Negative Regulator Of Mammalian Target Research Articles

Abstract P3194: Redd1 Is Required For Glucose Metabolism And Insulin Sensitivity In Cardiomyocytes

Type II diabetes (TIID) is characterized by insulin resistance (IR) and doubles a patient’s risk for cardiovascular disease (CVD). The pathogenesis of CVD in patients with TIID, or diabetic cardiomyopathy (DC), is unclear. Regulated in development and DNA damage (REDD)1 is a negative regulator of mammalian target of rapamycin complex (mTORC)1. Notably, deletion of REDD1 leads to systemic IR in mice. REDD1 is, therefore, necessary for insulin sensitivity, however the mechanisms required remain unclear. We hypothesized that REDD1 is required for glucose oxidation and, therefore, insulin sensitivity in cardiomyocytes. To examine the role of REDD1 in cardiomyocyte glucose metabolism and energetics, we cultured wild type (WT) AC16 cardiomyocytes or those with REDD1 deletion with 5.5 mM glucose and examined cellular respiration. The data demonstrate compromised respiration, as indicated by reduced maximal respiratory capacity in REDD1-null versus WT cells (1.5-fold±0.214). Importantly, western blotting confirmed the absence of REDD1 in cells with REDD1 deletion, as well as increased phosphorylation of eukaryotic translation initiation factor 4E-binding protein (p4E-BP)1 (Thr37/46) (1.3-fold±0.085), a direct target of mTORC1. Thus, REDD1 inhibits mTORC1 signaling and enhances glucose-driven respiration in cardiomyocytes. To examine the mechanisms by which REDD1 supports glucose utilization, we examined levels of phosphorylated pyruvate dehydrogenase (pPDH) (Ser293). Our data show that pPDH is increased in REDD1-null cells versus WT controls (1.2-fold±0.054), as well as in hearts from REDD1-deficient mice (1.2-fold±0.013), suggesting reduced glucose oxidation. We also observed enhanced extracellular acidification, an indirect measure of glycolysis, in our REDD1-null cells versus WT controls (1.7-fold±0.136). Pyruvate dehydrogenase kinases (PDKs) phosphorylate PDH. In our REDD1-null cells, we observe a significant increase in PDK4 mRNA versus WT controls (8-fold±0.593), as assessed by qPCR and consistent with the diabetic heart. Together, these data suggest that REDD1 inhibits mTORC1 activity and the expression of PDK4, activating PDH, and glucose oxidation as the mechanism by which cardiomyocytes remain insulin sensitive.

NPRL2 Inhibition of mTORC1 Controls Sodium Channel Expression and Brain Amino Acid Homeostasis.

Genetic mutations in nitrogen permease regulator-like 2 (NPRL2) are associated with a wide spectrum of familial focal epilepsies, autism, and sudden unexpected death of epileptics (SUDEP), but the mechanisms by which NPRL2 contributes to these effects are not well known. NPRL2 is a requisite subunit of the GAP activity toward Rags 1 (GATOR1) complex, which functions as a negative regulator of mammalian target of rapamycin complex 1 (mTORC1) kinase when intracellular amino acids are low. Here, we show that loss of NPRL2 expression in mouse excitatory glutamatergic neurons causes seizures before death, consistent with SUDEP in humans with epilepsy. Additionally, the absence of NPRL2 expression increases mTORC1-dependent signal transduction and significantly alters amino acid homeostasis in the brain. Loss of NPRL2 reduces dendritic branching and increases the strength of electrically stimulated action potentials (APs) in neurons. The increased AP strength is consistent with elevated expression of epilepsy-linked, voltage-gated sodium channels in the NPRL2-deficient brain. Targeted deletion of NPRL2 in primary neurons increases the expression of sodium channel Scn1A, whereas treatment with the pharmacological mTORC1 inhibitor called rapamycin prevents Scn1A upregulation. These studies demonstrate a novel role of NPRL2 and mTORC1 signaling in the regulation of sodium channels, which can contribute to seizures and early lethality.

A Retrospective Comparison of In Silico Pharmaceutical Recommendations with Tumor Board Recommendations in Pediatric Oncology

Background and Hypothesis: 
 The objective of this study was to analyze available whole genome sequencing from an adolescent male patient diagnosed with osteosarcoma (OS) in 2014. OS is a primary bone malignancy that most commonly affects the pediatric population. Precision medicine techniques provide new opportunities to improve treatment of OS patients. Pharmaceutical annotation tools such as PharmacoDB and DGIdb can help indicate chemotherapy agents that may benefit patients based on their molecular profiles. We hypothesize that these tools can indicate genome-specific chemotherapy agents for OS after genomic data has been aligned and analyzed. 
 
 Project Methods: 
 A PDX pipeline and retrospective study were performed that identified and compared pharmaceutical treatment options from software tools with the chemotherapy provided. Gene alignment and variant calling were used to process and analyze DNA sequencing data; germline and somatic mutations were also identified. Ensembl VEP was used for variant annotation. PharmacoDB and DGIdb were then applied to identify potentially beneficial medications. 
 
 Results: 
 Gene variant annotation indicated 54 potentially high impact mutations. Of these, DGIdb identified 15 drug-gene interactions. PharmacoDB identified no drugs that target any of the genes containing the 54 high impact mutations. For the entire mutated gene list, DGIdb identified 398 drug-gene interactions. After gene set enrichment, DGIdb identified medications targeting genes of pathways such as “O-glycan processing” and “Diseases of glycosylation”. Potentially harmful variants in the NPRL3 gene were identified. Because NPRL3 is a component of the Gator1 complex that serves as a negative regulator of mammalian target of rapamycin complex 1 (mTORC1), the identified variants in NPRL3 could have played a role in the patient’s OS. 
 
 Potential Impact: 
 This study will foster future collaborations to evaluate the pharmaceutical tool recommendations for this patient’s derived cell lines. These efforts will determine the efficacy of and identify improvements for computational treatment recommendation systems.

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.

Hypoxia-induced regulation of mTOR signaling by miR-7 targeting REDD1.

Oxygen is an important factor mediating cell growth and survival under physiological and pathological conditions. Therefore, cells have well-regulated response mechanisms in the face of changes in oxygen levels in their environment. A subset of microRNAs (miRNAs) termed the hypoxamir has been suggested to be a critical mediator of the cellular response to hypoxia. Regulated in development and DNA damage response 1 (REDD1) is a negative regulator of mammalian target of rapamycin (mTOR) signaling in the response to cellular stress, and is elevated in many cell types under hypoxia, with consequent inhibition of mTOR signaling. However, the underlying posttranscriptional regulatory mechanism by miRNAs that contribute to this hypoxia-induced reduction in REDD1 expression remain unknown. Therefore, the aim of the current study was to identify the miRNAs participating in the hypoxic cellular response by scanning the 3'-untranslated region (3'-UTR) of REDD1 for potential miRNA-binding sites using a computer algorithm, TargetScan. miR-7 emerged as a novel hypoxamir that regulates REDD1 expression and is involved in mTOR signaling. miR-7 could repress REDD1 expression posttranscriptionally by directly binding with the 3'-UTR. Upon hypoxia, miR-7 expression was downregulated in HeLa cells to consequently derepress REDD1, resulting in inhibition of mTOR signaling. Moreover, overexpression of miR-7 was sufficient to reverse the hypoxia-induced inhibition of mTOR signaling. Therefore, our findings suggest miR-7 as a key regulator of hypoxia-mediated mTOR signaling through modulation of REDD1 expression. These findings contribute new insight into the miRNA-mediated molecular mechanism of the hypoxic response through mTOR signaling, highlighting potential targets for tumor suppression.

Tsc1 expression by dendritic cells is required to preserve T-cell homeostasis and response

Dendritic cells (DCs) are pivotal to the induction of adaptive T-cell immune responses. Recent evidence highlights a critical role of tuberous sclerosis complex 1 (Tsc1), a primarily upstream negative regulator of mammalian target of rapamycin (mTOR), in DC development, but whether and how Tsc1 directly regulate mature DC function in vivo remains elusive. Here we show that selective disruption of Tsc1 in DCs results in a lymphoproliferative disorder with the spontaneous activation of T cells. Tsc1 deficiency results in the activation of mTORC1-PPARγ pathway, which leads to the upregulation of neuropilin-1 (Nrp1) expression on DCs to stimulate naive T-cell proliferation. However, Tsc1-deficient DCs have defects in the ability to induce antigen-specific T-cell responses in vitro and in vivo owing to impaired survival during antigen transportation and presentation. Indeed, Tsc1 promotes DC survival through restraining independent mTORC1 and ROS-Bim pathways. Our study identifies Tsc1 as a crucial signaling checkpoint in DCs essential for preserving T-cell homeostasis and response.

Age-dependent changes in the protein expression levels of Redd1 and mTOR in the gerbil hippocampus during normal aging.

Redd1, also known as RTP801/Dig2/DDIT4, is a stress-induced protein and a negative regulator of mammalian target of rapamycin (mTOR). Redd1 is also closely associated with oxidative stress and DNA damage. In the present study, age-related changes in the protein expression levels of mTOR and Redd1 were investigated using immunohistochemistry and western blot in the gerbil hippocampus at postnatal month (PM) 3, 6, 12 and 24. No significant differences were identified in the levels of mTOR among the experimental groups, whereas, the levels of phosphorylated mTOR decreased with age. The protein expression levels of Redd1 were observed to gradually increase with age; in the PM 24 group, the level was significantly increased (~189.2%), compared with the PM 3 group. In addition, Redd1 immunoreactivity was significantly increased in the hippocampal principal neurons of the PM 24 group, including the pyramidal cells in the hippocampus proper and granule cells in the dentate gyrus, compared with the other experimental groups. These results demonstrated that the protein expression of Redd1 in the hippocampus was markedly increased during normal aging, indicating that the age-related increase in the expression of Redd1 may be closely associated with age-related hippocampal change.

MTORC1 inhibition delays growth of neurofibromatosis type 2 schwannoma

Neurofibromatosis type 2 (NF2) is a rare autosomal dominant genetic disorder, resulting in a variety of neural tumors, with bilateral vestibular schwannomas as the most frequent manifestation. Recently, merlin, the NF2 tumor suppressor, has been identified as a novel negative regulator of mammalian target of rapamycin complex 1 (mTORC1); functional loss of merlin was shown to result in elevated mTORC1 signaling in NF2-related tumors. Thus, mTORC1 pathway inhibition may be a useful targeted therapeutic approach. We studied in vitro cell models, cohorts of mice allografted with Nf2(-/-) Schwann cells, and a genetically modified mouse model of NF2 schwannoma in order to evaluate the efficacy of the proposed targeted therapy for NF2. We found that treatment with the mTORC1 inhibitor rapamycin reduced the severity of NF2-related Schwann cell tumorigenesis without significant toxicity. Consistent with these results, in an NF2 patient with growing vestibular schwannomas, the rapalog sirolimus induced tumor growth arrest. Taken together, these results constitute definitive evidence that justifies proceeding with clinical trials using mTORC1-targeted agents in selected patients with NF2 and in patients with NF2-related sporadic tumors.

Disruption of TSC1/2 signaling complex reveals a checkpoint governing thymic CD4 + CD25 + Foxp3 + regulatory T‐cell development in mice

Thymic-derived CD4(+)CD25(+)Foxp3(+) natural regulatory T (nTreg) cells are essential for the maintenance of peripheral immune tolerance. Signaling pathways that drive immature thymic progenitors to differentiate into CD4(+)CD25(+)Foxp3(+) nTreg cells need to be elucidated. The precise role of the TSC1/2 complex, a critical negative regulator of mammalian target of rapamycin (mTOR), in thymic CD4(+)CD25(+)Foxp3(+) nTreg-cell development remains elusive. In the present study, we found that the percentage and cell number of thymic CD4(+)CD25(+)Foxp3(+) nTreg cells were significantly increased in T-cell-specific TSC1-knockout (TSC1KO) mice. Nevertheless, the levels of CD4(+)CD25(+)Foxp3(-) nTreg precursors in TSC1KO thymus were indistinguishable from those in wild-type mice. TSC1KO CD4(+)CD25(+)Foxp3(+) nTreg cells showed normal cell death but enhanced proliferative response to IL-2 in a STAT5-dependent manner. Rapamycin (Rapa) treatment failed to rescue but rather increased the frequency of CD4(+)CD25(+)Foxp3(+) nTreg cells in TSC1KO and RictorKO mice. The percentage and cell number of thymic CD4(+)CD25(+)Foxp3(+) nTreg cells were significantly increased in T-cell-specific RictorKO mice but not in PtenKO mice. Collectively, our studies suggest that TSC1 plays an important role in regulating thymic CD4(+)CD25(+)Foxp3(+) nTreg-cell development via a Rapa-resistant and mTORC2-dependent signaling pathway.

MTORC1 enhancement of STIM1-mediated store-operated Ca2+ entry constrains tuberous sclerosis complex-related tumor development

The protein complex of tuberous sclerosis complex (TSC)1 and TSC2 tumor suppressors is a key negative regulator of mammalian target of rapamycin (mTOR). Hyperactive mTOR signaling due to the loss-of-function of mutations in either TSC1 or TSC2 gene causes TSC, an autosomal dominant disorder featured with benign tumors in multiple organs. As the ubiquitous second messenger calcium (Ca(2+)) regulates various cellular processes involved in tumorigenesis, we explored the potential role of mTOR in modulation of cellular Ca(2+) homeostasis, and in turn the effect of Ca(2+) signaling in TSC-related tumor development. We found that loss of Tsc2 potentiated store-operated Ca(2+) entry (SOCE) in an mTOR complex 1 (mTORC1)-dependent way. The endoplasmic reticulum Ca(2+) sensor, stromal interaction molecule 1 (STIM1), was upregulated in Tsc2-deficient cells, and was suppressed by mTORC1 inhibitor rapamycin. In addition, SOCE repressed AKT1 phosphorylation. Blocking SOCE either by depleting STIM1 or ectopically expressing dominant-negative Orai1 accelerated TSC-related tumor development, likely because of restored AKT1 activity and enhanced tumor angiogenesis. Our data, therefore, suggest that mTORC1 enhancement of store-operated Ca(2+) signaling hinders TSC-related tumor growth through suppression of AKT1 signaling. The augmented SOCE by hyperactive mTORC1-STIM1 cascade may contribute to the benign nature of TSC-related tumors. Application of SOCE agonists could thus be a contraindication for TSC patients. In contrast, SOCE agonists should attenuate mTOR inhibitors-mediated AKT reactivation and consequently potentiate their efficacy in the treatment of the patients with TSC.

Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

The nuclear factor of activated T-cell (NFAT) proteins are a family of transcription factors (NFATc1-c4) involved in the regulation of cell differentiation. We identified REDD1, a negative regulator of mammalian target of rapamycin (mTOR) through the tuberous sclerosis complex (TSC1/2 complex), as a new molecular target of NFATc3. We show that treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus ionophore A23187 (Io), which induces NFAT activation, increased REDD1 mRNA and protein expression and inhibited mTOR signaling; pretreatment with the calcineurin inhibitor cyclosporin A (CsA), an antagonist of NFAT signaling, decreased REDD1 induction and mTOR inhibition. Knockdown of NFATc3, not NFATc1, NFATc2, or NFATc4, attenuated PMA/Io-induced REDD1 expression. Treatment with PMA/Io increased REDD1 promoter activity and increased NFATc3 binding to the REDD1 promoter. Overexpression of NFATc3 increased REDD1 mRNA and protein expression and increased PMA/Io-mediated REDD1 promoter activity. Treatment with PMA/Io increased expression of the goblet cell differentiation marker MUC2; these changes were attenuated by pretreatment with CsA or knockdown of REDD1 or NFATc3. Overexpression of NFATc3 increased, while knockdown of TSC2 decreased, MUC2 expression. We provide evidence showing NFATc3 inhibits mTOR via induction of REDD1. Our results suggest a role for the NFATc3/REDD1/TSC2 axis in the regulation of intestinal cell differentiation.

Sustained axon regeneration induced by co-deletion of PTEN and SOCS3

A formidable challenge in neural repair in the adult central nervous system (CNS) is the long distances that regenerating axons often need to travel in order to reconnect with their targets. Thus, a sustained capacity for axon regeneration is critical for achieving functional restoration. Although deletion of either Phosphatase and tensin homolog (PTEN), a negative regulator of mammalian target of rapamycin (mTOR), or suppressor of cytokine signaling 3 (SOCS3), a negative regulator of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, in adult retinal ganglion cells (RGCs) individually promoted significant optic nerve regeneration, such regrowth tapered off around two weeks after the crush injury1,2. Remarkably, we now find that simultaneous deletion of both PTEN and SOCS3 enables robust and sustained axon regeneration. We further show that PTEN and SOCS3 regulate two independent pathways that act synergistically to promote enhanced axon regeneration. Gene expression analyses suggest that double deletion not only results in the induction of many growth-related genes, but also allows RGCs to maintain the expression of a repertoire of genes at the physiological level after injury. Our results reveal concurrent activation of mTOR and STAT3 pathways as a key for sustaining long-distance axon regeneration in adult CNS, a crucial step toward functional recovery.

The Tuberous Sclerosis Complex–Mammalian Target of Rapamycin Pathway Maintains the Quiescence and Survival of Naive T Cells

Naive T cells receive stimulation from the positive selecting ligand in the periphery for their survival. This stimulation does not normally lead to overt activation of T cells, as the T cells remain largely quiescent until they receive either antigenic or lymphopenic stimuli. The underlying mechanism responsible for survival and quiescence of the naive T cells remains largely unknown. In this study, we report that T cell-specific deletion of Tsc1, a negative regulator of mammalian target of rapamycin, resulted in both spontaneous losses of quiescence and cellularity, especially within the CD8 subset. The Tsc1-deficient T cells have increased cell proliferation and apoptosis. Tsc1 deletion affects the survival and quiescence of T cells in the absence of antigenic stimulation. Loss of quiescence but not cellularity was inhibited by rapamycin. Our data demonstrate that tuberous sclerosis complex-mammalian target of rapamycin maintains quiescence and survival of T cells.

Frequent homozygous deletion of the LKB1/STK11 gene in non-small cell lung cancer

LKB1/STK11 is a tumor suppressor and a negative regulator of mammalian target of rapamycin signaling. It is inactivated in 30% of lung cancer cell lines but only 5-15% of primary lung adenocarcinomas. There is evidence that homozygous deletion (HD) of chromosome 19p at the LKB locus contributes to the inactivation of the gene in primary human lung cancers. Here, we used several complementary genetic approaches to assess the LKB1 locus in primary non-small cell lung cancers (NSCLCs). We first analyzed 124 NSCLC cases for allelic imbalance using eight microsatellite markers on chromosome 19p, which revealed an overall rate of 65% (80 of 124) loss of heterozygosity (LOH). We next used chromogenic in situ hybridization (CISH) to directly examine the chromosomal status of the LKB1 locus. In all, 65 of 124 LOH tested samples were available for CISH and 58 of those (89%) showed either loss of one copy of chromosome 19p (LOH, 40 of 65 cases, 62%) or both copies (HD 18 of 65 cases, 28%). The occurrence of HD was significantly more frequent in Caucasian (35%) than in African-American patients (6%) (P=0.04). A total of 62 of 124 samples with LOH at one or both markers immediately flanking the LKB1 gene were further analyzed by directly sequencing the complete coding region, which identified 7 of 62 (11%) tumors with somatic mutations in the gene. Jointly, our data identified total inactivation of the LKB1 gene by either HD or LOH with somatic mutation in 39% of tested samples, whereas loss of chromosome 19p region by HD or LOH at the LKB1 region occured in 90% of NSCLC.

Role of the TSC1-TSC2 Complex in the Integration of Insulin and Glucose Signaling Involved in Pancreatic β-Cell Proliferation

Tuberous sclerosis complex proteins 1-2 (TSC1-TSC2) complex integrates both nutrient and hormonal signaling and is a critical negative regulator of mammalian target of rapamycin (mTOR) complex 1. The use of different β-cell lines expressing or not the insulin receptor (IR+/+ and IR−/−) or with a reconstituted expression of IR isoform A or B (Rec A and Rec B) revealed that both phosphatidylinositol 3-kinase/Akt/TSC/mTOR complex 1 and MAPK kinase/ERK pathways mediate insulin signaling in IR+/+-, IRA-, or IRB-expressing cells. However, glucose signaling was mediated by MAPK kinase/ERK and AMP-activated protein kinase pathways as assessed in IR−/− cells. The effect of insulin on Akt phosphorylation was completely inhibited by the use of the phosphatidylinositol 3-kinase inhibitor wortmannin in IR+/+ and Rec B cells, a partial inhibitory effect being observed in Rec A cell line. The knockdown of TSC2 expression up-regulated the downstream basal phosphorylation of 70-kDa ribosomal protein S6 kinase (p70S6K) and mTOR. More importantly, upregulation of p70S6K signaling impaired insulin-stimulated phosphorylation of Akt Ser473 and p70S6K in IR+/+ and Rec B but not in Rec A cell lines. In fact, insulin receptor substrate-1 Ser307 phosphorylation signal in Rec B was stronger than in Rec A cell line during insulin action. Rec A cells induced a higher proliferation rate compared with Rec B or IR+/+ during serum stimulation. Thus, we propose that the regulation of TSC2 phosphorylation by insulin or glucose independently integrates β-cell proliferation signaling, the relative expression of IRA or IRB isoforms in pancreatic β cells playing a major role.

Role of the TSC1-TSC2 Complex in the Integration of Insulin and Glucose Signaling Involved in Pancreatic β-Cell Proliferation

Tuberous sclerosis complex proteins 1-2 (TSC1-TSC2) complex integrates both nutrient and hormonal signaling and is a critical negative regulator of mammalian target of rapamycin (mTOR) complex 1. The use of different beta-cell lines expressing or not the insulin receptor (IR(+/+) and IR(-/-)) or with a reconstituted expression of IR isoform A or B (Rec A and Rec B) revealed that both phosphatidylinositol 3-kinase/Akt/TSC/mTOR complex 1 and MAPK kinase/ERK pathways mediate insulin signaling in IR(+/+)-, IRA-, or IRB-expressing cells. However, glucose signaling was mediated by MAPK kinase/ERK and AMP-activated protein kinase pathways as assessed in IR(-/-) cells. The effect of insulin on Akt phosphorylation was completely inhibited by the use of the phosphatidylinositol 3-kinase inhibitor wortmannin in IR(+/+) and Rec B cells, a partial inhibitory effect being observed in Rec A cell line. The knockdown of TSC2 expression up-regulated the downstream basal phosphorylation of 70-kDa ribosomal protein S6 kinase (p70S6K) and mTOR. More importantly, upregulation of p70S6K signaling impaired insulin-stimulated phosphorylation of Akt Ser(473) and p70S6K in IR(+/+) and Rec B but not in Rec A cell lines. In fact, insulin receptor substrate-1 Ser(307) phosphorylation signal in Rec B was stronger than in Rec A cell line during insulin action. Rec A cells induced a higher proliferation rate compared with Rec B or IR(+/+) during serum stimulation. Thus, we propose that the regulation of TSC2 phosphorylation by insulin or glucose independently integrates beta-cell proliferation signaling, the relative expression of IRA or IRB isoforms in pancreatic beta cells playing a major role.

Inhibition of mTORC1 activity by REDD1 induction in myeloma cells resistant to bortezomib cytotoxicity

The combination of bortezomib and dexamethasone is becoming the reference induction treatment for multiple myeloma patients younger than 65 years. Despite its advantage over vincristin adryamicin dexamethasone induction treatment, bortezomib does not benefit all patients. We hypothesize that heterogeneity of the response experienced by myeloma patients is, at least in part, due to genomic variations in the malignant plasma cells. To test this hypothesis we used gene expression profiling to identify early responsive genes induced by bortezomib in resistant myeloma cells. Our study revealed: (i) a dramatic induction of REDD1, a negative regulator of mammalian target of rapamycin kinase complex 1 (mTORC1) activity, in these cells; (ii) a transient cell size decrease associated with REDD1 overexpression; and (iii) partial restoration of bortezomib sensitivity in REDD1 knockdown bortezomib-resistant myeloma cells. Together, these results identify a possible novel mechanism of bortezomib resistance in myeloma patients mediated by REDD1 overexpression involving inhibition of mTORC1 activity and suggest that the use of mammalian target of rapamycin inhibitors in myeloma patients could be deleterious.

Signaling Events Downstream of Mammalian Target of Rapamycin Complex 2 Are Attenuated in Cells and Tumors Deficient for the Tuberous Sclerosis Complex Tumor Suppressors

Mutations in the TSC1 and TSC2 tumor suppressor genes give rise to the neoplastic disorders tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis. Their gene products form a complex that is a critical negative regulator of mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and cell growth. We recently found that the TSC1-TSC2 complex promotes the activity of mTOR complex 2 (mTORC2), an upstream activator of Akt, and this occurs independent of its inhibitory effects on mTORC1. Loss of mTORC2 activity in cells lacking the TSC1-TSC2 complex, coupled with mTORC1-mediated feedback mechanisms, leads to strong attenuation of the growth factor-stimulated phosphorylation of Akt on S473. In this study, we show that both phosphatidylinositol 3-kinase-dependent and phosphatidylinositol 3-kinase-independent mTORC2 substrates are affected by loss of the TSC1-TSC2 complex in cell culture models and kidney tumors from both Tsc2(+/-) mice (adenoma) and TSC patients (angiomyolipoma). These mTORC2 targets are all members of the AGC kinase family and include Akt, protein kinase Calpha, and serum and glucocorticoid-induced protein kinase 1. We also show that the TSC1-TSC2 complex can directly stimulate the in vitro kinase activity of mTORC2. The interaction between these two complexes is mediated primarily through regions on TSC2 and a core component of mTORC2 called Rictor. Hence, loss of the TSC tumor suppressors results in elevated mTORC1 signaling and attenuated mTORC2 signaling. These findings suggest that the TSC1-TSC2 complex plays opposing roles in tumor progression, both blocking and promoting specific oncogenic pathways through its effects on mTORC1 inhibition and mTORC2 activation, respectively.

Signal Transducer and Activator of Transcription 3 Is Required for Abnormal Proliferation and Survival of TSC2-Deficient Cells: Relevance to Pulmonary Lymphangioleiomyomatosis

Tumor suppressor complex TSC1/TSC2 represents a key negative regulator of mammalian target of rapamycin (mTOR)-S6 kinase 1 signaling. Mutational inactivation of TSC1 or TSC2, linked to a rare lung disease, lymphangioleiomyomatosis (LAM), manifests as neoplastic growth of smooth-muscle (SM)-like cells and cystic destruction of the lungs that induces loss of pulmonary function. However, the precise mechanisms of abnormal cell growth in LAM remain uncertain. Here, we demonstrate increased signal transducer and activator of transcription (STAT) 3 expression, phosphorylation, and nuclear localization in SM-like cells in LAM lungs and in TSC2-null xenographic tumors. Treatment of TSC2-null tumors with mTOR inhibitor rapamycin attenuated STAT3 expression and phosphorylation. Increased STAT3 level and activation were also observed in LAM-dissociated (LAMD) cell cultures compared with normal human bronchus fibroblasts (HBFs) from LAM patients. Although interferon (IFN)-gamma inhibited proliferation of HBFs, IFN-gamma treatment had little effect on proliferation of LAMD and TSC2-null cells. Re-expression of TSC2 or treatment with rapamycin inhibited IFN-gamma-induced STAT3 phosphorylation and synergized with IFN-gamma in inhibiting TSC2-null and LAMD cell proliferation. Reduction of STAT3 protein levels or activity using specific small interfering RNA or inhibitory peptide, respectively, decreased proliferation and induced apoptosis in TSC2-null and LAMD cells and sensitized cells to growth-inhibitory and proapoptotic effects of IFN-gamma. Collectively, our data demonstrate that STAT3 activation is required for proliferation and survival of cells with TSC2 dysfunction, that STAT3 impedes growth-inhibitory and proapoptotic effects of IFN-gamma, and that TSC2- and rapamycin-dependent inhibition of STAT3 restores antiproliferative effects of IFN-gamma. Thus, STAT3 may provide a novel therapeutic target for diseases associated with TSC1/TSC2 dysfunction.

MTORC1-dependent and -independent regulation of stem cell renewal, differentiation, and mobilization

The Tuberous Sclerosis Complex component, TSC1, functions as a tumor suppressor via its regulation of diverse cellular processes, particularly cell growth. TSC1 exists in a complex with TSC2 and functions primarily as a key negative regulator of mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis, although the TSC1/TSC2 complex also shows mTORC1-independent outputs to other pathways. Here, we explored the role of TSC1 in various aspects of stem cell biology and dissected the extent to which TSC1 functions are executed via mTORC1-dependent versus mTORC1-independent pathways. Using hematopoietic stem cells (HSCs) as a model system, we demonstrate that somatic deletion of TSC1 produces striking stem cell and derivative effector cell phenotypes characterized by increased HSC cell cycling, mobilization, marked progressive depletion, defective long-term repopulating potential, and hematopoietic lineage developmental aberrations. On the mechanistic level, we further establish that TSC1 regulation of HSC quiescence and long-term repopulating potential and hematopoietic lineage development is mediated through mTORC1 signaling. In contrast, TSC1 regulation of HSC mobilization is effected in an mTORC1-independent manner, and gene profiling and functional analyses reveals the actin-bundling protein FSCN1 as a key TSC1/TSC2 target in the regulation of HSC mobilization. Thus, TSC1 is a critical regulator of HSC self-renewal, mobilization, and multilineage development and executes these actions via both mTORC1-dependent and -independent pathways.