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Protein kinase B (also known as AKT) and the mechanistic target of rapamycin (mTOR) are central regulators of T cell differentiation, proliferation, metabolism, and survival. Here, we show that during chronic murine lymphocytic choriomeningitis virus infection, activation of AKT and mTOR are impaired in antiviral cytotoxic T lymphocytes (CTLs), resulting in enhanced activity of the transcription factor FoxO1. Blockade of inhibitory receptor programmed cell death protein 1 (PD-1) in vivo increased mTOR activity in virus-specific CTLs, and its therapeutic effects were abrogated by the mTOR inhibitor rapamycin. FoxO1 functioned as a transcriptional activator of PD-1 that promoted the differentiation of terminally exhausted CTLs. Importantly, FoxO1-null CTLs failed to persist and control chronic viral infection. Collectively, this study shows that CTLs adapt to persistent infection through a positive feedback pathway (PD-1?FoxO1?PD-1) that functions to both desensitize virus-specific CTLs to antigen and support their survival during chronic viral infection., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aims: We have shown that autophagy and mitophagy are required for preconditioning. While statin's cardioprotective effects are well known, the role of autophagy/mitophagy in statin-mediated cardioprotection is not. In this study, we used HL-1 cardiomyocytes and mice subjected to ischemia/reperfusion to elucidate the mechanism of statin-mediated cardioprotection., Results: HL-1 cardiomyocytes exposed to simvastatin for 24 h exhibited diminished protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling, increased activation of unc-51-like kinase 1, and upregulation of autophagy and mitophagy. Similar findings were obtained in hearts of mice given simvastatin. Mevalonate abolished simvastatin's effects on Akt/mTOR signaling and autophagy induction in HL-1 cells, indicating that the effects are mediated through inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Simvastatin-treated HL-1 cells exhibited mitochondrial translocation of Parkin and p62/SQSTM1, fission, and mitophagy. Because Parkin is required for mitophagy and is expressed in heart, we investigated the effect of simvastatin on infarct size in Parkin knockout mice. Simvastatin reduced infarct size in wild-type mice but showed no benefit in Parkin knockout mice. Inhibition of HMG-CoA reductase limits mevalonate availability for both cholesterol and coenzyme Q10 (CoQ) biosynthesis. CoQ supplementation had no effect on statin-induced Akt/mTOR dephosphorylation or macroautophagy in HL-1 cells, but it potently blocked mitophagy. Importantly, CoQ supplementation abolished statin-mediated cardioprotection in vivo., Innovation and Conclusion: Acute simvastatin treatment suppresses mTOR signaling and triggers Parkin-dependent mitophagy, the latter which is required for cardioprotection. Coadministration of CoQ with simvastatin impairs mitophagy and cardioprotection. These results raise the concern that CoQ may interfere with anti-ischemic benefits of statins mediated through stimulation of mitophagy.

The metabolic benefits of lixisenatide as an anti-diabetic agent are recognized but potential extra-pancreatic effects of this glucagon-like peptide-1 (GLP-1) mimetic in the brain are less well known. This study examines actions within the hippocampus following chronic 40-day peripheral administration of lixisenatide to high-fat fed mice with established obesity, insulin resistance and impaired cognition. Lixisenatide (50 nmol/kg bw, twice-daily) resulted in marked improvements in glycemic status, insulin secretion and insulin sensitivity. Examination of pancreatic tissue revealed decreased islet area, increased islet number, and increased insulin content, with no evidence of pancreatic inflammation. Lixisenatide improved recognition memory during a novel object recognition task and this was associated with up-regulation of hippocampal expression of neurotrophic tyrosine kinase receptor type 2 (NTRK2) and mammalian target of rapamycin (mTOR) genes involved in modulating synaptic plasticity and long-term potentiation. Lixisenatide also enhanced progenitor cell proliferation and increased the number of immature neurons in the hippocampal dentate gyrus. These data indicate that lixisenatide is not only a new efficacious drug for treatment of diabetes but it also exerts favorable neuroprotective effects, reversing memory impairment in obesity-diabetes. Further clinical studies are necessary to fully assess potential beneficial actions of lixisenatide in the hippocampus and cognition in man., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recent literature suggests that sEcad exerts pro-oncogenic effects, possibly acting as a ligand for the human epidermal growth factor family. Here we show that sEcad is a novel candidate protein for drug targeting since it is increased in human and mouse HER2-positive (HER2+) breast tumors, MMTV-PyMT bodily fluids and human cell culture systems. Mechanistically, we show that endogenous sEcad, and to a lesser extent membrane-bound E-cadherin, associates with HER1, HER2, and HER3 in human and MMTV-PyMT mouse HER2+ tumors and with HER1 in triple negative breast cancer (TNBC) specimens. Furthermore, addition of exogenous recombinant human E-cadherin/Fc chimeric protein (rhEcad/Fc; sEcad) to HER2+ MCF-7, SKBR3, and HER2-negative MDA-MB-231 TNBC cells, resulted in sEcad-HER receptor family interactions, activation of HER1-4 and downstream pro-survival signaling, including the MAPK-PI3K/Akt/mTOR pathways and IAP family members. Lastly, we demonstrate that sEcad exerts pro-oncogenic effects via HER signaling, and acts additively with the HER ligand EGF to promote HER2+ breast cancer proliferation and migration, as well as TNBC invasion. Because sEcad associates and activates many of the oncogenic pathways that tumors utilize for growth and survival and serum levels in patients correlates with clinical response, suggests that targeted therapy against sEcad in combination with other therapies may potentially offer a novel therapeutic strategy for the treatment of breast cancers., (© 2013 Wiley Periodicals, Inc.)

Isoegomaketone (IK) is a major biologically active component of Perilla frutescens. In this study, we investigated the contribution of reactive oxygen species (ROS) to IK-induced apoptosis in human melanoma SK-MEL-2 cells. We found that IK inhibited the proliferation of SK-MEL-2 human melanoma cells in a dose-dependent manner. IK also induced sub-G1 DNA accumulation, formation of apoptotic bodies, nuclear condensation, and a DNA ladder in SK-MEL-2 cells. IK also induced activation of caspase-3 and -9, whereas caspase‑8 was unaffected. Further, N-acetyl-L-cysteine (NAC, ROS scavenger) treatment to SK-MEL-2 cells significantly reduced IK-induced cell death. Pretreatment of NAC to SK-MEL-2 cells followed by 100 µM IK reduced the protein levels of Bax and cytochrome c as well as PARP cleavage, whereas the protein level of Bcl-2 increased. Moreover, IK inhibited the phosphorylation of AKT/mTOR protein and cell proliferation induced by LY294002, a PI3K inhibitor. In conclusion, IK-induced ROS generation regulates cell growth inhibition and it induces apoptosis through caspase‑dependent and -independent pathways via modulation of PI3K/AKT signaling in SK-MEL-2 cells.

Liposarcoma is one of the most common histologic types of soft tissue sarcoma and is frequently an aggressive cancer with poor outcome. Hence, alternative approaches other than surgical excision are necessary to improve treatment of well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). For this reason, we performed a two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry/mass spectrometry (MALDI-TOF/MS) analysis to identify new factors for WDLPS and DDLPS. Among the selected candidate proteins, gankyrin, known to be an oncoprotein, showed a significantly high level of expression pattern and inversely low expression of p53/p21 in WDLPS and DDLPS tissues, suggesting possible utility as a new predictive factor. Moreover, inhibition of gankyrin not only led to reduction of in vitro cell growth ability including cell proliferation, colony-formation, and migration, but also in vivo DDLPS cell tumorigenesis, perhaps via downregulation of the p53 tumor suppressor gene and its p21 target and also reduction of AKT/mTOR signal activation. This study identifies gankyrin, for the first time, as new potential predictive and oncogenic factor of WDLPS and DDLPS, suggesting the potential for service as a future LPS therapeutic approach.

Background: The PI3K pathway plays a significant role in the progression of prostate cancer (PCa) to an advanced stage. Mouse models suggest that the downstream effector molecule of the PI3K pathway, mTOR, is also important in the development of PCa, where it plays a pivotal role in forming precursor lesions such as high grade prostatic intraepithelial neoplasia (HGPIN). This study was conducted to determine the status of phosphorylated-mTOR (p-mTOR the activated state of mTOR) across the PCa progression model by looking at expression in normal prostate tissue, proliferative inflammatory atrophy (PIA), HGPIN, and PCa., Methods: Expression of p-mTOR was evaluated by immunohistochemistry on tissue microarrays constructed from 120 archival formalin-fixed paraffin embedded radical prostatectomy tissue specimens. Levels of expression were recorded as the percentage of positive epithelial cells multiplied by the intensity of staining scored as 0-3., Results: p-mTOR expression was found to increase across the progression model with mean staining in non-neoplastic samples of 40 compared to 98 in PIA, 107 in HGPIN, and 136 in cancer (P < 0.001), but without significant increase between HGPIN and PIA. Correlation of high p-mTOR expression with outcome in PCa showed a trend towards worse prognosis, but this was not statistically significant., Conclusions: This study demonstrates that p-mTOR signaling has a potential role in both the initiation and progression of PCa. These data provide support for further research into the possible use of rapamycin analogues in the treatment of PCa, and raise the possibility that mTOR might be a potential target for chemoprevention., (© 2014 Wiley Periodicals, Inc.)

We found that restoration of miR-100 expression resulted in accumulation of LC3B-II and decrease of p62 in hepatocellular carcinoma (HCC) cells, whereas antagonism of miR-100 reduced the level of LC3B-II. Moreover, a significant correlation between miR-100 downregulation and p62 upregulation was observed in human HCC tissues, suggesting an autophagy-promoting effect of miR-100. Subsequent investigations disclosed that knockdown of Atg7 but not Beclin-1 attenuated the miR-100-induced LC3B-II elevation. Furthermore, miR-100 overexpression caused massive cell death, which was abrogated by both the Atg7 silencing and chloroquine treatment. Simultaneously, miR-100 expression led to increased fraction of cells with Annexin V-staining and loss of mitochondrial potential, implying that miR-100 may promote the Atg7-dependent autophagy and subsequent apoptotic cell death. Consistently, mouse xenograft models revealed that miR-100 inhibited the in vivo growth of HCC cells. We further showed that miR-100 suppressed the expression of mTOR and IGF-1R by binding to their 3' untranslated region, and knockdown of mTOR or IGF-1R phenocopied the pro-autophagy effect of miR-100, indicating that miR-100 may promote autophagy by reducing mTOR and IGF-1R level. Collectively, our data uncover a new regulatory mechanism of autophagy and a novel function of miR-100, and provide a potential therapeutic target for HCC.

Chronic myelogenous leukemia is a neoplasm of myeloid progenitor cells. We recently found that rapamycin could induce G0/G1 phase arrest and apoptosis and inhibit proliferation of K562 cells through inhibiting mammalian target of rapamycin (mTOR) pathway. However, whether rapamycin has synergistic effects with other drugs in chronic myelogenous leukemia (CML) therapies remain unclear. Therefore, we examined the effect of rapamycin combined with celecoxib on K562 cells in vitro. The survival rates showed a significant decrease in rapamycin + celecoxib treatment group. The combination treatment also increased the G0/G1 phase cells as compared to rapamycin or celecoxib treatment alone (P < 0.05), accompanied with the decreased population of S phase cells. Meanwhile, the rate of apoptosis was 15.87 ± 2.21 % in rapamycin + celecoxib treatment group, significantly higher than that in mono treatment group (P < 0.05). Western blot and reverse transcription PCR (RT-PCR) analysis showed that the expressions of mTOR, 4E-BP1, and p70S6K were all significantly decreased in K562 cells after rapamycin + celecoxib treatment (P < 0.05). In conclusion, rapamycin combined with celecoxib could induce cell cycle arrest and apoptosis and decrease the expressions of mTOR, 4E-BP1, and p70S6K. It suggested that the combination could enhance the antitumor effects of mono treatment on CML cells through downregulating mTOR pathway.

Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.

Drug resistance is one of the most formidable obstacles for treatment of glioma. Eukaryotic initiation factor 4E-binding protein (4E-BP1), a key component in the rate-limiting step of protein translation initiation, is closely associated with poor prognosis in multiple tumor types. However, it is unclear whether 4E-BP1 is involved in the drug resistance of human glioma. Herein we show that the expression of 4E-BP1 in human SWOZ2-BCNU drug-resistant glioma cells is significantly lower than that of the parent SWOZ2 cell line. Moreover, down-regulation of 4E-BP1 by short interfering RNA significantly impaired the sensitivity of SWOZ2 and U251 cells to carmustine (BCNU). Furthermore, overexpression of 4E-BP1 with plasmid transfection regained this sensitivity. Clinical studies showed that the expression levels of 4E-BP1 in primary glioma tissues were markedly higher than those of recrudescent glioma tissues. Taken together, our results suggest that 4E-BP1 is a novel protein that contributes to acquired drug resistance and it may be a potential target for reversing drug resistance in human glioma., (© 2013 Japanese Society of Neuropathology.)

Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Introduction: A phosphorylation score for estrogen receptor-alpha (ERα), called P7 score, was shown previously to be an independent prognostic factor in breast cancer patients treated with tamoxifen. Since mechanistic target of rapamycin (mTOR) activation is implicated in resistance to endocrine therapy in breast cancer we determined whether mechanistic target of rapamycin complex 1 (mTORC1) activation, measured by phosphorylation on S2448 (p-mTOR), was associated with the P7-score and/or clinical outcome in the same cohort., Methods: mTOR phosphorylation status was determined at S2448 residue in vivo by immunohistochemistry in a cohort of more than 400 well-characterized ERα positive breast tumors. MCF7 cells were treated with estrogen and activation of mTOR pathway was determined by Western blotting., Results: Contrary to earlier reports, p-mTOR expression, measured by immunohistochemistry, was negatively associated with size and nodal status. Additionally, p-S2448 mTOR expression was positively correlated with p-S118- ERα, p-S167-ERα and p-S282-ERα but negatively correlated with p-T311- ERα. Consistent with these, p-S2448 mTOR was negatively associated with P7-score and was significantly associated with overall survival (OS) (hazard ratio (HR) = 0.61, P = 0.028, 95% confidence interval (CI) 0.39 to 0.95, n = 337) and relapse-free survival (HR = 0.58, P = 0.0032, 95% CI 0.41 to 0.83, n = 337) following univariate but not multivariate analysis. Furthermore, we show that estrogen can regulate phosphorylation of mTOR and its down stream target p70S6 kinase. Additionally, recombinant mTOR can phosphorylate ERα in vitro., Conclusions: These data suggest that in breast tumors where there is intact estrogen regulated signaling, mTOR is regulated by estrogen and therefore associated with an increased likelihood of responsiveness to endocrine therapy.

The Securinega alkaloids are a class of natural products isolated from plants of the Euphorbiaceae family. L-securinine induces apoptosis in the human promyelocytic leukemia cell line HL-60 indicating its potential as an efficient natural antitumor drug with low toxicity. The aim of the present study was to investigate the apoptotic effects of L-securinine on HL-60 cells and to explore its potential underlying molecular mechanism(s) as an antitumor agent. HL-60 cells were cultured with L-securinine. The proliferation and changes in cell morphology were evaluated by cell counting Kit-8 (CCK-8) assay and electron microscopy, respectively. Induction of apoptosis and cell cycle progression were investigated by flow cytometry. The PI3K/AKT/mTOR pathway gene expression was measured by quantitative PCR (qPCR). L-securinine decreased the viability of HL-60 cells in a dose- and time-dependent manner, with IC50 values at 24, 48 and 72 h post-treatment of 47.88, 23.85 and 18.87 µmol/l, respectively. Numerous apoptotic bodies were observed in the HL-60 cells treated with 25 µmol/l L-securinine for 48 h. L-securinine at 12.5, 25 and 50 µmol/l increased the rate of apoptosis in HL-60 cells, and G1/S phase progression was retarded. Furthermore, L-securinine induced downregulation of PI3K, AKT and mTOR gene expression and upregulation of PTEN gene expression in a dose-dependent manner. In conclusion, L-securinine induces apoptosis and inhibition of cell cycle progression in HL-60 cells by modulation of the PI3K/AKT/mTOR pathway gene expression. These observations indicate the potential of L-securinine as an antitumor agent.

Purpose: Temsirolimus (TEM) is a novel, water-soluble mammalian target of rapamycin (mTOR) inhibitor that has shown activity against a wide range of cancers in preclinical models, but its efficacy against colorectal cancer (CRC) has not been fully explored., Methods: We evaluated the antitumor effect of TEM in CRC cell lines (CaR-1, HT-29, Colon26) in vitro and in vivo. In vitro, cell growth inhibition was assessed using a MTS assay. Apoptosis induction and cell cycle effects were measured using flow cytometry. Modulation of mTOR signaling was measured using immunoblotting. Antitumor activity as a single agent was evaluated in a mouse subcutaneous tumor model of CRC. The effects of adding chloroquine, an autophagy inhibitor, to TEM were evaluated in vitro and in vivo., Results: In vitro, TEM was effective in inhibiting the growth of two CRC cell lines with highly activated AKT, possibly through the induction of G1 cell cycle arrest via a reduction in cyclin D1 expression, whereas TEM reduced HIF-1α and VEGF in all three cell lines. In a mouse subcutaneous tumor model, TEM inhibited the growth of tumors in all cell lines, not only through direct growth inhibition but also via an anti-angiogenic effect. We also explored the effects of adding chloroquine, an autophagy inhibitor, to TEM. Chloroquine significantly potentiated the antitumor activity of TEM in vitro and in vivo. Moreover, the combination therapy triggered enhanced apoptosis, which corresponded to an increased Bax/Bcl-2 ratio., Conclusions: Based on these data, we propose TEM with or without chloroquine as a new treatment option for CRC.

Prostaglandin E2 (PGE2) has been shown to influence cell invasion and metastasis in several types of cancer, including hepatocellular carcinoma (HCC). however, the molecular mechanisms underlying it remain to be further elucidated. Snail, as one of key inducers of epithelial-mesenchymal transition (EMT), plays pivotal roles in HCC invasion and metastasis. The present study was designed to evaluate the possible signaling pathways through which PGE2 regulates Snail protein expression in HCC cell lines. PGE2 markedly enhanced Huh-7 cell invasion and migration ability by upregulating the expression level of Snail protein, and EP2 receptor played an important role in this process. Src, EGFR, Akt and mTOR were all activated and involved in the regulation of snail protein expression. Our findings suggest that PGE2 could upregulate the expression level of Snail protein through the EP2/Src/EGFR/Akt/mTOR pathway in Huh-7 cells, which promotes HCC cell invasion and migration.

Background: Vascular calcification is a major risk factor for cardiovascular diseases. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is a key step in vascular calcification, but the molecular mechanisms driving the differentiation remain elusive. In this study, the involvement of mammalian target of rapamycin (mTOR) signalling in osteoblastic differentiation of VSMCs is investigated., Methods: Calcification of VSMCs was induced in vitro using β-glycerophosphate (β-GP). Real-time polymerase chain reaction was used to measure messenger RNA (mRNA) expression, and Western blot was used to detect protein expression. Inhibition of mTOR expression was established by small interfering RNA (siRNA) and mTOR inhibitors., Results: The model for osteoblastic differentiation of VSMCs was established in vitro by treating mouse VSMCs with 10 mM β-GP for 3-15 days. Overexpression of mTOR was observed in differentiated VSMCs. Downregulation of mTOR by siRNA or rapamycin significantly inhibited osteoblastic differentiation of VSMCs and decreased the expression and phosphorylation of mTOR and P70 ribosomal S6 kinase in a time- and concentration-dependent manner. Furthermore, adiponectin inhibited the mRNA and protein expression of mTOR in β-GP-treated VSMCs in a time- and concentration-dependent manner., Conclusions: mTOR signalling plays a crucial role in the osteoblastic differentiation of VSMCs. Rapamycin and adiponectin might inhibit vascular calcification through regulation of the mTOR pathway., (Copyright © 2014 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

The role of LeuRS, an aminoacyl-tRNA synthetase, as an intracellular l-leucine sensor for the mTORC1 pathway has been the subject of much research recently. Despite this, the association between LeuRS and lactation in dairy cow mammary epithelial cells (DCMECs) remains unknown. In this study, we found that LeuRS expression in mammary gland tissue was significantly higher during lactation than pregnancy. Moreover, our data demonstrates that LeuRS is localized in the cytoplasm. Treatment with leucine increased DCMECs viability and proliferation, as well as mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase 1 (S6K1), p-S6K1, β-Casein, sterol regulatory element binding protein 1c (SREBP-1c), glucose transporter 1 (GLUT1), and Cyclin D1 mRNA and protein expression. Secretion of lactose and triglyceride were also increased. siRNA-mediated knockdown of LeuRS led to reduction in all of these processes. Based on these data, LeuRS up-regulates the mTOR pathway to promote proliferation and lactation of DCMECs in response to changes in the intracellular leucine concentration.

Diacylglycerol kinase (DGK) is a lipid kinase converting diacylglycerol to phosphatidic acid, and regulates many enzymes including protein kinase C, phosphatidylinositol 4-phosphate 5-kinase, and mTOR. To date, ten mammalian DGK subtypes have been cloned and divided into five groups, and they show subtype-specific tissue distribution. Therefore, each DGK subtype is thought to be involved in respective cellular responses by regulating balance of the two lipid messengers, diacylglycerol and phosphatidic acid. Indeed, the recent researches using DGK knockout mice have clearly demonstrated the importance of DGK in the immune system and its pathophysiological roles in heart and insulin resistance in diabetes. Especially, most subtypes show high expression in brain with subtype specific regional distribution, suggesting that each subtype has important and unique functions in brain. Recently, neuronal functions of some DGK subtypes have accumulated. Here, we introduce DGKs with their structural motifs, summarize the enzymatic properties and neuronal functions, and discuss the possibility of DGKs as a therapeutic target of the neuronal diseases.

Renal cell carcinoma is a heterogeneous disease, and tissue investigations provide clues that may predict treatment response. Oncogenomic analysis of five outliers, who achieved a sustained response with rapalogs, implicates alterations of the TSC1 and mTOR genes and reveals insights into the conserved evolution of tumors., (©2014 AACR.)

Background: Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway dysregulation has been implicated in the development of various human cancers. However, expression of mTOR cascade components in pancreatic neuroendocrine tumors (PNETs) has not been fully explored. The aim of this study was to assess the expression of mTOR pathway in PNETs using immunohistochemistry., Methods: From December 1984 to April 2012, we surgically treated 42 patients with PNETs. We used immunohistochemistry to evaluate expression of mTOR, phosphorylated mTOR (p-mTOR), p70S6 kinase (S6K), phosphorylated S6 ribosomal protein (p-S6rp), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and phosphorylated 4E-BP1 (p-4E-BP1) in the resected specimens. The relation between the expression of these molecules and clinicopathological characteristics was investigated., Results: We identified the expression of mTOR (28.6%), p-mTOR (52.4%), S6K (52.4%), p-S6rp (40.5%), 4E-BP1 (81.0%), and p-4E-BP1 (26.2%) in PNETs. The expression of mTOR, p-mTOR, S6K, and p-S6rp was significantly associated with tumor invasion, proliferation, and an advanced-stage. Particularly, the expression of p-mTOR was related to clinically relevant factors such as tumor size, vascular invasion, extrapancreatic invasion, lymph node and/or distant metastasis, mitotic count, and European Neuroendocrine Tumor Society TNM staging as well as the 2004 and 2010 World Health Organization (WHO) classification. In addition, p-S6rp expression was related to vascular invasion, extrapancreatic invasion, lymph node and distant metastasis, mitotic count, and the 2010 WHO classification. In contrast, no significant relation between 4E-BP1 activation and clinicopathological factors was observed. The expression of p-mTOR was strongly correlated with that of p-S6rp (r = 0.474, P = 0.002)., Conclusions: Our results suggest that activation of the mTOR/S6K signaling pathway plays a significant role in tumorigenesis and progression of PNET., (© 2013 Japanese Society of Hepato-Biliary-Pancreatic Surgery.)

Purpose: Rapalogs are allosteric mTOR inhibitors and approved agents for advanced kidney cancer. Reports of clonal heterogeneity in this disease challenge the concept of targeted monotherapy, yet a small subset of patients derives extended benefit. Our aim was to analyze such outliers and explore the genomic background of extreme rapalog sensitivity in the context of intratumor heterogeneity., Experimental Design: We analyzed archived tumor tissue of 5 patients with renal cell carcinoma, who previously achieved durable disease control with rapalogs (median duration, 28 months). DNA was extracted from spatially separate areas of primary tumors and metastases. Custom target capture and ultradeep sequencing was used to identify alterations across 230 target genes. Whole-exome sequence analysis was added to investigate genes beyond this original target list., Results: Five long-term responders contributed 14 specimens to explore clonal heterogeneity. Genomic alterations with activating effect on mTOR signaling were detected in 11 of 14 specimens, offering plausible explanation for exceptional treatment response through alterations in two genes (TSC1 and MTOR). In two subjects, distinct yet functionally convergent alterations activated the mTOR pathway in spatially separate sites. In 1 patient, concurrent genomic events occurred in two separate pathway components across different tumor regions., Conclusions: Analysis of outlier cases can facilitate identification of potential biomarkers for targeted agents, and we implicate two genes as candidates for further study in this class of drugs. The previously reported phenomenon of clonal convergence can occur within a targetable pathway which might have implications for biomarker development beyond this disease and this class of agents., (©2014 AACR.)

Background: mTOR, which can form mTOR Complex 1 (mTORC1) or mTOR Complex 2 (mTORC2) depending on its binding partners, is frequently deregulated in the pulmonary neoplastic conditions and interstitial lung diseases of the patients treated with rapalogs. In this study, we investigated the relationship between mTOR signaling and epithelial mesenchymal transition (EMT) by dissecting mTOR pathways., Methods: Components of mTOR signaling pathway were silenced by shRNA in a panel of non-small cell lung cancer cell lines and protein expression of epithelial and mesenchymal markers were evaluated by immunoblotting and immunocytochemistry. mRNA level of the E-cadherin repressor complexes were evaluated by qRT-PCR., Results: IGF-1 treatment decreased expression of the E-cadherin and rapamycin increased its expression, suggesting hyperactivation of mTOR signaling relates to the loss of E-cadherin. Genetic ablation of rapamycin-insensitive companion of mTOR (Rictor), a component of mTORC2, did not influence E-cadherin expression, whereas genetic ablation of regulatory-associated protein of mTOR (Raptor), a component of mTORC1, led to a decrease in E-cadherin expression at the mRNA level. Increased phosphorylation of AKT at Ser473 and GSK-3β at Ser9 were observed in the Raptor-silenced NSCLC cells. Of the E-cadherin repressor complexes tested, Snail, Zeb2, and Twist1 mRNAs were elevated in raptor-silenced A549 cells, and Zeb2 and Twist1 mRNAs were elevated in Raptor-silenced H2009 cells. These findings were recapitulated by treatment with the GSK-3β inhibitor, LiCl. Raptor knockdown A549 cells showed increased expression of N-cadherin and vimentin with mesenchymal phenotypic changes., Conclusions: In conclusion, selective inhibition of mTORC1 leads to hyperactivation of the AKT/GSK-3β pathway, inducing E-cadherin repressor complexes and EMT. These findings imply the existence of a feedback inhibition loop of mTORC1 onto mTORC2 that plays a role in the homeostasis of E-cadherin expression and EMT, requiring caution in the clinical use of rapalog and selective mTORC1 inhibitors.

Mechanistic target of rapamycin (mTOR) controls global translation of mRNA into protein by phosphorylating p70 S6 kinase (S6K) and eIF4E-binding protein-1. Akt and Rheb, a GTP-binding protein, regulate mTOR protein kinase activity. Molting in crustaceans is regulated by ecdysteroids synthesized by a pair of molting glands, or Y-organs (YOs), located in the cephalothorax. During premolt, the YOs hypertrophy and increase production of ecdysteroids. Rapamycin (1μM) inhibited ecdysteroid secretion in Carcinus maenas and Gecarcinus lateralis YOs in vitro, indicating that ecdysteroidogenesis requires mTOR-dependent protein synthesis. The effects of molting on the expression of four key mTOR signaling genes (mTOR, Akt, Rheb, and S6K) in the YO was investigated. Partial cDNAs encoding green crab (C. maenas) mTOR (4031bp), Akt (855bp), and S6K (918bp) were obtained from expressed sequence tags. Identity/similarity of the deduced amino acid sequence of the C. maenas cDNAs to human orthologs were 72%/81% for Cm-mTOR, 58%/73% for Cm-Akt, and 77%/88% for Cm-S6K. mTOR, Akt, S6K, and elongation factor 2 (EF2) in C. maenas and blackback land crab (G. lateralis) were expressed in all tissues examined. The two species differed in the effects of molting on gene expression in the YO. In G. lateralis, Gl-mTOR, Gl-Akt, and Gl-EF2 mRNA levels were increased during premolt. By contrast, molting had no effect on the expression of Cm-mTOR, Cm-Akt, Cm-S6K, Cm-Rheb, and Cm-EF2. These data suggest that YO activation during premolt involves up regulation of mTOR signaling genes in G. lateralis, but is not required in C. maenas., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The trophocytes and fat cells of honeybees (Apis mellifera) have been used in cellular senescence studies, but the changes of cellular degradation activity with aging in workers are unknown. In this study, cellular degradation activity was evaluated in the trophocytes and fat cells of young and old workers reared in a field hive. The results showed the following: (1) 20S proteosome activity decreased with aging, whereas its expression increased with aging; (2) the expression of microtubule-associated protein 1 light chain 3-II (LC3-II) and the 70 kD heat shock cognate protein (Hsc70) decreased with aging; (3) the size and number of autophagic vacuoles decreased with aging; (4) p62/SQSTM1 and polyubiquitin aggregate expression decreased with aging; (5) lysosomal efficiency decreased with aging; and (6) molecular target of rapamycin (mTOR) expression increased with aging. These results indicate that young workers have higher levels of cellular degradation activity than old workers and that aging results in a decline in the cellular degradation activity in worker honeybees., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This study evaluated the effects of an mTOR inhibitor everolimus alone or in combination with letrozole on MCF-7/Aro (MCF-7 cells stably transfected with CYP19) in vitro and in vivo. In vitro studies, full-length CYP19 (aromatase) was cloned in a plasmid transfer vector pH ß-Aro and then transfected into MCF-7 stem cells which were ESA(+)CD44(+)CD24(-/low) sorted by flow cytometry. MTT assays were used to quantify the inhibitory effect of the drugs on MCF-7/Aro stem cells (SCs) and non-stem cells (NSCs). Apoptosis and the cell cycle distributions of stem cells were examined by flow cytometry. The tumorigenicity of stem cells after treatment was investigated by soft agar colony formation assays. In vivo studies, the BALB/c mice were injected with MCF-7/Aro SCs, and the different treatments were administered. After necropsy, the expression of KI67, CD31, AKT1, phospho-AKT (Thr308), and mTOR was analyzed by immunohistochemistry. In vitro, compared with MCF-7/Aro NSCs, there were greater resistance to the standard treatment doses of letrozole and everolimus in MCF-7/Aro SCs (17- and 15-fold, respectively). Treatment with everolimus or letrozole resulted in growth inhibition of SCs in a dose-dependent manner. Compared with single-agent therapy, the combination of everolimus with letrozole was more effective in the inhibition of cell growth (P < 0.001) and tumorigenicity (P < 0.01). In addition, an increase in G1 cell cycle arrest and increases in early apoptosis were observed in the combination treatment group compared with either single-agent group. In vivo, the xenograft tumor sizes were significantly decreased in everolimus alone group compared to control group, and everolimus plus letrozole therapy was much more effective compared with either single agent alone (P < 0.01). Compared with everolimus alone, the combination of everolimus and letrozole reduced the expression of KI67, mTOR, and phospho-AKT (Thr308; P < 0.01). Everolimus has effective inhibition on aromatase-overexpressing stem cell in vitro and in vivo. The combination everolimus and letrozole could be more effective than either drug alone.

Introduction: The Runt-related transcription factor Runx2 is critical for skeletal development but is also aberrantly expressed in breast cancers, and promotes cell growth and invasion. A de-regulated serine/threonine kinase Akt signaling pathway is implicated in mammary carcinogenesis and cell survival; however, the mechanisms underlying Runx2 role in survival of invasive breast cancer cells are still unclear., Methods: The phenotypic analysis of Runx2 function in cell survival was performed by gene silencing and flow cytometric analysis in highly invasive MDA-MB-231 and SUM-159-PT mammary epithelial cell lines. The expression analysis of Runx2 and pAkt (serine 473) proteins in metastatic breast cancer specimens was performed by immunohistochemistry. The mRNA and protein levels of kinases and phosphatases functional in Akt signaling were determined by real-time PCR and Western blotting, while DNA-protein interaction was studied by chromatin immunoprecipitation assays., Results: The high Runx2 levels in invasive mammary epithelial cell lines promoted cell survival in Akt phosphorylation (pAkt-serine 473) dependent manner. The analysis of kinases and phosphatases associated with pAkt regulation revealed that Runx2 promotes pAkt levels via mammalian target of rapamycin complex-2 (mTORC2). The recruitment of Runx2 on mTOR promoter coupled with Runx2-dependent expression of mTORC2 component Rictor defined Runx2 function in pAkt-mediated survival of invasive breast cancer cells., Conclusions: Our results identified a novel mechanism of Runx2 regulatory crosstalk in Akt signaling that could have important consequences in targeting invasive breast cancer-associated cell survival.

WNT signaling has been implicated in both embryonic and postnatal bone formation. However, the pertinent WNT ligands and their downstream signaling mechanisms are not well understood. To investigate the osteogenic capacity of WNT7B and WNT5A, both normally expressed in the developing bone, we engineered mouse strains to express either protein in a Cre-dependent manner. Targeted induction of WNT7B, but not WNT5A, in the osteoblast lineage dramatically enhanced bone mass due to increased osteoblast number and activity; this phenotype began in the late-stage embryo and intensified postnatally. Similarly, postnatal induction of WNT7B in Runx2-lineage cells greatly stimulated bone formation. WNT7B activated mTORC1 through PI3K-AKT signaling. Genetic disruption of mTORC1 signaling by deleting Raptor in the osteoblast lineage alleviated the WNT7B-induced high-bone-mass phenotype. Thus, WNT7B promotes bone formation in part through mTORC1 activation.

Mammalian adult hematopoietic stem cells (HSCs) reside in the hypoxic bone marrow microenvironment and display a distinct metabolic phenotype compared with their progenitors. It has been proposed that HSCs generate energy mainly through anaerobic glycolysis in a pyruvate dehydrogenase kinase (Pdk)-dependent manner. Cited2 is an essential regulator for HSC quiescence, apoptosis, and function. Herein, we show that conditional deletion of Cited2 in murine HSCs results in elevated levels of reactive oxygen species, decreased cellular glutathione content, increased mitochondrial activity, and decreased glycolysis. At the molecular level, Cited2 deficiency significantly reduced the expression of genes involved in metabolism, such as Pdk2, Pdk4, and lactate dehydrogenases B and D (LDHB and LDHD). Cited2-deficient HSCs also exhibited increased Akt signaling, concomitant with elevated mTORC1 activity and phosphorylation of FoxOs. Further, inhibition of PI3/Akt, but not mTORC1, partially rescued the repression of Pdk4 caused by deletion of Cited2. Altogether, our results suggest that Cited2 is required for the maintenance of adult HSC glycolytic metabolism likely through regulating Pdk2, Pdk4, LDHB, LDHD, and Akt activity.

Aims and Background: Ginsenoside Rh2, which exerts the potent anticancer action both in vitro and in vivo, is one of the most well characterized ginsenosides extracted from ginseng. Although its effects on cancer are significant, the underlying mechanisms remain unknown. In this study, we sought to elucidate possible links between ginsenoside Rh2 and phosphoglucose isomerase/autocrine motility factor (PGI/AMF)., Methods: KG1α, a leukemia cell line highly expressing PGI/AMF was assessed by western blot analysis and reverse transcription- PCR (RT-PCR) assay after transfection of a small interfering (si)-RNA to silence PGI/AMF. The effect of PGI/ AMF on proliferation was measured by typan blue assay and antibody array. A cell counting kit (CCK)-8 and flow cytometry (FCM) were adopted to investigate the effects of Rh2 on PGI/AMF. The relationships between PGI/AMF and Rh2 associated with Akt, mTOR, Raptor, Rag were detected by western blot analysis., Results: KG1α cells expressed PGI/AMF and its down-regulation significantly inhibited proliferation. The antibody array indicated that the probable mechanism was reduced expression of PARP, State1, SAPK/JNK and Erk1/2, while those of PRAS40 and p38 were up-regulated. Silencing of PGI/AMF enhanced the sensibility of KG1α to Rh2 by suppressing the expression of mTOR, Raptor and Akt., Conclusion: These results suggested that ginsenoside Rh2 suppressed the proliferation of KG1α, the same as down-regulation of PGI/AMF. Down-regulation of PGI/ AMF enhanced the pharmacological effects of ginsenoside Rh2 on KG1α by reducing Akt/mTOR signaling.

Objectives: To evaluate the expression of multiple molecular markers involved in mammalian target of rapamycin (mTOR) signaling pathway in renal cell carcinoma (RCC) to determine the prognostic significance of these markers following radical nephrectomy., Material and Methods: The expression 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 nephrectomy specimens from 137 patients with nonmetastatic RCC by immunohistochemical staining., Results: During the follow-up period of this series (median, 63.5 mo), disease recurrence occurred in 59 of the 137 patients (43.0%), with a 5-year recurrence-free survival rate of 58.3%. On Univariate analysis, expression levels of p-mTOR and p-4E-BP1, in addition to the C-reactive protein level, pathological stage, and microvascular invasion, were identified as significant predictors for disease recurrence. Of these factors, the expression of p-4E-BP1, C-reactive protein level, and pathological T stage appeared to be independently related to recurrence-free survival on multivariate analysis. Moreover, significant differences were observed in recurrence-free survival according to the positive numbers of these 3 independent factors; that is, disease recurrence developed in 5 of 42 patients with negative results for any risk factor (11.9%), 23 of 50 patients with positive results for a single risk factor (46.0%), and 31 of 45 patients with positive results for 2 or 3 risk factors (68.8%)., Conclusions: The combined evaluation of the expression levels of potential markers in the mTOR signaling pathway, particularly p-4E-BP1, in RCC specimens with conventional prognostic parameters would contribute to the accurate prediction of disease recurrence following radical nephrectomy for nonmetastatic RCC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Unlabelled: The mammalian target of rapamycin complex 1 (mTORC1) is hyperactive in many human cancers and in tuberous sclerosis complex (TSC). Autophagy, a key mTORC1-targeted process, is a critical determinant of metabolic homeostasis. Metabolomic profiling was performed to elucidate the cellular consequences of autophagy dysregulation under conditions of hyperactive mTORC1. It was discovered that TSC2-null cells have distinctive autophagy-dependent pentose phosphate pathway (PPP) alterations. This was accompanied by enhanced glucose uptake and utilization, decreased mitochondrial oxygen consumption, and increased mitochondrial reactive oxygen species (ROS) production. Importantly, these findings revealed that the PPP is a key autophagy-dependent compensatory metabolic mechanism. Furthermore, PPP inhibition with 6-aminonicotinamide (6-AN) in combination with autophagy inhibition suppressed proliferation and prompted the activation of NF-κB and CASP1 in TSC2-deficient, but not TSC2-proficient cells. These data demonstrate that TSC2-deficient cells can be therapeutically targeted, without mTORC1 inhibitors, by focusing on their metabolic vulnerabilities., Implications: This study provides proof-of-concept that therapeutic targeting of diseases with hyperactive mTORC1 can be achieved without the application of mTORC1 inhibitors., (©2013 AACR.)

Background/aims: Glucagon-like peptide-1 (GLP-1), which counteracts insulin resistance in humans with type 2 diabetes, has been shown to ameliorate diabetic nephropathy in experimental models. However, the mechanisms through which GLP-1 modulates renal function remained illdefined. The present study investigated the putative mechanisms underlying effects of exendin-4, a GLP-1 analog, on mesangial cell proliferation and fibronectin., Methods: Rat mesangial cells (MCs) were treated with exendin-4 under high glucose conditions. AMP-activated protein kinase (AMPK) inhibitors (compound C) and agonists (AICAR) were used to analyze the role of this kinase. Cell proliferation was measured using a MTT assay. Fibronectin expression and AMPK-signaling pathway activity were assessed using ELISA and Western blotting, respectively. The production of matrix metalloproteinase (MMP)-2 and tissue inhibitors of metalloproteinases (TIMP)-2 was evaluated using quantitative real-time RT-PCR., Results: Exendin-4 inhibited cell proliferation and fibronectin secretion in high glucose-induced MCs. It also caused phosphorylation of AMPK and subsequently increased the ratio of MMP-2 to TIMP-2, which resulted in the degradation of fibronectin. Exendin-4 reversed extracellular signal-regulated kinase (ERK) phosphorylation and enhanced expression of mammalian target of rapamycin (mTOR) in MCs. Moreover, the activation of the AMPK pathway by exendin-4 was induced by AICAR, which was inhibited by compound C., Conclusion: Exendin-4 exerts an inhibitory effect on cell proliferation and fibronectin secretion in rat MCs, partly through AMPK activation. These results may explain some of the beneficial effects of exendin-4 on the kidney., (© 2014 S. Karger AG, Basel.)

Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-β42 signals the intraneuronal accumulation of PC(O-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(O-16:0/2:0) is also toxic to Saccharomyces cerevisiae, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)-binding protein, was relocalized in response to PC(O-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(O-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P2 leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(O-16:0/2:0) on the distribution of PM PtdIns(4,5)P2 pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(O-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression.

In mammals, resting female oocytes reside in primordial ovarian follicles. An individual primordial follicle may stay quiescent for a protracted period of time before initiating follicular growth, which is also termed “activation.” Female reproductive capacity is sustained by the gradual, streamlined activation of the entire population of primordial follicles, but this process also results in reproductive senescence in older animals. Based on the recent findings that genetically triggered, excessive mammalian target of rapamycin complex 1 (mTORC1) activation in mouse oocytes leads to accelerated primordial follicle activation, we examined the necessity of mTORC1 signaling in physiological primordial follicle activation. We found that induction of oocyte mTORC1 activity is associated with early follicular growth in neonatal mouse ovaries. Pharmacological inhibition of mTORC1 activity in vivo by rapamycin treatment leads to a marked, but partial, suppression of primordial follicle activation. The suppressive effect of rapamycin on primordial follicle activation was reproduced in cultured ovaries. While rapamycin did not apparently affect several plausible cellular targets in neonatal mouse ovaries, such as mTORC2, AKT, or cyclin-dependent kinase (CDK) inhibitor p27-KIP1, its inhibitory effect on Cyclin A2 gene expression implies that mTORC1 signaling in oocytes may engage a Cyclin A/CDK regulatory network that promotes primordial follicle activation. The current work strengthens the concept that mTORC1-dependent events in the oocytes of primordial follicles may represent potential targets for intervention in humans to slow the depletion of the ovarian reserve., (© 2013 Wiley Periodicals, Inc.)

This study aimed to determine whether the expression of various tumor biomarkers of the mTOR pathway predicts tumor response to everolimus in metastatic recurrent endometrial cancer. Tumor blocks from 44 patients of a phase II clinical trial receiving everolimus until progression or toxicity were collected and evaluated at 3 and 6 months for response. Thirty-six blocks were available for analysis of ER, PR, HER2, LKB1, PI3K, PTEN, pAKT, 4E-BP1, p4E-BP1, and S6RP expression by immunohistochemistry, PTEN deletion by FISH, and mutational status of K-RAS, PIK3CA, PTEN, and AKT1 genes. Twelve of 34 evaluable patients had partial response or stable disease (PR, SD) and 22 had progressive disease (PD). Immunohistochemistry showed that no protein expression could predict response to everolimus. Neither could loss of PTEN expression or PTEN deletion or PTEN mutation predict patient outcome. Thirty-one samples were assessable for K-RAS mutations (ten for PR+SD and 21 for PD). There are only four patients with K-RAS mutations and none of them responded to treatment. Median progression-free survival (PFS) and overall survival (OS) were longer in patients without K-RAS mutations (PFS 3.12 ± 1.7 months versus 1.05 ± 0.4 months, p < 0.001; OS 9.28 ± 2.0 months versus 2.30 ± 1.4 months, p = 0.034). In conclusion, the level of expression of proteins of the PI3K/mTOR pathway tested in this study cannot predict response to everolimus. However, endometrial cancer patients with K-RAS mutations do not seem to derive benefit from everolimus treatment.

Purpose: Penile squamous cell carcinoma is a rare neoplasm associated with a high risk of metastasis and morbidity. There are limited data on the role of the mTOR signaling pathway in penile squamous cell carcinoma carcinogenesis and tumor maintenance. We assessed a possible role for mTOR signaling pathway activation as a potential predictive biomarker of outcome and a therapeutic target for penile cancer., Material and Methods: A cohort of 67 patients diagnosed with invasive penile squamous cell carcinoma from 1987 to 2010 who had known HPV status were selected for study. Tissue microarrays were constructed with 67 primary penile squamous cell carcinomas, matched normal tissues and 8 lymph node metastases. Immunohistochemical staining was performed for p53, pmTOR, pERK, p4E-BP1, eIF4E and peIF4E. Expression was evaluated using a semiquantitative H-score on a scale of 0 to 300., Results: Expression of pmTOR, p4E-BP1, eIF4E and peIF4E was increased in penile tumors compared with matched adjacent normal tissues, indicating activation of the mTOR signaling pathway in penile tumorigenesis. Over expression of pmTOR, peIF4E and p53 was significantly associated with lymph node disease. peIF4E and p53 also correlated with a poor outcome, including recurrence, metastasis or disease specific death. In contrast, pERK and p4E-BP1 were associated with lower pT stages. pmTOR and intense p53 expression was associated with HPV negative tumors., Conclusions: Activation of mTOR signaling may contribute to penile squamous cell carcinoma progression and aggressive behavior. Targeting mTOR or its downstream signaling targets, such as peIF4E, may be a valid therapeutic strategy., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the effects of advanced age on whole-body protein synthesis and activation of the mechanistic target of rapamycin (mTOR) signaling pathway in skeletal muscle of horses., Animals: Six 22- to 26-year-old (aged) and six 7- to 14-year-old (mature) horses., Procedures: Whole-body protein synthesis was measured with a 2-hour primed constant infusion of (13)C sodium bicarbonate, followed by a 4-hour primed constant infusion of 1-(13)C phenylalanine. After the infusions, a biopsy specimen was obtained from a gluteus medius muscle and activation of protein kinase B (Akt), p70 riboprotein S6 kinase (S6K1), riboprotein S6 (rpS6), and eukaryotic initiation factor 4E binding protein 1 (4EBP1) was determined with western immunoblot analysis. For all horses, inflammatory cytokine expression in muscle and blood samples was measured with quantitative real-time PCR analysis., Results: Advanced age had no effect on whole-body protein synthesis or the phosphorylation of Akt, rpS6, and 4EBP1; however, muscle specimens of aged horses had 42% lower phosphorylation of S6K1 than did those of mature horses. Aged and mature horses had similar inflammatory cytokine expression in muscle and blood samples., Conclusions and Clinical Relevance: The lower S6K1 activation for aged horses, compared with that for mature horses, could be indicative of low rates of muscle protein synthesis in aged horses. However, advanced age had no effect on any other indicators of whole-body or muscle protein synthesis or on measures of systemic or muscle inflammation, which suggested that protein metabolism and subsequently requirements may not differ between healthy mature and aged horses.

The peroxisome proliferator-activated receptor-γ (PPAR-γ) coactivator-1β (PGC-1β) is a well-established regulator of mitochondrial biogenesis. However, the underlying mechanism of PGC-1β action remains elusive. This study reveals that knockdown of endogenous PGC-1β by short-hairpin RNA (shRNA) leads to a decrease in the expression of mammalian target of rapamycin (mTOR) pathway-related genes in MDA-MB-231 cells. After knockdown of PGC-1β, phosphorylation of AMP-activated protein kinase (AMPK), phosphorylation of Rictor on Thr1135, Raptor and S6 protein was inhibited. However, Akt phosphorylation on Ser473 was upregulated and cell apoptosis occurred. In particular, we demonstrate that the levels of PGC-1β and mTOR correlated with overall mitochondrial activity. These results provide new evidence that cell apoptosis is orchestrated by the balance between several signaling pathways, and that PGC-1β takes part in these events in breast cancer cells mediated by the mTOR signaling pathway.

Mantle cell lymphoma (MCL) is one of the most aggressive B-cell non-Hodgkin lymphomas with a median survival of approximately five years. Currently, there is no curative therapy available for refractory MCL because of relapse from therapy-resistant tumor cells. The NF-κB and mTOR pathways are constitutively active in refractory MCL leading to increased proliferation and survival. Targeting these pathways is an ideal strategy to improve therapy for refractory MCL. Therefore, we investigated the in vitro and in vivo antilymphoma activity and associated molecular mechanism of action of a novel compound, 13-197, a quinoxaline analog that specifically perturbs IκB kinase (IKK) β, a key regulator of the NF-κB pathway. 13-197 decreased the proliferation and induced apoptosis in MCL cells including therapy-resistant cells compared with control cells. Furthermore, we observed downregulation of IκBα phosphorylation and inhibition of NF-κB nuclear translocation by 13-197 in MCL cells. In addition, NF-κB-regulated genes such as cyclin D1, Bcl-XL, and Mcl-1 were downregulated in 13-197-treated cells. In addition, 13-197 inhibited the phosphorylation of S6K and 4E-BP1, the downstream molecules of mTOR pathway that are also activated in refractory MCL. Further, 13-197 reduced the tumor burden in vivo in the kidney, liver, and lungs of therapy-resistant MCL-bearing nonobese diabetic severe-combined immunodeficient (NOD/SCID) mice compared with vehicle-treated mice; indeed, 13-197 significantly increased the survival of MCL-transplanted mice. Together, results suggest that 13-197 as a single agent disrupts the NF-κB and mTOR pathways leading to suppression of proliferation and increased apoptosis in malignant MCL cells including reduction in tumor burden in mice., (©2013 AACR.)

Purpose: Clinical studies have implicated the mechanistic target of rapamycin (serine/threonine kinase; MTOR) pathway in the regulation of neuroendocrine tumor (NET) growth. We explored whether expression of MTOR pathway components has prognostic significance in NET patients., Patients and Methods: We evaluated immunohistochemical expression of MTOR and phospho (p) -MTOR; its downstream targets RPS6KB1, RPS6, and EIF4EBP1; and its upstream regulators, in a cohort of 195 archival neuroendocrine tumors. We correlated expression levels with clinical outcomes, after adjusting for other prognostic variables., Results: We observed anticipated correlations between expression of upstream components of the MTOR pathway and their downstream targets. Expression of PIK3CA, MTOR, or p-EIF4EBP1 was associated with high MKI67 (Ki-67) labeling index. We failed to identify clinical correlations associated with expression of the upstream regulators TSC1, TSC2, AKT, p-AKT, PDPK1, PTEN, PIK3R1, or PIK3CA. In contrast, high expression of MTOR or its activated downstream targets p-RPS6KB1, p-RPS6, or p-EIF4EBP1 was associated with adverse clinical outcomes., Conclusion: Our observations suggest that expression of MTOR or its downstream targets may be adverse prognostic factors in neuroendocrine tumors.

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc(13) localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc(13) localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal.

We analyzed aging parameters using a mechanistic target of rapamycin (mTOR) hypomorphic mouse model. Mice with two hypomorphic (mTOR(Δ/Δ)) alleles are viable but express mTOR at approximately 25% of wild-type levels. These animals demonstrate reduced mTORC1 and mTORC2 activity and exhibit an approximately 20% increase in median survival. While mTOR(Δ/Δ) mice are smaller than wild-type mice, these animals do not demonstrate any alterations in normalized food intake, glucose homeostasis, or metabolic rate. Consistent with their increased lifespan, mTOR(Δ/Δ) mice exhibited a reduction in a number of aging tissue biomarkers. Functional assessment suggested that, as mTOR(Δ/Δ) mice age, they exhibit a marked functional preservation in many, but not all, organ systems. Thus, in a mammalian model, while reducing mTOR expression markedly increases overall lifespan, it affects the age-dependent decline in tissue and organ function in a segmental fashion., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Flavonoids have been intensively explored for their anticancer activity. In this study, a total synthetic flavonoid protoapigenone, known as WYC02, was analysed for its potential anticancer activity on human cervical cancer cells as well as the underlying mechanisms for these effects. The site-moiety maps are used to explore the binding site similarity, pharmacophore and docking pose similarity. The effect of WYC02 on cell viability, migration, invasion and apoptosis as well as the underlying mechanisms was analysed in vitro using human cervical cancer cells. The effect of WYC02 on in vivo tumour growth was assessed in a tumour xenograft study. WYC02 inhibited cell proliferation, MMPs activity, migration and invasion in cervical cancer cells. We speculated that WYC02 might inhibit the activities of PIK3 family proteins, including PIK3CA, PIK3CB, PIK3CD and PIK3CG. Indeed, WYC02 decreased the expression of PIK3 family proteins, especially PIK3CG, through ubiquitination and inhibited the activities of PIK3CG and PIK3 downstream molecules AKT1 and MTOR in cervical cancer cells. Furthermore, PIK3 signalling pathway was involved in the inhibitory effect of WYC02 on cervical cancer cell proliferation and tumour growth in vitro and in vivo. WYC02 inhibits cervical cancer cell proliferation and tumourigenesis via PIK3 signalling pathway and has the potential to be developed as a chemotherapeutic agent in cervical cancer., (© 2013 Nordic Pharmacological Society. Published by John Wiley & Sons Ltd.)

Clinically, electroacupuncture is proved to be an effective therapy for vascular dementia; however, their mechanisms remain uncertain. The aim of the current study was to investigate the mechanism of electroacupuncture therapy for vascular dementia. One month after a vascular dementia animal model was established by bilateral occlusion of common carotid arteries, electroacupuncture treatment was given at "Baihui" (DU20), "Dazhui" (DU14), and "Shenshu" (BL23). Morris water maze was used to assess the learning and memory ability of rats. Western blot assay was performed to detect the expression of mammalian target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E (eIF4E) in hippocampus of rats. Morris water maze test showed that electroacupuncture improved the learning ability of vascular dementia rats. Western blot assay revealed that the expression level of mTOR and eIF4E in the electroacupuncture group and sham-operated group was higher than that in the vascular dementia group (P < 0.05). In conclusion, the decreasing expression of mTOR and eIF4E plays important roles in the pathogenesis of vascular dementia. Electroacupuncture improves learning and memory ability by up-regulating expression of mTOR and eIF4E in the hippocampus of vascular dementia rats.

Purpose: Although targeted therapies against HER2 have been one of the most successful therapeutic strategies for breast cancer, patients eventually developed acquired resistance from compensatory upregulation of alternate HERs and mitogen-activated protein kinase-phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling. As we and others have shown that the soluble ectodomain fragment of E-cadherin exerts prooncogenic effects via HER1/2-mediated binding and activation of downstream prosurvival pathways, we explored whether targeting this ectodomain [DECMA-1 monoclonal antibody (mAb)] was effective in the treatment of HER2-positive (HER2(+)) breast cancers., Experimental Design: MMTV-PyMT transgenic mice and HER2(+)/E-cadherin-positive MCF-7 and BT474 trastuzumab-resistant (TtzmR) cells were treated with the DECMA-1 mAb. Antitumor responses were assessed by bromodeoxyuridine incorporation, apoptosis, and necrosis. The underlying intracellular prooncogenic pathways were explored using subcellular fractionation, immunoprecipitation, fluorescence microscopy, and immunoblotting., Results: Treatment with DECMA-1 mAb significantly delayed tumor onset and attenuated tumor burden in MMTV-PyMT mice by reducing tumor cell proliferation and inducing apoptosis without any detectable cytotoxicity to mice or end-organs. In vitro treatment of MCF-7 and BT474 TtzmR cells reduced proliferation and induced cancer cell apoptosis. Importantly, this inhibition of breast tumorigenesis was due to concomitant downregulation, via ubiquitin-mediated degradation through the lysosome and proteasome pathways, of all HER family members, components of downstream PI3K/Akt/mTOR prosurvival signaling and suppression of inhibitor of apoptosis proteins., Conclusions: Our results establish that the E-cadherin ectodomain-specific mAb DECMA-1 inhibits Ecad(+)/HER2(+) breast cancers by hindering tumor growth and inducing apoptosis via downregulation of key oncogenic pathways involved in trastuzumab resistance, thereby establishing a novel therapeutic platform for the treatment of HER2(+) breast cancers.

Background: Uterine leiomyosarcoma (LMS) has an unfavorable response to standard chemotherapeutic regimens. Two natural occurring compounds, curcumin and epigallocatechin gallate (EGCG), are reported to have anti-cancer activity. We previously reported that curcumin reduced uterine LMS cell proliferation by targeting the AKT-mTOR pathway. However, challenges remain in overcoming curcumin's low bioavailability., Methods: The human LMS cell line SKN was used. The effect of EGCG, curcumin or their combination on cell growth was detected by MTS assay. Their effect on AKT, mTOR, and S6 was detected by Western blotting. The induction of apoptosis was determined by Western blotting using cleaved-PARP specific antibody, caspase-3 activity and TUNEL assay. Intracellular curcumin level was determined by a spectrophotometric method. Antibody against EGCG cell surface receptor, 67-kDa laminin receptor (67LR), was used to investigate the role of the receptor in curcumin's increased potency by EGCG., Results: In this study, we showed that the combination of EGCG and curcumin significantly reduced SKN cell proliferation more than either drug alone. The combination inhibited AKT, mTOR, and S6 phosphorylation, and induced apoptosis at a much lower curcumin concentration than previously reported. EGCG enhanced the incorporation of curcumin. 67LR antibody partially rescued cell proliferation suppression by the combination treatment, but was not involved in the EGCG-enhanced intracellular incorporation of curcumin., Conclusions: EGCG significantly lowered the concentration of curcumin required to inhibit the AKT-mTOR pathway, reduce cell proliferation and induce apoptosis in uterine LMS cells by enhancing intracellular incorporation of curcumin, but the process was independent of 67LR.

The mammalian target of rapamycin (mTOR) plays a key role in the regulation of cellular metabolism, growth and proliferation. It forms two multi-protein complexes known as complex 1 (mTORC1) and 2 (mTORC2). Raptor and Rictor are the core proteins for mTORC1 and mTORC2, respectively. This study examines the relationship between mTORC1, Rictor and Raptor mRNA expression and human breast cancer. Furthermore, the correlation between mTORC1 and hTERT was investigated. Breast cancer tissues (n=150) and normal tissues (n=31) were analysed using reverse transcription and quantitative PCR. Transcript levels were correlated with clinicopathological data. Higher mTOR expression was noted in breast cancer tissue (P=0.0018), higher grade tumours (grade 2 vs. 3, P=0.047), in ductal tumours (P=0.0014), and was associated with worse overall survival (P=0.01). Rictor expression was significantly higher in background breast tissues compared with tumours and was inversely related to the Nottingham Prognostic Index (NPI1 vs. 2, P=0.03) and tumour grade (grade 1 vs. 3, P=0.01) and was associated with better overall (P=0.037) and disease-free survival (P=0.048). The mRNA expression of Raptor was higher in tumours compared with normal tissues. Furthermore, the expression of Raptor was associated with a higher tumour grade (grade 1 vs. 3, P=0.027). A highly significant positive correlation between mTOR and hTERT (P<0.00001) was observed. These observations are consistent with the role of mTORC1 in the anti-apoptosis pathway and suggest that selective inhibitors of mTORC1 may be more efficacious in human breast cancer. Our findings support the hypothesis that mTORC1 is an important upregulator of telomerase in breast cancer.

Tissue microarray technology enables us to evaluate the pattern of protein expression in large numbers of samples. However, manual data acquisition and analysis still represent a challenge because they are subjective and time-consuming. Automated analysis may thus increase the speed and reproducibility of evaluation. However, the reliability of automated analysis systems should be independently evaluated. Herein, the expression of phosphorylated AKT and mTOR was determined by ScanScope XT (Aperio; Vista, CA) and ACIS III (Dako; Glostrup, Denmark) and compared with the manual analysis by two observers. The percentage of labeled pixels or nuclei analysis had a good correlation between human observers and automated systems (κ = 0.855 and 0.879 for ScanScope vs. observers and κ = 0.765 and 0.793 for ACIS III vs. observers). The intensity of labeling determined by ScanScope was also correlated with that found by the human observers (correlation index of 0.946 and 0.851 for pAKT and 0.851 and 0.875 for pmTOR). However, the correlation between ACIS III and human observation varied for labeling intensity and was considered poor in some cases (correlation index of 0.718 and 0.680 for pAKT and 0.223 and 0.225 for pmTOR). Thus, the percentage of positive pixels or nuclei determination was satisfactorily performed by both systems; however, labeling intensity was better identified by ScanScope XT.

Background: The objective of the present study was to investigate whether mTOR is involved in cardiac fibrosis evident in dilated cardiomyopathy, and whether rapamycin provides therapeutic potential for cardiac fibrosis., Methods: Forty-five rats were divided into three groups. Fifteen rats in the Adriamycin group underwent 8 weeks of Adriamycin treatment (2.5 mg/kg, twice per week; i.v.) to induce cardiac fibrosis and dilated cardiomyopathy. Fifteen rats in the rapamycin group received rapamycin (2 mg/kg, per day, orally) and i.v. Adriamycin simultaneously for 8 weeks. Fifteen untreated rats served as controls. Cardiac morphology and function were quantified using echocardiography. mTOR and p70S6K1 mRNA expression were assessed using reverse transcription-PCR., Results: Collagen volume fraction (CVF) was significantly elevated in the adriamycin group (3.36 ± 0.75) compared with controls (1.51 ± 0.31), whereas mTOR and p70S6K mRNA expression were significantly increased in the adriamycin group (0.68 ± 0.03 and 0.69 ± 0.03) compared with controls (0.38 ± 0.03 and 0.34 ± 0.02). The Adriamycin group was associated with cardiac dilation and decreased contractile function. The rapamycin group showed significantly decreased CVF (1.87 ± 0.45), accompanied with a significant decrease in mTOR and p70S6K mRNA expression (0.42 ± 0.05 and 0.45 ± 0.04) relative to the Adriamycin group. In addition, treatment with rapamycin recovered impairments in cardiac morphology and function., Conclusion: The mTOR/p70S6K pathway plays an important role in adriamycin-induced cardiac fibrosis resulting from dilated cardiomyopathy. Rapamycin is a potential therapeutic treatment that can be used to attenuate cardiac fibrosis and improve cardiac function., (Georg Thieme Verlag KG Stuttgart · New York.)