In this study with the use

In this study, with the use of 13C515N2-labeled glutamine, we observed that 13C515N2-labeled glutamine produces a wide range of NAAG isotopologues and its precursors: NAA and glutamate. The NAAG production from glutamine is consistently more elevated in MYC-ON lymphoma and OVCAR4 compared to MYC-OFF lymphoma and primary OVCA. We analyzed this metabolite in different grades of glioma and meningioma from patients and uncovered that NAAG concentrations in glioma grade IV (GBM) are significantly higher than in gliomas grade II or III and meningioma. These findings corroborate previous reports showing that high expression of N-acetyl-aspartate L-glutamate ligase or N-acetyl-aspartyl-glutamate synthetase A and B (enzyme to produce NAAG) is associated with a shorter survival time of cancer patients (Gyorffy et al., 2012). Furthermore, we uncovered a prognostic potential of NAAG due to its consistently elevated concentrations in both tumors and plasma of patients with higher grade 5-Hydroxy-CTP tumor. Interestingly, NAAG concentration was also found to strongly reflect tumor size in vivo, and the increase in NAAG concentration occurred before the increased tumor sizes were detected. These findings suggest that a non-invasive assessment of NAAG in plasma has the potential for use as a metabolite monitor for cancer progression, whereby NAAG concentration spikes can be detected in advance of any surges in tumor growth. Our next question was why higher grade or more aggressive cancer cells produced more NAAG, which prompted us to investigate the mechanistic action of NAAG. In order to grow substantially, higher grade cancer cells must be able to supply additional metabolites for bioenergy and synthesize the necessary biosynthetic precursors of proteins, nucleic acids, and membrane lipids. Previous studies by us and others have found that glycogen production is increased in many tumor cells, providing these cells with the ability to store glucose and thus resist metabolic deprivation in the harsh tumor microenvironment (Elgogary et al., 2016, Pelletier et al., 2012). In this study, we revealed that, similarly, NAAG production is consistently elevated in higher grade cancers to store glutamate for later use when glutamate production from other sources is limited. Specifically, in the context of adding exogenous NAAG, we found that NAAG increased tumor weights and tumor glutamate concentrations in vivo. Importantly, using 15N2-labeled NAAG via i.p. injection, we confirmed that 15N2-NAAG indeed was present in the tumors and directly hydrolyzed to 15N1-glutamate in vivo in the 15N2-NAAG-supplemented group. In the context of endogenous NAAG degradation, we showed evidence that NAAG production serves to store glutamate for later use when glutamate production from other sources is limited. Using BPTES, a specific glutaminase inhibitor that inhibits the conversion of glutamine to glutamate in vitro (Elgogary et al., 2016) as a way to reduce glutamate production, we found the greatest decreases in cell numbers were shGCPII-KD cells treated with BPTES. Lower cell numbers and glutamate concentrations in shGCPII-KD cells treated with BPTES compared to those in shControl treated with BPTES suggests a growth advantage when cells have GCPII to partially compensate for the decrease in glutamate caused by glutaminase inhibition. Similarly, we found a greater decrease in tumor weights and glutamate concentrations in 2-PMPA (GCPII inhibitor) and CB-839 (glutaminase inhibitor in vivo) treatment compared to either treatment alone in vivo. Moreover, we were able to restore cell numbers in the GCPII-KD group by glutamate, providing further evidence that GCPII supports OVCAR4 cell growth by supplying glutamate. Glutamate, in turn, provides nitrogen for nucleobase synthesis and carbon for TCA cycle processing, lipid synthesis, and nucleotide synthesis necessary for cell growth (Hensley et al., 2013). Moreover, as mentioned above, the excessive availability of glutamate would intoxicate cells (Whelan, 2000), explaining the need to store glutamate in NAAG, which can later be broken down, when needed, to satisfy the high energy demand of higher grade cancers. In light of these findings, we revealed that NAAG can provide glutamate to a subgroup of cancers that express GCPII, supporting tumor growth. We recognized that the hydrolysis of NAAG also provides NAA, which in turn generates aspartate and acetyl-CoA. These also play important roles in cancer growth but were not the focus of our current study.