Abstract
The role and function of a novel human AlkB homologue, ALKBH3, in human urothelial carcinoma development were examined. Biologic roles of ALKBH3 were examined by gene silencing analysis using in vitro and in vivo siRNA transfection. Immunohistochemical analyses of ALKBH3 and the related molecules using human bladder cancer samples were conducted to estimate the association with clinicopathologic or prognostic parameters. 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 phenotypes. ALKBH3 contributes to development of urothelial carcinomas by accelerating their survival, angiogenesis, and invasion through NOX-2-ROS and Tweak/Fn14-VEGF signals.
Highlights
In Escherichia coli, the AlkB protein, a repair enzyme of methylation-induced DNA damage, is implicated in preventing carcinogenesis [1, 2]
Protein expression was strongly reduced in both the human urothelial carcinoma cell lines UMUC2 and UMUC3, when transfected with 100 nmol/L of ALKBH3 siRNA for up to 72 hours (Fig. 1A)
Given that ALKBH8, an ALKBH family members, ALKBH8, contribute to reactive oxygen species (ROS) generation through NOX1 in human urothelial carcinoma cells [4], we examined whether ROS levels in cancer cells were affected by ALKBH3 gene expression
Summary
In Escherichia coli, the AlkB protein, a repair enzyme of methylation-induced DNA damage, is implicated in preventing carcinogenesis [1, 2]. Human homologues of the AlkB family include the genes ALKBH1-8 [3]. We have tried to identify the functional roles of ALKBH members in the progression of several types of human cancer. Human bladder cancer development [4]. ALKBH3 plays an important role in human pulmonary adenocarcinoma or prostatic adenocarcinoma cell survival and may be a therapeutic target [5, 6]. These reports show that ALKBH members are involved in a variety of biologic functions, including DNA repair, and may be of high-therapeutic value
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