Combined Inhibition Of Glycolysis Research Articles

Activation of TRPV4 channels promotes the loss of cellular ATP in organotypic slices of the mouse neocortex exposed to chemical ischemia.

The vertebrate brain has an exceptionally high energy need. During ischemia, intracellular ATP concentrations decline rapidly, resulting in the breakdown of ion gradients and cellular damage. Here, we employed the nanosensor ATeam1.03YEMK to analyse the pathways driving the loss of ATP upon transient metabolic inhibition in neurons and astrocytes of the mouse neocortex. We demonstrate that brief chemical ischemia, induced by combined inhibition of glycolysis and oxidative phosphorylation, results in a transient decrease in intracellular ATP. Neurons experienced a larger relative decline and showed less ability to recover from prolonged (>5min) metabolic inhibition than astrocytes. Blocking voltage-gated Na+ channels or NMDA receptors ameliorated the ATP decline in neurons and astrocytes, while blocking glutamate uptake aggravated the overall reduction in neuronal ATP, confirming the central role of excitatory neuronal activity in the cellular energy loss. Unexpectedly, pharmacological inhibition of transient receptor potential vanilloid 4 (TRPV4) channels significantly reduced the ischemia-induced decline in ATP in both cell types. Imaging with Na+ -sensitive indicator dye ING-2 furthermore showed that TRPV4 inhibition also reduced ischemia-induced increases in intracellular Na+ . Altogether, our results demonstrate that neurons exhibit a higher vulnerability to brief metabolic inhibition than astrocytes. Moreover, they reveal an unexpected strong contribution of TRPV4 channels to the loss of cellular ATP and suggest that the demonstrated TRPV4-related ATP consumption is most likely a direct consequence of Na+ influx. Activation of TRPV4 channels thus provides a hitherto unacknowledged contribution to the cellular energy loss during energy failure, generating a significant metabolic cost in ischemic conditions. KEY POINTS: In the ischemic brain, cellular ATP concentrations decline rapidly, which results in the collapse of ion gradients and promotes cellular damage and death. We analysed the pathways driving the loss of ATP upon transient metabolic inhibition in neurons and astrocytes of the mouse neocortex. Our results confirm the central role of excitatory neuronal activity in the cellular energy loss and demonstrate that neurons experience a larger decline in ATP and are more vulnerable to brief metabolic stress than astrocytes. Our study also reveals a new, previously unknown involvement of osmotically activated transient receptor potential vanilloid 4 (TRPV4) channels to the reduction in cellular ATP in both cell types and indicates that this is a consequence of TRPV4-mediated Na+ influx. We conclude that activation of TRPV4 channels provides a considerable contribution to the cellular energy loss, thereby generating a significant metabolic cost in ischemic conditions.

Targeting Glucose Metabolism Sensitizes Pancreatic Cancer to MEK Inhibition.

Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal. A critical unmet need exists to explore essential susceptibilities in PDAC and to identify druggable targets to improve PDAC treatment. KRAS mutations dominate the genetic landscape of PDAC and lead to activation of multiple downstream pathways and cellular processes. Here, we investigated the requirement of these pathways for tumor maintenance using an inducible KrasG12D -driven PDAC mouse model (iKras model), identifying that RAF-MEK-MAPK signaling is the major effector for oncogenic KRAS-mediated tumor maintenance. However, consistent with previous studies, MEK inhibition had minimal therapeutic effect as a single agent for PDAC in vitro and in vivo. Although MEK inhibition partially downregulated transcription of glycolysis genes, it failed to suppress glycolytic flux in PDAC cells, which is a major metabolic effector of oncogenic KRAS. Accordingly, an in vivo genetic screen identified multiple glycolysis genes as potential targets that may sensitize tumor cells to MEK inhibition. Inhibition of glucose metabolism with low-dose 2-deoxyglucose in combination with a MEK inhibitor induced apoptosis in KrasG12D -driven PDAC cells in vitro. The combination also inhibited xenograft PDAC tumor growth and prolonged overall survival in a genetically engineered PDAC mouse model. Molecular and metabolic analyses indicated that co-targeting glycolysis and MAPK signaling results in apoptosis via induction of lethal endoplasmic reticulum stress. Together, our work suggests that combined inhibition of glycolysis and the MAPK pathway may serve as an effective approach to target KRAS-driven PDAC. SIGNIFICANCE: This study demonstrates the critical role of glucose metabolism in resistance to MAPK inhibition in KRAS-driven pancreatic cancer, uncovering a potential therapeutic approach for treating this aggressive disease.

Marizomib suppresses triple-negative breast cancer via proteasome and oxidative phosphorylation inhibition.

Purpose: Lacking effective targeted therapies, triple-negative breast cancer (TNBCs) is highly aggressive and metastatic disease, and remains clinically challenging breast cancer subtype to treat. Despite the survival dependency on the proteasome pathway genes, FDA-approved proteasome inhibitors induced minimal clinical response in breast cancer patients due to weak proteasome inhibition. Hence, developing effective targeted therapy using potent proteasome inhibitor is required.Methods: We evaluated anti-cancer activity of a potent proteasome inhibitor, marizomib, in vitro using breast cancer lines and in vivo using 4T1.2 murine syngeneic model, MDA-MB-231 xenografts, and patient-derived tumor xenografts. Global proteome profiling, western blots, and RT-qPCR were used to investigate the mechanism of action for marizomib. Effect of marizomib on lung and brain metastasis was evaluated using syngeneic 4T1BR4 murine TNBC model in vivo.Results: We show that marizomib inhibits multiple proteasome catalytic activities and induces a better anti-tumor response in TNBC cell lines and patient-derived xenografts alone and in combination with the standard-of-care chemotherapy. Mechanistically, we show that marizomib is a dual inhibitor of proteasome and oxidative phosphorylation (OXPHOS) in TNBCs. Marizomib reduces lung and brain metastases by reducing the number of circulating tumor cells and the expression of genes involved in the epithelial-to-mesenchymal transition. We demonstrate that marizomib-induced OXPHOS inhibition upregulates glycolysis to meet the energetic demands of TNBC cells and combined inhibition of glycolysis with marizomib leads to a synergistic anti-cancer activity.Conclusions: Our data provide a strong rationale for a clinical evaluation of marizomib in primary and metastatic TNBC patients.

PO-241 Targeting fatty acid oxidation and glycolysis to overcome drug resistance to MAPK inhibitors

IntroductionMalignant melanoma is an aggressive tumour characterised by poor prognosis and therapeutic resistance. It is partly driven by the MAPK signalling pathway, providing a rationale for the development of inhibitors (MAPKi) targeting key components of the activated MAPK pathway. MAPKi induce tumour regression and improve progression-free and overall survival of metastatic melanoma patients. However, due to the development of drug resistance, even in patients displaying complete response, eventually the tumour progress. Both genetic and non-genetic resistance mechanisms have been described during MAPK inhibition. A better understanding of these resistance mechanisms could uncover novel cancer cell vulnerabilities to target in combination strategies for enforcing the efficacy and long-term response to MAPKi.Material and methodsTo identify mechanisms and biomarkers of adaptive MAPKi-resistance in BRAF-mutated melanoma cells, we performed a cell surface marker screening in A375P cells. A panel of 361 antibodies was used to analyse membrane antigens by flow cytometry at various time points following treatment with MAPKi. Candidates were validated by FACS and qPCR in established cell lines, in a panel of primary patient-derived melanoma cells and in xenograft tumours.Results and discussionsOur screening approach identified 42 membrane epitopes changing expression in A375P in response to MAPKi treatment at 36 and 60 hour. In vitro and in vivo validations highlighted CD36, a fatty acid transporter involved in fatty acid oxidation (FAO), as the most consistently up-regulated protein during treatments. Analysis of RNAseq and qPCR data revealed increased expression of FAO genes and unveiled MAPKi-induced metabolic reprogramming involving PPARα and CPT1α. Functional assays confirmed metabolic reprogramming in multiple human melanoma cell lines as well as association between increased FAO rates and enhanced resistance to MAPKi. However, while pharmacological inhibition of FAO failed to boost cell death during MAPKi treatments due to compensatory up-regulation of glycolysis, inhibition of both, FAO and glycolysis, was sufficient.ConclusionOur study identified CD36 as the most consistent cell surface protein marking BRAF-mutated melanoma cells in the early stage of the development of resistance. Results implicate adaptive metabolic reprogramming as a potential resistance mechanism and indicate that combined inhibition of glycolysis and FAO are critical to induce cell death and overcome MAPKi resistance in melanoma cells.

MTORC1-Dependent Metabolic Reprogramming Underlies Escape from Glycolysis Addiction in Cancer Cells

Although glycolysis is substantially elevated in many tumors, therapeutic targeting of glycolysis in cancer patients has not yet been successful, potentially reflecting the metabolic plasticity of tumor cells. In various cancer cells exposed to a continuous glycolytic block, we identified a recurrent reprogramming mechanism involving sustained mTORC1 signaling that underlies escape from glycolytic addiction. Active mTORC1 directs increased glucose flux via the pentose phosphate pathway back into glycolysis, thereby circumventing a glycolysis block and ensuring adequate ATP and biomass production. Combined inhibition of glycolysis and mTORC1 signaling disrupted metabolic reprogramming in tumor cells and inhibited their growth in vitro and in vivo. These findings reveal novel combinatorial therapeutic strategies to realize the potential benefit from targeting the Warburg effect.

Combined inhibition of glycolysis and AMPK induces synergistic breast cancer cell killing.

Targeting glycolysis for cancer treatment has been investigated as a therapeutic method but has not offered a feasible chemotherapeutic strategy. Our aim was to examine whether AMP-activated protein kinase (AMPK), a conditional oncogene, rescues the energetic stress and cytotoxicity induced by 2-deoxyglucose (2-DG), a glycolytic inhibitor, and the related mechanisms. Luciferin/luciferase adenosine triphosphate (ATP) determination, Western analysis, qRT-PCR analyses, MTT growth assay, clonogenic assay, and statistical analysis were performed in this study. 2-DG decreased ATP levels and subsequently activated AMPK, which contribute to intracellular ATP recovery in MCF-7 cells thus exhibiting no apparent cytotoxicity. Compound C, an AMPK inhibitor, further potentiates 2-DG-induced decrease in ATP levels and inhibits their recovery. 2-DG, via AMPK activation, stimulated cAMP response element-binding protein (CREB) phosphorylation and activity and promoted nuclear peroxisome proliferator-activated receptor gamma coactivator-1-beta (PGC-1β) and estrogen-related receptor α (ERRα) protein expression, leading to augmented mitochondrial biogenesis and expression of fatty acid oxidation (FAO) genes including PPARα, MCAD, CPT1C, and ACO. This metabolic adaptation elicited by AMPK counteracts the ATP-depleting and cancer cell-killing effect of 2-DG. However, 2-DG in combination with AMPK antagonists or small interfering RNA caused a dramatic increase in cytotoxicity in MCF-7 but not in MCF-10A cells. Similarly, when combined with inhibition of CREB/PGC-1β/ERRα pathway, 2-DG saliently suppressed mitochondrial biogenesis and the expression of FAO genes, depleted ATP production, and enhanced cytotoxicity in cancer cells. Collectively, the combination of 2-DG and AMPK inhibition synergistically enhanced the cytotoxic potential in breast cancer cells with a relative nontoxicity to normal cells and may offer a promising, safe, and effective breast cancer therapeutic strategy.

Combined inhibition of glycolysis, the pentose cycle, and thioredoxin metabolism selectively increases cytotoxicity and oxidative stress in human breast and prostate cancer

Inhibition of glycolysis using 2-deoxy-d-glucose (2DG, 20mM, 24–48h) combined with inhibition of the pentose cycle using dehydroepiandrosterone (DHEA, 300µM, 24–48h) increased clonogenic cell killing in both human prostate (PC-3 and DU145) and human breast (MDA-MB231) cancer cells via a mechanism involving thiol-mediated oxidative stress. Surprisingly, when 2DG+DHEA treatment was combined with an inhibitor of glutathione (GSH) synthesis (l-buthionine sulfoximine; BSO, 1mM) that depleted GSH>90% of control, no further increase in cell killing was observed during 48h exposures. In contrast, when an inhibitor of thioredoxin reductase (TrxR) activity (Auranofin; Au, 1µM), was combined with 2DG+DHEA or DHEA-alone for 24h, clonogenic cell killing was significantly increased in all three human cancer cell lines. Furthermore, enhanced clonogenic cell killing seen with the combination of DHEA+Au was nearly completely inhibited using the thiol antioxidant, N-acetylcysteine (NAC, 20mM). Redox Western blot analysis of PC-3 cells also supported the conclusion that thioredoxin-1 (Trx-1) oxidation was enhanced by treatment DHEA+Au and inhibited by NAC. Importantly, normal human mammary epithelial cells (HMEC) were not as sensitive to 2DG, DHEA, and Au combinations as their cancer cell counterparts (MDA-MB-231). Overall, these results support the hypothesis that inhibition of glycolysis and pentose cycle activity, combined with inhibition of Trx metabolism, may provide a promising strategy for selectively sensitizing human cancer cells to oxidative stress-induced cell killing.

Abstract C152: Characterizing rhabdomyosarcoma and osteosarcoma cellular metabolism.

Abstract Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. A renewed interest in the metabolic properties of cancer cells, as well as the recognition of altered cellular metabolism as a critical hallmark of cancer, has led to an exploration of targeting metabolic dependencies of cancer cells as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human rhabdomyosarcoma and osteosarcoma cell lines in order to identify potential differences in sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Glycolytic and mitochondrial bioenergetic profiles were generated using Seahorse extracellular flux analysis to measure extracellular acidification rates and oxygen consumption rates, and significant differences in glycolytic and respiratory bioenergetics between rhabdomyosarcoma and osteosarcoma cell lines were identified. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely-used diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells versus embryonal rhabdomyosarcoma, osteosarcoma, and normal cells, whereas inhibition of mitochondrial respiration by oligomycin or metformin did not significantly affect viability. Combined inhibition of glycolysis and respiration decreased cell growth more effectively than either treatment alone in all sarcoma lines tested. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels in all sarcoma lines tested, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy. Funded by NCI Contract No. HHSN261200800001E. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C152. Citation Format: Sameer Issaq, Beverly Teicher, Anne Monks. Characterizing rhabdomyosarcoma and osteosarcoma cellular metabolism. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C152.

Mitochondria-Targeted Drugs Synergize with 2-Deoxyglucose to Trigger Breast Cancer Cell Death

Cancer cells are long known to exhibit increased aerobic glycolysis, but glycolytic inhibition has not offered a viable chemotherapeutic strategy in part because of the systemic toxicity of antiglycolytic agents. However, recent studies suggest that a combined inhibition of glycolysis and mitochondrial function may help overcome this issue. In this study, we investigated the chemotherapeutic efficacies of mitochondria-targeted drugs (MTD) in combination with 2-deoxy-d-glucose (2-DG), a compound that inhibits glycolysis. Using the MTDs, termed Mito-CP and Mito-Q, we evaluated relative cytotoxic effects and mitochondrial bioenergetic changes in vitro. Interestingly, both Mito-CP and Mito-Q synergized with 2-DG to decrease ATP levels in two cell lines. However, with time, the cellular bioenergetic function and clonogenic survival were largely restored in some cells. In a xenograft model of human breast cancer, combined treatment of Mito-CP and 2-DG led to significant tumor regression in the absence of significant morphologic changes in kidney, liver, or heart. Collectively, our findings suggest that dual targeting of mitochondrial bioenergetic metabolism with MTDs and glycolytic inhibitors such as 2-DG may offer a promising chemotherapeutic strategy.

Abstract 1136: Synergistic inhibition of bioenergetic pathways in pancreatic cancer cells by mitochondria-targeted nitroxide and glycolytic inhibitors

Abstract Pancreatic adenocarcinoma is one of the most aggressive cancers with a high fatality rate. Currently available chemotherapy for treating pancreatic cancers is ineffective. Emerging research is focused on drugs targeting bioenergetic pathways in tumor cells. Despite decades of evidence that cancer cells have increased aerobic glycolysis, glycolytic inhibition has failed as a chemotherapeutic approach, due in part to enhanced systemic toxicity of antiglycolytic agents. We hypothesized that a combined inhibition of glycolysis and mitochondrial function may represent an alternate therapeutic approach that can potentially overcome the insufficiency of glycolytic inhibition alone. In this study we investigated the chemotherapeutic efficacy of combining mitochondria-targeted nitroxide, Mito-CP (a nitroxide, carboxy proxyl conjugated to a triphenylphosphonium (TPP+) moiety), and inhibitors of glycolysis, 2-deoxy-D-glucose (2-DG) or 3-bromopyruvate (3-BP) in pancreatic cancer cell lines, PANC-1 and MiaPaCa-2. The relative cytotoxicity and mitochondrial bioenergetic changes in pancreatic cancer cells were assessed. We observed that Mito-CP synergized with glycolytic inhibitors (2-DG or 3-BP) in inducing cell death. Using a Seahorse XF24 extracellular flux analyzer, we determined the combined effects of Mito-CP and 2-DG or 3-BP on cellular mitochondrial function and glycolytic function. The combined treatments induced fast and irreversible inhibition of mitochondrial functions and intracellular ATP depletion. Collectively, these results suggest that targeting mitochondrial bioenergetic metabolism with Mito-CP and glycolytic inhibitors such as 2-DG and 3-BP may be a promising chemotherapeutic strategy in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1136. doi:1538-7445.AM2012-1136

Synergistic Enhancement of 2-Deoxy-D-Glucose-Mediated Breast Cancer Cell Death by Mitochondria-Targeted Drugs

Despite decades of evidence that cancer cells have increased aerobic glycolysis, glycolytic inhibition has failed as a chemotherapeutic strategy, due in part to enhanced systemic toxicity of antiglycolytic agents. Recent research suggests that the combined inhibition of glycolysis and mitochondrial function may represent a possible therapeutic approach to overcome the toxicity of antiglycolytics. In this study we investigated the chemotherapeutic efficacies of mitochondria-targeted drugs (MTDs) in combination with 2-DG(an inhibitor of glycolysis) in breast cancer cell lines. Two MTDs were employed in this study: Mito-CP and MitoQ. Synergistic enhancement of 2-deoxy-D-glucose-mediated breast cancer cell death by mitochondria-targeted drugs.

Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437.

An increasing number of experimental chemotherapeutic agents induce apoptosis by directly triggering mitochondrial membrane permeabilization (MMP). Here we examined MMP induced by lonidamine, arsenite, and the retinoid derivative CD437. Cells overexpressing the cytomegalovirus-encoded protein vMIA, a protein which interacts with the adenine nucleotide translocator, were strongly protected against the MMP-inducing and apoptogenic effects of lonidamine, arsenite, and CD437. In a cell-free system, lonidamine, arsenite, and CD437 induced the permeabilization of ANT proteoliposomes, yet had no effect on protein-free liposomes. The ANT-dependent membrane permeabilization was inhibited by the two ANT ligands ATP and ADP, as well as by recombinant Bcl-2 protein. Lonidamine, arsenite, and CD437, added to synthetic planar lipid bilayers containing ANT, elicited ANT channel activities with clearly distinct conductance levels of 20+/-7, 100+/-30, and 47+/-7 pS, respectively. Altering the ATP/ADP gradient built up on the inner mitochondrial membrane by inhibition of glycolysis and/or oxidative phosphorylation differentially modulated the cytocidal potential of lonidamine, arsenite, and CD437. Inhibition of F(0)F(1)ATPase without glycolysis inhibition sensitized to lonidamine-induced cell death. In contrast, only the combined inhibition of glycolysis plus F(0)F(1)ATPase sensitized to arsenite-induced cell death. No sensitization to cell death induction by CD437 was achieved by glucose depletion and/or oligomycin addition. These results indicate that ANT is a target of lonidamine, arsenite, and CD437 and unravel an unexpected heterogeneity in the mode of action of these three compounds.

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [ 14C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2′-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5′-nucleotidase (EC 3.1.3.5), reflecting the markedly higher V max/ K m ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher V max/ K m ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.