Inhibition Of mTOR Complex Research Articles

Clinical features, epidemiology, and therapy of lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a multisystem disease of women, characterized by proliferation of abnormal smooth muscle-like LAM cells, leading to the formation of lung cysts, fluid-filled cystic structures in the axial lymphatics (eg, lymphangioleiomyomas), and renal angiomyolipomas. LAM is caused by mutations of the TSC1 or TSC2 genes, which encode, respectively, hamartin and tuberin, two proteins with a major role in control of the mammalian target of rapamycin (mTOR) signaling pathway. LAM occurs sporadically or in association with tuberous sclerosis complex, an autosomal-dominant syndrome characterized by widespread hamartomatous lesions. LAM may present with progressive dyspnea, recurrent pneumothorax, or chylothorax. Pulmonary function tests show reduced flow rates (forced expiratory volume in the first second) and diffusion capacity. Exercise testing may reveal gas exchange abnormalities, ventilatory limitation, and hypoxemia. The severity and progression of disease may be assessed by lung histology scores, quantification of computed tomography, pulmonary function testing, 6-minute walk tests, cardiopulmonary exercise testing, and measurement of serum vascular endothelial growth factor D levels. Sirolimus and everolimus, two mTOR inhibitors, are effective in stabilizing lung function and reducing the size of chylous effusions, lymphangioleiomyo-mas, and angiomyolipomas. However, inhibition of mTOR complex 1 increases autophagy, possibly enhancing LAM cell survival. Inhibition of autophagy with hydroxychloroquine, in combination with sirolimus, has been proposed as a possible treatment for LAM. Deficiency of tuberin results in increased RhoA GTPase activity and cell survival, an effect that is mediated through mTOR complex 2 signaling. Because sirolimus and everolimus only affect the activity of mTOR complex 1, therapies targeting RhoA GTPases with simvastatin, which inhibits Rho GTPases and promotes apoptosis, are being investigated. As in the case of cancer, LAM may be best treated with multiple drugs targeting signaling pathways considered important in the pathogenesis of disease.

Differences in the Responses to Chronic Rapamycin Treatment by Healthy and Insulin Resistant Rats.

The immunosuppressant rapamycin (Rap) inhibits mTOR Complex 1 and promotes longevity. However, Rap also induces type 2 diabetes (T2DM). Since DM is an independent predictor for cardiovascular disease, we investigated whether chronic Rap treatment that exacerbates diabetes modulates cardiac pathology differently in healthy and insulin resistant individuals. Young (8‐20 weeks) healthy Zucker lean (ZL) and insulin resistant Zucker obese (ZO) rats were subjected to 12‐week Rap treatment via subcutaneous implantation of Rap pellets delivering Rap at a concentration of 750μg/kg/day. Rap significantly suppressed body weight, heart weight, fasting plasma levels of cholesterol, triglycerides, and LDL in ZO rats, but worsened diabetes as indicated by severe hyperglycemia, polydipsia and polyuria (p<0.05). Echocardiography showed that 14‐week old ZO rats exhibited diastolic dysfunction evidenced by decreases in the tissue doppler E'/A' ratio, mitral inflow propagation velocity (Vp), and increases in LV filling pressure (E/E' and E/Vp ratios), isovolumic relaxation time (IVRT) and myocardial performance index (Tei index of global cardiac function) compared to age‐matched ZL rats(p<0.05). With the exception of IVRT, these abnormalities were improved in ZO rats treated for 6 weeks with Rap. However, an additional 6 weeks of Rap treatment reversed this effect. Histopathology showed that Rap induced significant cardiomyofibril disarray in both ZL and ZO rats (p<0.05), damaged the healthy circular morphology of pancreatic islets in ZL and ZO rats and reduced fibrosis in ZO rats, but not in ZL rats. Thus, the heart and pancreas of healthy and insulin resistant rats respond differently to chronic Rap treatment.

Differential Roles of Mammalian Target of Rapamycin (mTOR) in Phrenic Long‐Term Facilitation

Phrenic long‐term facilitation (pLTF) is a persistent increase in phrenic nerve activity after acute intermittent hypoxia (AIH). This form of spinal plasticity is expressed via distinct cell signaling cascades that depend on the severity of hypoxia during intermittent exposures. Each mechanism requires spinal protein synthesis, though the newly synthesized proteins differ (e.g. BDNF vs immature TrkB). Translational regulation of new protein synthesis is often regulated by mammalian Target of Rapamycin (mTOR)‐dependent translation. Here we tested the hypothesis that mTOR is necessary for severe AIH (sAIH; PaO2 25‐30mmHG), but not moderate AIH (mAIH; PaO2 35‐45mmHG) induced spinal protein synthesis. Phrenic nerve activity was recorded in anesthetized, paralyzed and ventilated male Sprague‐Dawley rats before, during and 60 min after mAIH or sAIH. Rats were pre‐treated with intrathecal injections of vehicle or Rapamycin (0.1mM, 12µl), a highly selective inhibitor of mTOR complex 1. Consistent with our hypothesis, Rapamycin blunted sAIH, but not mAIH induced pLTF. Thus, spinal activation of mTOR and/or its down‐stream targets are necessary for sAIH‐, but not mAIH‐induced pLTF. These results provide further evidence that mAIH and sAIH elicit pLTF via distinct cellular cascades despite differing only in the severity of hypoxemia within hypoxic episodes.

MP36-10 MIR-99A ACTS AS TUMOR SUPPRESSOR VIA TARGETING TO MTOR IN HUMAN BLADDER CANCER CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2015MP36-10 MIR-99A ACTS AS TUMOR SUPPRESSOR VIA TARGETING TO MTOR IN HUMAN BLADDER CANCER CELLS Te-Fu Tsai, Ji-Fan Lin, Yi-Chia Lin, Kuang-Yu Chou, Hung-En Chen, and Thomas I.S. Hwang Te-Fu TsaiTe-Fu Tsai More articles by this author , Ji-Fan LinJi-Fan Lin More articles by this author , Yi-Chia LinYi-Chia Lin More articles by this author , Kuang-Yu ChouKuang-Yu Chou More articles by this author , Hung-En ChenHung-En Chen More articles by this author , and Thomas I.S. HwangThomas I.S. Hwang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.738AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES mTOR is recognized as an important target in many cancer types. The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be insensitive to RAD001 and was shown to regulate the prosurvival kinase AKT by phosphrylation. We previously showed that the expression level of miR-99a was down-regulated in human bladder tumor tissues compared to their adjacent normal tissues. Since mTOR is predicted to be a potential target of miR-99a, we investigate the anti-cancer activity of miR-99a in bladder cancer cells. METHODS Vectors that express miR-99a were transfected into human bladder, prostate, embryonic kidney and cervical cancer cells. Expression level of miR-99a was monitored by miRNA Q-PCR. 3'-UTR of mTOR was constructed downstream of a luciferase gene, and luciferase reporter assays were performed to verify the direct binding of miR-99a to mTOR transcripts. The mRNA and protein expression level of mTOR were measured by Q-PCR and Western blot, respectively. Cell viability of miR-99a transfected cells was detected by WST-1 and colony formation assays. Inhibition of mTOR complex 1 and 2 (mTORC1 and mTORC2) signaling was monitored by detecting the phosphrylation of S6K and Akt using Western blot. Induction of autophagy was accessed by the expression of LC3-II marker protein. RESULTS Transfection of miR-99a expressing vector elevated the expression level of miR-99a up to 4.5-fold in cells compared to vector-only control. The function of matured miR-99a was confirmed by luciferase reporter assays. The level of mTOR RNA and protein were decreased in miR-99a transfected cells. Dual inhibition of mTORC1 and mTORC2 was confirmed by immunoprecipitation (IP) of mTOR associated Rictor and Raptor, and the decreased phosphrylation of S6K and Akt in miR-99a transfected cells. The LC3-II protein was accumulated in miR-99a transfected cells compared to monk transfected control, suggesting that inhibition of mTOR by miR-99a induces autophagy in bladder cancer cells. CONCLUSIONS This is the first study showed that miR-99a markedly inhibits bladder and prostate cancer cell growth via dual inhibition of mTORC1 and mTORC2. miR-99a treatment also induces autophagy through mTOR inhibition. However, the role of miR-99a induces autophagy remains further investigation. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e432 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Te-Fu Tsai More articles by this author Ji-Fan Lin More articles by this author Yi-Chia Lin More articles by this author Kuang-Yu Chou More articles by this author Hung-En Chen More articles by this author Thomas I.S. Hwang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

P5.03 - Control of cell fate by the mammalian target of rapamycin (mTOR) under stress depends on eIF2α serine 51 phosphorylation

ABSTRACT The mammalian/mechanist target of rapamycin (mTOR) plays an essential role in cell growth and metabolism by regulating protein and RNA stability, transcription as well as mRNA translation. Phosphorylation of the a subunit of translation initiation factor eIF2 at serine 51 (eIF2aS51P) is a key regulator of mRNA translation and an important determinant of cell fate under stress. Some studies have provided evidence for a cross-talk between mTOR and eIF2aS51P under stress through poorly defined mechanisms. Herein, we demonstrate that genetic as well as pharmacological inhibition of mTOR complex 2 (mTORC2) induces eIF2aS51P via the activation of the endoplasmic reticulum (ER)-resident kinase PERK/PEK as a result of Akt inactivation. Analysis of the functional interplay between eIF2aS51P and mTOR in tuberous sclerosis complex (TSC)-deficient cells showed that mTORC2 inactivation induces the PERK-eIF2aS51P arm to substitute for the loss of Akt and promote survival in response to ER stress (see Figure above). On the other hand, TSC-deficient cells decrease PERK activity and increase the activity of the double-stranded RNA-activated protein kinase PKR to promote death via eIF2aS51P, JNK activation and increased autophagy in response to oxidative stress (see attached Figure). The balance between PERK inactivation and PKR activation in TSC-mutant cells under oxidative stress is controlled by mTORC1 and ribosomal S6 kinase 1, 2 (S6K1, 2) activity. Impaired eIF2aS51P accelerates TSC-mutant tumor initiation in mice, which is associated with an impaired production of reactive oxygen species (ROS) compared to eIF2aS51P-proficient TSC-mutant tumors. Our study reveals that eIF2aS51P acts as a double-edged sword downstream of mTOR to promote either survival or death in response to distinct forms of stress with implications in tumor development and treatment with chemotherapeutic drugs. Download : Download full-size image P5.03 FIGURES .

MTOR inhibition in epilepsy: rationale and clinical perspectives.

Despite a large number of available medical options, many individuals with epilepsy are refractory to existing therapies that mainly target neurotransmitter or ion channel activity. A growing body of preclinical data has uncovered a molecular pathway that appears crucial in many genetic and acquired epilepsy syndromes. The mammalian target of rapamycin (mTOR) pathway regulates a number of cellular processes required in the growth, metabolism, structure, and cell-cell interactions of neurons and glia. Rapamycin and similar compounds inhibit mTOR complex 1 and decrease seizures, delay seizure development, or prevent epileptogenesis in many animal models of mTOR hyperactivation. However, the exact mechanisms by which mTOR inhibition drives decreased seizure activity have not been completely determined. Nonetheless, these preclinical data have led to limited use in humans with epilepsy due to tuberous sclerosis complex and polyhydramnios, megalencephaly, and symptomatic epilepsy with promising results. Currently, larger controlled studies are underway using mTOR inhibitors in individuals with tuberous sclerosis complex and intractable epilepsy.

Significance of 4E-binding protein 1 as a therapeutic target for invasive urothelial carcinoma of the bladder

BackgroundTo evaluate the expression of multiple molecular markers involved in the mammalian target of rapamycin (mTOR) signaling pathway in human muscle-invasive bladder cancer (BC) and to assess the therapeutic efficacies of mTOR inhibitors in human BC KoTCC-1 cells. MethodsExpression levels of 5 markers, including PTEN, phosphorylated (p)-Akt, p-mTOR, p-p70 ribosomal S6 kinase, and p-4E-binding protein 1 (4E-BP1), were measured in radical cystectomy specimens from 49 patients with muscle-invasive BC by immunohistochemical staining. We then analyzed the effects of treatment with temsirolimus or Ku-0063794, a dual inhibitor of mTOR complex 1 (C1) and mTOR complex 2 (C2), on changes in the growth and expression profiles of 5 mTOR-associated markers in KoTCC-1 cells. ResultsDuring the follow-up period of this study, disease recurred in 27 patients (55.1%), and of several factors examined, the expression level of p-4E-BP1 in addition to the pathological T stage was independently related to recurrence-free survival on multivariate analysis. Although the growth of KoTCC-1 cells was inhibited by both temsirolimus and Ku-0063794 in dose-dependent manners, treatment with Ku-0063794 resulted in a marked decrease in the expression of p-4E-BP1 in KoTCC-1 cells compared with that with temsirolimus. Furthermore, the growth-inhibitory effect of both mTOR inhibitors was shown to be proportional to the expression levels of p-4E-BP1. ConclusionsThe phosphorylation status of 4E-BP1 appeared to be correlated with the prognosis of patients with muscle-invasive BC following radical cystectomy as well as the sensitivities of BC cells to mTOR inhibitors; therefore, the inactivation of 4E-BP1 using Ku-0063794 may be a promising novel approach for muscle-invasive BC.

Resistance exercise-induced S6K1 kinase activity is not inhibited in human skeletal muscle despite prior activation of AMPK by high-intensity interval cycling.

Combining endurance and strength training in the same session has been reported to reduce the anabolic response to the latter form of exercise. The underlying mechanism, based primarily on results from rodent muscle, is proposed to involve AMPK-dependent inhibition of mTORC1 signaling. This hypothesis was tested in eight trained male subjects who in randomized order performed either resistance exercise only (R) or interval cycling followed by resistance exercise (ER). Biopsies taken from the vastus lateralis before and after endurance exercise and repeatedly after resistance exercise were assessed for glycogen content, kinase activity, protein phosphorylation, and gene expression. Mixed muscle fractional synthetic rate was measured at rest and during 3 h of recovery using the stable isotope technique. In ER, AMPK activity was elevated immediately after both endurance and resistance exercise (∼90%, P < 0.05) but was unchanged in R. Thr(389) phosphorylation of S6K1 was increased severalfold immediately after exercise (P < 0.05) in both trials and increased further throughout recovery. After 90 and 180 min recovery, S6K1 activity was elevated (∼55 and ∼110%, respectively, P < 0.05) and eukaryotic elongation factor 2 phosphorylation was reduced (∼55%, P < 0.05) with no difference between trials. In contrast, markers for protein catabolism were differently influenced by the two modes of exercise; ER induced a significant increase in gene and protein expression of MuRF1 (P < 0.05), which was not observed following R exercise only. In conclusion, cycling-induced elevation in AMPK activity does not inhibit mTOR complex 1 signaling after subsequent resistance exercise but may instead interfere with the hypertrophic response by influencing key components in protein breakdown.

SDF-1/CXCL12 induces directional cell migration and spontaneous metastasis via a CXCR4/Gαi/mTORC1 axis.

Multiple human malignancies rely on C-X-C motif chemokine receptor type 4 (CXCR4) and its ligand, SDF-1/CXCL12 (stroma cell-derived factor 1/C-X-C motif chemokine 12), to metastasize. CXCR4 inhibitors promote the mobilization of bone marrow stem cells, limiting their clinical application for metastasis prevention. We investigated the CXCR4-initiated signaling circuitry to identify new potential therapeutic targets. We used HeLa human cancer cells expressing high levels of CXCR4 endogenously. We found that CXCL12 promotes their migration in Boyden chamber assays and single cell tracking. CXCL12 activated mTOR (mechanistic target of rapamycin) potently in a pertussis-sensitive fashion. Inhibition of mTOR complex 1 (mTORC1) by rapamycin [drug concentration causing 50% inhibition (IC50) = 5 nM] and mTORC1/mTORC2 by Torin2 (IC50 = 6 nM), or by knocking down key mTORC1/2 components, Raptor and Rictor, respectively, decreased directional cell migration toward CXCL12. We developed a CXCR4-mediated spontaneous metastasis model by implanting HeLa cells in the tongue of SCID-NOD mice, in which 80% of the animals develop lymph node metastasis. It is surprising that mTORC1 disruption by Raptor knockdown was sufficient to reduce tumor growth by 60% and spontaneous metastasis by 72%, which were nearly abolished by rapamycin. In contrast, disrupting mTORC2 had no effect in tumor growth or metastasis compared with control short hairpin RNAs. These data suggest that mTORC1 may represent a suitable therapeutic target in human malignancies using CXCR4 for their metastatic spread. .

Abstract 13566: Pitavastatin Regulates mTOR Complex 1 Signaling Through Inhibition of Rheb in T Cells

Accumulated evidence has shown that statins have anti-inflammatory and immunomodulatory effects; however, the mechanism has not been fully elucidated. Here we report that pitavastatin inhibited mTOR complex 1 (mTORC1) activity by inhibiting the farnesylation of the small GTPase Rheb in murine T cells. As a result of The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibition, statins regulate the biosynthesis of cholesterol and isoprenoid farnesylpyrophosphate (farnesyl-PP) that mediates the farnesylation and membrane association of Rheb. Pitavastatin-treated T cells failed to differentiate into T helper (Th)1 and Th17 cells, and this failure was rescued by adding farnesyl-PP. Pitavastatin and a farnesyltransferase inhibitor affected the transcription of T-bet and RORγT which have critical roles in the development of Th1 and Th17 cells respectively. In a mouse model of experimental autoimmune myocarditis, a CD4+ T cell-mediated disease, pitavastatin treatment ameliorated the pathophysiological severity of myocarditis associated with reduced Th1 and Th17 responses and mTORC1 signaling. We conclude that statins reduce the inflammatory functions and pathogenic activity of T cells through inhibition of mTORC1 signaling, and that this pathway may be a potential therapeutic target for Th1- and Th17-related diseases.

Orexin/Hypocretin Activates mTOR Complex 1 (mTORC1) via an Erk/Akt-independent and Calcium-stimulated Lysosome v-ATPase Pathway

The lack of the neuropeptide orexin, also known as hypocretin, results in narcolepsy, a chronic sleep disorder characterized by frequent sleep/cataplexy attacks and rapid eye movement sleep abnormalities. However, the downstream pathways of orexin signaling are not clearly understood. Here, we show that orexin activates the mTOR pathway, a central regulator of cell growth and metabolism, in the mouse brain and multiple recombinant cell lines that express the G protein-coupled receptors (GPCRs), orexin 1 receptor (OX1R) or orexin 2 receptor (OX2R). This orexin/GPCR-stimulated mTOR activation is sensitive to rapamycin, an inhibitor of mTOR complex 1 (mTORC1) but is independent of two well known mTORC1 activators, Erk and Akt. Rather, our studies indicate that orexin activates mTORC1 via extracellular calcium influx and the lysosome pathway involving v-ATPase and Rag GTPases. Moreover, a cytoplasmic calcium transient is sufficient to mimic orexin/GPCR signaling to mTORC1 activation in a v-ATPase-dependent manner. Together, our studies suggest that the mTORC1 pathway functions downstream of orexin/GPCR signaling, which plays a crucial role in many physiological and metabolic processes.

AT-30 * EFFECTS OF EVEROLIMUS ON MENINGIOMA GROWTH IN PATIENTS WITH NEUROFIBROMATOSIS TYPE 2

BACKGROUND: Vestibular schwannomas (VS) and meningiomas are associated with biallelic loss of NF2, and may arise sporadically or in the context of the tumor predisposition syndrome neurofibromatosis type 2. Loss of NF2 activates the mammalian target of rapamycin (mTOR) pathway, and published in vitro and in vivo data indicate that mTOR inhibition may be effective in the treatment of NF2-deficient VS and meningiomas. Everolimus is an oral inhibitor of mTOR complex 1 (mTORC1) with anti-tumor activity in a variety of tumors. METHODS: We conducted a retrospective review of patients with NF2 and progressive vestibular schwannomas treated with everolimus on our prospective phase II clinical trial (ClinicalTrials.gov NCT01419639). Everolimus was administered at a daily dose of 10 mg in continuous 28-day courses for up to 12 courses. In this retrospective study, we included patients with at least one volumetrically measurable meningioma (>0.5 cc) who received at least six 28-day courses of therapy. Tumor response was assessed with brain MRI every three months using three-dimensional volumetric analysis. RESULTS: Three patients met criteria and had 10 evaluable meningiomas, with a total combined volume of 11.84 cc at baseline (median 1.20 cc, range 0.53–3.00 cc). Median time on therapy was 9 months (range 6–12 months). Total combined meningioma volume remained stable during treatment (-1.8%). Correspondingly, none of the individual tumors analyzed met criteria for volumetric response or progression during the treatment period (range -10% to +9.4%). CONCLUSIONS: Neither objective response nor progression was appreciated in meningiomas patients during a median treatment duration of 6 months. Further clinical studies will be required to determine whether everolimus affects meningioma growth velocity in patients with NF2. To better understand pharmacologic mTOR signaling pathway modulation in human VS and meningiomas in vivo, we are currently conducting a multi-center pharmacodynamic/pharmacokinetic (“phase 0”) study with everolimus (NCT01880749).

The nuclear import of ribosomal proteins is regulated by mTOR.

Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins.

Depletion of Rictor, an essential protein component of mTORC2, decreases male lifespan.

Rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR), robustly extends the lifespan of model organisms including mice. We recently found that chronic treatment with rapamycin not only inhibits mTOR complex 1 (mTORC1), the canonical target of rapamycin, but also inhibits mTOR complex 2 (mTORC2) in vivo. While genetic evidence strongly suggests that inhibition of mTORC1 is sufficient to promote longevity, the impact of mTORC2 inhibition on mammalian longevity has not been assessed. RICTOR is a protein component of mTORC2 that is essential for its activity. We examined three different mouse models of Rictor loss: mice heterozygous for Rictor, mice lacking hepatic Rictor, and mice in which Rictor was inducibly deleted throughout the body in adult animals. Surprisingly, we find that depletion of RICTOR significantly decreases male, but not female, lifespan. While the mechanism by which RICTOR loss impairs male survival remains obscure, we find that the effect of RICTOR depletion on lifespan is independent of the role of hepatic mTORC2 in promoting glucose tolerance. Our results suggest that inhibition of mTORC2 signaling is detrimental to males, which may explain in part why interventions that decrease mTOR signaling show greater efficacy in females.

Cordycepin down-regulates multiple drug resistant (MDR)/HIF-1α through regulating AMPK/mTORC1 signaling in GBC-SD gallbladder cancer cells.

Gallbladder cancer is the most common malignancy of the bile duct, with low 5-year survival rate and poor prognosis. Novel effective treatments are urgently needed for the therapy of this disease. Here, we showed that cordycepin, the bioactive compound in genus Cordyceps, induced growth inhibition and apoptosis in cultured gallbladder cancer cells (Mz-ChA-1, QBC939 and GBC-SD lines). We found that cordycepin inhibited mTOR complex 1 (mTORC1) activation and down-regulated multiple drug resistant (MDR)/hypoxia-inducible factor 1α (HIF-1α) expression through activating of AMP-activated protein kinase (AMPK) signaling in gallbladder cancer GBC-SD cells. Contrarily, AMPKα1-shRNA depletion dramatically inhibited cordycepin-induced molecular changes as well as GBC-SD cell apoptosis. Further, our results showed that co-treatment with a low concentration cordycepin could remarkably enhance the chemosensitivity of GBC-SD cells to gemcitabine and 5-fluorouracil (5-FU), and the mechanism may be attributed to AMPK activation and MDR degradation. In summary, cordycepin induces growth inhibition and apoptosis in gallbladder cancer cells via activating AMPK signaling. Cordycepin could be a promising new drug or chemo-adjuvant for gallbladder cancer.

TSC1 is critical for iNKT-cell terminal maturation and controls iNKT-1/17 balance (IRM7P.490)

Abstract iNKT-cells terminal maturation from CD44+NK1.1- stage 2 to CD44+NK1.1+ stage 3 is accompanied by functional acquisition of predominant IFNγ-producing (iNKT-1) but not IL-17-producing (iNKT-17) capability. iNKT-17-cells are rare and restricted to the CD44+NK1.1- subset. The mechanisms that control iNKT terminal maturation and predominance of iNKT-1 over iNKT-17 remain poorly understood. The tumor suppressor tuberous sclerosis 1 (TSC1) is an important negative regulator of mTOR signaling. We demonstrate here that deficiency of the TSC1 causes iNKT-cell developmental block at stage 2 and functional alteration from iNKT-1 predominance to iNKT-17 predominance, leading to enhanced airway hypersensitivity. TSC1 promotes T-bet but downregulates ICOS expression in iNKT-cells by inhibiting mTOR complex 1. Decreased T-bet contributes to both terminal maturation defect and iNKT-17 predominance over iNKT-1 while increased ICOS expression is required for iNKT-17 predominance in TSC1-deficient iNKT-cells. Thus, TSC1and its proper control of mTORC1 are crucial for iNKT-cell terminal maturation and effector lineage decision for iNKT-1 predominance over iNKT-17.

The SDF‐1/CXCR4 axis utilizes mTOR signal transduction to promote angiogenesis (278.8)

Pathological angiogenesis, which occurs during chronic inflammation or tumor growth, results when positive and negative angiogenic regulators are disrupted. The chemokine stromal‐cell derived factor‐1 (SDF‐1) and its receptor CXCR4 promote endothelial cell (EC) migration and tube formation. PI3K is a key downstream regulator of CXCR4‐mediated chemotaxis and mammalian target of rapamycin (mTOR) is at the center of PI3K‐mediated signal transduction. We have hypothesized that the SDF‐1/CXCR4/mTOR signaling axis contributes to the molecular mechanism of angiogenesis. This study illustrates that SDF‐1 stimulation activates mTOR signal transduction in EC grown in monolayer cell culture. The pharmacological inhibition of CXCR4 or mTOR blocks sprouting in an in vitro 3D angiogenesis model, and also abolishes EC migration in a 3D angiogenic invasion assay. Specific inhibition of mTOR complex 1 or mTOR complex 2 (mTORC2) reveals that SDF‐1‐induced angiogenesis occurs via mTORC2 signal transduction. An analysis of targets downstream of mTOR indicates that 6‐phosphofructo‐2‐kinase (PFKFB3), which is a key regulator of glycolysis, is downregulated in EC in which mTORC2 is inhibited. Taken together these data indicate that the SDF‐1/CXCR4 axis activates angiogenesis by utilizing an mTORC2 signal transduction pathway. In addition, these data present a potential link between mTORC2, metabolism and angiogenesis, which deserves further exploration.Grant Funding Source: The project described was supported by Grant Number T32CA009054 from the National Cancer Institute.

Everolimus Inhibits Anti-HLA I Antibody-Mediated Endothelial Cell Signaling, Migration and Proliferation More Potently Than Sirolimus

Antibody (Ab) crosslinking of HLA I molecules on the surface of endothelial cells triggers proliferative and pro-survival intracellular signaling, which is implicated in the process of chronic allograft rejection, also known as transplant vasculopathy (TV). The purpose of this study was to investigate the role of mammalian target of rapamycin (mTOR) in HLA I Ab-induced signaling cascades. Everolimus provides a tool to establish how the mTOR signal network regulates HLA I-mediated migration, proliferation and survival. We found that everolimus inhibits mTOR complex 1 (mTORC1) by disassociating Raptor from mTOR, thereby preventing class I-induced phosphorylation of mTOR, p70S6K, S6RP and 4E-BP1, and resultant class I-stimulated cell migration and proliferation. Furthermore, we found that everolimus inhibits class I-mediated mTORC2 activation (1) by disassociating Rictor and Sin1 from mTOR; (2) by preventing class I-stimulated Akt phosphorylation and (3) by preventing class I-mediated ERK phosphorylation. These results suggest that everolimus is more effective than sirolimus at antagonizing both mTORC1 and mTORC2, the latter of which is critical in endothelial cell functional changes leading to TV in solid organ transplantation after HLA I crosslinking. Our findings point to a potential therapeutic effect of everolimus in prevention of chronic Ab-mediated rejection.

INKT cells require TSC1 for terminal maturation and effector lineage fate decisions

Terminal maturation of invariant NKT (iNKT) cells from stage 2 (CD44+NK1.1-) to stage 3 (CD44+NK1.1+) is accompanied by a functional acquisition of a predominant IFN-γ-producing (iNKT-1) phenotype; however, some cells develop into IL-17-producing iNKT (iNKT-17) cells. iNKT-17 cells are rare and restricted to a CD44+NK1.1- lineage. It is unclear how iNKT terminal maturation is regulated and what factors mediate the predominance of iNKT-1 compared with iNKT-17. The tumor suppressor tuberous sclerosis 1 (TSC1) is an important negative regulator of mTOR signaling, which regulates T cell differentiation, function, and trafficking. Here, we determined that mice lacking TSC1 exhibit a developmental block of iNKT differentiation at stage 2 and skew from a predominantly iNKT-1 population toward a predominantly iNKT-17 population, leading to enhanced airway hypersensitivity. Evaluation of purified iNKT cells revealed that TSC1 promotes T-bet, which regulates iNKT maturation, but downregulates ICOS expression in iNKT cells by inhibiting mTOR complex 1 (mTORC1). Furthermore, mice lacking T-bet exhibited both a terminal maturation defect of iNKT cells and a predominance of iNKT-17 cells, and increased ICOS expression was required for the predominance of iNKT-17 cells in the population of TSC1-deficient iNKT cells. Our data indicate that TSC1-dependent control of mTORC1 is crucial for terminal iNKT maturation and effector lineage decisions, resulting in the predominance of iNKT-1 cells.

Activation of the PI3K/mTOR Pathway Is Involved in Cystic Proliferation of Cholangiocytes of the PCK Rat

The polycystic kidney (PCK) rat is an animal model of Caroli’s disease as well as autosomal recessive polycystic kidney disease (ARPKD). The signaling pathways involving the mammalian target of rapamycin (mTOR) are aberrantly activated in ARPKD. This study investigated the effects of inhibitors for the cell signaling pathways including mTOR on cholangiocyte proliferation of the PCK rat. Cultured PCK cholangiocytes were treated with rapamycin and everolimus [inhibitors of mTOR complex 1 (mTOC1)], LY294002 [an inhibitor of phosphatidylinositol 3-kinase (PI3K)] and NVP-BEZ235 (an inhibitor of PI3K and mTORC1/2), and the cell proliferative activity was determined in relation to autophagy and apoptosis. The expression of phosphorylated (p)-mTOR, p-Akt, and PI3K was increased in PCK cholangiocytes compared to normal cholangiocytes. All inhibitors significantly inhibited the cell proliferative activity of PCK cholangiocytes, where NVP-BEZ235 had the most prominent effect. NVP-BEZ235, but not rapamycin and everolimus, further inhibited biliary cyst formation in the three-dimensional cell culture system. Rapamycin and everolimus induced apoptosis in PCK cholangiocytes, whereas NVP-BEZ235 inhibited cholangiocyte apoptosis. Notably, the autophagic response was significantly induced following the treatment with NVP-BEZ235, but not rapamycin and everolimus. Inhibition of autophagy using siRNA against protein-light chain3 and 3-methyladenine significantly increased the cell proliferative activity of PCK cholangiocytes treated with NVP-BEZ235. In vivo, treatment of the PCK rat with NVP-BEZ235 attenuated cystic dilatation of the intrahepatic bile ducts, whereas renal cyst development was unaffected. These results suggest that the aberrant activation of the PI3K/mTOR pathway is involved in cystic proliferation of cholangiocytes of the PCK rat, and inhibition of the pathway can reduce cholangiocyte proliferation via the mechanism involving apoptosis and/or autophagy.