Rapamycin: One Drug, Many Effects

The mammalian target of rapamycin (mTOR) signaling pathway is a master regulator of cell growth and metabolism. Deregulation of the mTOR pathway has been implicated in a number of human diseases such as cancer, diabetes, obesity, neurological diseases, and genetic disorders. Rapamycin, a specific inhibitor of mTOR, has been shown to be useful in the treatment of certain diseases. Here we discuss its mechanism of action and highlight recent findings regarding the effects and limitations of rapamycin monotherapy and the potential utility of combination therapy with rapamycin

Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade

AbstractBackground: Fas (APO-1/CD95) is a member of the tumor necrosis factor receptor (TNF-R) family and induces apoptosis when crosslinked with either Fas ligand or agonistic antibody (Fas antibody). The Fas–Fas ligand system has an important role in the immune system where it is involved in the downregulation of immune responses and the deletion of peripheral autoreactive T lymphocytes. The intracellular domain of Fas interacts with several proteins including FADD (MORT-1), DAXX, RIP, FAF-1, FAP-1 and Sentrin. The adaptor protein FADD can, in turn, interact with the cysteine protease caspase-8 (FLICE/MACH/Mch5).Results: In a genetic screen for essential components of the Fas-mediated apoptotic cascade, we isolated a Jurkat T lymphocyte cell line deficient in caspase-8 that was completely resistant to Fas-induced apoptosis. Complementation of this cell line with wild-type caspase-8 restored Fas-mediated apoptosis. Fas activation of multiple caspases and of the stress kinases p38 and c-Jun NH2-terminal kinase (JNK) was completely blocked in the caspase-8-deficient cell line. Furthermore, the cell line was severely deficient in cell death induced by TNF-α and was partially deficient in cell death induced by ultraviolet irradiation, adriamycin and etoposide.Conclusions: This study provides the first genetic evidence that caspase-8 occupies an essential and apical position in the Fas signaling pathway and suggests that caspase-8 may participate broadly in multiple apoptotic pathways

Tuberous Sclerosis Complex Gene Products, Tuberin and Hamartin, Control mTOR Signaling by Acting as a GTPase-Activating Protein Complex toward Rheb

AbstractBackground: Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear.Results: We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently.Conclusions: We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells

Appetite for destruction: the inhibition of glycolysis as a therapy for tuberous sclerosis complex-related tumors

The elevated metabolic requirements of cancer cells reflect their rapid growth and proliferation and are met through mutations in oncogenes and tumor suppressor genes that reprogram cellular processes. For example, in tuberous sclerosis complex (TSC)-related tumors, the loss of TSC1/2 function causes constitutive mTORC1 activity, which stimulates glycolysis, resulting in glucose addiction in vitro. In research published in Cell and Bioscience, Jiang and colleagues show that pharmacological restriction of glucose metabolism decreases tumor progression in a TSC xenograft model

Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signaling to suppress tumorigenesis

The mechanistic target of rapamycin (mTOR) functions as a critical regulator of cellular growth and metabolism by forming multi-component, yet functionally distinct complexes mTORC1 and mTORC2. Although mTORC2 has been implicated in mTORC1 activation, little is known about how mTORC2 is regulated. Here we report that phosphorylation of Sin1 at T86 and T398 suppresses mTORC2 kinase activity by dissociating Sin1 from mTORC2. Importantly, Sin1 phosphorylation, triggered by S6K or Akt, in a cellular context-dependent manner, inhibits not only insulin/IGF-1-mediated, but also PDGF or EGF-induced Akt phosphorylation by mTORC2, demonstrating a negative regulation of mTORC2 independent of IRS-1 and Grb10. Lastly, a cancer patient-derived Sin1-R81T mutation impairs Sin1 phosphorylation, leading to hyper-mTORC2 activation via bypassing this negative regulation. Together, our work reveals a Sin1 phosphorylation-dependent mTORC2 regulation, providing a potential molecular mechanism by which mutations in the mTORC1/S6K/Sin1 signaling axis might cause aberrant hyper-activation of mTORC2/Akt that facilitates tumorigenesis

Estradiol and mTORC2 cooperate to enhance prostaglandin biosynthesis and tumorigenesis in TSC2-deficient LAM cells

Lymphangioleiomyomatosis (LAM) is a progressive neoplastic disorder that leads to lung destruction and respiratory failure primarily in women. LAM is typically caused by tuberous sclerosis complex 2 (TSC2) mutations resulting in mTORC1 activation in proliferative smooth muscle–like cells in the lung. The female predominance of LAM suggests that estradiol contributes to disease development. Metabolomic profiling identified an estradiol-enhanced prostaglandin biosynthesis signature in Tsc2-deficient (TSC−) cells, both in vitro and in vivo. Estradiol increased the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis, which was also increased at baseline in TSC-deficient cells and was not affected by rapamycin treatment. However, both Torin 1 treatment and Rictor knockdown led to reduced COX-2 expression and phospho-Akt-S473. Prostaglandin production was also increased in TSC-deficient cells. In preclinical models, both Celecoxib and aspirin reduced tumor development. LAM patients had significantly higher serum prostaglandin levels than healthy women. 15-epi-lipoxin-A4 was identified in exhaled breath condensate from LAM subjects and was increased by aspirin treatment, indicative of functional COX-2 expression in the LAM airway. In vitro, 15-epi-lipoxin-A4 reduced the proliferation of LAM patient–derived cells in a dose-dependent manner. Targeting COX-2 and prostaglandin pathways may have therapeutic value in LAM and TSC-related diseases, and possibly in other conditions associated with mTOR hyperactivation

EGFR feedback-inhibition by Ran-binding protein 6 is disrupted in cancer

Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway.This research was supported by the National Brain Tumor Society (I.K.M.), the National Institutes of Health grants 1R01NS080944-01 (I.K.M.), 1 R35 NS105109 01 (I.K.M.), and P30CA008748 (MSKCC Core Grant), the Geoffrey Beene Cancer Research Foundation (I.K.M.), the Cycle of Survival (I.K.M.), and the Seve Ballesteros Foundation (M.S.). B.O. was supported by an American–Italian Cancer Foundation fellowship and a MSKCC Brain Tumor Center grant. W.-Y.H. is the recipient of a FY15 Horizon Award from the U.S. Department of Defense (W81XWH-15-PRCRP-HA). A.C.-G. is the recipient of the Severo-Ochoa PhD fellowship. Further support was provided by the Sontag Foundation (B.S.T.). We thank all members of the Mellinghoff laboratory for helpful suggestions. We thank Dr. Fiona Ginty (Diagnostic Imaging and Biomedical Technologies, GE Global Research Center, Niskayuna, New York, USA) for assistance with multiplexed immunofluorescence. We thank A.J. Schuhmacher and C.S. Clemente-Troncone for assistance with the in vivo experiments, M. Kaufmann for assistance in the luciferase assays and N. Yannuzzi for assistance in cloning.S

Metformin Decreases Glucose Oxidation and Increases the Dependency of Prostate Cancer Cells on Reductive Glutamine Metabolism

Metformin inhibits cancer cell proliferation, and epidemiology studies suggest an association with increased survival in patients with cancer taking metformin; however, the mechanism by which metformin improves cancer outcomes remains controversial. To explore how metformin might directly affect cancer cells, we analyzed how metformin altered the metabolism of prostate cancer cells and tumors. We found that metformin decreased glucose oxidation and increased dependency on reductive glutamine metabolism in both cancer cell lines and in a mouse model of prostate cancer. Inhibition of glutamine anaplerosis in the presence of metformin further attenuated proliferation, whereas increasing glutamine metabolism rescued the proliferative defect induced by metformin. These data suggest that interfering with glutamine may synergize with metformin to improve outcomes in patients with prostate cancer.German Science Foundation (Grant FE1185)National Institutes of Health (U.S.)Glenn Foundation for Medical ResearchNational Institutes of Health (U.S.) (Grant 5-P50-090381-09)National Institutes of Health (U.S.) (Grant 5-P30-CA14051-39)Burroughs Wellcome FundSmith Family FoundationDamon Runyon Cancer Research FoundationNational Institutes of Health (U.S.) (Grant 1R01DK075850-01)National Institutes of Health (U.S.) (Grant 1R01CA160458-01A1