Abstract 4012: Upregulation and sustained activation of Stat1 are essential for IFN-γ-induced Duox2/DuoxA2 expression in the human pancreatic cancer cell line BxPc3

Abstract

Abstract Pancreatic inflammation accelerates the development of pancreatic cancer, possibly due to cytokine release and the generation of reactive oxygen species (ROS). Recently, it has been documented that pancreatic tumor cells frequently produce higher concentrations of ROS than their normal cellular counterparts. Although ROS at high concentration may be cytotoxic, physiologic ROS levels play a critical role in the control of tumor cell proliferation and signal transduction. Membrane-bound, epithelial NADPH oxidase (NOX) flavoproteins are major sources of ROS in tumor cells. Dual oxidase 2 (DUOX2) is one member of the NOX gene family; it was originally identified in the thyroid gland as the H2O2-generating enzyme responsible for thyroid hormone biosynthesis. Recently, DUOX2 also has been found in upper airway mucosa and throughout the gastrointestinal tract. Duox2 is a membrane glycoprotein with a unique N-terminal heme peroxidase-like domain as well as a membrane-spanning NAD(P)H oxidase domain, with an additional cytosolic segment containing two calcium-binding domains that generate H2O2 in the extracellular milieu. The associated dual oxidase maturation factor 2 (DUOXA2), an ER-resident protein, is absolutely necessary for posttranslational processing and translocation of DUOX2 to the plasma membrane to produce functional enzymatic activity. Because of the proposed growth promotion and proinflammatory role of DUOX2, we screened a panel of cytokines to determine whether they modulate DUOX2-mediated ROS production. Among several pancreatic cancer cell lines evaluated, BxPc3 is specifically and highly responsive to IFN-γ for the induction of both Duox2 and DuoxA2 mRNA, and Duox2 protein, in a time-dependent as well as concentration-dependent manner. Redox-sensitive assays for both intra- and extracellular ROS production confirmed that IFN-γ-induced Duox2 and DuoxA2 mRNA expression correlates with DUOX2 enzymatic activity. This enzymatic activity is calcium- and flavin dehydrogenase-dependent; both the calcium chelator BAPTA-AM and the flavoprotein inhibitor DPI (200nM) significantly decreased IFN-γ-mediated ROS production. BxPc3 cell exposure to actinomycin D or cyclohexamide demonstrated that IFN-γ-mediated Duox2 and DuoxA2 expression were both transcriptionally regulated; new protein synthesis also appeared to be absolutely necessary for the upregulation of Duox2 by IFN-γ. siRNA blockade of stat1 signaling revealed that upregulation and sustained activation of stat1 plays an important role in IFN-γinduced Duox2 expression in BxPc3 cells. In summary, these experiments suggest that upregulation of DUOX2 by IFN-γ-related stimulation of the stat1 pathway may play an important role in the generation of high local concentrations of extracellular ROS that could contribute to a pro-inflammatory milieu in the pancreas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4012.