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The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) senses and relays environmental signals from growth factors and nutrients to metabolic networks and adaptive cellular systems to control the synthesis and breakdown of macromolecules. However, beyond inducing de novo lipid synthesis, the role of mTORC1 in controlling cellular lipid content remains poorly understood. Here we show that inhibition of mTORC1 via small molecule inhibitors or nutrient deprivation leads to the accumulation of intracellular triglycerides in both cultured cells and a mouse tumor model. The elevated triglyceride pool following mTORC1 inhibition stems from the lysosome-dependent, but autophagy independent, hydrolysis of phospholipid fatty acids. The liberated fatty acids are available for either triglyceride synthesis or beta-oxidation. Distinct from the established role of mTORC1 activation in promoting de novo lipid synthesis, our data indicate that mTORC1 inhibition triggers membrane phospholipid trafficking to the lysosome for catabolism and an adaptive shift in the use of constituent fatty acids for storage or energy production.

5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug’s efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug’s DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.

Production of oxidized biomass, which requires regeneration of the cofactor NAD+, can be a proliferation bottleneck that is influenced by environmental conditions. However, a comprehensive quantitative understanding of metabolic processes that may be affected by NAD+ deficiency is currently missing. Here, we use computational and experimental approaches to demonstrate that in addition to requiring oxygen for lipid desaturation reactions, de novo lipid biosynthesis can impose a substantial NAD+ consumption cost in proliferating cancer cells. Our results reveal that when electron acceptors are limited environmental lipids become crucial for proliferation because NAD+ is required to generate precursors for fatty acid biosynthesis. We find that both oxidative and even net reductive pathways for lipogenic citrate synthesis are gated by reactions that depend on NAD+ availability. We also show that access to acetate can relieve lipid auxotrophy by bypassing the NAD+ consuming reactions involved in de novo lipid synthesis. Consistent with biochemical results, analysis of gene expression across diverse tumor types demonstrates that lipid biosynthesis strongly anticorrelates with expression of hypoxia markers, whereas lipid uptake genes are consistently upregulated in hypoxia. Overall, our results define a requirement for oxidative metabolism to support de novo lipid biosynthesis and provide a mechanistic explanation for cancer cell dependence on lipid uptake in electron acceptors limited conditions such as hypoxia.

The coordination of metabolic processes to allow increased nutrient uptake and utilization for macromolecular synthesis is central for cell growth. Although studies of bulk cell populations have revealed important metabolic and signaling requirements that impact cell growth on long time scales, whether the same regulation influences short-term cell growth remains an open question. Here we investigate cell growth by monitoring mass accumulation of mammalian cells while rapidly depleting particular nutrients. Within minutes following the depletion of glucose or glutamine, we observe a growth reduction that is larger than the mass accumulation rate of the nutrient. This indicates that if one particular nutrient is depleted, the cell rapidly adjusts the amount that other nutrients are accumulated, which is consistent with cooperative nutrient accumulation. Population measurements of nutrient sensing pathways involving mTOR, AKT, ERK, PKA, MST1, or AMPK, or pro-survival pathways involving autophagy suggest that they do not mediate this growth reduction. Furthermore, the protein synthesis rate does not change proportionally to the mass accumulation rate over these time scales, suggesting that intracellular metabolic pools buffer the growth response. Our findings demonstrate that cell growth can be regulated over much shorter time scales than previously appreciated.

Mammalian pyruvate kinase catalyzes the final step of glycolysis, and its M2 isoform (PKM2) is widely expressed in proliferative tissues. Mutations in PKM2 are found in some human cancers; however, the effects of these mutations on enzyme activity and regulation are unknown. Here, we characterized five cancer-associated PKM2 mutations, occurring at various locations on the enzyme, with respect to substrate kinetics and activation by the allosteric activator fructose-1,6-bisphosphate (FBP). The mutants exhibit reduced maximal velocity, reduced substrate affinity, and/or altered activation by FBP. The kinetic parameters of five additional PKM2 mutants that have been used to study enzyme function or regulation also demonstrate the deleterious effects of mutations on PKM2 function. Our findings indicate that PKM2 is sensitive to many amino acid changes and support the hypothesis that decreased PKM2 activity is selected for in rapidly proliferating cells.

The Warburg effect, the propensity of some cells to metabolize glucose to lactate in the presence of oxygen (also known as aerobic glycolysis), has long been observed in cancer and other contexts of cell proliferation, but only in the past two decades have significant gains been made in understanding how and why this metabolic transformation occurs. In 2004, Cancer Research published a study by Elstrom and colleagues that provided one of the first connections between a specific oncogene and aerobic glycolysis. Studying hematopoietic and glioblastoma cell lines, they demonstrated that constitutive activation of AKT promotes an increased glycolytic rate without altering proliferation or oxygen consumption in culture. They proposed that it is this effect that allows constitutive AKT activation to transform cells and found that it sensitizes cells to glucose deprivation. In the years since, mechanistic understanding of oncogenic control of metabolism, and glycolysis specifically, has deepened substantially. Current work seeks to understand the benefits and liabilities associated with glycolytic metabolism and to identify inhibitors that might be of clinical benefit to target glycolytic cancer cells. See related article by Elstrom and colleagues, Cancer Res 2004;64:3892–9

Many metabolic phenotypes in cancer cells are also characteristic of proliferating non-transformed mammalian cells, and attempts to distinguish between phenotypes resulting from oncogenic perturbation from those associated with increased proliferation are limited. Here, we examined the extent to which metabolic changes corresponding to oncogenic KRAS expression differed from those corresponding to epidermal growth factor (EGF)-driven proliferation in human mammary epithelial cells (HMECs). Removal of EGF from culture medium reduced growth rates and glucose/glutamine consumption in control HMECs despite limited changes in respiration and fatty acid synthesis, while the relative contribution of branched-chain amino acids to the TCA cycle and lipogenesis increased in the near-quiescent conditions. Most metabolic phenotypes measured in HMECs expressing mutant KRAS were similar to those observed in EGF-stimulated control HMECs that were growing at comparable rates. However, glucose and glutamine consumption as well as lactate and glutamate production were lower in KRAS-expressing cells cultured in media without added EGF, and these changes correlated with reduced sensitivity to GLUT1 inhibitor and phenformin treatment. Our results demonstrate the strong dependence of metabolic behavior on growth rate, and provide a model to distinguish the metabolic influences of oncogenic mutations and non-oncogenic growth.

The mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, its broader roles as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon insulin-stimulated mTORC1 signaling to those activated by the ISR. In multiple mouse embryo fibroblast and human cancer cell lines, the mTORC1-ATF4 pathway stimulated expression of only a subset of the ATF4 target genes induced by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis., eLife, 10, ISSN:2050-084X

Production of oxidized biomass, which requires regeneration of the cofactor NAD

The mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the adaptive stress-responsive transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, how ATF4 functions in response to these distinct modes of regulation and its broader role as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon activation of mTORC1 or the ISR. The mTORC1-ATF4 pathway stimulated the expression of only a subset of the ATF4 target genes stimulated by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis.

It is not well understood how physiological environmental conditions and nutrient availability influence cancer cell proliferation. Production of oxidized biomass, which requires regeneration of the cofactor NAD+, can limit cancer cell proliferation1-5. However, it is currently unclear which specific metabolic processes are constrained by electron acceptor availability, and how they affect cell proliferation. Here, we use computational and experimental approaches to demonstrate that de novo lipid biosynthesis can impose an increased demand for NAD+ in proliferating cancer cells. While some cancer cells and tumors synthesize a substantial fraction of their lipids de novo6, we find that environmental lipids are crucial for proliferation in hypoxia or when the mitochondrial electron transport chain is inhibited. Surprisingly, we also find that even the reductive glutamine carboxylation pathway to produce fatty acids is impaired when cancer cells are limited for NAD+. Furthermore, gene expression analysis of 34 heterogeneous tumor types shows that lipid biosynthesis is strongly and consistently negatively correlated with hypoxia, whereas expression of genes involved in lipid uptake is positively correlated with hypoxia. These results demonstrate that electron acceptor availability and access to environmental lipids can play an important role in determining whether cancer cells engage in de novo lipogenesis to support proliferation.

Cell growth and division require nutrients, and proliferating cells use a variety of sources to acquire the amino acids, lipids, and nucleotides that support macromolecule synthesis. Lipids are more reduced than other nutrients, whereas nucleotides and amino acids are typically more oxidized. Cells must therefore generate reducing and oxidizing (redox) equivalents to convert consumed nutrients into biosynthetic precursors. To that end, redox cofactor metabolism plays a central role in meeting cellular redox requirements. In this Minireview, we highlight the biosynthetic pathways that involve redox reactions and discuss their integration with metabolism in proliferating mammalian cells.

Tumor genetics guides patient selection for many new therapies, and cell culture studies have demonstrated that specific mutations can promote metabolic phenotypes. However, whether tissue context defines cancer dependence on specific metabolic pathways is unknown. Kras activation and Trp53 deletion in the pancreas or the lung result in pancreatic ductal adenocarinoma (PDAC) or non-small cell lung carcinoma (NSCLC), respectively, but despite the same initiating events, these tumors use branched-chain amino acids (BCAAs) differently. NSCLC tumors incorporate free BCAAs into tissue protein and use BCAAs as a nitrogen source, whereas PDAC tumors have decreased BCAA uptake. These differences are reflected in expression levels of BCAA catabolic enzymes in both mice and humans. Loss of Bcat1 and Bcat2, the enzymes responsible for BCAA use, impairs NSCLC tumor formation, but these enzymes are not required for PDAC tumor formation, arguing that tissue of origin is an important determinant of how cancers satisfy their metabolic requirements., National Institutes of Health (U.S.) (Grant F30CA183474), National Institutes of Health (U.S.) (Grant T32GM007753)

The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap−/− mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap−/− mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis., National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant P30DK034854), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant 1R01DK090311), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant R24OD017870), National Institutes of Health (U.S.) (Grant P30DK034854), National Institutes of Health (U.S.) (Grant 1R01DK090311), National Institutes of Health (U.S.) (Grant 1R01DK105198), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant 1R01DK105198), National Institutes of Health (U.S.) (Grant R24OD017870), National Institute of General Medical Sciences (NIGMS) (Grant T32GM007753), NHMRC (Grant 1146558), National Cancer Institute (U.S.) (Grant 5P01CA120964), National Cancer Institute (U.S.) (Grant 5P30CA006516)

Monoallelic point mutations in the gene encoding the cytosolic, NADP(+)-dependent enzyme isocitrate dehydrogenase 1 (IDH1) cause increased production of the oncometabolite 2-hydroxyglutarate (2-HG) in multiple cancers. Most IDH1 mutant tumors retain one wildtype (WT) IDH1 allele. Several studies have proposed that retention of this WT allele is protumorigenic by facilitating substrate channeling through a WT–mutant IDH1 heterodimer, with the WT subunit generating a local supply of α-ketoglutarate and NADPH that is then consumed by the mutant subunit to produce 2-HG. Here, we confirmed that coexpression of WT and mutant IDH1 subunits leads to formation of WT–mutant hetero-oligomers and increases 2-HG production. An analysis of a recently reported crystal structure of the WT–R132H IDH1 heterodimer and of in vitro kinetic parameters for 2-HG production, however, indicated that substrate channeling between the subunits is biophysically implausible. We also found that putative carbon-substrate flux between WT and mutant IDH1 subunits is inconsistent with the results of isotope tracing experiments in cancer cells harboring an endogenous monoallelic IDH1 mutation. Finally, using a mathematical model of WT–mutant IDH1 heterodimers, we estimated that the NADPH:NADP(+) ratio is higher in the cytosol than in the mitochondria, suggesting that NADPH is unlikely to be limiting for 2-HG production in the cytosol. These findings argue against supply of either substrate being limiting for 2-HG production by a cytosolic IDH1 mutant and suggest that the retention of a WT allele in IDH1 mutant tumors is not due to a requirement for carbon or cofactor flux between WT and mutant IDH1.

The role of pyruvate kinase M2 (PKM2) in cell proliferation is controversial. A unique function of PKM2 proposed to be important for the proliferation of some cancer cells involves the direct activity of this enzyme as a protein kinase; however, a detailed biochemical characterization of this activity is lacking. Using [32P]-phosphoenolpyruvate (PEP) we examine the direct substrates of PKM2 using recombinant enzyme and in vitro systems where PKM2 is genetically deleted. Labeling of some protein species from [32P]-PEP can be observed; however, most were dependent on the presence of ADP, and none were dependent on the presence of PKM2. In addition, we also failed to observe PKM2-dependent transfer of phosphate from ATP directly to protein. These findings argue against a role for PKM2 as a protein kinase., Howard Hughes Medical Institute (International Student Research Fellowship), Vertex Pharmaceuticals Incorporated. Vertex Scholars Program, Massachusetts Institute of Technology. Department of Biology (Grant T32GM007287), David H. Koch Institute for Integrative Cancer Research at MIT (Graduate Student Fellowship), National Cancer Institute (U.S.) (Grants R01CA168653 and P30CA14051), Burroughs Wellcome Fund

SummaryMitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis.

Defining the metabolic limitations of tumour growth will help to develop cancer therapies1. Cancer cells proliferate slower in tumours than in standard culture conditions, indicating that a metabolic limitation may restrict cell proliferation in vivo. Aspartate synthesis can limit cancer cell proliferation when respiration is impaired2–4; however, whether acquiring aspartate is endogenously limiting for tumour growth is unknown. We confirm that aspartate has poor cell permeability, which prevents environmental acquisition, whereas the related amino acid asparagine is available to cells in tumours, but cancer cells lack asparaginase activity to convert asparagine to aspartate. Heterologous expression of guinea pig asparaginase 1 (gpASNase1), an enzyme that produces aspartate from asparagine5, confers the ability to use asparagine to supply intracellular aspartate to cancer cells in vivo. Tumours expressing gpASNase1 grow at a faster rate, indicating that aspartate acquisition is an endogenous metabolic limitation for the growth of some tumours. Tumours expressing gpASNase1 are also refractory to the growth suppressive effects of metformin, suggesting that metformin inhibits tumour growth by depleting aspartate. These findings suggest that therapeutic aspartate suppression could be effective to treat cancer.

Studying lipid metabolism in cultured cells is complicated by the fact that cells are typically cultured in the presence of animal serum, which contains a wide, variable, and undefined variety of lipid species. Lipid metabolism can impact cell physiology, signaling, and proliferation, and the ability to culture cells in the absence of exogenous lipids can reveal the importance of lipid biosynthesis pathways and facilitate the generation of media with defined lipid species. We have adapted a protocol to remove lipids from serum without eliminating its ability to support the proliferation of cells in culture. This method requires di-isopropyl ether and butanol and can be used to generate small batches of lipid-stripped serum in four days. The resulting serum supports proliferation of many cell lines in culture and can be used to compare the metabolism of cells in lipid replete and depleted conditions.

The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells., National Institutes of Health (U.S.) (Grant R01CA168653), National Institutes of Health (U.S.) (Grant 5P01CA117969), National Institutes of Health (U.S.) (Grant P30CA147882), National Institutes of Health (U.S.) (Grant 5P30CA14051), National Institutes of Health (U.S.) (Grant 5K08CA136983), National Institutes of Health (U.S.) (Grant DK059635), National Institutes of Health (U.S.) (Grant R01DK092606), National Institutes of Health (U.S.) (Grant R00CA131472), National Institutes of Health (U.S.) (Grant R01GM056203), American Diabetes Association (Grant 7-12-BS-09), Smith Family Foundation, Burroughs Wellcome Fund, Damon Runyon Cancer Research Foundation, Stern Family

Changes to the biophysical properties of lymphocytes are identified as an adaptive response to acute nutrient stress that occurs before the induction of autophagy., Cytokine regulation of lymphocyte growth and proliferation is essential for matching nutrient consumption with cell state. Here, we examine how cellular biophysical changes that occur immediately after growth factor depletion promote adaptation to reduced nutrient uptake. After growth factor withdrawal, nutrient uptake decreases, leading to apoptosis. Bcl-xL expression prevents cell death, with autophagy facilitating long-term cell survival. However, autophagy induction is slow relative to the reduction of nutrient uptake, suggesting that cells must engage additional adaptive mechanisms to respond initially to growth factor depletion. We describe an acute biophysical response to growth factor withdrawal, characterized by a simultaneous decrease in cell volume and increase in cell density, which occurs before autophagy initiation and is observed in both FL5.12 Bcl-xL cells depleted of IL-3 and primary CD8+ T cells depleted of IL-2 that are differentiating toward memory cells. The response reduces cell surface area to minimize energy expenditure while conserving biomass, suggesting that the biophysical properties of cells can be regulated to promote survival under conditions of nutrient stress.

Cells must duplicate their mass in order to proliferate. Glucose and glutamine are the major nutrients consumed by proliferating mammalian cells, but the extent to which these and other nutrients contribute to cell mass is unknown. We quantified the fraction of cell mass derived from different nutrients and found that the majority of carbon mass in cells is derived from other amino acids, which are consumed at much lower rates than glucose and glutamine. While glucose carbon has diverse fates, glutamine contributes most to protein, suggesting that glutamine's ability to replenish tricarboxylic acid cycle intermediates (anaplerosis) is primarily used for amino acid biosynthesis. These findings demonstrate that rates of nutrient consumption are indirectly associated with mass accumulation and suggest that high rates of glucose and glutamine consumption support rapid cell proliferation beyond providing carbon for biosynthesis., National Institutes of Health (U.S.) (Grant U54CA143874)

The coordination of metabolic processes to allow increased nutrient uptake and utilization for macromolecular synthesis is central for cell growth. Although studies of bulk cell populations have revealed important metabolic and signaling requirements that impact cell growth on long time scales, whether the same regulation influences short-term cell growth remains an open question. Here we investigate cell growth by monitoring mass accumulation of mammalian cells while rapidly depleting particular nutrients. Within minutes following the depletion of glucose or glutamine, we observe a growth reduction that is larger than the mass accumulation rate of the nutrient. This indicates that if one particular nutrient is depleted, the cell rapidly adjusts the amount that other nutrients are accumulated, which is consistent with cooperative nutrient accumulation. Population measurements of nutrient sensing pathways involving mTOR, AKT, ERK, PKA, MST1, or AMPK, or pro-survival pathways involving autophagy suggest that they do not mediate this growth reduction. Furthermore, the protein synthesis rate does not change proportionally to the mass accumulation rate over these time scales, suggesting that intracellular metabolic pools buffer the growth response. Our findings demonstrate that cell growth can be regulated over much shorter time scales than previously appreciated., National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051), National Cancer Institute (U.S.). Physical Sciences Oncology Center (U54CA143874), National Institutes of Health (U.S.) (Contract R01GM085457), National Cancer Institute (U.S.) (Fellowship F31CA167872), National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Program 5T32GM008334)

Metformin use is associated with reduced cancer mortality, but how metformin impacts cancer outcomes is controversial. Although metformin can act on cells autonomously to inhibit tumor growth, the doses of metformin that inhibit proliferation in tissue culture are much higher than what has been described in vivo. Here, we show that the environment drastically alters sensitivity to metformin and other complex I inhibitors. We find that complex I supports proliferation by regenerating nicotinamide adenine dinucleotide (NAD)+, and metformin's anti-proliferative effect is due to loss of NAD+/NADH homeostasis and inhibition of aspartate biosynthesis. However, complex I is only one of many inputs that determines the cellular NAD+/NADH ratio, and dependency on complex I is dictated by the activity of other pathways that affect NAD+ regeneration and aspartate levels. This suggests that cancer drug sensitivity and resistance are not intrinsic properties of cancer cells, and demonstrates that the environment can dictate sensitivity to therapies that impact cell metabolism. Keywords: cancer metabolism; metformin; biguanide; NAD+/NADH ratio; drug sensitivity; complex I; mitochondria; aspartate, National Institutes of Health (U.S.) (Grant P30CA1405141), National Institutes of Health (U.S.) (Grant GG006413), National Institutes of Health (U.S.) (Grant R01 CA168653), National Institutes of Health (U.S.) (Grant R01 CA201276)

Acetyl-CoA has diverse fates in metabolism and can be derived from a variety of nutrients. In a recent study published in Nature, Wong et al. (2016) show that endothelial cells oxidize fatty acids to produce acetyl-CoA for epigenetic modifications critical to lymphangiogenesis.

Here, the authors identify a previously unknown regulatory strategy used by Escherichia coli to control end-product levels of the pyrimidine biosynthetic pathway: this involves feedback regulation of the near-terminal pathway enzyme UMP kinase, with accumulation of UMP prevented by its degradation to uridine through UmpH, a phosphatase with a previously unknown function. The control of the concentrations and fluxes of the thousand or so metabolites in a living cell such as Escherichia coli occurs via regulation of enzyme concentrations, activities and substrate occupancies. De novo pyrimidine biosynthesis has been reported to be regulated at the first committed pathway step (catalysed by aspartate transcarbamoylase) and at the previous (carbamoyl phosphate synthetase) step. Here the authors identify a novel regulatory strategy — an overflow pathway from UMP to uracil — that E. coli cells use to avoid the accumulation of an excess of the end products of pyrimidine biosynthesis. The process is analogous to that seen in central carbon metabolism, where excessive sugar catabolism leads to buildup of pyruvate that can be excreted as lactate, ethanol or acetate. Cellular metabolism converts available nutrients into usable energy and biomass precursors. The process is regulated to facilitate efficient nutrient use and metabolic homeostasis. Feedback inhibition of the first committed step of a pathway by its final product is a classical means of controlling biosynthesis1,2,3,4. In a canonical example, the first committed enzyme in the pyrimidine pathway in Escherichia coli is allosterically inhibited by cytidine triphosphate1,4,5. The physiological consequences of disrupting this regulation, however, have not been previously explored. Here we identify an alternative regulatory strategy that enables precise control of pyrimidine pathway end-product levels, even in the presence of dysregulated biosynthetic flux. The mechanism involves cooperative feedback regulation of the near-terminal pathway enzyme uridine monophosphate kinase6. Such feedback leads to build-up of the pathway intermediate uridine monophosphate, which is in turn degraded by a conserved phosphatase, here termed UmpH, with previously unknown physiological function7,8. Such directed overflow metabolism allows homeostasis of uridine triphosphate and cytidine triphosphate levels at the expense of uracil excretion and slower growth during energy limitation. Disruption of the directed overflow regulatory mechanism impairs growth in pyrimidine-rich environments. Thus, pyrimidine homeostasis involves dual regulatory strategies, with classical feedback inhibition enhancing metabolic efficiency and directed overflow metabolism ensuring end-product homeostasis.

Monoallelic point mutations in the cytosolic, NADP+-dependent enzyme isocitrate dehydrogenase 1 (IDH1) are associated with elevated production of the oncometabolite 2-hydroxyglutarate (2-HG) in multiple cancers. Production of 2-HG by mutant R132H IDH1 requires activity of a retained wild-type (WT) allele, suggesting possible channeling of α-ketoglutarate and NADPH in WT/R132H IDH1 heterodimers [1]. We report a mechanistic characterization of IDH1 2-HG production using a combination of mathematical modeling, in vivo kinetic measurements, and diffusion calculations. Numerical simulation of a biochemically realistic mathematical model of the IDH1 heterodimer indicated that substrate channeling would have a large impact on the dynamics of 2-HG generation. We then examined conversion of 13C labeled glucose and 13C labeled glutamine into labeled 2-HG in HT1080 cells and found that the dynamics are nearly identical in both cases. This result provides strong evidence against channeling because the production of 2-HG from glucose, but not glutamine, is dependent on WT IDH1. Calculations of substrate diffusion between the WT and mutant active sites of both isolated and agglomerated IDH1 heterodimers further support the absence of channeling. We propose instead that 2-HG production is sensitive to the global availability of substrates in the appropriate intracellular compartment, consistent with prior observations that 2-HG production by structurally analogous mutants of the mitochondrial isoform IDH2 is not WT-dependent [1]. Building on this observation, we report a strategy for inferring compartment-specific NADPH/NADP+ ratios using our model and steady-state measurements of 2-HG production by native IDH1 and by IDH1 artificially targeted to the mitochondrion.[1] Ward, P.S., C. Lu, J.R. Cross, O. Abdel-Wahab, R.L. Levine, G.K. Schwartz, and C.B. Thompson. 2013. J. Biol. Chem. 288: 3804-3815

It has been established that de novo serine biosynthesis is important for some cancers. We have previously shown that phosphoglycerate dehydrogenase (PHGDH), the enzyme responsible for catalyzing the first step in glycolytic serine biosynthesis, is amplified in certain cancers, including breast cancer and melanoma. Knockdown of PHGDH results in decreased proliferation of PHGDH-amplified cells, demonstrating a dependence on PHDGH in these cells; however, it has not been possible to rescue this defect with exogenous serine, suggesting that glycolytic serine biosynthesis has an alternate function in cells. Here we have investigated four mutually exclusive hypotheses each consistent with the inability of serine to rescue decreased glycolytic serine biosynthesis: 1: There exists an alternative function of PHGDH; 2: Pathway activity is important for TCA cycle anapleurosis; 3: Metabolic channeling of de novo serine; and 4: A unique product of this pathway other than serine is critical for proliferation. Using a combination of genetic and biochemical approaches, we show that flux through the serine biosynthesis pathway is required for cytosolic serine-glycine conversion via serine hydroxymethyltransferase 1 (SHMT1). In many cells flux through SHMT1 occurs in the glycine-to-serine direction, including PHDGH-dependent cell lines, and loss of PHGDH activity results in wasting of folates from the one-carbon pool to make serine. This reduces availability of one-carbon units in the folate pool for nucleotide biosynthesis, resulting in S-phase arrest and eventual cell death. These data link serine biosynthesis from glucose with the regulation of one-carbon folate metabolism, and provide an explanation for why cancer cells are dependent on de novo serine production even when serine is abundant. Citation Format: Caroline A. Lewis, Brian P. Fiske, Michael E. Pacold, Aaron M. Hosios, Katherine R. Mattaini, David M. Sabatini, Matthew G. Vander Heiden. Serine biosynthesis from glucose regulates folate availability. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr PR10.

To facilitate growth and division, proliferating cells must duplicate their mass over the cell cycle. Glucose and glutamine are the most consumed nutrients by proliferating mammalian cells, but the extent to which these and other nutrients contribute directly to cell mass has not been investigated. Using isotope labeled nutrients, we quantified the fraction of cell mass derived from different fuels and find that surprisingly neither glucose nor glutamine provide the majority of carbon present in cells. Instead, amino acids, which are consumed at substantially lower rates, together account for the majority of cell mass. While glucose carbon has diverse fates, glutamine is overwhelmingly diverted into protein and its incorporation into cell mass greatly exceeds that of other amino acids, suggesting that glutamine-driven anapleurosis is primarily used for amino acid biosynthesis. Our findings demonstrate that the most heavily consumed nutrients do not make a proportionately large contribution of carbon to macromolecule biosynthesis. Citation Format: Aaron M. Hosios, Vivian C. Hecht, Marc Johnson, Jeffrey C. Rathmell, Scott R. Manalis, Matthew G. Vander Heiden. Amino acids rather than glucose accounts for the majority of cell mass in rapidly proliferating mammalian cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A36.

Macromolecule biosynthesis is required to duplicate cell components and support proliferation. Studies examining the nutrients used by cancer cells have focused on the contribution of glucose and glutamine carbon for biosynthesis, but the importance of other metabolic fuels is becoming apparent. Labeling of two-carbon units in newly synthesized lipids has been used to infer the nutrients that contribute to the acetyl-CoA pools in cells. Glucose- and glutamine-derived carbon are known to contribute extensively to de novo lipid biosynthesis, and in this issue Kamphorst et al. find that extracellular acetate can also contribute substantially to this process [1]. In normoxia, glucose and glutamine together account for the majority of lipogenic acetyl-CoA, but this fraction falls substantially in hypoxia [2,3]. In hypoxia, flux from glucose to citrate as a source of acetyl-coA decreases, and cells utilize alternative carbon sources to generate this metabolite. Glutamine can provide some of this carbon through reductive carboxylation of glutamine-derived α-ketoglutarate to citrate [2,4], but a substantial amount of acetyl-CoA is labeled by neither glucose nor glutamine tracing. Breakdown of lipids scavenged from the environment is one alternate source of acetyl-CoA, but mammalian serum also contains acetate, and the authors found that exogenous acetate could be incorporated into acetyl-CoA and become available for lipid biosynthesis. Surprisingly, for some cell lines in hypoxia, acetate may be a major contributor to acetyl-CoA. Acetate is transported into cells by members of the monocarboxylate transporter family [5] where acetyl-CoA synthetases (ACSS) catalyze conversion to acetyl-CoA. Mammalian cells express mitochondrial and cytosolic forms of ACSS, and the authors cite emerging data from other groups that this enzyme can be important for growth of some tumors. Kamphorst et al. observe that acetate labels acetyl-CoA in hypoxic cells only, and it is interesting to consider whether this is due to increased acetate uptake or increased ACSS activity in low oxygen. Alternatively, it is possible that reduced synthesis of acetyl-CoA from glucose permits other sources of two-carbon units such as acetate to equilibrate with acetyl-CoA and have a higher fractional contribution to pools in cells. For the labeling experiments acetate was provided to cells exogenously, but Kamphorst et al.’s findings raise the question of what sources of acetate exist in vivo. Acetate is present in human and murine blood at concentrations ranging from 50–200 μM [6-9] but has been estimated to be as high as 500–600 μM by some [10,11]. There can be substantial variability of acetate concentrations across species with higher levels observed in cows relative to human, perhaps explaining why Kamphorst et al. measure high levels of acetate in bovine serum [12]. Acetate in plasma is supplied by both exogenous and endogenous sources. Exogenous acetate delivered to tumors via the blood may derive from the diet, with a substantial portion generated by gut microbiome metabolism of intestinal contents [13]. Fasting induces the liver to generate ketones for metabolism by other tissues. Starvation itself reduces circulating acetate [14], presumably because dietary sources of acetate are lacking; however when ketones are consumed by cells they generate acetyl-CoA. Finally, exogenous acetate can be generated from the oxidation of ethanol. In some individuals, this can be a major source of acetate, and chronic drinkers may have circulating acetate concentrations as high as the millimolar range [10,15]. In the hours following ethanol consumption, blood acetate has also been observed to reach similar levels [16], but it has also been suggested that ethanol-derived acetate is primarily trapped in the liver by high ACSS activity [13]. Acetate may also be generated in a cell’s microenvironment. Protein deacetylases and acetyl-CoA hydrolase both generate acetate via hydrolysis reactions [14], suggesting that release of acetyl groups from endogenous sources may contribute to the acetate pool, and raising the possibility that some cells could produce acetate used by neighboring cells. Kamphorst et al. find that acetate’s contribution to lipid biosynthesis is greatest when cells are hypoxic, suggesting that substantial acetate utilization occurs when exogenous sources delivered by the blood are not available. This argues that acetate produced locally in the microenvironment would be the major source available in this context. Future work should clarify the use of circulating acetate by tumors, or whether the contribution of locally generated acetate predominates. It will also determine the importance of acetate use for proliferation and tumor progression, and elucidate why the use of acetate might be advantageous to cancer cells.