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Anti-Cancer Drugs:
doi: 10.1097/CAD.0000000000000035
Review Articles

Emerging combination therapies to overcome resistance in EGFR-driven tumors

Ratti, Margherita; Tomasello, Gianluca

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Medical Oncology Unit, Azienda Istituti Ospitalieri Di Cremona, Cremona, Italy

Correspondence to Gianluca Tomasello, MD, Medical Oncology Unit, Azienda Istituti Ospitalieri Di Cremona, Viale Concordia 1, 26100 Cremona, Italy Tel: +39 0372 405248; fax: +39 0372 405702; e-mail: g.tomasello@ospedale.cremona.it

Received May 14, 2013

Accepted September 10, 2013

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Abstract

The epidermal growth factor receptor (EGFR) is responsible for the growth and progression of tumor cells; its overexpression and deregulation of its downstream signaling pathway have been found in many different neoplasms. These characteristics make it an ideal target for cancer treatment. Two classes of EGFR inhibitors, which bind to different parts of this molecule, have been developed and studied: monoclonal antibodies, such as cetuximab and panitumumab and tyrosine kinase inhibitors, including erlotinib and gefitinib. The effectiveness of these new drugs is considerably reduced by a number of mechanisms of resistance developed by tumor cells. Hence, there is a clear need for better characterization of these processes and finding new therapeutic strategies to make the action of these drugs more incisive. Here, we describe some of the mechanisms of resistance to EGFR inhibitors and review the main innovations attempting to overcome these drawbacks.

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Introduction

The epidermal growth factor receptor (EGFR) belongs to a family of transmembrane tyrosine kinase receptors involved in important transductional signal cascades regulating cellular processes such as proliferation, survival, adhesion, migration, and differentiation 1. The correct functioning of this receptor is basic for many cellular functions and activities and its aberrant expression plays an essential role in the development and growth of tumor cells.

The EGFR family includes four receptors: the epidermal growth factor itself (also known as ErbB1/HER1), ErbB2 (HER2/neu), ErB3 (HER3), and ErbB4 (HER4). They are key molecules that generate complex signaling pathways consisting of multileveled and cross-connected networks with different results and wide actions derived by their activation. The signaling cascade leads to the recruitment and phosphorylation of numerous intracellular substrates involved in proliferation control and other crucial activities for cell survival (Fig. 1) 2.

Fig. 1
Fig. 1
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Receptor overexpression, gene amplification, activating mutations, higher synthesis of receptor ligands, and loss of negative regulatory mechanisms are a few examples of the aberrant receptor’s activation, which may eventually lead to an alteration of signaling pathways and in turn cause the development of malignancies.

The signaling pathways start on the cell surface, where ligand–receptor and receptor–receptor relations occur. ErbB receptors are composed of an extracellular ligand-binding region, a transmembrane segment, and an intracellular protein kinase domain with a regulatory carboxyl terminal segment (Fig. 2). The extracellular part is composed of two types of domains: the L domain and the cysteine domain. They are arranged in a sequence of four domains: L1–CR1–L2–CR2. The ligand-binding site comprises domains I (L1) and III (L2). The binding of a ligand to domains I and III causes a rearrangement of these domains, which allows a stabilized conformation of the entire ectodomain, promoting homodimerization and heterodimerization. Dimerization activates the intrinsic EGFR protein tyrosine kinase and its autophosphorylation; this event leads to the recruitment and phosphorylation of numerous intracellular substrates 3.

Fig. 2
Fig. 2
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An important target of this signaling cascade is the Ras-Raf-MAP-kinase pathway. The activation of Ras gives rise to a multistep phosphorylation cascade that leads to the activation of mitogen-activated protein kinases (MAPKs), such as ERK1 and ERK2. ERK is involved in the transcription of molecules linked to the control of the cellular cycle. Another important pathway activated by EGFR is associated with the phosphatidylinositide 3-kinase (PI3K) and the protein serine-threonine-kinase Akt because of its role in cell growth, proliferation, survival, and motility.

The third pathway involved in this process is the stress-activated protein kinase pathway involving protein kinase C and Jak/Stat; their activation enables the expression of different nuclear transcriptional programs involved in the control of numerous cellular responses, such as division, survival, death, motility, invasion, adhesion, and DNA repair 4.

For all these reasons, it is easy to understand the high value of this receptor family in the control of cellular life. Several studies have been carried out on this subject in the last few years. Taking into consideration its basic role in cellular activities and its high levels of expression in a variety of tumor types, EGFR is emerging as an important target for cancer therapy.

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EGFR expression in cancer

EGFR regulates different aspects of cellular life and is normally expressed by many cell types, including those of epithelial and mesenchymal lineages; nevertheless, EGFR family signaling is deregulated in a number of subtypes of human cancers. Recent studies suggest that one-third of epithelial malignancies express high levels of EGFR, and some less conservative estimates report a deregulation of EGFR in at least 50% of human epithelial tumors, hence the evidence that the most common human epithelial cancers widely express EGFR 5.

Many studies have reported EGFR overexpression and deregulation in cancers of the head and neck, brain, bladder, ovary, cervix, esophagus, stomach, colon, endometrium, breast, glioma, lung [particularly non-small-cell lung cancers (NSCLCs)], and bile ducts or pancreas 6–9.

EGFR is frequently amplified and overexpressed in almost half of all glioblastomas (GBMs). In these cancers, gene amplification is frequently associated with deletion mutations involving the extracellular domain of EGFR with the so-called EGFRvIII as the most common variant. These mutations result in the constitutive activation of EGFR and are frequently associated with additional mutations in the cell cycle regulatory gene INK4a-ARF 10.

EGFR is also altered through point mutations or deletion mutations in the kinase domain in 10–15% of NSCLCs in the USA and in 30–50% of NSCLCs in Asia 11–13. These mutations are clustered within four exons encoding the kinase domain and produce a spectrum of biochemical effects ranging from increased kinase activity to ligand-independent constitutive activity. Destabilization of the self-inhibiting (non-autoinhibited) conformation of the EGFR intracellular domain (ICD) has been proposed to explain the constitutive activation of these EGFR mutants. On the basis of the solved EGFR ICD structure, the L858R mutation in the A-loop and the deletion Del (746–750) in the α-C helix are predicted to disrupt the self-inhibiting ICD 13.

EGFR is overexpressed without mutation in a much larger subset of NSCLCs; however, its etiologic role in these scenarios is not well established.

Conversely, the pathogenetic role of at least some of the common EGFR-mutant alleles in tumorigenesis has been confirmed by in-vitro and in-vivo experiments. These EGFR mutants are transforming in fibroblast and epithelial cell models, and induce lung tumors when expressed in mice lung epithelia 13.

Recent studies attempted to answer an important question: are the high levels of EGFR expression in cancer a predictor of response to therapy?

Preclinical studies on 60 cell lines provided by the USA National Cancer Institute Anticancer Drug Screen were carried out with a panel of 11 selective erbB inhibitors 14. Cell lines expressing high levels of EGFR could be divided into two groups on the basis of their sensitivity to EGFR inhibitors. The level of EGFR expression for the specific tumors appeared to be less important than the degree of activation of EGFR in predicting the response to targeted therapy. Specifically, factors related to activation status include receptor mutations, heterodimerization, and increased expression of ligands. In conclusion, these evidences show that there is no clear relationship between the level of EGFR expression and EGFR activation, thus complicating the prediction of the clinical efficacy of targeted therapies.

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EGFR inhibitors

Kawamoto et al. 15 first reported that a series of monoclonal antibodies directed to the EGF-binding site inhibit the growth of cancer cells expressing high levels of EGFRs and prevent the activation of the receptor tyrosine kinase. In addition, they discovered the antiproliferative effect caused by an agent targeting a growth factor receptor and obtained the same results by an agent targeting a protein kinase. After this first description of the event, their hypothesis was confirmed by numerous studies on the activity of two classes of anti-EGFR agents for cancer treatment. These are represented by monoclonal antibodies directed against the extracellular domain of the receptor and low-molecular-weight, ATP competitive inhibitors of the receptor’s tyrosine kinase (TKIs) (Table 1).

Table 1
Table 1
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The different mechanisms of action and toxicity profile of these agents compared with traditional cytotoxic therapies make these molecules the ideal drugs to be evaluated in combination with standard chemotherapy, attempting to achieve additive or synergistic anticancer activity.

Monoclonal antibodies show greater specificity for the EGFR compared with TKIs and receptor inhibition can be achieved at lower concentrations. However, monoclonal antibodies may also be less effective or ineffective against altered forms of EGFR, being unable to recognize and bind to an altered form of the receptor 16.

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Monoclonal antibodies

Monoclonal antibodies (mAbs) directed against EGFR present the following mechanisms of action:

extracellular binding;

internalization of receptor–antibody complex;

prevention of tyrosine kinase activation; and

inhibition of EGFR signaling pathways.

Here, we describe the most diffused and effective molecules of this class currently used in cancer treatment.

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Cetuximab

Cetuximab (also known as IMC-225 or C225, erbitux) is an immunoglobulin G1 chimeric mouse–human monoclonal antibody that targets the extracellular domain of EGFR. It was developed by combining the variable regions of the precursor mouse antibody (mAb 225) with human immunoglobulin G1 constant regions to reduce the possibility of an anti-mouse immunological reaction in patients.

Its therapeutic effect consists of blocking endogenous ligand binding to the extracellular domain of EGFR and in enhancing receptor internalization and degradation.

Cetuximab binds to the EGFR with high affinity, competes with ligand binding, and blocks the subsequent activation of receptor tyrosine kinase. It interacts with domain III of the EGFR, occluding the ligand-binding region on this domain 17. Cetuximab prevents the receptor from adopting the extended and stabilized conformation required for dimerization. In addition, it induces antibody-mediated receptor downregulation, an important effect for its growth-inhibitory capacity, and it can also induce antibody-dependent cell-mediated cytotoxicity (ADCC). Important antitumor activity was found in clinical trials as both monotherapy and in combination with chemotherapy and/or radiation, particularly in colorectal cancer (CRC) and head and neck squamous cell carcinoma (HNSCC) 18.

On the basis of the results of the pivotal ‘BOND’ trial 19, in 2004, for the first time, the FDA approved cetuximab use alone or in combination with irinotecan in irinotecan-refractory patients with metastatic CRC.

Different studies have been carried out in the last few years evaluating the use of cetuximab in the treatment of CRC. Cetuximab was subsequently approved for the treatment of KRAS mutation-negative (wild type) or as a single agent in patients who had failed oxaliplatin-based and irinotecan-based chemotherapy 20–22.

In contrast to these findings, the results of randomized trials evaluating the benefit of adding cetuximab to an oxaliplatin-based regimen are discordant: the multicenter European phase II ‘OPUS’ trial showed that combined therapy was associated with a significantly higher response rate 23.

The ‘COIN’ trial from the UK and the ‘NORDIC VII’ trial failed to show any benefit in either the response rate or progression-free survival (PFS) from the addition of cetuximab in patients with KRAS wild-type tumors 24,25. Thus, unlike irinotecan-based regimens, the benefit of adding cetuximab to a first-line oxaliplatin-based regimen remains uncertain.

It is now well established that KRAS mutation status is the stronger predictor of response to cetuximab therapy for patients with metastatic CRC 26. It is also clear that activating mutations in the KRAS gene, which result in constitutive activation of the RAS-RAF-ERK pathway, confer resistance to anti-EGFR therapy. KRAS activating mutations are found in ∼40% of all metastatic CRCs. For all these reasons, panitumumab and cetuximab are approved only for patients with wild-type KRAS tumors 27. The use of molecular testing to select patients for treatment with these agents is therefore essential.

Cetuximab has also been approved for the treatment of HNSCC as a single agent for recurrent or metastatic disease after platinum-based chemotherapy failure and in combination with radiation therapy as the initial treatment of locally or regionally advanced disease 28.

In a recent study, the addition of cetuximab to a platinum-based doublet regimen in metastatic HNSCC resulted in a significant increase in overall survival (OS) compared with doublet chemotherapy alone. In this phase III trial, 442 patients were assigned to a first-line regimen of cisplatin or carboplatin plus fluorouracil every 3 weeks with or without cetuximab. Chemotherapy plus cetuximab significantly prolonged the median OS compared with chemotherapy alone [10.1 vs. 7.4 months, hazard ratio (HR) for death 0.80, 95% confidence interval (CI) 0.64–0.99]. Significant improvements were also observed in the PFS and objective response rates 29. For these reasons, cetuximab is registered for first-line use in combination with platinum-based chemotherapy in both Europe and the USA.

Cetuximab activity was also evaluated in advanced NSCLC, showing contradictory results. The ‘FLEX’ and ‘BMS-099’ phase III trials tested the effects deriving from the combination of cetuximab with standard chemotherapy doublets. In the FLEX trial, 1125 previously untreated patients with advanced NSCLC were assigned randomly to receive cisplatin plus vinorelbine plus cetuximab or chemotherapy alone. The addition of cetuximab to chemotherapy significantly increased OS (median 11.3 vs. 10.1 months, HR 0.87, 95% CI 0.76–0.99) and the objective response rate (36 vs. 29%). On the basis of these results, the addition of cetuximab to a platinum-based combination chemotherapy regimen represents an option for patients with advanced or metastatic EGFR-expressing NSCLC 30.

In the second trial, 676 patients were randomized to carboplatin plus a taxane±weekly cetuximab. The addition of cetuximab to chemotherapy did not significantly increase PFS compared with chemotherapy alone (median 4.4 vs. 4.2 months with chemotherapy alone, HR 0.90, 95% CI 0.76–1.07) 31.

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Panitumumab

Panitumumab (ABX-EGF, vectibix) is a fully humanized immunoglobulin G2 monoclonal antibody with a high affinity for EGFR.

In vitro, panitumumab blocks the binding of both EGF and transforming growth factor-α (TGF-α) to the receptor, inhibits EGF-activated EGFR tyrosine phosphorylation, and activation and proliferation of tumor cells. It causes EGFR internalization in tumor cells and blocks activation of the EGFR tyrosine kinase. In vivo, it mediates therapeutic elimination of established tumors in mouse and acts cooperatively with chemotherapeutic agents to cause regression of tumors 32.

Panitumumab received FDA approval in 2006 for the treatment of patients with EGFR-expressing metastatic CRC whose disease progressed following chemotherapy treatments containing fluoropyrimidine, oxaliplatin, and irinotecan 33.

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Tyrosine kinase inhibitors

TKIs, originating from quinazoline, are low-molecular-weight synthetic molecules that block the magnesium-ATP-binding of the intracellular tyrosine kinase domain. Gefitinib and erlotinib target EGFR specifically, whereas other molecules belonging to the same class (i.e. lapatinib or vandetanib) have a wider range of action, targeting other receptors such as HER2 and vascular endothelial growth factor receptor-2 (VEGFR2) in addition to EGFR. The action of TKIs consists of blocking ligand-induced receptor autophosphorylation by binding to the tyrosine kinase domain and disrupting tyrosine kinase activity. This mechanism of action produces the abrogation of intracellular downstream signaling in the end 34. The FDA initially approved gefitinib in May 2003 as monotherapy for the treatment of patients with locally advanced or metastatic NSCLC after the failure of both platinum-based and docetaxel chemotherapies 35. The same regulatory agency required demonstration of a survival benefit in a subsequent clinical trial.

Three important prospective studies (‘INTACT 1’, ‘INTACT 2,’ and ‘ISEL’) showed no improvement in OS 36–38. This evidence led to a change in the original FDA approval in 2005, with the result that the indication was limited to patients with cancer who were currently benefiting or have benefited previously from gefitinib treatment 39. Erlotinib was originally approved in November 2004 as monotherapy for the treatment of NSCLC patients who did not respond to at least one previous chemotherapy 40. In November 2005, erlotinib was approved in combination with gemcitabine for patients with advanced pancreatic cancer who had not received previous chemotherapy 41.

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Gefitinib

This molecule is an orally active aminoquinazoline that inhibits EGFR and other receptor tyrosine kinases. It prevents EGFR autophosphorylation in a variety of human cancers by competing with ATP for its binding site on the ICD and interfering with the EGFR signaling pathway. At high dose levels, gefitinib may induce inhibition of other receptors, such as HER2. An important aspect of this molecule is that its additive or synergistic effects in combination therapy do not require high levels of EGFR expression by cancer cells 42.

Gefitinib has some of the same intracellular targets observed using mAb therapy contributing to the inhibition of the growth of human cancer cells in vitro and in vivo, probably inducing cycle arrest and apoptosis. It indirectly inhibits angiogenesis, reducing VEGF, basic fibroblast growth factor (bFGF) and TGF-α production, and tumor microvessel density 34. An interesting study shows that gefitinib prevents EGF-induced upregulation of VEGF and IL-8, and decreases endothelial cell migration and neovascularization in vivo (mouse corneal cells). In human squamous carcinoma cells, it was also shown to inhibit processes involved in cell migration and invasiveness, preventing extracellular matrix invasion 43. Combined treatment of gefitinib with cytotoxic chemotherapy produced a synergistic and additive effect, further inhibiting the growth of tumor cells and stimulating the death of apoptotic cells. In human colon, ovarian, NSCLC, and breast cancer lines, a cooperative antiproliferative and proapoptotic effect was obtained when cancer cells were treated with radiation, followed by gefitinib 14.

In two randomized phase 2 studies, gefitinib played a significant role in patients with NSCLC nonresponsive to previous chemotherapy regimens (platinum-based and docetaxel-based therapy included). Therefore, in May 2003, the FDA approved gefitinib as a third-line treatment for patients with locally advanced or metastatic NSCLC after failure of both platinum-based and docetaxel-based chemotherapy 44. A subsequent placebo-controlled randomized phase 3 trial (‘ISEL’ trial) showed that gefitinib was not effective in improving OS, except in patients of Asian origin and nonsmokers, who achieved a significant survival benefit 38. On the basis of these results, the FDA, in June 2005, restricted the use of gefitinib to patients participating in a clinical trial or continuing to benefit from treatment already started.

More recently, gefitinib was compared with platinum-based chemotherapy in patients with advanced NSCLC eligible for first-line treatment in various Asian randomized phase III trials.

In patients harboring the EGFR mutation and showing particular clinical characteristics (adenocarcinoma histology, female sex, nonsmokers, or light smokers), the use of gefitinib as first-line treatment resulted in longer PFS, higher objective response rate, lower toxicity, and better quality of life compared with standard chemotherapy 45.

In July 2009, the European Medicines Agency (EMA) approved gefitinib for the treatment of locally advanced or metastatic EGFR-mutated NSCLC across all lines of therapy. So far, this drug represents the best first-line treatment option in patients with these features 39.

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Erlotinib

This molecule is a quinazoline that inhibits human EGFR tyrosine kinase. It exerts antiproliferative effects, cell cycle arrest in the G1 phase, and induction of apoptosis. In-vitro and in-vivo studies indicated that erlotinib shows activity against human colorectal, head and neck, NSCLC, and pancreatic tumor cells. Preclinical studies suggest that this molecule may be active against cancer cells expressing HER2 and dependent on this factor for growth and survival 46.

The recently published ‘OPTIMAL’ study verified the efficacy and tolerability of erlotinib as first-line treatment compared with standard chemotherapy in patients with advanced EGFR-mutated NSCLC. The median PFS was significantly prolonged in erlotinib-treated patients (13.1 vs. 4.6 months). Compared with standard chemotherapy, erlotinib was also associated with a more favorable tolerability. OPTIMAL is the first reported prospective phase III study to confirm the role of erlotinib in advanced NSCLC patients with EGFR activating mutations 47.

In addition, in November 2005, the FDA approved erlotinib in combination with gemcitabine for the treatment of locally advanced, unresectable, or metastatic pancreatic cancer, on the basis of the results of a large phase III study 48.

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Mechanisms of resistance to EGFR inhibitors

EGFR mutations

EGFR mutations can be found in different human cancers such as breast, gliomas, prostate, lung, and ovarian. One of the most well-known mutation is the EGFR variant III (EGFRvIII), containing a deletion in the extracellular domain of EGFR, which prevents the mutated receptor from binding of ligands and allows a constitutive EGFR activation. We can observe this genetic alteration in particular in GBMs, but it has also been reported in breast cancer. GBM cell lines expressing EGFRvIII are relatively resistant to gefitinib; higher doses and longer exposure to this agent are necessary to produce the expected effect. In cells expressing EGFRvIII, gefitinib cannot prevent AKT phosphorylation 49.

Interestingly, a study of HNSCC tumors showed that up to 42% can express EGFRvIII, a molecular feature that correlates with increased proliferation in vitro and increased tumor growth in vivo. To determine whether this variant could contribute toward cetuximab resistance, HNSCC cells were engineered to overexpress EGFRvIII. These tumors showed increased proliferation in response to cetuximab treatment compared with vector-only controls, thus confirming that the presence of this variant is crucial in developing resistance to anti-EGFR Abs 50.

The most common mechanism of resistance to EGFR TKIs consists of secondary mutations of the EGFR gene in exon 20 related to the substitution of methionine for threonine at position 790 (T790M). This mutation confers resistance to gefitinib and erlotinib; in fact, the threonine residue is located in the hydrophobic ATP-binding pocket of the catalytic region and this is basic for the binding of small-molecule TKIs. Substitution of the threonine with a bulkier amino acid, such as methionine, can sterically interfere with the binding of gefitinib or erlotinib 51. This mutation has been found in 50% of the tissue samples from patients with acquired gefitinib resistance, but it may be present before treatment with TKIs, also contributing toward primary resistance 52. During treatment with a first-generation TKI, clonal selection may allow the T790M-expressing cells to overgrow 53,54.

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Altered VEGF/VEGFR expression

Different studies show that bFGF, VEGF, and TGF-β secreted by cancer cells are involved in angiogenesis regulation and contribute toward the growth of new tumoral vessels. EGFR plays an important role in this scenario; in fact, its autocrine pathway is involved in the production of several proangiogenic growth factors, including VEGF and bFGF 55.

The inhibition of EGFR activity by selective anti-EGFR agents often results in downregulation of VEGF and other angiogenic factors and of tumor-induced, VEGF-mediated angiogenesis.

Experimental studies by Viloria-Petit and colleagues attempted to show that altered angiogenesis could represent a potential mechanism of resistance to cetuximab therapy. They examined the highly EGFR-expressing A431 cell line in mouse xenografts, treated with three different EGFR-blocking antibodies (mR3, hR3, or cetuximab). All treatments produced evident regression of the tumor mass and long-lasting growth inhibition. At the time of tumor progression, the antibody therapy was ineffective. The authors concluded that anti-EGFR antibodies inhibited EGFR-mediated VEGF production, contributing toward the limitation of angiogenesis and to a decrease in tumor growth. They also hypothesized that the failure of the second administration of antibodies was related to an escape mechanism of this angiogenic inhibition and this assumption was confirmed by the evidence that the resistant variants showed increased VEGF expression. The researchers transfected A431 parental cells with VEGF, resulting in resistance to anti-EGFR antibodies in vivo, showing that resistance could appear in tumors that increase their VEGF production 56–58.

An increase in VEGF expression was also found in human GEO colon cancer, chronically treated with gefitinib. After 11–12 weeks of continuous therapy, a dimensional increase occurred and the lesion reached a growth comparable with that of untreated controls after a further 10 weeks. It was verified that the resistant cells showed 5–10-fold increases in VEGF expression compared with wild-type GEO-cells, and showed sensitivity to vandetanib 58–60.

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MET amplification

The MET pathway plays an important role in developing resistance to EGFR TKIs. In fact, it was shown that MET amplification causes gefitinib resistance by driving HER3-dependent activation of the PI3K/AKT/mTOR pathway 61.

MET amplification may be found in ∼20% of NSCLC patients developing resistance to EGFR TKIs, after an initial response to treatment 61–63.

An analysis of tumor samples from 51 NSCLC patients who underwent therapy with gefitinib showed that membrane expression of activated c-MET was related to shorter time to disease progression 62. This shows that MET may also be a potential marker of primary resistance to TKIs in NSCLC patients.

Alteration in MET pathways was also found in CRC 64. Its overexpression is described in most CRCs (KRAS wild type and KRAS mutant tumors). The role of MET consists of a coadjuvant action with EGFR to promote the growth of tumor cells. Higher expressions of the MET receptor and of its ligand [hepatocyte growth factor (HGF)] are associated with advanced stage and poor prognosis 65.

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IGF-1R and its role in anti-EGFR drugs’ resistance

The insulin-like growth factor 1 receptor (IGF-1R), a membrane tyrosine kinase receptor, is expressed in all cells and involved in different processes such as tumor cell proliferation, differentiation, apoptosis, and metastasis 66,67. IGF-1 activity is predominantly mediated through the interaction with the IGF-1R, which results in the activation of Ras/Raf/MAP kinase and the PI3K-AKT pathways. Because of the ability of IGF-1R to heterodimerize with EGFR, in the presence of dysregulated IGF-1R pathways, PI3K signaling is permanently activated. IGF1-R is upregulated in 50–90% of CRC, representing a poor prognostic factor 68. Overexpression of IGF-1R was recently shown to be related to resistance to cetuximab in KRAS wild-type CRC 69. In this subgroup of tumors, the suppression of the downstream biologic cascade through the inhibition of EGFR is basic for the therapeutic effect of anti-EGFR treatment strategies. In-vitro inhibition of the EGFR with monoclonal antibodies in colon cancer cells suppresses the Ras/Raf/MAP kinase pathway and leads to high levels of AKT expression, which is also modulated by IGF-1. When the IGF-1 cascade is active, tumor cells may escape anti-EGFR-mediated cell death as a consequence of the IGF-1-driven PI3K-AKT overstimulation 18,36,70. This mechanism is one of the potential explanations for the lack of efficacy of anti-EGFR antibodies in KRAS wild-type colorectal tumors.

Similar evidences were found in other neoplasms. Following treatment with EGFR TKIs, upregulation of IGF-1R expression was observed in human GBM cells’ primary resistance to therapy 70.

Furthermore, in androgen-independent prostate cancer, increased expression and activation of IGF-1R was reported, causing acquired resistance to gefitinib. The mechanism of resistance was related to the production of high levels of the IGF-2 ligand 71.

Finally, IGF-1R is highly expressed in lung cancer cells, in particular, in squamous cell carcinoma. The activation of IGF-1R by amphiregulin, an EGFR ligand, causes a positive feedback, giving rise to further release of both amphiregulin and IGF-1 72.

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Future strategies to overcome resistance in EGFR-driven tumors

Irreversible EGFR inhibitors

We previously showed how the treatment with a first-generation TKI can induce a clonal selection of mutant cells clusters and, in particular for T790M-expressing cells, promoting tumor growth.

Among new-generation EGFR TKIs, there are the irreversible inhibitors (Table 2). The quality of their binding increases their effectiveness, extending the duration of EGFR inhibition and avoiding the development of resistance. These drugs may prevent and overcome primary and acquired resistance to first-generation EGFR TKIs, because of their covalent binding to EGFR, and possibly delay the acquisition or growth of mutant cells.

Table 2
Table 2
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Afatinib (BIBW 2992) is an oral irreversible ErbB family inhibitor that binds to the ATP-binding pocket of EGFR, HER2, and ErbB4, thus blocking their pathways 73. It inhibits the kinase activity of wild type and mutant EGFR. It showed promising efficacy against a variety of EGFR mutants, including T790M. It also showed 100-fold greater activity against L858R/T790M EGFR double mutants than gefitinib, yielding promising results in inhibiting human NSCLC cell lines harboring wild-type EGFR or the L858R/T790M double mutant, being much more effective than erlotinib, gefitinib, and lapatinib 89. Afatinib was evaluated in a LUX-Lung 2 phase II trial, in which 129 adenocarcinoma patients with activating EGFR mutations, previously untreated, received afatinib as a single agent. An overall response rate of 60%, a median PFS of 14 months, and a median OS of 24 months were reported 90. In the subsequent phase III LUX-lung 3 trial, afatinib was compared with chemotherapy (cisplatin plus pemetrexed) as the initial therapy for advanced NSCLC in patients whose tumors contained activating mutations. PFS, the primary endpoint of the trial, was significantly increased with afatinib compared with chemotherapy (11.1 vs. 6.9 months) and the objective response rate was also considerably higher using the new TKI 73.

When compared with placebo in 585 patients who had progressed after at least 12 weeks of treatment with either erlotinib or gefitinib, afatinib prolonged PFS (median 3.3 vs. 1.1 months), without a significant difference in OS (LUX-Lung 1 trial) 91.

Preclinical studies in T790M transgenic murine models showed that combined EGFR targeting with afatinib and cetuximab can induce almost all complete responses 92. The association of cetuximab and afatinib yielded more than 30% confirmed partial responses in 22 evaluable patients (T790M mutation included) 93.

Dacomitinib (PF-00299804) is an irreversible pan-HER TKI that inhibits the kinase activity of wild-type EGFR, HER2, and HER4; it also showed a significant effect against NSCLC cell lines with double mutations: EGFR exon 19 deletion and L858R mutation and L858R/T790M mutations 74–76. In an NSCLC cell line with the T790M mutation, dacomitinib, unlike gefitinib, completely inhibited the HER3 signaling pathway and induced the expected apoptosis 74. Data from a phase II study showed that dacomitinib is associated with a superior PFS when compared with erlotinib in patients with chemotherapy-resistant NSCLC, not selected according to EGFR mutation status 94. In advanced NSCLC patients molecularly confirmed or clinically selected for EGFR mutations, the new TKI reported an 85% 9-month PFS rate 95. A recent randomized phase II study by Ramalingam et al. 96 compared dacomitinib with erlotinib in 188 patients with advanced NSCLC, already treated with one/two previous chemotherapy regimens. Dacomitinib showed significantly improved PFS versus erlotinib, with an acceptable toxicity profile. PFS benefit was observed in most clinical and molecular subsets, notably KRAS wild type/EGFR any status, KRAS wild type/EGFR wild type, and EGFR mutants. A phase III study is now ongoing 97.

Neratinib (HKI-272) is an irreversible inhibitor of EGFR and HER2, being the first agent explored in the setting of mutant EGFR-cell clones 77. It did not show significant efficacy against EGFR-mutant cells resistant to erlotinib/gefitinib. These disappointing results are probably related to the adverse effects that limited its administration 78.

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Proangiogenic growth factors

As mentioned previously, many studies showed strong connections between EGFR and VEGFR pathways. Furthermore, the increased VEGF and VEGFR expression is a key factor for the development and maintenance of anti-EGFR drug resistance.

This observation justifies the strategic combination of new agents targeting both signal transduction pathways.

Vandetanib (zactima, ZD6474) is an oral inhibitor of VEGFR, EGFR, and RET. Several studies tested the combination of this drug with standard chemotherapy, particularly in NSCLC. The results of the phase III ‘ZODIAC’ study of second-line vandetanib plus docetaxel showed that this association significantly improves PFS versus docetaxel alone (4.0 vs. 3.2 months) 79.

Results from the ‘ZEAL’ phase III trial showed that the addition of vandetanib to pemetrexed significantly improved the objective response rate (RR), but was not associated with an improvement in OS or PFS compared with chemotherapy alone 98. The phase III ‘ZEPHYR’ trial tested vandetanib following chemotherapy and treatment with an EGFR TKI in patients with recurrent NSCLC. Both PFS and objective RR were improved, but OS, which was the primary endpoint, was not significantly prolonged 99. The ‘ZEST’ trial, which evaluated vandetanib versus erlotinib in advanced NSCLC patients, did not show any significant difference in RR, PFS, or OS 100. On the basis of these data, vandetanib use in NSCLC has not been approved.

Preclinical studies showed that VEGF activation is an important factor involved in the development of resistance to first-generation EGFR TKIs. Therefore, many clinical trials evaluated the combined inhibition of EGFR and VEGF pathways. The association of the antiangiogenic drugs bevacizumab or sunitinib to erlotinib in unselected patients with chemorefractory NSCLC led to a modest improvement in PFS, without any benefit in OS 101,102. Another phase II trial, combining erlotinib with sorafenib in chemorefractory NSCLC, showed a non-negligible clinical benefit with the use of these combined agents in EGFR wild-type tumors 103. Additional studies are certainly warranted, but the large studies carried out to date show that the dual targeting of EGFR and VEGFR is not a good strategy to overcome resistance to EGFR inhibitors in terms of NSCLC. Similar results were obtained in CRC using this approach. In the ‘BOND-2’ phase II trial 21, the combination of bevacizumab plus cetuximab and irinotecan showed favorable results when compared with controls in bevacizumab-naive chemorefactory patients. Conversely, when anti-EGFR therapy was added to bevacizumab-based first-line chemotherapy in advanced CRC, no benefits were achieved in phase III trials 104. Specifically, the combination of panitumumab with bevacizumab-based and oxaliplatin-based chemotherapy, as tested in the ‘PACCE’ trial, was associated with an inferior PFS in the group of patients carrying wild-type KRAS 105. Similarly, the addition of cetuximab to XELOX plus bevacizumab, as verified in the ‘CAIRO-2’ study, had no effect on PFS among patients with KRAS wild-type cancer 106. Probably, the reasons for these results are related to a negative interaction between these two types of drugs when associated with chemotherapy 104. A new molecule, brivanib, a TKI that binds VEGFR strongly, has been tested recently in a phase III trial in combination with cetuximab, administered to patients with metastatic refractory wild-type KRAS CRC. Despite improvements in PFS and RR, this association did not show any survival benefit 80,107.

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Inhibition of the MET pathway

Increased MET expression occurs in about 25–75% of NSCLC and is related to a poor prognosis. A number of MET inhibitors are being tested in NSCLC 108,109.

Tivantinib is one of the most advanced MET inhibitors; this agent blocks the MET receptor in its inactive conformation through an unknown mechanism 81. When combined with erlotinib, it was associated with an improvement in PFS in a randomized phase II trial 110. A phase III trial comparing erlotinib plus tivantinib with erlotinib plus placebo has been completed and results are awaited 111.

Onartuzumab is a monovalent monoclonal antibody designed to bind MET and inhibit HGF binding. The monovalent structure of onartuzumab inhibits HGF binding without causing MET dimerization 83,84. A recent phase II trial proved the poor prognostic value of MET in NSCLC, but the combination of onartuzumab with erlotinib improved PFS and OS in patients with MET immunohistochemistry-positive tumors. A phase III trial is currently under way comparing erlotinib plus onartuzumab with erlotinib plus placebo 112. In addition to blocking abnormal anaplastic lymphoma kinase (ALK) gene and ROS1, crizotinib is also a potent MET inhibitor 82.

This important drug (xalcori) is already in clinical use, as it was approved in 2011 by the FDA for the treatment of locally advanced or metastatic ALK amplified NSCLCs.

Moreover, the results of a phase III trial (‘PROFILE 1007’) showed that crizotinib alone is more effective than standard chemotherapy (docetaxel or pemetrexed) in patients with advanced, ALK-positive NSCLC, treated previously with first-line, platinum-based therapy 113. A phase I/II trial is ongoing to verify the safety, efficacy, and pharmacokinetics of erlotinib alone or associated with crizotinib in patients with advanced NSCLC of adenocarcinoma histology 114.

In CRC, as anticipated previously, MET signaling collaborates with EGFR to promote the growth of CRC cells 64. New agents have been developed recently and evaluated in this malignancy. Preliminary results of a randomized phase I/II trial in KRAS wild-type chemorefractory patients showed a significant improvement in overall RR when rilotumumab was added to panitumumab. Rilotumumab is an investigational fully human monoclonal antibody designed to inhibit the hepatocyte growth factor/scatter factor (HGF/SF) involved in the MET pathway 85.

In addition, standard agents such as irinotecan and cetuximab are being tested in advanced CRC in combination with tivantinib in a phase I/II study 111.

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Targeting IGF-1R

A number of different monoclonal antibodies and TKIs have been developed and evaluated in the last few years, in NSCLC and CRC, yielding disappointing results in most cases. Figitumumab, an anti-IGF-1R IgG2 human monoclonal antibody, reached an advanced stage of development 86. A randomized, first-line phase II study comparing paclitaxel–carboplatin with or without figitumumab in NSCLC showed higher RR and longer PFS in figitumumab-treated patients 115. However, the phase III trial of carboplatin–paclitaxel with or without figitumumab, at the time of the planned interim analysis, was discontinued prematurely for futility 116. A synergistic antitumor effect of combining anti-EGFR with anti-IGF-1R agents was observed in CRC cell lines, but this did not translate into a real advantage when tested in clinical studies 117. The first drug studied was cixutumumab in patients refractory to anti-EGFR agents. Cixutumumab selectively targets the IGF-1R, thereby preventing the binding of its natural ligand IGF-1 and the subsequent activation of the PI3K/AKT signaling pathway 87. In a randomized phase II trial, clinical benefit was observed only in one of 64 patients treated with this mAb in association with cetuximab 118.

Furthermore, the combination of panitumumab and ganitumab, an anti-IGFR1 monoclonal antibody, was not shown to improve RRs, compared with panitumumab plus placebo in KRAS wild-type metastatic CRC 88. Unfortunately, the early closure of the ganitumab phase III ‘GAMMA’ study 119 for futility in metastatic pancreatic cancer has been announced recently. In fact, the addition of ganitumab to gemcitabine did not lead to a statistically significant improvement in the primary endpoint of OS compared with gemcitabine alone 120.

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Conclusion

EGFR is a key signaling pathway involved in several physiologic and pathologic processes.

It is now clear that targeting this network is very important but not sufficient to definitely block cancer growth because of the emergence of resistance to anti-EGFR agents. In addition to the presence of primary and acquired resistance related to specific mutations, one of the most important mechanisms of escape to EGFR inhibitors is represented by the activation of alternative signaling pathways. Furthermore, multiple strategies of resistance may occur at the same time.

Hence, developing and testing multitargeted drugs or, alternatively, combining different agents directed against multiple pathways, became necessary. A number of mechanisms of resistance have been elucidated in the last few years, but there are still many pathological strategies not yet individuated.

To develop more and more effective drugs, a better understanding of EGFR signaling pathway biology and of its multiple cross-talk networks is required.

There is no doubt that personalized cancer medicine based on molecular characterization of tumors is the strategy to pursue.

Hopefully, in the future, well-designed translational studies focused on this subject will help in accomplishing this difficult goal.

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Acknowledgements
Conflicts of interests

There are no conflicts of interest.

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Keywords:

epidermal growth factor receptor; resistance; targeted agents

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