Abstract 4205: Regulation of Hif-1α expression by NADPH oxidase 5 (Nox5) in human cancer cell lines

Abstract

Abstract Reactive oxygen species (ROS) produced by NADPH oxidases (Noxs) participate in signaling cascades regulating proliferation. However, the relative contribution to tumor cell growth by the seven Nox homologues (Nox1-5 and Duox 1 and 2) remains unclear. Several studies have proposed that Nox5 plays a role in promoting cell proliferation in Hairy cell leukemia, Barrett's esophageal adenocarcinoma cells, and in prostate cancer; however, little is known about the signaling pathways mediated by Nox5. We explored the functional role of Nox5 in a variety of tumor cell lines, including the NCI-60 cell panel; real time RT-PCR revealed that several melanoma lines express Nox4 and Nox5, but no Nox1 mRNA; and that prostate cell lines express Nox5 mRNA. Of the Nox5- expressing NCI-60 human cancer cell lines, we studied UACC-257 melanoma and PC-3 prostate cancer cells. These lines were characterized for variations in their N-terminus Ca2+ binding domains; RT-PCR revealed that both cell lines expressed the Nox5α and Nox5γ forms. The functional role of Nox5 in UACC-257 and PC-3 cells was studied by generating stable Nox5-overexpressing clones, and by transiently silencing Nox5 that was expressed endogenously, as well as that in clones stably overexpressing Nox5. Redox-sensitive assays for both intra- and extracellular ROS production confirmed that Nox5 was functional and generated superoxide in UACC-257 and PC-3 clones overexpressing Nox5. Nox5 could be activated by the phorbol ester PMA and by the Ca2+ ionophore ionomycin in overexpressing clones. This enzymatic activity was Ca2+- and flavin dehydrogenase-dependent; the calcium chelator BAPTA-AM and the flavoprotein inhibitor DPI (200nM) significantly decreased Nox5-mediated ROS production. Overexpression of Nox5 in UACC-257 cells resulted in increased normoxic Hif-1α expression that was also Ca2+-, flavin dehydrogenase- and ROS-dependent. Silencing Nox5 by RNAi significantly inhibited both endogenous and PMA-stimulated ROS production, confirming that ROS production was Nox5-mediated. Knockdown of both endogenous and overexpressed Nox5 in both UACC-257 and PC-3 cells resulted in decreased Hif-1α expression. Studies to characterize the mechanisms by which Nox5 modulates cell proliferation through the HIF-1α pathway are ongoing. Our experiments suggest that Nox5 expression may play an important role in the generation of high local concentrations of ROS that could contribute to activation of Hif-1α and promote cell proliferation in human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4205. doi:10.1158/1538-7445.AM2011-4205