Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism

Cancer cells exhibit increased glycolysis for ATP production due, in part, to respiration injury (the Warburg effect). Because ATP generation through glycolysis is less efficient than through mitochondrial respiration, how cancer cells with this metabolic disadvantage can survive the competition with other cells and eventually develop drug resistance is a long-standing paradox. We report that mitochondrial respiration defects lead to activation of the Akt survival pathway through a novel mechanism mediated by NADH. Respiration-deficient cells (rho(-)) harboring mitochondrial DNA deletion exhibit dependency on glycolysis, increased NADH, and activation of Akt, leading to drug resistance and survival advantage in hypoxia. Similarly, chemical inhibition of mitochondrial respiration and hypoxia also activates Akt. The increase in NADH caused by respiratory deficiency inactivates PTEN through a redox modification mechanism, leading to Akt activation. These findings provide a novel mechanistic insight into the Warburg effect and explain how metabolic alteration in cancer cells may gain a survival advantage and withstand therapeutic agents.     

Novel role of NOX in supporting aerobic glycolysis in cancer cells with mitochondrial dysfunction and as a potential target for cancer therapy

Elevated aerobic glycolysis in cancer cells (the Warburg effect) may be attributed to respiration injury or mitochondrial dysfunction, but the underlying mechanisms and therapeutic significance remain elusive. Here we report that induction of mitochondrial respiratory defect by tetracycline-controlled expression of a dominant negative form of DNA polymerase γ causes a metabolic shift from oxidative phosphorylation to glycolysis and increases ROS generation. We show that upregulation of NOX is critical to support the elevated glycolysis by providing additional NAD+. The upregulation of NOX is also consistently observed in cancer cells with compromised mitochondria due to the activation of oncogenic Ras or loss of p53, and in primary pancreatic cancer tissues. Suppression of NOX by chemical inhibition or genetic knockdown of gene expression selectively impacts cancer cells with mitochondrial dysfunction, leading to a decrease in cellular glycolysis, a loss of cell viability, and inhibition of cancer growth in vivo. Our study reveals a previously unrecognized function of NOX in cancer metabolism and suggests that NOX is a potential novel target for cancer treatment.     

Tyr-94 Phosphorylation Inhibits Pyruvate Dehydrogenase Phosphatase 1 and Promotes Tumor Growth

Many cancer cells rely more on aerobic glycolysis (the Warburg effect) than mitochondrial oxidative phosphorylation and catabolize glucose at a high rate. Such a metabolic switch is suggested to be due in part to functional attenuation of mitochondria in cancer cells. However, how oncogenic signals attenuate mitochondrial function and promote the switch to glycolysis remains unclear. We previously reported that tyrosine phosphorylation activates and inhibits mitochondrial pyruvate dehydrogenase kinase (PDK) and phosphatase (PDP), respectively, leading to enhanced inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) and consequently inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. In particular, Tyr-381 phosphorylation of PDP1 dissociates deacetylase SIRT3 and recruits acetyltransferase ACAT1 to PDC, resulting in increased inhibitory lysine acetylation of PDHA1 and PDP1. Here we report that phosphorylation at another tyrosine residue, Tyr-94, inhibits PDP1 by reducing the binding ability of PDP1 to lipoic acid, which is covalently attached to the L2 domain of dihydrolipoyl acetyltransferase (E2) to recruit PDP1 to PDC. We found that multiple oncogenic tyrosine kinases directly phosphorylated PDP1 at Tyr-94, and Tyr-94 phosphorylation of PDP1 was common in diverse human cancer cells and primary leukemia cells from patients. Moreover, expression of a phosphorylation-deficient PDP1 Y94F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at different tyrosine residues inhibits PDP1 through independent mechanisms, which act in concert to regulate PDC activity and promote the Warburg effect.     

Metabolic interplay between glycolysis and mitochondrial oxidation: The reverse Warburg effect and its therapeutic implication

Aerobic glycolysis, i.e., the Warburg effect, may contribute to the aggressive phenotype of hepatocellular carcinoma. However, increasing evidence highlights the limitations of the Warburg effect, such as high mitochondrial respiration and low glycolysis rates in cancer cells. To explain such contradictory phenomena with regard to the Warburg effect, a metabolic interplay between glycolytic and oxidative cells was proposed, i.e., the "reverse Warburg effect". Aerobic glycolysis may also occur in the stromal compartment that surrounds the tumor; thus, the stromal cells feed the cancer cells with lactate and this interaction prevents the creation of an acidic condition in the tumor microenvironment. This concept provides great heterogeneity in tumors, which makes the disease difficult to cure using a single agent. Understanding metabolic flexibility by lactate shuttles offers new perspectives to develop treatments that target the hypoxic tumor microenvironment and overcome the limitations of glycolytic inhibitors.     

Genome-scale metabolic modeling elucidates the role of proliferative adaptation in causing the Warburg effect

The Warburg effect--a classical hallmark of cancer metabolism--is a counter-intuitive phenomenon in which rapidly proliferating cancer cells resort to inefficient ATP production via glycolysis leading to lactate secretion, instead of relying primarily on more efficient energy production through mitochondrial oxidative phosphorylation, as most normal cells do. The causes for the Warburg effect have remained a subject of considerable controversy since its discovery over 80 years ago, with several competing hypotheses. Here, utilizing a genome-scale human metabolic network model accounting for stoichiometric and enzyme solvent capacity considerations, we show that the Warburg effect is a direct consequence of the metabolic adaptation of cancer cells to increase biomass production rate. The analysis is shown to accurately capture a three phase metabolic behavior that is observed experimentally during oncogenic progression, as well as a prominent characteristic of cancer cells involving their preference for glutamine uptake over other amino acids.     

Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.     

Tumor Cells Switch to Mitochondrial Oxidative Phosphorylation under Radiation via mTOR-Mediated Hexokinase II Inhibition - A Warburg-Reversing Effect

A unique feature of cancer cells is to convert glucose into lactate to produce cellular energy, even under the presence of oxygen. Called aerobic glycolysis [The Warburg Effect] it has been extensively studied and the concept of aerobic glycolysis in tumor cells is generally accepted. However, it is not clear if aerobic glycolysis in tumor cells is fixed, or can be reversed, especially under therapeutic stress conditions. Here, we report that mTOR, a critical regulator in cell proliferation, can be relocated to mitochondria, and as a result, enhances oxidative phosphorylation and reduces glycolysis. Three tumor cell lines (breast cancer MCF-7, colon cancer HCT116 and glioblastoma U87) showed a quick relocation of mTOR to mitochondria after irradiation with a single dose 5 Gy, which was companied with decreased lactate production, increased mitochondrial ATP generation and oxygen consumption. Inhibition of mTOR by rapamycin blocked radiation-induced mTOR mitochondrial relocation and the shift of glycolysis to mitochondrial respiration, and reduced the clonogenic survival. In irradiated cells, mTOR formed a complex with Hexokinase II [HK II], a key mitochondrial protein in regulation of glycolysis, causing reduced HK II enzymatic activity. These results support a novel mechanism by which tumor cells can quickly adapt to genotoxic conditions via mTOR-mediated reprogramming of bioenergetics from predominantly aerobic glycolysis to mitochondrial oxidative phosphorylation. Such a “waking-up” pathway for mitochondrial bioenergetics demonstrates a flexible feature in the energy metabolism of cancer cells, and may be required for additional cellular energy consumption for damage repair and survival. Thus, the reversible cellular energy metabolisms should be considered in blocking tumor metabolism and may be targeted to sensitize them in anti-cancer therapy.     

Malate–aspartate shuttle promotes l-lactate oxidation in mitochondria

Metabolism in cancer cells is rewired to generate sufficient energy equivalents and anabolic precursors to support high proliferative activity. Within the context of these competing drives aerobic glycolysis is inefficient for the cancer cellular energy economy. Therefore, many cancer types, including colon cancer, reprogram mitochondria-dependent processes to fulfill their elevated energy demands. Elevated glycolysis underlying the Warburg effect is an established signature of cancer metabolism. However, there are a growing number of studies that show that mitochondria remain highly oxidative under glycolytic conditions. We hypothesized that activities of glycolysis and oxidative phosphorylation are coordinated to maintain redox compartmentalization. We investigated the role of mitochondria-associated malate–aspartate and lactate shuttles in colon cancer cells as potential regulators that couple aerobic glycolysis and oxidative phosphorylation. We demonstrated that the malate–aspartate shuttle exerts control over NAD⁺/NADH homeostasis to maintain activity of mitochondrial lactate dehydrogenase and to enable aerobic oxidation of glycolytic l -lactate in mitochondria. The elevated glycolysis in cancer cells is proposed to be one of the mechanisms acquired to accelerate oxidative phosphorylation.     

Catabolic efficiency of aerobic glycolysis: The Warburg effect revisited

Background  

Cancer cells simultaneously exhibit glycolysis with lactate secretion and mitochondrial respiration even in the presence of oxygen, a phenomenon known as the Warburg effect. The maintenance of this mixed metabolic phenotype is seemingly counterintuitive given that aerobic glycolysis is far less efficient in terms of ATP yield per moles of glucose than mitochondrial respiration.     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

The Warburg effect: Evolving interpretations of an established concept

Metabolic reprogramming and altered bioenergetics have emerged as hallmarks of cancer and an area of active basic and translational cancer research. Drastically upregulated glucose transport and metabolism in most cancers regardless of the oxygen supply, a phenomenon called the Warburg effect, is a major focuses of the research. Warburg speculated that cancer cells, due to defective mitochondrial oxidative phosphorylation (OXPHOS), switch to glycolysis for ATP synthesis, even in the presence of oxygen. Studies in the recent decade indicated that while glycolysis is indeed drastically upregulated in almost all cancer cells, mitochondrial respiration continues to operate normally at rates proportional to oxygen supply. There is no OXPHOS-to-glycolysis switch but rather upregulation of glycolysis. Furthermore, upregulated glycolysis appears to be for synthesis of biomass and reducing equivalents in addition to ATP production. The new finding that a significant amount of glycolytic intermediates is diverted to the pentose phosphate pathway (PPP) for production of NADPH has profound implications in how cancer cells use the Warburg effect to cope with reactive oxygen species (ROS) generation and oxidative stress, opening the door for anticancer interventions taking advantage of this. Recent findings in the Warburg effect and its relationship with ROS and oxidative stress controls will be reviewed. Cancer treatment strategies based on these new findings will be presented and discussed.     

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.     

Akt and c-Myc Differentially Activate Cellular Metabolic Programs and Prime Cells to Bioenergetic Inhibition

The high glucose consumption of tumor cells even in an oxygen-rich environment, referred to as the Warburg effect, has been noted as a nearly universal biochemical characteristic of cancer cells. Targeting the glycolysis pathway has been explored as an anti-cancer therapeutic strategy to eradicate cancer based on this fundamental biochemical property of cancer cells. Oncoproteins such as Akt and c-Myc regulate cell metabolism. Accumulating studies have uncovered various molecular mechanisms by which oncoproteins affect cellular metabolism, raising a concern as to whether targeting glycolysis will be equally effective in treating cancers arising from different oncogenic activities. Here, we established a dual-regulatable FL5.12 pre-B cell line in which myristoylated Akt is expressed under the control of doxycycline, and c-Myc, fused to the hormone-binding domain of the human estrogen receptor, is activated by 4-hydroxytamoxifen. Using this system, we directly compared the effect of these oncoproteins on cell metabolism in an isogenic background. Activation of either Akt or c-Myc leads to the Warburg effect as indicated by increased cellular glucose uptake, glycolysis, and lactate generation. When cells are treated with glycolysis inhibitors, Akt sensitizes cells to apoptosis, whereas c-Myc does not. In contrast, c-Myc but not Akt sensitizes cells to the inhibition of mitochondrial function. This is correlated with enhanced mitochondrial activities in c-Myc cells. Hence, although both Akt and c-Myc promote aerobic glycolysis, they differentially affect mitochondrial functions and render cells susceptible to the perturbation of cellular metabolic programs.     

Understanding the central role of citrate in the metabolism of cancer cells

Cancers cells strongly stimulate glycolysis and glutaminolysis for their biosynthesis. Pyruvate derived from glucose is preferentially diverted towards the production of lactic acid (Warburg effect). Citrate censors ATP production and controls strategic enzymes of anabolic and catabolic pathways through feedback reactions. Mitochondrial citrate diffuses in the cytosol to restore oxaloacetate and acetyl-CoA. Whereas acetyl-CoA serves de novo lipid synthesis and histone acetylation, OAA is derived towards lactate production via pyruvate and / or a vicious cycle reforming mitochondrial citrate. This cycle allows cancer cells to burn their host's lipid and protein reserves in order to sustain their own biosynthesis pathways. In vitro, citrate has demonstrated anti-cancer properties when administered in excess, sensitizing cancer cells to chemotherapy. Understanding its central role is of particular relevance for the development of new strategies for counteracting cancer cell proliferation and overcoming chemoresistance.     

Combination of Sulindac and Dichloroacetate Kills Cancer Cells via Oxidative Damage

Sulindac is an FDA-approved non-steroidal anti-inflammatory drug with documented anticancer activities. Our recent studies showed that sulindac selectively enhanced the killing of cancer cells exposed to oxidizing agents via production of reactive oxygen species (ROS) resulting in mitochondrial dysfunction. This effect of sulindac and oxidative stress on cancer cells could be related to the defect in respiration in cancer cells, first described by Warburg 50 years ago, known as the Warburg effect. We postulated that sulindac might enhance the selective killing of cancer cells when combined with any compound that alters mitochondrial respiration. To test this hypothesis we have used dichloroacetate (DCA), which is known to shift pyruvate metabolism away from lactic acid formation to respiration. One might expect that DCA, since it stimulates aerobic metabolism, could stress mitochondrial respiration in cancer cells, which would result in enhanced killing in the presence of sulindac. In this study, we have shown that the combination of sulindac and DCA enhances the selective killing of A549 and SCC25 cancer cells under the conditions used. As predicted, the mechanism of killing involves ROS production, mitochondrial dysfunction, JNK signaling and death by apoptosis. Our results suggest that the sulindac-DCA drug combination may provide an effective cancer therapy.     

Oncosecretomics coupled to bioenergetics identifies α-amino adipic acid, isoleucine and GABA as potential biomarkers of cancer: Differential expression of c-Myc, Oct1 and KLF4 coordinates metabolic changes

Bioenergetic profiling of tumors is a new challenge of cancer research and medicine as therapies are currently being developed. Meanwhile, methodological means must be proposed to gather information on tumor metabolism in order to adapt these potential therapies to the bioenergetic specificities of tumors. Studies performed on tumors and cancer cell lines have shown that cancer cells bioenergetics is highly variable. This profile changes with microenvironmental conditions (eg. substrate availability), the oncogenes activated (and the tumor suppressors inactivated) and the interaction with the stroma (i.e. reverse Warburg effect). Here, we assessed the power of metabolic footprinting (MFP) to unravel the bioenergetics and associated anabolic changes induced by three oncogenes, c-Myc, KLF4 and Oct1. The MFP approach provides a quantitative analysis of the metabolites secreted and consumed by cancer cells. We used ultra performance liquid chromatography for quantifying the amino acid uptake and secretion. To investigate the potential oncogene-mediated alterations in mitochondrial metabolism, we measured oxygen consumption rate and ATP production as well as the glucose uptake and lactate release. Our findings show that c-Myc deficiency initiates the Warburg effect along with a reduction of mitochondrial respiration. KLF4 deficiency also stimulated glycolysis, albeit without cellular respiration impairment. In contrast, Oct1 deficiency reduced glycolysis and enhanced oxidative phosphorylation efficiency. MFP revealed that c-Myc, KLF4 and Oct1 altered amino acid metabolism with specific patterns. We identified isoleucine, α-aminoadipic acid and GABA (γ-aminoisobutyric acid) as biomarkers related. Our findings establish the impact of Oct1, KLF4 and c-Myc on cancer bioenergetics and evidence a link between oncosecretomics and cellular bioenergetics profile.     

Effect of soluble nickel on cellular energy metabolism in A549 cells

Iron is an essential nutrient to most organisms, and is actively involved in oxygen delivery, electron transport, DNA synthesis, and many other biochemical reactions important for cell survival. We previously reported that nickel (Ni) ion exposure decreases cellular iron level and converts cytosolic aconitase (c-aconitase) to iron-regulatory protein-1 in A549 cells (Chen H, Davidson T, Singleton S, Garrick MD, Costa M. Toxicol Appl Pharmacol 206:275-287, 2005). Here, we further investigated the effect of Ni ion exposure on the activity of mitochondrial iron-sulfur (Fe-S) enzymes and cellular energy metabolism. We found that acute Ni ion treatment up to 1 mM exhibits minimal toxicity in A549 cells. Ni ion treatment decreases the activity of several Fe-S enzymes related to cellular energy metabolism, including mitochondrial aconitase (m-aconitase), succinate dehydrogenase (SDH), and NADH:ubiquinone oxidoreductase (complex I). Low doses of Ni ion for 4 weeks resulted in an increased cellular glycolysis and NADH to NAD+ (NADH/NAD+) ratio, although glycolysis was inhibited at higher levels. Collectively, our results show that Ni ions decrease the activity of cellular iron (Fe)-containing enzymes, inhibit oxidative phosphorylation (OxPhos), and increase cellular glycolytic activity. Since increased glycolysis is one of the fundamental alterations of energy metabolism in cancer cells (the Warburg effect), the inhibition of Fe-S enzymes and subsequent changes in cellular energy metabolism caused by Ni ions may play an important role in Ni carcinogenesis.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.