Rapamycin-mediated Inhibition Research Articles

Rapamycin Abrogates Aggregation of Human α-Synuclein Expressed in Fission Yeast via an Autophagy-Independent Mechanism.

Aggregation of alpha-synuclein (α-Syn) is implicated in the pathogenesis of several neurodegenerative disorders, such as Parkinson's disease and Dementia with Lewy bodies, collectively termed synucleinopathies. Thus, tremendous efforts are being made to develop strategies to prevent or inhibit α-Syn aggregation. Here, we genetically engineered fission yeast to express human α-Syn C-terminally fused to green fluorescent protein (GFP) at low and high levels. α-Syn was localized at the cell tips and septa at low-level expression. At high-level expression, α-Syn was observed to form cytoplasmic aggregates. Notably, rapamycin, a natural product that allosterically inhibits the mammalian target of rapamycin (mTOR) by forming a complex with FKBP12, and Torin1, a synthetic mTOR inhibitor that blocks ATP binding to mTOR, markedly reduced the number of cells harboring α-Syn aggregates. These mTOR inhibitors abrogate α-Syn aggregation without affecting α-Syn expression levels. Rapamycin, but not Torin1, failed to reduce α-Syn aggregation in the deletion cells of fkh1+, encoding FKBP12, indicating the requirement of FKBP12 for rapamycin-mediated inhibition of α-Syn aggregation. Importantly, the effect of rapamycin was also observed in the cells lacking atg1+, a key regulator of autophagy. Collectively, rapamycin abrogates human α-Syn aggregation expressed in fission yeast via an autophagy-independent mechanism mediated by FKBP12.

Role of FMRP in AKT/mTOR pathway-mediated hippocampal autophagy in fragile X syndrome

Fragile X syndrome (FXS) is caused by epigenetic silencing of the Fmr1 gene, leading to the deletion of the coding protein FMRP. FXS induces abnormal hippocampal autophagy and mTOR overactivation. However, it remains unclear whether FMRP regulates hippocampal autophagy through the AKT/mTOR pathway, which influences the neural behavior of FXS. Our study revealed that FMRP deficiency increased the protein levels of p-ULK-1 and p62 and decreased LC3II/LC3I level in Fmr1 knockout (KO) mice. The mouse hippocampal neuronal cell line HT22 with knockdown of Fmr1 by lentivirus showed that the protein levels of p-ULK-1 and p62 were increased, whereas LC3II/LC3I was unchanged. Further observations revealed that FMRP deficiency obstructed autophagic flow in HT22 cells. Therefore, FMRP deficiency inhibited autophagy in the mouse hippocampus and HT22 cells. Moreover, FMRP deficiency increased reactive oxygen species (ROS) level, decreased the co-localization between the mitochondrial outer membrane proteins TOM20 and LC3 in HT22 cells, and caused a decrease in the mitochondrial autophagy protein PINK1 in HT22 cells and Fmr1 KO mice, indicating that FMRP deficiency caused mitochondrial autophagy disorder in HT22 cells and Fmr1 KO mice. To explore the mechanism by which FMRP deficiency inhibits autophagy, we examined the AKT/mTOR signaling pathway in the hippocampus of Fmr1 KO mice, found that FMRP deficiency caused overactivation of the AKT/mTOR pathway. Rapamycin-mediated mTOR inhibition activated and enhanced mitochondrial autophagy. Finally, we examined whether rapamycin affected the neurobehavior of Fmr1 KO mice. The Fmr1 KO mice exhibited stereotypical behavior, impaired social ability, and learning and memory impairment, while rapamycin treatment improved behavioral disorders in Fmr1 KO mice. Thus, our study revealed the molecular mechanism by which FMRP regulates autophagy function, clarifying the role of hippocampal neuron mitochondrial autophagy in the pathogenesis of FXS, and providing novel insights into potential therapeutic targets of FXS.

Parallels between oncogene-driven cardiac hyperplasia and heart regeneration in zebrafish.

The human heart is poorly regenerative and cardiac tumors are extremely rare. Whether the adult zebrafish myocardium is responsive to oncogene overexpression and how this condition affects its intrinsic regenerative capacity remains unknown. Here, we have established a strategy of inducible and reversible expression of HRASG12V in zebrafish cardiomyocytes. This approach stimulated a hyperplastic cardiac enlargement within 16 days. The phenotype was suppressed by rapamycin-mediated inhibition of TOR signaling. As TOR signaling is also required for heart restoration after cryoinjury, we compared transcriptomes of hyperplastic and regenerating ventricles. Both conditions were associated with upregulation of cardiomyocyte dedifferentiation and proliferation factors, as well as with similar microenvironmental responses, such as deposition of nonfibrillar Collagen XII and recruitment of immune cells. Among the differentially expressed genes, many proteasome and cell-cycle regulators were upregulated only in oncogene-expressing hearts. Preconditioning of the heart with short-term oncogene expression accelerated cardiac regeneration after cryoinjury, revealing a beneficial synergism between both programs. Identification of the molecular bases underlying the interplay between detrimental hyperplasia and advantageous regeneration provides new insights into cardiac plasticity in adult zebrafish.

Constitutive activation of TORC1 signalling attenuates virulence in the cross-kingdom fungal pathogen Fusarium oxysporum.

The filamentous fungus Fusarium oxysporum causes vascular wilt disease in a wide range of plant species and opportunistic infections in humans. Previous work suggested that invasive growth in this pathogen is controlled by environmental cues such as pH and nutrient status. Here we investigated the role of Target Of Rapamycin Complex 1 (TORC1), a global regulator of eukaryotic cell growth and development. Inactivation of the negative regulator Tuberous Sclerosis Complex 2 (Tsc2), but not constitutive activation of the positive regulator Gtr1, in F.oxysporum resulted in inappropriate activation of TORC1 signalling under nutrient-limiting conditions. The tsc2Δ mutants showed reduced colony growth on minimal medium with different nitrogen sources and increased sensitivity to cell wall or high temperature stress. Furthermore, these mutants were impaired in invasive hyphal growth across cellophane membranes and exhibited a marked decrease in virulence, both on tomato plants and on the invertebrate animal host Galleria mellonella. Importantly, invasive hyphal growth in tsc2Δ strains was rescued by rapamycin-mediated inhibition of TORC1. Collectively, these results reveal a key role of TORC1 signalling in the development and pathogenicity of F.oxysporum and suggest new potential targets for controlling fungal infections.

Disruption of mTORC1 rescues neuronal overgrowth and synapse function dysregulated by Pten loss.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of AKT/mTOR signaling pathway. Mutations in PTEN are found in patients with autism, epilepsy, or macrocephaly. In mouse models, Pten loss results in neuronal hypertrophy, hyperexcitability, seizures, and ASD-like behaviors. The underlying molecular mechanisms of these phenotypes are not well delineated. We determined which of the Pten loss-driven aberrations in neuronal form and function are orchestrated by downstream mTOR complex 1 (mTORC1). Rapamycin-mediated inhibition of mTORC1 prevented increase in soma size, migration, spine density, and dendritic overgrowth in Pten knockout dentate gyrus granule neurons. Genetic knockout of Raptor to disrupt mTORC1 complex formation blocked Pten loss-mediated neuronal hypertrophy. Electrophysiological recordings revealed that genetic disruption of mTORC1 rescued Pten loss-mediated increase in excitatory synaptic transmission. We have identified an essential role for mTORC1 in orchestrating Pten loss-driven neuronal hypertrophy and synapse formation.

Short-term rapamycin treatment increases life span and attenuates aortic aneurysm in a murine model of Marfan-Syndrome

BackgroundMarfan syndrome (MFS) is a genetic disorder leading to medial aortic degeneration and life-limiting dissections. To date, there is no causal prevention or therapy. Rapamycin is a potent and selective inhibitor of the mechanistic target of rapamycin (mTOR) protein kinase, regulating cell growth and metabolism. The mgR/mgR mice represent an accepted MFS model for studying aortic pathologies to understand the underlying molecular pathomechanisms. This study investigated whether rapamycin inhibits the development of thoracic aortic aneurysms and dissections in mgR/mgR mice. MethodsIsolated primary aortic smooth muscle cells (mAoSMCs) from mgR/mgR mice were used for in vitro studies. Two mg kg/BW rapamycin was injected intraperitoneally daily for two weeks, beginning at 7–8 weeks of age. Mice were sacrificed 30 days post-treatment. Histopathological and immunofluorescence analyses were performed using adequate tissue specimens and techniques. Animal survival was evaluated accompanied by periodic echocardiographic examinations of the aorta. ResultsThe protein level of the phosphorylated ribosomal protein S6 (p-RPS6), a downstream target of mTOR, was significantly increased in the aortic tissue of mgR/mgR mice. In mAoSMCs isolated from these animals, expression of mTOR, p-RPS6, tumour necrosis factor α, matrix metalloproteinase-2 and -9 was significantly suppressed by rapamycin, demonstrating its anti-inflammatory capacity. Short-term rapamycin treatment of Marfan mice was associated with delayed aneurysm formation, medial aortic elastolysis and improved survival. ConclusionsShort-term rapamycin-mediated mTOR inhibition significantly reduces aortic aneurysm formation and thus increases survival in mgR/mgR mice. Our results may offer the first causal treatment option to prevent aortic complications in MFS patients.

Epstein-Barr virus lytic replication activates and is dependent upon MAPK-interacting kinase 1/2 in a cell-type dependent manner.

Epstein-Barr virus (EBV) is known to manipulate its cellular environment to enhance viral replication, which can lead to dysregulation of cellular machinery, setting the stage for potential future disease. Previous research showed that under rapamycin-mediated inhibition of mTORC1, EBV lytic protein production was altered in a cell-type specific manner, suggesting that EBV differentially activates or utilizes signaling pathways in B versus epithelial cells. Here we correlated activation of the mTORC1, ERK1/2, and p38 pathways in relation to EBV lytic replication and discovered that activation of MAPK-interacting kinase 1/2 (Mnk1/2) was strongly associated with EBV lytic replication. Inhibition studies revealed that Mnk1/2 activated lytic replication in epithelial cells yet acted as an inhibitor of lytic replication in B cells. The ability of lytic epithelial cells to migrate, a potentially important aspect of metastasis, was also demonstrated to be dependent upon Mnk1/2 activity.

TTK inhibition increases cisplatin sensitivity in high-grade serous ovarian carcinoma through the mTOR/autophagy pathway

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. However, the molecular mechanisms underlying HGSOC development, progression, chemotherapy insensitivity and resistance remain unclear. Two independent GEO datasets, including the gene expression profile of primary ovarian carcinoma and normal controls, were analyzed to identify genes related to HGSOC development and progression. A KEGG pathway analysis of the differentially expressed genes (DEGs) revealed that the cell cycle pathway was the most enriched pathway, among which TTK protein kinase (TTK) was the only gene with a clinical-grade inhibitor that has been investigated in a clinical trial but had not been studied in HGSOC. TTK was also upregulated in cisplatin-resistant ovarian cancer cells from two other datasets. TTK is a regulator of spindle assembly checkpoint signaling, playing an important role in cell cycle control and tumorigenesis in various cancers. However, the function and regulatory mechanism of TTK in HGSOC remain to be determined. In this study, we observed TTK upregulation in patients with HGSOC. High TTK expression was related to a poor prognosis. Genetic and pharmacological inhibition of TTK impeded the proliferation of ovarian cancer cells by disturbing cell cycle progression and increasing apoptosis. TTK silencing increased cisplatin sensitivity by activating the mammalian target of rapamycin (mTOR) complex to further suppress cisplatin-induced autophagy in vitro. In addition, the enhanced sensitivity was partially diminished by rapamycin-mediated inhibition of mTOR in TTK knockdown cells. Furthermore, TTK knockdown increased the toxicity of cisplatin in vivo by decreasing autophagy. These findings suggest that the administration of TTK inhibitors in combination with cisplatin may lead to improved response rates to cisplatin in patients with HGSOC presenting high TTK expression. In summary, our study may provide a theoretical foundation for using the combination therapy of cisplatin and TTK inhibitors as a treatment for HGSOC in the future.

Transcriptional control of ribosome biogenesis in yeast: links to growth and stress signals.

Ribosome biogenesis requires prodigious transcriptional output in rapidly growing yeast cells and is highly regulated in response to both growth and stress signals. This minireview focuses on recent developments in our understanding of this regulatory process, with an emphasis on the 138 ribosomal protein genes (RPGs) themselves and a group of >200 ribosome biogenesis (RiBi) genes whose products contribute to assembly but are not part of the ribosome. Expression of most RPGs depends upon Rap1, a pioneer transcription factor (TF) required for the binding of a pair of RPG-specific TFs called Fhl1 and Ifh1. RPG expression is correlated with Ifh1 promoter binding, whereas Rap1 and Fhl1 remain promoter-associated upon stress-induced down regulation. A TF called Sfp1 has also been implicated in RPG regulation, though recent work reveals that its primary function is in activation of RiBi and other growth-related genes. Sfp1 plays an important regulatory role at a small number of RPGs where Rap1–Fhl1–Ifh1 action is subsidiary or non-existent. In addition, nearly half of all RPGs are bound by Hmo1, which either stabilizes or re-configures Fhl1–Ifh1 binding. Recent studies identified the proline rotamase Fpr1, known primarily for its role in rapamycin-mediated inhibition of the TORC1 kinase, as an additional TF at RPG promoters. Fpr1 also affects Fhl1–Ifh1 binding, either independently or in cooperation with Hmo1. Finally, a major recent development was the discovery of a protein homeostasis mechanism driven by unassembled ribosomal proteins, referred to as the Ribosome Assembly Stress Response (RASTR), that controls RPG transcription through the reversible condensation of Ifh1.

Soluble epoxide hydrolase inhibitor, TUPS, attenuates isoproterenol/angiotensin II-induced cardiac hypertrophy through mammalian target of rapamycin-mediated autophagy inhibition.

To investigate the potential role and mechanism of TUPS, a soluble epoxide hydrolase inhibitor, in cardiac hypertrophy. Rat and H9C2 cell models of cardiac hypertrophy were induced by isoproterenol and angiotensin II, respectively, followed by TUPS treatment. The expression of hypertrophic markers, ANP and BNP, was determined by quantitative real-time PCR. The abundance of Beclin-1, LC3, p-AMPK and phosphorylated-mammalian target of rapamycin (p-mTOR) proteins was analysed by Western blot and immunohistocytology. Cell morphology and viability were evaluated by F-actin staining and MTS. H9C2 cells were transfected with GFP-LC3 to evaluate autophagy flux. TUPS significantly inhibited rat heart size, heart weight-to-body weight ratio, heart wall thickness, hypertrophic H9C2 cell swelling and viability suppression as well as the expression of ANP and BNP genes in hypertrophic models. In addition, autophagic markers Beclin-1 and LC3 were elevated in both cellular and animal models, which were suppressed by TUPS, with corresponding changes of autophagy flux. The abundance of p-AMPK was increased, while p-mTOR was decreased in hypertrophic cells, which were abolished by TUPS. Rapamycin decreased p-mTOR level, increased Beclin-1 and LC3 expression and induced cell size enlargement and cell viability inhibition in hypertrophic H9C2 cells treated with TUPS. TUPS inhibits cardiac hypertrophy by regulating mTOR/autophagy axis.

Rapamycin-mediated mTOR inhibition impairs silencing of sex chromosomes and the pachytene piRNA pathway in the mouse testis.

Mechanistic target of rapamycin (mTOR) controls cell growth and metabolism in response to environmental and metabolic signals. Rapamycin robustly extends the lifespan in mammals and has clinical relevance in organ transplantation and cancer therapy but side effects include male infertility. Here, we report that chronic rapamycin treatment causes spermatogenic arrest in adult male mice due to defects in sex body formation and meiotic sex chromosome inactivation (MSCI). Many sex chromosome-linked genes were up-regulated in isolated pachytene spermatocytes from rapamycin-treated mice. RNA-Seq analysis also identified mRNAs encoding the core piRNA pathway components were decreased. Furthermore, rapamycin treatment was associated with a drastic reduction in pachytene piRNA populations. The inhibitory effects of rapamycin on spermatogenesis were partially reversible, with restoration of testis mass and sperm motility within 2 months of treatment cessation. Collectively, we have defined an essential role of mTOR in MSCI and identified a novel function as a regulator of small RNA homeostasis in male germ cells.

Integrated actions of mTOR complexes 1 and 2 for growth and development of Dictyostelium.

Multi-protein complexes mTORC1 and mTORC2 are required for growth and development of eukaryotes. mTORC1 is a nutrient sensor that integrates metabolic signals and energy state to regulate cell growth/proliferation, whereas, mTORC2 primarily regulates developmental processes. Dictyostelium proliferate in rich growth media, but initiate development upon nutrient depletion. Both mTOR complexes play essential roles in Dictyostelium, where growth and developmental cycles independently require, respectively, mTORC1 or mTORC2. Many protein associations and regulatory pathways for mTORC1 and mTORC2 in Dictyostelium have context similarity to mammalian cells and specificity to inhibition by the immunosuppressive drug rapamycin. In Dictyostelium, mTORC1 function is inactivated upon starvation-induced development, but development is directly induced through rapamycin-mediated inhibition of mTORC1 activity, even in the absence of nutrient withdrawal. Pharmacologic inhibition of mTORC1, in the absence of nutrient loss, has allowed the identification of a class of essential up-regulated, developmentally-associated signaling genes and down-regulated, growth genes. We also review functional pathway regulations that integrate mTORC1/mTORC2 activities and emphasize complexity of small GTPase regulation of mTORC2 activity. Finally, epistases experiments have suggested novel upstream pathway cross-talk in Dictyostelium that requires mTORC1 and mTORC2, but for separate and independent downstream functions.

Rapamycin-mediated mTOR inhibition uncouples HIV-1 latency reversal from cytokine-associated toxicity.

Current strategies for HIV-1 eradication require the reactivation of latent HIV-1 in resting CD4+ T cells (rCD4s). Global T cell activation is a well-characterized means of inducing HIV-1 transcription, but is considered too toxic for clinical applications. Here, we have explored a strategy that involves a combination of immune activation and the immunosuppressive mTOR inhibitor rapamycin. In purified rCD4s from HIV-1-infected individuals on antiretroviral therapy, rapamycin treatment downregulated markers of toxicity, including proinflammatory cytokine release and cellular proliferation that were induced after potent T cell activation using αCD3/αCD28 antibodies. Using an ex vivo assay for HIV-1 mRNA, we demonstrated that despite this immunomodulatory effect, rapamycin did not affect HIV-1 gene expression induced by T cell activation in these rCD4s. In contrast, treating activated rCD4s with the immunosuppressant cyclosporin, a calcineurin inhibitor, robustly inhibited HIV-1 reactivation. Importantly, rapamycin treatment did not impair cytotoxic T lymphocyte (CTL) recognition and killing of infected cells. These findings raise the possibility of using rapamycin in conjunction with T cell-activating agents in HIV-1 cure strategies.

Regulation of skeletal muscle insulin-stimulated signaling through the MEK-REDD1-mTOR axis

Recent findings in adipocytes suggest that mitogen-activated protein kinase (MAPK)/extracellular-regulated signaling kinase (ERK) kinase 1/2 (MEK1/2) signaling regulates regulated in development and DNA damage 1 (REDD1) protein expression. Similarly, our previous work show that a lack of REDD1 protein expression, and associated hyperactive basal mechanistic target of rapamycin (mTOR) signaling, limits skeletal muscle's response to insulin. Therefore, we sought to determine: 1) if MEK1/2 inhibition is sufficient to reduce REDD1 protein expression and subsequently insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation via negative feedback of hyperactive mTOR in REDD1 wild-type (WT) mice and 2) if rapamycin-mediated mTOR inhibition is sufficient to improve IRS-1 tyrosine phosphorylation in REDD1 knockout (KO) mice. REDD1 WT mice were injected with 10 mg/kg BW of the MEK1/2 non-competitive inhibitor, PD184352, 3 h prior to acute insulin treatment. In separate studies, REDD1 KO mice were injected with 5 mg/kg BW of the mTOR inhibitor, rapamycin, 3 h prior to acute insulin treatment. Following the inhibitor treatment period, markers of insulin signaling activation (IRS-1 Y1222, MEK1/2 S217/221, ERK1/2 T202/Y204), REDD1, and mTOR signaling activation (S6K1 T389, rpS6 S240/244) were examined in skeletal muscle collected before and after a 10 min insulin treatment. PD184352 treatment reduced MEK/ERK phosphorylation and REDD1 protein expression, independent of insulin. This reduction in REDD1 protein expression was associated with elevated basal S6K1 and rpS6 phosphorylation and reduced insulin stimulated IRS-1 phosphorylation. Conversely, rapamycin inhibited S6K1 and rpS6 activation, and significantly improved insulin –stimulated activation of IRS-1 and MEK1/2 in KO mice. These data support that REDD1 is required for normal insulin-stimulated signaling, and that a subtle balance exists between MEK1/2, REDD1, and mTOR for the proper regulation of insulin signaling.

Abstract A14: 13C metabolomic and fluxomic study of human B-cell lymphoma metabolic network: Quantifying glutaminolysis and reductive carboxylation

Abstract Intracellular 13C LC-MS and MRS isotopomer data coupled with metabolic modeling allows quantitative determination of metabolic fluxes and other metabolic parameters in vivo/in situ (1). Utilizing these methods, we have determined how an mTOR inhibitor, rapamycin, alters glycolysis, the TCA cycle, the pentose phosphate pathway (PPP) and glutamine metabolism in B-cell lymphoma cell lines. This analysis has revealed previously unknown pharmacological activities for rapamycin. DLCL2 (germinal-center type of diffuse large B-cell lymphoma) and Ramos (Burkitt’s lymphoma resembling activated ABC type of B-cells) cells were incubated in glucose and glutamine free DMEM containing 10% fetal bovine serum, 5 mM unlabeled or 13C-labelled glucose, 2 mM unlabeled or [U-13C5, 15N2] glutamine and rapamycin for 6 h. The steady-state labeling patterns of TCA cycle metabolites (LC-MS) and temporal profiles for labeled lactate and glutamate (NMR) were monitored. Data were analyzed with compartmentalized metabolic network models that included glucose, lactate and glutamine transport, glycolysis, TCA cycle, PPP, α-ketoglutarate-glutamate and oxaloacetate-aspartate exchange, pyruvate carboxylase activity, anaplerosis at the succinyl-CoA level, pyruvate recycling through malic enzyme, lactate dehydrogenase activity, glutaminolysis, reductive carboxylation and de novo fatty acids synthesis. Isotope balance equations were derived for every network metabolite. The resulted sets of differential equations were solved to determine flux through various metabolic pathways by isotopomer/cumomer analysis (2). Statistics for the obtained parameters/fluxes was calculated by Monte Carlo simulations including experimental noise levels. We analyzed changes in expression of ~10,000 metabolic genes in DLCL2 and Ramos cells induced by rapamycin (by RNA-seq). Anticipated related alterations indicate that mTOR inhibition diminishes glycolysis, TCA cycle activity, de novo fatty acid biosynthesis, pyruvate cycling and PPP. Glutaminolisys was also affected due to gene PPAT expression (responsible for enzymatic glutamate production from glutamine). Metabolic Flux Analysis revealed that in DLCL2 rapamycin produced dramatic metabolic changes in reductive carboxylation (41% decrease), glutaminolysis (95% decrease), mitochondrial pyruvate carrier (50% increase) and pyruvate carboxylase (520% increase) activities. Further study of these effects is required, yet these data strongly suggest that multiple pathways are affected and, potentially, these metabolic signatures may serve as key biomarkers of effective mTOR inhibition. Our genomic and proteomic analysis indicates that the expression level of a large number of enzymes in human lymphoma cells has been modified by rapamycin-mediated mTOR inhibition. These affected enzymes are involved in eight metabolic pathways: glycolysis, pyruvate cycling/metabolism, TCA cycle, glutaminolysis, fatty acid metabolism, cholesterol metabolism, phospholipid metabolism and the pentose shunt. Information available from 13C multiplets in 13C spectra and MID of numerous metabolites significantly increases precision of calculated metabolic fluxes and allows estimation of contributions of different pathways to particular metabolite labeling patterns. The new modeling approaches developed here provide more effecient methods for investigation of detailed mechanisms of cancer metabolism and therefore enable effective identification of targets for combinatorial therapy. Dynamic high-resolution MR spectra and LC-MS mass-isotopomers are very sensitive to changing/adding biochemical pathways and flux values, and metabolic modeling allows one to check precisely the feasibility of assumed general bionetworks and alterations of particular metabolic pathways.

MicroRNA 100 sensitizes luminal A breast cancer cells to paclitaxel treatment in part by targeting mTOR.

Luminal A breast cancer usually responds to hormonal therapies but does not benefit from chemotherapies, including microtubule-targeted paclitaxel. MicroRNAs could play a role in mediating this differential response. In this study, we examined the role of micro RNA 100 (miR-100) in the sensitivity of breast cancer to paclitaxel treatment. We found that while miR-100 was downregulated in both human breast cancer primary tumors and cell lines, the degree of downregulation was greater in the luminal A subtype than in other subtypes. The IC50 of paclitaxel was much higher in luminal A than in basal-like breast cancer cell lines. Ectopic miR-100 expression in the MCF-7 luminal A cell line enhanced the effect of paclitaxel on cell cycle arrest, multinucleation, and apoptosis, while knockdown of miR-100 in the MDA-MB-231 basal-like line compromised these effects. Similarly, overexpression of miR-100 enhanced the effects of paclitaxel on tumorigenesis in MCF-7 cells. Rapamycin-mediated inhibition of the mammalian target of rapamycin (mTOR), a target of miR-100, also sensitized MCF-7 cells to paclitaxel. Gene set enrichment analysis showed that genes that are part of the known paclitaxel-sensitive signature had a significant expression correlation with miR-100 in breast cancer samples. In addition, patients with lower levels of miR-100 expression had worse overall survival. These results suggest that miR-100 plays a causal role in determining the sensitivity of breast cancers to paclitaxel treatment.

The mTOR Kinase Inhibitor INK128 Blunts Migration of Cultured Retinal Pigment Epithelial Cells.

Retinal pigment epithelium (RPE) cell migration in response to disease has been reported for age-related macular degeneration, proliferative vitreoretinopathy, and proliferative diabetic retinopathy. The complex molecular process of RPE cell migration is regulated in part by growth factors and cytokines, and activation of the PI3/AKT/mTOR signaling pathway. Rapamycin, an allosteric mTOR inhibitor, has been shown to block only one of the primary downstream mTOR effectors, p70 S6 kinase 1, in many cell types. INK128, a selective mTOR ATP binding site competitor, blocks both p70 S6 kinase 1 and a second primary downstream effector, 4E-BP1. We performed scratch assays using differentiated ARPE-19 and primary porcine RPE cells to assess the effect of mTOR inhibition on cell migration. We found that INK128-mediated blocking of both p70 S6 kinase 1 and 4E-BP1 was much more effective at preventing RPE cell migration than rapamycin-mediated inhibition of p70 S6 kinase 1 alone.

Additive effects of rapamycin and aspirin on dendritic cell allostimulatory capacity

Acting as antigen presenting cells, mature dendritic cells (DCs) initiate both innate and adaptive alloimmune responses. However, immature DCs are weak immunostimulators and mediate tolerogenic effects under certain conditions. Tolerogenic activities of immature DCs can be enhanced by pharmacological agents. Here, we compared pharmacological DC preconditioning with rapamycin and aspirin, applied alone or in combination, on LPS-induced DC maturation and T-cell allostimulatory capacity. Preconditioning with aspirin but not rapamycin tended to reduce the number of mouse bone marrow-derived immature DCs expressing CD40 and major histocompatibility complex class II molecules upon LPS stimulation. Conversely, DC preconditioning with rapamycin, but not aspirin, reduced T-cell alloproliferative responses. A combination of rapamycin and aspirin was more effective than either drug applied alone with respect to inhibition of T-cell alloproliferation. The two agents in combination reduced numbers of CD4+IFN-γ+ Th1 and CD4+IL-17+ Th17 effector cells while maintaining Foxp3+ regulatory T cells. These results suggest aspirin may moderately enhance rapamycin-mediated inhibition of DC allostimulatory capacity.

Abstract PR04: mTOR mutations in cancer

Abstract Genes in the PI3K-Akt-mTOR signaling axis are frequently mutated in cancer but relatively few mutations have been observed in the MTOR gene itself. By mining publicly available cancer sequencing data, we recently reported that MTOR is commonly mutated in cancer, with more than 35 previously uncharacterized pathway activating mutations. Most were recurrent, localized within distinct clusters in the C-terminal half of the protein, and induced activation of mTOR Complex 1 (mTORC1), mTORC2, or both. The mutations did not prevent mTOR inhibition by rapamycin or ATP-competitive inhibitors, although several conferred resistance to nutrient withdrawal. Importantly, cancer cell lines with activating mTOR mutations are especially sensitive to rapamycin both in vitro and in vivo, indicating pathway dependency. In a companion report, we also demonstrated that the tumor of a bladder cancer patient with exquisite sensitivity to a rapamycin analog (rapalog) harbored two activating mTOR mutations, likely driving mTOR pathway dependency. However, after 14 months of rapalog response, the patient displayed progressive disease, indicating that the tumor had acquired resistance to the targeted therapy. Here, we demonstrate two mechanisms by which cancers may display resistance to rapamycin through mTOR mutation. In the first example, we acquired a tumor biopsy from an anaplastic thyroid carcinoma patient before they were enrolled on a rapalog clinical trial. The patient displayed a durable 18 month response to the rapalog before progressing, at which time we acquired a second biopsy. Whole exome sequencing of the biopsies revealed the presence of a loss-of-function mutation in the mTORC1 negative regulator Tuberin (TSC2), likely driving pathway addiction and thus sensitivity to the rapalog. Sequencing of the post-resistance biopsy revealed that the tumor had acquired a mutation in the rapamycin-binding domain of mTOR. Cells made to exogenously express this mutation displayed potent resistance to rapamycin-mediated inhibition of cell proliferation and phosphorylation of the mTORC1 substrate S6K1. Importantly, the cells remained sensitive to an ATP-competitive mTOR inhibitor because the two classes of mTOR inhibitors rely upon completely different mechanisms of action. Thus, we show that as patients acquire resistance to rapalogs, they may still be effectively treated with ATP-competitive mTOR inhibitors. The rapalog-resistant mutation described above is an example of an acquired mutation, although due to the genetic instability of cancer cells, a tumor may harbor pre-existing rapamycin-resistant mutations. To determine the prevalence of such mutations, we again turned to publicly available cancer sequencing data, identifying approximately 30 mutations in the rapamycin-binding domain of mTOR. The majority these mutations did not alter mTOR pathway activation and remained rapamycin sensitive. Importantly, one breast cancer sample harbored a rapamycin-resistant mTOR mutation, although it remained sensitive to an ATP-competitive mTOR inhibitor. This rare event is highly relevant given that rapalogs are currently approved for the treatment of breast cancer. Had this patient been treated with a rapalog, they would have only experienced the side-effects and none of the benefits of treatment. Collectively, we demonstrate that mTOR mutations are common in cancer, many of which confer pathway activation and addiction. Additionally, both acquired and pre-existing mTOR mutations can confer resistance towards rapamycin treatment, although this resistance can be overcome with ATP-competitive inhibitors. This work has important ramifications for the treatment of cancer patients with mTOR inhibitors, by identifying both those patients likely to respond or display resistance to such treatments. This abstract is also being presented as Poster A44. Citation Format: Brian Grabiner, Nikhil Wagle, Eliezer Van Allen, Levi Garraway, Jochen Lorch, David Sabatini. mTOR mutations in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr PR04.

Angiotensin II blockade: how its molecular targets may signal to mitochondria and slow aging. Coincidences with calorie restriction and mTOR inhibition

Caloric restriction (CR), renin angiotensin system blockade (RAS-bl), and rapamycin-mediated mechanistic target of rapamycin (mTOR) inhibition increase survival and retard aging across species. Previously, we have summarized CR and RAS-bl's converging effects, and the mitochondrial function changes associated with their physiological benefits. mTOR inhibition and enhanced sirtuin and KLOTHO signaling contribute to the benefits of CR in aging. mTORC1/mTORC2 complexes contribute to cell growth and metabolic regulation. Prolonged mTORC1 activation may lead to age-related disease progression; thus, rapamycin-mediated mTOR inhibition and CR may extend lifespan and retard aging through mTORC1 interference. Sirtuins by deacetylating histone and transcription-related proteins modulate signaling and survival pathways and mitochondrial functioning. CR regulates several mammalian sirtuins favoring their role in aging regulation. KLOTHO/fibroblast growth factor 23 (FGF23) contribute to control Ca(2+), phosphate, and vitamin D metabolism, and their dysregulation may participate in age-related disease. Here we review how mTOR inhibition extends lifespan, how KLOTHO functions as an aging suppressor, how sirtuins mediate longevity, how vitamin D loss may contribute to age-related disease, and how they relate to mitochondrial function. Also, we discuss how RAS-bl downregulates mTOR and upregulates KLOTHO, sirtuin, and vitamin D receptor expression, suggesting that at least some of RAS-bl benefits in aging are mediated through the modulation of mTOR, KLOTHO, and sirtuin expression and vitamin D signaling, paralleling CR actions in age retardation. Concluding, the available evidence endorses the idea that RAS-bl is among the interventions that may turn out to provide relief to the spreading issue of age-associated chronic disease.