Stéphane J. H. Ricoult

mTORC1 induces purine synthesis through control of the mitochondrial tetrahydrofolate cycle

Controlling supplies for DNA and RNA synthesis The mTORC1 protein kinase complex regulates anabolic metabolism and coordinates cellular signals that promote growth with availability of required precursor metabolites. Signaling through mTORC1 controls pyrimidine synthesis. Ben-Sahra et al. found that mTORC1 also functions by a different mechanism to regulate purine biosynthesis, thus generating precursors for the synthesis of RNA and DNA (see the Perspective by Ma and Jones). Signaling by mTORC1 caused accumulation of the transcription factor ATF4, which enhances production of the enzyme methylenetetrahydrofolate dehydrogenase 2, thus leading to increased production of the purine nucleotides needed for cell growth. Science, this issue p. 728; see also p. 670 Coupling cellular growth signals to synthesis of RNA and DNA. [Also see Perspective by Ma and Jones] In response to growth signals, mechanistic target of rapamycin complex 1 (mTORC1) stimulates anabolic processes underlying cell growth. We found that mTORC1 increases metabolic flux through the de novo purine synthesis pathway in various mouse and human cells, thereby influencing the nucleotide pool available for nucleic acid synthesis. mTORC1 had transcriptional effects on multiple enzymes contributing to purine synthesis, with expression of the mitochondrial tetrahydrofolate (mTHF) cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) being closely associated with mTORC1 signaling in both normal and cancer cells. MTHFD2 expression and purine synthesis were stimulated by activating transcription factor 4 (ATF4), which was activated by mTORC1 independent of its canonical induction downstream of eukaryotic initiation factor 2α eIF2α phosphorylation. Thus, mTORC1 stimulates the mTHF cycle, which contributes one-carbon units to enhance production of purine nucleotides in response to growth signals.

Coordinated regulation of protein synthesis and degradation by mTORC1

Eukaryotic cells coordinately control anabolic and catabolic processes to maintain cell and tissue homeostasis. Mechanistic target of rapamycin complex 1 (mTORC1) promotes nutrient-consuming anabolic processes, such as protein synthesis. Here we show that as well as increasing protein synthesis, mTORC1 activation in mouse and human cells also promotes an increased capacity for protein degradation. Cells with activated mTORC1 exhibited elevated levels of intact and active proteasomes through a global increase in the expression of genes encoding proteasome subunits. The increase in proteasome gene expression, cellular proteasome content, and rates of protein turnover downstream of mTORC1 were all dependent on induction of the transcription factor nuclear factor erythroid-derived 2-related factor 1 (NRF1; also known as NFE2L1). Genetic activation of mTORC1 through loss of the tuberous sclerosis complex tumour suppressors, TSC1 or TSC2, or physiological activation of mTORC1 in response to growth factors or feeding resulted in increased NRF1 expression in cells and tissues. We find that this NRF1-dependent elevation in proteasome levels serves to increase the intracellular pool of amino acids, which thereby influences rates of new protein synthesis. Therefore, mTORC1 signalling increases the efficiency of proteasome-mediated protein degradation for both quality control and as a mechanism to supply substrate for sustained protein synthesis.

The multifaceted role of mTORC1 in the control of lipid metabolism

The mechanistic target of rapamycin is a protein kinase that, as part of the mechanistic target of rapamycin complex 1 (mTORC1), senses both local nutrients and, through insulin signalling, systemic nutrients to control a myriad of cellular processes. Although roles for mTORC1 in promoting protein synthesis and inhibiting autophagy in response to nutrients have been well established, it is emerging as a central regulator of lipid homeostasis. Here, we discuss the growing genetic and pharmacological evidence demonstrating the functional importance of its signalling in controlling mammalian lipid metabolism, including lipid synthesis, oxidation, transport, storage and lipolysis, as well as adipocyte differentiation and function. Defining the role of mTORC1 signalling in these metabolic processes is crucial to understanding the pathophysiology of obesity and its relationship to complex diseases, including diabetes and cancer.

A growing role for mTOR in promoting anabolic metabolism

mTOR [mammalian (or mechanistic) target of rapamycin] is a protein kinase that, as part of mTORC1 (mTOR complex 1), acts as a critical molecular link between growth signals and the processes underlying cell growth. Although there has been intense interest in the upstream mechanisms regulating mTORC1, the full repertoire of downstream molecular events through which mTORC1 signalling promotes cell growth is only recently coming to light. It is now recognized that mTORC1 promotes cell growth and proliferation in large part through the activation of key anabolic processes. Through a variety of downstream targets, mTORC1 alters cellular metabolism to drive the biosynthesis of building blocks and macromolecules fundamentally essential for cell growth, including proteins, lipids and nucleic acids. In the present review, we focus on the metabolic functions of mTORC1 as they relate to the control of cell growth and proliferation. As mTORC1 is aberrantly activated in a number of tumour syndromes and up to 80% of human cancers, we also discuss the importance of this mTORC1-driven biosynthetic programme in tumour growth and progression.

Oncogenic PI3K and K-Ras stimulate de novo lipid synthesis through mTORC1 and SREBP

An enhanced capacity for de novo lipid synthesis is a metabolic feature of most cancer cells that distinguishes them from their cells of origin. However, the mechanisms through which oncogenes alter lipid metabolism are poorly understood. We find that expression of oncogenic PI3K (H1047R) or K-Ras (G12V) in breast epithelial cells is sufficient to induce de novo lipogenesis, and this occurs through the convergent activation of the mechanistic target of rapamycin complex 1 (mTORC1) downstream of these common oncogenes. Oncogenic stimulation of mTORC1 signaling in this isogenic setting or a panel of eight breast cancer cell lines leads to activation of the sterol regulatory element-binding proteins (SREBP1 and SREBP2) that are required for oncogene-induced lipid synthesis. The SREBPs are also required for the growth factor-independent growth and proliferation of oncogene-expressing cells. Finally, we find that elevated mTORC1 signaling is associated with increased mRNA and protein levels of canonical SREBP targets in primary human breast cancer samples. These data suggest that the mTORC1/SREBP pathway is a major mechanism through which common oncogenic signaling events induce de novo lipid synthesis to promote aberrant growth and proliferation of cancer cells.

Tuberous Sclerosis Complex 2 Loss Increases Lysophosphatidylcholine Synthesis in Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a destructive lung disease affecting women. LAM is caused by mutations in the tuberous sclerosis complex (TSC) genes. The TSC protein complex inhibits the mechanistic/mammalian target of rapamycin complex 1 (mTORC1), which is a master regulator of cellular metabolism. Using mass spectrometry-based lipid profiling, we analyzed plasma from patients with LAM and discovered elevated levels of four lysophosphatidylcholine (LPC) species (C16:0, C18:0, C18:1, and C20:4) compared with those in healthy control women. To investigate whether these lipids are generated in a TSC2-dependent manner, we profiled in vitro preclinical models of TSC/LAM and found significant LPC accumulation in TSC2-deficient cells relative to TSC2-expressing control cells. These lysoglycerophospholipid changes occurred alongside changes in other phospholipid and neutral lipid species. Treatment with rapamycin or torin1 or down-regulation of sterol regulatory element-binding protein (SREBP), a lipogenic transcription factor, did not suppress LPC in TSC2-deficient cells. Inhibition of distinct isoforms of phospholipase A2 decreased the proliferation of TSC2-deficient cells. Collectively, these results demonstrate that TSC2-deficient cells have enhanced choline phospholipid metabolism and reveal a novel function of the TSC proteins in choline lysoglycerophospholipid metabolism, with implications for disease pathogenesis and targeted therapeutic strategies.

Sterol Regulatory Element Binding Protein Regulates the Expression and Metabolic Functions of Wild-Type and Oncogenic IDH1

ABSTRACT Sterol regulatory element binding protein (SREBP) is a major transcriptional regulator of the enzymes underlying de novo lipid synthesis. However, little is known about the SREBP-mediated control of processes that indirectly support lipogenesis, for instance, by supplying reducing power in the form of NAPDH or directing carbon flux into lipid precursors. Here, we characterize isocitrate dehydrogenase 1 (IDH1) as a transcriptional target of SREBP across a spectrum of cancer cell lines and human cancers. IDH1 promotes the synthesis of lipids specifically from glutamine-derived carbons. Neomorphic mutations in IDH1 occur frequently in certain cancers, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG). We found that SREBP induces the expression of oncogenic IDH1 and influences 2-HG production from glucose. Treatment of cells with 25-hydroxycholesterol or statins, which respectively inhibit or activate SREBP, further supports SREBP-mediated regulation of IDH1 and, in cells with oncogenic IDH1, carbon flux into 2-HG.

Abstract B06: TSC2 loss induces aberrant choline lysoglycerophospholipid metabolism

Abstracts: AACR Special Conference: Metabolism and Cancer; June 7-10, 2015; Bellevue, WA Tuberous Sclerosis Complex (TSC) is an autosomal dominant disease characterized by multi-organ proliferation of TSC2-deficient cells with aberrant activation of the mechanistic/mammalian Target of Rapamycin Complex 1 (mTORC1), a master regulator of cell growth and metabolism. Up to 80% of women with TSC develop lymphangioleiomyomatosis (LAM), which consists of diffuse proliferation of TSC2-deficient smooth muscle-like cells with progressive cystic destruction of the lung. LAM can also occur as a sporadic disorder of women. A downstream effector of mTORC1, the Sterol Regulatory Element-Binding Protein (SREBP), regulates the transcription of de novo fatty acid synthesis enzymes in TSC2-deficient cells. The impact of loss of the TSC proteins on the lipidome and how complex lipid species are affected by rapamycin and its analogs are unknown. Here, we report the first systematic study of the TSC lipidome, revealing unexpected findings. Using mass spectrometry, we profiled 131 complex lipid species discovered elevated levels (p<0.05) of 4 lysophosphatidylcholine (LPC) species (C16:0, C18:0, C18:1, C20:4) in the plasma of LAM patients compared with healthy control women. LPC are a class of bioactive lipids generated by phospholipase A (PLA) activity. To investigate whether these lipids are generated in a TSC2-dependent manner, we profiled in vitro pre-clinical models of TSC/LAM and found significant LPC accumulation in TSC2-deficient cells relative to TSC2-expressing control cells. These lysoglycerophospholipid changes occurred alongside changes in other phospholipid, including the LPC precursors phosphatidylcholines (PC) and sphingolipids, and neutral lipid species, including triacylglycerols and cholesterol esters. To determine whether TSC2-deficient cells generate LPC using de novo synthesized PC, TSC2-deficient and TSC2-expressing cells were labeled with deuterated choline (choline chloride 1,1,2,2-d4) for 6 hours, and lipids isolated to measure choline incorporation into two saturated LPC (C16:0 and C18:0) and two parent PC (C32:0 and C34:0) species. Incorporation of deuterated choline into PC and LPC was greater than 2 fold higher in TSC2-deficient cells compared to TSC2-expressing controls (p<0.05). Surprisingly, treatment with rapamycin, the mTOR catalytic inhibitor Torin1, or downregulation of the mTORC1-regulated lipogenic transcription factor SREBP (Sterol Regulatory Element-Binding Protein) did not suppress LPC in TSC2-deficient cells, while they suppressed other phospholipid species. Finally, pharmacologic and genetic approaches revealed that inhibition of distinct isoforms of PLA2 may decrease the proliferation of TSC2-deficient but not TSC2-expressing cells. Collectively, these results demonstrate for the first time that TSC2-deficient cells have enhanced choline phospholipid metabolism, revealing a novel function of the TSC proteins in choline lysoglycerophospholipid metabolism, with implications for disease pathogenesis and targeted therapeutic strategies. Citation Format: Carmen Priolo, Stephane J. H. Ricoult, Damir Khabibullin, Harilaos Filippakis, Jane Yu, Brendan Manning, Clary Clish, Elizabeth P. Henske. TSC2 loss induces aberrant choline lysoglycerophospholipid metabolism. [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 B06.

Oncogenic signaling upstream of mTORC1 drives lipogenesis and proliferation through SREBP

Background The mechanistic target of rapamycin (mTOR) is a central regulator of cell growth and proliferation, and its aberrant activation is frequent in cancer [1]. We have previously shown that sterol regulatory element binding protein (SREBP) is a major transcriptional effector of mTOR complex 1 (mTORC1) signaling [2]. SREBP is a transcription factor that stimulates the expression of genes involved in the de novo synthesis of lipids [3]. Since mTORC1 is commonly activated in cancer through upstream oncogenic signaling pathways and enhanced lipogenesis is a metabolic hallmark of tumor cells, we hypothesize that the mTORC1-SREBP pathway is important for tumor metabolism and growth.

mTORC1 stimulates nucleotide synthesis through both transcriptional and post-translational mechanisms

Background Cellular growth signals stimulate anabolic processes. The mechanistic target of rapamycin (mTOR), as part of mTORC1, is a protein kinase that senses growth signals to regulate anabolic growth and proliferation. mTORC1 stimulates protein synthesis through effects on mRNA translation and ribosome biogenesis [1]. mTORC1 signaling also promotes de novo lipid and sterol synthesis through the activation of the sterol-response element-binding protein (SREBP) transcription factors, which stimulate the expression of the enzymes driving this biosynthetic process [2].