Background and objective: Bevacizumab is an antibody of VEGF and suppresses tumor angiogenesis. Bevacizumab has exhibited some therapeutic efficacy for metastatic breast cancer in combination with chemotherapy, but its impact on overall survival has not been proved. VEGF is a multi-function molecule targeting cell members in tumor microenvironment. We aimed to reveal VEGF-related molecular mechanisms on breast cancer cells itself.

Methods and Results: VEGF-expressing 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). VEGFA-KO 231 cells showed small and rounded morphology. Migration assay demonstrated the impaired cell migration of VEGFA-KO 231 cells. Bevacizumab treatment did not induce this morphologic change and impaired migration. Exogenous VEGF addition to VEGF-KO 231 cells recovered WT 231 cell like morphology and induced cell migration. Next, we generated soluble neuropilin-1 (sNRP1) overexpressed 231 cells. sNRP1 traps VEGFA to function as an antagonist. sNRP1-231 cells exhibited small and rounded morphology and impaired cell migration similar to that of VEGFA-KO 231 cells. Knock down of NRP1 by using siRNA in 231 cells also caused impaired cell migration. We evaluated the cell morphology using scanning electron microscope. It revealed diminished filopodia formation in VEGF-KO 231 cells compared to WT-231 cells. Exogenous rhVEGF addition to VEGF-KO 231 cells recovered filopodia formation. Filopodia is a fingerlike protrusion that comes out from the cell periphery by an actin polymerization. Filopodia is required for cell development and functions such as cell migration. Our results indicate, because of the absence of VEGF/NRP1 signaling, filopodia formation is abrogated. As a result of diminished filopodia formation, VEGFA-KO 231 cells showed small and rounded shape and reduced migration. 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 filopodia formation via NRP1 for 231 cells, and that bevacizumab could not block the VEGFA/NRP1 signal.

Discussion NRP1 is a receptor of VEGF, Semaphorine and other growth factors, and expressed in variety of cancer cells. Intracellular portion of NRP1 lacks kinase domain. Previous study showed that the VEGF stimulated to form a complex between NRP1 and GIPC1/Syx/RhoA, and promoted tumor cell proliferation independently from VEGFRs (Yoshida et al, 2015). However, the precise mechanisms regarding NRP1 signaling remain unknown. In conclusion, inhibition of VEGFA/NRP1 signaling caused rounded morphology with diminished filopodia formation, which impaired cell migration of 231 cells. These findings would provide profound insights regarding molecular mechanisms in resistance to bevacizumab treatment.

Citation Format: Marina Kiso, Sunao Tanaka, Masakazu Toi, Fumiaki Sato. VEGFA/NRP1 signal contributes to filopodia formation in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1885. doi:10.1158/1538-7445.AM2017-1885