glutaminase and Neoplasms

Kidney-type glutaminase (GLS1), a key enzyme controlling the hydrolysis of glutamine to glutamate to resolve the 'glutamine addiction' of cancer cells, has been shown to play a central role in supporting cancer growth and proliferation. Therefore, the inhibition of GLS1 as a novel cancer treating strategy is of great interest.. This review covers recent patents (2019-present) involving GLS1 inhibitors, which are mostly focused on their chemical structures, molecular mechanisms of action, pharmacokinetic properties, and potential clinical applications.. Currently, despite significant efforts, the search for potent GLS1 inhibitors has not resulted in the development of compounds for therapeutic applications. Most recent patents and literature focus on GLS1 inhibitors IPN60090 and DRP104, which have entered clinical trials. While other patent disclosures during this period have not generated any drug candidates, the clinical update will inform the potential of these inhibitors as promising therapeutic agents either as single or as combination interventions.

Topics: Enzyme Inhibitors; Glutaminase; Glutamine; Humans; Neoplasms; Patents as Topic

Rare subpopulations of cancer stem cells (CSCs) have the ability to self-renew and are the primary driving force behind cancer metastatic dissemination and the preeminent hurdle to cancer treatment. As opposed to differentiated, non-malignant tumor offspring, CSCs have sophisticated metabolic patterns that, depending on the kind of cancer, rely mostly on the oxidation of major fuel substrates such as glucose, glutamine, and fatty acids for survival. Glutaminolysis is a series of metabolic reactions that convert glutamine to glutamate and, eventually, α-ketoglutarate, an intermediate in the tricarboxylic acid (TCA) cycle that provides biosynthetic building blocks. These building blocks are mostly utilized in the synthesis of macromolecules and antioxidants for redox homeostasis. A recent study revealed the cellular and molecular interconnections between glutamine and cancer stemness in the cell. Researchers have increasingly focused on glutamine catabolism in their attempt to discover an effective therapy for cancer stem cells. Targeting catalytic enzymes in glutaminolysis, such as glutaminase (GLS), is achievable with small molecule inhibitors, some of which are in early-phase clinical trials and have promising safety profiles. This review summarizes the current findings in glutaminolysis of CSCs and focuses on novel cancer therapies that target glutaminolysis in CSCs.

Topics: Glucose; Glutamic Acid; Glutaminase; Glutamine; Humans; Neoplasms; Neoplastic Stem Cells

Glutamine metabolism is reprogrammed during tumorigenesis and has been investigated as a promising target for cancer therapy. However, efforts to drug this process are confounded by the intrinsic metabolic heterogeneity and flexibility of tumors, as well as the risk of adverse effects on the anticancer immune response. Recent research has yielded important insights into the mechanisms that determine the tumor and the host immune responses to pharmacological perturbation of glutamine metabolism. Here, we discuss these findings and suggest that, collectively, they point toward patient stratification and drug combination strategies to maximize the efficacy of glutamine metabolism inhibitors as cancer therapeutics.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Benzeneacetamides; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Clinical Trials as Topic; Disease Models, Animal; Drug Resistance, Neoplasm; Glutaminase; Glutamine; Humans; Neoplasms; NF-E2-Related Factor 2; Oxidative Stress; Thiadiazoles; Tumor Escape; Tumor Microenvironment

Glutamine metabolism is one of the hallmarks of cancers which is described as an essential role in serving as a major energy and building blocks supply to cell proliferation in cancer cells. Many malignant tumor cells always display glutamine addiction. The "kidney-type" glutaminase (GLS1) is a metabolism enzyme which plays a significant part in glutaminolysis. Interestingly, GLS1 is often overexpressed in highly proliferative cancer cells to fulfill enhanced glutamine demand. So far, GLS1 has been proved to be a significant target during the carcinogenesis process, and emerging evidence reveals that its inhibitors could provide a benefit strategy for cancer therapy. Herein, we summarize the prognostic value of GLS1 in multiple cancer type and its related regulatory factors which are associated with antitumor activity. Moreover, this review article highlights the remarkable reform of discovery and development for GLS1 inhibitors. On the basis of case studies, our perspectives for targeting GLS1 and development of GLS1 antagonist are discussed in the final part.

Topics: Apoptosis; Benzophenanthridines; Cell Proliferation; Diazooxonorleucine; Disease Progression; Drug Resistance, Neoplasm; Genes, myc; Glutaminase; Glutamine; Humans; MicroRNAs; Neoplasm Proteins; Neoplasms; NF-kappa B; Oxidation-Reduction; Phosphates; Prognosis; Retinoblastoma Protein; Sulfides; Thiadiazoles

Increased glutamine metabolism (glutaminolysis) is a hallmark of cancer and is recognised as a key metabolic change in cancer cells. Breast cancer is a heterogeneous disease with different morphological and molecular subtypes and responses to therapy, and breast cancer cells are known to rewire glutamine metabolism to support survival and proliferation. Glutaminase isoenzymes (GLS and GLS2) are key enzymes for glutamine metabolism. Interestingly, GLS and GLS2 have contrasting functions in tumorigenesis. In this review, we explore the role of glutaminase in cancer, primarily focusing on breast cancer, address the role played by oncogenes and tumour suppressor genes in regulating glutaminase, and discuss current therapeutic approaches to targeting glutaminase.

Topics: Animals; Glutaminase; Humans; Neoplasms

Targeted therapies against cancer have improved both survival and quality of life of patients. However, metabolic rewiring evokes cellular mechanisms that reduce therapeutic mightiness. Resistant cells generate more glutathione, elicit nuclear factor erythroid 2-related factor 2 (NRF2) activation, and overexpress many anti-oxidative genes such as superoxide dismutase, catalase, glutathione peroxidase, and thioredoxin reductase, providing stronger antioxidant capacity to survive in a more oxidative environment due to the sharp rise in oxidative metabolism and reactive oxygen species generation. These changes dramatically alter tumour microenvironment and cellular metabolism itself. A rational design of therapeutic combination strategies is needed to flatten cellular homeostasis and accomplish a drop in cancer development. Context-dependent glutaminase isoenzymes show oncogenic and tumour suppressor properties, being mainly associated to MYC and p53, respectively. Glutaminases catalyze glutaminolysis in mitochondria, regulating oxidative phosphorylation, redox status and cell metabolism for tumour growth. In addition, the substrate and product of glutaminase reaction, glutamine and glutamate, respectively, can work as signalling molecules moderating redox and bioenergetic pathways in cancer. Novel synergistic approaches combining glutaminase inhibition and redox-dependent modulation are described in this review. Pharmacological or genetic glutaminase regulation along with oxidative chemotherapy can help to improve the design of combination strategies that escalate the rate of therapeutic success in cancer patients.

Topics: Animals; Antineoplastic Agents; Antioxidants; Energy Metabolism; Enzyme Inhibitors; Glutamic Acid; Glutaminase; Glutamine; Glutathione; Humans; Mitochondria; Neoplasms; Oxidative Stress; Signal Transduction; Tumor Microenvironment

Metabolic reprogramming of tumours is a hallmark of cancer. Among the changes in the metabolic network of cancer cells, glutaminolysis is a key reaction altered in neoplasms. Glutaminase proteins control the first step in glutamine metabolism and their expression correlates with malignancy and growth rate of a great variety of cancers. The two types of glutaminase isoenzymes, GLS and GLS2, differ in their expression patterns and functional roles: GLS has oncogenic properties and GLS2 has been described as a tumour suppressor factor.. We have focused on glutaminase connections with key oncogenes and tumour suppressor genes. Targeting glutaminase isoenzymes includes different strategies aimed at deactivating the rewiring of cancer metabolism. In addition, we found a long list of metabolic enzymes, transcription factors and signalling pathways dealing with glutaminase. On the other hand, a number of chemicals have been described as isoenzyme-specific inhibitors of GLS and/or GLS2 isoforms. These molecules are being characterized as synergic and therapeutic agents in many types of tumours.. This review states the metabolic pathways that are rewired in cancer, the roles of glutaminase isoforms in cancer, as well as the metabolic circuits regulated by glutaminases. We also show the plethora of anticancer drugs that specifically inhibit glutaminase isoenzymes for treating several sets of cancer.

Topics: Carcinogenesis; Glutaminase; Humans; Isoenzymes; Neoplasms

Metabolic reprogramming in cancer targets glutamine metabolism as a key mechanism to provide energy, biosynthetic precursors and redox requirements to allow the massive proliferation of tumor cells. Glutamine is also a signaling molecule involved in essential pathways regulated by oncogenes and tumor suppressor factors. Glutaminase isoenzymes are critical proteins to control glutaminolysis, a key metabolic pathway for cell proliferation and survival that directs neoplasms' fate. Adaptive glutamine metabolism can be altered by different metabolic therapies, including the use of specific allosteric inhibitors of glutaminase that can evoke synergistic effects for the therapy of cancer patients. We also review other clinical applications of in vivo assessment of glutaminolysis by metabolomic approaches, including diagnosis and monitoring of cancer.

Topics: Antineoplastic Agents; Cell Proliferation; Cellular Reprogramming; Enzyme Inhibitors; Glutaminase; Glutamine; Humans; Neoplasms

Besides fast glucose catabolism, many types of cancers are characterized by elevated glutamine consumption. Medical oncology pursuits to block specific pathways, mainly glycolysis and glutaminolysis, in tumor cells to arrest cancer development. This strategy frequently induces adaptive metabolic resistance that must be countered. Combination therapy is an anticancer synergistic tool to overcome both cancer growth and resistance mechanisms. Dysregulation of glutaminase and glutamine synthetase are key events that allow anabolic adaptation of tumors. Several specific drugs that inhibit metabolic enzymes dealing with glutamine metabolism have been able to eliminate some neoplasms. Targeting the tumor microenvironment can be also another essential factor to be taken into account when single or combined cancer metabolic therapy fails.

Topics: Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Glycolysis; Humans; Neoplasms; Tumor Microenvironment

Metabolic alterations are established as a hallmark of cancer. Such hallmark changes in cancer metabolism are characterized by reprogramming of energy-producing pathways and increases in the generation of biosynthetic intermediates to meet the needs of rapidly proliferating tumor cells. Various metabolic phenotypes such as aerobic glycolysis, increased glutamine consumption, and lipolysis have also been associated with the process of metastasis. However, in addition to the energy and biosynthetic alterations, a number of secondary functions of enzymes and metabolites are emerging that specifically contribute to metastasis. Here, we describe atypical intracellular roles of metabolic enzymes, extracellular functions of metabolic enzymes, roles of metabolites as signaling molecules, and epigenetic regulation mediated by altered metabolism, all of which can affect metastatic progression. We highlight how some of these mechanisms are already being exploited for therapeutic purposes, and discuss how others show similar potential.

Topics: ATP Citrate (pro-S)-Lyase; Disease Progression; Energy Metabolism; Fatty Acids; Glucose; Glucose-6-Phosphate Isomerase; Glutaminase; Glutamine; Humans; Isocitrate Dehydrogenase; Neoplasm Metastasis; Neoplasms

Glutaminase (GLS), which is responsible for the conversion of glutamine to glutamate, plays a vital role in up-regulating cell metabolism for tumor cell growth and is considered to be a valuable therapeutic target for cancer treatment. Based on this important function of glutaminase in cancer, several GLS inhibitors have been developed in both academia and industry. Most importantly, Calithera Biosciences Inc. is actively developing the glutaminase inhibitor CB-839 for the treatment of various cancers, and it is currently being evaluated in phase 1 and 2 clinical trials. In this review, recent efforts to develop small molecule glutaminase inhibitors that target glutamine metabolism in both preclinical and clinical studies are discussed. In particular, more emphasis is placed on CB-839 because it is the only small molecule GLS inhibitor being studied in a clinical setting. The inhibition mechanism is also discussed based on X-ray structure studies of thiadiazole derivatives present in glutaminase inhibitor BPTES. Finally, recent medicinal chemistry efforts to develop a new class of GLS inhibitors are described in the hopes of providing useful information for the next generation of GLS inhibitors.

Topics: Antineoplastic Agents; Enzyme Inhibitors; Glutaminase; Humans; Neoplasms; Small Molecule Libraries; Thiadiazoles

Targeting cancer metabolism for therapy has received much attention over the last decade with various small molecule inhibitors entering clinical trials. The present review highlights the latest strategies to target glucose and glutamine metabolism for cancer therapy with a particular emphasis on novel combinatorial treatment approaches.. Inhibitors of glucose, lactate, and glutamine transport and the ensuing metabolism are in preclinical to clinical trial stages of investigation. Recent advances in our understanding of cell-intrinsic and cell-extrinsic factors that dictate dependence on these targets have informed the development of rational, synthetic lethality-based strategies to exploit these metabolic vulnerabilities.. Cancer cells exhibit a number of metabolic alterations with functional consequences beyond that of sustaining cellular energetics and biosynthesis. Elucidating context-specific metabolic dependencies and their connections to oncogenic signaling and epigenetic programs in tumor cells represents a promising approach to identify new metabolic drug targets for cancer therapy.

Topics: Amino Acid Transport System ASC; Antineoplastic Combined Chemotherapy Protocols; Clinical Trials, Phase I as Topic; Glucose; Glucose Transport Proteins, Facilitative; Glutaminase; Glutamine; Humans; Lactic Acid; Minor Histocompatibility Antigens; Molecular Targeted Therapy; Monocarboxylic Acid Transporters; Neoplasms; Pyruvate Dehydrogenase Complex

Altered cellular metabolism is a hallmark of cancer. Cancer cells express isoforms of metabolic enzymes that may constitute therapeutic targets. Glutaminase controls glutamine metabolism and their expression correlate with malignancy of tumours. The two types of glutaminase isoenzymes, GLS and GLS2, differ in their expression patterns and functional roles: GLS has oncogenic properties and GLS2 has been described as a tumour suppressor factor. Selective genomic and epigenomic intervention over glutaminase affects the metabolic reprogramming of cancer. This review highlights the molecular metabolic vulnerabilities in various types of cancer, to be used for biomarker development, drug design, and in personalized oncology.

Topics: Animals; Biomarkers, Tumor; Drug Design; Glutaminase; Humans; Isoenzymes; Molecular Targeted Therapy; Neoplasms; Precision Medicine

The kidney-type glutaminase (GLS) controlling the first step of glutamine metabolism is overexpressed in many cancer cells. Targeting inhibition of GLS shows obvious inhibitory effects on cancer cell proliferation. Therefore, extensive research and development of GLS inhibitors have been carried out in industrial and academic institutions over the past decade to address this unmet medical need.. This review covers researches and patent literatures in the field of discovery and development of small molecule inhibitors of GLS for cancer therapy over the past 16 years.. The detailed ligand-receptor interaction information from their complex structure not only guides the rational drug design, but also facilitates in silico structure-based virtual ligand screening of novel GLS inhibitors. Multi-drug combination administration is of great significance both in terms of safety and efficacy.

Topics: Animals; Antineoplastic Agents; Cell Proliferation; Drug Design; Enzyme Inhibitors; Glutaminase; Humans; Ligands; Neoplasms; Patents as Topic

Reliance on glutamine has long been considered a hallmark of cancer cell metabolism. However, some recent studies have challenged this notion

Topics: Glutaminase; Glutamine; Humans; Molecular Targeted Therapy; Neoplasms

Many cancer cells exhibit an altered metabolic phenotype, in which glutamine consumption is upregulated relative to healthy cells. This metabolic reprogramming often depends upon mitochondrial glutaminase activity, which converts glutamine to glutamate, a key precursor for biosynthetic and bioenergetic processes. Two isozymes of glutaminase exist, a kidney-type (GLS) and a liver-type enzyme (GLS2 or LGA). While a majority of studies have focused on GLS, here we summarize key findings on both glutaminases, describing their structure and function, their roles in cancer and pharmacological approaches to inhibiting their activities.

Topics: Carcinogenesis; Enzyme Inhibitors; Glutaminase; Humans; Neoplasms; Phenotype

Metabolomics, the newest of the omics sciences that also include genomics, transcriptomics and proteomics, has matured into a reliable high-throughput technology. Gas chromatography combined with time-of-flight mass spectrometry (GC-TOFMS) is a suitable method to analyze the central metabolism in fresh frozen tumor tissue samples. Bioinformatics methods, including the PROFILE clustering developed by us, permit integrated analysis and fast interpretation of metabolomics data in the context of enzymatic reactions and metabolic pathways. The metabolome analyses of three solid tumor types presented here, together with the results of other authors, show that metabolites are suitable as biomarkers and provide diverse options for translation into the clinical setting.

Topics: 4-Aminobutyrate Transaminase; beta-Alanine; Biomarkers, Tumor; Breast; Breast Neoplasms; Colonic Neoplasms; Female; Gas Chromatography-Mass Spectrometry; Glutamic Acid; Glutaminase; Glutamine; Humans; Metabolome; Neoplasms; Neoplasms, Hormone-Dependent; Ovarian Neoplasms; Ovary

Glutamine metabolism has been proved to be dysregulated in many cancer cells, and is essential for proliferation of most cancer cells, which makes glutamine an appealing target for cancer therapy. In order to be well used by cells, glutamine must be transported to cells by specific transporters and converted to glutamate by glutaminase. There are currently several drugs that target glutaminase under development or clinical trials. Also, glutamine metabolism restriction has been proved to be effective in inhibiting tumor growth both in vivo and vitro through inducing apoptosis, growth arrest and/or autophagy. Here, we review recent researches about glutamine metabolism in cancer, and cell death induced by targeting glutamine, and their potential roles in cancer therapy.

Topics: Animals; Apoptosis; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; Humans; Hypoxia-Inducible Factor 1; Neoplasms; Proto-Oncogene Proteins c-myc; ras Proteins; Tumor Suppressor Protein p53

Tumor cells suffer a metabolic reprogramming which allows them to use metabolic fuels (glucose, glutamine, lipids) through anabolic fates to support their enhanced proliferation and other carcinogenesis-related features. The present review tries to address and summarize the broad and growing information available about this reprogramming, whose pieces, put together, make up a complex scheme that encompasses different complexity scales, from cells to systemic networks.

Topics: Ammonia; Animals; Citric Acid Cycle; Glucose; Glutaminase; Glutamine; Glycolysis; Humans; Neoplasms; Oxidative Phosphorylation; Retinoblastoma Protein

Metabolism is the process by which cells convert relatively simple extracellular nutrients into energy and building blocks necessary for their growth and survival. In cancer cells, metabolism is dramatically altered compared with normal cells. These alterations are known as the Warburg effect. One consequence of these changes is cellular addiction to glutamine. Because of this, in recent years the enzyme glutaminase has become a key target for small molecule therapeutic intervention. Like many oncotargets, however, glutaminase has a number of upstream partners that might offer additional druggable targets. This review summarizes the work from the current decade surrounding glutaminase and its regulation, and suggests strategies for therapeutic intervention in relevant cases.

Topics: Animals; Antineoplastic Agents; Glutaminase; Glutamine; Humans; Neoplasms

Cancer cells must carefully regulate their metabolism to maintain growth and division under varying nutrient and oxygen levels. Compelling data support the investigation of numerous enzymes as therapeutic targets to exploit metabolic vulnerabilities common to several cancer types. We discuss the rationale for developing such drugs and review three targets with central roles in metabolic pathways crucial for cancer cell growth: pyruvate kinase muscle isozyme splice variant 2 (PKM2) in glycolysis, glutaminase in glutaminolysis, and mutations in isocitrate dehydrogenase 1 and 2 isozymes (IDH1/2) in the tricarboxylic acid cycle. These targets exemplify the drugging approach to cancer metabolism, with allosteric modulation being the common theme. The first glutaminase and mutant IDH1/2 inhibitors have entered clinical testing, and early data are promising. Cancer metabolism provides a wealth of novel targets, and targeting allosteric sites promises to yield selective drugs with the potential to transform clinical outcomes across many cancer types.

Topics: Antineoplastic Agents; Carrier Proteins; Citric Acid Cycle; Enzyme Inhibitors; Glutaminase; Glycolysis; Humans; Isocitrate Dehydrogenase; Isoenzymes; Membrane Proteins; Neoplasms; Neoplastic Stem Cells; Thyroid Hormone-Binding Proteins; Thyroid Hormones

The metabolic properties of cancer cells diverge significantly from those of normal cells. Energy production in cancer cells is abnormally dependent on aerobic glycolysis. In addition to the dependency on glycolysis, cancer cells have other atypical metabolic characteristics such as increased fatty acid synthesis and increased rates of glutamine metabolism. Emerging evidence shows that many features characteristic to cancer cells, such as dysregulated Warburg-like glucose metabolism, fatty acid synthesis and glutaminolysis are linked to therapeutic resistance in cancer treatment. Therefore, targeting cellular metabolism may improve the response to cancer therapeutics and the combination of chemotherapeutic drugs with cellular metabolism inhibitors may represent a promising strategy to overcome drug resistance in cancer therapy. Recently, several review articles have summarized the anticancer targets in the metabolic pathways and metabolic inhibitor-induced cell death pathways, however, the dysregulated metabolism in therapeutic resistance, which is a highly clinical relevant area in cancer metabolism research, has not been specifically addressed. From this unique angle, this review article will discuss the relationship between dysregulated cellular metabolism and cancer drug resistance and how targeting of metabolic enzymes, such as glucose transporters, hexokinase, pyruvate kinase M2, lactate dehydrogenase A, pyruvate dehydrogenase kinase, fatty acid synthase and glutaminase can enhance the efficacy of common therapeutic agents or overcome resistance to chemotherapy or radiotherapy.

Topics: Drug Resistance, Neoplasm; Fatty Acid Synthases; Glucose Transport Proteins, Facilitative; Glutaminase; Hexokinase; Humans; Isoenzymes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Neoplasms; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Pyruvate Kinase

The metabolic adaptations that support oncogenic growth can also render cancer cells dependent on certain nutrients. Along with the Warburg effect, increased utilization of glutamine is one of the metabolic hallmarks of the transformed state. Glutamine catabolism is positively regulated by multiple oncogenic signals, including those transmitted by the Rho family of GTPases and by c-Myc. The recent identification of mechanistically distinct inhibitors of glutaminase, which can selectively block cellular transformation, has revived interest in the possibility of targeting glutamine metabolism in cancer therapy. Here, we outline the regulation and roles of glutamine metabolism within cancer cells and discuss possible strategies for, and the consequences of, impacting these processes therapeutically.

Topics: Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; Glutaminase; Glutamine; Humans; Models, Molecular; Molecular Targeted Therapy; Neoplasms; Signal Transduction

Cancer cells require a robust supply of reduced nitrogen to produce nucleotides, non-essential amino acids and a high cellular redox activity. Glutamine provides a major substrate for respiration as well as nitrogen for the production of proteins, hexosamines, and macromolecules. Therefore, glutamine is one of key molecules in cancer metabolism during cell proliferation. The notion of targeting glutamine metabolism in cancer, originally rationalized by the number of pathways fed by this nutrient, has been reinforced by more recent studies demonstrating that its metabolism is regulated by oncogenes. Glutamine can exert its effects by modulating redox homeostasis, bioenergetics, nitrogen balance or other functions, including by being a precursor of glutathione, the major nonenzymatic cellular antioxidant. Glutaminase (GA) is the first enzyme that converts glutamine to glutamate, which is in turn converted to alpha-ketoglutarate for further metabolism in the tricarboxylic acid cycle. Different GA isoforms in mammals are encoded by two genes, Gls and Gls2. As each enzymatic form of GA has distinct kinetic and molecular characteristics, it has been speculated that the differential regulation of GA isoforms may reflect distinct functions or requirements in different tissues or cell states. GA encoded by Gls gene (GLS) has been demonstrated to be regulated by oncogenes and to support tumor cell growth. GA encoded by Gls2 gene (GLS2) reduces cellular sensitivity to reactive oxygen species associated apoptosis possibly through glutathione-dependent antioxidant defense, and therefore to behave more like a tumor suppressor. Thus, modulation of GA function may be a new therapeutic target for cancer treatment.

Topics: Glutaminase; Humans; Isoenzymes; Neoplasms; Oxidative Stress

Recently, the small molecule 968 was found to block the Rho GTPase-dependent growth of cancer cells in cell culture and mouse xenografts, and when the target of 968 was found to be the mitochondrial enzyme glutaminase (GLS1), it revealed a surprising link between Rho GTPases and mitochondrial glutamine metabolism. Signal transduction via the Rho GTPases, together with NF-κB, appears to elevate mitochondrial glutaminase activity in cancer cells, thereby helping cancer cells satisfy their altered metabolic demands. Here, we review what is known about the mechanism of glutaminase activation in cancer cells, compare the properties of two distinct glutaminase inhibitors, and discuss recent findings that shed new light on how glutamine metabolism might affect cancer progression.

Topics: Animals; Enzyme Activation; Enzyme Inhibitors; Glutaminase; Humans; Mitochondria; Neoplasms; rho GTP-Binding Proteins; Signal Transduction; Transport Vesicles

Over 80 years ago, Warburg discovered that cancer cells generate ATP through the glycolytic pathway, even in the presence of oxygen. The finding of this phenomenon, termed the "Warburg effect," stimulated much research on tumorigenesis, but few explanations were forthcoming to explain the observation. Recently, advanced developments in molecular biology and high-throughput molecular analyses have revealed that many of the signaling pathways altered by gene mutations regulate cell metabolism in cancer. Furthermore, mutations in isocitrate dehydrogenase 1 and 2 were shown to elevate 2-hydroxyglutarate, which led to changes in α-ketoglutarate-dependent dioxygenase enzyme activity, resulting in an increased risk of malignant tumors. Although these findings led to a renewed interest in cancer metabolism, our knowledge on the specifics of tumor metabolism is still fragmented. This paper reviews recent findings related to key transcription factors and enzymes that play an important role in the regulation of cancer metabolism.

Topics: Basic Helix-Loop-Helix Transcription Factors; Cell Transformation, Neoplastic; Fumarates; Genes, p53; Glutaminase; Glycolysis; Humans; Isocitrate Dehydrogenase; Mutation; Neoplasms; Proto-Oncogene Proteins c-myc; Pyruvate Kinase

The antitumour enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1, ASNase), which catalyses the deamidation of L-asparagine (Asn) to L-aspartic acid and ammonia, has been used for many years in the treatment of acute lymphoblastic leukaemia. Also NK tumours, subtypes of myeloid leukaemias and T-cell lymphomas respond to ASNase, and ovarian carcinomas and other solid tumours have been proposed as additional targets for ASNase, with a potential role for its glutaminase activity. The increasing attention devoted to the antitumour activity of ASNase prompted us to analyse recent patents specifically concerning this enzyme. Here, we first give an overview of metabolic pathways affected by Asn and Gln depletion and, hence, potential targets of ASNase. We then discuss recent published patents concerning ASNases. In particular, we pay attention to novel ASNases, such as the recently characterised ASNase produced by Helicobacter pylori, and those presenting amino acid substitutions aimed at improving enzymatic activity of the classical Escherichia coli enzyme. We detail modifications, such as natural glycosylation or synthetic conjugation with other molecules, for therapeutic purposes. Finally, we analyse patents concerning biotechnological protocols and strategies applied to production of ASNase as well as to its administration and delivery in organisms.

Topics: Animals; Antineoplastic Agents; Asparaginase; Asparagine; Drug Delivery Systems; Glutaminase; Glutamine; Humans; Metabolic Networks and Pathways; Neoplasms; Precursor Cell Lymphoblastic Leukemia-Lymphoma

Cancer cells re-program their metabolic machinery in order to satisfy their bioenergetic and biosynthetic requirements. A critical aspect of the re-programming of cancer cell metabolism involves changes in the glycolytic pathway (referred to as the "Warburg effect"). As an outcome of these changes, much of the pyruvate generated via the glycolytic pathway is converted to lactic acid, rather than being used to produce acetyl-CoA and ultimately, the citrate which enters the citric acid cycle. In order to compensate for these changes and to help maintain a functioning citric acid cycle, cancer cells often rely on elevated glutamine metabolism. Recently, we have found that this is achieved through a marked elevation of glutaminase activity in cancer cells. Here we further consider these findings and the possible mechanisms by which this important metabolic activity is regulated.

Topics: Animals; Cell Transformation, Neoplastic; Energy Metabolism; Enzyme Inhibitors; Glutaminase; Humans; Metabolic Networks and Pathways; Models, Biological; Neoplasms; rho GTP-Binding Proteins

Glutamine is a multifaceted amino acid that plays key roles in many metabolic pathways and also fulfils essential signaling functions. Although classified as non-essential, recent evidence suggests that glutamine is a conditionally essential amino acid in several physiological situations. Glutamine homeostasis must therefore be exquisitely regulated and mitochondria represent a major site of glutamine metabolism in numerous cell types. Glutaminolysis is mostly a mitochondrial process with repercussions in organelle structure and dynamics suggesting a tight and mutual control between mitochondrial form and cell bioenergetics. In this review we describe an updated account focused on the critical involvement of glutamine in oxidative stress, mitochondrial dysfunction and tumour cell proliferation, with special emphasis in the initial steps of mitochondrial glutamine pathways: transport into the organelle and hydrolytic deamidation through glutaminase enzymes. Some controversial issues about glutamine catabolism within mitochondria are also reviewed.

Topics: Animals; Brain; Glutaminase; Glutamine; Homeostasis; Humans; Mitochondria; Neoplasms; Oxidation-Reduction; Oxidative Stress

Glutamine is the most abundant free amino acid in the human body; it is essential for the growth of normal and neoplastic cells and for the culture of many cell types. Cancer has been described as a nitrogen trap. The presence of a tumor produces great changes in host glutamine metabolism in such a way that host nitrogen metabolism is accommodated to the tumor-enhanced requirements of glutamine. To be used, glutamine must be transported into tumor mitochondria. Thus, an overview of the role of glutamine in cancer requires not only a discussion of host and tumor glutamine metabolism, but also its circulation and transport. Because glutamine depletion has adverse effects for the host, the effect of glutamine supplementation in the tumor-bearing state should also be studied. This communication reviews the state of knowledge of glutamine and cancer, including potential therapeutic implications.

Topics: Biological Transport; Clinical Trials as Topic; Cloning, Molecular; Dietary Supplements; Gene Expression Regulation, Enzymologic; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Humans; Neoplasms; Nitrogen; Nutritional Status; Transglutaminases; Tumor Cells, Cultured

Topics: Acidosis; Animals; Aspartate Aminotransferases; Aspartic Acid; Brain; Cells, Cultured; Energy Metabolism; Glutamate Dehydrogenase; Glutamate-Ammonia Ligase; Glutamates; Glutaminase; Glutamine; Hepatic Encephalopathy; Humans; Intestine, Small; Intracellular Membranes; Kidney; Liver; Mitochondria; Mitochondria, Liver; Muscles; Neoplasms; Organ Specificity; Oxidation-Reduction; Rats

Topics: Acid Phosphatase; Alcohol Oxidoreductases; Aldehyde Oxidoreductases; Alkaline Phosphatase; Animals; Carcinoma, Hepatocellular; DNA Nucleotidyltransferases; Esterases; Fructose-Bisphosphatase; Fructose-Bisphosphate Aldolase; Glucosyltransferases; Glutaminase; Glycogen Synthase; Hexokinase; Humans; Isocitrate Dehydrogenase; Isoenzymes; L-Lactate Dehydrogenase; Liver Neoplasms; Malate Dehydrogenase; Mice; Neoplasms; Phosphotransferases; Pyruvate Kinase; Rats; Terminology as Topic; Thymidine Kinase; Transaminases; tRNA Methyltransferases; Uridine

Topics: Animals; Aspartate Aminotransferases; Carbamates; Cell Transformation, Neoplastic; DNA Nucleotidyltransferases; Fructose-Bisphosphatase; Fructose-Bisphosphate Aldolase; Fructosephosphates; Glutaminase; Glycerolphosphate Dehydrogenase; Hexokinase; Isocitrate Dehydrogenase; Isoenzymes; Kinetics; L-Lactate Dehydrogenase; Malate Dehydrogenase; Molecular Weight; NAD; NADP; Neoplasms; Phosphotransferases; Pyruvate Kinase; Transferases

Topics: Adult; Aminobutyrates; Animals; Asparaginase; Azaserine; Azo Compounds; Child; Diazooxonorleucine; Drug Combinations; Drug Interactions; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Humans; Hydroxylysine; Leukemia; Leukemia L1210; Liver; Methionine Sulfoximine; Mice; Neoplasms; Rats; RNA, Transfer

Topics: Animals; Asparaginase; Asparagine; Glutaminase; Guinea Pigs; Humans; Leukemia; Mice; Neoplasms; Neoplasms, Experimental; Rabbits; Rats

To evaluate the in vitro activity of polyethylene glycol-conjugated L-asparaginase (PEG-Lasparaginase) against fresh human tumor specimens, using the human tumor clonogenic assay (HTCA), and to perform a phase I dose-escalation clinical trial of PEG-L-asparaginase. The goal of the clinical study was to determine the toxicity and optimum biologic dose of PEG-L-asparaginase based on depletion of serum L-asparagine in patients with advanced solid tumors.. A modified method for determination of serum L-asparagine is described. PEG-L-asparaginase was administered by intramuscular injection every 2 weeks to 28 patients with various types of advanced solid tumor malignancies. At least 3 patients were evaluated at each dose level: 250 IU/m2, 500 IU/m2, 1,000 IU/m2, 1,500 IU/m2, 2,000 IU/m2.. The in vitro HTCA studies suggested good antitumor activity against malignant melanoma and multiple myeloma. Serum L-asparagine was most consistently and profoundly depleted (up to 4 weeks) in patients treated with 2,000 IU/m2. Patients receiving this dose level also showed more frequent grade 1, grade 2, and occasional grade 3 toxicities of fatigue/weakness, nausea/vomiting, and anorexia/ weight loss. Three patients developed hypersensitivity reactions, but these were not dose related. Two patients developed deep vein thromboses. We saw no episodes of clinical pancreatitis, but there were minor fluctuations of serum amylase and lipase. We saw no partial or complete responses in patients treated in this study, including 11 patients with malignant melanoma.. We conclude that PEG-L-asparaginase is generally well tolerated in patients with advanced solid tumors, and a dosage of 2,000 IU/m2 by intramuscular injection every 2 weeks results in significant depletion of serum L-asparagine.

Topics: Adult; Antineoplastic Agents; Asparaginase; Asparagine; Carcinoma, Non-Small-Cell Lung; Drug Screening Assays, Antitumor; Glutaminase; Humans; Lung Neoplasms; Melanoma; Neoplasm Proteins; Neoplasms; Polyethylene Glycols; Skin Neoplasms

Topics: Amino Acids; Animals; Asparaginase; Carcinoma, Ehrlich Tumor; Child; Clinical Trials as Topic; Diet; Dietary Proteins; Female; Glutaminase; Humans; Leukemia, Lymphoid; Mice; Mice, Inbred Strains; Neoplasms; Nitrogen

Competitive consumption of nutrients between rapidly proliferating cancer cells and T cells results in an immunosuppressive tumor microenvironment (TME) and nutrient deprivation of T cells, which can cause low response rate and resistance to immunotherapies. In this study, we proposed a dual-mechanism based nutrient partitioning nanoregulator (designated as DMNPN), which can simultaneously regulate the immunosuppressive TME and enhance T cell nutrient availability. DMNPN consists of a charge-reversal biodegradable mesoporous silica, encapsulating glycolysis inhibitor lonidamine, and small interfering RNA against glutaminase. Through inhibiting glycolysis to decrease the lactic acid production and downregulating glutaminase expression to reduce the uptake of glutamine by tumor cells, DMNPN enables effective remodeling of metabolism and nutrient partitioning, which alleviates the immunosuppressive TME and boosts nutrient availability for T cells with enhanced antitumor immunity. Such a nutrient partitioning nanoregulator can effectively inhibit the growth of anti-programmed death receptor 1 (anti-PD-1) resistant tumors and prevent tumor metastasis and recurrence. Overall, this dual-mechanism based nutrient reallocation strategy provides a promising approach for cancer therapy.

Topics: Cell Line, Tumor; Glutaminase; Humans; Immunosuppressive Agents; Immunotherapy; Neoplasms; Nutrients; T-Lymphocytes; Tumor Microenvironment

Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the "Warburg effect") and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this 'glutamine addiction' of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook.

Topics: Crystallography; Enzyme Inhibitors; Glutaminase; Glutamine; Neoplasms; Sulfides; Temperature; Thiadiazoles

Glutaminase 1 (GLS1) is overexpressed in multiple types of malignant tumors and is viewed as a promising target in cancer therapy. Thus, the discovery for small-molecule GLS1 inhibitors is being urgent. Based on our previous study of C147, a potent GLS1 allosteric inhibitor yet with a limited metabolic stability, a structure-based optimization was carried out, with a series of novel GLS1 allosteric inhibitors rationally designed, synthesized and biologically evaluated. Such endeavor has culminated in the identification of 41e, a promising GLS1 allosteric inhibitor with 4-piperidinamine linker and aromatic heterocycles. 41e displayed robust GLS1 binding affinity, superior liver microsome metabolic stability, and moderate anti-tumor activity (TGI: 47.5%) in HCT116 xenograft model with no considerable toxicity in vivo. The mechanism underlying the anti-proliferative effect of 41e on HCT116 cells was studied, revealing that the compound blocked the glutamine metabolism, induced the production of ROS, and triggered apoptosis. These findings merit further investigation of 41e as a targeted cancer therapeutic.

Topics: Animals; Apoptosis; Glutaminase; HCT116 Cells; Humans; Neoplasms

Cancer cells change their glucose and glutamine (GLU) metabolism to obtain the energy required to continue growing. Glutaminase (GLS) plays a crucial role in promoting cell metabolism for cancer cell growth; targeting GLU metabolism by inhibiting GLS has attracted interest as a potential cancer management strategy. Herein, we employed a sequential screening of traditional Chinese medicine (TCM) database followed by drug-likeness and molecular dynamics simulations against the active site of GLS. We report 12 potent compounds after screening the TCM database against GLS, followed by a drug-likeness filter with Lipinski and Veber rule criteria. Among them, ZINC03978829 and ZINC32296657 were found to have higher binding energy (BE) values than the control compound 6-Diazo-5-Oxo-L-Norleucine, with BEs of -9.3 and -9.7 kcal/mol, respectively, compared to the BE of 6-Diazo-5-Oxo-L-Norleucine (-4.7 kcal/mol) with GLS. Molecular dynamics simulations were used to evaluate the results further, and a 100 ns MD simulation revealed that the hits form stable complexes with GLS and formed 2-5 hydrogen bond interactions. This study indicates that these hits might be employed as GLS inhibitors in the battle against cancer. However, more laboratory tests are a prerequisite to optimize them as GLS inhibitors.

Topics: Diazooxonorleucine; Early Detection of Cancer; Glutaminase; Humans; Molecular Docking Simulation; Molecular Dynamics Simulation; Neoplasms; Neoplastic Processes

Topics: Glutaminase; Humans; Neoplasms; Thiadiazoles

Tumors can use metabolic reprogramming to survive nutrient stress. Epigenetic regulators play a critical role in metabolic adaptation. Here we screened a sgRNA library to identify epigenetic regulators responsible for the vulnerability of colorectal cancer (CRC) cells to glucose deprivation and found that more EZH2-knockout cells survived glucose deprivation. Then, we showed that EZH2 expression was significantly downregulated in response to glucose deprivation in a glucose-sensitive CRC cell line, and EZH2-knockdown cells were more resistant to glucose deprivation. Mechanistically, EZH2 deficiency upregulated the expression of glutaminase (GLS) and promoted the production of glutamate, which in turn led to increased synthesis of intracellular glutathione (GSH) and eventually attenuated the reactive oxygen species (ROS)-mediated cell death induced by glucose deprivation. Although EZH2 functioned as an oncogene in cancer progression and EZH2 knockout abolished colorectal cancer development in a mouse model, here we revealed a mechanistic link between EZH2 and metabolic reprogramming via the direct regulation of GLS expression and observed a negative correlation between EZH2 and GLS expression in colorectal cancer tissues. These findings further confirmed the importance of heterogeneity, provided an explanation for the clinical tolerance of cancer cells to EZH2 inhibitors from the perspective of metabolism, and proposed the possibility of combining EZH2 inhibitors and glutamine metabolism inhibitors for the treatment of cancer.

Topics: Enhancer of Zeste Homolog 2 Protein; Glutaminase; Humans; Neoplasms

The inhibition of glutaminase 1 (GLS1) represents a potential treatment of malignant tumors. Structural analysis led to the design of a novel series of macrocyclic GLS1 allosteric inhibitors. Through extensive structure-activity relationship studies, a promising candidate molecule

Topics: Allosteric Site; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Crystallography, X-Ray; Drug Design; Enzyme Inhibitors; Glutaminase; Glycolysis; Half-Life; Humans; Macrocyclic Compounds; Mice; Mice, Nude; Molecular Dynamics Simulation; Neoplasms; Oxidative Phosphorylation; Rats; Structure-Activity Relationship

According to data from the World Health Organization, cardiovascular diseases and cancer are the two leading causes of mortality in the world [1]. Despite the immense effort to study these diseases and the constant innovation in treatment modalities, the number of deaths associated with cardiovascular diseases and cancer is predicted to increase in the coming decades [1]. From 2008 to 2030, due to population growth and population aging in many parts of the world, the number of deaths caused by cancer globally is projected to increase by 45%, corresponding to an annual increase of around four million people [1]. For cardiovascular diseases, this number is six million people [1]. In the United States, treatments for these two diseases are among the most costly and result in a disproportionate impact on low- and middleincome people. As the fight against these fatal diseases continues, it is crucial that we continue our investigation and broaden our understanding of cancer and cardiovascular diseases to innovate our prognostic and treatment approaches. Even though cardiovascular diseases and cancer are usually studied independently [2-12], there are some striking overlaps between their metabolic behaviors and therapeutic targets, suggesting the potential application of cardiovascular disease treatments for cancer therapy. More specifically, both cancer and many cardiovascular diseases have an upregulated glutaminolysis pathway, resulting in low glutamine and high glutamate circulating levels. Similar treatment modalities, such as glutaminase (GLS) inhibition and glutamine supplementation, have been identified to target glutamine metabolism in both cancer and some cardiovascular diseases. Studies have also found similarities in lipid metabolism, specifically fatty acid oxidation (FAO) and synthesis. Pharmacological inhibition of FAO and fatty acid synthesis have proven effective against many cancer types as well as specific cardiovascular conditions. Many of these treatments have been tested in clinical trials, and some have been medically prescribed to patients to treat certain diseases, such as angina pectoris [13, 14]. Other metabolic pathways, such as tryptophan catabolism and pyruvate metabolism, were also dysregulated in both diseases, making them promising treatment targets. Understanding the overlapping traits exhibited by both cancer metabolism and cardiovascular disease metabolism can give us a more holistic view of how important metabolic dysregulat

Topics: Cardiovascular Diseases; Glutamic Acid; Glutaminase; Glutamine; Humans; Neoplasms

Altered metabolic pathways in cancer such as exacerbated glycolytic flux and increased glutamine metabolism are promising targets for anti-cancer therapies. While commonly observed in glycolytic tumors, extracellular acidosis has never been considered as a potential modulator of anti-metabolic drug activity such as dichloroacetate (DCA). Using cancer cells from various origins selected for their ability to proliferate under acidic conditions, we found that DCA exerts greater inhibitory effects on the growth of these acid-adapted cells than in parental cells. Moreover, daily DCA administration to mice led to a significant decrease in tumor growth from acid-adapted cells but not from parental cells. 13C-tracer studies revealed that DCA induced a double metabolic shift, diminishing glycolysis and increasing intracellular glutamine in acid-adapted cells. As a consequence, DCA reduced the pentose phosphate pathway activity more extensively and increased apoptosis in acid-adapted cells. Finally, the combination of DCA with a glutaminase inhibitor significantly enhanced the cytotoxic effects of DCA. Overall, the interplay between acidosis and DCA exposure leads to metabolic reprogramming that considerably alters cellular fitness.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dichloroacetic Acid; Drug Synergism; Female; Glutaminase; Glutamine; Glycolysis; Humans; Hydrogen-Ion Concentration; Mice; Neoplasms; Pentose Phosphate Pathway; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Sulfides; Thiadiazoles; Xenograft Model Antitumor Assays

Imported across the plasma membrane by SLC1A5, glutamine has emerged as a metabolic fuel that is catabolized by mitochondrial glutaminase to support tumor growth. The missing link between cytoplasmic and mitochondrial glutamine metabolism is now provided by Yoo et al., identifying the mitochondrial glutamine importer as a variant of SLC1A5.

Topics: Amino Acid Transport System ASC; Animals; Cell Proliferation; Cytoplasm; Glutaminase; Glutamine; Humans; Mitochondria; Neoplasms

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.

Topics: Animals; Carcinogenesis; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chlorocebus aethiops; COS Cells; Glutaminase; Hep G2 Cells; Humans; Neoplasms

Glucose metabolism is remodeled in cancer, but the global pattern of cancer-specific metabolic changes remains unclear. Here we show, using the comprehensive measurement of metabolic enzymes by large-scale targeted proteomics, that the metabolism both carbon and nitrogen is altered during the malignant progression of cancer. The fate of glutamine nitrogen is shifted from the anaplerotic pathway into the TCA cycle to nucleotide biosynthesis, with this shift being controlled by glutaminase (GLS1) and phosphoribosyl pyrophosphate amidotransferase (PPAT). Interventions to reduce the PPAT/GLS1 ratio suppresses tumor growth of many types of cancer. A meta-analysis reveals that PPAT shows the strongest correlation with malignancy among all metabolic enzymes, in particular in neuroendocrine cancer including small cell lung cancer (SCLC). PPAT depletion suppresses the growth of SCLC lines. A shift in glutamine fate may thus be required for malignant progression of cancer, with modulation of nitrogen metabolism being a potential approach to SCLC treatment.

Topics: Amidophosphoribosyltransferase; Animals; Biosynthetic Pathways; Cell Line, Tumor; Disease Progression; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; Humans; Metabolomics; Mice, Nude; Models, Biological; Molecular Targeted Therapy; Neoplasms; Nitrogen; Prognosis

LncRNAs have been found to be involved in various aspects of biological processes. In this study, we aimed to uncover the molecular mechanisms of lncRNA EPB41L4A-AS1 in regulating glycolysis and glutaminolysis in cancer cells.. The expression of EPB41L4A-AS1 in cancer patients was analyzed in TCGA and GEO datasets. The level of cellular metabolism was determined by extracellular flux analyzer. The relationship between p53 and EPB41L4A-AS1 was explored by qRT-PCR, luciferase assay and ChIP assay. The interactions between EPB41L4A-AS1 and HDAC2 or NPM1 were determined by RNA immunoprecipitation, RNA pull-down assay and RNA-FISH- immunofluorescence.. EPB41L4A-AS1 was a p53-regulated gene. Low expression and deletion of lncRNA EPB41L4A-AS1 were found in a variety of human cancers and associated with poor prognosis of cancer patients. Knock down EPB41L4A-AS1 expression triggered Warburg effect, demonstrated as increased aerobic glycolysis and glutaminolysis. EPB41L4A-AS1 interacted and colocalized with HDAC2 and NPM1 in nucleolus. Silencing EPB41L4A-AS1 reduced the interaction between HDAC2 and NPM1, released HDAC2 from nucleolus and increased its distribution in nucleoplasm, enhanced HDAC2 occupation on VHL and VDAC1 promoter regions, and finally accelerated glycolysis and glutaminolysis. Depletion of EPB41L4A-AS1 increased the sensitivity of tumor to glutaminase inhibitor in tumor therapy.. EPB41L4A-AS1 functions as a repressor of the Warburg effect and plays important roles in metabolic reprogramming of cancer.

Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleus; Glutaminase; Glycolysis; HeLa Cells; Hep G2 Cells; Histone Deacetylase 2; Humans; Mice; Mice, Nude; Mitochondrial Membrane Transport Proteins; Neoplasms; Nucleophosmin; RNA, Long Noncoding

Bcl-2 associated athanogene 3 (BAG3) is an important molecule that maintains oncogenic features of cancer cells via diverse mechanisms. One of the important functions assigned to BAG3 is implicated in selective macroautophagy/autophagy, which attracts much attention recently. However, the mechanism underlying regulation of autophagy by BAG3 has not been well defined. Here, we describe that BAG3 enhances autophagy via promotion of glutamine consumption and glutaminolysis. Glutaminolysis initiates with deamination of glutamine by glutaminase (GLS), by which yields glutamate and ammonia in mitochondria. The current study demonstrates that BAG3 stabilizes GLS via prohibition its interaction with SIRT5, thereby hindering its desuccinylation at Lys158 and Lys164 sites. As an underlying molecular mechanism, we demonstrate that BAG3 interacts with GLS and decreases SIRT5 expression. The current study also demonstrates that occupation by succinyl at Lys158 and Lys164 sites prohibits its Lys48-linked ubiquitination, thereby preventing its subsequent proteasomal degradation. Collectively, the current study demonstrates that BAG3 enhances autophagy via stabilizing GLS and promoting glutaminolysis. For the first time, this study reports that succinylation competes with ubiquitination to regulate proteasomal GLS degradation.

Topics: Adaptor Proteins, Signal Transducing; Ammonia; Apoptosis Regulatory Proteins; Autophagy; Enzyme Stability; Glutaminase; Glutamine; Hep G2 Cells; Humans; MCF-7 Cells; Mitochondria; Neoplasms; Proteasome Endopeptidase Complex; Proteolysis; Sirtuins; Transfection; Ubiquitination

Tumors have evolved a variety of methods to reprogram conventional metabolic pathways to favor their own nutritional needs, including glutaminolysis, the first step of which is the hydrolysis of glutamine to glutamate by the amidohydrolase glutaminase 1 (GLS1). A GLS1 inhibitor could potentially target certain cancers by blocking the tumor cell's ability to produce glutamine-derived nutrients. Starting from the known GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide, we describe the medicinal chemistry evolution of a series from lipophilic inhibitors with suboptimal physicochemical and pharmacokinetic properties to cell potent examples with reduced molecular weight and lipophilicity, leading to compounds with greatly improved oral exposure that demonstrate in vivo target engagement accompanied by activity in relevant disease models.

Topics: Animals; Antineoplastic Agents; Biological Availability; Cell Line, Tumor; Drug Discovery; Glutaminase; Humans; Male; Mice, SCID; Molecular Docking Simulation; Neoplasms; Pyridazines; Thiadiazoles

Cancer cells sustain growth by altering their metabolism to accelerated aerobic glycolysis accompanied by increased glucose demand and employ glutamine as additional nutrient source. This metabolic adaptation induces upregulation of glucose transporters GLUT-1 and -3, and simultaneous targeting of both transporters and of glutamine metabolism may offer a promising approach to inhibit cancer cell growth. We describe the discovery of the very potent glucose uptake inhibitor Glutor, which targets glucose transporters GLUT-1, -2, and -3, attenuates glycolytic flux and potently and selectively suppresses growth of a variety of cancer cell lines. Co-treatment of colon cancer cells with Glutor and glutaminase inhibitor CB-839 very potently and synergistically inhibits cancer cell growth. Such a dual inhibition promises to be particularly effective because it targets the metabolic plasticity as well as metabolic rescue mechanisms in cancer cells.

Topics: Benzeneacetamides; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Citric Acid Cycle; Female; Glucose; Glucose Transport Proteins, Facilitative; Glucose Transporter Type 1; Glucose Transporter Type 3; Glutaminase; Glutamine; Glycolysis; Humans; Male; Neoplasms; Thiadiazoles

Optimization of experimental conditions is critical in ensuring robust experimental reproducibility. Through detailed metabolomic analysis we found that cell culture conditions significantly impacted on glutaminase (GLS1) sensitivity resulting in variable sensitivity and irreproducibility in data. Baseline metabolite profiling highlighted that untreated cells underwent significant changes in metabolic status. Both the extracellular levels of glutamine and lactate and the intracellular levels of multiple metabolites changed drastically during the assay. We show that these changes compromise the robustness of the assay and make it difficult to reproduce. We discuss the implications of the cells' metabolic environment when studying the effects of perturbations to cell function by any type of inhibitor. We then devised 'metabolically rationalized standard' assay conditions, in which glutaminase-1 inhibition reduced glutamine metabolism differently in both cell lines assayed, and decreased the proliferation of one of them. The adoption of optimized conditions such as the ones described here should lead to an improvement in reproducibility and help eliminate false negatives as well as false positives in these assays.

Topics: Animals; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Glutaminase; Glutamine; Humans; Metabolomics; Neoplasms; Reproducibility of Results; Research Design; Thiadiazoles

Suppressing glutaminolysis does not always induce cancer cell death in glutamine dependent tumors because cells may switch to alternative energy sources. To reveal compensatory metabolic pathways, we investigated the metabolome-wide cellular response to inhibited glutaminolysis in cancer cells. Glutaminolysis inhibition with C.968 suppressed cell proliferation but was insufficient to induce cancer cell death. We found that lipid catabolism was activated as a compensation for glutaminolysis inhibition. Accelerated lipid catabolism, together with oxidative stress induced by glutaminolysis inhibition, triggered autophagy. Simultaneously inhibiting glutaminolysis and either beta oxidation with trimetazidine or autophagy with chloroquine both induced cancer cell death. Here we identified metabolic escape mechanisms contributing to cancer cell survival under treatment and we suggest potentially translational strategy for combined cancer therapy, given that chloroquine is an FDA approved drug. Our findings are first to show efficiency of combined inhibition of glutaminolysis and beta oxidation as potential anti-cancer strategy as well as add to the evidence that combined inhibition of glutaminolysis and autophagy may be effective in glutamine-addicted cancers.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Benzophenanthridines; Cell Line, Tumor; Cell Proliferation; Chloroquine; Glutaminase; Glutamine; Humans; Lipolysis; Metabolomics; Neoplasms; Oxidative Stress

The mitochondrial phosphate-activated glutaminase C (GAC) is produced by the alternative splicing of the GLS gene. Compared to the other GLS isoform, the kidney-type glutaminase (KGA), GAC is more enzymatically efficient and of particular importance for cancer cell growth. Although its catalytic mechanism is well understood, little is known about how post-translational modifications can impact GAC function. Here, we identified by mass spectrometry a phosphorylated serine at the GLS N-terminal domain (at position 95) and investigated its role on regulating GAC activity. The ectopic expression of the phosphomimetic mutant (GAC.S95D) in breast cancer cells, compared to wild-type GAC (GAC.WT), led to decreased glutaminase activity, glutamine uptake, glutamate release and intracellular glutamate levels, without changing GAC sub-cellular localization. Interestingly, cells expressing the GAC.S95D mutant, compared to GAC.WT, presented decreased migration and vimentin level, an epithelial-to-mesenchymal transition marker. These results reveal that GAC is post-translationally regulated by phosphorylation, which affects cellular glutamine metabolism and glutaminase-related cell phenotype.

Topics: Amino Acid Substitution; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Glutaminase; Humans; Mutation, Missense; Neoplasm Proteins; Neoplasms; Phosphorylation

Kr-POK (ZBTB7c) is a kidney cancer-related POK transcription factor that not only represses transcription of CDKN1A but also increases expression of FASN. However, precisely how Kr-POK affects cell metabolism by controlling gene expression in response to an energy source in rapidly proliferating cells remains unknown. In this study, we characterized the molecular and functional features of Kr-POK in the context of tumor growth and glutamine metabolism. We found that cells expressing Kr-POK shRNA exhibited more severe cell death than control cells in glucose-deprived medium, and that knockdown of Kr-POK decreased glutamine uptake. Glutamine is critical for tumor cell proliferation. Glutaminase (GLS1), which is activated by p-STAT1, catalyzes the initial reaction in the pathway of glutaminolysis. Kr-POK interacts with PIAS1 to disrupt the interaction between PIAS1 and p-STAT1, and free p-STAT1 can activate GLS1 transcription through an interaction with p300. Kr-POK can be also sumoylated by PIAS1, facilitating Kr-POK degradation by the ubiquitin-mediated proteasomal pathway. Finally, we showed that repression of Kr-POK inhibited tumor growth in vivo in a xenograft model by repressing GLS1 expression. Taken together, our data reveal that Kr-POK activates GLS1 transcription and increases glutamine uptake to support rapid cancer cell proliferation.

Topics: Animals; Cell Death; Cell Line; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Proteins; RNA, Small Interfering; STAT1 Transcription Factor; Transcription Factors; Transcription, Genetic

While glutamine is a nonessential amino acid that can be synthesized from glucose, some cancer cells primarily depend on glutamine for their growth, proliferation, and survival. Numerous types of cancer also depend on asparagine for cell proliferation. The underlying mechanisms of the glutamine and asparagine requirement in cancer cells in different contexts remain unclear. In this study, we show that the oncogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) accelerates the glutamine metabolism of glucose-independent proliferation of cancer cells by upregulating the expression of numerous critical enzymes, including glutaminase 2 (GLS2), glutamate dehydrogenase 1 (GLUD1), and glutamic-oxaloacetic transaminase 2 (GOT2), to support cell proliferation. Surprisingly, cell crisis is rescued only completely by supplementation with asparagine but minimally by supplementation with α-ketoglutarate, aspartate, or glutamate upon glutamine deprivation, implying an essential role of γ-nitrogen in glutamine and asparagine for cell proliferation. Specifically, glutamine and asparagine provide the critical γ-nitrogen for purine and pyrimidine biosynthesis, as knockdown of four rate-limiting enzymes in the pathways, including carbamoylphosphate synthetase 2 (CAD), phosphoribosyl pyrophosphate amidotransferase (PPAT), and phosphoribosyl pyrophosphate synthetases 1 and 2 (PRPS1 and PRPS2, respectively), suppresses cell proliferation. These findings indicate that glutamine and asparagine are shunted to the biosynthesis of nucleotides and nonessential amino acids from the tricarboxylic acid (TCA) cycle to support the anabolic proliferation of KSHV-transformed cells. Our results illustrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.

Topics: Asparagine; Aspartate Aminotransferases; Aspartic Acid; Cell Proliferation; Glutamate Dehydrogenase; Glutamic Acid; Glutaminase; Glutamine; Herpesvirus 8, Human; Humans; Metabolic Networks and Pathways; Neoplasms; Nitrogen; Nucleotides

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

Topics: AlkB Enzymes; AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; Alkylation; Animals; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; DNA Damage; Enzyme Inhibitors; Glutaminase; Humans; Male; Mice; Mice, Nude; Neoplasm Proteins; Neoplasms; Random Allocation; RNA Interference; Tumor Burden; Xenograft Model Antitumor Assays

Oxygenated cancer cells have a high metabolic plasticity as they can use glucose, glutamine and lactate as main substrates to support their bioenergetic and biosynthetic activities. Metabolic optimization requires integration. While glycolysis and glutaminolysis can cooperate to support cellular proliferation, oxidative lactate metabolism opposes glycolysis in oxidative cancer cells engaged in a symbiotic relation with their hypoxic/glycolytic neighbors. However, little is known concerning the relationship between oxidative lactate metabolism and glutamine metabolism. Using SiHa and HeLa human cancer cells, this study reports that intracellular lactate signaling promotes glutamine uptake and metabolism in oxidative cancer cells. It depends on the uptake of extracellular lactate by monocarboxylate transporter 1 (MCT1). Lactate first stabilizes hypoxia-inducible factor-2α (HIF-2α), and HIF-2α then transactivates c-Myc in a pathway that mimics a response to hypoxia. Consequently, lactate-induced c-Myc activation triggers the expression of glutamine transporter ASCT2 and of glutaminase 1 (GLS1), resulting in improved glutamine uptake and catabolism. Elucidation of this metabolic dependence could be of therapeutic interest. First, inhibitors of lactate uptake targeting MCT1 are currently entering clinical trials. They have the potential to indirectly repress glutaminolysis. Second, in oxidative cancer cells, resistance to glutaminolysis inhibition could arise from compensation by oxidative lactate metabolism and increased lactate signaling.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Glutaminase; Glutamine; HeLa Cells; Humans; Lactic Acid; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Monocarboxylic Acid Transporters; Neoplasms; Oxidation-Reduction; Symporters

Most cancer cells depend on enhanced glucose and glutamine (Gln) metabolism for growth and survival. Oncogenic metabolism provides biosynthetic precursors for nucleotides, lipids, and amino acids; however, its specific roles in tumor progression are largely unknown. We previously showed that distal-less homeobox-2 (Dlx-2), a homeodomain transcription factor involved in embryonic and tumor development, induces glycolytic switch and epithelial-mesenchymal transition (EMT) by inducing Snail expression. Here we show that Dlx-2 also induces the expression of the crucial Gln metabolism enzyme glutaminase (GLS1), which converts Gln to glutamate. TGF-β and Wnt induced GLS1 expression in a Dlx-2-dependent manner. GLS1 shRNA (shGLS1) suppressed in vivo tumor metastasis and growth. Inhibition of Gln metabolism by shGLS1, Gln deprivation, and Gln metabolism inhibitors (DON, 968 and BPTES) prevented Dlx-2-, TGF-β-, Wnt-, and Snail-induced EMT and glycolytic switch. Finally, shDlx-2 and Gln metabolism inhibition decreased Snail mRNA levels through p53-dependent upregulation of Snail-targeting microRNAs. These results demonstrate that the Dlx-2/GLS1/Gln metabolism axis is an important regulator of TGF-β/Wnt-induced, Snail-dependent EMT, metastasis, and glycolytic switch.

Topics: Adenosine Triphosphate; Apoptosis; Blotting, Western; Cell Proliferation; Chromatin Immunoprecipitation; Epithelial-Mesenchymal Transition; Fluorescent Antibody Technique; Glutaminase; Glutamine; Glycolysis; HeLa Cells; Hep G2 Cells; Homeodomain Proteins; Humans; MCF-7 Cells; Neoplasms; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.

Topics: Alleles; Alternative Splicing; Energy Metabolism; Glutaminase; HCT116 Cells; Humans; mRNA Cleavage and Polyadenylation Factors; Neoplasms; RNA Precursors; RNA, Long Noncoding; RNA, Messenger

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

Topics: Adenosine Triphosphate; Animals; Carcinoma; Cell Line, Tumor; Citric Acid Cycle; Combined Modality Therapy; Cytokines; Deoxyglucose; Drug Resistance, Neoplasm; Drug Synergism; Energy Metabolism; Everolimus; Female; Glucose-6-Phosphate Isomerase; Glutaminase; Glutamine; Glycolysis; Hep G2 Cells; Humans; Mechanistic Target of Rapamycin Complex 1; Metabolomics; Mice; Mice, Nude; Molecular Targeted Therapy; Multiprotein Complexes; Neoplasm Proteins; Neoplasms; Ovarian Neoplasms; Pentose Phosphate Pathway; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Small Interfering; TOR Serine-Threonine Kinases; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Many transformed cells exhibit altered glucose metabolism and increased utilization of glutamine for anabolic and bioenergetic processes. These metabolic adaptations, which accompany tumorigenesis, are driven by oncogenic signals. Here we report that the transcription factor c-Jun, product of the proto-oncogene JUN, is a key regulator of mitochondrial glutaminase (GLS) levels. Activation of c-Jun downstream of oncogenic Rho GTPase signalling leads to elevated GLS gene expression and glutaminase activity. In human breast cancer cells, GLS protein levels and sensitivity to GLS inhibition correlate strongly with c-Jun levels. We show that c-Jun directly binds to the GLS promoter region, and is sufficient to increase gene expression. Furthermore, ectopic overexpression of c-Jun renders breast cancer cells dependent on GLS activity. These findings reveal a role for c-Jun as a driver of cancer cell metabolic reprogramming, and suggest that cancers overexpressing JUN may be especially sensitive to GLS-targeted therapies.

Topics: Animals; Base Sequence; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Embryo, Mammalian; Fibroblasts; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; Humans; MCF-7 Cells; Mice; Molecular Sequence Data; Neoplasms; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Mas; Proto-Oncogene Proteins c-jun; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA Interference

Glutamine-derived carbon becomes available for anabolic biosynthesis in cancer cells via the hydrolysis of glutamine to glutamate, as catalyzed by GAC, a splice variant of kidney-type glutaminase (GLS). Thus, there is significant interest in understanding the regulation of GAC activity, with the suggestion being that higher order oligomerization is required for its activation. We used x-ray crystallography, together with site-directed mutagenesis, to determine the minimal enzymatic unit capable of robust catalytic activity. Mutagenesis of the helical interface between the two pairs of dimers comprising a GAC tetramer yielded a non-active, GAC dimer whose x-ray structure displays a stationary loop ("activation loop") essential for coupling the binding of allosteric activators like inorganic phosphate to catalytic activity. Further mutagenesis that removed constraints on the activation loop yielded a constitutively active dimer, providing clues regarding how the activation loop communicates with the active site, as well as with a peptide segment that serves as a "lid" to close off the active site following substrate binding. Our studies show that the formation of large GAC oligomers is not a pre-requisite for full enzymatic activity. They also offer a mechanism by which the binding of activators like inorganic phosphate enables the activation loop to communicate with the active site to ensure maximal rates of catalysis, and promotes the opening of the lid to achieve optimal product release. Moreover, these findings provide new insights into how other regulatory events might induce GAC activation within cancer cells.

Topics: Animals; Cell Line, Tumor; Enzyme Activation; Glutaminase; Glutamine; Humans; Mice; Neoplasm Proteins; Neoplasms; NIH 3T3 Cells; Protein Multimerization; Protein Structure, Secondary

There is a strong rationale for targeting the metabolic alterations of cancer cells. The most studied of these are the higher rates of glycolysis, glutaminolysis and de novo synthesis of fatty acids (FAs). Despite the availability of pharmacological inhibitors of these pathways, no preclinical studies targeting them simultaneously have been performed. In the present study it was determined whether three key enzymes for glycolysis, glutaminolysis and de novo synthesis of FAs, hexokinase-2, glutaminase and fatty acid synthase, respectively, were overexpressed as compared to primary fibroblasts. In addition, we showed that at clinically relevant concentrations lonidamine, 6-diazo-5-oxo-L-norleucine and orlistat, known inhibitors of the mentioned enzymes, exerted a cell viability inhibitory effect. Genetic downregulation of the three enzymes also reduced cell viability. The three drugs were highly synergistic when administered as a triple combination. Of note, the cytotoxicity of the triple combination was low in primary fibroblasts and was well tolerated when administered into healthy BALB/c mice. The results suggest the feasibility and potential clinical utility of the triple metabolic targeting which merits to be further studied by using either repositioned old drugs or newer, more selective inhibitors.

Topics: Animals; Apoptosis; Cell Survival; Diazooxonorleucine; Drug Synergism; Fatty Acid Synthases; Fatty Acids; Fibroblasts; Gene Expression Regulation, Neoplastic; Glutaminase; Glycolysis; Hexokinase; Humans; Indazoles; Lactones; Metabolic Networks and Pathways; Mice; Neoplasms; Orlistat

Topics: Animals; Enzyme Inhibitors; Glutaminase; Glutamine; Humans; Mice; Neoplasms

Resisting cell death, reprogrammed metabolism and immune escape are fundamental traits of hard-to-treat cancers. Therapeutic improvement can be expected by designing drugs targeting all three aspects. 5'-Triphosphate RNA (ppp-RNA), a specific ligand of the pattern recognition receptor retinoic acid-inducible gene I (RIG-I), has been shown to trigger intrinsic apoptosis of malignant cells and to activate antitumor immune responses via type I interferons (IFNs). In our study, we designed a ppp-modified siRNA specifically silencing glutaminase (ppp-GLS), a key enzyme of glutaminolysis that is indispensable for many cancer types. Bifunctional ppp-GLS induced more prominent antitumor responses than RNA molecules that contained either the RIG-I ligand motif or GLS silencing capability alone. The cytopathic effect was constrained to tumor cells as nonmalignant cells were not affected. We then analyzed the mechanisms leading to the profound antitumor efficacy. First, ppp-GLS effectively induced intrinsic proapoptotic signaling. In addition, GLS silencing sensitized malignant cells to RIG-I-induced apoptosis. Moreover, disturbed glutaminolysis by GLS silencing contributed to enhanced cytotoxicity. Finally, RIG-I activation blocked autophagic degradation leading to dysfunctional mitochondria and reactive oxygen species (ROS) generation, whereas GLS silencing severely impaired ROS scavenging systems, leading to a vicious circle of ROS-mediated cytotoxicity. Taken together, ppp-GLS combines cell death induction, immune activation and glutaminase inhibition in a single molecule and has high therapeutic efficacy against cancer cells.

Topics: Apoptosis; Cell Line, Tumor; Cell Survival; DEAD Box Protein 58; DEAD-box RNA Helicases; Enzyme Activation; Female; Glioma; Glutaminase; HeLa Cells; Humans; Lung Neoplasms; Mitochondria; Neoplasms; Pancreatic Neoplasms; Reactive Oxygen Species; Receptors, Immunologic; RNA Interference; RNA, Small Interfering; Uterine Cervical Neoplasms

Here, for the first time, we report an NMR spectroscopy study of l-Glutamine (Gln) conversion by Glutaminase (Glnase), which shows that the reaction involves two distinct steps. In the first step, Glnase rapidly hydrolyzes Gln to Glutamate (Glu) (∼16.87 μmol of Gln/min/mg of Glnase) and in the second step, Glu generated in the first step is decarboxylated into gamma-amino butyric acid (GABA) with a much slower rate (∼0.185 μmol/min/mg). When Glnase was added to the sample containing l-Glu alone, it was also converted to GABA, at a similar rate as in the second step mentioned above. The rate of Glu decarboxylation into GABA by Glnase is about an order of magnitude lower than that by commonly known enzyme, Glutamate decarboxylase. Potential impact of these findings, on the mechanistic aspects of Gln-Glu shuttle in neuroscience and glutaminolysis in tumors, is discussed.

Topics: Brain; gamma-Aminobutyric Acid; Glucose; Glutamate Decarboxylase; Glutamic Acid; Glutaminase; Glycolysis; Humans; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Biological; Neoplasms

L-asparaginase having low glutaminase has been a key therapeutic agent in the treatment of acute lymphpoblastic leukemia (A.L.L). In the present study, an extracellular L-asparaginase with low glutaminase activity, produced by Bacillus licheniformis was purified to homogeneity. Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine. The activity of purified L-asparaginase enhanced in presence of cations including Na+ and K+, whereas it was moderately inhibited in the presence of divalent cations and thiol group blocking reagents. The purified enzyme was maximally active over the range of pH 6.0 to 10.0 and temperature of 40°C and enzyme was stable maximum at pH 9.0 and -20°C. CD spectra of L-asparaginase predicted the enzyme to consist of 63.05% α-helix and 3.29% β-sheets in its native form with T222 of 58°C. Fluorescent spectroscopy showed the protein to be stable even in the presence of more than 3 M GdHCl. Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10(-5) M, 4.03 IU and 2.68×10(3) s(-1), respectively. The purified L-asparaginase had cytotoxic activity against various cancerous cell lines viz. Jurkat clone E6-1, MCF-7 and K-562 with IC50 of 0.22 IU, 0.78 IU and 0.153 IU respectively. However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug.

Topics: Animals; Antineoplastic Agents; Asparaginase; Bacillus; Cell Line, Tumor; CHO Cells; Cricetulus; Enzyme Stability; Glutaminase; Humans; Neoplasms; Substrate Specificity

Glutaminase catalyzes the hydrolysis of glutamine to glutamate and plays a central role in the proliferation of neoplastic cells via glutaminolysis, as well as in the generation of excitotoxic glutamate in central nervous system disorders such as HIV-associated dementia (HAD) and multiple sclerosis. Both glutaminase siRNA and glutaminase inhibition have been shown to be effective in in vitro models of cancer and HAD, suggesting a potential role for small molecule glutaminase inhibitors. However, there are no potent, selective inhibitors of glutaminase currently available. The two prototypical glutaminase inhibitors, BPTES and DON, are either insoluble or non-specific. In a search for more drug-like glutaminase inhibitors, we conducted a screen of 1280 in vivo active drugs (Library of Pharmacologically Active Compounds (LOPAC(1280))) and identified ebselen, chelerythrine and (R)-apomorphine. The newly identified inhibitors exhibited 10 to 1500-fold greater affinities than DON and BPTES and over 100-fold increased efficiency of inhibition. Although non-selective, it is noteworthy that the affinity of ebselen for glutaminase is more potent than any other activity yet described. It is possible that the previously reported biological activity seen with these compounds is due, in part, to glutaminase inhibition. Ebselen, chelerythrine and apomorphine complement the armamentarium of compounds to explore the role of glutaminase in disease.

Topics: AIDS Dementia Complex; Apomorphine; Azoles; Benzophenanthridines; Cell Proliferation; Dose-Response Relationship, Drug; Drug Design; Drug Evaluation, Preclinical; Glutaminase; Humans; Inhibitory Concentration 50; Isoindoles; Neoplasms; Organoselenium Compounds; RNA, Small Interfering; Sensitivity and Specificity

Topics: Alkanesulfonic Acids; Ammonia; Cell Proliferation; Glutaminase; Glutamine; HeLa Cells; Humans; Hydrogen-Ion Concentration; Kidney; Liver; MCF-7 Cells; Neoplasms; Piperazines

Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.

Topics: Cell Line, Tumor; Crystallization; Crystallography, X-Ray; Fluorescent Antibody Technique; Glutaminase; Humans; Immunoblotting; Immunohistochemistry; Mitochondria; Models, Molecular; Neoplasms; Phosphates; Protein Binding; Scattering, Small Angle

The glutaminase interacting protein (GIP) is composed of a single PDZ domain that interacts with a growing list of partner proteins, including glutaminase L, that are involved in a number of cell signaling and cancer pathways. Therefore, GIP makes a good target for structure-based drug design. Here, we report the solution structures of both free GIP and GIP bound to the C-terminal peptide analogue of glutaminase L. This is the first reported nuclear magnetic resonance structure of GIP in a complex with one of its binding partners. Our analysis of both free GIP and GIP in a complex with the glutaminase L peptide provides important insights into how a promiscuous binding domain can have affinity for multiple binding partners. Through a detailed chemical shift perturbation analysis and backbone dynamics studies, we demonstrate here that the binding of the glutaminase L peptide to GIP is an allosteric event. Taken together, the insights reported here lay the groundwork for the future development of a specific inhibitor for GIP.

Topics: Amino Acid Sequence; Binding Sites; Glutaminase; Humans; Intracellular Signaling Peptides and Proteins; Models, Molecular; Neoplasms; Nuclear Magnetic Resonance, Biomolecular; PDZ Domains; Peptides; Protein Binding; Protein Conformation; Signal Transduction; Solutions

The MYC oncogene encodes a transcription factor, c-Myc (Myc), which is a master regulator of cell metabolism and proliferation. Myc directly influences the expression of thousands of genes, of which, subsets are coordinately regulated with other transcription factors under specified conditions. Myc regulates entry into S phase by stimulating glucose and glutamine metabolism and mitochondrial biogenesis that are coupled to the regulation of E2F1 expression. As a transcription factor, Myc regulates genes involved in these pathways either transcriptionally or post-transcriptionally through direct regulation of microRNA expression. Myc's de-regulation of the expression of glutamine transporters and miR-23a/b that targets glutaminase, triggers an addiction to glutamine, which is required for bioenergetics, nucleotide biosynthesis and redox homeostasis in cancer cells. The induction of the miR-17 cluster by Myc attenuates E2F1 protein expression, such that interruption of this regulatory loop results in DNA replication stress. Hence, deregulated Myc expression in cancers is accompanied by key nodal points that could be exploited for therapeutic purposes.

Topics: E2F1 Transcription Factor; Glutaminase; Glutamine; Humans; MicroRNAs; Neoplasms; Proto-Oncogene Proteins c-myc; S Phase

The pattern of expression of glutaminase isoenzymes in tumour cells has been investigated to clarify its role in the malignant transformation and the prospect of its use as a clinically relevant factor. Using leukaemia cells from medullar blood of human patients and several established human cancer cell lines, we have developed a competitive RT (reverse transcriptase)-PCR assay to quantify simultaneously K-type (kidney-type) and L-type (liver-type) glutaminase mRNAs. Co-expression of both transcripts and higher amounts of L-type mRNA were always found in all cancer cell types analysed. However, mature lymphocytes from the medullar blood of a patient suffering aplasia did not express the K-type transcript and showed a 15-fold increase of L-type transcript. Co-expression was also confirmed at the protein level using isoform-specific antibodies; nevertheless, it did not correlate with the relative abundance of glutaminase transcripts and strong K-type protein signals were detected. On the other hand, marked differences were found with regard to glutamate inhibition and phosphate activation of tumour glutaminase activity. Taken together, the protein data suggest that K isoform would account for the majority of glutaminase activity in these human tumour cells. The results confirm that simultaneous expression of both isoenzymes in human cancer cells is a more frequent event than previously thought. Furthermore, the present work and other previous data suggest that K isoform is up-regulated with increased rates of proliferation, whereas prevalence of the L isoform seems to be related with resting or quiescent cell states.

Topics: Biomarkers; Blotting, Western; Brain; Cell Proliferation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glutamic Acid; Glutaminase; Humans; Isoenzymes; Kidney; Kinetics; Leukemia; Liver; Neoplasms; Phosphates; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

In this study the enzyme glutaminase, purified from the ascites fluid of ovarian cancer patients, was analysed for its antiangiogenic activity. Intraperitoneal administration of this enzyme reduces the number of tumor directed capillaries in solid and ascites tumor bearing Swiss mice induced by transplantation of Ehrlich ascites cells. The enzyme has a critical role in regulating the secretion of vascular endothelial growth factor (VEGF) from tumor cell and in turn tumor growth. Glutamine analogue like 6-diazo, 5- oxo L-norleucine (DON) is also found to be effective in regulating vascular endothelial growth factor (VEGF) secretion from tumor cells in vitro. Treatment with enzyme reduced serum VEGF levels of the tumor induced animals. In vitro VEGF production by EAC cells was reduced in a concentration dependent manner in presence of glutamine analogue.

Topics: Animals; Antineoplastic Agents; Body Weight; Carcinoma, Ehrlich Tumor; Cell Line, Tumor; Cell Proliferation; Diazooxonorleucine; Dose-Response Relationship, Drug; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; Mice; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Ovarian Neoplasms; Time Factors; Vascular Endothelial Growth Factor A

Topics: Alcaligenes; Ammonia-Lyases; Animals; Asparaginase; Carboxypeptidases; Carcinoma; Carcinoma 256, Walker; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Cell Line; Enzyme Therapy; Glutaminase; Humans; Leukemia, Experimental; Liver Neoplasms; Mice; Neoplasms; Neoplasms, Experimental; Sarcoma, Experimental

Topics: Adult; Chronic Disease; Female; Glutaminase; Glutamine; Humans; Male; Middle Aged; Neoplasms; Pregnancy; Transaminases

Topics: Adenocarcinoma; Adenoma; Animals; Carcinoma 256, Walker; Carcinoma, Squamous Cell; Fibrosarcoma; Glutaminase; Growth; Mammary Neoplasms, Experimental; Mathematics; Neoplasm Transplantation; Neoplasms; Neoplasms, Experimental; Osteosarcoma; Rats; Time Factors

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Glutaminase; Leukemia; Leukemia, Experimental; Lymphoma; Lymphoma, Non-Hodgkin; Mercaptopurine; Mice; Neoplasms; Neoplasms, Experimental; Pharmacology; Research; Sarcoma 180; Toxicology

Topics: Amidohydrolases; Glutaminase; HeLa Cells; Humans; Neoplasms

Topics: Amidohydrolases; Animals; Cytoplasm; Glutaminase; Liver; Lung Neoplasms; Neoplasms; Rats