Gene 366 (2006) 2–16www.elsevier.com/locate/gene

Review

Epidermal growth factor receptor (EGFR) signaling in cancer

Nicola Normanno a,⁎, Antonella De Luca a, Caterina Bianco b, Luigi Strizzi b, Mario Mancino a,Monica R. Maiello a, Adele Carotenuto a, Gianfranco De Feo a, Francesco Caponigro c,

David S. Salomon b

a Cell Biology and Preclinical Models Unit, INT-Fondazione Pascale, 80131 Naples, Italyb Tumor Growth Factor Section, Mammary Biology and Tumorigenesis Laboratory, NCI, NIH, Bethesda, MD, United States

c Medical Oncology B Unit, INT-Fondazione Pascale, 80131 Naples, Italy

Received 11 October 2005; accepted 15 October 2005Available online 27 December 2005

Received by A.J. van Wijnen

Abstract

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteinsare activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in thepathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, andin vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutationsof the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterationsof the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complexsystem in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derivefrom receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showingdifferent affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors andcognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by anetwork of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFRagents might depend on the total level of expression of ErbB receptors and ligands in tumor cells.Published by Elsevier B.V.

Keywords: ErbB; EGF; Growth factors; Signal transduction

Abbreviations: EGF, epidermal growth factor; RTK, receptor tyrosinekinases; EGFR, epidermal growth factor receptor; TGF-α, transforming growthfactor-α; AR, amphiregulin; BTC, betacellulin; HB-EGF, heparin binding-EGF;EPR, epiregulin; NRG, neuregulin; NSCLC, non small cell lung cancer; SH2,Src homology 2; PTB, phosphotyrosine binding; MAPK, mitogen-activatedprotein kinase; PI3K, phosphatidylinositol 3-kinase; PLCγ, phospholipase Cγ;STAT, signal transducer and activator of transcription; GH, growth hormone;GPCR, G-protein coupled receptor; Prl, prolactin; Jak2, Janus tyrosine kinase 2;Fz, frizzeled; DN, dominant-negative; K, keratin; MT, metallothionein; WAP,whey acidic protein; MMTV, mouse mammary tumor virus; GBM, glioblastomamultiforme; BAC, bronchioloalveolar carcinoma; ECD, extracellular domain.⁎ Corresponding author. Tel./fax: +39 081 5903826.E-mail address: nicnorm@yahoo.com (N. Normanno).

0378-1119/$ - see front matter. Published by Elsevier B.V.doi:10.1016/j.gene.2005.10.018

1. Introduction

The role of growth factors-driven signaling in the pathogen-esis of human cancer has been long established. Almost twentyyears ago Mike Sporn and Anita Roberts (Sporn and Roberts,1985), following the seminal observations of Joseph deLarcoand George Todaro (De Larco and Todaro, 1978), elaborated thetheory of autocrine secretion: cancer cells generally exhibit areduced requirement for exogenously supplied growth factors tomaintain a high rate of proliferation. This relaxation in growthfactor dependency is due in part to the ability of tumor cells toproduce high levels of peptide growth factors. Since thisseminal observation, an enormous amount of literature hasconfirmed the role of growth factor driven signaling in the

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pathogenesis of human cancer. It has been recognized thatdifferent mechanisms might contribute to amplify the signaldriven by growth factors. For example, expression of a highnumber of receptors on the surface of tumor cells can increasetheir sensitivity to low concentrations of host- or tumor-derivedgrowth factors. A direct correlation also exists between growthfactors and cellular proto-oncogenes (Aaronson, 1991; Goustinet al., 1986). In fact, several proto-oncogenes code for proteinsthat are either growth factors, or growth factor receptors, orproteins that are involved in the intracellular signal transductionpathway for growth factors. In addition, activated cellular proto-oncogenes may also control the endogenous production and/orthe response of tumor cells to peptide growth factors. Morerecently, the involvement of growth factors in sustaining thesurvival of cancer cells and in promoting tumor-inducedangiogenesis has been demonstrated, suggesting that growthfactors contribute to tumor progression through differentmechanisms.

Different families of growth factors and growth factorreceptors have been shown to be involved in the autonomousgrowth of cancer cells. Among these, the epidermal growthfactor receptor (EGFR) and the EGF-family of peptide growthfactor have a central role in the pathogenesis and progression ofdifferent carcinoma types (Salomon et al., 1995; Normanno etal., 2001). The EGF ligand/receptor system is also involved inearly embryonic development and in the renewal of stem cells innormal tissues such as the skin, liver and gut (Salomon et al.,1990; Campbell and Bork, 1993). However, it is important toemphasize that the EGFR belongs to a family of receptors thatencompasses three additional proteins, ErbB-2, ErbB-3 andErbB-4. These proteins and the growth factors of the EGF-

Fig. 1. Mechanisms of action of ErbB receptors in tumor cells. ErbB receptors are actsurrounding stromal cells. Binding of ligands to the extracellular domain of ErbBphosphorylation (P). The activated ErbB receptors are able to interact with different

family form an integrated system in which a signal that hits anindividual receptor type is often transmitted to other receptors ofthe same family. This mechanism leads to amplification anddiversification of the initial signal, a phenomenon that isimportant for cell transformation, as we will discuss later.Therefore, the role of EGFR signaling cannot be discussedwithout taking in account the complex interactions existingwithin the ErbB family of receptors and growth factors. Suchinteractions might also be important to develop more efficienttherapeutic approaches aimed to block EGFR signaling incancer patients.

Several different review articles have been published on therole of EGFR in the pathogenesis of human carcinoma. In thepresent article, we will describe the role of EGFR signaling incancer with a special focus on the cooperation between differentErbB receptors in cancer pathogenesis and progression.Furthermore, we will revise the most recent knowledge on themechanisms involved in EGFR activation in different types ofcancer, and their relevance to novel therapeutic approaches.

2. The EGFR family of receptors and cognate ligands:structure and functional organization

2.1. The ErbB receptors and their cognate ligands

The ErbB family of receptor tyrosine kinases (RTK)comprises four distinct receptors: the EGFR (also known asErbB-1/HER1), ErbB-2 (neu, HER2), ErbB-3 (HER3) andErbB-4 (HER4) (Ferguson et al., 2003; Yarden, 2001). Allproteins of this family have an extracellular ligand-bindingdomain, a single hydrophobic transmembrane domain and a

ivate by binding to specific ligands that are produced by either tumor cells or byreceptors results in receptor dimerization, tyrosine kinase activation and trans-signaling molecules that transmit the signal in the cell.

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cytoplasmic tyrosine kinase-containing domain (Olayioye et al.,2000). The intracellular tyrosine kinase domain of ErbBreceptors is highly conserved although the kinase domain ofErbB-3 contains substitutions of critical amino acids andtherefore lacks kinase activity (Guy et al., 1994). In contrast,the extracellular domains are less conserved among the fourreceptors, suggesting that they have different specificity inligand binding (Olayioye et al., 2000; Yarden, 2001; Yarden andSliwkowski, 2001). ErbB receptors are activated by binding togrowth factors of the EGF-family that are produced by the samecells that express ErbB receptors (autocrine secretion) or bysurrounding cells (paracrine secretion) (Fig. 1) (Olayioye et al.,2000; Yarden and Sliwkowski, 2001). Proteins that belong tothis family are characterized by the presence of an EGF-likedomain composed of three disulfide-bonded intramoleculargroups, which confers binding specificity, and additionalstructural motif such as immunoglobulin-like domains, hepa-rin-binding sites and glycosylation sites. With respect to ErbB-receptor binding, EGF-related growth factors can be dividedinto three groups (Table 1) (Normanno et al., 2003a; Yarden andSliwkowski, 2001). The first group includes EGF, transforminggrowth factor α (TGF-α) and amphiregulin (AR) which bindspecifically to the EGFR. The second group includes betacellu-lin (BTC), heparin-binding growth factor (HB-EGF) andepiregulin (EPR), which show dual specificity by bindingboth EGFR and ErbB-4. The third group is composed of theneuregulins (NRGs) and can be divided in two subgroups basedupon their capacity to bind ErbB-3 and ErbB-4 (NRG-1 andNRG-2) or only ErbB-4 (NRG-3 and NRG-4) (Carraway et al.,1997; Chang et al., 1997; Harari et al., 1999; Zhang et al.,1997). None of the EGF family of peptides binds ErbB-2.

2.2. Receptor activation

Binding of ligands to the extracellular domain of ErbBreceptors induces the formation of receptor homo- or hetero-dimers, and subsequent activation of the intrinsic tyrosinekinase domain (Fig. 1) (Olayioye et al., 2000). All possiblehomo-and heterodimeric receptor complexes between membersof the ErbB family have been identified in different systems(Olayioye et al., 2000; Gullick, 2001; Schlessinger, 2000).Receptor activation leads to phosphorylation of specifictyrosine residues within the cytoplasmic tail (Fig. 1). Thesephosphorylated residues serve as docking sites for proteinscontaining Src homology 2 (SH2) and phosphotyrosine binding

Table 1The ErbB receptors and their cognate ligands

ErbB Receptors EGFR ErbB-2 ErbB-3 ErbB-4

Cognate ligands EGF None NRG 1 NRG 1TGF-α NRG 2 NRG 2AR NRG 3EP NRG 4BTC TomoregulinHB-EGF HB-EGF

BTCEP

(PTB) domains, the recruitment of which leads to activation ofintracellular signaling pathways. Studies on the crystalstructures of EGFR, ErbB-2 and ErbB-3’s extracellular domainshave led to new insights in the process of ligand-inducedreceptor dimerization (Cho and Leahy, 2002; Cho et al., 2003;Garrett et al., 2003; Garrett et al., 2002; Ogiso et al., 2002). Theextracellular domain of each ErbB receptor consists of foursubdomains (I–IV). Subdomains I and III (also called L1 andL2) have a beta helical fold and are important for ligandbinding. Moreover, direct receptor–receptor interaction ispromoted by a beta hairpin (also termed dimerization loop) insubdomain II. In the crystal structure of the extracellular domainof EGFR bound to EGF, the dimerization loop protrudes fromEGFR and mediates interaction with another EGFR moleculeleading to the formation of a dimer composed of two 1 :1receptor/ligand complexes (Garrett et al., 2002; Ogiso et al.,2002). In contrast, the structure of inactive EGFR or ErbB-3 ischaracterized by intramolecular interactions between domains IIand IV (Cho and Leahy, 2002; Ferguson et al., 2003). Thestructure of ErbB-2 extracellular region differs significantlyfrom that of EGFR and ErbB-3. In the absence of a ligand,ErbB-2 has a conformation that resembles the ligand-activatedstate with a protruding dimerization loop (Cho et al., 2003;Garrett et al., 2003). In this conformation, domains L1 and L2are very close and this interaction makes ligand bindingimpossible, explaining why ErbB-2 has no ligand (Garrett et al.,2003). Furthermore, these findings explain why ErbB-2 hasenhanced capacity of heterodimerization and why it is thepreferred dimerization partner for the other activated ErbBreceptors (Graus-Porta et al., 1997; Tzahar et al., 1996). Amongall possible ErbB-2-contaning heterodimeric receptor com-plexes, the most potent signaling module in terms of cellproliferation and in vitro transformation is represented by ErbB-2/ErbB-3 heterodimers (Citri et al., 2003). The remarkablesignaling potency of ErbB-2/ErbB-3 heterodimers derives fromthe fact that this dimer has the capacity to signal very potentlyboth through the ras/raf/mitogen-activated protein kinase(MAPK) pathway for proliferation and through the phosphati-dylinositol 3-kinase (PI3K)/Akt pathway for survival (Ben-Levy et al., 1994; Citri et al., 2003; Prigent and Gullick, 1994).In addition, ErbB-2/ErbB-3 heterodimers evade downregulationmechanisms leading to prolonged signaling (Lenferink et al.,1998; Sorkin and Waters, 1993; Worthylake et al., 1999).

2.3. ErbB mediated signaling

Signal transduction pathways are initiated when activatedErbB tyrosine kinase receptors recruit signaling proteins, suchas Shc, Grb7, Grb2, Crk, Nck, the phospholipase Cγ (PLCγ),the intracellular kinases Src and PI3K, the protein tyrosinephosphatases SHP1 and SHP2 and the Cbl E3 ubiquitin ligase(Fig. 1) (Marmor and Yarden, 2004; Yaffe, 2002). All ErbBligands and receptors induce activation of the ras/raf/MEK/MAPK pathway through either Grb2 or Shc adaptor proteins(Carpenter, 2003; Citri et al., 2003; Jorissen et al., 2003). ErbBreceptors also activate PI3K by recruitment of the p85regulatory subunit to the activated receptors (Soltoff and

Table 2Summary illustrating receptor or ligand mutation and consequential organ defect

Mutation target Localization of relevant defects

EGFR Epidermis, mammary gland, lung, pancreas,intestine, central nervous system

ErbB-2 Mammary gland, heart, central nervous systemErbB-3 Heart, central nervous systemErbB-4 Mammary gland, heart, central and peripheral

nervous systemEGF Prostate, central and peripheral nervous systemTGF-α Epidermis, prostate, eyeHB-EGF Central nervous systemAR/EGF/TGF-α Gastrointestinal tract

Mice with mutations in genes that encode ErbB receptors develop multiorganfailure leading to embryonic or perinatal death, whereas mutations of ligands donot produce a lethal phenotype.

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Cantley, 1996). However, PI3K signaling is induced withdiffering potencies and kinetics through the four ErbBreceptors. While p85 binding to EGFR and ErbB-2 is indirectthrough adaptor proteins, ErbB-3 and ErbB-4 contain 6 and 1putative p85 binding sites, respectively (Elenius et al., 1999;Fedi et al., 1994; Prigent and Gullick, 1994). PhospholipasePLCγ, Eps15 and Cbl, which are EGFR-specific substrates, areother examples of signaling molecules that bind preferentiallyone member of the ErbB family (Chattopadhyay et al., 1999;Fazioli et al., 1993; Levkowitz et al., 1999; Waterman et al.,1999). EPS15, by binding to the clathrin adaptor proteincomplex AP-2, mediates clathrin-coated pits endocytosis of theEGFR (Torrisi et al., 1999). Furthermore, the E3 ubiquitin ligaseCbl targets the EGFR to the lysosomal compartment bypromoting receptor ubiquitination (Levkowitz et al., 1998).Finally, ErbB receptors activate various transcription factorssuch as c-fos, c-Jun, c-myc, signal transducer and activator(s) oftranscription (STAT), NF-kB, zinc finger transcription factorand Ets family members (Cutry et al., 1989; Quantin andBreathnach, 1988; Biswas et al., 2000; Olayioye et al., 1999;O'Hagan and Hassell, 1998).

Different factors might affect the type and the duration ofErbB signaling. The identity of the ligand, composition of thereceptor complex and specific structural determinants of thereceptors will determine the engagement of specific signalingpathways. The impaired internalization of ErbB-2 leads toprolonged signaling of receptor complexes that contain thisreceptor, whereas dimers containing ErbB-3 or ErbB-4 candirectly activate PI3K. However, signaling is also affected byligands. For example, the interaction between EGF and EGFR isstable at endosomal pH and results in lysosomal degradation(French et al., 1995). On the contrary, interaction of TGF-α andNRG-1 with their respective receptors is pH sensitive, resultingin dissociation in the endosome and recycling to the cellmembrane of receptors that will be available for a new cycle ofactivation by ligands (Waterman et al., 1998).

2.4. Transactivation of ErbB receptors

Another mechanism that induces ErbB receptor tyrosinephosphorylation and subsequent stimulation of intracellularsignaling pathways is known as ErbB transactivation. Forexample, cytokines, such as growth hormone (GH) andprolactin (Prl), can indirectly activate ErbB receptors throughJanus tyrosine kinase 2 (Jak2), which phosphorylates specifictyrosine residues in the cytoplasmic domains of EGFR or ErbB-2 (Yamauchi et al., 1997a, 2000). Similarly, Src phosphorylatesvarious residues on EGFR, leading to enhanced receptorsignaling (Biscardi et al., 1999). ErbB transactivation has alsobeen particularly well studied for G-protein coupled receptor(GPCR) agonists, such as endothelin-1, bombesin, thrombinand lysophosphatidic acid (Carpenter, 2000; Gschwind et al.,2001). In cells treated with a receptor agonist, GPCRsstimulates metalloproteinases, which induce cleavage of EGF-like ligand precursors, leading to phosphorylation of ErbBreceptors (Gschwind et al., 2002; Prenzel et al., 1999). Morerecently, ErbB transactivation has been shown to involve Wnt,

which binds to frizzled (Fz) receptors and stimulates EGFRtyrosine kinase activity through metalloproteinase-mediatedcleavage of EGF-like ligands (Civenni et al., 2003). Therefore,the ErbB receptors function as signal integrators, throughinteraction with different signaling proteins and membranereceptors.

3. The EGFR receptor/ligand system in development

3.1. Role of ErbB receptors in development

Observations made in knockout animal models havedemonstrated the importance of ErbB receptors for thedevelopment of different organs (Table 2). Geneticallyengineered mice with mutations in genes that encode ErbBreceptors develop multiorgan failure leading to embryonic orperinatal death. Null mutations for EGFR cause developmentaldefects in the epithelial structures of the skin, lung, pancreas,gastrointestinal tract and central nervous system (Miettinen etal., 1995; Sibilia and Wagner, 1995; Threadgill et al., 1995;Sibilia et al., 1998). Oligodendrocyte development and myelinformation is arrested in ErbB-2 knockout mice (Park et al.,2001). Mice deficient in ErbB-2 or ErbB-4 have alterations incardiac and neural structures that cause lethality (Lee et al.,1995; Gassmann et al., 1995; Leu et al., 2003).

In addition, ErbB-4 deficient mice show altered motor andbehavioral activities suggesting a role for this receptor not onlyin neuronal development but also function (Golub et al., 2004).Mice embryos deficient in ErbB-3 expression, in addition to theneural defects also seen in ErbB-2 or ErbB-4 null mice,succumb due to altered cardiac valve formation (Erickson et al.,1997). Conditional mutant mice with cardiac ventricle-restrict-ed deletion of ErbB-2 showed increased susceptibility fordeveloping dilated cardiomyopathy suggesting a role for ErbB-2 signaling in the prevention of pathologic heart dilatation(Crone et al., 2002).

ErbB receptors also play a role during the development of themammary gland as demonstrated by experiments using theexpression of a dominant-negative (DN) EGFR (Xie et al.,1997) or reconstitution experiments employing EGFR knockout(−/−) neonatal mammary gland that demonstrated the role of

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EGFR in mammary ductal development (Wiesen et al., 1999).Despite strain-specific patterns observed in side-branchingdensity during the development of the mouse mammary gland(Naylor and Ormandy, 2002), most strains exhibit defects inmammary ductal growth associated with defects in expressionof ErbB receptors. For instance, experiments with DN ErbB-2or ErbB-4, show normal ductal outgrowth but alterations inlobulo-alveolar development and milk protein production(Jones and Stern, 1999; Jones et al., 1999). Expression ofErbB-3 receptor has also been shown to be required for normallobulo-alveolar development (Troyer and Lee, 2001). Theseresults suggest that in the mouse mammary gland EGFR isnecessary for ductal morphogenesis, while ErbB-2 and ErbB-4receptors, and to a lesser extent ErbB-3, play an important roleduring normal mammary lobuloalveolar development and milksecretion.

3.2. Role of EGF-like peptides in development

Information on specific biological roles of ErbB receptorligands in different organs and tissues has been obtained bystudying animal models with the relative null mutations (Table2). In mice lacking the expression of EGF and TGF-α theprostate gland does not develop normally (Abbott et al., 2003).TGF-α knockout mice (TGF-α−/−) showed increased prolifer-ation of anterior, dorsal and lateral prostatic buds compared towild type. The increase in formation of buds did not occur inEGF knockout mice (EGF−/−). Mice with null mutations forboth EGF and TGF-α did not have prostatic bud formation. Itappears, therefore, that both EGF and TGF-α are necessary forprostate development and that perhaps TGF-α may benecessary for the control of possible EGF-dependentoverstimulation.

Different members of the EGF-family of growth factors, likeEGF, HB-EGF and TGF-α, are expressed throughout the centraland peripheral nervous system in which regulate cellularactivities such as proliferation, migration and differentiation(Xian and Zhou, 2004). Although knockout mice for TGF-αgene present with abnormalities in the skin, hair and eye (Mannet al., 1993), the absence of the TGF-α gene does notsignificantly affect the development or function of the nervoussystem in these animals even though they show reductions inthe number of neurons in areas of the midbrain and forebrain(Blum, 1998; Tropepe et al., 1997). In addition, TGF-αknockout mice did not show alteration in peripheral nerveregeneration (Xian et al., 2001). These results suggest that otherErbB ligands may compensate for defects in TGF-α function inthe nervous system. In mice carrying individual null mutationsfor TGF-α, EGF or AR no significant alteration in themorphology of gastrointestinal mucosa are observed (Luettekeet al., 1993; Mann et al., 1993; Luetteke et al., 1999). Incontrast, mice with triple null mutations lacking AR, EGF andTGF-α showed growth retardation presumably due to altera-tions in the gastrointestinal tract such as decreased duodenalmucin production, formation of short, fragile villi in the ileumand reduced DNA synthesis of the cryptic cells of the intestinalmucosa (Troyer et al., 2001). However, triple knockouts lacking

AR, EGF and TGF-α survive to adulthood with mild growthretardation, suggesting that other members of the EGF-familymay activate ErbB receptors leading to normal gastrointestinaldevelopment and physiology. In agreement with these findings,null mutations for EGF, AR and TGF-α did not affectproliferation or apoptosis within the mammary gland terminalend bud, and pubescent mice lacking AR showed deficientductal development but were still capable of nursing theiryoungs (Luetteke et al., 1999). However, triple knockout micelacking expression of AR, EGF and TGF-α showed aberrantmammary alveolar growth and reduced milk productionsuggesting an important role for these growth factors foralveolar development and lactogenesis. Taken together, thesestudies demonstrate that ErbB ligands may play an importantrole in the development and function of specific organs.However, complimentary signaling pathways resulting in totalor partial compensation from the lack of signaling from mutatedErbB ligand can occur as long as ErbB receptors are expressed.

4. The EGFR system in neoplastic transformation

4.1. The ErbB receptors and their ligands are transforminggenes in vitro and in vivo

Different studies have shown that overexpression of eitherEGFR or ErbB-2 leads to in vitro transformation of mouse NIH-3T3 cells. Transformation of these cells by EGFR is dependenton exogenous EGF that is required to activate the receptor,whereas ErbB-2-induced transformation is directly related tothe levels of expression of the oncogene (Di Fiore et al., 1987a,b). Overexpression of the EGFR ligand TGF-α also transformedRat-1 and NRK fibroblasts, as assayed by colony formation insoft agar and tumorigenicity in nude mice (Rosenthal et al.,1986; Watanabe et al., 1987). In contrast, NIH-3T3 cells weretransformed by expression of TGF-α only with concomitantexpression of EGFR (Di Marco et al., 1989). Finally,transformation by overexpression of TGF-α of human andmouse mammary epithelial cells that co-express a sufficientcomplement of functional EGFR has been demonstrated(Ciardiello et al., 1990; Shankar et al., 1989). Taken together,these data suggest that overexpression of EGFR is able toinduce transformation in presence of appropriate levels ofligands. In addition, the role of EGFR in the autonomousproliferation of carcinoma cells has been formally demonstratedby using different approaches such as retroviral antisenseexpression vectors, antisense oligonucleotide, or neutralizingantibodies. In fact, blockade of EGFR results in a significantinhibition of the in vitro and in vivo growth of several differentcell lines derived from human carcinoma of various histologicaltypes (Normanno et al., 2003a). Analogously, the anti-tumoractivity of anti-ErbB-2 monoclonal antibodies in differentcancer cell lines is well established (Normanno et al., 2003a).

The ability of ErbB receptors and their ligands to induce invivo transformation has been also investigated in differenttransgenic mice models (Table 3). Virgin transgenic mice inwhich EGFR expression was driven in the mammary gland byeither the mouse mammary tumor virus (MMTV) or the β-

Table 3Summary of transgenic studies with ErbB receptors and their ligands

Transgene Target tissue Tumor type

EGFR Breast Mammary adenocarcinomas inlactating animals

Urothelium NoneGlia NoneEsophagus None

Activated c-neu Breast Mammary carcinomas in virgin femalesand in males

c-neu Breast Mammary carcinomas after long latencyRat ErbB-2 Skin Squamous cell carcinomasErbB-3 Breast Lung adenocarcinoma after long latencyTGF- α Multiple* Liver carcinoma** or mammary

carcinoma in multiparous females***Breast Mammary carcinoma in virgin and

multiparous femalesSkin None

AR Skin NoneHRG Breast Mammary carcinoma in multiparous

females

*: MT promoter; **: in FVB/N mice; ***: in CD-1 mice.

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lactoglobulin promoter developed mammary hyperplasias thatprogressed to dysplasias and tubular adenocarcinomas inlactating animals (Brandt et al., 2000). In contrast, over-expression of the EGFR in different tissues includingurothelium, glial cells or esophageal keratinocytes by usingspecific promoters resulted in increased proliferation levels, butnot in tumor formation (Cheng et al., 2002; Ding et al., 2003;Andl et al., 2003). The occurrence of mammary carcinomasonly after pregnancy also suggests that overexpression of theEGFR by itself is not able to induce in vivo transformation, andthat other events such as activation of protooncogenes orinactivation of tumor suppressor genes are required for thisphenomenon to occur.

Generally speaking, ErbB-2 seems to have a higher in vivotransforming ability as compared with EGFR. Several studieshave sown that when an activated form of the ErbB-2 murineanalogue neu, which contains mutations within the transmem-brane domain, is overexpressed in transgenic mice under thecontrol of the MMTV promoter, mammary adenocarcinomasdevelop (Pattengale et al., 1989; Muller et al., 1988; Guy et al.,1996). These tumors arose rapidly in a synchronous, single stepmanner, suggesting that overexpression of the activated neu issufficient for malignant transformation of mammary epithelium(Muller et al., 1988; Guy et al., 1996). In contrast, expression ofwild-type neu in the mammary epithelium of transgenic miceusing the MMTV promoter resulted in the development of focalmammary tumors after a long latency (Guy et al., 1992).Appearance of tumors in these mice was not strictly dependenton pregnancy, since virgin transgenic mice also developedmammary tumors. Interestingly, it has been demonstrated thatthese tumors contain activating somatic mutations of neu(Siegel et al., 1994). Such mutations are mainly small in-framedeletions in the extracellular domain of neu that promotedimerization of the neu receptor through the formation ofdisulfide bonds, thus resulting in its constitutive activation(Siegel and Muller, 1996). Transgenic mice carrying these

altered neu receptors under the trascriptional control of MMTVdevelop multiple mammary tumors that frequently metastasizeto the lung (Siegel et al., 1999). Mammary carcinomas wereobserved in virgin animals carrying altered neu receptors,although pregnancy reduced the latency period.

Transgenic mice engineered to overexpress the activatedforms of rat ErbB-2 in the skin have been also developed byusing the keratin 14 (K14) or the K5 promoters (Bol et al., 1998;Xie et al., 1998). In both cases, expression of the transgenebefore birth resulted in high incidence of perinatal death due todefects in the skin and esophagus. Postnatal expression of thewild type rat ErbB-2 in transgenic animals was obtained byusing a doxycycline inducible expression vector and the bovineK5 promoter. These animals developed spontaneous papillo-mas, some of which converted to squamous cell carcinomaswith N90% incidence by 6 months (Kiguchi et al., 2000).Increased expression of both EGFR and ErbB-2 proteins as wellas an increase in ErbB-2/EGFR and ErbB-2/ErbB-3 hetero-dimers were observed in skin of the ErbB-2 transgenic mice.

Several studies in transgenic mice have also shown thetransforming properties of TGF-α. Metallothionein-directedTGF-α expression in transgenic mice (MT-TGF-α) determineda uniform epithelial hyperplasia of liver, pancreas andgastrointestinal tract and the occurrence of hepatocellular andbreast carcinoma (Jhappan et al., 1990; Sandgren et al., 1990).Development of different neoplasias in MT-TGF-α mice seemsto be related to the genetic background. In fact, MT drivenoverexpression of TGF-α leads to development of mammaryadenocarcinomas when operating on a FVB/N genetic back-ground, whereas it induces liver tumors in CD-1 mice (Jhappanet al., 1990; Sandgren et al., 1990). Liver adenomas andcarcinomas appeared stochastically and at multiple sites,suggesting that chronic exposure to TGF-α is not a sufficientstep in liver tumorigenesis. In agreement with these observa-tions, mammary adenocarcinomas developed in MT-TGF-αmice in the postlactational mammary gland (Sandgren et al.,1990). In addition, transformation of mammary epithelium inMT-TGF-α transgenic mice occurs despite a low level of TGF-α expression in this organ, suggesting that the mammaryepithelium is particularly sensitive to the effects of TGF-α invivo. Higher levels of TGF-α expression in the mammary glandwere observed in MMTV-TGF-α transgenic mice (Matsui et al.,1990). In these mice, overexpression of TGF-α in the mammaryepithelium of virgin females leads to widespread hyperplasia ofthe terminal ducts and alveolar glands. Although mammaryadenocarcinomas were mainly observed in multiparous femalemice, occurrence of mammary adenocarcinomas in virginMMTV-TGF-α mice has also been described (Halter et al.,1992). However, tumors appeared in virgin mice at a mucholder age as compared with multiparous transgenic mice (325days vs 210 days). Overexpression of TGF-α under the controlof the whey acidic protein (WAP) promoter induced largenumbers of hyperplastic alveolar nodules within the mammarygland (Sandgren et al., 1995). Non-virgin female WAP-TGF-αtransgenic mice developed relatively well differentiated adeno-mas and adenocarcinomas. These tumors displayed inductionof cyclin D1 mRNA, suggesting that expression of this gene

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may complement that of TGF-α during mammary tumordevelopment.

Targeted expression of TGF-α in the epidermis of transgenicmice by using either the human K1 or K14 promoter inducedspontaneous papillomas in regions of mechanical irritation orwounding but no carcinomas (Vassar and Fuchs, 1991;Dominey et al., 1993). In agreement with these findings,overexpression of AR in the basal keratinocytes of transgenicmice by using a K14 promoter-driven AR gene, inducedoccurrence of psoriatic lesions, but it did not induce theoccurrence of papillomas or carcinomas (Cook et al., 1997).Finally, overexpression of HRG in the mammary gland oftransgenic mice by using the MMTV promoter inducesmammary adenocarcinomas in multiparous female at a medianage of 12 months (Krane and Leder, 1996). Since these tumorsarise in a solitary fashion, it is conceivable that HRGoverexpression is not sufficient for tumor formation.

Taken together these results clearly demonstrate that ErbBreceptors and EGF-like peptides display different ability toinduce cell transformation in transgenic mice models. Inparticular, TGF-α is the only EGF-like growth factor that hasbeen demonstrated to induce transformation in different tissues.Analogously, ErbB-2 is the receptor that has shown the highesttransforming power, although mutational activation of thisreceptor seems to be necessary for tumor induction, at least inbreast tissue. Finally, these data also suggest that the mammarygland is the organ in which overexpression of ErbB receptorsand/or ligands is more efficient in inducing a tumor phenotype,as compared with other tissues. However, the incidence andlatency of these mammary carcinomas was markedly affectedby pregnancy, suggesting that other events, such as hormonalinfluences, are required for neoplastic transformation ofmammary epithelium by ErbB receptors and ligands.

4.2. Cooperation of ErbB receptors and EGF-like peptides inoncogenic transformation

Evidence suggests that the transforming ability of ErbBproteins is greatly enhanced when different receptors are co-expressed. In this regard, Kokai and co-workers (1989)demonstrated that contemporary expression of rodent p185c-neu and EGFR in NIH-3T3 cells was necessary to induce fulltransformation of these cells. Cooperation of ErbB-2 andErbB-3 in inducing in vitro transformation has also beendemonstrated. Under conditions in which neither gene aloneinduced transformation, ErbB-2 and ErbB-3 transformed NIH-3T3 cells if co-expressed (Alimandi et al., 1995). In addition,at high expression levels of ErbB-2, which cause transfor-mation, co-expression of ErbB-3 enhanced formation of fociby one order of magnitude. The relevance of ErbB-2/ErbB-3heterodimers in cell transformation was also demonstrated byWallasch et al. (1995). These authors found that co-expressionof the two receptors produced HRG-dependent transformationof NIH-3T3 cells. In agreement with these findings, HRGinduced proliferation, but not transformation, of cells expres-sing either ErbB-3 or ErbB-4 alone (Zhang et al., 1996).However, HRG-induced cell transformation was observed

when EGFR or ErbB-2 was coexpressed with ErbB-3 orErbB-4. Finally, Cohen et al. (1996) have shown that invitro transformation of a clone of NIH-3T3 devoid ofdetectable endogenous ErbB receptors occurred only whentwo different ErbB receptors were expressed in presence ofan appropriate ligand. Any combination of ErbB receptorswas able to induce in vitro transformation. However, theEGFR/ErbB-2 heterodimer was the only receptor pair able toefficiently induce in vivo transformation ( i.e., a tumorigenicphenotype).

Overexpression of ErbB-2 appears to be an amplifier of thesignaling stimuli that are carried by other ErbB receptors. Theimportance of the cooperation of ErbB-2 and other ErbBreceptors in inducing in vivo transformation is indeed suggestedby different observations. For example, mammary tumorsderived from transgenic mice engineered to overexpress neualso exhibit overexpression of endogenous EGFR (DiGiovannaet al., 1998). Similarly, transgenic mice carrying an activatedneu show high levels of tyrosine phosphorylation of both neuand ErbB-3 (Siegel et al., 1999). A synergistic interaction of theneu proto-oncogene and TGF-α in inducing transformation inthe mammary epithelium of transgenic mice has been alsodemonstrated (Muller et al., 1996). Bitransgenic female miceco-expressing TGF-α and neu developed multifocal mammarytumors with significantly shorter latency period as comparedwith either parental strain alone. Finally, treatment of ErbB-2transgenic mice with an EGFR tyrosine kinase inhibitor such asgefitinib efficiently prevents the formation of mammary tumors(Lu et al., 1991).

Taken together, the above-mentioned findings suggest thatco-expression of different ErbB receptors is necessary to inducefull transformation in vitro and in vivo. As we will discuss in thenext paragraphs, this hypothesis is confirmed by studies inhuman primary carcinomas. In addition, this observation hasimportant insights for assessment of prognosis and fordevelopment of therapeutic approaches.

5. EGFR expression and genetic alterations in humancarcinomas

5.1. The ErbB receptors and their ligands are frequentlyexpressed in human carcinoma

The role of ErbB receptors and their ligands in thepathogenesis of human carcinomas is confirmed by a numberof studies that have shown overexpression of these proteins inthe majority of solid neoplasms. Our group has previouslypublished exhaustive reviews on the expression of the ErbBreceptors and their cognate ligands in human carcinomas, andthis is beyond the purpose of this review article (Salomon et al.,1995; Normanno et al., 2003a, 2005a). Briefly, expression ofEGFR and ErbB-3 has been described to occur in the majorityof human carcinomas at high frequency. On average, 50% to70% of lung, colon and breast carcinomas have been found toexpress EGFR or ErbB-3 (Fig. 2) (reviewed in Normanno et al.,2003a; Abd El-Rehim et al., 2004). In contrast, ErbB-2expression is generally more restricted, with approximately

Fig. 2. Average protein expression of ErbB receptors and cognate ligands in breast, lung and colon carcinomas.

9N. Normanno et al. / Gene 366 (2006) 2–16

30% of human primary breast carcinomas expressing thisreceptor (Fig. 2). The expression of ErbB-4 has been mainlyinvestigated in breast carcinoma, where this receptor isexpressed in approximately 50% of the tumors (Fig. 2)(reviewed in Normanno et al., 2003a; Abd El-Rehim et al.,2004). Expression of ErbB-4 has been recently demonstrated tooccur in 22% of human primary colon carcinomas (Lee et al.,2002).

The prognostic significance of EGFR and ErbB-2 has beeninvestigated in numerous studies. Although expression of theseproteins has been often reported to be associated with a worseprognosis, the power of these receptors to predict the outcomeof individual patients has not been formally proven. Forexample, several clinical studies have suggested that EGFRmay be an important independent prognostic factor in humanbreast cancer, while others have found only a tendency or nosignificant relationship between these two parameters (reviewedin Klijn et al., 1992). Studies following patients for over 5 yearsgenerally do not show a significant effect on relapse freesurvival or overall survival, while those analysing patients at 1–2 years do. This might indicate that EGFR status defines asubgroup of early relapsing, poor prognosis patients. Inaddition, the power of ErbB-2 to predict clinical outcome inbreast cancer patients has long been investigated. In this respect,there is little doubt that ErbB-2 gene amplification and/orprotein overexpression in node positive breast cancer patients ispredictive of a poor outcome independently of other prognosticindicators (Gullick, 1990; Ravdin and Chamness, 1995).However, analysis of clinical studies in which node negativepatients were examined, suggests little clinical utility of ErbB-2expression assessment in this group (Ravdin and Chamness,1995).

It is important to underline that due to the high frequency ofexpression of individual ErbB receptor types in humancarcinoma, co-expression of different receptors occurs in themajority of tumors. As we have above discussed, thisphenomenon might be important for tumor pathogenesis.Indeed, tumors that co-express different ErbB receptors areoften associated with a more aggressive phenotype and a worseprognosis. For example, an elegant paper by DiGiovanna et al.(2005) has recently found EGFR expression in only 15% of 807invasive breast cancers. However, the majority of EGFR-

positive tumors (87%) were found to co-express ErbB-2. Moreimportantly, almost all tumors that expressed the phosphory-lated form of ErbB-2, co-expressed EGFR. Expression ofphosphorylated ErbB-2 or co-expression of ErbB-2 and EGFRwas associated with the shortest survival in cancer patients. Incontrast, patients with tumors that were negative for all threemarkers, or that expressed only EGFR or only non-phosphor-ylated ErbB-2 had a relatively good outcome. Taken together,these data clearly establish a link in vivo between expression ofEGFR and activation of ErbB-2 in breast cancer patients.

In agreement with these findings, co-expression of EGFR,ErbB-2 and ErbB-3 was found to have a negative synergisticeffect on patient outcome, independent of tumor size or lymphnode status, in a cohort of 242 patients with invasive breastcarcinomas with a median 15-year follow-up (Wiseman et al.,2005). A direct correlation was found between the number ofErbB receptors expressed within the tumor and patients’outcome. Breast cancer patients whose tumours co-expressedErbB-2 and ErbB-3, as well as those whose tumors co-expressed EGFR, ErbB-2 and ErbB-4, showed an unfavourableoutcome compared with other groups, while combined ErbB-3and ErbB-4 expression was associated with a better outcome(Abd El-Rehim et al., 2004). Interestingly, Brabender et al.(2001) found that lung cancer patients with high levels ofexpression of both ErbB-2 and EGFR transcripts showed aworse prognosis as compared with patients expressing a singlereceptor. Additional studies have shown that co-expression ofdifferent ErbB receptors is associated with worse outcome ascompared with expression of a single receptor in coloncarcinoma, transitional cell carcinoma of the urinary bladderand oral squamous cell carcinoma patients (Lee et al., 2002;Chow et al., 2001; Xia et al., 1999).

The redundancy of expression in human carcinomas is notlimited to the ErbB receptors. In fact, a number of studies havedemonstrated that co-expression of different EGF-like peptidesoccurs in a majority of human carcinomas. For example, wehave demonstrated that co-expression of TGF-α, AR and/orNRG occurs in human colon, breast, lung, ovarian and gastriccarcinomas, suggesting that co-expression of different EGF-likegrowth factors is a common phenomenon in human carcino-genesis (Fig. 2) (reviewed in Normanno et al., 2001). In thisrespect, it is important to underline that both tumor cells and

Table 5Incidence of EGFR tyrosine kinase domain mutations in NSCLC patients

Characteristics of tumors N. oftumors

Tumors with EGFRmutation

Number Rate (%)

Smoking historySmokers or former smokers 1.387 99 7.1Never smokers 504 231 45.8

SexMale 1.424 142 10Female 498 193 38.7

Histologic typeAdenocarcinoma 1.127 331 29.4Non-adenocarcinoma 916 17 1.8

EthnicityEast-Asian 843 282 33.4Non-East-Asian 1.200 66 5.5

TOTAL 2.043 348 17

10 N. Normanno et al. / Gene 366 (2006) 2–16

surrounding stromal cells might represent a source of ErbBligands.

Taken together, these findings suggest that a network ofErbB receptors and cognate ligands is involved in theprogression of human carcinoma. This hypothesis has importantimplications for both prognostic and therapeutic applications. Itis conceivable that the prognosis of patients might be related tothe expression of all the receptors and ligands of this family,rather than the expression of a single molecule. In other words,the dependence of a tumor from ErbB receptors for its growthcan be assessed only evaluating the expression of all thepotential components of the above described network. In thisrespect, this hypothesis implies that contemporary blockade ofdifferent signaling pathways driven by ErbB receptors might benecessary in order to obtain a significant tumor growthinhibition. Indeed, our group has demonstrated that simulta-neous blockade of different growth factors and/or receptors ofthe ErbB family produces a more significant growth inhibitionas compared with inhibition of a single component of thisnetwork (Normanno et al., 2001,2003a).

5.2. Genetic alterations of ErbB receptors in human carcinoma

The genetic alterations of ErbB receptors found in humancancer are summarized in Table 4. Gene amplification of EGFRhas been demonstrated to occur in different tumor types and it isusually associated with EGFR protein overexpression, althoughoverexpression of EGFR in absence of gene amplification hasbeen described (Salomon et al., 1995; Normanno et al., 2003a).The frequency of this phenomenon varies among the differenttumor types. In glioblastoma multiforme (GBM), EGFR geneamplification has been found in 37% to 58% of the tumors(reviewed in Wikstrand et al., 1998). However, the majority ofGBM with EGFR gene amplification have also mutations of theEGFR gene that might be relevant for the transforming power ofthis receptor (Frederick et al., 2000). The mutations found inGBM are deletion or tandem duplication of the gene, and someof these genomic alterations are associated with ligand

Table 4Genetic alterations of ErbB receptors in human carcinoma

Receptor Genetic alteration Ligand dependence

EGFR Gene amplification +N-terminal truncation (EGFRvI) −Deletion exons 14–15 (EGFRvII) +Deletion exons 2–7 (EGFRvIII)Deletion exons 25–27 (EGFRvIV) +C-terminal truncation (EGFRvV) +Tandem duplication exons 2–7 +Tandem duplication exons 18–25 −Tandem duplication exons 18–26 −Small in frame deletion or pointmutations in the kinase domain(exons 18–21)

+

ErbB-2 Gene amplification NASplice variants resembling neu mutants NASmall in frame insertions or missensesubstitutions in the kinase domain(exons 18–24)

NA

independent activity of the EGFR (Table 4) (Kuan et al.,2001). Interestingly, a significant fraction of GBM (33%) withEGFR amplification showed multiple types of EGFR mutations(Frederick et al., 2000). The most frequent variant of EGFR isthe EGFRvIII, which accounts for more than 50% of thegenomic alteration of EGFR observed in GBM (Kuan et al.,2001). The EGFRvIII mutant receptor contains an in-framedeletion of exons 2-7 from the extracellular region. The 145 kDEGFR protein encoded by this variant is constitutivelyactivated. Several studies have addressed the prognosticsignificance of EGFR amplification in GBM, with contrastingresults. A recent study suggested that EGFRvIII overexpressionin the presence of EGFR amplification is the strongest indicatorof a poor survival prognosis in GBM patients (Shinojima et al.,2003).

Expression of EGFRvIII has been also described to occur incarcinomas such as breast, ovarian and lung cancer (Pedersen etal., 2001). However, the frequency of this mutation and thepercentage of tumor cells carrying the mutated EGFR in tumorsother than gliomas need to be addressed in larger studies. Morerecently, several studies have shown that EGFR geneamplification and mutations of the tyrosine kinase domainoccur in carcinoma patients. In particular, EGFR geneamplification or high polysomy has been demonstrated in22% to 32% of primary NSCLC (Hirsch et al., 2003; Cappuzzoet al., 2005). In addition, small in frame deletions or pointmutations occurring in the tyrosine kinase domain (exons 18through 21) of the EGFR have been found in NSCLC patients(Lynch et al., 2004; Paez et al., 2004; Pao et al., 2004). Severalstudies have addressed the frequency of expression of thesemutations in patients with NSCLC (Table 5) (Lynch et al., 2004;Paez et al., 2004; Pao et al., 2004; Huang et al., 2004;Shigematsu et al., 2005a; Marchetti et al., 2005; Tokumo et al.,2005; Mitsudomi et al., 2005; Chou et al., 2005; Han et al.,2005). Overall, EGFR tyrosine kinase mutations have beendescribed up to now in 17% of NSCLC patients (Table 5).However, these mutations are far more frequent in East-AsianNSCLC patients (33.4%) as compared with Non-East-Asianpatients (5.5%) (Table 5). Furthermore, the EGFR mutations are

11N. Normanno et al. / Gene 366 (2006) 2–16

more frequent in female patients as compared with male (38.7%vs 10%); in adenocarcinoma, often with features of bronchio-loalveolar carcinoma (BAC), as compared with other histologictypes (29.4% vs 1.8%); and in non-smokers as compared withsmokers or former smokers (45.8% vs 7.1%) (Table 5). Takentogether, these characteristics identify a peculiar, uncommonsubtype of lung cancer with defined clinical and pathologicalfeatures. These findings suggest that the growth of this type oftumor is strictly dependent on the activation of the EGFRpathway, which might represent the leading pathway ininducing cellular transformation in these selected patients.

The biochemical characteristics of the mutated forms ofEGFR that have been isolated in NSCLC patients have beenstudied. However, discordant results have been reported up tonow. The initial findings suggesting an increased kinase activityof the mutated EGFR in response to exogenous EGF (Lynch etal., 2004; Paez et al., 2004) have not been confirmed in a morerecent study (Pao et al., 2004). Nevertheless, the above-described mutations are associated with an increased responserate to EGFR tyrosine kinase inhibitors in all the above reportedstudies. Indeed, these mutations have been discovered bystudying patients that responded to EGFR tyrosine kinaseinhibitors such as gefitinib or erlotinib. Interestingly, it has beenreported that a patient with NSCLC that initially responded togefitinib and that became resistant to this treatment developed asecond mutation of the EGFR (Kobayashi et al., 2005). Thetumor of this patient was found to contain a secondary mutationin exon 20, which leads to substitution of methionine forthreonine at position 790 (T790M) in the kinase domain.

Biochemical studies demonstrated that such mutation pro-duces resistance of the EGFR to gefitinib. The same secondaryEGFR mutation was found in three of six individuals whosedisease progressed on either gefitinib or erlotinib (Pao et al.,2005a). These findings suggest that growth of a subgroup ofNSCLC is strictly dependent on the activation of EGFR, andthat this type of tumors might escape from the anti-tumoractivity of EGFR tyrosine kinase inhibitors by developing novelmutations.The presence of a mutation of the EGFR is alsoassociated with a better survival among patients that initiallyrespond to anti-EGFR agents in NSCLC (Han et al., 2005).However, a recent study suggested that EGFR gene amplifica-tion or high polysomy is associated with higher response rate,longer time to progression and improved survival in NSCLCpatients treated with gefitinib (Cappuzzo et al., 2005). Incontrast, EGFR mutations showed a statistically significantcorrelation with response and time to progression, but not withsurvival. Furthermore, in multivariate analysis, only high EGFRgene copy number remained significantly associated with bettersurvival. In this respect, it has been recently reported that 8/9colon carcinoma patients with objective responses to the anti-EGFR monoclonal antibodies cetuximab or panitumumab hadan increased EGFR copy number as assessed by FISH (Moroniet al., 2005). By contrast, only 1 /21 non-responders assessableby FISH had an increased EGFR copy number.

These findings suggest that EGFR gene amplification or highpolysomy might be associated to increased response to anti-EGFR agents in different carcinoma types, and that it might

represent a more reliable marker of response to such agents ascompared with EGFR tyrosine kinase domain mutations. Thisinformation might be relevant to select patients that are likely torespond to anti-EGFR drugs. Mutations of the EGFR tyrosinekinase domain have not been found in different carcinomatypes, including breast and colon cancer, whereas a recent studyhas shown such mutations to occur in approximately 7% of headand neck cancer patients (Lee et al., 2005). A study in East-Asian patients has recently suggested that mutations of theEGFR might also occur in colon carcinoma (Nagahara et al.,2005). However, these findings need to be confirmed in largercohorts of patients, in order to assess the frequency of thisphenomenon, and whether any correlation between ethnicityand mutations might exist within colon cancer patients, aspreviously described for NSCLC patients. Finally, EGFR geneamplification has been recently reported in 6% of primary breastcarcinomas (Bhargava et al., 2005). A good correlation wasfound in these tumors between gene amplification andoverexpression of EGFR. The correlation between EGFRgene amplification and response to anti-EGFR agents in breastcarcinoma has not been assessed yet.

Overexpression of ErbB-2 is frequently associated with geneamplification, although overexpression in absence of geneamplification has also been observed (Salomon et al., 1995;Normanno et al., 2003a). The ErbB-2 gene is not frequentlymutated in carcinomas. However, a recent study has demon-strated the occurrence of in frame insertion or missensesubstitutions located in the kinase domain of the ErbB-2receptor (Stephens et al., 2004). In this initial report, ErbB-2mutations were only detected in NSCLC patients withadenocarcinoma with a frequency close to 10% (5 /51), andwere more frequent in current or ex-smokers. However, a morerecent study found mutations of the ErbB-2 tyrosine kinasedomain in 1.6% of 671 NSCLC (Shigematsu et al., 2005b).These mutations were more frequent in patients with adeno-carcinoma, female and non-smoker, i.e., the same phenotype ofpatients carrying EGFR mutations.

Interestingly, ErbB-2 mutations were found in patients thatdid not carry mutations of EGFR. However, the kinase activityand the transforming potential of this mutated form of ErbB-2have not yet been evaluated. Finally, both EGFR and ErbB-2mutations were found in patients that did not carry rasmutations, suggesting that these proteins are involved indifferent processes of transformation. Mutations of K-ras havebeen shown to be associated with resistance to the EGFRtyrosine kinase inhibitors gefitinib and erlotinib in NSCLC (Paoet al., 2005b).

Interestingly, alternative splice variants of ErbB-2 havebeen found in human breast cancers that most closelyresemble the neu deletion mutants detected in the wild-typeneu transgenic mice. These findings suggest that these ErbB-2splice variants like the neu mutants may form constitutivelyactive dimers that may play an important role in the developmentof human breast cancer (Siegel and Muller, 1996; Siegel et al.,1999). Finally, activation of ErbB-2 in breast cancer might alsooccur through post-translational modifications. Full-lengthErbB-2 (p185ErbB-2) undergoes proteolytic cleavage, shedding

12 N. Normanno et al. / Gene 366 (2006) 2–16

its extracellular domain (ECD), which is detectable in cellculture medium and in the sera of ErbB-2 positive patients (Linand Clinton, 1991; Pupa et al., 1993). Elevated levels of ErbB-2ECD in sera of breast cancer patients correlate with a poorerresponse to therapy with the anti-ErbB-2 monoclonal antibodyHerceptin (Yamauchi et al., 1997b; Colomer et al., 2000). Thetruncated ErbB-2 receptor (p95ErbB-2) resulting from theproteolytic cleavage showed an increased kinase activity andenhanced transforming efficiency as compared with p185ErbB-2, implicating that the ECD might act as a negative regulator ofErbB-2 kinase (Segatto et al., 1988). In this respect, expressionof p95ErbB-2 correlates with positive lymph node metastasis inErbB-2-overexpressing breast cancer patients (Christianson etal., 1998; Molina et al., 2002).

6. Conclusions

The findings described in this review article strongly supportthe hypothesis that a network formed by the ErbB receptors andtheir cognate ligands is involved in tumor pathogenesis andprogression. In fact, these proteins function as an integratedsystem that is able to regulate cellular functions that areimportant for tumor development, such as growth, differenti-ation, survival and angiogenesis. The type and the amount ofreceptors and ligands expressed on tumor cells and within thetumor environment will affect the “quality” and the “quantity”of ErbB signaling within each individual cancer. Furthermore,this network of ErbB receptors and EGF-like peptides is able tointeract with different signaling pathways. This hypothesis isimportant for clinical approaches of human carcinoma withtarget-based agents directed against these molecules. In fact,several studies have demonstrated that response to anti-EGFRagents is not related to the levels of expression of the targetreceptor in cell lines and in primary carcinomas (Normanno etal., 2003a,b; Campiglio et al., 2004). This phenomenon is notsurprising, since low levels of EGFR can be sufficient to turn onother ErbB receptors. It is possible that response to anti-EGFRagents might depend on the total levels of ErbB receptors andligands expressed in each tumor. Indeed, correlations betweenresponse to anti-EGFR agents and levels of expression of eitherErbB-2 or ErbB-3 have been demonstrated in in vitro studies(Moasser et al., 2001; Moulder et al., 2001; Normanno et al.,2002; Campiglio et al., 2004; Anido et al., 2003; Engelman etal., 2005; Hirata et al., 2005). In addition, the observation thatco-expression of different ErbB receptors and EGF-like growthfactors occurs in the majority of human carcinoma types, allowsus to hypothesize that a more efficient blockade of tumorgrowth might be obtained by using combinations of moleculesdirected against different targets.

We have shown that treatment of tumor cells withcombinations of antisense oligonucleotides directed againstdifferent EGF-like growth factors results in synergistic anti-tumor effects in different tumor types (reviewed in Normanno etal., 2003a). More recently, we and other groups found thatcombined treatment of breast cancer cells with the EGFRtyrosine kinase inhibitor gefitinib and the anti-ErbB-2 mono-clonal antibody herceptin produces a synergistic anti-tumor

effect (Normanno et al., 2002; Moulder et al., 2001). Theseresults have indicated a novel approach for the treatment ofcancer patients that might lead to an efficient and prolongedcontrol of tumor growth by using combinations of agentsdirected against the ErbB receptors and their ligands.

The recent discovery of the role of EGFRmutations and geneamplification in predicting the response to anti-EGFR agentsmight help to select patients that are likely to respond totreatment with these agents. However, the observation that co-expression of ErbB receptors and other tyrosine kinasereceptors frequently occurs in human carcinoma (Singer et al.,2004), also suggests that combinations of molecules directedagainst different classes of growth factor receptors are necessaryto efficiently block tumor growth. Finally, recent findings fromour group suggest that the EGFR might have importantbiological functions in non-cancer cell types of the neoplasticmicroenvironment. We have shown that the EGFR regulates theability of bone marrow stromal cells to induce osteoclastdifferentiation, suggesting that activation of EGFR in bonemarrow stromal cells might be involved in the generation andprogression of bone metastases (Normanno et al., 2005b). Thesefindings highlight the importance to address the role of EGFR inthe stromal compartment of the tumor that is directly involvedin tumor growth and progression.

Acknowledgments

This work was supported by grants from the AssociazioneItaliana per la Ricerca sul Cancro (A.I.R.C.) (national andregional grants), and from Ministero della Salute (Grant 110/FSN2004) to N. Normanno.

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