Abstract 683: VEGFA/NRP1 signal contributes to cell adhesion and motility in breast cancer cells

Abstract

Abstract Background and objective: Bevacizumab suppressed tumor angiogenesis by blocking the VEGFA signal into endothelial cells. Although, bevacizumab, in combination with chemotherapy, has exhibited some therapeutic efficacy for metastatic breast cancer, its impact on overall survival has not been proved. According to recent studies, VEGF is a multi-function molecule targeting not only endothelial cells but also other cell members in tumor microenvironment, including cancer cells, fibroblasts, immune cells, and so on. In this study, we aimed to reveal VEGF-related molecular mechanisms on breast cancer cells itself. Methods and Results: A VEGF-expressing basal-like type breast cancer cell line, MDA-MB-231 cells (231 cells) were used. VEGFA of 231 cell was knocked out using Crisper-Cas9 system (VEGFA-KO). Compared to the wild type (WT) 231 cells, VEGFA-KO 231 cells showed rounded and weakened adhesive morphology. Scratch assay and motion picture assay demonstrated the VEGFA-KO 231 cells had impaired cell motility. Bevacizumab treatment to WT 231 cells did not induce this morphologic change. Next, we generated soluble neuropilin-1 (sNRP1) overexpressed 231 cells. sNRP1 consists of the NRP1 extracellular domain that includes a VEGFA-binding site, traps VEGFA to function as an antagonist. sNRP1-231 cells exhibited rounded and lower adhesive morphology similar to that of VEGFA-KO 231 cells. Bevacizumab and NRP1 bind VEGFA at amino acid motifs from exon 3-4 and exon 7 of VEGFA, respectively. Thus, our findings suggested that VEGFA could play a role in cell morphology and motility via NRP1 for 231 cells, and that bevacizumab could not block the VEGFA/NRP1 signal. Discussion NRP1 is a transmembrane protein that has been identified as a VEGF-A receptor. NRP1 is expressed by endothelial cells and functions as a co-receptor of VEGFRs, enhancing VEGF-A binding and promoting downstream signaling. NRP1 is also expressed in variety of cancer cells including breast cancer. Since NRP1 molecule lacks kinase activity, there have been efforts to find the mechanisms of NRP1 signaling. To date, NRP1 expression of tumor cells has been shown to contribute to proliferative signal transduction from VEGF-A. Previous study showed VEGF-A/NRP1 signals induced the phosphorylation of Akt leading to breast cancer cell survival (Bachelder et al., 2001). And another study showed that the NRP1 cytoplasmic region enhanced the interaction between NRP1 and GIPC1/Syx/RhoA, and promoted tumor cell proliferation. However, the precise mechanisms regarding NRP1 signaling remains unknown. In conclusion, knocking out of VEGFA and trapping of VEGFA by sNRP1 induced weakened adhesive morphology and impaired cell motility in 231 cells. Because cell adhesion and motility are important factors for tumor metastasis, VEGFA /NRP1 signaling in breast cancer might be related to breast cancer metastasis, and can be a useful therapeutic/preventive target for breast cancer. Citation Format: Marina Kiso, Fumiaki Sato, Sunao Tanaka, Masakazu Toi. VEGFA/NRP1 signal contributes to cell adhesion and motility in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 683.