VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells

Ayumi Yoshida; Akio Shimizu; Hirotsugu Asano; Tetsuya Kadonosono; Shinae Kizaka Kondoh; Elena Geretti; Akiko Mammoto; Michael Klagsbrun; Misuzu Kurokawa Seo

doi:10.1242/bio.010918

VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells

Biol Open. 2015 Jul 24;4(9):1063-76. doi: 10.1242/bio.010918.

Authors

Ayumi Yoshida¹, Akio Shimizu², Hirotsugu Asano², Tetsuya Kadonosono³, Shinae Kizaka Kondoh³, Elena Geretti⁴, Akiko Mammoto⁴, Michael Klagsbrun⁴, Misuzu Kurokawa Seo⁵

Affiliations

¹ Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kyoto 603-8555, Japan.
² Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8047, Japan.
³ Biofunctional Engineering, Graduate School of Bioscience & Biotechnology, Tokyo Institute of Technology, Tokyo 226-8503, Japan.
⁴ Vascular Biology Program, Boston Children's Hospital, Departments of Surgery and Pathology and Harvard Medical School, Boston, MA 02115, USA.
⁵ Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kyoto 603-8555, Japan Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8047, Japan mseo@cc.kyoto-su.ac.jp.

Abstract

Neuropilin-1 (NRP1) has been identified as a VEGF-A receptor. DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1. In the present study, the RNA interference of VEGF-A or NRP1 suppressed DJM-1 cell proliferation. Furthermore, the overexpression of the NRP1 wild type restored shNRP1-treated DJM-1 cell proliferation, whereas NRP1 cytoplasmic deletion mutants did not. A co-immunoprecipitation analysis revealed that VEGF-A induced interactions between NRP1 and GIPC1, a scaffold protein, and complex formation between GIPC1 and Syx, a RhoGEF. The knockdown of GIPC1 or Syx reduced active RhoA and DJM-1 cell proliferation without affecting the MAPK or Akt pathway. C3 exoenzyme or Y27632 inhibited the VEGF-A-induced proliferation of DJM-1 cells. Conversely, the overexpression of the constitutively active form of RhoA restored the proliferation of siVEGF-A-treated DJM-1 cells. Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor. A cell-penetrating oligopeptide that targeted GIPC1/Syx complex formation inhibited the VEGF-A-induced activation of RhoA and suppressed DJM-1 cell proliferation. In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

Keywords: Cancer; Neuropilin; RhoA; Syx; VEGF-A.