Crosstalk Between Epidermal Growth Factor Receptor - Ingenta Connect

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Current Cancer Drug Targets, 2009, 9, 748-760

Crosstalk Between Epidermal Growth Factor Receptor- and Insulin-Like Growth Factor-1 Receptor Signaling: Implications for Cancer Therapy J. van der Veeken1, S. Oliveira1,2, R.M. Schiffelers1, G. Storm1, P.M.P. van Bergen en Henegouwen2 and R.C. Roovers*,2 1

Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Science Faculty, Utrecht University, Utrecht, The Netherlands; 2Cellular Dynamics, Science Faculty, Department of Biology, Utrecht University, Padualaan 8, 3584CH Utrecht, The Netherlands Abstract: Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) can contribute to tumor development and -progression through their effects on cell proliferation, inhibition of apoptosis, angiogenesis, anchorage-independent growth and tumor-associated inflammation. EGFR-targeting monoclonal antibodies and small molecule tyrosine kinase inhibitors are currently in clinical use for the treatment of several types of cancer. However, primary and acquired resistance to these agents often occurs and thereby limits the clinical efficacy of monospecific targeted therapy. Results from both in vitro and in vivo studies indicate that cross-talk between EGFR and IGF-1R can lead to acquired resistance against EGFR-targeted drugs. This review describes the interface between the EGFR and IGF-1R signaling networks and the implications of the extensive cross-talk between these two receptor systems for cancer therapy. EGFR and IGF-1R interact on multiple levels, either through a direct association between the two receptors, by mediating the availability of each others ligands, or indirectly, via common interaction partners such as G protein coupled receptors (GPCR) or downstream signaling molecules. This multi-layered cross-talk and its involvement in the induction of resistance to targeted therapies provide a clear rationale for dual targeting of EGFR and IGF-1R. We discuss several (potential) strategies to simultaneously inhibit EGFR and IGF-1R signaling as promising novel therapeutic approaches.

Keywords: Epidermal growth factor receptor, insulin-like growth factor-1 receptor, cross-talk, cancer therapy, dual targeting, resistance. INTRODUCTION The interest in the role of the epidermal growth factor (EGF) receptor (EGFR) in cancer development arose from the discovery that malignant transformation of cells by the oncogenic avian erythroblastosis virus resulted from the acquisition of a truncated form of the EGFR [1]. Overexpression of EGFR was later shown to induce the malignant transformation of NIH-3T3 cells [2]. Also, expression of truncated forms of the receptor (e.g. the constitutively active EGFRvIII, or de2-7 [3]) and activating mutations in the kinase domain of the protein [4] are commonly seen in different cancers. Overexpression of EGFR has been reported for a number of epithelial malignancies, including squamous cell carcinoma of the head and neck (HNSCC), non-small cell lung cancer (NSCLC), breast cancer, and ovarian cancer [5, 6]. Another activating mechanism that frequently contributes to aberrant EGFR signaling in cancer is the coexpression of EGFR and its ligand transforming growth factor- (TGF- ), creating an autocrine activation loop. Activation of EGFR can contribute to all aspects of tumor development, including cell proliferation, -survival, metastasis, angiogenesis, and tumor-associated inflammation. Similarly, activation of the insulin-like growth factor-1 receptor (IGF1R) pathway can contribute to tumor development by stimulating signaling cascades comparable to those that mediate the (patho-) physiological effects of EGFR. Both data from *Address correspondence to this author at the Cellular Dynamics, Science Faculty, Department of Biology, Utrecht University, Padualaan 8, 3584CH Utrecht, The Netherlands; Tel: +31-30-2539328; Fax: +31-30-2513655; E-mail: [email protected] 1568-0096/09 $55.00+.00

epidemiological studies and in vivo carcinogenesis models have linked increased levels of circulating IGF-1 with a higher risk of several types of cancer, including prostate-, colorectal-, lung-, and premenopausal breast cancer [7, 8]. Expression of IGF-1R is regulated by p53, one of the most commonly mutated genes in human cancer [9]. IGF-1R upregulation has been shown to convey invasive and metastatic potential in a transgenic mouse model, and has been demonstrated in a variety of primary human cancers [10-14]. Interestingly, expression of an intact IGF-1R is a prerequisite for the malignant transformation of cells by many cellular and viral oncogenes [15-17]. In fact, a functional copy of the IGF-1R gene is required for EGF-induced growth and transformation of EGFR overexpressing mouse embryonic fibroblasts [18]. Moreover, there is an increasing body of evidence that suggests that activation of IGF-1R signaling mediates resistance to EGFR targeted therapies. This review will discuss the mechanisms that underlie the interactions between EGFR and IGF-1R and their collaborative contribution to the malignant phenotype. The implications of this receptor cross-talk for mono- and dual-specific receptor targeting anti-cancer strategies are reviewed. Finally, we discuss the possibilities for the development of novel dual targeting therapeutics, which we are currently exploring experimentally. To gather relevant literature on EGFR, IGF-1R, the signaling pathways they activate and on receptor cross-talk, we searched the Scopus and Pubmed online databases for research manuscripts and review articles that were published in high impact journals and/or were highly cited over the years, as well as recent publications on these topics. We selected publications that describe novel mechanisms of receptor © 2009 Bentham Science Publishers Ltd.

Crosstalk Between Epidermal Growth Factor Receptor

cross talk, as well as studies that show a clear anti-tumor effect using innovative therapeutic approaches aimed at inhibiting multiple signaling pathways simultaneously. EGFR ACTIVATION The EGFR is a class 1 receptor tyrosine kinase (RTK) belonging to the ErbB family of receptors, which comprises ErbB-1 (EGFR/HER1), ErbB-2 (HER2), ErbB-3 (HER3), and ErbB-4 (HER4). EGFR integrates extracellular signals from a variety of ligands, including epidermal growth factor (EGF), transforming growth factor- (TGF-), amphiregulin (AR), betacellulin (BTC), heparin binding-EGF (HB-EGF), epiregulin (EPR), and the most recently identified ligand epigen, to yield diverse intracellular responses [19, 20]. EGFR ligands are produced as membrane-anchored precursors, ectodomain shedding of which results in the release of soluble growth factors. Cleavage of EGFR pro-ligands is mediated by the proteolytic activity of the disintegrin and metalloprotease (ADAM) protein family [21, 22]. ADAM 10 is thought to be the principal releasing enzyme for EGF and BTC, while shedding of EPR, TGF-, AR, HB-EGF, and epigen precursors is thought to be mediated by ADAM 17 [23, 24]. EGFR ligands bind to the N-terminal extracellular portion of the receptor (ectodomain), which can be further divided into four sub-domains: ligand binding domain 1 (L1 or domain I), cysteine rich domain 1 (CR1 or domain II), ligand binding domain 2 (L2 or domain III), and cysteine rich domain 2 (CR2 or domain IV). The transmembrane (TM) domain links the ectodomain to the intracellular portion of the receptor, the latter containing the juxtamembrane domain (JM, implicated in receptor sorting and trafficking), the catalytic kinase domain, and the C-terminal domain (containing the auto-phosphorylation sites involved in signal transduction, endocytosis, and receptor degradation) [25]. The reports describing the crystal structures of human EGF or TGF in a 2:2 complex with the EGFR extracellular domain have provided valuable insights in the process of ligand binding and subsequent receptor dimerization [26, 27]. According to the current model of EGFR functioning, EGFR monomers on the cell surface most likely exist in both a tethered, autoinhibited conformation stabilized by an intra-molecular interaction between domains II and IV, as well as in (possibly multiple) untethered, extended conformations [28]. In the ligand-bound EGFR dimer, a dimerization arm protruding from the back of domain II, exposed in the ligand-bound conformation, interacts with a pocket at the base of domain II on its partner EGFR, thus stabilizing the receptor-ligand dimer in a back-to-back orientation with ligands distant from each other [26, 27]. Both untethered monomers and predimerized receptors occur in the absence of ligand; however, ligand binding is necessary for activation of the kinase domain and subsequent initiation of receptor signaling [29, 30]. Ligand binding to the extracellular portion of a preformed receptor dimer was shown to induce a rotation of the JM and intracellular regions, positioning the kinase domains in close proximity to one another [31]. A recent study by Zhang et al. has begun to elucidate the structural intricacies underlying activation of the kinase domain of EGFR. It is proposed that the kinase domain is auto-inhibited and can be allosterically

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activated through the formation of an asymmetric kinase dimer in which the C-lobe of one kinase domain contacts the N-lobe of its partnering kinase, resulting in its activation [32]. Activation of the kinase domain allows trans(auto)phosphorylation of several tyrosine residues in the Cterminal cytoplasmic tail of the receptor [33]. Additional phosphorylations can occur through the activity of intracellular non-receptor tyrosine kinases such as c-Src, Janus kinase2 (JAK2), and Abelson leukemia virus (Abl) [34-38]; kinases which can, interestingly, also act downstream of activated EGFR. Receptor phosphorylation mediates the recruitment of a plethora of intracellular signal transducers and adaptors, establishing a scaffold for integrative signaling and receptor cross-talk. Recent quantitative proteomics studies have aimed to provide a comprehensive analysis of EGFR phosphorylation sites and interaction partners [39, 40]. EGFR SIGNALING The major signal transduction pathway through which EGFR signals is the Ras- mitogen-activated protein kinase (MAPK) mitogenic signaling cascade. Activation of this signaling pathway is initiated by the recruitment of growth factor receptor bound protein 2 (Grb2), which can occur either through a direct interaction between the Src homology 2 (SH2) domain of Grb2 and phosphotyrosines on the EGFR, or through an indirect interaction mediated by EGFR associated tyrosine-phosphorylated Shc [41-43]. Relocation of Grb2 to the EGFR at the plasma membrane induces the activation of the membrane associated small GTPase Ras by the Grb2-bound guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS). Activated Ras subsequently activates the serine/threonine kinase Raf (MAPKKK), which in turn phosphorylates MEK1/2 (MAPK/extracellular regulated kinase (ERK) kinase). MEK1/2 phosphorylates ERK1/2 (MAPK), which activates a variety of transcription factors including c-Myc, NF-IL6, Tal-1, Ets-2, and Elk that promote the transcription of anti-apoptotic Bcl-2 family members and inhibitor of apoptosis proteins (IAPs) [25, 44]. Moreover, ERK1/2 activates cytosolic phospholipase A2 (cPLA2), p90rsk, and MSK-1, which promote cell proliferation, differentiation, and survival through their effects on a variety of signaling molecules and transcription factors [45, 46]. MAPK signaling is also critically involved in tumor cell-host environment interactions, contributing to inhibition of anoikis and to tumor-associated inflammation and angiogenesis [47-49]. Importantly, MAPK activity can have widespread effects on cytoskeletal dynamics, epithelial to mesenchymal transition (EMT), and cell migration through crosstalk with other major signaling pathways, including NF-B, TGF-, Rac-1, and Notch signaling [50-53]. Interestingly, the MAPK pathway incorporates multiple positive and negative feedback loops. MAPK activation promotes transcription of several EGFR ligands, including TGF- and HBEGF, contributing to autocrine activation of EGFR [54]. Conversely, inherent negative control of MAPK signaling is established through transcriptional upregulation of dual specificity MAPK phosphatases (MKPs) following MAPK activation [55]. Phosphorylation and consequent deactivation of SOS, both by MAPK itself, as well as by p90rsk, is an example of one of many additional mechanisms that provide negative feedback on MAPK signaling [56, 57].

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Another essential signaling cascade through which EGFR (as well as many other RTKs) and G-protein coupled receptors (GPCRs) exert their physiological effects is the phosphatidylinositol-3 kinase (PtdIns3-K)/Akt pathway [58, 59]. Growth factor receptors signal primarily through the Ia subclass of PtdIns3-Ks, although there is also evidence that suggests signaling through the much less studied class II PtdIns3-Ks [60]. EGFR can activate the PtdIns3-K pathway via Grb2-dependent recruitment of the docking protein Gab1. The latter protein binds the p85 regulatory subunit of PtdIns3-K and recruits it to the activated receptor which results in the activation of the p110 catalytic subunit [61]. However, EGFR-dependent PtdIns3-K activation occurs primarily through the formation of EGFR/ErbB3 heterodimers, ErbB3 being the main p85 binding partner (containing six YxxM PtdIns-3K binding motifs) [62]. Activation of the catalytic p110 subunit of PtdIns3-K results in phosphorylation of the hydroxyl group on the 3’ position of the inositol ring of phosphatidylinositol-4,5-biphosphate (PtdIns(4,5)P2) to yield PtdIns(3,4,5)P3. A subset of signaling molecules, including Akt and its activating enzyme phosphoinositidedependent kinase 1 (PDK-1), are brought in close proximity to one another in specific plasma membrane regions through their pleckstrin homology (PH) domain-mediated recruitment to PtdIns(3,4,5)P3 [63, 64]. However, Akt can also be activated through mechanisms independent of PDK-1, for instance by the mammalian target of rapamycin complex 2 (mTOR2) [65]. Downstream effectors of Akt include protein kinases, transcription factors and pro-apoptotic proteins that are inhibited by Akt-mediated phosphorylation. Akt phosphorylates and de-activates the constitutively active glycogen synthase kinase-3 (GSK-3), resulting in activation of signaling pathways that are normally suppressed by GSK-3 [66]. mTOR is another downstream target of Akt, which is activated upon phosphorylation. Interestingly, mTOR inhibition can promote Akt phosphorylation, indicative of a negative feedback loop mediated by mTOR [67]. Transcription factors activated by Akt include NF-B, cAMP response element binding protein (CREB), and hypoxia inducible factor 1 (HIF-1). Other transcription factors are inhibited by Akt-dependent phosphorylation, including p53 and the forkhead box (FOX) family of transcription factors [44, 68]. Akt also directly promotes survival through phosphorylation and thereby inhibition of pro-apoptotic factors, such as caspase-9 and the pro-apoptotic Bcl-2 family member BAD. Cross-talk between the MAPK and PtdIns3-K pathways can occur on multiple levels; for instance, Ras can bind and activate the p110 subunit of PtdIns3-K [69]. However, PtdIns3-K activity can also suppress the MAPK pathway through Akt dependent inhibition of Raf-1 [70]. Moreover, protein levels of phosphatase and tensin homolog (PTEN), one of the pivotal regulatory components of PtdIns3-K signaling, and thereby de-phosphorylation of PtdIns(3,4,5)3-K by PTEN can be downregulated through a Ras/MAPK dependent mechanism [71]. In addition, negative feedback of Akt signaling is mediated by members of the protein kinase C (PKC) family, such as PKC [72]. EGFR is also involved in STAT (signal transducer and activator of transcription) signaling: the receptor is capable of directly binding and activating STAT proteins, as well as promoting c-Src-dependent STAT activation [73]. Moreover,

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in addition to PtdIns3-K, EGFR signaling can promote the activation of several other phospholipid metabolizing enzymes, including phospholipase A2 (PLA2), phospholipase C- (PLC- ), and phospholipase D2 (PLD2), both through direct and indirect mechanisms [46, 74, 75]. The physiological functions of these enzymes are detailed elsewhere [7678]. Interestingly, PLD2 has recently been implicated in the Grb-2 independent membrane translocation of SOS, providing an additional mechanism for EGFR-mediated Ras activation [79]. TERMINATION OF EGFR SIGNALING The primary mechanism responsible for termination of EGFR signaling is ligand-induced receptor sequestration and subsequent downregulation (i.e. degradation) of activated receptor. Without ligand binding, EGFR at the cell surface is internalized and recycled at a basal rate. Receptor occupancy augments the receptor’s internalization rate and alters its trafficking pattern, resulting in increased receptor degradation [80]. Receptor internalization can occur through both clathrin-dependent and clathrin-independent mechanisms. Receptor dimerization can initiate clathrin-mediated endocytosis (CME) of EGFR, which requires the presence of an AP2 complex-binding double-leucine motif in the intracellular domain. Alternatively, tyrosine phosphorylation of one (Y1045) or more (Y1068 and Y1086) tyrosine residues is responsible for the stimulation of EGFR internalization via the clathrin-coated route. These sites are involved in the direct (via Y1045) and indirect (Grb2-mediated) recruitment of the E3-ligase c-Cbl to the receptor, which results in the mono- and poly-ubiquitination of the EGFR [81, 82]. Recent studies suggest that the concentration of extra-cellular ligand controls the level of receptor ubiquitination and that this is what commits the receptor to either clathrin-mediated endocytosis or non-clathrin endocytosis (NCE) [83]. At low ligand concentrations, the AP2 cargo adaptor directs the receptor to clathrin coated pits. At high ligand concentrations, EGFR is internalized through both CME and NCE, the latter of which is mediated by receptor ubiquitination and subsequent recruitment of epsin and EGFR pathway substrate 15/related (Eps15/R) via their ubiquitin interacting motifs (UIMs). Ubiquitination of the endocytic machinery is required for CME; however, receptor ubiquitination is dispensable. Regardless of the internalization route of the EGFR, internalized receptor ends up in the early endosome, where sorting of ubiquitinated and non-ubiquitinated receptor takes place by the different endosomal sorting complex required for transport (ESCRT) complexes which include UIMdomain containing proteins as Eps15/R, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) and signal transducing adaptor molecule (STAM). Ubiquitinated receptor finally ends up in lysosomes while non-ubiquitinated EGFR is recycled back to the surface. Intracellular receptor trafficking following endocytosis is a complex and multifaceted process, an in-depth analysis of which is beyond the scope of this review. However, one aspect that should be noted here is that receptor sequestration not only attenuates signaling, but also contributes to it. Because ligand-induced EGFR internalization is much faster than receptor degradation, actively signaling receptor-ligand


complexes may accumulate in pre-degradative intracellular compartments. Cell surface- or endosomal receptor signaling may differ qualitatively by differentially activating certain pathways depending on the relative availability of EGFR adaptors or substrates [84]. IGF-1R ACTIVATION AND SIGNALING The central players in IGF-1R signaling are the receptor itself, its ligands IGF-1 and IGF-2, and the IGF binding proteins (IGFBPs). The IGF-2R competes with the IGF-1R for IGF-2 binding but lacks a kinase domain; therefore, it has no signaling capacity and can thus be regarded as a ‘decoy receptor’ or ‘sink’ for IGF-2. Indeed, overexpression of IGF2R is associated with suppression of tumor growth [85]. The IGF-1R is a type 2 RTK belonging to the insulin receptor family: one IGF-1R comprises two disulfide-linked heterodimers, each made up of an extracellular -chain and a transmembrane -chain. Additionally, one IGF-1R heterodimer can couple to an insulin receptor heterodimer to form a hybrid receptor, which can bind both IGF-1 and IGF2. However, both IGF-1R and hybrid receptors have relatively low affinity for insulin [86]. IGFBPs have a dual role in IGF-1R signaling. IGFBPs compete with receptors for IGF-1 binding and are therefore generally regarded as inhibitors of IGF-1R signaling. However, there seems to be a duality in their functioning: IGFBPs prolong ligand circulation time and conversely may, under some conditions, promote IGF-1R signaling [87]. Proteases in the tumor microenvironment may promote IGF-1R activation by releasing ligands from IGFBPs. Ligand binding to the receptor induces conformational changes that lead to the trans(auto)-phosphorylation of three tyrosine residues (Y1131, Y1135, Y1136) in the activation loop of the -chain kinase domain [88]. These tyrosine residues are sequentially phosphorylated (Y1135, followed by Y1131, then Y1136) and enzyme activity increases with each phosphorylation [89]. In the non-phosphorylated kinase domain, the activation loop sterically shields the enzymatic cleft from ATP and peptide substrates. This autoinhibitory conformation is destabilized by phosphorylation of Y1135 and Y1131; subsequent phosphorylation of Y1136 plays a key role in the stabilization of the active conformation [90]. Allosteric activation of the kinase domain in an asymmetric dimer, as described above for EGFR, has not been reported for IGF-1R. Activation of the kinase domain results in phosphorylation of several cytoplasmic tyrosine residues in the receptor, providing docking sites for adaptors and signal transducers [91]. Recruited proteins include the insulin receptor substrates (IRS1-4), Shc, and the p85 regulatory subunit of PtdIns3-K. Phosphorylated Shc provides a docking site for Grb2 which in turn binds the Ras-GEF SOS, leading to activation of the MAPK pathway. Both the MAPK pathway, as well as the PtdIns3-K pathway can also be activated via phophorylated IRS-1, which provides docking sites for Grb2 and the PtdIns3-K regulatory subunit p85 (among others). IRS proteins can be phosphorylated on multiple residues (up to 20 for IRS1), providing a powerful and versatile platform for adaptor recruitment and signal transduction [92]. In addition to IRS molecules and Shc, activated IGF1R may recruit various other adaptors. However, the func-


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tion of these proteins, particularly in tumor development has not yet been clearly defined. These include Crk family members, GTPase activating protein (GAP), Syp/SHP2, CSK, JAK-1 and -2, SOCS-1 and -2, 14.3.3 molecules, and RACK1 [91, 93]. Most of the important cellular functions of IGF-1R are mediated by MAPK and PtdIns3-K activation, and the many signal transducing molecules and transcription factors downstream of these central kinases (as described above). However, MAPK/PtdIns3-K-independent functions of IGF-1R that may contribute to tumor development and -progression have also been reported: e.g. it was shown that IGF-1R activation can contribute to EMT through direct interactions with cell surface adhesion molecules. IGF-1R can be included in large supra-molecular membrane complexes containing E-cadherin and -catenin. IGF-II can induce the internalization of these complexes leading to the degradation of E-cadherin and the re-location of -catenin to the nucleus [94]. Both the downregulation of E-cadherin as well as the transcriptional up-regulation of -catenin target genes have been associated with EMT, which can mediate sensitivity to EGFR inhibitors [95]. Similarly, it was recently shown that -catenin can function as a scaffold for the formation of IGF1R, E-cadherin, and v-integrin-containing membrane complexes. IGF-1 induced the disruption of these complexes and relocation of v-integrin from cell-cell contacts to focal contact sites, which correlated with an increase in cell motility [96]. The mechanisms involved in internalization, intracellular trafficking and degradation of IGF-1R have been much less studied than those of EGFR. It was recently reported that ubiquitination of IGF-1R is mediated by two E3 ubiquitin ligases with distinct functions: Mdm2 and c-Cbl [97]. Which ligase mediates the ubiquitination of the receptor may depend on the concentration of ligand: low dose IGF-1 stimulation induces Mdm2-mediated receptor ubiquitination, while high dose stimulation results in c-Cbl-dependent ubiquitination. Mdm2 is recruited to the receptor via the adaptor protein -arrestin [98], which also regulates CME of IGF-1R [99]. Conversely, tyrosine phosphorylated c-Cbl and c-Cblassociated protein are recruited to lipid rafts [100]. It has therefore been hypothesized that Mdm2-mediated ubiquitination induces IGF-1R internalization via CME, while c-Cbl dependent ubiquitination induces internalization via NCE [97]. RECEPTOR CROSS-TALK Receptor cross-talk encompasses the influence/effects of one receptor and the signal transduction pathways it activates on a second receptor system. In the case of EGFR and IGF1R, the cross-talk greatly expands the EGFR signaling network by simultaneously increasing the number of ligands that signal (indirectly) through the EGFR, as well as the downstream effectors of the receptor. Receptor cross-talk does not necessarily have to involve the physical interaction between receptors of a different family, but can also occur through shared components in the signaling pathways downstream of the receptor and through regulation of the expression of the other receptor or of its ligands.


One of the most common and obvious mechanisms for EGFR cross-talk, occurring at the receptor level, is receptor heterodimerization with one of its family members [101]. Heterodimerization with ErbB-3, for instance, greatly augments signaling through the PtdIns3-K pathway, a feature attributable to the p85 binding sites on the cytoplasmic tail of ErbB-3. Heterodimerization of EGFR with its preferred binding partner ErbB-2 (which conformation is always in the dimerization-competent state [102]) reduces ligand dissociation rates and potentiates signaling through the MAPK pathway [103]. Both EGFR/ErbB-2 and EGFR/ErbB3 heterodimers dissociate more readily in early endosomes, and are more efficiently recycled back to the cell surface following ligand induced internalization than EGFR homodimers [104]. EGFR also commonly engages in physical interactions with various non-ErbB family receptors at the cell surface. These interactions cause both trans-activation of EGFR, as well as EGFR-dependent trans-activation of various other receptors. For instance, a study by Reinehr et al has shown that FAS ligand, but not EGF, can induce FAS receptor/EGFR association and subsequent FAS receptor tyrosine phosphorylation, likely mediated by EGFR kinase activity [105]. In addition, EGFR may form heterodimers with other growth factor receptors, including platelet derived growth factor receptor (PDGFR) and IGF-1R [106, 107]. Trans-activation of EGFR can be mediated by several types of other receptors and often doesn’t involve direct physical interactions between the two receptors. For instance, agonist activation of GPCRs can stimulate ADAMdependent cleavage of membrane anchored EGFR proligands (e.g. HB-EGF, TGF-, or AR) and subsequent receptor activation [108]. It was recently shown that GPCRinduced trans-activation of EGFR by lysophosphatic acid or carbechol can regulate the proliferation and motility of HNSCC cells [109, 110]. A similar mechanism for Wntinduced EGFR trans-activation by frizzled (Fz) receptors has been shown in mammary cells [111]. Growth hormone binding to its receptor can also induce EGFR phosphorylation via activation of JAK-2 [35]. Moreover, chemokine receptors CXCR1, CXCR2 and CXCR4 can trans-activate EGFR upon binding of their respective ligands CXCL8/interleukin-8 (IL8) and stromal cell derived factor 1 (SDF-1)/CXCL12 [112, 113]. Given that EGFR activation can induce expression of IL-8 [114], this suggest a possible autocrine activation loop involving EGFR, IL-8, and CXCR1/CXCR2. An entirely different mechanism of EGFR cross-talk is the receptor-mediated inhibition of the tumor necrosis factor (TNF) family of death receptors (DR), that includes FAS receptor, DR4, DR5, and TNFR [44]. EGFR signaling results in an upregulation of IAPs, inhibition of pro-apoptotic Bcl-2 family members, and a downregulation of transcription of both death receptors and their ligands through inhibition of FOX transcription factors and p53. In this indirect mechanism of receptor cross-talk, the EGFR receptor system regulates the availability of ligands and receptors of a different receptor system. Given the fact that EGFR can also participate in the Fas-induced activation of Fas receptor through direct association with the receptor [105], it illustrates how EGFR may differentially regulate death receptor signaling,


depending on the presence or absence of a specific (nonEGFR) ligand. EGFR- AND IGF-1R CROSS-TALK RATIONALE FOR DUAL TARGETING

AND

THE

In addition to EGFR/IGF-1R heterodimerization (Fig. 1a), cell surface interactions between these two receptors can occur indirectly, mediated by the activity of GPCRs. As mentioned above, activation of GPCRs can induce metalloprotease (ADAM-)-dependent shedding of EGFR proligands, leading to receptor activation. Conversely, a role for IGF-1R has been described in the trans-activation of many GPCRs [115]. For instance, in breast cancer cells, CXCR4 can be trans-activated by IGF-1, independent of its ligand CXCL12. It was shown that IGF-1R forms a complex with CXCR4, and treatment with IGF-1 induces the release of activated G protein subunits from this complex [116]. Interestingly, EGFR can again be trans-activated by CXCR4 [112]. This suggests that an indirect cross-talk between IGF1R, GPCRs, and EGFR may exist, where IGF-1R-mediated trans-activation of GPCRs induces the activity of G proteins that stimulate metalloprotease dependent release of EGFR pro-ligands, thereby activating EGFR (Fig. 1b). This hypothesis is supported by the observation that EGFR transactivation by IGF-1R can occur in a metalloprotease- and HB-EGF dependent manner [117]. This illustrates that EGFR and IGF-1R may cross-talk/communicate through both direct- and indirect interactions at the cell surface. Cross-talk between EGFR and IGF-1R not only occurs at the cell surface: the interface between the two receptor systems is further extended by interactions between shared signaling components downstream of the activated receptors. For instance, EGF can induce the expression of IRS1 and IRS2 in breast cancer cells, which can be blocked by treatment with an EGFR tyrosine kinase inhibitor (TKI) [118]. Moreover, it was recently shown that in tamoxifen-resistant breast cancer cells, EGFR can recruit and phosphorylate IRS1. Treatment with the EGFR TKI gefitinib (“Iressa”) inhibited this interaction, but promoted the association between IRS1 and IGF-1R, establishing a mechanism for resistance. Importantly, gefitinib resistance in this model could be overcome by co-administration of an IGF-1R TKI [119]. There are several studies that support a role for EGFR-IGF1R cross-talk in the development of tamoxifen resistance in breast cancer cells. Knowlden et al have described an indirect uni-directional cross-talk mechanism between EGFR and IGF-1R that occurs in tamoxifen-resistant MCF7 and T47D breast cancer cells, but not in non-resistant cells [120]. In these cell lines, IGF-II-dependent activation of IGF-1R leads to the recruitment and activation of c-Src, which subsequently induces the phosphorylation of Y845 and Y1068 on EGFR, leading to activation of the MAPK signaling pathway. Additionally, a similar linear pathway leading to EGFR activation can be initiated by ligation of the estrogen receptor- (ER) with estradiol: the estradiol-ER complex interacts with IGF-1R and induces the metalloprotease dependent shedding of HB-EGF and activation of EGFR. This pathway was reported to be activated during the development of tamoxifen resistance [121]. In tamoxifen resistant cells, ER was shown to relocate to extra-nuclear sites in a



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Fig. (1). EGFR/IGF-1R interactions at the cell surface. Receptor crosstalk at the cell surface can occur through heterodimerization of IGF1R with EGFR or ErbB2 (a), or through GPCR mediated interactions (b). IGF-1R mediated transactivation of GPCRs induces the activity of G-proteins that mediate the metalloprotease dependent release of EGFR pro-ligands.

c-Src-dependent manner, promoting the association of cytoplasmic ER with growth factors on the cell surface and facilitating receptor cross-talk [122]. These studies demonstrate how multiple c-Src mediated mechanisms can stimulate EGFR-IGF-1R cross-talk dependent cell proliferation and -survival in tamoxifen resistant breast cancer cells. Similarly, IGF-1R can mediate resistance to EGFR targeted therapy in glioblastoma cells [123]. Differential responses to EGFR TKIs in two primary glioblastoma multiforme cell lines expressing equivalent levels of EGFR were shown to be mediated by IGF-1R expression. Dual inhibition

of both receptors enhanced both spontaneous- and radiation induced-apoptosis in the EGFR TKI resistant cell line [123]. In another study it was shown that treatment of NSCLC cells with the EGFR TKI erlotinib (“Tarceva”) induced EGFR/IGF-1R heterodimerization and IGF-1R activation. Through this mechanism, erlotinib treatment induced mTOR-mediated EGFR upregulation and expression of survivin. Dual targeting and inhibition of both receptors abolished erlotinib resistance and inhibited tumor growth in vivo [107]. Moreover, acquired resistance to gefitinib in squamous cell carcinoma cells has been attributed to the loss


of IGFBPs [124]. Gefitinib resistant cells showed increased IGF-1R activity, constitutive association of IRS1 with PtdIns3-K, and markedly reduced IGFBP3 and IGFBP4 expression. Treatment with rhIGFBP3 reversed gefitinib resistance in vitro. Dual targeting of IGF-1R and EGFR prevented tumor recurrence in a xenograft model, whereas targeting of either receptor alone failed to do so [124]. Similarly, dual targeting of EGFR and IGF-1R with a bispecific antibody strongly inhibited the growth of colorectal cancer xenografts, and was more efficient than targeting either receptor alone [125]. Finally, small interfering RNA- (siRNA-) mediated silencing of both EGFR and IGF-1R was shown to induce chemo-sensitization of liver cancer cells [126]. IGF-1R signaling can also influence ErbB2 targeted therapy. Nahta et al reported that acquired resistance to the antiErbB2 antibody trastuzumab (“Herceptin”) in breast cancer cells could be mediated by heterodimerization of ErbB2 with IGF-1R. In resistant cells, phosphorylation of ErbB2 was mediated by IGF-1R and could be overcome by treatment with an IGF-1R TKI [127]. Recent evidence suggests that even non-overexpressing tumors may benefit from dual targeting of ErbB2 and IGF-1R. Chakraborty et al studied the effects of dual targeting in two human breast cancer cell lines: BT474 (ErbB2 positive and low expressing IGF-1R) and MCF7 (low expressing ErbB2 and high expressing IGF1R). ErbB2 and IGF-1R could be co-immunoprecipitated in both cell lines and inhibition of one receptor cross-inhibited the other. Growth of BT474 cells and MCF7 cells could be inhibited by targeting ErbB2 or IGF-1R respectively, but not by inhibition of the non-overexpressed receptor alone. However, dual targeting of both receptors induced apoptosis and dramatically increased treatment efficacy in both cell lines [128]. Consistent with a role for IGFBP3 in inhibiting IGF1R activation by IGF-1, recombinant human IGFBP3 (rhIGFBP3) was shown to potentiate the in vitro and in vivo antitumor activity of trastuzumab by inhibiting signaling through the PtdIns3-K /Akt and MAPK pathways [129]. Interestingly, the beneficial effects of combined therapy with curcumin and 5-fluorouracil plus oxaliplatin (FOLFOX) in colorectal cancer have been attributed to modulation of ErbB and IGF-1R signaling. Combination therapy with curcumin and FOLFOX reduced the expression and activation of EGFR, ErbB2, ErbB3, IGF-1R, Akt, and cyclooxygenase-3, while increasing the expression of IGFBP3 [130]. These studies clearly demonstrate how resistance to EGFR targeted therapies can be mediated by signaling through, and cross-talk with the IGF-1R pathway (Fig. 2), and provide a clear rationale for dual targeting of EGFR and IGF-1R. Not surprisingly, several strategies aimed at simultaneous inhibition of both receptor systems are currently under investigation. Results from several ongoing clinical trials may provide additional insights in the therapeutic potential of EGFR/IGF-1R dual targeting. The fully human anti-IGF-1R monoclonal antibody IMC-A12, for instance, is currently being evaluated in phase II clinical trials for the treatment of metastatic head and neck cancer as a single agent, as well as in combination with the anti-EGFR monoclonal antibody cetuximab (“Erbitux”) [http://clinicaltrials.gov]. Another ongoing clinical trial is evaluating IMCA12, either as a single agent or in combination with cetuximab, for the treatment of metastatic colorectal cancer pa-


tients who have experienced disease progression on EGFRtargeted therapy. Notably, the variable regions of IMC-A12 have been combined with those of the anti-EGFR antibody IMC-11F8 in a bispecific antibody, which has shown promising results in an in vivo colorectal cancer model [125]. Several other IGF-1R inhibitors are currently undergoing phase I or II clinical trials [131, 132], and it would be interesting to determine if these agents could contribute to the clinical efficacy of approved EGFR or ErbB2 targeting drugs. EGFR-IGF-1R TARGETING: FUTURE PERSPECTIVES Recent insights in mechanisms of receptor cross-talk have redefined the borders of the EGFR signaling network and provide new opportunities for cancer therapy. As discussed, the EGFR and IGF-1R pathways can communicate on multiple levels. IGF-1R signaling may contribute to the autocrine production of EGFR ligands, while EGFR signaling, in turn, may regulate the availability of IGF-1 through its effects on IGFBPs. Receptor cross-talk can also be mediated by IRS proteins, GPCRs, or simply by direct interactions between IGF-1R and EGFR or ErbB2. These interactions can mediate and cause resistance to EGFR targeting therapies in vitro, as well as in tumor xenograft models, and provide a strong rationale for simultaneous targeting and inhibition of both receptors. Future dual targeting strategies may focus on interfering with multiple layers of EGFR and IGF-1R signaling. For instance, by combined targeting of ligands (e.g. with ligand neutralizing antibodies, possibly combined with rhIGFBP3), and receptors (e.g. with antagonistic antibodies or small molecule inhibitors). We have already reported the successful use of anti-EGFR single domain antibody fragments (derived from camelid heavy chain antibodies [133], also called VHH or ‘nanobodies’ [134]) in the inhibition of tumor outgrowth [135]. These nanobodies are useful building blocks to generate multi-specific, multivalent fragments to simultaneously block receptor and ligand, or two receptors, by linking the fragments together in a single polypeptide chain [135, 136] (for review see [137]). Such dual specificity (DS) nanobodies may well circumvent resistance problems associated with monospecific targeting strategies (as has been discussed). Another approach for dual targeting is the use of dual specific TKIs. For instance, INSM-18, a dual specificity IGF-1R/ErbB2 inhibitor is being developed by the University of California at San Francisco and Insmed and is currently undergoing phase I/II clinical trials. However, since most TKIs are ATP analogs and thus bind in the active site of the respective kinase, specificity and off-target effects of these inhibitors are an important issue. Therefore the effects may be partially due to the inhibition of kinases different from the targeted RTKs. Drugs interfering with each level of signaling may be combined as a cocktail, or, preferentially, incorporated in a single new therapeutic entity. The latter may be envisaged as e.g. multi-specific antibody formats (as described above), antibody-TKI conjugates (a concept that we are also explor-



755

Fig. (2). EGFR and IGF-1R signaling through shared pathways regulates the expression of ligands, receptors and IGFBPs, and can contribute to resistance against EGFR targeted therapies.

ing with nanobodies), or as immuno-liposomes (antibody(fragment)-coupled liposomes) targeting one receptor and carrying small molecule inhibitors or siRNA molecules directed to a second receptor. We have recently developed anti-EGFR nanobody-liposomes with the remarkable ability to induce sustained downregulation of EGFR, even without a payload, resulting in the in vitro inhibition of tumor cell proliferation (Oliveira et al., manuscript submitted). Because of their inherent effects on EGFR, these nanobody-liposomes have great potential as a scaffold for the further development of combination therapies aimed at interfering with multiple levels of EGFR and IGF-1R signaling. E.g. nanobody-

liposomes targeting (and inhibiting) one receptor may be loaded with other nanobodies recognizing the intracellular kinase of the same, or a different receptor. Since the specificity of antibodies (including nanobodies) for a particular kinase is expected to be superior to that of small molecule TKIs, unwanted side-effects and toxicity are expected to be far less of a problem. Cross-talk between different receptor systems, as has been discussed for EGFR and IGF-1R in this review, can occur through a variety of mechanisms. It is expected that identifying these routes of communication and determining in which way they contribute to disease initiation and pro-



gression, will further stimulate the development of novel therapeutics and contribute to more efficient cancer therapy.

JM

= juxtamembrane

L

= ligand-binding

ACKNOWLEDGEMENTS

MAPK

= mitogen-activated protein kinase

MEK

= MAPK/ERK kinase

MKP

= MAPK phosphatase

R.C. Roovers is financially supported by STW grant nr. 10074.

mTOR2

= mammalian target of rapamycin complex 2

ABBREVIATIONS

NCE

= non-clathrin endocytosis

Ab1

= Abelson leukemia virus

NSCLC

= non small-cell lung carcinoma

ADAM

= disintegrin and metalloprotease

PDGFR

= platelet derived growth factor receptor

AR

= amphiregulin

PDK-1

= phosphoinositide dependent kinase-1

BTC

= betacellulin

PH

= pleckstrin homology

CME

= clathrin-mediated endocytosis

PKC

= protein kinase C

CR

= cystein-rich

PLA2

= phospholipase A2

CRE

= cAMP response element

PLC-

= phospholipase C-

CREB

= cAMP response element binding protein

PLD2

= phospholipase D2

cPLA2

= cytosolic phospholipase A2

PtdIns(4,5)P2 = phosphatidylinositol-4,5-biphosphate

DR

= death receptor

PtdIns3-K

= phosphatidylinositol-3 kinase

EGF

= epidermal growth factor

PTEN

= phosphatase and tensin homolog

EGFR

= epidermal growth factor receptor

rhIGFBP3

= recombinant human IGFBP3

EMT

= epithelial to mesenchymal transition

RTK

= receptor tyrosine kinase

EPR

= epiregulin

SDF-1

= stromal cell derived factor 1

Eps15/R

= epidermal growth factor receptor pathway substrate 15/related

SH2

= Src homology 2

siRNA

= small interfering RNA

ERK

= extracellular regulated kinase

SOS

= son of sevenless

ESCRT

= endosomal sorting complex required for transport

STAM

= signal transducing adaptor molecule

FOX

= forkhead box

STAT

= signal transducer and activator of transcription

Fz

= frizzled

TGF-

= transforming growth factor-

GEF

= guanine nucleotide exchange factor

TKI

= tyrosine kinase inhibitor

GPCR

= G-protein coupled receptor

TM

= transmembrane

Grb2

= growth factor receptor bound protein 2

TNF

= tumor necrosis factor

GSK-3

= glycogen synthase kinase 3

TNFR

= tumor necrosis factor receptor

HB-EGF

= heparin-binding EGF

UIM

= ubiquitin interacting motif

HER

= human epidermal growth factor receptor

VHH

HIF-1

= hypoxia inducible factor 1

= variable domain of the heavy chain of heavy chain-only antibodies

HNSCC

= head and neck squamous cell carcinoma

IAP

= inhibitor of apoptosis protein

IGF

= insulin-like growth factor

IGF-1R

= insulin-like growth factor-1 receptor

IGFBP

= insulin-like growth factor binding protein

IL-8

= interleukin-8

IRS

= insulin receptor substrate

JAK

= Janus kinase

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Accepted: June 19, 2009