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ErbB receptors in the biology and pathology of the aerodigestive tract

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Review ErbB receptors in the biology and pathology of the aerodigestive tract Sarah Morgan, Jennifer R. Grandis Department of Otolaryngology, University of Pittsburgh School of Medicine, 200 Lothrop Street; Suite 500, Pittsburgh, PA 15213, USA ARTICLE INFORMATION ABSTRACT Article Chronology: Received 8 August 2008 Accepted 12 August 2008 Available online 23 August 2008 The most common sites of malignancies in the aerodigestive tract include the lung, head and neck and the esophagus. Esophageal adenocarcinomas (EA), esophageal squamous cell carcinomas (ESCC), and squamous cell carcinomas of the head and neck (SCCHN) are the primary focus of this review. Traditional treatment for aerodigestive tract cancers includes primary chemoradiotherapy (CRT) or surgical resection followed by radiation (or CRT). Recent developments in treatment have focused increasingly on molecular targeting strategies including cetuximab (a monoclonal antibody against epidermal growth factor receptor (EGFR)). Cetuximab was FDA approved in 2006 for treatment of SCCHN, underscoring the importance of understanding the biology of these malignancies. EGFR is a member of the ErbB family of growth factor receptor tyrosine kinases. The major pathways activated by ErbB receptors include Ras/Raf/MAPK; PI3K/AKT; PLCγ and STATs, all of which lead to the transcription of target genes that may contribute to aerodigestive tumor progression. This review explores the expression of ErbB receptors in EA, ESCC and SCCHN and the signaling pathways of EGFR in SCCHN. © 2008 Elsevier Inc. All rights reserved. Keywords: ErbB1 ErbB2 ErbB3 ErbB4 EGFR Head and neck cancer Esophageal cancer Aerodigestive cancer ErbB signaling Contents Introduction ................................... .......................... 573 Biology of ErbB receptors in the aerodigestive tract .............. .......................... 573 ErbB receptors in aerodigestive tract development ............. .......................... 573 ErbBs in aerodigestive tract cancer ....................... .......................... 574 ErbB family expression ........................... .......................... 574 EGFR................................... .......................... 574 ErbB2 .................................. .......................... 574 ErbB3 .................................. ......................... 575 ErbB4 .................................. ......................... 575 ErbB alterations ............................... ......................... 575 ErbB dimerization .............................. ......................... 575 ErbB signaling ................................ ......................... 575 MAPK .................................. ......................... 575 EXPERIMENTAL CELL RESEARCH 315 (2009) 572 582 Corresponding author. Fax: +1 412 383 5409. E-mail address: [email protected] (J.R. Grandis). 0014-4827/$ see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.08.009 available at www.sciencedirect.com www.elsevier.com/locate/yexcr
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E X P E R I M E N T A L C E L L R E S E A R C H 3 1 5 ( 2 0 0 9 ) 5 7 2 – 5 8 2

ava i l ab l e a t www.sc i enced i rec t . com

www.e l sev i e r. com/ loca te /yexc r

Review

ErbB receptors in the biology and pathology of theaerodigestive tract

Sarah Morgan, Jennifer R. Grandis⁎

Department of Otolaryngology, University of Pittsburgh School of Medicine, 200 Lothrop Street; Suite 500, Pittsburgh, PA 15213, USA

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Fax: +1 412 383 5409.E-mail address: [email protected] (J.R. Grand

0014-4827/$ – see front matter © 2008 Elseviedoi:10.1016/j.yexcr.2008.08.009

A B S T R A C T

Article Chronology:

Received 8 August 2008Accepted 12 August 2008Available online 23 August 2008

The most common sites of malignancies in the aerodigestive tract include the lung, head and neckand the esophagus. Esophageal adenocarcinomas (EA), esophageal squamous cell carcinomas(ESCC), and squamous cell carcinomas of the head and neck (SCCHN) are the primary focus of thisreview. Traditional treatment for aerodigestive tract cancers includes primary chemoradiotherapy(CRT) or surgical resection followed by radiation (or CRT). Recent developments in treatment havefocused increasingly on molecular targeting strategies including cetuximab (a monoclonalantibody against epidermal growth factor receptor (EGFR)). Cetuximab was FDA approved in2006 for treatment of SCCHN, underscoring the importance of understanding the biology of thesemalignancies. EGFR is a member of the ErbB family of growth factor receptor tyrosine kinases. Themajor pathways activated by ErbB receptors include Ras/Raf/MAPK; PI3K/AKT; PLCγ and STATs, all

of which lead to the transcription of target genes that may contribute to aerodigestive tumorprogression. This review explores the expression of ErbB receptors in EA, ESCC and SCCHN and thesignaling pathways of EGFR in SCCHN.

© 2008 Elsevier Inc. All rights reserved.

Keywords:

ErbB1ErbB2ErbB3ErbB4EGFRHead and neck cancer

Esophageal cancerAerodigestive cancerErbB signaling

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573Biology of ErbB receptors in the aerodigestive tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573

ErbB receptors in aerodigestive tract development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573ErbBs in aerodigestive tract cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574

ErbB family expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574EGFR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574ErbB2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574ErbB3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575ErbB4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575

ErbB alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575ErbB dimerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575ErbB signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575

MAPK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575

is).

r Inc. All rights reserved.

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STATs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576Src family kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577PLCγ-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577PI3K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577

ErbB crosstalk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577Concluding remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578

Introduction

The aerodigestive tract encompasses the lungs, esophagus, oralcavity, nasal cavity, paranasal sinuses, pharynx and larynx. The threemost commonsiteswheremalignancies arise include the lung, headand neck and esophagus. This review will focus on esophagealadenocarcinomas/squamous cell carcinomas and squamous cellcarcinomaof the head and neck (SCCHN). Theprimary risk factors inSCCHN include tobacco and alcohol use [1,2]. A subset of SCCHN hasbeen shown to be caused by the human papillomavirus, primarilytypes 16 and 18 [2,3]. There is a high incidence of synchronous andmetachronous esophageal squamous cell carcinoma (ESCC) inpatients diagnosed with SCCHN, indicating the common biology ofthese aerodigestive tract neoplasms [4–7]. A recent report notedthat ESCC accounts for approximately 38% of esophageal cancers inthe United States (1998–2003) [8]. Established risk factors for ESCCalso include tobacco and alcohol use [9] with esophagitis/inflam-mation as a possible contributing variable [10].

Esophageal adenocarcinoma (EA) is responsible for ~56% ofesophageal cancers in the United States (1998–2003) [8] and hasseveral established risk factors including Barrett's esophagus [11,12],gastro-esophageal reflux [13,14] and obesity (independent of reflux)[15–17].Medications that relax the loweresophageal sphinctermayalsocontribute but the current evidence is inconclusive [18]. Some reportssuggest that Helicobacter pylori and the regular use of non-steroidalanti-inflammatory drugs may contribute to a reduced risk of EA [18].

Treatment for aerodigestive tract cancers including SCCHN andESCC/EA has traditionally included primary chemoradiotherapy(CRT) or surgical resection followed by radiation (or CRT). Cetuximabis amonoclonal antibodyagainst EGFR thathasbeen shown to reducepatient mortality and increase locoregional control of the tumorwhen combinedwith radiotherapy in SCCHN[19]. In 2006 cetuximabbecamethe firstmolecular targeting strategyapprovedby theFDA forSCCHN. Preliminary work in ESCC has shown that cetuximab caninduce antibody-dependent cell cytotoxicity in ESCC cell lines [20]. Arecent phase II clinical trial reported that cetuximab can be safelyadministered in combination with chemotherapy and radiotherapyin esophageal carcinomas without increased mucosal toxicity [21]. Aphase III clinical trial is currently underway todetermine if cetuximabin combination with CRT treatment will increase survival comparedto CRT alone [21]. The success of this molecular targeting strategy inSCCHN and esophageal carcinomas underscores the importance ofunderstanding the biology of these malignancies.

Biology of ErbB receptors in the aerodigestive tract

ErbB receptors are members of the ErbB growth factor receptortyrosine kinase family and are generally found on the cell surface.

ErbB receptors contain an extracellular ligand binding domain, atransmembrane region and an intracellular domain with tyrosinekinase activity (except ErbB3). Upon ligand binding, the receptorsdimerize and autophosphorylate thereby initiating a signalingcascade downstream of the dimer. Ligand binding induces aconformation change of the receptor ectodomain (creating anextended and stabilized conformation, except for ErbB2 whichconstitutively maintains the stabilized conformation and has noknown ligand [22]) to facilitate receptor dimerization [23]. ErbBligands are produced as transmembrane precursors and theectodomains are processed via proteolysis leading to the sheddingof soluble growth factors [24]. In normal tissues this signalingcascade is tightly controlled and regulates processes that includeepithelial development and injury response. The major pathwaysactivated by ErbB receptors include Ras/Raf/MAPK; PI3K/AKT;PLCγ and STATs, all of which lead to the transcription of targetgenes that may contribute to aerodigestive tumor progression [25].Regulation of ErbB receptor signaling occurs through temporal andspatial expression of receptor ligands and through receptorendocytosis. Endocytic trafficking leads to receptor recycling orubiquitination and lysosomal degradation of the receptor [26].

EGFR activation can be induced through autocrine or paracrineligands. There are six major EGFR ligands that are expressed at themRNA level in some, but not all, SCCHN cell lines including:heparin binding EGF (HB-EGF), transforming growth factor alpha(TGF-α), betacellulin, amphiregulin (AR), heregulin, and epidermalgrowth factor (EGF) [27]. TGF-α and AR are the primary ligandsimplicated in autocrine growth signaling [28]. EGFR can homo-dimerize or heterodimerize with other members of the ErbBreceptor family [29].

ErbB2 has no known exogenous ligands that directly bind to it.If ErbB2 is highly overexpressed it can spontaneously dimerize andautoactivate, but it is most commonly activated via heterodimer-ization with other ErbB family members [22]. ErbB3 has nointrinsic tyrosine kinase activity but is transactivated by EGFR andErbB2. ErbB3 ligands include neuregulins, heregulin and neudifferentiation factor [30]. ErbB4 can homodimerize or hetero-dimerize with other members of the ErbB receptor family. ErbB4ligands include neuregulins, epiregulin, heregulin, neu differentia-tion factor, and betacellulin [30].

ErbB receptors in aerodigestive tract development

Knockout of each of the ErbB family members in mouse modelsleads to early stage lethality, limiting the study of ErbBs in thedevelopment of the aerodigestive tract. ErbB1 null mice had ageneralized epithelial immaturity and multiorgan failure [31];these mice have a short postnatal survival period in whichimpaired epithelial development in various organs is observed,

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including the skin, lungs and gastrointestinal tract. It was noted inEGFR−/− mice that the epithelium of the tongue also appeared tobe immature, was thinner, and less organized with no fungiformtaste papillae, indicating a role for EGFR in mucosal epithelialdevelopment [31]. ErbB2 and ErbB4 null mice die mid-gestationdue to malformations of the heart and central nervous system[32,33]. Most erbB3 null mice die before birth due to grossmalformations of the nervous system and Schwann cells and thosethat do survive through birth die shortly after from respiratoryinsufficiency [34]. These cumulative results suggest that ErbB1likely contributes primarily to epithelial development while ErbB2,ErbB3 and ErbB4 seem to regulate the development of neural andmuscle tissues. Interestingly, ErbB receptor null mice phenotypesare distinct depending on the precise genetic background,demonstrating that there are genetic factors that also influencethe role of ErbB receptors in development.

ErbBs in aerodigestive tract cancer

ErbB family expression

Expression of EGFR, ErbB2, ErbB3 and ErbB4 has been reported inSCCHN [28], although data on ErbB4 are conflicting [35–39]. Theentire ErbB receptor family has been found to be expressed atincreased levels in invasive carcinoma and ErbB1, ErbB2 and ErbB4have been identified as overexpressed in in situ carcinoma [28].Most cases of overexpression demonstrated elevated expression ofmultiple receptors simultaneously, providing indirect evidence forthe formation of ErbB receptor heterodimers [40].

EGFREGFR is the most well studied of the ErbB receptors in cancers ofthe aerodigestive tract. Overexpression of EGFR in oral dysplasiascompared to normal mucosa has been demonstrated [41,42].However, EGFR upregulation in the normal-appearing epitheliumadjacent to malignant tissue in SCCHN has also been reported,supporting the idea of field cancerization in SCCHN [43]. EGFRexpression has been demonstrated to be lower in laryngeal tumorsas compared to tumors of the pharynx and oral cavity [44]indicating that EGFR expression may differ between SCCHNanatomic sites [28]. In SCCHN tumors, 92% have elevated EGFRmRNA levels and 87% have elevated mRNA for its ligand TGF-α[43]. Additionally, EGFR protein overexpression has been reportedin over 80% of SCCHN cases [28] and EGFR gene amplification hasbeen demonstrated in up to 15% of SCCHN tumors [45]. In ESCC,EGFR protein overexpression has been detected in 60–70% [46,47].Gene amplification in ESCC was evaluated using FISH and found tobe present in approximately 28% of tumors [46]. In EA EGFR isfrequently expressed and may contribute to the progression ofBarrett's metaplasia to EA [48,49]. A recent study in esophagealcarcinomas indicates that TGF-α and EGFR are both expressed in88% of tumors [50]. High levels of EGFR protein expression havebeen correlated with lower patient survival in esophagealcarcinomas as well as SCCHN [47,51–53]. EGFR overexpression inESCC has also been significantly correlated with increased tumorinvasion [46].

EGFR dysregulation appears to result from several potentialmechanisms, including gene amplification and transcriptionalactivation [54–56]. Additionally, EGFR mRNA overexpression may

result from dysregulated p53, which directly increases EGFR genetranscription in SCCHN [57]. Abnormalities in dinucleotide repeatsin the first intron of the EGFR gene have also been implicated inaltering the efficiency of EGFR gene transcription. In 12 SCCHN celllines, those with lower numbers of dinucleotide repeats hadincreased levels of EGFRmRNA and protein [56,58]. Transcriptionalactivation appears to be a common cause of EGFR overexpressionin SCCHN, although the precise mechanisms leading to increasedgene transcription are incompletely understood [59].

Another possible mechanism of cellular dysregulation by EGFRis through nuclear localization of EGFR where it can act as atranscription factor. Nuclear localization of EGFR in SCCHN hasbeen reported and is associated with STAT3 interaction andtranscriptional activation of inducible nitric oxide synthase inSCCHN [28]. While the precise role of nuclear EGFR is incompletelyunderstood, localization of EGFR to the nucleus suggests that genesmay be transactivated by EGFR independent of direct EGFRdownstream signaling.

ErbB2ErbB2 is overexpressed in SCCHN and in esophageal cancerscompared to levels detected in corresponding normal mucosa.Studies have shown that ErbB2 is overexpressed in ~20–40% oftumors of SCCHN with gene amplification present in approxi-mately 5–10% of cases [37,60–64]. In ESCC, reports of IHC stainingindicate that ErbB2 is overexpressed in ~26–64% of cases [65–69].Increased expression of ErbB2 in EA is considered to be morecommon with ~10–70% of patients showing overexpression[70,71]. The large range of incidence of ErbB2 overexpression inthese studies is likely due to the variety of methods used to detectErbB2 that are currently in use. While there is no consensusregarding the optimal assay for ErbB2 detection, immunohisto-chemistry (IHC) and FISH have both been approved by the FDA andare commonly used [70]. IHC is only semi-quantitative and doesnot always accurately reflect ErbB2 status. ErbB2 gene amplifica-tion is found in few cases of ESCC (~5%) but is more common in EA(~15%) [72].

Studies in SCCHN indicate that ErbB2 overexpression maycorrelate with survival. IHC staining has shown that ErbB2overexpression is significantly correlated with a decrease indisease free survival indicating a possible prognostic value ofErbB2 expression in this cancer [64]. Gene amplification was alsostudied using a semi-quantitative PCR technique, but genomicamplifications of ErbB2 are not common and did not significantlycorrelate with patient survival in SCCHN [73]. ErbB2 may also beinvolved in resistance to 5-fluorouracil, cisplatin and the EGFRinhibitor gefitinib [36,74]. However, ErbB2 targeting strategieshave not demonstrated clinical efficacy to date in SCCHN [75–77].While ErbB2 may heterodimerize with other ErbB family membersand contribute to SCCHN tumor progression, the role of hetero-dimers in SCCHN is incompletely understood [36].

A correlation between patient survival and ErbB2 overexpres-sion or gene amplification has not been clearly elucidated inesophageal carcinomas. In ESCC, some reports indicate an associa-tion between ErbB2 overexpression and poor prognosis [69,78,79]while others have indicated no correlation [65–67]. Results of onestudy indicated a possible association between ErbB2 overexpres-sion and chemoradioresistance in ESCC [68]. Studies in EA are alsoinconclusive regarding the correlation of ErbB2 expression and/oramplification with patient prognosis [80,81].

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ErbB3ErbB3 is overexpressed at the mRNA and protein levels in a subsetof SCCHN cell lines [82] and is overexpressed in 21–81% of SCCHNtumors [37,60–62]. However, studies vary and are limited by thepoor quality of the antibodies available for IHC and immunoblot-ting. To date, ErbB3 gene amplification has not been detected inSCCHN [83]. ErbB3 appears to be related to the malignantprogression of SCCHN in clinical specimens [82] and ErbB3overexpression in SCCHN is correlated with survival and metas-tasis in several cohorts [84]. ErbB3 expression and signaling hasbeen correlated with resistance to the EGFR inhibitor gefitinib inlung cancer [36,85] and the limited SCCHN data suggests that asimilar mechanism may also be important in this cancer [36].

ErbB3 has not been studied specifically in ESCC and data in EA islimited. Some results suggest that ErbB3 is upregulated in ~40–50%of EA compared to normal tissue as detected by both quantitativeRT-PCR and IHC [86]. Further investigation of ErbB3 biology inesophageal carcinomas is warranted.

ErbB4Expression of ErbB4 is detected in SCCHN (26–69%) [37,39,60,61]but the role of this ErbB family member in these cancers is unclear.Expression of ErbB4 did not correlate with invasion, angiogenesis,metastasis or SCCHN tumor progression [87], although expressionof all four ErbB receptors in oral SCC was significantly associatedwith decreased patient survival [88]. Other evidence indicates thatErbB4 expression may be lost in vitro [39]. These findings provideindirect evidence of cooperation among ErbB receptors in SCCHNcancer progression.

In ESCC membranous, cytoplasmic and nuclear staining arenoted in more than 80% of ESCC samples demonstrating ErbB4expression in both the cell membrane and cytoplasm [89].Cytoplasmic and nuclear staining of ErbB4 expressing cells mayresult from ErbB4 cleavage by tumor necrosis factor alphaconverting enzyme (TACE) and γ-secretase producing an intracel-lular domain fragment that can be translocated to the nucleuswhere it functions as a transcription factor [90–92]. In ESCC, thefull-length membrane spanning ErbB4 receptor may contribute toinhibition of tumor progression while the transcription factorErbB4, when localized in the nucleus, may contribute to tumorprogression [89].

ErbB alterations

Unlike non-small cell lung carcinoma, SCCHN is not characterizedby mutations of the EGFR kinase domain [93]. In esophagealcarcinomas, EGFR mutations remain relatively uncharacterizedwith a few reports indicating that EGFR tyrosine kinase mutationsare rare in North America [94–96]. Expression of the EGFR variantIII has been identified in up to 40% of SCCHN tumors [97] but hasnot been studied, to date, in esophageal carcinomas. EGFRvIII wasoriginally characterized in glioblastomawhere it was found to be acommon somatic mutation associated with EGFR gene amplifica-tion leading to gene rearrangement [98,99]. EGFRvIII lacks exons2–7 with a novel glycine residue at the exon 1/8 junction. Exons2–7 comprise the majority of the extracellular ligand bindingdomain so that cells that express EGFRvIII are likely to bind to EGFRmonoclonal antibodies with reduced affinity. Thus, EGFRvIIIrepresents a possible mechanism of cetuximab resistance [97].EGFRvIII expression is lost in vitro; consequently SCCHN cells must

be stably transfected with an EGFRvIII construct to establish amodel for preclinical investigations. We have shown that EGFRvIIIexpression in SCCHN cell lines leads to increased cell proliferationin vitro and increased tumorigenicity in vivo as compared to vector-transfected control cells [97].

ErbB dimerization

Heterodimerization of ErbB receptors allows for potent transduc-tion of various signals. Heterodimerization of other members ofthe ErbB family with EGFR is implicated in early SCCHNcarcinogenesis where co-expression has been detected in pre-malignant lesions [100]. ErbB2 heterodimers have increasedsignaling potency compared to any other ErbB dimers. This iscaused by an increased ligand affinity through a decelerated rate ofligand dissociation, efficient coupling to signaling pathways and adecreased rate of receptor downregulation through endocytosis[101]. ErbB2–ErbB3 heterodimers have been shown to induce thePI3K pathway, most likely because ErbB3 can directly bind the PI3Kp85 subunit [83].

ErbB signaling

Upon ErbB receptor autophosphorylation, a variety of proteinsignaling molecules are recruited to the plasma membraneincluding growth factor receptor bound protein 2 (Grb2) and Shc.Activation of these proteins initiates the ErbB signaling cascadesthat lead to transcriptional regulation of target genes. In SCCHNmembers of the EGFR signaling pathway have been found atincreased levels, including MAPK, AKT, STAT3 and STAT5 (Fig. 1)[28]. Studies in esophageal carcinomas are more limited butindicate that theMAPK, AKTand STAT pathways are involved in theoncogenic signaling through EGFR overexpression [102–107]. Moremechanistic studies are needed to clearly define the signalingcascades involved with ErbB signal transduction and esophagealcarcinoma progression.

MAPKFollowing EGFR activation, Grb2 and Sos (adaptor proteins) bindEGFR directly at Y1092 and Y1110 or through Shc (which binds atEGFR Y1172 and Y1197) [108]. This activates Raf-1 initiating acascade that results in phosphorylated MAPK, which is thentranslocated to the nucleus where it activates cell proliferationtranscription factors [23]. The Ras-Raf-MEK-ERK pathway is theprimary MAPK pathway downstream of ErbBs in SCCHN and leadsto upregulation of cyclin D1, which induces cell cycle progression(Fig. 1) [83]. Activated MAPK in SCCHNwas found to correlate withEGFR and TGF-α overexpression [109]. The formation of anE-cadherin-EGFR intercellular complex between tumor cells isthought to contribute to SCCHN invasion and metastasis. Thiscomplex leads to ligand independent activation of EGFR, whichactivates the MAPK pathway and transcription of Bcl-2 allowingthe cell to escape apoptosis induced by loss of the extra cellularmatrix [110].

In esophageal carcinomas, MAPK has been implicated as a keymediator in the downstream signaling of EGFR activation. Use ofthe pan ErbB tyrosine kinase inhibitor CI-1033 in ESCC linesresulted in the inhibition of phosphorylation of MAPK andinhibition of cell proliferation [111]. A separate study combinedradiation treatment with the MEK inhibitor PD98059 and found

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synergistic effects on cell killing in esophageal cancer cell linesindicating that MAPK signaling may be a contributing factor in cellsurvival in esophageal cancer [106].

STATsSTAT1, STAT3, and STAT5 [100,112,113] are activated in SCCHN andcontribute to the transduction of EGFR signaling in the cell. STAT3and STAT5 are transcription factors and oncogenes that help toregulate cell cycle progression, angiogenesis and apoptosis inhibi-tion through their target genes. STATs can be activated by

Fig. 1 – EGFR signaling in SCCHN. EGFR is activated by ligand bindhomodimerization which leads to receptor autophosphorylation. E(GPCR). GRPR activates Src leading to activation of PDK1 and PI3K,EGFR can also be transactivated by cell adhesion molecules such ascomplex activates EGFR and leads to MAPK signaling allowing for cactivation leads to five primary signaling cascades. 1) MAPK: EGFRthat bind EGFR at Y1092 and Y1110 or alternately Grb2 and Sos binsignaling cascade is activated resulting in cell survival, proliferatiointeracting directly with EGFR or through Src-mediated EGFR signaheterodimerize and are translocated to the nucleus where they indprogression, angiogenesis, and apoptotic inhibition; 3) Src: Can beand can also activate EGFR by phosphorylating EGFR at Y845. Downpathway. Activation of Src is implicated in cell proliferation, migraBinds directly to EGFR and activates the MAPK pathway through PKin migration and invasion; and 5) PI3K: Involved in many pathwayresistance, cell growth, invasion and migration.

interacting directly with EGFR through SH2 domains or indirectlythrough Src-mediated EGFR signaling [114]. After activation, STATsdimerize and are then translocated to the nucleus where theyinduce the transcription of target genes (Fig. 1) [114].

Constitutive activation of STAT3 has been reported in SCCHN[114] and STAT3 has been shown to be an oncogene and amediatorof cellular transformation [23,115]. STAT3 is likely activated inSCCHN through autocrine activation of EGFR by TGF-α [114]. Theidentification of constitutive STAT3 activity in normal mucosaindicates that STAT3 activation may have an early role in SCCHN

ing and subsequent receptor heterodimerization orGFR can be transactivated by G-protein-coupled receptorswhereby TACE cleaves EGFR proligand and activates EGFR.E-cadherin from neighboring tumor cells. The EGFR-E-cadherinell survival during the early stages of metastasis. EGFRphosphorylation recruits Grb2 and Sos (adaptor proteins)d via Shc at Y1172 and Y1197. Raf-1 is activated and the MAPKn and differentiation; 2) STATs: STATs can be activated byling. Once phosphorylated STATs homodimerize oruce the transcription of target genes that lead to cell cycleactivated by binding directly to EGFR at Y915 and Y944stream of EGFR, Src can activate the STAT pathway or the PI3Ktion, adhesion, angiogenesis and immune function; 4) PLCγ:C and the PI3K pathway leading to AKT activation. This resultss and results in activation of AKT that leads to apoptotic

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progression. STAT3 has been demonstrated to be upregulated bothwith and independent of EGFR upregulation [114]. In SCCHN,targeting STAT3 inhibited cell growth in vivo and in vitro [116,117].Additionally, combined targeting of STAT3 and EGFR producesenhanced antitumor effects in vitro and in vivo as compared toEGFR targeting alone [118]. These findings indicate that STAT3overexpressionmay contribute to cancer progression. STAT5 is alsooverexpressed and activated in SCCHN and an antisense blockadeof STAT5b inhibited tumor growth [119].

In esophageal carcinoma cell lines, proliferation via autocrinesignaling of EGFR was attributed to TGF-α stimulation leading toSTAT3 activation [120]. In esophageal keratinocytes, EGFR stimula-tion leads to STAT1 phosphorylation and dimerization with STAT3and subsequent formation of the STAT–JAK complex. This pathwayresults in keratinocyte migration as well as MMP1 activity [103].

Src family kinasesSrc family kinases (SFKs) are involved in cell proliferation,migration, adhesion, angiogenesis, and immune function [23]. Srcis a signal transducer of EGFR signaling and is also independentlyactivated and leads to the activation of many pathways includingSTATs and PI3K [23]. In SCCHN cell lines Src family kinases areactivated by TGF-α stimulation via direct binding to EGFR at Y915and Y944 [108,121]. Corresponding normal epithelial cells did notshow activation of the SFKs (cSrc, cYes, Fyn, Lyn) [121]. Activatedlevels of STAT3 and STAT5 were highly correlated with Srcphosphotyrosine (activation) levels and coimmunoprecipitationof STAT3 or STAT5 showed interaction with cSrc. cSrc blockadedemonstrated reduced STAT3 and STAT5 activation in addition toreduced cell growth in SCCHN cell lines. This indicates that SFKsmediate STAT growth pathways in SCCHN [121]. In SCCHN Src canbe activated independently of EGFR and transphosphorylate EGFRat Y845, leading to EGFR receptor activation [122].

PLCγ-1PLCγ-1 likely contributes to SCCHN invasion andmigration. PLCγ-1can interact directly with EGFR and hydrolyses phosphatidylinosi-tol 4,5-bisphosphate (PIP2) to inositol 1,3,5-triphosphate (involvedin intracellular calcium release) and 1,2-diacylglycerol (a cofactor inPKC activation). PKC activates Raf, which leads to MAPK activation(Fig. 1) [23,123]. PLCγ-1 is downstream of EGFR and may mediatethe invasive and metastatic mechanisms of SCCHN [124]. PLCγ-1contributes to tumor cell invasion in in vitro SCCHN experimentswhen activated by EGFR [125]. Activation of PLCγ through EGFRstimulation with EGF promotes SCCHN migration [125]. In humanSCCHN tumor samples, IHC staining showed that the tumor stainedhigher for phosphorylated PLCγ-1 than the normal mucosa [125]demonstrating an increase in PLCγ-1 activity in SCCHN. Otherin vitro experiments on SCCHN cells demonstrated that chemo-taxis and invasion of metastatic SCCHN cells were dependent onPI3K and its substrate PLCγ-1 although this pathway can be acti-vated through EGFR or chemokine receptor 7 [126].

PI3KPI3K can be activated by EGFR through EGFR heterodimerizationwith ErbB3, which contains a docking site for the p85 subunit ofPI3K, or alternately through Gab-1 binding EGFR. Once the p85subunit is docked, the p110 subunit of PI3K (containing catalyticactivity) generates phosphatidylinositol 3,4,5-triphosphate (PIP3),which phosphorylates and activates AKT. Activated AKT was found

to be overexpressed in 57–81% of SCCHN tumors [125]. Thispathway is involved in resistance to apoptosis, cell growth, invasionandmigration [23]. Cell migrationmay also bemediated by the Rhofamily of GTP-binding proteins [127] and PI3K through activation ofRas andRac [128]. Other non-receptor tyrosine kinases appear to bepossiblemediators downstream of EGFR and upstream of PI3K. Thenon-receptor tyrosine kinase Sykmayoperate downstreamof EGFRto participate in mediating signaling through the PI3K and PLCγpathways in SCCHN causing increased cell motility [129].

The PI3K pathway downstream of the ErbB receptors is also amajor mechanism of apoptosis evasion in head and neck cancer.PI3K catalyzes the conversion of PIP2 to a lipid second messengerPIP3, which results in recruiting and activating of PKB and AKTthrough PDK. In head and neck cancers EGFR can lead to PI3Kactivation directly or through Ras. PI3K then induces downstreamamplification of PDK1, which in turn phosphorylates AKT. InSCCHN, AKT regulates cell survival by affecting several down-stream targets including the FOXO family of forkhead transcriptionfactors, Bad, caspase 9 and by activating NF-kB. Studies in SCCHNhave shown that PTEN mutations are extremely rare indicatingthat PI3K activation through loss of PTEN function is not a majorfactor in dysregulated PI3K signaling in SCCHN [83].

The PI3K/AKT pathway appears to also be involved inesophageal neoplasms. Esophageal cancer cells stimulated withEGF showed induction of AKT phosphorylation with differentialactivation between AKT isoforms [107]. In primary and immorta-lized esophageal organotypic cultures, AKT overexpression andactivation is permissive for differentiation of primary andimmortalized esophageal epithelial cells [104]. These resultsindicate a role for the PI3K/AKT pathway in the context of EGFRstimulation. Irradiation combined with the inhibitor of PI3KLY294002 resulted in a synergistic increase in radiation-inducedcell killing as compared to radiation alone in esophageal cancer celllines [106]. These studies indicate that the PI3K pathway isinvolved in esophageal cancer cell survival and may be anappropriate future target for molecular therapeutics.

ErbB crosstalk

EGFR can be transactivated by G-protein-coupled receptors(GPCRs) through ligand-dependent and independent pathways.GPCRs can directly activate the tyrosine kinase domain of EGFR orcan induce cleavage of EGFR ligand precursors. Several GPCRs havebeen implicated in EGFR activation in SCCHN including gastrin-releasing peptide (GRP), prostaglandin E2 (PGE2), bradykinin,thrombin and lysophosphatidic acid (LPA) [130]. GPCR-EGFRcrosstalk has been shown to contribute to lung and head andneck cancer progression [130]. GPCR ligands induce EGFRphosphorylation and downstream MAPK activation throughcleavage of EGFR proligands, including TGF-α and amphiregulin,by TNF α converting enzyme (TACE) (Fig. 1) [131]. EGFR and MAPKappear to be key mediators in the mitogenic effects of GPCR astheir inhibition eliminated SCCHN proliferation induced by GPCR[132]. Further investigation demonstrated a role for Src familykinases and the PDK1 subunit of PI3K in this process in SCCHN[133,134]. Combined blockade of GPCR and EGFR pathwayssignificantly inhibited proliferation, invasion and colony formationof SCCHN cell lines but not immortalized mucosal epithelial cells[135]. GPCRs can also activate EGFR via Src-mediated phosphor-ylation of EGFR at Y845 in SCCHN [28].

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Transactivation of EGFR by cell adhesion molecules such as E-cadherin from neighboring tumor cells has been proposed toprevent apoptosis in the early stages of metastasis as the tumor celldetaches from the extracellular matrix [110]. Cell adhesionmolecules such as E-cadherin and integrins in SCCHN can form acomplex with EGFR and transactivate EGFR (Fig. 1) [83,110]. Thistransactivation of EGFR by E-cadherin leads to activation of theMAPK pathway which leads to transcription of the anti-apoptoticprotein Bcl-2 [110] allowing for SCCHN tumor cell survival in theabsence of the extracellular matrix. EGFR activation can also alterthe expression of integrins and E-cadherin by phosphorylatingbeta-catenin which leads to the internalization of E-cadherin [83]allowing decreased tumor cell aggregation and increased motilityon extra cellular matrix proteins in SCCHN. In esophagealcarcinomas, an inverse correlation between E-cadherin and EGFRhas been reported where expression of these proteins may serve asprognostic markers in this malignancy [136–138].

In SCCHN, EGFR has also been shown to be involved in crosstalkwith platelet-derived growth factor receptor and hormonereceptors [26]. Overexpression of the urokinase-type plasminogenactivator receptor can also lead to ligand independent activation ofEGFR [23]. Insulin-like growth factor 1 receptor can transactivateEGFR [83], while EGFR is known to transactivate cMET in SCCHN.These cumulative results suggest that there are several potentialpathways to transactivate EGFR in this cancer.

Concluding remarks and future directions

Overexpression of EGFR is relatively common in both SCCHN andesophageal carcinomas where expression levels have beencorrelated with decreased survival [47,51–53]. Preclinical targetingof EGFR inhibited tumor growth in SCCHN and esophagealcarcinomas leading to the FDA-approval of the EGFR monoclonalantibody cetuximab in SCCHN in 2006. EGFR tyrosine kinaseinhibitors are under investigation and have shown efficacy inphase II studies in SCCHN [139] and advanced esophagealcarcinomas [140] but phase III data are lacking. EGFR signalingmechanisms likely contribute to the response to EGFR targetingagents. Increased understanding of the role of the other ErbBfamily members in aerodigestive tract cancers and the down-stream signaling consequences of EGFR homodimerization andheterodimerizationwith other ErbB family members may improveour ability to therapeutically target ErbB signaling in aerodigestivetract cancers by identifying those patients who are most likely torespond.

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