Human Cancer Biology ALKBH3 Contributes to Survival and Angiogenesis of Human Urothelial Carcinoma Cells through NADPH Oxidase and Tweak/Fn14/VEGF Signals

Purpose: The role and function of a novel human AlkB homologue, ALKBH3, in human urothelial carcinoma development were examined. Experimental design: Biologic roles of ALKBH3 were examined by gene silencing analysis using in vitro and in vivo siRNA transfection. Immunohistochemical analyses of ALKBH3 and the relatedmolecules using human bladder cancer samples were conducted to estimate the association with clinicopathologic or prognostic parameters. Results: ALKBH3 knockdown induced cell cycle arrest at the G1 phase through downregulation of NAD(P)H oxidase-2 (NOX-2)–mediated generation of reactive oxygen species (ROS). ALKBH3 knockdown reduced VEGF expression by reducing expression of tumor necrosis factor-like weak inducer of apoptosis (Tweak) and its receptor, fibroblast growth factor-inducible 14 (Fn14). Silencing of ALKBH3 or Tweak significantly suppressed invasion and angiogenesis of urothelial carcinoma in vivo as assessed both by a chorioallantoic membrane assay and in an orthotopic mouse model. Interestingly, not only urothelial carcinoma cells but also vascular endothelial cells within cancer foci expressed Fn14, which was strongly reduced by ALKBH3 and Tweak knockdown in vivo, suggesting that ALKBH3-dependent expression of Tweak stabilizes Fn14. Immunohistochemical examination showed high expression of ALKBH3, Tweak, and Fn14 in urothelial carcinoma, especially in high-grade, superficially, and deeply invasive carcinomas; moreover, Fn14-positive vessel counts within cancer foci were increased in invasive