Molecular and Cellular Pathobiology NADPH Oxidase NOX4 Supports Renal Tumorigenesis by Promoting the Expression and Nuclear Accumulation of HIF2a Jennifer L. Gregg 1 , Robert M. Turner II 1 , Guimin Chang 1 , Disha Joshi 1 , Ye Zhan 2 , Li Chen 1 , and Jodi K. Maranchie 1 Abstract Most sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia- inducible factor-2a (HIF2a), a master transcriptional regulator of genes that drive diverse processes, including angiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2a 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 HIF2a 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. Cancer Res; 74(13); 3501–11. Ó2014 AACR. Introduction Renal cell carcinoma (RCC) is a common adult malignancy with an estimated 65,150 new cases and 13,680 deaths in the United States in 2013 (1). Localized disease can be treated by surgical resection alone, but advanced RCC is notoriously resistant to cytotoxic therapy or radiation. Immunotherapy, the mainstay of treatment for several decades, is curative in fewer than 15% (2). Advances in the molecular genetics of kidney cancer have led to FDA-approval of targeted agents with good clinical response rates (3). However, complete, durable responses are rare, and novel therapeutic approaches are still desperately needed. More than 80% of clear cell RCCs have lost or mutated both alleles of the von Hippel Lindau (VHL) tumor suppressor. VHL is the binding subunit of an E3 ubiquitin ligase complex that targets the a subunits of hypoxia-inducible transcription factors 1 and 2 (HIFa) for ubiquitin-mediated, proteasomal degradation. In the absence of VHL, HIFa accumulate in the cell, leading to increased transcription of more than 100 HIF- regulated genes involved in angiogenesis, anaerobic metabo- lism, proliferation, and other cell survival pathways. We and others have shown that HIF2a is the relevant oncogenic target of VHL degradation. Forced accumulation of HIF2a is suffi- cient to support xenograft growth of RCC cells despite rein- troduction of wild-type VHL, (4, 5) and specific HIF2a inhibi- tion suppresses tumor growth (6). In contrast, forced expres- sion of HIF1a suppresses xenograft growth, (4, 7), and specific HIF1a shRNA enhances xenograft growth (8). HIF1a and HIF2a have unique, nonoverlapping regulatory profiles sug- gesting a more proapoptotic rather than proproliferative role for the former (7, 9). In short, specific activation of HIF2a seems to be critical for renal tumorigenesis. We previously reported that HIF2a expression and trans- activation are dependent upon expression of the NADPH oxidase 4 (Nox4; ref. 10). In the adult human, Nox4 is most highly expressed in the distal renal tubule where it generates intracellular superoxide and is implicated in oxygen sensing for regulation of erythropoietin, a HIF-dependent gene (11). In contrast to other Nox isoforms, Nox4 requires only p22 phox for coactivation (12). In renal cancer cells, Nox4 is a major source of intracellular reactive oxygen species (ROS; ref. 13). We hypothesized that this heightened oxidative state might pro- mote HIF2a transactivation under normal oxygen conditions. Authors' Affiliations: 1 University of Pittsburgh, Department of Urology and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania; 2 Depart- ment of Surgery, University of Massachusetts, Worcester, Massachusetts Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). J.L. Gregg and R.M. Turner II contributed equally to this work. Current address for Y. Zhan: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts, Massachusetts Corresponding Author: Jodi K. Maranchie, University of Pittsburgh, 5200 Centre Avenue, Suite 209, Pittsburgh, PA 15232. Phone: 412-623-3442; Fax: 412-605-3030; E-mail: maranchiejk@upmc.edu doi: 10.1158/0008-5472.CAN-13-2979 Ó2014 American Association for Cancer Research. Cancer Research www.aacrjournals.org 3501 Downloaded from http://aacrjournals.org/cancerres/article-pdf/74/13/3501/2700488/3501.pdf by guest on 19 June 2022