Understanding Resistance to EGFR Inhibitors—Impact on Future Treatment Strategies

Deric L. Wheeler; Emily F. Dunn; Paul M. Harari

In This Article

Mechanisms of Resistance to EGFR TKIs

Several mechanisms of resistance to erlotinib and gefitinib have been described in laboratory-based models (Figure 3).

Figure 3.

Mechanisms of resistance to EGFR TKIs. a | A mutant form of EGFR termed EGFR variant III has an in-frame deletion mutation that produces a truncated 150 kDa protein, which is constitutively phosphorylated in a ligand-independent manner.[161]b | EGFR-dependent tumors that are initially sensitive to EGFR TKIs acquire a mutation at threonine 790. Substitution of this residue in EGFR with a bulky methionine may cause resistance by steric interference with binding of TKIs, including gefitinib and erlotinib.[77–79]c | Tumors can become resistant when individual tumor cells undergo an oncogenic shift, which has been noted with several other RTKs, including MET,[148] AXL and IGF-1R. d | In addition to IGF-1R as a mechanism of escape, downregulation of the IGF-binding proteins IGFBP3 and IGFBP4, have been implicated in resistance to TKIs. These proteins are crucial for regulating the levels of IGF-1R ligands, and loss leads to overactivation of the receptor.[152]e,f | Mutations in both PTEN and Ras have been implicated in impaired response to TKI therapy.[134,135]g | Cells that developed acquired resistance to gefitinib in vivo were shown to have increased VEGF production, leading to altered angiogenesis and enhanced escape from cetuximab therapy.[116]h | VEGFR1 has also been implicated in the contribution to resistance to EGFR TKIs.[120] Abbreviations: AXL, tyrosine-protein kinase receptor UFO (AXL oncogene); EGFR, epidermal growth factor receptor; IGF-1R, insulin-like growth factor 1 receptor; IGFBP, insulin-like growth factor-binding protein; MAPK, mitogen-activated protein kinase; PTEN, phosphatase and tensin homolog; RTK, receptor tyrosine kinase; TKI, tyrosine kinase inhibitor, VEGF, vascular endothelial growth factor; VEGFR1, vascular endothelial growth factor receptor 1.


One identified mechanism of resistance involves the mutant, constitutively active form of EGFR termed EGFRvIII, which is commonly found in glioblastoma multiforme (GBM). GBM is a highly malignant primary brain tumor that accounts for >50% of all brain cancers diagnosed. EGFR is amplified in 50% of all GBM cases, and 40% of these cases express EGFRvIII. GBM cell lines that express EGFRvIII are resistant to gefitinib and, therefore, require higher drug doses and prolonged exposure to decrease the activity of EGFRvIII.[139] Studies analyzing the cell cycle in EGFR-expressing lines versus EGFRvIII lines revealed that DNA synthesis in EGFR cell lines is inhibited by gefitinib in a dose-dependent manner whereas it is unchanged in EGFRvIII cell lines. In addition, cells expressing EGFRvIII have higher activation of AKT, which is not affected by gefitinib treatment.

Role of Oncogenic Shift

One of the prevalent biological themes underlying intrinsic or acquired resistance involves 'oncogenic shift', which occurs when other membrane-bound RTK signaling pathways are involved in resistance. For example, HER2 and HER3 have been linked to gefitinib resistance. Erjala et al.[140] investigated molecular predictors of gefitinib response in HNSCC and measured key proteins in the EGFR signaling pathway. They reported an association between EGFR copy number and gefitinib sensitivity. Gefitinib-resistant cells had increased expression levels of HER2 and total HER3 protein. To determine if this increased activity of HER2 could contribute to gefitinib resistance, gefitinib was combined with pertuzumab, an antibody that targets HER2 heterodimerization.[140] This study resulted in an additive growth-inhibitory effect compared with gefitinib alone in gefitinib-resistant HNSCC cell lines. The authors concluded that EGFR amplification may predict sensitivity to gefitinib and that HER2 and HER3 may contribute to gefitinib resistance. Other studies of acquired resistance to gefitinib or erlotinib have suggested that ADAM17 (disintegrin and metalloproteinase domain-containing protein 17) can mediate the release of heregulin, leading to autocrine loop activation of HER2 and HER3, and thus provide a mechanism of escape from gefitinib.[141] HER2 and HER3 may serve as potential predictive markers and as therapeutic targets for combination therapy in the treatment of HNSCC with gefitinib.[140]

Activation of the AKT/mTOR Pathway

Another established finding in EGFR inhibitor resistance is the activation of the AKT/mTOR pathway, leading to enhanced cell survival. PI3K phosphorylates phosphatidylinositol (4,5)-disphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which serves as a docking site for AKT where it is activated by PDK1 and PDK2. Phosphatase and tensin homolog (PTEN) dephosphorylates PIP3 back to PIP2. Mutations in or loss of PTEN expression[142,143] may serve as a marker of primary resistance to gefitinib and erlotinib.[144,145] However, in a cohort of gefitinib-treated NSCLC, no correlation between PTEN and response to gefitinib treatment was observed, and researchers have questioned the role of these proteins in mediating primary insensitivity to gefitinib.[146] Engelman et al.[147] observed that gefitinib reduced AKT only in NSCLC cell lines where it inhibits growth. To better understand this observation, immunoprecipitates of PI3K from gefitinib-resistant and sensitive lines were analyzed. PI3K was exclusively associated with HER3 in gefitinib-sensitive cell lines, and gefitinib could dissociate this activity. These results suggested that HER3 couples EGFR to the PI3K/AKT pathway in gefitinib-sensitive NSCLC cell lines, but not gefitinib-resistant lines.[147] Further studies implicating HER3 have centered on gefitinib-resistant EGFR-mutant lung cancer lines. These cells lines displayed amplification of MET, which prolonged activation of the HER3/PI3K/AKT axis. Abrogation of MET activity restored sensitivity to gefitinib. To assess the clinical relevance of MET as a mechanism of resistance, Engelman et al.[148] also examined whether MET amplification could be detected in NSCLCs with mutant EGFR that had become resistant to gefitinib. MET amplification was detected in 4 out of 18 (22%) gefitinib/erlotinib-resistant tumor specimens.[148] This study was one of the first to suggest that oncogenic shift beyond the HER family, may contribute to resistance to EGFR-targeted therapies.[148] In addition to MET amplification, overexpression of hepatocyte growth factor (HGF), which is a ligand of MET, has been implicated in the development of acquired resistance to gefitinib. Yano et al.[149] reported that lung adenocarcinoma patients harboring EGFR-activating mutations, but no T790M mutation or MET amplification, showed a dramatic increase in HGF and activation of MET. This finding suggests that increased production of HGF represents a novel mechanism of gefitinib resistance in lung adenocarcinoma with EGFR-activating mutations.[149]

Role of IGF-1R in Resistance to EGFR Inhibitors

The insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) is ubiquitously expressed in cancer cells. This membrane-bound RTK has a role in tumor cell proliferation, differentiation, apoptosis, and metastasis.[150] It has also been strongly implicated in mediating resistance to the EGFR inhibitor AG1478 (A.G. Scientific, San Diego, CA). Analyzing two primary GBM cell lines with equal EGFR protein expression levels, but with distinct sensitivities to the TKI AG1478, indicated an upregulation of IGF-1R that resulted in sustained signaling to PI3K/AKT and ribosomal protein S6 kinase.[151] In addition to IGF-1R being implicated in resistance to gefitinib, IGF-binding proteins enhance acquired resistance to gefitinib. In these studies, A431 squamous-cell carcinoma lines were used to develop acquired resistance to gefitinib. Gefitinib-resistant clones exhibited hyperphosphorylation of IGF-1R and constitutive association of insulin-receptor substrate-1 with PI3K. Blockade of IGF-1R signaling disrupted this complex and restored the ability of gefitinib to downregulate PI3K/AKT signaling and cell growth. Gene profiling of the gefitinib-resistant clones indicated that resistant cells had markedly decreased expression of IGFBP3 and IGFBP4. These proteins are crucial for modulating the levels of the IGF-1R ligands, IGF-1 and IGF-2. Loss of these two regulatory proteins led to an increased availability of IGF-1 and IGF-2 and, therefore, constitutive activation of IGF-1R. Experiments using recombinant IGFBP-3 restored sensitivity of resistant cells to gefitinib.[152] Despite these reports in laboratory-based models of the role of IGF-1R and its associated regulatory elements, clinical analysis of IGF-1R expression in tumors from NSCLC patients indicated a lack of association with resistance to gefitinib.[146]