Aerobic Glycolysis In Cancer Cells Research Articles

A H2S-Nampt dependent energetic circuit is critical to survival and cytoprotection from damage in cancer cells.

We recently demonstrated that cancer cells that recover from damage exhibit increased aerobic glycolysis, however, the molecular mechanism by which cancer cells survive the damage and show increased aerobic glycolysis remains unknown. Here, we demonstrate that diverse cancer cells that survive hypoxic or oxidative damage show rapid cell proliferation, and develop tolerance to damage associated with increased production of hydrogen sulfide (H2S) which drives up-regulation of nicotinamide phosphoribosyltransferase (Nampt). Consistent with existence of a H2S-Nampt energetic circuit, in damage recovered cancer cells, H2S, Nampt and ATP production exhibit a significant correlation. Moreover, the treatment of cancer cells with H2S donor, NaHS, coordinately increases Nampt and ATP levels, and protects cells from drug induced damage. Inhibition of cystathionine beta synthase (CBS) or cystathionase (CTH), enzymes which drive generation of H2S, decreases Nampt production while suppression of Nampt pathway by FK866, decreases H2S and ATP levels. Damage recovered cells isolated from tumors grown subcutaneously in athymic mice also show increased production of H2S, Nampt and ATP levels, associated with increased glycolysis and rapid proliferation. Together, these data show that upon recovery from potential lethal damage, H2S-Nampt directs energy expenditure and aerobic glycolysis in cancer cells, leads to their exponential growth, and causes a high degree of tolerance to damage. Identification of H2S-Nampt as a pathway responsible for induction of damage tolerance in cancer cells may underlie resistance to therapy and offers the opportunity to target this pathway as a means in treatment of cancer.

Regulation of glucose metabolism in hepatocarcinogenesis by microRNAs.

In the past decade, considerable effort has been made in elucidating the mechanism underlying the high level of aerobic glycolysis in cancer cells. While some recent studies have attempted to address this issue, the potential role of microRNAs in this process has not been explored until recently. These studies have demonstrated involvement of just five deregulated miRNAs in glucose metabolism in hepatocarcinogenesis. This review discusses the metabolic significance of these miRNAs in hepatoceullular carcinoma, their targets in glycolysis, gluconeogenesis, and pentose phosphate pathways, and provides an insight into the therapeutic potential of targeting specific miRNAs.

Oleanolic Acid Suppresses Aerobic Glycolysis in Cancer Cells by Switching Pyruvate Kinase Type M Isoforms

Warburg effect, one of the hallmarks for cancer cells, is characterized by metabolic switch from mitochondrial oxidative phosphorylation to aerobic glycolysis. In recent years, increased expression level of pyruvate kinase M2 (PKM2) has been found to be the culprit of enhanced aerobic glycolysis in cancer cells. However, there is no agent inhibiting aerobic glycolysis by targeting PKM2. In this study, we found that Oleanolic acid (OA) induced a switch from PKM2 to PKM1, and consistently, abrogated Warburg effect in cancer cells. Suppression of aerobic glycolysis by OA is mediated by PKM2/PKM1 switch. Furthermore, mTOR signaling was found to be inactivated in OA-treated cancer cells, and mTOR inhibition is required for the effect of OA on PKM2/PKM1 switch. Decreased expression of c-Myc-dependent hnRNPA1 and hnRNPA1 was responsible for OA-induced switch between PKM isoforms. Collectively, we identified that OA is an antitumor compound that suppresses aerobic glycolysis in cancer cells and there is potential that PKM2 may be developed as an important target in aerobic glycolysis pathway for developing novel anticancer agents.

Cancer cell metabolism: implications for therapeutic targets

Cancer cell metabolism is characterized by an enhanced uptake and utilization of glucose, a phenomenon known as the Warburg effect. The persistent activation of aerobic glycolysis in cancer cells can be linked to activation of oncogenes or loss of tumor suppressors, thereby fundamentally advancing cancer progression. In this respect, inhibition of glycolytic capacity may contribute to an anticancer effect on malignant cells. Understanding the mechanisms of aerobic glycolysis may present a new basis for cancer treatment. Accordingly, interrupting lactate fermentation and/or other cancer-promoting metabolic sites may provide a promising strategy to halt tumor development. In this review, we will discuss dysregulated and reprogrammed cancer metabolism followed by clinical relevance of the metabolic enzymes, such as hexokinase, phosphofructokinase, pyruvate kinase M2, lactate dehydrogenase, pyruvate dehydrogenase kinase and glutaminase. The proper intervention of these metabolic sites may provide a therapeutic advantage that can help overcome resistance to chemotherapy or radiotherapy.

Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells

BackgroundMost normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA.MethodsHigh throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment.Results3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu398 cells.ConclusionsRapid chemical inhibition of LDHA by these quinoline 3-sulfonamids led to profound metabolic alterations and impaired cell survival in carcinoma cells making it a compelling strategy for treating solid tumors that rely on aerobic glycolysis for survival.

Energy Management by Enhanced Glycolysis in G1-phase in Human Colon Cancer Cells In Vitro and In Vivo

Activation of aerobic glycolysis in cancer cells is well known as the Warburg effect, although its relation to cell- cycle progression remains unknown. In this study, human colon cancer cells were labeled with a cell-cycle phase-dependent fluorescent marker Fucci to distinguish cells in G1-phase and those in S + G2/M phases. Fucci-labeled cells served as splenic xenograft transplants in super-immunodeficient NOG mice and exhibited multiple metastases in the livers, frozen sections of which were analyzed by semiquantitative microscopic imaging mass spectrometry. Results showed that cells in G1-phase exhibited higher concentrations of ATP, NADH, and UDP-N-acetylglucosamine than those in S and G2-M phases, suggesting accelerated glycolysis in G1-phase cells in vivo. Quantitative determination of metabolites in cells synchronized in S, G2-M, and G1 phases suggested that efflux of lactate was elevated significantly in G1-phase. By contrast, ATP production in G2-M was highly dependent on mitochondrial respiration, whereas cells in S-phase mostly exhibited an intermediary energy metabolism between G1 and G2-M phases. Isogenic cells carrying a p53-null mutation appeared more active in glycolysis throughout the cell cycle than wild-type cells. Thus, as the cell cycle progressed from G2-M to G1 phases, the dependency of energy production on glycolysis was increased while the mitochondrial energy production was reciprocally decreased. These results shed light on distinct features of the phase-specific phenotypes of metabolic systems in cancer cells.

Effective inhibition of nasopharyngeal carcinoma in vitro and in vivo by targeting glycolysis with oxamate

Elevated aerobic glycolysis in cancer cells (Warburg effect) has been observed in many tumor types including nasopharyngeal carcinoma (NPC), which can often be detected clinically using FDG-PET. However, the role of glycolysis in supporting the growth of NPC cells and its therapeutic implications still remain to be investigated. In the present study, we showed that the LDH inhibitor oxamate significantly suppressed NPC cell proliferation in vitro and tumor growth in vivo, yet exhibited minimum toxicity to normal nasopharyngeal epithelial cells in vitro and was well tolerated in mice. Moreover, oxamate exhibited cytotoxic effect in NPC cells under hypoxia. Mechanistic study showed that oxamate significantly inhibited LDH activity, leading to a substantial decrease in glucose uptake and lactate production. Combination of oxamate with a mitochondrial respiratory complex I inhibitor resulted in a significant depletion of cellular ATP and a synergistic killing of cancer cells. Our results suggest that inhibition of glycolysis by oxamate is an effective therapeutic strategy for treatment of NPC and that combination of this compound with mitochondrial-targeted agents may improve the therapeutic activity.

Tumor necrosis factor alpha induces Warburg‐like metabolism and is reversed by anti‐inflammatory curcumin in breast epithelial cells

The reprogramming of cellular metabolism in cancer cells is a well-documented effect. It has previously been shown that common oncogene expression can induce aerobic glycolysis in cancer cells. However, the direct effect of an inflammatory microenvironment on cancer cell metabolism is not known. Here, we illustrate that treatment of nonmalignant (MCF-10a) and malignant (MCF-7) breast epithelial cells with low-level (10 ng/ml) tumor necrosis factor alpha (TNF-α) significantly increased glycolytic reliance, lactate export and expression of the glucose transporter 1 (GLUT1). TNF-α decreased total mitochondrial content; however, oxygen consumption rate was not significantly altered, suggesting that overall mitochondrial function was increased. Upon glucose starvation, MCF7 cells treated with TNF-α demonstrated significantly lower viability than nontreated cells. Interestingly, these properties can be partially reversed by coincubation with the anti-inflammatory agent curcumin in a dose-dependent manner. This work demonstrates that aerobic glycolysis can be directly induced by an inflammatory microenvironment independent of additional genetic mutations and signals from adjacent cells. Furthermore, we have identified that a natural dietary compound can reverse this effect.

Abstract 1869: DCA and metformin synergistically induce apoptosis in breast cancer cells.

Abstract The unusual metabolism of aerobic glycolysis in cancer cells suggests that this phenomenon can be exploited for improved therapies. Metformin, a drug used to treat type 2 diabetics, has clinical evidence of cancer prevention, and some studies show that cancer patients have a better response when it is used with neoadjuvant therapy. One of the cellular targets of metformin is the inhibition of Complex I in the mitochondrial electron transport chain. This leads to an accumulation superoxide species that can cause oxidative damage to cells and eventually lead to cell death. Based on this, we hypothesized that drugs which promote mitochondrial metabolism will synergize with metformin to increase superoxide production and promote cell death of cancer cells. Cancer cells using aerobic glycolysis display a high level of glucose uptake that is processed through glycolysis to pyruvate, which is then converted to lactate and secreted from the cells. Diverting pyruvate to mitochondrial metabolism rather than its conversion to lactate was expected to enhance metformin cytotoxicity. Dichloroacetate (DCA) is well established drug that has been tested in humans for the treatment of certain metabolic diseases. DCA works through inhibition of pyruvate dehydrogenase kinase (PDK), thus reducing entry of pyruvate into mitochondrial oxidative phosphorylation pathways. Consequently DCA is expected to promote mitochondrial metabolism and reduce lactate secretion by cancer cells. We demonstrate that DCA and metformin synergistically kill cultured breast cancer cells although the drug combination has no effect on non-transformed MCF10A breast epithelial cells. The enhanced cell death of the breast cancer cells displayed is through apoptosis, as shown by caspase-dependent increases in cleaved PARP. DCA treatment was associated with reduced lactate secretion by the cells and increased pH of the growth medium which suggested it promoted oxidative phosphorylation. The combination of two drugs also promoted increased mitochondrial superoxide production compared to metformin alone which was associated with increased oxidative damage. These findings indicate that, as expected, DCA promotes metformin-mediated cell death by enhancing mitochondrial metabolism of pyruvate. Therefore, it is possible that the combination of metformin with DCA, or similar compounds, could enhance cancer therapy. Citation Format: Allison B. Haugrud, Yongxian Zhuang, Wilson K. Miskimins. DCA and metformin synergistically induce apoptosis in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1869. doi:10.1158/1538-7445.AM2013-1869

Abstract 4612: Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth.

Abstract The current understanding of the Warburg effect consists of an increase in aerobic glycolysis in cancer cells. The connection between glycolysis and PPP/biosynthesis is based upon a model in which glycolytic intermediates can be diverted into PPP and biosynthesis pathways as precursors. However, it remains unclear how cancer cells coordinate glycolysis and biosynthesis to fulfill the request of rapidly growing tumors. We found that glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers due to loss of TP53, regulates anabolic biosynthesis by controlling intracellular levels of its substrate 3-phosphoglycerate (3-PG) and product 2-phosphoglycerate (2-PG). Our co-crystal structure based analysis revealed that 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP) as a competitive inhibitor, while 2-PG activates 3-phosphoglycerate dehydrogenase (PHGDH) to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA results in increased 3-PG and decreased 2-PG levels in cancer cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth. These findings uncover that PGAM1 controls the intracellular 3-PG and 2-PG levels to serve as a novel link between glycolysis, biosynthesis, cancer cell proliferation and tumor development. Moreover, we screened and developed novel small molecule PGAM1 inhibitor (PGMI)-004A, which effectively inhibits PGAM1 enzyme activity, resulting in aberrant cancer cell metabolism with reduced glycolysis and PPP/anabolic biosynthesis, as well as attenuated cell proliferation in diverse human cancer cell lines, but not control normal human proliferating cells including human dermal fibroblasts and human foreskin fibroblasts. PGMI-004A treatment attenuates tumor growth in xenograft nude mice with minimal toxicity in vivo. Furthermore, PGMI-004A inhibits cell proliferation of primary leukemia cells from human AML, CML and B-ALL patients, but not control CD34+ cells isolated from bone marrow samples or mononucleocytes isolated from peripheral blood samples from healthy donors, suggesting minimal toxicity of PGMI-004A in human cells.These results together provide “proof of principle” for the development of PGAM1 inhibitors as anti-cancer agents. Citation Format: Taro Hitosugi, Lu Zhou, Martha Arellano, Hanna Khoury, Titus Boggon, Sumin Kang, Chuan He, Jing Chen. Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4612. doi:10.1158/1538-7445.AM2013-4612

Metabolic signature genes associated with susceptibility to pyruvate kinase, muscle type 2 gene ablation in cancer cells.

Pyruvate kinase, muscle type 2 (PKM2), is a key factor in the aerobic glycolysis of cancer cells. In our experiments, liver cancer cell lines exhibited a range of sensitivity to PKM2 knockdown-mediated growth inhibition. We speculated that this differential sensitivity is attributable to the variable dependency on glycolysis for the growth of different cell lines. Transcriptome data revealed overexpression of a glucose transporter (GLUT3) and a lactate transporter (MCT4) genes in PKM2 knockdown-sensitive cells. PKM2 knockdown-resistant cells expressed high levels of the lactate dehydrogenase B (LDHB) and glycine decarboxylase (GLDC) genes. Concordant with the gene expression results, PKM2 knockdown-sensitive cells generated high levels of lactate. In addition, ATP production was significantly reduced in the PKM2 knockdown-sensitive cells treated with a glucose analog, indicative of dependency of their cellular energetics on lactate-producing glycolysis. The PKM2 knockdown-resistant cells were further subdivided into less glycolytic and more (glycolysis branch pathway-dependent) glycolytic groups. Our findings collectively support the utility of PKM2 as a therapeutic target for high lactate-producing glycolytic hepatocellular carcinoma (HCC).

Organometallic anticancer agents that interfere with cellular energy processes: a subtle approach to inducing cancer cell death

Two hybrid compounds comprising an antimetastatic ruthenium-arene fragment tethered to an indazole-3-carboxylic acid derivative that inhibits aerobic glycolysis in cancer cells have been prepared and evaluated in a variety of cancer cell lines, including highly relevant human glioblastoma cells, with an apparent synergistic action between the two components observed.

Metabolic control of respiration and glycolysis of tumoral cells

The contribution of the author’s groups on the control of energy metabolism of cancer cells has been concisely reviewed. It is proposed that Otto Warburg’s “respiratory defect” of tumoral cells resides in an alteration of the recurrent filamentation cycle of mitochondria. These give those organelles an affinity lower for ADP than the affinity of the tumor cell isoenzyme of pyruvate kinase. These two findings may explain the essentials of the intimate mechanism of the aerobic glycolysis of cancer cells.

Galloflavin prevents the binding of lactate dehydrogenase A to single stranded DNA and inhibits RNA synthesis in cultured cells

Lactate dehydrogenase A (LDH-A) binds single stranded DNA (ssDNA) and stimulates cell transcription. Binding is prevented by NADH, suggesting that the coenzyme site is involved in the interaction LDH-A/ssDNA. We recently identified an inhibitor of LDH-A enzymatic activity (Galloflavin, GF) which occupies the NADH site. In the experiments reported here we studied whether GF can also hinder the binding of LDH-A to ssDNA and investigated its effects on RNA synthesis in cultured cells. Using a filter binding assay we observed that 4μM GF inhibited the binding of human LDH-A to a single stranded [3H]DNA sample by 50%. After only 0.5–1h, 50–100μM GF inhibited RNA synthesis in SW620 cells maintained in a medium in which galactose substituted glucose. In these culture conditions, SW620 cells did not produce lactic acid and effects caused by the inhibition of the enzymatic activity of LDH-A could be excluded.Novel LDH-A inhibitors which hinder aerobic glycolysis of cancer cells are at present actively searched. Our results suggest that: (i) inhibitors which bind the NADH site can exert their antiproliferative activity not only by blocking aerobic glycolysis but also by causing an inhibition of RNA synthesis independent from the effect on glycolysis; (ii) GF can be a useful tool to study the biological role of LDH-A binding to ssDNA.

The Histone Deacetylase SIRT6 Is a Tumor Suppressor that Controls Cancer Metabolism

Reprogramming of cellular metabolism is a key eventduring tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a tumor suppressor thatregulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, whereas transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. By using a conditional SIRT6 allele, we show that SIRT6 deletion invivo increases the number, size, and aggressiveness of tumors. SIRT6 also functions as a regulator of ribosome metabolismby corepressing MYC transcriptional activity. Lastly,Sirt6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancermetabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism.

Lineage-Specific Functions of LKB1 in CML and B Lymphoid Blast Crisis

Abstract 34 Background:The serine-threonine liver kinase B1 (LKB1, also called STK11) acts as negative regulator of aerobic glycoloysis, a metabolic pathway that is typically used in cancer cells (commonly referred to as ‘Warburg effect'). LKB1, together with AMPKs, shifts the metabolic balance from aerobic glycolysis to oxidative phosphorylation and thereby reverses the metabolic program of cancer cells and functions as tumor suppressor. Recently, it has been shown that LKB1 plays a critical role in the maintenance of quiescence and metabolic homeostasis of hematopoietic stem cells (HSCs). Hypothesis:In the present study, we focused on the roles of LKB1 in BCR-ABL1-driven leukemias including CML and B lymphoid blast crisis/Ph+ ALL (LBC). While LKB1 is widely seen as tumor suppressor in solid tumors, we found that high expression levels of LKB1 at diagnosis predict poor clinical outcome in patients with high risk acute lymphoblastic leukemia (n=207; COG P9906 trial; p=0.0204). In addition, high levels of LKB1 expression correlate with positive minimal residual disease (MRD+, p=0.0323) status in patients. These findings were unexpected and seem to contradict the common notion of LKB1 as a tumor suppressor. Results:To study the function of LKB1 in CML and B lymphoid blast crisis/Ph+ ALL (LBC), we developed a mouse model for inducible ablation of Lkb1 in BCR-ABL1-transformed hematopoietic stem and progenitor cells (CML-like) and B cell progenitors (LBC). To this end, Lkb1-fl/fl bone marrow hematopoietic stem and progenitor cells and B cell precursor cells were transformed with BCR-ABL1 and transduced with tamoxifen-inducible Cre. Unexpectedly, Cre-mediated deletion of Lkb1 had opposite effects in CML and LBC. While Lkb1-deletion in CML results in an initial proliferative burst of the leukemia cells, the vast majority of B cell lineage LBC cells undergo rapid cell cycle arrest. These findings are consistent with changes of cell cycle checkpoint proteins in response to Lkb1 deletion in CML and B cell lineage LBC. While Lkb1 deletion in CML cells results in downregulation of Arf, p53 and p27 levels, Lkb1 deletion in B lineage LBC cells resulted in upregulation of Arf and p27. In addition, Lkb1 deletion in CML resulted in inactivation of AMPK, a known substrate of LKB1, as well as enhanced activation of mTORC1. By contrast, while deletion of Lkb1 in B cell lineage LBC cells resulted in inactivation of AMPK as shown by reduced phosphorylation of AMPKα T172, there was reduction in mTORC1 activity based on diminished levels of phospho-p70 S6 kinase and S6 following LKB1 deletion.The effects of LKB1 on sensitivity of BCR-ABL1 CML and B lineage LBC cells to Imatinib were also examined. Lkb1-deficient ALL cells became more sensitive to Imatinib treatment. On the other hand, initial Lkb1 deletion rendered CML cells more resistant to Imatinib treatment. When primary patient-derived Ph+ ALL cells (n = 3) were treated with Imatinib, upregulation of phospho-LKB1 (S428) was observed. Finally, LKB1 was also shown to regulate energy homeostasis in CML and B cell lineage LBC in different manners, as measured by monitoring ATP and lactate production. Conclusions:Here we show that Lkb1 plays critical roles in mediating proliferation and cell growth in BCR-ABL1-driven leukemias. While LKB1 is widely seen as a tumor suppressor that limits aerobic glycolysis in cancer cells according to the Warburg effect, our findings demonstrate that LKB1 has lineage-specific functions in BCR-ABL1 driven leukemias. While LKB1 function in CML resembles its tumor suppressor function in solid tumors, LKB1 is critical for survival and proliferation on B cell lineage CML blast crisis and Ph+ ALL. The finding of a divergent role of Lkb1 in CML and B cell lineage LBC/Ph+ ALL is relevant because small molecule inhibitors of AMPK and mTORC1 are currently under development for the treatment of BCR-ABL1-driven leukemias. Disclosures:No relevant conflicts of interest to declare.

Phosphoglycerate Mutase 1 Promotes Leukemia Metabolism by Coordinating Glycolysis and Biosynthesis, and Represnts a Therapuetic Target in Leukemia Treatment

AbstractAbstract 860The current understanding of the Warburg effect consists of an increase in aerobic glycolysis in cancer cells. The connection between glycolysis and PPP/biosynthesis is based upon a model in which glycolytic intermediates can be diverted into PPP and biosynthesis pathways as precursors. However, it remains unclear how cancer cells coordinate glycolysis and biosynthesis to fulfill the request of rapidly growing tumors. We found that glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers including leukemias due to loss of TP53, regulates anabolic biosynthesis by controlling intracellular levels of its substrate 3-phosphoglycerate (3-PG) and product 2-phosphoglycerate (2-PG). Our co-crystal structure based analysis revealed that 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP) as a competitive inhibitor, while 2-PG activates 3-phosphoglycerate dehydrogenase (PHGDH) to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA results in increased 3-PG and decreased 2-PG levels in cancer and leukemia cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth. These findings uncover that PGAM1 controls the intracellular 3-PG and 2-PG levels to serve as a novel link between glycolysis, biosynthesis, cancer/leukemia cell proliferation and tumor development.Moreover, we found that PGAM1 protein expression and enzyme activity levels are commonly upregulated in diverse human leukemia cell lines, as well as in primary leukemia cells from human AML, CML and B-ALL patients. We screened and developed novel small molecule PGAM1 inhibitor (PGMI)-004A, which effectively inhibits PGAM1 enzyme activity, resulting in aberrant leukemia cell metabolism with reduced glycolysis and PPP/anabolic biosynthesis, as well as attenuated cell proliferation in diverse human leukemia cell lines, but not control normal human proliferating cells including human dermal fibroblasts and human foreskin fibroblasts. In addition, PGMI-004A treatment attenuates tumor growth in xenograft nude mice with minimal toxicity in vivo. Furthermore, PGMI-004A inhibits cell proliferation of primary leukemia cells from human AML, CML and B-ALL patients, but not control CD34+ cells isolated from bone marrow samples or mononucleocytes isolated from peripheral blood samples from healthy donors, suggesting minimal toxicity of PGMI-004A in human cells.Together, our findings demonstrate that PGAM1 regulates the concentrations of glycolytic metabolites 3-PG and 2-PG, which function as signaling molecules to directly affect the catalytic activity of enzymes involved in PPP and biosynthesis, representing a novel, additional link between glycolysis, PPP and biosynthesis. PGAM1 is commonly upregulated in human leukemia cells and important for cell metabolism and proliferation, representing a promising therapeutic target in treatment of leukemias. Small molecule inhibitor of PGAM1, PGMI-004A exhibits promising efficacy and minimal toxicity in treatment of xenograft nude mice and human primary leukemia cells, providing “proof of principle” for the development of PGAM1 inhibitors as anti-leukemia agents. Disclosures:No relevant conflicts of interest to declare.

Overexpression of Ecdysoneless in Pancreatic Cancer and Its Role in Oncogenesis by Regulating Glycolysis

To study the expression and function of a novel cell-cycle regulatory protein, human ecdysoneless (Ecd), during pancreatic cancer pathogenesis. Immunohistochemical expression profiling of Ecd was done in nonneoplastic normal pancreatic tissues and pancreatic ductal adenocarcinoma lesions (from tissue microarray and Rapid Autopsy program) as well as precancerous PanIN lesions and metastatic organs. To analyze the biological significance of Ecd in pancreatic cancer progression, Ecd was stably knocked down in pancreatic cancer cell line followed by in vitro and in vivo functional assays. Normal pancreatic ducts showed very weak to no Ecd expression compared to significant positive expression in pancreatic cancer tissues (mean ± SE composite score: 0.3 ± 0.2 and 3.8 ± 0.2 respectively, P < 0.0001) as well as in PanIN precursor lesions with a progressive increase in Ecd expression with increasing dysplasia (PanIN-1-PanIN-3). Analysis of matched primary tumors and metastases from patients with pancreatic cancer revealed that Ecd is highly expressed in both primary pancreatic tumor and in distant metastatic sites. Furthermore, knockdown of Ecd suppressed cell proliferation in vitro and tumorigenicity of pancreatic cancer cells in mice orthotopic tumors. Microarray study revealed that Ecd regulates expression of glucose transporter GLUT4 in pancreatic cancer cells and was subsequently shown to modulate glucose uptake, lactate production, and ATP generation by pancreatic cancer cells. Finally, knockdown of Ecd also reduced level of pAkt, key signaling molecule known to regulate aerobic glycolysis in cancer cells. Ecd is a novel tumor-promoting factor that is differentially expressed in pancreatic cancer and potentially regulates glucose metabolism within cancer cells.

Metabolic reprogramming and two-compartment tumor metabolism

Hypoxia-inducible factor (HIF) 1α and 2α are transcription factors responsible for the cellular response to hypoxia. The functional roles of HIF1α and HIF2α in cancer are distinct and vary among different tumor types. The aim of this study was to evaluate the compartment-specific role(s) of HIF1α and HIF2α in breast cancer. To this end, immortalized human fibroblasts and MDA-MB-231 breast cancer cells carrying constitutively active HIF1α or HIF2α mutants were analyzed with respect to their metabolic function(s) and ability to promote tumor growth in an in vivo setting. We observed that activation of HIF1α, but not HIF2α, in stromal cells promotes a shift toward aerobic glycolysis, with increased L-lactate production and a loss of mitochondrial activity. In a xenograft model, HIF1α-activated fibroblasts promoted the tumor growth of co-injected MDA-MB-231 cells without an increase in angiogenesis. Conversely, HIF2α-activated stromal cells did not favor tumor growth and behaved as the empty vector controls. Similarly, activation of HIF1α, but not HIF2α, in MDA-MB-231 cells promoted a shift toward aerobic glycolysis, with increased glucose uptake and L-lactate production. In contrast, HIF2α activation in cancer cells increased the expression of EGFR, Ras and cyclin D1, which are known markers of tumor growth and cell cycle progression. In a xenograft model, HIF1α activation in MDA-MB-231 cells acted as a tumor suppressor, resulting in an almost 2-fold reduction in tumor mass and volume. Interestingly, HIF2α activation in MDA-MB-231 cells induced a significant ~2-fold-increase in tumor mass and volume. Analysis of mitochondrial activity in these tumor xenografts using COX (cytochrome C oxidase) staining demonstrated elevated mitochondrial oxidative metabolism (OXPHOS) in HIF2α-tumors. We conclude that the role(s) of HIF1α and HIF2α in tumorigenesis are compartment-specific. HIF1α acts as a tumor promoter in stromal cells but as a tumor suppressor in cancer cells. Conversely, HIF2α is a tumor promoter in cancer cells. Mechanistically, HIF1α-driven aerobic glycolysis in stromal cells supports cancer cell growth via the paracrine production of nutrients (such as L-lactate) that can “feed” cancer cells. However, HIF1α-driven aerobic glycolysis in cancer cells inhibits tumor growth. Finally, HIF2α activation in cancer cells induces the expression of known pro-oncogenic molecules and promotes the mitochondrial activity of cancer cells.

Stat3-mediated activation of microRNA-23a suppresses gluconeogenesis in hepatocellular carcinoma by down-regulating Glucose-6-phosphatase and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha

Considerable effort has been made in elucidating the mechanism and functional significance of high levels of aerobic glycolysis in cancer cells, commonly referred to as the Warburg effect. Here we investigated whether the gluconeogenic pathway is significantly modulated in hepatocarcinogenesis, resulting in altered levels of glucose homeostasis. To test this possibility, we used a mouse model (mice fed a choline-deficient diet) that develops nonalcoholic steatohepatitis (NASH), preneoplastic nodules, and hepatocellular carcinoma (HCC), along with human primary HCCs and HCC cells. This study demonstrated marked reduction in the expressions of G6pc, Pepck, and Fbp1 encoding the key gluconeogenic enzymes glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, fructose-1,6-phosphatase, respectively, and the transcription factor Pgc-1α in HCCs developed in the mouse model that correlated with reduction in serum glucose in tumor-bearing mice. The messenger RNA (mRNA) levels of these genes were also reduced by ≈80% in the majority of primary human HCCs compared with matching peritumoral livers. The expression of microRNA (miR)-23a, a candidate miR targeting PGC-1α and G6PC, was up-regulated in the mouse liver tumors as well as in primary human HCC. We confirmed PGC-1α and G6PC as direct targets of miR-23a and their expressions negatively correlated with miR-23a expression in human HCCs. G6PC expression also correlated with tumor grade in human primary HCCs. Finally, this study showed that the activation of interleukin (IL)-6-Stat3 signaling caused the up-regulation of miR-23a expression in HCC. Based on these data, we conclude that gluconeogenesis is severely compromised in HCC by IL6-Stat3-mediated activation of miR-23a, which directly targets PGC-1α and G6PC, leading to decreased glucose production.