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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.

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. Glycolysis is elevated in many cancers. In this study, the authors show that lactoylglutathione, a by-product of methylglyoxal produced from increased glycolysis, is elevated in lung cancer in mouse models and humans, arguing reactive metabolite production can be a liability for cancers.

Defining the metabolic limitations of tumour growth will help to develop cancer therapies 1 . 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 impaired 2 – 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 asparagine 5 , 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. Garcia-Bermudez et al. and Sullivan et al. show that endogenous aspartate is a limiting metabolite for cancer cell proliferation under hypoxia and in tumours, and that metformin depletes aspartate to limit tumour growth.

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 Beati 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.

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. A metabolic purine safety valve 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 biosynthesis 1 , 2 , 3 , 4 . In a canonical example, the first committed enzyme in the pyrimidine pathway in Escherichia coli is allosterically inhibited by cytidine triphosphate 1 , 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 kinase 6 . 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 7 , 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.

Rag GTPases facilitate mTORC1 activation by recruiting it to Rheb at the lysosome when amino acids are abundant. A study now shows that the amino acid-induced change in the GTP/GDP-binding state of the Rag heterodimer paradoxically increases its dynamic release from the Ragulator at the lysosome and may limit mTORC1 activation.

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. Production of oxidized biomass is necessary to support cancer cell proliferation. In this work, Li et al. provide a quantitative analysis of the cellular needs and biochemical bottlenecks in lipid biosynthesis arising from the requirement to regenerate the cofactor NAD + .

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 β-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. Hosios et al. demonstrate that inhibition of mechanistic target of rapamycin complex 1 in cells and in tumors in mice leads to a lysosome-dependent but autophagy-independent shift in membrane lipid metabolism, resulting in increased intracellular triglyceride pools.

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

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.