Mediating EGFR-TKI Resistance by VEGF/VEGFR Autocrine Pathway in Non-Small Cell Lung Cancer

SIMPLE SUMMARY: Non-small cell lung cancer (NSCLC) patients acquire resistance to tyrosine kinase inhibitors (TKIs) via EGFR mutations or overexpression of vascular endothelial growth factor receptor-2 (VEGFR-2). In this study, we elucidated the mechanism of EGFR-TKI resistance mediated by VEGF/VEGFR in EGFR-mutated NSCLC cell lines and Erlotinib-resistant cell lines as compared to parental cell lines. Increased expression of VEGF, VEGFR-2, and NP1 was observed in Erlotinib-resistant cell lines. Furthermore, we observed an increased efficacy of Erlotinib in combination with a VEGFR-2 inhibitor in Erlotinib-resistant cell lines. Late-stage NSCLC patients with high expression of VEGFR-2 had shorter survival times compared to patients with low VEGFR-2 expression. These results indicate that VEGFR-2 may play a key role in EGFR-TKI resistance that can be overcome with a combination treatment of Erlotinib and a VEGFR-2 inhibitor, which may serve as an effective treatment option for NSCLC patients with EGFR mutations. ABSTRACT: NSCLC treatment includes targeting of EGFR with tyrosine kinase inhibitors (TKIs) such as Erlotinib; however, resistance to TKIs is commonly acquired through T790M EGFR mutations or overexpression of vascular endothelial growth factor receptor-2 (VEGFR-2). We investigated the mechanisms of EGFR-TKI resistance in NSCLC cell lines with EGFR mutations or acquired resistance to Erlotinib. These studies showed upregulated gene and protein expression of VEGF, VEGFR-2, and a VEGF co-receptor neuropilin-1 (NP-1) in Erlotinib-resistant (1.4–5.3-fold) and EGFR double-mutant (L858R and T790M; 4.1–8.3-fold) NSCLC cells compared to parental and EGFR single-mutant (L858R) NSCLC cell lines, respectively. Immunofluorescence and FACS analysis revealed increased expression of VEGFR-2 and NP-1 in EGFR-TKI-resistant cell lines compared to TKI-sensitive cell lines. Cell proliferation assays showed that treatment with a VEGFR-2 inhibitor combined with Erlotinib lowered cell survival in EGFR double-mutant NSCLC cells to 9% compared to 72% after treatment with Erlotinib alone. Furthermore, Kaplan–Meier analysis revealed shorter median survival in late-stage NSCLC patients with high vs. low VEGFR-2 expression (14 mos vs. 21 mos). The results indicate that VEGFR-2 may play a key role in EGFR-TKI resistance and that combined treatment of Erlotinib with a VEGFR-2 inhibitor may serve as an effective therapy in NSCLC patients with EGFR mutations.