of angiogenic effect by anti-BDNF antibodies. Of note also is that NGF is angiogenic (Campos et al., 2007; Lazarovici et al., 2006; Park et al., 2007). Campos et al. (2007) reported that NGF could induce the expression of three isoforms of VEGF. However, Lazarovici et al. (2006) could not inhibit the induction of angiogenesis with VEGF antagonists and suggested a direct NGF/TrkA-mediated stimulation of angiogenesis in their experimental setup. It is apparent from the above that in most investigations the expression of NT ligands and their respective receptors has influenced cell proliferation, inhibition of apoptosis and induction of cell migration, which is generally compatible with the presumed outcome. However, it is difficult to desist from the thought that the choice of NTs and receptors has tended to be somewhat tentative.

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8 Insulin-Like Growth Factors

Insulin-like growth factors (IGFs) regulate growth in various stages of development. They are involved in many facets of biological function such as tissue repair and regeneration, and in many disease processes including tumour cell growth, survival, angiogenesis and metastasis. The natural focus in this chapter is on IGF signalling in the development and dissemination of cancer.

Insulin-Like Growth Factors and Their Receptors Insulin-Like Growth Factor Receptors IGFs are polypeptide growth factors, structurally similar to insulin, composed of two subunits linked by disulphide bonds. IGF-I and IGF-II are two principal forms but several variants are known. Three receptors, IGFR1, IGFR2 and insulin receptor, mediate the biological effect of IGFs. There are characteristic differences in receptor affinities. IGFR1 binds both IGFs with high affinity. However, IGFR2 displays marked differences in its binding. It binds IGF-II with high affinity but IGF-I with only low affinity. IGF-I also binds insulin receptors at 100-fold lower affinity than insulin. There is recent evidence that IGF-II is also able to activate the insulin receptor. IGF-I is induced in the liver by somatotropin and it is a major effector of the biological effects of somatotropin. Another important component of IGF function is their association with the so-called IGF-binding proteins (IGFBP), which subserve the function of regulating the biological activity of IGFs by interfering with their interaction with IGFRs. The formation of the IGF/IGFBP complex also increases the half-life of IGFs. The IGFBP family is made up of six members, all capable of inhibiting IGF function, but IGFBPs 1, 3 and 5 might stimulate IGF action (Baxter, 2000).

Insulin Receptor Substrate Proteins The transduction of growth factor signals begins with the activation of appropriate tyrosine kinase receptors. New intermediaries have been discovered in the form of Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00008-6 © 2011 Elsevier Inc. All rights reserved.

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substrates for these kinases, which have been termed as insulin receptor substrates (IRSs) as they were identified with activation and downstream function of the insulin receptor. IRS-1 and IRS-2 are adaptor proteins. They translocate into the nucleus and can regulate genetic transcription. IRS-3 shows both membrane and nuclear localisation. Its pleckstrin homology domain is involved in this localisation process (Maffucci et al., 2003). IRS proteins have been attributed with a variety of biological responses elicited by growth factors, cell proliferation, migration and survival and in cancer development and metastasis. They have often been described as oncogenes, an epithet that has been applied to many genes on a severely misconceived thesis. IRS proteins are activated by phosphorylation on serine residues and on serine/threonine sites. IRS proteins are overexpressed in many forms of human cancer. It has been postulated that IRS-1 might suppress, and IRS-2 might promote, metastasis (see Gibson et al., 2007). IRS proteins and structural and functional homologues Gab1, Grb2 and DOS (Daughter of Sevenless) are conserved in evolution, which emphasises their importance in the mechanisms relating to the transduction of growth factor signals (reviewed by Yenush and White, 2005). DOS is involved in the activation of Sevenless receptor TRK and possesses amino (N)-terminal pleckstrin homology domain phosphorylation sites and functions as an adaptor protein (Raabe et al., 1996). The Gab family docking proteins are able to amplify growth factor signals. Gab1 participates in EGFR signalling, binds to the carboxy (C)-terminal region of EGFR and, when overexpressed, it enhances EGFR activation of JNK/PI3K/MAPK and promotes cell proliferation (Rodrigues et al., 2000). IRS-1 acts as a docking protein for SH domain-containing signalling molecules, such as PI3K and activates PI3K-mediated signalling. FRS2-α and FRS2-β are docking proteins associated with signalling by FGF, NGF and GDNF. FRS2-α and FRS2-β also bind to Grb2 and SH2 domain-containing proteins (Hadari et al., 1998; Kouhara et al., 1997).

IGF Signalling Pathways The binding of IGF to its receptors leads to their dimerisation and autophosphorylation in a conventional manner common to peptide growth factors. Signalling pathways activated downstream generate the phenotypic changes of promotion of cell proliferation and survival, and inhibition of apoptosis. IGF-I seems to transduce its signals through the canonical ERK and PI3K/Akt, but it can also activate JAK/STAT pathways. The signalling pathway called mTOR (mammalian target of rapamycin) is the serine–threonine kinase S6K1 (40S ribosomal S6 kinase1), which is activated by Akt (Asnaghi et al., 2004). Its importance in cancer has been highlighted by S6K1 overexpression in breast cancer and is associated with poor prognosis. Because S6K1 functions downstream of Akt, obviously mTOR would mediate cell proliferation. Besides, S6K1 regulates ER signalling by phosphorylating the receptor and in this way also promotes cell proliferation (Yamnik et al., 2009). EGF and IGF-1 signalling interact with the mTOR pathway. It has also been implicated in angiogenesis.

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The JAK/STAT pathway might be subject to negative feedback regulation by CIS (cytokine-induced SH2 protein inhibitor of signalling) and SOCS (suppressors of cytokine signalling). These regulate STAT function. Indeed, they modulate IGF-I-induced activation of JAK/STAT and possibly also other pathways interacting with IGF signalling. The PI3K/Akt and ERK1-2/MAPK pathways might transduce the IGF signals, because the effects of IGF on migration are inhibited by blocking both signalling systems (Saikali et al., 2008). The PI3K/Akt pathway is regulated by IRSs and by PTEN. IGF-1 downregulates PTEN expression. PTEN suppression leads to the activation of PI3K/Akt (Fernandez et al., 2008). Furthermore IGFs might be capable of recruiting the anti-apoptosis genes Bcl2/Bcl-XL and thus promote cell proliferation (Singh et al., 2008). IGFBPs are generally inhibitory of IGF signalling. They can form complexes with and sequester IGFs from interacting with IGFRs. However, some can stimulate IGF function (Figure 8.1). It ought to be stated here that IGFR1 overexpression has been linked with the induction of cyclin D1.

Figure 8.1  Promotion of cell proliferation and migration by IGF/IGFR. The consensus view is that IGF-I signals through the canonical pathways ERK and PI3K/Akt. The JAK/ STAT is a third mode of signalling. The JAK/STAT pathway might be subject to negative feedback regulation by CIS (cytokine-induced SH2 protein inhibitor of signalling) and SOCS (suppressors of cytokine signalling). This is not shown here. These regulate STAT function. Indeed, they do modulate IGF-I-induced activation of JAK/STAT and possibly other pathways interacting with IGF signalling. Cyclin D1 has been partly implicated in the induction of cell proliferation and tumour growth. The IRS docking proteins are involved in the activation of PI3K/Akt signalling. PTEN regulates this pathway. The anti-apoptosis genes Bcl2 and Bcl-XL have also implicated as influencing PI3K/Akt. IGF might promote cell survival in co-operation with AREG, but in this case signalling might occur through PKC and independently of PI3K/ Akt and MAPK. IGFBPs are generally inhibitory of IGF function. They can form complexes with and sequester IGFs from interacting with IGFRs. However, some can stimulate IGF effects. So IGFBPs can be viewed as a mode of regulation of both pathways. IGFBPs might also function independently of IGF but using Wnt signalling. This is discussed on pp. 96 and 97 in the text but not shown here.

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Jones et al. (2008) used a primary murine mammary tumour line with doxocylininducible IGFR1 and showed that IGFR1 regulates cell proliferation through cyclin D1 induction. Tumours developed more rapidly (approximately 3.5-fold) in murine hosts in the presence of doxycyclin than in its absence.

IGF Signalling Interacts with Other Signalling Systems The biological function of IGFs is often amplified or subdued by interaction with signalling by other growth factors or signalling molecules. Here the discussion is restricted to the major contributors to or negative regulators of the biological effects of IGF (Table 8.1).

The Interaction of Insulin and Growth Hormone with IGF Signalling A natural starting point would be the interaction of insulin signalling with the IGF signal. A brief prelude to the structure and receptor binding by insulin would be appropriate here. Insulin is a peptide hormone produced by pancreatic beta cells, which is actively involved in intermediate metabolism and mitosis of normal and tumour cells. It functions by binding to specific membrane receptors, promotes receptor phosphorylation, and activates signalling pathways, a feature it shares with growth factors in general. The monomeric form of insulin is made up of two amino acid chains held together by two disulphide bridges. The amino acid residues involved in receptor binding have been identified forming a conserved classical binding surface. A C-subdomain (B24–26) of this site is regarded as an imperative in insulin activation, and in negative co-operativity and receptor binding. This subdomain is sequestered in insulin dimerisation with the loss of negative co-operativity. Table 8.1  Signalling Systems that Interact with IGF Biological Function

Insulin Somatotropin (growth hormone) TGF GF TNF

VEGF Notch Wnt Leptin Oestrogen/Progesterone Androgen Glucocorticoids Note: The interaction of IGF signalling with oestrogen/progesterone, androgen and glucocorticoids is discussed in chapters 17 and 21 being classed as nuclear or cytoplasmic receptors.

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Receptor binding is dependent upon exposing these sites by a change of conformation (De Meyts et al., 1976, 1978; Mirmira et al., 1991; Xu et al., 2004). Further amino acid residues have been identified as describing a primary binding surface, because mutations of this region interfere with receptor binding. Each insulin molecule functions as a bivalent molecule and interacts with other insulin-receptor complexes, leading to a clustering of receptors that might enhance biological activity (Jeffrey, 1992). Insulin receptors (IRs) are plasma membrane-associated proteins. Each receptor is composed of two α- and two β-subunits held by disulphide bonds. The ligand binding α-subunit is extracellular but the β-subunit traverses the plasma membrane. Insulin binding leads to the activation of RTKs and autophosphorylation of the β-subunit in the conventional manner. The activated receptor phosphorylates several substrate proteins including IRSs. The IRSs are adaptor proteins and can translocate into the nucleus and regulate genetic transcription. They act as a docking protein for SH domain-containing signalling molecules, such as PI3K, and activate PI3Kmediated signalling. Insulin receptors are overexpressed in many human cancers. In particular, the isoform IR-A binds both insulin and IGF-II. This is further contributed by the fact that IR can form hybrid complexes with IGF1R (Frasca et al., 2008). This transactivation and co-operative hybridisation of receptors can lead to the amplification of the total biological effects. Further evidence of interaction comes at the IRS activation level. The induction of phosphorylation of IRS by insulin can be blocked by inhibiting IGFR1 (Rakatzi et al., 2006). Diaz et al. (2007) produced evidence that IGF-II activates IR in choriocarcinoma. The IGF-II-induced invasive ability and chemotaxis could be inhibited by inhibiting the IR; clearly this indicates that the biological effects occurred by this route. Growth hormone (GH) (somatotropin) is a peptide hormone that controls differentiation, growth and metabolism. There is much evidence of GH involvement in the induction of angiogenesis and lymphangiogenesis. There is a close link between the signalling systems operated by GH and insulin (Figure 8.2). One pathway of GH signalling is JAK-2 activation of the PIP2/DAG/PKC cascade, leading to the activation of STAT transcription factors and ultimately generating the phenotypic effects. GH can induce the liver to produce IGF-I and by this route induce proliferation of chondrocytes and bone growth. GH seems to induce IGF-I through the JAK-2/STAT5b pathway (Rosenfeld and Hwa, 2009). JAK-2 can function also by IRS phosphorylation. Insulin/IR activates IRS and PI3K/Akt signalling, possibly also independently of IRS. IR can also act using Grb2 docking protein and signal through the Ras/Raf/MEKMAPK pathway. The cross-talk between the insulin/IR pathway and GH signalling is further supported by the fact that brief exposure to insulin augments the activation of MEK/ERK but sustained exposure leads to an inhibition of GH signalling (Xu and Messina, 2009). On the other hand, IGF-I can negatively regulate GH production. High levels of IGF-I in the blood decrease the production of GH. IGF-I might achieve this by stimulating the secretion of somatostatin, an inhibitor of GH secretion by the pituitary gland.

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Figure 8.2  Co-operative signalling between IGFs and GH. GH activates JAK-2 (Janus kinase 2) and signals downstream through PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysed by phospholipase into the second messengers PIP3 and DAG, leading to activation of the STAT transcription factors generating phenotypic responses. GH also can induce IGF-I, interact with IRS and activate the PI3K/Akt signalling, possibly also independently of IRS. IR can also act using Grb2 docking protein and signal through the Ras/Raf/MEK-MAPK pathway (see also Figure 8.1).

IGF Cross-Talk with Cell Proliferation and Migration Signalling Other growth factors of note that interact with IGF signalling are EGF, TGF-β, VEGF, TNF and HGF/MET. IGFR1 interaction with EGFR occurs at various levels of signalling on account of shared signalling systems and interaction with other receptors. IGFBP can modulate cell migration by interacting with and influencing integrin-mediated signalling. IGFBPs can interact with IGF itself and inhibit receptormediated functions. Thus IGF seems to be able to interact with several factors that determine and modulate biological processes of cell proliferation, invasion and angiogenesis that are key components of cancer progression. There is increasing evidence of cross-communication between EGFR and IGFR1 in breast cancer cells. Riedemann et al. (2007) demonstrated a direct interaction between these two receptors. They showed co-precipitation in breast cancer cells as well as in breast cancer tissue, which disappeared upon abolition of either receptor. The removal of EGFR led to the ubiquitination and degradation of IGFR1, which supports the view that the EGF/IGFR1 complex might stabilise IGFR1 and promote IGFR1 signalling. Overexpression of both EGFR and IGFR1 has been encountered in nasopharyngeal carcinomas of patients with lymph node or distant metastases. Overexpression is also correlated with disease recurrence and inversely with survival (Yuan et al., 2008). Although the parallelism is convincing, one cannot infer any interaction in signalling. Saxena et al. (2008) found that IGF-I and leptin synergistically activated EGFR. Erlotinib and Lapatinib, which inhibit EGFR, seemed to inhibit induced invasion

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and migration of breast cancer cells. Two relevant considerations here are that it is difficult to assess the individual contributions of IGF-I and leptin in EGFR activation and, secondly, EGF itself can stimulate cell migration. Also, due consideration has to be accorded to the involvement of oestrogens with insulin and IGF-1 signalling, and of ER/PR with EGFR signalling as well as IGFR1 and EGFR interaction in tamoxifen resistance. The proliferation and survival of cells and apoptosis are either ends of a spectrum of biological function. Although IGF and EGF are promoters of cell proliferation, TNF generates an apoptosis response. It would be reasonable to postulate that these factors could jointly regulate and determine the state of cell proliferation and survival. TNF-α induces apoptosis in the neuronal cell line SH-SY5Y. IGF-1 is able to counteract this effect. IGF-1 was found to inhibit the JNK signalling pathway which mediates the apoptotic effect of TNF-α (Kenchappa et al., 2004). This has been shown also in another cell culture system where the presence of IGF-I markedly reduced apoptosis induced by TNF-α (Saini et al., 2008). Saini et al. (2008) also apply a pro-apoptosis role to IGF-I with the finding that IGF-I induced apoptosis when TNF-α was present in cultures in non-apoptotic concentrations, a reasoning that is insufficiently persuasive. Compatible with this thought, Storz et al. (2000) showed that TNF was able to downregulate IR and inhibit the downstream MAPK/STAT5 signalling cascade. One aspect of IGF cross-talk is its ability to influence invasion indirectly. As noted earlier, IGFBPs can and do interact with IGF and inhibit receptor-mediated function. IGFBP1 has a C-terminal RGD (Arg–Gly–Asp) domain and so potentially might bind integrin-α5β1. This raises the question of the potential of IGF to interact with integrins and link up with IGFBP/IGF signalling. Integrin-α5β1 is an essential element in cell migration. IGFBP1 induces cell migration. Its interaction with the integrin results in FAK and ERK1-2/MAPK activation (Gleeson et al., 2001). Here is a virtually unequivocal demonstration of how IGF cross-talk alters the migratory behaviour of cells.

IGF Induces VEGF Expression and Vascularisation With the revelation of the influence IGFs can bring to bear on cell proliferation and migration, it is natural to inquire into their potential influence on the angiogenic process. There is a considerable body of evidence that IGFs can induce VEGF expression. IGF-mediated induction of VEGF was demonstrated in colon carcinoma cells many years ago (Akagi et al., 1998; Warren et al., 1996). Luteinised granulosa cells in culture derived from polycystic ovarian disease produce VEGFA upon exposure to insulin or IGFs I and II (Stanek et al., 2007). In contrast, IGFBPs seem to inhibit angiogenesis by inhibiting VEGF expression. Moreno et al. (2006) used a somewhat elaborate experimental setup, which showed that U87MG glioma cells induced to differentiate using dibutyryl cyclic AMP (cAMP) responded with reduced cell proliferation and invasive behaviour, together with a loss of ability to induce angiogenic responses from endothelial cells in Matrigel cultures despite the fact that they were secreting VEGF into the conditioned medium. In parallel, these cells expressed many angiogenesis-related genes. Among them was IGFBP4, which

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seemed to be the reason for the loss of angiogenic response even in the presence of VEGF. Moreno et al. (2006) confirmed this by using recombinant IGFBP4, which inhibited angiogenic response induced by the reconditioned medium; furthermore, this could be reversed by anti-IGFBP4 antibodies. These findings contrast markedly with the demonstration some years ago that VEGF expression induced by IGF-I was not inhibitable by IGFBPs (Akagi et al., 1998). HIF-1 (hypoxia-inducible factor) is induced under hypoxic conditions. It is a heterodimeric bHLH (helix–loop–helix) transcription factor. HIF-1 activates transcription of hypoxia inducible genes and is known to activate transcription of VEGF. HIF-1 expression is associated with vascularisation of many human neoplasms. It is phosphorylated, binds to VEGF promoter and promotes transcription of the gene leading to angiogenesis. The signalling modes that IGFs use have been scrutinised recently and it would appear that IGFs activate the ERK1-2/MAPK pathway to activate HIF-1 (Sutton et al., 2007). IGF-II might activate both ERK1-2/MAPK and the PI3K systems in the induction of VEGF (Kim and Kim, 2005). According to Fukuda et al. (2002), IGF-I might activate both PI3K and MAPK pathways. Lewis lung carcinoma cells transfected with IGF-I express VEGFC messenger RNA (mRNA) and secrete VEGFC precursor protein. Here also IGF-I seems to have adopted mainly the PI3K pathway (Tang et al., 2003). Stearns et al. (2005) have suggested that IGF-1 induces VEGF by Ras functioning through IRS1 leading to PI3K signalling (Figure 8.3). VEGF occurs as several splice variants. Of these, variants that arise from exon 8 splicing at proximal sites promote angiogenesis, but the ‘b’ variants generated by splice at distant sites of the exon are competitively anti-angiogenic. Hence it is of considerable significance that IGF seems to regulate the generation of splice variants. IGF-I can not only enhance the expression of total VEGF but also decrease the

Figure 8.3  IGF signalling using ERK1-2/MAPK to phosphorylate HIF-1 transcription factor, which promotes VEGF gene transcription, leading to angiogenesis. IGFs are also postulated to act through IRS to activate the Ras/PI3K system to bring about angiogenesis.

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generation of anti-angiogenic ‘b’ variants (Nowak et al., 2006). Recently BrunetDunand et al. (2009) have described the ability MCF-7 breast cancer cells transfected with the GH gene to produce GH and enhance the expression of VEGFA and VEGFA receptor mRNAs, and induce tubule formation including enhanced tubule length as well as tubule density in human endothelial cell HMEC-1 cultures. The induction by GH of tubule formation in HMEC-1 is inhibited by Avastin, supporting the role attributed to VEGFA. Xenografts of GH secreting MCF-7 cells were associated with higher microvessel density and greater vascularisation. As stated earlier, IGF is regulated by GH. Hence the possibility that the effects of GH on VEGFA induction, together with the induction of tubule formation, might have been a consequence of GH/IGF cross-talk needs to be investigated. As discussed below, the IGF/IGFBP axis is a key regulator of cancer progression, which inevitably would embrace cell migration and angiogenesis. Continuing with the theme of angiogenesis, HGF (hepatocyte growth factor)/ MET receptor has been attributed with tumour invasion and angiogenesis. Bauer et al. (2006) downregulated potential endogenous IGF-I in the human pancreatic cancer cell line L3.6pl using MET ribozyme. Subsequent treatment of these cells with IGF-I stimulated migration, which was compounded by HGF. These authors believe that MET is needed for both HGF- and IGF-I signalling. However, HGF phosphorylates FAK which is also deeply involved in cell migration. So it is possible that HGF might be promoting migration in consort with IGFs in a FAK/ integrin-mediated manner. IGF and HGF activate both PI3K and MAPK pathways. It was suggested some while ago that IGF might preferentially activate PI3K, although HGF usually had recourse to MAPK. This is based on findings relating to the differential regulation of PI3K and MAPK signalling in the differentiation and proliferation of myogenic cells, which has led to the postulate that PI3K activation is related to differentiation and MAPK to cell proliferation (Halevy and Cantley, 2004). With IGF and HGF being involved in both invasion and cell proliferation, it is inevitable that some teleological thinking would enter into experimental interpretation.

Integration of Notch Signalling by IGF IGF/IGFR signalling is highly diverse, versatile and integrates developmental signalling pathways such as Notch, Wnt and Hedgehog. Notch is a major signalling pathway in normal development, morphogenesis and growth. It is closely associated with neuronal differentiation, mesoderm segmentation, the formation of blood vessels, haemo- and lymphopoiesis and determination of cell fate. Deregulation of Notch signalling leads to many disease states and developmental abnormalities. The Notch receptor is a transmembrane protein of which the extracellular domain characterised by EGF-like repeats is believed to be involved in ligand binding. The cytoplasmic section of the receptor possesses a domain that interacts with the transcriptional complex RBP-Jk/CBF1 (CSL family of DNA-binding proteins), a domain with ankyrin repeats and a PEST sequence. DSL (Delta, Serrate and LAG-2) binds and activates Notch. This results in proteolytic cleavage and release, and nuclear

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translocation of the intracellular Notch component. This activated component now binds to and activates RBP-Jk/CBF1. The activated RBP-Jk/CBF1 transcribes its primary target, namely the bHLH factors, which in turn regulate the appropriate target genes (reviewed by Chiaramonte et al., 2006). Notch signalling can interact with or act independently of the transduction of signals by several growth factors, for example BMP, FGF, VEGF and IGF-I, among others. Notch ligand can induce the secretion of these growth factors. Provisional reports indicate that Notch upregulates IGFR1 and sets in motion IGF signalling in the growth and survival of T-ALL (acute lymphoblastic leukaemia) (Medyouf et al., 2008), and in hepatocellular carcinoma it is said to be involved in cell migration (Cantarini et al., 2005). Overexpression of the Notch ligand Jagged2 has been detected in malignant plasma cells from patients with multiple myeloma (MM). The Jagged2 promoter is hypomethylated, which causes the overexpression. In vitro, Jagged2 overexpression was associated with the induction of IL-6, VEGF and IGF-1 in stromal cells, and by implication Notch signalling is active in this system (Houde et al., 2004). On the other hand, Notch is not an imperative. FGF, for instance, induces the expression of Jagged1 and activates Notch2 using ERK/MAPK and the Notch effector HES5 in the differentiation of lens fibre cells in the eye. IGF-I, however, does not induce Jagged1 (Saravanamuthu et al., 2009). Insulin/IGFR signalling operates in the production of gametes by germline proliferation in Caenorhabditis elegans, but again this occurs independently of Notch (Michaelson et al., 2010).

IGF and the Morphogenetic Function of Wnt Proteins The interaction of notch signalling with other pathways of signal transduction could be a mechanism of mutual regulation. The Wnt family of morphogenetic proteins is associated with segmentation in Drosophila, cell proliferation and migration as well as carcinogenesis. Notch and Wnt proteins exert opposing or co-operative effects in different systems. The Wnt signalling system modulates the expression of β-cateninlinked cadherins and regulates intercellular adhesion (Sherbet, 2001, 2003). Thus, overall, Notch would inevitably modulate cell motility or invasive behaviour, cell proliferation and apoptosis. The Wnt proteins appear prominently in embryonic development, cell proliferation and apoptosis, differentiation and pattern formation. They also take part in tumorigenesis and modulate invasive cell behaviour. They bind to the Frizzled family of protein receptors and LRP (low density lipoprotein receptor-related protein) Lrp5/Lrp6 co-receptors. The Wnt-Fizzled receptor complex activates several signalling systems, the canonical/β-catenin, and the non-canonical and calcium pathways. Essentially, the ligand/receptor complex releases β-catenin from the multi-protein complex, which then enters the nucleus and activates transcription factors. In the canonical pathway, β-catenin forms a complex with the transcription factor TCF (T-cell factors) or the Lef (lymphoid-enhancing factors), leading to the transcription of responsive genes. Upstream of Wnt signalling is the effector phosphoprotein dishevelled, which transduces the signal into the three pathways. Among those identified are the calcium/calmodulin-dependent kinase II (CamKII) and protein kinase

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C (PKC), the phospholipase C (PLC) and phosphodiesterase (PDE) activated by the recruitment of heterotrimeric GTP-binding proteins, the JNK (planar cell polarity) pathway and the conventionally recognised one with β-catenin as the main mediator. These pathways are integrated to specify developmental patterns and cell fate. All these pathways use calcium signalling systems as second messengers. Furthermore, S100A4, MMP25, RUNX2 and CD14 have all been implicated strongly in the Wnt signalling pathway (see Sherbet, 2008). The overlap of communication of IGF signalling with the Wnt pathway is supported by a significant mass of evidence. Insulin and IGF can stimulate nuclear translocation and binding of β-catenin/TCF transcription complex to the Wnt promoter (Jin et al., 2008). IGF-I induces proliferation of ER-positive MCF7 breast cancer cells and upregulates CCN5 (the Wnt-induced signalling protein). Oestrogen enhances this effect. CCN5 siRNA (short interference RNA) abolishes the effect. Inhibition of ER-mediated function blocks the upregulation of CCN5 and suppresses PI3K/Akt signalling activated by IGF-I. It seems therefore that oestrogen/ER and PI3K activation are integrated with IGF function (Dhar et al., 2007). However, dissension has been expressed that although IGF might activate PI3K, this does not occur with β-catenin/TCF in some experimental systems (Kiely et al., 2007). Much evidence has been adduced in support of the contribution of IGF-II in tumours induced by PLAG1 (pleomorphic adenoma gene 1). In pleomorphic adenomas of the salivary glands and breast adenomyoepitheliomas, IGF-II is upregulated and a putative link has been established between the IGF and PLAG1 in experimentally induced pleomorphic adenomas of salivary glands in transgenic mice. In this system, IGF-II inactivation led to delayed tumour development. However, the absence of a complete inhibition of tumour development has suggested the possibility of other genes such as Wnt being involved. Both IGF-II and Wnt contribute to tumour development (Declercq et al., 2008). Conventionally, IGFBP is regarded as a regulator of IGF function, but it has been attributed with independent functions. IGFBP4 promotes cardiomyocyte differentiation, which seems to occur without involving its conventional IGF-binding activity. Also, cardiomyogenesis was stimulated by the inhibition of Wnt signalling, apparently by the binding of IGFBP4 to the Wnt receptor Frizzled 8 and an LRP Wnt co-receptor (Zhu et al., 2008).

Leptin Synergy with IGF Function Leptin (LEP) is a 16-kilodalton (kDa) peptide hormone produced mainly by white adipocyte tissue (Zhang et al., 1994). It is a key participant in many physiological and metabolic processes; apart from regulation of body weight, it is also involved with haemopoiesis (Benett et al., 1996), angiogenesis (Bouloumie et al., 1998), immune processes (Loffreda et al., 1998) and reproduction (Hoggard et al., 1998). Leptin functions through its receptor LR, a class I cytokine receptor that is widely distributed, although initially identified in the hypothalamus and associated mainly with the regulation of food intake and body weight. LR occurs as many isoforms, of which LRb is thought to be the functional receptor (Briscoe et al., 2001; Tartaglia, 1997) because it is the only receptor that associates with signalling molecules.

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The linkage of LEP and LR with cell proliferation and angiogenesis has aroused much interest about its possible relevance in cancer development and progression. LEP has been shown to promote cell proliferation in cancer cell lines and tumour growth (Hu et al., 2002; Laud et al., 2002). It influences invasive behaviour or migration (Attoub et al., 2000). It is expressed in breast cancer (Caldefie-Chézet et al., 2005; Ishikawa et al., 2004) and might be present at increased levels in sera of patients (see Chen et al., 2006; Coskun et al., 2003; Tessitore et al., 2004). Interestingly, Chen et al. (2006) found that LEP levels correlated inversely with adiponectin, which influences insulin response. A subsequent report related high serum levels of LEP and LR in high-grade breast cancers (Liu et al., 2007). Kim et al. (2006) found no correlation between LEP and LR with any clinical features, tumour stage, nuclear grade, HER2 or ER expression, and disease-free survival of patients with early breast cancer. They found most (83%) breast cancers expressed LEP and approximately 40% were LR-positive. However, the data presented do not allow one to compare the various features when both LEP and LR were simultaneously expressed. LEP and LR are expressed in most gliomas, and their expression has correlated with tumour grade. They were overexpressed in glioblastomas and anaplastic astrocytomas compared with less malignant counterparts (Riolfi et al., 2010). Gliomas do not metastasise to sites outside the CNS and often the dissemination noticed might be iatrogenic in nature. So the significance of some of these findings for metastatic spread has to be considered as not established beyond reasonable doubt. However, there are reports that LEP can regulate VEGF expression. Insulin has been reported to increase the expression of LEP and in parallel increase HIF1-α and Sp1. Both bound the LEP promoter. Inhibition of ERK and PI3K pathways inhibited insulinmediated induction of LEP and reduced HIF1-α and Sp1 association with LEP promoter (Bartella et al., 2008). HIF1-α is known to activate VEGF transcription. So these events would be conducive to angiogenesis and metastatic dissemination. Three signalling pathways activated by LEP can be identified: the ERK mediated by SHP-2 (SH2-containing tyrosine phosphatase), STAT3 and JAK/PI3K pathways (Fruhbeck, 2006; Myers, 2004). SHP-2 was shown some time ago to be able to regulate negatively STAT3-mediated transcription of LR-responsive genes (Carpenter et al., 1998).The overlap of functional pathways is extensive and LEP shares many signalling pathways with other biological response modifiers. The commonality between LEP, insulin and IGFs has led to the exploration of potential interaction and synergy between the functions of these physiologically and metabolically important growth factors. As shown in Figure 8.4, there is likely to be much transactivation of receptors and intersection of the signalling systems. Of significance among the shared biological effects of consequence to cancer are promotion of cell proliferation and cell motility. Saxena et al. (2008) found that LEP and IGF-I in combination significantly increased proliferation, invasion and migration of breast cancer cells. This co-operative effect seems to be due to the ability of IGF-I to transactivate LRb, and in reciprocation LEP can phosphorylate IGFR1 and IRS1 and IRS2. Obviously this involves PI3K/Akt. JAK itself is able to activate IRS (see Figure 8.4) and collaborate with the activation of the P13K/Akt system. IRS activation also provides a potential relationship to angiogenesis, again an important attribute of

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Figure 8.4  The inter-relationship between LEP/LR and IGF/IGFR signalling, emphasising the postulated transactivation and collaborative function of the two growth factors.

leptin, especially with the association of the IRSs with invasion and metastasis. The cross-talk between IGF-I and LEP has been confirmed in that IGF-I does phosphorylate and activate LR and downstream signalling, but there is no indication of reciprocity in that LEP does not phosphorylate IGFR1 (Ozbay and Nahta, 2008). Saxena et al. (2008) also found that leptin was able to transactivate EGFR, a finding of much relevance in terms of the contribution to the promotion of cell proliferation. It may be recalled here that EGFR signalling activates PI3K/PTEN/Akt, JAK/STAT, Ras/MAP-kinase and PLC-γ/PKC pathways. So, it is little wonder that EGFR was found to overlap functionally with LEP and IGF. LEP might also activate HER2. Treatment of MCF-7 cells with LEP increased HER2 phosphorylation as a consequence of LR activation (Fiorio et al., 2008). Leptin is said to stimulate the growth of ER-positive breast cancer cells. This has been attributed to its ability to increase oestrogen levels by aromatisation of androgens and in turn function through its own receptors (see Cirillo et al., 2008). LEP is indeed able to enhance aromatase activity (Pino et al., 2006). Both LEP and LR have been detected in primary breast cancer and their presence correlated with the expression of ER but not with PR. LEP and LR expression also correlated with tumour size but not with the proliferation marker Ki67 (Jarde et al., 2008). However, with this apparent incongruity, perhaps using a second proliferation marker or even the mitotic index from histology would have been supportive of any conclusions. On the other hand, tumour size is a sum total of cell proliferation and apoptotic loss of cells. Maintenance of equilibrium between the opposing sway is evident from the fact that the expression of both pro- and anti-apoptosis genes is influenced by LEP and LR (Koda et al., 2007). One might also insert a comment here that Fiorio et al. (2008) found LEP and LR were associated more frequently with larger breast cancers. Using a xenograft MCF-7 breast tumour model, Yu et al. (2010) have shown that LEP treatment greatly enhances ER-α mRNA and protein expression, but ER-β is lower in the LEP treated group of hosts. A similar differential correlation of LEP with ER-α and ER-β was

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also reported in 3T3-L1 adipocytes (Yi et al., 2008). The significance of this differential expression in relation to LEP functions remains to be elucidated. However, it would be worthwhile recalling here the suggestion that ER-β and its isoforms might regulate and suppress ER-α function and in this way regulate oestrogen signalling (Hayashi et al., 2003). ER-α and ER-β seem to recruit co-activators preferentially. Ligands can bind to either or both receptors, but specifically activate a given receptor type with little or no effect of the other type (Wong et al., 2001), thus ensuring specificity of signalling outcome. Finally, further evidence for the promotion of proliferation by LEP comes from the study of LEP effects on cell-cycle regulators. LEP upregulated cyclin D1 and c-myc, but downregulated p21waf1/cip1 and p53 (Chen et al., 2006). Thus a final justifiable judgement would be that LEP and LR do relate to cell proliferation. It would also be amply justifiable to say that insulin, IGF and LEP signalling are cogently and closely integrated to generate their phenotypic functions.

IGF Expression in Cancers The basic approach to determining whether IGFs and their receptors influence cancer progression would be to examine the expression of the ligands and receptors and look for correlations with the state of tumour progression using clinico-pathological parameters together with assessments of prognosis. Experimental metastasis models have a part to play in unravelling this aspect of IGFs, to the extent that they can throw some light on specific features of cancer spread. Tang et al. (2003) demonstrated that Lewis lung carcinoma cells transfected with IGF-I produced VEGF-C mRNA and VEGF-C precursor protein, and furthermore that the transfectant cells tended to metastasise to the lymph nodes in vivo. Patients with prostate carcinoma have shown higher serum levels of IGF-I than normal subjects. Although IGF-I did not appear to relate to disease stage, there appears to be a significant direct correlation between IGF-I levels and cancer risk (Wolk et al., 1998). IGFR1 and IGF-II have been detected in most oesophageal squamous cell carcinomas. Expression was correlated with tumour stage, depth of tumour invasion, presence of metastatic disease and recurrence. Often either IGFR1 or R2 is expressed. This could make a difference in the light of the differences in the trans-binding activity of IGF-II, which binds IGFR1 with comparatively low affinity. Patients expressing both IGFR1 and IGF-II simultaneously had shorter survival than those who were positive to either on their own (Imsumran et al., 2007). Here IGF-II seems to be acting through IGFR1. EGFR and IGFR1 expressions have been studied in nasopharyngeal carcinomas. Both are highly expressed in carcinomas of patients with lymph node or distant metastases. Overexpression corresponded with disease recurrence. The 5-year survival rates were vastly superior in patients with receptor-negative tumours compared with those who were receptor positive (Yuan et al., 2008). It is difficult to assess the significance of either receptor individually; possibly this could have been a composite outcome. On the other hand, IGFBPs can interfere with the binding of IGFs to their receptor and in this way negate

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IGFs’ effects. This is obvious from the finding that IGFBP3, when co-administered with IGF-I, leads to a total inhibition of IGFR1 phosphorylation and the downstream activation of ERK1-2 and Akt signalling in hepatocellular carcinomas cells. Furthermore, low IGFBP3 expression seems to be conducive to invasive behaviour and poor prognosis (Aishima et al., 2006). Compatible with this notion, metastatic colorectal cancer progresses more slowly when plasma levels of IGFBP3 are high (Fuchs et al., 2008). IGFBP5 expression is downregulated in the progression of head and neck squamous cell carcinomas, and again low IGFBP5 is correlated with nodal spread (Hung et al., 2008). IGFBP3 introduction into melanoma cells not expressing the protein led to apoptosis; in agreement with this, inhibition of IGFBP3 in cells that expressed the protein led to reduction in cell proliferation. Also, in two cell lines the loss of IGFBP3 expression was due to methylation of the promoter of the IGFBP3 gene. When expressed, IGFBP3 is regulated by PI3K and ERK1-2/MAPK signalling (Oy et al., 2010). While interpreting the significance of IGFBP expression, it is necessary to keep in mind that IGFBPs in general are inhibitory of IGF function, but IGFBP1, IGFBP3 and IGFBP5 can stimulate IGF function. That IGFBPs might support the metastatic process is supported by investigators. One aspect of IGFBPs that has significant bearing on the invasive behaviour of cells is the possibility that they might influence integrin-mediated promotion of invasion. IGFBP1 has a C-terminal RGD (Arg–Gly– Asp) domain and so potentially might bind integrin-α5β1. This raises the question of the potential of IGF to interact with integrins and link up with IGFBP/IGF signalling. Integrin-α5β1 is an essential element in cell migration. IGFBP1 induces cell migration, but this does not occur when its RGD domain is mutated, which clearly demonstrates the interaction of IGFBP1 with integrin-α5β1. This interaction results in FAK and ERK1-2/MAPK activation (Gleeson et al., 2001). IGFBP2 was found to do this in gastric carcinomas. Not only did IGFBP2 expression differ between tumour tissue and normal mucosa, but it correlated with invasion, clinical stage and lymph node metastases (Zhang et al., 2007). As noted earlier, IGFBP regulates the biological activity of IGFs by interfering with their interaction with IGFRs, and the IGF/IGFBP complex is thought to increase the half-life of IGFs. Therefore, there is some basis for the postulate that IGFBPs might regulate IGF sensitivity in tumour development. In the initial phases of growth, murine renal carcinoma implants were found to be IGF sensitive and responded with enhanced cell proliferation and vascularisation together with increases in IGFBP3 expression and Smad2 proteins; however, in later development, tumours were refractory to IGF treatment and showed less proliferation and IGFBP3 and Smad expression (Rosendahl et al., 2008). This might simply reflect the fact that in the early stages of growth IGF function is accentuated by IGFBP3, although low expression of IGFBP3 in the later stages aids progression. The presence of Smads indicates possible collusion with TGF-β. Nonetheless, these snippets of information might be of value in potential therapeutic deployment of IGFs/IGFRs, but no differential expression of IGFBPs or differential proliferation has been reported in human neoplasms. Indeed, oral squamous cell carcinoma expresses IGFBP3 at much higher levels than corresponding normal epithelial tissue (Zhong et al., 2008).

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As noted earlier, IGFBPs are generally inhibitory of IGF signalling. They can form complexes with and sequester IGFs from interacting with IGFRs. However, some can stimulate IGF function (Figure 8.1). IGFBP4 seems to inhibit angiogenesis by inhibiting VEGF expression and influencing function of other growth factors by virtue of the presence of IGFBP modules. So a divergence of views is seen in regard to the function of IGFs as well as IGFBPs. Negative findings of IGF functions are few and far between, but one cannot be dismissive of the demonstration some time ago of a total lack of correlation between IGF-I and IGF-II to any pathological or clinical criteria. The expression of these ligands was low in both primary gall bladder tumours and corresponding metastatic tumours in the lymph node or liver (Kornprat et al., 2006). One can envisage the operation of other controlling factors. Okada (2007) showed that ADAM28 might release IGF from its complex with IGFBP. TGF-β is most certainly able to modulate IGF signalling. TGF-β greatly augmented the increase in expression induced by IGF-I (Rosendahl and Forsberg 2006). Also, eminently worthy of exploration is whether there might be some reciprocal mitigation of effects of the various IGFBPs or whether contributory signalling might be involved; this is an open question.

Modulation of Migration by IGFs and Their Receptors Cell migration is regulated by ECM components, integrins, laminins and cadherincytoskeletal dynamics. Proteoglycans form a major mediator of migration. They occur as soluble components of the ECM or as integral transmembrane molecules. They interact with other ECM components. The binding of these to proteoglycans occurs at specific sites with defined structural motifs. Heparan sulphate can bind to extracellular matrix components such as laminin, fibronectin and collagen. Proteoglycans can also interact with hyaluronic acid and modulate proteoglycan conformation. Growth factors such as PDGF, TGF-β, EGF and FGF can modulate proteoglycan expression. Heparan sulphate is an important link in FGF signalling. Finally, TGF-β type III receptor is a chondroitin/heparan sulphate proteoglycan. Molecular interactions of this kind are important events in cell adhesion and signal transduction. Modulation of cell migration and acquisition of invasive ability is a salient feature of cancer metastasis. IGFs and their receptors induce cell proliferation and migration. In evidence is the demonstration that inhibition of IGFR1 expression in A549 cells inhibits cell proliferation, enhances apoptosis and at the same time inhibits cell migration (Ma et al., 2007). However, both IGFs do not necessarily conform to IGFR mediation. IGF-I and IGF-II stimulate invasive behaviour of choriocarcinoma cells in culture, but IGF-I seems to function through its conventional receptor, whereas IGF-II is said to act through the insulin receptor. IGF-I can also do this, but it binds insulin receptor with greatly reduced affinity. However, inhibition of insulin receptor function also reduces IGF-II-induced effects. So even though IGF-I might contribute through insulin receptor, the effects of IGF-II seem to be brought about mainly by the activation of insulin receptor (Diaz et al., 2007). Some glioma cell lines, for instance, express the receptors and IGFBPs, but not the IGFs. Exposure of these

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cells to IGF-I, but not IGF-II, enhanced their proliferation and migration (SchlenskaLange et al., 2008). This is somewhat intriguing because IGFR1 can bind both IGFs. However, it is possible that this is due to interference by some of the endogenous IGFBPs expressed in the cells. Some IGFBPs can inhibit IGF function but probably not selectively that of IGF-II. IGFs markedly influence cell motility, dependent upon their expression and the presence of the appropriate receptors. ECM components seem conspicuouly to impinge upon this function. IGFs and their receptors appear to resort to this to bring changes in cell behaviour. IGFR1 forms a ternary complex with integrinαv and E-cadherin. This complex is disrupted when IGFR1 is bound by the ligand. This causes a rearrangement of integrin-αv distribution, resulting in cell migration (Canonici et al., 2008). Girdin is another component of the cytoskeletal machinery. It binds actin filaments together. Girdin is phosphorylated by Akt. The phosphorylated protein localises in the leading edge of migrating cells and so it could be a key component in cell migration (Enomoto et al., 2005). With its wide distribution in many forms of cancer, the relevance of Girdin in cancer invasion has received some attention. IGF-I seems to phosphorylate Girdin by signalling Akt activation. Indeed IGF-Istimulated migration of MDA-MB-231 breast cancer cells is dependent upon Girdin. In experimental metastasis models, inhibition of Girdin led to the inhibition of metastasis (Jiang et al., 2008). The adhesive interactions between invading cells and target tissues of invasion come into the picture and are implicated in invasion and metastasis. Neuroblastoma cells expressing IGFR1 at high levels firmly adhere to endothelial cells of the bone marrow. This possibly aids extravasation into bone marrow, where it can interact with the bone stroma to develop into secondary tumours (van Golen et al., 2006). Among other ECM components of note in adhesive interactions is laminin. Integrin-β1C upregulates IGF-II expression and increases cell adhesion to laminin-1 (Goel et al., 2006). Goel et al. (2006) postulate that in the absence of laminin-1 in prostate cancer, the adhesion link is disrupted, leading to the acquisition of invasive faculty. A recent study using an experimental metastasis model has shown that IGFR1 antibodies inhibited metastatic spread without affecting tumour growth. The diapedesis of cancer cells into the vascular system was inhibited, as indicated by the reduction in the numbers of circulating cells. The antibodies also inhibited pulmonary localisation of cells injected through the tail vein (Sachdev et al., 2010). The introduction of cells through the tail vein is a procedure that circumvents certain initial processes of cell adhesion (see Sherbet, 2006; Sherbet and Lakshmi, 2006). The observations of Sachdev et al. (2010) suggest that both extravasation of tumour cells at pulmonary sites and adhesion of circulating tumour cells in the lung parenchyma might be inhibited by the antibodies. Another group of ECM components of relevance in the context of cell migration are the MMPs. When IGFR1 is inhibited, MMP2 expression is also alleviated together with changes in cell motility (Ma et al., 2007). IGFR1-mediated upregulation of MMPs together with corresponding alterations in TIMP2 has been encountered in DU145 prostate cancer cells (Saikali et al., 2008). Similar results have been obtained by using a different experimental setup. Simvastatin inhibits IGFR1 expression in trophoblast cells and at the same time suppresses MMP2

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(Tartakover-Matalon et al., 2007). The view expressed in some quarters is ambivalent. Overexpression of IGFR1 was found in some studies to upregulate MMPs through PKC/PI3K signalling in cell cultures and in vivo experimental systems. Saikali et al. (2008) showed that IGF-1 used both PI3K/Akt and MAPK signalling in enhancing cell migration, and that this could be inhibited by blocking these pathways. They could function independently or possibly synergistically. MMP11 (Stromelysin-3) is upregulated in response to IGF in breast cancer cell lines, again involving both pathways (Kasper et al., 2007). However, the synthesis of MMPs regulated by PMA (phorbol 12-myristate 13-acetate) was inhibited in the same cell line (Li et al., 2009). It would be well to recall here that phorbol esters can directly activate or inhibit PKC, but Li et al. (2009) found that the effects were reversed in cells carrying C-terminal domain mutants of IGFR1, which emphasises that PKC inhibition was accomplished by IGFR1. Focal adhesion kinases (FAKs) could be another route that IGFs might adopt. Liu et al. (2007) found that inhibition of FAKs and IGFR1 simultaneously using NVPTAE226 inhibited the induction of cell motility by IGF-I. Also, NVP-TAE226 was able to inhibit IGFR1 phosphorylation, the activation downstream of MAPK/Akt signalling and cell migration in Matrigel analyses. FAK is activated by IGFR1 tyrosine kinase-mediated phosphorylation. Interestingly, insulin phosphorylated FAK in cells not attached to the substratum, although it is dephosphorylated in adherent cells (Baron et al., 1998). The implication of this is that activation by FAK of the cytoskeletal machinery goes hand in hand with adhesion-related phosphorylation of FAK. This has been borne out by subsequent work relating to the role of integrinmediated signalling by FAK. FAK binds by its C-terminal domain to, and activates, STAT1 during cell adhesion. In the absence of FAK, STAT activation is attenuated, adhesion is enhanced and migration of cells is reduced (Bing et al., 2001). One should recall that IGFBP1 can induce cell migration by interacting with integrinα5β1. This interaction results in the activation of FAK and ERK1-2/MAPK signalling (Gleeson et al., 2001). This brings together the IGF/IGFBP axis in cross-talk with integrin-mediated modulation of cell migration.

9 Connective Tissue Growth Factor Connective tissue growth factor (CTGF) is a cystine-knot protein of the CCN family. CTGF (also often referred to as CCN2) interacts with a variety of biological response modifiers and participates in several biological processes, for example cell proliferation, migration, differentiation and development of bone, cartilage and angiogenesis. CTGF is also associated with many pathological conditions. It seems to function as a mediator of fibrosis in atherosclerosis, myocardial infarction and hypertension. The fibrosis-promoting effects of endothelin have been attributed to CTGF (Recchia et al., 2009a). CTGF has also been implicated in the development of cancer. These wideranging pleiotropic effects and the versatility of CTGF are remarkably attested by the fact that the processed RNA has five exons encoding a protein composed of four modules: the IGFBP module that can interact with IGFs, a VWC (Von Willebrand type C repeat) module that can interact with members of TGF-β family, and the TSP1 and cystine-knot modules that interact with VEGF (Figure 9.1). The inevitable conclusion from this would be that these domains might participate in the activation of different signalling pathways. Thus, the VWC, TSP and CT modules together with the fulllength CTGF activate ERK, whereas the IGFBP module seems to activate JNK signalling. In contrast, all four modules or a combination have been implicated in MAPK signalling in some biological processes (Kubota, 2006).

CTGF in Wound Healing and Fibrosis CTGF functions downstream of TGF-β1 in the promotion and development of fibrosis as well as in differentiation and morphogenesis. It is known to take part in wound healing and fibrosis. Aberrant levels of CTGF have been reported in sera of patients with hepatitis B, which corresponded with the degree of hepatic fibrosis (Guo-Qiu et al., 2010). TGF-β-mediated CTGF involvement has been advocated to result in fibrosis associated with muscular dystrophy (Sun et al., 2008). TGF-β upregulates CTGF expression in chondrogenesis and osteogenesis (Oka et al., 2007). It initiates the mesenchymal cell condensation that occurs preparatory to cartilage formation, and cells stimulated in vitro by TGF-β1 show a concomitant upregulation of CTGF (Song et al., 2007). Luo et al. (2004) demonstrated an upregulation of CTGF in the early stages of stimulation of mesenchymal stem cells with BMP9, a member of the TGF-β family, and Wnt3A. Wnt was able to exert osteogenic stimulus independently of BMP9; however, when constitutively expressed, CTGF led to the inhibition of both BMP9 and Wnt3A. This suggests a close linkup between BMP and Wnt signalling pathways. CTGF is said directly to interact with Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00009-8 © 2011 Elsevier Inc. All rights reserved.

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Figure 9.1  The interaction of several factors that modify cellular responses with CTGF. Here the emphasis is on factors that interact by virtue of the occurrence of specific modules on CTGF. These wide-ranging pleiotropic effects and the versatility of CTGF are related in the sense that the processed RNA has five exons encoding a protein composed of four modules, namely the IGFBP module that can interact with IGFs, a VWC (Von Willebrand type C repeat) module that can interact with members of TGF-β family, and the TSP1 and cystineknot modules that interact with VEGF.

BMP and in this complex appears to regulate the proliferation and differentiation of chondrocytes (Maeda et al., 2009).

CTGF Expression Correlates with Cancer Progression and Prognosis CTGF expression has been tied in with tumour growth, invasion and metastasis. It shows aberrant expression in many forms of cancer, such as prostate, breast and oesophageal cancers, and in gliomas and melanomas. There was a close association of expression with tumour grade in gliomas and breast cancer. In the latter, CTGF expression was related to stage, tumour size and lymph node metastasis (Xie et al., 2001, 2004). A relationship with tumour grade was reported for oesophageal tumours (Deng et al., 2007) and has been shown to relate to poor prognosis (Zhou et al., 2009). Similar conclusions have been reached in investigations of patients with gastric cancer where high expressers showed higher lymph node metastases and poorer 5-year survival than patients with low CTGF expression (Liu et al., 2008). Both CTGF protein and mRNA are highly expressed in the stroma, tumour cells and endothelial cells in head and neck squamous cell carcinomas (Mullis et al., 2008). CTGF correlated with degree of fibrosis in Reed–Sternberg cells and macrophages in classical Hodgkin’s lymphoma and some specimens with the variant nodular lymphocytes, but malignant cells of non-Hodgkin’s lymphoma did not stain for CTGF (Birgersdotter et al., 2010).

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CTGF expression correlated with osteolytic invasion by mandibular oral squamous cell carcinomas and breast cancers (Shimo et al., 2008, 2006).

Is CTGF Involved in Osteotropic Metastasis? Some forms of cancer such as those of the breast, lung and myelomas show a propensity to disseminate to the bone and form osteolytic metastases. Others, for example tumours of the prostate, may also form osteoblastic metastases. These osteotropic metastases are believed to be due to growth factors produced by tumours. Given that several growth factors can interact with CTGF, one can legitimately inquire whether CTGF is directly or indirectly in collaboration with other growth factors that can promote osteotropism of cancer cells and osteolytic metastasis. The molecular mechanisms involved in osteotropism are increasingly being elucidated. The cytokine RANKL (receptor/activator of NF-κB ligand) is a member of the TNF family. The interaction of RANKL with its receptor RANK (receptor/activator of NF-κB) induces osteotropic migration of breast cancer and melanoma cells (Jones et al., 2006). RANKL occurs on osteoblasts and stromal cells of the bone marrow. It is involved with the differentiation of osteoclast progenitor cells into mature osteoclasts and it signals through membrane RANK expressed on precursor and mature osteoclasts. Osteoclast formation can occur independently of RANKL, for example, by the mediation of TNF-α and interleukins. The effects of inhibiting RANKL function or RANK on osteolysis and the formation of osteolytic metastasis have been investigated in tumour models in vitro and in vivo. Osteoprotegerin, an antagonist of RANKL, effectively inhibits tumour growth and the formation of osteolytic lesions in SCID-immunodeficient mice injected with PC3 prostate cancer cells (Armstrong et al., 2008). Inhibition of IKK, a component of NF-κB, has been shown to inhibit osteoclast activity and also prevent osteolytic metastasis of experimental tumours in vivo (Idris et al., 2009). CTGF possesses the IGFBP and VWC modules and therefore can interact with IGFs and members of TGF-β family. The participation of CTGF in osteolytic metastasis flows naturally from the fact that cancer cells produce Wnt proteins. It might be recalled here that growth factors such as IGFs and TGF-β occur in the osteolytic setting. Wnt proteins can induce CTGF expression. Prostate cancer cells do so and induce osteoblastic bone metastasis. Wnt function here is regulated by natural antagonists of frizzled receptors and DKKs (dickkopfs). The latter inhibit Wnt/ β-catenin signalling. Wnt signalling is promoted with the loss of DKKs (Hall et al., 2006). In fact, this might be promoted by a feedback loop wherein CTGF overexpression activates important elements in canonical signalling, namely β-catenin/TCF/ Lef (Deng et al., 2007). Now the parathyroid-related protein (PTHRP) from tumours is known to be upregulated by TGF-β. PTHRP is able to enhance the expression of RANKL that can activate osteoclasts. Interleukins have been implicated in PTHRPindependent osteolysis (see Kozlow and Guise, 2005). In animal tumour models the inhibition of CTGF using specific antibodies has led to a reduction in osteolytic

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metastases and to reduced angiogenesis. CTGF expression occurs in cells that produce PTHRP and the appropriate receptor. These cells are capable of invading the bone marrow (Shimo et al., 2006).

Pro-angiogenic Effect of CTGF The virtually unequivocal reflection of CTGF expression in cancer progression and prognosis finds a rational basis in its pro-angiogenic effects. Aikawa et al. (2006) studied the pancreatic cell line PANC-1 in vitro. This cell line expressed high levels of CTGF and responded to exposure to TGF-β with enhanced CTGF expression. The tumorigenicity of these cells and metastatic spread of the tumours formed when implanted as xenograft was inhibited by intraperitoneal introduction of CTGF-specific antibodies. Tumour size and microvessel density associated with tumours were markedly reduced. According to Deng et al. (2007), overexpression of CTGF in oesophageal squamous cell carcinoma correlated with tumour grade and metastasis status. Although correlations with tumour grade are quite obvious, this study was rather deficient in providing clinical information about the determination of metastasis status. The presence of the VHL (von Hippel–Lindau) gene presaged its interaction and participation in angiogenesis and the genesis of the autosomal dominant condition VHL syndrome with characteristic cystic tumours. Sporadic development of the tumour is due to inactivating mutations of both copies of the VHL gene, whereas subjects with VHL syndrome require one somatic mutation of the unmutated allele to lead to tumour development. Haemangioblastomas, which are benign, highly vascular tumours, exemplify the syndrome. This is a consequence of enhanced VEGF expression (see Urena and Gahbauer, 2008). VHL therefore seems to function as a negative regulator of angiogenesis; indeed it negatively regulates the HIF-1α subunit of HIF-1 transcription factor. VHL-mutant renal cell carcinoma cells in vitro show an increased expression of CTGF and Cyr61 (CCN1) and increased VEGF production. The effects of Cyr61 and CTGF were supplementary, suggesting they could be acting independently of each other (Chintalapudi et al., 2008). The demonstration that CTGF can upregulate the expression of VEGF has lent much support to the view that it can promote metastatic spread. Hypoxia has been shown to induce VEGF production by the human chondrocytic cell line HCS-2/8. Upregulation of VEGF and HIF-1 transcription occurred in cells that were transfected with CTGF-carrying expression vector. VEGF promoter activity was enhanced in the transfectants. In summary, these findings suggest that CTGF activates HIF-1 and by its mediation upregulates VEGF expression (Nishida et al., 2009). However, a diametrically opposite view has been expressed, wherein hypoxia reduced CTGF expression and this effect occurred through HIF-1. Furthermore, hypoxia reduced TGF-β-induced CTGF expression. These effects were recorded in HK-2 and HKC-8 (human kidney proximal tubular epithelial) cell lines and renal cell carcinoma cells (Kroening et al., 2009). In corneal epithelial cells with epithelial-to-mesenchymal transition possibly activated by TGF-β after injury, TGF-β does upregulate CTGF (Secker et al., 2008). So

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a final verdict should still be considered and it might be premature to pronounce on the potential outcome.

Regulation of CTGF by TGF-β In hepatocellular carcinoma (HCC) grown as xenograft, CTGF was upregulated in the stromal component by TGF-β1. The inhibition of such upregulation resulted in diminished tumour growth, vascular invasion and development of metastatic disease. Non-invasive HCC cells expressed CTGF at low levels, but these acquired invasive ability upon the induction of CTGF by exposure of the cells to TGF-β1. Inhibition of TGF-β1 using LY2109761 led to a loss of this acquired invasive and metastatic ability. Furthermore, patients who were high expressers of CTGF had poor prognosis (Mazzocca et al., 2010). CTGF does increase cell migration, and in human chondrosarcoma cells this has been found to be accompanied by MMP-13 expression; CTGF signals seem to be transduced through FAK and ERK and NF-κB signalling. Blocking these pathways inhibits both the enhancement in migration and MMP-13 expression (Tan et al., 2009). There is substantial agreement with this in the findings that TGF-β signals through the Ras/MEK/ERK MAPK system to upregulate CTGF in corneal epithelium (Secker et al., 2008), albeit the phenotypic function subserved here is epithelium–mesenchyme transition. Pi et al. (2008) have proposed a new site on CTGF which binds the ECM protein fibronectin and have suggested this as a possible means by which CTGF can promote cell migration. Chen et al. (2007) presented an interesting set of findings wherein overexpression of CTGF in MCF-7 cells in culture resulted in enhanced migratory behaviour and changes in cell morphology accompanied by actin polymerisation and promotion of focal adhesion. The cells reverted to epithelial morphology with the loss of migratory faculty upon inhibition of CTGF expression. Inhibition of integrin-αvβ3 also inhibited CTGF-induced cell migration and ERK1-2 activation by CTGF. Thus, CTGF seems to function in this system through integrin/ERK signalling. Of additional interest is the involvement of the pro-metastasis S100A4, which is known to deregulate cytoskeletal dynamics (Sherbet, 2001). S100A4 has been shown to reposition adhesion-mediating ECM proteins into a focal pattern that aids in promoting cell migration (Lakshmi et al., 1997). Also, it brings about changes in the cytoskeletal machinery and facilitates cell migration (Lakshmi et al., 1993). Chen et al. (2007) added a new element in the possible mechanism of CTGF function by demonstrating that inhibition of S100A4 blocked CTGF-induced mobility of MCF-7 cells, which was reversed by restoring S100A4 function. Indeed, CTGF upregulated S100A4 expression by αvβ3/ERK1-2 activation. TGF-β activates the canonical Smad signalling system in osteoblasts (Arnott et al., 2008). Besides, ERK inhibition also blocks activation of CTGF promoter in these cells. Arnott et al. (2008) have also implicated src activation upstream of ERK in the regulation of CTGF expression. Although the activation of some of these signalling systems might be cell-type specific, one can envisage a generalised picture in which CTGF so regulated could in its turn activate Akt signalling to promote cell proliferation.

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Cell-type specific expression might be an important factor in the promotion of growth, and of invasive and metastatic properties, because the general experience is that it occurs in a variety of component cells of the tumour. One might emphasise here CTGF overexpression is predominantly encountered in B-cell ALL (Sala-Torra et al., 2007). Indeed, Boag et al. (2007) have emphasised that it is expressed exclusively in B-cell and not T-cell lineage ALL. This indicates the potential relevance of CTGF in the disease process. Also, production of CTGF by pre-B cells might have significant overtones for treatment, because pre-B ALL is regarded as having a more favourable prognosis than T- and B-ALL. Apart from the various modes of regulation, CTGF might share some functional elements with Wnt signalling in promoting cell proliferation and tumorigenesis. Deng et al. (2007) encountered CTGF overexpression in oesophageal squamous cell carcinomas (ESCC), and the degree of expression was related to tumour grade. Experimentally forced expression of CTGF in ESCC cells resulted in increased cell proliferation, and transfected cells xenografted into nude mice displayed enhanced tumorigenicity. In the same in vitro system, CTGF overexpression led to the activation of β-catenin/TCF/Lef, which are important elements in canonical signalling by Wnt proteins; they upregulated the transcription of c-myc and cyclin D1 together with promotion of cell proliferation. As noted earlier, BMP9, a member of the TGF-β family, and Wnt3A can upregulate CTGF. Indeed, CTGF is able to interact directly with BMP and regulate cell proliferation and differentiation. Therefore, it is conceivable that cross-talk might occur between CTGF, BMP/TGF-β and Wnt.

10 Thrombospondins To follow the interactions of CTGF with several biologically active molecules such as the TGF-β family proteins, response to hypoxia with the activation of HIF-1 leading to VEGF expression and the generation of angiogenic response, it is relevant to state here that most of these interactions with other biologically active molecules are ascribable to the presence of the TSP module in CTGF. Besides CTGF, the TSP domain occurs in many molecules such as the ADAMTS proteins, CCN family members, for example CCN1, and WISPs (Wnt inducible signalling pathway protein), among others. The possibility of the presence of TSP domains as potential interacting sites for other biologically active molecule derives from the well-established anti-angiogenic properties of TSP and to its participation in cell–ECM interaction and cell-to-cell signalling. This inevitably leads one to examine the likely relevance in cancer invasion and metastasis. TSPs are not only anti-angiogenic (Lawler, 2002) but they also inhibit cell proliferation and cell migration. The proliferation inhibitory property might be an intrinsic ability of TSPs, but equally it might be a consequence of its regulation, positively by cell cycle control and tumour suppressor p53, and negatively by Ras/myc signalling (Dameron et al., 1994; Watnick et al., 2003). In the presence of mutated p53, TSP-1 expression is diminished (Iddings et al., 2007). TGF-β is able to activate TSP-1. TSPs use a variety of receptors; among them are CD36, CD47, proteoglycans and integrins. The inhibition of angiogenesis has been ascribed to the induction of apoptosis of endothelial cells and to the downregulation of VEGF function. TSP interacts with HGF, EGF, TGF-β and FGF-2, besides VEGF (Margosio et al., 2003; Rusnati and Presta, 2006; Taraboletti et al., 1997). Therefore it is a pre-eminently important molecule in cancer development and progression. TSPs are a family composed of five highly conserved structurally related ECM proteins. TSP-1 and -2 are homotrimers, whereas TSPs-3, -4 and -5 form pentamers. The oligomeric assembly of the TSP was demonstrated as an early event in ontogeny (Adams et al., 2003). Besides, oligomerisation also has functional implications. The TSP-1 monomer is deficient in cell attachment and spreading properties compared with the TSP-1 integral unit. These activities require the carboxy (C)-terminal TSP repeats (Anilkumar et al., 2002) (see below). The monomeric TSP-1 has several domains: an amino (N)-terminal globular domain, an oligomerisation domain, von Willebrand factor type C pro-collagen homology domain and three types of TSR (thrombospondin repeat) domain. There are three properdin type 1 domains, which possess specific ligand-binding property, three EGF-like type 2, 13 calcium binding type 3 repeats, and a globular domain at the C terminus (Adams and Lawler, 2004; Carlson et al., 2005, 2008; Lawler and Hynes, 1986; Lawler, 2000; Lawler and Detmas, 2004). Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00010-4 © 2011 Elsevier Inc. All rights reserved.

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TSR domains occur with varying numbers of copies in many proteins. They occur in the secreted or in the extracellular region of the protein. They seem to be essential for the functions of cell migration, tissue remodelling and signalling subserved by TSPs. They are believed to act as adhesion sites, and protein- and glycosaminoglycanbinding site receptors; they also participate in the anti-angiogenic function of TSPs (Chen et al., 2000). A specific TSR sequence (CSVTCG, cys–ser–val–thr–cys–gly) and possibly sequences flanking this have been identified with the inhibition of angiogenesis and implicated with TSP-1 binding to CD36 receptors that occur on endothelial cells (Iruela-Arispe et al., 1999; Tolsma et al., 1993; Dawson et al., 1997, 1999), a process that might bring about endothelial-cell apoptosis leading to the inhibition of angiogenesis. Angiocidin binds to the CSVTCG sequence of TSR. It inhibits endothelial cell proliferation, induces apoptosis and inhibits tumour growth (Zhou et al., 2004).

TSPs in Cancer The role of TSPs in cancer progression is still being debated fiercely. Indeed, evidence has often been proffered, some indicating promotion and others indicating inhibition of progression. Possibly there is a via media implicating a dual function for TSP, with its effects related to the specifically biological requirement related to the state of progression. Early experiments relating to the role TSP might play in cancer development and metastasis involved investigating its effects in experimental metastasis assays. Tuszynski et al. (1987) treated hosts with TSP and then introduced mouse sarcoma T241 cells intravenously. They observed that pre-treatment with TSP resulted in a markedly reduced lung colonisation by the tumour cells. However, this assay is not a spontaneous metastasis assay because lung offers the first target site the injected cells would encounter. No other possible metastatic sites were tested for secondary tumour from the lung deposits. Nonetheless, these experiments suggest some change might have occurred in the adhesion-related properties of the tumours cells, leading them to localise and form tumour deposits in the lungs. Tumour cells did show enhanced adhesion to substratum in vitro. Recently, much evidence has emerged that supports the view that TSPs indeed inhibit cancer progression. For instance, Iddings et al. (2007) found that in colorectal cancer absence of tumour in sentinel nodes closely correlated with high TSP-1 expression. TSP is said to be inactivated in penile cancers by hypermethylation. Approximately half of the specimens tested showed TSP promoter hypermethylation. Moreover, hypermethylation status was associated with high-grade invasive tumours and correlated with poor prognosis (Guerrero et al., 2008). Bai et al. (2009) compared TSP-1 expression, with PTEN and other genes, in supraglottis squamous cell carcinoma with and without lymphatic metastasis. Both TSP-1 and PTEN were found to be downregulated in carcinomas with lymphatic dissemination compared with those that showed no spread to the lymphatics. PTEN downregulation would be expected to allow signalling through Akt, which is activated downstream by VEGF. So one can

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Figure 10.1  Reconciliation of the opposing putative functions of TSP, postulating a direct route to metastasis inhibition through the inhibition of both angiogenesis and invasive and motile activity of cells. This inhibition can be overcome by TSP function through VEGF to activate Akt signalling, which in turn is conducive to endothelial cell survival and angiogenesis leading up to successful metastatic spread of cancer. Akt activation can allow cell proliferation to occur, resulting in tumour growth. PTEN would naturally be a key element in the regulation of this bypass route. Inhibition of cell migration can probably also be mitigated by an indirect route. TSP has been shown to transactivate EGF receptor and is indeed upregulated by EGF, which suggests a collaborative outcome in terms of cell proliferation and migration.

visualise an alternative or bypass pathway by which angiogenesis can be induced by VEGF/Akt signalling which promotes endothelial cell survival (Figure 10.1). The ability of TSPs to promote cell proliferation, and yet inhibit cell migration, invasion and metastasis, has been the subject of much study and speculation. Using an experimental mammary transgenic tumour (Pyt) model, Yee et al. (2009) have shown that tumours were larger in TSP-1 null mice and associated with better vasculature. In other words, TSP-1 suppresses tumour growth. However, under conditions where TSP-1 promoted tumour cell migration, it also enhanced metastatic behaviour. TSP-1 is capable of transactivating EGFR. Recombinant proteins containing the EGF-like TSR phosphorylate EGFR efficiently (Liu et al., 2009). Also, EGF is able to upregulate TSP expression. This mutual activation results in enhanced cell migration. EGFmediated upregulation of TSP has been shown to activate PI3K/MAPK signalling and enhance TIMP expression; this effect is controlled by PTEN (Soula-Rothhut et al., 2005). Signalling through PI3K has been confirmed recently. Because both TSP and hyaluronic acid activate EGFR and cell migration, they implicate CD44 in the induction of migration (Maier et al., 2009). It can be argued that because TSP-1 can alter the MMP/TIMP balance, it could be influencing cell motility. TSP-1 is said to increase TIMP protein and messenger RNA (mRNA) expression (John et al., 2009) and might thus effectively reduce MMP expression. But then TIMP is able to inhibit angiogenesis, and the enhancement of TIMP expression might adversely affect angiogenesis and metastasis. Moreover, Angiocidin

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which binds the TSR CSVTCG repeat, has been found to downregulate MMP-2 expression and in parallel inhibit the invasive behaviour of cancer cells (Yang et al., 2006). Compatible with this is the inverse relationship that seems to subsist between the expressions of TSP-1 on the one hand and VEGF and MMP-9 on the other in urothelial carcinomas. VEGF and MMP expressions were greater in high-grade than in low-grade tumours. Also, the depth of invasion is correlated with the high expression compared with invasive tumours (Donmez et al., 2009). TSP-2 has also been attributed with the ability to inhibit invasion by downregulating MMP-9 and uPA (Nakamura et al., 2008). Apart from modulating MMP expression and in this way altering invasion, MMPs such as ADAMTS1 might regulate TSP-mediated inhibition of angiogenesis and metastasis by influencing TSP cleavage. TSP-1 is cleaved by ADAMTS1, which can be blocked by using antibodies and short interference RNA (siRNA)-targeted against ADAMTS1 (Lee et al., 2010). One ought to bear in mind in this context ADAMTS possesses TSRs and might also directly inhibit angiogenesis and metastasis. Nevertheless, it is reasonable to postulate that the property of inhibition of growth and angiogenesis can be uncoupled from the promotion adhesion-mediated migration, which could then lead to metastatic spread. Neovascularisation is required for tumour growth; therefore, TSP-1-mediated inhibition of tumour growth could be due to inadequate angiogenesis and loss of tumour cells in the core of the growth by apoptosis and necrosis. There is general agreement that TSP does inhibit angiogenesis. Hypoxia-induced VEGF-A and consequent angiogenesis is inhibited by TSP-1; this it seems to achieve by interfering with downstream Akt signalling (Sun et al., 2009). Another facet of the potential mode of TSP-1 function has been suggested wherein the latter promotes VEGFR2 activation but decreases vascular permeability. This does explain the apparent contradictions in the effects on cell migration and metastasis. The implication here is that the differential function of TSPs might be related to which ECM receptor associates with VEGFR2. Although this is an attractive and a plausible possibility, it must await further confirmation (see Figure 10.1). The possibility that stromal cells might produce TSP and with its aid influence the rate of tumour growth has been advocated. Experimentally forced TSP-1 expression has been shown to reduce the growth of MDA-MB-231/B02 tumour, occurring in parallel with increased VEGF expression in the tumour cells, negating the growth inhibition by TSP-1 (Fontana et al., 2005). In the clinical setting, however, breast cancer stromal cells do produce TSP-1 but also decrease vascularisation. Furthermore, metastatic tumour in the lymph nodes produced more VEGF and TSP-1. This blurs the overall picture. This approach simply emphasises the problems of translating experimental findings to interpret clinical observations. It is needless to reiterate that the stromal and tumour cell interactions are a factor that must enter the equation. Nonetheless, the dangers of over-interpretation of data cannot be over looked. Okada et al. (2010) found in minimally invasive and invasive pancreatic cancers about one-third of the stroma displayed stronger expression of TSP-1 compared with non-invasive cancers. In contrast, a natural inference one can draw from the study of Wei et al. (2010) is that HGF-induced invasion of ovarian carcinoma cells is a result of the downregulation of TSP-1. In this process, HGF seems to signal through the MAPK pathway because, when it is blocked, HGF-mediated increase of invasive behaviour is also blocked (Wei et al., 2010).

11 Cytokines

Cytokines form a group of proteins that participate in cell signalling, intercellular communication and in many cellular and immunological functions. They are prominently involved in inflammatory responses and defence against viral infections. Here the focus is on their function of cell signalling and their activities akin to growthfactor regulators. The overall basis of discussion is that many cytokines function as growth factors, emphasising the importance of tumour necrosis factors (TNFs), the lymphokines, interleukins and interferons (IFNs) in the regulation of cell proliferation, apoptosis, differentiation, and development and progression of cancer with particular emphasis on their signalling pathways.

Tumour Necrosis Factors TNF-α is produced by activated macrophages and TNF-β by cytotoxic T lymphocytes. Both cytokines are associated with many disease states. They are linked with many autoimmune conditions such as inflammatory bowel disease, rheumatoid arthritis, lupus erythematosus, multiple sclerosis and ankylosing spondylitis. Of much interest here is that they are known to aid growth factor signalling, stimulate cell proliferation and tumour growth, and promote angiogenesis, invasion and metastasis. Several TNF family ligands are known. These function through binding to TNF/NGF (TNFR) family receptors. There are some 30 members in this family. TNFRs are type I (single pass) transmembrane proteins (Ware, 2003). The TNF receptor, TNFR1, undergoes trimerisation on ligand binding. The intracellular death domain also shows clustering and leads to the binding of the adaptor molecule TRADD (TNFR-associated death domain). TRADD can now bind to a variety of proteins, for example the TRAF2 (TNF-associated factor 2) adaptor protein. TRAF2 is a ubiquitin protein ligase E3 with a RING domain. E2 recruits cIAP-1 and -2 (cellular inhibitors of apoptosis) that are important participants of downstream signalling (Rothe et al., 1995; Yang et al., 2000). Binding of TRAF2 together with interactions between TRAF and cIAPs activates NF-κB and JNK signalling pathways, leading to cell survival. JNK activation can also lead to Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00011-6 © 2011 Elsevier Inc. All rights reserved.

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apoptosis. On the other hand, cross-talk between NF-κB and JNK signalling can switch between cell survival and apoptosis to generate a response to specific needs of the cell population. The binding of a different element, the FADD (fas-associated death domain), induces apoptosis through caspase activation (Gupta et al., 2006; Kimberley et al., 2007) (Figure 11.1). Signalling by TNF-α through TNFR1 and TNFR2, together with RANKL/RANK and IL-1/IL-1R in osteoclast differentiation and activation, is shown in Figure 11.2. Again, different TRAF elements are recruited (Udagawa et al., 2002).

Figure 11.1  The activation of TNF-α signalling cascade resulting in apoptosis or cell survival and in some cases in the induction of angiogenesis. Upon ligand binding the TNF receptor trimerises. Trimerisation of the intracellular death domain and recruitment of FADD and TRAF leads downstream to the activation of genes responsible for the phenotypic outcomes. (Based on Kimberley et al., 2007, Gupta et al., 2006, and references cited in the text.)

Figure 11.2  Signalling by TNF-α through TNFR1 and TNFR2, together with RANKL (RANK ligand)/RANK (receptor activator of NF-κB) and IL-1/IL-1R in osteoclast differentiation and activation. See also Figure 11.1. Again, different TRAF elements are recruited. (Based on Udagawa et al., 2002.)

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TNF, Cell Proliferation and Apoptosis The ability of TNF to regulate growth depends on the balanced outcome of promotion of cell proliferation and induction of apoptosis, both properties being established attributes of TNFs. Following up on this, there have been many attempts to define the signalling systems that produce these biological effects. The activation of NF-κB by TNFs was demonstrated some time ago, which was also shown to be involved in the production of other cytokines as a consequence of TNF function. TNF seems able to promote cell survival through NF-κB activation, again a view advocated for some time. Along with this it has also been noticed that TNF might induce apoptosis by taking recourse to JNK-mediated signalling and that cross-talk between these two pathways might determine the outcome in terms of tissue or tumour growth (Wang and Lin, 2008; Wu and Zhou, 2010). TNF can also activate the PI3K/Akt/mTOR pathway and in this way regulate cell proliferation, as shown in Figure 11.3.

TNF, Angiogenesis, Cancer Invasion and Metastasis Among genes activated by TNF are those associated with angiogenesis, invasion and metastasis. TNFs have been described as possessing angiogenic as well as antiangiogenic properties. Induction of angiogenesis might arise from stimulation of expression of VEGF and other factors such as FGF, and activation of NF-κB and JNK. TNF-α induces angiogenesis by activating NF-κB. For example, Kim et al. (2010) examined the effects of the anti-angiogenic NBBA [(Z)-N-(3-(7-nitro-3-oxobenzo[d] [1,2]selenazol-2(3H)-yl)benzylidene)propan-2-amine oxide]. This compound inhibited

Figure 11.3  TNF and other cytokines can engage JAK signalling. In this route, JAK or Tyk2 will phosphorylate certain but not all STATs. The phosphorylated STATs dimerise and the dimer enters the nucleus, recognises the responsive elements and initiates gene transcription. JAK can also activate the PI3K/Akt/mTOR pathway, where the complex enters the nucleus and initiates genetic transcription.

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angiogenesis of the chorioallantoic membrane of chick embryos and the invasive ability of HUVEC cells in vitro. These effects were accompanied by the inhibition of both NF-κB and JNK. The TNF-like growth factor (TWEAK) is a member of the TNF family. It is a weak inducer of apoptosis and functions by activating TNFR1, but only moderately activates NF-κB and JNK (Schneider et al., 1999). TWEAK can also induce the expression of VEGF by activating NF-κB through its own receptor (TWEAKR; CD266), a member of the TNFR family (Dai et al., 2009). Members of the TNF family are important modulators of cell migration and invasion. The Snail transcription factors have been identified with epithelial mesenchymal interactions on account of their ability to modulate intercellular adhesion (Thiery, 2003). They have also been implicated in TNF-α-induced cell migration. Not only does TNF-α induce cell migration, but enhanced migration is also associated with reduced expresssion of E-cadherin and upregulated expression of vimentin (Chuang et al., 2008), features that characterise epithelial mesenchymal transition (EMT). It is well documented that E-cadherin is prominent among adhesion-related proteins associated with cancer invasion. This functions as a suppressor of invasion and its loss leads to the acquisition of cell motility. Several factors that repress its transcription have been identified; Snail is one of them. Also, here one recalls that loss of cadherin goes hand in hand with enhanced expression of MMPs. Snail expression seems to correlate with NF-κB activation in cancer cells and metastatic tumour samples (Wu et al., 2009; Wu and Zhou, 2010). The TNF family ligand TRAIL (TNF-related apoptosis-inducing ligand) binds to its receptors, DR4 and DR5, to induce apoptosis in many cell types. Treatment of pancreatic cancer cell lines with TRAIL has led to increased cell migration and signalling through MEK1-2, PKC and NF-κB activation. These effects were accompanied by enhanced expression of uPA, IL-8, MMP-7 and MMP-9 (Zhou et al., 2008). TNF-α in fact activates the MMP promoter (Hohberger et al., 2008). Further support has come from the demonstration that MMP inhibition blocks TNF-α-induced invasion and that MMP regulation occurs through MAPK (Lee et al., 2008). It might be of interest here to recall that DR4 expression has shown correlation with tumour grade in invasive ductal carcinoma of the breast. HER2positive cancers showed higher expression of DR5 and TRAIL (Sanlioglu et al., 2007). However, a mixed message is presented by Loebinger et al. (2009), who transfected bone-marrow-derived mesenchymal stem cells (MSC) with inducible constructs of TRAIL. In vitro, the transfectants induced apoptosis of breast and lung cancer cell lines and of HeLa cells. However, TRAIL expression was associated in experimental metastasis models with reduced lung colonisation. Although induced apoptosis is compatible with the general view, lung colonisation in experimental tumour assays might be merely indicating changes in the adhesion-related features and not spontaneous metastasis. Investigations of TNF expression in relation to metastatic disease are few and far between. It was reported some time ago that TNF-α and TNFRII were more frequently detected in breast carcinomas in situ than in benign tumours. Infiltrating tumours showed more intense staining for TNF-α than did the benign tumour or in situ tumours; however, receptor expression seemed to be higher in infiltrating and in situ tumours,

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in that order, than in benign tumours (Garcia-Tunon et al., 2006). This could suggest that, given the appropriate receptors are expressed, higher levels of TNF-α might be conducive to tumour infiltration. In contrast, in another study, high levels of TNF-α correlated negatively with tumour cell proliferation. Also, high levels of the cytokine have been linked with improved survival (Evans et al., 2006). IL-6, IL-1β, and TNF-α have been shown to correlate with increased risk of cancer incidence and survival (Trompet et al., 2009). Metastasis suppressor genes have been identified as targets of TNF-α signalling, supporting the view that it promotes progression. The metastasis suppressor KiSS inhibits breast cancer cell migration and cell attachment to fibronectin-layered substratum by blocking TNF-α-mediated activation of RhoA/NF-κB signalling (Cho et al., 2009). The metastasis promoter S100A4 has been implicated with TNF-α function in rheumatoid arthritis. Blocking TNF-α has led to reduction in function of S100A4 (Oslejskova et al., 2009). This might be a fruitful approach to adducing further evidence for a potential role for TNF-α in cancer metastasis.

Targeting TNF-α Function through MicroRNAs Despite the variety of its function, TNF-α is not viewed as a tool in cancer therapy. As briefly indicated elsewhere, most biological effects of TNF-α are mediated by microRNAs (miRNAs). Induction of cell adhesion, invasion and apoptosis by TNF-α are mediated by miRNAs. As noted earlier, TNF-α induces cell migration in parallel with reduced E-cadherin expression and upregulation of vimentin (Chuang et al., 2008), suggestive of induction of EMT. The endothelial adhesion molecules E-selectin and ICAM-1 are targeted for regulation by miRNA induced by TNF-α (Suarez et al., 2010). E-selectin is known to influence metastatic behaviour by promoting the adhesion of tumour cells to the endothelium. ICAM (CD54) expression is conducive to metastasis. Downregulation of ICAM1 suppresses human breast-cell invasion in vitro. ICAM1 expression correlates with the metastatic potential of certain breast cancer cell lines (Rosette et al., 2005). VCAM1 (CD106) is also known to be inhibited by miRNAs (Harris et al., 2008). MicroRNAs regulate apoptosis signalling activated by TNF-α. Incoronato et al. (2010) reported that miR212 negatively regulated the anti-apoptosis PED protein, which inhibits apoptosis mediated by FAS and TNFR1, and that forced expression of miRNA increased TRAIL-induced apoptosis in NSCLC cells. Earlier, Garofalo et al. (2008) noticed increased expression of miRNA-221 and -222 led to inhibition of TRAIL-mediated apoptosis but transfection with anti-miRNA-221 and -222 restored TRAIL sensitivity. Indeed, TRAIL-related activation of caspases was linked with several miRNAs some time ago, and death receptors and apoptosis family genes were identified as targets of these miRNAs (Ovcharenko et al., 2007).

Interleukins Interleukins (ILs) are modulators of immune responses; besides, they possess the ability to regulate differentiation of lymphocytes and haemopoietic stem cells, cell

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proliferation and motility. On account of immune stimulatory action, some interleukins have been deployed in cancer therapy. A general perception of the relevance of cytokines to cancer is that immune cells present in the tumour microenvironment respond to stimuli from cancer cells, and that this interaction involves cytokines that have been implicated with a role in the development and progression of cancer. Interleukins exert a paracrine effect on the cells of the microenvironment and stimulate transcription of target genes. They activate TRKs, which as noted elsewhere (pp. 144 and 145) in this book, are the class of receptors mediating the function of many growth factors. The activation of another class of receptors, the GPCRs (seven-pass transmembrane G-protein-coupled receptors), sets in motion the conventional JAK/STAT pathway of signal transduction. JAK activation leads to the phosphorylation of STAT proteins, which eventually initiate genetic transcription. JNK mediation has also been implicated in some functions of cytokines, including interleukins and TNF. Many but not all cytokines interact with GPCR. Interleukin-1β signals through GPCR (Billington et al., 2000; Pascual et al., 2001).

Interleukins Induce Cell Motility Cellular interactions between tumour cells and stromal cells, including immune cells, seem to lead to a mutual regulation of cell behaviour (Figure 11.4). Monocytes and mature macrophages are both known to secrete cytokines when exposed to media in which tumour cells are cultivated. Zhu et al. (2008) detected IL-17 in macrophages associated with breast cancers. Jedinak et al. (2010) reported that cultured medium conditioned by activated macrophages enhanced tumour cell proliferation and migration. The conditioned medium contained IL-1β, IL-6 and TNF-α. Interestingly, the medium was said to stimulate the secretion of VEGF and induction of capillary

Figure 11.4  The interactive relationship between tumour and its stroma leading to cell proliferation, migration and angiogenesis, possibly with the intervention of MMPs, PA, cathepsins and VEGF. Like TNF and TWEAK, interleukins can activate JNK and NF-κB, with JNK signalling leading to apoptosis. In contrast, NF-κB/IκB would lead to inhibition of apoptosis and cell survival.

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formation in aortic endothelial cell cultures. However, TNF-α is capable of altering cell motility and of activating apoptosis as well as survival pathways. A similar effect has been shown to occur with monocytes that are induced to express MMPs by media conditioned by breast tumour cells. Here the active component seemed to be IL-6 (Mohamed et al., 2010). The MMPs could be aiding the invasion process. In some systems, IL-6 occurs with vimentin, again a protein that promotes cell adhesion and migration, in tumour as well as stromal cells (Kayamori et al., 2010). Gocheva et al. (2010) reported that IL-4 induced the production of cathepsins in macrophages and by this means stimulated tumour growth, invasion and angiogenesis. Cethepsin B expression correlated with invasion of the perineural space by pancreatic adenocarcinomas (Niedergethmann et al., 2000). IL-13 signalling through its receptor has also been credited with promotion of invasion in Matrigel assays and in vivo to the promotion of lymphatic spread. Consistent with the findings discussed above, these changes were accompanied by enhanced expression of MMPs (Fujisawa et al., 2009). The signalling pathways adopted by interleukins have been reasonably well elucidated. IL-6 induces uPA expression by signalling through a sequence of activation of PKC-α–JNK–NIK (NF-κB-inducing kinase)–IKK-β (β-subunit of the inhibitor of NF-κB kinase) -NF-κB, and this sequence of signalling might be involved in the modulation of cell migration (Cheng et al., 2009). Melisi et al. (2009) have confirmed that NF-κB is constitutively activated in non-metastasizingpancreatic cancer cells stably transfected with IL-1. The enforced presence of IL-1 then led downstream to the expression of uPA, VEGF and IL-8. The transfected cells also showed enhanced cell migration. When implanted into animals, the cells formed tumours with liver metastases. In contrast, certain other interleukins are found to inhibit cell migration and tumorigenesis. IL-11 has been found to inhibit the invasive behaviour of trophoblast cells in vitro, but it exerted no effects on MMPs or other proteinases (Paiva et al., 2009). Murine Lewis lung carcinoma cells transfected with IL-27 have shown reduced cell invasion and tumorigenesis. The invasion inhibitory effect seems to be mediated by the inhibition of vimentin. Interesting also is the finding that the transfectants showed decreased expression of the angiogenic mediator COX-2 (Ho et al., 2009); presumably, therefore, it would affect metastatic spread. IL-17 induced migration of some breast cancer cell lines but without modulating MMP expression; however, these cells showed altered MMP expression when exposed to TNF (Zhu et al., 2008). Furthermore, IL-17 has no effect on TNF expression, suggesting the effect exerted by the interleukin occurred independently of TNF. However, as we noted above, ILs can induce the expression of proteolytic systems other than MMP, and are capable of altering the ECM (extracellular matrix) and promoting cell migration.

Angiogenic Effects of Interleukins The attribution of migration- and angiogenesis-inducing properties appears to involve the activation of JNK and NF-κB signalling (Figure 11.4). IL-18 was shown to be the active angiogenic ingredient in the pathogenesis of rheumatoid arthritis (Park et al.,

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2001). Recently Amin et al. (2007) showed that IL-18 induces MCP-1/CCL2, VEGF in rheumatoid synovial tissue fibroblasts, and signalling by JNK, p38 MAPK, PI3K and NF-κB. JAK inhibitors exerted no effect. Recombinant IL-1α has been used to show that it induces the proliferation and invasive behaviour of HUVEC cells. HUVEC cell growth, migration and tubule formation was enhanced when they were co-cultivated with the malignant colon-carcinoma cell line WiDr, which produced IL-1α (Matsuo et al., 2009). Although one can see the relevance of these findings overall to HUVEC cell proliferation and migration, the experimental model is not sufficiently persuasive in terms of the interaction of stromal and tumour cells, which the authors sought to implicate based mainly on co-cultivation assays of tumour and stromal cells.

Interferons Interferons (IFNs) are produced by lymphocytes in response to the presence of pathogens as a defensive measure. They are also produced against non-self entities like cancer, inhibiting their growth, invasive faculty and neovascularisation. In this way IFNs probably provide a total defence against cancer progression. Apart from components of infectious pathogens, IFNs are induced by many mitogens and cytokines. In line with the objectives adopted here, the discussion will centre around tumour mediation of IFN induction and the activation of IFN signalling that triggers their inhibitor functions. IFNs are classified as type I or type II to accord with the receptors being bound. Type I IFNs include IFN-α -β and -ω; they engage IFNR-α, whereas type II IFN-γ binds IFNR-γ. IFN-α and -β are produced in response to viral infections, which lead to the destruction of the infected cells. IFN-γ is produced by lymphocytes activated by immunological challenge or mitogen stimulation and by NK cells. The promoter regions of type I IFN-inducible genes contain response elements, which interact with ISGFs (IFN-stimulated gene factors), namely ISGF1, ISGF2 and ISGF3. Of these, ISGF3 is thought to be the major primary transcriptional activator. Binding of the receptors by IFNs leads to the activation of JAK/STAT, in turn leading to the formation of ISGF3, a trimeric transcription factor composed of STAT1/STAT2/IRF9. IRFs (IFN regulatory factors) have the nuclear localisation signal, so the complex is translocated to the nucleus. ISGF3 recognises IFN response elements of target genes and activates their transcription. IFNs also function through an SH (src homology domain) containing adaptor protein CRK for STAT5, and signal through C3G (RAP guanine nucleotide exchange factor 1), activating the Ras protein RAP1A which is inhibitory of proliferation. IFN receptor activation can also transduce the signal through PI3K to regulate growth (Figure 11.5). IFNs generate a diversity of biological effects. Different IFNs might induce immunological functions and other features of biological behaviour related to the development and spread of cancer, for example proliferation, apoptosis and invasion by a differential engagement of signalling systems. Besides, IFNs induce the expression of many proteins. Many chemokines, often pleiotropic in nature, are induced by IFNs. Often the same IFN might induce chemokines possessing diametrically

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Figure 11.5  The generally established IFN signalling pathways. Upon IFN binding, the receptor activates JAK and in turn STATs 1 and 2, leading to the formation of the ISGF3 complex composed of STAT1/STAT2/IRF. ISGF3 recognises the IFN-responsive genes and activates their transcription. IFNs also function through a CRK adaptor for STAT5 protein and signal through C3G (Rap guanine nucleotide exchange factor 1), activating the Ras protein RAP1A which is inhibitory of proliferation. IFN receptor activation can also transduce the signal through PI3K. This leads to the promotion of cell proliferation. It is appropriate to mention that CRK adaptors are also involved in cell migration signalling. This figure is based on Alsayed et al. (2000) and Platanias (2005).

opposite biological effects (see below). Apart from the differential signalling activated by cytokines, the diversity of phenotypic function might arise from the induction of these proteins. So, overall, there is a stable platform on which to propose a mechanistic framework for cytokine function in general.

IFNs in Cancer Progression Although the influences of IFNs on tumour growth, cell proliferation and migration have led to the investigation of them as a mode of treatment, there are numerous queries about their overall effectiveness, specificity of action and their link with progression of cancer. IFN is used in the treatment of some forms of cancer, albeit with several caveats about the perceived relationship between response rates, arrest of disease progression, mechanisms of action and optimisation of administration of immunotherapy. Also, there are interacting and complicating factors such as the differential expression of IFN-regulating proteins (IRFs) and the type and nature of IFN-induced proteins which have different biological properties. IRFs appear to be a significant factor because of their influence on apoptosis and cytotoxicity; therefore, they are of great consequence in tumour growth and progression (Abrams, 2010). There are grey areas over the differences between IFN types in their expression in cancer. IFN-γ is said to be highly expressed more frequently than IFN-α in pancreatic cancer. However, this is not related to tumour size, invasion or the spread of the tumour

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to lymph nodes. However, IFN-α expression is related more favourably to survival than IFN-γ (Saidi et al., 2006). Using experimental tumour models, IL-12 administration has been shown to lead to a sustained production of IFN-γ, and in parallel to NK cell activation and a total inhibition of metastasis of CT-26 colon carcinoma cells. This did not occur in IFN-γ knockout mice, thus strongly implicating IFN-γ in the process (Uemura et al., 2010). IFN-γ and IFN-α promoted apoptotic loss of lymphatic endothelial cells in culture and were able to inhibit cell migration (Shao and Liu, 2006). That the effects of IFN-γ might be mediated by membrane-associated adhesion determinants has emerged from a study of prostate cancer cells. IFN-γ reduced annexin 2 expression and invasive behaviour of the prostate cancer cell line 1542CP3TX, whereas the annexin-2-negative LNCap displayed an increase in annexin expression and invasive behaviour. The effects seemed to be mediated by calpain (Hastie et al., 2008). Here, one has to consider if the expression of annexin is only secondary to the modulation of invasion. Annexin-2 is known to bind PA and tPA, so it can influence cell adhesion and migration. Another valid consideration is whether the two cell lines differ in the expression of chemoattractant N-formyl peptide receptors which also bind annexin. Besides, annexin-2 is known to be able to transactivate insulin and IGF receptors (Zhao et al., 2003). In the B16 murine melanoma system, the effects of IFN-γ on cell migration are dependent upon integrin-αvβ3 signalling, FAK and MMPs (Gong et al., 2008). Indeed, the modulation of cell migration by IFNs seems to be somewhat complex, and the attribution of the ability to inhibit is fraught with difficulties. IFN-γ probably does inhibit angiogenesis, because it downregulates VEGF. This effect has been attributed to anti-angiogenic IP-10 induced by IFN-γ (Adam et al., 2007). Moriai et al. (2009) found that IFN-γ promoted invasion and exerted no effects on the proliferation of SNK-6, SNT-8 (EB nasal NK/T-cell lymphoma lines) and tumour biopsy material. Strict comparisons of this type are probably not fully warranted as one is not comparing like with like. Xenografts of metastatic hepatocellular carcinoma exposed to IFN-α showed growth inhibition, enhanced apoptosis and reduction in microvessel density together with regularisation of tumour-associated blood vessels (Zhang et al., 2010). These effects are bound to affect metastatic spread. They lend support to the findings of Saidi et al. (2006) that IFN-α expression is an indicator of favourable prognosis. In the context of this discussion, attention might be directed to the recent reports that IFN-γ inhibits the expression of the metastasis promoter gene S100A4 (Andersen et al., 2003; Boye et al., 2007). We have noted elsewhere that S100A4 profoundly alters the biological behaviour of cancer cells and the changes that it induces are conducive to cell migration, proliferation, angiogenesis and metastasis (Sherbet, 2001, 2008). Although at a preliminary stage, the effects of IFNs on metastasis promoter and suppressor genes seem to be an area eminently worthy of further exploration.

Cytokines and Myeloid Differentiation Myeloid differentiation generates monocytes, macrophages and dendritic cells. This differentiation process is directed by the cytokines, interleukins and

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granulocyte–macrophage colony-stimulating factors GM-CSF, M-CSF and G-CSF. The function of these cytokines is regulated in a temporal fashion by the activation of different signalling sytems and further in a lineage-specific manner by genetic activation involving lineage-specific transcription factors.

Granulocyte–Macrophage (GM-CSF), Granulocyte (G-CSF) and Macrophage (M-CSF) Colony-Stimulating Factors GM-CSF is a 14 kilodalton (kDa) cytokine. It is also known by the rather cumbersome name molgramostim; the simpler and familiar term GM-CSF (granulocyte–macrophage colony-stimulating factor) is used here. The molecular weight is dependent upon the degree of glycosylation. GM-CSF contains 127 amino-acid residues with two glycosylation sites. The precursor protein has 144 residues, with an amino (N)-terminal hydrophobic secretory signal sequence that is cleaved from the precursor. The secretory sequence is found in many secreted cytokines and growth factors. GM-CSF can remain in the ECM in the form of a complex with proteoglycans and its activity can be regulated in this way within the ECM (Roberts et al., 1988). GM-CSF promotes clonal proliferation and then the differentiation of progenitor cells into mature macrophages and/or granulocytes. The haemopoietic CSF, granulocyte CSF, ILs 1, 3, 6 and 11 and SCF (stem cell factor/the Steel factor) subserve a similar function. GM-CSF is produced by many cell types, granulocytes, macrophages, endothelial and mast cells, T lymphocytes, smooth muscle cells and fibroblasts. Many cytokines induce GM-CSF. The effects of GM-CSF are mediated by its binding to heterodimeric receptors composed of a cytokine-specific α-subunit and a βc-subunit shared with IL-3 and IL-5 (Miyajima et al., 1993). The receptors are assembled into a complex dodecamer quaternary structure. The assembly of the dodecamer complex seems to be essential for receptor activation and signalling (Hansen et al., 2008). The MAPK/ERK, MAPK/p38 and the JNK pathways have been implicated in generating the diversity of phenotypic effects (Platanias, 2003; Tanoue and Nishida, 2003). PI3K/Akt might regulate the processes of apoptosis and cell proliferation (Vanhaesebroeck and Waterfield, 1999). Signalling through the shared βc-subunit might involve JAK/ STAT. IL-3, IL-6 and erythropoietin also activate JAK/STAT signalling (Kisseleva et al., 2002). As noted previously, IL-3 is known to function through the βc-subunit and possibly IL-6 functions in this way. As mentioned earlier, myeloid differentiation is a result of sequential signalling by ILs, GM-CSF, M-CSF and G-CSF. The early steps in the process involve ILs 3 and 5, and GM-CSF-mediated activation of STAT5A and STAT5B, which is followed again by STAT5 activation by M-CSF and G-CSF (Ilaria et al., 1999; Piazza et al., 2000; Rosen et al., 1996). Macrophage colony-stimulating factor (M-CSF) is a cytokine with 4α-helices. Not unlike GM-CSF, it is also produced by monocytes, macrophages, granulocytes and fibroblasts, and is involved in the regulation of cell proliferation, differentiation,

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apoptosis and survival of monocytes and macrophages. Like GM-CSF, M-CSF activates the MAPK/ERK pathway. Apart from myeloid differentiation, this pathway might be active in the promotion of tumour progression attributed to M-CSF. Indeed, it induces the expression of VEGF and, in vivo, the formation of vascular structures (Curry et al., 2008). G-CSF has also been found to promote angiogenesis (Shojaei et al., 2009). In addition, M-CSF seems to take the PI3K, Ras and PLC-γ route in producing some phenotypic effects. M-CSF initiates its effects on the growth and development of myeloid progenitor cells, mononuclear phagocytes and trophoblasts by activating its receptor. The c-fms typrosine kinase is the most likely receptor for M-CSF, because fms is known to interact with STAT (Novak et al., 1996), PI3K (Reedijk et al., 1992), PLC-γ (Bourette et al., 1997) and p120 RasGAP (GTPase activating protein)/p190RhoGAP (Trouliaris et al., 1995), which are the major pathways M-CSF activates to influence cell differentation, survival and to modulate cell morphology. Mutation of c-fms has been linked with the pathogenesis of myeloid leukaemias. Its inactivation leads to the disruption of the maturation of phagocytes, indicating that it mediates the effects of M-CSF (Dai et al., 2002). Granulocyte-stimulating factor (G-CSF) is also a prominent participant in differentiation of myeloid progenitor cells committed to the neutrophil and granuloctyte lineage, their proliferation and survival. It is produced by activated monocytes, macrophages and neutrophils, sources it shares with M-CSF and GM-CSF. Fibrolasts and endothelial cells, and several tumours and tumour-derived cells, also form a source of the cytokine. G-CSF is a 19 kDa glycoprotein, biologically active in the monomeric form. It binds with high affinity to its receptor G-CSFR (CD114). G-CSFR is a transmembrane molecule with conventional structural features, including a ligand-binding extracellular domain, and a transmembrane and cytoplasmic domain. Signalling by G-CSF binding to G-CSFR leads to its interaction with two protein kinases, JAK2 and the Src family Lyn kinase. Downstream the STATs pathway is activated. The Lyn kinase phosphorylates adaptor molecules, leading to the association of G-CSFR with PI3K and MAPK/ERK signalling (Rane and Reddy, 2002; Sampson et al., 2007).

Cytokines in Cancer Therapy Many cytokines have been investigated for potential anti-cancer effects although, in general, the results are not promising. Among those studied are IFN-α, ILs, TNF and the CSFs. Some years ago they were considered to hold much promise in the treatment of haematological malignancies, and IFN-α was regarded as a most useful and effective agent. IFN is used in the treatment of multiple myeloma, some forms of lymphomas and leukaemias, and melanoma. Clinical trials are still taking place. The absence of a cohesive outcome is probably a consequence of the fact that often these cytokines are not identifiable as individual entities but represent a family of molecules whose pleiotropic effects might vitiate clinical outcome.

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Cytokines possess anti-angiogenic properties, but there are indications that GM-CSF, M-CSF and G-CSF might be conducive to the differentiation of endothelial progenitor cells, proliferation and angiogenesis. GM-CSF has been the focus of investigation into its potential as an anti-tumour agent. A recent clinical trial of patients with Mullerian tumours has shown some response in tumour mitigation and reduction of the CA-125 (Roche et al., 2010). Eubank et al. (2009) reported that GM-CSF suppressed VEGF of stromal macrophages, which they attributed to the induction of soluble VEGF receptors (sVEGFR). Using a murine tumour model, they showed that GM-CSF treatment inhibited tumour growth and metastatic spread. It is possible that enhanced angiogenesis claimed in some studies might aid immunological defence against the tumour. Targeted delivery of GM-CSF into experimental tumours has resulted in the recruitment of CD40-activated B cells and dendritic cells, which are cellular elements of immunological defence (Gordon et al., 2008). Given that cytokines exert pleiotropic effects, one can visualise a conflict between cell survival and apoptosis, which could determine the outcome in terms of tumour growth or regression. Colluding or conflicting influences of other cytokines such as TGF-β need to be taken into account. For instance, GM-CSF promotes the proliferation of bone marrow endothelial progenitor cells (Wang et al., 2009). TGF-β seems able to inhibit GM-CSF signalling (Montenegro et al., 2009). Alteration in plasma VEGF levels might result from a balance between pro-angiogenic angiotensin-2 and sVEGFR, which inhibits angiogenesis. GM-CSF seems to restrict the former and enhance the latter, thus resulting in the inhibition of angiogenesis (Kumara et al., 2009). Chang et al. (2007) found GM-CSF had no apparent anti-tumour activity, whereas IL-2 did on its own or in combination with GM-CSF. They noted IFN-γ was produced by intra-tumoural injection of either or both GM-CSF and IL-2, but a combination of the two was more effective. This raises the possibility of mediation by IFN-γ in the perceived anti-tumour effects and host-cell infiltration into the tumours. Cells from head and neck squamous-cell carcinomas expressing GM-CSF, G-CSF and/or their receptors show stimulation of proliferation and motility in vitro, presumably attributable to autocrine or paracrine effects. Tumours xenografted into immune-compromised murine hosts were associated with increased angiogenesis and recruitment of inflammatory cells (Gutschalk et al., 2006). Inflammatory cells are a source of many cytokines, including ILs, IFN-γ and pro-angiogenic growth factors. These cytokines could be mediating the effects. IL-15 and GM-CSF conjugated with the EDB (extracellular domain B) sequence of fibronectin (FN) have been used to test their anti-tumour activity. The conjugates targeted in this way to the experimental subcutaneous as well as metastatic tumour showed marked anti-tumour effects (Kaspar et al., 2007). The EDB isoform of FN occurs mainly in tumour vasculature, from which is derived the specific localisation of the EDB/IL-15 and EDB/GM-CSF conjugates to the tumour. In the context of potential clinical deployment of these conjugates, it would be helpful to bear in mind that the EDB isoform occurs in retinal vascular component (Khan et al., 2004). Also, the EDB seems to be able to induce endothelial proliferation and tubule formation in vitro (Khan et al., 2005).

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Chemokines Chemokines are cytokines with chemoattractant properties that subserve several biological functions mediating the recruitment of leukocytes into tissues in inflammatory response. They modulate migration of monocytes, NK and dendritic cells, and some elicit angiogenic responses. They are closely connected with tumorigenesis and tumour progression, as evidenced by the fact that both primary and secondary tumours and stromal components produce them. The chemokines are sub-grouped into CC chemokines (β-chemokines), which possess two adjoining cyteine residues at the N terminus. When an intervening amino-acid residue occurs between these two cysteines, they are referred to as CXC chemokines. Chemokine receptors are G-protein-coupled transmembrane receptors. The chemokines function by binding to respective receptors, of which there are two major subfamilies, namely the CCRs and CXCRs. Around nine CCRs and seven CXCRs have been identified. CCRs and CXCRs occur in many cell types. Chemokines might signal by binding to more than one receptor (Baggiolini, 1998). This promiscuous binding might produce the pleiotropism of cytokines and the variety of targets of their action. The CC chemokines are around 27 in number and enumerated as chemokine ligands CCL-1 to CCL-27; they function through receptors referred to as CCR1 and so on. A notable member of the CC family is MCP1 (macrophage chemoattractant protein 1), referred to also as CCL-2. The CXC chemokines (CXCLs) are characterised by an intervening amino-acid residue and not infrequently more than one residue between the N-terminal cyteines. IL-8 (CXCL8), GROα (growth-related oncogene α) (CXCL1), ENA-78 (epithelial neutrophil acting protein) (CXCL5) and GCP2 (the granulocyte attractant protein 2) (CXCL6) are CXC chemokines. CXC chemokine receptors are known as CXCR1, CXCR2, etc. Chemokines are produced as a response to many stimuli such as the cytokines TNF-α, IFN-γ and IL-1β, and LPS. The angiogenic properties of chemokines and their potential role in tumorigenesis were recognised some years ago (Strieter, 1992). The CXC chemokines containing the ELR (glutamic acid-leucine-arginine) motif (ELR    CXC) have been found to promote tumorigenesis and induce endothelial migration and angiogenesis in animal tumour models of Lewis lung carcinoma (LLC) and C57BL/6 mice. The expression of ELR  CXC correlated with the growth and metastasis of LLC, which required the presence of the receptor CXCR2. Tumours growing in CXCR2-deficient animals showed reduced angiogenesis (Keane et al., 2004). CXCL1 and CXCL5 expression has been investigated in colon cancer. Both chemokines showed higher expression in cancer tissue than in adenomas. The expression of their messenger RNA (mRNA) was greater in primary cancer and metastatic tumour from liver than in corresponding normal tissue. These findings suggest that both chemokines might be associated with progression of colon cancer (Rubie et al., 2008). This study incorporated 46 primary and 16 liver secondary tumours. The link between expression levels and progression would have been very persuasive if the expression levels for these

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chemokines for the 16 metastatic tumours and their corresponding primaries had been made available. Nonetheless, these are highly significant findings.

IFN-Induced Chemokines in Cancer Invasion IFNs induce the expression of a multitude of genes. Identified from among them are several IFN-inducible (IP) proteins. Some induced proteins have been examined for possible effects on cell migration, proliferation and angiogenesis (Table 11.1). IP-10 induced by IFN-γ was identified as a CXC chemokine. It serves as a chemoattractant for activated T cells, stimulates monocytes and the maturation of NK cells. It is also known to affect cell adhesion, invasion and angiogenesis (Neville et al., 1997). IP-10 has strong links with Th-1-type inflammatory disease and autoimmune conditions, wherein T-cell infiltration of diseased tissues has corresponded with the levels of IP-10 and CXCR3, indicating T-cell mobilisation (Dufour et al., 2002). Much evidence has accumulated about the stimulation by IP-10 of changes in cancer cell behaviour. It promoted invasion without affecting the proliferation of SNK-6 and SNT-8 (Epstein-Barr virus EBV nasal NK/T-cell lymphoma lines) and tumour biopsy material (Moriai et al., 2009). IP-10 downregulates VEGF expression and clearly is able to inhibit angiogenesis. Several integrin receptors and their ligands appear to be involved in this process. As noted earlier, the IFN effects on cell adhesion and migration also involve membrane-associated determinants of cell adhesion. IFN induces certain transmembrane proteins (IFITMs) that show markedly altered expression in some forms of cancer. IFITMs have been found to be overexpressed in colonic tumours, and their upregulation is mediated by the activation of Wnt/β-catenin signalling (Andreu et al., 2006). In murine and human colon carcinoma cells, inhibition of adenomatous polyposis coli (APC)-activated β-catenin signalling markedly upregulated IFITMs. This is significant because inactivation of APC by mutation is an important element of development of FAP (familial adenomatous polyposis) and colon cancer. APC gene inactivation results in an increase of β-catenin in the nucleus and the transcription of responsive genes that lead to tumour progression. In head and neck squamous cell carcinomas (HNSCC), IFITM1 was detected at the invasive front of early invasive tumour and was markedly overexpressed in the invasive tumours. The stimulation of invasion was confirmed in vitro (Hatano et al., 2008). Seyfried et al. (2008) found three IFITMs upregulated in murine astrocytes and astrocytoma cells. Of these, IFITM1 and IFITM3 showed greater upregulation in astrocytomas than in terminally differentiated astrocytes. Another IFN-inducible protein of interest is IFIX-α. It is a member of the HIN-200 family of IFN-inducible proteins with the characteristic 200 amino-acid domains. They occur in many tissues although initially they were believed to be restricted to haemopoietic tissue (Ludlow et al., 2005). IFIX-α was identified as being able to inhibit cell proliferation (Asefa et al., 2004). This appears to be because it is able to bind and downregulate HDM2, the human homologue of the mdm2 gene (Ding et al., 2006). HDM2 is a negative regulator of p53 function, so the downregulation of the negative regulator leads to the activation of p53, which in turn leads to

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Table 11.1  IFN-Induced Proteins and Their Biological Effects IFN

Induced Protein

Biological Effect/Cell Line/Tumour Type

Reference

IFN

IFIX-α

Inhibit cell proliferation

Asefa et al. (2004); Ding et al. (2006)

Reduced expression in breast cancer

Ding et al. (2004)

Invasion ↓; MDA-MB-468 breast cancer cells

Yamaguchi et al. (2008)

Upregulated in colon carcinomas

Andreu et al. (2006)

Overexpressed in astrocytoma cells

Seyfried et al. (2008)

IFITM

IFITM 1 and 3 IFN-α, -β

Mx family

Boehm et al. (1998)

GTPases

IFN-γ

IFN-γ

MxA

Invasion↓PC3M prostate ca cells

Mushinski et al. (2009)

IP-10

Invasion ↑; no effect on proliferation; SNK-6 and SNT-8 (EBV nasal NK/T-cell lymphoma lines); biopsy material

Moriai et al. (2009)

VEGF ↓; angiogenesis inhibited

Adam et al. (2007)

47-kDa family GTPases; GBP guanylate-binding proteins

Boehm et al. (1998)

Rho GTPase activation T84 epithelial cells

Utech et al. (2005)

the suppression of cell proliferation. However, earlier, Ding et al. (2004) suggested that IFIX-α1 might be functioning independently of p53. They found that its expression was reduced in most breast cancers. Forced expression of the protein in breast cancer xenografts led to suppression of growth. Yamaguchi et al. (2008) reported that IFIX-α inhibited the invasive behaviour of MDA-MB-468 breast cancer cells. GTPases are a remarkable class of proteins which are effective in cell transformation and produce marked effects on cell adhesion and migration. Mushinski et al. (2009) studied IFN-inducible MxA (myxovirus resistance) GTPase in prostate carcinoma cells. MxA was found in PC-3 cells but not in the high-metastasis variant PC-3M. Forced expression of MxA in the latter decreased the ability of PC-3M

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to metastasize to the liver. MxA also inhibited the invasive behaviour of LOX melanoma cells. These authors tie in their findings with the deletion of MxA in prostate cancer. RhoA kinases, which are regarded as less robust transforming agents, might also be indirectly associated with the modulation of cell behaviour. IFN-γ is known to activate RhoA (Utech et al., 2005). Zhang et al. (2009) looked at the expression of IRF-4-binding protein (IRFBP), which has been identified as an activator of RhoA. IRFBP expression was high in colorectal cancer and correlated with clinical stage, but it was not expressed in normal tissue or in adenomas.

Monocyte Chemoattractant Protein (MCP) MCP-1 is a CCL class protein designated as CCL2. It is induced by pro-inflammatory cytokines. It is a key component in the pathogenesis of rheumatoid conditions. It has been linked with autoimmune conditions such as IgA (immunoglobulin A) nephropathy. MCP-1 promotes recruitment and activation of monocytes to target sites. Two MCP-1 receptors have been identified, generated by alternative splicing causing differences in the carboxy (C)-terminal region (Charo et al., 1994; Yoshimura and Leonard, 1990). The receptor CCR2 binds MCP-1 as well as other isoforms of MCP-1 that possess high sequence homology. CCR2 is expressed on monocytes, activated T-cells, NK cells, dendritic cells and basophils. Signalling is activated by the coupling of CCR2 to G-protein subtypes. Many pathways are activated, of which some have been implicated in the chemotactic behaviour of MCP-1. These include PLC/IP, ERK1/2, the MAPK pathway, PKC, PI3K and JAK, and NF-κ systems (Richmond, 2002; Rossi and Zlotnik, 2000).

MCP-1 Expression in Cancers The inflammatory responses associated with cancer have led to the investigation of MCP-1 expression and whether the degree of this might indicate the state of cancer progression. MCP-1 is one of the cytokines found in large amounts in prostatic hyperplasia (BPH) produced by stromal cells. It is said to induce proliferation of the epithelial component of BPH but not of the stromal cells, although both possess the required receptors (Fujita et al., 2010). Studies of prostate cancer cells in culture have revealed high levels of MCP-1 expression and of other chemokines such as IL-6, IL-8, GRO-α, ENA-78 and CXCL-16 (Lu et al., 2006, 2007a). Expression of CCR2 also corresponded with tumour aggressiveness, and higher expression was detected in metastatic than primary tumour. Expression in primary tumours correlated with Gleason grade and stage, and was higher than normal prostate tissue (Lu et al., 2007b). Two important caveats need to be considered here. One is that although MCP-1 functions predominantly through CCR2, other CCL ligands might also bind CCR2. Secondly, given the intrinsic heterogeneity of tumours, one can suggest the possibility that the tumour component that expresses the chemokines might be the metastasizing element. These

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authors have gone on to demonstrate, using conditioned medium and recombinant MCP-1, that MCP-1 and IL-8 might promote osteoclast activity and bone resorption, suggesting a potential lead to the effects of metastatic spread of prostate cancer to the bone. A most interesting prospect is that MCP-1 levels in patients might predict the likelihood of progression to metastasize to the bone. Lu et al. (2009) reported elevated levels of MCP-1 in patients with prostate carcinoma with bone metastases compared with those with localised disease. MCP-1 does seem able to induce invasive behaviour in vitro. It would be necessary to demonstrate target-specific invasion, which is a somewhat complex query to answer using in vitro assays of migration. Both stromal and tumour cells of papillary thyroid carcinoma express MCP-1, and the expression levels correlated with the spread of the tumour to the lymph nodes. MCP-1 was also predictive of recurrence (Tanaka et al., 2009). Patients with colorectal cancer expressing high levels of MCP-1 also showed multiple liver metastases. Significantly, high expression correlated with high microvessel density and Angiopoietin-2 levels. Moreover, MCP-1 expression seemed to be a good predictor of disease-free survival (Yoshidome et al., 2009). Niu et al. (2008) have reported that MCP-1 induces the expression of the transcription factor MCPIP and that the latter activates the N-cadherin cdh12 and cdh19 genes. N-cadherins activate the β-catenin signalling pathway and promote in vitro trans-endothelial adhesion and migration (Qi et al., 2005). N-cadherin also enhances migration in vitro and confers significant metastatic ability in experimental metastasis assays (Hazan et al., 2000). One might recall here that increases in macrophage infiltration are often accompanied by enhanced expression of both angiopoietin and MCP-1. A dominant negative mutant MCP-1 has shown a loss of its chemoattractant ability and a partial loss of the ability to induce angiogenesis (Koga et al., 2008). Van Golen et al. (2008) believe that MCP-1 activates Rac GTPase, which alters the morphology of tumour cells and aids their diapedesis across the endothelium into the metastatic site. Rho GTPases also regulate cell adhesion mediated by cadherin. The promotion of invasion, angiogenesis and correlation with metastatic spread strongly support a positive role for MCP-1 in disease progression. To what extent the stromal cells contribute to the levels of MCP-1 in altering the biological parameters and promoting tumour progression is yet to be ascertained. In experimental systems, obliteration of stroma cell function does diminish tumour aggressiveness.

12 Growth Factor and Hedgehog Signalling Pathways Meet in Developmental Systems

Growth factors participate and drive a wide spectrum of cellular processes such as cell proliferation and apoptosis, differentiation, intercellular adhesion, cell migration and chemotaxis. They transduce their signals through an array of channels to bring about these phenotypic changes in physiology, morphology and cell behaviour. Signalling by growth factors, hormones and vitamins such as the steroid sex hormone oestrogen, vitamin D3 (VD3) and retinoids is mediated by their respective receptors. Growth factor receptors are transmembrane in location, whereas others occur in the nucleus. Corticosteroid or glucocorticoid receptors are cytoplasmic receptors. Not only are the signalling systems highly complex but the integrity of individual components of the entire pathway is also of the utmost importance in the life of the cell. Signalling pathways in differentiation and morphogenesis are highly conserved systems. Several pathways can be implicated; they converge, cross-react, mutually regulate and function in combination, for example the TGF-β pathway, the hedgehog (Hh), Wnt, Notch, fibroblast growth factor (FGF) and EGF pathways. This situation is also in evidence in pathogenesis. So an analysis of the pattern of convergence and the modes of working of growth factors as a co-ordinated system is indispensable for understanding normal development, differentiation and pathogenesis of human diseases, especially in cancer development where normal signalling systems that operate in development and differentiation appear to be awry.

The Hedgehog (Hh) Proteins The Hh genes encode small secreted proteins that participate in intercellular signal transduction associated with a variety of cellular processes. Of the prototypal Drosophila Hh protein, three mammalian homologues have been identified. The Hh family is composed of three homologues: Sonic Hh (Shh), Indian Hh (Ihh) and Desert Hh (Dhh). These occur in the form of precursor proteins. The carboxy (C) terminus of the preprotein has an autocatalytic domain and a signalling domain. The preprotein goes through a process of autocatalytic cleavage resulting in the elimination of the autocatalytic domain (Bumcrot et al., 1995; Lee et al., 1994). Simultaneously with this truncation, cholesterol is covalently added to the C terminus of the signalling domain. The resultant increase in hydrophobicity of the Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00012-8 © 2011 Elsevier Inc. All rights reserved.

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Figure 12.1  The autocatalytic processing of Hedgehog preproprotein into mature functional protein. The autocatalytic domain is cleaved, cholesterol is added to the C terminus of the resulting signalling domain, and palmitate to the N terminus. SS contains the signal sequence. References are provided in the text.

signalling domain is believed to influence its spatial and subcellular distribution (Porter et al., 1996a, b). The protein is further modified by signal peptide cleavage and palmitoylation of amino (N)-terminal cysteine residues to generate the mature form of Hh (Figure 12.1). The mature protein is thought to be 30 times more active than corresponding unmodified Hh (Pepinsky et al., 1998). Palmitoylation is mediated by an acyltransferase encoded by the ski (Skinny hedgehog) gene (Chamoun et al., 2001). Most functional Hh occurs as soluble multimeric complexes. Chen et al. (2004) showed that palmitoylation is essential for Hh activity. This modification produces the soluble multimeric complex, which is the freely diffusible physiologically functional form of Hh protein (Goetz et al., 2006).

The Hh Signalling Cascade Much early work in Drosophila elucidated the Hh pathway and its association with growth factor signalling. The Hh system is a major signalling pathway in human embryonic development and differentiation. Hh signalling, which is accurately and closely conserved in the evolutionary timescale, is associated with the transduction of signals relating to cell proliferation, embryonic development and pattern formation. Therefore, it is unsurprising that deregulation or inappropriate activation of Hh signalling results in developmental abnormalities and in the pathogenesis of many forms of human cancer. Besides, not only are the growth factor and hormone signalling pathways interlinked and intimately integrated, they may function in conjunction with the Hh pathway, more often than not as downstream signalling systems. This integration of different pathways enables the expression of the vast variety of cellular function and phenotypes to be generated in development and differentiation. Many developmental and congenital abnormalities as well as the incidence of some forms of cancer have been said to arise when the integrity of these pathways is compromised. Loss of integrity of signalling can now be directly linked with the pathogenesis of many human diseases.

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The Hh family proteins are secreted proteins and function in a paracrine mode on their target cells. Hh signalling occurs with the binding of Hh to the glycoprotein called Patched (PTCH1). PTCH has been demonstrated to be an Hh receptor (Chen et al., 1996; Marigo et al., 1996). It is a 12-pass (with 12 transmembrane domains) transmembrane protein, with two extracellular loops and intracellular N and C termini and a sterol-sensing domain (see Atlas of Genetics and Cytogenetics in Oncology and Haematology, 1997, 2000). PTCH binds to the cholesterol-modified N-terminal fragment of Hh. Another human homologue, PTCH2, has been cloned. PTCH1 and PTCH2 bind to Hh proteins with similar affinity. Furthermore, PTCH2 is demonstrably a participant in Hh (Dhh) signalling (Carpenter et al., 1998). A second component involved in Hh signalling is called Smoothened (SMO), a seven-pass G-protein-coupled receptor, which essentially is the signalling entity. SMO functions downstream of PTCH (Marigo et al., 1996). SMO may be physically linked with PTCH; they are co-expressed in many tissues, but SMO may not directly interact with Hh (Stone et al., 1996). It can regulate the activity of the Gli family of zinc finger family transcription factors. It is repressed by PTCH, but it can regulate Gli activity when released from PTCH-mediated repression by the binding of Hh to PTCH. A tetrameric signalling complex is made up in vertebrates of Gli, Costal 2, serine/threonine kinase Fused and Suppressor of Fused. This complex is linked up with SMO through Cos 2 (Cohen, 2003). The latter binds to PKA (protein kinase A), and the kinases CK1 and GSK3 (glycogen synthase kinase 3). These kinases potentially intervene in the signalling process. This complex occurs downstream of SMO. In the absence of Hh, SMO is repressed. Cos2 and Suppressor of Fused bind to Gli and promote its retention in the cytoplasm (Dunaeva et al., 2003), so it becomes available for phosphorylation by kinases. The binding of Hh to PTCH leads to SMO activation and the inhibition of proteolytic processing of Gli; the uncleaved full-length Gli retains its transcription activator function. Not being subject to Suppressor-of-Fused-mediated inhibition, the uncleaved Gli enters the nucleus to induce the transcription of responsive target genes (Cohen, 2003; Hooper and Scott, 2005). In the presence of SMO, Gli phosphorylation is prevented. PKA phosphorylates Gli at several sites, followed by further phosphorylation of interspersed sites by GSK3β (Jia et al., 2002; Price et al., 2002). Hyper-phosphorylated Gli undergoes proteasomal degradation to form a repressor form. The cleft Gli forms a transcription repressor. Consistent with this, PKA inhibits Hh signalling (Epstein et al., 1996; Wang et al., 2000). Three members of the Gli family have been identified. These are Gli1, Gli2 and Gli3. Gli1 activates transcription, whereas Gli2 and Gli3 suppress transcription (Tsanev et al., 2009). Also, when Hh is absent, Gli2 and Gli3 show significant levels of expression; they are phosphorylated by PKA, CK1 and GSK3β and ubiquitinated and processed by proteasomes (Sheng et al., 2006, Tempe et al., 2006; Wang and Li, 2006). It has been recognised that the activity of Gli is liable to modification by other signalling pathways, such as MEK/ERK and PI3K/AKT, although some have argued that the ERK but not the PI3K/Akt might modulate Gli. As discussed elsewhere, this would integrate the Hh signalling pathway with that of growth factors such as the EGF, FGF, TGF-β family members and Wnt family proteins. This co-operative signalling between Hh, EGFR and Wnt is illustrated in the context of EGFR function (Figure 12.2). Hh is said to

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Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy Hh ligands [Indian, Sonic, Desert Hh] GSK3/PKA/SMO

Hh Receptor TGF-βs, Activin, Nodal BMPs, GDFs Retinoids FGF

Wnt ← ← Gli ←

p53 ↔ Bcl

myc

DNA replication/repair

↓ ↓ ↓

Differentiation Pattern formation Cell motility

Figure 12.2  The convergence of the TGF-β family and retinoid signalling with the Hh signalling pathway in generating phenotypic features of differentiation, pattern formation, cell motility, and DNA replication and repair processes. The portrayal here is not intended to suggest that TGF-β family members and other growth factors function downstream of Hh, but function in collusion with them. For instance, Nodal is said to act upstream of Hh in ventral telencephalon (details in the text). Hh interacts with EGFR pathway. Hh can activate EGFR and conversely EGFR can interact through Raf/MEK/ERK signalling with Gli and modulate genetic transcription as shown in Figure 13.5.

be able to activate EGFR. Reciprocally, EGFR can influence Gli-mediated transcription of target genes by activating the Raf/MEK/ERK pathway. GSK3β and PKA also can inhibit Gli. The reasoning behind this, as stated before, is that in the presence of SMO, Gli phosphorylation is prevented. PKA and GSK3β sequentially phosphorylate Gli. This hyperphosphorylated form of Gli undergoes proteasomal degradation to generate a repressor form. Consistent with this, PKA inhibits Hh signalling (Epstein et al., 1996; Wang et al., 2000). Growth factors can activate SMO by the mediation of PKA/Akt (Riobó et al., 2006), but the precise mechanism is not yet clear. In Figure 12.2 a putative link with Wnt signalling is indicated because GSK3β activation is also regulated by Wnt. Not only is PTCH a receptor but its expression in the cell membrane is upregulated by Hh. The consequence of this would be sequestration of free Hh and restriction of its mobilisation to the responsive cells (Chen and Struhl, 1996; Cohen, 2003). Thus there is a PTCH-mediated equilibrium between Hh production and its paracrine function. An important element in this PTCH-mediated sequestration and maintenance of equilibrium signalling is another transmembrane protein called Dispatched (Disp). Disp is required for the release of the secreted Hh protein. Disp has a cholesterol sensing domain and is able to regulate the release of Hh that has been modified with the cholesterol insert. Its role is obvious from the finding that cholesterolmodified Hh is retained by the secreting cells in the absence of Disp, i.e. Disp releases Hh that is anchored to the secreting cell by cholesterol (Burke et al., 1999).

Hh Homologues and Their Role in Morphogenesis Three Hh homologues, Shh, Ihh and Dhh, have been identified with specific developmental functions. Shh has been associated with the development of limb, somites and

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neural tube. In embryonic development the early notochord, composed of mesodermal tissue, induces the specialised differentiation of the overlying ventral midline of the neural tube into the floor plate. The notochord together with the floor plate functions as an important signalling centre leading to the differentiation of oligodendrocytes, serotonergic neurons of the raphé nuclei of the brainstem and cranial motor neurons. Shh is found in the node, notochord, floor plate and in posterior limb bud mesenchyme, and is suggested to be able to induce derivative cells from the floor plate (Krauss et al., 1993; Roelink et al., 1994). The notochord and the floor plate express Shh. This link-up is supported by its ability to activate genes that are related to floor-plate differentiation (Echelard et al., 1993). Ericson et al. (1995) demonstrated the induction of ventral neuronal cell types in explants of prospective forebrain regions of the neural plate exposed in vitro to Shh. The neurons induced from prospective diencephalic and telencephalic regions displayed features characteristic of their in vivo counterparts. The LIM homeodomain protein Isl-1 was expressed in the induced neurons. This LIM protein is a marker of differentiated phenotype for motor but not sensory neurons (Thor et al., 1991). Shh induces midbrain dopaminergic neurons and other neuronal phenotypes in vitro (Hynes et al., 1995; Wang et al., 1995). The expression of Shh could extend to regions outside the neural plate into ventro-lateral regions from which some motor neuron precursors of the central nervous system arise. Indeed, using neural explants, Shh has been shown to induce formation of motor neurons (Marti et al., 1995a, b). Hu and Marcucio (2009) recently showed that activation of the Shh pathway in the brain affects craniofacial morphogenesis of chick and murine embryo models. The paraxial mesoderm condenses into somites during the 4–5 weeks of embryonic development. The somite differentiates and becomes identifiable as the ventro-medial sclerotome, the dorso-lateral dermatome and the myotome. Component cells of these migrate from the somite into their respective final destinations to differentiate into tissue according to their signalling specification. Cells from the sclerotome migrate medially to form vertebrae and bones, dermatome cells migrate to form the connective tissue of the dermis, and myotome cells migrate to form skeletal muscle. Shh produced by the notochord and floor plate induces somite differentiation and seems to preferentially support the development of the sclerotome to the detriment of dermatome development (Fan and Tessier-Lavigne, 1994; Johnson et al., 1994). However, Shh can also regulate myosin expression during myotome formation (Sacks et al., 2003). In other words, the segmentation of the somite into the three regions itself is a process of determination of the developmental fate. Which signalling systems specify the developmental fate of cells of the dermatome and the myotome is uncertain at present. In the sclerotome, Shh signalling is said to be through the activation of Pax1 and Pax9 genes that are expressed in the developing sclerotome (Balling et al., 1996). The expression of the Pax1 and myotome-specific QmyoD genes differs markedly in the sclerotome and the myotome (Borycki et al., 1997). Furumoto et al. (1999) found that apart from Pax1, Foxc2 (also referred to as Mfh1) is expressed in the sclerotome and indeed is activated by Shh and associated with the proliferation of sclerotome cells. Some have argued that Foxc2 is not expressed in the dermatome but there is little evidence for this. However, it is possible that Foxc1 is inhibited by interaction with other proteins in the dermatome. The Pbx1 homeodomain protein is said to inactivate Foxc1 (Berry et al., 2005). We know that limb patterning might be dependent upon Pbx1 and 2, which in

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turn seem to be regulated by Shh (Capellini et al., 2006). These findings suggest that potential specification of the differentiation pathway could be by the activation of different target genes by Shh or other signalling ligands. Shh is also involved with antero–posterior patterning in the limb. Extraneous Shh expression by Shh-expressing implants leads to limb duplication (Riddle et al., 1993). Furthermore, Shh induces the expression of FGF-4 and BMP2 in regulating the growth and patterning processes in limb bud development (Laufer et al., 1994). The development of the skeletal system, with the formation of bone tissue, the development of long bones and fracture repair, involves the process called endochondral ossification in which cartilage is replaced by bone tissue. This developmental process involves a progressive differentiation of chondrocytes from a proliferative to prehypertrophic and then into a hypertrophic state. Ihh is expressed in the prehypertrophic chondrocytes and appears to be able to control hypertrophic differentiation (Vortkamp et al., 1996). Ihh regulates chondrocyte differentiation by regulating the production of PTHRP (parathyroid hormone-related protein), which is involved in endochondral bone development (Kronenberg et al., 1998; Lanske et al., 1996). Dhh is expressed in the testis. Its expression begins in precursors of Sertoli cells after the SRY (Sex-determining Region Y) gene is activated. SRY encodes a transcription factor that is responsible for male sex determination and so is closely implicated with spermatogenesis. Dhh expression then continues into adulthood (Bitgood et al., 1996). Bitgood and McMahon (1995) reported that both Shh and Ihh are associated with the development of the gut. They also drew attention to the possibility of Hh proteins functioning in conjunction with BMPs as shown earlier by Laufer et al. (1994).

Co-operative Signalling by Hh and Growth Factors TGF-β Family Ligands and Hh Signalling Hh ligands function in conjunction with many growth factors. So far, several TGF-β family members have been identified as being involved with Hh in development and pattern formation. The Nodal specifies left–right (LR) symmetry in vertebrates and has been shown to operate with Shh. In chick embryos a well-defined pattern of operation of ActRIIa, Shh and the mouse Nodal gene cNR-1 was described some years ago. Their expression was asymmetric during and after gastrulation. Furthermore, they appeared to regulate the expression of one another in a specified sequence. Experimental alteration of sidedness of expression of activin or Shh altered heart situs or location (Levin et al., 1995). The association of Hh with Nodal-related patterning of the central nervous system has been studied in some detail. The dorso-ventral patterning of the central nervous system is dependent upon both Nodal and Hedgehog signalling. Nodal signalling can induce the NK2 homeobox transcription factor genes nk2.1a in the ventral diencephalon, but the induction of nk2.1b in the ventral telencephalon requires Nodal as well as Hh signalling, with Nodal possibly acting upstream of Hh (Rohr et al., 2001). Mutation in the cyclops locus leads to defective ventral forebrain development and

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cyclopia. On the other hand, cognisance has to be taken of the view that Shh might not be as relevant as purported to be and that other determinants might be involved. Gutin et al. (2006) believe that FGFs might subserve an important function in the development of ventral telencephalon. This proposal is based on their finding that simultaneous deletion of Fgfr1 and Fgfr3 in the telencephalon leads to loss of differentiated ventromedial cells, and further that double mutants of FGFR1 and FGFR2 lead to the loss of ventral precursor cells, as observed with loss of Shh. Shh and Gli1 were still expressed in their experimental system, suggesting the phenotypic influence of FGF can occur independently of Shh. Rebagliati et al. (1998a) showed that the cyclops locus codes for Ndr2, which is related to Nodal. Nodal signalling regulates cyclops and one-eyed-pinhead (the EGFCFC co-factor discussed in pp. 8, 32–35) regulates Squint (Schier and Talbot, 2001). Rebagliati et al. (1998b) showed further that it functions with Shh and one-eyedpinhead, itself a regulator of cyclops in ventral midline patterning of the embryonic central nervous system. Nodal and Hh are both involved with individual roles as well as co-operative functions in the development and maintenance of the hypothalamus (Mathieu et al., 2002). One might briefly mention here that activin B and FGF2 have been found to inhibit Shh in presumptive embryonic pancreatic endoderm, and this regulation of Shh expression determines the expression of the homeobox PDx1 gene that characterises pancreatic differentiation (also see below).

FGF and Hh Signalling Shh was reported some time ago to function in an integrated manner with FGF4 in the development, growth and patterning of the limb. It was able to induce the expression of FGF4 in the ectoderm and BMP2 in the mesoderm. Shh signalling induced FGF4, which is required in mesoderm differentiation (Laufer et al., 1994). Activation of Shh signalling in the chick and the mouse brain has led to exaggerated growth within the avian fronto-nasal process but to growth impairment in the medial regions. The alterations in craniofacial morphogenesis were accompanied by the downregulation of FGF8 (Hu and Marcucio, 2009). The effects were not totally attributable to FGF downregulation because other signalling systems were also found to be downregulated. Organogenesis in the gastrointestinal tract occurs as a result of signalling interactions between the three germ layers. Epithelial and mesodermal interactions and intraepithelial signalling are essential in normal organogenesis associated with the gastrointestinal tract. Among the most important factors involved in this are Hh ligands, BMPs as well as Notch, Sox and Wnt (Barbara et al., 2003). Ihh is expressed in the gut and cartilage (Bitgood and McMahon, 1995). Ramalho-Santos et al. (2000) found that both Shh and Ihh are expressed in the gut endoderm with the expression of target genes in the mesenchyme. Both these Hh ligands are required for the normal organogenesis of the gastrointestinal tract, as indicated by intestinal malformations associated with mice carrying mutant Shh and Ihh. The islet cells of the pancreas, and pancreatic morphogenesis and growth, are regulated by Ihh expressed in pancreatic or adjacent tissue. Furthermore, defective Hh signalling is believed to produce congenital pancreatic malformations and glucose intolerance (Hebrok et al., 2000). Kim et al. (2000) reported

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that ActRIIA and ActRIIB, the receptors for activin and other TGF-β family growth factors, are essential for the normal development of posterior foregut derivatives, for example the stomach, pancreas and spleen. Earlier, Hebrok et al. (1998) had shown that Shh and Ihh were both subject to regulation by FGF2 (bFGF) and activin B from the notochord. They suggest that FGF2 and activin B inhibit Shh in the presumptive pancreatic endoderm. This allows the expression of Pdx1, a homeobox gene that plays an important part in the development of the pancreas and insulin expression. Shh inhibition using antibodies inhibits the expression of these genes. The embryonic patterning role played by RA (retinoic acid) seems to recruit both Hh and FGF signals. Investigating early mouse caudal patterning, Ribes et al. (2009) have shown that RA is required so that mesodermal and neural progenitors can respond to the Shh signal at late gastrulation. RA repressed Gli2 expression and equilibrated with FGF expression. On similar lines is their finding that the regulation of the proneural neurogenin-2 gene during early mouse spinal neurogenesis is closely linked with signalling by Shh and FGF. FGF2 upregulates the expression of bHLH (basic helix– loop–helix) containing neurogenin (Vergano-Vera et al., 2009). Neurogenins inhibit neuronal stem cells from differentiating into glial cells by sequestering transcription factors required for glial differentiation and promote neural differentiation by functioning as a transcriptional promoter (Sun et al., 2001). Neurogenins and NeuroD, another bHLH transcription factor that regulates neurogenesis, also influence cytoskeletal dynamics and migration of neurons (Seo et al., 2007). A 798-base-pair enhancer element (Neurog2-798) upstream of the Neurogenin-2 coding sequence directs the early caudal expression of neurogenin-2; this is targeted by RA, Shh and FGF. Furthermore, two RA response elements and a Gli-binding site occur in the Neurog2-798 enhancer element (Ribes et al., 2008). On the other hand, RA and FGF might counterbalance their functions by indirect means. FGF2, BMP2 and BMP4 have been found to bind to cellular RA-binding proteins (CRABP) or RARs. In 3T3 fibroblasts, FGF2 increased the level of CRABP1 RNA, whereas BMP2 and BMP4 reduced the level of CRABP-I RNA as well as that of CRABP-II and RAR-β mRNA (Means and Gudas, 1996). This is also obvious from the basic facts of mutual regulation of FGF and RA, with FGF inhibiting caudal differentiation, but RA participating in the rostral region for neuronal differentiation and in the establishment of ventral neural pattern. In this way FGF and RA might determine rostro-caudal axis patterning (Diez del Corral and Storey, 2004). Another mechanism involved in this process of co-regulation would be of RAR mediation of FGF-binding protein. RAR post-transcriptionally regulates the mRNA levels of FGF-binding protein (Boyle et al., 2000). One should also preserve the possibility in this situation that RA could exert differential effects on different stem cells. These observations lend considerable support to the view that the signalling systems directly interact and effectively regulate the flow of information relating to the spatial requirements of patterning.

13 Epidermal Growth Factors and Their Signalling Systems

Growth factors have a long and chequered history. They were discovered over a century ago. Erythropoietin enjoys the reputation of being the first growth factor to be discovered, in 1906. The identification of several important mediators of cell proliferation, apoptosis, and tissue-specific growth and differentiation began with the discovery of nerve growth factor (NGF) and epidermal growth factor (EGF). Many more were identified in later years. However, the elucidation of the subtle nuances of their cellular function has been a continuum of discoveries. Most importantly, the unravelling of their function has gone hand in hand with the discovery and function of their receptors. Thus, although the obvious format would have been to describe the characteristics and characterisation of growth factors first, what follows here is the simple rationale that receptor activation or transactivation and downstream signalling systems form such important an aspect of growth factor function that the receptor families and signalling systems have taken precedence over the growth factors themselves.

Growth Factor Signal Transduction Epidermal Growth Factor (EGF) Family Ligands and Receptors The EGF family is composed of growth factors of much relevance to cell proliferation, apoptosis, and differentiation and tumorigenesis. Several cognate ligands have been identified, among them are EGF itself, TGF-α, neuregulins (heregulins, HRGs), amphiregulin (AREG), epiregulin (EREG), HB-EGF (heparin-binding EGF-like growth factor) and β-cellulin (BTC) (Yarden and Sliwkowski, 2001). Most of these ligands are transmembrane proteins with an extracellular amino (N)-terminal region possessing the EGF module, a transmembrane domain and a carboxy (C)-terminal intracellular region. The extracellular region with the EGF module is released by proteolytic cleavage as the growth factor that can function in autocrine, paracrine or endocrine modes. These ligands between them activate the EGFR (erbB) family, namely EGFR (erbB1/HER1), HER2 (erbB2), erbB3 (HER3) and erbB4 (HER4) (Hynes et al., 2001). The EGFR family receptors are one-pass transmembrane proteins and share many structural features: for example, they possess an extracellular ligand-binding domain, a transmembrane region and a cytoplasmic tyrosine–kinase domain (Holbro et al., 2003). A fairly consistent pattern of EGF family ligand–receptor interaction has emerged over the years (Figure 13.1). These ligands activate EGFRs by inducing them to Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00013-X © 2011 Elsevier Inc. All rights reserved.

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Figure 13.1  EGF family ligands and the EGFR family receptors that they activate. EGF, TGF-α and AREG function through binding to EGFR. AREG is said to bind specifically to homodimers of EGFR. Recently it has been shown that the spectrum of specificity of the ligands to the receptors can be altered by inserting amino acid sequences that are compatible with the binding process. Other ligands can transactivate the receptors or recruit them as co-receptors. IL-6 signalling is often mediated by HER2. In the present work the epidermal growth factor receptor is referred to as EGFR, erbB2 either as erbB2 or HER2 on account of consistent use of the latter in the cancer field, and erbB3 and erbB4 as such. The last two are often referred to as HER3 and HER4, respectively.

dimerise. Many facts about receptor dimerisation and subsequent activation of its tyrosine kinase function have been well established. This tyrosine kinase activity leads to the autophosphorylation of the receptor itself. The C-terminal phosphorylation domain of the receptor has been ascribed with the regulation of protein kinase activity and further downstream to the phosphorylation of cytoplasmic target proteins and signal transduction. In the inactive state the extracellular domain appears to adopt an auto-inhibited configuration, which prevents the intramolecular interactions seen in the activated form. So the process of activation involves domain rearrangement that exposes the dimerisation interface (Ferguson et  al., 2003). In the rearranged configuration, EGF displays distinctly superior receptor binding compared with the autoinhibited monomeric receptor (Klein et al., 2004). The signals transmitted by the several ligands activate EGFR and bring about specific phenotypic changes. Furthermore, many of the signalling pathways also are shared. These features are a continuing conundrum (see Table 13.1 and Figures 13.1– 13.6). There are many potential answers. Different growth factors might be expressed in different levels, thus introducing a competitive element in activation. One can envisage differential affinity and specificity of the ligands to the receptor. Also the ligand expression might have a temporal dimension. Indeed the receptors might also be expressed in a definable temporal and spatial pattern. Membrane fluidity might have a bearing on receptor density and a consequent effect on activation occasioned by factors of proximity. Phenotypic perception of signalling might be an outcome of crosstalk or interaction between different signalling systems. Also, one cannot exclude the possibility of negative regulation of receptor function through interactive signalling.

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Table 13.1  EGFs and Their Biological and Signalling Attributes Growth factor

Biological function

Signalling attributes

EGF

Normal development, differentiation, cell proliferation, invasion

EGFR, ERK

TGF-α

Normal development and differentiation, proliferation, EGFR↑ angiogenesis

EGFR, MAPK, CD44, MMPs, GPCRs, NFκB

HRGs

Differentiation (neural), proliferation, apoptosis, growth regulation, cancer progression

ErbB2 (HER2)-erb B2-B3-B4; ERK1/ERK2/ PI3-K/Akt; STAT; EGFR

Amphiregulin

Mammary morphogenesis, influences cell proliferation, adhesion, motility, cancer progression

EGFR; ERK1/2 Elk1, cyclin D1; Wnt/β-catenin

Epiregulin

Mitogenic, upregulates TGF-α, AREG, HB-EGF

EGFR/erbB4; MAPK, PI3K/ Akt

HB-EGF

Normal and tumour tissue, expressed in ovarian, gastric, and breast cancer, melanoma and glioblastoma, invasion metastasis, wound healing

EGFR/erbB4

β-cellulin

Cell proliferation

EGFR, HER2/erbB3 when co-expressed, erbB4 PI3K/ Akt/GSK/FoxO/β-catenin; cyclin D1

The signalling pathways are delineated in detail in Figures 13.2–13.6. References are provided in the text.

The ligands display differential binding to the EGFR family receptors. EGF, TGFα, AREG, EREG and HB-EGF bind and activate EGFR. AREG is said to bind specifically to homodimers of EGFR. Other ligands can transactivate the receptors or recruit them as co-receptors. HER2 (erbB2), which is regarded and probably functions as a co-receptor, forms heterodimers with other members of the family. HER2 is believed to differ from other EGFRs in that it seems to lack the auto-inhibition mechanism in the extracellular domain. The constraints on conformational changes are probably imposed by inter-domain interaction and binding (Cho and Leahy, 2002). Structural studies have revealed that HER2 presents a static conformation of the dimerisation arm that resembles ligand-activated conformation constitutively open to ligand binding and dimerisation (Burgess et  al., 2003; Cho et  al., 2003). The specificty as well as transactivation ability of the ligands can be modified by insertion of the amino acid sequence YYDLL in their C-terminal linear region. Such an insertion in EREG extends its binding ability to include erbB3 together with an increase in its affinity to erbB4. Introduction of this sequence into neuregulin-1 greatly enhanced its affinity for erbB3 (van der Woning et al., 2009).

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Figure 13.2  Overview of signalling pathways that transduce signalling by cytokines, growth factors and G-protein-coupled receptor (GPCR) ligands. EGFR signalling pathways include the Ras/MAP-kinase, PI3K/PTEN/Akt and the Jak/Stat systems. The Src and Abl family tyrosine kinases are also able to phosphorylate EGFRs. Phosphorylation leads to the binding of proteins through the SH2 domains activating these downstream signalling pathways. Not all the components of these signalling systems are portrayed in the figure. The PLC-γ/PKC pathway is not shown here. Nor is the perceived involvement of NF-κB shown. According to Merkhofer et al. (2009), HER2 mediates the activation of the NF-κB pathway, which induces cell invasion but not proliferation, whereas HER2 can activate the PI3K/Akt pathway to stimulate cell proliferation. This differential functional activation of HER2 is discussed in some detail in a later section (pp. 160–184) and in the context of NF-κB and EGFR signalling (see Figure 13.7).

EGF Signalling Pathways Signalling by the receptors of the EGFR family uses several pathways of flow of information: the Ras/MAP-kinase, PI3K/PTEN/Akt, Jak/Stat and PLC-γ/PKC pathways. The binding of the ligands to the receptors leads to the formation of homoand heterodimer receptor complexes, to the activation of the receptor and the autophosphorylation of their intracellular domains, which in turn lead to the activation of several signalling systems. The phosphorylated tyrosine residues provide recognition and docking sites for the SH2 (src homology) and PTB (phosphotyrosine-binding) domain-containing proteins, which include the p85 regulatory subunit of PI3K, PLC-γ, src kinases, SH2 domain-containing tyrosine phosphatases, adaptor proteins of SHC family and GRB2, GRB7 and GRB10 (growth factor receptorbound proteins) and others, which can promote or attenuate the signal. Among the pathways activated are Ras/Raf/MEK and downstream MEKK/MAPK/MKK. Activation of the p70 S6 kinase cascade also takes place. These result eventually in the activation of genes related to cell proliferation. Activation of PI3K/Akt leads to anti-apoptotic and pro-survival signalling. Growth factor signalling can be compounded by EGFR-mediated phosphorylation and activation of FAK (focal adhesion kinase) EGFR family receptors. FAK can also be activated by integrin receptors

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Figure 13.3  A more detailed scheme of the pathways that transduce growth factor signals. The phosphorylated tyrosine residues provide recognition and docking sites for SH2 and PTB domain-containing proteins, which include the p85 regulatory subunit of PI3K, PLC-γ, src kinases, SH2 domain-containing tyrosine phosphatases, SHC family, GRB2, GRB7 and GRB10 (growth factor receptor-bound proteins) and others, which can promote or attenuate the signal. These are not shown in this figure, but see Figures 13.4–13.6. Activation of the ERK2 pathway (extracellular signal-regulated kinase) also promotes cell proliferation (see Figure 13.4). The Wnt signalling system is also shown here and its participation in cellular function is discussed elsewhere (pp. 150, 151) in the text. Hh and EGFR markedly influence each other. Signals from the ECM are channelled through integrin receptors and src/FAK to influence cell adhesion and proliferation. This could accentuate EGFR signalling.

Figure 13.4  Elements of the ERK signalling pathway. EGFR signals through the docking proteins complex SHC/GRB2/SOS (Son of Sevenless, a guanine nucleotide-exchange factor), which activates the membrane-bound GTPase Ras and downstream Raf/MEK/ERK to activate genetic transcription.

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Figure 13.5  Co-operative signalling between Hh, EGFR and Wnt. Hh is said to be able to activate EGFR. Reciprocally, EGFR can influence Gli-mediated transcription of target genes by activating the Raf/MEK/ERK pathway. GSK3β and PKA can also inhibit Gli. The reasoning is that in the presence of Smo, Gli phosphorylation is prevented. PKA phosphorylates Gli at several sites, followed by further phosphorylation of interspersed sites by GSK3β (Jia et al., 2002; Price et al., 2002). Hyper-phosphorylated Gli undergoes proteasomal degradation to form a repressor form. Consistent with this, PKA inhibits Hh signalling (Epstein et al., 1996; Wang et al., 2000). Growth factors can activate SMO by the mediation of PKA/Akt (Riobó et al., 2006), but the precise mechanism is not yet clear. In this figure, a putative link with Wnt signalling is indicated because GSK3β activation is also regulated by Wnt.

Figure 13.6  Amplified vision of G-protein-mediated signalling through the PLC/PKC and PKA pathways leading to genetic transcription and promotion of cell proliferation. GPCR also regulates cell survival or apoptosis through PI3K, with the mediation of genes such as apoptosis-inducing Bax and inhibitory Bcl2 genes.

upon their activation by binding of ECM ligands. Activated FAK signals operate downstream along the Ras/Raf/MEK pathway. The phenotypic effects of signalling are also attributable in a significant degree to the interaction of these pathways with other signalling systems such as the Wnt

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pathway and G-protein-mediated signalling. The endpoint of signalling is the activation of specific transcription factors. It is hardly surprising therefore that activation of some transcription factors is closely linked with EGFR signalling, for example NF-κB. These pathways are delineated in Figures 13.2–13.6. NF-κB is singled out here for discussion because it exemplifies a unique feature whereby two distinct phenotypic features are the outcome of activation of one species of EGFR by a growth factor ligand. The role of NF-κB in EGFR signalling has received much attention lately. NF-κB is a dimeric transcription factor possessing DNA-binding Rel regions and regions that bind the regulatory IκB kinase (IKK). NF-κB participates, with the regulatory IKK, in many biological processes such as cell proliferation, apoptosis and cell motility. EGF is an activator of NF-κB signalling. In ER-negative breast cancer cells it enhances NF-κB levels through EGFR, which then is inhibited by antibodies against EFGR. The activation of NF-κB results in the activation of cyclin D and an increase in Rb protein phosphorylation (Biswas et al., 2000). Interestingly, these authors demonstrate that PI3K might be involved in this phosphorylation process. One cannot glean much information about EGFR signalling on cell motility, whether EGFR signalling can be dissected out into proliferation and invasion directing pathways. This appears to take place in HER2-mediated signal transduction. According to Merkhofer et  al. (2010), HER2 also mediates the activation of NF-κB pathway which induces cell invasion but not proliferation, whereas it can activate the PI3K/Akt pathway and stimulate cell proliferation. HER2-mediated NF-κB signalling has no influence on cell proliferation. This seems to be due to the participation of another regulatory molecule, IKK (IκB kinase). IKK is made up of IKK-α and IKK-β. IKK-β activates NF-κB, whereas IKK-α inhibits it. Thus, when NF-κB is inhibited by IKK-α, cell motility is blocked but cell proliferation is unaffected (Merkhofer et al., 2009). This is compatible with the finding that inhibition of NF-κB signalling downregulates MMP-9 expression and inhibits cell motility in vitro (Huang et al., 2001). IKK-α is an inhibitor of cell proliferation (Descargues et al., 2008). So in the context of Merkhofer et al. (2009), IKK-α has no effect because HER2 uses the PI3K/Akt pathway. By the same line of reasoning, one might suggest that EGFR stimulation of NF-κB could lead to activation of PI3K/Akt and cell proliferation, as reported by Biswas et al. (2000), owing to the absence of IKK-α intervention in the signalling process (Figure 13.7).

EGF Signalling through CD44 The phenomenon of cell motility and cancer cell invasion is a complex process that involves ECM remodelling, modulation of expression and spatial organisation of ECM components that mediate intercellular adhesion, modulation of cytoskeletal dynamics, and cytoskeletal linkage to cadherin/catenin complex. CD44 glycoprotein is a major cell surface component intricately involved with cell motility and, by being associated with angiogenesis, with cancer metastasis. It is the major receptor for hyaluronic acid. Many CD44 isoforms resulting from alternative splicing have been described. CD44 isoforms subserve many functions including adhesion, growth factor activation, invasion and migration. The expression of these splice variants has

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Figure 13.7  Differentiation between EGFR and HER2 in terms of the phenotypic effects induced by the appropriate ligands. HER2-mediated signalling into induction of cell invasion or cell proliferation is depicted here. (This is based on references cited in the text.)

been associated with tumour progression, especially CD44v6, which has been attributed with the ability to confer metastatic ability on tumour cells (McDonnell et al., 2006). The ability of CD44 to induce cell motility was attributed to the redistribution, and clustering of CD44v6 receptors on the cell surface and aiding cell locomotion by facilitating intercellular and cell to substratum adhesion, which in turn is induced by metastasis promoting gene expression (Lakshmi et al., 1997). The molecular mechanisms involved in the perceived enhancement of metastatic ability by CD44 variants are unclear at present. EGF and CD44 are both known to enhance the invasive ability of certain human cancers such as the gliomas, which has afforded some movement in this direction. Monaghan et  al. (2000) found that EGF enhanced cell motility in vitro in hyaluronan-fortified Matrigel and upregulated CD44 messenger RNA (mRNA) expression in gliomas. EGF seems to achieve this by activating the MAPK pathway. It has been suggested that the enhancement of migration by EGF follows cleavage of CD44 by ADAM10. The release of the soluble ectodomain region of CD44 causes the cleavage and release of the intracellular domain of CD44. The latter translocates to the nucleus and activates the transcription of responsive genes (Okamoto et  al., 2002). ADAM10 does indeed cleave the ectodomain of CD44. Now EGF and other growth factors do upregulate the expression of the soluble forms of MMPs (Rooprai et al., 2000), and further some membrane type MMPs are also known to cleave CD44 and promote migration. However, Anderegg et al. (2009) believe that MMP14 does not mediate CD44 ectodomain release. CD44 is cleaved through Rac1 and MAPK activation (Murai et al., 2006). Gliomas expressed the standard form CD44s and EGF did not induce the expression of any splice variants (Murai et al., 2006). However, the generation of splice variants is stimulated by the Ras/Raf/MEK/MAPK pathway (Cheng and Sharp, 2006; Matter et al., 2002; Weg-Remers et  al., 2001), which is known to be activated by EGF. Besides, CD44v6-mediation of Ras signalling itself might require growth factor RTKs

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(Orian-Rousseau et  al., 2002). Overall, these reports suggest that EGF can conceivably modify the migratory behaviour of cells by having recourse to CD44 and the MAPK pathway; however, it would not be too conservative a comment that there has been no significant progress in the search for the mechanisms that might be involved in resolving the influence of CD44/EGF on cell migration. Perhaps one could venture the view that it is possible to differentiate between induction of cell motility and induction of proliferation as being actuated by markedly distinctive pathways. Signalling through the nuclear receptor superfamily composed of AR, ER, PR and retinoids also forms a major pathway that interacts with, modulates and is modulated by growth factor signalling. This aspect is discussed in detail elsewhere (chapters 17–21) in this book.

GPCRs in Growth Factor Signalling Another signalling system that can apparently function synergistically with growth factors involves GPCRs (Figure 13.6). G proteins are heterotrimeric proteins made up of three subunits, α, β and γ. G proteins participate in the transduction of signals imparted by growth factors, hormones and neurotransmitters by receptor binding. The G-protein family is divided into four subfamilies: Gs, Gi,o, Gq and G12. Gα12 and Gα13 belong to the G12 subfamily. These subfamilies have gained much prominence because these receptors are activated by many important ligands, among them are endothelin, TSH, lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and angiotensin. Of much relevance in the present context is the finding that G12 family proteins have been shown to activate tyrosine kinases, for example EGFR (Gohla et  al., 1998). Both Gα12 and Gα13 function by activating the JNK pathway (Collins et al., 1996; Prasad et al., 1995). Gα13 has been associated with angiogenesis because its deletion leads to abnormalities in the formation of blood vessels. Also, the GCPR agonists thrombin and angiotensin enhance VEGF expression (Richard et  al., 2000; Williams et  al., 1995). S1P released by platelets induces migration, proliferation of endothelial cells and brings about cytoskeletal changes in these cells. S1P binds to GPCRs of the endothelial differentiation gene (EDG) family (Lee MJ et  al., 1998, 1999; Lee OH et  al., 1999; Wang et al., 1999). S1P has been closely linked with angiogenesis. The signalling downstream involves PI3-K and Akt. S1P can also function through the Ca2/ calmodulin pathway. VEGF also activates eNOS through VEGFR-2, PI3K and Akt (Tanimoto et  al., 2002). S1P is also able to transactivate VEGFR and activate the PI3K/Akt signalling system (Figure 13.8). GPCRs have various functions in differentiated cells as well as in transducing mitogenic signalling through RTKs. They have been closely identified with cell proliferation and cell transformation and have often been labelled as oncoproteins. Ligands that activate GPCRs function in synergy with growth factors such as EGF and PDGF, among others, provide a powerful integration of GPCR with RTK in the regulation of cell proliferation. In breast cancer, oestrogens are also capable of transactivating EGFR and the MAPK pathway by means of GCPR30 (Filardo et al., 2002). This is clinically highly significant because a proportion of ER-negative breast cancers tend to be high expressers of EGFR.

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Figure 13.8  VEGF and S1P signalling pathways leading to angiogenesis, showing the transactivation of VEGFR mediated by SIP1R involving EDG/S1P2 and src.

GPCRs function with EGFR to promote cell proliferation and migration. The presence of ADAM (A Disintegrin And Metalloprotease domain)-containing protein relates to disease processes such as arthritis, inflammation, and tumour development and progression (see below). Of considerable interest here is that they can activate GPCRs and co-ordinate GPCR-mediated transactivation of EGFR. We know from the work of Schafer et  al. (2004) that ADAM-17 (TNF-α-converting enzyme with the acronym TACE) is required for transactivation of EGFR. They showed that inhibition of ADAM inhibits EGFR transactivation and cell proliferation. AREG-imposed cellular responses also involve the participation of ADAM-17 in pro-AREG cleavage and GPCR signalling. Inhibition of AREG by short interfering RNA (siRNA) or by AREG antibodies blocks EGFR activation, cell proliferation and migration together with the Akt-mediated cell survival signalling (Gschwind et  al., 2003). The importance of ADAMs in cell signalling is emphasised by their ability to cleave neuregulins, PDGFR, TNFR, MET (HGF receptor) and erbB4, among others (Mishra-Gorur et al., 2002).

The Wnt/β-catenin Pathway in Growth Factor Signalling The Wnt family genes feature prominently in a wide variety of biological functions including embryonic development, cell proliferation and apoptosis, differentiation and pattern formation, tissue repair and remodelling. They are also involved in cell transformation, tumorigenesis and cancer invasion. The faculty of promotion of cell motility and invasion flows from the ability of Wnt proteins to modulate cell adhesion. So it is easy to appreciate the magnitude of their involvement in cancer development and dissemination. The Wnt proteins are secreted cysteine-rich proteins that are structurally conserved and exhibit similar biological biochemical features (Logan and Nusse, 2004; Nusse and Varmus, 1992; Nusse, 2005; also reviewed by Sherbet and Lakshmi, 1997; Sherbet, 2008).

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The Wnt signalling system has been elucidated in much detail. The activation of Wnt signalling begins with the binding of Wnt proteins to the Frizzled family of protein receptors and co-receptors. The Wnt/Fizzled receptor complex activates the canonical β-catenin, and the non-canonical and calcium pathways. β-Catenin is localised in the membrane as a part of a complex with E-cadherin, APC protein and the actin cytoskeleton (Aberle et  al., 1994). The ligand/receptor complex releases β-catenin from the multi-protein complex. The glycogen synthase kinase-3β (GSK3) is an important participant in the signalling system. It phosphorylates β-catenin, which leads to its degradation. With the activation of Wnt signalling, GSK3 is inactivated and unphosphorylated β-catenin accumulates, enters the nucleus and activates the transcription factors (Barth et al., 1997) (Figures 13.3 and 13.5). In the canonical pathway, β-catenin forms a complex with the transcription factor TCF (T-cell factors) or the Lef (lymphoid enhancing factors), leading to the transcription of responsive genes. Upstream of Wnt signalling is the effector phosphoprotein Dishevelled, which transduces the signal into the three pathways (Harland and Gerhart, 1997; Logan and Nusse, 2004; Wodarz and Nusse, 1998). Among them are Ca2/calmodulin-dependent kinase II (CamKII) and PKC, PLC (phospholipase) and PDE (phosphodiesterase), which are activated by the recruitment of G-proteins, the JNK (Jun N-terminal kinase) in the non-canonical planar cell polarity pathway, and the canonical one with β-catenin. They are integrated to specify developmental patterns and cell fate. These pathways use calcium signalling systems as second messengers (Kestler and Kuhl, 2008; Kohn and Moon, 2005; Whitaker and Smith, 2008). The operation of Wnt signalling in human disease processes has received much attention. Regulation of cell proliferation is an essential element in differentiation as well as cancer. Ye et  al. (2007) noted that canonical Wnt signalling is repressed in senescent human cells. Aberrant Wnt signalling has been encountered in FAP (familial adenomatous polyposis) and colorectal cancer (Behrens and Lustig, 2004; Hlubek et al., 2007; Nathke, 2004; Stein et al., 2006), osteosarcomas and Ewing’s sarcoma (Hoang et al., 2004; Uren et al., 2004) (Figures 13.2 and 13.5). AREG has been identified as a potential target of the Wnt/β-catenin signalling pathway because the 5 region of its promoter possesses three TCF/LEF-binding sites, so raising the possibility that activation of Wnt signalling could upregulate AREG expression.

Hedgehog Signalling in EGFR Function A close and complex integration of several signalling pathways is a hallmark of the processes of differentiation and morphogenesis; they converge, cross-react, mutually regulate and collaborate with one another. TGF-β, BMP, Wnt, Notch, the FGF pathway and the EGF signalling system also overlap with the Hedgehog (Hh) signalling system, which is implicated in many facets of cell proliferation, embryonic development and pattern formation. In embryonic stem cells, Shh appears to be able to activate most of these pathways, possibly reflecting their pluripotency (Heo et al., 2007). The mediation of Hh in EGFR signalling was recognised and described a decade ago. Hh was shown to activate the expression of the Vn (Vein) gene that encodes a

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Drosophila homologue of EGFR ligand (Amin et  al., 1999). Activation of EGFR caused many morphogenetic abnormalities in limb development and patterning. Although the evidence is circumstantial, abnormalities in the expression of Shh and related signalling genes, BMP, FGF4 and other known participant genes were implicated (Omi et  al., 2005). Confirmation is also available that Shh indeed activates EGFR (Heo et al., 2007). Inhibition of Shh signalling by the steroidal alkaloid cyclopamine, which binds SMO, produced growth inhibition by apoptosis in cancer cell lines and inhibited cell proliferation (Sui et  al., 2004). Hu et  al. (2007) found that inhibition of Shh was accompanied by downregulation of EGFR in pancreatic carcinoma cells. Although it is generally accepted that cyclopamine inhibits the Hh pathway by altering the equilibrium between the active and inactive forms of SMO, whether there are other specific molecular targets yet to be identified is not known. Conversely, EGFR has been found to modulate the expression of Gli transcription factor functioning downstream of SMO activation (Figure 13.5) and furthermore that EGFR activates the RAF/MEK/ERK pathway to transcribe Gli target genes (Kasper et al., 2006).

Negative Regulation of EGFR Signalling There are two facets to the interaction of EGFR signalling with other systems of signal transduction. One is collaborative signalling in which the outcome of EGFR activation is accentuated by positive regulation by the cross-talk between the signalling systems. It has also been recognised that EGF and TGF-α can themselves upregulate the expression of EGFR. There also exist many checks and balances designed to maintain homeostasis and operate negative regulation of the flow of information. Several feedback inhibitors of EGFR signalling have been identified, among them are MIG6, LRIG1 (leucine-rich repeats and immunoglobulin-like domains protein 1), SOCS4 and SOCS5. The MIG6 (mitogen-induced gene 6 located on chromosome 1p36) gene product is ERRFI1 (erbB receptor feedback inhibitor 1). MIG6 is a 58-kilodalton (kDa) adaptor protein, which contains several domains that participate in protein–protein interaction; the N-terminal CRIB (Cdc42-Rac interactive binding) domain that can interact with Cdc42 and IκBα, a proline-rich motif that binds to SH3 domain containing proteins and a 14-3-3 protein interaction domain. An EGFRbinding domain occurs in the C-terminal AH (arfaptin homology) domain (Zhang, 2008). It is able to inhibit EGFR autophosphorylation and inhibit EGF-mediated activation of Ras, ERK, JNK, Akt/PKB and Rb protein (Xu et  al., 2005). MIG6 inhibits tyrosine kinase activity of EGFR (Anastasi et  al., 2007). It appears that EGFR is capable of inducing the MIG6, indicating a negative self-regulatory loop in the mode of EGFR signalling function (Zhang et al., 2007). Indeed, it has been viewed as a protective regulator of high levels of kinase activity of EGFR (Nagashima et  al., 2009) (Figure 13.9). Deletion of MIG6 results in inordinate activation of EGFR and activation of MAPK signalling leading to enhanced cell proliferation (Ferby et  al., 2006). More recently, MIG6 has been directly implicated in endometrial carcinogenesis. It has been shown to be a downstream target of progesterone receptor. MIG6 is required

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Figure 13.9  The negative self-regulatory loop involving MIG6. LRGI1 functions in a fashion similar to that of MIG6 but is not known to be regulated by activated EGFR. Progesterone receptor (PR) activates MIG6 and RTK inhibition. HGF is known to exert its effects through the MET receptors. HGF and MET have been reported to show mutual regulation and in this way regulate cell motility and proliferation. (This figure is based on references cited in the text.)

for progesterone function. Using a murine model, Jeong et  al. (2009) have demonstrated that in its absence progesterone cannot inhibit oestrogen-responsive genes and that mice developed endometrial hyperplasia. Furthermore, MIG6 was expressed at greatly reduced levels in endometrial carcinomas from women. MIG6 was among many genes that showed downregulated expression in carcinomas of the breast, lung and kidneys (Amatschek et al., 2004), and hence it is regarded as a cancer suppressor gene. It is worthy of note that MIG6 also inhibits HGF/MET receptor kinasemediated signalling, leading to cell motility and proliferation (Pante et al., 2005). Among other EGFR regulators is LRIG1, which also interacts with EGFR receptor tyrosine kinases and functions as a negative regulator of EGFR (Gur et al., 2004; Shattuck et al., 2007) by degrading the receptor by augmentated ubiquitination. Kiyokawa et  al. (1997) found that EGFR signalling was suppressed in the M-phase of the cell cycle and attributed this to a marked decrease in affinity of the ligand to the receptor and consequent loss of dimerisation of the receptor. This suppression was negated by overexpression of the receptor. Mattila et al. (2005) showed that the integrin-α1β1 interacts with and activates a tyrosine phosphatase and can lead to reduced EGFR activation. They also showed that introduction of the integrin into cells resulted in the inhibition of EGF-mediated stimulation of proliferation and anchorage-independent growth of tumour cells. These findings clearly emphasise the negative regulatory effects of other interactive signalling pathways.

Preferential Receptor Activation and Transactivation Many examples can be cited of the presence of negative regulation of EGFR function. This can occur either by preferential receptor activation or the intervention of

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other agents in normal receptor function. HRG seems to induce heterodimerisation of erbB2/erbB3 and promote cell proliferation. In certain systems HRG appears to activate erbB4. This could happen if erbB2 and/or erbB3 are suppressed. For instance, the activation of PKC signalling has been found to regulate erbB2 negatively and possibly in this way leads to inhibition of cell proliferation by HRG (Marte et al., 1995). AREG is another example of differential regulation of signalling. AREG has the ability to regulate cell growth positively or negatively (Plowman et al., 1990; Shoyab et al., 1989), in co-operation with other EGF family ligands. It is possible that negative regulation might result from the cross-talk of AREG with other cell signals by acting as a downstream effector of other biological response modifiers. This is exemplified by vitamin D3 (VD3, 1,25-dihydroxyvitamin D-3 cholecalciferol), an inhibitor of cell proliferation, which can induce AREG expression (Akutsu et al., 2001). Here AREG could be seen as negatively regulating cell proliferation.

Metastasis Suppressor Genes and Growth Factor Signalling Several molecular events occur during disease progression, which appear to be causally and temporally related. Genetic programmes related to cell proliferation and apoptosis, differentiation, adhesion and motility are often deregulated in cancer initiation, development and metastasis. Tumour progression can be viewed as occurring in distinct stages, and distinct genetic profiles can be identified with the different stages of dissemination and with the acquisition of phenotypic properties. Thus tumour initiation can be attributed to genetic damage, environmental and occupational factors, inherited susceptibility to cancer, and deregulation of differentiation. This is followed by deregulated cell proliferation and apoptosis, and formation of an in situ tumour. Acquisition of invasive behaviour represents a stage of induction of angiogenesis leading to the formation of metastatic tumour (Sherbet, 2006). Genes that regulate and control the changes that lead to metastasis all have the potential to inhibit progression to metastatic disease. It is little wonder, therefore, that numerous genes have been identified as metastasis suppressors; however, attempts are being made to redefine metastasis suppressors as genes that allow metastatic dissemination but suppress growth of the tumour at the metastatic site. Conceptually this requires the demonstration that these genes are expressed in a temporal affiliation with progression. The multiplicity of metastasis suppressors identified seems to support the argument that these are indeed linked with the many physical and phenotypic features associated with growth, invasion, ECM remodelling and alterations in adhesive property, diapedesis across the vascular endothelia and simply with cell proliferation. Nasopharyngeal carcinomas (NPCs) provide a good example, wherein the expression of many genes has been linked with different stages of the development and dissemination. The NGX6 gene is an inhibitor of intercellular adhesion, cell proliferation, invasion and metastasis, so is putatively a tumour suppressor. Its expression is reduced in many human cancers, for example NPC, and lung and colorectal cancer (Fan et al., 2008). An important feature is that its expression is low in the early stages of cancer and in cancers that have already metastasised. Also, it was reported that the degree

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of downregulation of the gene was far greater in patients with lymph node or distant dissemination than in those without metastatic disease (Zhang et al., 2003). In colon cancer, reduced expression was encountered in primary tumour tissue as well as in metastatic tumour in the lymph nodes (Peng et al., 2009). Indeed, together with LTF and Ezrin, NGX6 is regarded as an indicator of invasive behaviour. Peng et al. (2007) found ezrin expressed in high levels both in nasopharyngeal carcinoma tissues and in nasopharyngeal carcinoma 5–8F cells. Blocking of ezrin using siRNA reduced invasion of 5–8F in vitro. They also showed that NGX6 overexpression led to reduction in invasive behaviour. Of much significance is their further finding that NGX6 associated with the cytoplasmic region of ezrin and by this association NGX6 seemed to be able to downregulate ezrin expression and its effects on cell migration. In these early stages of tumour development, NGX6 seems to inhibit cell proliferation by inhibiting the Raf/MEK/ERK pathway (Wang et al., 2005). In colorectal cancers, NGX6 overexpressed and inhibited the activation and nuclear translocation of JNK1 but not ERK1/2. It also inhibited AP-1 (c-jun and c-fos) and Ets-1, markedly downregulated cyclin D1, and inhibited cell cycle progression. This would naturally have led to such inhibition (Peng et al., 2009). In HT-29, NGX6 downregulated the expression of cyclin E and cyclin D1, and upregulated the cdk inhibitor p27 (Wang et al., 2006). Cyclin D1 is a downstream effector of AP1/Jun-mediated signalling, which is consistent with AP-1/Jun/cyclin signalling reported by others. The lactoferrin (LTF) gene might be a potentially significant factor in the growth and invasive behaviour of cancer. It is downregulated in many cancer cell lines in vitro and has been associated with enhanced cell motility. This gene is lost in a deletion involving the chromosomal region containing the gene locus at 3p21.3, and its expression is downregulated, apparently by methylation, in several small cell lung cancer and NSCLC cell lines (Iijima et al., 2006). LTF is an iron-binding glycoprotein that has been attributed with several biological functions, especially immunomodulation and in the regulation of cell proliferation. Xiao et al. (2004) showed that LTF blocked cell cycle progression at the G0–G1 checkpoint, and involved regulatory control by Rb (retinoblastoma susceptibility) protein and suppression of cyclin E as well as inhibiting the anti-apoptosis Akt gene function. Son et al. (2006) have subsequently confirmed the increase of hypophosphorylated Rb in the inhibition by LTF of cell proliferation. LTF is also described as being capable of altering cell motility in vitro (Ishii et al., 2009; Vecchi et al., 2008). The possibility of interference by LTF in cytoskeletal dynamics has been raised by the findings of Close et al. (1997). However, the role of LTF in cell invasion is not fully established. NAG7 is another gene that is downregulated in NPC but probably not in gastric or colorectal cancer (Zhang et al., 2003). NAG7 was cloned and characterised to be a transmembrane protein containing a PKC phosphorylation site and a myristyle site (Xie et al., 2001). It influences the expression of many genes; it upregulates the expression of some and downregulates the expression of others (Tan et al., 2003). Its relevance in tumorigenesis can be highlighted by citing its inhibitory effect on ER (oestrogen receptor) (Huang et al., 2009). Oestrogens are mitogenic. NAG7-mediated inhibition of ER can lead to inhibition of cell proliferation. This is clearly another example of a suppressor gene functioning by interacting with another signalling system.

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The point to ponder over is that some of the suppressor functions are encountered in the early stages of tumorigenesis and at any rate before secondary dissemination. Although the activity of these genes in actual metastatic tumours has not been the subject of much study for logistical reasons, one would expect that their expression would be suppressed in metastatic tumours. These comments apply equally to the more extensively investigated metastasis suppressor nm23 (see below). The process of progression in colon cancer as well as in ovarian cancers is characterised by a temporal gene expression pattern that closely relates to proliferation, invasion and metastasis (Sherbet and Patil, 2006; see also Sherbet and Lakshmi (1997) for a review of colon cancer progression). It is essential that such a relationship is established before one can conceptualise the findings, however proven and appealing they might appear. As things stand, cell proliferation is the most convincingly demonstrated feature that is influenced by both metastasis suppressor and promoter genes; as shown in Figure 13.10, they might directly impart cell proliferation inhibitory or stimulatory signals, or equally they could affect other signalling pathways that can regulate cell proliferation.

Nm23 Interaction with Growth Factor Function Easily deserving the epithet as the foremost among metastasis suppressors are the nm23 genes. The nm23 genes are famously involved in metastasis suppression, counteracting metastasis promoter genes, and in the regulation of cell proliferation and cell motility. Although we have a wealth of information on the biological effects of nm23, the mechanism of action of Nm23 protein is not yet truly understood, let alone established. It was originally identified as an NDPK (nucleoside diphosphate kinase). It is yet unclear how NDPK action results in metastasis suppression.

Figure 13.10  How signalling by metastasis suppressor genes leads to the inhibition of cell proliferation. The figure also shows activation by EGFR of Ras-MEK-ERK to promote cell proliferation, which is inhibited by metastasis suppressor genes, thus counteracting the effects of EGFR activation. However, nm23 overexpression may not be invariably inhibitory. Also depicted here is NAG7 inhibition of ER and consequent inhibition of cell proliferation.

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Subsequently histidine kinase activity was attributed to it. This was thought to be responsible for the suppression of motility, postulated to be by serine phosphorylation of the kinase suppressor of Ras and consequent suppression of Ras-mediated signalling (Hartsough et  al., 2002; Wagner and Vu, 1995), but probably not under all experimental conditions. Bago et al. (2009) found that EGF-mediated activation of Ras signalling is unaffected by the overexpression of nm23-H1. Ma et al. (2004) have ascribed the nm23-H1 protein with a 3–5 exonuclease activity, which is implicated in monitoring the integrity of DNA repair and replication. However, its association with metastasis suppressor function is yet to be established. Despite the varied cellular functions subserved by nm23 proteins, the focus has been on metastasis suppression. This function is reasonably well established in many tumour types, but is unproven in some forms of cancer. This is probably because the significance of its function has been evaluated without reference to other suppressors and without consideration that nm23 effects might be counterbalanced by genetic activity that promotes tumour growth and metastatic spread (Sherbet, 2001). One can accept without much reservation that nm23 acts as a suppressor of metastasis. To comply with the postulate discussed earlier that suppressor genes would allow dissemination of the primary tumour to metastatic sites and then enforce dormancy on the metastatic tumour, it would be necessary to examine if there is a pattern of loss of expression of the gene in the early stages of the disease and emergence of expression upon dissemination. Nm23 shows some signs of stage-related methylation and hypomethylation. Enhancement of nm23-H1 expression using inhibitors of methylation is accompanied by decreases in cell motility in vitro in 5 out of 11 cell lines tested, but in these particular experiments cell proliferation is unaffected (Hartsough et al., 2001). Recently Bago et al. (2009) have reported that CAL 27 oral squamous cell carcinoma overexpressing the subtype nm23-H1 showed greater adhesion to substratum and reduced migratory and invasive potential. A similar correlation was found in MDA-MB-435 and MDA-MB-231 cells (McDermott et  al., 2008) and in nasopharyngeal carcinoma cells (Liu et al., 2008). In the last group of cells, experimental upregulation of nm23-H1 led to a decrease in cell motility. However, it would be far fetched from this even to suggest that downregulation of nm23-H1 expression might be associated with intracranial invasion of nasopharyngeal carcinoma, because the expression of several other genes relevant in the promotion of invasive behaviour was found to be altered in biopsy materials. Also, there is a consensus view that downregulation of expression of nm23 and the invasion suppressor gene E-cadherin correlates with invasion and the presence of nodal metastasis. This happens in colorectal cancer. Expression of both genes correlates with absence of metastatic spread, and a relationship subsists in early stages of the disease, so much so that the loss of nm23 and E-cadherin expression could be an early event in the progression of colorectal cancer, as reported some time ago (Garinis et al., 2003). This applies equally to other metastasis suppressors such as KiSS1 of melanomas (Lee et al., 1996, 1997; Shirasaki et al., 2001), which appears to be most effective in its ability to inhibit growth and invasion at the primary locus (Nash and Welch, 2006). The methylation status of KiSS1 is related to loss of expression. In bladder cancer, methylation was related to both tumour stage

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and grade, with increases in gene transcripts with increasing stage (Mendez et  al., 2009). BRMS1 (breast cancer metastasis suppressor 1) is silenced by methylation of its promoter; also, primary breast cancers as well as corresponding metastatic tumours showed BRMS1 methylation (Metge et al., 2008). The interaction of oestrogen/progesterone signalling with growth factor signalling has been discussed in the context of other growth factors: one may recall here that NAG7 (Huang et al., 2009) and BRMS1 seem potentially able to interact and exert their inhibitory effects on cell proliferation. BRMS1 was found to be highly expressed in PR breast cancers (Zhang et  al., 2006) and so possibly counteracts the effects of PR on proliferative signalling, in a fashion roughly analogous with NAG7-mediated inhibition of ER. The function of nm23 as a growth suppressor has not received the degree of attention it deserves. Several early observations indicated the involvement of nm23 in the regulation of cell proliferation. Its expression was inversely related to the metastasis promoter gene S100A4, which promoted cell proliferation by abrogating the cell cycle checkpoint control mechanisms (Cajone and Sherbet, 1999; Parker et al., 1994; Sherbet, 2001; Sherbet and Lakshmi, 2006). A direct mode of growth inhibition has been attributed wherein nm23 inhibits EGF- and Ras-induced cell proliferation through the Raf/MEK/ERK pathway (Lee et  al., 2009). However, as stated elsewhere, EGF-mediated activation of Ras signalling does not appear to be affected by overexpression of nm23-H1 in Cal 27 oral carcinoma cells. Zhang et  al. (2009) found that Verapamil downregulated EGFR and in parallel upregulated nm23 expression. However, modulation of intracellular Ca2 using Verapamil or Thapsigargin downregulated nm23 and upregulated S100A4 expression (Parker and Sherbet, 1992b). Furthermore, S100A4 but not nm23 expression correlated with the presence of EGFR (Parker et  al., 1992b; Sherbet et  al., 1995). These findings might provide some help in interpretating the findings of Zhang et al. (2009). It is not unlikely that the inhibition of EGFR and upregulation of nm23 might have links with the effects of Verapamil on the expression of S100A4, which, as pointed out, is itself expressed in an inverse relationship with nm23. The findings of Hartsough et al. (2001) that enhancing the expression of nm23 by inhibiting methylation affected only cell motility but cell proliferation may suggest interaction of nm23 with other signalling systems, for example, other growth factors or concurrent function of other suppressor genes that are known to affect growth factor or ER signalling (see below). In this context, it might be noteworthy that oestrogen appears to downregulate nm23-HI expression together with promotion of cell migration and invasion by activating the PIK3/AKT pathway. Progesterone exerts an opposing effect (Hua et al., 2008). One can see in this discussion the germ of the thought that nm23 might function by distinctly different pathways in generating its effects on cell proliferation, migration and invasion (Figure 13.11). Possibly this is just the beginning of the story; for complementary DNA (cDNA) microassay analyses have revealed many genes that could be implicated in direct signalling by nm23 or suggest signalling systems that could interact with nm23 signalling. Importantly, however, many of these genes relate to ECM components, adhesion mediating proteins and metalloproteinases, all of them involved in ECM restructuring and potentially capable of determining cell motility and invasion. It is not difficult to visualise the

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Figure 13.11  Postulated differential signalling by nm23 modulating, and being modulated by, EGF and oestrogen/progesterone in bringing about its effects on cell proliferation, migration and invasion.

induction and promotion of motility and invasion as a multistep process with the concerted operation of more than one signalling pathway. Equally, one has to add the rider that parallel changes in cohorts of genes, with nm23 being one of them, do not allow any firm conclusions to be drawn about interactive signalling. Notwithstanding these demonstrations of nm23-mediated effects compatible with its tumour/metastasis suppressor function, some ambiguity has been created by contrary views. Keim et  al. (1992) stated that high nm23 expression was found in advanced stage neuroblastomas of large size with metastatic spread compared with localised counterparts. The expression of nm23 also correlated with N-myc amplification. High expression of nm23 was also associated with enhanced cell proliferation. Mitotic stimulation of normal lymphocytes increased nm23 expression, and treatment of mitotically stimulated lymphocytes with cyclosporin prevented the upregulation of its expression. In human prostate cancer tissue, nm23-H1 immunostaining was intense in more than 70% of patients with stage D disease compared with 23.1% in stage B and 18.7% in stage C. Similarly intensive staining occurred in a vastly greater proportion of poorly differentiated compared with well-differentiated tumours. Furthermore, in prostate cancer cell lines, nm23-Hl mRNA levels declined in serum starvation and were restored with the addition of serum (Igawa et al., 1994). Although the mRNA expression in this study was unequivocal, some unease has to be expressed with the staining ‘intensity’ data. What the authors used was the proportion of cells in a tumour that stained intensely to grade the staining, not the measured intensity of staining. Cipollini et al. (1997) found that inhibition of nm23 using antisense oligonucleotides weakly inhibited cell proliferation; also transfection of vectors carrying antisense nm23-H1 mRNA similarly reduced proliferative rate. The course of differentiation of the trophoblast from the first trimester to second trimester and term placenta is accompanied by a decrease in nm23-H1 expression. In other words, nm23 expression decreased with higher differentiated, less proliferative state (Okamoto et  al., 2002). However, more recently, Jin et  al. (2009) who investigated

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two human CML cell lines reported that overexpression of nm23-H1 enhanced cell proliferation. Knockdown of the gene is reported to have led to G0–G1 arrest. Nonetheless one should reiterate strongly that nm23 does indeed inhibit cell proliferation, but the ambiguities lie in whether there is stage specificity in the inactivation and activation of nm23 expression.

Growth Factor Receptor Expression in Cancer A close link has emerged over the years between EGFR expression and cancer development and its progression to the metastatic state. There is general recognition that EGFR overexpression could facilitate closer proximity of the receptors, ease of dimerisation and activation. Increase in copies of the EGFR gene has been found in pancreatic cancer with enhanced EGF expression and heightened EGFR signalling (Tzeng et al., 2007). Maiden et al. (1988) reported the amplification of the cytoplasmic domain of the EGFR gene, but not the EGF-binding domain in a few gliomas. Amplification is not invariably accompanied by increased EGFR protein expression (Bredel et  al., 1999; Dacic et  al., 2006). EGFR family members are overexpressed in many epithelial tumours. Amplification has also been found of HER2 with amplification of EGFR in many epithelial tumours. Because EGFR itself is activated by TGF-α, the frequent expression of this ligand is greatly conducive to autocrine growth of tumour cell populations. HER2 is expressed in a significant proportion of breast cancers, and the occurrence of this receptor in a perpetually activated conformation markedly enhances its potential in the transduction of growth factor signals. The EGFRs are not only aberrantly expressed but also expressed differentially between normal and tumour tissues. To take a historical perspective, it may be recalled that EGFR is overexpressed in several human cancers, for example of the breast (Sainsbury et  al., 1985), bladder and lung (Berger et  al., 1987), brain (Libermann et al., 1985) and ovary (Gullick et al., 1986), among others. EGFR mutation also led to cell transformation in vitro (Khazaie et  al., 1988; Massoglia et  al., 1990). Prostate cancers show a higher expression of EGFR compared with benign epithelial tissues (Ching et al., 1993; Glynne-Jones et al., 1996). Most prostate cancer cell lines overexpress EGFR (Glynne-Jones et al., 1996; Liu et al., 1993; Zhau et al., 1996). Furthermore, overexpression of EGFR might be correlated with increased invasive and metastatic ability and poorer prognosis (Gullick, 1991; Modjtahedi and Dean, 1994; Zhau et al., 1996). The expression of erbB3 and erbB4 in breast cancer has been reported to correlate inversely with EGFR (Bieche et al., 2003). Also apparent is the notion of specificity of ligand–receptor interaction. Thus EGF, TGF-α and AREG bind EGFR but no others. However, β-cellulin reputedly binds all four receptors (Shin et al., 2003). Wang et al. (1998) showed that β-cellulin activated erbB4, but probably prominently both EGFR and erbB4. The ligands display different degrees of affinity to the receptors. Different ligands could bind and differentially phosphorylate the receptor (Riese et al., 1998). Much co-operative signalling can also be envisaged. Wang et al. (1998) demonstrated rather elegantly the co-operative functions of HER2 and erbB4. They constructed murine 3D transfectants carrying human erbB4 alone (3D-erbB4) or

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both HER2 and erbB4 (3D-HER2/erbB4) and tested them for proliferative response upon exposure to EGF family ligands. Neuregulin-1 and β-cellulin produced a strong proliferative response from 3D-erbB4. EGF and TGF-α showed only a slight response, HB-EGF no response at all. However, when HER2 was present with erbB4, the proliferative response evoked by EGF and TGF-α was greatly enhanced. These varied options could conceivably contribute to functional differences. It has been proposed that such perceived functional selectivity might arise from conformational changes in the ligand/receptor complex that could lead to variation in sites of tyrosine phosphorylation of the receptor and to interaction with downstream effectors (Wilson et  al., 2009); so the information might be conveyed by different signalling pathways. Although EGFRs transduce signals that determine important cell functions in normal and pathological states, there exist many checks and balances exerted by negative regulation of signalling which regulate information flows through pathways that interact with signalling by ligands of the EGF family.

EGF Family Receptors in Cancer Therapy Herceptin (Trastuzumab) in Breast Cancer Treatment The relationship subsisting in the EGFR family to cancer progression and prognosis has provided a most valuable route for targeted treatment and management of patients. Targeting growth factor function to control breast cancer development and secondary spread has rightly received much attention (Sherbet, 2009). There has been a sharp focus on HER2 receptors that are amplified in around 25% of breast cancers, which correlates with aggressive behaviour and poor prognosis (Natali et  al., 1990; Slamon et  al., 1987, 1989; Venter et  al., 1987). Monoclonal antibodies (Herceptin) raised against the external domain of HER2 have provided a successful way of treating metastatic breast cancers with high HER2 expression and gene amplification (Cobleigh et  al., 1999; Shepard et  al., 2008; Vogel et  al., 2002). A review of recurrence in women with low grade and node-negative, HER2-positive breast cancer has revealed higher risk of recurrence of the disease and poorer survival. These constitute a group of patients who are regarded as having good prognosis and who might benefit from adjuvant Herceptin treatment (Gonzalez-Angulo et al., 2009; Tovey et al., 2009). HER2 expression in breast cancer and cell lines derived from it has shown correlation with the expression of the angiogenesis-promoting protein angiopoietin-2 (Ang2), which seems to involve activation of Akt and ERK/MAPK signalling. Herceptin downregulated angiopoietin expression. Also, inhibition of Akt and ERK signalling inhibited angiopoietin upregulation (Niu and Carter, 2007). Other angiogenic growth factors such as FGFs and TGF-β are also inhibited with angiopoietins by Herceptin (Gong et al., 2010), and we know that many angiogenic signals as well as the EGFR family activate the PI3K/Akt pathway, leading to the upregulation of VEGF expression (Sherbet, 2003). The anti-angiogenic effects of Herceptin have been known for some time. Herceptin decreases microvessel density associated with tumours in vivo, and inhibits endothelial cell migration (Izumi et al., 2002; Klos et al., 2003). HER2 enhances the

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expression of VEGF and IL-8 in MCF-7 and T-47D breast cancer cells and decreases the expression of thrombospondin-1, suggestive of its being able to alter the outcome by modulating the balance between the pro- and anti-angiogenic factors (Wen et  al., 2006). Recently it has been reported that Herceptin inhibits tumour growth and reduces VEGF expression, thus effectively inhibiting microvascular density and vascular permeability (Le et  al., 2008). It also inhibits experimentally induced corneal neovascularisation (Guler et al., 2009). One should recall here the correlation of HER2 with VEGF-C expression reported for ovarian carcinoma (Hsieh et al., 2004). VEGF-C is a promoter of lymphangiogenesis and might be involved in lymphatic spread of the carcinoma. Indeed, overexpression of HER2 was associated in MCF-7 cells with an enhancement of VEGF-C, which could be reduced by Herceptin (Tsai et  al., 2003). Contrary views have also been expressed that Herceptin can increase angiogenesis and vascular density (Hardee et  al., 2009). However, it would be premature to suggest that the clinical benefits of inhibiting HER2 function might be less persuasive in early-stage tumours than in advanced disease. The remarkable success achieved with Herceptin in patients with metastatic and early breast cancer has led to the exploration modes of potentiating the efficacy of HER2 inhibitors. Combination of Herceptin with chemotherapy has yielded considerable benefits in terms of reduction of recurrence and mortality. So HER2 expression has come into the reckoning for adjuvant chemotherapy. Combining Herceptin either with an anthracycline plus cyclophosphamide or with Paclitaxel, as first-line therapy for metastatic breast cancer overexpressing the HER2 receptor, has provided significant benefits in terms of objective response, duration of response and survival compared with chemotherapy alone. Furthermore, the benefits were related to the degree of HER2 overexpression (McKeage and Perry, 2002). The combination approach has been so successful that the efficacy of conjugates of anti-HER2 antibodies with cytotoxic drugs has been tested to achieve targeted delivery of the cytotoxic agents. Not all HER2-positive cancers respond to Herceptin, and many patients who initially respond to Herceptin develop resistance to it and show disease progression. Combination of Lapatinib (see below) has been found to be beneficial in treating tumours resistant to Herceptin (Collins et  al., 2009; Roy and Perez, 2009). On the other hand, many patients with tumours overexpressing HER2 do not respond to Herceptin.

Molecular Mechanisms of Herceptin Resistance Among the approximately 30% of patients overexpressing HER2 who respond to treatment, disease progression is encountered within a year of starting it. So the overall success of treatment in the long term requires an elucidation of the potential mechanisms involved in the development of resistance. A basic tenet of this treatment is the rationale that HER2 is a mediator of growth factor signalling and its blockade by HER2 antibodies would suppress proliferative signalling. As noted earlier, growth factors activate several signalling pathways. So the argument goes that possibly the signalling systems operating in these events might provide clues to Herceptin resistance. It might be noted here that HER2induced angiogenesis activates the Akt signalling system. Many angiogenic signals

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as well as the EGFR family activate PI3K/Akt (Sherbet, 2003). In fact, the Akt pathway is constitutively active when EGFR, ErbB2 and ErbB3 are overexpressed (Longva et al., 2005). Therefore, here one has to consider the possibility that other EGFRs such as erbB3 (HER3) receptor might be involved in proliferative signalling. It has been recognised that HER2 also shows heterologous dimerisation with erbB3. The suppressor gene PTEN regulates the pro-survival function of Akt. Abnormalities of PTEN could conceivably therefore influence the effects of Herceptin treatment. The evidence presented by Berns et al. (2007) for this is quite compelling. Mutations occur in the region coding for the catalytic subunit of PI3K (PIK3CA gene) or the gene is amplified in several human neoplasms, including breast cancer. These are gain-of-function mutations occurring in exons 9 and 20, and mainly involve the substitution of one of the three amino acids in the helical or kinase domains with the acquisition of enzymatic and signalling activity (Zhao and Vogt, 2008). Lai et  al. (2008) assessed 152 breast cancer patients, of whom 26% showed mutations; most mutations were in exon 20. They also stated that mutations in exon 20 correlated with poorer prognosis compared with those without this mutation. Lerma et  al. (2008) concurred that exon 20 mutations in HER2-positive breast cancer were more frequent than those in exon 9, but found no relationship with prognosis. Low PTEN expression and PI3KCA mutations conferred Herceptin resistance in breast cancer. Combination of these two events caused greater resistance than PTEN alone, emphasising the importance of monitoring PI3KCA mutations in assessing suitability of patients for Herceptin treatment. This is compatible with the general conclusion that Herceptin does indeed activate PTEN and inhibit PI3K/Akt signalling. Other molecular entities that can interact with the EGFR system could similarly affect Herceptin-mediated suppression of HER2 signalling. MUC-1 has been postulated to counteract Herceptin effects in this way. MUC-1 is known to participate in cell signalling. It is overexpressed in many tumours. The C-terminal domain of its β-subunit is involved in cell signalling by many pathways including ERK, Src, NF-κB and Ras/MAPK. MUC-1 interacts with the SH2 domain of GRB2 of the Ras/GRB2/ SOS signalling complex. Of much interest in the present context is that MUC-1 can bind EGFR, HER2, erbB3 and erbB4, all members of the EGFR family. Upon ligand binding, the receptor is ubiquitinated and degraded. It has been shown that MUC-1 binding protects EGFR from EGF-induced ubiquitination and degradation. In other words, it is essentially constitutively functional and effectively promotes EGFRdependent signalling (Pochampalli et  al., 2007). In a similar fashion, the MUC-1 C-terminal domain of the β-subunit seems to bind, stabilise and activate ER-α and so hold up ER-α-mediated promotion of tumour growth. It has emerged recently that MUC-1 antagonists can negate resistance to Herceptin and that Herceptin-resistant cells can be transformed into a Herceptin sensitive-state by MUC-1 treatment (Fessler et al., 2009). A direct effect of MUC-1 on cell cycle regulation has been adduced by the demonstration that it binds to the PE21 element of the p53 cell cycle regulator gene and can inhibit its regulatory function. The promoter region 96 to 41 of this gene harbours NF-κB, c-myc binding sites and the PE21 element. Growth inhibitors such as oncostatin seem to inhibit p53 transcription by binding to this element (Li et al.,

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2001). Cyclin-dependent kinase (cdk) inhibitors, p21(waf1/cip1), p27(Kip1) and p57(Kip2), are regarded as tumour suppressors. Kip1 and Kip2 regulate the entry of cells into the S-phase by influencing the phosphorylation of Rb protein by cdk inhibition (Lakshmi and Sherbet, 1997). A decrease in Kip1 has been encountered in Herceptin-resistant cells, and cdk2 activity increased. Resistant cells became Herceptin sensitive upon addition of exogenous Kip1 (Nahta et al., 2004). Although these effects are quite persuasive per se, here again the involvement of the PTEN/ Akt signalling is a distinct possibility. PTEN expression is associated with increased expression of Kip1, decreased expression of cyclins A and D3, and inhibition of cdk2 activity. In this setting the growth inhibition seems to result from the dephosphorylation of pRb and without p53 intervention. Furthermore, inhibition of Kip1 leads to the negation of growth arrest induced by PTEN (Gottschalk et al., 2001). The association of Herceptin with inhibition of cell proliferation comes from the involvement of NF-κB in its action. NF-κB occurs in an activated state in many forms of human cancer. Its activation has been associated with the overexpression of many growth factors and their receptors, for example EGF (Biswas et al., 2000), HER2 (Pianetti et  al., 2001), HGF (Fan et  al., 2005) and IL-1 (Wolf et  al., 2001). Both EGFR- and HER2-mediated signalling uses the PI3K pathway (Romieu-Mourez et al., 2001). HER2 induces NF-κB activation with the regulatory function of IκBα, which is inhibited by the suppressor gene PTEN implicating Akt activity. Indeed Herceptin has been found to activate PTEN and inhibit PI3k/Akt signalling. In the experimental setting, enforced overexpression of HER2 in MCF-7 cells results in increased activation of NF-κB. Herceptin is able to inhibit this (Guo et al., 2004). There might exist other indirect means by which Herceptin resistance could occur. Kang et al. (2008) used HER2-positive SK-BR-3 cells and the derivative Herceptinresistant SK-BR-3 (SK-BR-3 HR) cells. They found that suppression of HSP27 expression in the resistant SK-BR-3 HR cells using HSP27-specific siRNA rendered them sensitive to Herceptin. From co-immunoprecipitation experiments they suggest the possibility that HSP27 might bind to and stabilise HER2. However, it ought to be recognised that HSP27 inhibits cell proliferation by a variety of means, not least by inhibiting the function of tumour promoter genes (Albertazzi et al., 1998).

EGFR Inhibitor Lapatinib (Tykerb) in Breast Cancer Treatment EGFR is overexpressed in many forms of cancer. It is overexpressed also in a proportion of breast cancers that are ER-negative. Furthermore, EGFR expression correlates with the expression of metastasis-promoting genes. It has been known to function in conjunction with HER2. Therefore, the considerable potential clinical benefit of EGFR inhibitors for breast cancer treatment has been widely recognised. The strategy that has been evolved is to develop TRK inhibitors with marked pharmacological potential. Lapatinib is one such inhibitor; indeed it is a powerful dual inhibitor of EGFR and HER2. Lapatinib (a 4-anilinoquinazoline derivative) inhibits growth factor signalling by binding to the ATP-binding pocket of both EGFR and HER2 receptor proteins, so preventing autophosphorylation of the receptors and blocking the signalling

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cascade resulting in the suppression of growth of tumours and advanced or metastatic breast cancers that are resistant to Herceptin (Johnston and Leary, 2006; Nelson and Dolder, 2006). Lapatinib is clinically very effective, especially in advanced or metastatic breast cancer and in patients with brain metastases. Clinical trials combining Lapatinib with inhibitors of DNA synthesis such as Capecitabine have shown much promise in terms of disease progression (Bilancia et  al., 2007). Also promising has been its combination with Paclitaxel in the management of HER2-positive patients (see Di Leo et  al., 2008). The effectiveness of Lapatinib on EGFR overexpressing colorectal cancer and squamous cell carcinoma of the head and neck is not remarkable (Montemurro et al., 2007). So the line of thinking is that Lapatinib might be beneficial only to patients with HER2-overexpressing cancers (Press et al., 2008).

Treatment of Triple-negative Breast Cancer A major challenge is the treatment of patients with triple-negative breast cancer (TNBC) who are ER-/PR-/HER2- and so cannot respond to tamoxifen, aromatase inhibitors or to Herceptin. Indeed, more than 10% of breast cancers are triple negative. TNBC is highly aggressive, showing greater risk of recurrence and poor survival. TNBC patients carry an increased risk of metastatic disease and poor prognosis, possibly in the initial five years after diagnosis (Dent et al., 2007). A study in Nottingham involving 1944 invasive breast cancers showed that 16% were TNBC and that this phenotype was associated with recurrence, distant metastases and poor clinical outcome (Rakha et al., 2007). Several treatment options for TNBC have been clinically tested. These have included targeting of DNA homologous recombination repair, PARP inhibition, inhibition of EGFR and VEGFR functioning using antibodies and small RTK inhibitors alluded to earlier, and inhibitors of mTOR and src kinase signalling.

DNA Homologous Recombination Repair in Targeted Therapy Breast cancer subtypes representing the luminal and basal types of epithelial cells are identifiable. The basal-like subtype is mainly a result of mutations in the breast-cancersusceptibility genes BRCA1 and BRCA2, and often tend to display the TCNB phenotype (Hu et al., 2006; Nielsen et al., 2004; Perou et al., 2000; Sorlie et al., 2001). Among other novel areas under scrutiny is the base excision repair of DNA by poly(ADP)-ribose polymerase-1 (PARP-1), especially in TNBC not expressing the tumour suppressor gene BRCA. A link has been shown between BRCA loss-offunction mutation and the TNBC by the demonstration that most of BRCA1-mutated tumours tend to be triple negative and hence the therapeutic approach involving BRCA. BRCA proteins are closely associated with the double-strand break (dsb) repair by homologous recombination of DNA and possibly also non-homologous end-joining and nucleotide excision repair (Hartman and Ford 2002; Zhang and Powell, 2005). DNA repair defects make BRCA1-mutant cells sensitive to DNA cross-linking agents as well as to PARP inhibitors (Bryant et al., 2005; Farmer et al., 2005). Tumour cell lines with mutated BRCA1 and BRCA2 seemed susceptible to

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PARP-1 inhibitors. Many PARP inhibitors have been developed and found to be effective in cancers carrying BRCA mutation and TNBC. The possible sensitivity of TNBC to cisplatin in neoadjuvant settings has been recognised. Response to cisplatin was associated with low BRCA1 transcription, and promoter methylation (Siler et  al., 2010). Koshy et  al. (2010) reported that patients having TNBC with metastatic disease seemed to benefit in terms of reduced risk of disease progression from a combination of cisplatin with gemcitabine. The association of BRCA1 mutation appeared to render TNBC more sensitive to the combination. Winer and Iglehart (2009) have authoritatively reviewed the outcome of two PARP inhibitors, Iniparib (BSI-201) (BiPar Pharmaceuticals) and olaparib (AZD2281) (Astra Zeneca), in phase II clinical trials, which they described as promising. This was a randomised trial of carboplatin/gemcitabine with or without Iniparib (O’Shaughnessy et al., 2009), with the overall response rate being 16% in the group of patients treated with chemotherapy alone and 48% among the group who received chemotherapy plus Iniparib. Also evident was an improvement in progression-free and overall survival. Winer and Iglehart (2009) attribute the increased progressionfree overall response to the inhibition by Iniparib of DNA damage inflicted by carboplatin and gemcitabine. The outcome of trials involving patients with the loss of the wild-type allele of BRCA1 and BRCA2 and residual non-functional proteins has not been totally convincing. Iniparib on its own or in combination with gemcitabine and carboplatin has provided encouraging results in a phase I trial involving TNBC, with progression-free and overall survival. Clinical trials are also afoot with ovarian and uterine cancer, NSCLC and glioblastoma (Liang and Tan, 2010). In a phase 1 study, Fong et  al. (2009) and De Bono et  al. (2007) found olaparib to be active in individuals with advanced solid tumours carrying mutated BRCA and in patients with advanced ovarian cancer BRCA mutations. A further study by Tutt et al. (2009) has suggested a higher dose of olaparib as being more effective. However, it is difficult to draw firm conclusions from this non-randomised effort involving a few patients.

EGFR-mediated Targeting of TNBC The molecular patterns of expression of signalling components have revealed a few leads that can be pursued to develop modes of targeted therapy for TNBC. Several new approaches can be identified. A natural broadening of TNBC sensitivity would be the inclusion of the EGFR dimension. The potential value of EGFR in the treatment of TNBC has been recognised, and monoclonal anti-EGFR antibodies and TRK inhibitors have been in clinical use, although the clinical outcome is probably far from satisfactory. As often stated in this book, we showed many years ago that a proportion of ER-negative tumours have tended to be EGFR. Rakha et  al. (2007) reported that the TNBC phenotype correlated with EGFR expression. Many also carry p53 suppressor gene mutations (Farmer et al., 2005). Corkery et al. (2009) found overexpression of EGFR in TNBC compared with HER2  cell lines. The receptors were activated by exposure to EGF and phosphorylation was inhibited by gefitinib, which seemed to inhibit MAPK and Akt signalling leading to G1 arrest. Of much interest is the finding from this study that gefitinib enhanced the response of

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TNBC cells to both carboplatin and docetaxel, and that these three agents seemed to function synergistically. Indeed, more recently, high-grade, node-positive and ER breast cancers have been reported to express EGFR. Furthermore, phosphorylation of EGFR was inversely related to the presence of ER. However, no correlations were noticed with HER2 expression (Koletsa et al., 2010).

AR Expression in TNBC AR expression is low (10–35%) in TNBC, whereas HER2 tumours show a much higher incidence of AR (Gucalp and Traina, 2010; Niemeier et al., 2010; Park et al., 2010). AR was expressed in most (95%) of ER  tumours. Only 10% of TNBCs expressed ER, but 63% (5 out of 8) ER/HER2  expressed AR. Park et al. (2010) have found AR expression in 35% of TNBCs. Another study has found AR  breast cancers to be ER/PR. Thus a clear and significant correlation exists between hormonal receptor and HER2 and AR. These findings suggest possible interacting influences of other growth factors. Indeed AR interacts with growth factor signalling by EGF and HER2. HER2 activates AR signalling and promotes prostate cancer growth independently of androgen both in vitro and in vivo (Craft et al., 1999). It induces PSA through MAPK signalling mediated by AR (Yeh et al., 1999). Thus, although there is evidence for transactivation of AR, precisely how growth factors might affect AR expression is unclear. Nonetheless, the therapeutic potential of deploying AR deserves much thought.

Src Kinase Inhibitors in TNBC Src is a tyrosine kinase that is involved in the regulation of cell proliferation, angiogenesis, intercellular adhesion, invasion, motility, survival and metastasis (Hiscox et al., 2006, 2007; Rucci et al., 2006; Thomas and Brugge, 1997). The expression of src kinases often correlates with cell proliferation and invasion because src/Abl tyrosine kinases are able to phosphorylate EGFRs. Also, growth factors alter ECM characteristics by regulating the expression of uPA and MMPs, which possibly aids cell locomotion. EGF/EGFR and HER2 activate NF-κB signalling. Inhibition of NF-κB signalling has been found to downregulate MMP-9 expression and inhibit cell motility in vitro (Huang et al., 2001; Merkhofer et al., 2009). Signals from the ECM may be channelled through integrin receptors and src/FAK, influence cell adhesion, and proliferation could accentuate EGFR signalling. The src inhibitors dasatinib, bosutinib and saracatinib are currently undergoing clinical trials. Lyn tyrosine kinase of the src family was expressed in TNBC, which correlated with reduced overall survival. Interference-RNA-mediated knockdown of Lyn in breast cancer cell lines with EMT led to the inhibition of cell migration and invasion, but not cell proliferation. Dasatanib exerted similar effects on these cell lines (Choi et  al., 2010). Pichot et  al. (2009) found that dasatanib varied in its effects on breast cancer cell lines despite comparable levels of inhibition of src kinase. MDA-MB-231, which is triple negative, showed the highest sensitivity to dasatanib, markedly inhibiting cell migration and in vitro invasion, and inducing

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G1 arrest. The anthracycline antibiotic doxorubicin seemed to function synergistically with dasatanib. Equally, doxorubicin could be providing an additive effect by its inhibition of DNA replication. There is now evidence that dasatanib might exert its effects through EGFR signalling. Nautiyal et  al. (2009) found that it reduced EGFR phosphorylation and downstream Akt and ERK signalling. Src inhibitors do inhibit ERK signalling. EGFR activation leading to ERK signalling can inhibit the TGF-β pathway and inhibit the induction of EMT and invasive behaviour. Dasatinib has been found to inhibit STAT5 signalling and downregulate expression of STAT5 target genes, including Bcl-x, Mcl-1 and cyclin D1, and thus induce apoptosis and inhibit cell proliferation (Nam et al., 2007). In other words, the therapeutic effects of dasatinib and related compounds might be achieved through more than one signalling system.

Targeting of Microtubules Microtubules have been viewed as a therapeutic target. Many antimicrotubule agents, for example taxanes, have been found to be effective in the treatment of cancer. Nontaxanes such as the epothilones have been shown to possess cytotoxic properties; these have been attributed to their ability to stabilise microtubules leading to the inhibition of G2M transition in the cell cycle traverse. Perez et al. (2010) studied the effects of ixabepilone (Aza-epothilone B), an analogue of epothilone B, in TNBC and noticed a complete response in 26% of patients with TNBC compared with 15% of those without it. When combined with capecitabine in the phase II study, ixabepilone gave an overall response of 23% in triple-negative patients. A similar response (31%) was obtained in the phase III trials. Overall, the combination therapy resulted in improvements in median progression-free survival for patients with TNBC treated with ixabepilone plus capecitabine compared with those treated with capecitabine alone.

Inhibitors of mTOR in TNBC The mTOR (mammalian target of rapamycin) signalling inhibitors might offer potential new devices for TNBC management. TNBC is said to resist Rapamycin. However, in animal tumour models, a combination of Rapamycin with cyclophosphamide seemed to reduce the growth of tumours from MDA-MB-231 breast cancer cells (Zeng et al., 2010). Lee et al. (2008) studied the transcription factor YB-1, which is highly expressed in breast cancer. YB-1 might induce tumour growth by its ability to induce the dimerisation of HER2 with EGFR and thus activate signalling. It has another function of promoting cell survival through mTOR/Akt mediation. Inhibition of YB-1 effectively blocked the proliferation of breast cancer cell lines in which HER2 was overexpressed and also in cell lines that were triple negative. Yb-1 inhibition induced apoptosis in cell lines with the amplification. It also led to a decrease in mTOR with decreased phosphorylation of STAT3S727, ERK1/2T202/ Y204 and mTOR-S2448 (phosphorylated at serine 2448). Fujii et  al. (2009) have confirmed these findings. The mTOR signalling pathway involves the effector serine–threonine kinase S6K1 (40S ribosomal S6 kinase 1), which is activated by Akt (Asnaghi et al., 2004).

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Its importance in cancer has been highlighted by S6K1 overexpression in breast cancer and its association with poor prognosis. Because S6K1 functions downstream of Akt, obviously mTOR would mediate cell proliferation and cell survival. Notably mTOR integrates ER, EGFR and IGFR signalling and is implicated in angiogenesis. The operation of mTOR can be considered with its involvement in the angiogenic process. Everolimus and gefitinib seem to inhibit EGFR-related signalling of growth and VEGF production (Bianco et  al., 2008). The flavanoid silibinin, which inhibits tumour growth, is known to downregulate iNOS. It inhibited hypoxia-induced HIF-1α and VEGF production. The angiogenic and lymphangiogenic effects exerted by FGF-2 have been attributed to mTOR activation (Matsuo et al., 2007). These events correlated with the inactivation of mTOR signalling (Garcia-Maceira and Mateo, 2009). It would seem, therefore, that there are solid grounds on which to conclude that mTOR inhibitors might be a viable approach for combination therapy.

GPCRs in TNBC The cross-talk that takes place between GPCRs, EGFR and other signalling system has been discussed at length in an earlier section (pp. 146, 149, 150). This occurs in many human neoplasms. Furthermore, under continual stimulation, besides the conventional route, GPCR signalling can lead to the activation of GPCR kinase, leading in turn to the engagement of other proteins resulting in activation of alternative pathways. Cross-talk with EGFR provides new avenues of approach for treating TNBC. Oestrogen-mediated induction of cell proliferation can occur independently of the canonical form of ER signalling. The identification of mER (membrane-bound ER) led to two postulated optional signalling modes: (1) that mER is the relocated nuclear ER (ER-α and ER-β); and (2) that GPCR30 is indeed the mER. This second mode, which involves the activation of GPR30, the so-called orphan GPCR, has been called the non-genomic pathway (Couse and Korach, 1999; Filardo et al., 2000; Mueller and Korach, 2001). Oestrogens generate a variety of effects in some systems with the activation of GPCRs. So it would not be totally tenable to argue that these effects are mediated by conventional ER signalling in all settings because these systems might often express GPR30 (Rae and Johnson, 2005). GPCR ligands activate the RhoA/ CCN1/integrin cascade to influence cell migration and proliferation (Walsh et  al., 2008). Identification of GPCR30 as mER accentuates the importance of looking at GPCR30 status in TNBC. As alluded to before, not infrequently ER-negative breast cancers express EGFR; in this context it is relevant to know that oestrogen can transactivate EGFR through GPCR30. It is critical therefore to establish the expression status of GPCR30 in TNBC.

Cell Cycle Checkpoint Inhibitors The maintenance of genomic integrity requires that cells are prevented from entering into mitotic or meiotic division when DNA replication is incomplete. DNA replication checkpoints control cell cycle progression. Three DNA damage surveillance checkpoints have been identified and defined. These are the G1–S transition checkpoint, which restrains cells with damaged DNA from entering the S-phase. The second

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checkpoint, namely the S-phase checkpoint, monitors the progress of cells through the S-phase and regulates the rate of DNA synthesis. The third checkpoint monitors the G2M boundary (see Sherbet, 2003). DNA damage response involves many signalling pathways that regulate cell cycle checkpoints, DNA repair and apoptosis programmes; these help to maintain genomic integrity. The checkpoint kinases Chk1 and Chk2 function downstream in DNA damage response. They are important participants in the network that maintain genomic integrity. Chk2 has an essential role in p53-dependent apoptosis. Indeed, checkpoint kinases have been regarded as tumour suppressor genes. These modes of genetic defence would be counterproductive to cancer management by chemo- and radiotherapy. Chk1 inhibition is expected to lead to chemo-sensitisation of tumours. Hence designing agents to target Chk1 among others, which contributes to cycle checkpoints, at G1/S, S-phase, G2/M transition and the mitotic spindle checkpoint, has come into the forefront of research (Dai and Grant, 2010). AZD7762 is a checkpoint kinase inhibitor which is being tested in clinical trials alone or in combination. Morgan et al. (2010) found that in vitro AZD7762 alone or in combination with gemcitabine significantly sensitised MiaPaCa2 pancreatic carcinoma cells to radiation. Also AZD7762 significantly reduced tumour growth in response to gemcitabine and radiation of both MiaPaCa2 and tumour xenografts. On the basis of these findings they have proposed the use of AZD7762 in combination with gemcitabine and radiation for patients with locally advanced pancreatic cancer. Some preliminary studies have shown that Chk1 is overexpressed in TNBC of high-grade compared with non-TNBC of the same grade (Verlinden et al., 2007).

New, Unexplored Avenues Several unexplored avenues worthy of attention can be identified. Much work has been described using conventional genetic profiling to identify signalling components that might be amenable to targeting. Andersen et  al. (2010) treated prostatic cancer cells with PI3K inhibitors of Akt, PDK1 (phosphoinositide-dependent kinase 1), or both PI3K and mTOR pathways, and identified PI3K-relevant phosphopeptides targets that were the most modulated by treatment. They then raised antibodies against selected target peptides and assessed their suitability as biomarkers to predict the activation of PI3K pathway and hence the susceptibility of cancer cells to AKT inhibitor. There was a differential downregulation of phosphopeptides by the different PI3K inhibitors. Identification of specific targets in this way might be clinically useful. Moritz et al. (2010) have similarly used the proteomics approach and identified many phosphorylation targets in MET, EGFR and PDGR signalling. However, it is more important to identify the links in the signalling cascade than the general channel of signalling, especially because the considerable cross-talk and mutual regulation occurs in signal transduction. As evident from these studies, the large number of differentially phosphorylated targets identified would make it a massive undertaking in drug development. It is undeniable that only a very small proportion of potential drugs identified progress to clinical trials for evaluation. This is a novel and intellectually appealing approach, and further work along these lines could provide clues

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to the identification of potentially exploitable targets in the many signalling systems activated by growth factors and other biological response modifiers. With the focus here on the signalling systems, one would enquire instinctively if there are EGFR-signalling components that might be amenable to targeting in the management of TNBC. Several pathways activated by growth factors and known to influence cell proliferation as well as angiogenesis have been discussed in these pages, among them are TGF-β, FGF, Notch, ER, IGF, Wnt and others. All these would provide a fertile ground in targeting drug-modulated signalling links and designing appropriate modes of inhibiting their function. Efforts are being made. For instance Wang et al. (2010) found increased expression of phosphorylated JNK (c-Jun NH2-terminal kinase) in both basal-like and TNBC, but it is difficult to visualise how specificity can be achieved by targeting certain systems that function ubiquitously. TGF-β and members of the TGF-β family, indeed most of the cystine-knot group of growth factors, need to be focused upon. The TGF-β family impinges upon virtually all phenotypic features that define neoplasia, and it exerts marked bivalent function by signalling through Smad and non-Smad pathways. So dissecting out this signalling system is an onerous task, but nonetheless needs to be tackled. Some preliminary work has shown that mouse models of the TNBC phenotype have shown marked inhibition of metastatic dissemination upon exposure to antibodies against TGF-β (Tan et al., 2009).

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14 The Epidermal Growth Factor (EGF) Family

It would have been conventional to discuss the molecular features, patterns of distribution in normal and disease tissue, operation and behavioural features of growth factors and to follow that with the signalling systems. However, the discovery and function of their receptors have occurred virtually in parallel with the discovery of the growth factors themselves. So the departure from convention is based on the reasoning that receptor activation or transactivation and downstream signalling systems are central to the theme and relevant in the evolution of targeted therapies. However, this has to be evaluated in the backdrop of the degree and patterns of the expression of the growth factors. These last aspects are discussed in this chapter.

Structure, Function and Expression of Epidermal Growth Factor (EGF) and EGF Receptor Epidermal growth factor (EGF) is a 6.0-kilodalton (kDa) peptide comprising 53 amino-acid residues. It is formed from the 128-kDa precursor pro-EGF. There are six cysteine residues that are involved in three disulphide bridges (Cys 6–20, Cys 14–31 and Cys 33–42) (Carpenter and Cohen, 1990) to form the EGF module of three loops, A, B and C, which are essential for stable molecular structure and for receptor binding and biological activity (Harris et al., 2003). EGF and TGF-α both are closely involved in both normal and aberrant development and differentiation. In the present context of development and progression of tumours, it will suffice to focus on the effects of these growth factors of deregulating cell proliferation and promoting tumour development and progression. EGF induces density-independent growth, reduces serum requirement for cell growth and enhances the ability of cells to grow in soft agar, all features characteristic of cellular transformation. Indeed, EGF has been known for a long time to enhance proliferation, induce cell migration and invasion both in vitro and in vivo, and EGF-activated signalling has been encountered in many human neoplasms. Not only do cancers produce more EGF, but it is released into the circulatory system in some cases. High serum levels of EGF have been found in patients with colon cancer, and this is said to relate to prognosis (Tomoichiro et al., 2002). Zhang et al. (2008) investigated normal oesophageal tissue, oesophageal dysplasia and oesophageal squamous cell carcinoma for EGF, EGF receptor (EGFR) and nucleostemin expression. Nucleostem is a nucleolar protein found in many types of stem cell and in tumour, but not in differentiated Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00014-1 © 2011 Elsevier Inc. All rights reserved.

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tissues. Nucleostemin is believed to modulate the stability of the cell cycle regulator p53. EGF and EGFR expressions were markedly greater in carcinomas than in dysplastic and normal oesophageal tissues. However, the difference in expression levels between dysplasia and normal tissue was less remarkable. Zhang et  al. (2008) also looked at nucleostemin levels in these tissues. One would have thought that the transition of normal oesophageal tissue to dysplasia would have significantly affected the expression of nucleostemin, but apparently not. Perhaps monitoring of the expression of more conventional cell proliferation markers would have been more informative and supportive of their findings. Also, this study concentrated on messenger RNA (mRNA) expression and does not appear to have checked the protein levels. The possibility of post-translational regulation should be borne in mind at all times.

Single Nucleotide Polymorphism of EGF and Its Significance Genetic alterations that influence the expression of growth factors and their effects on cancer risk, progression and prognosis have been described. Genetic polymorphisms of EGF as well as of EGFR can conceivably affect their expression and signalling and so could be associated with susceptibility to cancer and the nature and outcome of its progression. Single nucleotide polymorphisms (SNPs) have been detected in the promoter regions of the EGF gene, and in the coding as well as noncoding regions. There is a distinct possibility that the different SNPs might function synergistically. SNPs occurring in different growth factors too can produce a synergistic effect, but these aspects have not so far received much attention. So there has been a spurt of activity in this field. SNPs have been reported in respect of EGF and EGFR. An SNP at EGF position  61 (A/G) in the 5-untranslated region of the EGF gene has been linked with enhanced and probably prolonged EGF expression. Vauleon et al. (2007) demonstrated that the SNP was not only functional but that the G-allele was more active than the A-allele. EGF with the 61G allele has longer half-life (1.6) than the A-allele and EGF secretion is markedly (twice) higher in hepatocellular carcinomas carrying the G/G genotypes than corresponding A/A carriers (Tanabe et  al., 2008). Higher levels of EGF have been found in patients with gastroesophageal reflux disease but free of oesophageal carcinoma (Lanuti et  al., 2008). Certain other polymorphisms of the EGF promoter, for example, in patients with breast cancer, EGF-1380AA carriers, had significantly higher plasma EGF levels than EGF-1380GG carriers, but this was unrelated to cancer risk (Wang et al., 2008). Prostate cancers are known to express higher levels of EGF and TGF-α than benign hyperplasia or normal epithelium (Miguel et al., 1999). However, Teixeira et al. (2008) do not suggest such a relationship. The presence of G/A or G/G phenotype was reported in melanoma and glioblastoma (Bhowmick et al., 2004; Shahbazi et al., 2002). However, it is not regarded as a risk factor in the predisposition to, or pathogenesis of, melanoma (Casula et al., 2009; McCarron et  al., 2003). But Okamoto et  al. (2006) believe that the polymorphism reflected disease-free survival. Kang et  al. (2007) found no association between EGF61A/A and A/G genotypes with the risk of lung cancer compared

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with 61G/G genotype carriers. Furthermore, G/G homozygous polymorphism has been attributed with a reduced risk of developing breast and ovarian cancer, and G/G carriers showed a later onset of disease than those with the A/A genotype (Araujo et al., 2009a, 2009b). The EGF61AS/G SNP showed no links with susceptibility to glioma development (Liu et  al., 2009). Earlier, Costa et  al. (2007) found no association between polymorphism and overall survival of patients with glioblastoma or oligodendroglioma. The onset of neurofibromatosis has been linked with the presence of this SNP but apparently not with the progression to the pathogenesis of neural tissue tumours (Ribeiro et  al., 2007). Hamai et  al. (2005) reported it in gastric cancer, but Goto et al. (2005) found none. Spindler et al. (2007) looked for this SNP in colon cancer. Roughly half of the carcinoma samples showed the A/G phenotype, approximately a third were A/A and the remainder were G/G. In normal colon tissue, EGF expression varied between the three genotypes, but not in adenocarcinoma tissue. It is also worth noting that median EGF expression was lower in carcinoma than normal tissue. Kovar et al. (2009) have stated that patients with the G/G genotype carry a relatively greater risk of developing hepatic metastases than those with the A-allele. Also of interest here is that the G/G genotype correlated significantly with clinical stage. It is encountered also in prostate cancer and has been described as being able to influence disease relapse interval (Teixeira et  al., 2008). Higher risk of developing oesophageal cancer was linked with this SNP and this has been suggested to be predictive of risk of carcinoma in gastroesophageal reflux disease and Barrett’s disease (Lanuti et  al., 2008). There was no difference in the frequency of the polymorphism in cervical cancer, but lymph node involvement seemed to increase from the A/A homozygous genotype to the G/G homozygous genotype (Kang et al., 2007). These authors insert the caveat that it was a trend rather than a statistically significant correlation. No correlation has been seen in SNPs EGF61G/A and EGFR2073A/T in nasopharyngeal carcinoma (Gao et  al., 2008). Inagaki et  al. (2007) found no link between the incidence of these SNPs in EGF and EGFR in endometriosis for disease stage or risk factor. As stated earlier, in malignant melanoma the presence of SNPs has been described as reflecting disease-free survival. Overall, probably it is premature to pronounce on the clinical significance of this, in the light of the divergent views being expressed; also, it is yet unclear how the polymorphism affects the expression of EGF, or the nature of its relationship to progression. Although the original postulate was that this genetic alteration might enhance EGF expression; this is yet to be firmly established. TGF-β levels in plasma have shown to relate to two polymorphisms (G  A at position 800 base pairs (bp) and C  T at position 509 bp) in the promoter of TGF-β1 gene and so is obviously under genetic control (Grainger et  al., 1999). Patients with chronic HBV (hepatitis B virus) infection who carry the 509 C  T polymorphism are reported to be at greater risk of developing hepatocellular carcinoma (Kim et  al., 2003). A common polymorphism (936 C/T) encountered in VEGF has been linked with reduced VEGF expression and reduced risk of developing breast cancer (Krippl et al., 2003).

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However, this genotype and other polymorphisms did not seem to have any perceived relationship with tumour stage, grade or nodal spread of colorectal cancer (Hofmann et  al., 2008). In cutaneous melanomas, some single-base polymorphism genotypes and haplotypes showed noteworthy association with early-stage disease (Howell et al., 2002). On the other hand, certain other EGF polymorphism genotypes have not been found to be as relevant or significant in the disease process. One can discern significant dissensions in terms of the relevance of SNPs to cancer susceptibility, disease onset and progression. The studies with different ethnicity and demographic distribution of populations investigated might be an important factor of which one needs to take cognizance. Liu et al. (2009) emphasised the need to look carefully at the distribution of SNPs in Caucasian and Asian populations. For populations might harbour different SNPs and differ in the context of susceptibility to disease or response to treatment. Indeed, there might be differences between demographic regions within the same ethnic group. For instance, Casula et al. (2009) noted marked differences in the distribution of the G/G genotype between subjects from northern and southern Italy. SNPs might be related to differences in susceptibility to different forms of cancer within the same ethnic population. This could be the reason why Korean populations display marked differences in risk factor to lung cancer compared with cervical cancer (Kang et  al., 2007; Kang et  al., 2007); here, of course, the gender factor comes into focus. Finally, it is of much further interest and would notably deserve study whether breast and ovarian cancers that are linked by heritable genetic factors might display the same SNPs and similar susceptibility, onset and progression. A promising advance is represented by Antoniou et al. (2010), who identified five SNPs on 19p13 that were associated with breast cancer risk, and ER-negative as well as triple-negative breast cancer (TNBC) in BRCA1 carriers. However, these SNPs were not associated with risk of ovarian cancer.

Function of Transforming Growth factor (TGF)-α TGF-α is one of the transforming peptides discovered over a couple of decades ago. Its role as a transforming agent was demonstrated using temperature-sensitive mutants of murine sarcoma virus. TGF-α was released only when cells were grown at temperatures permissive for transformation. Cells transformed by myc and Ras oncogenes secrete excessive amounts of this growth factor. Furthermore, TGF-α is capable of inducing angiogenesis (see Sherbet, 1987). TGF-α is a small mitogenic peptide with 50 amino-acid residues and three disulphide bridges (Moy et al., 1993). Its secondary structure closely resembles that of EGF and bears 40% sequence homology with EGF. The molecular conformation of TGF-α and EGF is determined by the disulphide bridges making provision for the formation of three loops, A, B and C (Campbell et  al., 1989; Prestrelski et  al., 1992; Simpson et  al., 1985). By virtue of its structural homology to EGF, TGF-α can competitively bind to EGFR (Carpenter, 1987; Korc and Finman, 1989; Lax et al., 1991). McInnes et al. (1996) have shown that residues of the A- and C-loops of TGF-α provide sites for receptor binding, with the B-loop functioning as a scaffold.

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As its name implies, TGF-α is produced by transformed cells, embryonic cells and tumour cells. EGF and TGF-α are potent stimulators of cell migration and proliferation. Therefore, conventional wisdom would suggest that they are involved in cancer progression. As stated earlier, src and Abl family tyrosine kinases are able to phosphorylate EGFRs and the expression of src kinases often correlates with cell invasion. Also, there is a considerable body of early evidence that both of these growth factors alter ECM characteristics by regulating the expression of uPA (Urokinase-type plasminogen activator) and MMPs, which possibly aids cell locomotion. EGF is an activator of NF-κB signalling, it enhances NF-κB levels and this is inhibited by antibodies against EGFR (Biswas et al., 2000). Inhibition of NF-κB signalling has been found to downregulate MMP-9 expression and inhibit cell motility in vitro (Huang et al., 2001). HER2 also can mediate NF-κB signalling to induce cell migration without influencing cell proliferation (Merkhofer et al., 2009). EGF and TGF-α both bind to a common receptor, EGFR, with similar affinity and equivalent ability to activate it (see Sherbet, (1987) for a review). A novel thought has been emerging that temporally differential expression of growth factors might drive tumours along the route of progression. Definable patterns of gene expression in close association with processes related to cancer progression have been described before for the progression of colon and ovarian cancers (see Sherbet and Patil, 2006). However, there is no overwhelming evidence that EGF and TGF-α display temporally differential expression. Some time ago, investigating endometrial carcinomas, Yokoyama et al. (1996) found EGF and EGFR expressed in a sizeable proportion of cancers but the expression was unrelated to tumour grade or stage. In contrast, in most samples investigated TGF-α expression correlated with histological grade and stage. Oestradiol was detected in a small proportion of specimens of low-grade and early-stage tumours. Furthermore, oestradiol expression was inversely related to TGF-α expression in advance-stage tumours. This leads one to speculate whether, in the absence of oestradiol, growth regulation switches to TGF-α in the later stages of progression of endometrial carcinomas. In colorectal cancer the expression of both EGFR and TGF-α was higher in patients who had metastatic disease at presentation (Tampellini et  al., 2007). Curiously Tampellini et al. (2007) used S6 kinase (S6K) as a marker of downstream regulation of cell adhesion and migration. The problem with this is that S6K is implicated in many signalling pathways. It is highly expressed in some cancers and involved in the modulation of cytoskeletal dynamics. Unfortunately, its targets are neither fully established nor fully explored. In NSCLC both EGFR and TGF-α were overexpressed but unrelated to tumour stage (Rusch et al., 1997). However, a recent study indicates TGF-α overexpression in a third of NSCLC samples. It was co-expressed with EGFR again in a third of the specimens. Furthermore, their expression correlated strongly with the activation of anti-apoptosis gene Akt (Mukohara et al., 2004). Among other human cancers studied from this viewpoint are bladder cancers. In these tumours TGF-α was co-expressed with EGFR in advance-stage tumours showing muscle invasion (Thogersen et al., 1999). According to one report, hepatocellular carcinomas show no relationship between TGF-α expression and disease stage (Nalesnik et al., 1998). These authors found no correlation between growth factor expression and degree of cell proliferation either. So some uncertainty rightly arises in relation to this report.

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The prostate cancer cell line LNCaP is a non-metastasising cell line from which the C4-2B cell line has been isolated. The latter possesses the ability to metastasise. Interestingly, these cell lines display markedly different response to EGF and TGF-α. Although TGF-α induces proliferation in C4-2B, it exerts no such effect on LNCaP. This has been attributed to the differential activation of MAPK proliferative and antiapoptotic Akt survival signalling in these cell lines (DeHaan et  al., 2009). On the other hand, EGF is said to induce LNCaP to produce more osteoprotegerin (OPG) than TGF-α can in C4-2B cells. However, in the latter cells, TGF-α increased the expression of RANK ligand (RANKL, receptor activator of nuclear factor-κB ligand). RANKL is a regulator of bone remodelling. OPG functions as a decoy receptor for, and binds to, RANKL, and in this way inhibits osteoclast development. Significantly in these experiments, OPG is induced in LNCaP cells. Upon exposure to TGF-α, C4-2B cells induce the expression of an activator of bone remodelling. These findings do suggest a correlation between TGF-α and metastasis, because these are established markers of the presence of metastatic lesions in the bone. Nonetheless, the involvement of other growth factors such as TGF-β cannot be excluded, for osteoclasts do promote cell proliferation through release of TGF-β. TGF-α, TGF-β and EGF were shown some time ago to induce colony formation of LNCaP cells (Hara, 1992). The possibility of an auto-regulation of the TGF-α effect is emphasised by the upregulation of EGFR by both EFG and TGF-α in prostate cancer cell lines including LNCaP (Seth et  al., 1999). Now, Runx2, a bone-specific transcriptional regulator, shows low expression in LNCaP cells but high expression is related to metastatic disease. Inhibition of Runx2 decreases in vitro invasion by PC3 cells which possess high metastatic potential (Akech et al., 2009). Runx3-expressing cells also respond to TGF-β with inhibition of proliferation and induction of apoptosis. Some of these findings relating to the intervention of other growth factors signalling using other pathways need to be borne in mind while interpreting experimental data relating to TGF-α function in prostate cancer cells. Furthermore, there are no intermediate stages of progression between non-metastasising LNCaP and their metastasising counterparts, so one can justifiably call for an extension of these studies to tumour samples at different stages of progression.

Neuregulins (Heregulins) Neuregulins are members of the EGF family. They are secreted soluble proteins that participate in cell proliferation, survival, differentiation and neoplastic development. In general terms, one can say that these growth factors activate the erbB2-3/MAPK/ PI3K/Akt signalling cascade. Four neuregulins and some isoforms of neuregulin-1 have been discovered. Neuregulins 1–4 are encoded by four different genes. Several isoforms of neuregulin-1 are generated by alternative splicing of the pro-mRNA transcript (Marchionni et al., 1993). The isoform I of neuregulin-1 is known by the alternative names of Heregulin (HRG), and Neu differentiation factor, and isoform II as Glial Growth Factor-2. Wen et al. (1992) demonstrated the release of a biologically

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active peptide, which they called the Neu differentiation factor. This neuregulin is a transmembrane protein with an extracellular region containing an EGF domain, which they suggested might function as a receptor-recognition site. HRG binds to and activates HER2 receptor (Wen et al., 1992), but according to Wang et al. (1998), both neuregulin-1 and β-cellulin elicit a strong proliferative response from murine 3D cells transfected with human erbB4. There is some evidence of HRG transactivating EGFR. HRG seems to function by activating the Rac1/ERK pathway, leading to the upregulation of the expression of cyclin D1 and p21waf1/cip1 and promoting cell proliferation (Yang et al., 2008). The involvement of the Rho family Rac1 GTPase also potentially links HRG to signalling the cells into apoptosis through MAPK/ERK and p53 mediation. This could partly explain the growth inhibitory properties attributed to HRG. Following their apparent association with the central nervous system, much early work focused on the functional links of HRG with the central nervous system. HRGs have been found to subserve diverse functions in the nervous system. The role they play in neuronal differentiation is amply illustrated by Ghashghaei et  al. (2006), who showed that HRG2 promotes proliferation of neuronal progenitor cells in the subventricular zone through the erbB4 receptor and thus leads to the generation of neurons in vivo. They also suggest that different HRG isoforms might influence the differentiation of distinct precursor populations. Neuregulins provide the signal that stimulates transcription of AchR (acetylcholine receptor) genes at the synaptic site (Burden et  al., 1993). They have been found to stimulate Schwann cell proliferation, increase the rate of AChR synthesis, and are expressed in motor neurons (Falls et al., 1993; Marchionni et al., 1993). Neuregulins have also been attributed with the ability to increase α7 nicotinic acetylcholine receptors in neurons (Liu et al., 2001). Incidentally, α7 have also been found in non-neuronal cells and implicated in cellular function. The α7 receptors are expressed in bronchial epithelial and endothelial cells (Wang et al., 2001), and in immune cells, so are linked with the regulation of immune responses and inflammation (Tracey, 2002; Wang et al., 2003). The influence of HRGs on differentiation and the potential pathways activated in producing these responses have also been investigated using epithelial cell systems. Chausovsky et al. (1998) claimed that neuregulins exerted marked morphogenetic effects in epithelial cells in culture, which included the formation of epithelial islands and the formation of internal lumina in the larger ring-shaped structures. These were said to have adherens junctions containing cadherins and catenins. Interestingly Chausovsky et al. (1998) go on to describe an apparent increase in cell motility in the process of scattering and ring formation. However, these results are intriguing because the presence of cadherins is not compatible with enhanced cell motility. Nonetheless, it is not unlikely that neuregulins influence cell motility. The exposure of AU565 breast carcinoma cells in culture to HRG can induce differentiation or proliferation depending upon the dose of HRG used. At high doses, treatment with HRG-β1 has been reported to induce differentiation together with a sustained activation of ERK, but at concentrations that were conducive to the induction of proliferation ERK activation was only brief. However, HRG might not have been the initiator of ERK activation, because transfection of a constitutively active ERK

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construct can also induce differentiation in the absence of HRG (Lessor et al., 1998). Possibly this provides an example of how HRG might collude with other growth factors in generating phenotypic responses. The conferment of cellular motility has unsurprisingly invited investigations into the possible roles that HRGs might serve in tumour development and invasive behaviour. Liu et al. (2001) have reported overexpression of neuregulins in some cancers. All four forms of neuregulin are expressed in the cytoplasm or nuclei of in situ ductal carcinoma of the breast (Marshall et al., 2006). MTLn3 breast cancer cells overexpressing ErbB3 receptor display increased invasive behaviour in in vitro assays when exposed to HRG-β1 (Hernandez et al., 2009). HRG-β1 seems to induce invasive behaviour of SKBR3 breast cancer cells (Cheng et al., 2009). HRG has been found to upregulate MMP-7 expression (Yuan et al., 2008). There is much evidence that the expression of MMPs is associated with enhanced invasive and metastasis-promoting ability. It is possible that this could be one of the mechanisms by which HRG alters cell behaviour by facilitating their release and initiation. MMPs such as MMP1, ADAM-17 and ADAMTS1 might also be involved in the release of EGF ligands from their membrane-bound location (Lu et al., 2009; Merchant et al., 2008) and facilitate signalling. Kansra et al. (2004) showed that activation of EGFR and the ERK signalling system coincided with the appearance of soluble amphiregulin (AREG), which could be inhibited by MMP inhibitors and anti-AREG antibodies. HRG induces phosphorylation of ErbB3, consequent upregulation of MMP-9 and enhancement of invasive ability of B16-BL6 cell lines together with an increase in metastatic ability in experimental assays (Ueno et al., 2008). Unfortunately the latter does not provide a realistic assessment of metastatic ability and the in vivo metastasis assay of tumours in the footpad is even less desirable. HRGs are distinctly mitogenic growth factors. They activate the HER2/erbB3 heterodimer complex and downstream activate the PI3K pathway in human mammary epithelial cell lines. HRG-β is a powerful activator of PI3K, indeed more so than EGF, IGF or HRG-α (Ram et al., 2000). These findings have been confirmed using a rat tumour model; HRGs not only induce proliferation but promote tumour development. These phenotypical changes are mediated by HER2/erbB3, which are frequently co-expressed. HRG-induced heterodimerisation of these receptors activates signalling. Apart from Akt signalling, HRG seems to invoke the MEK/MAPK pathway and induce tumour cell proliferation (Kim et al., 2005; Marte et al., 1995). Both FGF1 and HRG-β1 can activate MEK/ERK1-2 signalling in ERMCF7 cell lines even in the presence of anti-oestrogens (Thottassery et  al., 2004). However, Estes et al. (2006) found no substantial effects from MEK deletion on HRG-β1, FGF1 or oestrogen-induced proliferation, opening up an opposite view that MEK activation might play only a limited role. There are many reports that HRG is able to inhibit cell proliferation and indeed MMP- or uPA- (urokinase type plasminogen activator) mediated enhancement of invasion. This inhibition of cell proliferation seems to be an outcome of the interaction between HRG-mediated signalling and other signalling pathways. Marte et al. (1995) found that the activation of the PKC pathway seemed to regulate HER2 negatively and so lead to a negation of stimulation of proliferation by HRG. Inhibition of HRG protein

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levels using short interfering RNA (siRNA) reduces HER2/erbB3 activation together with reduced proliferation. This loss of proliferation could be reversed by exogenous HRG (Schmitt et al., 2006). Tang et al. (1998) provided significant evidence that HRGs are mitogenic and functioned through erbB4 receptor. In cells transfected with hammerhead ribozymes raised against erbB4 mRNA, the mitogenic effect of HRG was not evident. However, more recently, erbB4 has been implicated in inhibition of proliferation of growth by HRG of breast cancer cells expressing it. Here activation of erbB4 is said to have inhibited proliferation that occurred from delays in G2M transition of cells. Furthermore, in parallel there was an increase in the expression of BRCA1, which also occurred in an erbB4-dependent fashion (Muraoka-Cook et al., 2006). The genes BRCA1 and BRCA2 are tumour suppressor genes. Loss-of-function mutations of these genes are associated with the development of breast cancer. The expression of the wild-type genes is co-ordinately regulated during cell proliferation and differentiation of mammary epithelial cells, and BRCA genes are recognised as being able to inhibit cell proliferation. In MCF-7 and ZR-75-1 cells, oestradiol has been found to activate ERK/MAPK and promote cell proliferation. Both responses are prevented by the presence of wild-type BRCA1 (Razandi et al., 2004). Grimm et  al. (1998) recognised the antithesis in relation to proliferation. They found that overexpression of HRG can lead to apoptosis. This ability extended to several HRG isoforms. They have suggested that the secreted form might be mitogenic, whereas the cytoplasmic section of HRG might be inducing apoptosis. Tang et  al. (1998) have argued that the response to HRGs is dependent upon the level of expression and activation of the receptor. High levels of erbB4 expression, activation and dimerisation are required for the mitogenic effect. This possibly also requires higher ligand levels. On the other hand, overexpression of HRG leads to the downregulation of apoptosis inhibitor Bcl-2. Furthermore, transfection of Bcl-2 into MCF-7 cells prevented HRG-induced apoptosis (Weinstein et al., 1998). Indeed, HRGs can induce differentiation or proliferation depending upon the dose of HRG exposure. A valid and highly relevant alternative explanation can be found in the earlier discussion that HRG might collude with other growth factors in generating phenotypic responses. Lessor et al. (1998) had reported that HRG activates ERK signalling, but under conditions where ERK signalling is constitutively active, cell differentiation can occur even in the absence of HRG, which suggests that HRGs can augment differentiation processes initiated by other biological response modifiers. HRGs have been shown to transactivate EGFR and the Rac1/ERK pathway, leading to regulated expression of cyclin D1 and p21 and promoting cell proliferation (Yang et  al., 2008). The involvement of the Rho family Rac1 GTPase links HRG also to potential signalling of cells into apoptosis through MAPK/ERK and p53 mediation. This could partly explain the growth inhibitory properties attributed to HRG. Nonetheless, one should not lose sight of the possible consequences of the fact that the expression of BRCA1 can inhibit cell proliferation on its own. BRCA1 and BRCA2 display a cell cycle-dependent expression and appear to function in the S-phase. A large proportion of familial breast cancer has been attributed to the presence of germ line mutation of BRCA genes (Nathanson et al., 2001). Tumours with BRCA germ line mutations also possess a high growth fraction.

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Other suggested mechanisms implicate the integrins and focal adhesion kinase, which are an important factor in cancer cell invasion. HRG1 is expressed in the central nervous system and is associated with cell migration in the developing brain. Ritch et al. (2003) showed that glioma cell motility is closely linked with HRG-1. The effects seemed to be mediated through HER2/erbB3 and downstream by β1-integrin and FAK, which showed co-localisation. Folgiero et  al. (2007) have suggested that the formation of HER2/erbB3 heterodimer promotes α6β4-dependent activation of PI3K/Akt, possibly signalling to regulate apoptosis and invasion. The activation of the PI3K/Akt pathway has been shown to be conducive to tumour cell invasion mediated by MMPs (Rychahou et al., 2004). HRG1 induces the adhesive faculty of lymphoblasts and this is inhibited by HER2, PI3K/Akt inhibitors. The uPA and uPAR (uPA receptor) system has also been seen as a potential participant in invasion. HRG-β1 has been reported to be able to induce both uPA and uPAR transcription from their respective promoters in MCF-7 breast cancer cells and in parallel induce cell motility (Mazumdar et  al., 2001). So also do AREG and TGF-α, both members of the EGF family of growth factors (discussed elsewhere in this book, pp. 177, 186), influence uPA expression (Giusti et al., 2003). As alluded to earlier in relation to BRCA1, there are clear indications of the interaction of HRG signalling with the function of other genetic factors. Thus it would appear that HRG stimulation seems to delay G2M transition and in this way effectively reduce cell proliferation (Tang et al., 1998). In this situation, BRCA1 might have influenced cell proliferation independently of HRG or might have interacted with and disrupted HRG signalling. In breast cancer cells, ER and PR are active determinants of cell proliferation. Functionally active ER can induce the expression of PR (Horowitz and McGuire, 1975). That HRG interacts with ER/PR signalling was recognised by Balañá et al. (1999), who showed that MPA (medroxyprogesterone acetate, a synthetic progestin) upregulated the expression of HRG mRNA in progestin-dependent tumour cell lines. HRG and MPA were mitogenic in these cells. Antisense oligodeoxynucleotides generated against HRG inhibited MPA-induced proliferation. Both HRG- and MPA-activated HER2 and erbB3 receptors and their mitogenic effects were negated by inhibition of HER2 mRNA by antisense oligonucleotides. The apparent cross-talk between these signalling systems has been analysed in greater detail in C4HD murine mammary tumour line and T47D cells. In these cells HRG can induce PR to bind to PRE (progesterone response element). Inhibition of HER2 using antisense oligodeoxynucleotides abolished the ability of HRG to induce the binding of PR to PRE (Labriola et  al., 2003). In another study with MCF-7 cells, HRG-β1 has been shown to phosphorylate Akt, mediated by PI3K. Akt phosphorylation is blocked by anti-oestrogens and inhibitors of HER2 and anti-HER2 ribozyme. HRG treatment does not lead to Akt phosphorylation in ER-negative cells, but this is reversed by transient transfection of ER-α into these cells. In other words, both ER and HER2 are essential components of Akt phosphorylation by HRG-β1 (Stoica et al., 2003). It would appear that the two signalling systems interact immediately downstream of receptor activation by HRG. The situation is probably more complex than meets the eye. In a murine mammary carcinoma that grows in vitro independently of steroid hormones, FGF-2 is not only overexpressed but also activates PR and stimulates cell proliferation (Giulianelli et al., 2008).

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Amphiregulin (AREG) Amphiregulin (AREG) is a single-pass transmembrane glycoprotein. The AREG gene is located on chromosomal region 4q13.3 and is related to EGF and TGF-α. The mature AREG protein, with 84 amino-acid residues, is generated from its transmembrane precursor with 252 amino-acid residues. The C-terminal region (residues 46–84) exhibits striking homology to the EGF family of proteins. AREG binds to EGFR and can function as a replacement, albeit less effectively, for EGF and TGF-α have been attributed with a dual function of inhibition as well as promotion of proliferation of tumour and normal cells in culture (Plowman et al., 1990; Shoyab et al., 1989). The chromosomal region of the AREG locus harbours break points associated with acute lymphoblastic leukaemia. It also contains many genes of interest in terms of cell proliferation, cell invasion and motility, and cancer metastasis suppressor genes. Of note here is SIP1 (Smad-interacting, multi-zinc finger transcription factor). SIP1 downregulates the expression of the invasion suppressor gene E-cadherin transfection. It is expressed at a high level in E-cadherin-negative human carcinoma cell lines. Furthermore, TGF-β enhances SIP1 expression (Comijn et al., 2001). BMP2, a growth factor involved in osteoblast differentiation, is able to induce BMP2 kinase. The BMP2K gene, which encodes this kinase, occurs at 4q21 (NCBI Entrez Gene database, 2009). CCN1/Cyr61 of the CCN family of genes (the cysteine-rich 61/ connective tissue growth factor/nephroblastoma overexpressed) participates in cell differentiation, proliferation, cell adhesion, migration and survival. CCN1 occurs at 4q21.1. The HNRPDL (heterogeneous nuclear ribonucleoprotein D-like) gene, which encodes a transcription regulator, also occurs at 4q21.22. Loss of 4q13–q21 was reported in association with ALK (anaplastic lymphoma kinase)-positive anaplastic large-cell lymphomas (Salaverria et  al., 2008). Some genes of the cytokine family, namely the neutrophil-activating protein NAP-1/IL-8, the interferon-inducible C-X-C cytokine IP-10, the human platelet factor 4 (C-X-C ligand 4) gene, and the gene GRO that encodes the mitogenic polypeptide melanoma growth stimulatory activity (MGSA) related to the platelet-derived beta-thromboglobulin, are located at 4q12–21 (Griffin et al., 1987; Luster et al., 1987; Richmond et al., 1988). IP-10 functions not only as a chemoattractant for cells of the immune system, but it is also known to promote the adhesion of T-cells to endothelia, and possesses anti-tumour and anti-angiogenic activity (Angiolillo et al., 1995; Dufour et al., 2002).

Differential Signalling by AREG There is at present a clear perception of differential AREG signalling. This can be viewed from the outcome in the form of the ability of AREG to regulate cell growth negatively or positively (Plowman et al., 1990; Shoyab et al., 1989), in the collaboration of AREG with other ligands and with the cross-talk with other signalling pathways. These differing modes of AREG function throw some light on the mechanisms that might be leading to the perceived differential effects of AREG on proliferation and differentiation. Because EGFRs are shared, a competitive element emerges,

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especially because the binding affinity of AREG to EGFR is lower than natural ligand. Thus AREG and TGF-α differ markedly in their effects on cell morphology, which can be attributed to potential differences in their mode of action (Chung et al., 2005). Furthermore, EGF itself can upregulate AREG expression but this occurs at vastly higher levels in cancer cells than normal cells (Silvy et al., 2001). Johnson et al. (1993) reported that AREG phosphorylates EGFR and HER2 exclusively, but the activation of HER2 might indicate potential cross-talk between the signalling systems. Equally, one can envisage differences in the outcome arising from a differential activation of signalling pathways. However, whereas EGF can promote both cell proliferation and cell motility, the effects of AREG can be uncoupled (Solic and Davies, 1997). This would suggest that although EGF does upregulate AREG expression, it might activate different signalling pathways downstream. One indication of this is that inhibition of AREG probably switches cells into the apoptosis pathway (Castillo et al., 2006; Yotsumoto et al., 2008). A switch of signalling pathways could be happening in MCF10A cells exposed to AREG and SUM149 breast cancer cells with forced AREG expression, wherein AREG but not EGF activates NF-κB and through its mediation upregulates the expression of IL-α/IL-β (Streicher et al., 2007), in this way regulating the invasive and proliferative capacity of the cells. Heparin-binding growth factors including AREG are expressed in primary myeloma cells at higher levels than in normal bone marrow plasma cells. It is said to stimulate IL-6 production and cell proliferation (Mahtouk et al., 2005). At the other end of the proliferative outcome, AREG could function as downstream effector of other response modifiers. For example, vitamin D3 (VD3, 1,25-dihydroxyvitamin D-3 cholecalciferol) is a known inhibitor of cell proliferation. VD3 has been shown to induce AREG expression (Akutsu et al., 2001) and it is reasonable to suggest that here AREG is negatively regulating cell proliferation. The intricate nuances of AREG function were highlighted some time ago by the effects of the cytokines IL-1α and TNF-α on the proliferation of normal cervical epithelial cells, HPV (human papilloma virus)-transformed cells and cell lines derived from cervical carcinomas. Although both IL-1α and TNF-α inhibited proliferation of normal epithelial cells, both stimulated proliferation of HPV-transformed cells and cervical carcinoma cells, which was accompanied by AREG expression (Woodworth et al., 1995). It might be worthwhile noting here that TNF-α enhances the production of IL-6 and inhibits IL-6 signalling through STAT3 in cervical cancer cells, but in normal epithelial cells the STAT3 pathway is not active as indicated by the lack of response IL-6. This implicates TNF-α/IL-6 signalling in HPV (Gage and Martinez-Maza, 1997), a signalling pathway activated by the EGF family of growth factors and interleukins.

AREG in Cell Proliferation AREG is distributed widely in normal tissues (Plowman et  al., 1990). It is virtually ubiquitously distributed in normal human tissues and many forms of cancer, for example carcinoma of the colon (Saeki et  al., 1992), breast, bladder, prostate,

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pancreas, lung, ovary, squamous cell carcinomas, renal cell carcinoma, cholangiocarcinoma (Yotsumoto et al., 2008), hepatocellular carcinoma, cirrhosis of the liver, but not in normal liver (BeRasain et al., 2009) and meningiomas (Laurendeau et al., 2009). Notwithstanding the dual function of promotion of cell proliferation and apoptosis subserved by AREG engendered by cross communication with other signalling systems, it is relevant and worthwhile to focus on its potential involvement in growth and progression of cancer. It was shown to stimulate proliferation of colon carcinoma Geo cells through EGFR. Antibodies against EGFR prevented the stimulation of proliferation by AREG. The Geo cells constitutively expressed AREG at high levels but it is expressed also in the normal colon (Johnson et al., 1992). AREG stimulates proliferation in human keratinocytes independently of other growth factors (Cook et al., 1992; Tsai et  al., 2006). Breast cancer cells overexpressing AREG can also grow independently of other growth factors (Willmarth and Ethier, 2006). Inhibition of AREG in hepatocellular carcinoma SK-Hep1 cells has led to the inhibition of EGFR, activation and inhibition of cell proliferation and enhanced apoptosis, whereas overexpression increased cell proliferation and significantly led to enhancement of tumorigenesis in vivo (Castillo et al., 2006). AREG-mediated inhibition of apoptosis might involve IGF receptor and IGF might indeed co-operate with AREG in the inhibition of apoptosis. This is achieved through a PKC-dependent pathway that leads to the inactivation of Bad and Bax pro-apoptosis genes. However, survival signalling was independent of PI3K and MAPK (Hurbin et  al., 2002, 2005). Recently Shigeishi et  al. (2009) found that AREG induced proliferation in two cell lines derived from the periodontal human osseous dysplasia, a benign fibro-ossesous condition of the jawbone characterised by disproportionate proliferation of fibrous connective tissue. There are suggestions that AREG might differentially influence proliferation in normal and tumour cells. Its expression seemed to be related to the state of differentiation in colonic tumours. Here 71% of well-differentiated tumours expressed AREG compared with only 18% of poorly differentiated tumours. By implication this would suggest AREG expression was inversely related to cell proliferation. Paradoxically, however, all normal tissues also expressed AREG (Saeki et al., 1992). Since the original postulate that AREG subserved dual function, there has been very little substantive evidence in support of the view that AREG inhibited cell proliferation. Stoll et al. (2007, 2009) have reported amphiregulin-mediated inhibition of keratinocyte proliferation. AREG-specific hairpin RNA inhibited AREG mRNA as well as the protein with consequential reduction (80%) in proliferation. The growth inhibition with AREG mRNA inhibition reported here is quite convincing, but it is somewhat intriguing that this occurred even in the presence of other EGFR ligands. One can adduce some indirect evidence for growth inhibition by AREG. Vitamin D3, a known inhibitor of cell proliferation, has been shown to induce AREG expression (Akutsu et al., 2001).

AREG in Invasion and Metastasis As noted earlier, AREG participates in cell proliferation and adhesion-related biological function, namely cell motility and invasion. There is ample evidence that AREG

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is aberrantly expressed in many human neoplasms and that suggests correlations of AREG expression both in disease progression and prognosis. There have been many efforts to examine possible links between AREG expression and cancer invasion and metastasis. Some attempts have been made to relate AREG expression and function with potential for metastasis. Zvibel et al. (1999) appeared to argue rather unconvincingly towards a differential growth stimulatory effect of AREG in two colon cancer cell lines differing in metastatic ability. Their study did not reveal any effect on differential adhesion to hepatocyte-derived ECM or any enhancement of cell migration or metastatic ability in the low metastasis cell line. Nevertheless, differences between tumour cells and their normal counterparts in the effects of AREG on proliferation have emerged. Overexpression of AREG seems to enhance cell motility in some experimental systems, and attempts have also been made to unravel possible molecular mechanisms that might be involved. Willmarth and Ethier (2006) found that breast cancer cells grown in the presence of exogenous AREG, and MCF10 cells overexpressing AREG, showed greater invasive ability; AREG upregulated the expression of some genes associated with cell adhesion and motility. Of these, an MMP gene comes easily to mind in relation to cell motility. Silvy et al. (2001) have reported stimulation of proliferation in normal breast epithelial cells but not in MCF-7 and MDA-MB231 breast cancer cells. To contrast with this, AREG enhanced the expression of uPA and uPA inhibitor in the tumour cells but not in the normal epithelial cells. Together with this, AREG enhanced cell motility, as demonstrated in Matrigel assays of breast cancer cells; this enhanced mobility appears to follow an upregulation of uPA. This AREG effect was blocked by anti-uPA receptor antibodies (Silvy et al., 2001). Giusti et  al. (2003) have subsequently confirmed that both AREG and TGF-α influence uPA expression. They have reported that reduction of AREG in breast cancer cell lines led to a decrease in uPA and TGF-α expression. When cells transfected with antisense-AREG were treated with AREG, the expression of uPA and TGF-α was stimulated. Thus both of them could be functioning in consort in the upregulation of uPA expression. Ando and Jensen (1996) found upregulation of uPA expression in migrating keratinocytes, but this did not appear to be due to AREG. HRG-β1 has been reported to induce both uPA and uPAR transcription from their respective promoters in MCF-7 breast cancer cells and in parallel induce cell motility (Mazumdar et  al., 2001). It may be recalled here that plasminogen activators have been linked with tissue remodelling and cell migration (Sherbet and Lakshmi, 1997). Highspecificity uPA receptors occur in breast cancers. Urokinase bound the receptors by the inactive ‘A’ chain, with the active site of the enzyme being presented to the tumour stroma (Needham et al., 1987). AREG and EGF seem to induce MMPs in MCF7 breast cancer cells but not in cell lines that possess no metastasising ability (Kondapaka et  al., 1997). This has been demonstrated in another transformed breast epithelial cell line by Menashi et  al. (2003), who found that the expression of MMP-2 and MMP-9 was upregulated by AREG, which was inhibited by antisense-AREG and antisense-EGFR. So, overall, there seems to be little doubt about the upregulation of both uPA and MMPs

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by AREG. Although the role of MMPs in cancer invasion is well documented, it is unclear if AREG-mediated induction of MMPs in this system led to greater invasive ability. Another mode of MMP function is to cleave and release the soluble ligand for binding to EGFR and initiate signalling. Several MMPs containing the ADAM domain have been identified as being associated with inflammation (ADAM-10 and ADAM-17). Structurally homologous to ADAM proteins are the ADAMTS proteins. These contain additional TSP1 (thrombospondin type 1) repeats at the C-terminal end (Porter et  al., 2005). The ADAMTS proteins are secreted by the cells and are not membrane bound like ADAMs. ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase-2) have been implicated in the pathogenesis of osteo- and rheumatoid arthritis. The presence of ADAMs in endothelial cells was reported; their potential role in angiogenesis, compatible with the angiogenesis-promoting ability of other MMPs, was recognised some years ago. It was shown that inhibitors of ADAMs inhibited endothelial cell migration and adhesion in models of angiogenesis in vitro (Trochon et  al., 1998). ADAM-8 is found in new microvasculature formed after spinal injury (Mahoney et  al., 2009). Retinal neovascularisation requires ADAM9, for in mutant ADAM-9 (/) angiogenesis is inhibited (Guaiquil et  al., 2009). Puxeddu et al. (2008) showed the soluble fragment of ADAM-33 carrying the catalytic domain was capable of inducing endothelial cell differentiation in vitro. Also, it induced neovascularisation in human embryonic/foetal lung explants and in vitro chorioallantoic membrane assays. Using Matrigel-supported fibroblast scaffold, ADAM-17 has been shown to induce the formation of capillary-type structures out of HUVEC cells. In vitro invasion is also enhanced by ADAM-17. Furthermore, inhibition of ADAM-17 resulted in reduced expression of VEGF and MMP-2 activation (Gooz et al., 2009). These in vitro findings without doubt provide an insight into the possible involvement of ADAM proteins in angiogenesis. However, most are correlative in nature and causal connections are being inferred. The ADAMTS1 protein might possess anti-angiogenic properties. ADAMTS1 binds to and sequesters VEGF, resulting in a lack of VEGFR phosphorylation and the resultant inhibition of endothelial cell proliferation (Luque et al., 2003). On the other hand, VEGF can upregulate ADAMTS1 (Xu et  al., 2006), thus completing a regulatory loop, which might tightly control neovascularisation. Nonetheless it shows widespread tissue distribution (Günther et al., 2005). The effects of ADAMTS1 are probably more complex than these findings would suggest. When overexpressed, the full-length ADAMTS1 promotes angiogenesis and invasion and metastasis. It can also cleave itself to form NH2- and CO-OH-terminal cleavage fragments; the overexpression of these fragments leads to inhibition of metastasis (Liu et al., 2006). Several ADAM proteins, for example ADAM-9, ADAM-12, ADAM-15 and ADAM-17, have been implicated in tumour development. ADAM-17, for instance, is expressed at higher levels in early as well as advanced ovarian cancer than in normal ovaries. Its contribution seems to be the shedding of membrane-bound EGF family ligands (Tanaka et al., 2005) and, by their participation in angiogenesis, the ADAMs probably also promote cancer progression and metastasis. Indeed, ADAM proteins have been proposed as potential therapeutic targets. Equally, the ADAMTS proteins found to possess anti-angiogenic properties could be clinically relevant.

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ADAM-17 expression in breast cancer cell lines closely correlated with in vitro invasion and proliferation. Its expression in breast cancer correlated with tumour grade, and the levels of active ADAM-17 increased progressively with progression to metastatic disease. Furthermore, high expression of ADAM-17 was associated with a significantly shorter overall survival compared with those with low expression (see McGowan et al., 2007, 2008). ADAM-17 is overexpressed in primary and metastatic colonic cancers compared with normal colonic mucosa. Inhibition of EGFR activation using RTK inhibitors and inhibition of ADAM-17 inhibits cell proliferation, and downstream inhibits activation of MAPK (Merchant et al., 2008). Lu et al. (2009) demonstrated that not only do MMP1 and ADAMTS1 release AREG, TGF-α and HB-EGF from their membranebound location, but that this process is linked with the development of metastatic disease in the bone in patients with breast cancer. This provides a rational explanation for the propensity of breast cancer to metastasise to the bone. EGF stimulates AREG expression, but the induced expression is five- to tenfold greater in breast cancer cells than in corresponding normal cells (Silvy et al., 2001). EGF induces both cell proliferation and enhances cell motility. In two colon carcinoma cell lines these faculties can be uncoupled. Mitomycin C-mediated inhibition of proliferation had no effect on the morphological epithelial–mesenchymal transition of cells or on motility. PI3K inhibitors inhibited proliferation but not morphological alterations. On the other hand, AREG induced cell proliferation without bringing about any changes in morphology or adhesive faculties (Solic and Davies, 1997). This suggests that although EGF does upregulate AREG expression, it might also activate different signalling pathways downstream. Inhibition of AREG by using interference RNA seems to switch cells into the apoptosis pathway (Yotsumoto et al., 2008). Another aspect of AREG function in cancer is exemplified by the observation that it is co-expressed with CD44 and EFGR (Nylander 1998). Several CD44 splicevariant isoforms act as receptors for the ECM component hyaluronan, a non-sulphated glycosaminoglycan. CD44 is closely identified with cell adhesion and invasion, and is strongly implicated in the promotion of cancer metastasis. CD44 enhances invasive ability (Merzak et al., 1994; Radotra et al., 1994). Metastasis promoter genes such as S100A4 alter the distribution of CD44 from uniform pattern into a patchy focal pattern (Lakshmi et al., 1997). This enhanced lateral mobility of CD44 has been attributed to cytoskeletal depolymerisation. These dense CD44 foci could provide discrete adhesive footholds for traction, thus aiding invasion. It has been postulated that hyaluronanCD44-mediated adhesion might be important in peritoneal dissemination of ovarian cancers. Hyaluronan present on mesothelial cells could mediate adhesion of the tumour cells to the mesothelium, as a prelude to invasion. Thus borderline ovarian tumours, which possess the capacity for adhesion to the mesothelium, represent a transitional type between benign tumours and carcinomas (Sherbet and Patil, 2006). How AREG signalling through EFGR might be aided by CD44 is yet unclear. Proteoglycan components of the ECM have been known to participate in and facilitate ligand-membrane receptor binding. Sulphation of CD44 is required for hyaluronan binding and for CD44-mediated cell adhesion to occur. Inhibition of sulphation of CD44 blocks hyaluronan binding and the adhesion process (Brown et al.,

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2001; Delcommenne et  al., 2002). Esford et  al. (1998) found, in a murine mutant cell line defective in heparan sulphate synthesis, that addition of heparan sulphate and chondroitin sulphate was necessary to induce hyaluronan binding of CD44. Heparan sulphate modifies CD44 isoforms containing alternatively spliced exon V3 and facilitates its binding by some growth factors but not AREG (Bennett et al., 1995). Heparan-mediated modification of CD44 possibly blocks the binding of CD44 by AREG. This could be a reason why polysaccharides such as heparin sulphate, dextran and sulphated chondroitins inhibit the AREG-mediated induction of proliferation of human keratinocyte cultures (Cook et al., 1992). Equally, sulphation of glycosaminoglycan residues is essential for AREG and HB-EGF to activate the RTK receptors. The sulphatase HSulf-1 inhibits the EGFR-ERK pathway, which is activated by the EGF family of growth factors (Narita et al., 2007). Separately, antiAREG and anti HB-EGF antibodies also inhibited the activation of this pathway. Although no direct link seems to have been established, reading between the lines the sulphatase could be obstructing signalling by the two heparan-binding growth factors. Thus, with this mode of participation of CD44 in cancer invasion and metastasis, its co-localisation with AREG functionally acquires an added significance. The putative activity of AREG in cancer growth and progression is further supported by the demonstration that it seems to transmit its signal by activating EGFR and possibly HER2 (Johnson et  al., 1993). The expression of both these receptor types closely reflects cancer progression. We showed some years ago that breast cancers that are ER-negative tend to express EGFR (Sainsbury et  al., 1984, 1985). However, in ER  non-proliferating cells, oestrogen can take recourse to enhancing the expression of AREG mRNA through ER-α (Ciarloni et al., 2007; Mallepell et  al., 2006). Most ERα-positive breast cancers have been found to overexpress AREG (Johnston, 2006). In early hyperplasia with malignant potential progressing from hormone-dependent breast cancer, Lee et al. (2007) found enhanced ERα expression and in parallel a marked increase in AREG. The enhancement of AREG expression corresponded with a marked decrease in EGF but with an unchanged EGFR re-expression. This would suggest that switching of signalling could occur in breast cancers that are EGFR-negative. Given that oestrogen is not the mediator of AREG upregulation, alternative mechanisms for this to happen need to be evoked. Martinez-Lacaci et al. (1995) found that oestradiol induced AREG expression in breast cancer cells. Miceli et  al. (2009) found higher levels of aromatase in some hepatocellular carcinoma cell lines which corresponded with increases in AREG expression. Because aromatase catalyses the conversion of testosterone to oestradiol, it seems possible that the enhanced expression of AREG is a result of the production of oestrogen by these cells. However, here the AREG-mediated stimulation of proliferation is seen as an outcome occurring independently of EGFR. Martinez-Lacaci et al. (1995) showed that oestradiol as well as TPA (12-O-tetradecanoylphorbol-13acetate) induce AREG expression in breast carcinoma cells. TPA is a tumour promoter that functions by activating PKC function. Furthermore, loss of PKC promotes apoptosis: in other words, this kinase serves as a cell survival signal (Whelan and Parker, 1998). It is of interest to note in this situation that AREG can activate IGF-1 receptor and that in co-operation with IGF, AREG activates the PKC-dependent

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pathway and inhibits apoptosis by inactivating the pro-apoptosis genes Bad and Bax, quite independently of MAPK and PI3K (Hurbin et al., 2005). PKC has been found to upregulate the expression of uPA and uPAR in migrating keratinocyte cultures subjected to mechanical injury. These effects occurred independently of AREG or other growth factors, but they were blocked by PKC inhibitors. Another potential effector that needs to be considered is the MMP system. MMPs are seen as successful effectors of growth factor function. As alluded to earlier, HRG can regulate MMP-7 expression (Yuan et  al., 2008). AREG and EGF do indeed induce MMPs in MCF7 breast cancer cells (Kondapaka et  al., 1997). Admittedly, MMPs, ADAM-17 and ADAMTS1 are involved with the release of EGF ligands from their membrane-bound location. Nonetheless, MMP mediation of the proliferation promoting effects of growth factors, including AREG, cannot be excluded. A summary of postulated alternative pathways of AREG signalling to achieve cell proliferation is presented in Figure 14.1, which gives the impression, not unreasonably, of ‘putting the cart before the horse’. However, it is clear from the above that AREG can function independently of its own receptor. For there is prima facie evidence for the possibility of the involvement of PKC signalling, which needs to be established by further, definitive experimental studies. Direct activation of the PKC pathway is not unknown, as seen in signalling by parathyroid hormone in promoting osteoblast differentiation; it does so by activation of PKC-δ through a PLC (phospholipase C)-independent mechanism. Wnt signalling is also associated with AREG function and one cannot exclude the possibility of the Wnt/G-protein/PLC/PKC cascade being integrated in AREG function. Approximately 25% of breast cancers show amplification of the HER2 gene, which correlates with the aggressive cancer and reflects poor prognosis. A humanised chimaeric antibody against HER2 has been approved for the treatment of

Figure 14.1  The possible integration of AREG signalling independent of EGFR in EGF/ER  breast cancers. This is based on references cited in the text. The possible integration of these pathways with the Wnt signalling cascade or those involving uPA and MMPs to promote cell proliferation is not shown here.

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patients with breast cancers which overexpress HER2 and are resistant to tamoxifen (Sherbet, 2009). AREG can bind to phosphorylate both EGFR and HER2. This is of some consequence in devising treatment modalities. In the light of these observations, no further iteration of the potential significance of activation of EGFR and HER2 is necessary.

AREG Expression in Human Neoplasms AREG is expressed in a wide spectrum of human cancers. Among them are breast cancer, NSCLC, colorectal, liver, gastric, prostate and oesophageal cancers, and myeloma, on which many studies have focused. AREG has been known for some time to play an important part in the morphogenesis and differentiation of the mammary gland. Using a murine experimental system, Sternlicht et  al. (2005) demonstrated that ADAM-17 was essential for the release of AREG in the mammary epithelium, and that AREG was then instrumental in promoting the differentiation of mammary ducts. However, of the EGF family ligands expressed in the epithelium and promoting growth, only AREG appeared to be able to promote ductal morphogenesis. It was reported earlier that AREG is expressed together with EGF, TGF-α and Cripto-1 (Kenney et  al., 1995), and that their expression was also more frequent in primary infiltrating ductal and infiltrating lobular breast carcinomas than in normal breast tissue (Qi et al., 1994). In another of these early studies that followed the discovery of AREG, LeJeune et al. (1993) found that AREG expression is upregulated in breast cancer. Its expression was restricted to tumour epithelium and none was found in tumour stroma or normal tissue. Furthermore, AREG was detected in 47% of primary cancer that had metastasised to the regional lymph nodes, compared with 26% of cancers that were lymph node negative. However, AREG was expressed in only 40 of 111 cancers investigated. In the absence of any information about EGF or TGF-α expression, it is difficult to attribute much significance to the correlation noticed between AREG expression and spread of the tumour to the lymph nodes. Salomon et al. (1995) also found that AREG mRNA and protein are expressed in breast cancer as well as in breast cancer cell lines. They also found AREG expressed more frequently and at higher levels in invasive carcinoma than in situ ductal cancer and normal epithelium. The findings of Ma et  al. (2001) make one feel indecisive about the significance of the state of expression of AREG and EGFR. They found both AR and EGFR in non-malignant breast tissues as well as in adjoining normal tissue in all samples tested. In comparison, only half of invasive epithelial tumours expressed AREG and around a fifth of them EGFR. Nonetheless, expression of AREG and EGFR in invasive carcinoma closely correlated with tumour size, nodal involvement and absence of oestrogen receptors. Low EGFR expression is not incompatible with highly effective ligand function. In invasive carcinomas, expression of the ligand and receptor correlated with poor prognosis. This is possibly due to the presence of both AREG and EGFR in the stromal component, albeit this was only seen in around 12% of the invasive carcinoma samples. It would not be imprudent to throw an isolated spanner into the works. In sharp contrast, Visscher et  al. (1997) found AREG and HRG expression

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only in the stromal and not the epithelial component of breast cancers, and both correlated with recurrence of the disease. So it is a gross oversimplification to attribute the malignant properties to AREG and its receptor in either the tumour epithelial cells or the stromal cell. Quite obviously the stromal component makes a sizeable contribution to the degree of malignancy. Of considerable interest is the finding that oestrogens increased AREG expression. Aromatase catalyses the conversion of testosterone to oestradiol, and recently Miceli et al. (2009) found higher aromatase expression in some hepatocellular carcinoma cell lines, which corresponded with increases in AREG expression. However, LeJeune et al. (1993) found no relationship between ER and AREG levels in breast carcinoma. An investigation of NSCLC primary tumours and their metastasis in the brain for the presence of EGF family ligands and their receptors has revealed that, on the whole, metastatic deposits displayed higher levels of EGF, TGF-α, neuregulins and a prominent difference in activated EGFR than the primary tumours (Sun et al., 2009). However, we have no indication here as to how individual primaries and their corresponding metastases fared in the degree of enhanced expression of these ligands and the receptors. AREG and epiregulin are detectable in adenomas and carcinomas of the colon but are not expressed in normal colonic mucosa. Weak immunostaining was also detected in mesenchymal cells from the tumour tissues. These also occur in the active mucosa ulcerative colitis and Crohn’s disease. Here AREG is found mainly in epithelial cells, with some distribution also in the surrounding mesenchymal cells (Nishimura et al., 2008). A moderate expression of AREG has been described in benign prostatic epithelium, prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma (Bostwick et al., 2004). These authors found nuclear staining in benign epithelium, but encountered cytoplasmic or nuclear staining in PIN and adenocarcinoma. They ascribed this to differences in the pathways of signalling. However, a simpler explanation is that this subcellular distribution reflects only a phase of signalling and the transition of the ligands from cytoplasmic to nuclear location; this is a situation analogous to the movement of androgen receptors upon growth factor activation.

Epiregulin (EREG) EREG is an EGF family growth factor that was isolated from conditioned medium in which NIH3T3 cells were grown. It is a small peptide of 46 amino-acid residues and bears 24–50% sequence homology with other EGF family members (Toyoda et  al., 1995a, 1995b). The EREG gene encodes a precursor peptide of 163 amino-acid residues. EREG is secreted as a 5-kDa protein. The EREG gene is located at 4q13.3. Early work by Toyoda et  al. (1995a, 1997) also showed the EREG to function as an active mitogen in many normal and tumour cells. EREG activates EGFR and erbB4. So it is of much interest in terms of its outcome that it can upregulate the expression of TGF-α, AREG and HB-EGF. Also, it can itself be upregulated by many of the growth factors (Komurasaki et al., 2002; Shirakata et al., 2000), and in this way

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EREG might significantly amplify its full proliferative influence. Apart from the work of Toyoda et  al. (1995a, 1995b), there are reports that EREG stimulates the proliferation of many types of normal and tumour cells. EGFR activation and enhanced proliferation is encountered in rabbit gastric cells in primary culture (Sasaki et  al., 1997). EGFR signalling uses several pathways of flow of information, namely the Ras/MAPK, PI3K/PTEN/Akt, the Jak/Stat and the PLC-γ/PKC pathways. In primary hepatocyte cultures, EREG seemed to activate MAPK signalling, apparently more efficiently than EGF (Komurasaki et al., 2002). In cell cultures of the human corneal epithelium, cell proliferation is accompanied by EREG-mediated activation of EGFR and ERK1-2 (Morita et al., 2007). In primary cultures of renal proximal tubular cells, EREG showed proliferation- as well as migration-inducing potential similar to EGF, and activated EGFR, PI3K/Akt and ERK1-2 pathways (Zhuang et al., 2007). The activation of these pathways with enhancement of invasion has been confirmed recently (Hu et  al., 2009). Benzo(a)pyrene markedly stimulated proliferation of A549 lung adenocarcinoma cell lines in vitro. Here exposure of the carcinogen seemed to upregulate the expression of EREG and EGFR together with increased phosphorylation of EGFR and activation downstream of ERK1-2 signalling (Kometani et al., 2009). The activation of Ki-ras signalling was identified with enhanced EREG expression some time ago (Baba et al., 2000). In Ki-ras-transformed cells, the RAF/MEK/ERK pathway was constitutively active and led to the activation of Ets-1 transcription factor and to increased EREG expression (Cho et al., 2008). These data are compatible with the findings that colorectal cancer patients with higher EREG, AREG and EGFR are more responsive to EGFR inhibitor cetuximab treatment than those who display lower levels of the growth factors, and further that response to cetuximab therapy is far superior in patients who have no Ki-ras mutations than in those who do carry mutations in this gene (see Khambata-Ford et al., 2007).

EREG Expression in Cancers Early findings about EREG indicated its possible association with embryonic development. EREG may be involved with blastocyst implantation (Das et  al., 1997). EREG and β-cellulin expression is switched on in uterine tissue around the implanting blastocyst. Both temporally and spatially these events are co-ordinated; nonetheless, the nature of its involvement in the interaction of the blastocyst with uterine tissue is not clear at present. Toyoda et al. (1995b) reported that EREG was detectable in early embryos but underwent a gradual reduction and was not found in normal adult tissue. Extraneously administered EREG and TGF-α affect brain development and influence other developmental processes such as eyelid opening and tooth eruption in neonates (see Tsuda et  al., 2008). These findings support the view that the expression of growth factors, such as EREG, may be developmentally regulated and activated. EREG expression has been detected in many cell lines derived from human cancers, tumour cells experimentally selected for enhanced metastatic ability and so on. However, investigations of EREG in human cancer tissue and its potential relationship to tumour grade, stage and metastatic progression are few and far between. Both

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EREG and AREG are found, in adenomas and colon carcinomas but not in normal mucosa. Ulcerative colitis and Crohn’s disease are forms of inflammatory bowel disease. Both EREG and AREG are found in epithelial cells of ulcerative colitis and Crohn’s disease (Nishmura et al., 2008). EREG and EGFR were invariably detected in human malignant fibrous histiocytoma (Yamamoto et al., 2004). Das et al. (2008) investigated the expression of genes associated with wound healing. Among those upregulated in expression was EREG together with VEGF-B and angiomotin, a protein expressed in endothelial cells that enhances endothelial cell migration. Also upregulated was Nogo. Nogo is a membrane protein that inhibits neurite outgrowth. It is found in endothelial cells, smooth muscle cells and blood vessels. Nogo B has been linked with the migration of endothelial cells but not cells of the vascular smooth muscle (Acevedo et al., 2004). Hu et al. (2009) detected increased levels of cyclooxygenase (Cox)-2 as a result of EREG treatment. Cox-2 shows positive correlation with angiogenesis and VEGF and PDGF expression (Tatsuguchi et al., 2004). These observations do suggest a potential involvement of EREG in angiogenic processes. Simultaneous expression of EREG and Cox-2 would appear to facilitate breast cancer metastasis, as indicated by the induction of neovascularisation and the entry of tumour cells into the circulation preparatory to metastatic deposition in the lungs (Gupta et  al., 2007). So it is remarkable that nearly a decade ago higher levels of EREG, HB-EGF and TGF-α were reported in more advanced invasive T2–T4 stage bladder cancers than early-stage superficial tumours, and furthermore that EREG expression was related to poor survival (Thogersen et al., 2001). Although statistics might indicate the relative importance of these individual markers in progression, it is difficult to determine how much co-operative functioning might have led to the perceived link with disease progression and patient survival.

β-Cellulin (BTC) β-cellulin (BTC) was identified as a growth factor by Dunbar et al. (1999). It belongs to the EGF family. It is a 32-kDa (Sasada et al., 1993) transmembrane protein. The extracellular domain is released by proteolytic cleavage as a mature growth factor. The BTC gene is located at 4q13.3 with AREG and EREG (Katoh and Katoh, 2006b). Early reports suggested that BTC phosphorylated EGFR, less effectively than EREG, but not HER2 or erbB3 (Riese et  al., 1996, 1998; Watanabe et  al., 1994). Alimandi et al. (1997) had reported that EGF and BTC bound HER2 and erbB3 when both are expressed simultaneously, but not individually. Shin et al. (2003) found BTC could phosphorylate all four EGFR family receptors. It seems to induce phosphorylation of Akt, GSK3α/β and two FoxO factors, FKHR and AFX. The activation of PI3K/Akt pathways increases cyclin D1 and stimulates DNA synthesis. AREG produced the same proliferation effects, but by activating Ras/Raf and MEK1 to ERK1 and MEK2 to ERK2. They imply that this could be because AREG acts through EGFR (erbB1), whereas BTC could be activating one of the other EGFR family receptors (Shin et al., 2003). The answer could lie in the ability of BTC to bind

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heretodimers of various combinations (Dunbar and Goddard, 2000). These studies could be valuable in using BTC-directed antibodies as a therapeutic tool.

BTC Expression in Cancers BTC gene was cloned some time ago, but very little work has been done with regard to its function, or its role in normal physiology, embryonic development, differentiation and cancer. BTC is produced by pancreatic beta cells; indeed it was first isolated from conditioned medium of beta cell murine tumours (Shing et al., 1993). We know nothing about its physiological function in pancreatic beta cells. Neither BTC nor the related growth factor TGF-α appears to exert any control over insulin production by these cells (Sjoholm and Kindmark, 1999). BTC is expressed in some glandular and epithelial components of the stomach and the intestines, but again we know little about their function in these tissues (Kallincos et al., 2000). Preliminary indications are that BTC is involved both in embryonic development and in cancers. The differentiation of ovarian follicle preparatory to ovulation is a complex process involving LH-mediated induction of growth factors such as BTC, AREG and EREG, which activate appropriate receptors, leading to oocyte nuclear maturation (Conti et al., 2006). Das et al. (1997) found that BTC is required in successful implantation of the blastocyst. It is expressed, together with TGF-α and EGFR, in a high proportion of hepatocellular carcinomas and at higher levels compared with normal tissue. BTC expression strongly correlated with that of EGFR in tumour endothelial cells, suggesting a potential role in aiding angiogenesis (Moon et  al., 2006). In mesenchymal malignancy fibrous histiocytoma, BTC is detected only in a relatively small proportion of tumours compared with EREG and HB-EGF (Yamamoto et al., 2004).

Heparin-Binding Epidermal Growth Factor-Like Growth Factor HB-EGF (heparin-binding epidermal growth factor-like growth factor) markedly promotes growth in normal cells. It is overexpressed in a variety of human tumours and has been studied in much greater detail than BTC, largely also because of its angiogenic properties. The soluble sHB-EGF is a 22-kDa protein (Higashiyama et al., 1991). It is proteolytically cleaved and released from the membrane-associated precursor molecule called proHB-EGF.

HB-EGF Induces Angiogenesis HB-EGF has been reported to induce the expression of eNOS mRNA, stimulate eNOS protein production, increase NO release from HUVEC cells and initiate cell migration and in vitro angiogenesis. HB-EGF phosphorylates eNOS through PI3K mediation to stimulate in vitro angiogenesis. Inhibition of eNOS inhibited HG-EGF-induced cell migration and angiogenesis. Furthermore, inhibition of MAPK signalling enhanced the

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induction of cell migration and angiogenesis by HG-EGF. It would appear therefore that HB-EGF functions through EGFR to activate eNOS and NO production by activating PI3K signalling (Mehta et  al., 2008). HB-EGF can phosphorylate EGFR and rapidly induce p42 MAPK phosphorylation. It phosphorylates PI3K/Akt and eNOS less rapidly. The activation of eNOS is blocked by MAPK and PI3K inhibitors. Also, VEGF antagonists suppress HB-EGF-induced activation of Akt and eNOS, suggesting the involvement of endogenous VEGF in the process (Nakai et  al., 2009). However, Mehta et al. (2008) and Mehta and Besner (2007) found both HB-EGF and EGF were effective and seemed to act independently of VEGF. What is intriguing about the work of Mehta and colleagues is that they found that HB-EGF and EGF did not influence cell proliferation. HB-EGF is mitogenic to fibroblasts and not to endothelial cells, but EGF is mitogenic to both. Presumably this is because HB-EGF can bind EGFR with greater affinity than EGF itself. It is far more mitogenic for smooth muscle cells than EGF (Higashiyama et al., 1991). Ongusaha et al. (2004) studied both the membranebound and soluble forms of sHB-EGF in bladder cancer cells. sHB-EGF increased growth rate and colony-forming ability, and activated cyclin D1, which is probably reflected in enhanced tumour growth in vivo. Two other important findings that emerged from this study are the induction of VEGF itself by sHB-EGF. This would certainly detract from perceived ability of direct induction of angiognesis by sHB-EGF. Also, sHB-EGF led to the production of MMP9 and MMP-3, which most certainly would promote cell migration and even aid in angiogenesis independently of HB-EGF.

Influence of HB-EGF on Cell Motility and Invasion Perhaps the exclusive focus on the soluble ligand has eclipsed the potential of the residual membrane-bound proHB-EGF. Not all of this is cleaved to release sHBEGF. It was recognised many years ago that both proHB-EGF and sHB-EGF may have distinct functions. sHB-EGF participates in proliferation and migration signalling through EGFR family receptors (Faull et  al., 2001; Piepkorn et  al., 1998; Tokumaru et al., 2000). ProHB-EGF, on the other hand, has been attributed with the ability to interact with ECM components such as integrins and to influence intercellular cohesion. Lagaudriere-Gesbert et al. (1997) noticed that CD9 could upregulate proHB-EGF. CD9, a member of the tetrapanin (transmembrane 4) superfamily, is a cell-surface glycoprotein that is actively involved in cell proliferation and motility. Peria et al. (1999) argued that HB-EGF bound only weakly to EGFR but quite markedly bound heparin sulphate proteoglycans. Because CD44, especially CD44v6, is closely associated with metastasis, the HB-EGF-mediated induction of intercellular adhesion and migration acquires additional significance. However, Bennett et  al. (1995) have attributed this to the CD44v3 isoform. ProHB-EGF could infuence cell behaviour by a different mechanism than noticed in the case of sHB-EGF. ProHBEGF interacted with DRAP27/CD9 and integrin-α3β1. Interestingly DRAP27/ CD9 and α3β1 occurred at intercellular cohesion sites together with α-catenin and vinculin (Nakamura et  al., 1995). CD9-associated vascular invasion leading to the deposition of lymph metastasis is indicated by its enhanced expression in gastric carcinoma (Hori et  al., 2004). This is consistent with the view that it upregulates

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HB-EGF (Lagaudriere-Gesbert et al., 1997). CD9 trasfection can lead to an upregulation of MMP-2 expression, which seems to result from the activation of the p38 MAPK/JNK signalling pathway (Hong et al., 2005). So possibly CD9 could be promoting invasion by increasing MMP expression through upregulation of HB-EGF. In contrast, however, CD9 transfection has also been reported to reduce cell motility (Ikeyama et  al., 1993; Miyake et  al., 2000). In some forms of cancer, reduced CD expression has been correlated with poor prognosis (Funakoshi et al., 2003; Hashida et al., 2003; Huang et al., 1998).

HB-EGF in Cancer Progression HB-EGF has been associated with many biological processes, such as blastocyst implantation, wound healing, and atherosclerosis, among others. With its ability to markedly influence adhesion-dependent processes, cell motility, invasion and angiogenesis, it is natural to inquire if it might play a role in tumour progression. Matsuzaki et  al. (2005) investigated the expression of the transmembrane heparan sulphate proteoglycan syndecan-1 (CD138) in ovarian carcinomas. They found its expression was threefold greater in early-stage ovarian cancer than in advanced ovarian cancer. They also showed that enforced inhibition of syndecan-1 in HRA ovarian cancer cells by transfection with a syndecan-1 antisense complementary DNA (cDNA) increased the invasive behaviour of the transfectants when exposed to HB-EGF (Matsuzaki et  al., 2005). Another approach to this has been to inhibit HB-EGF directly. Again in ovarian carcinoma cells, reduction of HB-EGF expression led to a reduction in the expression of MMP-2 and VEGF, together with a reduction in invasive ability and angiogenesis. Quite significantly, the metastatic tumour burden appeared to relate to the level of HB-EGF. Yagi et  al. (2008) used SKOV3 cells transfected with proHB-EGF cDNA and showed that inhibition of HB-EGF expression using CRM197, a mutated diphtheria toxin regarded as a specific inhibitor of HB-EGF, significantly reduced metastatic burden in spontaneous metastasis assays. They also looked at the expression of HG-EGF in normal and peritoneal tissues of patients with ovarian carcinoma and found its expression was higher in tissues of those with carcinoma than those without. Also elevated were AREG and EREG. Bladder carcinomas stained for HB-EGF in the cytoplasm and nuclei. Patients with nuclear staining had much poorer prognosis than those who showed only cytoplasmic HB-EGF staining (Kramer et al., 2007). However, one should recall here that HB-EGF was detected infrequently in normal and hyperplastic pancreatic duct epithelium. It was detected in approximately half (22 out of 40) of pancreatic adenocarcinoma samples. It was also expressed in samples with low cell proliferation index and in well-differentiated early-stage tumours of small size and without lymph node involvement. These tumours also expressed EGFR at low levels (Ito et  al., 2001a). It is possible that here HB-EGF might be activating receptors other than EGFR. Interestingly, this group of investigators reported virtually identical findings in hepatocellular carcinomas (Ito et al., 2001b). A rather ubiquitous receptor that comes to mind is the leptin receptor OBR. OBR functions by activating several signalling systems. It possesses intracellular motifs

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required for activation of the JAK/STAT signalling transduction pathway. Also activated is PI3K signalling (Baumann et  al., 1996; Tartaglia, 1997). These are also major signalling pathways activated by EGFR ligand binding. Leptin signals through the PKA and MAPK pathways and induces NO (Mehebik et al., 2005); in this way it might be associated with angiogenesis. The Rho GTPases that modulate cytoskeletal dynamics have also been suggested to be liable to activation by OBR. Now, leptin is able to enhance the expression of HB-EGF and TGF-α genes and stimulate cell proliferation (Ogunwobi and Beales, 2008). It would appear, then, that leptins might accentuate the proliferative and invasion-inducing effects of HB-EGF, as well as actuating the OBR-responsive genes, leading to the same phenotypic outcome.

15 The Fibroblast Growth Factor Family

The fibroblast growth factor (FGF) family constitutes a large family of approximately 20 structurally related growth factors (Blaber et  al., 1996; Finklestein and Plomaritoglou, 2001; Ornitz and Itoh, 2001). Although structurally related, they do display different functional modes. FGFs 1–10 function as paracrine factors and bind FGF receptors (FGFRs), but the intracellular FGFs 11–14 (also called FGF homologous factors 1–4) do not bind FGFRs besides possessing different functions (Itoh and Ornitz, 2008; Olsen et al., 2003). Some FGFs, for example FGF15/FGF19, FGF21 and FGF23, possess an endocrine mode of function. They exert systemic effects: FGF15/19 from the intestine inhibits bile acid synthesis, liver FGF21 in carbohydrate and lipid metabolism, and FGF23 is produced in the bone and is involved in its metabolism. FGF1 (acidic FGF) and FGF2, regarded as the prototype growth factors of the family, participate in many biological processes. Their overriding significance in the discussion here arises from their ability to influence endothelial cell proliferation and migration, and to induce tumour growth and invasion angiogenesis. FGF1 binds to FGFR; the specificity of this interaction is attributed to molecular features of the ligand. Members of the FGF family are structurally similar, with approximately 14% sequence homology and a core region containing conserved amino acid sequences and structural motifs. The core structure represents the 18-kilodalton (kDa) FGF2. This core represents a region showing the greatest sequence identity. Essentially, the core contains regions required for receptor binding, nuclear localisation and signal peptide. The hydrophobic signal sequence occurs at the amino (N) terminus of many FGFs, and this seems to direct the growth factor into the secretory pathway, although FGFs lacking this signal peptide are also secreted. From the viewpoint of FGF–FGFR interaction most important are the Ig-domains, D2 and D3, and the linker sequence between D2 and D3 (Ornitz and Itoh, 2001). Four FGFRs carrying substantial sequence homology have been identified. The transmembrane receptors have an extracellular domain with the IgG-like domains, a transmembrane domain and a cytoplasmic domain with a tyrosine kinase domain. Further isoforms of these FGFRs are generated by alternative splicing. FGFs require heparan sulphate to activate the receptors. The growth factors bind heparan sulphate polysaccharides and this complex facilitates binding to and activation of FGFRs. Many molecular features of the polysaccharides enter into the interaction with growth factors. Heparin sequences required for and participating in this complex formation have been identified, and these occur in two conformational states that are in equilibrium with each other. These conformational states Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00015-3 © 2011 Elsevier Inc. All rights reserved.

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are relevant in terms of specificity of growth factor binding and receptor activation (Guerrini et al., 2007; Guglieri et al., 2008). It has emerged recently that FGF1/FGF2 chimaeric growth factor can bind all the receptor isoforms even in the absence of heparin (Motomura et al., 2008). FGFs that lack the signal peptide use a non-classical means by forming a multi-protein complex including S100A13 and the calcium sensor protein synaptotagmin 1 (Mohan et al., 2010; Mouta et al., 1998). The systemic effects exerted by some FGFs require the transmembrane protein klotho or β-klotho, besides the conventional FGFRs for their function (Fukumoto, 2008). Klotho plays an important role in bone and mineral metabolism. FGF23 binds FGFR, and klotho is believed to facilitate this process by functioning as a co-receptor (Kurosu and Kuro-o, 2009). Vitamin D (calcitriol) regulates the levels of calcium and phosphorus, and participates in bone mineralisation. Klotho is actively involved here by regulating FGF23 function (Prie et al., 2009a,b). The crystal structure of the complex between FGF1 and FGF2 with the ligand-binding domains D2 and D3 (immunoglobulin IgG-like domains) of FGFR1 and FGFR2 has revealed that the interfaces between FGF-D2 and FGF-linker (between D2 and D3) determine the specificity of interaction between FGF and FGFR. The specificity of interaction is subject to modulation by changes in primary structure and alternative splicing (Plotnikov et al., 2000). TGF-β, IL-6 and FGF1 itself can modulate the expression of FGFR isoforms. FGF2 mainly binds FGFR1 and FGFR2, but FGF1 binds and activates all FGFRs 1–4, and the specificities of interactions could alter the biological responses. Alternative splicing can lead to ligand-specific binding to FGFR isoforms (Yu and Ornitz, 2001) and indeed to a wider range of biological activity. FGFs target a variety of cell types and regulate an array of biological processes such as cell differentiation, motility, proliferation, apoptosis, angiogenesis in wound healing, regulation of metabolism and several pathological conditions. A reconciliation of this phenotypic diversity with the array of FGF isomers and their receptors has necessitated the elucidation of the modes of their signalling and the potential of interactive signalling in achieving the diversity of their perceived functions. Of especial interest in this regard are TGF-β family ligands, Hh and Wnt, which have considerable bearing on all these phenotypic features in embryonic development, differentiation as well as in pathogenesis. The best-characterised members of the family are FGF1 (acidic FGF) and FGF2 (basic FGF). The discussions in this chapter predominantly address FGF1 and FGF2, but other FGFs are referred to as appropriate. FGF effects are mediated by specific receptor tyrosine kinases and facilitated by heparan sulphate; indeed, they were initially described as heparan-binding growth factors. FGFs 1 and 2 are said to bind heparan sulphate with greater affinity than to their respective receptors. FGFs serve in both endocrine and paracrine modes. The endocrine function might be facilitated by binding to heparan sulphate, and in this way FGFs might be localised at the cell surface and allow them to bind to the appropriate receptors. The paracrine function is supported by protective mechanisms. FGFs are tethered tightly to heparan sulphate proteoglycans and to the ECM. They are mobilised and activated by binding to FGF-binding protein (FGF-BP). These are secreted proteins and bind FGF reversibly, protect them from degradation and function as chaperones (Abuharbeid et al., 2006;

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Czubayko et al., 1994; Wu et al., 1991). FGF-BP possesses distinct domains through which it interacts with FGF and heparan sulphate proteoglycans. The heparin-binding domain is a conserved domain encompassing amino acids 110–143. The FGF-binding domain occurs in the carboxy (C) terminus of FGF-BP (Xie et al., 2006).

FGFs in the Development of the Central Nervous System and Neuronal Differentiation The morphogenesis of the central nervous system essentially involves the regulation of developmental programmes. Much effort has been expended on the differentiation of the cerebral cortex from the rostral regions of the neural plate and the formation from the epithelial cells of the walls of the neural tube of neurons and the glial component. The induction of ventral neuronal cell types in prospective forebrain regions of the neural plate and the differentiation of neuronal precursors have been studied in much molecular detail. The notochord together with the floor plate functions as an important signalling centre leading to the differentiation of oligodendrocytes, serotonergic neurons of the raphe nuclei of the brainstem and cranial motor neurons. The genetic expression profile of proliferation and differentiation of neuronal precursors and neuronal development in the cerebral cortex demonstrates the involvement of many genes, which is compatible with the occurrence of inordinate neuronal diversity. Notable among ligands involved in the cortical areas are growth factor genes. The expression of these genes and their participation in developmental programmes is determined by the expression of homeodomain, helix–loop–helix and T-box transcription factors, with distinctive topographical localisation. Among growth factors of note in this context are FGFs, which are essential elements in the development of the cerebral cortex. Experimental modulation of FGF signalling leads to changes in structure of the cortex (Iwata and Hevner, 2009). Also, FGF8 regulates the growth and polarity of dopaminergic axons of the midbrain (Yamauchi et al., 2009). The involvement of FGF in morphogenesis and in the affirmation of dorsoventral and rostrocaudal patterning, etc. is quite spectacular. Mutation in the Brachyury gene leads to abnormal notochord development attributed to defects in mesoderm formation and the differentiation of the notochord. FGF2 has been shown to induce mesoderm formation, notochord differentiation and Brachyury expression (Nakatani et al., 1996; Schulte-Merker and Smith, 1995). FGF in reciprocation maintains Brachyury expression, thus completing a regulatory loop (Schulte-Merker and Smith, 1995). FGF3 is required for proper morphogenesis to take place. Using the Ciona intestinalis model, Shi et al. (2009) have shown that FGF receptor mutation or inhibition of FGF3 activity leads to defective notochord cell intercalation. The Notch signalling pathway also appears to activate the Brachyury gene (Corbo et al., 1998). Early embryonic developmental processes such as gastrulation and morphogenesis of the neural tube and other organs involve the functioning of a family of T-box transcription factors of the Brachyury family. A considerable body of evidence about regulation of the expression of T-box genes, their interaction with other signalling

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pathways and the differential phenotypic effects that they exert in the developmental processes has accumulated in recent years. It has been known for some time that Brachyury gene activation is an early response to signals that specify induction of the embryonic axis (Kispert et al., 1995a). Brachyury appears to accomplish morphogenetic movements called the convergent extension of notochord cells. Several notochord specification genes act downstream of Brachyury and are involved in convergence/extension of the embryo. Suppression of some of these genes results in the failure of notochord cells to converge and complete or partial intercalation (Hotta et al., 2007). Indeed, as stated earlier, Brachyury encodes a tissue-specific transcription factor (T-box protein) that regulates expression of genes required for normal development of the mesoderm. Furthermore, activin A induces ectopic expression of the Brachyury transcription factor in the pre-primitive streak of chick blastoderms (Kispert et  al., 1995a,b). Nodal also upregulates the expression of Brachyury and Goosecoid. Inhibition of Alk4/5/7 receptors in turn inhibits Nodal upregulation. The involvement of Nodal is further confirmed by the requirement of Cripto for the Nodal-mediated enhancement of Brachyury expression (Nakaya et al., 2008). Subsequently, both FGF and activin at low concentrations have been shown to activate the Brachyury promoter, and activation is suppressed by paired-type homeodomain proteins Goosecoid, Mix.1 and Xotx2 (Latinkić et al., 1997). Some recent work has added more detail to the perceived inhibition by Goosecoid of Brachyury promoter activation. Messenger et al. (2005) found that N-terminal domain of Brachyury interacts with the C-terminal MH2 domain of Smad1, a component of BMP signalling pathway. When this interaction is interrupted, Goosecoid activation occurs, which in turn inhibits the activation of the Brachyury promoter. The association of the TGF-β family with FGF signalling also comes from the finding that BMP2-mediated induction of chondrogenic differentiation is accompanied by an upregulation of FGFR3 (FGF receptor 3) and a somewhat tardy FGFR2 response (Hoffman et  al., 2002). Overall, an inescapable conclusion is that T-box proteins might interact with other signalling proteins, for example TGF-β family members, to promote a specification course of morphogenesis. The interaction of FGF and TGF-β signalling can conceivably be regulated by the intervention of the FGF-BPs. As stated earlier, FGFs bind heparan sulphate proteoglycans and are anchored to the ECM. They are mobilised and activated by FGF-BP. FGF-BP protects FGFs from degradation and possibly chaperones them (Abuharbeid et  al., 2006; Czubayko et al., 1994; Wu et al., 1991). TGF-β represses the transcription of FGF-BP1 gene by binding to a short 3-base-pair (bp) sequence in the FGF-BP1 promoter. Downstream, TGF-β seems to use the conventional Smad2/3 pathway (Briones et  al., 2006). Therefore, TGF-β seems eminently capable of negating effective FGF signalling. Indeed, TGF-β is a known inhibitor of FGF-induced angiogenesis and there are indications that the inhibition of angiogenesis occurs through TGF-β type III (Bandyopadhyay et al., 2002). FGF interacts with the TGF-β family in cell fate determination, for instance in the induction of endoderm. Endoderm induction fails if the FGF/ERK pathway is activated, possibly thus negating Tar/activin A receptor IB (see below). When FGF signalling is inhibited, endoderm precursor cells increase in number together with

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enhancement of the activin receptor. BMP appears to regulate this process negatively (Poulain et  al., 2006). Tar (TARAM-A) is a Nodal receptor of the zebrafish. Tar does not seem to mediate BMP signalling (Aoki et al., 2002). From this the conclusion follows that FGF interacts with the signalling of both BMP and Nodal in the regulation of endoderm induction. Nie (2005) reported considerable overlap between BMP and FGFR expression. Early embryonic stages of differentiation of the tongue showed BMPs (2, 4 and 5) in mesenchymal tissue, and BMP3 and BMP7 in the epithelium. These BMPs were co-expressed with FGFRs, FGFR1c and FGFR2c in the mesenchyme and FGFR2b was in the epithelium. Thus, it might be reasonable to suggest that FGF and BMPs could jointly regulate differentiation and cell proliferation. Embryonic stem (ES) cells are derived from the inner cell mass of trophoblasts. These are pluripotent, undifferentiated cells. FGF2 can sustain the undifferentiated state of the stem cells. It seems to recruit activin and Nodal in the maintenance of this pluripotent state, and these act through the canonical Smad2/3 signalling pathway (Honda et al., 2009). The maintenance of the undifferentiated state of murine ES cells requires LIF (leukaemia inhibitory factor), which activates STAT3 signalling. Human ES cannot maintain pluripotency, even when the LIF-STAT3 signalling is active. Indeed, human ES cells remain pluripotent without displaying LIF-STAT3 activation (Dahéron et al., 2004). It is possible that the maintenance of self-renewing and undifferentiated state could be due to the provision of FGF signalling. On the other hand, ES cells can be induced to differentiate into specific cell types with the appropriate signalling molecules. Hansson et al. (2009) investigated the differentiation of mouse ES cells by monitoring the expression of markers of the primitive streak and its derivatives. Activin increased the numbers of cells that expressed neural-specific transcription factors Sox17 and Goosecoid, but this was inhibited by another TGF-β family ligand, namely BMP4. One might as well bear in mind in this context that FGF2 does not affect Goosecoid expression. So TGF-β family members might be involved as part of a complex regulatory mode in the maintenance of the undifferentiated status of embryonic stem cells. FGF signalling incorporates intermediates such as FLRT3 (fibronectin-leucinerich transmembrane protein). The FLRT family comprises three proteins that contain 10 leucine-rich repeats, a fibronectin-type domain and an intracellular tail (Lacy et al., 1999). These proteins possess two distinctive functions: promoting intercellular adhesion and participating in FGF signalling. Individual FLRTs show topographically distinctive distribution patterns (Haines et  al., 2006), which might possibly be related to the function they subserve. The leucine-rich repeat domain appears to mediate Ca2-dependent intercellular adhesion, and the intracellular domain in the cytoplasmic tail region mediates FGF signalling (Karaulanov et  al., 2006). FGF might function with activin and Nodal in the cell adhesion process. These last TGF-β family members induce the expression of FLRT3 as well as that of the small GTPase. Rnd1 might modulate intercellular adhesion by controlling the levels of cadherin at the cell surface (Ogata et al., 2007). FLRT3 is expressed at the cell surface and seems to promote neurite extension in vitro (Robinson et  al., 2004; Tsuji et al., 2004). Induction of neurite extension in PC12 cells seems to recruit SH2B1β. SH2B1β is an SH2B family adaptor protein that binds many receptor tyrosine

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kinases. This seems to activate the MEK-ERK1/2-STAT3-Egr1 signalling pathway (Lin et al., 2009). FGFs are closely involved in the induction of the endoderm and delineating the zone of mesoderm formation. The cells of the anterior visceral endoderm produce FLRT3, which is regarded as a positive regulator of FGF signalling. FLRT3 seems to regulate mesodermal marker genes such as Eomes, Brachyury/T and FGF8, because the expression of these is upregulated in FLRT3(/) mutants (Egea et  al., 2008). FLRT3 expression has been reported to occur during chick embryonic limb development (Smith and Tickle, 2006). These authors suggest that FLRT3 may regulate cellular adhesion between the apical epithelial ridge and the underlying mesenchyme, and establish the dorsoventral position of the ridge. Apart from the cell adhesive function, the co-expression of FLRT3 with FGF in the apical epithelial ridge (Haines et  al., 2006) suggests, albeit in a correlative fashion, its involvement in FGF signalling. Equally, FGF itself might function on its own. The presence of FGFR has been known to provide the required environment for neural crest cell migration in FGF-mediated specification of the differentiation of pharyngeal ectoderm (Trokovic et al., 2005). FLRTs display a defined pattern of expression in the differentiating somite. The somite differentiates into the ventromedial sclerotome, the dorsolateral dermatome and the myotome. Component cells of these migrate from the somite into their respective final destinations to differentiate into tissues according to their signalling specification. Cells from the dermatome migrate to form the connective tissue of the dermis and myotomal muscle precursor cells. FLRT3 is found to be co-expressed with FGF in muscle precursor cells of the dermatome that migrate to the myotome, and then in the muscle precursor myotomal cells (Haines et al., 2006).

FGF2 (Basic FGF) The FGF2 gene encodes four FGF2 polypeptides of 18–24.2 kDa. FGF2 participates in diverse cell functions and is strongly implicated in cell proliferation, apoptosis, cell motility, embryonic development, differentiation, wound healing and prominently in angiogenesis (Gospodarowicz et al., 1986; Rifkin and Moscatelli, 1989; Shing et al., 1984; Thomas and Gimenez-Gallego, 1986). FGF2 has been implicated in the growth and maturation of ovarian follicles (Schams et  al., 2009). Exposure of human ovarian tissue in vitro to FGF2 has shown an increase in the number of developing follicles and markedly enhanced oestradiol production (Garor et al., 2009). FGF also acts by negating growth arrest and inducing apoptosis. FGF2 is not only mitogenic but it can also induce the expression of pS2 (Garnier et  al., 2003), and possibly therefore its effects could be mediated by oestradiol production. FGF and other growth factors such as PDGF and IGF-1 (insulin-like growth factor-1) regulate FOXO (Forkhead box O transcription factor) at the transcriptional level. FOXO inhibits cell growth and induces apoptosis. However, FOXO is phosphorylated in response to these growth factors and in this way counteracts these effects (Essaghir et al., 2009). Many of these features are highly relevant in the growth, invasion and metastasis of cancer. A close correlation has been seen between the presence of FGFs and their

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receptors with metastatic ability of many forms of cancer. FGF1, FGF2, FGFR1 and FGFR2 have been reported to be overexpressed in colon cancer, and the overexpression of FGFR1 has been found to correlate with the presence of metastases in the liver (Sato et al., 2009).

FGF2-Mediated Promotion of Intercellular Adhesion The ability of FGF to promote adhesion to growth substratum and cell proliferation in vitro is well documented. The potential of interactive FGF signalling with other growth factors has been under scrutiny, especially for potential cross-talk between growth factor receptors in relation to cancer growth. FGF2 and FGFR1 IIIc and/or FGFR2 IIIc are co-expressed in NSCLC (non-squamous cell lung carcinoma) cell lines. Suppression of FGF2 inhibited anchorage-independent growth, and inhibition of the receptors inhibited basal FRS2 (FGF receptor substrate 2) adaptor protein and ERK phosphorylation (Marek et al., 2009). The phosphorylated form of FRS2 needs to associate with substrates to activate FGFR signalling. Possibly FRS2 might transmit FGF signals through subsidiary growth factor receptors to kinase signalling cascades. It is needless to reiterate that indeed there are a host of receptors possibly implicated in substratum attachment, mobility and cell proliferation.

FGF2 and Promotion of Neurite Extension The development of the nervous system incorporates several events such as the formation of neurons, neurite extension and neuronal guidance, migration of neuronal cells and the formation of synapses. These processes involve many signalling mechanisms. The process of neurite extension is a complex phenomenon encompassing the modification of adhesive interactions, motility-related features such as cytoskeletal dynamics and membrane transport. Heparin-binding proteins (Sakiyama et al., 1999), N-cadherin, NCAM (neural cell adhesion molecule) and integrins promote neurite extension (Bixby and Zhang, 1990; Colman, 1997; Doherty et  al., 1991; Drazba and Lemmon, 1990; Hansen et  al., 2008; Neugebauer et  al., 1988; Oblander et  al., 2007). Cadherins might function alongside other ECM components such as integrins, and their participation might relate to the stage of axonal development (Tomaselli et al., 2008). The neuronal cell adhesion molecule L1 induces neurite outgrowth, and Stallcup et al. (2000) identified the third fibronectin type III repeat occurring in L1 as being responsible for the ability of L1 to promote neurite extension. These are adhesion-mediating molecules. They also participate in downstream signalling systems. Cadherins are transmembrane proteins with an N-terminal extracellular Ca2-binding domain. The intracellular C-terminal domain binds several proteins, for example αand β-catenins and plakoglobin. The N-terminal domain of catenins links it to the actin cytoskeleton (Sherbet and Lakshmi, 1997). Over a decade ago, Lom et al. (1998) demonstrated experimentally that FGFR activation was required for N-cadherin to stimulate neurite outgrowth of retinal neurons.

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They showed that forced expression of dominant-negative FGFR in retinal neurons led to shorter neurite extension than a control group when the cells had been plated on N-cadherin substratum. Both FGF2- and N-cadherin-induced neurite extension was blocked by inhibitors of DAG lipase or Ca2-CAMKII (calmodulin kinase II). In confirmation of this early work, Boscher and Mège (2008) reported that cadherin-11 induces neurite extension, which is dependent upon FGFR activity and activation downstream of DAG lipase/CAM kinase and PI3-K, but not MAPK signalling. The possibility of other signalling systems interacting with the cadherin/catenin cascade cannot be excluded. β-Catenin has been described to regulate neurite extension negatively (Ouchi et  al., 2005), which would appear prima facie not compatible with induction of neurite extension by cadherins. However, Ouchi et  al. (2005) state that the negative regulation by β-catenin is mediated by the transcription factor LEF-1 (lymphoid enhancer factor-1). Now LEF1 is known to suppress the transcription of E-cadherin (Jamora et al., 2003). The β-catenin component is shared by Wnt signalling and the cadherin system (Nelson and Nusse, 2004). Furthermore, LEF has been closely identified with the Wnt/β-catenin signalling system (Kim et  al., 2002). This not only reconciles the apparent negative regulatory effect of β-catenin but also quite clearly implicates other signalling ligands in the process of neurite extension. The Hes1 transcription factor that figures prominently in Notch signalling seems to inhibit NCAM-induced neurite outgrowth in PC12 cells (Jessen et al., 2003). This could be viewed as interaction with the Notch signalling system that is actively involved in cell fate determination through Delta-dependent activation of Hes1. FGF2 is synthesised and secreted by astrocytes and functions in both a paracrine and an endocrine manner. Neural stem cells in culture show neuronal differentiation and migration when grown in the presence of FGF2, which upregulates the expression of bHLH (basic helix–loop–helix transcription factor neurogenin (Vergano-Vera et al., 2009). Ribes et al. (2008) have identified a 798-bp enhancer element upstream of the neurogenin-2 coding sequence that directs the early caudal expression of neurogenin-2; this is targeted by FGF, RA and Shh. Neurogenins inhibit stem cells from differentiating into glial cells by sequestering transcription factors required for glial differentiation and promotes neural differentiation by functioning as a transcriptional promoter (Sun et al., 2001). Neurogenins and NeuroD, another bHLH transcription factor that regulates neurogenesis, also influence cytoskeletal dynamics and migration of neurons (Seo et al., 2007). FGF2 promotes neurite extension in an autocrine mode of function. It seems to upregulate its own synthesis by astrocytes (Delgado-Rivera et al., 2009). FGF2 production seems to occur upon activation of classical dopamine receptor D-1 or D-2. Apomorphine, an agonist of these receptors, induces FGF2 expression which seems to occur from activation of cAMP/PKA and MEK/MAPK signalling (Li et al., 2006). Following this, Zhang et al. (2009) suggest that IP3/Ca2-CAMK might regulate the synthesis of FGF2 in astrocytes. FGF2 has been reported to upregulate N-cadherin protein but not E-cadherin expression in human calvaria osteoblasts and increase intercellular adhesion as determined in vitro by cell aggregation assays. The cell adhesion was inhibited by N-cadherin antibodies but not antibodies against E-cadherin. PKC inhibitors and

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src-family tyrosine kinase inhibitors counteracted these effects, suggesting the operation of these pathways in FGF2-promoted cadherin function (Debiais et  al., 2001). Although this study does suggest N-cadherin to be a target of FGF2, the alterations in the relative expression of N- and E-cadherin are totally opposite to that found in the induction of EMT by TGF-β. The loss of E-cadherin is virtually synonymous with loss of intercellular adhesion and acquisition of invasive ability. Indeed in TGF-β-mediated activation of EMT, there is a loss of E-cadherin and an increase in N-cadherin expression (see below). N-Cadherin supports growth cone extension. In this it seems to directly interact with FGFR occurring in the growth cones, and the MAPK pathway might be activated in the generation of axon extension (Doherty et  al., 2000). N-Cadherin and FGF2 have been found to activate certain genes associated with invasive behaviour of cancer cells. Here the activation of the MAPK/ERK pathway has been suggested as a possible signalling mode. In this experimental system, FGF2 seemed to bind to FGFR1, leading to the stabilisation of the receptor with consequent continued signalling by FGF2 (Suyama et al., 2002). Debiais et al. (2001) found neither MAPK nor MEK inhibitors affected FGF2-induced N-cadherin gene transcription. The conclusion from this is that different pathways might possibly be activated by the same ligands in different experimental systems and pathways of differentiation. There is also significant localisation of cadherin signalling cascade functions at synapses, and one might infer from this that cadherins could be involved in the formation of synapses by their ability to bind together adjacent neurons, possibly by altering cytoskeletal dynamics and Ca2 influx (Brusés, 2006). Following from the implication of FGF in promoting axonal extensions, the idea that FGF as well as other signalling ligands might be involved has taken root but awaits de facto establishment.

FGF2 and Osteogenesis Evidence has accumulated of the importance of FGF signalling in osteogenesis. Frenkel et al. (1992) showed that in the chick embryo, persistent treatment with FGF results in the formation both of osteogenic and chondrogenic cells. Inhibition of endogenous FGF-2 activity prevents cranial osteogenesis (Moore et al., 2002). FGF2 and heparan sulphate proteoglycans control osteoblast growth by regulating the activity of Runx2/Cbfa1, which is known to be associated with osteoblast proliferation and maturation. Runx2 has been found to upregulate the expression FGFRs and proteoglycans, which possibly indicates that mediation by proteoglycans is essential for the osteogenic process and is regulated by FGF2 and Runx2 (Teplyuk et al., 2009). The thought of FGF participation in the osteogenic process also comes from the finding that BMP2-mediated induction of chondrogenic differentiation is accompanied by an upregulation of FGFR3 (FGF receptor 3) and a somewhat tardy FGFR2 response (Hoffman et  al., 2002). T-box transcription factors have been implicated here on account of their function in signalling by growth factors of the TGF-β family.

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FGFs 1 and 2 Regulate Cell Proliferation It has been known for some time that FGF1 and FGF2 induce cell proliferation and differentiation in a wide variety of cells and are associated with angiogenesis. FGFs induce proliferation of mesodermal and epithelial cells. They markedly stimulate proliferation of endothelial cells. One of the modes of the proliferative effects has been known to involve interference in apoptosis signalling. Bryckaert et al. (1999) showed that FGF2 markedly inhibited caspase-dependent apoptosis of retinal pigment cells. Levels of apoptosis increased when cells were treated with antisense FGF1, suggesting its involvement in the regulation of FGF2-mediated cell survival. FGF2 effects were mediated by the activation of FGFR1 and ERK2 signalling. Furthermore, FGF2 increased the expression of the pro-apoptotic Bcl-x gene. In confirmation, inhibition of ERK2 activation and downregulation of Bcl-x expression inhibited promotion of survival by FGF2. Endogenous FGF1 has been found to inhibit p53-dependent apoptosis. In the presence of FGF1, an enhancement of mdm2 expression both at messenger RNA (mRNA) and protein levels has been reported in rat embryonic fibroblasts, which led to enhanced degradation of p53. It was also noticed that when FGF1 was present the pro-apoptotic bax was not transactivated by p53 (Bouleau et  al., 2005). Bouleau et al. (2007) have demonstrated an FGF1-mediated negation of p53-dependent apoptosis in PC12 cells. The experimental work described by Wang et al. (2005) on foetal lung transplants in vitro suggests the operation of PI3K-Akt signalling. FGF1 treatment increased the number of terminal buds representing lung primordia in the explants and increased proliferation, but this was inhibited by PI3K and MAPK inhibitors, which obviously led to cell survival. As noted earlier, FGF and other growth factors such as PDGF and IGF-1 induce Akt-mediated phosphorylation of FOXO, which inhibits apoptosis and growth arrest induced by the transcription factor (Essaghir et al., 2009). FGF1 can signal through membrane integrin components. Mori et  al. (2008) showed that FGF1 binds directly to αvβ3-integrin. They also showed that a mutant FGF1 called R50E was defective in integrin binding and was unable to elicit the usual effects inducing cell proliferation, DNA synthesis and cell motility normally exerted by wild-type FGF1. However, R50E did not affect AKT and ERK1/2 signalling, which suggests the mutant affected further downstream events.

FGFs 1 and 2 and the Induction of Angiogenesis Angiogenesis is an important requirement for several biological processes such as embryonic development, wound healing and chronic inflammation. It is also a key prerequisite for the successful establishment, growth and dissemination of tumours. Tumour growth needs adequate vascularisation. In the early avascular phase, tumour growth is self-limiting, but in the vascular phase tumours grow rapidly (see Sherbet and Lakshmi, 1996). FGF1 and FGF2 have both been linked with the process of angiogenesis. Both are capable of inducing endothelial cell proliferation and angiogenesis

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associated with wound healing. FGFs have been implicated in angiogenesis associated with endometriosis. S100A13 is overexpressed in endometriosis, and the association of this calcium-binding protein with FGF function has led to the suggestion that it might be involved in the induction of angiogenesis in endometriosis through FGF function (Hayrabedyan et al., 2008). The angiogenic function of FGF2 encompasses the basic processes of endothelial cell proliferation, migration and extracellular proteolysis (Gospodarowicz et al., 1986, Montesano et al., 1986, Tsuboi 1990). Tumours produce several factors with angiogenic ability; among them are FGF2, VEGF, TGF-β and hepatocyte growth factor. However, the modes of signalling involved are not well understood. Chen et al. (2007) showed that the induction of angiogenesis by FGF1 is blocked by fumagillin. The latter downregulated PI3K/Akt signalling. The inhibition seems to have been brought about by binding of fumagillin to the cytoplasmic domain of FGFR. FGF2 is also produced by normal cells. It can act in an autocrine fashion on endothelial cells, stimulate their proliferation and induce angiogenesis. It can also act synergistically with VEGF by upregulating the expression of the latter (Mattern et al., 1997; Pepper et al., 1992; Stavri et al., 1995). On the other hand, VEGF might be required in FGF2-mediated induction of angiogenesis. Tille et al. (2001) found that the effect of FGF2 is negated by VEGF receptor inhibitors (see section on cystine knot growth factors for a detailed discussion of the biological properties, the signalling pathways and the relevance of VEGF in the context of cancer spread).

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16 Intracellular Receptor Binding Growth Factors

Intracellular receptor binding ligands are identifiable as: (1) nuclear receptor binding growth factors, for example oestrogen, progesterone, VD3 and retinoids, and (2) cytoplasmic receptor binding ligands such as the glucocorticoids. The physiological effects of several hormones, androgen, vitamin D3, thyroid hormones, retinoids, and oestrogen and progesterone are mediated by the activation of a large family of nuclear receptors. Activated receptors dimerise and bind to responsive elements in the regulatory regions of target genes and induce or repress their transcription (White and Parker, 1998) of responsive genes, resulting in appropriate physiological function. Thyroid hormone receptors (TRs), for instance, repress transcription of target gene in the absence of the ligand but activate transcription when the ligand is present (Koenig, 1998). TRs associate and form complexes with co-repressors such as N-CoR and SMRT (McKenna et al., 1999; Xu et al., 1999). When bound to the ligand, TRs complex with co-activators (Yao et al., 1996). Because many of the co-repressors and co-activators have histone acetyl transferase activity, the processes of genetic repression and activation are believed to involve remodelling of chromatin (Chen et al., 1997; Spencer et al., 1997). Steroid hormone can often function in a non-genomic mode of action through receptors located in the cell membrane and mitochondria (Alexaki et al., 2006; Benten et al., 1997; Lösel et al., 2003; Scheller et al., 2003). The androgens regulate the growth and differentiation of the normal prostate and are closely implicated in the pathogenesis of prostate cancer. Androgens enter into interactive regulation with growth factors, often influencing mutual expression and so contribute to the progression of prostate cancer. Steroid hormones as well as glucocorticoids are known to modulate the biological function of IGFs. Oestrogens can harness insulin and IGF signalling, and oestrogen and progesterone receptors are known to engage with signalling through EGFR, and indeed not insignificantly with IGFR and EGFR interaction in tamoxifen resistance. Glucocorticoid receptors on the other hand bind to and activate cytoplasmic receptors. They then translocate into the nucleus to activate genetic transcription. As proposed at the beginning of this book, certain basic features such as the structure of the growth factors, the nature of receptors that they bind and the signalling cascade they activate have formed the focus of discussion. In this section, growth factors and hormones that function through intracellular, nuclear and cytoplasmic receptors have been discussed.

Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00016-5 © 2011 Elsevier Inc. All rights reserved.

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17 The Androgens and Androgen

Receptors in Development, Differentiation and Neoplasia

The androgens are steroid hormones that regulate the development of male sex organs and secondary sexual characteristics. Dihydrotestosterone, which is responsible for most androgenic functions, and oestradiol are conversion products of testosterone. The latter can convert to androstenedione and in turn into the oestrogen oestrone. Androgens also regulate the growth and development of the prostate, are closely related to the development of benign prostatic hyperplasia (BPH) and are involved in the development and progression of prostate cancer. The progression of prostate cancer is also accompanied by transition from an androgen-dependent state to an androgen-independent state. Several factors could be operating in bringing about this transition. The expression and functional integrity of androgen receptor (AR) are an obvious source. Interaction with other signalling systems could conspicuously alter the physiological outcome. Modulation and switching of signalling pathways can be seen as an important aspect of this transition. Progression to the androgen-independent state could comprise the transactivation of AR by other ligands. Structural modifications of AR resulting from mutation could provide another possible route to the altered state of responsiveness.

Androgen Receptor and Androgen-Responsive Elements Androgen receptor is a member of the steroid and nuclear hormone receptor family, which includes glucocorticoid and mineralocorticoid receptors, ER, PR and the vitamin D3 receptor (Mangelsdorf et al., 1995). The activation of AR, a nuclear transcription factor, begins with ligand binding, leading to the transcription of several responsive target genes that delineate its role related to its physiological functions as well as development and progression of cancer. Several target genes may be cited, among them are those coding for PSA (prostate-specific antigen, a kallikrein-like serine protease that is wholly restricted to the luminal epithelial cells of the prostate) and NKX3.1, a suppressor protein that is downregulated in prostate cancer (Kim and Coetzee, 2004), MMPs (Li et al., 2006), Ezrin, which is involved in the regulation of the actin cytoskeleton (Chuan et al., 2006) and p21 cyclin-dependent kinase inhibitor (Lu et al., 1999). The signalling process begins with the dimerisation of the receptor, which leads to the binding to cognate response elements and the recruitment of co-regulators, Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00017-7 © 2011 Elsevier Inc. All rights reserved.

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identifiable as co-activators or co-repressors, which modify and stabilise the chromatin and make it conducive to transcription. Both tightly control the transcription of target genes. Androgen response elements (AREs) occur in the promoter regions of other genes that are regulated by androgen and in genes where androgens function as co-regulators. Androgens regulate the expression of PSA through AR, which interacts with elements in the regulatory regions of target genes (Reigmen et al., 1991). Several AREs have been identified; they are AR specific but differ in their affinity (Claessens et al., 2001). In PSA, which is regarded as a marker of prostate cancer, AREs occur in the proximal promoter. Control elements also occur outside the proximal promoter. The classical steroid receptors AR and PR, and gluco- and mineralocorticoid receptors, recognise an element made up of two hexameric 5-AGAACA-3 motifs as inverted repeats with a three-nucleotide spacer. As opposed to these, selective AREs have been identified, which consist of two hexameric motifs occurring as direct repeats with a three-nucleotide spacer. This organisation and orientation of the motifs is seen as a means that confers AR-specific gene activation. Co-activators are recruited to bind to the promoters of target genes at AF-1 and AF-2, the two transcription activation domains that form a familiar feature of nuclear receptors (Beato and Sanchez-Pacheco, 1996; McKenna and O’Malley, 2002; McKenna et al., 1989). The SRC (steroid receptor co-activators) are structurally related and possess a basic helix–loop–helix and two PAS (Per–Arnt–Sim) domains. The helix–loop–helix/PAS domain participates in DNA binding and is a protein dimerisation motif of many transcription factors (Leo et al., 2000). Notable SRC family members are SRC-1 (nuclear receptor co-activators 1), SRC-2 (TIF2/ transcription intermediary 2) and SRC-3 (p300/E1A binding protein) (Auboeuf et al., 2002; Hittelman et al., 1999; McKenna et al., 1999; Onate et al., 1995; Roy et al., 2001; Torchia et al., 1997). SRC-1 is required for growth, differentiation and organogenesis. SRC-2 interacts with glucocorticoid receptors and SRC-3 is involved as an activator of thyroid and retinoid receptors. SRC-3 is involved in growth regulation, cell division and differentiation. Several co-repressors have also been identified. Cyclin D1, a regulator of G1–S transition of the cell cycle (Petre et al., 2002), RAD proteins involved in DNA replication, repair and recombination (Wang et al., 2004), N-Cor (nuclear receptor co-repressor) (Wu et al., 2006) and ARA70α function as co-repressors. ARA70α is a co-repressor, of which the full-length isoform ARA70α inhibits prostate cancer cell proliferation and tumour growth, whereas the spliced isoform ARA70β promotes prostate cancer cell growth and invasion (Ligr et al., 2010). These co-regulators subserve diverse phenotype-related functions. Thus, different co-activators may be required for the transcription of specific genes and individual co-repressors might be associated with the regulation of specific genes. In this way, the co-regulators might be responsible for determining the course of AR function.

AR-Activated Signalling Pathways Two major pathways are activated by AR. AR uses the Ras/RAF/ERK1/MAPKmediated signalling to drive prostate cancer progression. It is expressed in breast

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cancer, and the ERK/MAPK system has been found to be most active in many breast cancers. The second one is the PI3K/Akt pathway, which is operated by many growth factors and has also been implicated in AR signalling. AR is phosphorylated at serine residues 213/210 and 791/790 by the PI3K/Akt pathway, which is also activated by growth factors such as IGF-1 (Lin et al., 2003). The involvement of PI3K/ Akt is suggested by the loss of PTEN expression. Inhibitors of PI3K can inhibit the transcription activity of AR (Li et al., 2001). The Akt gene is not amplified or Akt protein expression upregulated in prostate cancers compared with normal tissue, but poorly differentiated tumours do show Akt activation which contrasts with normal tissue or well-differentiated prostate cancers (Ghosh et al., 2003). There are many checks and balances in the flow of information along the PI3K/ Akt pathway. The mammalian target of rapamycin (mTOR) is a downstream element of PI3K/Akt signalling and has turned out to be a meeting point of several signalling systems. In this way mTOR could facilitate cross-talk between growth factor signalling pathways, including signalling by androgens. The classical pathway involves PI3K-mediated activation of Akt, leading to cell survival. PI3K can also activate mTOR, which can function through two further downstream effectors, namely the eukaryotic initiation factor 4E (eIF4E) and the p70 ribosomal S6 kinase 1 (S6K1). These effectors enable mTOR to control cell growth and to promote cell cycle progression and proliferation effectively (Fingar et al., 2002, 2004). Signalling through mTOR has also been linked with the MAPK cascade (Ras/Raf/ MEK/ERK) pathway, often implicated in tumorigenesis. Inhibition of mTOR leads to activation of ERK and signalling along the MAPK pathway (Figure 17.1). PTEN, an inhibitor of Akt, would therefore be able to regulate both the classical Akt signalling as well as mTOR-mediated cell survival signalling (Sawyers, 2008). AR and EGFR cross-talk upregulates cyclin D1 in prostate cancer cells exposed to EGF and

Figure 17.1  The integration of PI3K/Akt and the MAPK signalling cascade by mTOR. In addition, PI3K can influence cell growth and proliferation through mTOR-mediated activation of S6K1. IRS, insulin-receptor substrate; S6K1, ribosomal protein S6 kinase, 70-kilodalton (kDa), polypeptide 1.

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5α-dihydrotestosterone. Inhibition of p38 MAPK and mTOR inhibits the upregulation of cyclin D1, suggesting AR and EGFR signalling for the stimulation of cell growth is integrated by mTOR (Recchia et al., 2009b). This integrating function of mTOR, the recognition of the frequent upregulation of PI3K/Akt/mTOR, possibly with an associated loss of PTEN, and the effects of this upregulation on the growth and progression of prostate cancer have inevitably led to proposals towards the deployment of mTOR inhibitors in therapy. Kobayashi et al. (2010) have reported that treatment of PC3 and DU145 cells with the mTOR inhibitor rapamycin reduced cell proliferation together with a decrease in the activation of the downstream effector S6 kinase. Combination of mTOR inhibitors with PI3K/Akt inhibitors has proved to be effective in inhibiting cell proliferation of prostate cancer cell lines in vitro, with confirmation from in vivo studies of tumour growth in compatible murine hosts (see Kinkade et al., 2008).

AR Expression in Cancer Progression Androgen receptors are found in BPH and in all histological types and clinical stages of prostate cancer. Hence androgen function might be involved in the progression of the disease. ARs are not restricted to prostate as a responsive tissue. Because androgens are involved in the development of the prostate itself, it is reasonable to suppose that AR occurs in the developing prostate as well. Androgen binds to AR and stimulates the transcription of androgen-responsive genes, including those that regulate the growth of prostate cells. AR has been detected in approximately 15% of renal cell carcinomas (RCCs), where its expression was significantly associated with early-stage pT1 compared with pT3 and low-grade carcinomas. Also, AR RCCs showed better prognosis than AR-negative cases (Langner et al., 2004). Breast cancers express AR frequently (70–80%), mainly in ER cancers (Castellano et al., 2010; Niemeier et al., 2010). According to Castellano et al. (2010), AR expression is an independent indicator of prognosis in patients with ER breast cancers. It is associated with small tumour size (Niemeier et al., 2010), so could relate to favourable prognosis because some regard small tumours to be less prone to metastasise. However, with the presence of ER the treatment modality has to come into the reckoning. AR expression is low (10–35%) in triple-negative (i.e. not expressing ER, PR and HER2) tumours, whereas HER2 tumours show a much higher incidence of AR (Gucalp and Traina, 2010; Niemeier et al., 2010; Park et al., 2010), suggesting possible interacting influences of other growth factors. One might recall that Schuurmans et al. (1989) showed that EGF stimulates the growth of LNCaP cells and increases their EGFR expression. Indeed, AR interacts with growth factor signalling by EGF and HER2. HER2 activates AR signalling and promotes prostate cancer growth independently of androgen both in vitro and in vivo (Craft et al., 1999). HER2 induces PSA through MAPK signalling mediated by AR (Yeh et al., 1999). Thus, although there is evidence for transactivation of AR, precisely how growth factors might affect AR expression is unclear. Nonetheless, the therapeutic potential of deploying AR deserves much thought.

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Functional Integrity of AR and AR-Mediated Signalling The structural integrity of receptors and the functional integrity that flows from it are a basic requirement of successful signalling. If this is compromised, deregulation of signalling is the inevitable outcome. Therefore, it is important to address the question of whether genomic changes of AR could be a cause of progression of cancer. Two isoforms of AR of 110 and 87 kDa are found in normal colon, but only the 87-kDa isoform is detectable in colon carcinoma. CAG repeat tracts occur in exon 1 of AR. CAG tracts have been linked with many genetic diseases (see Sherbet, 2003). The potential significance of these to cancer risk, progression and prognosis has been scrutinised in many tumours, for example prostate, endometrial and breast cancers and hepatocellular carcinomas. A diminution of CAG repeat length is believed to lead to cancer development. Deletion of the repeats generates truncated isoforms. The truncation occurs in the amino (N)-terminal region corresponding to the diminished CAG repeat region (Ferro et al., 2002). Short repeats in AR have been associated with higher risk of developing prostate cancer (Giovannucci et al., 1997; Ingles et al., 1997). A correlation between the incidence of shorter repeat tracts and tumour grade has been reported but not with prognosis (Cude et al., 2002). However, there are reports that shorter CAG tracts might protect from cancer risk (Elhaji et al., 2001; Giguere et al., 2001; Rebbeck et al., 1999). Potential links between tract lengths and cancer risk have been reported in familial cancers, suggesting other genetic factors might control the outcome in association with the CAG repeat state. BRCA1 and BRCA2 are suppressor genes of which mutation or loss markedly correlates with familial breast cancer. Li et al. (2010) have found that in patients with BRCA2 mutations, shorter CAG tracts corresponded with poorer prognosis than in patients with longer repeat tracts; but tract lengths per se did not reflect prognosis. Among other relevant considerations is the configuration of the tracts, which is said to determine their stability and that of the DNA. Their polymorphism might therefore influence the responses that AR elicits from responsive tissues.

Interaction of Androgen and Growth Factor Signalling Apart from changes in the hormonal milieu, interaction of androgen signalling with other growth factors has been implicated in disease processes. AR interacts with the signalling by many growth factors, for example EGF, FGF, IGF, VEGF and TGF-β, especially in the development and progression of prostate cancer. Indeed, EGF, ILs and IGF-1 appear to be able to transactivate AR in a physiologically androgen-independent environment. The expression of AR messenger RNA (mRNA) has been reported to correlate with HER2 mRNA levels in the invasive stage of prostate cancer (Brys et al., 2004). The interaction of androgen and growth factor signalling has come into prominence because the progression of prostate cancer is associated with a change from an androgen-dependent to an androgen-independent state, analogous to some steroid receptor (ER/PR) negative breast cancers becoming responsive to growth factor

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stimulation. Carcinomas of the prostate have notably been described as being androgen dependent at early stages of their development, but become independent of this hormone during progression of the disease. Androgen signalling might be able to activate growth factor signalling, which could be the mechanism in the development of the androgen-independent cancer phenotype. Androgens regulate the expression of the prostate-specific antigen (PSA). They regulate PSA gene transcription by means of the androgen-responsive elements that occur in the promoter region of the PSA gene (Riegman et al., 1991). Probably cisacting elements are also involved in the androgen receptor mediated transcription of PSA (Zhang et al., 1997). The PSA-related protein, human glandular kallikrein-1, is also regulated by androgens. The gene that encodes it contains an androgen-responsive element (Murtha et al., 1993). Some recent experimental work has shown that PSA can inhibit endothelial cell proliferation and the angiogenic effects of FGF and VEGF. In the mouse model, PSA has been found to reduce metastatic deposition of tumour cells in the lungs (Fortier et al., 1999). Also, its expression has been reported to increase from benign epithelium through intraepithelial neoplasia to carcinomas (Darson et al., 1997). In the mouse model, PSA has been found to reduce metastatic deposition of tumour cells in the lungs (Fortier et al., 1999). Contrary to this, there are reports that its expression increases from benign epithelium through intraepithelial neoplasia to carcinomas (Darson et al., 1997). As mentioned in an earlier section, breast cancers express AR frequently (70–80%). Mainly in ER cancers, AR expression is an independent indicator of prognosis in patients with ER breast cancers. AR expression is low (10–35%) in triple-negative tumours, whereas HER2 tumours show a much higher incidence of AR. This suggests possible interacting influences of other growth factors. EGF stimulates the growth of LNCaP cells and increases the levels of EGFR. AR can interact with factor signalling by EGF, IGF and HER2. HER2 is known to activate AR signalling and promote prostate cancer growth independently of androgen both in vitro and in vivo through activation of MAPK signalling mediated by AR. In summary, there is a large body of evidence indicating the transactivation of AR by growth factors.

18 Vitamin D

in Cell Proliferation, Apoptosis and Differentiation 3

Vitamin D3 (VD3) (1α,25-dihydroxyvitamin D3) is the metabolised active form derived from 7-dehydrocholesterol. VD3 subserves several physiological functions, for example calcium absorption, calcium mobilisation in bone, calcium reabsorption, cell differentiation, cell proliferation and apoptosis. The structure, metabolism and many of the diverse functions of VD3 have been reviewed in depth (DeLuca, 1983, 2004). Here, the focus is on cell proliferation, survival and differentiation, and on the function of VD3 and its receptor in cancer growth and progression. The effects are mediated by VD3 receptors (VDRs), which belong to the family of intracellular receptors alluded to earlier.

The VD3 Receptor, VDRs and VDR Response Elements The VDR is composed of a DNA-binding C-domain, a ligand-binding E-domain and an activating F-domain. VDR recognises and binds response elements (VDREs), which occur near the start site of responsive genes. VDREs represent six-nucleotide (PuG(G/T)TCA) repeats with three-nucleotide spacers (DeLuca, 2004; DeLuca and Schnoes, 1983). VDREs with direct repeats separated by three base pairs (bp) are called DR-3 type; those with direct repeats with four or six spacing nucleotides are the DR-4 and DR-6 types. IP-9 type VDREs are composed of inverted palindromes of the motifs with nine nucleotide spacers. VDREs may be described as negative and positive in function. Many genes are downregulated by VD3 and these target genes have been postulated to possess negative VDREs. The negative functional character, however, is dependent upon chromatin organisation, and occasionally by the presence of more than one promoter region that might be involved in the downregulation of expression. Target genes might contain both positive and negative VDREs. Human osteocalcin has been described to have DR-3 and DR-6 VDREs. Alterations in chromatin organisation as a consequence of ligand binding might lead to selective functioning of either. The VDR transcription complex, in common with events that occur with other nuclear receptors, might recruit co-activator and suppressor proteins to facilitate or inhibit the transcription of genes. Several co-activators of the SRC (steroid receptor co-activator) family have been identified, for example SRC-1/NCoA-1, Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00018-9 © 2011 Elsevier Inc. All rights reserved.

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TIF2/NcoA-2, ACTR/pCIP/AIB1/RAC3 TRAM-1, CBP/300, and DRIP/TRAP (vitamin D interacting protein/thyroid hormone-associated protein). Many co-activators possess a consensus motif at the receptor interaction sites (Torchia et al., 1997). A co-activator NCoA62/SKIP not belonging to the SRC family has been described. This binds promoters of VDR target genes of ROS17/2.8 osteosarcoma cells in a VD3-dependent manner (Zhang et al., 2003). Transcription factors such as the retinoic acid receptors (RARs) can be viewed as partners of heterodimeric co-activation. VDR can also show dimerisation after interaction with the co-activators SRC-1 and TRAM-1 on VDRE and enhance ligand-induced gene transcription (Takeshita et al., 2000). Despite the consensus of the receptor-interacting motif, the co-activators subserve different functions (Oda et al., 2007; Rachez et al., 2000) and therefore are capable of determining the specificity of target gene transcription. Oda et al. (2007) have gone on to suggest they might be involved with sequential regulation of genes in transition of the cell from the proliferative to the differentiated state.

VDR Signalling in Cell Proliferation, Apoptosis and Differentiation VD3 elicits responses from a panel of genes that regulate cell cycle progression and the expression of ECM-associated macromolecules that determine cell shape, adhesion faculty and motility. VD3-mediated induction of differentiation and modulation of cell proliferation is accompanied by the upregulation of the expression of cdk inhibitors p21waf1 and p27kip1 (Campbell et al., 1997; Matsumoto et al., 1998), so markedly controlling cell proliferation. The participation of VDR in the inhibition of cell proliferation and promotion of differentiation is supported by the fact that VDR induces the cdk inhibitors p21 and p27 and in this way suppresses cell proliferation (Figure 18.1). Indeed, the promoter of the p21 gene has VDR-binding sites, of which two also bind p53 (Saramaki et al., 2006). A link with p53 functioning is worth recording here. The p53 protein and other members of the p53 family, namely the homologue p63, are able to induce VDR expression, which could indeed lead to the inhibition of cell proliferation. The other potentially important interacting p63 isoforms δ-Np63 (amino-deleted) and TAp63 (transactivation-active) would no doubt influence the outcome in terms of cell proliferation and apoptosis by modulating VDR expression. The isoform TAp63 is a pro-apoptosis protein; δ-Np63 is anti-apoptosis (Candi et al., 2007). Besides, δ-Np63 protein binds to and suppresses VDR expression (Kommagani et al., 2009). Compatible with the inhibition of proliferation is the ability of VDR signalling to induce differentiation. For instance, VDR induces osteocalcin and osteopontin expression and promotes differentiation (Sherbet, 2001). Regulation of differentiation has been postulated to involve the induction by VD3 of hKSR-1 (kinase suppressor of Ras-1) and hKSR-2. VD3 has been found to upregulate the expression of the antiapoptotic hKSR-2 in HL60 leukaemic cells. Wang et al. (2006) have identified a VDR-binding element in the hKSR-1 promoter. Inhibition of apoptosis

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Figure 18.1  Possible routes by which VDR promotes differentiation and inhibits cell proliferation. Also shown is induction of cadherin expression by calcium-mediated induction of ERK/MAPK signalling and changes in cell adhesion properties. Of interest here is that cadherin is a known suppressor of cell motility, and VD3 might contribute to tumour suppression by this route, with the collaboration of Wnt/catenin signalling. Cell proliferation might be regulated by the induction of p21 and p27, which are inhibitors of cell cycle progression, and by the promotion of apoptosis by the PTEN/Akt pathway. The potential interaction of p53 and the p63 isoforms δ-Np63α and TAp63, leading to the modulation of VDR expression, is not shown here.

occurred by the involvement of Bcl-2 family genes and caspase-3. From these observations has arisen the postulate that there is an optimal cell survival requirement for the protection of differentiating cells (Wang et al., 2007, 2008). It is possible that the perceived inhibition of apoptosis in relation to the differentiation of HL60 leukaemic cells might be a specialised event associated with monocyte-like cell differentiation. On the other hand, VD3 seems able to induce PDF (prostate-derived factor). PDF promotes apoptosis and inhibits cell proliferation. For VD3 to induce PDF, functional p53 is obligatory (Lambert et al., 2006). The activation of caspases has also been implicated on a provisional basis; the intervening signalling events have not been elucidated. It is needless to reiterate that the ability of VD3 to inhibit proliferation and promote apoptosis might be regarded as well established. The Akt signalling system is a prominent player in the regulation of cell proliferation and apoptosis. That VD3 might activate the Akt apoptosis signalling is evident from some recent work demonstrating the promotion of apoptosis of gastric cancer cells by VD3 treatment. The induction of apoptosis was accompanied by PTEN upregulation (Pan et al., 2010) (Figure 18.1).

Modulation of Cell Adhesion and Motility The ECM has an important part in determining cell adhesion, tissue cohesion and cell motility. VDR signalling can restructure and remodel the ECM by targeting specific components of it. Genes coding for fibronectin (FN) and β3-integrin possess

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VDRE (Polly et al., 1996). VD3 analogues are known to upregulate the expression of the transmembrane protein E-cadherin, a suppressor of cell motility, in prostate cancer cell lines (Campbell et al., 1997). The effects of VD3 on the adhesion-mediated function of cells seem to be a result of the activation of MAPK signalling. VDR signalling by this pathway has been found to upregulate E-cadherin. The Wnt/catenin pathway, which also regulates intercellular adhesion, appears to contribute to this effect of VDR (OrdonezMoran et al., 2008). This is to be expected because cadherin is a component of the Wnt/β-catenin signalling complex. The activation of ERK/MAPK can also lead to the induction of VDR expression (Cañadillas et al., 2010), which could accentuate the physiological effects of VDR. Earlier, Cordes et al. (2006) showed that VD3 activated ERK/MAPK in two distinct phases: an early phase of brief exposure of breast cancer cells to VD3 and another phase of activation upon exposure of the cells to treatment for 2–24 hours. Interestingly, the early phase activation occurred even in VDR-negative cells. This finding could have a bearing on the possible induction of VDR by the initial phase of ERK/MAPK activation (summarised in Figure 18.1). Another adhesion-mediating molecule that is modulated is ICAM-1 (intercellular adhesion molecule). VD3 derivatives have been reported to downregulate ICAM expression in peripheral blood mononuclear cells as well as in HUVEC endothelial cells derived from normal subjects and from patients with inflammatory bowel disease (Martinesi et al., 2008). ICAM is a ligand for integrin-αL/-β2 of the ECM and is involved with diapedesis of leukocytes across the endothelium. Treatment of WEHI-3 and LLC cells with VD3 or its metabolic derivative 1,24-dihydroxy-VD3 reduced α(v)β(3)-integrin expression, and this loss corresponded with loss of cell migration (Wietrzyk et al., 2008). Finally, Bao et al. (2006a) had shown some time ago that VD3 reduced the invasive behaviour of LNCaP, PC-3 and DU 145 cells, with a concomitant reduction in the expression of MMP-9 and cathepsin with corresponding increases in the expression of their inhibitors. Bao et al. (2006b) have followed this up with the demonstration that VD3 inhibited endothelial cell migration and endothelial tubule formation. There is recent confirmation that VD3 indeed inhibits endothelial cell proliferation (Chung et al., 2009; Gonzalez-Pardo et al., 2010). Chung et al. (2009) also showed VD3 inhibits tumour vasculature in TRAMP-2 murine tumours. In short, VDR can signal the restructuring and remodelling of the ECM, alter the adhesive interactions between cells and diminish the expression of ECM components that are conducive to cell motility and invasion. VDR can also inhibit invasion when it is itself modulated by other means. As stated before, TP63 occurs in two isoforms, namely the pro-apoptosis TAp63 and antiapoptosis δ-Np63. The isoform δ-Np63 protein binds to and suppresses VDR expression (Kommagani et al., 2009). Despite the well-established inhibitory effects of VD3 on biological parameters related to cancer progression, the evidence about how it affects prognosis is still ambivalent. Although in vitro assays are a good indicator of the potential of VD3 to regulate these features, direct inquiries using human tumour material are required, with adequate provision of histopathological and clinical data and supporting information about the expression of the various modifiers of biological responses.

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Collaborative Function of VDR and RA Receptors VDR signal determining cell differentiation is amplified by its concerted and combined function with retinoic acid receptors (RARs). RARs function as transcription factors in articulation of the transcription of genes required for cell differentiation, morphogenesis and neoplasia. They are known to heterodimerise with other transcription factors. VD3 induces VDR to form a heterodimeric transcription complex with RXR, a form of RAR, which then binds to the VDREs, leading to the regulation of cell proliferation (Carlsberg and Polly, 1998; Sherbet, 1997, 2001). The 5 region of VDRE binds the RXR receptor component; the 3 arm binds the VDR component of the transcription complex. There is a thought that RXR might function as a co-activator of VDR.

wwwwwww

19 Retinoids in Development and Pattern Formation

Developing systems involve multiple pathways of signalling that interact and co-ordinate the flow of information, leading to the unfolding of morphogenetic events and pattern formation. The versatility of the modes of retinoid function is further reflected by the interaction and cross-talk between retinoid signalling and signalling by growth factors and hormones, and the activation or alteration of expression of a variety of responsive genes. The responsive genes that are recognised include oncogenes, metastasis suppressor and promoter genes, and genes involved with the remodelling of the extracellular matrix. Retinoids also modulate cytoskeletal dynamics. Retinoids were recognised many years ago as being able to alter cellular responses to epidermal growth factor, thyroid hormones, granulocyte colony-stimulating factor, interleukin secretion, etc. (Parker and Sherbet, 1992; Sherbet, 1997). The active participation of retinoid signalling begins with early embryonic development, which takes place in conjunction with other biological response modifiers. The embryonic epiblast cells contain RA-responsive cells. RA-deficient cells show defects in early caudal patterning arising from alterations in the expression of genes involved with neurogenesis and morphogenesis of the embryonic spinal cord (Ribes et al., 2009).

Retinoic Acid Receptors Signalling by retinoids leading to the regulation of cell proliferation, apoptosis and differentiation is mediated by retinoic acid receptors (RARs). The RARs possess highly conserved domains that determine their ligand- and DNA-binding properties. Three RARs identified initially were RAR-α, RAR-β and RAR-γ (Krust et al., 1989; Lehmann et al., 1991). RXR is another member of the RAR family. RXR-α, RXR-β and RXR-γ are RXR products of three genes. Distinctive functions have been attributed to the different receptors. RXR functions as a heterodimer with RAR, which takes part in the differentiation of myeloid cells.

RAR-Mediated Biological Effects RARs generate a diversity of phenotypic effects by transactivation of a spectrum of genetic determinants. Among them are cell proliferation, apoptosis, differentiation Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00019-0 © 2011 Elsevier Inc. All rights reserved.

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and left–right symmetry (Chazaud et al., 1999; Edward, 1997). The left–right asymmetry of early embryonic development is influenced by RA by modulating the expression of Nodal, Lefty and downstream the homeodomain transcription factor Pitx2 genes. RA receptor inhibitors prevent the expression of these genes, which is reversed by the addition of RA. However, both treatments have led to changes in left–right situs of the heart (Chazaud et al., 1999). Retinoids have been directly or indirectly implicated in the mediation of signalling by several growth factors (see Parker and Sherbet, 1992; Sherbet and Lakshmi, 1997). RARs often form heterodimers with other transcription factors. As stated earlier, RAR and vitamin D3 receptor (VDR) heterodimers signal many biological functions. The cell differentiation signal imparted by VDR is amplified by heterodimerisation with RAR. In fact, VD3 induces VDR to form a heterodimeric transcription complex with RXR, which binds vitamin D3 response element (VDRE) and regulates cell proliferation (see Carlsberg and Polly, 1998; Sherbet, 1997, 2001). The 5 region of VDRE binds the RXR component; the 3 arm binds the VDR component of the transcription complex. It has been suggested that RXR might function as a co-activator of VDR. RARs also influence phenotypic outcome in other ways. A most characteristic feature of APL (acute promyelocytic leukaemia) is the occurrence of the PML–RAX-α fusion protein, a recombination of PRKAR1A–RAR-α, often due to t(15;17)(q22;q21) translocation. This causes a reduction in the sensitivity of RAR to retinoids and leads to a block of myeloid differentiation (de Thé et al., 1990; Melnick and Licht, 1999). The PML protein activates apoptosis and possesses growth inhibitory and tumour suppressor properties. The PRKAR1A–RAR-α fusion protein can homodimerise or heterodimerise with RXR-α, and the PRKAR1A–RAR-α/RXR-α ratio alters the interaction of the fusion protein dimer or the heterodimer with RAR response elements (Qiu et al., 2010). Thus, in the chimaeric form with RAR, PML seems unable to exert its normal function in cell proliferation and differentiation. RAR forms a chimaera with the PLZF (PML zinc finger) protein, again as consequence of the t(11;17)(q23;q21) translocation. The PLZF protein is known to regulate cell cycle progression negatively in association with cdk2. PLZF–RAR-α promotes cell proliferation, and this is associated with the repression of DUSP6 (a negative regulator of MAPK and cell proliferation) and CdkN2D (cyclin-dependent kinase 4 inhibitor D), which negatively regulates cell cycle progression and induction of c-myc expression. The PLZF–RAR-α binding site on c-myc promoter overlaps the PLZF binding site, which abolishes the cell growth inhibitory effect of PLZF (Rice et al., 2009). Both PML–RAR-α and PLZF–RAR-α fusion proteins are said to increase cyclin A1 expression, but RAR-α itself negatively regulates cyclin A1 expression. This clearly indicates a close collaboration between them. Furthermore, PLZF–RAR-α protein has been implicated in vitamin D3 and G-CSF signalling pathways (see Sherbet, 2003). RARs are regarded as tumour suppressors. Methylation of RAR-β2 promoter occurs frequently in renal cell, breast and ovarian carcinomas, and methylation has been found to correlate with progression (Khodyrev et al., 2008). Miasaki et al. (2008) investigated the methylation status of RAR-β promoter in human thyroid cancer cell lines. Demethylation of the promoter led to a marked inhibition of cell growth.

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RA can also bind to the nuclear receptor PPAR (peroxisome proliferator-activated receptor). RXR-α forms a heterodimer with PPAR-α, but the biological significance of this is yet uncertain. PPARs are nuclear receptors and function as transcription factors, of which three isoforms (PPAR-α, PPAR-β and PPAR-γ) are known. PPARs are mainly involved in metabolism, but they have also been implicated in tumorigenesis and might negatively regulate tumour growth and invasion. Some synergistic inhibition of growth of osteosarcoma cells has been reported when PPAR-γ, RXR-α and RAR-α, individually or in combination, are overexpressed (He et al., 2010). RA/RARs are involved in interactions with other proliferation signalling pathways such as ER/PR signalling. In breast cancer, ERα and RAR are said to compete for binding sites, thus generating opposing regulatory effects on cell proliferation (Hua et al., 2009). This may be so, and this information could be valuable in the clinical context of managing ER breast cancers. However, difficulties of management could be related to ER-negative cancers, of which a subgroup tends to be HER2- and EGFRpositive. Possibly there are hidden complications in the ER/RAR scheme. Ross-Innes et al. (2010) have confirmed the earlier findings of Hua et al. (2009) and have shown that RAR-α and ER share regulatory regions. This would result in mutual transcriptional regulation. Of further interest is that this occurs with oestrogen exposure and oestrogen/ER-mediated induction of RAR expression. The induced RARs are potentially able to regulate ER-mediated cell proliferation. However, the activation of other pathways needs to be examined too. Oestrogen can upregulate PTEN (Jeong et al., 2010) and thus inhibit Akt signalling and diminish cell proliferation. Jeong et al. (2010) also found PPAR-γ upregulated in their experiments, which could also have a bearing on cell proliferation. RA/RAR involvement in cross-talk that might impinge upon angiogenesis has also received much attention. In this context, one should recall the finding that alltrans retinoic acid (RA) and 9-cis RA can induce receptor RAR-α-mediated expression of FGF2 in bovine aortic endothelial cells and generate a biphasic increase of cell proliferation and induction of angiogenesis (Gaetano et al., 2001). Iriyama et al. (2008) showed that the lipofuscin component A2E is able to activate RAR and induce VEGF expression and vascularisation in the human retinal pigment epithelial (RPE) cell line ARPE-19, which could be counteracted by the inhibition of RAR activation. According to Saito et al. (2007), human umbilical vein endothelial cells (HUVECs)/normal human dermal fibroblast co-culture can be induced with all-trans RA to form capillary-like structures; this effect is negated by RAR antagonists. They also demonstrated stimulation by RA of transcription from VEGF gene promoter in dermal fibroblasts and have implicated RAR overexpression in the process. One should insert a caveat here that the relationship between retinoids and vascularisation is still subject to debate. Roughly over a decade ago it was demonstrated that retinoids possessed anti-angiogenic properties, and similar findings have been reported in more recent times. It was also obvious from some early work that angiogenesis can be inhibited by RA and that the inhibition is mediated by RAR-α (Majewski et al., 1995). Noonan et al. (2007) found that the synthetic retinoid 4-hydroxyfenretinide has anti-angiogenic properties. RA treatment has been found to inhibit VEGF-induced retinal neovascularisation in Wistar albino rats (see Ozkan et al., 2006). Kim et al. (2006)

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showed that RA abolishes the upregulation of VEGF induced in the human epidermal cell line HaCaT by exposure to ultraviolet light. In parallel, they showed that in human skin the increase in vascularisation associated with VEGF upregulation resulting from a single exposure to ultraviolet light was also inhibited. The synthetic retinoid Am80, which functions specifically through RAR-α/β, has been reported to inhibit VEGFinduced phosphorylation of the VEGF receptor (Sanda et al., 2005). RA appears to decrease production of the angiogenic NO with a concomitant decrease in the expression of VEGF and its receptor KDR/Flk-1, although these changes occurred independently of RA response elements (Cho et al., 2005). Using squamous cell carcinoma cells xenografted in athymic nude mice, Liaudet-Coopman (1997) showed that RA inhibited angiogenesis, possibly through inhibition of the expression of FGF- and FGF-binding protein. A recent report has claimed that in superficial bladder cancer, ketoconazole all-trans retinoic acid, which inhibits RA catabolysing cytochrome, inhibited VEGF and TGF-α expression and improved survival time (Hameed and El-Metwally, 2008). It would have been of interest to know what proportion of the patients in this study had shown recurrence, which does not infrequently tend to be invasive. It can be argued that the rationale for the treatment of psoriasis with retinoids is that they inhibit the VEGF production that occurs at high levels in plaques of psoriasis. However, therapeutic agents such as Tazarotene seem to fulfil other criteria of outcome than inhibition of angiogenesis. Tazarotene is a RAR-specific topical retinoid that is highly effective in the treatment of plaque psoriasis and acne vulgaris. It seems to upregulate the expression of TIG (tazarotene-induced gene)-1, -2 and -3 (Duvic et al., 1997), which could be mediating its antiproliferative effect. As yet there is no evidence that this drug functions through VEGF inhibition.

20 Oestrogens and Progesterone in

Normal Physiology and Neoplasia

Oestrogens are known to stimulate the growth of a variety of tissues. Oestrogens and progesterone are associated with the differentiation and development of breast tissue. By virtue of their proliferation-stimulating ability, oestrogens affect the progression of breast cancer. Progesterone, in contrast, is regarded by some as a stimulator of proliferation and as an inhibitor by others. The progesterone receptor (PR) status is said to inversely relate to the mitotic index of tumours. If this were the case, progestins might have a protective role in cancer development. Patients with PR-negative breast cancers seem to have a poorer prognosis than patients with PR-positive tumours. On the other hand, the perceived stimulation of growth by progestins might promote tumorigenicity by synergy with oestrogens. A randomised clinical trial sponsored by the US National Institutes of Health-sponsored Women’s Health Initiative that has focused on assessing the risk by a combination of oestrogen and progesterone has noticed a 26% increase of breast cancer risk, but reduction in risk for colorectal (37%) and endometrial (17%) cancers. The oestrogen receptor (ER) and PR are closely related to breast cancer progression. They are important factors in cancer management because the presence of the receptors has considerable bearing on response to therapy. Of breast cancers, around 40% are ER/PR. Patients with tumours expressing both receptors have the best prognosis and are more likely to respond to hormone treatment. ER/PR are well-differentiated tumours that show less proliferation (Osborne, 1998). This could be due to the opposing effects of progestins on cell proliferation. Chen et al. (2005) showed that progesterone does indeed block oestrogen-mediated activation of PI3K/Akt signalling and inhibits cell proliferation. Around 50% of ER  tumours do not express PR, and these as well as ER/PR do not respond to hormonal therapy (Lapidus et al., 1998). Also, because functional ER can induce PR, the diminished response of ER/PR patients to hormonal therapy might suggest that ER does not function properly in these tumours (Horwitz and McGuire, 1975). Loss of both ER and PR is predominantly due to gene silencing by methylation (Cui et al., 2005; Liu et al., 2003; Maeda and Tsuda, 2009; Mirza et al., 2007).

Receptor Mediation of Oestrogen and Progesterone Function Oestrogen receptors ERα and ERβ function as transcription factors; they bind to oestrogen response elements (EREs). ERα and ERβ differ in cellular distribution, and Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00020-7 © 2011 Elsevier Inc. All rights reserved.

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might target different genes and often exert effects contradictory to each other. HSP90 is known to bind and inactivate ERα when oestrogen is not present in the ambient environment. However, oestrogen binding produces a conformational change in the receptor that leads to the release of HSP90, receptor activation and genetic transcription. Oestrogens as well as anti-oestrogens bind to ERα. ERα becomes a transcription repressor when tamoxifen binds it, which forms the basis of tumour response to tamoxifen. ERα is also repressed by its splice variant ERβcx, and when ERα and ERβcx are simultaneously expressed ERα loses its ability to function. ERβ is also expressed in normal and neoplastic breast tissue, but its role is yet undefined (see Palmieri et al., 2002). ERα, ERβ and PR share a typical domain structure with other receptors of the family, for example thyroid hormone receptor, glucocorticoid receptor and RA receptor. They possess an amino (N)-terminal transcription activation function domain AF1, a conserved DNA-binding domain of about 68 amino acid residues, a hinge region and a carboxy (C)-terminal ligand-binding domain with 225–285 residues, which includes AF2 domain and a dimerisation domain (Moggs and Orphanides, 2001). AF2 is said to be the key activation domain (Tora et al., 1989). ER recruits co-regulators for the transcription of target genes. The regulators are activators or suppressors. The major co-activators are histone acetyltransferases (HAT) such as SRC-1 and SRC-3 (ACTR), which interact with AF-2 and might recruit other HATs, namely p300/CBP (CREBP-binding protein) and pCAF (p300/CBP-associated factor). The SRC family members might behave differently. For example, in MCF7 breast cancer cells, depletion of SRC-2 and SRC-3 inhibits cell cycle progression and enhances apoptosis and transcriptional signalling by ERα. The transcription of TFF1, but not c-myc, was affected by the loss of any one of them, which exemplifies differential target gene activation with the mediation of the co-activators (Karmakar et al., 2009). In contrast, histone deacetylases (HDACs) function as repressors or recruit other repressors such as NcoR and SMRT to the promoters of oestrogen-responsive genes (see Moggs and Orphanides, 2001). PR occurs as two isoforms, PRα and PRβ. In general, PRβ mediates gene transcription. In contrast, PRα could function as a repressor of transcription in some biological conditions (Pieber et al., 2001). Expression of PR is regulated by oestrogen, hence the suggestion that the presence of PR indicates that ER is functional.

ER/PR in Cell Adhesion, Proliferation and Cross-talk with Growth Factor Signalling ER Signalling and Cell Motility The ER targets numerous genes including many genes associated with cell adhesion and motility, cell proliferation and apoptosis. The transcription of these target genes leads to significant changes to cellular behaviour. Oestrogen alters MMP expression in many tissues. Endometrial carcinoma cells transfected with ERα display MMP upregulation accompanied by acquisition of invasive ability together with enhanced cyclin D1 expression (Mizumoto et al., 2002). In contrast, transfection of the PR gene inhibits the invasive behaviour of breast cancer cells (Lin et al., 2001). MMP upregulation has also

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been shown to occur in other disease states. In cells derived from the lung disease called lymphangioleiomyomatosis, activation of ER has led to transcriptional enhancement of MMP-2 expression and an increase in invasive behaviour of the cells in vitro. The invasive behaviour could be inhibited by MMP inhibitors (Glassberg et al., 2008). It is needless to say that there are opposing views expressed as well. MMP-2 was reported to have been downregulated by oestradiol in an ER-dependent fashion in rat cardiac fibroblasts and in a human fibroblast cell line. The inhibition of MMP seemed to occur through activation of MAPK signalling because inhibition of the latter also negated the downregulation of MMP (Mahmoodzadeh et al., 2010). However, one should consider the possible transcription of the endogenous inhibitor TIMP gene, which could negatively regulate MMP gene transcription. A possible explanation might be found in the demonstration of a disparity of effect exerted by the two transcription activation domains AF-1 and AF-2 of ER. In the absence of AF-2, an increase in MMP-13 activity occurred compared with the wild-type ER. In contrast, deletion of AF-1 had the opposite effect (Achari et al., 2009). Oestrogens do bring about ECM changes apart from altering MMPs. Oestrogen affects another ECM enzyme, heparanase. Oestrogen upregulates the expression of the heparanase gene. Four EREs have been identified in the promoter of heparanase gene (Elkin et al., 2003).

ER and Cell Proliferation Oestrogens and ER markedly influence cell cycle progression and modulate the kinetics of cell proliferation. They interact with and alter the expression of many of the cell cycle regulatory genes and their downstream effectors. The tumour suppressor p53 does engage ER in interactive cross-talk and it has been implicated in the oestrogen/ ER-mediated modulation of cell cycle progression. Collaborative functioning of ER and p53 is also evident from some recent work using murine models genetically engineered to provide a genetic environment with deregulated ERα and p53 haploinsufficiency. Each lesion or a combination of both led to the development of pre-neoplastic changes and cell proliferation, but maximum change accrued with the presence of both lesions wherein enhanced proliferation was encountered (Diaz-Cruz and Furth, 2010). Compatible with its classical opposing influence, progesterone inhibits cell cycle progression by the upregulation of Cdk inhibitors. PTEN is a tumour suppressor gene. Loss of PTEN function by mutation or loss of heterozygosity is conducive to tumorigenesis. PTEN promotes apoptosis with concomitant downregulation of Akt. It is also capable of altering Rb gene function and in this way regulating cell cycle progression. The PTEN/Akt route has been implicated, albeit without consensus, in ER/PR function. Low levels of PTEN have been correlated with high ER and low PR in some cancers. This opposing correlation of ER and PR with PTEN is compatible with ER status being related to clinically highly aggressive disease and the well-documented opposing effect of oestrogen and progesterone on cell proliferation. The loss of ER is associated with poor prognosis in breast and endometrial cancers. ER-positive tumours respond to tamoxifen whereas ER-negative tumours are resistant, but these continue to grow rapidly with resulting unfavourable prognosis.

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It has been postulated therefore that tumour progression might involve a transition from a hormone-dependent to a hormone-independent state by virtue of proliferative signalling imparted by growth factors such as EGFR, HER2 or other growth factor receptors. ER-positive breast epithelium shows higher proliferation than ER-negative epithelium. Not infrequently, ER-negative breast cancers have been found to express EGFR (see Sherbet, 2005). It was reported more than a decade ago that oestradiol upregulated EGF/EGFR (DiAugustine et al., 1988; Huet-Hudson et al., 1990), so also TGF-α and IGF-1 (Murphy and Ghahary, 1990; Nelson et al., 1992). MAPK signalling is constitutively active in ER  MCF-7 breast cancer cells which correlates with ER/ PR-mediated genetic transcription (Atanaskova et al., 2002). But then EGFR can be activated independently of ER (Couse and Korach, 1999; Mueller and Korach, 2001). On the other hand, ER signalling is said to repress HER2 transcriptionally and vice versa (Newman et al., 2000). The suppression has been attributed to the downregulation or competitive sequestration of the transcription factor AP2 or SRC1 (Newman et al., 2000; Perissi et al., 2000). An inverse relationship between ER and HER2 has been found in breast tumours (Bershtein et al., 2003; Horiguchi et al., 2003). Also, signalling by both EGFR and the HER2 receptor is said to activate ER and its co-activator AIB1 (Osborne and Schiff, 2003). More recently, Zhu et al. (2006) showed that oestrogen induces ErbB4 and the translocation of its intracellular domain into the nucleus as a part of the mechanism by which it can enhance ER activation. So the transition from hormone dependence to growth factor dependence might be a more complex phenomenon than would appear and remains to be fully resolved. Apoptosis is an inseparable part of tumour growth and there have been efforts to determine the nature of the involvement of oestrogen/ER signalling. Pro-apoptosis genes might be downregulated in the ER environment (Frasor et al., 2003). Also, the expression of the Bcl-2 apoptosis inhibitor gene has been correlated with high expression corresponding to an ER-positive state (Linjawi et al., 2004). However, no definitive evidence to substantiate a causal link-up is available at present. Oestrogen-mediated induction of cell proliferation can occur independently of canonical ER signalling. The identification of mER (membrane-bound ER) had led to two optional signalling modes: (1) that mER is the relocated nuclear ER (ERα and ERβ); and (2) GPR30, the so-called orphan GPCR (G-protein-coupled receptor), is indeed the mER. This second mode, which involves the activation of GPR30, has been called the non-genomic pathway (Couse and Korach, 1999; Filardo et al., 2000; Mueller and Korach, 2001). Oestrogens generate a variety of effects which occur in other systems with the activation of GPCRs. So it would not be totally tenable to argue that these effects are mediated by conventional ER signalling in all settings because these systems often might express GPR30 (Rae and Johnson 2005).

ER/PR Cross-talk with Tumour Suppressor and Promoter Signalling A striking example of cross-talk with suppressor gene signalling is the suppressor function of BRCA1 and BRCA2 and ER/PR signalling. An indirect link can be

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suggested between oestrogen and BRCA. BRCA bears partial sequence homology to the granin family proteins, with the presence of a granin motif at the C-terminus. Now, granins are regulated by oestrogen, which can downregulate the expression of both Granin A and BRCA (Gudas et al., 1995). Early reports indicated that Granin A is synthesised by prostate cancer cells in vitro, and high levels of the protein are detected in the serum of patients with prostate cancer (Deftos, 1998). However, this enhanced expression was found even in the absence of elevated PSA, which raises some ambiguities about the perceived correlation. Indeed, Granin A expression in invasive ductal carcinoma of the breast has been reported to correlate with PR and to be inversely related to histological grade of the tumour. Expression of BRCA1 influenced Granin A expression. Furthermore, lack of its expression correlated with poor prognosis. Similarly, absence of Granin B also portended poor prognosis (Yoshida et al., 2002). This potential link-up, or the lack of it, has not been pursued recently. In any event, the relationship appears rather tenuous. The metastasis suppressor nm23 has been studied for potential links with ER/PR signalling. Oestradiol seemed to increase nm23-H1 expression and this was related to the levels of ER in human breast cancer cells. The upregulation correlated with the levels of ERα in the cells. Furthermore, an ERE element was also identified in the nm23 promoter (Lin et al., 2002). Confirmatory evidence has come from the work of Curtis et al. (2007), who found that when nm23-H1 is inhibited the expression of several genes that promote cell proliferation, Bcl-2, cyclin D1 and cathepsin D among them, are upregulated. Also altered is the responsiveness of PR to oestrogen. However, expression of the trefoil factor TFF1 was not affected. More recently, similar association has been found between nm23-H2 and ERβ. Oestrogen-induced upregulation of nm23-H2 correlated with a reduction in cell migration (Rayner et al., 2008). Quite clearly ER is able to signal through nm23 to inhibit cell migration. However, the increase of cell motility induced by the oestrogen/ER through the trefoil TFF1 route occurs independently of, and is not influenced by, nm23. Curtis et al. (2007) showed this earlier. Incidentally, the Kiss-1 metastasis suppressor gene is also upregulated by oestrogen (Richard et al., 2008). The metastasis promoter S100A4, in contrast, has shown a negative correlation with ER/PR expression. Breast cancers that expressed S100A4 at low levels were ER/PR positive (Albertazzi et al., 1998), which can be interpreted as indicating that in the absence of S100A4 expression tumour growth is determined by ER/PR signalling. An alternative explanation that also arises from that work is that when S100A4 expression is low, nm23 might induce an upregulation of ER/PR in these tumours. The MTA family genes, of which three genes (MTAs 1, 2 and 3) and some isoforms have been identified, are metastasis-associated genes. The MTAs have been linked with chromatin remodelling and transcriptional regulation. They have also been associated with cancer progression. MTA1 is overexpressed in many human tumours. The MTA proteins function as co-repressors in the fashion of HDACs. MTA1 seems to convert cancer cells into a more aggressive phenotype, which has been attributed to deacetylation of chromatin in the ERE of oestrogen-responsive genes. Furthermore, oestrogen induces the expression of MTA3 and suppresses epithelial mesenchyme transition, which is a significant feature of epithelial tumorigenesis. This has been found to be

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due to E-cadherin expression (Toh and Nicolson, 2009). MTA function involves the BRCA1 suppressor as well. The MTA1–nucleosome remodelling and deacetylating (NuRD) complex associates with the ERE of the BRCA1 promoter and downregulates its expression. The recruitment of the MTA1 to the ERE is ERα dependent (Molli et al., 2008), possibly suggesting a co-repressor function to MTA.

The Trefoil Protein TFF1 The TFF1 (pS2) gene is an oestrogen-responsive gene. It is expressed in oestrogenresponsive, ER-positive breast cancer cells. The pS2 is a small (7–12 kDa) secreted cysteine-rich trefoil protein. The peptides TFF1, TFF2 (the spasmolytic peptide SP) and TFF3 (the intestinal trefoil peptide) share a consensus domain which has three disulphide bridges that form a three-loop structure; hence they are known as trefoil peptides. Apart from oestrogen, several growth factors are known to regulate the trefoil peptides. TFF1 gene possesses an ERE in its 5-flanking region, an AP1 response element in the neighbourhood of ERE and an EGF response motif (Nunez et al., 1989).

TFF1, Cell Proliferation and Invasion Much early work claimed that TFF1 was inhibitor of cell proliferation and tumour growth. TFF1, both in monomeric and dimeric forms, reduced cell proliferation in vitro of the gastric adenocarcinoma cell line AGS. Dimeric TFF1 showed a higher ability to reduce proliferation than monomeric TFF1 (Calnan et al., 1999). However, in interval breast cancers, namely those that develop during the interval between screenings, TFF1 was expressed at high levels. In the screening stage, TFF1 correlated with ER, but not so in the interval cancers (Crosier et al., 2001). Recently, Amiry et al. (2009) have found that forced expression of TFF1 increased proliferation and migration of breast cancer cells in vitro. Obviously, as a consequence of this increased cell proliferation, these cells produced larger tumours when implanted as xenografts in murine hosts. Constitutive expression of TFF1 by transfection of TFF1 cDNA into cells derived from adenomatous polyposis increased anchorage-independent growth and enhanced the growth of these cells as xenografts in immune-compromised mice. In these cells, CDC25A and CDC25B, the dual specificity phosphatases, which positively regulate CDKs (cyclin-dependent kinases), were upregulated (Rodrigues et al., 2006). Despite the early evidence to the contrary, TFF1 has been linked with cancer progression because it is able to regulate cell proliferation in response to oestrogen/ ER and it induces cell migration. TFF1 induces cell migration in vitro (Prest et al., 2002). The TFF1 protein is normally expressed in some regions of the gastrointestinal tract and might be involved in the repair of damaged tissues. However, studies using mutant forms of TFF have led to the re-emergence of contradictions. Yio et al. (2006) investigated wild-type wtTFF1 and two mutant TFF1s forms, A10D and E13K, from human gastric carcinoma cells with a point mutation affecting their ability to influence cell proliferation, apoptosis and cell invasion. Recombinant wtTFF1

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inhibited cell proliferation but enhanced cell motility. The mutant TFF1 had lost the ability to suppress proliferation, but did induce cell motility to a far greater extent than wtTFF1. The induction of invasion by both A10D and E13K could be inhibited by inhibiting PLC/PI3K signalling, but inhibition of Rock kinase inhibited induction of invasion only by the E13K mutant (Yio et al., 2006). Possibly a major cause of dissensions could arise from the techniques of transfection, which can bring about collateral genetic changes. This criticism would apply equally to experiments using vectors carrying inducible gene constructs.

TFFs in Progression and Prognosis TFFs have been detected in many human tumours. The probability that they might be involved in inhibiting the proliferative response elicited by oestrogens would contribute to good prognosis. If this were the case, TFF expression relates inversely to cancer progression. There are many reports supporting this view, but equally the higher TFF expression has been correlated with tumour progression (see Sherbet, 2005). More recent findings also are not totally conclusive either way. Higher plasma levels of TFFs were reported in patients with advanced prostate cancer compared with those with localised disease, and TFF3 levels were higher in patients with bone metastases (Vestergaard et al., 2006). In gastric cancer, however, similar levels of TFF1 and TFF2 have been found in the pre-cancerous state and in overt carcinomas (Shi et al., 2006). This might in fact suggest a differential targeting of genes; possibly, in this case, it suggests the transcription of genes assisting metastasis to the bone. The major problem with attribution of TFF expression in progression is that although TFFs are oestrogen induced, oestrogen/ER reaches a wide spectrum of target genes that are variously associated with invasion and metastasis. It is difficult to judge the significance of TFFs in isolation. It is well to bear in mind that trefoil proteins are expressed in most ER/ PR-positive, and not infrequently in receptor-negative, breast cancer. The perceived ability of TFFs to induce cell motility could be a crucial finding in relation to progression. Significant also is their expression in the invasion front of tumours (Hackel et al., 1998). Equally, the induction of invasion by TFFs could be occurring independently of oestrogen and its receptors, for example through EGFR activation (Couse and Korach, 1999; Mueller and Korach, 2001; Rodrigues et al., 2003). This has been called the nongenomic pathway, in which conventional ER is not required, but activation of the signalling system requires GPR30, the orphan GPCR. Furthermore, ER is able to induce ErbB4 and translocation of its signalling domain into the nucleus; thus ErbB4 could function as a co-regulator of transcription of ER target genes.

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21 Glucocorticoid Signalling in

Normal and Aberrant Physiology

Glucocorticoids are steroid hormones synthesised in the adrenal cortex. Most glucocorticoid activity is from cortisol (also known as hydrocortisone). Glucocorticoids occur ubiquitously in animal cells and perform diverse functions. They participate in many physiological functions, for example carbohydrate metabolism, embryonic or foetal development and regulation of immune function and inflammation. Glucocorticoids are also able to induce apoptosis. On account of these properties, they have been used in the treatment of lung diseases (Pujols et al., 2004), inflammatory bowel disease (Orii et al., 2002) and rheumatoid arthritis (Chikanza, 2002). Glucocorticoid forms an important mode of treatment of haematological malignancies, for example acute lymphocytic leukaemia (ALL), Hodgkin’s lymphoma, multiple myeloma, etc. (Alexanian and Dimopoulos, 1994; Gaynon and Carrel, 1999). The rationale of treatment is the ability of these steroids to induce apoptosis (Distelhorst, 2002; Greenstein, 2002; Smets, 1999).

Glucocorticoid Receptors in Signalling Glucocorticoid function is mediated by glucocorticoid receptors (GRs). These are often referred to as belonging to the superfamily of nuclear receptors. However, receptors that are not bound by ligands occur in the cytoplasm. GRs share a typical domain structure with other receptors of the family, for example thyroid hormone receptor, ER, PR and RA receptor. GRs possess a ligand-binding carboxy (C)-terminal domain, essential for dimerisation and gene transactivation, nuclear localisation signal domain and a zinc finger DNA-binding domain. The amino (N)-terminus has a domain that participates in gene transactivation independently of ligand binding. The gene has nine exons with the 5 untranslated region composed of exon 1 and part of exon 2; exons 2–9 are coding exons with the 3 untranslated region in exon 9 (Encio and Detera-Wadleigh, 1991). Human GR gene expression is regulated by three promoters, 1A, 1B and 1C, of which 1A is the main regulator in haemopoietic cells, whereas 1B and 1C are expressed in most cells, so they could be housekeeping promoters. 1B and 1C do not possess GREs (glucocorticoid response elements) (Breslin et al., 2001; Schaaf and Cidlowski, 2002). GR can bind AP-1 and NF-κB independently of GRE and regulate gene expression. Some of these ideas are summarised in Figure 21.1. Two isoforms of GR, namely GRα and GRβ, have been identified. These are generated by alternative splicing of exon 9 of the GR transcript (Hollenberg et al., 1985; Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy. DOI: 10.1016/B978-0-12-387819-9.00021-9 © 2011 Elsevier Inc. All rights reserved.

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Figure 21.1  Differential regulation of target genes by the GR receptor. Transcription of target genes is repressed by interaction with transcription factors such as c-jun/c-fos/NF-κB, or their expression is regulated through negative or positive GREs.

Weinberg et al., 1985; Yudt et al., 2002). GRβ is a dominant negative inhibitor of GRα function by the formation of a GRα/GRβ heterodimer (Oakley et al., 1999a,b). Glucocorticoid resistance or insensitivity has been attributed to the relatively high levels of GRβ compared with GRα (Hamid et al., 1999; Hamilos et al., 2001; Honda et al., 2000; Orii et al., 2002). The ligand-free GR is cytoplasmic and translocates to the nucleus upon ligand binding. Essentially, GR functions as a transcription factor upon being activated by ligand binding. Nuclear translocation requires that GR binds to the heat shock protein HSP90 to form a heterocomplex. The formation of the heterocomplex plays an important part in ligand binding because the heat shock protein maintains GR in an appropriate folded and competent state. The C-terminal third of GR constitutes the hormone-binding domain, but the latter cannot bind the ligand unless GR is bound to HSP90 (Bresnick et al., 1989; Davies et al., 2005; Scherrer et al., 1990; Simons, 1994, 1997). This monomer–ligand complex can activate specific transcription factors to suppress the transcription of genes such as the cytokine genes. The activated or ligand-bound receptors dimerise, and the dimers translocate to the nucleus where they can recognise and bind to GRE in the promoter regions of target genes. It has been known for a while that the activated GR can activate or repress gene function (Beato et al., 1995; Jonat et al., 1990). Positive and negative GREs have been identified (Schaaf et al., 2002; Tsai and O’Malley, 1994). So, depending upon whether the negative or positive GREs are recognised, specific target genes are suppressed or transcribed (Pelaia et al., 2003; Ruegg et al., 2004). Another layer of specificity could be inherent in the recruitment of co-regulators. GR recruits many co-activators and co-repressors, which are shared with other members of the nuclear receptor superfamily. References to these have been made in other locations in relation to target gene transcription mediated by other nuclear receptors. These coregulators subserve diverse phenotype-related functions. Thus, different co-activators

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may be required for the transcription of specific genes and individual co-repressors might be associated with the regulation of specific genes. The activated receptor transcription complex can facilitate or inhibit the transcription of genes. The co-activators bind to the promoters of target genes at the transcription activation domains AF-1 and AF-2 (Beato and Sanchez-Pacheco, 1996; McKenna and O’Malley, 2002; McKenna et al., 1989). Several SRC family co-activators have been identified, for example SRC-1/ NCoA-1, SRC-2 (TIF2/NcoA-2) and SRC-3 (NcoA-3) (also known variously and somewhat confusingly as CBP/300, ACTR, pCIP, AIB1, RAC3, TRAM-1). DRIP, also called TRAP (vitamin D interacting protein/thyroid hormone-associated protein), is another transcription co-activator complex that forms a bridge between transcription factors and RNA polymerase II (Auboeuf et al., 2002; Hittelman et al., 1999; Torchia et al., 1997). Often the co-activators possess a consensus motif at the receptor interaction sites. A coactivator NCoA62/SKIP, which does not belong to the SRC family, has been described (Zhang et al., 2003). SRC-2 interacts with GR, and SRC-3 is involved as an activator of thyroid and retinoid receptors. Several co-repressors have also been described. The nuclear receptor co-repressor N-Cor (Wu et al., 2006) and ARA70α function as corepressors. ARA-70α is a co-repressor, of which the full-length isoform ARA-70α inhibits prostate cancer cell proliferation and tumour growth, whereas the spliced isoform ARA-70β promotes prostate cancer cell growth and invasion (Ligr et al., 2010).

Effects of Glucocorticoids on Cell Function Glucocorticoids influence the patterns of cell behaviour besides alleviating inflammatory processes, inducing apoptosis and inhibiting cell proliferation. The latter are important features, especially in evaluating glucocorticoids in the management of neoplasia. However, the cellular faculties that are influenced by glucocorticoids are much wider in expanse and include extracellular matrix remodelling, cell motility, invasion and angiogenic signalling. The following discussion is therefore restricted to the regulation of these biological features by glucocorticoids.

Regulation of Cell Proliferation The inhibition of inflammatory responses involves inhibition of cell proliferation. Glucocorticoids prevent the proliferation of T-cells in response to exposure to alloantigens by inhibiting the expression of cytokines. Glucocorticoids inhibit the proliferation of many cell types by preventing cell cycle progression. They produce cell cycle arrest and induce apoptosis in immature thymocytes, T-cells, leukaemia cell lines and other tumour cells and many epithelial cell types. As would be expected, the inhibition of proliferation is accompanied in some instances by the induction of differentiation. Glucocorticoids induce stage-dependent cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p21waf1/cip1 and inhibiting the expression of cyclin-D1. The dephosphorylation of Rb leading to the prevention of G1–S transition has also been implicated in glucocorticoid-mediated cell cycle regulation.

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The inhibitory effects on cell proliferation should be viewed in the background of the effects on apoptosis. Induction of apoptosis by glucocorticoids in leukaemic and lymphoma cells was shown some while ago to involve Bcl-2 family genes (CaronLeslie et al., 1994; Greenstein et al., 2002; Smets and van den Berg, 1996; Torigoe et al., 1994). More recently, the apoptotic effects have been shown to be mediated by GR. When GRs are overexpressed in human SCLC cell lines, a significant increase in apoptosis occurs, which is blocked by GR antagonists (see Sommer et al., 2007). It ought to be recognised that glucocorticoids regulate apoptosis, that is, they induce or inhibit apoptosis as required in the physiological and differentiating environment. The Bcl-2 family genes are involved in lymphoid homeostasis (Rathmell et al., 2002). Neutrophil homeostasis is an essential element of physiological function, which is achieved by regulating apoptosis of the cells. This is a crucial mechanism in bacterial infection and inflammatory conditions associated with autoimmune diseases. In neutrophils, glucocorticoids delay apoptosis by delaying caspase activation and altering apoptosis-related gene expression. Dexamethasone increased A1, a Bcl-2 related protein that is anti-apoptotic, but decreased the expression of pro-apoptotic Bak (Madsen-Bouterse et al., 2006).

Modulation of Extracellular Matrix Enzyme Systems Whether glucocorticoids affect cell motility and invasion is an essential part of the investigation of wound healing, tissue repair and reconstruction, as well as cancer invasion and the development of metastatic disease. The cytoskeletal organisation and adhesive functions are an integral component of cell motility. Several extracellular matrix components such as fibronectin, collagens, cell adhesion molecules and integrins participate in cell motility. The extracellular matrix is also remodelled by several enzyme systems, notably MMPs and TIMP. Hydrocortisone markedly alters MMP expression in gingival fibroblasts in a concentration-dependent fashion. At high concentrations it upregulated MMP, but at lower concentrations downregulated or reduced the degree of upregulation of MMP without much effect on TIMP expression (Cury et al., 2007). MMPs might be selectively modulated. For example, in the zebrafish, MMP-13 is affected by glucocorticoids (Hillegass et al., 2007), whereas Cury et al. (2007) found alterations in MMP-1, MMP-2, MMP-3, MMP-7 and MMP-11. Dexamethasone, which exhibits more powerful binding to GR than cortisol, seemed to have the opposite effect. It markedly upregulated TIMP-1 expression in murine vascular endothelial cell lines (Forster et al., 2007). Several TIMPs occur in vascular endothelium. Hartmann et al. (2009) found TIMP-3 expression was upregulated by hydrocortisone, but TIMPs 1, 2 and 4 were in fact downregulated. The regulation of MMP expression is essential in the maintenance of the integrity of the blood–brain barrier. Increase of MMP expression has been linked with the disruption of this crucial barrier (Lohmann et al., 2004). Therefore, one should countenance MMP activity as equilibrated or counterbalanced by endogenous inhibitors. Changes in, or remodelling of, the extracellular matrix are bound to have repercussions in terms of cell motility. Cortisol, but not testosterone or oestradiol, was

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reported to enhance α5-integrin expression of synovial fibroblasts from rheumatoid arthritis. Also enhanced was adhesion of cells to fibronectin. In parallel, the cells showed inhibition of invasion of the synovial fibroblasts into cartilage in vivo in a murine model. Inhibition of α5-integrin using antibodies increased cell motility (Lowin et al., 2009). The MAPK pathway is negatively regulated by activated GRs (Lowin et al., 2009; Piette et al., 2009). Dexamethasone seems to achieve inhibition of motility and invasion by inhibiting ERK/MAPK signalling mediated through GR. Furthermore, MIF (the macrophage migration inhibitory factor) enhanced cell migration and ERK/ MAPK signalling. The inhibitory effects of dexamethasone are indeed accentuated by MIF inhibitors (Piette et al., 2009). Zhang et al. (2008) found that inhibition of JNK, another arm of the MAPK system, rather than ERK, effectively inhibited macrophage migration.

Glucocorticoids and Cancer Progression The anti-proliferative and apoptosis promotion and inhibition of invasion have led legitimately to exploration of the potential of glucocorticoids to suppress not only the growth but also the progression of cancer. The inhibition of invasion when viewed together with their ability to maintain the integrity of vascular endothelium does provide grounds for the attribution of a potential role for glucocorticoids as a metastasis inhibitor. VEGF is a major stimulator of endothelial cell proliferation and induction of angiogenesis, and thus is an important contributor to the process of cancer cell dissemination. Recently, dexamethasone was found to inhibit VEGF production. Of two studies of head and neck cancer cell lines, one showed VEGF inhibition. In parallel, STAT3 was downregulated in this cell line. When induced to overexpress STAT3, the inhibitory effect of dexamethasone was overcome and VEGF production was restored (Shim et al., 2010). These findings are important per se. The divergence in the behaviour of two cells of head and neck cancers raises the possible operation of other factors. As discussed earlier in discussions of VEGF function, singlenucleotide polymorphisms in the VEGF gene have been related to disease state of certain forms of cancer. In fact, higher expression of VEGF detected in patients with some cancers has been provisionally attributed by some investigators to singlenucleotide polymorphisms. Also vitiating the situation is the possible interaction of VEGF with other angiogenic factors. Both hydrocortisone and dexamethasone were reported earlier to inhibit VEGF as well as IL-8 expression in vitro. The VEGF-C isoform was particularly downregulated. The effects were repeated in xenografts of the cells concerned, with tumour displaying reduction of size and associated vascular density (Yano et al., 2006a,b). Other factors related to the regulation of angiogenesis such as NO (nitric oxide), iNOS (inducible nitric oxide synthase) and COX (cyclooxygenase)-2 are among those that are affected by glucocorticoids. NO is known to enhance, and NOS antagonists inhibit, VEGF synthesis (Dulak et al., 2000; Jozkowicz et al., 2001). NO also upregulates COX-2, so a strong link has been established between NO, VEGF and angiogenesis. Glucocorticoids seem to activate different signalling pathways in

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achieving this outcome. COX-2 expression is related to inducible NOS and VEGF, and is associated with high microvascular density. Glucocorticoids functioning through GR suppress COX-2 by inhibiting p36/MAPK signalling (Brewer et al., 2003). However, Zhu et al. (2009) found cortisol activated GR and enhanced COX-2 expression in human amniotic fibroblasts. It increased phosphorylated CREB (CREbinding protein)-1 binding to CRE (cAMP response element) of the COX promoter and enhanced COX-2 transcription. In cardimyocytes, glucocorticoids activate the p38/MAPK pathway, leading to CREB phosphorylation. This pathway is activated only in cardiomyocytes and not in cardiac fibroblasts, even with GR in the microenvironment (Sun et al., 2008). Finally, NO itself is known to be reduced by glucocorticoids (Pudrith et al., 2010). Glucocorticoid use in the management of non-haematological malignancies seems to have no appreciable benefit in terms of response to treatment or survival outcome. Keith (2008) reviewed trial data relating to some forms of human neoplasms. The general conclusion is that glucocorticoids might afford some benefit in breast and prostate cancers. Both have shown some response, but in advanced breast cancer, combination with other therapy does not affect the outcome. Most of the reviewed data relate to old clinical trials and there are problems with comparison of findings per se, mainly owing to the modes of assessment used, so it is difficult to evaluate them in the present-day management setting. The paucity of data is also a complicating factor. This might have arisen from glucocorticoid-induced resistance encountered in solid tumours. Distinct molecular mechanisms, including differential function of the GR isoforms, were suggested some time ago (Schaaf and Cidlowski, 2002). The involvement of Bcl-2 family apoptosis regulatory genes has been viewed as a distinct complicating factor in the development of resistance to therapy. Herr and Pfitzenmaier (2006) have argued that disseminated tumour cells might resist apoptosis aided also by the downregulation of immune resistance. Glucocorticoid resistance could result from interaction of glucocorticoid signalling with other systems. Such interactions could switch glucocorticoid function to resist apoptosis, rendering combination therapy with glucocorticoid counterproductive.

Epilogue

Therapeutic targets for cancer therapy can be identified on the basis of three basic criteria: (1) physiological/differentiation-related features that target cell signalling pathways such as by modulation of hormonal and growth factor responses; (2) physical/morphology-related targets, motility factors, remodelling of the cell membrane and the extracellular matrix, and cytoskeletal targets; and (3) molecular targets related to tumour growth, cell proliferation and apoptosis, and targets differentially expressed in situ and invasive and metastatic disease, such as metastasis promoter and suppresser genes (Sherbet, 2008). Given that mutations or overexpression of growth factor receptors are associated with aggressive cancers and poor prognosis, growth factor receptors have become the inevitable focus of attention and have led the way to the targeting of growth factor function as a means of controlling breast cancer development and secondary spread. Monoclonal antibodies (Herceptin) raised against the external domain of human epidermal growth factor receptor (HER)-2 have provided a highly successful mode of treatment for metastatic breast cancer, showing high HER2 expression and HER2 gene amplification. Blocking receptor function with Herceptin had suppressed tumour growth and possibly also tumour-associated vascular density. Protein kinase inhibitors have offered another means of blocking growth factor receptor signalling. These hold much promise because they could be effective in managing patients with Herceptinresistant tumours. A strategy similar to that adopted with epidermal growth factor family growth factors has been used to target vascular endothelial growth factor receptor and inhibit signalling by VEGF. Avastin (bevacizumab), a humanised monoclonal antiVEGFA antibody, is such an inhibitor (see Sherbet, 2009). Aspects of combination therapy enter into patient management all the time. Herceptin inhibits not only tumour growth, but it has also been claimed to reduce VEGF expression and in this way inhibit microvascular density and vascular permeability. Another potentially significant putative effect of Herceptin is the possible upregulation of the anti-angiogenic thrombospondin (TSP)-1. This line of reasoning leads to its logical conclusion that Herceptin has the ability to compound its own anti-tumour effects arising from HER-2 inhibition by inhibiting neovascularisation. A seemingly viable form of combination therapy that deserves study is inhibition of the mTOR pathway, which integrates ER, EGFR, VEGF and IGFR signalling, and is implicated in cell proliferation as well as in angiogenesis. The use of mTOR (mammalian target of rapamycin) inhibitors on their own or in combination with EGFR inhibitors has been shown to be quite effective in EGFR inhibitor-resistant cancer cell lines. EGFR inhibitors could regain effectiveness when combined with

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mTOR inhibitors. Furthermore, experimental studies appear to suggest that mTOR and EGFR inhibitors are quite effective in blocking tumour growth and VEGF production. So one arrives at a virtually inescapable conclusion that mTOR inhibition might be an approach that is eminently suitable for further study. The challenge posed by triple-negative breast cancers (TNBCs), which are ER-/ PR-/HER2-negative and so cannot respond to tamoxifen, aromatase inhibitors or Herceptin, has been highlighted in the relevant sections of this book. Indeed, more than 10% of breast cancers are triple negative. TNBC is highly aggressive, shows greater risk of recurrence, carries increased propensity to metastasise and is associated with poor survival. These considerations, as well as discussion relating to several novel approaches, has emphasised the significance of growth factor signalling, especially by EGFR, HER2 and TGF-β family ligands in identifying new therapeutic targets and designing appropriate new strategies of treatment. Among other novel areas under scrutiny are DNA homologous recombination repair targeted therapy, EGFR-mediated targeting and the potential of androgen receptor (AR) in TNBC. One cannot overemphasise the significance of AR because we know that it interacts with EGF and HER2 pathways. In fact, HER2 can activate AR signalling and promote the growth of prostate cancer. Much further work is needed to elucidate the transactivation of AR by growth factors, but the therapeutic potential of deploying AR deserves serious consideration. A question that many investigators are currently attempting to answer concerns the involvement of GPCRs (G-protein-coupled receptors) in TNBC. The so-called orphan receptor GPCR30 is indeed the mER. Oestrogens generate a variety of effects that occur in other systems with the activation of GPCRs. So one cannot overemphasise or overrate the value of looking at GPCR30 status in TNBC. Because not infrequently, ER-negative breast cancers express EGFR and in this context it is highly relevant to know that oestrogen can transactivate EGFR through GPCR30. It is critical therefore to establish the expression status of GPCR30 in TNBC. The conversion of epithelial cells into mesenchymal cells or EMT is a developmental programme that is accompanied by changes in cell morphology, reduced intercellular adhesion and enhanced cell migration. EMT is a significant component that drives tumour progression. Inevitably EMT is a major target for preventing progression. The focus on EMT has been severely sharpened because it is activated in the presence of EGFR and HER2. This provides heightened impetus to the deployment of Herceptin and EGFR inhibitors in the treatment of epithelial neoplasms. As noted elsewhere in this book, TGF-β exerts a bimodal effect on tumour growth and progression functioning as a tumour suppressor in early stages but promoting progression in the later stage of the disease. EMT is promoted by TGF-β and PDGF, but inhibited by BMP7, which seems to support the reverse process of mesenchymal epithelial transition. BMP7 and possibly other TGF-β ligands might be valuable targets. We also know that TNF-α and IFN can activate the inhibitory Smad7 by different routes and in that way inhibit EMT. Therefore, the need for deeper studies into the identification and investigation of the signalling channels adopted by the TGF-β family and related ligands cannot be overemphasised. A new avenue of approach being explored now relates to the discovery of microRNAs (miRNAs). These regulate gene expression and have been implicated with

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differentiation and many aspects of cell behaviour related to tumour growth and progression. They have been implicated in the regulation of EMT. Inevitably a powerful case is being made for the use of specific miRNAs to block EMT as a mode of treatment. Some miRNAs are downregulated with the induction of EMT by TGF-β and their expression has been found to block EMT. So re-expression of miRNAS has been advocated as a means of blocking cancer progression and as a possible means of anti-cancer therapy. Many miRNAs have been reported to function as tumour repressors, but equally others can promote tumorigenesis by enhancing cell proliferation and inhibiting apoptosis. miRNAs basically target numerous genes with varying outcomes. They interact with signalling by many important factors, which markedly influence biological behaviour of cancers; hence the complexity of interaction makes them difficult choices in terms of achieving specificity of function. They have been reported to interact with, and regulate signalling by, ER and AR, and in turn are themselves regulated by steroid receptors and other growth factors such as the BMPs. One would expect many pathways to be activated by these ligands and activated receptors; identification of specific molecular targets requires a colossal effort. miRNAs have recently been implicated in angiogenesis, some with promotion and others with its inhibition. Neoangiogenesis is a critical step in cancer dissemination. So it is a natural progression of events that with their ability to regulate cell proliferation, these studies, albeit at an early stage, have prompted the targeting of miRNAs to regulate angiogenesis as a means to control tumour growth and progression. Finally, certain miRNAs have been associated with drug resistance, thus adding a new dimension that might be highly relevant in the development of new drugs. However, it should be noted in any attempts at devising anti- or pro-miRNA strategy that miRNAs target numerous genes and collateral effects resulting from incidental interference with genes might be counterproductive. Furthermore, miRNAs do not display a consistent pattern of expression. Given the numerical abundance of miRNAs, the spectrum of positive and negative regulator species identified so far and the variety of biological effects that they exert, any attribution to individual miRNAs of regulatory properties with any degree of specificity would remain illusive. The major objective that inspired, motivated and stimulated the writing of this book was to group together rationally the wide panoply of growth factors, in the direct context of their ability to impinge upon and determine normal physiological function as well as aberrant morphogenesis, differentiation and the development of neoplasia and its progression, analyse the functions of growth factors, analyse their modes of signalling function and evaluate the various elements in the signalling cascades as potential targets of therapeutic deployment in the management of disease. So it is inevitable that it addresses not only how one can identify them but also how to scrutinise the constraints and limitations in their deployment for targeted therapy. The discussions in these pages bear ample testimony that many modes of growth factor signalling in cell cycle regulation, cell motility, ECM remodelling, induction of angiogenesis and vascular permeability have been unravelled to a substantial degree to link them to tumour growth and progression. Growth factor signalling occurs in the presence of the appropriate receptors to which they can bind and transduce their signal. These receptors would occur

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ubiquitously and in most cell types. We have noted the operation of mechanisms that might determine and direct the interaction between different pathways of signalling, different modes of interaction, and self- and inter-regulation of signal flow. As noted, several growth factors and other modifiers of biological responses modulate the cell phenotype by different routes or in collaboration with other growth factors. This constitutes another layer of complexity in the regulation of signalling and phenotype specification. Here we encounter not merely a staggering multiplicity of growth factors capable of controlling diverse cellular function but often also the sharing of receptors and downstream signalling elements, yet generating the same differentiated phenotype. Equally, other growth factors share the signalling system but can generate more than one phenotype. Growth factors can exert their influence in an autocrine or paracrine fashion. This is determined to a large degree upon the processing of the precursor ligand. In the TGF-β family, the processing of the precursor has a significant bearing on the mode of function. Thus, molecular modifications form an aspect of functional stability. Many proteins require activation by proteolytic processing. Prodomains of proteins subserve many functions. They can stabilise protein structure, inhibit the activity of proteins and besides they offer a target for protein stabilisation. Prodomains can bind to and regulate signalling by other proteins. Deletion of the prodomain has been found to alter the mode of protein functioning, that is, in an autocrine or paracrine fashion. This is especially the case in the TGF-β ligands. Furthermore, ligand function is facilitated by targeting them to appropriate receptors. The analyses here elucidate how this might occur, for example by differential affinity and specificity to the receptors, different means of targeting the ligands to the receptors, the recruitment of different signalling elements in the downstream cascade chain, specificity of activation of transcription factors, and signalling cross-talk that might enhance or inhibit signalling pathway. We have in this context noted how these determinants of specificity of signalling would affect the deployment of signalling elements as therapeutic targets. Here much effort has been expended on many molecular modules that attract, mediate and modulate the effects of growth factors. The TSP module might be cited as one of many domains that offers a potential interacting site for other biologically active molecules. The TSP domain has anti-angiogenic properties and participates in cell – extracellular matrix and cell-to-cell contact, so can be viewed as highly relevant in cancer invasion and metastasis, and can inhibit cell proliferation and cell migration. However, to focus on inhibition of angiogenesis, TSP induces endothelial cell apoptosis and downregulates VEGF. Besides, it can interact with HGF, EGF, TGF-β and FGF-2. These features endow a state of pre-eminence of molecular modules such as TSPs in cancer development and progression. With the revelation from wide-ranging and intensive study that many prominent pathways of signal transduction, for instance TGF-β, the hedgehog, Wnt, Notch, FGF and EGF family ligands, are able to alter, modify and amend the outcome of one another, a robust approach has been made and powerful emphasis placed in these pages on the highly conserved signalling systems in morphogenesis, differentiation and disease, to appraise their integrity as an entire entity as well as the integrity of

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individual elements, and the much evident cross-talk and convergence or interaction between relevant cascades of flow of information in mutual regulation and collaborative function. One has to take serious cognisance of proactive and counteractive aspects of interactive signalling while engaging in the search for appropriate targets. Cross-talk between growth factor signalling systems can be a positive force if the signalling collaboratively accentuates or inhibits signalling so that appropriate means of blocking signalling to provide a planned output. However, when a growth factor has too broad a range of interaction, cross-talk and inter-regulation, it would certainly pose an impediment to the development of a strategy of targeting the receptors of these ligands. IGFs are a standing example of why targeting them may be a debatable proposition. IGFs regulate normal development, tissue repair and regeneration, and many disease processes including tumour cell growth, survival, angiogenesis and metastasis. The biological function of IGFs is often amplified or subdued by interaction with signalling by other growth factors or signalling molecules. IGFBPs are generally inhibitory of IGF function. They can form complexes with and sequester IGFs from interacting with IGFRs. However, some can stimulate IGF effects. So IGFBPs can be viewed as a mode of regulation of both pathways. IGF signalling interacts with EGF, TGF-β, VEGF, TNF and HGF/MET, which modulate biological processes of cell proliferation, invasion and angiogenesis that are key components of cancer progression. IGF induces VEGF expression and vascularisation. IGF-I and LEP synergistically activate EGFR. Erlotinib and Lapatinib, which inhibit EGFR, also seem able to inhibit invasion and migration of breast cancer cells. The linkage of LEP and LR with cell proliferation and angiogenesis has aroused much interest about its possible relevance in cancer development and progression. LEP has been shown to promote cell proliferation in cancer cell lines and tumour growth. The inter-relationship between LEP/LR and IGF/IGFR signalling emphasises how the postulated transactivation and collaborative function of the two growth factors can affect IGF/IGFR targeting. The complexities of signalling interactions are only too obvious from this work. They sharply focus the need for an integrated and collective approach by molecular and cell biologists, together with clinicians, to unravel the outcome of signalling crosstalk, to interpret the data, to respond adequately to the state of the disease, to retard or inhibit progression and to promote remission to the benefit to the patient. An integrated approach of this kind to the study of several diseases is being made by the Centres of Integrated Systems Biology that have been established in the United Kingdom and around the world. An ardent appreciation of the interaction and mutual regulation of growth factor function in the development and secondary spread of cancer requires more than mere accumulation of technologies in a place; it requires cross-talk between the centres and more importantly with the clinical faculty of oncologists, pathologists and surgeons, together with modern technologies including molecular biology and artificial intelligence. Artificial neural networks are a powerful tool for analysing the complex interactions occurring between the host of growth factors that operate in a biological system and for predicting outcomes of relevance in the treatment of disease. The author’s team in the School of Electrical Electronic and Computer Engineering, University of Newcastle upon Tyne, has made a reasonable contribution

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to the application of artificial neural networks to predict cancer progression and prognosis, amply justifying the concept of integrated systems biology. However, one ought to be mindful that a great deal more needs to be done before laboratory findings can be applied in the clinics with stringent control and robust confidence. Systematic and systemic analyses have been attempted and pursued dynamically and vigorously to unravel many of the possible myriad of intricacies. Hopefully these might provide a deeper understanding of the patterns of co-ordinated function of growth factors and afford clues to the deployment of signalling components for developing modes of targeted therapy for human disease. How worthwhile has this effort been? We have traversed a full circle of argument and reasoning, and we have arrived at the beginning and at the opening Thirukkural in Tamil. We can affirm that the objective was indeed deemed worthwhile and significant enough to provide the purpose and resolve to undertake this tome, with the earnest hope that readers will also so judge this work.

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