bims-glucam 2022-02-06 papers

Amino Acids. 2022 Feb 02.

Excess glutamine does not alter myotube metabolism or insulin sensitivity.

Martina J Krone, Caroline N Rivera, Madison E Rivera, Rachel M Watne, Sarah E Lemonds, Andrew J Wommack, Roger A Vaughan.

Glutamine is an amino acid previously linked with improved skeletal muscle metabolism and insulin signaling, however, past observations often use cell culture models with only supraphysiological concentrations. Additionally, past reports have yet to simultaneously investigate both metabolic outcomes and insulin signaling. The present report utilized cell culture experiments and measured the effects of both physiological and supraphysiological levels of glutamine on myotube metabolism and insulin signaling/resistance. It was hypothesized the addition of glutamine at any level would increase cell metabolism and related gene expression, as well as improve insulin signaling versus respective control cells. C2C12 myotubes were treated with glutamine ranging from 0.25 mM-4 mM (or media control) for 24 h to capture a range of physiological and supraphysiological concentrations. qRT-PCR was used to measure metabolic gene expression. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Insulin sensitivity (indicated by pAkt:Akt) and metabolism following glucose/insulin infusion were also assessed. Glutamine treatment consistently increased mitochondrial and glycolytic metabolism versus true controls (cells treated with media void of glutamine), however, supraphysiological glutamine did not enhance metabolism beyond that of cells with physiological levels of glutamine. Neither physiological nor supraphysiological levels of glutamine altered insulin signaling regardless of insulin stimulation or insulin resistance when compared with respective controls. These data demonstrate excess glutamine does not appear to alter myotube metabolism or glucose disposal when base levels of glutamine are present. Moreover, glutamine does not appear to alter insulin sensitivity regardless of level of insulin resistance or presence of insulin stimulation.

Keywords: Insulin resistance; Mitochondrial biogenesis; Skeletal muscle; c2c12 myotubes; pAkt/Akt

DOI: https://doi.org/10.1007/s00726-022-03131-x

Adv Cancer Res. 2022 ;pii: S0065-230X(21)00081-6. [Epub ahead of print]153 267-304

Unraveling and targeting RAS-driven metabolic signaling for therapeutic gain.

Jonathan M DeLiberty, Ryan Robb, Claire E Gates, Kirsten L Bryant.

RAS mutations are among the most frequent oncogenic drivers observed in human cancers. With a lack of available treatment options, RAS-mutant cancers account for many of the deadliest cancers in the United States. Recent studies established that altered metabolic requirements are a hallmark of cancer, and many of these alterations are driven by aberrant RAS signaling. Specifically, RAS-driven cancers are characterized by upregulated glycolysis, the differential channeling of glycolytic intermediates, upregulated nutrient scavenging pathways such as autophagy and macropinocytosis, and altered glutamine utilization and mitochondrial function. This unique metabolic landscape promotes tumorigenesis, proliferation, survival in nutrient deficient environments and confers resistance to conventional cytotoxic and targeted therapies. Emerging work demonstrates how these dependencies can be therapeutically exploited in vitro and in vivo with many metabolic inhibitors currently in clinical trials. This review aims to outline the unique metabolic requirements induced by aberrant RAS signaling and how these altered dependencies present opportunities for therapeutic intervention.

Keywords: Autophagy; Glycolysis; Macropinocytosis; Metabolism; Mitochondria; RAS; Scavenging

DOI: https://doi.org/10.1016/bs.acr.2021.07.010

Proc Natl Acad Sci U S A. 2022 Feb 08. pii: e2120617119. [Epub ahead of print]119(6):

Loss of glucose 6-phosphate dehydrogenase function increases oxidative stress and glutaminolysis in metastasizing melanoma cells.

Arin B Aurora, Vishal Khivansara, Ashley Leach, Jennifer G Gill, Misty Martin-Sandoval, Chendong Yang, Stacy Y Kasitinon, Divya Bezwada, Alpaslan Tasdogan, Wen Gu, Thomas P Mathews, Zhiyu Zhao, Ralph J DeBerardinis, Sean J Morrison.

The pentose phosphate pathway is a major source of NADPH for oxidative stress resistance in cancer cells but there is limited insight into its role in metastasis, when some cancer cells experience high levels of oxidative stress. To address this, we mutated the substrate binding site of glucose 6-phosphate dehydrogenase (G6PD), which catalyzes the first step of the pentose phosphate pathway, in patient-derived melanomas. G6PD mutant melanomas had significantly decreased G6PD enzymatic activity and depletion of intermediates in the oxidative pentose phosphate pathway. Reduced G6PD function had little effect on the formation of primary subcutaneous tumors, but when these tumors spontaneously metastasized, the frequency of circulating melanoma cells in the blood and metastatic disease burden were significantly reduced. G6PD mutant melanomas exhibited increased levels of reactive oxygen species, decreased NADPH levels, and depleted glutathione as compared to control melanomas. G6PD mutant melanomas compensated for this increase in oxidative stress by increasing malic enzyme activity and glutamine consumption. This generated a new metabolic vulnerability as G6PD mutant melanomas were more dependent upon glutaminase than control melanomas, both for oxidative stress management and anaplerosis. The oxidative pentose phosphate pathway, malic enzyme, and glutaminolysis thus confer layered protection against oxidative stress during metastasis.

Keywords: glutaminolysis; melanoma; metastasis; oxidative stress; pentose phosphate pathway

DOI: https://doi.org/10.1073/pnas.2120617119

J Exp Clin Cancer Res. 2022 Feb 01. 41(1): 45

Pyrroline-5-Carboxylate Reductase 1: a novel target for sensitizing multiple myeloma cells to bortezomib by inhibition of PRAS40-mediated protein synthesis.

Inge Oudaert, Hatice Satilmis, Philip Vlummens, Wouter De Brouwer, Anke Maes, Dirk Hose, Elke De Bruyne, Bart Ghesquière, Karin Vanderkerken, Kim De Veirman, Eline Menu.

BACKGROUND: Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both MM cell survival and drug resistance. Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are two mitochondrial enzymes that facilitate the last step in the glutamine-to-proline conversion. Overexpression of PYCR1 is involved in progression of several cancers, however, its' role in hematological cancers is unknown. In this study, we investigated whether PYCR affects MM viability, proliferation and response to bortezomib.
METHODS: Correlation of PYCR1/2 with overall survival was investigated in the MMRF CoMMpass trial (653 patients). OPM-2 and RPMI-8226 MM cell lines were used to perform in vitro experiments. RPMI-8226 cells were supplemented with 13C-glutamine for 48 h in both normoxia and hypoxia (< 1% O2, by chamber) to perform a tracer study. PYCR1 was inhibited by siRNA or the small molecule inhibitor pargyline. Apoptosis was measured using Annexin V and 7-AAD staining, viability by CellTiterGlo assay and proliferation by BrdU incorporation. Differential protein expression was evaluated using Western Blot. The SUnSET method was used to measure protein synthesis. All in vitro experiments were performed in hypoxic conditions.
RESULTS: We found that PYCR1 and PYCR2 mRNA expression correlated with an inferior overall survival. MM cells from relapsed/refractory patients express significantly higher levels of PYCR1 mRNA. In line with the strong expression of PYCR1, we performed a tracer study in RPMI-8226 cells, which revealed an increased conversion of 13C-glutamine to proline in hypoxia. PYCR1 inhibition reduced MM viability and proliferation and increased apoptosis. Mechanistically, we found that PYCR1 silencing reduced protein levels of p-PRAS40, p-mTOR, p-p70, p-S6, p-4EBP1 and p-eIF4E levels, suggesting a decrease in protein synthesis, which we also confirmed in vitro. Pargyline and siPYCR1 increased bortezomib-mediated apoptosis. Finally, combination therapy of pargyline with bortezomib reduced viability in CD138+ MM cells and reduced tumor burden in the murine 5TGM1 model compared to single agents.
CONCLUSIONS: This study identifies PYCR1 as a novel target in bortezomib-based combination therapies for MM.

Keywords: Hypoxia; Multiple myeloma; PYCR1; Proline; Protein synthesis

DOI: https://doi.org/10.1186/s13046-022-02250-3

Proc Natl Acad Sci U S A. 2022 Feb 08. pii: e2114912119. [Epub ahead of print]119(6):

Leucine retention in lysosomes is regulated by starvation.

Urmi Bandyopadhyay, Pavlina Todorova, Natalya N Pavlova, Yuma Tada, Craig B Thompson, Lydia W S Finley, Michael Overholtzer.

Cells acquire essential nutrients from the environment and utilize adaptive mechanisms to survive when nutrients are scarce. How nutrients are trafficked and compartmentalized within cells and whether they are stored in response to stress remain poorly understood. Here, we investigate amino acid trafficking and uncover evidence for the lysosomal transit of numerous essential amino acids. We find that starvation induces the lysosomal retention of leucine in a manner requiring RAG-GTPases and the lysosomal protein complex Ragulator, but that this process occurs independently of mechanistic target of rapamycin complex 1 activity. We further find that stored leucine is utilized in protein synthesis and that inhibition of protein synthesis releases lysosomal stores. These findings identify a regulated starvation response that involves the lysosomal storage of leucine.

Keywords: leucine; lysosome; mTOR

DOI: https://doi.org/10.1073/pnas.2114912119

Cancer Discov. 2022 Jan 31.

NRF2: KEAPing Tumors Protected.

Ray Pillai, Makiko Hayashi, Anastasia-Maria Zavitsanou, Thales Papagiannakopoulos.

The Kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway plays a physiologic protective role against xenobiotics and reactive oxygen species. However, activation of NRF2 provides a powerful selective advantage for tumors by rewiring metabolism to enhance proliferation, suppress various forms of stress, and promote immune evasion. Genetic, epigenetic, and posttranslational alterations that activate the KEAP1/NRF2 pathway are found in multiple solid tumors. Emerging clinical data highlight that alterations in this pathway result in resistance to multiple therapies. Here, we provide an overview of how dysregulation of the KEAP1/NRF2 pathway in cancer contributes to several hallmarks of cancer that promote tumorigenesis and lead to treatment resistance. SIGNIFICANCE: Alterations in the KEAP1/NRF2 pathway are found in multiple cancer types. Activation of NRF2 leads to metabolic rewiring of tumors that promote tumor initiation and progression. Here we present the known alterations that lead to NRF2 activation in cancer, the mechanisms in which NRF2 activation promotes tumors, and the therapeutic implications of NRF2 activation.

DOI: https://doi.org/10.1158/2159-8290.CD-21-0922

Transl Cancer Res. 2020 Aug;9(8): 4906-4913

Glutaminase 2 functions as a tumor suppressor gene in gastric cancer.

Liang Xu, Dong Zhou, Fang Li, Lijiang Ji.

Background: Glutaminase 2 (GLS2) has been described as a tumor suppressor in hepatocellular carcinoma (HCC) and colon cancer. This study aimed to investigate the expression of GLS2 and its biological role in gastric cancer.
Methods: The expression of GLS2 was determined by quantitative Real-time PCR (qRT-PCR). Proliferation assay was performed by Cell Counting Kit-8 assay. Cell apoptosis assay was performed by Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit. Migration capability analysis was performed by Transwell chamber assay. The protein GLS2 and caspase 3 was determined by western blotting.
Results: Here, we demonstrated that GLS2 displayed a significant downregulation in gastric cancer tissues compared to adjacent non-cancer tissues, which suggested that the downregulation of GLS2 might possibly be associated with the development and progression of gastric cancer. We also found that GLS2 overexpression could significantly suppress gastric cancer cell proliferation and migration and enhance gastric cancer cell apoptosis via upregulating the expression of caspase 3.
Conclusions: These data taken together show that GLS2 functions as a tumor suppressor gene in gastric cancer. This study not only enriches the molecular mechanism of gastric cancer but also supplies a scientific basis for targeted treatment of gastric cancer.

Keywords: Glutaminase 2 (GLS2); gastric cancer; metastasis; proliferation

DOI: https://doi.org/10.21037/tcr-20-2246