Review Epidermal growth factor receptor (EGFR) signaling in cancer Nicola Normanno a,���, Antonella De Luca a, Caterina Bianco b, Luigi Strizzi b, Mario Mancino a, Monica R. Maiello a, Adele Carotenuto a, Gianfranco De Feo a, Francesco Caponigro c, David S. Salomon b a Cell Biology and Preclinical Models Unit, INT-Fondazione Pascale, 80131 Naples, Italy b Tumor Growth Factor Section, Mammary Biology and Tumorigenesis Laboratory, NCI, NIH, Bethesda, MD, United States c Medical Oncology B Unit, INT-Fondazione Pascale, 80131 Naples, Italy Received 11 October 2005 accepted 15 October 2005 Available online 27 December 2005 Received by A.J. van Wijnen Abstract The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutations of the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterations of the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complex system in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derive from receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showing different affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors and cognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFR agents might depend on the total level of expression of ErbB receptors and ligands in tumor cells. Published by Elsevier B.V. Keywords: ErbB EGF Growth factors Signal transduction 1. Introduction The role of growth factors-driven signaling in the pathogen- esis of human cancer has been long established. Almost twenty years ago Mike Sporn and Anita Roberts (Sporn and Roberts, 1985), following the seminal observations of Joseph deLarco and George Todaro (De Larco and Todaro, 1978), elaborated the theory of autocrine secretion: cancer cells generally exhibit a reduced requirement for exogenously supplied growth factors to maintain a high rate of proliferation. This relaxation in growth factor dependency is due in part to the ability of tumor cells to produce high levels of peptide growth factors. Since this seminal observation, an enormous amount of literature has confirmed the role of growth factor driven signaling in the Gene 366 (2006) 2���16 www.elsevier.com/locate/gene Abbreviations: EGF, epidermal growth factor RTK, receptor tyrosine kinases EGFR, epidermal growth factor receptor TGF-��, transforming growth factor-�� AR, amphiregulin BTC, betacellulin HB-EGF, heparin binding-EGF EPR, epiregulin NRG, neuregulin NSCLC, non small cell lung cancer SH2, Src homology 2 PTB, phosphotyrosine binding MAPK, mitogen-activated protein kinase PI3K, phosphatidylinositol 3-kinase PLC��, phospholipase C�� STAT, signal transducer and activator of transcription GH, growth hormone GPCR, G-protein coupled receptor Prl, prolactin Jak2, Janus tyrosine kinase 2 Fz, frizzeled DN, dominant-negative K, keratin MT, metallothionein WAP, whey acidic protein MMTV, mouse mammary tumor virus GBM, glioblastoma multiforme BAC, bronchioloalveolar carcinoma ECD, extracellular domain. ��� Corresponding author. Tel./fax: +39 081 5903826. E-mail address: nicnorm@yahoo.com (N. Normanno). 0378-1119/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.gene.2005.10.018

pathogenesis of human cancer. It has been recognized that different mechanisms might contribute to amplify the signal driven by growth factors. For example, expression of a high number of receptors on the surface of tumor cells can increase their sensitivity to low concentrations of host- or tumor-derived growth factors. A direct correlation also exists between growth factors and cellular proto-oncogenes (Aaronson, 1991 Goustin et al., 1986). In fact, several proto-oncogenes code for proteins that are either growth factors, or growth factor receptors, or proteins that are involved in the intracellular signal transduction pathway for growth factors. In addition, activated cellular proto- oncogenes may also control the endogenous production and/or the response of tumor cells to peptide growth factors. More recently, the involvement of growth factors in sustaining the survival of cancer cells and in promoting tumor-induced angiogenesis has been demonstrated, suggesting that growth factors contribute to tumor progression through different mechanisms. Different families of growth factors and growth factor receptors have been shown to be involved in the autonomous growth of cancer cells. Among these, the epidermal growth factor receptor (EGFR) and the EGF-family of peptide growth factor have a central role in the pathogenesis and progression of different carcinoma types (Salomon et al., 1995 Normanno et al., 2001). The EGF ligand/receptor system is also involved in early embryonic development and in the renewal of stem cells in normal tissues such as the skin, liver and gut (Salomon et al., 1990 Campbell and Bork, 1993). However, it is important to emphasize that the EGFR belongs to a family of receptors that encompasses three additional proteins, ErbB-2, ErbB-3 and ErbB-4. These proteins and the growth factors of the EGF- family form an integrated system in which a signal that hits an individual receptor type is often transmitted to other receptors of the same family. This mechanism leads to amplification and diversification of the initial signal, a phenomenon that is important for cell transformation, as we will discuss later. Therefore, the role of EGFR signaling cannot be discussed without taking in account the complex interactions existing within the ErbB family of receptors and growth factors. Such interactions might also be important to develop more efficient therapeutic approaches aimed to block EGFR signaling in cancer patients. Several different review articles have been published on the role of EGFR in the pathogenesis of human carcinoma. In the present article, we will describe the role of EGFR signaling in cancer with a special focus on the cooperation between different ErbB receptors in cancer pathogenesis and progression. Furthermore, we will revise the most recent knowledge on the mechanisms involved in EGFR activation in different types of cancer, and their relevance to novel therapeutic approaches. 2. The EGFR family of receptors and cognate ligands: structure and functional organization 2.1. The ErbB receptors and their cognate ligands The ErbB family of receptor tyrosine kinases (RTK) comprises four distinct receptors: the EGFR (also known as ErbB-1/HER1), ErbB-2 (neu, HER2), ErbB-3 (HER3) and ErbB-4 (HER4) (Ferguson et al., 2003 Yarden, 2001). All proteins of this family have an extracellular ligand-binding domain, a single hydrophobic transmembrane domain and a Fig. 1. Mechanisms of action of ErbB receptors in tumor cells. ErbB receptors are activate by binding to specific ligands that are produced by either tumor cells or by surrounding stromal cells. Binding of ligands to the extracellular domain of ErbB receptors results in receptor dimerization, tyrosine kinase activation and trans- phosphorylation (P). The activated ErbB receptors are able to interact with different signaling molecules that transmit the signal in the cell. 3 N. Normanno et al. / Gene 366 (2006) 2���16

cytoplasmic tyrosine kinase-containing domain (Olayioye et al., 2000). The intracellular tyrosine kinase domain of ErbB receptors is highly conserved although the kinase domain of ErbB-3 contains substitutions of critical amino acids and therefore lacks kinase activity (Guy et al., 1994). In contrast, the extracellular domains are less conserved among the four receptors, suggesting that they have different specificity in ligand binding (Olayioye et al., 2000 Yarden, 2001 Yarden and Sliwkowski, 2001). ErbB receptors are activated by binding to growth factors of the EGF-family that are produced by the same cells that express ErbB receptors (autocrine secretion) or by surrounding cells (paracrine secretion) (Fig. 1) (Olayioye et al., 2000 Yarden and Sliwkowski, 2001). Proteins that belong to this family are characterized by the presence of an EGF-like domain composed of three disulfide-bonded intramolecular groups, which confers binding specificity, and additional structural motif such as immunoglobulin-like domains, hepa- rin-binding sites and glycosylation sites. With respect to ErbB- receptor binding, EGF-related growth factors can be divided into three groups (Table 1) (Normanno et al., 2003a Yarden and Sliwkowski, 2001). The first group includes EGF, transforming growth factor �� (TGF-��) and amphiregulin (AR) which bind specifically to the EGFR. The second group includes betacellu- lin (BTC), heparin-binding growth factor (HB-EGF) and epiregulin (EPR), which show dual specificity by binding both EGFR and ErbB-4. The third group is composed of the neuregulins (NRGs) and can be divided in two subgroups based upon their capacity to bind ErbB-3 and ErbB-4 (NRG-1 and NRG-2) or only ErbB-4 (NRG-3 and NRG-4) (Carraway et al., 1997 Chang et al., 1997 Harari et al., 1999 Zhang et al., 1997). None of the EGF family of peptides binds ErbB-2. 2.2. Receptor activation Binding of ligands to the extracellular domain of ErbB receptors induces the formation of receptor homo- or hetero- dimers, and subsequent activation of the intrinsic tyrosine kinase domain (Fig. 1) (Olayioye et al., 2000). All possible homo-and heterodimeric receptor complexes between members of the ErbB family have been identified in different systems (Olayioye et al., 2000 Gullick, 2001 Schlessinger, 2000). Receptor activation leads to phosphorylation of specific tyrosine residues within the cytoplasmic tail (Fig. 1). These phosphorylated residues serve as docking sites for proteins containing Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains, the recruitment of which leads to activation of intracellular signaling pathways. Studies on the crystal structures of EGFR, ErbB-2 and ErbB-3���s extracellular domains have led to new insights in the process of ligand-induced receptor dimerization (Cho and Leahy, 2002 Cho et al., 2003 Garrett et al., 2003 Garrett et al., 2002 Ogiso et al., 2002). The extracellular domain of each ErbB receptor consists of four subdomains (I���IV). Subdomains I and III (also called L1 and L2) have a beta helical fold and are important for ligand binding. Moreover, direct receptor���receptor interaction is promoted by a beta hairpin (also termed dimerization loop) in subdomain II. In the crystal structure of the extracellular domain of EGFR bound to EGF, the dimerization loop protrudes from EGFR and mediates interaction with another EGFR molecule leading to the formation of a dimer composed of two 1:1 receptor/ligand complexes (Garrett et al., 2002 Ogiso et al., 2002). In contrast, the structure of inactive EGFR or ErbB-3 is characterized by intramolecular interactions between domains II and IV (Cho and Leahy, 2002 Ferguson et al., 2003). The structure of ErbB-2 extracellular region differs significantly from that of EGFR and ErbB-3. In the absence of a ligand, ErbB-2 has a conformation that resembles the ligand-activated state with a protruding dimerization loop (Cho et al., 2003 Garrett et al., 2003). In this conformation, domains L1 and L2 are very close and this interaction makes ligand binding impossible, explaining why ErbB-2 has no ligand (Garrett et al., 2003). Furthermore, these findings explain why ErbB-2 has enhanced capacity of heterodimerization and why it is the preferred dimerization partner for the other activated ErbB receptors (Graus-Porta et al., 1997 Tzahar et al., 1996). Among all possible ErbB-2-contaning heterodimeric receptor com- plexes, the most potent signaling module in terms of cell proliferation and in vitro transformation is represented by ErbB- 2/ErbB-3 heterodimers (Citri et al., 2003). The remarkable signaling potency of ErbB-2/ErbB-3 heterodimers derives from the fact that this dimer has the capacity to signal very potently both through the ras/raf/mitogen-activated protein kinase (MAPK) pathway for proliferation and through the phosphati- dylinositol 3-kinase (PI3K)/Akt pathway for survival (Ben- Levy et al., 1994 Citri et al., 2003 Prigent and Gullick, 1994). In addition, ErbB-2/ErbB-3 heterodimers evade downregulation mechanisms leading to prolonged signaling (Lenferink et al., 1998 Sorkin and Waters, 1993 Worthylake et al., 1999). 2.3. ErbB mediated signaling Signal transduction pathways are initiated when activated ErbB tyrosine kinase receptors recruit signaling proteins, such as Shc, Grb7, Grb2, Crk, Nck, the phospholipase C�� (PLC��), the intracellular kinases Src and PI3K, the protein tyrosine phosphatases SHP1 and SHP2 and the Cbl E3 ubiquitin ligase (Fig. 1) (Marmor and Yarden, 2004 Yaffe, 2002). All ErbB ligands and receptors induce activation of the ras/raf/MEK/ MAPK pathway through either Grb2 or Shc adaptor proteins (Carpenter, 2003 Citri et al., 2003 Jorissen et al., 2003). ErbB receptors also activate PI3K by recruitment of the p85 regulatory subunit to the activated receptors (Soltoff and Table 1 The ErbB receptors and their cognate ligands ErbB Receptors EGFR ErbB-2 ErbB-3 ErbB-4 Cognate ligands EGF None NRG 1 NRG 1 TGF-�� NRG 2 NRG 2 AR NRG 3 EP NRG 4 BTC Tomoregulin HB-EGF HB-EGF BTC EP 4 N. Normanno et al. / Gene 366 (2006) 2���16