Most sporadically occurring renal tumors include a functional loss of the tumor suppressor VHL. Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor HIF-2α, a master transcriptional regulator of genes that drive diverse processes including angiogenesis, proliferation and anaerobic metabolism. In determining the critical functions for HIF-2α expression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions of NOX4 to RCC have not been evaluated directly. Here we report that NOX4 silencing in VHL-deficient RCC cells abrogates cell branching, invasion, colony formation and growth in a murine xenograft model RCC. These alterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression of manganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficient to block nuclear accumulation of HIF-2α in RCC cells. Our results offer direct evidence that NOX4 is critical for renal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cells are genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade.

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PURPOSE Cell Adhesion Molecules (CADMs) family comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3 and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer. EXPERIMENTAL DESIGN CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by RT-PCR and an immunohistochemical tissue array respectively. Effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR (MSP). RESULTS We report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines LNCaP and DU145 expressed negligible CADM-2a relative to primary prostate tissue and cell lines RWPE-1 and PPC-1 while CADM-2b was maintained. Tissue array results from clinical specimens using immunohistochemistry demonstrated statistically significant decreased expression in prostate carcinoma compared to normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia (PIN), and normal tissue adjacent to tumor (P<0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and MSP. Accordingly, treatment of cells with the demethylating agent 5-aza-2′-deoxycytidine alone or in combination with the histone deacetylase inhibitor Trichostatin A resulted in reactivation of CADM-2a expression. CONCLUSIONS CADM-2 protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers.

Results show that G complemented recombinant VSV-DeltaG vectors, especially rVSV-DeltaG-SV5-F, are an effective oncolytic agent against mouse prostate cancer cells in vitro and in an in vivo immunocompetent mouse model system.