Gene silencing of indoleamine 2,3-dioxygenase 2 in melanoma cells induces apoptosis through the suppression of NAD+ and inhibits in vivo tumor growth

// Yanling Liu 1, 2, 3, 4 , Yujuan Zhang 1, 3 , Xiufen Zheng 4 , Xusheng Zhang 4 , Hongmei Wang 1 , Qin Li 1 , Keng Yuan 1, 3 , Nanjing Zhou 1, 3 , Yanrong Yu 1, 3 , Na Song 1 , Jiamin Fu 1 , Weiping Min 1, 3, 4 1 Institute of Immunotherapy of Nanchang University, and Jiangxi Academy of Medical Sciences, Nanchang, China 2 Jiangxi University of Technology, Nanchang, China 3 Jiangxi Provincial Key Laboratory of Immunotherapy, Nanchang, China 4 Department of Surgery, Pathology, and Oncology, University of Western Ontario, London, Canada Correspondence to: Yujuan Zhang, email: zyj900203@sina.com Weiping Min, email: mweiping@uwo.ca Keywords: IDO2, NAD+, apoptosis, neoplasm, siRNA Received: December 13, 2015      Accepted: March 14, 2016      Published: April 6, 2016 ABSTRACT Indoleamine 2,3-dioxygenase 2 (IDO2) is a newly discovered enzyme that catalyzes the initial and rate-limiting step in the degradation of tryptophan. As a homologous protein of IDO1, IDO2 plays an inhibitory role in T cell proliferation, and it is essential for regulatory T cell (Treg) generation in healthy conditions. Little is known about the immune-independent functions of IDO2 relevant to its specific contributions to physiology and pathophysiology in cancer cells. The purpose of this study was to assess the impact of IDO2 gene silencing as a way to inhibit B16-BL6 cancer cells in a murine model. Here, for the first time, we show that knockdown of IDO2 using small interfering RNA (siRNA) inhibits cancer cell proliferation, arrests cell cycle in G1, induces greater cell apoptosis, and reduces cell migration in vitro . Knockdown of IDO2 decreased the generation of nicotinamide adenine dinucleotide (NAD+) while increasing the generation of reactive oxygen species (ROS). We further demonstrate that cell apoptosis, induced by IDO2 downregulation, can be weakened by addition of exogenous NAD+, suggesting a novel mechanism by which IDO2 promotes tumor growth through its metabolite product NAD+. In addition to in vitro findings, we also demonstrate that IDO2 silencing in tumor cells using short hairpin RNA (shRNA) delayed tumor formation and arrested tumor growth in vivo . In conclusion, this study demonstrates a new non-immune-associated mechanism of IDO2 in vitro and IDO2 expression in B16-BL6 cells contributes to cancer development and progression. Our research provides evidence of a novel target for gene silencing that has the potential to enhance cancer therapy.