The type 1 (EGFR-related) family of growth factor receptors and their ligands.

Progress in Growlh Facror Research, Vol. 4, pp. l-24. Printed in Great Britain. All rights reserved.

0955-2235/92 SC 1992 Pergamon

1992

THE TYPE 1 (EGFR-RELATED) GROWTH FACTOR RECEPTORS LIGANDS

$15.00 Press Ltd

FAMILY OF AND THEIR

Sally A. Prigent and Nicholas R. Lemoine ICRF Oncology Group Royal Postgraduate Medical School, Hammersmith Hospital Du Cane Road, London WI2 ONN, U.K.

This review considers the biology of the type 1 growth factor receptor family which is increasingly recognised as important in the control of normal cellproltferation and in the pathogenesis of human cancer. The family currently comprises three closely related members: the epidermal growth factor (EGF) receptor, c-erbB-2 and c-erbB-3, all of which show abnormalities of expression in various human tumours. The family offactors related to EGF has also expanded recently and now includes transforming growth factor alpha, heparin-binding EGF, amphiregulin, cripto and heregulin, as well as several other potential ligands for the c-erbB2-2 receptor. The involvement of these receptors and growth factors in human cancer has implications for the design of novelforms of therap?, ,for cancer, and we review recent advances andfuture avenues for investigation. Keywords: EGF receptor, c-erbB-2 (HERyneu), c-erbB-3 (HER3). amphiregulin, heregulin, oncogene, growth factor.

EGF, TGF alpha,

INTRODUCTION Epidermal growth factor was first extracted from mouse submaxillary glands [ 1] and was subsequently shown to have growth promoting effects and to stimulate uptake of glucose and amino acids by cells. Isolation of this ligand made possible the purification of the epidermal growth factor receptor (EGFR) from the squamous carcinoma cell line A43 1 in which it is overexpressed [2], and some years later its cDNA sequence [3,4, 5, 61 and genomic structure [7] were determined. The techniques of molecular biology facilitated the identification of two related putative receptors, c-erbB-2 (HER2) and more recently c-erbB-3 (HER3), by low stringency probing of human cDNA libraries with sequences from the EGFR (c-erbB-1) gene or its avian viral homologue v-erbB. While the signalling mechanism of the EGF receptor has been carefully examined, the Acknowledgements-We arc grateful to Bill Gullick for his critical reading of the manuscript and his helpful comments, and to Zoe Redley for her excellent typing.

2

S.A. Prigent and N.R. Lemoine

biology of c-erbB-2 and c-erbB-3 has been poorly defined, mainly because their natural ligands have not been identified. It is now clear that EGF is just one member of an expanding family of related peptides, of which some are capable of binding to the EGF receptor and others are candidate ligands for the c-erbB-2 and c-erbB-3 receptors. This review will describe our current knowledge of the members of the c-erbB family of receptors, their ligands and their involvement in human neoplasia. THE EPIDERMAL

GROWTH

FACTOR

RECEPTOR

The EGF receptor is a 170 kDa glycoprotein consisting of three domains: a 621 amino acid extracellular domain responsible for ligand recognition which contains a high proportion of cysteine residues clustered in two distinct regions; a 23 amino acid hydrophobic transmembrane region; and a 542 amino acid intracellular region containing a highly conserved tyrosine kinase domain (Fig. 1). Binding of ligand leads to receptor dimerisation which increases the catalytic activity of its tyrosine kinase [8]. Receptor autophosphorylation on tyrosine residues occurs at three major sites towards the C-terminus: Y-1068, Y-l 148, Y-l 173 [3], although two other potential sites have been reported 19, lo]. Mutational analysis suggests that phosphorylation of the three major sites is important for maximum biological activity [l 1, 121, and may be a necessary prerequisite to permit access of substrates to the catalytic site of the EGFR. The C-terminal tail may also contain a more proximal region involved in negative regulation and internalisation [13, 141. Following activation, the EGFR kinase phosphorylates a number of cellular substrates including phospholipase C gamma, MAP kinase and the ras GTPase-activating protein GAP, which, at least in the case of phospholipase C gamma, leads to an increase in catalytic activity [ 15, 16, 17, 18, 191. It has recently been shown that a small soluble actin-binding protein called profilin binds to the substrate phosphatidylinositol 4, 5-biphosphate and inhibits its hydrolysis by phospholipase C-gamma unless this enzyme has been phosphorylated by the EGF receptor tyrosine kinase [20]. The involvement of profilin may be the link that could explain the effects induced by EGF or TGF alpha on cellular morphology and motility. The EGF receptor can itself be a substrate for phosphorylation on threonine residues. Phosphorylation of threonine-654 has been shown to inhibit EGFR tyrosine kinase activity [21]. Recently two additional growth factor-stimulated kinases have been isolated which phosphorylate the EGF receptor at threonine669 [22]. This site has been reported to regulate internalisation and substrate phosphorylation on tyrosine residues 1231. Phosphorylation of EGFR by serine/ threonine kinases may be the mechanism by which platelet-derived growth factor, phorbol esters and other factors can influence the activity of the EGF receptor ~241. The activated receptor/ligand complex is endocytosed and degraded within the lysosomes, except in some cell types such as hepatocytes, where the receptor is recycled to the cell membrane. This process of internalisation may be essential for control of normal mitogenic signalling since a truncated EGFR mutant with normal kinase activity is not internalised and possesses increased transforming activity u41.

rvpe I Growth Factor Receptors

% amino acid

EGFR c-erbB2 c-erbB3

EGFFU

sequence EGFFU

Identity c-tiB21

42

I

46

46

45

45

46

44

41

50

41

82

60

62

I

TK TK

FIGURE 1. Diagrammatic representation of the sequence similarities of ditfere-nt domains of the EGF receptor, c-erbBt and c-erbB3. The percentage figares indicate the proportion of identical residoes shared by the indicated pairs of receptors. (Reproduced by permission from Cancer Topics, 1992 Vol. 8.)

Abnormalities of EGFR Signal Transduction

In pathological conditions the signal transduction pathway involving the EGF receptor can be subverted in various ways. Overexpression of the stimulating ligands TGF alpha and EGF, and/or the EGF receptor itself, occurs in a wide variety of human tumour types. EGF receptor overexpression may be associated with amplification of the gene or may occur in the presence of the normal gene copy number, presumably due to increased transcription or increased stability of the transcribed messenger RNA. Amplification of the EGF receptor locus may involve large amplicons of DNA spanning up to 1000 kb, and the contribution of flanking sequences and co-amplified genes to cellular transformation in these cases is not yet clear. In addition, structural alterations have been reported affecting both extracellular and intracellular domains of the receptor in the A431 vulva1 carcinoma cell line and in malignant gliomas, one type of human brain tumour [25, 26, 27, 28, 291. Overexpression of either ligand or receptor in the absence of the other does not usually result in full neoplastic transformation in vitro [30], but high expression of both components together can lead to transformation of a variety of cell types [31, 32, 33, 34, 351. However, transgenic animal experiments suggest that overexpression of the EGF receptor does not transform cells in vivo even when ligand is available [36].

4


It is beyond the scope of this review to discuss the aberrant expression of EGFR in tumours and this topic has recently been reviewed in detail elsewhere [37]. In summary, the EGF receptor is overexpressed at high frequency in a wide range of tumours, commonly in squamous carcinomas of various sites and less commonly in adenocarcinomas (particularly in pancreatic and gastric cancers) [38, 391. Overexpression, sometimes associated with gene amplification, may be associated with poor prognosis in breast cancer, lung cancer and bladder cancer. THE c-e&El-2 PROTO-ONCOGENE The human c-erbB-2 proto-oncogene was identified independently by two groups [40,41] who probed human genomic libraries at low stringency with probes from the v-erbB oncogene (a truncated version of the EGF receptor from the avian erythroblastosis retrovirus). Amplification of the gene was found in a human breast cancer (MAC1 17) [42] and the MKN-7 gastric cancer cell line [43]. Subsequent work has shown that c-erbB-2 is amplified and overexpressed in a significant proportion of adenocarcinomas of various sites, but rarely in other types of malignant tumour. Analysis of the c-erbB-2 coding sequence shows that it has structural features of a growth factor receptor with close similarity to the EGF receptor protein [44] and the gene is sometimes referred to as HER-2, based on its homology to the human EGF Ieceptor. To determine whether c-erbB-2 can convey a mitogenic signal an elegant strategy has been employed [45, 461 to create a chimaeric protein consisting of the extracellular ligand binding domain of the EGF receptor fused to the cytoplasmic sequence of the human c-erbB-2 protein or the equivalent rat protein, neu. The chimaeric protein bound EGF, which stimulated autophosphorylation and mitogenesis, and was shown to induce increased transcription of fos, jun, the glucose transporter and ornithine decarboxylase genes [47]. A number of groups have shown that activation of the EGF receptor can result in cross-phosphorylation of the c-erbB-2 protein in cells that express both receptors [48, 49, 501. This might be achieved, at least in part, by heterodimerisation of the EGF receptor and the c-erbB-2 protein, which is induced by EGF binding and dramatically increases the capacity for self-phosphorylation of the dimerised receptor [51, 521. It seems likely that while there may be cellular substrates that are common to both receptor tyrosine kinases, there may be others that are specific for either the EGF receptor or c-erbB-2. In experiments with chimaeric receptors regions within the cytoplasmic domain have been identified that may determine this specificity [53, 541. The EGF receptor and c-erbB-2 protein can act in a synergistic fashion to transform rodent cells [55], but the mechanisms involved in this cooperation are still unclear although the capacity for formation of heterodimers is obviously a possibility [5 1,521. Mechanisms

of Activation

of c-erbB-2 to a Transforming

Oncogene

As with most proto-oncogenes c-erbB-2 can be activated to a transforming oncogene by several, quite different, mechanisms. The first indication of the role of the gene in cancer came from a rat experimental carcinogenesis model. Treatment of pregnant BDIX rats with the chemical carcinogen ethylnitrosourea leads to the development of

Type 1 Growth Factor Receptors

5

tumours of the CNS at high frequency. Bargmann and Weinberg [56] subsequently demonstrated that this was due to a single point mutation in the c-erbB-2 gene (in the rat referred to as the neu gene) causing a valine residue in the transmembrane region of the molecule to be converted to glutamic acid. This mutation stabilises receptors in a dimeric form in which they are catalytically active [57]. When the same mutation is introduced into the equivalent position (codon 659) of the human gene it will transform cells in culture [58] and the same mutation in the Drosophila EGF receptor (DER) greatly increases its tyrosine kinase activity [59]. It is intriguing that similar mutations in the transmembrane domain of the human EGF receptor do not apparently produce oncogenic activation [60,61]. We have used sensitive polymerase chain reaction (PCR) assays to detect such activating point mutations of c-e&B-2 and found no evidence of their occurrence in a large series of human tumours of various types [38,62,63,64,65]. Other groups have reached the same conclusion by sequence analysis in smaller numbers of human cancer DNAs [66, 671. Mutations at other codons in the transmembrane encoding region do not activate the transforming function of the neu or c-erhB-2 genes. There is some polymorphism of the surrounding sequence in normal individuals [68]. Expression of the mutant rat neu gene under the control of mouse mammary tumour virus long terminal repeat (MMTV LTR) causes the frequent development of breast cancers in transgenic mice [69,70] and introduction of the gene into mammary epithelial cells in the breast tissue of living rats produces tumours [71]. Expression of the wild-type or a mutant c-erbB-2 gene under the control of the MMTV promoter produced adenocarcinomas of several sites as well as B cell lymphomas [72]. The c-erbB-2 protein can be partially activated by truncation of the gene so that the extracellular portion of the molecule is deleted [58, 731. Such changes are observed in human tumours as rearrangements of the c-erbB-2 gene, but relatively few instances of this have been reported [see for example 74, 75, 76,771. The chief mechanism of activation of the c-erbB-2 gene in human cancers is by aberrantly high expression of the receptor protein. Gene amplification appears to be the predominant mechanism leading to elevated protein expression, at least in breast cancer. but overexpression by increased mRNA transcription alone does occur in a minority of cases [78, 791. In several breast cancer cell lines there is evidence of increased transcription of individual copies of c-erbB-2 contained in the amplicon, which suggests that increased transcription may precede amplification and be sustained after the increase in gene copy number [78]. The elements involved in the control of c-erbB-2 expression are under intensive study and it has recently been shown that oestrogen may have an inhibitory effect on c-erbB-2 transcription in breast cancer cells [80]. The role of abnormal c-erbB-2 expression in human tumours is currently uncertain, but it does not appear to be directly associated with cell proliferation. Attempts have been made to correlate c-erbB-2 overexpression with high rates of cell proliferation in invasive and in situ breast cancer [81,82,83] but experimental studies suggest that there may not be a simple relationship between expression of c-erbB-2 and human breast cancer cell proliferation in vivo and in vitro [80,84,85]. There is evidence that c-erbB-2 may contribute to the invasion and spread of tumours. Clinical studies have demonstrated that c-erbB-2 amplification and overexpression are correlated with aggressive tumour growth and poor prognosis in breast and ovarian cancer [66] and

6


experimental studies show that the rat neu oncogene can induce the metastatic phenotype in transfected cells [86]. Expression

of Human c-erbB-2 in Normal Tissues and Tumours

The c-erbB-2 protein is expressed widely on normal human and animal tissues, but characteristically on secretory epithelia [87, 88, 89,901. The expression of c-erbB-2 in human tumours is reviewed in detail elsewhere [91] but we will briefly outline the major points here. Overexpression of the c-erbB-2 growth factor receptor occurs, usually as a consequence of gene amplification, in a fifth of breast, stomach, pancreatic, bladder and ovarian cancers. Overexpression occurs less frequently in some other adenocarcinomas and in squamous tumours of the lung. Overexpression is associated with poor prognosis in breast and ovarian cancers and possibly non-small cell lung cancers. THE c-e&B-3 PROTO-ONCOGENE Two groups have recently cloned a third member of the erbB receptor tyrosine kinase family. Aaronson and colleagues [92] detected the new member of the erbB protooncogene family by reduced stringency hybridisation of a v-erbB probe to normal human genomic DNA. Todaro and colleagues cloned a gene of almost identical sequence from the MDA-MB-361 and A431 cell lines using hybridisation with degenerate oligonucleotides encoding a conserved 7 amino acid sequence in the tyrosine kinase domain of the EGF receptor [93]. The predicted structure of the c-erbB-3 protein is closely similar to that of both the EGF receptor and the c-erbB-2 growth factor receptor (Fig. 1). The highest degree of sequence identity is in the tyrosine kinase domain where 60% of the residues are identical in EGFR and c-erbB-3. The kinase domains of EGFR and c-erbB-2 are more similar to each other (82% homology) than to that of c-erbB-3. The mature protein is extensively glycosylated and has a molecular weight of 160 kDa. Expression of c-erbB-3 transcripts was demonstrated in cultured keratinocytes and melanocytes but not in stromal fibroblasts, and was detected in 36 of 38 carcinomas but in only 2 of 12 sarcomas and 0 of 7 haematopoietic malignant cell lines. Overexpression of c-erbB-3 mRNA (in the absence of gene amplification or gross rearrangement) was found in 6 of 17 breast cancer cell lines. The possibility that c-erbB-3 expression might be relatively restricted to cells of epithelial or neuroectodermal origin is confirmed by our own studies using a panel of specific antibodies to this protein [94]. We find that while it is widely expressed in normal epithelial and some mesenchymal tissues, it is not generally found in haematopoietic or lymphoid cells. THE EGF FAMILY

OF GROWTH

FACTORS

Purification and sequence analysis of EGF revealed the presence of six conserved cysteine residues which cross-bond to create three peptide loops [95]. More recently several other peptides which interact with the EGF receptor have been purified and cloned and contain the same generalised motif X,CX,CX,,,CX,,CXCX,GX,CX,, where X represents any non-cysteine amino acid, and n is a variable number. These include TGF-alpha, amphiregulin, heparin-binding EGF and some virally-encoded

/

Type I Growth Fuctor Receptors

peptides. An interesting feature of these peptides is that they are all synthesised as much larger membrane-bound, glycosylated precursors which (at least in the case of EGF and TGF-alpha) have been shown to possess biological activity. Very recently it has been reported that a ligand for c-erbB-2 called Heregulin also possesses the same general structure and is synthesised as a larger precursor. The predicted secondary structure of five members of the EGF ligand family is shown schematically in Fig. 2. EGF-like sequences are not confined to growth factors but have been observed in a variety of cell-surface and extracellular proteins which have interesting properties in cell adhesion, protein-protein interaction and development [96]. These proteins include blood coagulation factors (factors VI, IX, X, XII, protein C, protein S, protein Z, tissue plasminogen activator, urokinase); extracellular matrix components (laminin, cytotactin, entactin); cell surface receptors (LDL receptor, thrombomodulin receptor) and immunity-related proteins (complement Clr, uromodulin). Interestingly, the general structural pattern of EGF-like precursors is preserved through lower organisms as well as in mammalian cells. A number of genes with developmental significance have been identified in invertebrates with EGF-like repeats. Perhaps the best characterised of these is the Notch gene of Drosophila which encodes 36 tandemly arranged 40 amino acid repeats which show homology to EGF [97]. Hydropathy plots indicate a putative membrane spanning domain, with the EGFrelated sequences being located on the extracellular side of membrane. Other homeotic genes with EGF-like repeats include Delta, 95F and 5ZD which were identified using probes based on Notch, and the nematode gene U-I.2 which encodes a putative receptor for a developmental signal transmitted between two specified cells. The conservation of EGF-like motifs between invertebrates and mammals is intriguing. It is interesting to speculate that EGF-like growth factors and the many other molecules containing EGF-like sequences which participate in a wide variety of functional systems, may have evolved from an ancestral cell-to-cell communication system. This review will consider only those proteins with the characteristics of growth factors. Epidermal Growth Factor

The history of the discovery of EGF and its structural and functional properties have recently been reviewed by Laurence and Gusterson [96]. During studies of nerve growth factor, a peptide was detected in extracts of submaxillary glands which induced precocious eyelid opening and incisor eruption by direct stimulation of epidermal growth and keratinisation [98]. A 53 amino acid peptide was purified from mouse submaxillary glands and sequenced [l]. The human equivalent, urogastrone, was subsequently isolated from urine [99]. The mRNA for EGF is approximately 4750 base pairs and encodes an EGF-precursor of 1217 amino acids [loo, 1011. Contained within this long coding region are the EGF coding region itself and nine other sequences with a high degree of homology to EGF. The mature EGF unit is that closest to the carboxyl terminus in the precursor protein. The protein begins with a 29 amino acid signal sequence and the precursor structure resembles a transmembrane receptor with a hydrophobic alpha helical membrane spanning domain and a region of high homology to the low density lipoprotein receptor [ 1021. Several mouse tissues, particularly distal renal tubule cells, do not process the 1217 amino acid precursor [ 1031, suggesting that EGF and the nine additional EGF-like sequences in the precursor may exist as a cell

8


surface protein and have some recognition function. Studies using an EGF mini-gene expression system have recently shown that the membrane-anchored form of EGF is capable of stimulating EGF receptors on adjacent cells, similar to the situation for TGF-alpha [ 1041. The 3D structure of EGF has been determined by high resolution ‘H NMR and computational analysis, and functionally important residues have been located by a combination of site-directed mutagenesis and ‘H NMR [105]. EGF binds exclusively to the EGF receptor and not to c-erbB-2 or c-erbB-3. It is a potent stimulator of cell multiplication and a modulator of the differentiation and function of cells of various types [99, 106, 1071. EGF inhibits gastric acid secretion and protects the gastric mucosa from damage by the acid [IO& 1091 and has recently been associated with a novel cell lineage responsible for mucosal repair of the gastrointestinal tract [I 10, 111, 1121. It has been localised immunohistochemically to the submandibular salivary gland, serous glands of the nasal cavity, stomach, Brunner’s glands of the duodenum, anterior pituitary, bone marrow, sweat glands, mammary gland, ovary, uterus and placenta [ 113, 114, 115, 116, 1171. Synthesis of EGF in mouse salivary glands is strongly induced by both androgens and Padrenergic agonists [98]. It is detectable in saliva, milk and urine but, in man, circulates primarily in alpha granules in platelets [I 181, suggesting a role in wound healing analogous to PDGF and TGF alpha, which are also found in platelet granules [119]. EGF accelerates eyelid opening and tooth eruption in new born mice [ 1051, skin development in foetal lambs [120] and lung maturation in foetal rabbits [121] and lambs [122]. It also stimulates growth of gastric mucosa in suckling rats [ 1231 and of hypoproliferative intestinal epithelium in adult rats maintained on total parenteral nutrition 11241. Recently it has been shown that EGF antisense oligonucleotides can block the initiation of odontogenesis in murine mandibular explants [ 1251. Little is known of the involvement of this growth factor in neoplasia, but there are reports of elevated expression in 12% of pancreatic cancers [ 112],86% of pleomorphic adenomas of salivary glands [I 26],68% of prostatic carcinomas [ 1271, and around 20% of gastric carcinomas [39, 1281. Elevated levels of “EGF-like” factor have been described in about 30% of ovarian, endometrial and cervical carcinomas and 16% of breast carcinomas [ 1291. Transforming

Growth

Factor-Alpha

Transforming growth factor-alpha (TGF-alpha) is a potent mitogen [130]. TGFalpha structurally and functionally resembles epidermal growth factor [EGF] and induces a mitogenic response by binding to and activating the tyrosine kinase activity of the EGF receptor [131, 132, 1331. TGF-alpha does not bind c-erbB-2 and its interaction with c-erbB3 has not been examined. The mature form of TGF-alpha is synthesised as part of a 160 amino acid cell surface precursor from a 4.8kb mRNA [134, 1351. The mature 50 amino acid TGF-alpha is released from the extracellular domain by proteolytic cleavage between alanine-valine residues at both termini. Larger soluble forms of TGF-alpha representing incompletely-processed intermediates have also been identified [136, 1371. Such precursor molecules may accumulate on the surface of certain cells [138] and may be biologically active through interaction with EGF receptors on the surface of adjacent cells [138, 1391. TGF-alpha may therefore be biologically active as either a secreted peptide or a membrane-bound ligand.

9

rvpe I Growth Factor Receptors

There is now a great deal of evidence to implicate TGF-alpha in processes of normal development. Exogenously administered EGF or TGF-alpha can influence the development of several tissues. Exogenous TGF-alpha accelerates tooth eruption and eyelid opening in new born mice [140, 1411. TGF-alpha mRNA has been identified in preimplantation mouse embryos [142] and at low levels in several tissues of foetal day 9 and 10 mice [143]. In adults, TGF-alpha mRNA and/or protein have been detected in the epithelial cells of the anterior pituitary [144], maternal decidua [145], skin keratinocytes [ 1461, bronchus, intestine, kidney tubules, female genital tract [ 1471, brain [148] and activated macrophages [149]. TGF alpha has also been implicated in inflammation and wound healing [ 112, 1501, cell migration [ 1511, angiogenesis [ 1521. and bone resorption [ 153, 1541. Elevated expression of TGF-alpha has been frequently associated with neoplastic transformation. TGF-alpha activity was first described in the culture medium of retrovirally transformed cells by virtue of its ability to collaborate in the reversible transformation of cultured NRK cells [155, 156, 1571. Elevated production of TGFalpha is frequently found in human cancers [158] and malignantly transformed cultured cells [ 1591 including malignancies of the liver [ 160, 16 1] breast [ 1621, stomach [163], lung [164, 1651, colon [164], kidney [166], pancreas [112, 167, 1681, ovary [147] and tumours of the thyroid [169]. Overexpression of TGF-alpha under certain conditions can lead to hyperproliferation of cells in the absence of neoplasia as in the case of psoriasis [170]. Direct evidence to suggest that TGF-alpha can contribute to neoplastic transformation has been provided by reconstruction experiments in which recombinant TGF-alpha is expressed in cultured cells in vitro. Transfection of TGF-alpha plasmids into rat I [31] and NRK fibroblasts [32] and mouse mammary epithelial cells [33, 341 can produce full transformation as assayed by anchorage independence in soft agar and tumorigenicity in nude mice. However, other workers have failed to observe transformation of NRK fibroblasts [33] or NIH 3T3 fibroblasts [I711 expressing high levels of TGF-alpha. It has been shown that expression of membrane-anchored proTGF-alpha can be sufficient to produce this transformation, and that this form can interact with EGF receptors on adjacent cells without further processing [172]. Transformation of NIH 3T3 fibroblasts by expression of TGF-alpha may be dependent upon the simultaneous overexpression of EGF receptors [35, 171, 1731. Expression of TGF-alpha under the control of the metallothionein gene promoter in transgenic mice leads to disordered growth and differentiation in breast, liver and pancreas, and ultimately to neoplasia in liver and breast [174, 1751. Amphiregulin

Todaro and colleagues have identified a new growth factor which was isolated from the conditioned medium of MCF-7 cells treated with phorbol ester [176, 1771. This growth factor, termed amphiregulin, was found to be both structurally and functionally related to EGF and TGF-alpha. The mature protein of either 78 or 84 amino acids is embedded within a 252 amino acid transmembrane precursor, much like that found in TGF-alpha. In contrast to EGF and TGF alpha amphiregulin also has a hydrophilic 43 amino acid extension rich in lysine and arginine residues at its Nterminus (Fig. 2). This domain also includes motifs usually associated with nuclear localisation and DNA binding [I781 and specific antibodies have been used to

S.A. Prigent and N.R. Lemoinr

10

demonstrate nuclear immunoreactivity in both cultured cells and human biopsy material [179, and our own unpublished data]. High level expression of amphiregulin occurs in normal placentae, testis and ovaries and significant amounts in pancreas, colon, breast and some other tissues [178]. As its name suggests amphiregulin has both stimulatory and inhibitory effects on cell growth depending on the cell type [ 1761 and concentration of ligand [ 1791. Amphiregulin stimulates proliferation of normal human fibroblasts and keratinocytes but dramatically inhibits the growth of two human breast cancer cell lines that overexpress both TGF alpha and EGF receptor (MDA-MB-468 and MDA-MB-231). The expression of amphiregulin is inversely correlated with the expression of TGF-alpha in individual cell lines [l SO]. The mechanism by which amphiregulin produces its bifunctional effects on cell growth is not yet clear. Ciardiello et al. [ 18 l] report overexpression of amphiregulin (together with cripto) in 60-70% of primary or metastatic colorectal carcinomas compared with normal colonic mucosa. Recently heparin-affinity chromatography has been used to purify from the conditioned medium of human keratinocytes a peptide called keratinocyte autocrine factor (KAF) that is closely related or possibly identical to amphiregulin [182]. The function of this growth factor can be inhibited by heparin. Schwannoma-Derived

Growth Factor (SDGF)

This heparin-binding factor which has mitogenic activity on glial cells was isolated from the conditioned medium of the JSl cell line established from a rat Schwann ccl! tumour [183]. Screening of a cDNA library from JSl cells with degenerate oligonucleotides based on the amino acid sequence allowed cloning of nucleic acid sequence for a 243 residue prepro-protein and a 3’-untranslated sequence. It seems likely that SDGF is the rat homologue of amphiregulin since the amino acid sequence of SDGF is 76% identical to human amphiregulin, but there are distinct differences between the two molecules such as the greater molecular weight of the most abundant form of SDGF compared with amphiregulin. There are also differences in their biological activities, with amphiregulin being a less potent mitogen than SDGF for 3T3 cells and SDGF lacking any inhibitory effect on the growth of A43 1 cells. Heparin-Binding

EGF-Like

Growth

Factor (HB-EGF)

An apparently novel growth factor of the EGF family has been purified from the conditioned medium of the human U-937 histiocytic lymphoma cell line [184] and cDNA subsequently cloned from cells stimulated with phorbol ester. The primary translation product comprises 208 amino acids with a central domain, including an EGF-like motif rich in cysteine residues. HB-EGF most closely resembles amphiregulin, since these two polypeptides consist of a comparable number of amino acids and both have a notably hydrophilic domain upstream of the EGF-like motif [184]. These authors have shown that HB-EGF can bind to the EGF receptor on A43 1 cells and smooth muscle cells with high affinity, and is mitogenic for keratinocytes and postulate that it may be involved in wound healing.

Type I Growth Factor Receptors TGF-alpha

EGF

Cripto

FIGURE 2. Schematic representation of tive members of the EGF family. The secondary stroctore of hmnao EGF is redrawn from Hommel et aL, 1991 [lw aod contairrs two antiparallel /&sheets. Cysthe bridges are indicated by bars. Tke representethos of the other ECF-like ligaods are not based on NMR data, bat have been drawn to iaclade tke EGF core. Completely conserved residues are shown in black and positiveiy-ckarf@ residues in the N-termioal extensions of HB-EGF nod ampkiregolin are represented by shaded circles. Where indicated (?), N- and C-termini have not been determioed.

Viral Growth

Factors

DNA sequences encoding EGF-like peptides were first demonstrated in the genomes of several pox-viruses including Vaccinia virus [185], Shope fibroma virus [186], Molluscum contagiosum [187] and Myxoma virus [188] by searching genetic

12


databanks. The processed gene product of one of these sequences, Vaccinia virus growth factor (WGF) was subsequently purified from the medium of Vaccinia virus infected cells, and was found to be a 77 amino acid glycosylated peptide [189]. VVGF is structurally similar to EGF (37% amino acid homology) and TGF-alpha (30% homology) with an identical pattern of cysteine residues to that in TGF-alpha. It is derived from a large precursor protein and the glycosylated processed form has a molecular weight of 23 kDa. It stimulates proliferation of human diploid fibroblasts by binding to the EGF receptor. The putative growth factors encoded in the genomes of other pox-viruses have not been purified, however a synthetic 55 amino acid peptide encoding the ( trboxyl portion of the Shope fibroma virus growth factor is capable of inducing EGF like bioeffects [190]. The role of these growth factors in viral replication and cytopathology is unclear. Cripto

The cripto gene was cloned accidentally [ 19 l] from a human embryonal carcinoma cell line as a composite cDNA with an unrelated gene. The open reading frame of the gene encodes a protein of 188 amino acids with a central domain that includes a structural cysteine-rich motif common to other members of the EGF family including EGF, TGF-alpha and human amphiregulin. Distinct from other family members cripto does not possess the A loop which is created when cysteines Cl and C3 (numbered from the N-terminus) form a disulphide bond, and it does not appear to exist in a transmembrane form. The interaction of cripto with the EGF receptor, c-erbB-2 or c-erbB-3 receptors has not been investigated. It has recently been shown that expression of cripto under the control of the RSV long terminal repeat in immortalised mouse NOG-8 mammary epithelial cells produces partial transformation, characterised by anchorage independence but not tumorigenicity [192]. Expression of cripto, and also amphiregulin, is elevated in colorectal and gastric neoplasms compared to the normal surrounding mucosa [ 18 I, 1931, but other tumours have not yet been examined. POTENTIAL

LIGANDS

FOR THE c-erbB-2 RECEPTOR

Lupu et al. [194] purified a growth factor secreted by the MDA-MB-231 human breast cancer cell line and identified it as a 30kDa glycoprotcin. This growth factor, which they termed gp30, was able to stimulate phosphorylation of both c-erbB-2 p 185 and the EGF receptor directly and independently, and has been shown to specifically inhibit the growth and suppress the soft agar colony formation of SK-BR-3 cells overexpressing c-erbB-2. Treatment of cells with anti-EGFR antibody had no effect on the inhibition of SK-BR-3 cell colony formation produced by gp30, and direct binding of gp30 to c-erbB-2 growth factor receptor was confirmed by binding competition experiments with anti-c-erbB-2 antibody. It thus appears that gp30 is a ligand for c-erbB-2, and also for the EGF receptor. The gene encoding this protein has not yet been cloned. Recently M. Shepard’s group at Genentech (also working with the MDA-MB-231 cell line) have reported the cloning of a gene for a novel protein which they have christened Heregulin (H. M. Shepard, Cold Spring Harbor Symposium, 1991). This

13

Type I Growth Factor Receptors

protein appears to have the appropriate credentials to be a specific ligand for the cerbB-2 receptor. The mature glycosylated 45 kDa protein, structurally related to EGF. is derived from a larger 645 amino acid precursor. Heregulin binds to and activates c-erbB-2, but not the EGF receptor, and has properties that appear to be distinct from those of gp30. Yarden and Weinberg [195] found that conditioned medium from cells transformed with an activated ras oncogene could induce phosphorylation of the neu ~185 protein and stimulate proliferation in haemopoietic cells transfected with the rat neu oncogene. The active factor in this conditioned medium has been partially purified and identified as a 35kDa glycoprotein which is heat-stable but sensitive to reduction [196]. It is capable of stimulating the proto-oncogenic receptor but is ineffective on the oncogenic rat neu protein which is constitutively active. The factor is also active on the EGF receptor at a similar concentration to that required to activate c-erbB-2. The relationship of this factor and gp30 is not yet clear. Another activating factor for the neu protein has been termed NAF (for neuactivating factor) by Dobashi et al. [197]. NAF was partially purified from medium conditioned by the human T cell line ATL-2. The heat-stable peptide has a molecular weight of less than 30kDa and specifically binds to neu resulting in tyrosine kinase activation and receptor internalisation. The growth-promoting effects of NAF are reported to be specific to cells expressing the neu protein. Tarakhovsky et al. [198] have used a ligand-trapping assay to show that mouse peritoneal macrophages activated by muramyl dipeptide secrete a 25 kDa polypeptide that interacts with the rat neu receptor. The identity of the gene encoding this factor is not yet known. It is not presently clear to what extent these peptides represent distinct ligands, since in most cases they have not been purified to homogeneity and no sequence information is available. It is possible that several ligands exist for c-erbB-2, as is the case for the EGF receptor. IMPLICATIONS

FOR CANCER THERAPY

There is increasing evidence that genetic alterations in growth factor signalling pathways are closely linked to developmental abnormalities and to chronic diseases including cancer [ 1991. Members of the EGF receptor family are frequently implicated in human cancer. As already discussed amplification or overexpression of the genes for EGF receptor and c-erbB-2 are commonly observed in certain malignancies. Overexpression of either gene under appropriate conditions in mammalian cells confers the transformed phenotype [30,73]. Additionally there is one report to suggest that c-erbB-3 is overexpressed in certain breast carcinomas, although at present there is no evidence that this gene product is able to transform mammalian cells or that it possesses tyrosine kinase activity [92]. At least in the case of EGF receptor, transformation appears to be dependent on the presence of an activating ligand. TGFalpha is frequently detected in carcinomas expressing large amounts of EGF receptors thus establishing autocrine loops where the EGF receptor is chronically stimulated [35]. It is possible that such autocrine loops are more prevalent than current knowledge suggests, since any one of an increasing number of EGF-like peptides could act as the stimulating ligand. Similarly for c-erbB-2, autocrine stimulation may be achieved by as

14


yet unidentified peptides, although there is some evidence to suggest that the c-erbB-2 tyrosine kinase may possess a relatively high basal activity [200]. Potentially activating mutations in the genes for EGF-like growth factor receptors are not commonly observed in human malignancies, one notable exception being human gliomas where rearrangement of the EGF receptor gene frequently occurs resulting in the production of a truncated protein. Our present knowledge of the role of EGF receptors and ligands in cancer offers possibilities for improvements in diagnosis and prognosis, and opportunities for therapeutic intervention. Since gene amplification of EGF receptor and c-erbB-2 is confined to tumour tissue, identification of multiple copies of these genes by quantitative PCR could provide a diagnostic indicator. There is now strong evidence that the presence of large amounts of EGF receptor or c-erbB-2 protein is an indicator of poor prognosis in breast cancer, although for other cancers this relationship is less well defined. Immunocytochemical screening of tumour biopsies would therefore identify those patients for which more aggressive therapy would be appropriate. Perhaps the most challenging question is whether these growth factor signalling pathways could be manipulated therapeutically. Such intervention could theoretically be achieved by inhibiting ligand binding, receptor dimerisation, tyrosine kinase activation or protein expression of ligand or receptor. Monoclonal antibodies have been raised against both ligands and receptors for the EGF receptor system and against the extracellular domain of c-erbB-2. Neutralising antibodies against EGF and TGF alpha are effective in vitro, but have not been used successfully in vivo [201]. Recent reports of the existence of other stimulating ligands for EGF receptor may explain the lack of success of this approach. Antibodies against the extracellular domain of both receptors have however produced more promising results both in vitro and in vivo. Such antibodies could potentially be useful per se to down-regulate surface receptors, to inhibit ligand binding or as vehicles for localising toxic molecules to the site of the tumour. Perhaps the most success has been achieved with an antibody known as 4D5 which reacts with an extracellular epitope on c-erbB-2 and reversibly inhibits in vitro proliferation of human breast cancer cell lines expressing this oncogene product [202]. This antibody has been shown to increase the sensitivity of cancer cells to tumour necrosis factor alpha [202] and to enhance the cytotoxic effect of diammedichloroplatinum on breast cancer cell lines expressing high levels of c-erbB-2 both in vitro and in vivo [203]. It is currently being used in clinical trials for tumour localisation and possible efficacy in combination with CDDP [204]. The 4D5 antibody has recently been humanised, and bispecific antibodies containing Fv fragments of both 4D5 and anti-CD3 antibodies have been engineered which should promote T-cell recruitment to the tumour site [205]. In the case of human gliomas where gene rearrangement of the EGF receptor results in unique protein sequences at the splice junctions, antibodies recognising these sequences could provide very specific tools for tumour targeting. Another promising approach has been to synthesise fusion proteins of TGF-alpha and toxin sequences (such as Pseudomonas endotoxin) which bind to EGF receptors and have cytotoxic activity on tumour cells but not on normal tissues [206]. Other experimental approaches to interfere with receptor signalling have included the design of inhibitors of receptor dimerisation using either molecules which interpose within the cell membrane [201] or isolated domains of the receptor [207]. Inhibitors of tyrosine kinases have been developed which, though interesting, are probably insufficiently specific or of high enough affinity for clinical use [201].

IS

i’J+pe I Growth Fuctor Receptors

Clearly the EGF receptor family is potentially a very useful target for cancer therapy. However progress in this direction is limited by our fundamental understanding of the biochemical processes involved in normal receptor function. Transcription factors responsible for overexpression of these receptors in the absence of gene amplification might provide a site for intervention in malignancy. A knowledge of the threedimensional structure of these receptors will assist the design of peptides or other molecules capable of inhibiting dimerisation. An improved understanding of the mechanism of action of recently-described EGF-like ligands, particularly that of amphiregulin which is able to inhibit growth of some cells, might reveal new strategies for compromising receptor activity. The recent cloning of Heregulin, the ligand for cerbB-2 should greatly improve our understanding of the function of the c-erbB-2 protein. The c-erbB-3 protein meanwhile remains completely uncharacterised. It is hoped that our improved understanding of the role of these new molecules might provide rational new approaches for cancer therapy.

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20

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The nerve growth factor family of receptors.

ErbB receptors and their growth factor ligands in pediatric intestinal inflammation.

Expression of vascular endothelial growth factor receptors and their ligands in rat uterus during the postpartum involution period.

Ligands and receptors of the interleukin-1 family in immunity and disease.

Type I and type II insulin-like growth factor receptors and their function in human Ewing's sarcoma cells.

All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors.

Guanylyl cyclase receptors and their endocrine, paracrine, and autocrine ligands.

Type 1 Insulin-like Growth Factor Hybrid Receptors.

The nerve growth factor family.

Hematopoietic growth factor receptors.

Molecular cloning and characterization of the human and porcine transforming growth factor-beta type III receptors.

Human melanoma cells have both nerve growth factor and nerve growth factor-specific receptors on their cell surfaces.

Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification.

Recombinant pigment epithelium-derived factor PEDF binds vascular endothelial growth factor receptors 1 and 2.

Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro.

Fibroblast Growth Factor Receptor 1 and Related Ligands in Small-Cell Lung Cancer.

Epidermal growth factor receptors in the oesophagus.

Altered trafficking of mutated growth factor receptors and their associated molecules: implication for human cancers.

The magnificent seven: epidermal growth factor receptor ligands.

The transforming growth factor-beta family.

Molecular cloning and expression of the type 1 and type 2 murine receptors for tumor necrosis factor.

Ontogeny of hepatic type I insulin-like growth factor receptors in the rat.

Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands.

Genetic functions of the NAIP family of inflammasome receptors for bacterial ligands in mice.