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      Pancreatic cancers rely on a novel glutamine metabolism pathway to maintain redox balance

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          Abstract

          The metabolic requirements of a proliferating cell differ from those of a cell in homeostasis. 1 In order for a cell to duplicate, it must double its genome, protein content and lipid mass. This process requires energy in the form of ATP and NADPH. However, unlike ATP, the amount of NADPH required for biosynthesis is much greater than that needed for homeostasis, which makes the generation of NADPH rate limiting for cellular proliferation. NADPH is used for both macromolecular biosynthesis (e.g., lipids and deoxynucleotide triphosphates) and the maintenance of a reduced intracellular environment. 2 Given this dual role, when the demand for NADPH is high (e.g., during proliferation), moderate impacts on NADPH production challenge the maintenance of redox control. As such, the generation of reducing power in the form of NADPH is tightly regulated during proliferation to ensure that a sufficient amount is available to run biosynthetic reactions and protect against oxidative stress. Recently, we demonstrated that mutant Kras is required for maintenance of established pancreatic tumors, in part through the regulation of anabolic glucose metabolism. 3 Mutant Kras drives glucose uptake and its diversion into the non-oxidative arm of the pentose phosphate pathway (PPP) to generate ribose 5-phosphate, which is used in nucleic acid biosynthesis. This was a particularly surprising finding, as such metabolic rewiring bypasses the NADPH-generating oxidative arm of the PPP and suggests that an alternate mechanism for NADPH production must dominate in Kras-transformed pancreatic tumors. In a recent study, 4 we used an integrative genetic approach combined with metabolomic tracing experiments to examine this question. By assessing the role of the two primary anabolic carbon sources (i.e., glucose and glutamine; Gln) on the cellular redox state (a surrogate for NADP+/NADPH) in pancreatic cancers, we found that while both glucose and Gln were required for cell proliferation, only Gln deprivation dramatically increased redox stress. Metabolic rescues of pancreatic cancer cells grown in the absence of Gln revealed that the Gln carbon skeleton (α-ketoglutarate, αKG) was unable to rescue growth unless it was combined with a cocktail of non-essential amino acids (NEAA). These results illustrated an important finding, namely, that pancreatic cancer cells metabolize Gln in a manner that is distinct from the classical αKG-generating mitochondrial pathway that utilizes glutamate dehydrogenase (GLUD1; Fig. 1A). 5 , 6 Figure 1. Gln metabolism is rewired in pancreatic cancer to facilitate NADPH production. (A) Canonical anabolic Gln metabolism. Gln-derived Glu is processed into αKG through mitochondrial GLUD1, which is used for anaplerotic filling of the TCA cycle (green circle). The TCA cycle is coupled to the malate-aspartate shuttle (blue circle), which is used to bring reducing equivalents derived from glycolysis into the mitochondria for oxidative phosphorylation. (B) In pancreatic cancer, Gln metabolism is reprogrammed through the mutant Kras-mediated activation of GOT1 expression and repression of GLUD1. Repression of GLUD1 promotes the mitochondrial aspartate aminotransferase (GOT2)-mediated generation of Asp in the mitochondria. This Asp is released into the cytosol and converted through a series of reactions into pyruvate and reducing potential in the form of NADPH. This series of reactions decouples TCA cycle activity from the malate-aspartate shuttle. Enzymes that facilitate this pathway are presented in upper-case letters. Metabolites are presented in lower-case letters. Cit, citrate; Fum, fumarate; Pyr, pyruvate; Iso, isocitrate; Suc, succinate. The observation that NEAAs were required downstream of Gln metabolism suggested that transaminases may play a central role in pancreatic cancer. Indeed, we demonstrated that the cytosolic aspartate aminotransferase, GOT1, was required for the maintenance of redox control and for pancreatic cancer cell proliferation. By then tracing Gln metabolism in GOT1 knockdown cells using carbon-13 isotope-labeled Gln and mass spectrometry-based metabolomic profiling, 7 it became apparent that Gln metabolism through GOT1 was required for the maintenance of redox balance. Moreover, the altered metabolite distribution in GOT1 knockdown cells suggested that GOT1 functioned upstream of cytosolic malic enzyme (ME1), which we envisioned was required for the generation of reducing equivalents in the form of NADPH (Fig. 1B). This model was then validated by knocking down ME1 and again following the distribution of glutamine-derived carbon-13 into downstream metabolites. Subsequent analysis of the oxidized-to-reduced NADP ratio following knockdown of classical NADPH-generating cytosolic enzymes revealed that glucose 6-phosphate dehydrogenase (G6PD, the rate limiting enzyme in the oxidative PPP) or isocitrate dehydrogenase (IDH1) knockdown did not affect the NADP ratio. On the other hand, knockdown of ME1 or GOT1 increased the oxidized-to-reduced NADP ratio, providing clear evidence that this pathway is a major source of NADPH in pancreatic cancers for the maintenance of redox balance. A consequence of the redox imbalance that occurs by blocking Gln metabolism in pancreatic cancer is the inhibition of proliferation, where suppression of any component enzyme in this pathway impairs growth in a manner similar to that observed upon Gln withdrawal. In fact, this Gln metabolism-mediated redox maintenance is so central to the role of Gln in pancreatic cancer that the defects in proliferation observed upon Gln withdrawal or GOT1 knockdown can be rescued by solely restoring redox balance through media supplementation with a cell-permeable form of reduced glutathione or the antioxidant N-acetyl cysteine. Collectively, these results demonstrate that a principal function of Gln metabolism in pancreatic cancer is to generate reducing power in the form of NADPH, and that this is used, in part, to maintain redox homeostasis, which enables proliferation. Given the dependence of pancreatic cancer on this Gln metabolism pathway, a major question arises concerning its role in normal cells. Importantly, we demonstrated that GOT1 knockdown did not impair growth across a panel of normal cell lines. Moreover, we found that the signature transforming event in pancreatic cancer, Kras mutation, led to the reprogramming of Gln metabolism. This occurred in part through increasing GOT1 expression and repressing GLUD1 expression. Thus, by changing the ratio of expression of these two enzymes, mutant Kras shunts Gln flux through the aspartate aminotransferase pathway (Fig. 1B). The observation that this Gln metabolism pathway is downstream of mutant Kras provides clear rationale as to why pancreatic cancer exhibits this unique metabolic dependency. Finally, in addition to providing several new metabolic therapeutic targets in pancreatic cancer, the findings from this study also suggest that inhibition of Gln metabolism in pancreatic cancer may synergize with therapies that increase ROS, such as chemotherapy and radiation.

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          Most cited references 7

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          Understanding the Warburg effect: the metabolic requirements of cell proliferation.

          In contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed "the Warburg effect." Aerobic glycolysis is an inefficient way to generate adenosine 5'-triphosphate (ATP), however, and the advantage it confers to cancer cells has been unclear. Here we propose that the metabolism of cancer cells, and indeed all proliferating cells, is adapted to facilitate the uptake and incorporation of nutrients into the biomass (e.g., nucleotides, amino acids, and lipids) needed to produce a new cell. Supporting this idea are recent studies showing that (i) several signaling pathways implicated in cell proliferation also regulate metabolic pathways that incorporate nutrients into biomass; and that (ii) certain cancer-associated mutations enable cancer cells to acquire and metabolize nutrients in a manner conducive to proliferation rather than efficient ATP production. A better understanding of the mechanistic links between cellular metabolism and growth control may ultimately lead to better treatments for human cancer.
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            Aerobic glycolysis: meeting the metabolic requirements of cell proliferation.

            Warburg's observation that cancer cells exhibit a high rate of glycolysis even in the presence of oxygen (aerobic glycolysis) sparked debate over the role of glycolysis in normal and cancer cells. Although it has been established that defects in mitochondrial respiration are not the cause of cancer or aerobic glycolysis, the advantages of enhanced glycolysis in cancer remain controversial. Many cells ranging from microbes to lymphocytes use aerobic glycolysis during rapid proliferation, which suggests it may play a fundamental role in supporting cell growth. Here, we review how glycolysis contributes to the metabolic processes of dividing cells. We provide a detailed accounting of the biosynthetic requirements to construct a new cell and illustrate the importance of glycolysis in providing carbons to generate biomass. We argue that the major function of aerobic glycolysis is to maintain high levels of glycolytic intermediates to support anabolic reactions in cells, thus providing an explanation for why increased glucose metabolism is selected for in proliferating cells throughout nature.
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              Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction.

              Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Most of the remaining metabolites taken up by proliferating cells are not catabolized, but instead are used as building blocks during anabolic macromolecular synthesis. Investigations of phosphoinositol 3-kinase (PI3K) and its downstream effector AKT have confirmed that these oncogenes play a direct role in stimulating glucose uptake and metabolism, rendering the transformed cell addicted to glucose for the maintenance of survival. In contrast, less is known about the regulation of glutamine uptake and metabolism. Here, we report that the transcriptional regulatory properties of the oncogene Myc coordinate the expression of genes necessary for cells to engage in glutamine catabolism that exceeds the cellular requirement for protein and nucleotide biosynthesis. A consequence of this Myc-dependent glutaminolysis is the reprogramming of mitochondrial metabolism to depend on glutamine catabolism to sustain cellular viability and TCA cycle anapleurosis. The ability of Myc-expressing cells to engage in glutaminolysis does not depend on concomitant activation of PI3K or AKT. The stimulation of mitochondrial glutamine metabolism resulted in reduced glucose carbon entering the TCA cycle and a decreased contribution of glucose to the mitochondrial-dependent synthesis of phospholipids. These data suggest that oncogenic levels of Myc induce a transcriptional program that promotes glutaminolysis and triggers cellular addiction to glutamine as a bioenergetic substrate.
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                Author and article information

                Journal
                Cell Cycle
                Cell Cycle
                CC
                Cell Cycle
                Landes Bioscience
                1538-4101
                1551-4005
                01 July 2013
                10 June 2013
                10 June 2013
                : 12
                : 13
                : 1987-1988
                Affiliations
                [1 ]Department of Medicine; Weill Cornell Medical College; New York, NY USA
                [2 ]Division of Genomic Stability and DNA Repair; Department of Radiation Oncology; Dana-Farber Cancer Institute; Boston, MA USA
                Author notes
                [* ]Correspondence to: Lewis C. Cantley, Email: LCantley@ 123456med.cornell.edu and Alec C. Kimmelman, Email: Alec_Kimmelman@ 123456DFCI.HARVARD.EDU
                Article
                2013FT0967 25307
                10.4161/cc.25307
                3737294
                23759579
                Copyright © 2013 Landes Bioscience

                This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

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                Editorials: Cell Cycle Features

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