Leukemia (2015) 29, 1163–1176 © 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu ORIGINAL ARTICLE GAPDH enhances the aggressiveness and the vascularization of non-Hodgkin’s B lymphomas via NF-κB-dependent induction of HIF-1α J Chiche1,2, S Pommier1,2,3, M Beneteau1,2, L Mondragón1,2, O Meynet1,2, B Zunino1,2, A Mouchotte1,2, E Verhoeyen1,2, M Guyot2,4, G Pagès2,4, N Mounier5, V Imbert2,6, P Colosetti2,7, D Goncalvès2,7, S Marchetti2,7, J Brière8, M Carles1,2,3, C Thieblemont8 and J-E Ricci1,2,3 Deregulated expression of glycolytic enzymes contributes not only to the increased energy demands of transformed cells but also has non-glycolytic roles in tumors. However, the contribution of these non-glycolytic functions in tumor progression remains poorly defined. Here, we show that elevated expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), but not of other glycolytic enzymes tested, increased aggressiveness and vascularization of non-Hodgkin’s lymphoma. Elevated GAPDH expression was found to promote nuclear factor-κB (NF-κB) activation via binding to tumor necrosis factor receptor-associated factor-2 (TRAF2), enhancing the transcription and the activity of hypoxia-inducing factor-1α (HIF-1α). Consistent with this, inactive mutants of GAPDH failed to bind TRAF2, enhance HIF-1 activity or promote lymphomagenesis. Furthermore, elevated expression of gapdh mRNA in biopsies from diffuse large B-cell non-Hodgkin’s lymphoma patients correlated with high levels of hif-1α, vegf-a, nfkbia mRNA and CD31 staining. Collectively, these data indicate that deregulated GAPDH expression promotes NF-κB-dependent induction of HIF-1α and has a key role in lymphoma vascularization and aggressiveness. Leukemia (2015) 29, 1163–1176; doi:10.1038/leu.2014.324 INTRODUCTION glyceraldehyde-3-phosphate dehydrogenate (GAPDH) have linked Although cancer is a heterogeneous group of diseases with glycolytic enzymes to the control of cell growth and modifications 6–10 varying characteristics specific to each type of tumor, there are of signaling pathways. However, the contribution of these criteria common to all of these tumors, such as metabolic changes. functions to tumorigenesis in vivo remains poorly defined. Tumors in growth phase have a metabolic demand that exceeds The glycolytic pathway involves the conversion of glucose to the reduced oxygen tension (hypoxia)/nutrients they receive until lactate and the generation of ATP via a series of 10 enzymes. One new vessels are formed (angiogenesis). Low pressure of oxygen of them, GAPDH, which catalyzes the reaction of glyceraldehyde- + will stabilize the hypoxia-inducing factor-1α (HIF-1α) that will lead 3-phosphate (G3P)+NAD +Pi into 1,3-diphosphoglycerate+NADH+ + to the expression of genes involved in cell survival and adaptation, H , is a key enzyme of this metabolic pathway. Although GAPDH including vascular endothelial growth factor (VEGF) that will favor has long been considered to be an enzyme of seemingly little angiogenesis during cancer progression.1 Therefore, VEFG signal- interest, recent studies have demonstrated that in addition to its ing has emerged as a clinical attractive strategy in oncology. well-characterized glycolytic role in energy production, GAPDH is a However, insufficient efficacy and resistance limit its success.2 multifunctional protein with numerous significant non-glycolytic – Fundamental metabolic changes, controlled in part by HIF-1, are functions.7,10 12 Although the role of GAPDH in tumorigenesis is implemented by tumor cells to meet their high-energy still unclear, some studies have linked high levels of gapdh mRNA requirements.3 This increased dependence for glycolysis (Warburg to a poor prognosis, specifically in breast cancer and lung effect) that helps to meet the energy needs and to facilitate the cancer.13,14 However, how GAPDH expression could be related to uptake and incorporation of nutrients into the biomass of tumor aggressiveness is still elusive. developing tumors is a common feature of malignant tumors The present study was conducted to investigate whether and is hypothesized to be a key step in tumorigenesis.4,5 specific glycolytic enzymes could play a role in tumor aggressive- Investigation into the mechanisms driving the tumor metabolic ness. This study was performed using the Eμ-Myc transgenic shift has created interest in the identification of new functions of mouse model of non-Hodgkin’s lymphoma15 and results were glycolytic enzymes involved in cancer progression, whatever they confirmed using non-Hodgkin’s lymphoma patient biopsies. We are or not related to their enzymatic properties. Recently, the revealed that only GAPDH, but not other tested glycolytic non-metabolic activities of 6-phosphofructo-2-kinase/fructose-2,6- enzymes, contributes to an increase in lymphoma growth and biphosphatase 3, hexokinase II, pyruvate kinase M2 (PKM2) and vascularization. Mechanistic investigations revealed that GAPDH 1Inserm, U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), équipe ‘contrôle métabolique des morts cellulaires’, équipe 3, Nice, France; 2Université de Nice-Sophia- Antipolis, Faculté de Médecine, Nice, France; 3Centre Hospitalier Universitaire de Nice, Département d’Anesthésie Réanimation, Nice, France; 4Institute for Research on Cancer and Aging, CNRS UMR 7284/U INSERM 1081, Centre A. Lacassagne, Nice, France; 5Centre Hospitalier Universitaire de Nice, Département d’Onco-Hématologie, Nice, France.; 6Inserm, U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), équipe ‘inflammation, cancer et cellules souches cancéreuses’, Nice, France; 7Inserm, U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), équipe ‘mort cellulaire, différenciation, inflammation et cancer’, Nice, France and 8AP-HP-Hôpital Saint-Louis, Service d’hémato- Oncologie, Université Paris Diderot, Sorbonne Paris Cité, F-75010 Paris, France. Correspondence: Dr J-E Ricci, Inserm U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), équipe ‘contrôle métabolique des morts cellulaires’, équipe 3, 151 route de St Antoine de Ginestière, Batiment Archimed, BP 23194, Nice 06204, France. E-mail: [email protected] Received 3 July 2014; revised 4 November 2014; accepted 10 November 2014; accepted article preview online 14 November 2014; ; advance online publication, 16 December 2014 GAPDH accelerates lymphomagenesis via NF-κB/HIF1 J Chiche et al 1164 regulates HIF-1α by mediating nuclear factor-κB (NF-κB) activation, (Promega, Madison, WI, USA). The luciferase assay was performed as a process driven by the interaction of GAPDH with tumor necrosis previously described.10 factor receptor-associated factor-2 (TRAF2). NF-κB activity. HeLa cells stably expressing GAPDH-V5 or GAPDH double mutant (DM-V5) control (pMIG) were transiently co-transfected with a MATERIALS AND METHODS vector-encoding cyan fluorescent protein (0.5 μg) and a luciferase reporter μ κ Cell culture and hypoxic exposure gene (2 g) controlled by a minimal tk promoter and six reiterated B sites (κBx6 tk luc). Twenty-four hours after transfection, cells were exposed to HeLa cells were obtained from American Type Culture Collection and either normoxia or hypoxia for 24 h before analyzing by fluorescence- cultured as recommended. Mouse primary Eμ-Myc lymphomas were activated cell sorting for the transfection efficiency and harvesting and 16 isolated as described previously. Incubation in hypoxia was carried out at analyzing as previously described.19 For NF-κB activity in mouse primary B 1% O2 (as opposed to normoxic, 21% O2 incubation), 37 °C in 95% lymphomas, Eμ-Myc cells were stably transduced with the κB-luciferase- 20 19 humidity and 5% CO2/94% N2 in a sealed anaerobic workstation (Whitley IRES-GFP lentiviral construct and analyzed as previously described. hypoxystation H35, Shipley, UK). Patients and tissue sample preparation Reagents and antibodies A total of 13 patients who underwent biopsies at the diagnosis stage for Mouse anti-V5 was from Invitrogen (Carlsbad, CA, USA), rabbit anti-GAPDH diffuse large B-cell lymphoma (DLBCL) between May 2007 and May 2011 at was from (Abcam, Cambridge, UK), mouse anti-Erk2 was from Santa Cruz the Saint-Louis Hospital (Department of Onco-Hematology, Hospital Saint- Biotechnology (Santa Cruz, CA, USA). Mouse anti-GAPDH used for Louis, Paris, France) were selected. The patients received the necessary immunoprecipitation was from Santa Cruz Biotechnology. Rabbit anti- information concerning the study and consent was obtained. The main GAPDH used for immunohistochemistry was a ‘Prestige antibody’ from clinical and histopathological data of the patients are summarized in Sigma (St Louis, MO, USA). Rabbit anti-HIF-1α was a gift of Dr Pouysségur. Supplementary Table 1. The morphologic classification of the tumors was 21 Anti-CD19-PE was from BD Bioscience (Franklin Lakes, NJ, USA), Anti-CD31 carried out according to the World Health Organization criteria. Ann Arbor stage was assigned to the tumor extensions, and all patients were was from BD Bioscience. Other antibodies were from Cell Signaling 22 23 Technology (Beverly, MA, USA). GAPDH-specific inhibitor, Koningic acid scored by IPIaa. RNA was extracted as previously described. (KA), was from Euromedex (Souffelweyersheim, France). 2-Deoxyglucose was from Toronto Research Chemicals (Toronto, ON, Canada). Statistical analysis Pimonidazole- and HP-Red549-labeled anti-pimonidazole were purchased Continuous variables and binomial variables, expressed as mean (s.d.), from Hypoxyprobe Inc. (Burlington, MA,