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.

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.

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.

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.

IMPDH inhibitors for antitumor therapy in tuberous sclerosis complex.

Recent studies in distinct preclinical tumor models have established the nucleotide synthesis enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) as a viable target for antitumor therapy. IMPDH inhibitors have been used clinically for decades as safe and effective immunosuppressants. However, the potential to repurpose these pharmacological agents for antitumor therapy requires further investigation, including direct comparisons of available compounds. Therefore, we tested structurally distinct IMPDH inhibitors in multiple cell and mouse tumor models of the genetic tumor syndrome tuberous sclerosis complex (TSC). TSC-associated tumors are driven by uncontrolled activation of the growth-promoting protein kinase complex mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), which is also aberrantly activated in the majority of sporadic cancers. Despite eliciting similar immunosuppressive effects, the IMPDH inhibitor mizoribine, used clinically throughout Asia, demonstrated far superior antitumor activity compared with the FDA-approved IMPDH inhibitor mycophenolate mofetil (or CellCept, a prodrug of mycophenolic acid). When compared directly to the mTOR inhibitor rapamycin, mizoribine treatment provided a more durable antitumor response associated with tumor cell death. These results provide preclinical support for repurposing mizoribine, over other IMPDH inhibitors, as an alternative to mTOR inhibitors for the treatment of TSC-associated tumors and possibly other tumors featuring uncontrolled mTORC1 activity.

Collagen-derived proline promotes pancreatic ductal adenocarcinoma cell survival under nutrient limited conditions.

Tissue architecture contributes to pancreatic ductal adenocarcinoma (PDAC) phenotypes. Cancer cells within PDAC form gland-like structures embedded in a collagen-rich meshwork where nutrients and oxygen are scarce. Altered metabolism is needed for tumour cells to survive in this environment, but the metabolic modifications that allow PDAC cells to endure these conditions are incompletely understood. Here we demonstrate that collagen serves as a proline reservoir for PDAC cells to use as a nutrient source when other fuels are limited. We show PDAC cells are able to take up collagen fragments, which can promote PDAC cell survival under nutrient limited conditions, and that collagen-derived proline contributes to PDAC cell metabolism. Finally, we show that proline oxidase (PRODH1) is required for PDAC cell proliferation in vitro and in vivo. Collectively, our results indicate that PDAC extracellular matrix represents a nutrient reservoir for tumour cells highlighting the metabolic flexibility of this cancer.

Tissue of origin dictates branched-chain amino acid metabolism in mutant Kras-driven cancers.

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.

Circulating Metabolites and Survival Among Patients With Pancreatic Cancer.

BACKGROUND: Pancreatic tumors cause changes in whole-body metabolism, but whether prediagnostic circulating metabolites predict survival is unknown. METHODS: We measured 82 metabolites by liquid chromatography-mass spectrometry in prediagnostic plasma from 484 pancreatic cancer case patients enrolled in four prospective cohort studies. Association of metabolites with survival was evaluated using Cox proportional hazards models adjusted for age, cohort, race/ethnicity, cancer stage, fasting time, and diagnosis year. After multiple-hypothesis testing correction, a P value of .0006 or less (.05/82) was considered statistically significant. Based on the results, we evaluated 33 tagging single-nucleotide polymorphisms (SNPs) in the ACO1 gene, requiring a P value of less than .002 (.05/33) for statistical significance. All statistical tests were two-sided. RESULTS: Two metabolites in the tricarboxylic acid (TCA) cycle--isocitrate and aconitate--were statistically significantly associated with survival. Participants in the highest vs lowest quintile had hazard ratios (HRs) for death of 1.89 (95% confidence interval [CI] = 1.06 to 3.35, Ptrend < .001) for isocitrate and 2.54 (95% CI = 1.42 to 4.54, Ptrend < .001) for aconitate. Isocitrate is interconverted with citrate via the intermediate aconitate in a reaction catalyzed by the enzyme aconitase 1 (ACO1). Therefore, we investigated the citrate to aconitate plus isocitrate ratio and SNPs in the ACO1 gene. The ratio was strongly associated with survival (P trend < .001) as was the SNP rs7874815 in the ACO1 gene (hazard ratio for death per minor allele = 1.37, 95% CI = 1.16 to 1.61, P < .001). Patients had an approximately three-fold hazard for death when possessing one or more minor alleles at rs7874851 and high aconitate or isocitrate. CONCLUSIONS: Prediagnostic circulating levels of TCA cycle intermediates and inherited ACO1 genotypes were associated with survival among patients with pancreatic cancer.

Elevation of circulating branched-chain amino acids is an early event in human pancreatic adenocarcinoma development.

Most patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed with advanced disease and survive less than 12 months. PDAC has been linked with obesity and glucose intolerance, but whether changes in circulating metabolites are associated with early cancer progression is unknown. To better understand metabolic derangements associated with early disease, we profiled metabolites in prediagnostic plasma from individuals with pancreatic cancer (cases) and matched controls from four prospective cohort studies. We find that elevated plasma levels of branched-chain amino acids (BCAAs) are associated with a greater than twofold increased risk of future pancreatic cancer diagnosis. This elevated risk was independent of known predisposing factors, with the strongest association observed among subjects with samples collected 2 to 5 years before diagnosis, when occult disease is probably present. We show that plasma BCAAs are also elevated in mice with early-stage pancreatic cancers driven by mutant Kras expression but not in mice with Kras-driven tumors in other tissues, and that breakdown of tissue protein accounts for the increase in plasma BCAAs that accompanies early-stage disease. Together, these findings suggest that increased whole-body protein breakdown is an early event in development of PDAC.