Original Articles Hypoxia-induced IL-32β increases glycolysis in breast cancer cells Jeong Su Park a , Sunyi Lee a , Ae Lee Jeong a , Sora Han a , Hye In Ka a , Jong-Seok Lim a , Myung Sok Lee a , Do-Young Yoon b , Jeong-Hyung Lee c , Young Yang a, * a Department of Biosystems, Sookmyung Women’s University, Seoul 140-742, Republic of Korea b Department of Bioscience and Biotechnology, Konkuk University, Seoul, Republic of Korea c Department of Biochemistry, Kangwon National University, Chuncheon 200-701, Republic of Korea ARTICLE INFO Article history: Received 14 August 2014 Received in revised form 28 October 2014 Accepted 28 October 2014 Keywords: Interleukin-32 Hypoxia Mitochondrial biogenesis OXPHOS Glycolysis A B ST R AC T IL-32β is highly expressed and increases the migration and invasion of gastric, lung, and breast cancer cells. Since IL-32 enhances VEGF production under hypoxic conditions, whether IL-32β is regulated by hypoxia was examined. Hypoxic conditions and a mimetic chemical CoCl2 enhanced IL-32β production. When cells were treated with various inhibitors of ROS generation to prevent hypoxia-induced ROS func- tion, IL-32β production was suppressed by both NADPH oxidase and mitochondrial ROS inhibitors. IL- 32β translocated to the mitochondria under hypoxic conditions, where it was associated with mitochondrial biogenesis. Thus, whether hypoxia-induced IL-32β is associated with oxidative phosphorylation (OXPHOS) or glycolysis was examined. Glycolysis under aerobic and anaerobic conditions is impaired in IL-32β- depleted cells, and the hypoxia-induced IL-32β increased glycolysis through activation of lactate dehydrogenase. Src is also known to increase lactate dehydrogenase activity, and the hypoxia-induced IL-32β was found to stimulate Src activation by inhibiting the dephosphorylation of Src. These findings revealed that a hypoxia-ROS-IL-32β-Src-glycolysis pathway is associated with the regulation of cancer cell metabolism. © 2014 Elsevier Ireland Ltd. All rights reserved. Introduction Tumor cells are frequently exposed to low nutrient and oxygen levels, thus the cells stimulate angiogenesis to overcome hypoxic conditions [1,2]. The metabolism of cancer cells should also be altered in other ways to adapt to hypoxic conditions for survival and pro- gression [2]. Under hypoxic conditions, the hypoxia inducible factor HIF-l activates LDH-A, which catalyzes the conversion of pyruvate to lactate by utilizing NADH, leading to aerobic glycolysis [3,4]. HIF-1 also activates PDK1, resulting in the inhibition of pyruvate dehy- drogenase [4]. Thereby, pyruvate shunts to lactate, instead of being converted to acetyl-CoA [3,5]. In addition to the regulation of glycolysis-related enzymes by HIF-1, hypoxia also increases ROS, the by-products of rapid metabolism. The increased ROS inactivates PKM2 by modifying a critical sulfhydryl group, and also activates PFKFB4 through the stabilization of HIF-1 [2,6]. These two changes initiated by hypoxia shunt glycolysis to the PPP, which is needed for the anabolic pathway in fast growing tumor cells [7,8]. On the other hand, hypoxia also activates PFKFB3, which drives glycoly- sis. Thus, the balance of PFKFB4 and PFKFB3 determines the metabolic fate of tumor cells. IL-32 is known as a proinflammatory cytokine because it en- hances the production of IL-1β and TNFα [9–11]. In addition to its proinflammatory role in the immune system, many roles of IL-32 in cancer cells have recently been unveiled. IL-32 is overexpressed in many types of cancer [12–14]. However, the role of IL-32 is dif- ferent with respect to the type of cancer. IL-32α, for example, shows anti-cancer effects in human leukemia and colon cancer cells [15,16], whereas it enhances the proliferation of pancreatic cells [14]. In addition, IL-32γ inhibits cancer growth through inactivation of NF- κB and STAT3 signaling, and enhances TNF-α-induced cancer death in colon cancer [15]. Contrary to the controversial role of IL-32 in cancer cell proliferation and death, several reports have unambigu- ously shown that IL-32 enhances the migration and invasion of several cancers, such as breast, gastric and lung [13,17,18]. We re- ported previously that IL-32β increases breast cancer migration and the invasion of breast cancer cell lines through VEGF-STAT3 sig- naling [13]. Now we report that hypoxia-induced ROS increase the levels of IL-32β, resulting in enhanced glycolysis in breast cancer cells. Abbreviation: ECAR, extracellular acidification rate; HIF-1, hypoxia inducible factor- 1; IL-32, interleukin-32; LDH-A, lactate dehydrogenase A; OCR, oxygen consumption rate; OXPHOS, oxidative phosphorylation; PDK1, pyruvate dehydrogenase kinase; PFKFB4, fructose-2,6-bisphosphatase 4; PKM2, pyruvate kinase M2; PP2A, protein phosphatase 2A; PPP, pentose phosphate pathway; ROS, reactive oxygen species. * Corresponding author. Tel.: +82 2 710 9590; fax: +82 2 2077 7322. E-mail address: yyang@sm.ac.kr (Y. Yang). http://dx.doi.org/10.1016/j.canlet.2014.10.030 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved. Cancer Letters 356 (2015) 800–808 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet