Inhibition Of mTORC1 Activity Research Articles

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.

Abstract SP139: HER2 targeted therapy: Determinants of response and mechanisms of resistance

Abstract HER2-positive (+) breast cancer (BC), accounting for 15-20% of all BCs, is characterized by overexpression, mostly via gene amplification, of HER2. HER2 is a key member of the HER family of 4 tyrosine kinase receptors. Multiple clinically available HER2-targeted therapies, including monoclonal antibodies, tyrosine kinase inhibitors (TKIs), and antibody-drug conjugates have revolutionized the outcome of patients with HER2+ BC. Despite these effective therapies, intrinsic and acquired resistance still occurs, posing a major challenge in the clinical management of this disease. A better understanding of the determinants of response and mechanisms of resistance may help develop personalized treatment approaches and new strategies to overcome resistance. Tumors that are truly addicted to HER2, clinically reflected especially under chemotherapy-sparing HER2-targeted therapy regimens, are associated with high and homogeneous levels of HER2 gene amplification, protein, and activity. Even in these HER2-addicted tumors, the efficacy of anti-HER2 therapy can be jeopardized by deregulations in the downstream PI3K pathway (e.g., PIK3CA mutations), which can lead to constitutive activation of the PI3K/AKT pathway and resistance. Given the functional redundancy of signaling from multiple HER receptor dimers and compensatory signaling within the pathway, dual anti-HER2 therapy has proven superior to single agents in achieving a more comprehensive blockade of the entire HER receptor layer and in anti-tumor efficacy. Further, in the HER2+ tumors that co-express ER, an unblocked, re-expressed and/or reactivated ER signaling can provide alternative proliferative and survival signals to evade sustained HER2 blockade, thus underscoring the need for concurrent blockade of HER2 and ER signaling. Nevertheless, effective inhibition of HER2 might prove challenging in some cases due to molecular masking of the HER receptors (e.g., mucins) or due to the de novo presence or acquisition of genetic, epigenetic or post-translational alterations in HER2 itself, including activating HER2 mutations (e.g., L755S), and p95HER2. We recently reported that acquired resistance to HER2-targeted therapy, especially TKIs, is mediated by the common HER2 L755S mutation, the clinical importance of which is underscored by the observation that this and other HER mutations are further enriched in the metastatic lesions compared to primary HER2+ tumors. On the other hand, when HER2 does remain effectively inhibited under potent HER2-targeted therapy, resistance can arise due to the upregulation of alternative escape pathways that transmit proliferative stimuli. These include activation of other receptor tyrosine kinases (e.g., AXL, FGFR), other downstream/intracellular signaling (e.g., SRC, YES1), or metabolic pathways (e.g., FASN and mevalonate pathways). Our recent data suggest that the mevalonate pathway offers an escape mechanism by providing alternative signaling through the YAP/TAZ-mTORC1-survivin axis to activate a transcriptional program that promotes resistant cell proliferation and survival, which can be overcome using inhibitors of mevalonate pathway (e.g., statins). Importantly, activation of the key cell cycle regulator cyclin D1/CDK4 complex has been shown to mediate resistance to HER2-targeted therapy and that CDK4/6 inhibitors, at least partly by also inhibiting mTORC1 activity, can overcome this resistance. Finally, the role of tumor microenvironment, including host immune components (e.g., TILs) and extracellular matrix components signaling via integrins, have been shown to play a role in modulating tumor response to treatment and in resistance. Together, these findings suggest new strategies to enhance sensitivity and overcome resistance to HER2-targeted therapy, some of which are already under clinical development. Citation Format: R Schiff, J Veeraraghavan, C De Angelis, C Osborne, MF Rimawi. HER2 targeted therapy: Determinants of response and mechanisms of resistance [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP139.

Methionine controls insulin/mammalian target of rapamycin complex 1 activity by modulating tuberous sclerosis complex 2 stability

Tuberous sclerosis complex 2 (TSC2) is a tumor-suppressor protein that is partially regulated by insulin, energy, oxygen, and growth factors. Mutations in the TSC2 gene and loss of TSC2 promote cell growth by the mammalian target of rapamycin complex 1 (mTORC1) activation. Furthermore, S-adenosylmethionine (SAM) sensor upstream of mTORC1 indirectly inhibits mTORC1 activity via the methionine metabolite SAM. Here, we investigated the effects of methionine on insulin/TSC2/mTORC1 activity. Our results showed that methionine affected TSC2 stability and abolished TSC2 localization to the lysosome. Moreover, activation of insulin signaling contributed to TSC2 degradation in a methionine deprivation-dependent manner. Thus, methionine and insulin crosstalk occurred via TSC2.

The GATOR-Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids.

The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR-Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase-independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.

Abstract A16: Suppressor of morphogenesis in genitalia 1 (SMG1) is a novel negative regulator of mammalian target of rapamycin complex 2 (mTORC2)

Abstract SMG1 is a member of the PI-3 kinase-like kinase family of proteins, which includes ataxia telangiectasia mutated (ATM), Dna-dependent protein kinase (DNA-PK) and mTOR. Our previous work demonstrated that SMG1 loss in mice increased cancer development, particularly blood cancers. The mechanism by which SMG1 loss enhances cancer development is currently unknown. Here we show that SMG1 acts to negatively regulate mTORC2 activity. In cells from mice lacking one or both alleles of SMG1 or following siRNA knockdown of SMG1, phosphorylation of Akt or Protein kinase C alpha is significantly increased. Using downregulation of SMG1 followed by treatment with IGF1 or serum to induce signaling from mTORC2 and 1 respectively, downstream reporters of mTORC1 (P-S6 Kinase Thr389) and mTORC2 (P-AktSer473) were upregulated. Upregulation of mTORC1 is at least partly due to feedback from mTORC2 as P-S6KThr389 upregulation can be partially inhibited by Akt inhibition. Phosphorylation of both can be completely inhibited by the mTOR inhibitor INK128. Phosphorylation of mTOR at Ser2481, a marker of mTORC2 complex stability, increased at both baseline and upon growth factor stimulation after SMG1 knockdown. No effect of SMG1 knockdown was seen upstream of mTORC2. Additionally, overexpression of SMG1 but not a kinase dead mutant limited mTORC2 signaling. SMG1 can be immunoprecipitated with members of the mTORC2 complex including mTOR, Rictor and Sin1.1 but no Raptor or other Sin1 isoforms are detected in these immunoprecipitations. This indicates that SMG1 can inhibit mTORC2 activity by interacting with Sin1.1 containing complexes. The unique exon present in Sin1.1 contains a putative SMG1 phosphorylation site. We are currently investigating whether SMG1-mediated phosphorylation at this site mediates inhibition of mTORC2 activity. We have further examined the relationship between SMG1 expression and mTOR activity in patients with chronic lymphocytic leukemia (CLL). We recruited 70 patients from Liverpool Hospital and isolated circulating tumor cells from their blood. We analyzed SMG1 expression level and extent of Akt Ser473 phosphorylation in these cells. SMG1 expression inversely correlated with phosphorylation of Akt, indicating that SMG1 also acts to negatively regulate mTORC2 in cancer cells. Overall, we have identified a novel mechanism for regulation of mTORC2 activity by SMG1, which may explain why SMG1 loss enhances cancer development. Citation Format: Alexander James, Patricia Rebeiro, Uda Ho, C. Soon Lee, Nicole Caixeiro, Martin F. Lavin, Tara L. Roberts. Suppressor of morphogenesis in genitalia 1 (SMG1) is a novel negative regulator of mammalian target of rapamycin complex 2 (mTORC2) [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A16.

Abstract A10: Exploring the interplay between the purinosome, a multienzyme, purine biosynthetic machine, and the Rheb-mTORC1 signaling axis

Abstract Pharmacologic agents that interfere with nucleotide metabolism constitute an important class of anticancer agents. Recent studies demonstrated that mTOR complex 1 (mTORC1) inhibitors suppress de novo biosynthesis of pyrimidine and purine nucleotides. We have previously demonstrated that mTORC1 itself is suppressed by drugs that reduce intracellular purine nucleotide pools. Cellular treatment with AG2037, an inhibitor of the purine biosynthetic enzyme, GARFT, profoundly inhibits mTORC1 activity via a reduction in the level of GTP-bound Rheb, an obligate upstream activator of mTORC1, in part due to a reduction in intracellular guanine nucleotides. Purines are synthesized intracellularly in dynamic metabolic structures known as the purinosome. Our emerging data suggest that Rheb associates with the purinosome and treatment with AG2037 disrupts this interaction. We hypothesize that Rheb senses the availability of purines at the site of the purinosome and communicates purine sufficiency to activate mTORC1. The results of this project will increase our understanding of the regulation of purine biosynthesis and mTORC1 activation, two key processes in cell proliferation and cancer. Citation Format: Natasha Emmanuel, Elyssa Bernfeld, Shoba Ragunathan, Fang Wang, Qin Shan, Andreas Giannakou, Nanni Huser, Guixian Jin, Jeremy Myers, Keziban Unsal-Kacmaz, Robert T. Abraham. Exploring the interplay between the purinosome, a multienzyme, purine biosynthetic machine, and the Rheb-mTORC1 signaling axis [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A10.

Tacrolimus, a rapamycin target protein inhibitor, exerts anti-cystic echinococcosis effects both in vitro and in vivo

In view of a growing need for new treatment options for human cystic echinococcosis (CE), we aimed to investigate the efficacy of mTOR pathway inhibitors against CE in vitro and in vivo. Among the seven mTOR inhibitors evaluated, tacrolimus (TAC) showed significant dose- and time-dependent killing of cultured protoscoleces and cysts in vitro. Notably, the oral administration of TAC (4 mg/kg/day) to CE mice model highly effectively reduced both the weight and number of parasitic cysts. Transmission electron microscopy revealed that TAC destroys the ultrastructure of cysts, both in vitro and in vivo. Furthermore, TAC had no significant effect on blood glucose, body weight, liver, or kidney functions in mice. We further observed that the ATP levels and glucose content of cysts reduced upon TAC treatment, indicating that inhibiting mTORC1 activity possibly affects glucose metabolism in the cysts of mice. Based on our experimental data, TAC emerged as a promising anti-cyst drug that efficiently inhibits the growth of cysts.

Phagosome-regulated mTOR signalling during sarcoidosis granuloma biogenesis.

Sarcoidosis and tuberculosis are granulomatous pulmonary diseases characterised by heightened immune reactivity to Mycobacterium tuberculosis antigens. We hypothesised that an unsupervised analysis comparing the molecular characteristics of granulomas formed in response to M. tuberculosis antigens in patients with sarcoidosis or latent tuberculosis infection (LTBI) would provide novel insights into the pathogenesis of sarcoidosis. A genomic analysis identified differentially expressed genes in granuloma-like cell aggregates formed by sarcoidosis (n=12) or LTBI patients (n=5) in an established in vitro human granuloma model wherein peripheral blood mononuclear cells were exposed to M. tuberculosis antigens (beads coated with purified protein derivative) and cultured for 7 days. Pathway analysis of differentially expressed genes identified canonical pathways, most notably antigen processing and presentation via phagolysosomes, as a prominent pathway in sarcoidosis granuloma formation. The phagolysosomal pathway promoted mechanistic target of rapamycin complex 1 (mTORc1)/STAT3 signal transduction. Thus, granuloma formation and related immune mediators were evaluated in the absence or presence of various pre-treatments known to prevent phagolysosome formation (chloroquine) or phagosome acidification (bafilomycin A1) or directly inhibit mTORc1 activation (rapamycin). In keeping with genomic analyses indicating enhanced phagolysosomal activation and predicted mTORc1 signalling, it was determined that sarcoidosis granuloma formation and related inflammatory mediator release was dependent upon phagolysosome assembly and acidification and mTORc1/S6/STAT3 signal transduction. Sarcoidosis granulomas exhibit enhanced and sustained intracellular antigen processing and presentation capacities, and related phagolysosome assembly and acidification are required to support mTORc1 signalling to promote sarcoidosis granuloma formation.

Decanoic acid inhibits mTORC1 activity independent of glucose and insulin signaling

Low-glucose and -insulin conditions, associated with ketogenic diets, can reduce the activity of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, potentially leading to a range of positive medical and health-related effects. Here, we determined whether mTORC1 signaling is also a target for decanoic acid, a key component of the medium-chain triglyceride (MCT) ketogenic diet. Using a tractable model system, Dictyostelium, we show that decanoic acid can decrease mTORC1 activity, under conditions of constant glucose and in the absence of insulin, measured by phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). We determine that this effect of decanoic acid is dependent on a ubiquitin regulatory X domain-containing protein, mediating inhibition of a conserved Dictyostelium AAA ATPase, p97, a homolog of the human transitional endoplasmic reticulum ATPase (VCP/p97) protein. We then demonstrate that decanoic acid decreases mTORC1 activity in the absence of insulin and under high-glucose conditions in ex vivo rat hippocampus and in tuberous sclerosis complex (TSC) patient-derived astrocytes. Our data therefore indicate that dietary decanoic acid may provide a new therapeutic approach to down-regulate mTORC1 signaling.

Abstract 5853: Identification of TSC1 and TSC2 as potential determinants of sensitivity to trastuzumab emtansine

Abstract Trastuzumab emtansine (T-DM1, Kadcyla) is an antibody-drug conjugate used in the treatment of HER2-positive breast cancer. However, its use is limited by acquired and intrinsic resistance, the mechanisms of which are not well understood. Further knowledge of T-DM1 resistance may provide new combination strategies or therapeutic targets to overcome resistance or new predictive biomarkers to identify the patients most likely to benefit from T-DM1 therapy. To discover genes responsible for T-DM1 sensitivity and resistance in an unbiased manner, we have conducted CRISPR/Cas9 functional genomics screens by a two-stage process. Firstly, we performed whole genome screens in MDA-MB-361 and MDA-MB-453 cells transduced with Cas9 and the GeCKOv2 lentiviral library that were exposed to T-DM1 and its effector DM1 for 8-13 weeks. Gene knockouts enriched or depleted in response to T-DM1 or DM1 treatment in either cell line were identified by sequencing of genomic DNA and differentially expressed genes by RNA sequencing, revealing 599 candidate genes of T-DM1 sensitivity. For high-throughput validation of the 599 genes, we developed a custom library of 2539 guide RNAs (gRNAs) to target these 599 genes, plus non-targeting controls. Cas9-expressing MDA-MB-361 cells were transduced with the custom library and exposed to T-DM1 for 28 days. MAGeCK analysis of gRNA sequencing revealed 11 genes that were significantly enriched and one gene that was significantly depleted at a false discovery rate (FDR) of <0.1. Two of the top hits in the secondary screen were two genes whose loss is known to promote T-DM1 resistance: ERBB2 (HER2) and SLC46A3 (P<8 × 10−5; FDR <0.007). Other top hits were TSC1 and TSC2 (P<3 × 10−6; FDR= 0.0004); which are both tumor suppressor genes and negative regulators of mTOR complex 1 (mTORC1). For subsequent validation, we have generated TSC2 knockout cell pools, which were more resistant to T-DM1 than wildtype cells in a competition growth assay. Knockout clones have been isolated and are being tested for T-DM1 resistance. Since mTOR inhibitors can phenocopy TSC1 and TSC2 by inhibiting mTORC1 activity, we have also evaluated T-DM1 in combination with the mTOR inhibitor KU-0063794 in a sulforhodamine B assay in MDA-MB-361 and MDA-MB-453 cells. Each agent potently inhibited cell proliferation and demonstrated synergistic anti-proliferative activity in combination. Together, our results suggest that TSC1 and TSC2 knockout may promote T-DM1 resistance and that targeting mTOR may be an effective strategy to overcome T-DM1 resistance. Citation Format: Francis W. Hunter, Barbara A. Lipert, Kyla N. Siemens, Aziza Khan, Hilary R. Barker, Troy W. Ketela, William R. Wilson, Tet-Woo Lee, Stephen M. Jamieson. Identification of TSC1 and TSC2 as potential determinants of sensitivity to trastuzumab emtansine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5853.

Abstract 671: mTORC1 suppression in Paneth cells prevents tumors in a mouse model of intestinal cancer

Abstract The mechanistic (or mammalian) target of rapamycin, mTOR, is a highly conserved PI3K-related kinase that regulates cell growth, proliferation, survival, transcription, and protein synthesis. It forms two different complexes, mTOR complex-1 (mTORC1) and -2 (mTORC2). Rapamycin is an allosteric inhibitor specific to mTORC1 and extends life and health span with chronic use. Previous research from our lab shows that an enteric release formulation of rapamycin, eRapa, enhances the lifespan of mice mutated for adenomatous polyposis coli (Apc) tumor suppressor (ApcMin/+ mice), which normally develop intestinal cancer. These mice model familial adenomatous polyposis (FAP) in humans. eRapa restored a normal (and in 60% of the mice longer) life span in female ApcMin/+ mice. Mutations in Apc, a part of the Wnt/β-catenin pathway, accelerate the initiation of the adeno-carcinoma in FAP, resulting in numerous colorectal polyps at a young age, and if left untreated these polyps progress to colorectal cancer.eRapa treatment prevents polyposis in ApcMin/+ mice but the precise mechanism of action remains to be determined. We used a cross sectional and survival approach to test the preventive effects of eRapa and gain mechanistic insights. Four-week-old male and female animals were put on a diet containing either 42 ppm of eRapa or empty microcapsules. A subset of the animals was sacrificed to harvest tissue for analysis at 16 weeks of age and the rest were allowed to live out their lifespans.eRapa prevented polyposis in the small intestine of ApcMin/+ mice with only a few to no tumors in both males and females. In addition to adenomas in the intestine, ApcMin/+ mice also suffer from severe anemia. Hematocrits from the animals showed anemia in the control mice, however, eRapa treated animals did not have anemia. There was a reduction in phosphorylation of ribosomal protein S6 in the small intestine tissue, showing suppression of mTORC1 activity with eRapa. Histological analysis revealed that this suppression occurred in the Paneth cells of the small intestinal crypt. The Paneth cells, along with the stem cells at the base of the crypt form a niche and signaling between this niche maintains homeostasis in the crypt. Although the polyps are believed to originate from stem cells, our results suggest involvement of Paneth cells in tumor prevention perhaps by signaling changes in the niche. These are novel effects of rapamycin and help define its mechanisms of cancer prevention. Citation Format: Manish Parihar, Sherry G. Dodds, Paul Hasty, Zelton Dave Sharp. mTORC1 suppression in Paneth cells prevents tumors in a mouse model of intestinal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 671.

Treatment of ErbB2 breast cancer by mitochondrial targeting

BackgroundErbB2 breast cancer still remains an unmet need due to primary and/or acquired resistance to current treatment strategies. MEDICA compounds consist of synthetic long-chain α,ω-dicarboxylic acids previously reported to suppress breast cancer in PyMT transgenic mice.MethodsMEDICA efficacy and mode of action in the ErbB2 context was studied in ErbB2 transgenic mice and human breast cancer cells.ResultsMEDICA treatment is shown here to suppress ErbB2 breast tumors and lung metastasis in ErbB2/neu MMTV transgenic mice, to suppress ErbB2/neu xenografts in nod/scid mice, and to suppress survival of AU565 and BT474 human ErbB2 breast cancer cells. Suppression of ErbB2 breast tumors by MEDICA is due to lipid raft disruption with loss of ErbB family members, including EGFR, ErbB2, and ErbB3. In addition, MEDICA inhibits mTORC1 activity, independently of abrogating the ErbB receptors and their signaling cascades. The double hit of MEDICA in abrogating ErbB and mTORC1 is partly accounted for by targeting mitochondria complex I.ConclusionsMitochondrial targeting by MEDICA suppresses ErbB2 breast tumors and metastasis due to lipid raft disruption and inhibition of mTORC1 activity. Inhibition of mTORC1 activity by MEDICA avoids the resistance acquired by canonical mTORC1 inhibitors like rapalogs or mTOR kinase inhibitors.

The Gαh/phospholipase C-δ1 interaction promotes autophagosome degradation by activating the Akt/mTORC1 pathway in metastatic triple-negative breast cancer.

Lung metastasis (LM) is commonly found in triple-negative breast cancer (TNBC); however, the molecular mechanism underlying TNBC metastasis to lungs remains largely unknown. We thus aimed to uncover a possible mechanism for the LM of TNBC. Here we show that the phosphorylation of Akt and mTORC1 was positively but the autophagy activity was negatively correlated with endogenous Gαh levels and cell invasion ability in TNBC cell lines. Whereas the knockdown of Gαh, as well as blocking its binding with PLC-δ1 by a synthetic peptide inhibitor, in the highly invasive MDA-MB231 cells dramatically suppressed Akt/mTORC1 phosphorylation and blocked autophagosome degradation, the overexpression of Gαh in the poorly invasive HCC1806 cells enhanced Akt/mTORC1 phosphorylation but promoted autophagosome degradation. The pharmaceutical inhibition of autophagy initiation by 3-methyladenine was found to rescue the cell invasion ability and LM potential of Gαh-silenced MDA-MB231 cells. In contrast, the inhibition of mTORC1 activity by rapamycin suppressed autophagosome degradation but mitigated the cell invasion ability and LM potential of Gαh-overexpressing HCC1806 cells. These findings demonstrate that the induction of autophagy activity or the inhibition of Akt-mTORC1 axis provides a useful strategy to combat the Gαh/PLC-δ1-driven LM of TNBC.

Skeletal Muscle Raptor And Tsc1/2 Differential Responses To Activity And Fasting In The Tumor Environment

Cancer is a debilitating disease that is often accompanied by decreased physical activity and chronic energetic stress that disrupts muscle proteostasis. Skeletal muscle protein turnover is highly sensitive to changes in feeding and activity. Raptor serine 792 and TSC1/2 serine 1387 phosphorylation sites, when activated, can inhibit mTORC1 activation leading to suppressed anabolic signaling, and has been implicated in the regulation of skeletal muscle wasting with cancer. PURPOSE: To examine the effect of a 12-hour fast on the phosphorylation of Raptor and TSC1/2 in male ApcMin/+ mice, and if voluntary wheel activity can alter the fasting response. METHODS: Male C57BL/6 (B6, N=24) and ApcMin/+ (MIN, N=31) mice were either sacrificed under ad libitum conditions (B6-fed, M-fed), fasted for 12hrs (B6-fast, M-fast), or fasted for 12hrs following 4wks of voluntary wheel running (B6+W, M+W). TSC1/2 serine 1387 and Raptor serine 792 were measured in the gastrocnemius muscle as phosphorylation to total ratio by western blot. Protein synthesis was measured by puromycin incorporation. RESULTS: All MIN mice exhibited body weight loss (p<0.001) and reduced gastrocnemius mass (p<0.001) when compared to all B6 mice. Raptor phosphorylation (pRaptor) was induced in M-fast compared to M-fed (p=0.019), but there was no change in B6+fast compared to B6-fed (p=0.414). TSC1/2 phosphorylation was induced in M-fast compared to M-fed (p=0.001) and in B6-fast to B6-fed (p=0.039). Puromycin was trending to be reduced in M-fast compared to MIN-fed (p=0.070), but there was no change in B6-fast to B6-fed (p=0.323). Raptor phosphorylation was not different in M-fast compared to M+W (p=0.302), however pRaptor was reduced in B6+W compared B6-fast (p=0.028). TSC1/2 phosphorylation (p=0.001) and puromycin (p=0.016) were induced in MIN+W compared to M-fast, with no changes in the B6-fast to B6+W (p=0.360, p=0.196; respectively). CONCLUSIONS: Our results provide evidence that the cancer environment disrupts anabolic suppression of mTORC1 in skeletal muscle. Interestingly, TSC1/2 phosphorylation was sensitive to fasting independent of the tumor environment. Wheel activity induced protein synthesis independent of Raptor phosphorylation therefore, further studies are warranted to determine this specific mechanism.

Carbohydrate response element-binding protein regulates lipid metabolism via mTOR complex1 in diabetic nephropathy.

Lipid deposition caused by the disorder of renal lipid metabolism is involved in diabetic nephropathy (DN). Carbohydrate response element-binding protein (ChREBP) is a key transcription factor in high glucose-induced cellular fat synthesis. At present, the regulation and mechanism of ChREBP on fat metabolism in diabetic kidneys are still unclear. In this study, we showed that lack of ChREBP significantly improved renal injury, inhibited oxidative stress, lipid deposition, fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC) and thioredoxin-interacting protein (TXNIP) expression, as well as the activity of mammalian target of rapamycin complex 1 (mTORC1) in diabetic kidneys. Meanwhile, ChREBP deficiency upregulated the expression of peroxisome proliferator-activated receptor-α (PPARα), carnitine palmitoyltransferaser 1A (CPT1A) and acyl-coenzyme A oxidase 1 (ACOX1) in diabetic kidneys. In vitro, knockdown of ChREBP attenuated lipid deposition, mTORC1 activation, and expression of FASN and ACC, increased PPARα, CPT1A, and ACOX1 expression in HK-2 cells and podocytes under high glucose (HG) conditions. Moreover, HG-induced lipid deposition, increased expression of FASN and ACC and decreased expression of PPARα, CPT1A, and ACOX1 were reversed by rapamycin, a specific inhibitor of mTORC1, in HK-2 cells. These results indicate that ChREBP deficiency alleviates diabetes-associated renal lipid accumulation by inhibiting mTORC1 activity and suggest that reduction of ChREBP is a potential therapeutic strategy to treat DN.

Human herpesvirus 6A promotes glycolysis in infected T cells by activation of mTOR signaling.

Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus worldwide. However, whether and how HHV-6 infection influences the metabolic machinery of the host cell to provide the energy and biosynthetic resources for virus propagation remains unknown. In this study, we identified that HHV-6A infection promotes glucose metabolism in infected T cells, resulting in elevated glycolytic activity with an increase of glucose uptake, glucose consumption and lactate secretion. Furthermore, we explored the mechanisms involved in HHV-6A-mediated glycolytic activation in the infected T cells. We found increased expressions of the key glucose transporters and glycolytic enzymes in HHV-6A-infected T cells. In addition, HHV-6A infection dramatically activated AKT-mTORC1 signaling in the infected T cells and pharmacological inhibition of mTORC1 blocked HHV-6A-mediated glycolytic activation. We also found that direct inhibition of glycolysis by 2-Deoxy-D-glucose (2-DG) or inhibition of mTORC1 activity in HHV-6A-infected T cells effectively reduced HHV-6 DNA replication, protein synthesis and virion production. These results not only reveal the mechanism of how HHV-6 infection affects host cell metabolism, but also suggest that targeting the metabolic pathway could be a new avenue for HHV-6 therapy.

Dehydroepiandrosterone activates 5′-adenosine monophosphate-activated protein kinase and suppresses lipid accumulation and adipocyte differentiation in 3T3-L1 cells

Substantial evidence has linked dehydroepiandrosterone (DHEA) levels to the anti-obesity and anti-diabetic effects of exercise. While 5′-adenosine monophosphate-activated protein kinase (AMPK) is a negative regulator of adipocyte differentiation and lipid accumulation, activation of mammalian target of rapamycin complex 1 (mTORC1), which is inhibited by AMPK, is required for adipocyte differentiation and positively regulates lipid accumulation. DHEA treatment activates the AMPK pathway in C2C12 myotubes. Hence, DHEA addition to preadipocytes and adipocytes might activate AMPK and inhibit mTORC1, resulting in the inhibition of adipogenesis and lipid accumulation. Therefore, we investigated the effect of DHEA on the AMPK pathway, mTORC1 activity, adipocyte differentiation, and lipid accumulation in 3T3-L1 cells. DHEA suppressed lipid accumulation and adipogenic marker expression during differentiation. It also activated AMPK signaling in preadipocytes and adipocytes and suppressed mTORC1 activity during differentiation. These results suggest that the activation of the AMPK pathway and inhibition of mTORC1 activity may mediate the anti-obesity effect of DHEA, providing novel molecular-level insights into its physiological functions.

Extracellular Acidosis and mTOR Inhibition Drive the Differentiation of Human Monocyte-Derived Dendritic Cells

During inflammation, recruited monocytes can differentiate either into macrophages or dendritic cells (DCs); however, little is known about the environmental factors that determine this cell fate decision. Low extracellular pH is a hallmark of a variety of inflammatory processes and solid tumors. Here, we report that low pH dramatically promotes the differentiation of monocytes into DCs (monocyte-derived DCs [mo-DCs]). This process is associated with a reduction in glucose consumption and lactate production, the upregulation of mitochondrial respiratory chain genes, and the inhibition of mTORC1 activity. Interestingly, we also find that both serum starvation and pharmacological inhibition of mTORC1 markedly promote the differentiation of mo-DCs. Our study contributes to better understanding the mechanisms that govern the differentiation of monocytes into DCs and reveals the role of both extracellular pH and mTORC1 as master regulators of monocyte cell fate.

Infrared-A Irradiation-induced Inhibition of Human Keratinocyte Proliferation and Potential Mechanisms.

Infrared‐A (IRA), which can penetrate deeply into the human skin, is a major component of solar radiation and is recognized to promote photoaging of human dermis. To our knowledge, however, the cellular and molecular consequences of human epidermis exposure to IRA have not been clarified. Thus, we investigated whether IRA inhibits the proliferation of normal human epidermal keratinocytes (NHEKs). IRA irradiation ed in cell cycle arrest at G1 and a dose‐dependent reduction in the proliferation of NHEKs. We found that mechanistic target of rapamycin complex 1 (mTORC1) was initially inactivated during IRA irradiation due to the formation of stress granules (SGs), and this inactivation was maintained for at least 6 h after irradiation due to Akt dephosphorylation. Furthermore, repeated exposure of human skin equivalents to IRA led to marked thinning of the epidermal cell layer. In conclusion, IRA irradiation inhibits mTORC1 activity possibly through two molecular mechanisms involving SG formation in the early‐phase and subsequent Akt dephosphorylation. This sequential mechanism seems to cause G1 cell cycle arrest and a reduction in cell proliferation, supporting the hypothesis that the decreased proliferation of basal keratinocytes that occurs during skin aging might be partly attributable to IRA radiation.

Tuberous sclerosis 1 (Tsc1) mediated mTORC1 activation promotes glycolysis in tubular epithelial cells in kidney fibrosis

Energy reprogramming to glycolysis is closely associated with the development of chronic kidney disease. As an important negative regulatory factor of the mammalian target of rapamycin complex 1 (mTORC1) signal, tuberous sclerosis complex 1 (Tsc1) is also a key regulatory point of glycolysis. Here, we investigated whether Tsc1 could mediate the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells. We induced mTORC1 signal activation in tubular epithelial cells in kidneys with fibrosis via unilateral ureteral occlusion. This resulted in increased tubular epithelial cell proliferation and glycolytic enzyme upregulation. Prior incubation with rapamycin inhibited mTORC1 activation and abolished the enhanced glycolysis and tubular epithelial cell proliferation. Furthermore, knockdown of Tsc1 expression promoted glycolysis in the rat kidney epithelial cell line NRK-52E. Specific deletion of Tsc1 in the proximal tubules of mice resulted in enlarged kidneys characterized by a high proportion of proliferative tubular epithelial cells, dilated tubules with cyst formation, and a large area of interstitial fibrosis in conjunction with elevated glycolysis. Treatment of the mice with the glycolysis inhibitor 2-deoxyglucose notably ameliorated tubular epithelial cell proliferation, cystogenesis, and kidney fibrosis. Thus, our findings suggest that Tsc1-associated mTORC1 signaling mediates the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells.