Inhibition Of mTOR Activity Research Articles

Exogenous Restoration of TUSC2 Expression Induces Responsiveness to Erlotinib in Wildtype Epidermal Growth Factor Receptor (EGFR) Lung Cancer Cells through Context Specific Pathways Resulting in Enhanced Therapeutic Efficacy.

Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR.

Glutamine promotes ovarian cancer cell proliferation through the mTOR/S6 pathway.

Glutamine is one of the main nutrients used by tumor cells for biosynthesis. Therefore, targeted inhibition of glutamine metabolism may have anti-tumorigenic implications. In the present study, we aimed to evaluate the effects of glutamine on ovarian cancer cell growth. Three ovarian cancer cell lines, HEY, SKOV3, and IGROV-1, were assayed for glutamine dependence by analyzing cytotoxicity, cell cycle progression, apoptosis, cell stress, and glucose/glutamine metabolism. Our results revealed that administration of glutamine increased cell proliferation in all three ovarian cancer cell lines in a dose dependent manner. Depletion of glutamine induced reactive oxygen species and expression of endoplasmic reticulum stress proteins. In addition, glutamine increased the activity of glutaminase (GLS) and glutamate dehydrogenase (GDH) by modulating the mTOR/S6 and MAPK pathways. Inhibition of mTOR activity by rapamycin or blocking S6 expression by siRNA inhibited GDH and GLS activity, leading to a decrease in glutamine-induced cell proliferation. These studies suggest that targeting glutamine metabolism may be a promising therapeutic strategy in the treatment of ovarian cancer.

Intestinal mTOR regulates GLP-1 production in mouse L cells.

Glucagon-like peptide (GLP-1), an intestinal incretin produced in L cells through proglucagon processing, is released in response to meal intake. The intracellular mechanism by which L cells sense the organism energy level to coordinate the production of GLP-1 remains unclear. Mechanistic target of rapamycin (mTOR) is an intracellular fuel sensor critical for energy homeostasis. In this study, we investigated whether intestinal mTOR regulates GLP-1 production in L cells. The effects of mTOR on GLP-1 production were examined in lean- or high-fat diet (HFD) induced diabetic C57/BL6, db/db, Neurog3-Tsc1(-/-) mice, and STC-1 cells. GLP-1 expression was investigated by real-time PCR and western blotting. Plasma GLP-1 and insulin were detected by enzyme immunoassay and radioimmunoassay, respectively. Fasting downregulated mTOR activity, which was associated with a decrement of intestinal proglucagon and circulating GLP-1. Upon re-feeding, these alterations returned to the levels of fed animals. In HFD induced diabetic mice, ileal mTOR signalling, proglucagon and circulating GLP-1 were significantly decreased. Inhibition of mTOR signalling by rapamycin decreased levels of intestinal and plasma GLP-1 in both normal and diabetic mice. Activation of the intestinal mTOR signalling by L-leucine or Tsc1 gene deletion increased levels of intestinal proglucagon and plasma GLP-1. Overexpression of mTOR stimulated proglucagon promoter activity and GLP-1 production, whereas inhibition of mTOR activity by overexpression of tuberous sclerosis 1 (TSC1) or TSC2 decreased proglucagon promoter activity and GLP-1 production in STC-1 cells. mTOR may link energy supply with the production of GLP-1 in L cells.

Abstract P6-13-04: Metformin for the treatment of ductal carcinoma in situ (DCIS)

Abstract BACKGROUND: Each year about 60,000 new cases of breast ductal carcinoma in situ (DCIS) are diagnosed in the United States. DCIS significantly increases the risk of invasive breast cancer. To reduce this risk, tamoxifen is offered to patients with ER-positive DCIS in the adjuvant setting. In contrast, no adjuvant therapy exists for ER-negative DCIS. Metformin is a frontline therapy for type 2 diabetes and its safety and side effect profiles are well documented. Preclinical, epidemiologic, and retrospective data all support an antitumor effect of metformin in breast cancer. Metformin may act via activation of AMP kinase (AMPK), a sensor of cellular energy, which in turn inhibits mTOR activity and shuts down global protein synthesis. Previous data from our group showed that metformin treatment is associated with a higher rate of pathologic complete response in diabetic breast cancer patients receiving preoperative chemotherapy. In the current study, using preclinical models, we explored the effects of metformin in DCIS and its progression to invasive breast cancer. EXPERIMENTAL DESIGN AND METHODS: We tested the anti-tumor activity of metformin in terms of proliferation and invasion using both DCIS cell lines and a mouse intraductal (MIND) transplantation model previously developed by us. The latter involves intraductal injection of human DCIS cells into mouse mammary glands, and importantly recapitulates the progression of DCIS to invasive cancer seen in human breast cancer. Western blot and immunofluorescent staining for AMP kinase, mTOR, and other relevant targets, as well as flow cytometry, were used to investigate mechanism. RESULTS: Metformin significantly inhibited cell proliferation, migration, and invasion in all DCIS cell lines tested. In the mouse model, using metformin at a dose that is readily achievable in human patients, metformin significantly reduced the tumor burden in terms of median number of ducts filled, from 11 to 2 using DCIS.com cells (p < 0.001) and from 10 to 3 using SUM.225 cells (p = 0.004). In addition, importantly, metformin inhibited the progression of DCIS lesions to invasive breast cancer significantly, with a reduction in invasive lesions from 74 to 17% (p = 0.003). In mechanistic studies, metformin showed activation of AMPK and subsequent inactivation of mTOR and other downstream targets. Treatment with metformin did not lead to significant cell cycle arrest but rather to increased cell death. We found that cell death by apoptosis increased only modestly with metformin treatment, while cell death by necrosis increased significantly. CONCLUSION: Metformin inhibits the growth of DCIS and its progression to invasive breast cancer. To our knowledge, this is the first reported evidence of metformin’s anti-tumor activity in DCIS. Our data support the exciting prospect of developing metformin clinically for the treatment of DCIS. Citation Format: Atreyi Dasgupta, David G Edwards, Frances S Kittrell, Susan G Hilsenbeck, Daniel Medina, Sao Jiralerspong. Metformin for the treatment of ductal carcinoma in situ (DCIS) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-13-04.

Berberine inhibits Smad and non-Smad signaling cascades and enhances autophagy against pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative lung disorder of unknown aetiology. Transforming growth factor-β1 (TGF-β1)-mediated Smad and non-Smad signaling cascades are considered as central players in accelerating pulmonary fibrosis. We earlier reported berberine's amelioration against TGF-β1-mediated pro-fibrotic effects in bleomycin-induced pulmonary fibrosis. The present study aimed to determine the regulatory role of berberine on abrogated Smad 2/3 and FAK-dependent PI3K/Akt-mTOR signaling cascades in bleomycin-induced pulmonary fibrosis. Male Wistar rats were subjected to single intratracheal instillation of bleomycin (2.5 U/kg) on day 0, and berberine treatments were provided in either preventive or therapeutic modes, respectively. Berberine mitigated the elevated expression of fibrotic markers, α-smooth muscle actin (α-SMA), fibronectin, collagens I and III and reversed bleomycin-induced ultrastructural alterations in the lungs. Berberine inhibited the bleomycin-induced raise in p-Smad 2/3 and enhanced Smad 7 expression. Berberine blocked the activation of FAK and PI3K/Akt against bleomycin-induced dysregulation, with subsequent raise in PTEN expression. In addition, by inhibiting p-mTOR, berberine stimulated autophagy as evidenced by increase in Beclin-1, LC3-II levels with enhanced autophagosome formation. Cumulatively, through targeted inhibition of dysregulated Smad and FAK-dependent PI3K/Akt-mTOR signaling axis, berberine attenuated the fibrotic insults of bleomycin. Berberine inhibits Smad 2/3 activation and enhances Smad 7 in bleomycin-induced rat lungs. Bleomycin-induced activation of FAK is inhibited by berberine. Berberine inhibits bleomycin-induced activation of PI3K/Akt cascade. Berberine inhibits mTOR activation to enhance autophagy and suppresses fibrotic markers.

Che-1-induced inhibition of mTOR pathway enables stress-induced autophagy.

Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates cell growth, metabolism, and autophagy to maintain cellular homeostasis. Its activity is inhibited by adverse conditions, including nutrient limitation, hypoxia, and DNA damage. In this study, we demonstrate that Che-1, a RNA polymerase II-binding protein activated by the DNA damage response, inhibits mTOR activity in response to stress conditions. We found that, under stress, Che-1 induces the expression of two important mTOR inhibitors, Redd1 and Deptor, and that this activity is required for sustaining stress-induced autophagy. Strikingly, Che-1 expression correlates with the progression of multiple myeloma and is required for cell growth and survival, a malignancy characterized by high autophagy response.

Local therapeutic efficacy with reduced systemic side effects by rapamycin-loaded subcapsular microspheres

Kidney injury triggers fibrosis, the final common pathway of chronic kidney disease (CKD). The increase of CKD prevalence worldwide urgently calls for new therapies. Available systemic treatment such as rapamycin are associated with serious side effects. To study the potential of local antifibrotic therapy, we administered rapamycin-loaded microspheres under the kidney capsule of ureter-obstructed rats and assessed the local antifibrotic effects and systemic side effects of rapamycin. After 7 days, microsphere depots were easily identifiable under the kidney capsule. Both systemic and local rapamycin treatment reduced intrarenal mTOR activity, myofibroblast accumulation, expression of fibrotic genes, and T-lymphocyte infiltration. Upon local treatment, inhibition of mTOR activity and reduction of myofibroblast accumulation were limited to the immediate vicinity of the subcapsular pocket, while reduction of T-cell infiltration was widespread. In contrast to systemically administered rapamycin, local treatment did not induce off target effects such as weight loss. Thus subcapsular delivery of rapamycin-loaded microspheres successfully inhibited local fibrotic response in UUO with less systemic effects. Therapeutic effect of released rapamycin was most prominent in close vicinity to the implanted microspheres.

Azithromycin suppresses CD4(+) T-cell activation by direct modulation of mTOR activity.

Advanced macrolides, such as azithromycin (AZM) or clarithromycin (CLM), are antibiotics with immunomodulatory properties. Here we have sought to evaluate their in vitro influence on the activation of CD4+ T-cells. Isolated CD4+ T-cells were stimulated with agonistic anti-CD3/anti-CD28 monoclonal antibodies in the presence of 0.6 mg/L, 2.5 mg/L, 10 mg/L or 40 mg/L AZM or CLM. Cell proliferation, cytokine level in supernatants and cell viability was assessed. Intracellular signaling pathways were evaluated using reporter cell lines, FACS analysis, immunoblotting and in vitro kinase assays. AZM inhibited cell proliferation rate and cytokine secretion of CD4+ T-cells in a dose-dependent manner. Similarly, high concentrations of CLM (40 mg/L) also suppressed these T-cell functions. Analysis of molecular signaling pathways revealed that exposure to AZM reduced the phosphorylation of the S6 ribosomal protein, a downstream target of mTOR. This effect was also observed at 40 mg/L CLM. In vitro kinase studies using recombinant mTOR showed that AZM inhibited mTOR activity. In contrast to rapamycin, this inhibition was independent of FKBP12. We show for the first time that AZM and to a lesser extent CLM act as immunosuppressive agents on CD4+ T-cells by inhibiting mTOR activity. Our results might have implications for the clinical use of macrolides.

The PI3K/mTOR dual inhibitor P7170 demonstrates potent activity against endocrine-sensitive and endocrine-resistant ER+ breast cancer.

Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway has been implicated in anti-estrogen resistance in breast cancer. We tested the therapeutic potential of the novel PI3K/mTOR dual inhibitor P7170 in a panel of anti-estrogen-sensitive and anti-estrogen-resistant models of ER+ breast cancer. Estrogen receptor-positive (ER+) breast cancer cells were treated ±P7170. Fresh cores from primary ER+/HER2- tumors from two patients were treated ±P7170 ex vivo. Mice bearing breast cancer xenografts were randomized to treatment with vehicle, fulvestrant, P7170, or combinations, and tumor volumes were measured. Tissues and cells were analyzed for markers of pathway activity, cell viability, and apoptosis. In cell lines, P7170 exhibited IC50 values in the range of 0.9-7 nM and induced apoptosis. P7170 potently inhibited mTOR activity (≤ 25 nM) and inhibited PI3K at higher concentrations (≥ 200 nM). P7170 completely inhibited MCF-7 tumor growth, significantly inhibited growth of fulvestrant-resistant T47D tumors, and suppressed tumor cell proliferation but did not induce apoptosis. While P7170 inhibits PI3K and mTOR in ER+/HER2- human breast cancer cells and tumors ex vivo, in vivo data indicate that the primary mechanism of P7170 anti-tumor action is inhibition of mTOR and cell proliferation. P7170 is a novel agent worthy of further investigation for the treatment of ER+ breast cancer.

MTOR kinase inhibitors promote antibody class switching via mTORC2 inhibition.

The mammalian target of rapamycin (mTOR) is a kinase that functions in two distinct complexes, mTORC1 and mTORC2. In peripheral B cells, complete deletion of mTOR suppresses germinal center B-cell responses, including class switching and somatic hypermutation. The allosteric mTORC1 inhibitor rapamycin blocks proliferation and differentiation, but lower doses can promote protective IgM responses. To elucidate the complexity of mTOR signaling in B cells further, we used ATP-competitive mTOR kinase inhibitors (TOR-KIs), which inhibit both mTORC1 and mTORC2. Although TOR-KIs are in clinical development for cancer, their effects on mature lymphocytes are largely unknown. We show that high concentrations of TOR-KIs suppress B-cell proliferation and differentiation, yet lower concentrations that preserve proliferation increase the fraction of B cells undergoing class switching in vitro. Transient treatment of mice with the TOR-KI compound AZD8055 increased titers of class-switched high-affinity antibodies to a hapten-protein conjugate. Mechanistic investigation identified opposing roles for mTORC1 and mTORC2 in B-cell differentiation and showed that TOR-KIs enhance class switching in a manner dependent on forkhead box, subgroup O (FoxO) transcription factors. These observations emphasize the distinct actions of TOR-KIs compared with rapamycin and suggest that TOR-KIs might be useful to enhance production of class-switched antibodies following vaccination.

DEPTOR Is a Stemness Factor That Regulates Pluripotency of Embryonic Stem Cells

The mammalian target of rapamycin (mTOR) pathway regulates stem cell regeneration and differentiation in response to growth factors, nutrients, cellular energetics, and various extrinsic stressors. Inhibition of mTOR activity has been shown to enhance the regenerative potential of pluripotent stem cells. DEPTOR is the only known endogenous inhibitor of all known cellular mTOR functions. We show that DEPTOR plays a key role in maintaining stem cell pluripotency by limiting mTOR activity in undifferentiated embryonic stem cells (ESCs). DEPTOR levels dramatically decrease with differentiation of mouse ESCs, and knockdown of DEPTOR is sufficient to promote ESC differentiation. A strong decrease in DEPTOR expression is also observed during human ESCs differentiation. Furthermore, reduction in DEPTOR level during differentiation is accompanied by a corresponding increase in mTOR complex 1 activity in mouse ESCs. Our data provide evidence that DEPTOR is a novel stemness factor that promotes pluripotency and self-renewal in ESCs by inhibiting mTOR signaling.

Abstract SY42-01: Translational control and tumor stem cell radioresponse

Abstract SY42-01: Translational control and tumor stem cell radioresponse

Abstract 2789: Iron chelation inhibits mTOR activity in cancer cells

Abstract Mammalian target of rapamycin (mTOR) plays a critical role in regulating cell proliferation and survival through sensing environmental cues. mTOR functions as at least two complexes (mTORC1/2). In cancer cells, mTOR activity is often deregulated. Intracellular iron levels are higher in cancer cells than in normal cells mainly due to the increased requirement of iron for proliferation. Thus, cancer cells are more sensitive to iron chelation than normal cells. Iron chelators have been used as traditional treatment for iron overload diseases. Recent studies have shown that iron chelators possess potent anticancer activity. However, the underlying mechanism is not well understood. In this study, we found that ciclopirox olamine (CPX), an iron chelator and off-patent antifungal drug, inhibited cell proliferation and induced cell death in human head and neck cancer cells (PCI-13 and FaDu), breast adenocarcinoma (MDA-MB-231), human rhabdomyosarcoma (Rh30), and skin squamous cell carcinoma (SRB-M7) cells. Similar results were observed using Dp44mT, another iron chelator. Concurrently, CPX and Dp44mT inhibited mTORC1 signaling in the cells. This is evidenced by the findings that treatment with the iron chelators inhibited phosphorylation of 4E-BP1 and S6K1, two best known effector molecules of mTORC1, which could be blocked by addition of ferrous sulfate, but not cuprous sulfate. At the same time, the addition of ferrous sulfate prevented cancer cell death. The findings suggest that the inhibitory effect of CPX or Dp44mT on mTORC1 function was attributed to selective chelation of iron in the cells. Mechanistically, chelation of iron did not activate protein phosphatase 2A (PP2A), but induced raptor (S792) phosphorylation and disrupted mTORC1 integrity. Of note, the effect of iron chelation on mTORC2 was cell line dependent. Further research may enhance our understanding the role of iron in the regulation of mTOR signaling. Citation Format: Chaowei Shang, Hongyu Zhou, Shile Huang. Iron chelation inhibits mTOR activity in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2789. doi:10.1158/1538-7445.AM2014-2789

MiR-15/16 complex targets p70S6 kinase1 and controls cell proliferation in MDA-MB-231 breast cancer cells

BackgroundMicroRNAs are small non-coding RNAs that regulate post-transcriptional mRNA expression by binding to 3′ untranslated region (3′-UTR) of the complementary mRNA sequence resulting in translational repression and gene silencing. They act as negative regulators of gene expression and play a pivotal role in regulating apoptosis and cell proliferation. Studies have shown that miRNAs interact with p53 by regulating the activity and function of p53 through direct repression or its regulators. Mammalian target of rapamycin (mTOR) is an evolutionary conserved check point protein kinase that plays a major effect in the control of cell division via protein synthesis regulation. mTOR regulates protein synthesis through phosphorylation and inactivation of 4E-BP1 and through phosphorylation and activation of S6 kinase 1 (S6K1). These two downstream effectors of mTOR control cell growth and metabolism. In mammals, mTOR protein kinase is the central node in the nutrient and growth factor signaling and p53 plays a critical role in sensing genotoxic stress. Activation of p53 inhibits mTOR activity, which in turn regulates its downstream targets providing a cross talk among both the signaling machinery. MicroRNA-15 and 16 belong to a common precursor family and are highly conserved. Deletion or downregulation of these two microRNAs has been shown to accelerate cell division by modulating the expression of the genes involved in controlling cell cycle progression. These microRNAs may function as tumor suppressors and act on the downstream targets of p53 signaling pathway. To have a better insight of the role of miR-15/16 in regulating the cross talk of p53 and mTOR, we performed an in depth study in MDA-MB-231 breast cancer cells by performing a gain-of-function analysis with lentiviral plasmids expressing microRNA-15 and 16. MethodsThe effect of individual microRNAs on RPS6KB1 was examined by using 3′-UTR clones via luciferase based assays. The cell cycle effects were observed by flow-cytometric analysis. Reverse transcription PCR was used to explore the expression of mTOR and RPS6KB1 in cells transfected with miR-15/16. ResultsOverexpression of miR-15/16 led to inhibition of cell proliferation causing G1 cell cycle arrest as well as caspase-3 dependent apoptosis. Forced expression of miR-15/16 might lead to decrease in mRNA level of RPS6KB1, mTOR. The effect was a complete reversal after treatment with anti-miRs against miR-15/16 proving the specificity of the expression. In addition, the dual luciferase reporter assays indicated a clear decrease in luciferase gene expression in cells transfected with lentiviral based miR-15 and 16 plasmids indicating that miR-15/16 directly targets RPS6KB1 through its 3′-UTR binding. Further, these microRNAs also inhibit epithelial to mesenchymal transition (EMT) by targeting key proteins such as Twist1 and EZH2 clearly demonstrating its crucial role in controlling cell proliferation. ConclusionThis study suggests that exogenous microRNA-15/16 can target RPS6KB1, control cell proliferation and cause apoptosis in caspase-dependent manner even in the absence of functional p53.

Aurora kinase a suppresses metabolic stress-induced autophagic cell death by activating mTOR signaling in breast cancer cells.

Aberrant Aur-A signaling is associated with tumor malignant behaviors. However, its involvement in tumor metabolic stress is not fully elucidated. In the present study, prolonged nutrient deprivation was conducted into breast cancer cells to mimic metabolic stress in tumors. In these cells, autophagy was induced, leading to caspase-independent cell death, which was blocked by either targeted knockdown of autophagic gene ATG5 or autophagy inhibitor 3-Methyladenine (3-MA). Aur-A overexpression mediated resistance to autophagic cell death and promoted breast cancer cells survival when exposed to metabolic stress. Moreover, we provided evidence that Aur-A suppressed autophagy in a kinase-dependent manner. Furthermore, we revealed that Aur-A overexpression enhanced the mammalian target of rapamycin (mTOR) activity under metabolic stress by inhibiting glycogen synthase kinase 3β (GSK3β). Inhibition of mTOR activity by rapamycin sensitized Aur-A-overexpressed breast cancer cells to metabolic stress-induced cell death. Consistently, we presented an inverse correlation between Aur-A expression (high) and autophagic levels (low) in clinical breast cancer samples. In conclusion, our data provided a novel insight into the cyto-protective role of Aur-A against metabolic stress by suppressing autophagic cell death, which might help to develop alternative cell death avenues for breast cancer therapy.

Mammalian target of rapamycin cell signaling pathway contributes to the protective effects of ischemic postconditioning against stroke.

Whether the mammalian target of rapamycin (mTOR) pathway is protective against brain injury from stroke or is detrimental is controversial, and whether it is involved in the protective effects of ischemic postconditioning (IPC) against stroke is unreported. Our study focuses on the protective role of mTOR against neuronal injury after stroke with and without IPC. We used both an in vitro oxygen-glucose deprivation model with a mixed neuronal culture and hypoxic postconditioning, as well as an in vivo stroke model with IPC. Rapamycin, a specific pharmacological inhibitor of mTOR, and mTOR short hairpin RNA lentiviral vectors were used to inhibit mTOR activity. A lentiviral vector expressing S6K1, a downstream molecule of mTOR, was used to confirm the protective effects of mTOR. Infarct sizes were measured and protein levels were examined by Western blot. We report that stroke resulted in reduced levels of phosphorylated proteins in the mTOR pathway, including S6K1, S6, and 4EBP1, and that IPC increased these proteins. mTOR inhibition, both by the mTOR inhibitor rapamycin and by mTOR short hairpin RNA, worsened ischemic outcomes in vitro and in vivo and abolished the protective effects of hypoxic postconditioning and IPC on neuronal death in vitro and brain injury size in vivo. Overexpression of S6K1 mediated by lentiviral vectors significantly attenuated brain infarction. mTOR plays a crucial protective role in brain damage after stroke and contributes to the protective effects of IPC.

Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF's support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF's suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1β impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.

Activating mutations in p110δ cause human immunodeficiency and reveal important roles of PI3K in T cell senescence and homeostatic cell death (HUM2P.334)

Abstract The p110δ subunit of phosphatidylinositol-3-kinase (PI3K) is selectively expressed in leukocytes and plays critical roles in lymphocyte biology. We have identified 14 patients from 7 families who were heterozygous for one of three different germline, gain-of-function mutations in PIK3CD (which encodes p110δ) and suffered from sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia, and viremia due to cytomegalovirus and/or Epstein-Barr virus. Strikingly, hyperactivation of PI3K signaling caused significant skewing of peripheral blood CD8 T cells toward an effector phenotype with exaggerated effector characteristics in vitro. Mechanistically, T cell blasts harboring gain-of-function mutations in p110δ exhibited constitutively hyperactive Akt and mTORC1 signaling with enhanced glucose uptake. These changes promoted marked terminal effector cell differentiation and senescence of CD8 T cells. Notably, treatment of patients with rapamycin to inhibit mTOR activity in vivo partially restored the abundance of naive T cells, largely ‘rescued’ the in vitro T cell defects, and greatly improved clinical course. In addition to enhanced effector characteristics, in vitro analyses of patient T cell blasts also revealed a greater susceptibility to TCR restimulation-induced cell death. Indeed, in an independent chemical genetics screen, PI3K stood out as a critical pro-death kinase whose downstream signaling connected with caspase activation upon TCR restimulation.

Temsirolimus, the mTOR inhibitor, induces autophagy in adenoid cystic carcinoma: In vitro and in vivo

Temsirolimus acts as a mammalian target of rapamycin (mTOR)-dependent autophagic inhibitor. In order to clarify its effects and mechanisms on human salivary adenoid cystic carcinoma (ACC), we examined whether temsirolimus induced autophagy as the mTOR inhibitor in ACC, both in vitro and in vivo. In this study, MTT assay showed that the inhibition effect of temsirolimus assumed an obvious dose–response relationship on ACC-M cells, and the 50% inhibitory concentration (IC50) approached 20μmol/l; numerous autophagosomes were observed by the transmission electron microscopy (TEM) in temsirolimus treatment groups; notably, expression of LC3 and Beclin1 was significantly up-regulated by temsirolimus. More importantly, the xenograft model provided further evidence of temsirolimus-induced autophagy in vivo by inhibiting mTOR activation as well as up-regulation the expression of Beclin1. These results suggest that temsirolimus could act as an mTOR inhibitor to induce autophagy in adenoid cystic carcinoma both in vitro and in vivo.

Dihydromyricetin induces autophagy in HepG2 cells involved in inhibition of mTOR and regulating its upstream pathways

Dihydromyricetin (DHM), a bioactive flavonoid compound extracted from the stems and leaves of Ampelopsis grossedentata, has oxidation resistance, anti-tumor and free radical scavenging capabilities. In this study, we found that DHM-induced autophagy inhibited the cell proliferation in HepG2 cells. The transmission electron microscopy results showed that DHM induced significantly autophagosome characteristics like autophagolysosome containing degraded cellular content. GFP labled LC3 plasma transfection showed that LC3 largely diffused to punctate structures with DHM treatment, while lysosomal-rich/acidic compartments detected using LysoTracker Red staining. In addition, DHM promoted the expressions of LC3-II and Beclin-1 in a dose- and time-dependent manner. Further study showed that DHM suppressed the activation of mTOR (mammalian targets of rapamycin) involved in regulating its upstream signaling pathways including extracellular signal-regulated kinase 1/2 (ERK1/2), AMPK (AMP-activated kinase) and class III phosphatidylinositol 3-kinase/phosphoinositide-dependent protein kinase 1/protein kinase B (PI3K/PDK 1/Akt) pathways. Taken together, all the results demonstrated that DHM-induced autophagy inhibited the cell proliferation in HepG2 cells, the possible mechanism involved in inhibition of mTOR activation and regulating the related upstream signaling pathways.