Abstract 189: EGFR TKI resistance via role VEGFR2
Authors: Osude Chike , Lin Leo , Puri Neelu 
Published: August 2019

The treatment paradigm for late stage non-small cell lung carcinoma (NSCLC) has shifted towards molecular targeted therapy like epidermal growth factor receptor (EGFR). Tyrosine kinase inhibitors (TKIs) against EGFR have proven to be effective; however, TKI resistance is commonly acquired by the T790M mutation in the EGFR kinase domain. Very few studies suggest the role of vascular endothelial growth factor receptor (VEGFR) in mediating erlotinib resistance. We suggest that overexpression of VEGFR-2 in tumors creates an autocrine loop that mediates TKI resistance. Thus, our purpose is to investigate the VEGF/VEGFR autocrine loop as a potential mechanism of TKI resistance via alternative signaling.

H2170, H358, H1975 and H3255 NSCLC parental cells were obtained from ATCC. Parental cells were then treated with erlotinib to create resistant cells. DNA sequencing was used to exclude the presence of the T790M mutation. Quantitative real-time PCR (qPCR) was conducted to code for VEGF, VEGFR-2, and neuropilin-1 (NP-1) genes and immunoblotting (WB) was used to detect their protein expression. Fluorescence-activated cell sorting (FACS) analysis and immunofluorescence (IF) analysis was performed with antibodies against VEGFR-2 and NP-1. Secretion of VEGF from NSCLC cells was studied at 24/48 hours by ELISA. Cell proliferation assay (MTT) was conducted to assess the viability of resistant cells after treatment with VEGFR-2 inhibitor and VEGF neutralizing antibody in media with/without erlotinib. 48 and 31 tumor biopsies were immunostained (IHC) with anti-VEGFR-2 and anti-NP-1 antibodies, respectively.

qPCR showed a 1.4 to 3.5-fold increase in expression of VEGF, VEGFR-2 and NP-1 in H2170 resistant cells and a 2.2 to 3.6-fold increase in expression in H358 resistant cells in comparison to their parental cells. WB results showed an increase in protein expression in resistant cells compared to parental cells. FACS analysis showed a 1.3 to 6-fold increase in expression of VEGFR and NP-1 in resistant cells which was confirmed by IF, showing higher expression of both VEGFR-2 and NP-1 in the resistant cells. ELISA studies showed that VEGF secretion increases over time. MTT assays showed that the VEGFR-2 inhibitor (ZM HCl 323-881) lowered cell death to 10-57% (p=0.05) with erlotinib in resistant cells compared to the erlotinib alone (72-75%). Kaplan-Meier analysis revealed shorter median survival with high vs low expression of total VEGFR-2 expression (14mo vs 21mo, p=0.05) in patients NSCLC tumors.

Our results suggest that by upregulating the VEGF/VEGFR autocrine system as an alternative signaling pathway to promote proliferation, tumor cells may bypass the inhibitory effect of TKIs on the EGFR pathway, ultimately overcoming resistance. Furthermore, IHC demonstrated that high expression of VEGFR-2 is associated with poor prognosis in tumors, suggesting its potential as a prognostic biomarker for lung cancer.

Citation Format: Chike Osude, Leo Lin, Neelu Puri. EGFR TKI resistance via role VEGFR2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 189.