Abstract 2862: Long isoform of VEGF stimulates cell migration of breast cancer by filopodia formation via NRP1/ARHGAP17/Cdc42 regulatory network
Authors: Marina Kiso, Sunao Tanaka, Masakazu Toi, Fumiaki Sato
Journal: Cancer Research (2019)

Abstract Background and objective: Bevacizumab is an antibody of VEGF and suppresses tumor angiogenesis. It exhibited therapeutic efficacy for metastatic breast cancer, but impact on overall survival has not been proved. VEGF is a multi-function molecule. We revealed VEGF-related molecular mechanisms on breast cancer (BC). Methods and Results: A VEGF-expressing BC cell line, MDA-MB-231 cells (WT231) were used. VEGF of WT231 cells was knocked out using Crisper-Cas9 system (231VEGFKOex3). 231VEGFKOex3 cells showed small and rounded morphology. Migration assay demonstrated the impaired cell migration of 231VEGFKOex3 cells. Bevacizumab treatment did not induce this morphologic change and impaired migration. Next, we generated soluble neuropilin-1 (sNRP1) overexpressed 231 cells (231sNRP1). sNRP1 traps VEGF to function as an antagonist. 231sNRP1 cells exhibited small and rounded morphology and impaired cell migration similar to that of 231VEGFKOex3 cells. VEGF165 (contains a NRP-binding domain) addition to 231VEGFKOex3 cells recovered WT231 cell-like morphology and induced cell migration. On the other hand, VEGF121 (doesn’t contain NRP-binding domain) addition could not recover morphology and migration. As WT231 cells express almost no VEGFR, these results indicate that the interaction between NRP1 and long isoform of VEGF containing a NRP-binding domain regulates the morphology and migration ability of 231 cells. Genome-wide gene expression profiling identified ARHGAP17 as one of the target genes in the downstream of the VEGF/NRP1 signal. The expression of ARHGAP17 was upregulated in 231VEGFKOex3 cells. ARHGAP17 is a RhoGAP which inactivates Cdc42. Cdc42 is important for the formation of filopodia which required for cell functions such as migration. Scanning electron microscope revealed diminished filopodia formation in 231VEGFKOex3 cells compared to WT231 cells. As a result, 231VEGFKOex3 cells showed reduced migration. Our results indicate that VEGF/NRP1/ARHGAP17/Cdc42 signaling network controls filopodia formation and migration of the BC cells. Among 450 hormone-receptor-negative BC (HR(-)BC) cases from a public database, the ratio of VEGF and SEMA3A in primary tumor is associated with ARHGAP17 expression inversely, and with disease free survival. Discussion: Bevacizumab and NRP1 bind VEGF at amino acid motifs from exon 3-4 and exon 7 of VEGF, respectively. Thus, our findings suggested that long isoform of VEGF could play a role in filopodia formation via VEGF/NRP1/ARHGAP17/Cdc42 singaling network for WT231 cells, and that bevacizumab could not block the signal. In conclusion, long isoform of VEGF can generate a signal through NRP1 without VEGFR into BC cells. In addition, we identified a target molecule of this signal, ARHGAP17, that regulates Cdc42 activity. VEGF/NRP1/ARHGAP17/Cdc42 regulatory network may contribute to malignant behaviors of HR(-)BC. Citation Format: Marina Kiso, National Hospital Organization Himeji Medical Center, Sunao Tanaka, Masakazu Toi, Fumiaki Sato. Long isoform of VEGF stimulates cell migration of breast cancer by filopodia formation via NRP1/ARHGAP17/Cdc42 regulatory network [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 2862.