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

Table of Contents

Cover image

Front-matter

Dedication

Copyright

Preface

Abbreviations

Introduction

1. Convergence of Growth-Factor Signalling Pathways in Developmental Systems and Pathogenesis

2. Growth Factor Families

3. Growth Factors in Differentiation and Morphogenesis

4. Vascular Endothelial Growth Factor

5. Hepatocyte Growth Factor

6. The Platelet-Derived Growth Factor Family

7. Nerve Growth Factors

8. Insulin-Like Growth Factors

9. Connective Tissue Growth Factor

10. Thrombospondins

11. Cytokines

12. Growth Factor and Hedgehog Signalling Pathways Meet in Developmental Systems

13. Epidermal Growth Factors and Their Signalling Systems

14. The Epidermal Growth Factor (EGF) Family

15. The Fibroblast Growth Factor Family

16. Intracellular Receptor Binding Growth Factors

17. The Androgens and Androgen Receptors in Development, Differentiation and Neoplasia

18. Vitamin D3 in Cell Proliferation, Apoptosis and Differentiation

19. Retinoids in Development and Pattern Formation

20. Oestrogens and Progesterone in Normal Physiology and Neoplasia

21. Glucocorticoid Signalling in Normal and Aberrant Physiology

Epilogue

References

Front-matter

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

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

Dedication

To Shri Sai Baba

Copyright

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First edition 2011

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Preface

G.V. Sherbet

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 growth-factor-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.

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.

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

FLRT

Fibronectin-leucine-rich transmembrane protein

Fox

Forkhead box transcription factors

FSH

Follicle stimulating hormone

GDF

Growth and differentiation protein

GH

Growth hormone (Somatotropin)

GM-CSF

Granulocyte–macrophage colony-stimulating factor

GPCR

G-protein coupled receptors

GRE

Glucocorticoid receptor elements

GSK

Glycogen synthase kinase

HB-EGF

Heparin binding epidermal growth factor

HCG

Human chorionic gonadotropin

HER2

Human epidermal growth factor receptor (erbB2)

HESC

Human embryonic stem cell

HGF

Hepatocyte growth factor

Hh

Hedgehog proteins

HIF-1

Hypoxia-inducible factor 1 transcription activator

HLH

Helix-loop-helix transcription factors

HMG

High Mobility Group transcription factors encoded by Sox genes

HPV

Human papilloma virus

HRG

Heregulin

IFITMS

IFN-induced transmembrane protein

IFN

Interferon

IGF

Insulin-like growth factor

IGFBP

IGF binding proteins

IGFR

IGF receptor

Ihh

Indian hedgehog

IP

Interferon induced protein

IPT

Ig domain of HGF/MET receptor

IR

Insulin receptor

IRS

Insulin receptor substrate proteins

JAK

Janus kinase

JNK

Jun N-terminal kinase

LEF

Lymphoid enhancer factor

LH

Luteinising hormone

LIF

Leukaemia inhibitory factor

LIM

Homeodomain proteins with Cys-His motifs

LTF

Lactoferrin

MAD

MAX dimerisation protein

MAPK

Mitogen-activated protein kinase

MEK

A threonine and tyrosine kinase that phosphorylates MAPK

MEKK

MAPK kinase kinase

mER

Membrane located oestrogen receptor

MH

MAD homology domain

MCP

Monocyte chemoattractant protein

MMP

Matrix metalloproteinase

MPA

Medroxyprogesterone acetate, a synthetic progestin

mTOR

Mammalian target of rapamycin

NCAM

Neural cell adhesion molecule

NGF

Nerve growth factor

NO

Nitric oxide

NOS

Nitric oxide synthase

NSCLC

Non-small cell lung carcinoma

OPG

Osteoprotegerin

PACE

Paired basic amino acid cleaving enzyme

PDF

Prostate derived factor

PDGF

Platelet-derived growth factor

PDGFR

Platelet derived growth factor receptor

PI3K

Phosphoinositide-3 kinase

PIN

Prostatic intraepithelial neoplasia

PIP2

Phosphatidylinositol 4,5-bisphosphate

Pitx

Paired-like homeobox transcription factor

PKA

Protein kinase A

PKC

Protein kinase C

PLGF

Placental growth factor

PPAR

Peroxisome proliferator-activated receptor

PR

Progesterone receptor

PSA

Prostate-specific antigen

PSI

Plexin semophorin integrin domain of MET receptor

PTCH

Patched glycoprotein binding hedgehog proteins

PTHRP

Parathyroid hormone-related protein

RANK

Receptor/activator of NF-κB transcription factor

RANKL

The receptor/activator of NF-κB ligand

RAR

Retinoic acid receptors

Rb

Retinoblastoma protein

RCC

Renal cell carcinomas

RTK

Receptor tyrosine kinase

S1P

Sphingosine 1-phosphate, a GPCR ligand

Shh

Sonic hedgehog

siRNA

Small interference RNA

SMO

Smoothened, a component of Hh signalling

SRC

Steroid receptor co-activator

STAT

Signal transducers and activators of transcription

Tbx

T-box gene family of transcription factors

TFF1

Trefoil protein

TGF

Transforming growth factor

TIMP

Tissue inhibitor of metalloproteinase

TNBC

Triple-negative breast cancer

TNF

Tumour necrosis factor

TNFR

TNF receptor

TRADD

TNFR associated death domain

TRAIL

TNF associated apoptosis inducing ligand

TRAF

TNF associated factor

TSH

Thyroid stimulating hormone

TSP

Thrombospondin

TWEAK

TNF family type II transmembrane ligand

TWEAKR

TWEAK receptor

uPA

Urokinase-type Plasminogen Activator

uPAR

uPA receptor

VD3

1,25-dihydroxyvitamin D-3 cholecalciferol

VDR

Vitamin D 3 receptor

VDRE

Vitamin D 3 response element

VEGF

Vascular endothelial growth factor

Vg-1

TGF-β homologue from Xenopus laevis

VHL

Von Hippel-Lindau

VWC

Von Willebrand type C repeat module

WISPs

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 growth-factor-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 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.

1. Convergence of Growth-Factor Signalling Pathways in Developmental Systems and Pathogenesis

The dynamics of differentiation, growth and the initiation and progression of cancer requires a co-ordinated function of growth factors and hormones to achieve regulation of cell proliferation and survival, invasion and metastatic spread. Growth factors often share signal transduction systems, yet they can generate distinct phenotypic features by intricate information flow. The commonality or merger of signalling cascades in their function as signalling molecules is highlighted in this short chapter.

Key words: Biological response modifiers, cell motility, growth factors, metastasis, steroid hormones signal transduction

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.

2. Growth Factor Families

Growth factors are divisible into two families of ligands: (1) membrane-receptor binding family; (2) intracellular-receptor binding family. The membrane-receptor binding family can be differentiated into the cystine-knot growth factor group and the EGF and FGF family of growth factors. The intracellular-receptor binding ligands bind cytoplasmic receptors or nuclear receptors to transduce their message.

Key words: Cystine-knot growth factors, cytoplasmic receptors, membrane receptors, nuclear receptors, epidermal growth-factor group, fibroblast growth factor family, retinoids, steroid hormones, vitamin D3

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 sub-divided here into the cystine-knot group of growth factors, and the EGF- and FGF-family 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 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 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.

3. Growth Factors in Differentiation and Morphogenesis

This chapter focuses on the transforming growth factor-β (TGF-β) superfamily of ligands, namely the TGF-β isoforms, nodal and activin/inhibin, together with BMP, GDF, Vg-1 and anti-Müllerian hormone. TGF-β family growth factors are transcribed as precursor proteins and processed into mature functional proteins. TGF-β is a highly versatile ligand with diverse and pleiotropic physiological effects in both normal cellular function and in disease processes. In relation to cancer, TGF-β can reputedly suppress tumorigenesis in early stages of cell transformation and growth, and yet it can promote progression of advanced cancer. Members of the TGF-β family transduce their effects through the same transmembrane receptors and the canonical Smad signalling system, but generate widely diverse effects. This is the focal point of debate here, especially how the signals provided by the different ligands of the family interact, modulate and collude to achieve their physiological outcome.

Key words: Activin/Inhibin, BMP, GDF, nodal pleiotropic effects, TGF-β

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.

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 (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 and Kerhl et al., 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 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 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 ligand-dependent 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 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 non-conserved 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; (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 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-κxB 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 Smad-mediated 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 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, 2006 b). A full or normal complement of endoglin is necessary for maximal angiogenesis. 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., 2007 a). 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 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 contra-indications 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, 2002 b). 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