Anne Le

Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression

As the result of genetic alterations and tumor hypoxia, many cancer cells avidly take up glucose and generate lactate through lactate dehydrogenase A (LDHA), which is encoded by a target gene of c-Myc and hypoxia-inducible factor (HIF-1). Previous studies with reduction of LDHA expression indicate that LDHA is involved in tumor initiation, but its role in tumor maintenance and progression has not been established. Furthermore, how reduction of LDHA expression by interference or antisense RNA inhibits tumorigenesis is not well understood. Here, we report that reduction of LDHA by siRNA or its inhibition by a small-molecule inhibitor (FX11 [3-dihydroxy-6-methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylic acid]) reduced ATP levels and induced significant oxidative stress and cell death that could be partially reversed by the antioxidant N-acetylcysteine. Furthermore, we document that FX11 inhibited the progression of sizable human lymphoma and pancreatic cancer xenografts. When used in combination with the NAD+ synthesis inhibitor FK866, FX11 induced lymphoma regression. Hence, inhibition of LDHA with FX11 is an achievable and tolerable treatment for LDHA-dependent tumors. Our studies document a therapeutical approach to the Warburg effect and demonstrate that oxidative stress and metabolic phenotyping of cancers are critical aspects of cancer biology to consider for the therapeutical targeting of cancer energy metabolism.

Glucose-Independent Glutamine Metabolism via TCA Cycling for Proliferation and Survival in B Cells

Because MYC plays a causal role in many human cancers, including those with hypoxic and nutrient-poor tumor microenvironments, we have determined the metabolic responses of a MYC-inducible human Burkitt lymphoma model P493 cell line to aerobic and hypoxic conditions, and to glucose deprivation, using stable isotope-resolved metabolomics. Using [U-(13)C]-glucose as the tracer, both glucose consumption and lactate production were increased by MYC expression and hypoxia. Using [U-(13)C,(15)N]-glutamine as the tracer, glutamine import and metabolism through the TCA cycle persisted under hypoxia, and glutamine contributed significantly to citrate carbons. Under glucose deprivation, glutamine-derived fumarate, malate, and citrate were significantly increased. Their (13)C-labeling patterns demonstrate an alternative energy-generating glutaminolysis pathway involving a glucose-independent TCA cycle. The essential role of glutamine metabolism in cell survival and proliferation under hypoxia and glucose deficiency makes them susceptible to the glutaminase inhibitor BPTES and hence could be targeted for cancer therapy.

MYC-Induced Cancer Cell Energy Metabolism and Therapeutic Opportunities

Although cancers have altered glucose metabolism, termed the Warburg effect, which describes the increased uptake and conversion of glucose to lactate by cancer cells under adequate oxygen tension, changes in the metabolism of glutamine and fatty acid have also been documented. The MYC oncogene, which contributes to the genesis of many human cancers, encodes a transcription factor c-Myc, which links altered cellular metabolism to tumorigenesis. c-Myc regulates genes involved in the biogenesis of ribosomes and mitochondria, and regulation of glucose and glutamine metabolism. With E2F1, c-Myc induces genes involved in nucleotide metabolism and DNA replication, and microRNAs that homeostatically attenuate E2F1 expression. With the hypoxia inducible transcription factor HIF-1, ectopic c-Myc cooperatively induces a transcriptional program for hypoxic adaptation. Myc regulates gene expression either directly, such as glycolytic genes including lactate dehydrogenase A (LDHA), or indirectly, such as repression of microRNAs miR-23a/b to increase glutaminase (GLS) protein expression and glutamine metabolism. Ectopic MYC expression in cancers, therefore, could concurrently drive aerobic glycolysis and/or oxidative phosphorylation to provide sufficient energy and anabolic substrates for cell growth and proliferation in the context of the tumor microenvironment. Collectively, these studies indicate that Myc-mediated altered cancer cell energy metabolism could be translated for the development of new anticancer therapies. (Clin Cancer Res 2009;15(21):6479–83)

Reprogramming of proline and glutamine metabolism contributes to the proliferative and metabolic responses regulated by oncogenic transcription factor c-MYC.

In addition to glycolysis, the oncogenic transcription factor c-MYC (MYC) stimulates glutamine catabolism to fuel growth and proliferation of cancer cells through up-regulating glutaminase (GLS). Glutamine is converted to glutamate by GLS, entering the tricarboxylic acid cycle as an important energy source. Less well-recognized, glutamate can also be converted to proline through Δ1-pyrroline-5-carboxylate (P5C) and vice versa. This study suggests that some MYC-induced cellular effects are due to MYC regulation of proline metabolism. Proline oxidase, also known as proline dehydrogenase (POX/PRODH), the first enzyme in proline catabolism, is a mitochondrial tumor suppressor that inhibits proliferation and induces apoptosis. MiR-23b* mediates POX/PRODH down-regulation in human kidney tumors. MiR-23b* is processed from the same transcript as miR-23b; the latter inhibits the translation of GLS. Using MYC-inducible human Burkitt lymphoma model P493 and PC3 human prostate cancer cells, we showed that MYC suppressed POX/PRODH expression primarily through up-regulating miR-23b*. The growth inhibition in the absence of MYC was partially reversed by POX/PRODH knockdown, indicating the importance of suppression of POX/PRODH in MYC-mediated cellular effects. Interestingly, MYC not only inhibited POX/PRODH, but also markedly increased the enzymes of proline biosynthesis from glutamine, including P5C synthase and P5C reductase 1. MYC-induced proline biosynthesis from glutamine was directly confirmed using 13C,15N-glutamine as a tracer. The metabolic link between glutamine and proline afforded by MYC emphasizes the complexity of tumor metabolism. Further studies of the relationship between glutamine and proline metabolism should provide a deeper understanding of tumor metabolism while enabling the development of novel therapeutic strategies.

Therapeutic targeting of cancer cell metabolism

In 1927, Otto Warburg and coworkers reported the increased uptake of glucose and production of lactate by tumors in vivo as compared with normal tissues. This phenomenon, now known as the Warburg effect, was recapitulated in vitro with cancer tissue slices exhibiting excessive lactate production even with adequate oxygen. Warburgs in vivo studies of tumors further suggest that the dependency of tumors in vivo on glucose could be exploited for therapy, because reduction of arterial glucose by half resulted in a four-fold reduction in tumor fermentation. Recent work in cancer metabolism indicates that the Warburg effect or aerobic glycolysis contributes to redox balance and lipid synthesis, but glycolysis is insufficient to sustain a growing and dividing cancer cell. In this regard, glutamine, which contributes its carbons to the tricarboxylic acid (TCA) cycle, has been re-discovered as an essential bioenergetic and anabolic substrate for many cancer cell types. Could alterations in cancer metabolism be exploited for therapy? Here, we address this question by reviewing current concepts of normal metabolism and altered metabolism in cancer cells with specific emphasis on molecular targets involved directly in glycolysis or glutamine metabolism.

Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis

Glutaminase (GLS), which converts glutamine to glutamate, plays a key role in cancer cell metabolism, growth, and proliferation. GLS is being explored as a cancer therapeutic target, but whether GLS inhibitors affect cancer cell-autonomous growth or the host microenvironment or have off-target effects is unknown. Here, we report that loss of one copy of Gls blunted tumor progression in an immune-competent MYC-mediated mouse model of hepatocellular carcinoma. Compared with results in untreated animals with MYC-induced hepatocellular carcinoma, administration of the GLS-specific inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) prolonged survival without any apparent toxicities. BPTES also inhibited growth of a MYC-dependent human B cell lymphoma cell line (P493) by blocking DNA replication, leading to cell death and fragmentation. In mice harboring P493 tumor xenografts, BPTES treatment inhibited tumor cell growth; however, P493 xenografts expressing a BPTES-resistant GLS mutant (GLS-K325A) or overexpressing GLS were not affected by BPTES treatment. Moreover, a customized Vivo-Morpholino that targets human GLS mRNA markedly inhibited P493 xenograft growth without affecting mouse Gls expression. Conversely, a Vivo-Morpholino directed at mouse Gls had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cell-autonomous dependence on GLS for cancer therapy.

Quantitative determinants of aerobic glycolysis identify flux through the enzyme GAPDH as a limiting step

Aerobic glycolysis or the Warburg Effect (WE) is characterized by the increased metabolism of glucose to lactate. It remains unknown what quantitative changes to the activity of metabolism are necessary and sufficient for this phenotype. We developed a computational model of glycolysis and an integrated analysis using metabolic control analysis (MCA), metabolomics data, and statistical simulations. We identified and confirmed a novel mode of regulation specific to aerobic glycolysis where flux through GAPDH, the enzyme separating lower and upper glycolysis, is the rate-limiting step in the pathway and the levels of fructose (1,6) bisphosphate (FBP), are predictive of the rate and control points in glycolysis. Strikingly, negative flux control was found and confirmed for several steps thought to be rate-limiting in glycolysis. Together, these findings enumerate the biochemical determinants of the WE and suggest strategies for identifying the contexts in which agents that target glycolysis might be most effective. DOI: http://dx.doi.org/10.7554/eLife.03342.001

Alveolar cell apoptosis is dependent on p38 MAP kinase-mediated activation of xanthine oxidoreductase in ventilator-induced lung injury

Signaling via p38 MAP kinase has been implicated in the mechanotransduction associated with mechanical stress and ventilator-induced lung injury (VILI). However, the critical downstream mediators of alveolar injury remain incompletely defined. We provide evidence that high-tidal volume mechanical ventilation (HVt MV) rapidly activates caspases within the lung, resulting in increased alveolar cell apoptosis. Antagonism of MV-induced p38 MAP kinase activity with SB-203580 suppresses both MV-induced caspase activity and alveolar apoptosis, placing p38 MAP kinase upstream of MV-induced caspase activation and programmed cell death. The reactive oxygen species (ROS)-producing enzyme xanthine oxidoreductase (XOR) is activated in a p38 MAP kinase-dependent manner following HVt MV. Allopurinol, a XOR inhibitor, also suppresses HVt MV-induced apoptosis, implicating HVt MV-induced ROS in the induction of alveolar cell apoptosis. Finally, systemic administration of the pan-caspase inhibitor, z-VAD-fmk, but not its inactive peptidyl analog, z-FA-fmk, blocks ventilator-induced apoptosis of alveolar cells and alveolar-capillary leak, indicating that caspase-dependent cell death is necessary for VILI-associated barrier dysfunction in vivo.

Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HVT MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

Induction of ectopic Myc target gene JAG2 augments hypoxic growth and tumorigenesis in a human B-cell model

Ectopic Myc expression plays a key role in human tumorigenesis, and Myc dose-dependent tumorigenesis has been well established in transgenic mice, but the Myc target genes that are dependent on Myc levels have not been well characterized. In this regard, we used the human P493-6 B cells, which have a preneoplastic state dependent on the Epstein–Barr viral EBNA2 protein and a neoplastic state with ectopic inducible Myc, to identify putative ectopic Myc target genes. Among the ectopic targets, JAG2 that encodes a Notch receptor ligand Jagged2, was directly induced by Myc. Inhibition of Notch signaling through RNAi targeting JAG2 or the γ-secretase Notch inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT) preferentially inhibited the neoplastic state in vitro. Furthermore, P493-6 tumorigenesis was inhibited by DAPT in vivo. Ectopic expression of JAG2 did not enhance aerobic cell proliferation, but increased proliferation of hypoxic cells in vitro and significantly increased in vivo tumorigenesis. Furthermore, the expression of Jagged2 in P493-6 tumors often overlapped with regions of hypoxia. These observations suggest that Notch signaling downstream of Myc enables cells to adapt in the tumor hypoxic microenvironment.