Inhibition Of mTORC1 Signaling Research Articles

Abstract 4655: Iron chelation inhibits mTORC1 signaling in tumor cells

Abstract Iron chelators are commonly used for treatment of iron overload diseases. Recent studies have shown that iron chelators possess potent anticancer activities. Iron chelators (ciclopirox and 3-AP) are in clinical trials for cancer treatment. However, the molecular mechanism underlying the anticancer effect of iron chelation is not well understood. Mammalian/mechanistic target of rapamycin (mTOR) is a master kinase, regulating cell growth/proliferation and survival through sensing environmental cues. mTOR functions as at least two complexes (mTORC1/2). Here we show that two iron chelators, ciclopirox olamine (CPX) and Dp44mT, inhibit tumor cell proliferation by suppressing mTORC1 signaling in human rhabdomyosarcoma (Rh30) and lung cancer (A549 and A427) cells. This is evidenced by the findings that treatment with CPX or Dp44mT inhibited the phosphorylation of 4E-BP1 and S6K1, two best known effector molecules of mTORC1, which could be blocked by addition of ferrous sulfate. Addition of ferrous sulfate prevented the growth inhibitory effect of CPX. Ectopic expression of constitutively active mTOR also conferred resistance to CPX-induced growth inhibition. Mechanistically, iron chelation inhibition of mTORC1 was partially linked to the activation of AMPK, a negative regulator of mTORC1. Overexpression of a dominant-negative AMPK (DN-AMPK) in Rh30 cells attenuated the inhibitory effect of CPX or Dp44mT on the phosphorylation of S6K1 and 4E-BP1. In addition, iron chelation also transiently stabilized HIF-1α and further upregulated the expression of its downstream effector Bnip3, another negative regulator of mTORC1. Knockdown of Bnip3 partially attenuated the inhibitory effect of iron chelation on mTORC1. Furthermore, we found that iron chelation increased the translocation of mTOR from the cytoplasm to the nucleus. The co-localization of mTOR with the third negative regulator of mTOR, PML (promyelocytic leukemia), increased in the nucleus upon iron chelation. Moreover, iron chelation did not obviously alter the phosphorylation of IGF-1 receptor, PI3K or PTEN. The effect of iron chelation on mTORC2-mediated phosphorylation of Akt was cell line dependent. Taken together, the results suggest that the anticancer effect of iron chelation is in part related to inhibition of mTORC1 signaling, and iron chelation inhibits mTORC1 signaling via multiple mechanisms. (Supported by the FWCC, LSU Health Sciences Center) Citation Format: Chaowei Shang, Hongyu Zhou, Shile Huang. Iron chelation inhibits mTORC1 signaling in tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4655.

Ciclopirox olamine inhibits mTORC1 signaling by activation of AMPK

Ciclopirox olamine (CPX), an off-patent antifungal agent, has recently been identified as a potential anticancer agent. The mammalian target of rapamycin (mTOR) is a central controller of cell growth, proliferation and survival. Little is known about whether and how CPX executes its anticancer action by inhibiting mTOR. Here we show that CPX inhibited the phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), two downstream effector molecules of mTOR complex 1 (mTORC1), in a spectrum of human tumor cells, indicating that CPX inhibits mTORC1 signaling. Using rhabdomyosarcoma cells as an experimental model, we found that expression of constitutively active mTOR (E2419K) conferred resistance to CPX inhibition of cell proliferation, suggesting that CPX inhibition of mTORC1 contributed to its anticancer effect. In line with this, treatment with CPX inhibited tumor growth and concurrently suppressed mTORC1 signaling in RD xenografts. Mechanistically, CPX inhibition of mTORC1 was neither via inhibition of IGF-I receptor or phosphoinositide 3-kinase (PI3K), nor by activation of phosphatase and tensin homolog (PTEN). Instead, CPX inhibition of mTORC1 was attributed to activation of AMP-activated protein kinase (AMPK)-tuberous sclerosis complexes (TSC)/raptor pathways. This is supported by the findings that CPX activated AMPK; inhibition of AMPK with Compound C or ectopic expression of dominant negative AMPKα partially prevented CPX from inhibiting mTORC1; silencing TSC2 attenuated CPX inhibition of mTORC1; and CPX also increased AMPK-mediated phosphorylation of raptor (S792). Therefore, the results indicate that CPX exerts the anticancer effect by activating AMPK, resulting in inhibition of mTORC1 signaling.

LRH-1-dependent programming of mitochondrial glutamine processing drives liver cancer.

Various tumors develop addiction to glutamine to support uncontrolled cell proliferation. Here we identify the nuclear receptor liver receptor homolog 1 (LRH-1) as a key regulator in the process of hepatic tumorigenesis through the coordination of a noncanonical glutamine pathway that is reliant on the mitochondrial and cytosolic transaminases glutamate pyruvate transaminase 2 (GPT2) and glutamate oxaloacetate transaminase 1 (GOT1), which fuel anabolic metabolism. In particular, we show that gain and loss of function of hepatic LRH-1 modulate the expression and activity of mitochondrial glutaminase 2 (GLS2), the first and rate-limiting step of this pathway. Acute and chronic deletion of hepatic LRH-1 blunts the deamination of glutamine and reduces glutamine-dependent anaplerosis. The robust reduction in glutaminolysis and the limiting availability of α-ketoglutarate in turn inhibit mTORC1 signaling to eventually block cell growth and proliferation. Collectively, these studies highlight the importance of LRH-1 in coordinating glutamine-induced metabolism and signaling to promote hepatocellular carcinogenesis.

Gankyrin activates mTORC1 signaling by accelerating TSC2 degradation in colorectal cancer

Gankyrin is overexpressed in some malignancies. However its roles in colorectal carcinogenesis and underlying mechanisms remain largely unexplored. Here we report that gankyrin promotes the initiation and development of colorectal carcinogenesis by activating mTORC1 signaling through TSC/Rheb dependent mechanism. We further show that Gankyrin overexpression accelerated TSC2 degradation, while knockdown in a panel of colorectal cancer (CRC) cell lines, cell line derived xenografts and CRC patient derived xenograft (PDX) tumors delayed TSC2 degradation, restored the TSC2 protein level, and inhibited mTORC1 signaling and CRC growth. Our findings reveal a unique mechanism by which gankyrin promotes colorectal carcinogenesis and show that gankyrin is a potential therapeutic target to improve the clinical management of CRC.

Ubiquilin-mediated Small Molecule Inhibition of Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling

Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism, growth, and proliferation. mTORC1 has been implicated in many diseases such as cancer, diabetes, and neurodegeneration, and is a target to prolong lifespan. Here we report a small molecule inhibitor (Cbz-B3A) of mTORC1 signaling. Cbz-B3A inhibits the phosphorylation of eIF4E-binding protein 1 (4EBP1) and blocks 68% of translation. In contrast, rapamycin preferentially inhibits the phosphorylation of p70(S6k) and blocks 35% of translation. Cbz-B3A does not appear to bind directly to mTORC1, but instead binds to ubiquilins 1, 2, and 4. Knockdown of ubiquilin 2, but not ubiquilins 1 and 4, decreases the phosphorylation of 4EBP1, suggesting that ubiquilin 2 activates mTORC1. The knockdown of ubiquilins 2 and 4 decreases the effect of Cbz-B3A on 4EBP1 phosphorylation. Cbz-B3A slows cellular growth of some human leukemia cell lines, but is not cytotoxic. Thus Cbz-B3A exemplifies a novel strategy to inhibit mTORC1 signaling that might be exploited for treating many human diseases. We propose that Cbz-B3A reveals a previously unappreciated regulatory pathway coordinating cytosolic protein quality control and mTORC1 signaling.

Abstract P5-03-04: Identification of compounds from natural sources with selective activity against triple-negative breast cancer molecular subtypes

Abstract Triple negative breast cancers (TNBCs) lack expression of the estrogen and progesterone receptors (ER/PR) and do not have amplified HER2. While targeted therapies for ER+/PR+ and HER2-amplified breast cancers have greatly improved patient survival, there are no targeted therapies for TNBCs and no effective therapies to treat metastatic disease. There is a need to identify new therapeutic agents and molecular targets for treating TNBCs, but efforts have been limited by a lack of understanding of the subtypes of these heterogeneous diseases. However, gene expression profiling of TNBC patients recently identified 6 molecular subtypes of TNBC and representative cell lines, providing the first opportunity to identify subtype-specific leads for TNBC. We performed high-content screening to evaluate novel libraries of extracts from Texas plants and diverse fungal cultures for antiproliferative and/or cytotoxic activity in a panel of cell lines modeling five different TNBC molecular subtypes. The aim was to identify extracts with selective activity in a single cell line. We hypothesized that extracts found to have selective activity in one of these cell lines may target a protein or cellular process critical to the growth of that subtype. We identified 11 extracts with selective activity against cell lines representing four different TNBC molecular subtypes. From a fungal culture we identified a new compound called maximiscin, which was found to have selective cytotoxic efficacy against the MDA-MB-468 cell line of the basal-like 1 subtype. From a plant extract we isolated deguelin, which had selective activity in the MDA-MB-453 cell line, a model of the luminal androgen receptor (LAR) subtype. The molecular mechanisms of action of each compound were investigated in cell line models. Initial cell cycle studies using flow cytometry showed that maximiscin caused an accumulation of cells in G1 after 18h of treatment. Protein microarray studies indicated that maximiscin increased levels of phospho-p53, which was consistent with the observed G1 accumulation. Based on these findings, we hypothesized that maximiscin induces DNA damage and investigated the effects of maximiscin on the phosphorylation of several DNA damage response proteins. Maximiscin increased phosphorylation of Chk1, Chk2, p53 and H2A.X as soon as 2h after treatment, indicating an accumulation of DNA damage. Previous studies have shown that LAR TNBC cells are particularly sensitive to PI3K inhibitors in vitro compared to other TNBC subtypes. The effects of deguelin on PI3K-Akt-mTORC1 signaling were evaluated in both MDA-MB-453 and MDA-MB-231 cells. Phosphorylation of both ribosomal protein S6 and 4E-BP1 were dramatically reduced in MDA-MB-453 cells 2h after deguelin treatment. Interestingly this was not observed in MDA-MB-231 cells, suggesting inhibition of mTORC1 signaling may be involved in the selective activity of deguelin in MDA-MB-453 cells. Preliminary studies suggest deguelin may also decrease androgen receptor abundance in MDA-MB-453 cells, indicating multiple molecular mechanisms may be involved in its selective effects. These results demonstrate that compounds with selective activity against TNBC subtypes can be identified from nature. Citation Format: Robles AJ, Du L, Cai S, Risinger AL, Cichewicz RH, Mooberry SL. Identification of compounds from natural sources with selective activity against triple-negative breast cancer molecular subtypes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-04.

MTORC1-Activated Monocytes Increase Tregs and Inhibit the Immune Response to Bacterial Infections.

The TSC1/2 heterodimer, a key upstream regulator of the mTOR, can inhibit the activation of mTOR, which plays a critical role in immune responses after bacterial infections. Monocytes are an innate immune cell type that have been shown to be involved in bacteremia. However, how the mTOR pathway is involved in the regulation of monocytes is largely unknown. In our study, TSC1 KO mice and WT mice were infected with E. coli. When compared to WT mice, we found higher mortality, greater numbers of bacteria, decreased expression of coactivators in monocytes, increased numbers of Tregs, and decreased numbers of effector T cells in TSC1 KO mice. Monocytes obtained from TSC1 KO mice produced more ROS, IL-6, IL-10, and TGF-β and less IL-1, IFN-γ, and TNF-α. Taken together, our results suggest that the inhibited immune functioning in TSC1 KO mice is influenced by mTORC1 activation in monocytes. The reduced expression of coactivators resulted in inhibited effector T cell proliferation. mTORC1-activated monocytes are harmful during bacterial infections. Therefore, inhibiting mTORC1 signaling through rapamycin administration could rescue the harmful aspects of an overactive immune response, and this knowledge provides a new direction for clinical therapy.

TDP ‐43 loss of function increases TFEB activity and blocks autophagosome–lysosome fusion

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. In this study, we found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy-lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration.

System-L amino acid transporters play a key role in pancreatic β-cell signalling and function.

The branched-chain amino acids (BCAA) leucine, isoleucine and valine, are essential amino acids that play a critical role in cellular signalling and metabolism. They acutely stimulate insulin secretion and activate the regulatory serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1), a kinase that promotes increased β-cell mass and function. The effects of BCAA on cellular function are dependent on their active transport into the mammalian cells via amino acid transporters and thus the expression and activity of these transporters likely influence β-cell signalling and function. In this report, we show that the System-L transporters are required for BCAA uptake into clonal β-cell lines and pancreatic islets, and that these are essential for signalling to mTORC1. Further investigation revealed that the System-L amino acid transporter 1 (LAT1) is abundantly expressed in the islets, and that knockdown of LAT1 using siRNA inhibits mTORC1 signalling, leucine-stimulated insulin secretion and islet cell proliferation. In summary, we show that the LAT1 is required for regulating β-cell signalling and function in islets and thus may be a novel pharmacological/nutritional target for the treatment and prevention of type 2 diabetes.

Quantitative Proteomic and Transcriptomic Analysis Reveals Post-Transcriptional Regulation of Mitochondrial Biogenesis during Erythropoiesis

The differentiation and maturation of erythroid cells require highly regulated patterns of gene expression and metabolism. Mitochondria are critical for heme biosynthesis and iron metabolism in erythroid cells, yet their regulation during normal erythroid maturation remains largely unexplored. Here we measured global protein and mRNA expression in primary human fetal liver and adult bone marrow-derived CD34+ hematopoietic stem/progenitor cells (HSPCs) and differentiated erythroid precursors (proerythroblasts or ProEs) by mass-spectrometry-based quantitative proteomics and RNA-seq analysis, respectively. In-depth proteomic profiling resulted in identification and high-quality quantification of proteins encoded by 14,502 genes, accounting for 72.4% of the total annotated protein-coding genes in humans. Through unbiased and comprehensive comparison of the proteomic and transcriptomic dynamics between primary HSPCs and committed erythroid precursors, we discovered a number of previously unrecognized regulatory pathways essential for normal erythropoiesis.Importantly, we identified key pathways related to mitochondrial biogenesis, including ATP biosynthesis, electron transport chain, oxidative phosphorylation, and cellular respiration, whose protein expression was substantially induced during erythropoiesis. Strikingly, the increases in protein expression were not paralleled by changes in mRNA expression of these genes, suggesting that they are regulated through post-transcriptional mechanisms. Consistent with the enhanced mitochondrial protein expression, we observed substantial increases in mitochondrial membrane potential (MMP) and intracellular ATP level during early erythroid differentiation before clearance of mitochondria at terminal maturation stages. We next profiled metabolite levels in HSPCs and erythroid precursors at various differentiation stages, and observed progressive progressive alterations of metabolites from the tricarboxylic acid cycle and other mitochondrial pathways during erythroid maturation. Furthermore, we identified two principal mitochondria-associated transcription factors, mitochondrial transcription factor A (TFAM) and Prohibitin 2 (PHB2), whose protein but not mRNA expression was substantially increased during erythroid maturation. Depletion of TFAM or PHB2 expression markedly impaired erythroid differentiation from primary human CD34+ HSPCs. TFAM or PHB2-deficient cells displayed impaired erythroid differentiation, proliferation and hemoglobin expression, reduced mitochondrial mass, membrane potential and ATP synthesis, and increased apoptosis. Pharmacological inhibition of mitochondrial activities by targeting mitochondrial complex I (metformin, phenformin, and rotenone), complex III (antimycin A), or complex V (oligomycin) in CD34+ HSPCs impaired erythroid differentiation in a dose-dependent manner, consistent with an essential role of mitochondria for normal erythroid development.To elucidate the regulatory mechanisms underlying the developmental control of mitochondrial biogenesis, we measured protein synthesis rate in CD34+ HSPCs and differentiated erythroid cells by analyzing the incorporation of the methionine analogue HPG (L-homopropargylglycine). The rate of HPG incorporation was the lowest in undifferentiated CD34+, and increased by 1.8 and 2.5-fold in CD71+CD235a- and CD71+CD235a+ erythroid progenitor cells, respectively. Consistent with the progressive increase in protein synthesis, we observed a gradual increase of mTORC1 signaling, as measured by the phosphorylated eIF4-E binding protein (4E-BP1) and S6 kinase (p70S6K), during erythroid differentiation of CD34+ HSPCs. Inhibition of mTORC1 signaling by active-site mTOR inhibitors markedly impaired expression of mitochondria-associated proteins, mitochondrial mass and membrane potential, and erythroid differentiation of CD34+ HSPCs. Thus, our results support a model that mitochondrial biogenesis is highly regulated through mTORC1-mediated activation of protein translation during erythroid lineage specification. Our studies also suggest a novel mechanism for proper regulation of mitochondrial biogenesis by post-transcriptional machinery, and may have direct relevance to the hematological defects associated with human mitochondrial diseases and aging. DisclosuresDeBerardinis:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Peloton Therapeutics: Membership on an entity's Board of Directors or advisory committees.

ONC201 Induces p53-Independent Apoptosis and Abrogates Stem Cell Function in Hematological Malignancies By Induction of ATF4 through Integrated Stress Response

The clinical challenge posed by p53-deficiency in hematological malignancies needs novel therapeutic strategies. ONC201, a first-in-class small molecule, was discovered as a p53-independent activator of apoptosis with a benign preclinical safety profile (Allen et al, Sci Transl Med 2013). Here we report that ONC201 exerts anti-tumor effects in hematological malignancies and leukemia stem cells (LSC) via the induction of ATF4 in the integrated stress response (ISR).ONC201 induced p53-independent apoptosis in cell lines and primary patient samples from mantle cell lymphomas (MCL) and acute myeloid leukemias (AML), independent of genetic alterations that correlate with poor prognosis (e.g., TP53 mutation, FLT3-ITD, or complex karyotype). ONC201 also induced apoptosis in LSC, while sparing normal bone marrow progenitors, as measured in vitro and after transplantation. Specifically, we recovered unfractionated LSC-containing populations of AML cells (t(9;11)(p22; q23), CEBPA and ATM mutant) from secondarily-engrafted mice and cultured them in vitro for 48 hr in two groups, ONC201-treated (5 uM) or Control. For both groups, the same number of Trypan Blue-negative cells was then re-transplanted. The frequency of human CD45+ cells 4 weeks after transplantation was 38.09 ± 2.59 % of tibial BM cells in untreated mice and 0.10 ± 0.05% in treated mice (n = 3 for each, p < 0.01). The survival of the treated group was dramatically prolonged (Figure 1).Gene Set Enrichment Analysis (GSEA) of gene expression profiling (GEP) data of Jeko-1 and Z-138 cells treated with ONC201 implicated up-regulated Endoplasmic Reticulum (ER) stress-related genes, such as targets of the ER stress-induced transcription factor CHOP (DDIT3; FDR q = 0.016), and ER component proteins (FDR q = 0.039). We confirmed the ONC201-induced increased mRNA levels of DDIT3, GADD34, DR5 and TRIB3, and increased protein levels of CHOP, ATF4 and IRE1-a in Jeko-1 cells. Knockdown of ATF4 and IRE1-a revealed that ATF4 is an essential protein for ONC201-induced apoptosis in most AML cells and MCL cells, while IRE1-a is only necessary for apoptosis in MCL cells. However, unlike other ER stress inducers, ONC201 did not cause phosphorylation of an eIF2a kinase PERK, a hallmark of classical ER stress. Importantly, ONC201 was also effective in lymphoma cells resistant to bortezomib, an ER stress inducer, supporting the notion that ONC201 uses a unique mechanism to trigger the ISR. In addition, ONC201 inhibited mTORC1 signaling, likely secondary to ATF4 activation via the induction of DDIT4, a negative regulator of mTORC1, which presumably increases the cytotoxicity of ONC201 by global translation inhibition.Investigating the type of apoptosis induced by ONC201, we examined protein levels of anti-apoptotic BCL-2 family members (BCL-2, BCL-XL and MCL-1) after ONC201 treatment. MCL-1 was reduced most notably after 12 hr. We then tested the effect of ONC201 on cells with overexpression or knockdown of BCL-2 family proteins. MCL-1 knockdown in OCI-AML3 cells increased their sensitivity to ONC201 only slightly, but ONC201 efficacy was dramatically reduced in BCL-2-overexpressing HL-60 cells, even more so than in BCL-XL-overexpressing HL-60 cells. Therefore, we investigated whether ONC201 sensitivity could be increased by ABT-199, a small-molecule BH3 mimetic that specifically inhibits BCL-2, and is known to be ineffective in cell lines with MCL-1 overexpression; accordingly, the combination of ONC201 and BCL-2 antagonist ABT-199 was highly synergistic (Figure 2).In conclusion, ONC201 induces p53-independent apoptosis and abrogates LSC function by ATF4 induction, via the ISR. By suppressing MCL-1, ONC201 can also increase the effectiveness of the Bcl-2 inhibitor ABT-199. These findings suggest that ONC201 may provide promising novel therapeutic strategies for TP53 -wild type and TP53 -mutant hematological malignancies. Phase I/II clinical trials have been initiated at the MD Anderson Cancer Center in leukemias and lymphomas to determine safety, efficacy and further characterize mechanism of action. [Display omitted] [Display omitted] DisclosuresIshizawa:Karyopharm: Research Funding. Allen:Oncoceutics, Inc: Employment, Equity Ownership. Orlowski:Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; Forma Therapeutics: Consultancy; Onyx Pharmaceuticals: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Spectrum Pharmaceuticals: Research Funding. Wang:Celgene: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Andreeff:Oncoceutics, Inc.: Membership on an entity's Board of Directors or advisory committees.

Activation of AMPK Stimulates Neurotensin Secretion in Neuroendocrine Cells.

AMP-activated protein kinase (AMPK), a critical fuel-sensing enzyme, regulates the metabolic effects of various hormones. Neurotensin (NT) is a 13-amino acid peptide predominantly localized in enteroendocrine cells of the small bowel and released by fat ingestion. Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with an increased risk of diabetes, cardiovascular disease, and mortality; however, the mechanisms regulating NT release are not fully defined. We previously reported that inhibition of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) increases NT secretion and gene expression through activation of the MEK/ERK pathway. Here, we show that activation of AMPK increases NT secretion from endocrine cell lines (BON and QGP-1) and isolated mouse crypt cells enriched for NT-positive cells. In addition, plasma levels of NT increase in mice treated with 5-aminoimidazole-4-carboxamide riboside, a pharmacologic AMPK activator. Small interfering RNA-mediated knockdown of AMPKα decrease, whereas overexpression of the subunit significantly enhances, NT secretion from BON cells treated with AMPK activators or oleic acid. Similarly, small interfering RNA knockdown of the upstream AMPK kinases, liver kinase B1 and Ca(2+) calmodulin-dependent protein kinase kinase 2, also attenuate NT release and AMPK phosphorylation. Moreover, AMPK activation increases NT secretion through inhibition of mTORC1 signaling. Together, our findings show that AMPK activation enhances NT release through inhibition of mTORC1 signaling, thus demonstrating an important cross talk regulation for NT secretion.

Genetic deletion of Rhes or pharmacological blockade of mTORC1 prevent striato-nigral neurons activation in levodopa-induced dyskinesia

Ras homolog enriched in striatum (Rhes) is a small GTP-binding protein that modulates signal transduction at dopamine receptors, and also activates mammalian target of rapamycin complex 1 (mTORC1). Rhes binding to mTORC1 is hypothesized to play a role in motor disorders such as levodopa-induced dyskinesia. Here, we investigate the behavioral and in vivo neurocircuitry changes associated with genetic deletion of Rhes or inhibition of mTORC1 signaling in the mouse model of levodopa-induced dyskinesia. 6-Hydroxydopamine-hemilesioned Rhes knockout mice and wild-type littermates were chronically treated with levodopa. In parallel, 6-hydroxydopamine-hemilesioned naïve mice were chronically treated with levodopa or levodopa plus rapamycin, to block mTORC1 pathway activation. Dyskinetic movements were monitored during levodopa treatment along with motor activity on the rotarod. Finally, dyskinetic mice underwent microdialysis probe implantation in the dopamine-depleted striatum and ipsilateral substantia nigra reticulata, and GABA and glutamate levels were monitored upon acute challenge with levodopa. Both Rhes knockouts and rapamycin-treated mice developed less dyskinesia than controls, although only rapamycin-treated mice fully preserved rotarod performance on levodopa. Levodopa elevated nigral GABA and glutamate in controls but not in Rhes knockouts or rapamycin-treated mice. Levodopa also stimulated striatal glutamate in controls and Rhes knockouts but not in rapamycin-treated mice. We conclude that both genetic deletion of Rhes and pharmacological blockade of mTORC1 significantly attenuate dyskinesia development by reducing the sensitization of striato-nigral medium-sized spiny neurons to levodopa. However, mTORC1 blockade seems to provide a more favorable behavioral outcome and a wider effect on neurochemical correlates of dyskinesia.

Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes.

Adipose tissue glycogen accumulation is associated with obesity-linked inflammation in humans.

ObjectiveGlycogen metabolism has emerged as a mediator in the control of energy homeostasis and studies in murine models reveal that adipose tissue might contain glycogen stores. Here we investigated the physio(patho)logical role of glycogen in human adipose tissue in the context of obesity and insulin resistance.MethodsWe studied glucose metabolic flux of hypoxic human adipoctyes by nuclear magnetic resonance and mass spectrometry-based metabolic approaches. Glycogen synthesis and glycogen content in response to hypoxia was analyzed in human adipocytes and macrophages. To explore the metabolic effects of enforced glycogen deposition in adipocytes and macrophages, we overexpressed PTG, the only glycogen-associated regulatory subunit (PP1-GTS) reported in murine adipocytes. Adipose tissue gene expression analysis was performed on wild type and homozygous PTG KO male mice. Finally, glycogen metabolism gene expression and glycogen accumulation was analyzed in adipose tissue, mature adipocytes and resident macrophages from lean and obese subjects with different degrees of insulin resistance in 2 independent cohorts.ResultsWe show that hypoxia modulates glucose metabolic flux in human adipocytes and macrophages and promotes glycogenesis. Enforced glycogen deposition by overexpression of PTG re-orients adipocyte secretion to a pro-inflammatory response linked to insulin resistance and monocyte/lymphocyte migration. Furthermore, glycogen accumulation is associated with inhibition of mTORC1 signaling and increased basal autophagy flux, correlating with greater leptin release in glycogen-loaded adipocytes. PTG-KO mice have reduced expression of key inflammatory genes in adipose tissue and PTG overexpression in M0 macrophages induces a pro-inflammatory and glycolytic M1 phenotype. Increased glycogen synthase expression correlates with glycogen deposition in subcutaneous adipose tissue of obese patients. Glycogen content in subcutaneous mature adipocytes is associated with BMI and leptin expression.ConclusionOur data establish glycogen mishandling in adipose tissue as a potential key feature of inflammatory-related metabolic stress in human obesity.

HDAC5-mTORC1 Interaction in Differential Regulation of Ghrelin and Nucleobindin 2 (NUCB2)/Nesfatin-1.

Sodium valporate (VPA), a broad-spectrum inhibitor of histone deacetylases (HDACs), increased ghrelin whereas decreased nesfatin-1 in mice fed normal chow diet or high-fat diet. Alterations in ghrelin and nucleobindin 2/nesfatin-1 were mediated by HDAC5 but not HDAC4. Activation of mTORC1 significantly attenuated the effect of VPA on ghrelin and nesfatin-1 levels. HDAC5 coimmunoprecipitated with raptor. Inhibition of HDAC5 by VPA, trichostatin A, or siHDAC5 markedly increased acetylation of raptor Lys840 and subsequent phosphorylation of raptor Ser792, resulting in suppression of mTORC1 signaling. A raptor mutant lacking the Lys840 acetylation site showed a decrement in phosphorylation of raptor Ser792 and subsequent increase in mTORC1 signaling. These alterations were associated with reciprocal changes in ghrelin and nucleobindin 2/nesfatin-1 expression. These findings reveal HDAC5-mTORC1 signaling as a novel mechanism in the differential regulation of gastric ghrelin and nesfatin-1.

Blocking rpS6 Phosphorylation Exacerbates Tsc1 Deletion-Induced Kidney Growth.

The molecular mechanisms underlying renal growth and renal growth-induced nephron damage remain poorly understood. Here, we report that in murine models, deletion of the tuberous sclerosis complex protein 1 (Tsc1) in renal proximal tubules induced strikingly enlarged kidneys, with minimal cystogenesis and occasional microscopic tumorigenesis. Signaling studies revealed hyperphosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and increased phosphorylation of ribosomal protein S6 (rpS6) in activated renal tubules. Notably, knockin of a nonphosphorylatable rpS6 in these Tsc1-mutant mice exacerbated cystogenesis and caused drastic nephron damage and renal fibrosis, leading to kidney failure and a premature death rate of 67% by 9 weeks of age. In contrast, Tsc1 single-mutant mice were all alive and had far fewer renal cysts at this age. Mechanistic studies revealed persistent activation of mammalian target of rapamycin complex 1 (mTORC1) signaling causing hyperphosphorylation and consequent accumulation of 4E-BP1, along with greater cell proliferation, in the renal tubules of Tsc1 and rpS6 double-mutant mice. Furthermore, pharmacologic treatment of Tsc1 single-mutant mice with rapamycin reduced hyperphosphorylation and accumulation of 4E-BP1 but also inhibited phosphorylation of rpS6. Rapamycin also exacerbated cystic and fibrotic lesions and impaired kidney function in these mice, consequently leading to a premature death rate of 40% within 2 weeks of treatment, despite destroying tumors and decreasing kidney size. These findings indicate that Tsc1 prevents aberrant renal growth and tumorigenesis by inhibiting mTORC1 signaling, whereas phosphorylated rpS6 suppresses cystogenesis and fibrosis in Tsc1-deleted kidneys.

Abstract 968: Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma (LUAC) with distinct biology and therapeutic vulnerabilities

Abstract Introduction The development of more effective treatment strategies for LUAC bearing activating mutations in KRAS is hampered by the biological heterogeneity of KRAS-mutant tumors. The molecular underpinnings that drive this process are poorly characterized. Here, we implemented an integrated approach to the discovery of biologically distinct subsets of KRAS-mutant LUAC and explored their molecular vulnerabilities. Methods Our datasets consisted of 68 KRAS-mutant tumors from TCGA, 88 additional chemo-naive KRAS-mutant LUACs (PROSPECT and Chitale datasets) and 36 platinum-refractory LUACs from the BATTLE-2 clinical trial. Non-negative matrix factorization (NMF) consensus clustering was applied to RNASeq data as previously described. Signature enrichment was assessed using Gene Set Enrichment Analysis (GSEA). Results NMF consensus clustering identified three robust subsets of KRAS-mutant LUAC that were reproducible across diverse clinical datasets of early-stage, chemotherapy-naive and metastatic, chemo-refractory tumors. Distinct KRAS-mutant alleles were not differentially represented in the three subgroups (P = 0.3). In contrast, the subgroups were dominated, respectively, by co-occurring genetic events in STK11/LKB1 (termed the KL subgroup) (P = 1.03e−05), TP53 (KP) (P = 3.8e-06) and low expression of TTF1 coupled with frequent CDKN2A/B inactivation (KC) (P = 0.004 and P = 0.002). Distinct patterns of intracellular signaling were detected in the three subsets. KL tumors showed evidence of LKB1-AMPK pathway inactivation and adaptation to energetic, proteotoxic and oxidative stress, the latter exemplified by near ubiquitous inactivation of KEAP1 and up-regulation of a NRF2-driven antioxidant signature. KP tumors carried a higher somatic mutation load and were characterized by prominent inflammation and up-regulation of several immune checkpoint effector molecules, including PD-L1. KC tumors frequently displayed a GI-like differentiation program, suppression of MTORC1 signaling and elevated wild-type p53 transcriptional output. Using a large panel of KRAS-mutant NSCLC cell lines we detected co-mutation-dependent patterns of drug sensitivity. Specifically, KL cell lines showed enhanced sensitivity to several structurally distinct HSP90 inhibitors. These results were confirmed in panels of isogenic cell lines. Mechanistically, treatment with ganetespib resulted in concurrent degradation of several molecules with established role in supporting the fitness of LKB1-deficient cells. Conclusions Our work identifies three major subsets of KRAS-mutant LUAC - dominated by co-occurring genetic events - with distinct biology and therapeutic vulnerabilities. Citation Format: Ferdinandos Skoulidis, Lauren Byers, Lixia Diao, Vassiliki Papadimitrakopoulou, Pan Tong, Julie Izzo, Carmen Behrens, Humam Kadara, Edwin R. Parra, Jaime Rodriguez-Canales, Jianjun Zhang, Uma Giri, Jayanthi Gudikote, Maria Angelica Cortez, Chao Yang, You Hong Fan, Michael Peyton, Luc Girard, Kevin R. Coombes, Carlo Toniatti, Timothy P. Heffernan, Murim Choi, Garrett M. Frampton, Vincent Miller, John N. Weinstein, Roy S. Herbst, Kwok-Kin Wong, Jianhua Zhang, Padmanee Sharma, Gordon M. Mills, Waun Ki Hong, John D. Minna, James P. Allison, Andrew Futreal, Jing Wang, Ignacio Wistuba, John V. Heymach. Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma (LUAC) with distinct biology and therapeutic vulnerabilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 968. doi:10.1158/1538-7445.AM2015-968

Regulation of Human Trophoblast GLUT3 Glucose Transporter by Mammalian Target of Rapamycin Signaling.

Glucose transporter isoform-3 (GLUT3), one of the primary placental facilitative glucose transporters responsible for basal glucose transport, has a crucial role in glucose transport and fetal growth during early pregnancy. A GLUT3 mutation in mice has been reported to cause loss of early pregnancy or late-gestational fetal growth restriction. However, the underlying mechanisms that regulate the placental GLUT3 transporter in humans are largely unknown. In the present study, we used the JEG-3 human choriocarcinoma cell line, which resembles a first trimester placental model, to study the role of the mammalian target of rapamycin complex 1 (mTORC1) in the regulation of placental GLUT3. We combined rapamycin treatment and small interfering (si) RNA-mediated silencing approaches with mRNA and protein expression/localization studies to investigate the alteration of GLUT3 expression and localization following mTORC1 inhibition in JEG-3 trophoblasts. Inhibition of mTORC1 signaling by silencing raptor decreased GLUT3 mRNA expression (−41%) and protein expression (−50%). Similar effects were obtained in cells in which mTORC1 was inhibited by rapamycin. Immunofluorescence analysis revealed that GLUT3 expression was markedly reduced in the cell surface and cytoplasm of JEG-3 cells in response to mTORC1 silencing. Because placental mTORC1 activity and GLUT3 expression are decreased in human intrauterine growth restriction, our data suggested one possible mechanism for the abnormal fetal growth in this pregnancy complication.

Abstract 364: Pioglitazone Inhibition of Vascular Smooth Muscle Cell Proliferation is Associated with Activation of AMP-activated Protein Kinase but not Peroxisome Proliferator-Activated Receptor Gamma

Atherosclerosis and restenosis after angioplasty are associated with exaggerated proliferation of vascular smooth muscle cells (VSMCs). Pioglitazone (PIO), an antidiabetic drug that belongs to the family of peroxisome proliferator-activated receptor gamma (PPARg) agonists, reduces intimal hyperplasia after coronary angioplasty in diabetic and nondiabetic subjects. However, the molecular mechanisms by which PIO regulates VSMC phenotype remain unclear. The objective of the current study is to determine the role of AMP-activated protein kinase (AMPK, a cellular energy sensor) versus PPARg in PIO-induced changes in VSMC phenotype. Exposure of human aortic VSMCs to PIO followed by immunoblot analysis revealed a robust increase in the phosphorylation of AMPK (Thr172) and its downstream effector, Acetyl-CoA carboxylase (p &lt; 0.05, n = 3), as a function of concentration (3 to 30 μM) and time (1 to 48 hr). Notably, PIO (30 μM, 48 hr) activation of AMPK remained essentially the same after PPARg downregulation (~90%) by target-specific siRNA (500 pmoles), suggesting lack of dependency on PPARg. In addition, PIO activation of AMPK was accompanied by induction of mitochondrial membrane depolarization with a trend toward an increase in AMP/ATP ratio (~40%), as revealed by tetramethylrhodamine methyl ester (TMRM) fluorescent redistribution &amp; LC-MS/MS MRM analysis, respectively. Importantly, PIO pretreatment (30 μM, 30 min) resulted in significant inhibition of PDGF (30 ng/ml, 48 hr)-induced VSMC proliferation by ~85% (cell count) and the associated increases in proliferative markers (cyclin D1 and pRb) by &gt; 95% (p &lt; 0.05, n = 3). Downregulation of AMPKa1 (~80%) by target-specific AMPKa1 siRNA (500 pmoles) revealed that PIO-mediated AMPK activation inhibited mTORC1 signaling by &gt; 95% &amp; upregulated cell cycle inhibitor p27Kip1 by ~40%, thus accounting for PIO-mediated changes in VSMC phenotype. In vivo, PIO administration (10 mg/Kg/d, oral gavage) for 3 weeks in CJ57BL6 mice mitigated neointimal growth by ~60% in guide wire-injured femoral artery (p &lt; 0.05, n = 8). In conclusion, the present study provides the first direct evidence for VSMC-specific AMPK activation by PIO, which may in part contribute to its vasoprotective effects in nondiabetic state.