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1.  Mitochondrial metabolism in cancer metastasis 
Cell Cycle  2012;11(7):1445-1454.
We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TOMM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TOMM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TOMM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.
doi:10.4161/cc.19841
PMCID: PMC3350881  PMID: 22395432
caveolin-1; oxidative stress; MCT4; metabolic coupling; tumor stroma; SLC16A3; monocarboxylic acid transporter; two-compartment tumor metabolism; metastasis; TOMM20; complex IV; OXPHOS; mitochondria; inflammation
2.  Using the “reverse Warburg effect” to identify high-risk breast cancer patients 
Cell Cycle  2012;11(6):1108-1117.
We have recently proposed a new model of cancer metabolism to explain the role of aerobic glycolysis and L-lactate production in fueling tumor growth and metastasis. In this model, cancer cells secrete hydrogen peroxide (H2O2), initiating oxidative stress and aerobic glycolysis in the tumor stroma. This, in turn, drives L-lactate secretion from cancer-associated fibroblasts. Secreted L-lactate then fuels oxidative mitochondrial metabolism (OXPHOS) in epithelial cancer cells, by acting as a paracrine onco-metabolite. We have previously termed this type of two-compartment tumor metabolism the “reverse Warburg effect,” as aerobic glycolysis takes place in stromal fibroblasts, rather than epithelial cancer cells. Here, we used MCT4 immunostaining of human breast cancer tissue microarrays (TMAs; >180 triple-negative patients) to directly assess the prognostic value of the “reverse Warburg effect.” MCT4 expression is a functional marker of hypoxia, oxidative stress, aerobic glycolysis and L-lactate efflux. Remarkably, high stromal MCT4 levels (score = 2) were specifically associated with decreased overall survival (<18% survival at 10 years post-diagnosis). In contrast, patients with absent stromal MCT4 expression (score = 0), had 10-year survival rates of ∼97% (p-value < 10−32). High stromal levels of MCT4 were strictly correlated with a loss of stromal Cav-1 (p-value < 10−14), a known marker of early tumor recurrence and metastasis. In fact, the combined use of stromal Cav-1 and stromal MCT4 allowed us to more precisely identify high-risk triple-negative breast cancer patients, consistent with the goal of individualized risk-assessment and personalized cancer treatment. However, epithelial MCT4 staining had no prognostic value, indicating that the “conventional” Warburg effect does not predict clinical outcome. Thus, the “reverse Warburg effect” or “parasitic” energy-transfer is a key determinant of poor overall patient survival. As MCT4 is a druggable target, MCT4 inhibitors should be developed for the treatment of aggressive breast cancers, and possibly other types of human cancers. Similarly, we discuss how stromal MCT4 could be used as a biomarker for identifying high-risk cancer patients that could likely benefit from treatment with FDA-approved drugs or existing MCT-inhibitors (such as, AR-C155858, AR-C117977 and AZD-3965).
doi:10.4161/cc.11.6.19530
PMCID: PMC3335917  PMID: 22313602
caveolin-1; oxidative stress; pseudohypoxia; lactate shuttle; MCT4; metabolic coupling; tumor stroma; predictive biomarker; SLC16A3; monocarboxylic acid transporter; two-compartment tumor metabolism
3.  The Warburg effect in leukemia-stroma cocultures is mediated by mitochondrial uncoupling associated with uncoupling protein 2 activation 
Cancer research  2008;68(13):5198-5205.
In 1956 Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that bypassed the Pasteur effect, i.e. the inhibition of anaerobic fermentation by oxygen. Since then, permanent defects in oxygen consumption that could explain the dependence of cancer cells on aerobic glycolysis have not been identified. Here we demonstrate that under normoxic conditions exposure of leukemia cells to bone marrow derived mesenchymal stromal cells (MSC) promotes accumulation of lactate in the culture medium and reduces mitochondrial membrane potential (ΔΨM) in both cell types. Notably, the consumption of glucose was not altered in cocultures suggesting that the accumulation of lactate was the result of reduced pyruvate metabolism. Interestingly, the decrease in ΔΨM was mediated by mitochondrial uncoupling in leukemia cells and was accompanied by increased expression of uncoupling protein 2 (UCP2). HL60 cells fail to increase UCP2 expression, are not uncoupled after coculture, and do not exhibit increased aerobic glycolysis, whereas siRNA-mediated suppression of UCP2 in OCI-AML3 cells reversed mitochondrial uncoupling and aerobic glycolysis elicited by MSC. Taken together, these data suggest that microenvironment-activation of highly conserved mammalian uncoupling proteins may facilitate the Warburg effect in the absence of permanent respiratory impairment.
doi:10.1158/0008-5472.CAN-08-0555
PMCID: PMC2562568  PMID: 18593920
Warburg effect; uncoupling proteins; mitochondria; leukemia; mesenchymal stromal cells
4.  Mitochondrial dysfunction in breast cancer cells prevents tumor growth 
Cell Cycle  2013;12(1):172-182.
Metformin is a well-established diabetes drug that prevents the onset of most types of human cancers in diabetic patients, especially by targeting cancer stem cells. Metformin exerts its protective effects by functioning as a weak “mitochondrial poison,” as it acts as a complex I inhibitor and prevents oxidative mitochondrial metabolism (OXPHOS). Thus, mitochondrial metabolism must play an essential role in promoting tumor growth. To determine the functional role of “mitochondrial health” in breast cancer pathogenesis, here we used mitochondrial uncoupling proteins (UCPs) to genetically induce mitochondrial dysfunction in either human breast cancer cells (MDA-MB-231) or cancer-associated fibroblasts (hTERT-BJ1 cells). Our results directly show that all three UCP family members (UCP-1/2/3) induce autophagy and mitochondrial dysfunction in human breast cancer cells, which results in significant reductions in tumor growth. Conversely, induction of mitochondrial dysfunction in cancer-associated fibroblasts has just the opposite effect. More specifically, overexpression of UCP-1 in stromal fibroblasts increases β-oxidation, ketone body production and the release of ATP-rich vesicles, which “fuels” tumor growth by providing high-energy nutrients in a paracrine fashion to epithelial cancer cells. Hence, the effects of mitochondrial dysfunction are truly compartment-specific. Thus, we conclude that the beneficial anticancer effects of mitochondrial inhibitors (such as metformin) may be attributed to the induction of mitochondrial dysfunction in the epithelial cancer cell compartment. Our studies identify cancer cell mitochondria as a clear target for drug discovery and for novel therapeutic interventions.
doi:10.4161/cc.23058
PMCID: PMC3570509  PMID: 23257779
chemoprevention; Metformin; mitochondrial dysfunction; breast cancer; tumor growth; UCP; mitochondrial uncoupling proteins; autophagy; ketone body production; fatty acid beta-oxidation; ATP-rich vesicles
5.  Is cancer a metabolic rebellion against host aging? 
Cell Cycle  2012;11(2):253-263.
Aging drives large systemic reductions in oxidative mitochondrial function, shifting the entire body metabolically toward aerobic glycolysis, a.k.a, the Warburg effect. Aging is also one of the most significant risk factors for the development of human cancers, including breast tumors. How are these two findings connected? One simplistic idea is that cancer cells rebel against the aging process by increasing their capacity for oxidative mitochondrial metabolism (OXPHOS). Then, local and systemic aerobic glycolysis in the aging host would provide energy-rich mitochondrial fuels (such as L-lactate and ketones) to directly “fuel” tumor cell growth and metastasis. This would establish a type of parasite-host relationship or “two-compartment tumor metabolism,” with glycolytic/oxidative metabolic coupling. The cancer cells (“the seeds”) would flourish in this nutrient-rich microenvironment (“the soil”), which has been fertilized by host aging. In this scenario, cancer cells are only trying to save themselves from the consequences of aging by engineering a metabolic mutiny, through the amplification of mitochondrial metabolism. We discuss the recent findings of Drs. Ron DePinho (MD Anderson) and Craig Thomspson (Sloan-Kettering) that are also consistent with this new hypothesis, linking cancer progression with metabolic aging. Using data mining and bioinformatics approaches, we also provide key evidence of a role for PGC1a/NRF1 signaling in the pathogenesis of (1) two-compartment tumor metabolism and (2) mitochondrial biogenesis in human breast cancer cells.
doi:10.4161/cc.11.2.19006
PMCID: PMC3293377  PMID: 22234241
aging; mitochondria; cancer metabolism; autophagy; mitophagy; aerobic glycolysis; oxidative phosphorylation; Metformin; drug resistance; chemoresistance; Warburg effect; metabolic compartments; parasite; PGC1a; PGC1b; NRF1; two-compartment tumor metabolism
6.  Negative regulation of UCP2 by TGFβ signaling characterizes low and intermediate-grade primary breast cancer 
Cell Death & Disease  2010;1(7):e53-.
The histological manifestation of growth-regulating and differentiation-inducing signals in cancer cells is considered as a key component for clinical outcome prediction and commonly defined as tumor differentiation grade. However, the molecular and functional framework underlying this clinical parameter remains poorly understood. Our correlative data display a significant association (P>0.001) between mitochondrial uncoupling protein 2 (UCP2) and tumor grade in primary breast cancer (n=234). Through mechanistic analyses, we show a synergistic link between UCP2 and established cellular pathways in conferring grade-associated functional phenotypes. Here, the application of well to moderately differentiated primary tumor cell lines has enabled direct observation of SMAD recruitment to the UCP2 promoter underlying repression of gene transcription. In contrast, poorly differentiated tumor cells, known to be TGFβ resistant, displayed aberrant UCP2 regulation, and consequently, gene overexpression, which reduced mitochondrial calcium and facilitated the maintenance of mitochondrial membrane potential, thereby significantly decreasing oxidative stress and inhibiting cell death. Conversely, UCP2 silencing in such cells rapidly led to the induction of apoptosis and cell differentiation, concurrent with reduced cell survival and proliferation, confirming gene-specific effects. Demonstration of a biologically driven role for UCP2 dysregulation in promoting multiple characteristics of tumor aggressiveness strongly endorses assessment of gene expression at clinical presentation to augment therapeutic decision-making and improve patient outcome through personalized targeting approaches.
doi:10.1038/cddis.2010.30
PMCID: PMC3032562  PMID: 21364658
cancer aggressiveness; differentiation status; mitochondrial changes; apoptosis; proliferation
7.  SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization 
Cancer cell  2011;19(3):416-428.
Summary
Tumor cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of oxygen. This metabolic reprogramming, known as the Warburg effect, provides tumor cells with the substrates required for biomass generation. Here, we show that the mitochondrial NAD-dependent deacetylase SIRT3 is a crucial regulator of the Warburg effect. Mechanistically, SIRT3 mediates metabolic reprogramming by destabilizing hypoxia-inducible factor-1α (HIF1α), a transcription factor that controls glycolytic gene expression. SIRT3 loss increases reactive oxygen species production, leading to HIF1α stabilization. SIRT3 expression is reduced in human breast cancers, and its loss correlates with the upregulation of HIF1α target genes. Finally, we find that SIRT3 overexpression represses glycolysis and proliferation in breast cancer cells, providing a metabolic mechanism for tumor suppression.
doi:10.1016/j.ccr.2011.02.014
PMCID: PMC3065720  PMID: 21397863
8.  Impaired OXPHOS Complex III in Breast Cancer 
PLoS ONE  2011;6(8):e23846.
We measured the mitochondrial oxidative phosphorylation (mtOXPHOS) activities of all five complexes and determined the activity and gene expression in detail of the Complex III subunits in human breast cancer cell lines and primary tumors. Our analysis revealed dramatic differences in activity of complex III between normal and aggressive metastatic breast cancer cell lines. Determination of Complex III subunit gene expression identified over expression and co-regulation of UQCRFS1 (encoding RISP protein) and UQCRH (encoding Hinge protein) in 6 out of 9 human breast tumors. Analyses of UQCRFS1/RISP expression in additional matched normal and breast tumors demonstrated an over expression in 14 out of 40 (35%) breast tumors. UQCRFS1/RISP knockdown in breast tumor cell line led to decreased mitochondrial membrane potential as well as a decrease in matrigel invasion. Furthermore, reduced matrigel invasion was mediated by reduced ROS levels coinciding with decreased expression of NADPH oxidase 2, 3, 4 and 5 involved in ROS production. These studies provide direct evidence for contribution of impaired mtOXPHOS Complex III to breast tumorigenesis.
doi:10.1371/journal.pone.0023846
PMCID: PMC3162009  PMID: 21901141
9.  The Warburg Effect Is Genetically Determined in Inherited Pheochromocytomas 
PLoS ONE  2009;4(9):e7094.
The Warburg effect describes how cancer cells down-regulate their aerobic respiration and preferentially use glycolysis to generate energy. To evaluate the link between hypoxia and Warburg effect, we studied mitochondrial electron transport, angiogenesis and glycolysis in pheochromocytomas induced by germ-line mutations in VHL, RET, NF1 and SDH genes. SDH and VHL gene mutations have been shown to lead to the activation of hypoxic response, even in normoxic conditions, a process now referred to as pseudohypoxia. We observed a decrease in electron transport protein expression and activity, associated with increased angiogenesis in SDH- and VHL-related, pseudohypoxic tumors, while stimulation of glycolysis was solely observed in VHL tumors. Moreover, microarray analyses revealed that expression of genes involved in these metabolic pathways is an efficient tool for classification of pheochromocytomas in accordance with the predisposition gene mutated. Our data suggest an unexpected association between pseudohypoxia and loss of p53, which leads to a distinct Warburg effect in VHL-related pheochromocytomas.
doi:10.1371/journal.pone.0007094
PMCID: PMC2738974  PMID: 19763184
10.  Mitochondrial uncoupling and the Warburg effect: Molecular basis for the reprogramming of cancer cell metabolism 
Cancer research  2009;69(6):10.1158/0008-5472.CAN-08-3722.
The precise mitochondrial alterations that underlie the increased dependence of cancer cells on aerobic glycolysis for energy generation have remained a mystery. Recent evidence suggests that mitochondrial uncoupling – the abrogation of ATP synthesis in response to mitochondrial membrane potential – promotes the Warburg effect in leukemia cells, and may contribute to chemoresistance. Intriguingly, leukemia cells cultured on bone marrow-derived stromal feeder layers are more resistant to chemotherapy, increase the expression of uncoupling protein 2, and decrease the entry of pyruvate into the Krebs cycle – without compromising the consumption of oxygen, suggesting a shift to the oxidation of non-glucose carbon sources to maintain mitochondrial integrity and function. Since fatty acid oxidation has been linked to chemoresistance and mitochondrial uncoupling, it is tempting to speculate that Warburg’s observations may indeed be the result of the preferential oxidation of fatty acids by cancer cell mitochondria. Therefore, targeting fatty acid oxidation or anaplerotic pathways that support fatty acid oxidation may provide additional therapeutic tools for the treatment of hematopoietic malignancies.
doi:10.1158/0008-5472.CAN-08-3722
PMCID: PMC3822436  PMID: 19258498
Warburg effect; uncoupling proteins; mitochondria; leukemia; Krebs cycle
11.  Hypoxia-Inducible Factor-1α and the Glycolytic Phenotype in Tumors1 
Neoplasia (New York, N.Y.)  2005;7(4):324-330.
Abstract
Metastatic tumors generally exhibit aerobic glycolysis (the Warburg effect). The advent of [18F]fluorodeoxyglucose positron emission tomography imaging, coupled with recent findings linking hypoxia-inducible factor (HIF-1α) overexpression to aggressive cancers, has rekindled an interest in this aspect of tumor metabolism. These studies explore the role of HIF-1α in human breast cancer lines and its relationship to glycolytic regulation. Here we demonstrate that, under normal oxygen conditions, nonmetastatic cells consume less glucose and express low HIF-1α, whereas metastatic cells constitutively express high glycolysis and HIF-1α, suggesting that dysregulation of HIF-1α may induce the Warburg effect. This hypothesis was tested by renormalizing HIF-1α levels in renal carcinoma cells, leading to inhibition of aerobic glycolysis.
PMCID: PMC1501147  PMID: 15967109
Warburg effect; hypoxia-inducible factor; glucose consumption; lactate; glycolysis
12.  Evidence for a stromal-epithelial “lactate shuttle” in human tumors 
Cell Cycle  2011;10(11):1772-1783.
Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the “reverse Warburg effect,” which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, “energy transfer” or “metabolic-coupling” between the tumor stroma and epithelial cancer cells “fuels” tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.
doi:10.4161/cc.10.11.15659
PMCID: PMC3142461  PMID: 21558814
caveolin-1; oxidative stress; pseudohypoxia; lactate shuttle; MCT1; MCT4; metabolic coupling; tumor stroma; predictive biomarker; SLC16A1; SLC16A3; monocarboxylic acid transporter
13.  Energy metabolism of cancer: Glycolysis versus oxidative phosphorylation (Review) 
Oncology Letters  2012;4(6):1151-1157.
Metabolic activities in normal cells rely primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate ATP for energy. Unlike in normal cells, glycolysis is enhanced and OXPHOS capacity is reduced in various cancer cells. It has long been believed that the glycolytic phenotype in cancer is due to a permanent impairment of mitochondrial OXPHOS, as proposed by Otto Warburg. This view is challenged by recent investigations which find that the function of mitochondrial OXPHOS in most cancers is intact. Aerobic glycolysis in many cancers is the combined result of various factors such as oncogenes, tumor suppressors, a hypoxic microenvironment, mtDNA mutations, genetic background and others. Understanding the features and complexity of the cancer energy metabolism will help to develop new approaches in early diagnosis and effectively target therapy of cancer.
doi:10.3892/ol.2012.928
PMCID: PMC3506713  PMID: 23226794
cancer; glycolysis; oxidative phosphorylation
14.  Regulation of the Warburg Effect in Early-Passage Breast Cancer Cells12 
Neoplasia (New York, N.Y.)  2008;10(8):745-756.
Malignancy in cancer is associated with aerobic glycolysis (Warburg effect) evidenced by increased trapping of [18F]deoxyglucose (FdG) in patients imaged by positron emission tomography (PET). [18F]deoxyglucose uptake correlates with glucose transporter (GLUT-1) expression, which can be regulated by hypoxia-inducible factor 1 alpha (HIF-1α). We have previously reported in established breast lines that HIF-1α levels in the presence of oxygen leads to the Warburg effect. However, glycolysis and GLUT-1 can also be induced independent of HIF-1α by other factors, such as c-Myc and phosphorylated Akt (pAkt). This study investigates HIF-1α, c-Myc, pAkt, and aerobic glycolysis in low-passage breast cancer cells under the assumption that these represent the in vivo condition better than established lines. Similar to in vivo FdG-PET or primary breast cancers, rates of glycolysis were diverse, being higher in cells expressing both c-Myc and HIF-1α and lower in cell lines low or negative in both transcription factors. No correlations were observed between glycolytic rates and pAkt levels. Two of 12 cell lines formed xenografts in mice. Both were positive for HIF-1α and phosphorylated c-Myc, and only one was positive for pAkt. Glycolysis was affected by pharmacological regulation of c-Myc and HIF-1α. These findings suggest that c-Myc and/or HIF-1α activities are both involved in the regulation of glycolysis in breast cancers.
PMCID: PMC2481565  PMID: 18670636
15.  The Role of Uncoupling Proteins in Diabetes Mellitus 
Journal of Diabetes Research  2013;2013:585897.
Uncoupling proteins (UCPs) are anion carriers expressed in the mitochondrial inner membrane that uncouple oxygen consumption by the respiratory chain from ATP synthesis. The physiological functions of UCPs have long been debated since the new UCPs (UCP2 to 5) were discovered, and the role of UCPs in the pathogeneses of diabetes mellitus is one of the hottest topics. UCPs are thought to be activated by superoxide and then decrease mitochondrial free radicals generation; this may provide a protective effect on diabetes mellitus that is under the oxidative stress conditions. UCP1 is considered to be a candidate gene for diabetes because of its role in thermogenesis and energy expenditure. UCP2 is expressed in several tissues and acts in the negative regulation of insulin secretion by β-cells and in fatty acid metabolism. UCP3 plays a role in fatty acid metabolism and energy homeostasis and modulates insulin sensitivity. Several gene polymorphisms of UCP1, UCP2, and UCP3 were reported to be associated with diabetes. The progress in the role of UCP1, UCP2, and UCP3 on diabetes mellitus is summarized in this review.
doi:10.1155/2013/585897
PMCID: PMC3687498  PMID: 23841103
16.  Ketones and lactate “fuel” tumor growth and metastasis 
Cell Cycle  2010;9(17):3506-3514.
Previously, we proposed a new model for understanding the “Warburg effect” in tumor metabolism. In this scheme, cancer-associated fibroblasts undergo aerobic glycolysis and the resulting energy-rich metabolites are then transferred to epithelial cancer cells, where they enter the TCA cycle, resulting in high ATP production via oxidative phosphorylation. We have termed this new paradigm “The Reverse Warburg Effect.” Here, we directly evaluate whether the end-products of aerobic glycolysis (3-hydroxy-butyrate and L-lactate) can stimulate tumor growth and metastasis, using MDA-MB-231 breast cancer xenografts as a model system. More specifically, we show that administration of 3-hydroxy-butyrate (a ketone body) increases tumor growth by ∼2.5-fold, without any measurable increases in tumor vascularization/angiogenesis. Both 3-hydroxy-butyrate and L-lactate functioned as chemo-attractants, stimulating the migration of epithelial cancer cells. Although L-lactate did not increase primary tumor growth, it stimulated the formation of lung metastases by ∼10-fold. Thus, we conclude that ketones and lactate fuel tumor growth and metastasis, providing functional evidence to support the “reverse Warburg effect.” Moreover, we discuss the possibility that it may be unwise to use lactate-containing i.v. solutions (such as lactated Ringer's or Hartmann's solution) in cancer patients, given the dramatic metastasis-promoting properties of L-lactate. Also, we provide evidence for the upregulation of oxidative mitochondrial metabolism and the TCA cycle in human breast cancer cells in vivo, via an informatics analysis of the existing raw transcriptional profiles of epithelial breast cancer cells and adjacent stromal cells. Lastly, our findings may explain why diabetic patients have an increased incidence of cancer, due to increased ketone production, and a tendency towards autophagy/mitophagy in their adipose tissue.
doi:10.4161/cc.9.17.12731
PMCID: PMC3047616  PMID: 20818174
3-hydroxybutyrate (ketone bodies); L-lactate; stroma; tumor growth; metastasis; the Warburg effect; aerobic glycolysis; tumor microenvironment; cancer associated fibroblasts
17.  AAV3-mediated Transfer and Expression of the Pyruvate Dehydrogenase E1 alpha Subunit Gene Causes Metabolic Remodeling and Apoptosis of Human Liver Cancer Cells 
Molecular genetics and metabolism  2009;98(3):289-299.
Most cancers rely disproportionately on glycolysis for energy even in the presence of adequate oxygen supply, a condition known as “aerobic glycolysis”, or the Warburg effect. Pharmacological reversal of the Warburg effect has been shown to cause selective apoptosis of tumor cells, presumably by stimulating mitochondrial respiratory chain activity and production of reactive oxygen species that, in turn, induce a caspase-mediated series of reactions leading to cell death. We reasoned that a similar effect on tumor cells might result from up-regulation of the E1α subunit gene (pda1) of the pyruvate dehydrogenase complex (PDC) that catalyzes the rate-limiting step in aerobic glucose oxidation and thus plays a major role in the control of oxidative phosphorylation. To test this postulate, we employed a self-complementary adeno-associated virus (scAAV)-based delivery and expression system for targeting pda1 to the mitochondria of primary cultures of human hepatoblastoma (HB) and hepatocellular carcinoma (HCC) cells. Serotypes 1-10 scAAV vectors that included enhanced green fluorescent (egfp) reporter gene driven by either cytomegalovirus (CMV) or chicken beta-actin (CBA) promoters were analyzed for transduction ability of HB (Huh-6) and HCC (Huh-7 and HepG2) cell lines and primary cultures of normal human hepatocytes. Serotype 3 scAAV-egfp (scAAV3-egfp) vector was the most efficient and transduced up to 90% of cells. We limited the transgene expression primarily to liver cancer cells by generating scAAV3 vectors that contained the human alpha-fetoprotein promoter (AFP)-driven reporter gene (scAAV3.AFP-egfp) and the potentially therapeutic gene scAAV3.AFP-pda1. Infection of Huh-6 cells by the scAAV3.AFP-pda1 vector increased protein expression of E1α, PDC catalytic activity, and late-stage apoptotic cell death. Apoptosis was also associated with increased protein expression of Bcl-X/S, an early marker of apoptosis, and release of cytochrome c into the cytosol of infected HB cells. These data indicate that molecular targeting of mitochondrial oxidative metabolism in liver cancer cells by AAV3-mediated delivery of pda1 holds promise as a novel and effective therapeutic approach for human hepatic tumors.
doi:10.1016/j.ymgme.2009.05.010
PMCID: PMC2779054  PMID: 19586787
Adeno-associated viral vectors; apoptosis; hepatoblastoma; hepatocellular carcinoma; gene therapy; pyruvate dehydrogenase complex
18.  Thyroid Hormone Differentially Modulates Warburg Phenotype in Breast Cancer Cells 
Sustenance of cancer cells in vivo critically depends on a variety of genetic and metabolic adaptations. Aerobic glycolysis or Warburg effect has been a defining biochemical hallmark of transformed cells for more than five decades although a clear molecular basis of this observation is emerging only in recent years. In this study, we present our findings that thyroid hormone exerts its non-genomic and genomic actions in two model human breast cancer cell lines differentially. By laying a clear foundation for experimentally monitoring the Warburg phenotype in living cancer cells, we demonstrate that thyroid hormone-induced modulation of bioenergetic profiles in these two model cell lines depends on the degree of Warburg phenotype that they display. Further we also show that thyroid hormone can sensitize mitochondria in aggressive, triple-negative breast cancer cells favorably to increase the chemotherapeutic efficacy in these cells. Even though the role of thyroid hormone in modulating mitochondrial metabolism has been known, the current study accentuates the critical role it plays in modulating Warburg phenotype in breast cancer cells. The clinical significance of this finding is the possibility to devise strategies for metabolically modulating aggressive triple-negative tumors so as to enhance their chemosensitivity in vivo.
doi:10.1016/j.bbrc.2011.09.024
PMCID: PMC3207240  PMID: 21945435
Breast Cancer; Mitochondria; Warburg Effect; Thyroid Hormone; Bioenergetics; Chemotherapy
19.  Targeting metabolism with arsenic trioxide and dichloroacetate in breast cancer cells 
Molecular Cancer  2011;10:142.
Background
Cancer cells have a different metabolic profile compared to normal cells. The Warburg effect (increased aerobic glycolysis) and glutaminolysis (increased mitochondrial activity from glutamine catabolism) are well known hallmarks of cancer and are accompanied by increased lactate production, hyperpolarized mitochondrial membrane and increased production of reactive oxygen species.
Methods
In this study we target the Warburg effect with dichloroacetate (DCA) and the increased mitochondrial activity of glutaminolysis with arsenic trioxide (ATO) in breast cancer cells, measuring cell proliferation, cell death and mitochondrial characteristics.
Results
The combination of DCA and ATO was more effective at inhibiting cell proliferation and inducing cell death than either drug alone. We examined the effect of these treatments on mitochondrial membrane potential, reactive oxygen species production and ATP levels and have identified new molecular mechanisms within the mitochondria for both ATO and DCA: ATO reduces mitochondrial function through the inhibition of cytochrome C oxidase (complex IV of the electron transport chain) while DCA up-regulates ATP synthase β subunit expression. The potentiation of ATO cytotoxicity by DCA is correlated with strong suppression of the expression of c-Myc and HIF-1α, and decreased expression of the survival protein Bcl-2.
Conclusion
This study is the first to demonstrate that targeting two key metabolic hallmarks of cancer is an effective anti-cancer strategy with therapeutic potential.
doi:10.1186/1476-4598-10-142
PMCID: PMC3240126  PMID: 22093145
Dichloroacetate; breast cancer; electron transport chain; mitochondria; arsenic trioxide
20.  Phospholipase D-mTOR Requirement for the Warburg Effect in Human Cancer Cells 
Cancer letters  2010;299(1):72-79.
A characteristic of cancer cells is the generation of lactate from glucose in spite of adequate oxygen for oxidative phosphorylation. This property – known as the “Warburg effect” or aerobic glycolysis – contrasts with anaerobic glycolysis, which is triggered in hypoxic normal cells. The Warburg effect is thought to provide a means for cancer cells to survive under conditions where oxygen is limited and to generate metabolites necessary for cell growth. The shift from oxidative phosphorylation to glycolysis in response to hypoxia is mediated by the production of hypoxia-inducible factor (HIF) – a transcription factor family that stimulates the expression of proteins involved in glucose uptake and glycolysis. We reported previously that elevated phospholipase D (PLD) activity in renal and breast cancer cells is required for the expression of the α subunits of HIF1 and HIF2. We report here that the aerobic glycolysis observed in human breast and renal cancer cells is dependent on the elevated PLD activity. Intriguingly, the effect of PLD on the Warburg phenotype was dependent on the mammalian target of rapamycin complex 1 (mTORC1) in the breast cancer cells and on mTORC2 in the renal cancer cells. These data indicate that elevated PLD-mTOR signaling, which is common in human cancer cells, is critical for the metabolic shift to aerobic glycolysis.
doi:10.1016/j.canlet.2010.08.006
PMCID: PMC3010225  PMID: 20805015
Phospholipase D; Warburg effect; glycolysis; metabolic transformation; hypoxia-inducible factor
21.  Phosphoglycerate dehydrogenase is dispensable for breast tumor maintenance and growth 
Oncotarget  2013;4(12):2502-2511.
Cancer cells rely on aerobic glycolysis to maintain cell growth and proliferation via the Warburg effect. Phosphoglycerate dehydrogenase (PHDGH) catalyzes the first step of the serine biosynthetic pathway downstream of glycolysis, which is a metabolic gatekeeper both for macromolecular biosynthesis and serine-dependent DNA synthesis. Here, we report that PHDGH is overexpressed in many ER-negative human breast cancer cell lines. PHGDH knockdown in these cells leads to a reduction of serine synthesis and impairment of cancer cell proliferation. However, PHGDH knockdown does not affect tumor maintenance and growth in established breast cancer xenograft models, suggesting that PHGDH-dependent cancer cell growth may be context-dependent. Our findings suggest that other mechanisms or pathways may bypass exclusive dependence on PHGDH in established human breast cancer xenografts, indicating that PHGDH is dispensable for the growth and maintenance of tumors in vivo.
PMCID: PMC3926844  PMID: 24318446
PHGDH; breast cancer cells; in vivo
22.  Mitochondrial p32 Protein Is a Critical Regulator of Tumor Metabolism via Maintenance of Oxidative Phosphorylation ▿  
Molecular and Cellular Biology  2010;30(6):1303-1318.
p32/gC1qR/C1QBP/HABP1 is a mitochondrial/cell surface protein overexpressed in certain cancer cells. Here we show that knocking down p32 expression in human cancer cells strongly shifts their metabolism from oxidative phosphorylation (OXPHOS) to glycolysis. The p32 knockdown cells exhibited reduced synthesis of the mitochondrial-DNA-encoded OXPHOS polypeptides and were less tumorigenic in vivo. Expression of exogenous p32 in the knockdown cells restored the wild-type cellular phenotype and tumorigenicity. Increased glucose consumption and lactate production, known as the Warburg effect, are almost universal hallmarks of solid tumors and are thought to favor tumor growth. However, here we show that a protein regularly overexpressed in some cancers is capable of promoting OXPHOS. Our results indicate that high levels of glycolysis, in the absence of adequate OXPHOS, may not be as beneficial for tumor growth as generally thought and suggest that tumor cells use p32 to regulate the balance between OXPHOS and glycolysis.
doi:10.1128/MCB.01101-09
PMCID: PMC2832503  PMID: 20100866
23.  Tumor-Associated Mutant p53 Drives the Warburg Effect 
Nature communications  2013;4:2935.
Tumor cells primarily utilize aerobic glycolysis for energy production, a phenomenon known as the Warburg effect. Its mechanism is not well-understood. The tumor suppressor gene p53 is frequently mutated in tumors. Many tumor-associated mutant p53 (mutp53) proteins not only lose tumor suppressive function, but also gain new oncogenic functions that are independent of wild type p53, defined as mutp53 gain-of-function (GOF). Here we show that tumor-associated mutp53 stimulates the Warburg effect in cultured cells and mutp53 knock-in mice as a new mutp53 GOF. Mutp53 stimulates the Warburg effect through promoting GLUT1 translocation to plasma membrane, which is mediated by the activated RhoA and its downstream effector ROCK. Inhibition of the RhoA/ROCK/GLUT1 signaling largely abolishes mutp53 GOF in stimulating the Warburg effect. Furthermore, inhibition of glycolysis in tumor cells greatly compromises mutp53 GOF in promoting tumorigenesis. Thus, our results reveal a new mutp53 GOF and a mechanism for controlling the Warburg effect.
doi:10.1038/ncomms3935
PMCID: PMC3969270  PMID: 24343302
24.  Novel Role of NOX in Supporting Aerobic Glycolysis in Cancer Cells with Mitochondrial Dysfunction and as a Potential Target for Cancer Therapy 
PLoS Biology  2012;10(5):e1001326.
NAD(P)H oxidase plays a role in cancer metabolism by providing NAD+ to support increased glycolysis.
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.
Author Summary
Glycolysis is a cytoplasmic metabolic process that produces energy from glucose. In normal cells, the rate of glycolysis is low, and glycolysis products are further processed in the mitochondria via oxidative phosphorylation, a very efficient energy-producing process. Cancer cells, however, display higher levels of glycolysis followed by cytoplasmic fermentation, and reduced levels of oxidative phosphorylation. It was thought that increased glycolysis is associated with mitochondrial dysfunction, but how these phenomena are functionally linked was not known. Understanding how these processes are regulated will be essential for developing more effective anti-cancer therapies. Here, we show that induction of mitochondrial dysfunction by either genetic or chemical approaches results in a switch from oxidative phosphorylation to glycolysis. We further show that NADPH oxidase (NOX), an enzyme known to catalyze the oxidation of NAD(P)H, also plays a critical role in supporting increased glycolysis in cancer cells by generating NAD+, a substrate for one of the key glycolytic reactions. Inhibition of NOX leads to inhibition of cancer cell proliferation in vitro and suppression of tumor growth in vivo. This study reveals a novel function for NOX in cancer metabolism, explains the increased glycolysis observed in cancer cells, and identifies NOX as a potential anti-cancer therapeutic target.
doi:10.1371/journal.pbio.1001326
PMCID: PMC3348157  PMID: 22589701
25.  Glycolysis Inhibitor Screening Identifies the Bis-geranylacylphloroglucinol Protonophore Moronone from Moronobea coccinea 
Journal of natural products  2012;75(12):2216-2222.
Tumor cells exhibit enhanced glucose consumption and lactate production even when supplied with adequate oxygen (a phenomenon known as the Warburg effect, or aerobic glycolysis). Pharmacological inhibition of aerobic glycolysis represents a potential tumor-selective approach that targets the metabolic differences between normal and malignant tissues. Human breast tumor MDA-MB-231 cells were used to develop an assay system to discover natural product-based glycolysis inhibitors. The assay employed was based on hypersensitivity to glycolytic inhibition in tumor cells treated with the mitochondrial electron transport inhibitor rotenone. Under such conditions, ATP supply, and hence cell viability, depends exclusively on glycolysis. This assay system was used to evaluate 10,648 plant and marine organism extracts from the U.S. National Cancer Institute's Open Repository. Bioassay-guided isolation of an active Moronobea coccinea extract yielded the new bis-geranylacylphloroglucinol derivative moronone (1). Compound 1 exhibited enhanced antiproliferative/cytotoxic activity in the presence of rotenone-imposed metabolic stress on tumor cells. Surprisingly, mechanistic studies revealed that 1 did not inhibit glycolysis, but rather functions as a protonophore that dissipates the mitochondrial proton gradient. In the presence of rotenone, tumor cells may be hypersensitive to protonophores due to increased ATP utilization by the ATP synthase.
doi:10.1021/np300711e
PMCID: PMC3532528  PMID: 23245650

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