Role of GRM1 in altering glutamate metabolism and bioavailability
Creators: name: Shah, Raj D.
Publisher: name: No Publisher Supplied
Year: 2020

description: Altered metabolic activity has been implicated in several types of cancer including malignant melanoma. Previously, we have illustrated the role of a neuronal receptor, metabotropic glutamate receptor 1 (GRM1), in the neoplastic transformation of melanocytes in vitro and spontaneous development of metastatic melanoma in vivo. Glutamate, the natural ligand of GRM1, is one of the most abundant amino acids in humans and the predominant excitatory neurotransmitter in the central nervous system. The overall goal of this dissertation is to determine how ectopic GRM1 expression leads to the rewiring of metabolic processes, especially in glutamate metabolism, and how this may contribute to deregulated tumor cell proliferation. Using a set of isogenic melanoma cell lines, we demonstrated correlations between GRM1 and glutaminase (GLS) expression. Metabolomics revealed that GRM1+ melanoma cells exhibit elevated levels of glutaminolytic mitochondrial tricarboxylic acid (TCA) cycle-related amino acids and intermediates, especially glutamate. The increased intracellular pool size of glutamate could be a direct result of increased conversion of glutamine to glutamate via the activity of GLS. Furthermore, principle component analysis revealed that modulation of GRM1 in the aforementioned set of isogenic melanoma cells causes metabolic perturbations that overlap with GRM1 expression levels. It has been well known that glutaminolysis is primarily responsible for increased glutamate production in tumors. Using a rational drug-targeting strategy, we critically evaluate metabolic bottlenecks with the goal to cut off tumor glutamate bioavailability. In cultured GRM1+ melanoma cell lines, CB-839, a potent, selective, and orally bioavailable inhibitor of GLS suppressed cell proliferation while riluzole, an inhibitor of glutamate release, promoted apoptotic cell death in vitro and in vivo. Combined treatment with CB-839 and riluzole treatment proved to be superior to single agent treatment, restricting glutamate bioavailability and leading to severe suppression of tumor cell proliferation in vitro. Most importantly, disruption of GRM1 signaling through combined actions of CB-839 and riluzole significantly suppressed tumor growth in two independent xenograft mouse models of melanoma, with no obvious symptoms of toxicity detected. Molecular analysis of excised tumor specimens demonstrated enhanced suppression of ERK and AKT phosphorylation with the combination of CB-839 and riluzole. Using LC-MS analysis, we determined that the blood plasma concentration of unbound riluzole is substantially higher in male mice compared to females possibly clarifying why riluzole treatment displays a superior response in males. Finally, we established that GLS overexpression, in GRM1+ cell lines, ensues at least in part, through the deep-rooted mTORC axis, as seen through pharmacological inhibition of mTOR phosphorylation and subsequent downregulation of GLS. These insights, combined with our data, support the rationale to target glutamate bioavailability and may aid in the identification of novel metabolic targets to combat GRM1+ human neoplasia.