Abstract 4796: Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody.
Authors: Antony Smitha , Wu Yongzhong , Hewitt Stephen M. , Anver Miriam R. , Jiang Guojian , Meitzler Jennifer L. , Liu Han , Juhasz Agnes , Lu Jiamo , Roy Krishnendu K. , Doroshow James H. 
Published: August 2014

NADPH oxidase (Nox) family enzymes (Nox1-5 and Duox1-2) generate reactive oxygen species (ROS) and modulate cellular pathways that regulate tumorigenesis. Recent studies have proposed that Nox5 plays a role in the proliferative capacity of Hairy cell leukemia, Barrett's esophageal adenocarcinoma, and in prostate cancer cells. Although implicated in several malignancies, the role of Nox5 in tumor cell biology is still unclear. This impediment is due to a lack of reliable immunological probes as well as the fact that little is known regarding Nox5 expression in human tumors. In this study, we report for the first time the generation and characterization of a mouse monoclonal antibody against a recombinant Nox5 protein (600-746), and the expression profile of Nox5 in human tumors by tissue microarray analysis. We also report the detection of endogenous Nox5 protein in the UACC-257 human melanoma cell line using our novel Nox5 antibody. Specificity of the antibody for Nox5 was confirmed by generating stable Nox5 over-expressing UACC-257 cell clones, and by transiently silencing both endogenous Nox5 and stably overexpressed Nox5. Additionally, no cross-reactivity of the antibody with other Nox homologs was detected. Sub-cellular fractionation studies revealed that Nox5 was predominantly expressed in the membranes of UACC-257 cells and its Nox5 overexpressing clones. Immunofluorescence, confocal microscopy, and immunohistochemistry demonstrated that Nox5 localizes throughout parental UACC-257 cells and Nox5 overexpressing clones, with perinuclear enhancement. To evaluate the clinical significance of Nox5 in human cancer, we determined the expression of Nox5 protein in a representative set (382 samples) of human tumors. Tissue microarray analysis revealed substantial Nox5 overexpression in several human tumors of epithelial origin including breast, colon, lung, prostate, and ovarian cancers, as well as non-epithelial malignancies (glioblastoma multiforme, melanoma, and lymphoma). Expression was markedly increased in tumors compared to normal tissues of the same organ. Collectively, our study provides the first demonstration of Nox5 overexpression in human tumors by tissue microarray, and reports on the first mouse monoclonal antibody against Nox5. The elevated expression of Nox5 in tumors provides impetus to explore the functional significance of Nox5 in the context of tumor development and maintenance.

Citation Format: Smitha Antony, Yongzhong Wu, Stephen M. Hewitt, Miriam R. Anver, Guojian Jiang, Jennifer L. Meitzler, Han Liu, Agnes Juhasz, Jiamo Lu, Krishnendu K. Roy, James H. Doroshow. Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4796. doi:10.1158/1538-7445.AM2013-4796