glutaminase and Glioblastoma

Glutamine (Gln) and glutamate (Glu) play pivotal roles in the malignant phenotype of brain tumors via multiple mechanisms. Glutaminase (GA, EC 3.5.1.2) metabolizes Gln to Glu and ammonia. Human GA isoforms are encoded by two genes: GLS gene codes for kidney-type isoforms, KGA and GAC, whereas GLS2 codes for liver-type isoforms, GAB and LGA. The expression pattern of both genes in different neoplastic cell lines and tissues implicated that the kidney-type isoforms are associated with cell proliferation, while the liver-type isoforms dominate in, and contribute to the phenotype of quiescent cells. GLS gene has been demonstrated to be regulated by oncogene c-Myc, whereas GLS2 gene was identified as a target gene of p53 tumor suppressor. In glioblastomas (GBM, WHO grade IV), the most aggressive brain tumors, high levels of GLS and only traces or lack of GLS2 transcripts were found. Ectopic overexpression of GLS2 in human glioblastoma T98G cells decreased their proliferation and migration and sensitized them to the alkylating agents often used in the chemotherapy of gliomas. GLS silencing reduced proliferation of glioblastoma T98G cells and strengthen the antiproliferative effect evoked by previous GLS2 overexpression.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Glioblastoma; Glutaminase; Humans; Isoenzymes; Phenotype

Glioblastoma multiforme (GBM) is the most frequent, lethal and aggressive tumour of the central nervous system in adults. The discovery of novel anti-GBM agents based on the isocitrate dehydrogenase (IDH) mutant phenotypes and classifications have attracted comprehensive attention.. Diterpenoids are a class of naturally occurring 20-carbon isoprenoid compounds, and have previously been shown to possess high cytotoxicity for a variety of human tumours in many scientific reports. In the present study, 31 cassane diterpenoids of four types, namely, butanolide lactone cassane diterpenoids (I) (1-10), tricyclic cassane diterpenoids (II) (11-15), polyoxybutanolide lactone cassane diterpenoids (III) (16-23), and fused furan ring cassane diterpenoids (IV) (24-31), were tested for their anti-glioblastoma activity and mechanism underlying based on IDH1 mutant phenotypes of primary GBM cell cultures and human oligodendroglioma (HOG) cell lines.. We confirmed that tricyclic-type (II) and compound 13 (Caesalpin A, CSA) showed the best anti-neoplastic potencies in IDH1 mutant glioma cells compared with the other types and compounds. Furthermore, the structure-relationship analysis indicated that the carbonyl group at C-12 and an α, β-unsaturated ketone unit fundamentally contributed to enhancing the anti-glioma activity. Studies investigating the mechanism demonstrated that CSA induced oxidative stress via causing glutathione reduction and NOS activation by negatively regulating glutaminase (GLS), which proved to be highly dependent on IDH mutant type glioblastoma. Finally, GLS overexpression reversed the CSA-induced anti-glioma effects in vitro and in vivo, which indicated that the reduction of GLS contributed to the CSA-induced proliferation inhibition and apoptosis in HOG-IDH1-mu cells.. Therefore, the present results demonstrated that compared with other diterpenoids, tricyclic-type diterpenoids could be a targeted drug candidate for the treatment of secondary IDH1 mutant type glioblastoma through negatively regulating GLS.

Topics: Apoptosis; Brain Neoplasms; Cell Line, Tumor; Diterpenes; Glioblastoma; Glutaminase; Humans; Isocitrate Dehydrogenase; Mutation; Oxidative Stress

Glutaminase isoenzymes GLS and GLS2 play apparently opposing roles in cancer: GLS acts as an oncoprotein, while GLS2 (GAB isoform) has context specific tumour suppressive activity. Some microRNAs (miRNAs) have been implicated in progression of tumours, including gliomas. The aim was to investigate the effect of GLS and GAB expression on both miRNAs and oxidative status in glioblastoma cells.. Microarray profiling of miRNA was performed in GLS-silenced LN229 and GAB-transfected T98G human glioblastoma cells and their wild-type counterparts. Results were validated by real-time quantitative RT-PCR. Oxidative status and antioxidant enzymes were determined by spectrophotometric or fluorescence assays in GLS-silenced LN229 and T98G, and GAB-transfected LN229 and T98G.. MiRNA-146a-5p, miRNA-140-3p, miRNA-21-5p, miRNA-1260a, and miRNA-92a-3p were downregulated, and miRNA-1246 was upregulated when GLS was knocked down. MiRNA-140-3p, miRNA-1246, miRNA-1260a, miRNA-21-5p, and miRNA-146a-5p were upregulated when GAB was overexpressed. Oxidative status (lipid peroxidation, protein carbonylation, total antioxidant capacity, and glutathione levels), as well as antioxidant enzymes (catalase, superoxide dismutase, and glutathione reductase) of silenced GLS glioblastoma cells and overexpressed GAB glioblastoma cells significantly changed versus their respective control glioblastoma cells. MiRNA-1246, miRNA-1260a, miRNA-146a-5p, and miRNA-21-5p have been characterized as strong biomarkers of glioblastoma proliferation linked to both GLS silencing and GAB overexpression. Total glutathione is a reliable biomarker of glioblastoma oxidative status steadily associated to both GLS silencing and GAB overexpression.. Glutaminase isoenzymes are related to the expression of some miRNAs and may contribute to either tumour progression or suppression through certain miRNA-mediated pathways, proving to be a key tool to switch cancer proliferation and redox status leading to a less malignant phenotype. Accordingly, GLS and GAB expression are especially involved in glutathione-dependent antioxidant defence.

Topics: Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Glioblastoma; Glutaminase; Humans; Isoenzymes; MicroRNAs; Oxidative Stress; Up-Regulation

Cancer cells can metabolize glutamine to replenish TCA cycle intermediates, leading to a dependence on glutaminolysis for cell survival. However, a mechanistic understanding of the role that glutamine metabolism has on the survival of glioblastoma (GBM) brain tumor stem cells (BTSC) has not yet been elucidated. Here, we report that across a panel of 19 GBM BTSC lines, inhibition of glutaminase (GLS) showed a variable response from complete blockade of cell growth to absolute resistance. Surprisingly, BTSC sensitivity to GLS inhibition was a result of reduced intracellular glutamate triggering the amino acid deprivation response (AADR) and not due to the contribution of glutaminolysis to the TCA cycle. Moreover, BTSC sensitivity to GLS inhibition negatively correlated with expression of the astrocytic glutamate transporters EAAT1 and EAAT2. Blocking glutamate transport in BTSCs with high EAAT1/EAAT2 expression rendered cells susceptible to GLS inhibition, triggering the AADR and limiting cell growth. These findings uncover a unique metabolic vulnerability in BTSCs and support the therapeutic targeting of upstream activators and downstream effectors of the AADR pathway in GBM. Moreover, they demonstrate that gene expression patterns reflecting the cellular hierarchy of the tissue of origin can alter the metabolic requirements of the cancer stem cell population. SIGNIFICANCE: Glioblastoma brain tumor stem cells with low astrocytic glutamate transporter expression are dependent on GLS to maintain intracellular glutamate to prevent the amino acid deprivation response and cell death.

Topics: Amino Acids; Astrocytes; Benzeneacetamides; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Citric Acid Cycle; Excitatory Amino Acid Transporter 1; Excitatory Amino Acid Transporter 2; Glioblastoma; Glutamic Acid; Glutaminase; Humans; Neoplastic Stem Cells; Signal Transduction; Thiadiazoles

Topics: Adolescent; Biomarkers, Tumor; Brain Neoplasms; Child; China; Computational Biology; Databases, Genetic; Female; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Glioblastoma; Glutaminase; Humans; Male; NADPH Oxidases; Prognosis; Protein Interaction Maps; Transcriptome

Phosphate-activated glutaminase (GA), a ubiquitous glutamine-metabolizing enzyme, is encoded by two genes, GLS and GLS2. In mammalian cancers, GLS isoforms are perceived as molecules promoting cell proliferation and invasion, whereas the role of GLS2 isoforms seems to be more complex and cell type-specific. Previous studies have shown abundance of GLS and lack of GLS2 transcripts in T98G human glioblastoma (GBM) cell line and patient-derived GBM. Transfection with GAB sequence, the whole GLS2 cDNA transcript, suppressed malignant phenotype of T98G cells. Microarray analysis revealed upregulation of GATA3, the product of which has been implicated in suppressing growth of some peripheral cancers. In this study we confirmed a significant upregulation of GATA3 expression in the transfected cells both at mRNA and protein level. Considerable expression of GATA3 was also observed in GBM tissues (previously shown as not expressing GLS2), while only traces or no GATA3 was detected in (GLS2-expressing) non-tumorigenic brain samples. In conclusion, while mechanistic relation between GAB and GATA3 expression is evident following in vitro manipulation of GBM cell line, it does not appear to be an intrinsic property of GBM nor non-tumorigenic brain tissue.

Topics: Adult; Aged; Brain; Brain Neoplasms; Cell Line, Tumor; Female; GATA3 Transcription Factor; Glioblastoma; Glutaminase; Humans; Male; Middle Aged

Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) is now a widely-used modality for glioblastoma (GBM) treatment. However, intratumoral heterogeneity of fluorescence intensity may reflect different onco-metabolic programs. Here, we investigated the metabolic mechanism underlying the heterogeneity of 5-ALA fluorescence in GBM. Using an in-house developed fluorescence quantification system for tumor tissues, we collected 3 types of GBM tissues on the basis of their fluorescence intensity, which was characterized as strong, weak, and none. Expression profiling by RNA-sequencing revealed 77 genes with a proportional relationship and 509 genes with an inverse relationship between gene expression and fluorescence intensity. Functional analysis and in vitro experiments confirmed glutaminase 2 (GLS2) as a key gene associated with the fluorescence heterogeneity. Subsequent metabolite profiling discovered that insufficient NADPH due to GLS2 underexpression was responsible for the delayed metabolism of 5-ALA and accumulation of protoporphyrin IX (PpIX) in the high fluorescence area. The expression level of GLS2 and related NADPH production capacity is associated with the regional heterogeneity of 5-ALA fluorescence in GBM.

Topics: Aminolevulinic Acid; Brain Neoplasms; Cell Line, Tumor; Fluorescence; Fluorescent Dyes; Gene Expression Profiling; Glioblastoma; Glutaminase; Humans; Levulinic Acids; NADP; Prospective Studies; Protoporphyrins; Surgery, Computer-Assisted

Glioblastoma is the most aggressive manifestation of malignant gliomas and considered to be among the deadliest forms of human cancers. MicroRNAs are found to tightly regulate diverse biological processes and considered to play important roles in cancer etiology. In this study, we found that microRNA-153 was significantly downregulated in glioblastoma tissues compared to matched non-tumor tissues and in glioblastoma cell lines. To investigate the potential function of microRNA-153 in glioblastoma, we transfected glioblastoma cell line U87MG as well as U373MG with synthetic microRNA-153 oligos and observed decreased cell proliferation and increased apoptosis. We further found that microRNA-153 restrained glutamine utilization and glutamate generation. Bioinformatics analysis revealed that glutaminase, which catalyzed the formation of glutamate from glutamine, is the potential target of microRNA-153. Indeed, microRNA-153 cannot further reduce glutamine utilization when glutaminase was knocked down. Overexpression of glutaminase abrogates the effect of microRNA-153 on glutamine utilization. Furthermore, the relative expression of microRNA-153 and glutaminase in glioblastoma versus matched non-tumor tissues showed a reverse correlation, further indicating that microRNA-153 may negatively regulate glutaminase in vivo. These results demonstrate an unexpected role of microRNA-153 in regulating glutamine metabolism and strengthen the role of microRNA-153 as a therapeutic target in glioblastoma.

Topics: Brain Neoplasms; Cell Growth Processes; Cell Line, Tumor; Down-Regulation; Glioblastoma; Glutaminase; Glutamine; Humans; Immunohistochemistry; In Situ Hybridization; MicroRNAs

Human phosphate-activated glutaminase (GA) is encoded by two genes: GLS and GLS2. Glioblastomas (GB) usually lack GLS2 transcripts, and their reintroduction inhibits GB growth. The GLS2 gene in peripheral tumors may be i) methylation- controlled and ii) a target of tumor suppressor p53 often mutated in gliomas. Here we assessed the relation of GLS2 downregulation in GB to its methylation and TP53 status. DNA demethylation with 5-aza-2'-deoxycytidine restored GLS2 mRNA and protein content in human GB cell lines with both mutated (T98G) and wild-type (U87MG) p53 and reduced the methylation of CpG1 (promoter region island), and CpG2 (first intron island) in both cell lines. In cell lines and clinical GB samples alike, methylated CpG islands were detected both in the GLS2 promoter (as reported earlier) and in the first intron of this gene. CpG methylation of either island was absent in GLS2-expressing non-tumoros brain tissues. Screening for mutation in the exons 5-8 of TP53 revealed a point mutation in only one out of seven GB examined. In conclusion, aberrant methylation of CpG islands, appear to contribute to silencing of GLS2 in GB by a mechanism bypassing TP53 mutations. © 2015 Wiley Periodicals, Inc.

Topics: Brain; Brain Neoplasms; Cell Line, Tumor; CpG Islands; DNA Methylation; Down-Regulation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Genes, p53; Glioblastoma; Glutaminase; Humans; Point Mutation; Promoter Regions, Genetic; Tumor Suppressor Protein p53

Notch signaling can promote tumorigenesis in the nervous system and plays important roles in stem-like cancer cells. However, little is known about how Notch inhibition might alter tumor metabolism, particularly in lesions arising in the brain. The gamma-secretase inhibitor MRK003 was used to treat glioblastoma neurospheres, and they were subdivided into sensitive and insensitive groups in terms of canonical Notch target response. Global metabolomes were then examined using proton magnetic resonance spectroscopy, and changes in intracellular concentration of various metabolites identified which correlate with Notch inhibition. Reductions in glutamate were verified by oxidation-based colorimetric assays. Interestingly, the alkylating chemotherapeutic agent temozolomide, the mTOR-inhibitor MLN0128, and the WNT inhibitor LGK974 did not reduce glutamate levels, suggesting that changes to this metabolite might reflect specific downstream effects of Notch blockade in gliomas rather than general sequelae of tumor growth inhibition. Global and targeted expression analyses revealed that multiple genes important in glutamate homeostasis, including glutaminase, are dysregulated after Notch inhibition. Treatment with an allosteric inhibitor of glutaminase, compound 968, could slow glioblastoma growth, and Notch inhibition may act at least in part by regulating glutaminase and glutamate.

Topics: Brain Neoplasms; Cell Line, Tumor; Cyclic S-Oxides; Glioblastoma; Glutamic Acid; Glutaminase; Homeostasis; Humans; Metabolome; Receptors, Notch; Thiadiazoles

The mechanistic target of rapamycin (mTOR) is hyperactivated in many types of cancer, rendering it a compelling drug target; however, the impact of mTOR inhibition on metabolic reprogramming in cancer is incompletely understood. Here, by integrating metabolic and functional studies in glioblastoma multiforme (GBM) cell lines, preclinical models, and clinical samples, we demonstrate that the compensatory upregulation of glutamine metabolism promotes resistance to mTOR kinase inhibitors. Metabolomic studies in GBM cells revealed that glutaminase (GLS) and glutamate levels are elevated following mTOR kinase inhibitor treatment. Moreover, these mTOR inhibitor-dependent metabolic alterations were confirmed in a GBM xenograft model. Expression of GLS following mTOR inhibitor treatment promoted GBM survival in an α-ketoglutarate-dependent (αKG-dependent) manner. Combined genetic and/or pharmacological inhibition of mTOR kinase and GLS resulted in massive synergistic tumor cell death and growth inhibition in tumor-bearing mice. These results highlight a critical role for compensatory glutamine metabolism in promoting mTOR inhibitor resistance and suggest that rational combination therapy has the potential to suppress resistance.

Topics: Aged; Animals; Antineoplastic Combined Chemotherapy Protocols; Benzophenanthridines; Brain Neoplasms; Cell Line, Tumor; Citric Acid Cycle; Drug Resistance, Neoplasm; Drug Synergism; Energy Metabolism; Gas Chromatography-Mass Spectrometry; Glioblastoma; Glutamic Acid; Glutaminase; Glutamine; Glycolysis; Humans; Indoles; Ketoglutaric Acids; Magnetic Resonance Spectroscopy; Male; Metabolome; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Targeted Therapy; Neoplasm Proteins; Protein Kinase Inhibitors; Purines; RNA, Small Interfering; Rotarod Performance Test; Signal Transduction; Temporal Lobe; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Topics: Animals; Brain Neoplasms; Cell Survival; Gene Expression Regulation, Neoplastic; Genomics; Glioblastoma; Glutaminase; Homeostasis; Humans; Isoenzymes; Kidney; Liver; Mice; Phosphatidylinositol 3-Kinases; Proteomics; Signal Transduction; TOR Serine-Threonine Kinases

Glutamine (Gln) metabolism, initiated by its degradation by glutaminases (GA), is elevated in neoplastic cells and tissues. In malignant glia-derived tumors, GA isoforms, KGA and GAC, coded by the GLS gene, are overexpressed, whereas the GLS2-coded GAB and LGA isoforms, are hardly detectable in there. Our previous study revealed that transfection of T98G glioblastoma cells with GAB reduced cell proliferation and migration, by a yet unknown mechanism not related to Gln degradation. The question arose how simultaneous overexpression of GAB and inhibition of KGA would affect glioblastoma cell growth. Here, we used siRNA to silence the expression of Gls in T98G cells which were or were not stably transfected with GAB (TGAB cells). In both T98G and TGAB cell lines, silencing of Gls with siRNAs targeted at different sequences decreased cell viability and proliferation in a different, sequence-dependent degree, and the observed decreases were in either cell line highly correlated with increase of intracellular Gln (r > 0.9), a parameter manifesting decreased Gln degradation. The results show that combination of negative modulation of GA isoforms arising from GLS gene with the introduction of the GLS2 gene product, GAB, may in the future provide a useful means to curb glioblastoma growth in situ. At the same time, the results underscore the critical role of Gln degradation mediated by KGA in the manifestations of aggressive glial tumor phenotype.

Topics: Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Gene Silencing; Glioblastoma; Glutaminase; Humans; Isoenzymes; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Transfection

Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused (13)C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo.

Topics: Animals; Brain Neoplasms; Glioblastoma; Gluconeogenesis; Glucose; Glutamate-Ammonia Ligase; Glutamic Acid; Glutaminase; Glutamine; Glycolysis; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Mitochondria; Neoplasm Transplantation; Neostriatum; Oxidation-Reduction; Phenotype; Pyruvate Carboxylase; Statistics, Nonparametric; Tumor Cells, Cultured

Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in cancer. Precursors for lipids, nucleic acids, and proteins are generated in the tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many tumor cells use glutamine to feed anaplerosis. Here we studied how "glutamine-addicted" cells react to interruptions of glutamine metabolism. Silencing of glutaminase (GLS), which catalyzes the first step in glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by pyruvate carboxylase (PC), allowing the cells to use glucose-derived pyruvate rather than glutamine for anaplerosis. Although PC was dispensable when glutamine was available, forcing cells to adapt to low-glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic enzymes. Cells with high PC activity were resistant to GLS silencing and did not require glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated, glucose-dependent anaplerosis allows cells to achieve glutamine independence. Induction of PC during chronic suppression of glutamine metabolism may prove to be a mechanism of resistance to therapies targeting glutaminolysis.

Topics: Cell Line, Tumor; Cell Proliferation; Citric Acid Cycle; Glioblastoma; Glutaminase; Glutamine; Humans; Pyruvate Carboxylase; Pyruvic Acid