Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells.

Mitochondrial 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.

PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells.

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.

Lack of Evidence for PKM2 Protein Kinase Activity.

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 [(32)P]-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 [(32)P]-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.

Pyruvate kinase isoform expression alters nucleotide synthesis to impact cell proliferation.

Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2 deletion affects proliferation and metabolism in nontransformed, nonimmortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis.

Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth.

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.

The redox requirements of proliferating mammalian cells.

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.

Lack of evidence for substrate channeling or flux between wildtype and mutant isocitrate dehydrogenase to produce the oncometabolite 2-hydroxyglutarate.

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.

Pyrimidine homeostasis is accomplished by directed overflow metabolism.

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 biosynthesis. In a canonical example, the first committed enzyme in the pyrimidine pathway in Escherichia coli is allosterically inhibited by cytidine triphosphate. 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 kinase. 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 function. 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.

An RNA damage response network mediates the lethality of 5-FU in colorectal cancer.

5-fluorouracil (5-FU), a major anti-cancer therapeutic, is believed to function primarily by inhibiting thymidylate synthase, depleting deoxythymidine triphosphate (dTTP), and causing DNA damage. Here, we show that clinical combinations of 5-FU with oxaliplatin or irinotecan show no synergy in human colorectal cancer (CRC) trials and sub-additive killing in CRC cell lines. Using selective 5-FU metabolites, phospho- and ubiquitin proteomics, and primary human CRC organoids, we demonstrate that 5-FU-mediated CRC cell killing primarily involves an RNA damage response during ribosome biogenesis, causing lysosomal degradation of damaged rRNAs and proteasomal degradation of ubiquitinated ribosomal proteins. Tumor types clinically responsive to 5-FU treatment show upregulated rRNA biogenesis while 5-FU clinically non-responsive tumor types do not, instead showing greater sensitivity to 5-FU's DNA damage effects. Finally, we show that treatments upregulating ribosome biogenesis, including KDM2A inhibition, promote RNA-dependent cell killing by 5-FU, demonstrating the potential for combinatorial targeting of this ribosomal RNA damage response for improved cancer therapy.

An RNA Damage Response Network Mediates the Lethality of 5-FU in Clinically Relevant Tumor Types.

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.

mTORC1 regulates a lysosome-dependent adaptive shift in intracellular lipid species.

The mechanistic target of rapamycin 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 ß-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.

The non-essential TSC complex component TBC1D7 restricts tissue mTORC1 signaling and brain and neuron growth.

The tuberous sclerosis complex (TSC) 1 and 2 proteins associate with TBC1D7 to form the TSC complex, which is an essential suppressor of mTOR complex 1 (mTORC1), a ubiquitous driver of cell and tissue growth. Loss-of-function mutations in TSC1 or TSC2, but not TBC1D7, give rise to TSC, a pleiotropic disorder with aberrant activation of mTORC1 in various tissues. Here, we characterize mice with genetic deletion of Tbc1d7, which are viable with normal growth and development. Consistent with partial loss of function of the TSC complex, Tbc1d7 knockout (KO) mice display variable increases in tissue mTORC1 signaling with increased muscle fiber size but with strength and motor defects. Their most pronounced phenotype is brain overgrowth due to thickening of the cerebral cortex, with enhanced neuron-intrinsic mTORC1 signaling and growth. Thus, TBC1D7 is required for full TSC complex function in tissues, and the brain is particularly sensitive to its growth-suppressing activities.

Reciprocal effects of mTOR inhibitors on pro-survival proteins dictate therapeutic responses in tuberous sclerosis complex.

mTORC1 is aberrantly activated in cancer and in the genetic tumor syndrome tuberous sclerosis complex (TSC), which is caused by loss-of-function mutations in the TSC complex, a negative regulator of mTORC1. Clinically approved mTORC1 inhibitors, such as rapamycin, elicit a cytostatic effect that fails to eliminate tumors and is rapidly reversible. We sought to determine the effects of mTORC1 on the core regulators of intrinsic apoptosis. In TSC2-deficient cells and tumors, we find that mTORC1 inhibitors shift cellular dependence from MCL-1 to BCL-2 and BCL-XL for survival, thereby altering susceptibility to BH3 mimetics that target specific pro-survival BCL-2 proteins. The BCL-2/BCL-XL inhibitor ABT-263 synergizes with rapamycin to induce apoptosis in TSC-deficient cells and in a mouse tumor model of TSC, resulting in a more complete and durable response. These data expose a therapeutic vulnerability in regulation of the apoptotic machinery downstream of mTORC1 that promotes a cytotoxic response to rapamycin.

Cancer cells depend on environmental lipids for proliferation when electron acceptors are limited.

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 show that de novo lipid biosynthesis can impose a substantial NAD+ consumption cost in proliferating cancer cells. 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. Gene expression analysis demonstrates that lipid biosynthesis strongly anti-correlates with expression of hypoxia markers across tumor types. Overall, our results define a requirement for oxidative metabolism to support biosynthetic reactions and provide a mechanistic explanation for cancer cell dependence on lipid uptake in electron acceptor-limited conditions, such as hypoxia.

Cancer Signaling Drives Cancer Metabolism: AKT and the Warburg Effect.

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.

The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis downstream of growth signals.

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.

When building healthy tissue, the human body must carefully control the growth of new cells to prevent them from becoming cancerous. A core component of this regulation is the protein mTORC1, which responds to various growth-stimulating factors and nutrients, and activates the chemical reactions cells need to grow. Part of this process involves controlling 'nutrient-sensing transcription factors' ­ proteins that regulate the activity of specific genes based on the availability of different nutrients. One of these nutrient-sensing transcription factors, ATF4, has recently been shown to be involved in some of the processes triggered by mTORC1. The role this factor plays in how cells respond to stress ­ such as when specific nutrients are depleted, protein folding is disrupted or toxins are present ­ is well-studied. But how it reacts to the activation of mTORC1 is less clear. To bridge this gap, Torrence et al. studied mouse embryonic cells and human prostate cancer cells grown in the laboratory, to see whether mTORC1 influenced the behavior of ATF4 differently than cellular stress. Cells were treated either with insulin, which activates mTORC1, or an antibiotic that sparks the stress response. The cells were then analyzed using a molecular tool to see which genes were switched on by ATF4 following treatment. This revealed that less than 10% of the genes activated by ATF4 during cellular stress are also activated in response to mTORC1-driven growth. Many of the genes activated in both scenarios were involved in synthesizing and preparing the building blocks that make up proteins. This was consistent with the discovery that ATF4 helps mTORC1 stimulate growth by promoting protein synthesis. Torrence et al. also found that mTORC1's regulation of ATF4 stimulated the synthesis of glutathione, the most abundant antioxidant in cells. The central role mTORC1 plays in controlling cell growth means it is important to understand how it works and how it can lead to uncontrolled growth in human diseases. mTORC1 is activated in many overgrowth syndromes and the majority of human cancers. These new findings could provide insight into how tumors coordinate their drive for growth while adapting to cellular stress, and reveal new drug targets for cancer treatment.

Differential substrate use in EGF- and oncogenic KRAS-stimulated human mammary epithelial cells.

Many metabolic phenotypes in cancer cells are also characteristic of proliferating nontransformed 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 nononcogenic growth.

Cancer-associated mutations in human pyruvate kinase M2 impair enzyme activity.

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.

Author Correction: Cooperative nutrient accumulation sustains growth of mammalian cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reactive metabolite production is a targetable liability of glycolytic metabolism in lung cancer.

Increased glucose uptake and metabolism is a prominent phenotype of most cancers, but efforts to clinically target this metabolic alteration have been challenging. Here, we present evidence that lactoylglutathione (LGSH), a byproduct of methylglyoxal detoxification, is elevated in both human and murine non-small cell lung cancers (NSCLC). Methylglyoxal is a reactive metabolite byproduct of glycolysis that reacts non-enzymatically with nucleophiles in cells, including basic amino acids, and reduces cellular fitness. Detoxification of methylglyoxal requires reduced glutathione (GSH), which accumulates to high levels in NSCLC relative to normal lung. Ablation of the methylglyoxal detoxification enzyme glyoxalase I (Glo1) potentiates methylglyoxal sensitivity and reduces tumor growth in mice, arguing that targeting pathways involved in detoxification of reactive metabolites is an approach to exploit the consequences of increased glucose metabolism in cancer.