The multiple roles of amphiregulin in human cancer

Biochimica et Biophysica Acta 1816 (2011) 119–131

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Biochimica et Biophysica Acta

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Review

The multiple roles of amphiregulin in human cancer

Benoit Busser a,b,⁎, Lucie Sancey a, Elisabeth Brambilla a,c, Jean-Luc Coll a, Amandine Hurbin a

a INSERM, U823, Institut Albert Bonniot, Grenoble, France, Université Joseph Fourier, Grenoble, Franceb CHRU Grenoble, Hôpital Michallon, UF Cancérologie Biologique et Biothérapie, Grenoble, Francec CHRU Grenoble, Hôpital Michallon, Département d'Anatomie et Cytologie Pathologiques, Grenoble, France

Abbreviations: AREG, amphiregulin; TACE, tumor-neepidermal growth factor; EGFR, epidermal growth factorheparin-binding EGF-like growth factor; EREG, epiregulphosphoinositide 3-kinase; PLC, phospholipase C; SH2,regulated kinases; mTOR, mammalian Target of Rapaminsulin like growth factor 1; BM–MSC, bonemarrow–mesquamous cell carcinoma; NSCLC, non small cell lung cagrowth factor; ECM, extracellular matrix; MMP, matrix⁎ Corresponding author. Tel.: +33 4 76 76 63 74; fax

E-mail addresses: bbusser@chu-grenoble.fr (B. Busse(J.-L. Coll), Amandine.Hurbin@ujf-grenoble.fr (A. Hurbin

a b s t r a c t

Article history:Received 4 April 2011Received in revised form 20 May 2011Accepted 21 May 2011Available online 30 May 2011

Keywords:AmphiregulinCancerBiomarkerEpidermal growth factor receptor tyrosinekinase inhibitorAngiogenesisMetastasis

Amphiregulin (AREG) is one of the ligands of the epidermal growth factor receptor (EGFR). AREG plays acentral role inmammary gland development and branchingmorphogenesis in organs and is expressed both inphysiological and in cancerous tissues. Various studies have highlighted the functional role of AREG in severalaspects of tumorigenesis, including self-sufficiency in generating growth signals, limitless replicativepotential, tissue invasion and metastasis, angiogenesis, and resistance to apoptosis. The oncogenic activity ofAREG has already been described in the most common human epithelial malignancies, such as lung, breast,colorectal, ovary and prostate carcinomas, as well as in some hematological and mesenchymal cancers.Furthermore, AREG is also involved in resistance to several cancer treatments.In this review, we describe the various roles of AREG in oncogenesis and discuss its translational potential,such as the development of anti-AREG treatments, based on AREG activity. In the last decade, independentgroups have reported successful but sometimes contradictory results in relation to the potential of AREG toserve as a prognostic and/or predictive marker for oncology, especially with regard to anti-EGFR therapies.Thus, we also discuss the potential usefulness of using AREG as a therapeutic target and validated biomarkerfor predicting cancer outcomes or treatment efficacy.

crosis factor-alpha converting enzyme; ADAM, a disintegreceptor; EGFR-TKI, epidermal growth factor receptor tyrin; BTC, betacellulin; NRG, neuregulins; HER, human epidSrc homology domain 2; SOS, son of sevenless; MEK, mycin; PKC, protein kinase C; GPCR, G-protein coupled resenchymal stem cells; LIF, leukemia-inhibitory factor; PHDrcinoma; EMT, epithelial–mesenchymal transition; HIF, hmetalloproteinase; SiRNAs, small interfering RNAs: +33 4 76 54 94 13.r), lucie.sancey@ujf-grenoble.fr (L. Sancey), ebrambilla@).

ll rights reserved.

Contents

1. Structure and function of AREG in normal tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1201.1. Discovery and structure of AREG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1201.2. AREG and its receptor, the epidermal growth factor receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1201.3. Role of AREG in normal tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

2. Role of AREG in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1222.1. Self-sufficiency in growth signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1232.2. Evading apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1232.3. Limitless replicative potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1242.4. Sustained angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1242.5. Tissue invasion and metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1252.6. Resistance to cancer treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

3. AREG as a cancer biomarker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263.1. Prognostic marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263.2. Predictive marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

rin and metalloproteinase; CTF, carboxy-terminal fragment; EGF,osine kinase inhibitor; TGF-α, tumor growth factor-alpha; HB-EGF,ermoid receptors; MAPK, mitogen-activated protein kinase; PI3K,itogen-activated protein kinase kinase; ERK, extracellular signal-ceptor; TNF-α, tumor necrosis factor-alpha; KO, knock-out; IGF1,2, prolyl-4-hydroxylase domain enzyme 2; HNSCC, head and neckypoxia-inducible transcription factor; VEGF, vascular endothelial

chu-grenoble.fr (E. Brambilla), jean-luc.coll@ujf-grenoble.fr

120 B. Busser et al. / Biochimica et Biophysica Acta 1816 (2011) 119–131

3.2.1. AREG predicts sensitivity to anti-ErbB therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263.2.2. AREG predicts resistance to anti-ErbB therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

4. AREG as a therapeutic target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1275. Discussion/conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

1. Structure and function of AREG in normal tissues

1.1. Discovery and structure of AREG

Human amphiregulin (AREG) is an 84-amino acid glycoproteindiscovered and characterized in the late 1980s by Shoyab et al. [1].Murine AREG was also described in mice and named schwannoma-derived growth factor [2]. AREG was originally isolated from theconditioned medium of phorbol 12-myristate 13-acetate (PMA)-stimulated MCF-7 human breast carcinoma cells [1]. The humanAREG gene (geneID 374) spans about 10 kb of genomic DNA and islocated on the q13–q21 region of chromosome 4. It is composed of 6exons encoding a 1.4 kb pre-protein mRNA transcript. The AREGprotein is synthesized as a 252-amino acid transmembrane precursor,pro-AREG. At the plasma membrane, pro-AREG is subjected tosequential proteolytic cleavages within its ectodomain and is thenreleased as the soluble AREG protein (Fig. 1). Depending on the celltype and microenvironment, AREG can be produced in multiplecellular and mature forms using alternative pro-AREG cleavage sitesand glycosylation motifs, thus impacting the biological activity ofAREG [3]. AREG shedding is essentially mediated by tumor-necrosisfactor-alpha converting enzyme (TACE), a member of the disintegrinand metalloproteinase (ADAM) family (also known as ADAM-17) [4].

The mature, soluble AREG contains six cysteines involved indisulfide linkages, which confer the three-looped structure (Fig. 2).The amino-terminus domain (N-Ter) of AREG is hydrophilic andcontains an N-glycosylated heparin binding domain (Fig. 1). Thecarboxy-terminus domain (C-Ter) has an epidermal growth factor(EGF)-like domain, and it shows remarkable homology with all theother members of the EGF-like growth factor family (Fig. 2), including

Fig. 1.Human AREG gene and protein domains. The gene is shown 5′-to-3′ to scale. The exonsrepresented as well as the protein domains. TM represents the transmembrane domain. Scisspro-AREG (between amino acids 100–101 and 184–185) to produce mature AREG contaiuntranslated region. (Adapted from [27]).

EGF itself (38% homology), tumor growth factor-alpha (TGF-α) (32%homology), heparin-binding EGF-like growth factor (HB-EGF), epir-egulin (EREG), betacellulin (BTC), and the neuregulins (NRGs) [4,5].

1.2. AREG and its receptor, the epidermal growth factor receptor

Because AREG has been shown to bind the EGF receptor (EGFR) incompetition with EGF, logically, AREG was incorporated into the EGFfamily [6]. The EGFR is a tyrosine kinase receptor that is involved infundamental signaling pathways and is therefore a major target inoncology. The EGFR belongs to the ErbB/human epidermoid receptor(HER) family, which contains the following 4 members: the epithelialgrowth factor receptor (EGFR/HER1/ErbB1), HER2/neu (ErbB2), HER3(ErbB3) and HER4 (ErbB4). These receptors share important struc-tural homology and are composed of an intracellular domain withtyrosine kinase activity, a hydrophobic alpha-helix transmembranedomain, and an extracellular domain required for ligand recognitionand binding. The extracellular domain is the least conserved amongthe 4 ErbBmembers, allowing distinct specificities and selectivities forligands [7]. For example, EGF, TGF-α and AREG specifically bind to theEGFR, whereas BTC, HB-EGF and EREG bind to both EGFR and HER4.Finally, the NRGs, NRG1 and NRG2, bind to HER3 and HER4, whereasNRG3 and NRG4 only bind to HER4 (see [8] for review). After thebinding of a ligand, the EGFR homo- or heterodimerizes with HER2,HER3 or HER4 [9]. Dimerization leads to tyrosine kinase domainautotransphosphorylation and activation of a complex network ofpathways, including the Ras/MAPK, PI3K/AKT, PLCγ and STATpathways (see Fig. 3 and [10] for review).

Comparedwithother EGFR-ligands,AREG is remarkably less effectivein inducing EGFR and other ErbB receptors tyrosine phosphorylation at

are indicated in black boxes and the introns inwhite boxes. The correspondingmRNA isors represent the TACE/ADAM-17 protein, which exerts a double proteolytic cleavage ofning the N-Terminus hydrophilic domain and the C-Terminus EGF-like domain. UTR:

Fig. 2. AREG, EGF and TGF-α: Protein sequences and structural homologies. Secondary structures of the mature forms of EGF (A), TGF-α (B) and AREG (C). Orange amino acids (aa)are conserved between EGF and TGF-α, and yellow aa are conserved between EGF and AREG. The N-Terminus hydrophilic aa of AREG are colored green. The arrow indicates analternative cleavage site leading to the release of another functional 78 aa AREG protein. (Adapted from [52]).

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low concentrations [11], probably because of a lower affinity for thereceptor [6], but its biological activity is similar to other ligands at higherconcentrations [12]. Moreover, AREG fully replaces the requirement ofmurine keratinocytes for EGF or TGF-α [6]. The identity of the ligand,composition of the receptor complex and specific structural determi-nants of the receptors will determine the engagement of specificsignaling pathways [13]. The strength and duration of EGFR signals aredirectly, but not only, related to the EGFR availability at the cell surface.Little is known about how different EGFR ligands could serve distinctfunctions despite their shared interactions with the same receptor. It iswell established thatEGF, but not TGF-α, triggers efficientdegradationofthe EGFR [14]. In contrast, several reports demonstrated that AREG doesnot lead to EGFR degradation [15], but targets EGFR to a recyclingpathway [16], and favors accumulation of EGFR at the cell surface [17].Similarly to EGF and TGF-α [18,19], activation of the EGFR pathway byAREG via autocrine [20], paracrine [21] and/or juxtacrine [21,22]mechanismsresults inpleiotropic cellular effects, includingproliferation,invasiveness, motility, angiogenesis and inhibition of apoptosis [23].

1.3. Role of AREG in normal tissues

Numerous endogenous and exogenous stimuli are able to induceAREG synthesis (Table 1). Among various other cytokines and growthfactors, prostaglandin-E2, interleukin-1 β, TNF-α and EGF have beenshown to strongly induce AREG mRNA expression [24,25]. In addition,treatment with various molecules, such as lysophosphatidic acid,gastrin-releasing peptide, cigarette smoke and cannabinoid, increasesAREG production in an indirect manner through a process termed“EGFR transactivation.” These G-protein coupled receptor (GPCR)agonists bind to their respective receptors and stimulate pro-AREGcleavage at the cell membrane through TACE/ADAM-17 activation [26].

AREG is expressed in various tissues but mainly in the reproductiveand urinary systems (mammary glands, uterus/ovary, placenta andprostate) as well as in the pancreas, circulatory system (vascular wall,bone marrow, blood and lymph), and respiratory and gastrointestinaltracts (lung, spleen, kidney, trachea, esophagus, stomach, intestine andcolon) [27]. AREG knockout (KO) mice are viable and fertile, suggestingthat the function of AREG can be offset by the other EGFR ligands;however, at the adult stage, the livers of mice show signs of chronic

damage in the absence of any harmful treatment [28]. AREG KO micealso suffer frommammary gland immaturity and are onlyweakly able tosuckle their offspring. Consequently, the second generation animalssuffer and usually die from postpartum malnutrition [5,29]. AREGparticipates in a wide range of physiological processes, includingmammaryglanddevelopment, blastocyst implantation, bone formation,axonal outgrowth, and keratinocyte proliferation.

AREG plays a central role in the physiological development of themammary glands during puberty and gestation [30]. AREG inducesproliferation of normal mammary glands upon estrogen and proges-terone stimulation and activates ERK and AKT intracellular pathwaysto regulate cell proliferation [31]. Overexpression of AREG by breasttissue appears to correlate with the development of cell abnormalitiesin human adult breast cells, such as hyperplasia or breast cancer [32].During the proliferative phase of the endometrium, EGFR and itsligands, AREG and TGF-α, are expressed in a cyclic fashion [33]. Inresponse to estradiol and progesterone, the cytokine, leukemia-inhibitory factor (LIF), is highly expressed, and the endothelium ofmice becomes receptive to the attaching blastocyst. LIF up-regulatesAREG, immune response gene-1 and IGF-binding protein 3, therebycontributing to implantation and blastocyst attachment [34,35]. AREGis also involved in preparing the uterus for embryo implantation [36].In early pregnancy, ovarian prolactin receptor signaling induces LIFexpression, leading to the expression of implantation-specific genes,such as AREG and HB-EGF, which are expressed in a time- and cell-dependent manner [37,38]. Luteinizing hormone also locally andtransiently regulates the expression of various members of the EGFfamily, including AREG, EREG and BTC, that contribute to oocytematuration [39].

In addition to mammary gland development [21], AREG contrib-utes to the branching and tubulogenesis processes of other tissues,including prostate [40], kidneys [41,42] and lungs [43]. Both epithelialand mesenchymal cells secrete AREG and are required for lungbranching morphogenesis. The production of AREG by mesenchymalcells also contributes to epithelial growth through its interaction withheparan sulfate proteoglycan [43]. However, in healthy branchinglivers, AREG is not expressed, but it is highly induced in chronic oracute liver injury as well as in liver cancers [44]. A recent study hasalso indicated that AREG might be important in spermatogenesis.

Fig. 3. EGFR activation and signaling. The binding of a ligand such as AREG induces homo- or heterodimerization of the receptor and autophosphorylation of the tyrosine kinasedomain. This activation allows the recruitment of adaptative proteins that recognize phosphotyrosines of activated EGFR-tyrosine kinase with either SH2 domains (green-colored) orboth SH2 and PTB domains (orange-colored). Signal propagation and amplification follows through diverse and complementary signaling pathways (Ras/Raf/MEK/ERK, PI3K/AKT/mTOR, PLCγ/PKC and CaMK, and Src/STAT 3/5 pathways). These pathways lead to the activation of various transcription factors, which promote various biological effects. (Adaptedfrom [10].)

AREG, HB-EGF, and TGF-α are regulated by the gonadotropinhormones, luteinizing hormone-releasing hormone (LH-RH) andhuman chorionic gonadotropin (hCG) and are expressed locally tosupport spermatogenesis [45].

AREG also plays a role in the development of neuronal and bonetissues [46,47]. AREG is upregulated after neurogenic differentiationof bone marrow–mesenchymal stem cells (BM–MSC) concomitantlywith other genes involved in neurite outgrowth, early neuronal celldevelopment, neuropeptide and neuronal receptor signaling/synthesis[48], indicating a crucial role for AREG in neuronal development. Inagreementwith these data, AREGacts as amitogen for adult neural stemcells and is involved in the neurogenesis of the adult brain [49,50].

Cellular growth and differentiation are partly regulated by AREG[43], which stimulates the growth of hyperplasic epithelial cells [51],carcinomatosis cells [52], fibroblasts, epithelial mammary cells [20],hepatocytes [25], hepatic stellate cells [53] and keratinocytes [54–56],cells in which the pro-AREG transmembrane precursor also plays acrucial role [57].

2. Role of AREG in cancer

Historically, this growth factor was named Amphi-regulin because itcan either induce proliferation and differentiation of fibroblasts in

culture or can inhibit the growth of normal epithelial cells andaggressive carcinoma cell lines [1]. However, 20 years of basic researchhave mainly led to the discovery of AREG as an oncogenic factor. In thepresent section,wewill focus on themultifaceted role ofAREG inhumancancer. Depending on the cellular context, AREG can promote suchdiverse effects as self-sufficiency in growth signals, tissue invasion, andevasion of apoptosis, processes all involved in tumor development andprogression [58]. AREGgeneoverexpressionhas beendemonstrated in awide variety of human cancer tissues (Table 2). However, the genetic orepigenetic alterations responsible for this overexpression remainunknown despite numerous identified stimuli (Table 1).

In vitro studies performed in tumor cell lines treated with AREG orwith specific small interfering RNAs (SiRNAs) to silence AREG geneexpression have shown that AREG plays important roles in theproliferation and survival of transformed cells [59]. AREG alsoparticipates in the maintenance of the oncogenic and metastaticproperties of these cells as well as their resistance to chemotherapy[60]. The role of AREG in cancer development and progression is alsosupported by clinical data showing that AREG may serve as aprognostic [61] and/or a predictive [62] biomarker. We discusshereafter the intrinsic tumor-promoting activities of AREG, althoughits role in tumor initiation and/or progression is inextricably linked tothat of its receptor, EGFR.

Table 2Expression of AREG in cancers.

Epithelial carcinomas AREGoverexpression

Materials andsamples

Reference

Breast mRNA H [191]mRNA, Prot H [74]

Lung Prot H [125]Prot C, H [67,192]

Colon Prot H [73]mRNA, Prot H [103]mRNA H [193]

Liver mRNA, Prot C,X,H [66]Ovary mRNA, Prot H [132]Prostate Prot H [127]Pancreas mRNA, Prot C, H [128]

mRNA C [194]Stomach mRNA H [193]Bladder Prot C [195]Biliary cancer mRNA C, H [196]Skin Prot C, X, H [197]Head and Neck mRNA C [198]

Mesenchymalmalignancies

AREG overexpression Materials andsamples

Reference

Malignant fibroushistiocytoma

Prot H [199]

Hematologicalmalignancies

AREGoverexpression

Materials andsamples

Reference

Myeloma mRNA C, H [200]

Overexpression of AREG at the mRNA or protein (Prot) level in epithelial carcinoma,mesenchymal or hematological malignancies. Samples studied were from cancer celllines (C), xenografts (X), or human biopsies/resections/primary cells (H).

Table 1Endogenous and exogenous stimuli leading to AREG synthesis.

Endogenous stimuli Studied cell line/tissue Reference

D3 vitamin Mammary carcinoma [167]IL-1α and TNF-α Cervix epith. and carcinoma [168]TGF-β Lung adenocarcinoma A549 [169]Gastrin Rat stomach [170]

Head and neck carcinoma [171]Prostaglandins Intestinal epithelial cells [172]Androgens Prostate carcinoma [173]Progesterone Murine uterus [36,174]

Hamster uterus [175]Estrogens Mammary gland [29,176]

Mammary carcinoma [173,177,178]Luteinizing hormone Ovarian follicle [39]hCG Granulosa cells [179]Parathyroid hormone Osteoblasts [46]Adenosine 3',5'-monophosphate Ovary and breast carcinoma [180]Insulin Prostate carcinoma [181]IGF-1 Keratinocytes [182]Hypoxia Intestinal epithelial cells [96]Oxidative stress Rat gastric epithelial cells [183]

Exogenous stimuli Studied cell line/tissue Reference

Phorbol esters Breast carcinoma [1,178,184]Particulate matter Epithelial cells [185,186]Dioxine Mouse ureter [187]Tobacco smoke Oral epithelial cells [188]

Lung epithelial cells [189]Pervanadate NIH-3T3 keratinocytes [190]

Cell lines and tissues are of human origin unless otherwise specified.

2.1. Self-sufficiency in growth signals

Although some tumor cells develop the ability to proliferatethrough ligand-independent signaling, some cancer cells are alsoable to generate most of their own growth factors [63]. Tumorssensitive to AREG signaling overexpress and secrete AREG as a meansof reducing their dependence on the surrounding normal microenvi-ronment, creating an autocrine stimulation loop. Whereas it was firstthought that AREG functioned as an EGF-dependent autocrine growthfactor in c-Ha-Ras- or c-Neu-transformed mammary epithelial cells[64], it has further been demonstrated that AREG mediates the EGF-independent growth of human breast cancer cells and that the AREG-overexpressing cells survive even in serum-free conditions [65].Whencancer cells were cultured in the absence of serum, AREG productionand secretion were increased in hepatocellular [66] and lung cancercell lines [67], thus supporting the hypothesis that AREG is a typicalsurvival factor for these cells and is involved in growth signal self-sufficiency. Immunospecific removal of AREG from serum-freeconditioned medium in hepatocarcinoma, lung and colon carcinomacells inhibited cell growth [66–68].

Various studies have demonstrated that AREG stimulates its owngene expression in hepatocellular carcinoma cells [66], the metastaticcolon cancer cell line, KM12 [69], and vascular smooth muscle cells[70], indicating the existence of a positive feedback loop for AREGproduction. Other studies have revealed the existence of anautoregulated feedback loop for AREG activity in breast cancer[22,71] colon cancer [68] and pancreatic cancer [72]. AREG specificimmunodetection was confined to colorectal tumor cells and was notapparent in the surrounding stroma, smooth muscle, or capillaryendothelial cells, demonstrating that this ligand is secreted in anautocrine fashion by cancer cells [73] (Fig. 4). Similar to primarybreast cancer cells, AREGwas only expressed in tumor epithelium, butnot in stromal or inflammatory cells [74], and AREG, TGF-α, and Criptoexpression were significantly increased in malignant mammaryepithelium compared with normal epithelium [75]. Blocking therelease of EGFR ligands also strongly inhibited autocrine activation ofthe EGFR and reduced both the rate and the persistence of cellmigration [76]. The AREG autocrine loop was mainly dependent on

the cleavage of AREG bymetalloproteinases in breast cancer [22] sincemetalloproteinase inhibitors reduced cell growth. Moreover, inmammary epithelial cells, soluble AREG was a more potent mitogenicfactor than membrane-anchored AREG [76]. The latter two studiesillustrate the juxtacrine activity for AREG membrane-bound pre-cursors (pro-AREG) (Fig. 4). Recently, Stoll et al. showed that theAREG carboxy-terminal fragment (CTF) was absolutely required tomaintain the autocrine growth of cultured keratinocytes, thushighlighting the signaling activity of the CTF [77].

AREG-overexpressing cells can also sustain the growth of theirneighboring cells through paracrine stimulation (Fig. 4) [21]. BothcDNAmicroarray and quantitative RT-PCR analyses have revealed thatAREG gene expression, among several other paracrine acting factors,is elevated in the extracellular environment of senescent prostatefibroblasts and modulates the proliferation and potentially theneoplastic transformation of cocultured immortalized epithelial cells[78].

Last but not least, AREG is present at detectable levels in the bloodand is therefore considered a systemic (endocrine) growth factor(Fig. 4). AREG, EGF, and TGF-α serum levels have been evaluated in ahealthy population and compared with levels from head and necksquamous cell carcinoma (HNSCC) or non-small cell lung cancer(NSCLC) patients [79]. The serum AREG levels were significantlydecreased in HNSCC patients [79], and increased AREG correlatedwitha poor response to therapy in advanced NSCLC patients [62].

Thus, AREG activates signaling through its receptor in an autocrinemanner in tumor cells, via paracrine/juxtacrine signals from themicroenvironment and via endocrine signals from distant healthytissues secreting AREG into systemic circulation. AREG can thus beconsidered a multicrine signaling protein (Fig. 4).

2.2. Evading apoptosis

Resistance to apoptosis is acquired by cancer cells through variousmechanisms, such as the overexpression of antiapoptotic molecules(i.e., Bcl-2, Bcl-xL), the inactivation of proapoptotic molecules (i.e., Bax,

Fig. 4. AREG multicrine signaling. AREG is secreted as a membrane precursor (pro-AREG), which is cleaved by the matrix metalloproteinase, TACE (ADAM-17). The soluble AREG isreleased into the microenvironment in which AREG may stay in the extracellular matrix or reach the bloodstream. AREG may activate EGFR by an autocrine signaling feedback loop,both in primary tumor cancer cells and in metastatic cancer cells. Similarly, AREG may also be produced by the surrounding stromal cells and activates the EGFR from the cancer/metastatic cells by a paracrine signaling. A juxtacrine signaling is also possible when membrane pro-AREG from a cancer or a stromal cell directly activates EGFR from neighborcancer cell. Moreover, AREG may be secreted into the bloodstream by distant AREG-secreting organs. This circulating AREG will benefit the tumor through an endocrine signalingmechanism. Thus, a tumor/metastasis may produce its own AREG and/or use AREG from other cells through distinct, but not necessarily concomitant, activating strategies.

Bad), the disruption of Fas/Fas ligand signaling, and p53 inactivation.The growth factor, AREG, is a natural survival protein, and some studieshave shown AREG to be a mediator of anti-apoptotic signals. AREGadministration abrogated Fas-mediated liver injury in mice and haddirect anti-apoptotic effects in primary hepatocytes through AKT andsignal transducer and activator of transcription-3 (STAT-3) survivalpathways, as well as up-regulation of Bcl-xL expression [28]. Moreover,AREG protectedmice from lethal doses of Fas agonist antibodies [28]. Inagreement with the latter data, the depletion of shed AREG sensitizedcells to FAS-induced apoptosis in normal hepatocytes [80]. In livercancer, AREG downregulation increased the expression of the BH3-onlyproapoptotic protein, BIM, and led to increasedTGF-β induced apoptosis[66]. AREG-overexpressing breast cancer cells also demonstratedantiapoptotic properties, characterized by a p53 null phenotype, aswell as adecreasedp21expressionandelevatedBcl-2 levels [60]. In lungadenocarcinoma cell lines, AREG cooperated with insulin-like growthfactor type 1 (IGF-1) to prevent cells from serum-starved apoptosis [67].AREG conferred apoptosis resistance through a novel PI3K/MAPK-independent but protein kinase C-dependent pathway, leading to theinactivation of the proapoptotic proteins, Bad and Bax [81]. AREG hasalso been identified as the factor mediating resistance of glioma cells tocannabinoid-induced apoptosis [82].

2.3. Limitless replicative potential

Growth signal autonomy and resistance to apoptosis allow cells togrow uncontrollably. Despite this deregulation, mammalian cellspossess a finite replicative potential and limit their multiplicationafter a given number of mitotic cycles [83] through senescencefollowing the progressive shortening of telomeres. The acquisition oftelomeremaintenance via upregulation of telomerase enzyme activityor activation of alternative mechanisms of lengthening telomeres is a

hallmark of almost all types of malignant cells. The role of AREG in theacquisition of unlimited replicative potential has been demonstratedin an aging keratinocyte model [84]. AREG withdrawal from culturemedium reduced telomerase activity, suggesting that AREG sustainstelomerase activity and promotes limitless replication in epithelialcells [84]; nevertheless this specific role in cancer cells remains to bedemonstrated.

2.4. Sustained angiogenesis

Ischemia and hypoxic conditions, which initiate a cascade of highlycoordinated cellular functions and result in the establishment of newblood vessels for oxygen and nutrient supply, are major drivers oftumor neovascularization, also termed tumor angiogenesis [85].

As the growing mass of cells increases, many tumors experience aseverely hypoxic microenvironment and secrete factors to stimulateangiogenesis [86]. Hypoxia is a characteristic feature of most solidtumors and contributes to the malignant phenotype; it is alsoassociated with resistance to therapies and poor prognosis [87].Adaptive cellular responses to hypoxia are mainly mediated byheterodimeric hypoxia-inducible transcription factors (HIFs), andmany HIF target genes are involved in cancer progression [88].Vascular endothelial growth factor (VEGF) is one of the best studiedtargets of HIF and is considered a potent proangiogenic secreted factor[89]. Interestingly, AREG transcription has been found to befunctionally regulated by HIF-2 in a breast cancer model [90]. In thelatter study, the authors showed that AREG angiogenic activity wasregulated by the tumor suppressor, prolyl-4-hydroxylase domainenzyme 2 (PHD2), a regulator the stability of HIF-α that is involved intumor progression [91] and angiogenesis [92,93]. AREG mRNA andAREG levels increased after PHD2 downregulation and decreased afterPHD2 overexpression [90]. Interestingly, PHD2 suppression increased

tumor growth and reduced survival in MCF-7 xenografted mice andin breast cancer patients [90]. The mechanism by which AREGcontributes to neovascularization remains unknown. However, intransformed human breast cells, it has been shown that AREGsilencing through antisense treatment reduces tumor vascularization[71] and the expression of the proangiogenic factor, TGFβ1 [94],suggesting that AREG modulates angiogenesis in these cells. The roleof oxygen in AREG regulation is incompletely understood. Hyperoxiaincreased AREG in a bronchopulmonary dysplasia model [95],whereas hypoxia upregulated AREG gene expression in a CRE-bindingprotein dependent mechanism in intestinal epithelial cells [96].Hypoxia increased TACEmRNA and protein expression in the placentain the absence of gestation and correlated with AREG levels [97].Hypoxia also induced the release of endothelin-1 (ET-1), whichregulates vascular remodeling and angiogenic genes, including AREG,in pulmonary vascular smooth muscle cells [98]. AREG was also foundto be a very potent mitogen for vascular smoothmuscle cells [70], andneutralizing antibodies against AREG prevented cultured endothelialcells from forming tubes, suggesting that the presence of AREG isnecessary for the formation of neovessels [90].

Increased HOXB9 expression induced the upregulation of bothAREG and several angiogenic factors, such as VEGF and bFGF [99], andthe prostaglandin E2 induced the concomitant overexpression ofAREG and VEGF-A [100], highlighting the fact that AREG andproangiogenic growth factors have common regulators.

2.5. Tissue invasion and metastasis

Tumor cells migrate from their primary site to settle and colonizedistant organs. To achieve this, cancer cells mediate phenotypictransformations that lead to drastic changes in communication withtheir microenvironment and to the activation of extracellular pro-teases [63]. The central regulatory role of AREG in branching andoutgrowth of various organs, such as mammary glands, lungs, kidneysand prostate, illustrates the ability of AREG to stimulate cellularinvasion and motility [101]. In breast cancer cells, AREG upregulatedseveral genes known to be involved in motility and invasion [22,102].The transcription factor, HOXB9, is overexpressed in breast carcino-mas and promotes tumorigenicity and lung metastases and correlateswith high tumor grade. HOXB9 induced the expression of AREG andother ErbB ligands, resulting in increased cell motility [99], whereasEGFR inhibitors reduced invasion and migration. The knockdown ofpro-AREG expression by SiRNAs inhibited the GPCR-induced chemo-tactic migration of SCC-9 head and neck cancer cells towardfibronectin [26]. AREG mRNA is expressed in only 2–7% of normalhuman colonic mucosa, but it is expressed in 60–70% of primary andmetastatic colorectal cancers [103]. Mammary cancerous cells alsoexpress both TGF-α and AREG and require autocrine signalingthrough the EGFR for proliferation and migration [76].

Alterations in cellular adhesion interactions are required for tumorcells to invade and metastasize, and any factor affecting these changesmay be a key component of tumor progression. E-cadherin representsthe major protein responsible for intercellular adhesion and cell-to-environment interactions, and its function is frequently lost in epithelialcancers. AREGwas found tomarkedly reduce E-cadherin expression andprocessing in keratinocytes in AREG-transgenic mice, and it stimulatedneutrophil migration in an in vitro epithelial intact barrier model [56].Modification in integrin expression is also frequently described ininvasive cancers. AREG autocrine expression and integrin activationhave been shown to be involved in the adhesion, growth anddifferentiation of colon adenocarcinoma cells [104].

Proteases are required for the degradation of the extracellularmatrix (ECM)during invasion, andAREGhas been shown to induce theexpression of matrix metalloproteases, such as matrix metalloprotei-nase-2 (MMP-2) and gelatinase (MMP-9), as well as other factorsinvolved in matrix degradation, such as urokinase (uPA), extracellular

matrix metalloproteinase inducer (EMMPRIN), and plasminogenactivator inhibitor-1 (PAI-1) [101], in head and neck cancer cells[105], breast cancer [106,107] and malignant mesothelioma cell lines[108].

Tumor cells progress from a non-invasive to a malignant phenotypevia morphological changes, referred to as the epithelial–mesenchymaltransition (EMT), which is required for metastatic dissemination ofprimary-site cancer cells [109]. During EMT, destabilization of theepithelium increases cell motility, indicating that EMT promotesinvasion andmetastasis. In a transgenic mouse model of c-RAF inducedEMT, AREG was shown to be significantly upregulated in lungadenocarcinoma compared to precancerous dysplasia [110]. MiR-448suppressionwas found to be involved in chemotherapy induced EMTbyincreasing the levels of AREG, which initiated an EGFR signalingpathway in a complex feedback mechanism [111]. Yes-associatedprotein (YAP) is a pivotal effector of the Hippo pathway and atranscriptional co-activator that is amplified in mouse and humancancers to promote EMT and malignant transformation. Surprisingly,AREG is a transcriptional target of YAP and contributes to YAP-mediatedcell proliferation and migration, but this process does not occur in EMTin breast epithelial cells [112]. The involvement of AREG in invasive andmigratory phenotypes and in the subsequent EMT suggests that AREGmay be associated with the metastatic process. AREG promotes theability of transformed cells to survive and grow in anchorage-independent conditions [66,113,114], which is also a typical feature ofmetastatic cells. The AREG gene was upregulated and associated withlymphogenous metastases in rat pancreatic cell lines [115], and AREGwas found to belong to a 10-gene signature predicting liver metastasisfor colorectal cancer patients [116]. In addition, AREG serum levelswereassociated with vascular invasion in colorectal cancer, and an AREG-positive status significantly correlated with depth of tumor invasion,distant metastases, and nerve invasion [117]. Another study hasrevealed that AREG was also more highly expressed in NSCLC brainmetastases compared with the corresponding primary tumor [118].Interestingly, AREG expression in primary lesions of colorectal cancersignificantly correlates with liver metastasis [119]. Thus, the metastaticsite does not need to secrete AREG for being an attractive site for themigrating cancer cells, since AREG is hardly detectable in the healthyliver [53]. Consequently, many in vitro evidence indicate that AREG isinvolved in the acquisition of metastatic properties. Nevertheless, AREGspecific role in the complex microenvironment of metastatic sitesremains to be further characterized in more in vivo studies.

2.6. Resistance to cancer treatments

In addition to its participation in the five hallmarks of cancerdescribed above, AREG is also involved in resistance to cancertreatments. Several reports have suggested that AREG is involved inmechanisms leading to radioresistance. AREG was found to beoverexpressed in radiation-resistant pancreatic cancer cells, andboth cDNA microarray and quantitative RT-PCR revealed that AREGwas expressed approximately 2.5 fold higher once a cell line becameradioresistant [120]. In addition, two studies have demonstrated thatK-RAS mutated tumor cells develop an increased ability for autocrineAREG secretion, which activates the EGFR and downstream PI3K-AKTsurvival pathway, leading to radioresistance [121,122].

AREG is also involved in resistance to various chemotherapeuticagents. AREG was associated with cisplatin resistance in a panel of 12breast cancer cell lines [60] and a HepG2 hepatoma cell line [123] butnot in lung cancer cell lines [60]. Cisplatin-resistant MCF-7 breastcancer cells selectively upregulated AREG mRNA in a microarray andan RT-PCR assay [60]. The subsequent knockdown of AREG by SiRNAsresulted in a complete reversion of the resistant phenotype [60].

AREG has been found to be highly expressed by hormone therapy-resistant breast cancer cells [124]. Exemestane is an aromataseinhibitor that prevents estrogen synthesis from androgens and is

typically indicated for the treatment of postmenopausal womensuffering from estrogen receptor positive breast cancers. It has beenshown that exemestane induces AREG expression, which subsequent-ly activates EGFR and MAPK to promote cell proliferation inexemestane-resistant cells [124]. Recently, AREG has also beenfound to be involved in resistance to anti-ErbB targeted therapies,which will be described in more detail in Section 3.2.2.

3. AREG as a cancer biomarker

Both prognostic and predictive markers provide information onthe likely future behavior of a tumor. Prognostic factors provideinformation on the outcome of the disease, whereas predictive factorsare used to prospectively select responsiveness or resistance to aspecific treatment. AREG can have both prognostic and predictiveability depending on the treatment and the cancer type.

3.1. Prognostic marker

The prognostic role of AREG in lung cancer has been addressed inan extensive immunohistochemical analysis of the expression ofseveral ErbB receptors and ligands on resected NSCLC samples. AREGoverexpression was shown to be independently associated with areduced overall survival in amultivariate analysis of 195 patients withstages I–III NSCLC [125]. AREG was also more highly expressed inaggressive forms of lung tumors compared to dysplasia [110]. Morerecently, a retrospective investigation has revealed that elevatedAREG serum levels correlate with poor prognostic outcome in NSCLCpatients, as high AREG levels correlated significantly with poorerperformance status and anemia as well as with poorer overall survivaland progression-free survival [61]. Both AREG and TGF-α correlatedwith a poorer overall survival in an unselected population of NSCLCpatients in a univariate analysis [62]. AREG immunoreactivity wasobserved in 71% of neoplastic breast cancer cases but never in normalbreast tissues [126]. Furthermore, 85% of breast tumor tissues positivefor AREG staining relapsed in contrast to the 14% of AREG negativetumors [126], again suggesting that AREG serves as an indicator ofpoor outcome in breast cancer cases where the prognostic value ofAREG is associated with estrogen receptor (ER-α) status [59].

An immunohistochemical study of 93 radical prostatectomies hasrevealed that AREG expression increases progressively from benignepithelium to prostate intraepithelial neoplasia and adenocarcinoma[127], suggesting that increased expression of AREGmay contribute tothe development of prostate cancer. Moreover, cytoplasmic AREGwasassociated with a more advanced clinical stage in prostate cancers[128]. AREG and cripto expression may discriminate normal frommalignant colonic epithelium and may provide a selective growthadvantage for colorectal carcinomas [103]. Similarly, AREG and EREGwere not expressed in the normal colonic mucosa but were clearlydetectable in adenomas and carcinomas [129]. High serum and tissuelevels of AREG were predictors of poor prognosis [117] and wererelated to disease-free survival and hepatic metastasis-free survivalfor patients with colorectal carcinoma [119]. Expression of AREG andother EGFR-ligands was associated with a reduced life span forbladder cancer patients [130] and with enhanced tumor aggressive-ness and shorter survival periods following tumor resection inpancreatic ductal adenocarcinoma [131]. Surprisingly, in humanovarian carcinomas, AREG expression was significantly associatedwith both low-grade carcinoma and low proliferative activity [132].

3.2. Predictive marker

For any specific type of cancer, not all patients will respond to aparticular treatment, but nearly all are likely to suffer from adverseeffects. We focus here on the predictive role of AREG in determiningsensitivity/resistance to ErbB targeted therapies.

It has been shown that ErbB receptors are frequently over-expressed in various human malignancies [133], and their activationand/or mutations often correlate with poor prognosis. Accordingly,these receptors have been intensively studied, and many ErbBtargeting agents have been developed and have progressed to clinicalapplications [134], including EGFR tyrosine kinase inhibitors (EGFR-TKI, e.g., erlotinib and gefitinib), anti-EGFR monoclonal antibodies(e.g., cetuximab) and anti-ErbB2/HER2 antibodies (e.g., trastuzumab).AREG can behave as a resistance or sensitivity marker to anti-ErbBtreatment depending both on the drug and the molecular character-istics of the cancer.

3.2.1. AREG predicts sensitivity to anti-ErbB therapiesVarious independent studies have reported that elevated AREG

mRNA levels are predictors of the cetuximab response in colorectalcancer, whereas the mRNA expression levels of EGFR and its otherknown ligands EGF, TGF-α, BTC, and HB–EGF showed no correlationwith disease control under cetuximab. High AREG and EREG mRNAlevels strongly correlatedwith disease control, whereas low AREG andEREG mRNA levels were associated with unresponsiveness tocetuximab monotherapy in metastatic patients [135]. Interestingly,AREG and EREG colocalize on chromosome 4q13.3. High transcrip-tional levels of AREG and EREG are highly correlated with antitumoractivity, but serum protein levels are not, suggesting a post-transcriptional regulation of these genes. These results have beenconfirmed in metastatic colorectal cancer patients treated withcetuximab in combination with irinotecan [136] and in colorectalcancer patients harboring the K-RASwild-type genotype [137–139]. Astudy of 79 different patients derived tumor xenografts from variouslocalizations also found AREG and EREG to be predictors of cetuximabsensitivity in the majority of tumors, especially for lung, head andneck, and colon cancers, in the absence of negative predictors forsensitivity, such as mutations in KRAS or MET [140]. AREG wassecreted predominantly in gefitinib- and cetuximab-sensitive headand neck or NSCLC cell lines [141,142] and in mucoepidermoidcarcinoma cell lines [143]. Moreover, the immunohistochemicalanalysis of 24 NSCLC tumors from different histological subtypesexpressing wild-type EGFR revealed that patients with stable diseaseunder gefitinib or erlotinib treatment had high AREG expressionlocally in tumors, whereas patients with low AREG expression intumors had progressive disease [142]. Unfortunately, AREG serumlevels were not evaluated in the study.

Progression-free survival and overall survival were significantlybetter in AREG-positive NSCLC patients expressing the wild-typeEGFR and treated with gefitinib or erlotinib, suggesting that patientswith wild-type EGFR lung tumors and an AREG-positive status couldbenefit from EGFR-TKI treatment [144]. Finally, high AREG serumlevels were associated with disease-specific survival in EGFR-TKItreated patients with advanced NSCLC compared to control patientswithout EGFR-TKI treatment [145].

3.2.2. AREG predicts resistance to anti-ErbB therapiesResistance to treatment is a major barrier in the treatment of

cancer. In gastric and breast cancer cell lines, the EGFR ligands,including AREG, correlated with a decreased activity of specific EGFRand HER2 inhibitors [146–148]. A low-scale proteomic approach hasallowed the identification of AREG as a potential protein involved intrastuzumab resistance. The trastuzumab refractory cells were foundto secrete higher levels of several growth factors, including AREG,compared to trastuzumab-sensitive cells [147]. In NSCLC, the role ofAREG in resistance to EGFR-targeted treatment is a complex issue. Anincrease in the level of expression of AREG and other EGF-relatedgrowth factors correlated with lung cancer resistance to cetuximabtreatment [149]. AREG was overexpressed in NSCLC patients thatwere unresponsive to gefitinib treatment but undetectable ingefitinib-sensitive patients in a study that analyzed differences in

gene expression between gefitinib-responders and non-responders inlung tumor biopsies [150]. These data were also confirmed by RT-PCR,immunohistochemistry and ELISA for protein levels [150]. Thus, AREGwas suggested to be a predictive marker for resistance to gefitinib[151]. Elevated AREG serum levels have also been correlated withresistance to gefitinib in advanced NSCLC patients [62], suggesting apotential role for AREG in such resistance. Our previous work showedthat AREG-overexpressing H358 lung adenocarcinoma cells wereresistant to gefitinib-induced apoptosis and that inhibition of AREGexpression by SiRNAs restored gefitinib sensitivity [152]. AREGinduced the resistance to gefitinib treatment through the sequestra-tion of the proapoptotic protein, Bax, by Ku70 in an acetylation-dependent mechanism [153]. Moreover, we recently showed thatAREG overexpression in EGFR-TKI resistance may be strongly relatedto the mucinous lung adenocarcinoma subtype [154].

4. AREG as a therapeutic target

Due to its diverse functions in oncogenesis, AREG representsa promising target for cancer treatment, especially for AREG-overexpressing tumors. The belief that ligand targeting is less effectivethan receptor targeting in the EGF signaling network has delayed thedevelopment of suchmedicines, although increasing evidence suggeststhat this is not true [155]. Many strategies have been developed todisrupt AREG-mediated oncogenic pathways. The first option is toneutralize soluble AREG with antibodies. AREG-neutralizing antibodieshave been shown to attenuate the growth of hepatocellular carcinomacells [66] and abolish the anti-apoptotic activity of AREG in lungadenocarcinoma cells [67]. AREG also contains a heparin-bindingdomain, and addition of heparin has been demonstrated to neutralizeAREG and inhibit both autocrine growth of human keratinocytes [156]and proliferation of hepatocarcinoma cell lines [66]. Another strategy isto inhibit the shedding of AREG by using TACE inhibitors or anti-TACESiRNAs. This approach reverted the malignant phenotype in a breastcancer cell line by preventing themobilization of TGF-α and AREG [157]and sensitized colorectal cancer cells to chemotherapy [158]. Extensiveefforts in the downregulation of AREG have been directed toward thedesign of antisense oligonucleotides and SiRNAs. Normanno et al. werethe first to inhibit AREG activity through antisense oligodeoxynucleo-tides against AREG [114]. AREG antisense oligonucleotides inhibited thegrowth of human colon carcinomas [114] and pancreatic cancer cells[72] and reverted the malignant phenotype of highly tumorigenicimmortalized breast cells in vitro and in vivo[71]. More recently, AREGSiRNAs prevented EGFR activation, leading to reduced growth andaggressiveness of hepatocellular carcinoma cell lines [66]. This strategyrestored sensitivity to anti-EGFR treatments for resistant lung cancercells both in vitro and in vivo[152]. Most of the studies using anti-AREGapproaches highlight the ability of these treatments to restore tumorcell sensitivity to 5-fluorouracil (5-FU), adriamycin, mitomycin C, andcis-platinum [123,159,160], gemcitabine and paclitaxel [160], doxoru-bicin [66], anti-EGFR therapy [72,152], and histone deacetyltransferaseinhibitors [153].

5. Discussion/conclusion

AREG plays a central role in numerous physiological andpathological processes, especially in cancer progression and develop-ment and resistance to various cancer treatments, such as radio-,chemo-, and hormone-therapies, as well as new targeted treatments.

The mechanisms responsible for AREG upregulation in trans-formed cells are complex and not fully understood andmay vary fromone tumor subtype to another. In addition, it is difficult to assesswhether AREG mediates the acquisition of these various neoplasticphenotypes by itself or in cooperation with other growth factors and/or receptors. On one hand, the intrinsic role of AREG in tumorigenesishas been demonstrated in multiple types of cancer by direct and/or

indirect mechanisms without compensation by other EGF familymembers. On the other hand, even if the EGFR is the only receptor forAREG, some studies have shown that AREG is able to activate otherreceptors. Various molecules are known to crosstalk with the EGFR,such as cMET [161], integrins [162], G-protein coupled receptors[163], and the androgen receptor [164]. Moreover, direct interactionsor heterodimerization between the ErbB members have been wellcharacterized [7], as have interactions with other membrane re-ceptors, such as the PDGF receptor [165] and the IGF1 receptor [166].Thus, the AREG-induced oncogenic signaling network is dramaticallymore complex than previously thought. AREG has pleiotropic effectson cancer cells, and it may function as a stimulatory or inhibitorygrowth factor depending on the phenotype of and environmentsurrounding a given cell. Thus, we should acknowledge the possibilitythat the effects of AREG expression are not necessarily similar eitherin type or degree between different tumors, between a primary andmetastatic site or between distinct histological tumors.

Twodecades of basic and clinical investigationshave identifiedAREGas one of the few proteins to have entered the clinical oncology arenaboth as a potential therapeutic target for cancer treatment and as avaluable predictive and prognostic biomarker. The targeting of AREGexpression and/or activity as an anticancer treatment will requirefurther in vivo studies followed by relevant clinical trials using anti-AREG molecules. The clinical feasibility and relevance of such studies,although promising, remain uncertain. Further exploratory studies arealso required to assesswhetherAREGcan be considered an independentprognostic biomarker for a given tumor. Thus, AREG status should bedetermined in every patient, both in situ and in serum, because tumorsnot expressing AREG can benefit from systemic/endocrine AREGproduced at sites distant from the tumor. Moreover, these futurestudies will have to be carefully designed within homogeneous groupsof patients and in a prospective manner whenever possible.

Obviously, AREG is also a promisingmarker to identify patients likelyto benefit from various cancer treatments, but discrepant roles havebeen reported. Themost striking example is that of lungcancer, inwhichAREG can predict either sensitivity or resistance to EGFR-TKI. Theseresults were obtained from independent studies within heterogeneoushistological subgroups of patients and were often lacking the relevantmatched control groups. Moreover, different methods to detect AREGexpression locally in tumors or in systemic circulation were used,making it difficult to compare the predictive value of AREG betweenthese studies. It is worth noting that AREG predictive ability may bestrongly related to the subgroups of patients or cancer histology. AREGcould also belong to a set of markers that statistically enhance thepredictive power of each singlemarker when analyzed together, and soevaluation of AREG as a single predictive biomarker in an unselectedpopulation may not be relevant. Thus, clinicians will have to designstudies evaluating AREG within a panel of other relevant markers. Tovalidate AREG, further evaluations should be prospective and based onselected cancer patient populations that are randomly assigned totreated and non-treated groups.

Finally, AREG will definitely serve as a routine marker in oncologyonce the accurate assessment of histological subtype, geneticbackground and molecular characteristics of the tumor are carefullydefined, allowing the appropriate personalized treatment for eachcancer patient.

Acknowledgments

We thank la Ligue contre le Cancer, comités de l'Isère et du Puy deDôme for their financial support.

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The multiple roles of amphiregulin in human cancer

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