glutaminase and Neuroblastoma

Neuroblastoma is a malignancy [corrected] of childhood and accounts for 7-10% of childhood cancers, leading to approximately 15% of pediatric cancer deaths. MicroRNAs (miRNAs) are a family of short (about 18-25 nucleotides), noncoding and single stranded endogenous RNAs, which complementarily bind to the 3' untranslated regions of their target genes. Recently, glutamine metabolism has been recognized as an important nutrition source for tumor cells, and hence targeting glutamine metabolism could benefit to development of anti-cancer agents. In this study, we investigate the roles of miR-513c in human neuroblastoma. We report miR-513c is significantly downregulated in human neuroblastoma tissues compared with their adjacent normal tissues. Moreover, miR-513c is significantly downregulated in neuroblastoma cell lines compared with normal neuroblast cells. Overexpression of miR-513c suppresses neuroblastoma cells' migration, invasion, and proliferation. We demonstrate the glutaminase (GLS) is a direct target of miR-513c in human neuroblastoma cells. In addition, we found restoration of GLS expression recovered the neuroblastoma cells' migration, invasion, and proliferation. In summary, this study illustrates a miR-513c mediated neuroblastoma cells suppression, providing a new aspect on the miRNA-based therapeutic approach for the treatments of neuroblastoma.

Topics: 3' Untranslated Regions; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression; Glutaminase; Humans; MicroRNAs; Neuroblastoma; RNA Interference

Deamidation of glutamine to glutamate by glutaminase 1 (GLS1, also called GLS) and GLS2 is an essential step in both glutaminolysis and glutathione (GSH) biosynthesis. However, mechanisms whereby cancer cells regulate glutamine catabolism remains largely unknown. We report here that N-Myc, an essential Myc family member, promotes conversion of glutamine to glutamate in MYCN-amplified neuroblastoma cells by directly activating GLS2, but not GLS1, transcription. Abrogation of GLS2 function profoundly inhibited glutaminolysis, which resulted in feedback inhibition of aerobic glycolysis likely due to thioredoxin-interacting protein (TXNIP) activation, dramatically decreasing cell proliferation and survival in vitro and in vivo. Moreover, elevated GLS2 expression is significantly elevated in MYCN-amplified neuroblastomas in comparison with non-amplified ones, correlating with unfavorable patient survival. In aggregate, these results reveal a novel mechanism deciphering context-dependent regulation of metabolic heterogeneities, uncovering a previously unsuspected link between Myc, GLS2 and tumor metabolism.

Topics: Animals; Apoptosis; Blotting, Western; Cell Proliferation; Chromatin Immunoprecipitation; Enzyme Activation; Glutaminase; Glutamine; Glycolysis; Humans; Hydrolysis; Immunoenzyme Techniques; Mice; Mice, Inbred NOD; Mice, SCID; Neuroblastoma; Proto-Oncogene Proteins c-myc; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

HFE gene variants are relatively common genetic variants in Caucasians. The H63D HFE genetic variant has been repeatedly associated with a number of neurodegenerative diseases. We developed neuroblastoma cell lines expressing different HFE polymorphisms to explore the mechanisms behind these associations. Here we tested the hypothesis that cells with the H63D variant have a phenotype that promotes glutamate toxicity. In support of this hypothesis, expression of H63D HFE is associated with increased calcium-induced glutamate secretion and decreased cellular glutamate uptake. The polymorphism-associated changes in glutamate secretion were mimicked by altering cellular iron. Additionally, intracellular calcium is altered in a genotype-specific manner which could further impact glutamate secretion. HFE-dependent effects on glutamate uptake were confirmed in astrocytoma cell lines with endogenous expression of HFE. The ability of minocycline and the antioxidant Trolox to increase glutamate uptake differed by HFE genotype and implicate oxidative stress in glutamate regulation. This study demonstrates HFE cellular effects that extend beyond iron regulation, and suggests that H63D HFE may promote glutamate toxicity.

Topics: Analysis of Variance; Calcium; Cell Line, Tumor; Deferoxamine; Enzyme Inhibitors; Ferric Compounds; Gene Expression Regulation, Neoplastic; Glutamate Plasma Membrane Transport Proteins; Glutamic Acid; Glutaminase; Hemochromatosis Protein; Histocompatibility Antigens Class I; Humans; Intracellular Fluid; Iron; Membrane Proteins; Minocycline; Neuroblastoma; Polymorphism, Genetic; Quaternary Ammonium Compounds; Siderophores; Sodium; Tacrine; Transfection; Tritium; Vesicular Glutamate Transport Protein 1

A novel form of phosphate activated glutaminase (PAG), catalyzing the synthesis of glutamate from glutamine, has been detected in cultured astrocytes and SH-SY5Y neuroblastoma cells. This enzyme form is different from that of the kidney and liver isozymes. In these cells we found high enzyme activity, but no or very weak immunoreactivity against the kidney type of PAG, and no immunoreactivity against the liver type. PAG was also investigated in brain under pathological conditions. In patients with Down's syndrome the immunoreactivity in the frontoparietal cortex was significantly reduced. The findings leading to our conclusion of a functionally active PAG on the outer face of the inner mitochondrial membrane are discussed, and a model is presented.

Topics: Astrocytes; Brain; Brain Neoplasms; Cell Line, Tumor; Cells, Cultured; Glutaminase; Humans; Immunohistochemistry; Mitochondria; Mitochondrial Membranes; Neuroblastoma