Published OnlineFirst August 3, 2012; DOI: 10.1158/0008-5472.CAN-12-0979 Cancer Therapeutics, Targets, and Chemical Biology Research Inactivation of the HIF-1a/PDK3 Signaling Axis Drives Melanoma toward Mitochondrial Oxidative Metabolism and Potentiates the Therapeutic Activity of Pro-Oxidants Jerome Kluza1, Paola Corazao-Rozas1, Yasmine Touil1, Manel Jendoubi1, Cyril Maire1, Pierre Guerreschi1, Aurelie Jonneaux1, Caroline Ballot1,Stephane Balayssac4, Samuel Valable2,3, Aurelien Corroyer-Dulmont2,3, Myriam Bernaudin2,3, Myriam Malet-Martino4, Elisabeth Martin de Lassalle1, Patrice Maboudou5, Pierre Formstecher1, Renata Polakowska1, Laurent Mortier1, and Philippe Marchetti1,5 Abstract Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1a (HIF-1a). Pharmacologic or genetic blockades of the HIF-1a pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease. Cancer Res; 72(19); 5035–47. Ó2012 AACR. Introduction (ROS; refs. 2, 3). It is generally postulated that the cellular Over the last decades, huge efforts devoted to comprehend effects of ROS depend on the level at which ROS are produced. melanoma biology led to the identification of new targets for Controlled production of ROS participates in the promotion antimelanoma therapy (1). Apart from targeting the oncogenic and progression of melanoma (3), whereas higher ROS gener- mutations present in many (but certainly not all) melamomas, ation displays cell-damaging effects (4). Constitutive produc- another promising strategy is to exploit biochemical particu- tion of the ROS weakens melanoma cells that are closer to the larities of melanoma cells. One biochemical hallmark of mel- point where cell death can occur. Hence, melanoma cells show anoma is the generation of excessive reactive oxygen species increased sensitivity to ROS-induced death as compared with melanocytes and to other tumors (5). According to this view, elesclomol, a pro-oxidant molecule that targets the mitochon- Authors' Affiliations: 1Unit 837 Equipe 4 Inserm and FacultedeM edecine, drial electron transport chain (ETC; ref. 6), has been evaluated Universite de Lille II 1 Place, Verdun Cedex; 2CNRS, UMR ISTCT 6301, CERVOxy Group; GIP CYCERON, Caen Cedex; 3Universite de Caen in clinical trials for the treatment of metastatic melanomas and Basse-Normandie, Normandie; 4Laboratoire SPCMIB, UMR CNRS 5068 has shown encouraging clinical responses. Intriguingly, clinical Universite Paul Sabatier, Toulouse Cedex; and 5Centre de Bio-Pathologie, favorable responses occurred in a subset of patients distin- Plate-forme de Biotherapie, Banque de Tissus, CHRU Lille, France guished by low serum lactate dehydrogenase (LDH; ref. 7). Note: Supplementary data for this article are available at Cancer Research Thus, serum LDH can be considered as the predictor of Online (http://cancerres.aacrjournals.org/). response to elesclomol. At a cellular level, elesclomol requires J. Kluza and P. Corazao-Rozas contributed equally to this work. a functioning ETC to induce ROS-mediated melanoma cell Corresponding Author: Philippe Marchetti, INSERM U 837 Facultede death (6). In this context, unraveling the regulatory mechan- Medecine 1, Place Verdun F-59045, Lille Cedex, France. Phone: 33-3-20- isms essential for enhancing ROS production is fundamental 62-69-52; Fax: 33-3-20-62-68-84; E-mail: [email protected] for improving the efficiency of pro-oxidants in melanoma. doi: 10.1158/0008-5472.CAN-12-0979 Approximately, 60% to 90% of cancers display a metabolic Ó2012 American Association for Cancer Research. profile, the so-called Warburg phenotype, characterized by www.aacrjournals.org 5035 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst August 3, 2012; DOI: 10.1158/0008-5472.CAN-12-0979 Kluza et al. theirdependenceuponglycolysisasthemajorsourceof study was obtained from the local Person's Protection Com- energy, irrespective of the oxygen level (8). According to the mittee, and all patients provided informed consent. The sam- Warburg effect, pyruvate, the end product of glycolysis, is ples came from patients who had to be treated surgically for mainly converted into lactate by LDH-A that is upregulated cutaneous melanoma metastases. in transformed cells, rather than oxidized in mitochondria. It seems conceivable that melanoma cells mainly rely on Cell lines glycolysis for energetic needs based on the following reasons: HL60, A375, and SKMel-28 were obtained from American (i) the glycolytic phenotype of melanoma cell lines has been Type Culture Collection over the past 2 years. The human recently identified by metabolic profiling (9); (ii) metastatic melanoma cell lines HBL, LND, MM074, and MM079 were melanomas are characterized by their particularly high avidity established in the Laboratory of Oncology and Experimental for 2[18F]fluoro-2-deoxy-D-glucose (18FDG) clinically detect- Surgery (Institut Bordet, Universite libre de Bruxelles, Brussels, able on positron emission tomography (PET; ref. 10); (iii) the Belgium) from lymph node metastases of patients with mel- isoenzyme LDH-5, the more effective in the conversion of anoma and were kindly provided by G. Ghanem in 2009. Mel- pyruvate to lactate, is detected on histologic sections and in 4M was established in the laboratory by Dr. Polakowska in blood sera from patients with highly metastatic melanoma (for 2009. All cells were passaged within 6 months of thawing. The review; ref. 11); and (iv) hypoxia-inducible factor-1a (HIF-1a), a identity of cell lines was regularly examined by morphologic master regulator of metabolism required for the active adap- criteria and the presence of differentiation markers (last test in tation of cancer cells to hypoxic conditions, is overexpressed in March 2012). They were expressing at least one of the mela- human melanoma (12, 13). In addition to hypoxia, HIF-1a has nocyte differentiation markers (TYR, TYRP1, TYRP2/DCT, also been found stabilized in normoxic conditions in melano- Melan-A/MART-1, gp100/pmel17, S100B) as routinely assessed ma cells. For instance, melanoma antigen-11 regulates HIF-1 by Western blotting/immunofluorescence. The identity of accumulation by inhibition of prolyl hydroxylase activity (14). HBL, LND, and Mel-4M was also confirmed by karyotyping Endothelin or the antiapoptotic protein, Bcl-2, induces HIF-1a and array comparative genomic hybridization testing (IRCL accumulation (15, 16). Genetic alterations, such as Micro- and Laboratoire de genetique, CHRU, Lille, France). phthalmia-Associated Transcription Factor (MITF) germline All cell lines were cultured at 37 C under 5% CO2 in RPMI mutation (17) or the oncogenic V600E BRAF mutation (18) medium (Gibco-BRL, Life Technologies) supplemented with overexpress HIF-1a and enhance transcriptional activity of 10% fetal calf serum (Gibco-BRL), antibiotics, and 1 mmol/L downstream genes. sodium pyruvate (Gibco-BRL). Cells were transfected with However, recent studies indicate that melanomas do not siRNA targeting PDK-3 (sc-39029, Santa Cruz Biotechnology) adopt a bona fide Warburg phenotype, as glutamine stimulates or a nontargeting control siRNA (sc-37707) using Lipofecta- mitochondrial metabolism to favor melanoma growth (9, 19). It mine 2000 (Life Technologies). PDK3 overexpression in mel- is well established that, under hypoxic conditions, HIF-1a anoma cell lines was obtained by transfection with pJP1520- activates the expression of glycolytic enzymes and glucose PDK3 vector [provided through central repository DNASU transporters and downregulates mitochondrial activity and (http://dnasu.asu.edu; ref. 23)] DCA, and/or elesclomol were ROS production through several distinct mechanisms in a added 48 hours after transfection. HBL cells lacking mitochon- context-specific manner (20). HIF-1a has also been involved drial DNA (HBL r0) were generated as published (24). The A375 in the "glutamine addiction" of cancer cells (21). cell line, which harbors the BRAFV600E mutation, was treated Our objective