Regulation Of mTORC1 Research Articles

NANOS2 suppresses the cell cycle by repressing mTORC1 activators in embryonic male germ cells

SummaryDuring murine germ cell development, male germ cells enter the mitotically arrested G0 stage, which is an initial step of sexually dimorphic differentiation. The male-specific RNA-binding protein NANOS2 has a key role in suppressing the cell cycle in germ cells. However, the detailed mechanism of how NANOS2 regulates the cell cycle remains unclear. Using single-cell RNA sequencing (scRNA-seq), we extracted the cell cycle state of each germ cell in wild-type and Nanos2-KO testes and revealed that Nanos2 expression starts in mitotic cells and induces mitotic arrest. We identified Rheb, a regulator of mTORC1, and Ptma as possible targets of NANOS2. We propose that repression of the cell cycle is a primary function of NANOS2 and that it is mediated via the suppression of mTORC1 activity through the repression of Rheb in a post-transcriptional manner.

MTORC1 is a mechanosensor that regulates surfactant function and lung compliance during ventilator-induced lung injury.

The acute respiratory distress syndrome (ARDS) is a highly lethal condition that impairs lung function and causes respiratory failure. Mechanical ventilation (MV) maintains gas exchange in patients with ARDS but exposes lung cells to physical forces that exacerbate injury. Our data demonstrate that mTOR complex 1 (mTORC1) is a mechanosensor in lung epithelial cells and that activation of this pathway during MV impairs lung function. We found that mTORC1 is activated in lung epithelial cells following volutrauma and atelectrauma in mice and humanized in vitro models of the lung microenvironment. mTORC1 is also activated in lung tissue of mechanically ventilated patients with ARDS. Deletion of Tsc2, a negative regulator of mTORC1, in epithelial cells impairs lung compliance during MV. Conversely, treatment with rapamycin at the time MV is initiated improves lung compliance without altering lung inflammation or barrier permeability. mTORC1 inhibition mitigates physiologic lung injury by preventing surfactant dysfunction during MV. Our data demonstrate that, in contrast to canonical mTORC1 activation under favorable growth conditions, activation of mTORC1 during MV exacerbates lung injury and inhibition of this pathway may be a novel therapeutic target to mitigate ventilator-induced lung injury during ARDS.

A role for BDNF- and NMDAR-induced lysosomal recruitment of mTORC1 in the regulation of neuronal mTORC1 activity

Memory and long term potentiation require de novo protein synthesis. A key regulator of this process is mTORC1, a complex comprising the mTOR kinase. Growth factors activate mTORC1 via a pathway involving PI3-kinase, Akt, the TSC complex and the GTPase Rheb. In non-neuronal cells, translocation of mTORC1 to late endocytic compartments (LEs), where Rheb is enriched, is triggered by amino acids. However, the regulation of mTORC1 in neurons remains unclear. In mouse hippocampal neurons, we observed that BDNF and treatments activating NMDA receptors trigger a robust increase in mTORC1 activity. NMDA receptors activation induced a significant recruitment of mTOR onto lysosomes even in the absence of external amino acids, whereas mTORC1 was evenly distributed in neurons under resting conditions. NMDA receptor-induced mTOR translocation to LEs was partly dependent on the BDNF receptor TrkB, suggesting that BDNF contributes to the effect of NMDA receptors on mTORC1 translocation. In addition, the combination of Rheb overexpression and artificial mTORC1 targeting to LEs by means of a modified component of mTORC1 fused with a LE-targeting motif strongly activated mTOR. To gain spatial and temporal control over mTOR localization, we designed an optogenetic module based on light-sensitive dimerizers able to recruit mTOR on LEs. In cells expressing this optogenetic tool, mTOR was translocated to LEs upon photoactivation. In the absence of growth factor, this was not sufficient to activate mTORC1. In contrast, mTORC1 was potently activated by a combination of BDNF and photoactivation. The data demonstrate that two important triggers of synaptic plasticity, BDNF and NMDA receptors, synergistically power the two arms of the mTORC1 activation mechanism, i.e., mTORC1 translocation to LEs and Rheb activation. Moreover, they unmask a functional link between NMDA receptors and mTORC1 that could underlie the changes in the synaptic proteome associated with long-lasting changes in synaptic strength.

MycobacteriumtuberculosisProtein PE6 (Rv0335c), a Novel TLR4 Agonist, Evokes an Inflammatory Response and Modulates the Cell Death Pathways in Macrophages to Enhance Intracellular Survival.

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that exploits moonlighting functions of its proteins to interfere with host cell functions. PE/PPE proteins utilize host inflammatory signaling and cell death pathways to promote pathogenesis. We report that M. tb PE6 protein (Rv0335c) is a secretory protein effector that interacts with innate immune toll-like receptor TLR4 on the macrophage cell surface and promotes activation of the canonical NFĸB signaling pathway to stimulate secretion of proinflammatory cytokines TNF-α, IL-12, and IL-6. Using mouse macrophage TLRs knockout cell lines, we demonstrate that PE6 induced secretion of proinflammatory cytokines dependent on TLR4 and adaptor Myd88. PE6 possesses nuclear and mitochondrial targeting sequences and displayed time-dependent differential localization into nucleus/nucleolus and mitochondria, and exhibited strong Nucleolin activation. PE6 strongly induces apoptosis via increased production of pro-apoptotic molecules Bax, Cytochrome C, and pcMyc. Mechanistic details revealed that PE6 activates Caspases 3 and 9 and induces endoplasmic reticulum-associated unfolded protein response pathways to induce apoptosis through increased production of ATF6, Chop, BIP, eIF2α, IRE1α, and Calnexin. Despite being a potent inducer of apoptosis, PE6 suppresses innate immune defense strategy autophagy by inducing inhibitory phosphorylation of autophagy initiating kinase ULK1. Inversely, PE6 induces activatory phosphorylation of autophagy master regulator MtorC1, which is reflected by lower conversion of autophagy markers LC3BI to LC3BII and increased accumulation of autophagy substrate p62 which is also dependent on innate immune receptor TLR4. The use of pharmacological agents, rapamycin and bafilomycin A1, confirms the inhibitory effect of PE6 on autophagy, evidenced by the reduced conversion of LC3BI to LC3BII and increased accumulation of p62 in the presence of rapamycin and bafilomycin A1. We also observed that PE6 binds DNA, which could have significant implications in virulence. Furthermore, our analyses reveal that PE6 efficiently binds iron to likely aid in intracellular survival. Recombinant Mycobacterium smegmatis (M. smegmatis) containing pe6 displayed robust growth in iron chelated media compared to vector alone transformed cells, which suggests a role of PE6 in iron acquisition. These findings unravel novel mechanisms exploited by PE6 protein to subdue host immunity, thereby providing insights relevant to a better understanding of host–pathogen interaction during M. tb infection.

Abstract 1015: mTOR inhibitor induces non-apoptotic, autophagy dependent cell death (ADCD) in TP53 mutant HNSCC

Abstract TP53 is the most frequently mutated gene in Head and Neck Squamous Cell Carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis. A phase II multi-institutional clinical trial, determined that, adjuvant therapy with an mTOR inhibitor significantly increased 2-year progression free survival (PFS) in patients with p53 mutant tumors. Previous studies have shown that mutant p53 causes sustained activation of PI3K/AKT/mTOR pathway in cancer. We investigated the mechanism by which the mTOR inhibitor CCI-779 inhibits cell growth of TP53 mutant HNSCC in vitro. HPV-negative HNSCC cell lines harboring TP53 mutations at different positions were treated with CCI-779.CCI-779 significantly reduced cell viability and colony forming ability of all the cell lines tested. CCI-779 did not induce apoptosis or cell cycle arrest in these cells. However, the reduction in cell viability was associated with increase in autophagy as demonstrated by increased levels of LC3-II. The autophagy inhibitors, 3-methyl adenine (3-MA) and ULK-101 restored the cell viability by inhibiting CCI-779 induced autophagy. Therefore, we postulate that CCI-779 induces nonapototic autophagy dependent cell death (ADCD) in HPV-negative TP53 mutant HNSCC cell lines. CCI-779 induced autophagy is accompanied by increased level of ULK1 and Beclin1 as well as increased phosphorylation of ULK1 at Serine 555. However, ULK101 (ULK1 inhibitor) and 3-MA (PI3K inhibitor) when combined with CCI-779 reduced the level of both total and phosphorylated levels of ULK1, which resulted in inhibition of autophagy and improved cell viability. However, combination of CCI-779 and bafilomycin A1 treatment showed increased level of LC3-II, ULK1 and phospho-ULK1 (S555) with concomitant decrease in cell viability. Our data suggests that rapalog induces ADCD by facilitating autophagy initiation through the stabilization and activation of ULK1. Moreover our data demonstrated that CCI-779 mediated activation of ULK1 is associated with down regulation of mTORC1 but not mTORC2 signaling. This is a novel mechanism of how rapalogues induce ADCD through the stabilization/activation of ULK1 in HPV-negative TP53 mutant HNSCC cells. Together these data highlight the importance of how mTORi can be used to accelerate tumor cell death in HPV-negative TP53 mutant HNSCC. Citation Format: Md Maksudul Alam, Janmaris Marin Fermin, Patrick T. Spiller, Channing Dorr, Xiaohua Rong, Tara-Moore Medlin, Alok R. Khandelwal, Cherie-Ann Nathan. mTOR inhibitor induces non-apoptotic, autophagy dependent cell death (ADCD) in TP53 mutant HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1015.

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and invivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how invivo tissue-specific phenotypes emerge in ribosomopathies.

MTORC1 activation is not sufficient to suppress hepatic PPARα signaling or ketogenesis

The mechanistic target of rapamycin (mTOR) is often referred to as a master regulator of the cellular metabolism that can integrate the growth factor and nutrient signaling. Fasting suppresses hepatic mTORC1 activity via the activity of the tuberous sclerosis complex (TSC), a negative regulator of mTORC1, to suppress anabolic metabolism. The loss of TSC1 in the liver locks the liver in a constitutively anabolic state even during fasting, which was suggested to regulate peroxisome proliferator-activated receptor alpha (PPARα) signaling and ketogenesis, but the molecular determinants of this regulation are unknown. Here, we examined if the activation of the mTORC1 complex in mice by the liver-specific deletion of TSC1 (TSC1L−/−) is sufficient to suppress PPARα signaling and therefore ketogenesis in the fasted state. We found that the activation of mTORC1 in the fasted state is not sufficient to repress PPARα-responsive genes or ketogenesis. Furthermore, we examined whether the activation of the anabolic program mediated by mTORC1 complex activation in the fasted state could suppress the robust catabolic programming and enhanced PPARα transcriptional response of mice with a liver-specific defect in mitochondrial long-chain fatty acid oxidation using carnitine palmitoyltransferase 2 (Cpt2L−/−) mice. We generated Cpt2L−/−; Tsc1L−/− double-KO mice and showed that the activation of mTORC1 by deletion of TSC1 could not suppress the catabolic PPARα-mediated phenotype of Cpt2L−/− mice. These data demonstrate that the activation of mTORC1 by the deletion of TSC1 is not sufficient to suppress a PPARα transcriptional program or ketogenesis after fasting.

Sensitive period for rescuing parvalbumin interneurons connectivity and social behavior deficits caused by TSC1 loss

The Mechanistic Target Of Rapamycin Complex 1 (mTORC1) pathway controls several aspects of neuronal development. Mutations in regulators of mTORC1, such as Tsc1 and Tsc2, lead to neurodevelopmental disorders associated with autism, intellectual disabilities and epilepsy. The correct development of inhibitory interneurons is crucial for functional circuits. In particular, the axonal arborisation and synapse density of parvalbumin (PV)-positive GABAergic interneurons change in the postnatal brain. How and whether mTORC1 signaling affects PV cell development is unknown. Here, we show that Tsc1 haploinsufficiency causes a premature increase in terminal axonal branching and bouton density formed by mutant PV cells, followed by a loss of perisomatic innervation in adult mice. PV cell-restricted Tsc1 haploinsufficient and knockout mice show deficits in social behavior. Finally, we identify a sensitive period during the third postnatal week during which treatment with the mTOR inhibitor Rapamycin rescues deficits in both PV cell innervation and social behavior in adult conditional haploinsufficient mice. Our findings reveal a role of mTORC1 signaling in the regulation of the developmental time course and maintenance of cortical PV cell connectivity and support a mechanistic basis for the targeted rescue of autism-related behaviors in disorders associated with deregulated mTORC1 signaling.

Amino Acid Trafficking and Skeletal Muscle Protein Synthesis: A Case of Supply and Demand

Skeletal muscle protein synthesis is a highly complex process, influenced by nutritional status, mechanical stimuli, repair programs, hormones, and growth factors. The molecular aspects of protein synthesis are centered around the mTORC1 complex. However, the intricacies of mTORC1 regulation, both up and downstream, have expanded overtime. Moreover, the plastic nature of skeletal muscle makes it a unique tissue, having to coordinate between temporal changes in myofiber metabolism and hypertrophy/atrophy stimuli within a tissue with considerable protein content. Skeletal muscle manages the push and pull between anabolic and catabolic pathways through key regulatory proteins to promote energy production in times of nutrient deprivation or activate anabolic pathways in times of nutrient availability and anabolic stimuli. Branched-chain amino acids (BCAAs) can be used for both energy production and signaling to induce protein synthesis. The metabolism of BCAAs occur in tandem with energetic and anabolic processes, converging at several points along their respective pathways. The fate of intramuscular BCAAs adds another layer of regulation, which has consequences to promote or inhibit muscle fiber protein anabolism. This review will outline the general mechanisms of muscle protein synthesis and describe how metabolic pathways can regulate this process. Lastly, we will discuss how BCAA availability and demand coordinate with synthesis mechanisms and identify key factors involved in intramuscular BCAA trafficking.

Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress.

Appropriate regulation of the Integrated stress response (ISR) and mTORC1 signaling are central for cell adaptation to starvation for amino acids. Halofuginone (HF) is a potent inhibitor of aminoacylation of tRNAPro with broad biomedical applications. Here, we show that in addition to translational control directed by activation of the ISR by general control nonderepressible 2 (GCN2), HF increased free amino acids and directed translation of genes involved in protein biogenesis via sustained mTORC1 signaling. Deletion of GCN2 reduced cell survival to HF whereas pharmacological inhibition of mTORC1 afforded protection. HF treatment of mice synchronously activated the GCN2-mediated ISR and mTORC1 in liver whereas Gcn2-null mice allowed greater mTORC1 activation to HF, resulting in liver steatosis and cell death. We conclude that HF causes an amino acid imbalance that uniquely activates both GCN2 and mTORC1. Loss of GCN2 during HF creates a disconnect between metabolic state and need, triggering proteostasis collapse.

The Convergence of Two Signaling Pathways Within the Striatum Reveals Potential Mechanisms of Neuropsychiatric Disease

The serine-threonine kinase mTOR (mechanistic target of rapamycin) is the cornerstone of a signaling pathway that serves as an essential regulator of cellular processes such as protein synthesis and autophagy (1). Aberrant activity of mTOR complex 1 (mTORC1) has been identified both in neurodevelopmental disorders and in sporadic neurodegenerative disease (2). In particular, loss-of-function mutations in genes encoding negative regulators of mTORC1, such as TSC1, TSC2, and PTEN, result in syndromic neurodevelopmental disorders associated with a constellation of manifestations, which can include epilepsy, autism spectrum disorder, and cognitive impairments.

ArfGAP1 inhibits mTORC1 lysosomal localization and activation

The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid‐deficient conditions is not completely understood. Here, we identify ADP‐ribosylation factor GTPase‐activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature‐sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.

TSC1 binding to lysosomal PIPs is required for TSC complex translocation and mTORC1 regulation

The TSC complex is a critical negative regulator of the small GTPase Rheb and mTORC1 in cellular stress signaling. The TSC2 subunit contains a catalytic GTPase activating protein domain and interacts with multiple regulators, while the precise function of TSC1 is unknown. Here we provide a structural characterization of TSC1 and define three domains: a C-terminal coiled-coil that interacts with TSC2, a central helical domain that mediates TSC1 oligomerization, and an N-terminal HEAT repeat domain that interacts with membrane phosphatidylinositol phosphates (PIPs). TSC1 architecture, oligomerization, and membrane binding are conserved in fungi and humans. We show that lysosomal recruitment of the TSC complex and subsequent inactivation of mTORC1 upon starvation depend on the marker lipid PI3,5P2, demonstrating a role for lysosomal PIPs in regulating TSC complex and mTORC1 activity via TSC1. Our study thus identifies a vital role of TSC1 in TSC complex function and mTORC1 signaling.

Environmental Sensing by mTORC1

The mammalian target of rapamycin (mTOR) is an evolutionary conserved Ser/Thr kinase that can sense multiple upstream stimuli to regulate cell growth, metabolism, and autophagy. mTOR is the key component of a multi-protein complex termed mTOR complex 1 (mTORC1). Increased mTORC1 activation is common in many human diseases such as cancer. Small molecules like rapamycin that target and inhibit mTORC1, are currently used in the clinic with limited success. Thus, deciphering the molecular mechanisms involved in mTORC1 regulation is crucial in order to treat mTORC1-mediated disease. The majority of research has focused on stimuli that activate mTORC1, like growth factors and amino acids. In contrast, less is known about signaling pathways that can directly inhibit mTORC1 activity. We recently discovered that G-protein coupled receptor (GPCR) signaling potently inhibits mTORC1. GPCRs paired to Gas proteins activate Protein Kinase A (PKA) by increasing intracellular cyclic adenosine 3’5’ monophosphate (cAMP) levels. PKA phosphorylates the mTORC1 component Raptor resulting in decreased mTORC1 activity. Importantly, GPCRs are the largest family of drug targets and many compounds have been approved by the FDA to regulate GPCR signaling. We have identified an A-kinase anchoring protein as a crucial scaffold involved in GPCR-Gassignaling to mTORC1. Targeting this signaling pathway may be beneficial for human diseases with hyperactivated mTORC1.

Reciprocal regulation of mTORC1 and ribosomal biosynthesis determines cell cycle progression in activated T cells

Abstract Anabolic metabolism of antigen-stimulated T cells is coordinated by mTOR which is activated by TCR and IL-2 signals. We show that mTORC1 regulates both the exit of quiescence and later divisions as Raptor-deficient T cells proceed 3x slower through each cell division in vivo. We noticed that following stimulation the amount of RNA per cell increases dramatically (10–40x) and is reduced to ~50% in Raptor-, but not Rictor-deficient T cells. RNAseq data indicate that this applies to all RNA polymerase I- and II-dependent biotypes. Using Coulter volume measurements, RNA-, FISH- and imaging-flow cytometry we determined that following stimulation the cell volume increases five-fold. It consists of the increase of nuclear volume and nucleolar rRNA synthesis on day 1 and a further increase of cytoplasmic volume on day 2. Raptor-deficiency delays and diminishes the growth of these cellular compartments, decreases ribosomal biosynthesis and thus the cell volume. Inhibitors of rRNA transcription block cell cycle progression at two checkpoints, of which only the G1/S transition is regulated by Raptor. We found that mTORC1 activity depends on ongoing rRNA synthesis in early activation and regulates the further synthesis rate. Altogether our data show that mTOR-adjusted cell physiology and the biosynthesis of ribosomal ‘hardware’ are reciprocally regulated during clonal T cell expansion.

MTORC1 Activity Occurs Predominantly in the Periphery of Human Skeletal Muscle Fibers Following Anabolic Stimuli

The mechanistic target of rapamycin complex 1 (mTORC1) is regarded as the master regulator of protein synthesis in skeletal muscle and is essential for optimal responses to acute anabolic stimuli. We have shown mTORC1 to translocate toward the cell periphery where it colocalizes with upstream activators, downstream effectors and the microvasculature after protein feeding and muscle contraction. These events occur at similar times to elevated whole muscle mTORC1 kinase activity and protein synthesis; however, whether mTORC1 activity occurs in a region-specific pattern is yet to be studied. Therefore, the aim of the current study was to develop an immunofluorescent staining (IF) protocol to measure mTORC1 activation in a region-specific manner following anabolic stimuli in human skeletal muscle. Fourteen young, healthy males (22±4yrs, 14±4% body fat, mean±SD) participated in the study and were randomized to complete trials where a protein-carbohydrate beverage was ingested at rest (FED, n=7) or following a whole-body resistance exercise session (EXFED, n=7). Vastus lateralis biopsy samples were obtained prior to feeding/exercise (basal) and 120 and 300min following anabolic stimuli. Phosphorylated ribosomal protein S6Ser240/244 (p-RPS6Ser240/244) was chosen as the marker of mTORC1 activation as this phosphorylation event is rapamycin-sensitive. We first optimized and validated an IF protocol for p-RPS6Ser240/244 through the omission of primary and secondary antibodies. Using this protocol, we then showed that RPS6Ser240/244 phosphorylation was elevated at 120min (p<0.04) with EXFED being greater than FED (p=0.02). p-RPS6Ser240/244 remained elevated above basal values at 300min in EXFED only (p=0.04). Importantly, a strong positive correlation was observed between RPS6Ser240/244 phosphorylation measured by immunoblotting and IF (r=0.85, p<0.001) showing our novel IF method is a valid readout of mTORC1 activity. In central regions of skeletal muscle fibers, p-RPS6Ser240/244 phosphorylation was only elevated in EXFED at 120min. In the outer 5.5µm of fibers (periphery), mTORC1 activity increased at 120min in both conditions (p<0.03) but to a greater extent in EXFED (p=0.002), remaining elevated above basal levels at 300min only in EXFED (p=0.02). Similarly, the peripheral-to-central ratio of RPS6Ser240/244 phosphorylation was elevated above basal values in both conditions at 120min and remained elevated only in EXFED at 300min, suggesting mTORC1 activity occurs to a greater extent in the periphery of skeletal muscle fibers. In conclusion, we have optimized and validated an IF technique to investigate mTORC1 activity in a region-specific manner in human skeletal muscle. We show that, following physiological anabolic stimuli, mTORC1 activity occurs to a greater extent in the periphery of fibers, regions where mTORC1 is commonly visualized in humans and protein synthesis is observed in animal models. These findings extend our knowledge regarding the spatial regulation of mTORC1 in human skeletal muscle and provide a novel method to investigate mTORC1 dynamics in various populations.

SQSTM1/p62 Inhibits whereas Nrf2 Promotes Tumorigenesis by Inducing Cell Population Remodeling and Metabolic Reprograming in Mouse Livers with mTORC1 Activation and Defective Autophagy

Tsc1 (Tuberous Sclerosis 1) and Tsc2 are tumor suppressor genes and negative regulators of mTORC1, and the mutation of Tsc1 or Tsc2 is associated with various cancers including hepatocellular carcinoma (HCC). mTORC1 activation promotes anabolic protein synthesis and inhibits catabolic autophagy process. Either hepatic persistent activation of mTORC1 due to the loss of Tsc1 or impaired hepatic autophagy due to the loss of Atg5 leads to spontaneous liver tumorigenesis in mice. However, how autophagy impacts mTORC1-mediated liver metabolic changes and tumorigenesis is less clear. Here we show that deletion of Atg5 in liver-specific Tsc1 knockout (L-Tsc1 KO) mice inhibited liver tumorigenesis but increased mortality of L-Tsc1 KO mice. Histological and unbiased RNA-seq as well as metabolomic analysis revealed that lack of hepatic autophagy dramatically induced the remodeling of liver cell population and hepatic metabolic reprogramming in L-Tsc1 KO mice. RNA-seq analysis revealed increased cholangiocyte, hepatic stellate cell and macrophage but decreased hepatocyte gene signatures in liver-specific Tsc1 and Atg5 double knockout (DKO) mice. Histologically, Atg5 and Tsc1 DKO mice had increased hepatomegaly, ductular reactions, increased number of macrophage/Kupffer cells and infiltrated neutrophils as well as massive fibrosis, and the DKO mice died at the age of approximately 8-months-old with no detectable liver tumors. Metabolomic analysis revealed marked increased hepatic glutathione and glutathione metabolites but decreased hepatic levels of glucose and lipid with accumulation of TCA cycle intermediate metabolites and fatty acid oxidation intermediates in Atg5 and Tsc1 DKO mice. Deletion of hepatic p62 partially but deletion of Nrf2 almost completely rescued the liver cell type remodeling and metabolic reprogramming and the mortality of Atg5 and Tsc1 DKO mice. Deletion of p62 promoted whereas deletion of Nrf2 suppressed the liver tumorigenesis of Atg5 and Tsc1 DKO mice. These results reveal a critical role of Nrf2 in regulating liver cell type remodeling and metabolic reprogramming in mice lack of hepatic autophagy with mTORC1 activation. Our data may provide a mechanistic basis for a precision medicine to target Nrf2 pathway for treating as subset of HCC, especially in HCV- or alcohol-associated liver cancers that may have defective autophagy and mTOR activation.

Spatial Compartmentalization of mTORC1 to the Outer Mitochondrial Membrane to Regulate Mitochondrial Metabolism

mTOR complex 1 (mTORC1) integrates inputs from multiple pathways, such as PI3K/Akt, AMPK, Ca2+, and cAMP/PKA, and senses diverse signals to regulate cell growth, protein translation, and proliferation. Although the subcellular compartmentalization of signaling pathways can enhance signal specificity and efficiency, the spatial regulation of mTORC1 and its specific functions are still poorly understood. A pool of mTORC1 associated with the outer mitochondrial membrane has previously been identified but the function of mitochondrial mTORC1 is largely unknown. Previous studies have linked mTORC1 inhibition to changes in mitochondrial metabolism through its effect on transcription or translation, but a direct role for mTORC1 at the mitochondria is not yet elucidated. By targeting our FRET-based mTORC1 activity reporter, TORCAR, to the outer mitochondrial membrane, we found that treating cells with platelet-derived growth factor leads to a gradual increase in mitochondrial mTORC1 activity, and that this response is abrogated with rapamycin pre-treatment. In pursuit of identifying the function of mTORC1 at the outer mitochondrial membrane, we have also developed a genetically encodable peptide-based mTORC1 inhibitor, TerminaTOR, targeted to the outer mitochondrial membrane for specific subcellular inhibition of mTORC1. Using these molecular tools, we set out to examine whether mitochondrial mTORC1 activity is critical for mitochondria-governed cellular homeostasis. We hypothesize that mitochondrial mTORC1 plays a critical role in altering mitochondrial metabolism upon stimulation with growth factor. We aim to further elucidate the functions of this activity by selectively inhibiting mitochondrial mTORC1 using mitochondria-targeted TerminaTOR and quantifying changes in mitochondrial metabolism. We will also identify mitochondrial proteins that may interact with mTORC1 to contribute to these metabolic changes. This research will increase our understanding of the role that mTORC1 may play in diseases associated with altered metabolism or mitochondrial dysfunction.

Divergent transcriptional response to mTORC1‐mediated nutrient signaling in mouse liver and skeletal muscle

The mechanistic target of rapamycin complex 1 (mTORC1) has a central role in balancing nutrients and growth factors with metabolic processes. Many of its mediated processes can vary between tissues to meet specific metabolic demands. Here we examine this process in mouse liver and skeletal muscle to characterize the tissue specific transcriptional response to the mTORC1 inhibitor rapamycin. We performed RNA-seq analysis on fasted tissue over a refeeding time course with and without rapamycin. Since the canonical regulation of mTORC1 is active in response to nutrient abundance and inactive during nutrient deprivation, our goal was to capture the transcriptional elements that are regulated by mTORC1 activation in both tissues. We observed altered expression of major metabolic pathways in the liver response to rapamycin but not in skeletal muscle. In addition, our time course analysis revealed a distinct increase in expression shift duration in response to rapamycin. Together our data show a clear tissue specific transcriptional response to rapamycin and a differential expression pattern that's detection was made possible through our analysis of multiple time points.

A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling.

Amino acid transporters play a vital role in metabolism and nutrient signaling pathways. Typically, transport activity is investigated using single substrates and competing amounts of other amino acids. We used GC-MS and LC-MS for metabolic screening of Xenopus laevis oocytes expressing various human amino acid transporters incubated in complex media to establish their comprehensive substrate profiles. For most transporters, amino acid selectivity matched reported substrate profiles. However, we could not detect substantial accumulation of cationic amino acids by SNAT4 and ATB0,+ in contrast to previous reports. In addition, comparative substrate profiles of two related sodium neutral amino acid transporters known as SNAT1 and SNAT2, revealed the latter as a significant leucine accumulator. As a consequence, SNAT2, but not SNAT1, was shown to be an effective activator of the eukaryotic cellular growth regulator mTORC1. We propose, that metabolomic profiling of membrane transporters in Xe nopus laevis oocytes can be used to test their substrate specificity and role in intracellular signaling pathways.