the role of receptor tyrosine kinase inhibition in treating gastrointestinal malignancy

Review

Ashley Publicationswww.ashley-pub.com

1. Introduction

2. Phosphorylation in cellular

signalling

3. Receptor tyrosine kinases

4. The ErbB/type I receptor family

5. Insulin-like growth factor

receptor-1

6. The Met receptor

7. The vascular endothelial growth

factor receptors

8. Combination therapy

9. Kit inhibition: the success story

10. Expert opinion

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The role of receptor tyrosine kinase inhibition in treating gastrointestinal malignancyMR Anderson1† & JAZ Jankowski2

1†Department of Medical Sciences, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, B15 2TH, UK & 2Digestive Disease Centre, Windsor Building, Leicester Royal Infirmary, Leicester, LE1 5WW, UK

Tyrosine kinase receptors are proteins that transduce the signal from manygrowth factor and cytokine ligands to produce intracellular responses. Assuch they can activate multiple signalling cascade pathways and influence celldivision, migration and survival. Many show upregulation in certain malig-nancies, including those of the gastrointestinal tract, and are thought to playkey roles in carcinogenesis. This makes them attractive targets for drug ther-apy and in recent years many inhibitors have been developed. This review dis-cusses the current situation regarding the development of inhibitors withparticular reference to the erbB family, the insulin-like growth factor recep-tor, the Met receptor, the receptor for vascular endothelial growth factor andthe Kit receptor. The evidence will be related back to cancers of the gutlumen. Clinical effectiveness in this area seems to lie in using a combinatorialapproach that inhibits multiple key signalling points, and the reasons for thiswill be discussed.

Keywords: colorectal cancer, EGFR, gastric cancer, IGF-1R, Met, oesophageal cancer, tyrosine kinase, VEGFR

Expert Opin. Investig. Drugs (2003) 12(4):577-592

1. Introduction

Cells can only produce intracellular responses to extracellular changes if a signal istransduced across the cell membrane [1]. Although some chemical signals are mem-brane permeable (e.g., phospholipase C, steroid hormones), the majority of signalmolecules are membrane impermeable and are recognised by specific receptors. Sig-nals ranging from small molecules like acetylcholine to large proteins such as growthfactors all bind to membrane receptors which transduce the signal by several mecha-nisms. One of the most common mechanisms is to change the phosphorylation stateof a cellular protein, which in turn alters its properties. Kinases cause phosphoryla-tion and phosphatases cause dephosphorylation on serine, threonine and tyrosineresidues and together represent a method of altering the activity of intracellular pro-teins that is found throughout multicellular eukaryotic cell systems [2].

In each normal cell there are 200 – 300 tyrosine kinases [3], but the phosphoryla-tion on tyrosine residues constitutes only 0.01% of the total intracellular phosphor-ylation [2]. However, this vital mechanism helps to regulate protein synthesis, celldivision, cell differentiation and apoptosis [4]. Increased activity of tyrosine kinaseshas been implicated in many cancers and some are thought to be key proteins indriving the uncontrolled proliferation characteristic of malignancies. Inhibitors thatblock the action of specific tyrosine kinases have formed the basis of a therapeuticapproach that is now a rapidly developing area.

Malignancies of the gastrointestinal tract are common, in fact the main three can-cers of the lumen (oesophageal, gastric and colorectal cancers) together account for20% of all cancer incidence in the western world [201]. Curative treatment still

2003 © Ashley Publications Ltd ISSN 1354-3784 577

The role of receptor tyrosine kinase inhibition in treating gastrointestinal malignancy

depends on surgery being performed in the early stages of thedisease. In the year 2000, 29,800 people died of one of thesethree cancers in the UK. It is estimated that our lifetime riskof developing one of these cancers is 1 in 14 and bothoesophageal and colorectal cancers have shown a rising inci-dence in the last decade [201]. Such statistics have challengedresearchers to find novel therapeutic approaches.

Whilst much of the progress in using tyrosine kinase inhib-itors has been made in other diseases, it is becoming apparentthat many gastrointestinal cancers also display abnormal tyro-sine kinase activity. This review will discuss in more detailhow certain tyrosine kinases can influence cell behaviour andwill link the developing use of inhibitors to the treatment ofgastrointestinal malignancies.

2. Phosphorylation in cellular signalling

The phosphorylation of a protein represents a small modifica-tion of vital importance. The transfer of a phosphate groupfrom ATP to an amino acid residue is catalysed by a kinaseand causes conformational changes that can either activate orinhibit the protein concerned. This process can dictate theactivity of an enzyme site, the binding relationships withother proteins and even the subcellular location of a protein[1,5]. Similarly, dephosphorylation by phosphatases can reversethe change and this balance controls many intracellular proc-esses, with the dominance of kinase versus phosphatase actiondetermining the final phenotype [1].

The superfamily of protein kinases share homology in theirkinase catalytic domains [6] but historically have been splitinto two classes: kinases that phosphorylate serine and/or thre-onine residues and kinases that phosphorylate tyrosine resi-dues. Dual specificity kinases have also been recognised andmembers of the mitogen-activated protein kinase (MAPK)family cascade are probably the best known of these [7].

Hundreds of tyrosine kinases have been identified anddefined into 13 families of receptor tyrosine kinases and ninefamilies of non-receptor tyrosine kinases [6]. The receptortyrosine kinases are predominantly involved in the transduc-tion of growth and differentiation signals and, consequently,their increased activity is often implicated in malignancy.

The serine/threonine kinases are less commonly found asmembrane signal transducers but are more usually activatedupon release of second messengers (e.g., calcium/calmodulin-dependent kinases) [8] or act further downstream of signalcascades (e.g., cyclin-dependent kinases) [9]. The MAPKs area family of serine/threonine and dual specificity kinases thatare of particular interest in apoptosis regulation and cellcycling [10].

Even membrane receptors that lack intrinsic catalyticdomains use tyrosine phosphorylation to transduce their sig-nal. Cytokine receptors react to ligand binding by recruitingthe Janus kinases (JAKs), which are intracellular tyrosinekinases [11] that phosphorylate signal transducers and activa-tors of transcription (STATs).

Proteins in the extracellular matrix can also signal to theintracellular environment by binding to membrane boundintegrins, which by clustering can activate intracellular non-receptor tyrosine kinases such as Src [1,12].

It is apparent then that bridging the gap between extra-cellular signals and intracellular responses is the process ofphosphorylation.

3. Receptor tyrosine kinases

Although the receptor tyrosine kinases can be divided into13 families, they all share the same basic structure (Figure 1). Ingeneral, the extracellular domains begin with the N-terminusand contain the ligand binding site and one or more cysteinerich regions. There then follows a transmembrane domain andeither one or two intracellular tyrosine kinase regions. Once lig-ands bind to the receptor tyrosine kinases, most initially formdimers and then autophosphorylate their intracellular domains.The subsequent phosphotyrosine residues can then act as selec-tive binding sites for proteins containing SH2 (Src homologydomain 2) domains. These proteins are then also phosphor-ylated and the signal cascade is initiated. Several intracellularproteins can be activated in this way and one type of receptorcan activate multiple pathways, a phenomenon termed pleio-tropic signalling. Proteins activated by the receptors in this wayinclude phospholipase C (which activates the inositol triphos-phate pathway) [6], phosphatidylinositol 3 (PI3) kinase (leadingto protein kinase C activation) [1], proteins of the adherensjunction (such as β-catenin, which carries out a nuclear signal-ling role) [14] and members of the Ras-MAPK cascade viarecruitment of growth factor receptor-bound protein 2 (Grb2)[14]. It is evident from this list that a single genetic or physiolog-ical alteration in receptor tyrosine kinase signalling can haveprofound effects on cell growth and the list of kinases impli-cated in malignancies is increasing. The converse of this is thatinhibiting a receptor tyrosine kinase may downregulate multi-ple pathways and this has made them popular targets for drugdesign. One approach is to develop inhibitory antibodies oragents that prevent the ligands binding to the receptor. A sec-ond approach is to design specific inhibitors of the catalytickinase domain. This has been a productive method and manysmall molecule tyrosine kinase inhibitors have been designedand have reached clinical trials. Key examples of tyrosinekinases and their potential as therapeutic targets in gastrointes-tinal malignancy will be discussed below.

4. The ErbB/type I receptor family

The ErbB family or Type I receptor tyrosine kinase family, is aset of four homologous proteins that plays a role in controllingcell proliferation [15]. ErbB-1 is more commonly known as epi-dermal growth factor receptor (EGFR) and similarly ErbB-2has also been named Neu or Her-2. There are several ligandsthat can initiate signal transduction in this family by binding tothe cysteine-rich extracellular domains. Dimerisation of recep-

578 Expert Opin. Investig. Drugs (2003) 12(4)

Anderson & Jankowski

tors occurs and the intracellular kinase domains are then acti-vated. The ligands for these receptors are divided into threegroups; epidermal growth factor (EGF)-like growth factors(which include EGF, transforming growth factor-α [TGF-α]and amphiregulin), the neuregulins and betacellulin [15].

The specificity between ligands and receptors is complex(Figure 2). The EGF-like growth factors bind to EGF receptor(EGFR) [16,17], the neuregulins can bind to ErbB-3 andErbB-4, and betacellulin can bind to EGFR and ErbB-4 [18].

The relationship between ligand binding and activation isfurther complicated by the fact that receptors can also form het-erodimers with other members of the ErbB family. Certainly,ErbB-2 has no known ligand but it can form heterodimers withactivated EGFR and enhance the subsequent kinase activity[19]. Similarly, ErbB-3 and ErbB-4 are able to form heterodim-ers with both EGFR and ErbB-2. This process appears hierar-chical, with ErbB-2 being the preferred partner for all the othermembers [21]. Heterodimers are generally more active thanhomodimers [22]. Furthermore, crosstalk has been demon-strated where EGFR is activated by a ligand, forms heterodim-ers with ErbB-2 and the activated ErbB-2, then dissociates anddimerises with ErbB-3 or 4, thus propagating the signal [20].

The existence of multiple ligands, the capacity to form het-erodimers and the activation of multiple signal cascades allmakes ErbB receptor signalling a complex phenomenon witha wide range in signal output.

4.1 Epidermal growth factor receptor4.1.1 Epidermal growth factor receptor biologyEGFR is the prototypal member of this family and was thefirst to be sequenced [24]. Its expression has been found onmost epithelial and stromal cell types and is often restrictedto the basolateral regions, facilitating epithelial-stromalcommunication [23]. EGFR is a pleiotropic signaller and cantrigger multiple cascades (Figure 1). The biological responsesto activation include mitogenesis, migration and differenti-ation. Its importance in development has been demon-strated by EGFR knockouts which display malformation ofepithelial organs and die as neonates [23]. EGFR signallinginduces MAPK activation and proliferation in many celllines [26] and has been shown to cause altered expression ofadhesion proteins which facilitates cell migration and inva-sion [14]. It is apparent from this that EGFR activity may beimportant in malignant growth. Certainly studies on trans-

EGF, TGF-α Insulin, IGFs HGF VEGFsN N N N

C C C C

C

EGFR Insulin receptor subclass Met VEGFR

β-catenin

PLC-γ

Shc/Grb2

PI3K

IRS

PKC

Shc/Grb2

Src/FAK

PLC-γ

Shc/Grb2

PI3K

MAPK MAPK MAPK

N

Cysteine-rich region Ig-like binding domain Tyrosine kinase domain

Figure 1. Schematic diagram to show structures of four subclasses of receptor tyrosine kinases and the predominant signaltransduction pathways initiated upon dimerisation and activation. Structural similarities are seen with the presence ofextracellular cysteine-rich regions or immunoglobulin-like domains. Similarities are also seen in the signalling pathways activated. Note: Receptors dimerise first and then activate pathways, with the exception of insulin receptor subclass, which already exist as dimers.EGF: Epidermal growth factor; FAK: Focal adhesion kinase; Grb2: Growth factor receptor-bound protein 2; HGF: Hepatocyte growth factor; IGFs: Insulin-like growth factors; IRS: Insulin receptor substrate; MAPK: Mitogen-activated protein kinase; PLC: Phospholipase C; PI3K: Phosphatidylinositol 3 kinase;PKC: Protein kinase C; TGF-α; Transforming growth factor-α; VEGF: Vascular endothelial growth factor.

Expert Opin. Investig. Drugs (2003) 12(4) 579


genic mice show that overexpression of EGFR promotescarcinogenesis and many human epithelial tumours displayoverexpresion of EGFR, with or without gene amplification[27]. In addition, some display increased production ofEGFR ligands, causing activation of autocrine loops [28,29].Importantly, overexpression of EGFR has been associatedwith more aggressive tumour behaviour in some cancers. Arecent review of 200 studies on the relationship betweenEGFR expression and prognosis identified cancer types thatshow a consistent relationship [27]. EGFR was found to actas a strong prognostic indicator in ovarian, bladder,oesophageal and head and neck cancers and also gave mod-erate prognostic information in gastric, breast and colorec-tal cancers (Table 1).

A drawback of comparing studies of the levels of EGFRexpression (and indeed of any other receptor) is that there isno standardised assay for measuring levels and different tech-niques have been utilised by each research group. Some haveused assays of the mRNA level present and only recently hasthe quantitative reverse transcription-polymerase chain reac-tion (RT-PCR) assay begun to supercede in situ hybridisationtechniques. Such assays assume mRNA levels translate intoprotein levels, but even techniques that assay the protein itself,such as immunohistochemistry, vary widely in the antibodies,concentrations and scoring systems used. Even when such

studies are pooled [27] they may underestimate the significanceof EGFR as they only address total EGFR levels rather thanreceptor activity.

4.1.2 Epidermal growth factor receptor inhibitorsThe development of mAbs that inhibit ligand binding toEGFR was first suggested in the 1980s [30,31] and murine (e.g.,EMD 55900) and chimeric antibodies (e.g., IMC-C225cetuximab; ImClone Systems) have been produced [32]. APhase II trial in advanced head and neck cancer patients whowere treated with combined cisplatin and cetuximab therapyshowed 23% of patients exhibited a partial response [33]. APhase III trial also showed the response rate was higher in thecombined group than in cisplatin alone, although theimprovement in time-to-disease progression was small [34].One of the drawbacks of mAb therapy is that treatment mustbe given intravenously. Furthermore, around 2% of patientsdevelop an anaphylactic reaction, presumably due to themouse component, and even the use of chimericmurine:human antibodies causes patients to produce antibod-ies against the treatment [35].

To combat this immunogenicity, a fully human mAb(ABX-EGF; Abgenix) has been developed and is due toundergo Phase II testing [32]. However, much interest sur-rounds another approach; using specific kinase inhibitors.

EGF,TGF-α, HB-EGF,amphiregulin, epiregulin,

betacellulin NeuregulinsNeuregulins,betacellulin

(No known ligand)

EGFR ErbB-2 ErbB-3 ErbB-4

Figure 2. The ErbB (Type 1 receptor tyrosine kinase) family and their dimerisation options. The ligands for each of the fourreceptors in the family are shown. The double headed arrows below indicate possible heterodimers that can form following ligandactivation of one receptor. The thicker the arrow, the greater the affinity for each other. It should be noted that all members can formhomodimers as well.EFG: Epidermal growth factor; EFGR: Epidermal growth factor receptor; HB-EGF: Heparin-binding epidermal growth factor; TGF: Transforming growth factor.



Mutated EGFR that lacks tyrosine kinase function does notcause biological responses, implying that kinase activity is essen-tial [18,36]. The intracellular kinase domain is an attractive targetas its action does not depend on which ligands are activatingEGFR. The drugs tend to be of low molecular weight enablingoral administration and potentially better tumour penetrance.The discovery of naturally occuring tyrosine kinase inhibitorssuch as quercetin, genistein and lavendustin-A (which act bycompeting with ATP in the kinase reaction), prompted thedesign of the first generation tyrphostins [2]. However, thesecompounds exhibited poor specificity or lacked efficacy in vivo[32]. Progress has been made using molecular modeling studiesto design low molecular weight inhibitors specific for EGFRand the class of quinazoline inhibitors has emerged, whichcompete with ATP for the binding site and inhibit EGFR innanomolar concentrations. Two such drugs in clinical trials areZD1839 (Iressa™; AstraZeneca) and OSI-774 (erlotinib; OSIPharmaceuticals), and a further dual EGFR/ErbB-2 inhibitor,PKI166 (Novartis) has been produced (Table 2).

ZD1839 is arguably the most advanced EGFR inhibitor inclinical trials and it was recently awarded fast track status bythe US FDA [37]. Cell culture assays have shown it inhibitsreceptor autophosphorylation and blocks downstream effectson cell growth, protein phosphorylation, cyclin D1 levels andgrowth factor production [36,38-41]. It also reduces endothelialcell migration, suggesting it may reduce tumour angiogenesis[42]. Tests on cancer cell lines have shown it can inhibit cellproliferation and potentiate the effects of cytotoxic drugs [43].Lung, colon and epidermoid tumour xenografts in mice haveshown responses to ZD1839 [44]. Mice bearing colon cancerxenografts showed a more pronounced survival benefit whenthe treatment was combined with paclitaxel [43], and a similarresult was seen against the A459 adenocarcinoma cell tumourswhen combined with cisplatin or paclitaxel [46]. It thereforeappears that ZD1839 can enhance the antitumour effect of avariety of cytotoxic agents. Human tolerability studies showedthat the drug has a clean profile, with no serious adverse events

[47]. The dose-limiting toxicity appears to be the occurrence ofgrade 3 diarrhoea, with an acneiform rash as the other notableside effect [48]. Phase II trials have used ZD1839 to treat non-small cell lung cancer. Partial response rates were 10 – 19%[49,50], but in a trial of 24 patients, one developed a completeremission lasting > 8 months at the time of reporting [51].Larger, randomised Phase III trials are underway.

OSI-774 has shown similar developments. In vitro it inhib-its EGFR activation and slows tumour cell proliferation in thenanomolar range [52]. Phase II trials indicate a similar toxicityprofile to ZD1839 and clinical response rates of up to 12%have been reported in non-small cell lung cancer [53] andsquamous cell carcinoma of the head and neck [54]. Phase IIItrials of OSI-774 are also underway.

So how relevant is EGFR inhibition to gastrointestinal can-cers? In oesophageal cancer, studies have demonstrated astrong association between elevated EGFR levels and poorpatient outlook or increased metastasis [27,55,56]. One studyidentified a subgroup with low levels of EGF and EGFR witha 5 year survival rate of 69% compared to 14% in those withhigher levels [57]. It has been shown that the growth factor,TGF-α, and the EGFR are present in Barrett’s metaplasia andshow increasing expression as dysplasia progresses [58]. Thus,oesophageal cancers are highly appropriate candidates forEGFR inhibition. A Phase II trial using ZD1839 in oesopha-geal adenocarcinoma is underway in the UK and preliminaryresults suggest a favourable response rate (pers. commun.).

Gastric cancer may represent a similar candidate as severalstudies have linked EGFR expression to advanced clinicalstage [59-61], and EGFR inhibitory antibodies have been effec-tive against gastric tumours in nude mice [62].

In colorectal cancer EGFR expression has been associatedwith tumour grade and stage [63,64], but only with overall sur-vival in two thirds of studies [65-67]. Small molecule EGFRinhibitors have been shown to reduce polyp formation in theAPCmin mouse [68], but a conflicting report showed no effect

Table 1. The number of studies into EGFR expression that show an association with raised levels and poor overall survival.

Cancer type Studies associating EGFR with poor overall survival (%)

No. of studies

Head and neck 82 11

Oesophageal 69 13

Ovarian 67 9

Bladder 63 11

Colorectal 67 3

Breast 55 11

Gastric 50 6

Studies varied in the assay techniques used. Adapted from figures quoted in [27].EGFR: Epidermal growth factor receptor.

Table 2. Examples of low molecular weight tyrosine kinase inhibitors in recent clinical trials.

Company Inhibitor Main target

AstraZeneca ZD1839 (gefitinib, Iressa™)ZD6474

EGFRVEGFR

Novartis STI-571 (imatinib, Gleevec™)PTK-787

KitVEGFR

Roche/OSI OSI-774 (erlotinib, Tarceva™)

EGFR

SUGEN/Pharmacia SU-5416 (semaxanib)SU-6668

VEGFRVEGFR/FGFR

Pfizer CI-1033 ErbB-family

EGFR: Epidermal growth factor receptor; FGFR: Fibroblast growth factor receptor; VEGFR: Vascular endothelial growth factor receptor.



on polyp number using a different inhibitor in the samemodel [69]. Neither study examined the temporal requirementfor EGFR in polyp formation. However, a group using allelesfor inactive EGFR in the APCmin mouse studied the temporaldependency on EGFR signalling [70]. Their data suggests thata level of EGFR activity is transiently required after loss ofAPC for development of microadenomas but not for theexpansion to macroadenomas.

However, in vitro assays of EGFR inhibitors have stillshown efficacy in colon cancer cell lines. EGFR inhibition hasinduced apoptosis in colon cancer cells [71] whilst nude micebearing colon cancer xenografts have responded well to bothZD1839 therapy [43] and cetuximab inhibitory antibodies [73].Most of these studies showed that EGFR inhibition exhibitsmodest antitumour activity as a single agent but displays syn-ergism with cytotoxic chemotherapy. In a clinical trial,patients with colorectal cancer were placed on irinotecan andhad cetuximab therapy added if the disease progressed. Of120 patients who had cetuximab treatment, 22.5% achieved apartial response [74]. A similar combinatorial approach was touse both EGFR inhibition and cyclooxygenase (COX) inhibi-tion in the APCmin mouse. This provided striking protectionfrom tumour development with a 95% reduction in polypnumber [68] raising the exciting possibility that EGFR inhibi-tion may magnify the antineoplastic effects of non-steroidalanti-inflammatory drugs (NSAIDs) or vice versa. Certainlyprostaglandin-E can trigger EGFR [75] and NSAIDs have beenreported to inhibit EGFR signalling [76]; conversely EGF canstimulate COX-2 expression [77]. This all provides a rationalefor future trials combining EGFR inhibition with COX-2inhibition in colorectal cancer.

Critical questions still need to be resolved. First, mostprognostic studies have only addressed absolute EGFR levelsrather than levels of activation and as a consequence mayunderestimate prognostic relationships. Second, if we are tomaximise the effect of EGFR inhibition, future studiesshould address the wider EGFR network, both in terms ofits dimerisation partners and its crosstalk with other signal-ling pathways. An example of such synergism has beenshown using breast cancer cells. The combination ofZD1839 with the ErbB-2 inhibitory treatment, trastuzu-mab, produced enhanced antiproliferative and proapoptoticeffects [173]. This should not be surprising when we considerthat ErbB-2 forms more active heterodimers with EGFR.Such combinatorial approaches may well be the key tomanipulating EGFR signalling. Pharmaceutical companiesare already developing EGFR family inhibitors and Pfizerhas an orally available pan-erbB receptor inhibitor (CI-1033) that may block signal transduction through all fourmembers of the family [25].

4.2 ErbB-2ErbB-2 is a 185 kDa receptor tyrosine kinase that forms activeheterodimers which lead to enhanced signalling propertieswhen compared with homodimeric counterparts. Overexpres-

sion of ErbB-2 in cell lines can lead to a transformed pheno-type in the absence of a ligand [78].

One of the best examples of tyrosine kinase inhibitionbeing adopted as a mainstream clinical therapy comes fromErbB-2 manipulation in breast cancer. Slamon et al. [79]

observed ErbB-2 amplification in 30% of breast carcinomasand overexpression was found to be an independent prognos-tic indicator of survival and relapse [80,81]. Studies on MCF-7breast cancer cells showed that overexpressing ErbB-2 ren-dered them tamoxifen resistant [82,83] and subsequent clinicaltrials found an association between ErbB-2 overexpressionand tamoxifen failure [84]. A recombinant humanised mAbspecific for the ErbB-2 receptor was developed called trastuzu-mab (Herceptin™; Roche Pharmaceuticals) [85], whichin vitro, inhibited the growth of ErbB-2 overexpressing cells.Two pivotal clinical trials took place. The first compared tras-tuzumab with chemotherapy against chemotherapy alone in amulti-centre Phase III trial recruiting 469 patients withErbB-2 positive metastatic breast cancer [86]. The trastuzumabgroup had significantly longer time to tumour progressionand increased overall survival (25.4 months compared to20.3 months with chemotherapy alone). The second trialused trastuzumab as a single agent in over 200 patients whosemetastatic disease had progressed after conventional chemo-therapy and whose tumours crucially also displayed ErbB-2expression [87]. Objective responses were seen in 15% ofpatients. Following these two trials, trastuzumab is currentlyindicated for breast cancer patients who have received priorchemotherapy or as a combination with paclitaxel therapyand it is mandatory that patients have proven ErbB-2 expres-sion [85]. The National Surgical Adjuvant Breast Project(NSABP) in the US has begun a large Phase III trial (protocolB-31) of adjuvant ErbB-2 inhibition and ErbB-2 testing isreaching standard practice in the UK.

So, can this be applied to cancers of the gastrointestinaltract? ErbB-2 does not appear to be a factor in the develop-ment of colorectal cancer. An immunohistochemical study of250 tumours revealed that while 81% may express ErbB-2, inthe majority of cases equivalent levels were seen in adjacentnormal mucosa and no association with prognosis was seen[174]. Other studies have shown the level of ErbB-2 does notchange from adenoma to carcinoma [20] or with the develop-ment of liver metastases [88].

In the setting of cancer of the oesophagus, there is conflict-ing data. Studies on the level of protein report overexpressionranging between 10 and 73% of cancers [89-93], whereas geneamplification was shown in 15.4% of cases [94]. Evidence ofan association with prognosis has also been variable. A studyof 80 cases of oesophageal adenocarcinoma found that ErbB-2expression correlated with lymph node spread and metastasesbut not survival [91], and another study found no relationbetween ErbB-2 expression and prognosis [95]. This was alarger study, but it used a combined group of both squamouscell and adenocarcinomas, and both diseases arise along differ-ent pathways. Reduced expression of E-cadherin and



increased nuclear β-catenin are common features of Barrett’sdysplasia and adenocarcinoma [96]. ErbB-2 activity is associ-ated with β-catenin signalling [97,98], so it is tempting to spec-ulate that this may be a factor. However, in the absence ofstronger clinical data, oesophageal cancer does not seem astrong candidate for inhibitory therapy.

Studies on gastric cancer have given similar results withimmunohistochemical studies reporting frequencies ofErbB-2 expression in the range of 8 – 91% and gene amplifi-cation in 6 – 15% [85]. Such wide variance probably representsthe fact that different antibodies, different scoring systemsand different patient groups were all used, thus highlightingthe need for standardised assays. Prognostic studies are moreconsistent with several studies showing ErbB-2 overexpressionassociated with tumour invasion, metastasis and poorer sur-vival rates in intestinal type gastric cancer [99-101]. Interest inusing trastuzumab in gastric cancer has persisted [102]. Noclinical trials have yet been carried out, but gastric cancer celllines have been shown to respond to such intervention [103].

The EGFR family continues to represent a potentiallyeffective therapeutic target.

5. Insulin-like growth factor receptor-1

The ability of cancer cells to metastasise is a process thatinvolves cells breaking free from their attachment to the pri-mary tumour, migrating, invading tissues and continuing toproliferate. One tyrosine kinase implicated in this phenotypeis the receptor for insulin-like growth factor 1 (IGF-1R).

The insulin growth factor system is a complex one withmultiple ligands and receptors, but the insulin receptor andIGF-1R both act via a tyrosine kinase domain [15]. Insulin andinsulin-like growth factors display a weak cross affinity foreach other’s receptors [104], but the insulin receptor is criticallyinvolved in regulating metabolism, whilst IGF-1R can give amitogenic, antiapoptotic signal [105]. The IGFs are thought toplay a critical role in regulating rapidly renewing epithelial cellpopulations, such as those found in the gastrointestinal tract.Physiologically, the ligand IGF-1 is released from the liver as aresponse to growth hormone. IGF-2 may be expressed by thegut and aid regeneration of injured epithelium [106]. However,aberrant expression of both the growth factor IGF-1 and thereceptor IGF-1R have been reported in several cancers includ-ing those of lung, breast and the gastrointestinal tract [107],and constitutive activation of IGF-1R is associated withmalignant transformation [108].

The ligand with the highest affinity for IGF-1R is IGF-1.Receptor activation causes autophosphorylation of key tyrosineresidues which allows docking and phosphorylation of down-stream substrates, including insulin receptor substrate (IRS),PI-3 kinase, Grb2 and focal adhesion kinase [107]. It can bemitogenic, antiapoptotic, promigratory and appears to facilitatethe transformation effects induced by several other oncogenes[109]. The number of receptors determines the response to IGF-1. For instance, in one cell line: at 3 x 103 receptors/cell, there is

no response; at 15 x 103 receptors/cell DNA synthesis isinduced but not cell division; and at 30 x 103 receptors/cellthere is cell transformation [110]. IGF-1 can inactivate glycogensynthase kinase 3β (GSK-3β) and thus increase the transcrip-tional activity of the β-catenin pool [111]. It has also been shownto increase the stability of vascular endothelial growth factor(VEGF) mRNA [112] and recent studies show IGF-1R activa-tion mediates upregulated expression of COX-2 [113] and TGF-α [114]. These are all important mediators of a malignant phe-notype and IGF-1R is thought to be a facilitator of neoplasia.

This is reinforced by the fact that patients with acromegalyhave an 18-fold increased risk of colorectal cancer [105] andprospective clinical studies have associated high plasma IGF-1levels with increased risk of breast, prostate and colon cancer[107]. Increased expression of IGF-1R and its ligands has beenshown in colorectal cancer tissue compared with adjacent nor-mal tissue [115] and strong IGF-1R expression has correlatedwith higher grade or tumour stage [116] (although this is notborn out by all studies [117,118]). Mice with colon cancer trans-plants showed significantly increased rates of tumour growthand metastasis when injected with IGF-1 compared withthose that received saline injections [119].

IGF-1R activity has been suppressed using antisense oligo-nucleotides, antisense cDNA and peptide analogues [107]. Suchtargeting of IGF-1R can result in massive apoptosis andreduced anchorage-independent growth, although interestinglyhas little effect on growth in monolayer culture [109]. IGF-1Rkinase domain mutants can block the metastatic potential oftumour cells in in vivo models and it has been suggested thatliver metastases may be particularly suitable for IGF-1R-directed therapy [107]. However, such therapy relies on thedevelopment of vectors to deliver genetic information ormutant receptors into cells. This is a significant drawback, butcompanies are already developing specific IGF-1R antagonists[120] which may represent future treatments for colon cancer.

6. The Met receptor

The Met receptor was first identified as an oncogene in 1984[121]. It acts as a high affinity receptor for hepatocyte growthfactor (HGF), also known as scatter factor; a protein producedprimarily by mesenchymal cells to act on Met-expressing epi-thelial cells in a paracrine fashion [122]. Met is the prototypicmember of a subfamily of receptors that includes Sea and Ron,the receptor for macrophage-stimulating protein [122].

Met is expressed in the developing gastrointestinal tract byweek 7 in human fetuses and is implicated in the morphogen-esis of the digestive system [123]. Physiologically it plays a rolein epithelial cell growth [124] and evidence suggests it is pivotalin the healing of ulcerated gut mucosa [125]. In vivo HGF/Metsignalling likely plays a role in angiogenesis, wound healing,tumour invasion and metastasis [122,126].

As with other receptors, ligand binding induces dimerisa-tion and autophosphorylation of specific tyrosine residues inthe kinase domains [126]. Further residues in the C terminus



are also phosphorylated and may mediate substrate docking.The biological effects of HGF signalling are many. In vitrostudies have shown cells proliferate, dissociate and scatter bymoving into the extracellular matrix [127,128].

The process of cell scattering requires disassembly of cell-to-cell adhesions. The relationship between Met activity andadhesion has just begun to be explored. Reduced E-cadherinexpression is well known to increase cancer cell invasivenessand β-catenin can act to promote transcription of genesinvolved in cell cycling. So it is interesting to note that studieshave shown an association between HGF/Met stimulationand increased phosphorylation of β-catenin [129,130], nucleartranslocation of β-catenin [131] and even increased levels ofβ-catenin/T-cell factor (TCF) target oncogene proteins likec-Myc [132]. At the same time research suggests HGF/Met sig-nalling leads to decreased E-cadherin levels in melanoma [133],prostate [134] and gastric [135] cancer cell lines. There is alsoevidence of crosstalk between integrins and HGF/Met [122].Studies have shown that Met activation can induce invasive-ness by stimulating cell motility [136].

Transgenic mice that overexpress HGF develop a broadarray of epithelial and mesenchymal tumours [175]. A directconnection between the HGF/Met pathway and human can-cer was identified when germline and somatic mutations ofthe catalytic domain of Met were discovered to cause papillaryrenal carcinomas [126]. Since then increased Met or HGFexpression has been associated with higher grade or worseprognosis [122]. In breast cancer high Met expression has beencorrelated with lower 5-year survival rates [137,138] and immu-nohistochemical staining of Met protein is associated withhigher grade in prostate cancer [139] and poorer differentiationin hepatocellular carcinoma [140].

Given its role in gut development and physiology, we wouldexpect to see a significant role in gastrointestinal tumours. Stud-ies of the expression of Met in oesophageal malignancy showedincreased expression in tumours compared to normal mucosa[141,142]. A study predominantly of squamous cell carcinomasfound no correlation with clinical parameters [142], but an asso-ciation with poorer short-term survival may be seen in oesopha-geal adenocarcinomas (pers. data).

In gastric cancers, one study showed that 67% expressedMet [143], whilst another found that 70% of adenocarcinomashad elevated Met mRNA levels compared to normal gastricmucosa [144]. Whilst amplification of the c-Met gene has beenreported in 7% of intestinal type cancers [101], it appears morefrequently in diffuse type gastric cancer where it correlateswith prognosis [145].

Colorectal cancers have consistently shown overexpressionof Met, with a sixfold increase of Met mRNA compared tonormal colonic mucosa [146] and one third of tumours dis-playing overexpression of Met protein [147]. Patients withfamilial adenomatous polyposis show Met overexpression indysplastic aberrant crypt foci, the earliest neoplastic lesions ofcolorectal cancer [148]. However, Met expression is alsoenhanced in adenomas [146] and is not linked to patient sur-

vival [147], suggesting it plays a role in the early stages ofcolorectal carcinogenesis.

Researchers have attempted to apply methods of inhibitionto the Met receptor. Members of the geldanamycin antibioticfamily have been shown to inhibit HGF/Met signalling, inpart by downregulating Met protein expression [122]. Peptidesderived from the Met receptor tail can bind to the receptorand with an inhibitory effect, demonstrated on A549 cellmigration [149]. A truncated HGF variant that contains onlydomains of the alpha chain is termed the NK4 antagonist. Itretains Met binding but does not activate the receptor. It hasbeen shown to reduce motility and invasion of colorectal can-cer cells [150] and inhibit angiogenic behaviour of umbilicalvein endothelial cells [151]. However, none of these methodshave reached clinical testing and the development of smallmolecule inhibitors would be timely.

7. The vascular endothelial growth factor receptors

The growth of new blood vessels is an imperative step if a neo-plasm is to become an enlarging cancer with metastatic poten-tial. Tumours that lack adequate vasculature will becomenecrotic or apoptotic [152]. The development of new vascularnetworks is tightly regulated by specific growth factors, pre-dominantly VEGFs. An increased understanding of the proc-ess of angiogenesis has led to the development of therapiesthat inhibit the VEGF receptor (VEGFR) signalling pathway.

The best characterised and most potent proangiogenicfactor is VEGF, but the family has now been found to con-tain at least five other homologous members (VEGF-B to-E) [152]. Hypoxia is a stimulus for its production in bothmalignant and normal tissues [153,154], but VEGF mRNA isalso induced by growth factors and cytokines such as EGFand TNF-α [155]. Pathological levels of VEGF have beenimplicated in diseases of increased angiogenesis such as pso-riasis and retinopathy [152].

Three high-affinity endothelial receptors for VEGF havebeen identified and are single pass transmembrane receptorswith cytoplasmic tyrosine kinase activity [152]. The biologicaleffects of VEGFR activation include endothelial sprouting,increased vascular permeability and ultimately, endothelialcell migration and mitogenesis [152]. Thus, VEGF is pivotal inneovascularisation and recent data suggests it is vital to thesurvival of immature blood vessels forming in tumours [156].

The evidence implicating VEGFR and its receptors in solidtumour angiogenesis is accumulating and overexpression hasbeen demonstrated in breast, gastric, bladder and prostatecancers [157-160].

Studies on Barrett’s metaplasia have shown it to be highlyneovascularised and VEGF-A and the receptor VEGFR-2 areboth strongly expressed in the lesion [161]. The overexpressionof VEGF has been found in 46% of intestinal type gastric car-cinoma and correlates with lymph node and liver metastasis[145]. Work on the proliferation of lymphatic vessels into pri-



mary gastric cancers shows a link between VEGF-C andVEGFR-3 expression and the grade of lymphatic invasion,suggesting VEGF signalling may facilitate the process of lym-phatic spread [162]. In colon cancer the expression of VEGF bytumours has been shown to predict disease recurrence follow-ing surgery [163].

Several approaches to targeting VEGF signalling have beentaken. Blocking VEGF action by inhibiting its synthesis orneutralising it in the microcirculation are methods now inclinical testing. One study which randomised patients withcolorectal cancer to receive either mAbs against VEGF andchemotherapy or chemotherapy alone showed that responserates and time to progression were both improved by the addi-tion of the antiVEGF component [164]. However, most phar-maceutical companies have concentrated on developinginhibitors of the receptor VEGFR (Table 2).

SU-5416 (semaxanib; SUGEN/Pharmacia) has reachedPhase III clinical trials, but inhibitors by AstraZeneca(ZD6474) and Novartis (PTK-787) are also in clinical tests.

SU-5416 is a potent inhibitor of the VEGFR kinases, com-peting for the ATP-binding site, yet is relatively specific withno activity against other tyrosine kinases such as Met and Src[165]. It has been shown to inhibit tumour growth in vivo buthas little effect in vitro where cells are not angiogenesisdependent [165]. Activity against tumour xenografts in nudemice has been demonstrated with several tumour typesincluding lung, breast and prostate. In a study of humancolon cancer xenografts, SU-5416 inhibited microvessel for-mation, cell proliferation and metastatic spread [166]. Clinicaltrials have shown that the drug is well-tolerated and stable dis-ease has been demonstrated in cases of lung, colon and basalcell cancers [167,168]. Recently, the development of SU-5416has been halted in favour of its follow-up compound,SU-11248 which has a broad range of targets [176], and thesame company also has a drug with a similar structure(SU-6668) that is less selective and inhibits fibroblast growthfactor receptors. This lower selectivity may in fact make themmore potent, by preventing angiogenesis and inhibiting stro-mal support tissue growth [169].

The VEGFR inhibitor ZD6474 is another quinazolinemolecule. It has shown high oral bioavailability and has dem-onstrated broad antitumour efficacy in human tumourxenografts in mice [170]. Novartis have a VEGFR inhibitorfrom the phthalazine class (PTK-787) now reported to be inPhase II clinical trials, but they have also developed a follow-up compound (AAL-993) with an improved toxicity profile[37]. It is interesting that inhibiting VEGFR does not appearto impair wound healing as had been speculated for antiang-iogenic therapy [171].

As the results of clinical trials begin to accrue in the next fewyears, the future of VEGFR inhibition will become clearer.While it is certainly evident that commercial companies arewilling to invest heavily in this competitive area, clinicians tooare keen to develop strategies that combine antiangiogenicagents with cytotoxic chemotherapy. It is hoped that this dual

approach of reducing tumour burden alongside preventingneovascularisation, will be highly effective.

8. Combination therapy

The first phase of low molecular weight tyrosine kinaseinhibitors has passed into the setting of clinical trials. Inmany cases, specifically inhibiting one receptor appears toproduce clinical responses, but always in a small subgroupof patients. As more of the signalling pathways are revealedit has become apparent that the amount of crosstalkbetween classical ligand–receptor relationships is greaterthan first realised. It is no longer appropriate to consider asingle ligand and receptor acting in isolation. Instead weshould visualise the signalling pathways as an intercon-nected web of ligands, receptors and substrates, with thenet result on a cell representing the overall balance of sig-nalling activity. Given the number of pathways that can beupregulated in malignancy, it is likely that inhibiting onereceptor alone is always going to meet with limited success.Only by identifying and inhibiting the key points that fuelthe overall signalling activity, will a greater antineoplasticeffect be achieved.

Trials that have used combined therapies have consistentlyshown the greatest responses clinically, often suggesting synergis-tic effects. The most striking protection from colon polyp forma-tion was seen when Torrance used combined EGFR and COXinhibition [68] and the response to EGFR inhibition is alwaysgreater when combined with cytotoxic chemotherapy [74,172].

We need to address the interplay of multiple receptors andinvestigate receptor activity rather than simple absolute levelsif we are to discover prognostic links and make progress ininhibitory treatment.

Inhibiting both VEGFR and EGFR in conjunction has alsoshown increased efficacy, reducing both colon cancer growthand malignant ascites in mouse models [73]. Similarly, theblockade of both VEGFR and EGFR in nude mice with gas-tric tumours caused a greater decrease in tumour growth thanin mice given either therapy alone [62].

It also now seems likely that EGFR signalling and Meteffects are intricately linked with further evidence of cross-talk between ligand-receptor systems. In one study, HGFhas been shown to stimulate production of TGF-α (the lig-and for EGFR) [72] and another has demonstrated that TGF-α can activate Met even in the absence of its conventionalligand, HGF [45]. Such crosstalk between Met and EGFRmay have significant implications for how growth factorsinfluence tumorigenesis and may also limit the benefitsobtained from inhibiting one receptor only. This serves tohighlight again the importance of the concept of combinato-rial therapy.

The hypothesis that inhibiting multiple pathways may bemore effective has caused a change in the attitude of drugdesigners towards in vitro kinase selectivity. Specific selectivityof kinase inhibitors does not always mean better safety profiles



and drugs that inhibit more than one key point may actuallybe beneficial.

9. Kit inhibition: the success story

It is with intent that the best example has been saved untillast. Inhibition of the Kit receptor is transforming the man-agement of gastrointestinal stromal tumours (GISTs) and hasbeen hailed as a major breakthrough [177].

The standard treatment for GISTs has traditionally beensurgery. However, 40 – 80% will recur despite histopathologi-cally complete resection and systemic chemotherapy has beengenerally ineffective [177]. The discovery that mutations in theKit proto-oncogene [178] and increased Kit expression madeKit activation a ubiquitous feature of GISTs [179], providedresearchers with a logical molecular target for therapy.

The Kit protein is a transmembrane receptor for thecytokine called stem cell factor and acts via an intracellulartyrosine kinase domain. A competitive inhibitor called imat-inib (Gleevec™; Novartis) had been developed that targetedKit as well as other kinases including the Bcr-Abl fusion pro-tein found in some leukaemias. It is a low molecular weightcompound that can be given orally and acts by competingwith ATP for the kinase binding site [177]. Two clinical trialsused imatinib in the treatment of metastatic GISTs and haveshown remarkable success. The US-Finland study recruited147 patients and reported a response rate of 54% [180], andthe European Organisation for Research and Treatment ofCancer (EORTC) trial which included 36 patients withGISTs found that 90% of patients had symptomatic improve-ment and the disease only progressed in 11% [181].

It appears therefore, that imatinib is the first effective sys-temic therapy for GISTs and trials are now underway toassess the optimum duration of treatment, the role of com-bination therapies and the possible benefits in non-meta-static cases. However, it seems fair to say that this is proof ofconcept that selective inhibition of aberrant tyrosine kinaseactivity may provide effective anticancer therapy.

10. Expert opinion

It is beyond the scope of this review to commentate on all thetyrosine kinases currently implicated in gastrointestinal can-cers. Protein kinase A appears to be important in colon cancerprogression and many non-receptor tyrosine kinases are simi-larly being implicated. However, it is hoped that the examplesso far discussed will highlight the direction in which this ther-apeutic approach is heading.

GISTs are associated with c-Kit activity and Kit inhibi-tors are proving effective in this disease. The use of ErbB-2inhibition is falling into standard clinical practice in breastcancer treatment and the results of EGFR inhibitors insolid tumours are promising, especially when combinedwith cytotoxic chemotherapy. The blockade of VEGFR sig-nalling provides a key to preventing tumour growth andmay act in synergy with EGFR inhibition. As the biology ofMet and IGF-1R is elucidated, they too may act as thera-peutic targets.

As our understanding of the interplay of multiple receptorsincreases and the key molecules involved become apparent,then tyrosine kinase inhibition may finally change the way wetreat cancer.

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Website201. http://www.cancerresearchuk.org/

aboutcancer/statistics/29375/29902 Website for Cancer research UK. Incidence and mortality figures. Updated October 2002.

AffiliationMR Anderson1† & JAZ Jankowski2†Author for correspondence1†Department of Medical Sciences, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, B15 2TH, UK E-mail: [email protected] Disease Centre, Windsor Building, Leicester Royal Infirmary, Leicester, LE1 5WW, UK


the role of receptor tyrosine kinase inhibition in treating gastrointestinal malignancy

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