Primary and acquired resistance to anti-EGFR targeted drugs in cancer therapy

r 2007, Copyright the Authors Differentiation (2007) 75:788–799 DOI: 10.1111/j.1432-0436.2007.00200.x Journal compilation r 2007, International Society of Differentiation

RE VIEW

Floriana Morgillo . Maria Anna Bareschino . Roberto Bianco . Giampaolo Tortora . Fortunato Ciardiello

Primary and acquired resistance to anti-EGFR targeted drugs in cancer therapy

Received March 6, 2007; accepted in revised form April 23, 2007

Abstract In recent years, the epidermal growth factor receptor (EGFR) has been recognized as a central player and regulator of cancer cell proliferation, apoptosis and angiogenesis and, therefore, as a potentially relevant therapeutic target. Several strategies for EGFR targeting have been developed, the most succesful being represented by monoclonal antibodies, that directly interfere with ligand-receptor binding and small molecule tyrosine kinase inhibitors, that interfere with activation/phosphorylation of EGFR. These agents have been authorized in advanced chemorefractory cancers, including colorectal cancer, non-small-cell lung cancer and head and neck cancer. However, evidence of resistance to these drugs has been described and extensive studies have been performed to investigate whether resistance to EGFR-targeted therapy is primary or secondary. Cellular levels of EGFR do not always correlate with response to the EGFR inhibitors. Indeed, in spite of the over expression and efficient inhibition of EGFR, resistance to EGFR inhibitors may occur. Moreover, given the genetic instability of cancer cells, genetic modifications could enable them to acquire a resistant phenotype to anti-EGFR therapies. Taken together, these findings support the importance of understanding the molecular mechanisms affecting cancer cell sensitivity or resistance to such inhibitors. . )
Maria Anna Bareschino
Floriana Morgillo (* Fortunato Ciardiello Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale ‘‘F. Magrassi e A. Lanzara’’ Seconda Universita` degli Studi di Napoli Via Pansini 5 80131 Napoli, Italy E-mail: fl[email protected] Roberto Bianco
Giampaolo Tortora Dipartimento di Endocrinologia Molecolare e Clinica Universita` degli Studi diNapoli ‘‘Federico II’’ Via Pansini 5 80131 Napoli, Italy

This review will focus on the most relevant mechanisms contributing to the acquisition of sensitivity/resistance to EGFR inhibitors. Key words EGFR pathways
resistance
erlotinib
gefitinib
cetuximab

Introduction As a better understanding of the molecular basis of carcinogenesis has emerged, different cell-signaling pathways have been successfully targeted in various hematologic and solid malignancies. The HER family of receptors consists of HER1 (EGFR/ErbB1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4) (Bogdan and Kla¨mbt, 2001). The epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane glycoprotein, which consists of an extracellular domain that recognizes and binds to specific ligands, a hydrophobic transmembrane domain, involved in interactions between receptors within the cell membrane, and an intracellular domain that serves as the site of protein kinase activity. Multiple ligands are known to activate EGFR, including EGF, transforming growth factor (TGF-a), heparin-binding EGF-like growth factor, amphiregulin, betacellulin, and epiregulin. Once the ligand binds to the extracellular domain, EGFR undergoes homodimerization or heterodimerization. Dimerization induces the activation of the tyrosine kinase (TK) domain, which leads to autophosphorylation of critical tyrosine residues on the cytoplasmic terminal. These tyrosine residues serve as attachment sites for a range of cellular docking proteins, activating a variety of downstream signaling cascades to affect gene transcription (Yarden and Sliwkowski, 2001). Three downstream pathways of EGFR have been identified: the Ras/Raf mitogen-activated protein (MAP) kinase,

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PI3K/Akt, and Jak2/STAT3 pathways. Activation of these pathways affect from cell proliferation, tumor invasion, and metastasis to antiapoptotic signaling and tumor resistance to chemotherapy (Masuda et al., 2002; Vivanco and Sawyers, 2002; Kruger and Reddy, 2003). In addition, the EGFR pathway is implicated in angiogenesis, affects components associated with cell–cell adhesion in the integrin pathway, and promotes metastasis by stimulating tumor cell motility (Khazaie et al., 1993; Hazan and Norton, 1998; Ellerbroek et al., 2001). The EGFR is frequently expressed in a variety of epithelial tumors; non-small cell lung cancer (NSCLC) is among the epithelial cancers that are characterized by a generally high expression of ligands and receptors belonging to the EGF system (Normanno et al., 2003), with an expression of about 40%–80%. EGFR overexpression has also been demonstrated in colorectal cancer (CRC) (72%–82%), head and neck cancer (95%– 100%), breast cancer (14%–91%), and renal cell cancer (50%–90%) (Salomon et al., 1995). In an effort to apply this information to the treatment of human cancers, molecules that inhibit EGFR activity have been developed and tested; they include small molecules as well as monoclonal antibodies (Table 1). Monoclonal antibodies bind to the extracellular domain of EGFR and competitively inhibit ligand binding (Mendelson, 2000). The EGFR–monoclonal antibody complex is subsequently internalized, causing a transient decrease in EGFR expression, which prevents EGFR heterodimerization in a phosphorylation statusindependent manner. Cetuximab (Erbituxs, IMCC225; ImClone), a chimeric human-mouse anti-EGFR monoclonal immunoglobulin 1 antibody, has been shown to inhibit EGFR signaling by blocking the receptor and also recruiting immune cells, leading to antibody-dependent cell-mediated cytotoxicity. This antibody has been extensively studied and approved by the Food and Drug Administration (FDA) for the treatment of patients with advanced CRC whose disease

is refractory to CPT-11, and is now being tested in patients with either chemotherapy-naı¨ ve or refractory NSCLC. Unlike monoclonal antibodies, TK inhibitors (TKIs) do not affect internalization of the receptor and are often not specific for EGFR, affecting the kinase activity of other ErbB family receptors. EGFR TKIs block the ATP pocket of EGFR, thereby inhibiting EGFR phosphorylation and down-stream signal transduction. The two most developed agents in this class of drugs are erlotinib (Tarcevas, OSI-774; Genentech, South San Francisco, CA) and gefitinib (Iressas, AstraZeneca, Wilmington, DE), which have been investigated in combination with cytotoxic agents in first-line therapy or as single agents in the salvage setting. Erlotinib has been approved by the United States FDA in November 2004 for the treatment of locally advanced or metastatic NSCLC that has failed to respond to one chemotherapy regimen, whereas gefitinib’s use is approved only in cancer patients who have already received and are benefiting from gefitinib treatment. Both drugs demonstrated promising results in preclinical studies and in phases I/II trials, but separate large, multinational, randomized, double-blind, placebo-controlled phase III trials showed no significant survival benefit of either drug when utilized in combination with chemotherapy (Gatzemeier et al., 2004; Giaccone et al., 2004; Herbst et al., 2004, 2005a). It has become clear that therapies designed to inhibit oncogenic kinases are unable to eradicate tumors completely because of the development of resistance. This review summarizes the current state of what is known about mechanisms of resistance to EGFR inhibitors in solid tumors. Primary resistance refers to patients who either do not achieve stable disease or who progress within 6 months after an initial clinical response, whereas secondary or acquired resistance typically occurs after prolonged treatment. To begin with among several studies, we tried to identify a mechanism of resistance to

Table 1 Anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors in clinical development Drug

Target

Cancer type

Clinical development

Monoclonal antibodies Cetuximab (IMC-225; Erbitux) Panitumumab Matuzumab (EMD-72000)

erb-B1 erb-B1 erb-B1

CRC; NSCLC; HNSCC Renal cancer; NSCLC; prostate cancer CRC; esophageal cancer; HNSCC; ovarian cancer

Marketed Phase III Phase II

Tyrosine kinase inhibitors Erlotinib (OSI-774; Tarceva) Gefitinib (ZD1839; Iressa) Lapatinib (GW-2016) EKB-569 BMS-599626 Carnetinib (CI-1033) Vandetanib (ZD6474) AEE788

erb-B1 erb-B1 erb-B1; erb-B2 erb-B1 erb-B1; erb-B2 Pan-erb erb-B1; VEGFR-2 erb-B1; erb-B2; VEGFR-2

NSCLC NSCLC Breast cancer CRC — NSCLC; HNSCC NSCLC —

Marketed Marketed Phase III Phase II Phase I Phase II Phase III Phase I

CRC, colorectal cancer; NSCLC, non-small-cell lung cancer; HNSCC, head and squamous cell carcinoma.

790 Table 2 Mechanisms of resistance to EGFR inhibitors Primary resistance Host-related mechanisms EGFR mutations K-RAS mutations EGFR copy number Secondary resistance Secondary mutations Epithelial–mesenchimal transition Activation of alternative pathways Constitutive activation of downstream pathways

Defective immuno-response; rapid metabolism/poor absorption; ethnicity; sex; histology; smoking habit Extracellular domain: EGFRvIII (del exon 2 to 7) Tyrosine kinase domain: L858R (exon 21); deletion in exon 19

Tyrosine kinase domain: T790M (exon 20) IGF-1R; mTOR; VEGFR; Herb-B2 PI3K/Akt; MAPK; p27; cyclin D1; PTEN mutations

EGFR, epidermal growth factor receptor; IGF, insulin like growth factor; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin.

EGFR-targeted therapy (Table 2) by distinguishing between a primary or a secondary mechanism. However, despite the clear scholastic differentiation between these two mechanisms, the reality is quite different; some of the mechanisms, such as the co-expression of other erbB receptors or the constitutive activation of other downstream pathways, are unlikely to be located in one of the two types of resistance.

Primary resistance Host-related mechanisms, such as defective immunesystem activity, rapid metabolism, or poor absorption, are predominantly responsible for intrinsic or primary resistance. Furthermore, while overall data from the erlotinib and gefitinib trials were in unselected patient populations, subset analyses from the studies demonstrated a distinct survival advantage in particular patient populations (specifically, those with high tumor EGFR gene copy number, East Asian ancestry, and lifetime non-smokers), conferring to some genetic and clinical characteristics a reliable role in predicting response to anti-EGFR therapies (Hirsch and Witta, 2005). Molecular characterizations of EGFR gene abnormalities in human tumors have provided new avenues into the future of genotype-directed targeted therapy. Among the first genetic alterations of the EGFR that have been found, there is the EGFR variant III (EGFRvIII), which is the result of an in-frame deletion from exons 2 through 7 in the extracellular domain that prevents EGFRvIII from binding EGF and other ligands. The constitutive signaling by EGFRvIII and the resistance to EGFR-targeted therapy are thought to be because of structural changes in the EGFR protein that could affect the intracellular domain conformation and the ATP pocket (Learn et al., 2004; Li et al., 2004). This ligand-independent mutant protein is constitutively activated and has been observed in up to 40% of glioblastomas and in other human malignancies. Therefore, the presence of a large

number of these truncated receptors on the cell surface could affect the efficacy of EGFR targeting. Indeed, gefitinib can reduce EGFRvIII phosphorylation after several days of treatment, but it does not influence cell growth (Learn et al., 2004). In the same way, DNA synthesis is not affected in EGFRvIII-containing cells by increasing doses of gefitinib (Moscatello et al., 1995). This may be due, in part, to the permanent activation of Akt, which provides critical information for cell survival, proliferation, and mobility and is believed to cause a ‘‘pathway addiction.’’ On the other hand, loss of PTEN, a tumor-suppressor protein that is commonly lost in gliobastoma and that inhibits the PI3K signaling pathway, may promote resistance to EGFR kinase inhibitors, as described below. In a recent study, the analysis of the expression of EGFR deletion mutant variant III and PTEN in patients with recurrent malignant gliomas evidenced a positive correlation between responsiveness to EGFR kinase inhibitors and co-expression of EGFRvIII and PTEN (Mellinghoff et al., 2005). More recently, important findings have suggested a strong relationship between resistance to EGFR TKIs and the absence of an activating mutation in the intracellular domain of the receptor (Lynch et al., 2004; Paez et al., 2004). These EGFR kinase mutations enhance ligand-dependent activation of EGFR, and simultaneously increase sensitivity to TKIs. Approximately 90% of these EGFR gene mutations affect the small region of the gene within the exons (18–24) that code for the TK domain. The more common mutations are an in-frame deletion in exon 19 around codons 746–750 (45%–50% of all somatic EGFR mutations) and a missense mutation leading to leucine to arginine substitution at codon 858 (L858R) in exon 21 (35%–45% of mutations) (Shigematsu et al., 2005). A large number of retrospective analysis have evidenced a link between these somatic mutations in the EGFR gene and clinical characteristics associated with TKI response (adenocarcinomas histology, especially brochioloalveolar carcinoma, non-smokers, patients of Asian ethnicity, and female) (Janne et al., 2005). Several studies have dem-

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onstrated that patients with EGFR mutation-positive tumors showed an improved response rate and survival with TKIs compared with wild-type cases, with a median survival of up to 30 months in mutation-positive cases (Han et al., 2005; Mitsudomi et al., 2005), and therefore their absence can be considered to be a predictor of TKI resistance. Recent findings have also shown that mutations in K-ras predict a response to EGFR TKIs (Pao et al., 2005a). Tumors with K-ras exon 2 mutations (n 5 9) were associated with a lack of response to gefitinib or erlotinib (0% observed response rate; 95% (confidence interval [CI]): 0%–30%). Retrospective analysis of clinical studies with TKIs has evidenced a link between K-ras mutations and a significant decrease in time to progression and survival when compared with the wild types (Eberhard et al., 2005; Tsao et al., 2006). K-ras mutations status also appears to be predictive of lack of response to the mAbs cetuximab and panitumumab therapy in advanced CRC patients in which it is also associated with a worse prognosis (Lievre et al., 2006), suggesting a role for K-ras gene mutation as a useful marker for selecting those patients who will not benefit from anti-EGFR therapies. Increased EGFR gene copy number as determined by fluorescent in situ hybridization (FISH) and EGFR protein overexpression measured by immunohistochemistry (IHC) were recently reported to correlate with improved response and survival with gefitinib treatment. In a cohort of non-small cell lung cancer patients from Italy, 33% of the lung tissues had an increased EGFR gene copy number (including gene amplification and high-level polysomy) defined as FISH1, and achieved higher RR to gefitinib therapy (36%) compared with FISH patients (3%; P 5 0.001) and had increased median survival (18.7 versus 7.0 months; P 5 0.03). Within the same cohort, EGFR protein overexpression (IHC1) was demonstrated in 59% of the tumors, and was associated with increased response (21% versus 5%; P 5 0.03) and survival (11.5 versus 5.0 months; P 5 0.01) (Cappuzzo et al., 2005). Moreover, available samples from the randomized BR21 and ISEL studies were analyzed for FISH status (Tsao et al., 2005; Hirsch et al., 2006). In the BR21 trial, FISH positivity (approximately 45%) was predictive of improved survival with erlotinib (HR 5 0.44, P 5 0.01). The molecular subgroup analysis of the ISEL trial evidenced a FISH1 status in 30.8% of patients, which was associated with a non-significant trend toward improved survival with gefitinib treatment, suggesting that gene copy number was a predictor of survival benefit with gefitinib compared with placebo. Of interest, recently, a significant correlation has been reported between increased EGFR gene copy number by FISH and response in patients with CRC treated with anti-EGFR mAbs (cetuxmab and panitumumab) (Moroni et al., 2005).

Secondary resistance Secondary or acquired resistance typically occurs after prolonged treatment, and several molecular mechanisms have been suggested to contribute to the resistance phenotype. Given the genetic instability of cancer cells, the insurgence of several genetic modifications could induce acquired resistance and confer them with the ability to select a different molecular signaling and to quickly switch to an alternative survival pathway. As EGFR antagonists interfere with the activation of several intracellular pathways that control cell proliferation, survival, apoptosis, metastatic capability, invasion, and angiogenesis, the molecular mechanisms of acquired resistance can be due to several processes: 1. 2. 3. 4.

Insurgence of secondary mutation in EGFR. Epithelial-mesenchymal transition. Activation of alternative pathways. Constitutive activation of downstream pathway.

Development of secondary mutation in EGFR As described previously, about 90% of NSCLC-associated mutations occur as either multinucleotide inframe deletions in exon 19, involving elimination of four amino acids, Leu-Arg-Glu-Ala, or as a single nucleotide substitution at nucleotide 2573 (TfiG) in exon 21, resulting in substitution of arginine for leucine at position 858 (L858R) and both of these mutations are associated with sensitivity to the small-molecule KIs gefitinib or erlotinib. Unfortunately, nearly all patients who experience marked improvement on these drugs eventually develop progression of disease. While K-ras mutations have been associated with some cases of primary resistance to gefitinib or erlotinib, its role in determining ‘‘acquired’’ or ‘‘secondary’’ resistance is still unknown. Acquired resistance to kinase-targeted anticancer therapy has been most extensively studied with imatinib, an inhibitor of the aberrant BCR-ABL kinase, in chronic myelogenous leukemia (CML). Relapse during imatinib treatment in patients with CML is commonly caused by mutations in the kinase domain of Bcr-Abl that interfere with imatinib binding. The highest degree of resistance to imatinib is conferred by a single mutation (T315I) deep within the ATP-binding pocket of the Abl TK that causes a steric hindrance caused by the replacement of threonine with the bulkier isoleucine residue (Talpaz et al., 2006). The structural similarity between Abl and EGFR is fairly high because the threonine-to-isoleucine mutation at codon 315 corresponds to the threonine to methionine mutation at codon 790 (T790M) in EGFR. Indeed, in addition to a primary drug-sensitive mutation in EGFR, a secondary mutation in exon 20 (T790M) in the kinase domain of the EGFR gene occurs in tumors with acquired resis-

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tance to gefitinib and erlotinib (Pao et al., 2005b). The crystallographic structure of the EGFR protein and its kinase domain shows that a wild-type threonine residue is located in the hydrophobic ATP-binding pocket of the catalytic domain, where it forms a critical hydrogen bond with the drug (Stamos et al., 2002) and the substitution of a large methionine would result in a steric conflict with the drugs. Clinical studies have reported that the presence of the T790 M mutation was observed in approximately 50% of the cases in which biopsy was obtained at the time of relapse following gefitinib or erlotinib treatment in patients with the LREA deletion or L858R EGFR mutation (Pao et al., 2006). In contrast to the multiple resistance mutations in CML, no EGFR resistance mutations have been identified other than the T790 M.

Epithelial–mesenchymal transition Epithelial–mesenchymal transition (EMT) is a process characterized by a loss of polarity and cell–cell contacts by the epithelial cell layers, which undergo a dramatic remodeling of their cytoskeleton (Thiery, 2003). Along with a loss of epithelial cell adhesion and alterations in their cytoskeletal component, cells undergoing EMT acquire expression of mesenchymal components. A main feature of EMT is the loss of E-cadherin expression (Kang and Massague´, 2004). In the TRIBUTE (Tarceva responses in conjunction with paclitaxel and carboplatin) trial (Herbst et al., 2005a), among patients receiving erlotinib and chemotherapy, time to progression was longer for those with E-cadherin-positive staining (Yauch et al., 2005). Several important genes that induce EMT (Snail, Twist, SIP1 [Zeb-2]) have been shown to act as E-cadherin repressors (Kang and Massague´, 2004). Growth factors, including hepatocyte growth factor, TGF-a, and EGF, have also been found to induce EMT; the vascular endothelial growth factor receptor-1 itself plays a role in tumor progression through the induction of EMT (Yang et al., 2006). A mesenchymal phenotype has been linked to erlotinib resistance in NSCLC cell lines and xenografts (Thomson et al., 2005). Erlotinib-sensitive cell lines were E-cadherin positive and vimentine negative, while insensitive cell lines showed a pattern of EMT, with loss of E-cadherin and expression of vimentin or fibronectin. Interestingly, a cell line containing an EGFR deletion mutation (D746-750), sensitive to erlotinib, showed an E-cadherin-positive/vimentin-negative profile (Thomson et al., 2005). Measurement of RNA abundance for Snail, Twist, Zeb-1, and Zeb-2 (SIP1) showed that Zeb-1 was the most highly expressed of the genes involved in the induction of EMT in erlotinib-insensitive NSCLC lines that had undergone EMT (Thomson et al., 2005). In this regard, Witta et al. (2006) have shown that pre-treating resistant cell lines with a histone

deacetylase inhibitor induces E-cadherin expression and leads to a growth-inhibitory and apoptotic effect of gefitinib similar to that in gefitinib-sensitive cell lines, including those harboring EGFR mutations. Their rationale is that Zeb-1 inhibits E-cadherin expression by recruiting histone deacetylases. Whether this model can translate to the clinical setting remains to be seen.

Activation of alternative pathways An area of growing interest is the cross-talk among EGFR and other signal transduction molecules, which may represent an escape mechanism of survival in the presence of anti-EGFR drugs. In this regard, the molecular mechanism of acquired resistance could be due to activation of alternative signaling pathways that stimulate other pathways involved in cell survival. EGFR is not the only receptor on the cell surface that can sustain proliferating stimuli; when it is blocked other receptors can drive cell survival by replacing its main functions. Signaling through the insulin-like growth factor I receptor (IGF-IR) represents at least one mechanism of resistance to the blockade of EGFR. The IGF-IR is a transmembrane TK, the amino acid sequence of which is highly homologous to that of the insulin receptor (IR) (Ullrich et al., 1986). Two major pathways are thought to originate from IGF-IR: one through IR substrate-1 (IRS-1), which activates the PI3-K/Akt pathway, and the other through Shc, which activates the Ras/Raf/MEK/ERK pathway (Peruzzi et al., 1999). Elevated levels of IGF-IR have been observed in the breast (Merrill and Edwards, 1990), brain (Peruzzi et al., 1999), and lung and colon (Gschwind et al., 2001), and are associated with a poor prognosis (Merrill and Edwards, 1990). Activated IGF-IR plays a key role in cell growth, establishment, and maintenance of a transformed phenotype, cell survival, and differentiation (Sell et al., 1993; Baserga, 1997; O’Connor et al., 1997). It has been demonstrated that in breast and prostate cancer cells, acquired resistance to gefitinib is associated with an increasing signaling via the IGF-IR pathway, which also plays a role in the invasive phenotype of the gefitinib resistant cells (Jones et al., 2004). Similarly, Chakravarti et al. (2002) have suggested that glioma may gain resistance to anti-EGFR therapies through up-regulation and activation of IGFR and its downstream signal transduction pathway through the phosphatidylinositol 3-kinase/Akt pathway. The real nature of this crosstalk is still poorly understood. Recently, Ahmad et al. (2004) have suggested that IGF-IR forms a complex with EGFR and that IGF-I trans-phosphorylates EGFR, presumably through this interaction, which then leads to ERK activation. Recently, the first evidence has been provided that acquired resistance to EGFR antagonists can be caused by a heterodimerizat-

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ion between IGF-IR and EGFR in NSCLC (Morgillo et al., 2006). Using a panel of NSCLC cell lines, treatment with erlotinib increased the levels of EGFR/IGFIR heterodimers localized on the cell membrane and activated IGF-IR (Morgillo et al., 2006). Given the considerable similarity between EGFR and IGF-IR in the sequence of their extracellular domain (Garrett et al., 1998), it is plausible that erlotinib-mediated IGF-IR/EGFR heterodimerization can stimulate intracellular signaling components in a distinct pattern and allow NSCLC cells to resist anti-EGFR therapy. Indeed, erlotinib-resistant cells showed an increased activation of PI3K/Akt and mitogen-activated protein kinase (MAPK) pathways by erlotinib treatment, as a consequence of IGF-IR activation (Morgillo et al., 2006). Moreover, erlotinib treatment induced, in erlotinib-resistant cells, mammalian target of rapamycin (mTOR)-mediated de novo protein synthesis of survivin, a well-known anti-apoptotic protein, and of EGFR itself with no detectable change in their mRNA levels (Morgillo et al., 2006). It is plausible to suggest that increased expression of EGFR proteins further enhances the interplay between the EGFR and IGFR on the cell membrane, resulting in a further amplification of IGF-IR and mTOR signaling. In addition, the increased survivin proteins seem to provide survival potential to the NSCLC cells against the erlotinib treatment. Inhibition of IGF-IR activation, suppression of mTOR-mediated protein synthesis, or knockdown of surviving expression abolished resistance to erlotinib and induced apoptosis in NSCLC cells in vitro and in vivo, suggesting a role of IGF-IR expression in predicting erlotinib resistance in NSCLC (Morgillo et al., 2006). Importantly, unpublished data showed that clinical samples from NSCLC patients revealed that the majority of EGFR-overexpressing samples showed correlative increases in IGF-IR protein levels compared with their paired normal counterparts from the same patients. With this prospect, IGF-IR–targeting combination treatment may be required when erlotinib is considered as a therapeutic agent for NSCLC patients. Alternatively, mTOR inhibitors could confer benefit to erlotinib-resistant patients. In some experimental systems, overexpression of HER-2, the second member of the erbB family, counteracts the ability of EGFR kinase inhibitors to block EGFR activity (Christensen et al., 2001). Indeed, HER-2 can potentiate EGFR signaling (Karunagaran et al., 1996) and contribute to EGFR-mediated transformation and tumor progression (Muller et al., 1996). Cancers that co-overexpress both EGFR and HER-2 behave worse than those that overexpress either receptor alone (Tateishi et al., 1994; Xia et al., 1999), and inactivation of HER-2 is required to block EGFR-mediated transformation (Qian et al., 1994; Beerli et al., 1995; Graus-Porta et al., 1995).

Conversely, high levels of activated EGFR abrogate the efficacy of trastuzumab, an anti-erbB2 monoclonal antibody, against HER-2 amplified cancer cells and this resistance is reversed by EGFR inhibitors (Motoyama et al., 2002). Taken together, these results led to the hypothesis that HER-2 signaling may represent a reason of resistance to anti-EGFR therapies and combinations of EGFR and HER-2 inhibitors could be synergistic against EGFR-positive, HER-2 overexpressing tumors. Selective inhibitors of both EGFR and HER-2 TKs have been developed, such as lapatinib, which has demonstrated activity in vitro and in vivo, against a broad spectrum of breast cancer cell lines overexpressing HER-2 and revealing a synergistic interaction in combination with trastuzumab (Konecny et al., 2006). Actually, the drug is in clinical evaluation, and recently a phase I study has been reported evidencing promising clinical activity in heavily pre-treated patients with ErbB1—expressing and/or ErbB2-overexpressing metastatic cancers (Burris et al., 2005). On the other hand, a phase II study (Arteaga et al., 2004) of trastuzumab in combination with either gefitinib or erlotinib in patients with breast cancer was unexpectly disappointing because of failure to meet its pre-determined median time to progression end point, highlighting the fact that robust pre-clinical data do not always predict clinical trial results and this combination needs to be re-examined. The EGFR pathway has been known to have an important role in angiogenesis. Activation of erbB signaling by EGF, heregulin, or by TGF-a can up-regulate the production of vascular endothelial growth factor (VEGF) in human cancer cells (Goldman et al., 1993; Gille et al., 1997), suggesting that the oncogenic properties of EGFR may, at least in part, be mediated by stimulation of tumor angiogenesis by up-regulating potent angiogenesis growth factors. In this respect, EGFR blockade causes inhibition of the secretion of VEGF and of other angiogenic growth factors, including basic fibroblast growth factor, interleukin 8, and TGF-a (Bruns et al., 2000; Eatock et al., 2000; Ciardiello et al., 2001). EGFR and VEGFR share a common downstream pathway and exert their effect directly and indirectly on tumor cells. Recent studies have evidenced the important role of VEGF in the resistance to EGFR blockade (Viloria-Petit et al., 2001). Cancer cell lines established from resistant tumors after two consecutive cycles of therapy with one of three different anti-EGFR monoclonal antibodies: mR3, hR3, or cetuximab retained high-EGFR expression, normal sensitivity to anti-EGFR antibody or ligand, and an unaltered growth rate when compared with the parental line in vitro. In contrast, five of six resistant variants expressed increased levels of VEGF, which paralleled an increase in both angiogenic potential in vitro and tumor angiogenesis in vivo. In a recent study (Ciardiello et al., 2004), it has been reported that

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EGFR inhibitor-resistant gene expression omnibus (GEO) colon-cancer cells exhibit a 5–10-fold increase in VEGF production and secretion compared with wildtype GEO cells, suggesting that an increased angiogenic potential through enhanced endothelial cell proliferation and permeabilization could have contributed to increased GEO tumor growth escaping from chronic EGFR inhibition. This study, also observed in an EGFR inhibitor-resistant GEO cells model, a 5–10-fold increase in expression of COX-2 and phosphorylated, activated MAPK. Increasing evidence supports the role of COX-2 in promoting tumor cell growth and angiogenesis, probably through the activity of COX-2derived prostaglandins (PGE2) (Turini and DuBois, 2002). Moreover, COX-2-dependent promotion of neoangiogenesis has been associated with induction of basic fibroblast growth factor and VEGF (Masferrer et al., 2000; Turini and DuBois, 2002). On the other hand, activation of the MAPK cascade is associated with upregulation of VEGF expression in cancer cells (Milanini et al., 1998). Therefore, an increase in COX-2 and MAPK pathways could contribute to the enhanced production of VEGF in EGFR inhibitor-resistant GEO-cells. On the basis of all this information, several pre-clinical studies have been realized with the intent to discover the effects of a combined targeting of the erbB and VEGF pathways by using different approaches (Ciardiello et al., 2000; Li et al., 2002). The encouraging data have led to the initiation of a number of clinical studies evaluating the combination of EGFR TKI or monoclonal antibodies, such as erlotinib and cetuximab, with an anti-VEGF antibody, bevacizumab, in a range of tumor types (Hainsworth et al., 2005; Herbst et al., 2005b). In addition to the option of using anti-EGFR therapies in combination with anti-VEGF drugs, there are now available a series of TKs that block both the EGFR and the VEGF receptor TK, such as vandetanib, which has demonstrated significant activity as a single agent and in combination with traditional chemotherapeutics in several human tumor types. (Ciardiello et al., 2003; Heymach et al., 2006; Natale et al., 2006).

Constitutive activation of downstream pathways Persistent activation of downstream signaling steps such as PI3K and MAPK pathway has been implicated in causing resistance to EGFR-targeted therapy in solid tumors (Magne et al., 2002; Janmaat et al., 2003), leading to the hypothesis that in some tumors the activation of downstream elements of the EGFR pathway is EGFR-independent, and not sensitive to the EGFR inhibition. PI3K has multiple effects, including activation of Akt/PKB and of p70S6-kinase, thus promoting cell survival signals and G1 cell cycle transition, and of the

small G protein Rac, mediating cytoskeletal rearrangements (Wymann and Pirola, 1998). The MAPK/extracellular signal-regulated kinase pathway regulates fundamental cellular function such as cell proliferation, survival, differentiation, and motility (Kolch et al., 2005). In the Janmaat study (Janmaat et al., 2003), the intrinsic activity of at least one of these two pathways (PI3K/Akt and MAPK) defines a resistant phenotype unaffected by the treatment with ZD1839 and C225. In addition to their contribution to cell proliferation, persistently active Erk and Akt pathways may protect cells from apoptosis induced by EGFR-targeted agents. Earlier reports demonstrated that cells transfected with active mutants of members of the MEK/Erk or PI3K/ Akt pathways by passed apoptosis induced by ZD1839 or Herceptin (Gilmore et al., 2002; Yakes et al., 2002). The Akt pathway appears to have a major role in resistance to EGFR therapy. In one study, gefitinibresistant clones of human non-small cell lung carcinoma subline (PC-9/ZD) were generated and analyzed (Koizumi et al., 2005). Relative to the parental cell lines, the inhibition of phosphorylated Akt by gefitinib treatment was not significant in PC-9/ZD cells, suggesting persistent downstream signaling even in the presence of EGFR inhibitors. Increased and EGFR independent activity of PI3K could results from direct gene amplification, activating mutations of p85, overexpression of downstream effectors such as Akt/PKB, inactivating mutations or loss of molecular regulator such as PTEN. One of the most frequently inactivated genes in tumors is PTEN (Li et al., 1997). It encodes a 403-aminoacid phosphatase that antagonizes the activity of phosphatidylinositol-3 0 -OH kinase (PI3K) on phosphoinositide substrates (Maehama and Dixon, 1998). In PTEN-null cell lines derived from tumors, expression of PTEN induced a marked decrease in proliferation due to cell cycle arrest in the G1 phase (Furnari et al., 1998). This arrest has been attributed to a decrease in cyclin D1 expression and an increase in the levels of CKI p27Kip1 that have an important role in cell cycle regulation (Cheney et al., 1999). Cyclin D1 is frequently overexpressed in tumor cells and its expression has been studied in correlation with gefitinib sensitivity in head and squamous cell carcinoma (Kalish et al., 2004). Three of six cell lines displayed cyclin D1 amplification and/or overexpression, and these cell lines were resistant to gefitinib. Moreover, when treated with ZD1839, SCC9 cells, transfected with cyclin D1, continued to proliferate and to maintain their S-phase fraction, whereas parental SCC-9 cells or cells transfected with an empty vector displayed decreased proliferation and marked S-phase reduction. The regulation of the CKI p27Kip by PTEN has been explained by two mechanisms. A first mechanism is based on the activation of p27Kip gene transcription by Forkhead transcription factors (FKHR) (Medema

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et al., 2000; Nakamura et al., 2000). These factors are phosphorylated and inactivated by Akt/PKB (Brunet et al., 1999; Kops et al., 1999) and rendered active by inhibition of the PI3K/Akt pathway with PI3K inhibitors (Medema et al., 2000). A second mechanism consists of reduced degradation of p27Kip (Mamillapalli et al., 2001). Altogether, these findings indicate a potential role of PTEN in the resistance of cancer cells to EGFR TKIs. In fact, the lack of activity of PTEN has been considered to be one of the main reasons for gefinitib resistance in several studies. Bianco et al. (2003) recently found that ZD 1839 can inhibit autophosphorylation of EGFR in the MDA-468 breast cancer cell line, which carry a deletion and frame shift mutation at codon 70 of the PTEN protein at a concentration as low as 0.1 mM, but concentrations greater than 1 mM were required to suppress PI3K/Akt signaling (Bianco et al., 2003). By reintroducing PTEN expression through retroviral infection, the ZD1839 treatment inhibited Akt activation, induced relocalization of the forkhead factor FKHRL1 to the cell nucleus, and increased its transcription activity. ZD1839 induced a greater degree of apoptosis and cell cycle delay in PTEN-reconstituted cells showing, that a mutant PTEN status contributed to the resistance of Akt signaling to ZD1839. Although this study suggests that the expression of PTEN can sensitize certain PTEN-null tumor cell lines to ZD1839, this strategy has limited clinical implications because restoration of PTEN function in the tumors of patients is not realistic right now, although the evaluation of wild-type PTEN expression might potentially serve as a marker for prediction of response. Moreover, because one of the main targets of PTEN is the PI3K/Akt pathway, more balanced phosphoinositide signaling may also be achieved by pharmacological inhibition of the PI3K/Akt activity. The treatment of MDA-468 cells with low doses of LY294002, which reduced the basal activity of Akt and re-established the EGFR-dependent Akt signaling, induced sensitization of these cells to ZD1839 by four-fold (She et al., 2003). The problem is that there are no PI3K inhibitors as yet that can be safely used in the clinical practice. Furthermore, because of the role of PI3K in physiologic processes, these drugs could be limited by a wide range of toxic side effects and are not likely to be tumor specific. In addition, PI3K inhibitors block DNA-PK and ATM activities, involved in DNA repair and cell cycle checkpoint control, respectively. Chronic inhibition of the two enzymes can lead to undesirable toxicity (Stein, 2001), accelerated tumor progression, and hypersensitivity of normal cells to chemo- and radiotherapy. Nevertheless, PI3K inhibition remains an important target for the treatment of cancer because of its role in promoting cell survival and also because of its role in contributing to angiogenic signaling in endothelial cells (Humar et al., 2002). These characteristics might result

in a stronger antitumor effect of PI3K inhibitors and in enhanced therapeutic properties of EGFR antagonists. An alternative therapeutic approach to inhibiting PI3K is targeting its downstream molecular effectors such as mTOR, a member of the phosphoinositide kinase-related kinase family. Growing evidence from pre-clinical studies suggests that blockade of mTOR activity results in pronounced antitumor effects in the absence of significant toxicity (Neshat et al., 2001) and in an inhibition of VEGF production (Guba et al., 2002). A previous report (Rao et al., 2005) has evidenced the activation of 4EBP1 by an EGFR TKI, EKI-785, thus decreasing the inhibitory interaction of 4EBP1 to eIF4E, indicating that EGFR TKIs may paradoxically promote the translation of complex 5’UTR-containing transcripts, some of which encode for proteins involved in driving cell proliferation. Consistent with these data, combined treatment with mTOR inhibitors and anti-EGFR TKIs has shown promising activity (Di Cosimo et al., 2004; Morgillo et al., 2006).

Conclusions The activity of anti-EGFR compounds has been clearly demonstrated, and these agents are excellent candidates to incorporate into treatment of in human tumors. This field is rapidly evolving as new insights in receptor biology, as well as the results from a large number of clinical trials, are becoming available. The elucidation of the molecular mechanisms of resistance to antiEGFR therapies is providing rational targets to overcome the resistance. Knowledge of the underlying mutations that cause resistance to therapy and tumor progression during cancer treatment will assist in tailoring individualized therapy for cancer patients based on their mutation status. Certainly, the translation of tumor biology from bench studies to a heterogeneous group of patients with a heterogeneous group of tumors has limitations and will necessitate better identification and segregation of patient/tumor characteristics. Moreover, there is a strong rationale, supported by pre-clinical and clinical works, for using EGFR inhibitors in combination with antiangiogenic treatment modalities or with other drugs that target different pathways involved in survival. Early clinical trials showed that these combinations are feasible with manageable toxicity and have interesting clinical activity in different advanced, chemo-resistant cancer types. However, important clinical and pharmacological work needs to be carried out in the areas of patient’s selection, selection of appropriate dose and schedules with new agents, and implementation of strategies to study the appropriate combinations.

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