Understanding Resistance to EGFR Inhibitors—Impact on Future Treatment Strategies

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

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EGFR Biology

Aberrant expression or activity of EGFR has been identified as an important factor in many human epithelial cancers, including head and neck squamous-cell carcinoma (HNSCC), NSCLC, colorectal cancer (CRC), breast cancer, pancreatic cancer and brain cancer. EGFR is a member of the EGFR tyrosine kinase family, which consists of EGFR (ErbB1/HER1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). All family members contain an extracellular ligand-binding domain (domains I, II, III, IV), a single membrane-spanning region, a juxtamembrane nuclear localization signal, and a cytoplasmic tyrosine kinase domain. HER receptors are ubiquitously expressed in various cell types, but primarily in those of epithelial, mesenchymal and neuronal origin. Under homeostatic conditions, receptor activation is tightly regulated by the availability of ligands, which collectively form the EGF family.[8] This family is divided into three distinct groups. The first includes EGF, transforming growth factor alpha (TGF-α) and amphiregulin, which all bind specifically to EGFR. The second group includes betacellulin, heparin-binding EGF and epiregulin, which bind to both EGFR and HER4. The third group is composed of the neuregulins (NRG1–4), which is further subdivided based on their ability to bind HER3 and HER4 (NRG1 and NRG2), or only to HER4 (NRG3 and NRG4).[32] HER2 has no known ligand.[33] Ligand binding to domains I and III of the RTK induces major conformational changes that lead to the dimerization loop in domain II of the receptor being exposed.[34] This exposure of the dimerization loop allows receptor homodimerization or heterodimerization at the plasma membrane. This interaction activates the RTK, which causes autophosphorylation of the cytoplasmic tails of each dimer pair. HER3 is the only family member that lacks intrinsic kinase activity;[35] however, downstream signaling is readily achieved through heterodimerization.[36] Phosphorylated cytoplasmic tails serve as docking sites for numerous proteins that contain Src homology and phosphotyrosine-binding domains.

EGFR activation stimulates many complex intracellular signaling pathways that are tightly regulated by the presence and identity of the ligand, heterodimer composition, and the availability of phosphotyrosine-binding proteins. The two primary signaling pathways activated by EGFR include the RAS/RAF/MEK/ERK and the PI3K/AKT axes; however, Src tyrosine kinases, PLCγ, PKC, and STAT activation and downstream signaling have also been well documented (Figure 1).[8,9] Tumor cell proliferation, survival, invasion and angiogenesis can be promoted through activation of these pathways. In addition to traditional cytoplasmic signaling, EGFR also acts as a membrane-bound chaperone protein for the sodium/glucose cotransporter SGLT1.[37] These results point to a new kinase-independent role for EGFR in promoting metabolic homeostasis in cancer cells.

Figure 1.

EGFR biology. a | Ligand binding to EGFR causes receptor homodimerization or heterodimerization, which leads to transphosphorylation of the cytoplasmic tail tyrosine residues. Lysine 721 (K721) is the critical site for ATP-binding and kinase activity of EGFR (shown in yellow). Mutation of this amino acid causes the receptor to become inactive.[153,154] Tyrosine phosphorylation in the C-terminus includes Y974, Y992, Y1045, Y1068, Y1086, Y1148 and Y1173 (shown in orange), or SFKs can phosphorylate Y845 and Y1101 (shown in purple). Reported biological effects of phosphorylation of each tyrosine are noted.[155–158]b | EGFR has been consistently detected in the nuclei of cancer cells, primary tumor specimens and highly proliferative tissues.[38–42] EGFR binds to STAT3 to increase expression of iNOS,[47] E2F1 to increase expression of B-Myb,[46] and with STAT5 to increase expression of Aurora A.[52] It also increases the expression of cyclin D1.[40] EGFR has kinase-dependent activity within the nucleus of proliferating cells, which includes the phosphorylation of PCNA leading to its stability and enhancing cell proliferation,[53] and translocation and activation of DNA-PK.[159] Abbreviations: AP-2, transcription factor AP-2; B-Myb, Myb-related protein B; CBL, E3 ubiquitin-protein ligase CBL; DNA-PK, DNA-dependent protein kinase catalytic subunit; E2F1, transcription factor E2F1; EGFR, epidermal growth factor receptor; GRB2, growth factor receptor-bound protein 2; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; P, phosphorylation; PCNA, proliferating cell nuclear antigen; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLCγ, 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-1; SFK, Src family kinase; SGLT1, sodium/glucose cotransporter 1; SHP1, tyrosine-protein phosphatase non-receptor type 6; SRC, proto-oncogene tyrosine-protein kinase Src; STAT, signal transducer and activator of transcription. Permission obtained from NPG © Nyati, M. K. et al. Nat. Rev. Cancer6, 876–885 (2006).

EGFR has been consistently detected in the nuclei of cancer cells from primary tumor specimens and highly proliferative tissues.[38–42] Increased nuclear EGFR localization correlates with poor clinical outcome in patients with breast cancer,[43] oropharyngeal HNSCC,[44] and ovarian cancer.[45] Nuclear localization of EGFR is associated with increased expression of cyclin D1,[40] B-Myb,[46] inducible nitric oxide synthase[47] and COX-2,[48] all of which increase G1/S progression of the cell cycle and proliferation of cancer cells. A novel nuclear localization sequence for EGFR and its family members has been reported.[49] Furthermore, mechanisms of transport of EGFR to the nucleus have been reported.[50] These mechanisms involve interactions with dynamin, importins, Sec61, and exportin-1.[50,51] More importantly, reports have indicated a mechanism of EGFR-mediated kinase-independent gene regulation in the nucleus, which involves direct interaction with the transcription factors STAT3, STAT5 and E2F1.[46,47,52] In addition, nuclear EGFR functions as a tyrosine kinase in the nucleus, phosphorylating and stabilizing proliferating cell nuclear antigen and thus enhancing the proliferative potential of cancer cells.[53] As data accrues implicating the functional impact of nuclear EGFR, it becomes valuable to understand the extent to which this protein may contribute to cancer growth and progression, but also to the therapeutic response to EGFR-targeted therapies.

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