Oncogene (2012) 31, 4996 --5006 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Switching from aerobic glycolysis to oxidative phosphorylation modulates the sensitivity of mantle cell lymphoma cells to TRAIL GL Robinson, D Dinsdale, M MacFarlane and K Cain TRAIL (TNF (tumour necrosis factor)-related apoptosis-inducing ligand) a putative anti-cancer cytokine induces apoptosis through DISC (death-inducing signalling complex)-mediated activation of caspase-8 and/or cleavage of Bid. TRAIL is relatively specific for tumour cells but primary chronic lymphocytic leukaemia and mantle cell lymphoma (MCL) cells are resistant. Herein, we show that cellular metabolism influences cell death and that MCL cells (Z138 cell line) can survive/proliferate in glucose-free media by switching from aerobic glycolysis to ‘coupled’ oxidative phosphorylation. Extracellular flux analysis and mitochondrial inhibitors reveal that in the absence of glycolysis, Z138 cells have enhanced respiratory capacity coupled to ATP synthesis, similar to ‘classical’ state 3 mitochondria. Conversely, 2-deoxyglucose (2DG) blocked glycolysis and partially inhibited glycolytic- dependent oxidative phosphorylation, resulting in a 50% reduction in cellular ATP levels. Also, 2DG sensitised Z138 cells to TRAIL and induced a marked decrease in caspase-8, -3, cFLIPS, Bid and Mcl-1 expression but Bak remained unchanged, altering the Mcl-1/Bak ratio, facilitating cytochrome c release and cell death. Conversely, under glucose-free conditions, Z138 cells were less sensitive to TRAIL with reduced TRAIL-R1/R2 surface receptor expression and impaired DISC formation. Anti-apoptotic proteins Bcl-2 and XIAP were up-regulated while pro-apoptotic BAX was down-regulated. Additionally, mitochondria had higher levels of cytochrome c and ultrastucturally exhibited a condensed configuration with enhanced intracristal spaces. Thus, metabolic switching was accompanied by mitochondrial proteome and ultrastructural remodelling enabling enhanced respiration activity. Cytochrome c release was decreased in glucose-free cells, suggesting that either pore formation was inhibited or that cytochrome c was more tightly bound. Glucose-free Z138 cells were also resistant to intrinsic cell death stimuli (ABT-737 and ionising radiation). In summary, in MCL cells, the anti-glycolytic effects of 2DG and glucose restriction produced opposite effects on TRAIL-induced cell death, demonstrating that mitochondrial metabolism directly modulates sensitivity of tumour cells to apoptosis. Oncogene (2012) 31, 4996--5006; doi:10.1038/onc.2012.13; published online 6 February 2012 Keywords: TRAIL; apoptosis; oxidative phosphorylation; aerobic glycolysis; mitochondria; 2DG INTRODUCTION The extrinsic pathway is initiated by the extracellular binding of Otto Warburg1 first described how cancer cells utilise aerobic members of the tumour necrosis factor (TNF) ligand family, such glycolysis rather than oxidative phosphorylation---a phenomena as TNFa, CD95L/FasL or TNF-related apoptosis-inducing ligand now known as the ‘Warburg effect’. In cancer cells, even under (TRAIL), to their cognate transmembrane death receptors.7 TRAIL, normoxic conditions, there is enhanced conversion of glucose to unlike CD95L or TNFa, can be selectively toxic to tumour cells8--11 lactate with decreased mitochondrial respiration. This results and initiates apoptosis through the death-inducing signalling in increased glucose demand, which is used diagnostically with complex (DISC). TRAIL-R1/-R2 ligation leads to recruitment of the FDG-PET scanning whereby the non-metabolisable fluorodeoxy- adaptor molecule Fas-associated death domain (FADD), which gluocose analogue is used to image tumours.2 During neoplasia, then recruits procaspase-8 to form the DISC. Caspase-8, activated extensive metabolic reprogramming supports tumour cell growth within the DISC, initiates a caspase cascade activating the and proliferation3 and although aerobic glycolysis is an inefficient executioner caspases-3/-7.12 --14 Originally described for CD95, method of producing ATP, it allows cancer cells to utilise glucose, different cell types respond to death ligands as either; type I cells, which is in high concentration. This provides key intermediates which rapidly form large amounts of DISC and activate caspase-8 necessary for the pentose phosphate pathway, which provides and -3 directly or type II cells, where comparatively small amounts ribose sugars (nucleotide synthesis), glycerol and citrate (lipid of DISC are formed and the intrinsic pathway is activated by synthesis) and non-essential amino acids. Since the switch from cleaved Bid.15 Type I and II responses have also been described for mitochondrial respiration to glycolysis appears to be a hallmark of TRAIL where blockade of the mitochondrial amplification arm by 16 cancer cells, there is increasing interest in using and developing either over-expression of anti-apoptotic Bcl-2/Bcl-XL, or knock- glycolytic inhibitors to target tumour cells.4 out of caspase-917 or Bax18 attenuates TRAIL-induced apoptosis. Treatments for cancer largely work by inducing apoptosis Modulation of glucose metabolism has been reported to affect through either the extrinsic or intrinsic (mitochondrial) pathway. intrinsic and extrinsic apoptotic cell death. For example, inhibition The latter involves cytotoxic stress (for example, irradiation of doxorubicin-induced apoptosis by the anti-glycolytic analogue or chemical), cytochrome c release, Apaf-1 oligomerisation 2-deoxyglucose (2DG) leads to cellular depletion of ATP and and caspase-9 recruitment to form the apoptosome complex.5,6 abrogation of DNA cleavage.19 Similarly, oligomycin-induced ATP MRC Toxicology Unit, University of Leicester, Leicester, UK. Correspondence: Professor M MacFarlane or Professor K Cain, MRC Toxicology Unit, University of Leicester, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, UK. E-mail: [email protected] or [email protected] Received 4 July 2011; revised 6 December 2011; accepted 22 December 2011; published online 6 February 2012 Metabolism modulates MCL sensitivity to TRAIL GL Robinson et al 4997 depletion blocked the intrinsic pathway induced by anti-cancer residual ECAR, suggesting that this residual acidification is most agents but failed to inhibit CD95L-induced apoptosis,20 support- likely due to a rotenone-sensitive ETC-dependent ‘proton leak’ ing earlier studies showing that ATP levels determine whether a from the mitochondria (Figure 1b). cell dies by apoptosis or necrosis.21 ATP is needed for both Previous findings have shown that 2DG potentiates TNFa killing intrinsic and extrinsic-mediated apoptotic cell death, and apopto- in U937 cells23 and CD95 and TRAIL-induced killing in HeLa, MCF-7 tic cells appear to have elevated levels of ATP.22 However, some and melanoma cells.25,28 Similarly, Z138 cells treated with 2DG studies have shown that cells deprived of glucose have decreased were six- to eightfold (Figure 1g) more sensitive to TRAIL-induced ATP levels and are sensitised to death-receptor-mediated apop- cell killing (EC50 ¼ 25--30 ng/ml) compared with control cells 21,23 --26 tosis. We therefore decided to investigate the effect of (TRAIL EC50 ¼ B180--200 ng/ml). Cleavage of caspase-8, -3 and chronic glucose deprivation on cellular metabolism, ATP levels PARP, a caspase-3 substrate, was detectable with lower concen- and TRAIL-induced apoptosis. Using XF (extracellular flux) analysis, trations (25 ng/ml) in 2DG-treated cells compared with 100 ng/ml we have measured mitochondrial oxidative phosphorylation and for control cells (Figure 1h). 2DG alone increased the endogenous aerobic glycolysis in a mantle cell lymphoma (MCL) cell line (Z138). level of cell death from B10 to 20% (Figure 1g) and flow Prolonged exposure of Z138 cells to glucose-free media, cytometry showed that this was due to increased apoptotic and supplemented with pyruvate/glutamine, mediated a metabolic secondary necrotic cell death (Supplementary Figures S1a and b), switch from aerobic glycolysis to oxidative phosphorylation. Under which was largely insensitive to 20 mM zVAD.fmk (Supplementary these conditions, ATP levels were maintained and the cells were Figure S1c). This contrasted with TRAIL-induced caspase-8 significantly less sensitive to TRAIL and ABT-737. Conversely, activation and cell death, which was sensitive to zVAD.fmk inhibiting aerobic glycolysis with 2DG decreased ATP levels and (Figure 1g). TRAIL DISC formation, as determined by FADD and enhanced sensitivity to TRAIL. Glucose-free conditions induced caspase-8 recruitment to TRAIL-R1/R2 was rapid in both control significant changes in pro- and anti-apoptotic proteins. We and 2DG-treated cells (Figure 1i). Interestingly, TRAIL-R1 migrated propose that ATP levels regulate the levels of mitochondrially on SDS--PAGE (sodium dodecyl sulphate polyacrylamide gel associated pro- and anti-apoptotic proteins and suggest that, in electrophoresis) as two separate bands, which after 2DG some cells, oxidative phosphorylation can supply enough ATP to treatment migrated faster (Figure 1i), indicating decreased enable them to survive glucose-free conditions. molecular weight, possibly due to changes in post-translational modification (see later). The effects of 2DG on TRAIL induced caspase-mediated RESULTS apoptosis, but not on cellular ATP levels, were inhibited by the 2DG inhibits glycolysis and sensitises Z138 cells to TRAIL-induced poly caspase inhibitor zVAD.fmk (Figures 1f and g). Thus,