Dual oxidase 2 and pancreatic adenocarcinoma: IFN-γ-mediated dual oxidase 2 overexpression results in H 2 O 2 -induced, ERK-associated up-regulation of HIF-1α and VEGF-A

// Yongzhong Wu 1 , Jennifer L. Meitzler 1 , Smitha Antony 1 , Agnes Juhasz 1 , Jiamo Lu 1 , Guojian Jiang 1 , Han Liu 2 , Melinda Hollingshead 2 , Diana C. Haines 3 , Donna Butcher 3 , Michaela S. Panter 2 , Krishnendu Roy 2 , James H. Doroshow 1, 2 1 Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 2 Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD, USA 3 Pathology/Histotechnology Laboratory, Leidos, Inc./Frederick National Laboratory for Cancer Research, National Cancer Institute, Frederick, MD, USA Correspondence to: James H. Doroshow, email: doroshoj@mail.nih.gov Keywords: dual oxidase, NADPH oxidase, pancreatic cancer, hydrogen peroxide, angiogenesis Received: February 19, 2016      Accepted: September 01, 2016      Published: September 15, 2016 ABSTRACT Several NADPH oxidase family members, including dual oxidase 2 [DUOX2], are expressed in human tumors, particularly gastrointestinal cancers associated with long-standing chronic inflammation. We found previously that exposure of pancreatic ductal adenocarcinoma cells to the pro-inflammatory cytokine IFN-γ increased DUOX2 expression (but not other NADPH oxidases) leading to long-lived H 2 O 2 production. To elucidate the pathophysiology of DUOX2-mediated H 2 O 2 formation in the pancreas further, we demonstrate here that IFN-γ-treated BxPC-3 and CFPAC-1 pancreatic cancer cells (known to increase DUOX2 expression) produce significant levels of intracellular oxidants and extracellular H 2 O 2 which correlate with concomitant up-regulation of VEGF-A and HIF-1α transcription. These changes are not observed in the PANC-1 line that does not increase DUOX2 expression following IFN-γ treatment. DUOX2 knockdown with short interfering RNA significantly decreased IFN-γ-induced VEGF-A or HIF-1α up-regulation, as did treatment of pancreatic cancer cells with the NADPH oxidase inhibitor diphenylene iodonium, the multifunctional reduced thiol N-acetylcysteine, and the polyethylene glycol-modified form of the hydrogen peroxide detoxifying enzyme catalase. Increased DUOX2-related VEGF-A expression appears to result from reactive oxygen-mediated activation of ERK signaling that is responsible for AP-1-related transcriptional effects on the VEGF-A promoter. To clarify the relevance of these observations in vivo , we demonstrate that many human pre-malignant pancreatic intraepithelial neoplasms and frank pancreatic cancers express substantial levels of DUOX protein compared to histologically normal pancreatic tissues, and that expression of both DUOX2 and VEGF-A mRNAs is significantly increased in surgically-resected pancreatic cancers compared to the adjacent normal pancreas.