Abstract 2595: Role of VEGF/VEGFR autocrine in mediating EGFR TKI resistance in non-small cell lung cancer
Authors: Osude Chike , Lin Leo , Foster Brad , Puri Neelu 
Published: August 2018

The treatment paradigm for late stage non-small cell lung carcinoma (NSCLC) has shifted towards molecular targeted therapy like epidermal growth factor receptor (EGFR), one of the most studied targets. Tyrosine kinase inhibitors (TKIs) against EGFR have proven to be effective, however TKI resistance is common due to the acquisition of the T790M mutation in the EGFR kinase domain. There are currently very few studies suggesting the role of VEGFR in mediating erlotinib resistance. We suggest that over-expression of vascular endothelial growth factor receptors (VEGFRs) in tumors may create an autocrine loop that may mediate TKI resistance. The purpose of this study is to investigate the VEGF/VEGFR autocrine loop as a potential mechanism of TKI resistance via alternative signaling. H2170 NSCLC parental cells were obtained from ATCC. The resistant cells were obtained by treating parental cells with increasing concentrations of erlotinib. DNA sequencing was performed to exclude the presence of the T790M mutation. Quantitative real-time PCR was carried out for target genes coding for VEGF, VEGFR-2, and neuropilin-1 (NP-1). Fluorescence-activated cell sorting (FACS) analysis and immunofluorescence (IF) analysis were performed with antibodies against VEGFR-2 and NP-1. The viability of resistant cells was assessed after being treated with increasing concentrations of a VEGF inhibitor (VEGF neutralizing antibody) in medium with or without erlotinib using a cell proliferation assay (MTT). Thirty paraffin embedded tumor biopsies were immunostained (IHC) with an antibody against VEGFR-2. On the transcriptional level, real-time PCR revealed a 13.1, 3.4 and a 2.9 fold increase in gene expression for VEGF, VEGFR-2 and NP-1 respectively in the resistant cells compared to the parental cells. When looking at membrane bound receptors, FACS analysis showed a 2.1 and a 3.8 fold increase in the expression of VEGFR and NP-1 in resistant cells, respectively. IF studies correlated with the FACS results, showing significantly higher expression of both VEGFR-2 and NP-1 in the resistant cells. MTT assays demonstrated that the VEGF inhibitor was able to decrease cell death from 10 to 80% (0.1-10ug/ml) as evidenced by decreasing viability of resistant cells treated with increasing concentrations of VEGF inhibitor compared to the untreated control. Kaplan-Meier analysis revealed shorter median survival with high vs low expression of total VEGFR-2 expression (7mo vs 25mo, p=0.05) in NSCLC tumors in patients diagnosed with late stage NSCLC. Our results suggest that by up-regulating the VEGF/VEGFR autocrine system as an alternative signaling pathway to promote proliferation, tumor cells may bypass the inhibitory effect of TKI's on the EGFR pathway, leading to resistance. Furthermore, IHC demonstrated the presence of high expression of VEGFR-2 in tumors, suggesting its potential as a prognostic biomarker for lung cancer.

Citation Format: Chike Osude, Leo Lin, Brad Foster, Neelu Puri. Role of VEGF/VEGFR autocrine in mediating EGFR TKI resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2595.