Understanding Resistance to EGFR Inhibitors—Impact on Future Treatment Strategies

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

Disclosures
In This Article

Mechanisms of EGFR Antibody Resistance

EGFR Mutations

In 2004, a series of landmark papers identified EGFR mutations in the tyrosine kinase domain in patients with NSCLC that predicted response to the TKIs erlotinib and gefitinib.[74–76] These mutations included in-frame deletion of amino acids 746–750 in exon 19, and a point mutation in exon 21 (L858R). More importantly, these mutations led to gain-of-function and conferred dependence of the tumor cell on the mutated kinase. These mutations in EGFR rendered tumors dramatically more sensitive to the effects of erlotinib and gefitinib than tumors without these mutations. This important finding has stimulated a prolific body of preclinical and clinical research that has substantially advanced our understanding of EGFR mutations and their role in governing response to small-molecule TKIs directed against EGFR. However, no mutations in EGFR have been identified to date that are reliably predictive for response to antibody-based EGFR therapies.[112] This finding suggests that other molecular mechanisms may exist that modulate intrinsic (primary) or acquired (secondary) resistance to EGFR antibody-based therapies (Figure 2).

Figure 2.

Mechanisms of resistance to EGFR antibodies. a | One mechanism of resistance to cetuximab is overexpression of the EGFR ligand TGFα.[160]b | Overexpression of EGFR has also been implicated in the development of acquired resistance.[117]c | Ubiquitylation is important for mechanisms of escape to cetuximab therapy.[117,122]d | Modulation of EGFR by SFKs, and increased activity of SFKs in cetuximab-resistant lines have been reported.[122,123]e | The binding and activation of EGFR or HER2 to HER3 has been reported, which allows prolonged signals to the PI3K/AKT pathway.[117,123]f | Translocation of EGFR to the nucleus has a role in resistance to cetuximab.[125]g | Increased VEGF production leads to altered angiogenesis and enhanced escape from cetuximab therapy.[115,116]h | VEGFR1 also contributes to resistance to cetuximab.[120]i,j | Mutations in both PTEN and Ras have been implicated in impaired response to cetuximab therapy.[96]k | Mutations in KRAS keep it in a constant GTP-bound, active state, allowing it to send signals downstream independently from RTK activation. l | EGFRvIII a truncated form of EGFR that is constitutively phosphorylated in a ligand-independent manner.[121,161]m | MDGI alters trafficking of EGFR, leading to resistance to cetuximab therapy.[126] Abbreviations: B-Myb, Myb-related protein B; CBL, E3 ubiquitin-protein ligase CBL; E2F1, transcription factor E2F1; EGFR, epidermal growth factor receptor; MAPK, mitogen-activated protein kinase; MDGI, mammary derived growth inhibitor; P, phosphorylation; PCNA, proliferating cell nuclear antigen; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate PTEN, phosphatase and tensin homolog; RTK, receptor tyrosine kinase; SFK, Src family kinase; STAT3, signal transducer and activator of transcription 3; TGF-α, transforming growth factor alpha; Ub, ubiquitylation; VEGF, vascular endothelial growth factor; VEGFR1, vascular endothelial growth factor receptor 1.

Altered VEGF/VEGFR Expression

EGFR signaling can contribute to the production of several proangiogenic factors in tumors, including VEGF and basic fibroblast growth factor.[113,114] To investigate whether altered angiogenesis could serve as a potential mechanism of resistance to cetuximab therapy, Viloria-Petit et al.[115] examined the highly EGFR-expressing A431 cell line in mouse xenografts. Tumor xenografts were treated with three different EGFR-blocking antibodies (mR3, hR3 or cetuximab). Tumors treated with these three anti-EGFR antibodies led to prompt regression of the tumor followed by a long latency period. Once the tumors reappeared, they were refractory to a second round of antibody therapy. Several established cell lines from hR3 and mR3-treated tumors (cells from cetuximab-treated tumors could not be obtained) retained their sensitivity to these antibodies, whereas some variants exhibited accelerated growth rate and attenuated response to hR3 and mR3 in subsequent testing.[115] Owing to the reported suppressive effects of EGFR inhibitors on VEGF production, the researchers hypothesized that these anti-EGFR antibodies inhibited EGFR-mediated VEGF production, thereby decreasing angiogenesis and leading to decreased tumor growth. They further postulated that escaping this angiogenic inhibition might have contributed to anti-EGFR antibody resistance. Indeed, five of six resistant variants exhibited increased VEGF expression. Furthermore, A431 parental cells transfected with VEGF resulted in resistance to anti-EGFR antibodies in vivo. This report indicated that resistance could emerge in tumors that increase their VEGF production.

In 2004, Ciardiello et al.[116] reported that ZD6474 (vandetanib), a dual EGFR/VEGFR2 TKI, could overcome resistance to cetuximab. In this study, the investigators developed cetuximab-resistant GEO CRC cell lines in vivo by prolonged exposure to cetuximab. This treatment led to tumor control for 80–90 days followed by tumor growth, despite continuation of cetuximab therapy. Discontinuation of cetuximab and treatment of these resistant tumors with ZD6474 resulted in efficient tumor growth inhibition for up to an additional 150 days. Cell lines derived from this work showed a dramatic increase in phosphorylated MAPK, and increased COX-2 and VEGF protein expression compared with parental controls. The authors concluded that inhibition of VEGFR signaling in cetuximab-resistant tumor cells offered a potential anticancer strategy. In addition to cetuximab resistant clones, Ciardiello and colleagues also generated gefitinib-resistant GEO colon cancer cells.[116] Resistant clones resulting from these experiments also exhibited an increase in the expression of proteins COX-2 and VEGF. Collectively, these data suggest that challenge with both classes of EGFR inhibitors can alter VEGF production, and highlights neoangiogenesis as a potential shared mechanism of EGFR inhibitor escape. Although this work has indicated that ZD6474 may be a viable treatment for tumors that manifest resistance to prolonged cetuximab therapy, ZD6474 itself demonstrates anti-EGFR activity. Tumors with resistance to anti-EGFR antibody therapy may retain sensitivity to EGFR-based TKI therapies. Similar findings have since been reported.[117–119] Work from Bianco et al.[120] has further implicated the VEGF and VEGFR system in resistance to cetuximab. They reported that VEGFR1 was overexpressed in cells resistant to cetuximab. Experiments silencing VEGFR1 in cetuximab-resistant cells restored sensitivity to cetuximab, whereas exogenous overexpression of VEGFR1 in cetuximab-sensitive cells conferred resistance to cetuximab. A similar analysis of cells with resistance to gefitinib also exhibited increased expression of VEGFR1.[120]

EGFRvIII

Although no point mutations are known to be associated with resistance to cetuximab or panitumumab, preclinical models analyzing the EGFR variant III (EGFRvIII), which lacks the ligand-binding domain, have provided new information.[121] In a study of HNSCC tumors, 42% of tumors expressed EGFRvIII, which correlated with increased proliferation in vitro and increased tumor growth in vivo.[121] To determine if this variant could contribute to cetuximab resistance, HNSCC cells were engineered to overexpress EGFRvIII. These tumors showed increased proliferation in response to cetuximab treatment compared with vector-only controls. These findings suggest that a percentage of HNSCC tumors may express EGFRvIII and this protein contributes to cetuximab resistance.[121]

Ubiquitination of EGFR

Two papers have identified the potential role of EGFR ubiquitination as a mechanism of acquired resistance to cetuximab.[117,122] Wheeler et al.[117] developed cells with acquired resistance to cetuximab in vitro by prolonging and escalating dose exposure to cetuximab. Several resistant clones were derived from this work that had increased EGFR expression compared with parental controls, which was associated with dysregulation of EGFR internalization or degradation. This altered processing of EGFR led to sustained signaling from EGFR, which caused activation of HER3. Lu et al.[122] used a similar approach and found that EGFR had an increased association with the ubiquitin ligase CBL, leading to increased ubiquitination and downregulation of EGFR. Although lower levels of EGFR were expressed in these cetuximab-resistant cells, EGFR retained strong activity that seemed to be associated with cooperation with Src family kinases (SFKs). Similar to these findings, it was reported in 2008 that EGFR and SFKs cooperate in acquired resistance to cetuximab.[123] In this work, cells with acquired resistance to cetuximab exhibited robust expression of active SFKs, and this activity enhanced EGFR activation of HER3 and the PI3K/AKT pathway, leading to enhanced survival. Blockade of SFK activity using dasatinib could, therefore, resensitize tumors to cetuximab therapy.

Cellular Localization of EGFR

EGFR has been reported to function in the nucleus as a transcription factor as well as a tyrosine kinase that enhances cell proliferation.[53,124] Furthermore, nuclear EGFR is a prognostic factor in human disease.[43–45] Subcellular distribution of EGFR to the nucleus might have a role in resistance to cetuximab therapy.[125] Clones with acquired resistance to cetuximab expressed nuclear EGFR, which regulated the expression of several genes involved in G1/S progression. The authors reported that nuclear translocation of EGFR was mediated by SFKs and that abrogation of SFK activity led to loss of nuclear EGFR, increased membrane EGFR, and resensitization to cetuximab.[125]

Nevo et al.[126] investigated the role of mammary-derived growth factor inhibitor (MDGI) in conferring resistance to cetuximab. The authors reported that MDGI, a small cytosolic protein involved in fatty-acid binding, leads to the intracellular accumulation of EGFR where it remains active, and cannot be targeted by cetuximab therapy.[126] These data suggest that the subcellular distribution of EGFR may be an effective escape from cetuximab therapy.

Epithelial-mesenchymal Transition

Epithelial-mesenchymal transition has also been implicated in the resistance to both cetuximab and EGFR small-molecule inhibitors. Fuchs et al.[127] reported that in a series of 12 hepatocellular carcinoma cells classified as epithelial or mesenchymal (based on E-cadherin and vimentin expression), the cells exhibited variable sensitivity to EGFR inhibitors. Cells that were identified as epithelial had increased sensitivity to erlotinib, gefitinib and cetuximab compared with cells that were defined as mesenchymal. The authors further reported that mesenchymal cells had increased AKT and STAT3 activation associated with elevated expression of the integrin-linked kinase ILK, which led to resistance.[127]

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