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Sample records for akt mammalian target

  1. Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in cancer stem cells.

    PubMed

    Martelli, A M; Evangelisti, C; Follo, M Y; Ramazzotti, G; Fini, M; Giardino, R; Manzoli, L; McCubrey, J A; Cocco, L

    2011-01-01

    Cancer stem cells (CSCs) comprise a subset of hierarchically organized, rare cancer cells with the ability to initiate cancer in xenografts of genetically modified murine models. CSCs are thought to be responsible for tumor onset, self-renewal/maintenance, mutation accumulation, and metastasis. The existence of CSCs could explain the high frequency of neoplasia relapse and resistance to all of currently available therapies, including chemotherapy. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is a key regulator of physiological cell processes which include proliferation, differentiation, apoptosis, motility, metabolism, and autophagy. Nevertheless, aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences prognosis. Several lines of evidence indicate that this signaling system plays a key role also in CSC biology. Of note, CSCs are more sensitive to pathway inhibition with small molecules when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling transduction pathways between CSCs and healthy stem cells can be identified. Here, we review the evidence which links the signals deriving from the PI3K/Akt/mTOR network with CSC biology, both in hematological and solid tumors. We then highlight how therapeutic targeting of PI3K/Akt/mTOR signaling with small molecule inhibitors could improve cancer patient outcome, by eliminating CSCs.

  2. Grape polyphenols inhibit Akt/mammalian target of rapamycin signaling and potentiate the effects of gefitinib in breast cancer.

    PubMed

    Castillo-Pichardo, Linette; Dharmawardhane, Suranganie F

    2012-01-01

    We recently reported that a combination of dietary grape polyphenols resveratrol, quercetin, and catechin (RQC), at low concentrations, was effective at inhibiting metastatic cancer progression. Herein, we investigate the molecular mechanisms of RQC in breast cancer and explore the potential of RQC as a potentiation agent for the epidermal growth factor receptor (EGFR) therapeutic gefitinib. Our in vitro experiments showed RQC induced apoptosis in gefitinib-resistant breast cancer cells via regulation of a myriad of proapoptotic proteins. Because the Akt/mammalian target of rapamycin (mTOR) signaling pathway is often elevated during development of anti-EGFR therapy resistance, the effect of RQC on the mTOR upstream effector Akt and the negative regulator AMP kinase (AMPK) was investigated. RQC was found to reduce Akt activity, induce the activation of AMPK, and inhibit mTOR signaling in breast cancer cells. Combined RQC and gefitinib decreased gefitinib resistant breast cancer cell viability to a greater extent than RQC or gefitinib alone. Moreover, RQC inhibited Akt and mTOR and activated AMPK even in the presence of gefitinib. Our in vivo experiments showed combined RQC and gefitinib was more effective than the individual treatments at inhibiting mammary tumor growth and metastasis in nude mice. Therefore, RQC treatment inhibits breast cancer progression and may potentiate anti-EGFR therapy by inhibition of Akt/mTOR signaling.

  3. Phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathway in non-small cell lung cancer

    PubMed Central

    2015-01-01

    Non-small cell lung cancer (NSCLC) is a devastating disease with poor prognosis. Systemic chemotherapy has been the mainstay of treatment in advanced disease for many decades. Personalized targeted therapy such as epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) and crizotinib has significantly changed the treatment paradigm in NSCLC. The future success of development of molecular targeted therapy relies on the understanding of signal transduction pathways. The PI3K-Akt-mTOR pathway is commonly deregulated in human malignancy including NSCLC. Therefore, this pathway is a target for many therapeutic developments. This review will provide an overview of PI3K-Akt-mTOR signaling pathway, genetic alterations activating the pathway and clinical therapeutic development of pathway inhibitors. PMID:25870799

  4. Protection Against Epithelial Damage During Candida albicans Infection Is Mediated by PI3K/Akt and Mammalian Target of Rapamycin Signaling

    PubMed Central

    Moyes, David L.; Shen, Chengguo; Murciano, Celia; Runglall, Manohursingh; Richardson, Jonathan P.; Arno, Matthew; Aldecoa-Otalora, Estibaliz; Naglik, Julian R.

    2014-01-01

    Background. The ability of epithelial cells (ECs) to discriminate between commensal and pathogenic microbes is essential for healthy living. Key to these interactions are mucosal epithelial responses to pathogen-induced damage. Methods. Using reconstituted oral epithelium, we assessed epithelial gene transcriptional responses to Candida albicans infection by microarray. Signal pathway activation was monitored by Western blotting and transcription factor enzyme-linked immunosorbent assay, and the role of these pathways in C. albicans–induced damage protection was determined using chemical inhibitors. Results. Transcript profiling demonstrated early upregulation of epithelial genes involved in immune responses. Many of these genes constituted components of signaling pathways, but only NF-κB, MAPK, and PI3K/Akt pathways were functionally activated. We demonstrate that PI3K/Akt signaling is independent of NF-κB and MAPK signaling and plays a key role in epithelial immune activation and damage protection via mammalian target of rapamycin (mTOR) activation. Conclusions. PI3K/Akt/mTOR signaling may play a critical role in protecting epithelial cells from damage during mucosal fungal infections independent of NF-κB or MAPK signaling. PMID:24357630

  5. Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade.

    PubMed

    Rivera Rivera, Amilcar; Castillo-Pichardo, Linette; Gerena, Yamil; Dharmawardhane, Suranganie

    2016-01-01

    The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC), at equimolar concentrations, reduces breast cancer (BC) growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K) and 4E binding protein (4EBP1), were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15μM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic.

  6. Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade

    PubMed Central

    Rivera Rivera, Amilcar; Castillo-Pichardo, Linette; Gerena, Yamil; Dharmawardhane, Suranganie

    2016-01-01

    The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC), at equimolar concentrations, reduces breast cancer (BC) growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K) and 4E binding protein (4EBP1), were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15μM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic. PMID:27285995

  7. Role of the Phosphoinositide 3-Kinase-Akt-Mammalian Target of the Rapamycin Signaling Pathway in Long-Term Potentiation and Trace Fear Conditioning Memory in Rat Medial Prefrontal Cortex

    ERIC Educational Resources Information Center

    Sui, Li; Wang, Jing; Li, Bao-Ming

    2008-01-01

    Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt (also known as protein kinase B, PKB), mammalian target of rapamycin (mTOR), the 70-kDa ribosomal S6 kinase (p70S6k), and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), may play important roles in long-term synaptic plasticity and memory in many…

  8. Activation of phosphatidylinositol 3-kinase/Akt-mammalian target of Rapamycin signaling pathway in the hippocampus is essential for the acquisition of morphine-induced place preference in rats.

    PubMed

    Cui, Yue; Zhang, X Q; Cui, Y; Xin, W J; Jing, J; Liu, X G

    2010-11-24

    Hippocampus is a critical structure for the acquisition of morphine-induced conditioned place preference (CPP), which is a usual learning paradigm for assessing drug reward. However, the precise mechanisms remain largely unknown. Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt, mammalian target of Rapamycin (mTOR) and 70-kDa ribosomal S6 kinase (p70S6K), are critical molecules implicated in learning and memory. Here, we tested the role of PI3K/Akt-mTOR-p70S6K signaling pathway in morphine-induced CPP in the hippocampus. Our results showed that the acquisition of morphine CPP increased phosphorylation of Akt in the hippocampal CA3, but not in the nucleus accumbens (NAc), the ventral tegmental area (VTA) or the CA1. Moreover, the phosphorylated Akt exclusively expressed in the CA3 neurons. Likewise, levels of phosphorylated mTOR and p70S6K were significantly enhanced in the CA3 following morphine CPP. The alterations of these phosphorylated proteins are positively correlated with the acquisition of morphine CPP. More importantly, microinjection of PI3K inhibitor (LY294002) or mTOR inhibitor (Rapamycin) into the CA3 prevented the acquisition of CPP and inhibited the activation of PI3K-Akt signaling pathway. In addition, pre-infusion of β-FNA (β-funaltrexamine hydrochloride), a selective irreversible μ opioid receptor antagonist, into CA3 significantly prevented the acquisition of CPP and impaired Akt phosphorylation. All these results strongly implied that the PI3K-Akt signaling pathway activated by μ opioid receptor in hippocampal CA3 plays an important role in acquisition of morphine-induced CPP.

  9. All Akt Isoforms (Akt1, Akt2, Akt3) Are Involved in Normal Hearing, but Only Akt2 and Akt3 Are Involved in Auditory Hair Cell Survival in the Mammalian Inner Ear

    PubMed Central

    Brand, Yves; Levano, Soledad; Radojevic, Vesna; Naldi, Arianne Monge; Setz, Cristian; Ryan, Allen F.; Pak, Kwang; Hemmings, Brian A.; Bodmer, Daniel

    2015-01-01

    The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear—especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear. PMID:25811375

  10. A hexane fraction of guava Leaves (Psidium guajava L.) induces anticancer activity by suppressing AKT/mammalian target of rapamycin/ribosomal p70 S6 kinase in human prostate cancer cells.

    PubMed

    Ryu, Nae Hyung; Park, Kyung-Ran; Kim, Sung-Moo; Yun, Hyung-Mun; Nam, Dongwoo; Lee, Seok-Geun; Jang, Hyeung-Jin; Ahn, Kyoo Seok; Kim, Sung-Hoon; Shim, Bum Sang; Choi, Seung-Hoon; Mosaddik, Ashik; Cho, Somi K; Ahn, Kwang Seok

    2012-03-01

    This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography-mass spectrometry tentatively identified 60 compounds, including β-eudesmol (11.98%), α-copaene (7.97%), phytol (7.95%), α-patchoulene (3.76%), β-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), α-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer.

  11. Recent development of anticancer therapeutics targeting Akt.

    PubMed

    Morrow, John K; Du-Cuny, Lei; Chen, Lu; Meuillet, Emmanuelle J; Mash, Eugene A; Powis, Garth; Zhang, Shuxing

    2011-01-01

    The serine/threonine kinase Akt has proven to be a significant signaling target, involved in various biological functions. Because of its cardinal role in numerous cellular responses, Akt has been implicated in many human diseases, particularly cancer. It has been established that Akt is a viable and feasible target for anticancer therapeutics. Analysis of all Akt kinases reveals conserved homology for an N-terminal regulatory domain, which contains a pleckstrin-homology (PH) domain for cellular translocation, a kinase domain with serine/threonine specificity, and a C-terminal extension domain. These well defined regions have been targeted, and various approaches, including in silico methods, have been implemented to develop Akt inhibitors. In spite of unique techniques and a prolific body of knowledge surrounding Akt, no targeted Akt therapeutics have reached the market yet. Here we will highlight successes and challenges to date on the development of anticancer agents modulating the Akt pathway in recent patents as well as discuss the methods employed for this task. Special attention will be given to patents with focus on those discoveries using computer-aided drug design approaches.

  12. Isoflurane Promotes Non-Small Cell Lung Cancer Malignancy by Activating the Akt-Mammalian Target of Rapamycin (mTOR) Signaling Pathway

    PubMed Central

    Zhang, Wenhua; Shao, Xueqian

    2016-01-01

    Background Lung cancer is one of the leading causes of cancer mortalities worldwide, and non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer cases. Surgery remains one of the front-line treatment options for NSCLC, but events within the perioperative period were found to affect cancer prognosis, such as anesthesia procedures. Isoflurane, a commonly used volatile anesthetic, enhances the malignant potential of renal, prostate, and ovarian cancer cells, but its effects on NSCLC development have not been previously reported. Material/Methods CCK-8 and MTT cell proliferation assays were used to analyze NSCLC cell proliferation. Metastatic ability was examined by wound healing and transwell assays. We used Western blot analysis to study the mechanism of effect of Isoflurane in NSCLC development. Results We demonstrated that isoflurane promotes proliferation, migration and invasiveness of NSCLC cells, as well as upregulation of the Akt-mTOR signaling pathway in NSCLC cells. Pharmacological inhibition of Akt-mTOR signaling abolished the ability of isoflurane to promote proliferation, migration, and invasion of NSCLC cells, indicating that isoflurane promotes NSCLC cell malignancy by activating the Akt-mTOR signaling pathway. Conclusions Isoflurane promotes NSCLC proliferation, migration and invasion by activating the Akt-mTOR signaling pathway. PMID:27897153

  13. Emerging therapeutics for targeting Akt in cancer.

    PubMed

    Gdowski, Andrew; Panchoo, Marlyn; Treuren, Timothy Van; Basu, Alakananda

    2016-01-01

    The ultimate goal of cancer therapeutic research is to develop effective, targeted therapeutics that exploit the vulnerabilities of cancer cells. The three isoforms of Akt, also known as protein kinase B (PKB), are important mediators of various pathways that transmit mitogenic signals from the cell's exterior to the effector proteins of the cell's interior. Due to Akt\\\\\\\\\\\\\\'s importance in cell functions such as growth, proliferation and cell survival, many cancer cells rely on this pathway to aid in their survival. This dependence can lead to chemoresistance and selection of more adapted populations of cancer cells. Thus, it is important to understand the functional significance of isoform specificity and its relation to chemoresistance. In this review, we have summarized recent studies on Akt isoform specific regulation as well as each isoform's role in chemoresistance, emphasizing their potential as targets for cancer therapy. We have also condensed ongoing clinical studies involving various types of Akt inhibitors while highlighting the type of study, rationale and co-therapies involved in identifying Akt isoforms as promising therapeutic targets.

  14. Sann-Joong-Kuey-Jian-Tang induces autophagy in HepG2 cells via regulation of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and p38 mitogen-activated protein kinase pathways.

    PubMed

    Chuang, Wan-Ling; Su, Chin-Cheng; Lin, Ping-Yi; Lin, Chi-Chen; Chen, Yao-Li

    2015-08-01

    Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicine, was previously reported to induce autophagy and inhibit the proliferation of the human HepG2 hepatocellular carcinoma cell line via an extrinsic pathway. In the present study, the effects of SJKJT-induced autophagy and the cytotoxic mechanisms mediating these effects were investigated in HepG2 cells. The cytotoxicity of SJKJT in the HepG2 cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that the half-maximal inhibitory concentration of SJKJT was 2.91 mg/ml at 24 h, 1.64 mg/ml at 48 h and 1.26 mg/ml at 72 h. The results of confocal fluorescence microscopy indicated that SJKJT resulted in the accumulation of green fluorescent protein-LC3 and vacuolation of the cytoplasm. Flow cytometric analysis revealed the accumulation of acidic vesicular organelles. Furthermore, western blot analysis, used to determine the expression levels of autophagy-associated proteins, demonstrated that the HepG2 cells treated with SJKJT exhibited LC3B-I/LC3B-II conversion, increased expression levels of Beclin, Atg-3 and Atg-5 and reduced expression levels of p62 and decreased signaling of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and the p38 mitogen-activated protein kinase pathways. Taken together, these findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer.

  15. Sann-Joong-Kuey-Jian-Tang induces autophagy in HepG2 cells via regulation of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and p38 mitogen-activated protein kinase pathways

    PubMed Central

    CHUANG, WAN-LING; SU, CHIN-CHENG; LIN, PING-YI; LIN, CHI-CHEN; CHEN, YAO-LI

    2015-01-01

    Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicine, was previously reported to induce autophagy and inhibit the proliferation of the human HepG2 hepatocellular carcinoma cell line via an extrinsic pathway. In the present study, the effects of SJKJT-induced autophagy and the cytotoxic mechanisms mediating these effects were investigated in HepG2 cells. The cytotoxicity of SJKJT in the HepG2 cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that the half-maximal inhibitory concentration of SJKJT was 2.91 mg/ml at 24 h, 1.64 mg/ml at 48 h and 1.26 mg/ml at 72 h. The results of confocal fluorescence microscopy indicated that SJKJT resulted in the accumulation of green fluorescent protein-LC3 and vacuolation of the cytoplasm. Flow cytometric analysis revealed the accumulation of acidic vesicular organelles. Furthermore, western blot analysis, used to determine the expression levels of autophagy-associated proteins, demonstrated that the HepG2 cells treated with SJKJT exhibited LC3B-I/LC3B-II conversion, increased expression levels of Beclin, Atg-3 and Atg-5 and reduced expression levels of p62 and decreased signaling of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and the p38 mitogen-activated protein kinase pathways. Taken together, these findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer. PMID:25847489

  16. Targeting the Akt/mTOR pathway in Brca1-deficient cancers.

    PubMed

    Xiang, T; Jia, Y; Sherris, D; Li, S; Wang, H; Lu, D; Yang, Q

    2011-05-26

    The breast cancer susceptibility gene 1 (Brca1) has a key role in both hereditary and sporadic mammary tumorigenesis. However, the reasons why Brca1-deficiency leads to the development of cancer are not clearly understood. Activation of Akt kinase is one of the most common molecular alterations associated with human malignancy. Increased Akt kinase activity has been reported in most breast cancers. We previously found that downregulation of Brca1 expression or mutations of the Brca1 gene activate the Akt oncogenic pathway. To further investigate the role of Brca1/Akt in tumorigenesis, we analyzed Brca1/Akt expression in human breast cancer samples and found that reduced expression of Brca1 was highly correlated with increased phosphorylation of Akt. Consistent with the clinical data, knockdown of Akt1 by short-hairpin RNA inhibited cellular proliferation of Brca1 mutant cells. Importantly, depletion of Akt1 significantly reduced tumor formation induced by Brca1-deficiency in mice. The third generation inhibitor of mammalian target of rapamycin (mTOR), Palomid 529, significantly suppressed Brca1-deficient tumor growth in mice through inhibition of both Akt and mTOR signaling. Our results indicate that activation of Akt is involved in Brca1-deficiency mediated tumorigenesis and that the mTOR pathway can be used as a novel target for treatment of Brca1-deficient cancers.

  17. Targeting the Akt/mTOR pathway in Brca1-deficient cancers

    PubMed Central

    Xiang, T; Jia, Y; Sherris, D; Li, S; Wang, H; Lu, D; Yang, Q

    2011-01-01

    The breast cancer susceptibility gene 1 (Brca1) has a key role in both hereditary and sporadic mammary tumorigenesis. However, the reasons why Brca1-deficiency leads to the development of cancer are not clearly understood. Activation of Akt kinase is one of the most common molecular alterations associated with human malignancy. Increased Akt kinase activity has been reported in most breast cancers. We previously found that downregulation of Brca1 expression or mutations of the Brca1 gene activate the Akt oncogenic pathway. To further investigate the role of Brca1/Akt in tumorigenesis, we analyzed Brca1/Akt expression in human breast cancer samples and found that reduced expression of Brca1 was highly correlated with increased phosphorylation of Akt. Consistent with the clinical data, knockdown of Akt1 by short-hairpin RNA inhibited cellular proliferation of Brca1 mutant cells. Importantly, depletion of Akt1 significantly reduced tumor formation induced by Brca1-deficiency in mice. The third generation inhibitor of mammalian target of rapamycin (mTOR), Palomid 529, significantly suppressed Brca1-deficient tumor growth in mice through inhibition of both Akt and mTOR signaling. Our results indicate that activation of Akt is involved in Brca1-deficiency mediated tumorigenesis and that the mTOR pathway can be used as a novel target for treatment of Brca1-deficient cancers. PMID:21242970

  18. Chemokine CXCL12 uses CXCR4 and a signaling core formed by bifunctional Akt, extracellular signal-regulated kinase (ERK)1/2, and mammalian target of rapamycin complex 1 (mTORC1) proteins to control chemotaxis and survival simultaneously in mature dendritic cells.

    PubMed

    Delgado-Martín, Cristina; Escribano, Cristina; Pablos, José Luis; Riol-Blanco, Lorena; Rodríguez-Fernández, José Luis

    2011-10-28

    Chemokines control several cell functions in addition to chemotaxis. Although much information is available on the involvement of specific signaling molecules in the control of single functions controlled by chemokines, especially chemotaxis, the mechanisms used by these ligands to regulate several cell functions simultaneously are completely unknown. Mature dendritic cells (maDCs) migrate through the afferent lymphatic vessels to the lymph nodes, where they regulate the initiation of the immune response. As maDCs are exposed to chemokine CXCL12 (receptors CXCR4 and CXCR7) during their migration, its functions are amenable to be regulated by this ligand. We have used maDCs as a model system to analyze the mechanisms whereby CXCL12 simultaneously controls chemotaxis and survival in maDCs. We show that CXCL12 uses CXCR4, but not CXCR7, and the components of a signaling core that includes G(i)/Gβγ, PI3K-α/-δ/-γ, Akt, ERK1/2 and mammalian target of rapamycin complex 1 (mTORC1), which organize hierarchically to control both functions. Downstream of Akt, Forkhead box class O (FOXO) regulates CXCL12-dependent survival, but not chemotaxis, suggesting that downstream of the aforementioned signaling core, additional signaling molecules may control more selectively CXCL12-dependent chemotaxis or survival. Finally, the data obtained also show that CXCR4 uses a signaling signature that is different from that used by CCR7 to control similar functions.

  19. Deletion of MLIP (Muscle-enriched A-type Lamin-interacting Protein) Leads to Cardiac Hyperactivation of Akt/Mammalian Target of Rapamycin (mTOR) and Impaired Cardiac Adaptation*

    PubMed Central

    Cattin, Marie-Elodie; Wang, Jessica; Weldrick, Jonathan J.; Roeske, Cassandra L.; Mak, Esther; Thorn, Stephanie L.; DaSilva, Jean N.; Wang, Yibin; Lusis, Aldon J.; Burgon, Patrick G.

    2015-01-01

    Aging and diseases generally result from tissue inability to maintain homeostasis through adaptation. The adult heart is particularly vulnerable to disequilibrium in homeostasis because its regenerative abilities are limited. Here, we report that MLIP (muscle enriched A-type lamin-interacting protein), a unique protein of unknown function, is required for proper cardiac adaptation. Mlip−/− mice exhibited normal cardiac function despite myocardial metabolic abnormalities and cardiac-specific overactivation of Akt/mTOR pathways. Cardiac-specific MLIP overexpression led to an inhibition of Akt/mTOR, providing evidence of a direct impact of MLIP on these key signaling pathways. Mlip−/− hearts showed an impaired capacity to adapt to stress (isoproterenol-induced hypertrophy), likely because of deregulated Akt/mTOR activity. Genome-wide association studies showed a genetic association between Mlip and early response to cardiac stress, supporting the role of MLIP in cardiac adaptation. Together, these results revealed that MLIP is required for normal myocardial adaptation to stress through integrated regulation of the Akt/mTOR pathways. PMID:26359501

  20. Role of Akt signaling in resistance to DNA-targeted therapy

    PubMed Central

    Avan, Abolfazl; Narayan, Ravi; Giovannetti, Elisa; Peters, Godefridus J

    2016-01-01

    The Akt signal transduction pathway controls most hallmarks of cancer. Activation of the Akt cascade promotes a malignant phenotype and is also widely implicated in drug resistance. Therefore, the modulation of Akt activity is regarded as an attractive strategy to enhance the efficacy of cancer therapy and irradiation. This pathway consists of phosphatidylinositol 3 kinase (PI3K), mammalian target of rapamycin, and the transforming serine-threonine kinase Akt protein isoforms, also known as protein kinase B. DNA-targeted agents, such as platinum agents, taxanes, and antimetabolites, as well as radiation have had a significant impact on cancer treatment by affecting DNA replication, which is aberrantly activated in malignancies. However, the caveat is that they may also trigger the activation of repairing mechanisms, such as upstream and downstream cascade of Akt survival pathway. Thus, each target can theoretically be inhibited in view of improving the potency of conventional treatment. Akt inhibitors, e.g., MK-2206 and perifosine, or PI3K modulators, e.g., LY294002 and Wortmannin, have shown some promising results in favor of sensitizing the cancer cells to the therapy in vitro and in vivo, which have provided the rationale for incorporation of these novel agents into multimodality treatment of different malignancies. Nevertheless, despite the acceptable safety profile of some of these agents in the clinical studies, with regard to the efficacy, the results are still too preliminary. Hence, we need to wait for the upcoming data from the ongoing trials before utilizing them into the standard care of cancer patients. PMID:27777878

  1. Molecular targets for cancer therapy in the PI3K/AKT/mTOR pathway.

    PubMed

    Polivka, Jiri; Janku, Filip

    2014-05-01

    Aberrations in various cellular signaling pathways are instrumental in regulating cellular metabolism, tumor development, growth, proliferation, metastasis and cytoskeletal reorganization. The fundamental cellular signaling cascade involved in these processes, the phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K/AKT/mTOR), closely related to the mitogen-activated protein kinase (MAPK) pathway, is a crucial and intensively explored intracellular signaling pathway in tumorigenesis. Various activating mutations in oncogenes together with the inactivation of tumor suppressor genes are found in diverse malignancies across almost all members of the pathway. Substantial progress in uncovering PI3K/AKT/mTOR alterations and their roles in tumorigenesis has enabled the development of novel targeted molecules with potential for developing efficacious anticancer treatment. Two approved anticancer drugs, everolimus and temsirolimus, exemplify targeted inhibition of PI3K/AKT/mTOR in the clinic and many others are in preclinical development as well as being tested in early clinical trials for many different types of cancer. This review focuses on targeted PI3K/AKT/mTOR signaling from the perspective of novel molecular targets for cancer therapy found in key pathway members and their corresponding experimental therapeutic agents. Various aberrant prognostic and predictive biomarkers are also discussed and examples are given. Novel approaches to PI3K/AKT/mTOR pathway inhibition together with a better understanding of prognostic and predictive markers have the potential to significantly improve the future care of cancer patients in the current era of personalized cancer medicine.

  2. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers.

    PubMed

    Goda, Jayant S; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-02-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies.

  3. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers

    PubMed Central

    Goda, Jayant S.; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-01-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies. PMID:27121513

  4. Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor, ARQ 092

    PubMed Central

    Yu, Yi; Savage, Ronald E.; Eathiraj, Sudharshan; Meade, Justin; Wick, Michael J.; Hall, Terence; Abbadessa, Giovanni; Schwartz, Brian

    2015-01-01

    As a critical component in the PI3K/AKT/mTOR pathway, AKT has become an attractive target for therapeutic intervention. ARQ 092 and a next generation AKT inhibitor, ARQ 751 are selective, allosteric, pan-AKT and AKT1-E17K mutant inhibitors that potently inhibit phosphorylation of AKT. Biochemical and cellular analysis showed that ARQ 092 and ARQ 751 inhibited AKT activation not only by dephosphorylating the membrane-associated active form, but also by preventing the inactive form from localizing into plasma membrane. In endometrial PDX models harboring mutant AKT1-E17K and other tumor models with an activated AKT pathway, both compounds exhibited strong anti-tumor activity. Combination studies conducted in in vivo breast tumor models demonstrated that ARQ 092 enhanced tumor inhibition of a common chemotherapeutic agent (paclitaxel). In a large panel of diverse cancer cell lines, ARQ 092 and ARQ 751 inhibited proliferation across multiple tumor types but were most potent in leukemia, breast, endometrial, and colorectal cancer cell lines. Moreover, inhibition by ARQ 092 and ARQ 751 was more prevalent in cancer cell lines containing PIK3CA/PIK3R1 mutations compared to those with wt-PIK3CA/PIK3R1 or PTEN mutations. For both ARQ 092 and ARQ 751, PIK3CA/PIK3R1 and AKT1-E17K mutations can potentially be used as predictive biomarkers for patient selection in clinical studies. PMID:26469692

  5. Akt isoforms in vascular disease

    PubMed Central

    Yu, Haixiang; Littlewood, Trevor; Bennett, Martin

    2015-01-01

    The mammalian serine/threonine Akt kinases comprise three closely related isoforms: Akt1, Akt2 and Akt3. Akt activation has been implicated in both normal and disease processes, including in development and metabolism, as well as cancer and cardiovascular disease. Although Akt signalling has been identified as a promising therapeutic target in cancer, its role in cardiovascular disease is less clear. Importantly, accumulating evidence suggests that the three Akt isoforms exhibit distinct tissue expression profiles, localise to different subcellular compartments, and have unique modes of activation. Consistent with in vitro findings, genetic studies in mice show distinct effects of individual Akt isoforms on the pathophysiology of cardiovascular disease. This review summarises recent studies of individual Akt isoforms in atherosclerosis, vascular remodelling and aneurysm formation, to provide a comprehensive overview of Akt function in vascular disease. PMID:25929188

  6. Dual Targeting of Akt and mTORC1 Impairs Repair of DNA Double-Strand Breaks and Increases Radiation Sensitivity of Human Tumor Cells

    PubMed Central

    Holler, Marina; Grottke, Astrid; Mueck, Katharina; Manes, Julia; Jücker, Manfred

    2016-01-01

    Inhibition of mammalian target of rapamycin-complex 1 (mTORC1) induces activation of Akt. Because Akt activity mediates the repair of ionizing radiation-induced DNA double-strand breaks (DNA-DSBs) and consequently the radioresistance of solid tumors, we investigated whether dual targeting of mTORC1 and Akt impairs DNA-DSB repair and induces radiosensitization. Combining mTORC1 inhibitor rapamycin with ionizing radiation in human non-small cell lung cancer (NSCLC) cells (H661, H460, SK-MES-1, HTB-182, A549) and in the breast cancer cell line MDA-MB-231 resulted in radiosensitization of H661 and H460 cells (responders), whereas only a very slight effect was observed in A549 cells, and no effect was observed in SK-MES-1, HTB-182 or MDA-MB-231 cells (non-responders). In responder cells, rapamycin treatment did not activate Akt1 phosphorylation, whereas in non-responders, rapamycin mediated PI3K-dependent Akt activity. Molecular targeting of Akt by Akt inhibitor MK2206 or knockdown of Akt1 led to a rapamycin-induced radiosensitization of non-responder cells. Compared to the single targeting of Akt, the dual targeting of mTORC1 and Akt1 markedly enhanced the frequency of residual DNA-DSBs by inhibiting the non-homologous end joining repair pathway and increased radiation sensitivity. Together, lack of radiosensitization induced by rapamycin was associated with rapamycin-mediated Akt1 activation. Thus, dual targeting of mTORC1 and Akt1 inhibits repair of DNA-DSB leading to radiosensitization of solid tumor cells. PMID:27137757

  7. AKT is a therapeutic target in myeloproliferative neoplasms

    PubMed Central

    Khan, Irum; Huang, Zan; Wen, Qiang; Stankiewicz, Monika J.; Gilles, Laure; Goldenson, Benjamin; Schultz, Rachael; Diebold, Lauren; Gurbuxani, Sandeep; Finke, Christy M.; Lasho, Terra L.; Koppikar, Priya; Pardanani, Animesh; Stein, Brady; Altman, Jessica K.; Levine, Ross L.; Tefferi, Ayalew; Crispino, John D.

    2014-01-01

    The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K, and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with Ruxolitinib in suppressing the growth of JAK2V617F mutant SET2 cells. Importantly MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis (PMF) and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs. PMID:23748344

  8. Targeting the PI3K/AKT/mTOR Signaling Axis in Children with Hematologic Malignancies

    PubMed Central

    Barrett, David; Brown, Valerie I.; Grupp, Stephan A.; Teachey, David T.

    2014-01-01

    The phosphatidylinositiol 3-kinase (PI3K), AKT, mammalian target of rapamycin (mTOR) signaling pathway (PI3K/AKT/mTOR) is frequently dysregulated in disorders of cell growth and survival, including a number of pediatric hematologic malignancies. The pathway can be abnormally activated in childhood acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML), as well as in some pediatric lymphomas and lymphoproliferative disorders. Most commonly, this abnormal activation occurs as a consequence of constitutive activation of AKT, providing a compelling rationale to target this pathway in many of these conditions. A variety of agents, beginning with the rapamycin analogue (rapalog) sirolimus, have been used successfully to target this pathway in a number of pediatric hematologic malignancies. Rapalogs demonstrate significant preclinical activity against ALL, which has led to a number of clinical trials. Moreover, rapalogs can synergize with a number of conventional cytotoxic agents and overcome pathways of chemotherapeutic resistance for drugs commonly used in ALL treatment, including methotrexate and corticosteroids. Based on preclinical data, rapalogs are also being studied in AML, CML, and non-Hodgkin’s lymphoma. Recently, significant progress has been made using rapalogs to treat pre-malignant lymphoproliferative disorders, including the autoimmune lymphoproliferative syndrome (ALPS); complete remissions in children with otherwise therapy-resistant disease have been seen. Rapalogs only block one component of the pathway (mTORC1), and newer agents are under preclinical and clinical development that can target different and often multiple protein kinases in the PI3K/AKT/mTOR pathway. Most of these agents have been tolerated in early-phase clinical trials. A number of PI3K inhibitors are under investigation. Of note, most of these also target other protein kinases. Newer agents are under development that target both m

  9. The ketogenic diet inhibits the mammalian target of rapamycin (mTOR) pathway.

    PubMed

    McDaniel, Sharon S; Rensing, Nicholas R; Thio, Liu Lin; Yamada, Kelvin A; Wong, Michael

    2011-03-01

    The ketogenic diet (KD) is an effective treatment for epilepsy, but its mechanisms of action are poorly understood. We investigated the hypothesis that the KD inhibits mammalian target of rapamycin (mTOR) pathway signaling. The expression of pS6 and pAkt, markers of mTOR pathway activation, was reduced in hippocampus and liver of rats fed KD. In the kainate model of epilepsy, KD blocked the hippocampal pS6 elevation that occurs after status epilepticus. Because mTOR signaling has been implicated in epileptogenesis, these results suggest that the KD may have anticonvulsant or antiepileptogenic actions via mTOR pathway inhibition.

  10. Combined Targeting of mTOR and Akt Using Rapamycin and MK-2206 in The Treatment of Tuberous Sclerosis Complex

    PubMed Central

    Ji, Shuang; Lin, Wei; Wang, Li; Ni, Zhaofei; Jin, Fuquan; Zha, Xiaojun; Fei, Guanghe

    2017-01-01

    Tuberous sclerosis complex (TSC), caused by loss-of-function mutations in the TSC1 or TSC2 genes, is an autosomal dominant disease characterized by benign tumor formation in multiple organs. Hyperactivation of mammalian target of rapamycin (mTOR) is the primary alteration underlying TSC tumor. Thus, rapamycin, as an mTOR specific inhibitor, has been assumed as a potential drug for the treatment of TSC. However, its application in TSC patients has been limited due to side effects. By analyzing Tsc1- or Tsc2-null mouse embryonic fibroblasts (MEFs), we found that loss of TSC1 or TSC2 led to a decreased sensitivity to MK-2206, a novel allosteric Akt inhibitor. Ectopic expression of a constitutively activated Akt (myristoylated Akt-1, myrAkt-1) sensitized Tsc2-null and Tsc1-null MEFs to MK-2206. Furthermore, MK-2206 increased the cytotoxicity of rapamycin in Tsc1-/-or Tsc2-/- MEFs. Moreover, the benefit of the combinatorial treatment was also demonstrated in a TSC xenograft mouse model. We conclude that the combination of rapamycin and MK-2206 may be utilized as a new therapeutic regimen for TSC. PMID:28367235

  11. Ciliary transport regulates PDGF-AA/αα signaling via elevated mammalian target of rapamycin signaling and diminished PP2A activity.

    PubMed

    Umberger, Nicole L; Caspary, Tamara

    2015-01-15

    Primary cilia are built and maintained by intraflagellar transport (IFT), whereby the two IFT complexes, IFTA and IFTB, carry cargo via kinesin and dynein motors for anterograde and retrograde transport, respectively. Many signaling pathways, including platelet- derived growth factor (PDGF)-AA/αα, are linked to primary cilia. Active PDGF-AA/αα signaling results in phosphorylation of Akt at two residues: P-Akt(T308) and P-Akt(S473), and previous work showed decreased P-Akt(S473) in response to PDGF-AA upon anterograde transport disruption. In this study, we investigated PDGF-AA/αα signaling via P-Akt(T308) and P-Akt(S473) in distinct ciliary transport mutants. We found increased Akt phosphorylation in the absence of PDGF-AA stimulation, which we show is due to impaired dephosphorylation resulting from diminished PP2A activity toward P-Akt(T308). Anterograde transport mutants display low platelet-derived growth factor receptor (PDGFR)α levels, whereas retrograde mutants exhibit normal PDGFRα levels. Despite this, neither shows an increase in P-Akt(S473) or P-Akt(T308) upon PDGF-AA stimulation. Because mammalian target of rapamycin complex 1 (mTORC1) signaling is increased in ciliary transport mutant cells and mTOR signaling inhibits PDGFRα levels, we demonstrate that inhibition of mTORC1 rescues PDGFRα levels as well as PDGF-AA-dependent phosphorylation of Akt(S473) and Akt(T308) in ciliary transport mutant MEFs. Taken together, our data indicate that the regulation of mTORC1 signaling and PP2A activity by ciliary transport plays key roles in PDGF-AA/αα signaling.

  12. Novel withanolides target medullary thyroid cancer through inhibition of both RET phosphorylation and the mammalian target of rapamycin pathway

    PubMed Central

    Samadi, Abbas K.; Bazzill, Joseph; Zhang, Xuan; Gallagher, Rob; Zhang, Hauping; Gollapudi, Rao; Kindscher, Kelly; Timmermann, Barbara; Cohen, Mark S.

    2013-01-01

    Background Despite development of current targeted therapies for medullary thyroid cancer (MTC), long-term survival remains unchanged. Recently isolated novel withanolide compounds from Solanaceae physalis are highly potent against MTCs. We hypothesize that these withanolides uniquely inhibit RET phosphorylation and the mammalian target of rapamycin (mTOR) pathway in MTC cells as a mechanism of antiproliferation and apoptosis. Methods MTC cells were treated with novel withanolides and MTC-targeted drugs. In vitro studies assessed cell viability and proliferation (MTS; trypan blue assays), apoptosis (flow cytometry with Annexin V/PI staining; confirmed by Western blot analysis), long-term cytotoxic effects (clonogenic assay), and suppression of key regulatory proteins such as RET, Akt, and mTOR (by Western blot analysis). Results The novel withanolides potently reduced MTC cell viability (half maximal inhibitory concentration [IC50], 270–2,850 nmol/L; 250–1,380 nmol/L for vandetanib; 360–1,640 nmol/L for cabozantinib) with induction of apoptosis at <1,000 nmol/L of drug. Unique from other targeted therapies, withanolides suppressed RET and Akt phosphorylation and protein expression (in a concentration- and time-dependent manner) as well as mTOR activity and translational activity of 4E-BP1 and protein synthesis mediated by p70S6kinase activation at IC50 concentrations. Conclusion Novel withanolides from Physalis selectively and potently inhibit MTC cells in vitro. Unlike other MTC-targeted therapies, these compounds uniquely inhibit both RET kinase activity and the Akt/mTOR prosurvival pathway. Further translational studies are warranted to evaluate their clinical potential. PMID:23158190

  13. Targeting Akt in cell transfer immunotherapy for cancer.

    PubMed

    Crompton, Joseph G; Sukumar, Madhusudhanan; Restifo, Nicholas P

    2016-01-01

    Pharmacologic inhibitors of the serine/threonine kinase Akt, initially aimed at deranged oncogenic pathways in tumors, have recently been shown to act as immunomodulators that markedly enhance the antitumor properties of T cells. Repurposing Akt inhibitors to improve antitumor immunity may be viewed as a manifestation of a larger paradigmatic shift in which hallmark characteristics of cancer (e.g., immune evasion), rather than merely causal features (e.g., somatic mutations) can be exploited for therapeutic benefit.

  14. Electrostimulation during hindlimb unloading modulates PI3K-AKT downstream targets without preventing soleus atrophy and restores slow phenotype through ERK.

    PubMed

    Dupont, Erwan; Cieniewski-Bernard, Caroline; Bastide, Bruno; Stevens, Laurence

    2011-02-01

    Our aim was to analyze the role of phosphatidylinositol 3-kinase (PI3K)-AKT and MAPK signaling pathways in the regulation of muscle mass and slow-to-fast phenotype transition during hindlimb unloading (HU). For that purpose, we studied, in rat slow soleus and fast extensor digitorum longus muscles, the time course of anabolic PI3K-AKT-mammalian target of rapamycin, catabolic PI3K-AKT-forkhead box O (FOXO), and MAPK signaling pathway activation after 7, 14, and 28 days of HU. Moreover, we performed chronic low-frequency soleus electrostimulation during HU to maintain exclusively contractile phenotype and so to determine more precisely the role of these signaling pathways in the modulation of muscle mass. HU induced a downregulation of the anabolic AKT, mammalian target of rapamycin, 70-kDa ribosomal protein S6 kinase, 4E-binding protein 1, and glycogen synthase kinase-3β targets, and an upregulation of the catabolic FOXO1 and muscle-specific RING finger protein-1 targets correlated with soleus muscle atrophy. Unexpectedly, soleus electrostimulation maintained 70-kDa ribosomal protein S6 kinase, 4E-binding protein 1, FOXO1, and muscle-specific RING finger protein-1 to control levels, but failed to reduce muscle atrophy. HU decreased ERK phosphorylation, while electrostimulation enabled the maintenance of ERK phosphorylation similar to control level. Moreover, slow-to-fast myosin heavy chain phenotype transition and upregulated glycolytic metabolism were prevented by soleus electrostimulation during HU. Taken together, our data demonstrated that the processes responsible for gradual disuse muscle plasticity in HU conditions involved both PI3-AKT and MAPK pathways. Moreover, electrostimulation during HU restored PI3K-AKT activation without counteracting soleus atrophy, suggesting the involvement of other signaling pathways. Finally, electrostimulation maintained initial contractile and metabolism properties in parallel to ERK activation, reinforcing the idea of a

  15. Dysregulation of mammalian target of rapamycin pathway in upper tract urothelial carcinoma.

    PubMed

    Munari, Enrico; Fujita, Kazutoshi; Faraj, Sheila; Chaux, Alcides; Gonzalez-Roibon, Nilda; Hicks, Jessica; Meeker, Alan; Nonomura, Norio; Netto, George J

    2013-12-01

    Upper tract urothelial carcinoma (UTUC) accounts for 5% to 10% of all urothelial carcinomas. Despite many shared features, key clinical and molecular genetic differences between upper tract and bladder urothelial carcinomas are becoming apparent. We have previously demonstrated alterations of mammalian target of rapamycin (mTOR) pathway in bladder carcinoma with a potential impact on biological behavior. In the current study, we evaluated the expression status and prognostic significance of mTOR pathway members in UTUC. Archival formalin-fixed and paraffin-embedded tissues from 99 primary UTUCs were retrieved from one of the authors' institution. Tissue microarrays were constructed with triplicate tumor samples and paired nonneoplastic urothelium. Tissue microarrays were analyzed using immunohistochemistry for mTOR pathway members: PTEN, phos-AKT, phos-mTOR, phos-S6, phos-4EBP1, and related markers p27 and c-MYC; correlation with clinicopathologic parameters and outcome was performed. We found significantly lower expression of PTEN, phos-AKT, phos-mTOR, phos-S6, phos-4EBP1, p27, and c-MYC in UTUC compared with paired benign urothelium (P < .0005). We found a strong positive correlation between PTEN and phos-AKT. Moderate correlation was observed between phos-mTOR and phos-S6, PTEN and p27, phos-AKT and p27, phos-S6 and p27, phos-mTOR and c-MYC, phos-S6 and c-MYC, and p27 and c-MYC. None of the evaluated biomarkers were associated with increased hazard ratios for tumor recurrence or for cancer-specific mortality, when adjusting for relevant clinicopathologic variables. Dysregulation of the mTOR pathway was observed in UTUC compared with normal urothelium, implicating a potential pathogenic role in tumor development. In our cohort, expression of the evaluated biomarkers had no prognostic value.

  16. Mammalian circadian signaling networks and therapeutic targets.

    PubMed

    Liu, Andrew C; Lewis, Warren G; Kay, Steve A

    2007-10-01

    Virtually all cells in the body have an intracellular clockwork based on a negative feedback mechanism. The circadian timekeeping system in mammals is a hierarchical multi-oscillator network, with the suprachiasmatic nuclei (SCN) acting as the central pacemaker. The SCN synchronizes to daily light-dark cycles and coordinates rhythmic physiology and behavior. Synchronization in the SCN and at the organismal level is a key feature of the circadian clock system. In particular, intercellular coupling in the SCN synchronizes neuron oscillators and confers robustness against perturbations. Recent advances in our knowledge of and ability to manipulate circadian rhythms make available cell-based clock models, which lack strong coupling and are ideal for target discovery and chemical biology.

  17. Susceptibility of PTEN-positive metastatic tumors to small interfering RNA targeting the mammalian target of rapamycin.

    PubMed

    Koide, Hiroyuki; Asai, Tomohiro; Kato, Hiroki; Yonenaga, Norihito; Yokota, Masafumi; Ando, Hidenori; Dewa, Takehisa; Nango, Mamoru; Maeda, Noriyuki; Oku, Naoto

    2015-01-01

    PTEN-positive tumors are not susceptible to the treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR). Here, we determined the susceptibility of PTEN-positive cells to small interfering RNA for mTOR (si-mTOR) by using a novel liposomal delivery system. We prepared dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL) decorated with polyethylene glycol (PEG) grafting Ala-Pro-Arg-Pro-Gly (APRPG), a VRGFR-1-targeting peptide. APRPG-PEG-decorated TEPA-PCL carrying si-mTOR (APRPG-TEPA-PCL/si-mTOR) had an antiproliferative effect against B16F10 murine melanoma cells (PTEN-positive) and significantly inhibited both the proliferation and tube formation of mouse 2H-11 endothelial-like cells (PTEN-positive). APRPG-TEPA-PCL/si-mTOR treatment did not induce Akt phosphorylation (Ser473) in either B16F10 or 2H-11 cells although there was strong phosphorylation of Akt in response to rapamycin treatment. Intravenous injection of APRPG-TEPA-PCL/si-mTOR significantly suppressed the tumor growth compared with rapamycin treatment in mice bearing B16F10 melanoma. These findings suggest that APRPG-TEPA-PCL/si-mTOR is useful for the treatment of PTEN-positive tumors.

  18. Dysregulation of the mammalian target of rapamycin pathway in chromophobe renal cell carcinomas.

    PubMed

    Chaux, Alcides; Albadine, Roula; Schultz, Luciana; Hicks, Jessica; Carducci, Michael A; Argani, Pedram; Allaf, Mohamad; Netto, George J

    2013-10-01

    Targeted therapy in advanced clear cell renal cell carcinomas (RCC) is now an established modality. The latter is in stark contrast to non-clear cell subtypes. We explored the translational support for the use of antagonists of the mammalian target of rapamycin (mTOR) and the vascular endothelial growth factor pathways in chromophobe RCC. The immunoexpression of PTEN, phos-AKT, phosphorylated S6 (phos-S6), 4EBP1, p27, c-MYC, and HIF-1α was evaluated in 33 patients with chromophobe RCC who were treated by partial/radical nephrectomy without adjuvant therapy. PTEN was lower in tumor than in normal kidney (P<.001), and loss of PTEN expression was found in 67% of the tumors. In tumor tissues, phos-S6 and 4EBP1 were higher than in normal kidney (P≤.005). Conversely, scores of p27 were lower in tumor than in normal kidney (P<.001). Finally, scores of phos-AKT, c-MYC, and HIF-1α were not significantly different in tumor and in normal kidney. Overall mortality and cancer-specific mortality were 9% and 0%, respectively. Multifocal tumors had higher levels of PTEN, phos-AKT, and HIF-1α (P≤.01). None of the clinicopathologic variables (age, ethnicity, gender, pT stage, tumor size, multifocality, and positive surgical margins) was associated with outcome. Similarly, none of the tested biomarkers predicted overall mortality, either in unadjusted or adjusted Cox regression models. In summary, our study provides new evidence of dysregulation of the mTOR pathway in chromophobe RCC. Immunohistochemistry for mTOR pathway and hypoxia-induced pathway members lacked prognostic significance in our cohort.

  19. Recent advances in targeted genome engineering in mammalian systems.

    PubMed

    Sun, Ning; Abil, Zhanar; Zhao, Huimin

    2012-09-01

    Targeted genome engineering enables researchers to disrupt, insert, or replace a genomic sequence precisely at a predetermined locus. One well-established technology to edit a mammalian genome is known as gene targeting, which is based on the homologous recombination (HR) mechanism. However, the low HR frequency in mammalian cells (except for mice) prevents its wide application. To address this limitation, a custom-designed nuclease is used to introduce a site-specific DNA double-strand break (DSB) on the chromosome and the subsequent repair of the DSB by the HR mechanism or the non-homologous end joining mechanism results in efficient targeted genome modifications. Engineered homing endonucleases (also called meganucleases), zinc finger nucleases, and transcription activator-like effector nucleases represent the three major classes of custom-designed nucleases that have been successfully applied in many different organisms for targeted genome engineering. This article reviews the recent developments of these genome engineering tools and highlights a few representative applications in mammalian systems. Recent advances in gene delivery strategies of these custom-designed nucleases are also briefly discussed.

  20. Targeting the PI3K/Akt pathway in prostate cancer: challenges and opportunities (review).

    PubMed

    Toren, Paul; Zoubeidi, Amina

    2014-11-01

    The PI3K/Akt pathway is an actively pursued therapeutic target in oncology. In prostate cancer, the activation of this pathway appears to be characteristic of many aggressive prostate cancers. Further, activation of the PI3K/Akt pathway is more frequently observed as prostate cancer progresses toward a resistant, metastatic disease. Signalling from this pathway activates numerous survival, growth, metabolic and metastatic functions characteristic of aggressive cancer. Biomarkers of this pathway have correlated activation of this pathway to high grade disease and higher risk of disease progression. Therefore there is significant interest in developing effective strategies to target this pathway in prostate cancer. In this review, we discuss the pre-clinical and clinical data relevant to targeting of the PI3K/Akt pathway in prostate cancer. In particular, we review the rationale and relevance of co-targeting approaches against the PI3K/Akt pathway. It is anticipated that through an improved understanding of the biology of the PI3K/Akt pathway in prostate cancer, relevant biomarkers and rationale combination therapies will optimize targeting of this pathway to improve outcomes among patients with aggressive prostate cancer.

  1. miR-218 inhibits the invasion and migration of colon cancer cells by targeting the PI3K/Akt/mTOR signaling pathway.

    PubMed

    Zhang, Xiangliang; Shi, Huijuan; Tang, Hongsheng; Fang, Zhiyuan; Wang, Jiping; Cui, Shuzhong

    2015-05-01

    Colon cancer is one of the most common and lethal malignancies worldwide. Despite major advances in the treatment of colon cancer, the prognosis remains very poor. Thus, novel and effective therapies for colon cancer are urgently needed. In the present study, the expression status of miR-218 and the role of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway were investigated in colon cancer samples. Firstly, we observed that miR-218 expression was significantly reduced, while PI3K/Akt/mTOR pathway activity was enhanced. The overexpression of miR-218 suppressed the proliferation, migration and invasion of LoVo colon cancer cells, whereas the inhibition of miR-218 promoted these processes. Furthermore, the PI3K/Akt/mTOR signaling pathway was identified as a direct target of miR-218. The upregulation of miR-218 inhibited the activation of the PI3K/Akt/mTOR signaling pathway, as well as the expression of matrix metalloproteinase (MMP)9. The downregulation of miR-218 activated the PI3K/Akt/mTOR signaling pathway and promoted MMP9 expression. Taken together, our results demonstrate that miR-218 suppresses the proliferation, migration and invasion of LoVo colon cancer cells by targeting the PI3K/Akt/mTOR signaling pathway and MMP9. Our data indicate that miR-218 is a potential target in the treatment of colon cancer.

  2. Targeting the PI3K/AKT/mTOR and Raf/MEK/ERK pathways in the treatment of breast cancer.

    PubMed

    Saini, Kamal S; Loi, Sherene; de Azambuja, Evandro; Metzger-Filho, Otto; Saini, Monika Lamba; Ignatiadis, Michail; Dancey, Janet E; Piccart-Gebhart, Martine J

    2013-12-01

    Alterations of signal transduction pathways leading to uncontrolled cellular proliferation, survival, invasion, and metastases are hallmarks of the carcinogenic process. The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and the Raf/mitogen-activated and extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways are critical for normal human physiology, and also commonly dysregulated in several human cancers, including breast cancer (BC). In vitro and in vivo data suggest that the PI3K/AKT/mTOR and Raf/MEK/ERK cascades are interconnected with multiple points of convergence, cross-talk, and feedback loops. Raf/MEK/ERK and PI3K/AKT/mTOR pathway mutations may co-exist. Inhibition of one pathway can still result in the maintenance of signaling via the other (reciprocal) pathway. The existence of such "escape" mechanisms implies that dual targeting of these pathways may lead to superior efficacy and better clinical outcome in selected patients. Several clinical trials targeting one or both pathways are already underway in BC patients. The toxicity profile of this novel approach of dual pathway inhibition needs to be closely monitored, given the important physiological role of PI3K/AKT/mTOR and Raf/MEK/ERK signaling. In this article, we present a review of the current relevant pre-clinical and clinical data and discuss the rationale for dual inhibition of these pathways in the treatment of BC patients.

  3. Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

    PubMed

    Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

    2014-10-01

    Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers.

  4. Targeting nasopharyngeal carcinoma by artesunate through inhibiting Akt/mTOR and inducing oxidative stress.

    PubMed

    Li, Qin; Ni, Wei; Deng, Zhifeng; Liu, Minghe; She, Lazhi; Xie, Qiong

    2017-01-11

    Drug repurposing has become an alternative therapeutic strategy for cancer treatment given the known pharmacokinetics and toxicity. The inhibitory effects of artesunate have been reported in various cancers. In this work, we investigated the effects of artesunate in nasopharyngeal carcinoma (NPC). We demonstrate that artesunate significantly inhibits proliferation via arresting NPC cells at G2/M phase. It also induces apoptosis through caspase-dependent and mitochondria-independent pathways in multiple NPC cell lines. The combination of artesunate and cisplatin is synergistic in targeting NPC cells in in vitro cellular culture system and in vivo xenograft tumor models. Artesunate inhibits phosphorylation of essential molecules involved in Akt/mTOR pathway in NPC cells, such as Akt, mTOR, and 4EBP1, and its inhibitory effects are partially abolished by overexpression of constitutively active Akt. In addition, artesunate also induces mitochondrial dysfunction and oxidative stress via inhibiting mitochondrial respiration, increasing levels of mitochondrial superoxide and cellular reactive oxygen species (ROS), leading to decreased ATP levels. Two ROS scavengers partially abolish the inhibitory effects of artesunate in NPC cells. These data suggest that both inhibition of Akt/mTOR pathway and induction of ROS are required for the action of artesunate in NPC cells. Our work demonstrates that artesunate is a potential candidate for NPC treatment. Our work also highlights the critical roles of Akt/mTOR pathway and mitochondrial function in NPC cells.

  5. Efficacy of targeted AKT inhibition in genetically engineered mouse models of PTEN-deficient prostate cancer.

    PubMed

    De Velasco, Marco A; Kura, Yurie; Yoshikawa, Kazuhiro; Nishio, Kazuto; Davies, Barry R; Uemura, Hirotsugu

    2016-03-29

    The PI3K/AKT pathway is frequently altered in advanced human prostate cancer mainly through the loss of functional PTEN, and presents as potential target for personalized therapy. Our aim was to determine the therapeutic potential of the pan-AKT inhibitor, AZD5363, in PTEN-deficient prostate cancer. Here we used a genetically engineered mouse (GEM) model of PTEN-deficient prostate cancer to evaluate the in vivo pharmacodynamic and antitumor activity of AZD5363 in castration-naïve and castration-resistant prostate cancer. An additional GEM model, based on the concomitant inactivation of PTEN and Trp53 (P53), was established as an aggressive model of advanced prostate cancer and was used to further evaluate clinically relevant endpoints after treatment with AZD5363. In vivo pharmacodynamic studies demonstrated that AZD5363 effectively inhibited downstream targets of AKT. AZD5363 monotherapy significantly reduced growth of tumors in castration-naïve and castration-resistant models of PTEN-deficient prostate cancer. More importantly, AZD5363 significantly delayed tumor growth and improved overall survival and progression-free survival in PTEN/P53 double knockout mice. Our findings demonstrate that AZD5363 is effective against GEM models of PTEN-deficient prostate cancer and provide lines of evidence to support further investigation into the development of treatment strategies targeting AKT for the treatment of PTEN-deficient prostate cancer.

  6. The Mammalian target of rapamycin inhibitors in breast cancer: current evidence and future directions.

    PubMed

    Malaguti, Paola; Vari, Sabrina; Cognetti, Francesco; Fabi, Alessandra

    2013-01-01

    Mammalian target of rapamycin (mTOR) is a crucial mediator of tumor progression and may be a promising target in a significant proportion of patients with breast cancer. More specifically, the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mTOR pathway plays a critical role in multiple cellular functions including metabolism, proliferation, growth and survival. This pathway is higly active in many types of cancer and is linked to resistance to many types of therapy. Direct blockade of the mTOR pathway is a new area in breast cancer therapy, with the potential to modulate growth factor- and estrogen-dependent and estrogen-independent pathways, which contribute to the pathogenesis and progression of tumors. Thus, inhibitors of mTOR are of interest as potential therapeutic agents for patients with breast cancer, everolimus and temsirolimus being the main representatives of this category. This review of the literature analyzes the available data emerging from trials and evaluates the efficacy and safety of mTOR inhibitors in all subtypes of breast cancer.

  7. A spectrophotometric assay for routine measurement of mammalian target of rapamycin activity in cell lysates.

    PubMed

    Dekter, Hinke E; Romijn, Fred P H T M; Temmink, Wouter P M; van Pelt, Johannes; de Fijter, Johan W; Smit, Nico P M

    2010-08-01

    The mammalian target of rapamycin (mTOR) is an important mediator in the PI3K/AKT signaling pathway. mTOR is the target of immunosuppressive drugs, such as rapamycin and everolimus, that are used in transplant patients but also for the treatment of various cancers. We have developed a method for mTOR activity measurement in cell lysates that measures the phosphorylation of p70 S6 kinase by an enzyme linked immunosorbent assay (ELISA) protocol. Using an optimized lysis composition, activity could be measured in the peripheral blood mononuclear cells (PBMCs) isolated from blood. For the PBMCs, intra- and interassay variations of 7 and 10%, respectively, were found using one lot number of the kit. With different lot numbers, the interassay variation increased up to 21%. Activity remained constant for PBMC pool samples on storage for a period of more than 7 months. Activity could also be measured in CD3+ T-cells isolated from blood. In vitro experiments revealed maximum mTOR inhibition of 30% in PBMCs and 44% in T-cells. The in vitro inhibition in PBMCs could also be demonstrated by Western blotting. The mTOR activity measurements may be used to show in vivo inhibition in renal allograft patients during everolimus treatment and to study mTOR activity in various (tumor) cell types.

  8. Novel Kinase Inhibitors Targeting the PH Domain of AKT for Preventing and Treating Cancer | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute's Medical Oncology Branch is seeking statements of capability or interest from parties interested in licensing and co-development collaborative research to further develop, evaluate, or commercialize novel kinase inhibitors targeting the PH domain of AKT.

  9. MicroRNA-184 promotes differentiation of the retinal pigment epithelium by targeting the AKT2/mTOR signaling pathway

    PubMed Central

    Jiang, Chao; Qin, Bing; Liu, Guohua; Sun, Xiantao; Shi, Houxia; Ding, Sijia; Liu, Yuan; Zhu, Meidong; Chen, Xue; Zhao, Chen

    2016-01-01

    Dedifferentiation of retinal pigment epithelium (RPE) cells is a crucial contributing factor to the pathology of retinal degenerative diseases, including age-related macular degeneration (AMD). Herein, we aim to reveal the roles of microRNAs (miRNAs) in RPE dedifferentiation and seek for potential therapeutic targets. Based on the microarray data, miR-184 was sorted out as the most up-regulated signature along with the differentiation from human induced pluripotent stem cells (hiPSC) to RPE cells, suggesting its potential promotive role in RPE differentiation. In vitro study indicated that miR-184 insufficiency suppressed RPE differentiation, typified by reduction of RPE markers, and promoted cell proliferation and migration. The role of miR-184 in maintaining regular RPE function was further proved in zebrafish studies. We also noticed that miR-184 expression was reduced in the macular RPE-choroid from a donor with RPE dysfunction compared to a healthy control. We next demonstrated that RAC-beta serine/threonine-protein kinase (AKT2) was a direct target for miR-184. MiR-184 promoted RPE differentiation via suppression of AKT2/mammalian target of rapamycin (mTOR) signaling pathway. We also found that AKT2 was up-regulated in macular RPE-choroid of the donor with RPE dysfunction and dry AMD patients. Taken together, our findings suggest that miR-184 insufficiency is involved in the pathogenesis of dry AMD. MiR-184 promotes RPE differentiation via inhibiting the AKT2/mTOR signaling pathway. MiR-184 based supplementary therapeutics and mTOR blocker, like rapamycin, are prospective options for AMD treatment. PMID:27418134

  10. Healthy CD4+ T lymphocytes are not affected by targeted therapies against the PI3K/Akt/mTOR pathway in T-cell acute lymphoblastic leukemia

    PubMed Central

    Martelli, Alberto M.; Zauli, Giorgio; Ultimo, Simona; McCubrey, James A.; Gonelli, Arianna; Marisi, Giorgia; Ulivi, Paola; Capitani, Silvano; Neri, Luca M.

    2016-01-01

    An attractive molecular target for novel anti-cancer therapies is the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway which is commonly deregulated in many types of cancer. Nevertheless, the effects of PI3K/Akt/mTOR inhibitors on T lymphocytes, a key component of immune responses, have been seldom explored. In this study we investigated the effects on human CD4+ T-cells of a panel of PI3K/Akt/mTOR inhibitors: BGT226, Torin-2, MK-2206, and ZSTK474. We also assessed their efficacy against two acute leukemia T cell lines. T lymphocytes were stimulated with phytohemagglutinin. Inhibitor effects on cell cycle and apoptosis were analyzed by flow cytometry, while cytotoxicity was assessed by MTT assays. In addition, the activation status of the pathway as well as induction of autophagy were analyzed by Western blotting. Quiescent healthy T lymphocytes were unaffected by the drugs whereas mitogen-stimulated lymphocytes as well as leukemic cell lines displayed a cell cycle block, caspase-dependent apoptosis, and dephosphorylation of key components of the signaling pathway. Autophagy was also induced in proliferating lymphocytes and in JURKAT and MOLT-4 cell lines. When autophagy was inhibited by 3-methyladenine or Bafilomycin A1, drug cytotoxicity was increased, indicating that autophagy is a protective mechanism. Therefore, our findings suggest that PI3K/Akt/mTOR inhibitors preserve lymphocyte viability. This is a valuable result to be taken into account when selecting drugs for targeted cancer therapy in order to minimize detrimental effects on immune function. PMID:27494886

  11. Potential Molecular Targeted Therapeutics: Role of PI3-K/Akt/mTOR Inhibition in Cancer.

    PubMed

    Sokolowski, Kevin M; Koprowski, Steven; Kunnimalaiyaan, Selvi; Balamurugan, Mariappan; Gamblin, T Clark; Kunnimalaiyaan, Muthusamy

    2016-01-01

    Primary liver cancer is one of the most commonly occurring cancers worldwide. Hepatocellular carcinoma (HCC) represents the majority of primary liver cancer and is the 3rd most common cause of cancer-related deaths globally. Survival rates of patients with HCC are dependent upon early detection as concomitant liver dysfunction and advanced disease limits traditional therapeutic options such as resection or ablation. Unfortunately, at the time of diagnosis, most patients are not eligible for curative surgery and have a five-year relative survival rate less than 20%, leading to systemic therapy as the only option. Currently, sorafenib is the only approved systemic therapy; however, it has a limited survival advantage and low efficacy prompting alternative strategies. The inception of sorafenib for HCC systemic therapy and the understanding involved of cancer therapy have led to an enhanced focus of the PI3-k/Akt/mTOR pathway as a potential area of targeting including pan and isoform-specific PI3-K inhibitors, Akt blockade, and mTOR suppression. The multitude, expanding roles, and varying clinical trials of these inhibitors have led to an increase in knowledge and availability for current and future studies. In this review, we provide a review of the literature with the aim to focus on potential targets for HCC therapies as well as an in depth focus on Akt inhibition.

  12. The PI3K/AKT Pathway as a Target for Cancer Treatment.

    PubMed

    Mayer, Ingrid A; Arteaga, Carlos L

    2016-01-01

    Anticancer targeted therapies are designed to exploit a particular vulnerability in the tumor, which in most cases results from its dependence on an oncogene and/or loss of a tumor suppressor. Genes in the phosphoinositide 3-kinase (PI3K)/AKT pathway are the most frequently altered in human cancers. Aberrant activation of this pathway, as a result of these somatic alterations, is associated with cellular transformation, tumorigenesis, cancer progression, and drug resistance. Several drugs targeting PI3K/ATK are currently in clinical trials, alone or in combination, in both solid tumors and hematologic malignancies. These drugs are the focus of this review.

  13. Mammalian target of rapamycin signaling in diabetic cardiovascular disease.

    PubMed

    Chong, Zhao Zhong; Maiese, Kenneth

    2012-07-16

    Diabetes mellitus currently affects more than 170 million individuals worldwide and is expected to afflict another 200 million individuals in the next 30 years. Complications of diabetes as a result of oxidant stress affect multiple systems throughout the body, but involvement of the cardiovascular system may be one of the most severe in light of the impact upon cardiac and vascular function that can result in rapid morbidity and mortality for individuals. Given these concerns, the signaling pathways of the mammalian target of rapamycin (mTOR) offer exciting prospects for the development of novel therapies for the cardiovascular complications of diabetes. In the cardiovascular and metabolic systems, mTOR and its multi-protein complexes of TORC1 and TORC2 regulate insulin release and signaling, endothelial cell survival and growth, cardiomyocyte proliferation, resistance to β-cell injury, and cell longevity. Yet, mTOR can, at times, alter insulin signaling and lead to insulin resistance in the cardiovascular system during diabetes mellitus. It is therefore vital to understand the complex relationship mTOR and its downstream pathways hold during metabolic disease in order to develop novel strategies for the complications of diabetes mellitus in the cardiovascular system.

  14. Mammalian target of rapamycin inhibition in hepatocellular carcinoma

    PubMed Central

    Ashworth, René E; Wu, Jennifer

    2014-01-01

    Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. It is associated with a poor prognosis and has limited treatment options. Sorafenib, a multi-targeted kinase inhibitor, is the only available systemic agent for treatment of HCC that improves overall survival for patients with advanced stage disease; unfortunately, an effective second-line agent for the treatment of progressive or sorafenib-resistant HCC has yet to be identified. This review focuses on components of the mammalian target of rapamycin (mTOR) pathway, its role in HCC pathogenesis, and dual mTOR inhibition as a therapeutic option with potential efficacy in advanced HCC. There are several important upstream and downstream signals in the mTOR pathway, and alternative tumor-promoting pathways are known to exist beyond mTORC1 inhibition in HCC. This review analyzes the relationships of the upstream and downstream regulators of mTORC1 and mTORC2 signaling; it also provides a comprehensive global picture of the interaction between mTORC1 and mTORC2 which demonstrates the pre-clinical relevance of the mTOR pathway in HCC pathogenesis and progression. Finally, it provides scientific rationale for dual mTORC1 and mTORC2 inhibition in the treatment of HCC. Clinical trials utilizing mTORC1 inhibitors and dual mTOR inhibitors in HCC are discussed as well. The mTOR pathway is comprised of two main components, mTORC1 and mTORC2; each has a unique role in the pathogenesis and progression of HCC. In phase III studies, mTORC1 inhibitors demonstrate anti-tumor activity in advanced HCC, but dual mTOR (mTORC1 and mTORC2) inhibition has greater therapeutic potential in HCC treatment which warrants further clinical investigation. PMID:25429315

  15. Mammalian target of rapamycin (mTor) mediates tau protein dyshomeostasis: implication for Alzheimer disease.

    PubMed

    Tang, Zhi; Bereczki, Erika; Zhang, Haiyan; Wang, Shan; Li, Chunxia; Ji, Xinying; Branca, Rui M; Lehtiö, Janne; Guan, Zhizhong; Filipcik, Peter; Xu, Shaohua; Winblad, Bengt; Pei, Jin-Jing

    2013-05-31

    Previous evidence from post-mortem Alzheimer disease (AD) brains and drug (especially rapamycin)-oriented in vitro and in vivo models implicated an aberrant accumulation of the mammalian target of rapamycin (mTor) in tangle-bearing neurons in AD brains and its role in the formation of abnormally hyperphosphorylated tau. Compelling evidence indicated that the sequential molecular events such as the synthesis and phosphorylation of tau can be regulated through p70 S6 kinase, the well characterized immediate downstream target of mTor. In the present study, we further identified that the active form of mTor per se accumulates in tangle-bearing neurons, particularly those at early stages in AD brains. By using mass spectrometry and Western blotting, we identified three phosphoepitopes of tau directly phosphorylated by mTor. We have developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background. In these cellular systems, we not only confirmed the tau phosphorylation sites found in vitro but also found that mTor mediates the synthesis and aggregation of tau, resulting in compromised microtubule stability. Changes of mTor activity cause fluctuation of the level of a battery of tau kinases such as protein kinase A, v-Akt murine thymoma viral oncogene homolog-1, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and tau protein phosphatase 2A. These results implicate mTor in promoting an imbalance of tau homeostasis, a condition required for neurons to maintain physiological function.

  16. Akt targeting as a strategy to boost chemotherapy efficacy in non-small cell lung cancer through metabolism suppression

    PubMed Central

    Le Grand, Marion; Berges, Raphael; Pasquier, Eddy; Montero, Marie-Pierre; Borge, Laurence; Carrier, Alice; Vasseur, Sophie; Bourgarel, Veronique; Buric, Duje; André, Nicolas; Braguer, Diane; Carré, Manon

    2017-01-01

    Metabolic reprogramming is a hallmark of cancer development, mediated by genetic and epigenetic alterations that may be pharmacologically targeted. Among oncogenes, the kinase Akt is commonly overexpressed in tumors and favors glycolysis, providing a rationale for using Akt inhibitors. Here, we addressed the question of whether and how inhibiting Akt activity could improve therapy of non-small cell lung cancer (NSCLC) that represents more than 80% of all lung cancer cases. First, we demonstrated that Akt inhibitors interacted synergistically with Microtubule-Targeting Agents (MTAs) and specifically in cancer cell lines, including those resistant to chemotherapy agents and anti-EGFR targeted therapies. In vivo, we further revealed that the chronic administration of low-doses of paclitaxel - i.e. metronomic scheduling - and the anti-Akt perifosine was the most efficient and the best tolerated treatment against NSCLC. Regarding drug mechanism of action, perifosine potentiated the pro-apoptotic effects of paclitaxel, independently of cell cycle arrest, and combining paclitaxel/perifosine resulted in a sustained suppression of glycolytic and mitochondrial metabolism. This study points out that targeting cancer cell bioenergetics may represent a novel therapeutic avenue in NSCLC, and provides a strong foundation for future clinical trials of metronomic MTAs combined with Akt inhibitors. PMID:28332584

  17. MiR-194 Deregulation Contributes To Colorectal Carcinogenesis via Targeting AKT2 Pathway

    PubMed Central

    Zhao, Hui-Jun; Ren, Lin-Lin; Wang, Zhen-Hua; Sun, Tian-Tian; Yu, Ya-Nan; Wang, Ying-Chao; Yan, Ting-Ting; Zou, Weiping; He, Jie; Zhang, Yaou; Hong, Jie; Fang, Jing-Yuan

    2014-01-01

    Abstract: Recent studies have increasingly linked microRNAs to colorectal cancer (CRC). MiR-194 has been reported deregulated in different tumor types, whereas the function of miR-194 in CRC largely remains unexplored. Here we investigated the biological effects, mechanisms and clinical significance of miR-194. Functional assay revealed that overexpression of miR-194 inhibited CRC cell viability and invasion in vitro and suppressed CRC xenograft tumor growth in vivo. Conversely, block of miR-194 in APCMin/+ mice promoted tumor growth. Furthermore, miR-194 reduced the expression of AKT2 both in vitro and in vivo. Clinically, the expression of miR-194 gradually decreased from 20 normal colorectal mucosa (N-N) cases through 40 colorectal adenomas (CRA) cases and then to 40 CRC cases, and was negatively correlated with AKT2 and pAKT2 expression. Furthermore, expression of miR-194 in stool samples was gradually decreased from 20 healthy cases, 20 CRA cases, then to 28 CRC cases. Low expression of miR-194 in CRC tissues was associated with large tumor size (P=0.006), lymph node metastasis (P=0.012) and shorter survival (HR =2.349, 95% CI = 1.242 to 4.442; P=0.009). In conclusion, our data indicated that miR-194 acted as a tumor suppressor in the colorectal carcinogenesis via targeting PDK1/AKT2/XIAP pathway, and could be a significant diagnostic and prognostic biomarker for CRC. PMID:25285168

  18. MicroRNA-145 suppresses hepatocellular carcinoma by targeting IRS1 and its downstream Akt signaling

    SciTech Connect

    Wang, Yelin; Hu, Chen; Cheng, Jun; Chen, Binquan; Ke, Qinghong; Lv, Zhen; Wu, Jian; Zhou, Yanfeng

    2014-04-18

    Highlights: • MiR-145 expression is down-regulated in HCC tissues and inversely related with IRS1 levels. • MiR-145 directly targets IRS1 in HCC cells. • Restored expression of miR-145 suppressed HCC cell proliferation and growth. • MiR-145 induced IRS1 under-expression potentially reduced downstream AKT signaling. - Abstract: Accumulating evidences have proved that dysregulation of microRNAs (miRNAs) is involved in cancer initiation and progression. In this study, we showed that miRNA-145 level was significantly decreased in hepatocellular cancer (HCC) tissues and cell lines, and its low expression was inversely associated with the abundance of insulin receptor substrate 1 (IRS1), a key mediator in oncogenic insulin-like growth factor (IGF) signaling. We verified IRS1 as a direct target of miR-145 using Western blotting and luciferase reporter assay. Further, the restoration of miR-145 in HCC cell lines suppressed cancer cell growth, owing to down-regulated IRS1 expression and its downstream Akt/FOXO1 signaling. Our results demonstrated that miR-145 could inhibit HCC through targeting IRS1 and its downstream signaling, implicating the loss of miR-145 regulation may be a potential molecular mechanism causing aberrant oncogenic signaling in HCC.

  19. Immunoexpression status and prognostic value of mammalian target of rapamycin and hypoxia-induced pathway members in papillary cell renal cell carcinomas.

    PubMed

    Chaux, Alcides; Schultz, Luciana; Albadine, Roula; Hicks, Jessica; Kim, Jenny J; Allaf, Mohamad E; Carducci, Michael A; Rodriguez, Ronald; Hammers, Hans-Joerg; Argani, Pedram; Reuter, Victor E; Netto, George J

    2012-12-01

    Dysregulation of the mammalian target of rapamycin and hypoxia-induced pathways has been consistently identified in clear cell renal cell carcinomas. However, experience with non-clear cell renal cell carcinoma subtypes is scant. In this study, we evaluated the immunohistochemical expression of upstream (PTEN and phosphorylated AKT) and downstream (phosphorylated S6 and 4EBP1) effectors of the mammalian target of rapamycin pathway, as well as related cell-cycle proteins (p27 and c-MYC), and a member of the hypoxia-induced pathway (HIF-1α) in 54 patients with papillary renal cell carcinoma treated by nephrectomy. PTEN was lower in tumor than in normal kidney, and loss of PTEN expression was found in 48% of the patients. In tumor tissues, phosphorylated S6, 4EBP1, and HIF-1α were higher than in normal kidney. Conversely, scores of p27 were lower in tumor than in normal kidney. Finally, scores of c-MYC and phosphorylated AKT were similar in tumor and in normal kidney. Overall mortality and cancer-specific mortality were 24% and 11%, respectively. Tumor progression was observed in 17% of the patients. None of the tested biomarkers predicted cancer-specific mortality or tumor progression. As expected, patients with high T-stage tumors had higher hazard ratios for cancer-specific mortality (hazard ratio, 6.9) and tumor progression (hazard ratio, 6.7). Patients with higher Fuhrman grades also had higher risks for cancer-specific mortality (hazard ratio, 11.4) and tumor progression (hazard ratio, 4.5). In summary, our study provides evidence of dysregulation of the mammalian target of rapamycin and hypoxia-induced pathways in papillary renal cell carcinoma. Immunohistochemistry for members of the mammalian target of rapamycin pathway and for HIF-1α lacked prognostic significance in our cohort.

  20. P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration.

    PubMed

    Hernández-Negrete, Ivette; Carretero-Ortega, Jorge; Rosenfeldt, Hans; Hernández-García, Ricardo; Calderón-Salinas, J Victor; Reyes-Cruz, Guadalupe; Gutkind, J Silvio; Vázquez-Prado, José

    2007-08-10

    Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gbetagamma and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration.

  1. Pleckstrin Homology Domain of Akt Kinase: A Proof of Principle for Highly Specific and Effective Non-Enzymatic Anti-Cancer Target

    PubMed Central

    Joh, Eun-Ha; Hollenbaugh, Joseph A.; Kim, Baek; Kim, Dong-Hyun

    2012-01-01

    While pharmacological inhibition of Akt kinase has been regarded as a promising anti-cancer strategy, most of the Akt inhibitors that have been developed are enzymatic inhibitors that target the kinase active site of Akt. Another key cellular regulatory event for Akt activation is the translocation of Akt kinase to the cell membrane from the cytoplasm, which is accomplished through the pleckstrin homology (PH) domain of Akt. However, compounds specifically interacting with the PH domain of Akt to inhibit Akt activation are currently limited. Here we identified a compound, lancemaside A (LAN-A), which specifically binds to the PH domain of Akt kinase. First, our mass spectra analysis of cellular Akt kinase isolated from cells treated with LAN-A revealed that LAN-A specifically binds to the PH domain of cellular Akt kinase. Second, we observed that LAN-A inhibits the translocation of Akt kinase to the membrane and thus Akt activation, as examined by the phosphorylation of various downstream targets of Akt such as GSK3β, mTOR and BAD. Third, in a co-cultured cell model containing human lung epithelial cancer cells (A549) and normal human primary lung fibroblasts, LAN-A specifically restricts the growth of the A549 cells. LAN-A also displayed anti-proliferative effects on various human cancer cell lines. Finally, in the A549-luciferase mouse transplant model, LAN-A effectively inhibited A549 cell growth with little evident cytotoxicity. Indeed, the therapeutic index of LAN-A in this mouse model was >250, supporting that LAN-A is a potential lead compound for PH domain targeting as a safe anti-cancer Akt inhibitor. PMID:23189201

  2. Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

    SciTech Connect

    Zhang, Pengpeng; Shan, Tizhong; Liang, Xinrong; Deng, Changyan; Kuang, Shihuan

    2014-09-12

    Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor{sup flox/flox} mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor{sup flox/flox} mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.

  3. Role of Calcineurin, hnRNPA2 and Akt in Mitochondrial Respiratory Stress-Mediated Transcription Activation of Nuclear Gene Targets

    PubMed Central

    Guha, Manti; Tang, Weigang; Sondheimer, Neal; Avadhani, Narayan G.

    2010-01-01

    Pathophysiological conditions causing mitochondrial dysfunction and altered transmembrane potential (Δψm) initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a variety of mammalian cells. An increase in the cytosolic Ca2+ [Ca2+]c as part of this signaling cascade activates Ca2+ responsive phosphatase, Calcineurin (Cn). Activation of IGF1R accompanied by increased glycolysis, invasiveness, and resistance to apoptosis are phenotypic hallmarks of C2C12 rhabdomyoblast cells subjected to this stress. The signaling is associated with activation and increased nuclear translocation of a number of transcription factors including a novel NFκB (cRel: p50) pathway, NFAT, CREB and C/EBPδ. This culminates in the upregulation of a number of nuclear genes including Cathepsin L, RyR1, Glut4 and Akt1. We observed that stress regulated transcription activation of nuclear genes involves a cooperative interplay between NFκB (cRel:p50), C/EBPδ, CREB, NFAT. Our results show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein, hnRNPA2 as a transcriptional co-activator. We report here that mitochondrial stress leads to induced expression and activation of serine threonine kinase Akt1. Interestingly, we observe that Akt1 phosphorylates hnRNPA2 under mitochondrial stress conditions, which is a crucial step for the recruitment of this coactivator to the stress target promoters and culmination in mitochondrial stress-mediated transcription activation of target genes. We propose that mitochondrial stress plays an important role in tumor progression and emergence of invasive phenotypes. PMID:20153290

  4. Molecular neuro-oncology and development of targeted therapeutic strategies for brain tumors. Part 2: PI3K/Akt/PTEN, mTOR, SHH/PTCH and angiogenesis.

    PubMed

    Newton, Herbert B

    2004-02-01

    Brain tumors are a diverse group of malignancies that remain refractory to conventional treatment approaches. Molecular neuro-oncology has now begun to clarify the transformed phenotype of brain tumors and identify oncogenic pathways that might be amenable to targeted therapy. Activity of the phosphoinositide 3; kinase (PI3K)/Akt pathway is often upregulated in brain tumors due to excessive stimulation by growth factor receptors and Ras. Loss of function of the tumor suppressor gene PTEN also frequently contributes to upregulation of PI3K/Akt. Several compounds, such as wortmannin and LY-294002, can target PI3K and inhibit activity of this pathway. The mammalian target of rapamycin (mTOR) is an important regulator of cell growth and metabolism and is often upregulated by Akt. Clinical trials of CCI-779, an inhibitor of mTOR, are ongoing in recurrent malignant glioma patients. The sonic hedgehog/PTCH pathway is involved in the tumorigenesis of some familial and sporadic medulloblastomas. This pathway can be targeted by cyclopamine, which is under evaluation in preclinical studies. Angiogenesis is a critical process for development and progression of brain tumors. Targeted approaches to inhibit angiogenesis include monoclonal antibodies, receptor tyrosine kinase inhibitors, antisense oligonucleotides and gene therapy. Clinical trials are ongoing for numerous angiogenesis inhibitors, including thalidomide, CC-5103 and PTK 787/ZK 222584. Further development of targeted therapies and evaluation of these new agents in clinical trials will be needed to improve survival and quality of life of patients with brain tumors.

  5. Dysregulation of mammalian target of rapamycin pathway in plasmacytoid variant of urothelial carcinoma of the urinary bladder.

    PubMed

    Gonzalez-Roibon, Nilda D; Chaux, Alcides; Al-Hussain, Turki; Osunkoya, Adeboye O; Bezerra, Stephania Martins; Hicks, Jessica; Epstein, Jonathan I; Netto, George J

    2013-04-01

    Plasmacytoid urothelial carcinoma is a rare but aggressive variant of bladder cancer with no clear therapeutic guidelines. Dysregulation of the mammalian target of rapamycin (mTOR) pathway has been linked to oncogenesis in conventional bladder cancer. Several antineoplastic agents targeting mTOR pathway are currently available. This study assesses mTOR pathway status as well as c-myc and p27 expression. We retrieved 19 archival cases of plasmacytoid urothelial carcinoma from two institutions. Whole tissue sections were evaluated for immunoexpression of phosphatase and tensin homolog (PTEN), phosphorylated mTOR, phosphorylated protein kinase B (AKT), phosphorylated S6, c-myc, and p27. We evaluated intensity (0 to 3+) and extent (0%-100%) of expression for all markers. An H score was calculated as the sum of products of intensity and extent for each marker and used during analysis. In addition, PTEN loss was defined as absence of expression in >10% of tumor cells. We encountered PTEN loss in 28%. Higher H score for nuclear phosphorylated AKT and a lower H score for phosphorylated S6 was encountered in muscle invasive tumors compared to non-muscle invasive tumors (P = .007 and P = .009, respectively). Although a trend for negative prognostic impact on overall survival for higher phosphorylated mTOR expression was noted (P = .051), markers expression levels failed to predict survival in our cohort. We found dysregulation of mTOR pathway members in urinary bladder plasmacytoid urothelial carcinoma, suggesting that the use of mTOR pathway inhibitors might be beneficial for patients with this aggressive tumor.

  6. Target regulation of PI3K/Akt/mTOR pathway by cannabidiol in treatment of experimental multiple sclerosis.

    PubMed

    Giacoppo, Sabrina; Pollastro, Federica; Grassi, Gianpaolo; Bramanti, Placido; Mazzon, Emanuela

    2017-01-01

    This study was aimed to investigate whether treatment with purified cannabidiol (CBD) may counteract the development of experimental multiple sclerosis (MS), by targeting the PI3K/Akt/mTOR pathway. Although the PI3K/Akt/mTOR pathway was found to be activated by cannabinoids in several immune and non-immune cells, currently, there is no data about the effects of CBD in the PI3K/Akt/mTOR activity in MS. Experimental Autoimmune Encephalomyelitis (EAE), the most common model of MS, was induced in C57BL/6 mice by immunization with myelin oligodendroglial glycoprotein peptide (MOG)35-55. After EAE onset, which occurs approximately 14days after disease induction, mice were daily intraperitoneally treated with CBD (10mg/kg mouse) and observed for clinical signs of EAE. At 28days from EAE-induction, mice were euthanized and spinal cord tissues were sampled to perform immunohistochemical evaluations and western blot analysis. Our results showed a clear downregulation of the PI3K/Akt/mTOR pathway following EAE induction. CBD treatment was able to restore it, increasing significantly the phosphorylation of PI3K, Akt and mTOR. Also, an increased level of BNDF in CBD-treated mice seems to be involved in the activation of PI3K/Akt/mTOR pathway. In addition, our data demonstrated that therapeutic efficacy of CBD treatment is due to reduction of pro-inflammatory cytokines, like IFN-γ and IL-17 together with an up-regulation of PPARγ. Finally, CBD was found to promote neuronal survival by inhibiting JNK and p38 MAP kinases. These results provide an interesting discovery about the regulation of the PI3K/Akt/mTOR pathway by cannabidiol administration, that could be a new potential therapeutic target for MS management.

  7. CX-5461 induces autophagy and inhibits tumor growth via mammalian target of rapamycin-related signaling pathways in osteosarcoma

    PubMed Central

    Li, Leiming; Li, Yan; Zhao, Jiansong; Fan, Shuli; Wang, Liguo; Li, Xu

    2016-01-01

    Osteosarcoma (OS) is the most common primary bone tumor, but molecular mechanisms of the disease have not been well understood, and treatment of metastatic OS remains a challenge. Rapid ribosomal RNA synthesis in cancer is transcribed by RNA polymerase I, which results in unbridled cell growth. The recent discovery of CX-5461, a selective RNA polymerase I inhibitor, exerted its inhibitory effect of ribosomal RNA synthesis and antiproliferative potency. Here, we demonstrate that CX-5461 induces G2 arrest in the cell cycle and expression of microtubule-associated protein 1 light chain 3 II isoform in OS cell lines. Autophagic vacuoles could be observed in electron microscopy and 3-methyladenine prevented cell death mediated by CX-5461. Moreover, it significantly augmented phosphorylated AMP-Activated Protein Kinases α (p-AMPK α). (Thr172) expression in U2-OS cells and decreased p-Akt (Ser473) expression in MNNG cells, respectively, which repressed their downstream effector, mammalian target of rapamycin. On the other hand, CX-5461 increased p53 accumulation and messenger RNA level of its target genes, p21, MDM2, and Sestrin1/2 in U2-OS cells. Knockdown of p53 expression markedly impaired cell death as well as the expression of light chain 3-II and p21 induced by CX-5461. It also significantly enhanced doxorubicin-mediated cytotoxic effect in vitro and in vivo together with additive expression of p53, p21, and light chain 3-II in U2-OS cells. Our data indicate that CX-5461 might induce autophagy via mammalian target of rapamycin-associated signaling pathways dependent on p53 status and exert p53-dependent synergistic antitumor effect combined with doxorubicin in OS. These results suggest that CX-5461 might be promising in clinical therapy for OS, especially cases harboring wild-type p53. PMID:27729807

  8. Targeting hyperactivated DNA-PKcs by KU0060648 inhibits glioma progression and enhances temozolomide therapy via suppression of AKT signaling

    PubMed Central

    Qu, Yanming; Zhang, Mingshan; Wang, Haoran; Zhang, Zhihua; Zhou, Wei; Fan, Xinyi; Yu, Chunjiang; Zhan, Qimin; Song, Yongmei

    2016-01-01

    The overall survival remains undesirable in clinical glioma treatment. Inhibition of DNA-PKcs activity by its inhibitors suppresses tumor growth and enhances chemosensitivity of several tumors to chemotherapy. However, whether DNA-PKcs could be a potential target in glioma therapy remains unknown. In this study, we reported that the hyperactivated DNA-PKcs was profoundly correlated with glioma malignancy and observe a significant association between DNA-PKcs activation and survival of the glioma patients. Our data also found that inhibition of DNA-PKcs by its inhibitor KU0060648 sensitized glioma cells to TMZ in vitro. Specifically, we demonstrated that KU0060648 interrupted the formation of DNA-PKcs/AKT complex, leading to suppression of AKT signaling and resultantly enhanced TMZ efficacy. Combination of KU0060648 and TMZ substantially inhibited downstream effectors of AKT. The in vivo results were similar to those obtained in vitro. In conclusion, this study indicated that inhibition of DNA-PKcs activity could suppress glioma malignancies and increase TMZ efficacy, which was mainly through regulation of the of AKT signaling. Therefore, DNA-PKcs/AKT axis may be a promising target for improving current glioma therapy. PMID:27487130

  9. OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells

    PubMed Central

    Liu, Ta-Ming; Ling, Yonghua; Woyach, Jennifer A.; Beckwith, Kyle; Yeh, Yuh-Ying; Hertlein, Erin; Zhang, Xiaoli; Lehman, Amy; Awan, Farrukh; Jones, Jeffrey A.; Andritsos, Leslie A.; Maddocks, Kami; MacMurray, Jessica; Salunke, Santosh B.; Chen, Ching-Shih; Phelps, Mitch A.; Byrd, John C.

    2015-01-01

    Aberrant regulation of endogenous survival pathways plays a major role in progression of chronic lymphocytic leukemia (CLL). Signaling via conjugation of surface receptors within the tumor environmental niche activates survival and proliferation pathways in CLL. Of these, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway appears to be pivotal to support CLL pathogenesis, and pharmacologic inhibitors targeting this axis have shown clinical activity. Here we investigate OSU-T315, a compound that disrupts the PI3K/AKT pathway in a novel manner. Dose-dependent selective cytotoxicity by OSU-T315 is noted in both CLL-derived cell lines and primary CLL cells relative to normal lymphocytes. In contrast to the highly successful Bruton's tyrosine kinase and PI3K inhibitors that inhibit B-cell receptor (BCR) signaling pathway at proximal kinases, OSU-T315 directly abrogates AKT activation by preventing translocation of AKT into lipid rafts without altering the activation of receptor-associated kinases. Through this mechanism, the agent triggers caspase-dependent apoptosis in CLL by suppressing BCR, CD49d, CD40, and Toll-like receptor 9-mediated AKT activation in an integrin-linked kinase-independent manner. In vivo, OSU-T315 attains pharmacologically active drug levels and significantly prolongs survival in the TCL1 mouse model. Together, our findings indicate a novel mechanism of action of OSU-T315 with potential therapeutic application in CLL. PMID:25293770

  10. OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells.

    PubMed

    Liu, Ta-Ming; Ling, Yonghua; Woyach, Jennifer A; Beckwith, Kyle; Yeh, Yuh-Ying; Hertlein, Erin; Zhang, Xiaoli; Lehman, Amy; Awan, Farrukh; Jones, Jeffrey A; Andritsos, Leslie A; Maddocks, Kami; MacMurray, Jessica; Salunke, Santosh B; Chen, Ching-Shih; Phelps, Mitch A; Byrd, John C; Johnson, Amy J

    2015-01-08

    Aberrant regulation of endogenous survival pathways plays a major role in progression of chronic lymphocytic leukemia (CLL). Signaling via conjugation of surface receptors within the tumor environmental niche activates survival and proliferation pathways in CLL. Of these, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway appears to be pivotal to support CLL pathogenesis, and pharmacologic inhibitors targeting this axis have shown clinical activity. Here we investigate OSU-T315, a compound that disrupts the PI3K/AKT pathway in a novel manner. Dose-dependent selective cytotoxicity by OSU-T315 is noted in both CLL-derived cell lines and primary CLL cells relative to normal lymphocytes. In contrast to the highly successful Bruton's tyrosine kinase and PI3K inhibitors that inhibit B-cell receptor (BCR) signaling pathway at proximal kinases, OSU-T315 directly abrogates AKT activation by preventing translocation of AKT into lipid rafts without altering the activation of receptor-associated kinases. Through this mechanism, the agent triggers caspase-dependent apoptosis in CLL by suppressing BCR, CD49d, CD40, and Toll-like receptor 9-mediated AKT activation in an integrin-linked kinase-independent manner. In vivo, OSU-T315 attains pharmacologically active drug levels and significantly prolongs survival in the TCL1 mouse model. Together, our findings indicate a novel mechanism of action of OSU-T315 with potential therapeutic application in CLL.

  11. FAM83B-mediated activation of PI3K/AKT and MAPK signaling cooperates to promote epithelial cell transformation and resistance to targeted therapies

    PubMed Central

    Cipriano, Rocky; Miskimen, Kristy L.S.; Bryson, Benjamin L.; Foy, Chase R.; Bartel, Courtney A.; Jackson, Mark W.

    2013-01-01

    Therapies targeting MAPK and AKT/mTOR signaling are currently being evaluated in clinical trials for several tumor types. However, recent studies suggest that these therapies may be limited due to acquired cancer cell resistance and a small therapeutic index between normal and cancer cells. The identification of novel proteins that are involved in MAPK or AKT/mTOR signaling and differentially expressed between normal and cancer cells will provide mechanistically distinct therapeutic targets with the potential to inhibit these key cancer-associated pathways. We recently identified FAM83B as a novel, previously uncharacterized oncogene capable of hyperactivating MAPK and mTOR signaling and driving the tumorigenicity of immortalized human mammary epithelial cells (HMEC). We show here that elevated FAM83B expression also activates the PI3K/AKT signaling pathway and confers a decreased sensitivity to PI3K, AKT, and mTOR inhibitors. FAM83B co-precipitated with the p85α and p110α subunits of PI3K, as well as AKT, and increased p110α and AKT membrane localization, consistent with elevated PI3K/AKT signaling. In tumor-derived cells harboring elevated FAM83B expression, ablation of FAM83B decreased p110α and AKT membrane localization, suppressed AKT phosphorylation, and diminished proliferation, AIG, and tumorigenicity in vivo. We propose that the level of FAM83B expression may be an important factor to consider when combined therapies targeting MAPK and AKT/mTOR signaling are used. Moreover, the identification of FAM83B as a novel oncogene and its integral involvement in activating PI3K/AKT and MAPK provides a foundation for future therapies aimed at targeting FAM83B in order to suppress the growth of PI3K/AKT- and MAPK-driven cancers. PMID:23676467

  12. Computer-Aided Targeting of the PI3K/Akt/mTOR Pathway: Toxicity Reduction and Therapeutic Opportunities

    PubMed Central

    Li, Tan; Wang, Guanyu

    2014-01-01

    The PI3K/Akt/mTOR pathway plays an essential role in a wide range of biological functions, including metabolism, macromolecular synthesis, cell growth, proliferation and survival. Its versatility, however, makes it a conspicuous target of many pathogens; and the consequential deregulations of this pathway often lead to complications, such as tumorigenesis, type 2 diabetes and cardiovascular diseases. Molecular targeted therapy, aimed at modulating the deregulated pathway, holds great promise for controlling these diseases, though side effects may be inevitable, given the ubiquity of the pathway in cell functions. Here, we review a variety of factors found to modulate the PI3K/Akt/mTOR pathway, including gene mutations, certain metabolites, inflammatory factors, chemical toxicants, drugs found to rectify the pathway, as well as viruses that hijack the pathway for their own synthetic purposes. Furthermore, this evidence of PI3K/Akt/mTOR pathway alteration and related pathogenesis has inspired the exploration of computer-aided targeting of this pathway to optimize therapeutic strategies. Herein, we discuss several possible options, using computer-aided targeting, to reduce the toxicity of molecularly-targeted therapy, including mathematical modeling, to reveal system-level control mechanisms and to confer a low-dosage combination therapy, the potential of PP2A as a therapeutic target, the formulation of parameters to identify patients who would most benefit from specific targeted therapies and molecular dynamics simulations and docking studies to discover drugs that are isoform specific or mutation selective so as to avoid undesired broad inhibitions. We hope this review will stimulate novel ideas for pharmaceutical discovery and deepen our understanding of curability and toxicity by targeting the PI3K/Akt/mTOR pathway. PMID:25334061

  13. Akt isoform specific effects in ovarian cancer progression

    PubMed Central

    Linnerth-Petrik, Nicolle M.; Santry, Lisa A.; Moorehead, Roger; Jücker, Manfred

    2016-01-01

    Ovarian cancer remains a significant therapeutic problem and novel, effective therapies are needed. Akt is a serine-threonine kinase that is overexpressed in numerous cancers, including ovarian. Mammalian cells express three Akt isoforms which are encoded by distinct genes. Although there are several Akt inhibitors in clinical trials, most indiscriminately target all isoforms. Current in vitro data and animal knockout experiments suggest that the Akt isoforms may have divergent roles. In this paper, we determined the isoform-specific functions of Akt in ovarian cancer cell proliferation in vitro and in ovarian cancer progression in vivo. For in vitro experiments, murine and human ovarian cancer cells were treated with Akt inhibitors and cell viability was assessed. We used two different in vivo approaches to identify the roles of Akt isoforms in ovarian cancer progression and their influence on the primary tumor and tumor microenvironment. In one experiment, wild-type C57Bl6 mice were orthotopically injected with ID8 cells with stable knockdown of Akt isoforms. In a separate experiment, mice null for Akt 1-3 were orthotopically injected with WT ID8 cells (Figure 1). Our data show that inhibition of Akt1 significantly reduced ovarian cancer cell proliferation and inhibited tumor progression in vivo. Conversely, disruption of Akt2 increased tumor growth. Inhibition of Akt3 had an intermediate phenotype, but also increased growth of ovarian cancer cells. These data suggest that there is minimal redundancy between the Akt isoforms in ovarian cancer progression. These findings have important implications in the design of Akt inhibitors for the effective treatment of ovarian cancer. PMID:27533079

  14. Targeting hyperactivation of the AKT survival pathway to overcome therapy resistance of melanoma brain metastases.

    PubMed

    Niessner, Heike; Forschner, Andrea; Klumpp, Bernhard; Honegger, Jürgen B; Witte, Maria; Bornemann, Antje; Dummer, Reinhard; Adam, Annemarie; Bauer, Jürgen; Tabatabai, Ghazaleh; Flaherty, Keith; Sinnberg, Tobias; Beck, Daniela; Leiter, Ulrike; Mauch, Cornelia; Roesch, Alexander; Weide, Benjamin; Eigentler, Thomas; Schadendorf, Dirk; Garbe, Claus; Kulms, Dagmar; Quintanilla-Martinez, Leticia; Meier, Friedegund

    2013-02-01

    Brain metastases are the most common cause of death in patients with metastatic melanoma, and the RAF-MEK-ERK and PI3K-AKT signaling pathways are key players in melanoma progression and drug resistance. The BRAF inhibitor vemurafenib significantly improved overall survival. However, brain metastases still limit the effectiveness of this therapy. In a series of patients, we observed that treatment with vemurafenib resulted in substantial regression of extracerebral metastases, but brain metastases developed. This study aimed to identify factors that contribute to treatment resistance in brain metastases. Matched brain and extracerebral metastases from melanoma patients had identical ERK, p-ERK, and AKT immunohistochemistry staining patterns, but there was hyperactivation of AKT (p-AKT) and loss of PTEN expression in the brain metastases. Mutation analysis revealed no differences in BRAF, NRAS, or KIT mutation status in matched brain and extracerebral metastases. In contrast, AKT, p-AKT, and PTEN expression was identical in monolayer cultures derived from melanoma brain and extracerebral metastases. Furthermore, melanoma cells stimulated by astrocyte-conditioned medium showed higher AKT activation and invasiveness than melanoma cells stimulated by fibroblast-conditioned medium. Inhibition of PI3K-AKT signaling resensitized melanoma cells isolated from a vemurafenib-resistant brain metastasis to vemurafenib. Brain-derived factors appear to induce hyperactivation of the AKT survival pathway and to promote the survival and drug resistance of melanoma cells in the brain. Thus, inhibition of PI3K-AKT signaling shows potential for enhancing and/or prolonging the antitumor effect of BRAF inhibitors or other anticancer agents in melanoma brain metastases.

  15. MicroRNA-542-3p Suppresses Tumor Cell Invasion via Targeting AKT Pathway in Human Astrocytoma*

    PubMed Central

    Cai, Junchao; Zhao, JingJing; Zhang, Nu; Xu, Xiaonan; Li, Rong; Yi, Yang; Fang, Lishan; Zhang, Le; Li, Mengfeng; Wu, Jueheng; Zhang, Heng

    2015-01-01

    The molecular mechanism underlying constitutive activation of AKT signaling, which plays essential roles in astrocytoma progression, is not fully characterized. Increasing numbers of studies have reported that microRNAs are involved in the malignant behavior of astrocytoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we found that microRNA (miR)-542-3p expression was decreased in glioblastoma cell lines and astrocytoma tissues, and reduced levels of miR-542-3p expression correlated with high histopathological grades and poor prognosis of astrocytoma patients. Exogenous miR-542-3p suppressed glioblastoma cell invasion through not only targeting AKT1 itself but also directly down-regulating its two important upstream regulators, namely, integrin-linked kinase and PIK3R1. Notably, overexpressing miR-542-3p decreased AKT1 phosphorylation and directly and indirectly repressed nuclear translocation and transactivation activity of β-catenin to exert its anti-invasive effect. Furthermore, the miR-542-3p expression level negatively correlated with AKT activity as well as levels of integrin-linked kinase and PIK3R1 in human astrocytoma specimens. These findings suggest that miR-542-3p acts as a negative regulator in astrocytoma progression and that miR-542-3p down-regulation contributes to aberrant activation of AKT signaling, leaving open the possibility that miR-542-3p may be a potential therapeutic target for high grade astrocytoma. PMID:26286747

  16. In vitro and in vivo activity of novel small-molecule inhibitors targeting the pleckstrin homology domain of protein kinase B/AKT.

    PubMed

    Moses, Sylvestor A; Ali, M Ahad; Zuohe, Song; Du-Cuny, Lei; Zhou, Li Li; Lemos, Robert; Ihle, Nathan; Skillman, A Geoffrey; Zhang, Shuxing; Mash, Eugene A; Powis, Garth; Meuillet, Emmanuelle J

    2009-06-15

    The phosphatidylinositol 3-kinase/AKT signaling pathway plays a critical role in activating survival and antiapoptotic pathways within cancer cells. Several studies have shown that this pathway is constitutively activated in many different cancer types. The goal of this study was to discover novel compounds that bind to the pleckstrin homology (PH) domain of AKT, thereby inhibiting AKT activation. Using proprietary docking software, 22 potential PH domain inhibitors were identified. Surface plasmon resonance spectroscopy was used to measure the binding of the compounds to the expressed PH domain of AKT followed by an in vitro activity screen in Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. We identified a novel chemical scaffold in several of the compounds that binds selectively to the PH domain of AKT, inducing a decrease in AKT activation and causing apoptosis at low micromolar concentrations. Structural modifications of the scaffold led to compounds with enhanced inhibitory activity in cells. One compound, 4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide, inhibited AKT and its downstream targets in cells as well as in pancreatic cancer cell xenografts in immunocompromised mice; it also exhibited good antitumor activity. In summary, a pharmacophore for PH domain inhibitors targeting AKT function was developed. Computer-aided modeling, synthesis, and testing produced novel AKT PH domain inhibitors that exhibit promising preclinical properties.

  17. Temsirolimus in advanced leiomyosarcomas: patterns of response and correlation with the activation of the mammalian target of rapamycin pathway.

    PubMed

    Italiano, Antoine; Kind, Michèle; Stoeckle, Eberhard; Jones, Natalie; Coindre, Jean-Michel; Bui, Binh

    2011-06-01

    Preclinical data have indicated that alteration of PTEN and activation of the mammalian target of rapamycin (mTOR) pathway play a crucial role in the oncogenesis of leiomyosarcoma. The objective of this exploratory study was to assess the clinical role of mTOR inhibition in patients with advanced leiomyosarcoma refractory to standard chemotherapy. Patients with advanced leiomyosarcoma were treated with temsirolimus and consented to retrospective collection of data from their medical records and analysis of archival tumor specimens. Tumor response was determined according to the response evaluation criteria in solid tumor (RECIST) and Choi criteria. Tumors were assessed for immunohistochemical evidence of PTEN loss of expression and mTOR activation. Six patients participated in the study. According to the RECIST, three patients had stable disease and three patients had progressive disease. The three patients with RECIST stable disease had partial response according to the Choi criteria. Partial response according to the Choi criteria was associated with clinical improvement and biological signs of temsirolimus antitumor activity. The immunohistochemical status of PTEN and phosphorylated S6 ribosomal protein was not predictive of the outcome. This exploratory study indicates antitumor activity of temsirolimus in leiomyosarcoma, possibly through a mechanism involving aberration of the PTEN gene. Further investigations of the phosphoinositide 3-kinases/PTEN/Akt/mTOR pathway are needed to explore the role of mTOR inhibitors, either alone or in combination, in patients with advanced sarcoma.

  18. The mammalian target of rapamycin complex 1 (mTORC1) in breast cancer: the impact of oestrogen receptor and HER2 pathways.

    PubMed

    Jerjees, Dena A; Negm, Ola H; Alabdullah, M Layth; Mirza, Sameer; Alkaabi, Methaq; Hameed, Mohamed R; Abduljabbar, Rezvan; Muftah, Abir; Nolan, Chris C; Green, Andrew R; Tighe, Patrick J; Band, Vimla; Ellis, Ian O; Rakha, Emad A

    2015-02-01

    The mammalian target of rapamycin complex 1 (mTORC1) is a downstream of the PI3K/Akt pathway which affects cancer development. mTORC1 has many downstream signalling effectors that can enhance different cellular responses. This study aims to investigate the expression of mTORC1 in breast cancer (BC) and correlate it with key clinicopathological and molecular features of BC especially to proteins related to oestrogen receptor (ER) and HER2 pathways in different BC classes. Moreover, mTORC1 expression was assessed in 6 BC cell lines including ER+ and ER- cell lines with and without HER2 transfection. Immunohistochemistry was used to assess the expression of phospho (p) mTORC1 in a large (n = 1300) annotated BC series prepared as tissue microarray. Reverse phase protein array (RPPA) was used to assess its expression in the different BC cell lines. The expression of p-mTORC1 was cytoplasmic with moderate/high expression noted in 44 % of BC. p-mTORC1 expression was associated with clinicopathological variables characteristic of good prognosis. Positive correlation with ER, ER-related proteins AKT, PI3K and luminal differentiation markers were observed in the whole series and in the ER+HER2- subgroup. Association with HER2 was mainly observed in the ER-negative class. RPPA indicated that p-mTORC1 expression was mainly related to ER expression and with better outcome in the Akt positive tumours. p-mTORC1 is associated with good prognostic features. Its expression is related to ER and ER related proteins in addition to AKT and PI3K. Its relation with HER2 expression is mainly seen in the absence of ER expression.

  19. Targeting mammalian organelles with internalizing phage (iPhage) libraries

    PubMed Central

    Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

  20. The marine fungal metabolite, AD0157, inhibits angiogenesis by targeting the Akt signaling pathway.

    PubMed

    García-Caballero, Melissa; Cañedo, Librada; Fernández-Medarde, Antonio; Medina, Miguel Ángel; Quesada, Ana R

    2014-01-16

    In the course of a screening program for the inhibitors of angiogenesis from marine sources, AD0157, a pyrrolidinedione fungal metabolite, was selected for its angiosupressive properties. AD0157 inhibited the growth of endothelial and tumor cells in culture in the micromolar range. Our results show that subtoxic doses of this compound inhibit certain functions of endothelial cells, namely, differentiation, migration and proteolytic capability. Inhibition of the mentioned essential steps of in vitro angiogenesis is in agreement with the observed antiangiogenic activity, substantiated by using two in vivo angiogenesis models, the chorioallantoic membrane and the zebrafish embryo neovascularization assays, and by the ex vivo mouse aortic ring assay. Our data indicate that AD0157 induces apoptosis in endothelial cells through chromatin condensation, DNA fragmentation, increases in the subG1 peak and caspase activation. The data shown here altogether indicate for the first time that AD0157 displays antiangiogenic effects, both in vitro and in vivo, that are exerted partly by targeting the Akt signaling pathway in activated endothelial cells. The fact that these effects are carried out at lower concentrations than those required for other inhibitors of angiogenesis makes AD0157 a new promising drug candidate for further evaluation in the treatment of cancer and other angiogenesis-related pathologies.

  1. PI3K/AKT/PTEN pathway as a target for Crohn's disease therapy (Review).

    PubMed

    Tokuhira, Nana; Kitagishi, Yasuko; Suzuki, Miho; Minami, Akari; Nakanishi, Atsuko; Ono, Yuna; Kobayashi, Keiko; Matsuda, Satoru; Ogura, Yasunori

    2015-01-01

    The pathogenesis of inflammatory bowel disease (IBD), including Crohn's disease, is a subject of increasing interest. Loss-of-function mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) are strong genetic factors linked to Crohn's disease, which eventually leads to an excessive mucosal inflammatory response directed against components of normal gut microbiota. Reactive oxygen species (ROS) play an important role in inflammation processes, as well as in transduction of signals from receptors for several cytokines, such as tumor necrosis factor α (TNFα). ROS activate nuclear factor-κB (NF-κB) via IκB kinase (IKK) through the PI3K/AKT/PTEN pathway. Therefore, this pathway is recognized to play a key role in Crohn's disease. Loss of function has been demonstrated to occur as an early event in a wide variety of diseases. Given this prevalent involvement in a number of diseases, the molecular development that modulates this pathway has been the subject of several studies. In addition, it has been the focus of extensive research and drug discovery activities. A better understanding of the molecular assemblies may reveal novel targets for the therapeutic development against Crohn's disease.

  2. Inhibiting Cycloxygenase and Ornithine Decarboxylase by Diclofenac and Alpha-Difluoromethylornithine Blocks Cutaneous SCCs by Targeting Akt-ERK Axis

    PubMed Central

    Arumugam, Aadithya; Weng, Zhiping; Talwelkar, Sarang S.; Chaudhary, Sandeep C.; Kopelovich, Levy; Elmets, Craig A.; Afaq, Farrukh; Athar, Mohammad

    2013-01-01

    Non-melanoma skin cancer (NMSC) is the most common type of skin cancer in Caucasian populations. Its increasing incidence has been a major public health concern. Elevated expressions of ODC and COX-2 are associated with both murine and human NMSCs. Inhibition of these molecular targets singly employing their respective small molecule inhibitors showed limited success. Here, we show that combined blockade of ODC and COX-2 using their potent inhibitors, DFMO and diclofenac respectively abrogates growth of A431 epidermal xenograft tumors in nu/nu mice by more than 90%. The tumor growth inhibition was associated with a diminution in the proliferation and enhancement in apoptosis. The proliferation markers such as PCNA and cyclin D1 were reduced. TUNEL-positive apoptotic cells and cleaved caspase-3 were increased in the residual tumors. These agents also manifested direct target-unrelated effects. Reduced expression of phosphorylated MAPKAP-2, ERK, and Akt (ser473 & thr308) were noticed. The mechanism by which combined inhibition of ODC/COX attenuated tumor growth and invasion involved reduction in EMT. Akt activation by ODC+COX-2 over-expression was the key player in this regard as Akt inhibition manifested effects similar to those observed by the combined inhibition of ODC+COX-2 whereas forced over-expression of Akt resisted against DFMO+diclofenac treatment. These data suggest that ODC+COX-2 over-expression together leads to pathogenesis of aggressive and invasive cutaneous carcinomas by activating Akt signaling pathway, which through augmenting EMT contributes to tumor invasion. PMID:24260338

  3. Targeted disruption of TC-PTP in the proliferative compartment augments STAT3 and AKT signaling and skin tumor development

    PubMed Central

    Lee, Hyunseung; Kim, Mihwa; Baek, Minwoo; Morales, Liza D.; Jang, Ik-Soon; Slaga, Thomas J.; DiGiovanni, John; Kim, Dae Joon

    2017-01-01

    Tyrosine phosphorylation is a vital mechanism that contributes to skin carcinogenesis. It is regulated by the counter-activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Here, we report the critical role of T-cell protein tyrosine phosphatase (TC-PTP), encoded by Ptpn2, in chemically-induced skin carcinogenesis via the negative regulation of STAT3 and AKT signaling. Using epidermal specific TC-PTP knockout (K14Cre.Ptpn2fl/fl) mice, we demonstrate loss of TC-PTP led to a desensitization to tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA)-induced apoptosis both in vivo epidermis and in vitro keratinocytes. TC-PTP deficiency also resulted in a significant increase in epidermal thickness and hyperproliferation following exposure to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Western blot analysis showed that both phosphorylated STAT3 and phosphorylated AKT expressions were significantly increased in epidermis of TC-PTP-deficient mice compared to control mice following TPA treatment. Inhibition of STAT3 or AKT reversed the effects of TC-PTP deficiency on apoptosis and proliferation. Finally, TC-PTP knockout mice showed a shortened latency of tumorigenesis and significantly increased numbers of tumors during two-stage skin carcinogenesis. Our findings reveal that TC-PTP has potential as a novel target for the prevention of skin cancer through its role in the regulation of STAT3 and AKT signaling. PMID:28322331

  4. Co-operating STAT5 and AKT signaling pathways in chronic myeloid leukemia and mastocytosis: possible new targets of therapy.

    PubMed

    Bibi, Siham; Arslanhan, Melis Dilara; Langenfeld, Florent; Jeanningros, Sylvie; Cerny-Reiterer, Sabine; Hadzijusufovic, Emir; Tchertanov, Luba; Moriggl, Richard; Valent, Peter; Arock, Michel

    2014-03-01

    Chronic myeloid leukemia and systemic mastocytosis are myeloid neoplasms sharing a number of pathogenetic and clinical features. In both conditions, an aberrantly activated oncoprotein with tyrosine kinase activity, namely BCR-ABL1 in chronic myeloid leukemia, and mutant KIT, mostly KIT D816V, in systemic mastocytosis, is key to disease evolution. The appreciation of the role of such tyrosine kinases in these diseases has led to the development of improved therapies with tyrosine kinase-targeted inhibitors. However, most drugs, including new KIT D816V-blocking agents, have failed to achieve long-lasting remissions in advanced systemic mastocytosis, and there is a similar problem in chronic myeloid leukemia, where imatinib-resistant patients sometimes fail to achieve remission, even with second- or third-line BCR-ABL1 specific tyrosine kinase inhibitors. During disease progression, additional signaling pathways become activated in neoplastic cells, but most converge into major downstream networks. Among these, the AKT and STAT5 pathways appear most critical and may result in drug-resistant chronic myeloid leukemia and systemic mastocytosis. Inhibition of phosphorylation of these targets has proven their crucial role in disease-evolution in both malignancies. Together, these observations suggest that STAT5 and AKT are key drivers of oncogenesis in drug-resistant forms of the diseases, and that targeting STAT5 and AKT might be an interesting approach in these malignancies. The present article provides an overview of our current knowledge about the critical role of AKT and STAT5 in the pathophysiology of chronic myeloid leukemia and systemic mastocytosis and on their potential value as therapeutic targets in these neoplasms.

  5. Co-operating STAT5 and AKT signaling pathways in chronic myeloid leukemia and mastocytosis: possible new targets of therapy

    PubMed Central

    Bibi, Siham; Arslanhan, Melis Dilara; Langenfeld, Florent; Jeanningros, Sylvie; Cerny-Reiterer, Sabine; Hadzijusufovic, Emir; Tchertanov, Luba; Moriggl, Richard; Valent, Peter; Arock, Michel

    2014-01-01

    Chronic myeloid leukemia and systemic mastocytosis are myeloid neoplasms sharing a number of pathogenetic and clinical features. In both conditions, an aberrantly activated oncoprotein with tyrosine kinase activity, namely BCR-ABL1 in chronic myeloid leukemia, and mutant KIT, mostly KIT D816V, in systemic mastocytosis, is key to disease evolution. The appreciation of the role of such tyrosine kinases in these diseases has led to the development of improved therapies with tyrosine kinase-targeted inhibitors. However, most drugs, including new KIT D816V-blocking agents, have failed to achieve long-lasting remissions in advanced systemic mastocytosis, and there is a similar problem in chronic myeloid leukemia, where imatinib-resistant patients sometimes fail to achieve remission, even with second- or third-line BCR-ABL1 specific tyrosine kinase inhibitors. During disease progression, additional signaling pathways become activated in neoplastic cells, but most converge into major downstream networks. Among these, the AKT and STAT5 pathways appear most critical and may result in drug-resistant chronic myeloid leukemia and systemic mastocytosis. Inhibition of phosphorylation of these targets has proven their crucial role in disease-evolution in both malignancies. Together, these observations suggest that STAT5 and AKT are key drivers of oncogenesis in drug-resistant forms of the diseases, and that targeting STAT5 and AKT might be an interesting approach in these malignancies. The present article provides an overview of our current knowledge about the critical role of AKT and STAT5 in the pathophysiology of chronic myeloid leukemia and systemic mastocytosis and on their potential value as therapeutic targets in these neoplasms. PMID:24598853

  6. Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response

    PubMed Central

    Chan, Tung O.; Zhang, Jin; Tiegs, Brian C.; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M.; Armen, Roger S.; Rodeck, Ulrich; Penn, Raymond B.

    2015-01-01

    The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr308 in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr308 dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser473) increased phosphatase resistance of the phosphorylated activation loop (pThr308) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr308 phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. PMID:26201515

  7. Targeting the PI3K/Akt pathway in murine MDS/MPN driven by hyperactive Ras

    PubMed Central

    Akutagawa, Jon; Huang, Tannie Q.; Epstein, Inbal; Chang, Tiffany; Quirindongo-Crespo, Maricel; Cottonham, Charisa L.; Dail, Monique; Slusher, Barbara S.; Friedman, Lori S.; Sampath, Deepak; Braun, Benjamin S.

    2016-01-01

    Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation due to NRAS, KRAS, PTPN11, CBL, and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3´-OH kinase (PI3K)/Akt cascades. Mx1-Cre, KrasD12 and Mx1-Cre, Nf1flox/− mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, KrasD12 mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia, and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients. PMID:26965285

  8. MicroRNA-21 accelerates hepatocyte proliferation in vitro via PI3K/Akt signaling by targeting PTEN

    SciTech Connect

    Yan-nan, Bai; Zhao-yan, Yu; Li-xi, Luo; Jiang, Yi; Qing-jie, Xia

    2014-01-17

    Highlights: •miRNAs-expression patterns of primary hepatocytes under proliferative status. •miR-21 expression level peaked at 12 h after stimulated by EGF. •miR-21 drive rapid S phase entry of primary hepatocytes. •PI3K/Akt signaling was modulated via targeting PTEN by miR-21. -- Abstract: MicroRNAs (miRNAs) are involved in controlling hepatocyte proliferation during liver regeneration. In this study, we established the miRNAs-expression patterns of primary hepatocytes in vitro under stimulation of epidermal growth factor (EGF), and found that microRNA-21 (miR-21) was appreciably up-regulated and peaked at 12 h. In addition, we further presented evidences indicating that miR-21 promotes primary hepatocyte proliferation through in vitro transfecting with miR-21 mimics or inhibitor. We further demonstrated that phosphatidylinositol 3′-OH kinase (PI3K)/Akt signaling was altered accordingly, it is, by targeting phosphatase and tensin homologue deleted on chromosome 10, PI3K/Akt signaling is activated by miR-21 to accelerate hepatocyte rapid S-phase entry and proliferation in vitro.

  9. Targeted deletion of Kif18a protects from colitis-associated colorectal (CAC) tumors in mice through impairing Akt phosphorylation.

    PubMed

    Zhu, Houbao; Xu, Wangyang; Zhang, Hongxin; Liu, Jianbing; Xu, Haimin; Lu, Shunyuan; Dang, Suying; Kuang, Ying; Jin, Xiaolong; Wang, Zhugang

    2013-08-16

    Kinesins are a superfamily of molecular motors involved in cell division or intracellular transport. They are becoming important targets for chemotherapeutic intervention of cancer due to their crucial role in mitosis. Here, we demonstrate that the kinesin-8 Kif18a is overexpressed in murine CAC and is a crucial promoter during early CAC carcinogenesis. Kif18a-deficient mice are evidently protected from AOM-DSS-induced colon carcinogenesis. Kif18A is responsible for proliferation of colonic tumor cells, while Kif18a ablation in mice promotes cell apoptosis. Mechanistically, Kif18a is responsible for induction of Akt phosphorylation, which is known to be associated with cell survival regulation. In conclusion, Kif18a is critical for colorectal carcinogenesis in the setting of inflammation by mechanisms of increased PI3K-AKT signaling. Inhibition of Kif18A activity may be useful in the prevention or chemotherapeutic intervention of CAC.

  10. Hepatitis C virus inhibits AKT-tuberous sclerosis complex (TSC), the mechanistic target of rapamycin (MTOR) pathway, through endoplasmic reticulum stress to induce autophagy.

    PubMed

    Huang, He; Kang, Rongyan; Wang, Ji; Luo, Guangxiang; Yang, Wei; Zhao, Zhendong

    2013-02-01

    Hepatitis C virus (HCV) is able to induce autophagy via endoplasmic reticulum (ER) stress, but the exact molecular signaling pathway is not well understood. We found that the activity of the mechanistic target of rapamycin complex 1 (MTORC1) was inhibited in Huh7 cells either harboring HCV-N (genotype 1b) full-genomic replicon or infected with JFH1 (genotype 2a) virus, which led to the activation of UNC-51-like kinase 1 (ULK1) and thus to autophagy. We then analyzed activity upstream of MTORC1, and found that both protein kinase, AMP-activated, α (PRKAA, including PRKAA1 and PRKAA2, also known as AMP-activated protein kinase, AMPKα) and AKT (refers to pan AKT, including three isoforms of AKT1-3, also known as protein kinase B, PKB) were inhibited by HCV infection. The inhibition of the AKT-TSC-MTORC1 pathway contributed to upregulating autophagy, but inhibition of PRKAA downregulated autophagy. The net effect on autophagy was from AKT, which overrode the inhibition effect from PRKAA. It was further found that HCV-induced ER stress was responsible for the inhibition of the AKT pathway. Metformin, a PRKAA agonist, inhibited HCV replication not only by activating PRKAA as previously reported, but also by activating AKT independently of the autophagy pathway. Taken together, our data suggested HCV inhibited the AKT-TSC-MTORC1 pathway via ER stress, resulting in autophagy, which may contribute to the establishment of the HCV-induced autophagy.

  11. Enhanced Cardiac Akt/Protein Kinase B Signaling Contributes to Pathological Cardiac Hypertrophy in Part by Impairing Mitochondrial Function via Transcriptional Repression of Mitochondrion-Targeted Nuclear Genes

    PubMed Central

    Wende, Adam R.; O'Neill, Brian T.; Bugger, Heiko; Riehle, Christian; Tuinei, Joseph; Buchanan, Jonathan; Tsushima, Kensuke; Wang, Li; Caro, Pilar; Guo, Aili; Sloan, Crystal; Kim, Bum Jun; Wang, Xiaohui; Pereira, Renata O.; McCrory, Mark A.; Nye, Brenna G.; Benavides, Gloria A.; Darley-Usmar, Victor M.; Shioi, Tetsuo; Weimer, Bart C.

    2014-01-01

    Sustained Akt activation induces cardiac hypertrophy (LVH), which may lead to heart failure. This study tested the hypothesis that Akt activation contributes to mitochondrial dysfunction in pathological LVH. Akt activation induced LVH and progressive repression of mitochondrial fatty acid oxidation (FAO) pathways. Preventing LVH by inhibiting mTOR failed to prevent the decline in mitochondrial function, but glucose utilization was maintained. Akt activation represses expression of mitochondrial regulatory, FAO, and oxidative phosphorylation genes in vivo that correlate with the duration of Akt activation in part by reducing FOXO-mediated transcriptional activation of mitochondrion-targeted nuclear genes in concert with reduced signaling via peroxisome proliferator-activated receptor α (PPARα)/PGC-1α and other transcriptional regulators. In cultured myocytes, Akt activation disrupted mitochondrial bioenergetics, which could be partially reversed by maintaining nuclear FOXO but not by increasing PGC-1α. Thus, although short-term Akt activation may be cardioprotective during ischemia by reducing mitochondrial metabolism and increasing glycolysis, long-term Akt activation in the adult heart contributes to pathological LVH in part by reducing mitochondrial oxidative capacity. PMID:25535334

  12. [Regulative mechanisms of mammalian target of rapamycin signaling pathway in glomerular hypertrophy in diabetic nephropathy and interventional effects of Chinese herbal medicine].

    PubMed

    Yang, Jing-Jing; Huang, Yan-ru; Wan, Yi-gang; Shen, Shan-mei; Mao, Zhi-min; Wu, Wei; Yao, Jian

    2015-08-01

    Glomerular hypertrophy is the main pathological characteristic in the early stage of diabetic nephropathy (DN), and its regulatory mechanism is closely related to mammalian target of rapamycin (mTOR) signaling pathway activity. mTOR includes mTOR complex 1 (mTORC1) and mTOR complex 2(mTORC2), in which, the upstream pathway of mTORC1 is phosphatidylinositol-3-kinase (PI3K)/serine-threonine kinase(Akt)/adenosine monophosphate activated protein kinase(AMPK), and the representative signaling molecules in the downstream pathway of mTORC1 are 4E-binding proteins(4EBP) and phosphoprotein 70 S6Kinase(p70S6K). Some Chinese herbal extracts could improve cell proliferation via intervening the expressions of the key molecules in the upstream or downstream of PIK/Akt/mTOR signaling pathway in vivo. As for glomerular mesangial cells(MC) and podocyte, mTOR plays an important role in regulating glomerular inherent cells, including adjusting cell cycle, energy metabolism and matrix protein synthesis. Rapamycin, the inhibitor of mTOR, could suppress glomerular inherent cell hypertrophy, cell proliferation, glomerular basement membrane (GBM) thickening and mesangial matrix deposition in model rats with DN. Some Chinese herbal extracts could alleviate glomerular lesions by intervening mTOR signaling pathway activity in renal tissue of DN animal models or in renal inherent cells in vivo and in vitro.

  13. Myopathy caused by mammalian target of rapamycin complex 1 (mTORC1) inactivation is not reversed by restoring mitochondrial function.

    PubMed

    Romanino, Klaas; Mazelin, Laetitia; Albert, Verena; Conjard-Duplany, Agnès; Lin, Shuo; Bentzinger, C Florian; Handschin, Christoph; Puigserver, Pere; Zorzato, Francesco; Schaeffer, Laurent; Gangloff, Yann-Gaël; Rüegg, Markus A

    2011-12-20

    Mammalian target of rapamycin complex 1 (mTORC1) is central to the control of cell, organ, and body size. Skeletal muscle-specific inactivation of mTORC1 in mice results in smaller muscle fibers, fewer mitochondria, increased glycogen stores, and a progressive myopathy that causes premature death. In mTORC1-deficient muscles, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), which regulates mitochondrial biogenesis and glucose homeostasis, is strongly down-regulated. Here we tested whether induction of mitochondrial biogenesis pharmacologically or by the overexpression of PGC-1α is sufficient to reverse the phenotype of mice deficient for mTORC1. We show that both approaches normalize mitochondrial function, such as oxidative capacity and expression of mitochondrial genes. However, they do not prevent or delay the progressive myopathy. In addition, we find that mTORC1 has a much stronger effect than PGC-1α on the glycogen content in muscle. This effect is based on the strong activation of PKB/Akt in mTORC1-deficient mice. We also show that activation of PKB/Akt not only affects glycogen synthesis but also diminishes glycogen degradation. Thus, our work provides strong functional evidence that mitochondrial dysfunction in mice with inactivated mTORC1 signaling is caused by the down-regulation of PGC-1α. However, our data also show that the impairment of mitochondria does not lead directly to the lethal myopathy.

  14. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

    PubMed Central

    Santiago, Yolanda; Chan, Edmond; Liu, Pei-Qi; Orlando, Salvatore; Zhang, Lin; Urnov, Fyodor D.; Holmes, Michael C.; Guschin, Dmitry; Waite, Adam; Miller, Jeffrey C.; Rebar, Edward J.; Gregory, Philip D.; Klug, Aaron; Collingwood, Trevor N.

    2008-01-01

    Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural—but imperfect—DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR−/− cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2–3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR−/− cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production. PMID:18359850

  15. Fucoidan inhibits the migration and proliferation of HT-29 human colon cancer cells via the phosphoinositide-3 kinase/Akt/mechanistic target of rapamycin pathways.

    PubMed

    Han, Yong-Seok; Lee, Jun Hee; Lee, Sang Hun

    2015-09-01

    Fucoidan, a sulfated polysaccharide, has a variety of biological activities, including anti-cancer, anti-angiogenic and anti-inflammatory effects. However, the underlying mechanisms of fucoidan as an anti‑cancer agent remain to be elucidated. The present study examined the anti‑cancer effect of fucoidan on HT‑29 human colon cancer cells. The cell growth of HT29 cells was significantly decreased following treatment with fucoidan (200 µg/ml). In addition, fucoidan inhibited the migration of HT‑29 cells by decreasing the expression levels of matrix metalloproteinase‑2 in a dose‑dependent manner (0‑200 µg/ml). The underlying mechanism of these inhibitory effects included the downregulation of phosphoinositide 3‑kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) by treatment with fucoidan. Furthermore, fucoidan increased the expression of cleaved caspase‑3 and decreased cancer sphere formation. The present study suggested that fucoidan exerts an anti‑cancer effect on HT‑29 human colon cancer cells by downregulating the PI3K‑Akt‑mTOR signaling pathway. Therefore, fucoidan may be a potential therapeutic reagent against the growth of human colon cancer cells.

  16. KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1{alpha} survival pathways

    SciTech Connect

    Oommen, Deepu; Prise, Kevin M.

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer KNK437, a benzylidene lactam compound, is a novel radiosensitizer. Black-Right-Pointing-Pointer KNK437 inhibits AKT signaling and abrogates the accumulation of HIF-1{alpha} under hypoxia. Black-Right-Pointing-Pointer KNK437 abrogates hypoxia induced resistance to radiation. -- Abstract: KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1{alpha} (HIF-1{alpha}). HIF-1{alpha} is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1{alpha}. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1{alpha} in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1{alpha} levels in KNK437-treated cells. This suggested that the absence of HIF-1{alpha} in hypoxic cells was not due to the enhanced protein degradation. HIF-1{alpha} is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1{alpha} mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1{alpha} levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

  17. Targeting EMP3 suppresses proliferation and invasion of hepatocellular carcinoma cells through inactivation of PI3K/Akt pathway

    PubMed Central

    Lee, Chien-Hsing; Yang, Shun-Fa; Cheng, Chun-Wen; Tang, Meng-Ju; Lin, Chia-Liang; Lin, Chu-Liang; Chou, Ruey-Hwang

    2015-01-01

    Epithelial membrane protein-3 (EMP3), a typical member of the epithelial membrane protein (EMP) family, is epigenetically silenced in some cancer types, and has been proposed to be a tumor suppressor gene. However, its effects on tumor suppression are controversial and its roles in development and malignancy of hepatocellular carcinoma (HCC) remain unclear. In the present study, we found that EMP3 was highly expressed in the tumorous tissues comparing to the matched normal tissues, and negatively correlated with differentiated degree of HCC patients. Knockdown of EMP3 significantly reduced cell proliferation, arrested cell cycle at G1 phase, and inhibited the motility and invasiveness in accordance with the decreased expression and activity of urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) in HCC cells. The in vivo tumor growth of HCC was effectively suppressed by knockdown of EMP3 in a xenograft mouse model. The EMP3 knockdown-reduced cell proliferation and invasion were attenuated by inhibition of phosphatidylinositol 3-kinase (PI3K) or knockdown of Akt, and rescued by overexpression of Akt in HCC cells. Clinical positive correlations of EMP3 with p85 regulatory subunit of PI3K, p-Akt, uPA, as well as MMP-9 were observed in the tissue sections from HCC patients. Here, we elucidated the tumor progressive effects of EMP3 through PI3K/Akt pathway and uPA/MMP-9 cascade in HCC cells. The findings provided a new insight into EMP3, which might be a potential molecular target for diagnosis and treatment of HCC. PMID:26472188

  18. Metastasis and AKT activation.

    PubMed

    Sheng, Shijie; Qiao, Meng; Pardee, Arthur B

    2009-03-01

    Metastasis, responsible for 90% of cancer patient deaths, is an inefficient process because many tumor cells die. The survival of metastatic tumor cells should be considered as a critical therapeutic target. This review provides a new perspective regarding the role of AKT in tumor survival, and the rationale to target AKT in anti-metastasis therapies.

  19. FLZ protects dopaminergic neuron through activating protein kinase B/mammalian target of rapamycin pathway and inhibiting RTP801 expression in Parkinson's disease models.

    PubMed

    Bao, X-Q; Kong, X-C; Qian, C; Zhang, D

    2012-01-27

    The pathogenesis of Parkinson's disease is characterized by progressive degeneration of dopaminergic neurons in substantia nigra (SNpc). FLZ, a novel synthetic squamosamide derivative from a Chinese herb, has been shown to have neuroprotective effects in experimental Parkinson's disease (PD) models. However, it is still unclear whether FLZ protects against PD through regulating the function of dopaminergic system. In this study, we carried out a set of in vitro and in vivo experiments to address these questions. Oral administration of FLZ significantly improved motor dysfunction of mice challenged by MPTP. The beneficial effects of FLZ on motor behavior attributed to the elevation of dopamine level in striatum, tyrosine hydroxylase (TH)-positive cells, and TH activity in the middle brain of mouse. Mechanism study showed that treatment of FLZ increased the phosphorylation of activating protein kinase B (Akt) and mammalian target of rapamycin (mTOR). Using LY294002 to block phosphoinositide 3-kinases (PI3K)/Akt signaling pathway prevented the phosphorylation of mTOR and attenuated the neuroprotection of FLZ in MN9D cells challenged by MPP(+). In addition, FLZ reduced the expression of RTP801, an important protein in PD, in mice and cells intoxicated by MPTP/MPP(+). Taken together, these results revealed a novel role that FLZ elevated TH expression and activity in dopaminergic neuron through activation of Akt/mTOR survival pathway and inhibition of RTP801 in MPTP/MPP(+)-induced PD models. The data also provided evidence that FLZ had potent neuroprotecive effects and might become a new promising anti-PD drug.

  20. Trisubstituted-Imidazoles Induce Apoptosis in Human Breast Cancer Cells by Targeting the Oncogenic PI3K/Akt/mTOR Signaling Pathway

    PubMed Central

    Mervin, Lewis; Mohan, Surender; Paricharak, Shardul; Baday, Sefer; Li, Feng; Shanmugam, Muthu K.; Chinnathambi, Arunachalam; Zayed, M. E.; Alharbi, Sulaiman Ali; Bender, Andreas; Sethi, Gautam; Basappa; Rangappa, Kanchugarakoppal S.

    2016-01-01

    Overactivation of PI3K/Akt/mTOR is linked with carcinogenesis and serves a potential molecular therapeutic target in treatment of various cancers. Herein, we report the synthesis of trisubstituted-imidazoles and identified 2-chloro-3-(4, 5-diphenyl-1H-imidazol-2-yl) pyridine (CIP) as lead cytotoxic agent. Naïve Base classifier model of in silico target prediction revealed that CIP targets RAC-beta serine/threonine-protein kinase which comprises the Akt. Furthermore, CIP downregulated the phosphorylation of Akt, PDK and mTOR proteins and decreased expression of cyclin D1, Bcl-2, survivin, VEGF, procaspase-3 and increased cleavage of PARP. In addition, CIP significantly downregulated the CXCL12 induced motility of breast cancer cells and molecular docking calculations revealed that all compounds bind to Akt2 kinase with high docking scores compared to the library of previously reported Akt2 inhibitors. In summary, we report the synthesis and biological evaluation of imidazoles that induce apoptosis in breast cancer cells by negatively regulating PI3K/Akt/mTOR signaling pathway. PMID:27097161

  1. Computational modeling of novel inhibitors targeting the Akt pleckstrin homology domain.

    PubMed

    Du-Cuny, Lei; Song, Zuohe; Moses, Sylvestor; Powis, Garth; Mash, Eugene A; Meuillet, Emmanuelle J; Zhang, Shuxing

    2009-10-01

    Computational modeling continues to play an important role in novel therapeutics discovery and development. In this study, we have investigated the use of in silico approaches to develop inhibitors of the pleckstrin homology (PH) domain of AKT (protein kinase B). Various docking/scoring schemes have been evaluated, and the best combination was selected to study the system. Using this strategy, two hits were identified and their binding behaviors were investigated. Robust and predictive QSAR models were built using the k nearest neighbor (kNN) method to study their cellular permeability. Based on our in silico results, long flexible aliphatic tails were proposed to improve the Caco-2 penetration without affecting the binding mode. The modifications enhanced the AKT inhibitory activity of the compounds in cell-based assays, and increased their activity as in vivo antitumor testing.

  2. Chemical-biological characterization of a cruzain inhibitor reveals a second target and a mammalian off-target.

    PubMed

    Choy, Jonathan W; Bryant, Clifford; Calvet, Claudia M; Doyle, Patricia S; Gunatilleke, Shamila S; Leung, Siegfried S F; Ang, Kenny K H; Chen, Steven; Gut, Jiri; Oses-Prieto, Juan A; Johnston, Jonathan B; Arkin, Michelle R; Burlingame, Alma L; Taunton, Jack; Jacobson, Matthew P; McKerrow, James M; Podust, Larissa M; Renslo, Adam R

    2013-01-01

    Inhibition of the Trypanosoma cruzi cysteine protease cruzain has been proposed as a therapeutic approach for the treatment of Chagas' disease. Among the best-studied cruzain inhibitors to date is the vinylsulfone K777 (1), which has proven effective in animal models of Chagas' disease. Recent structure-activity studies aimed at addressing potential liabilities of 1 have now produced analogues such as N-[(2S)-1-[[(E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]amino]-3-(4-methylphenyl)-1-oxopropan-2-yl]pyridine-4-carboxamide (4), which is trypanocidal at ten-fold lower concentrations than for 1. We now find that the trypanocidal activity of 4 derives primarily from the inhibition of T. cruzi 14-α-demethylase (TcCYP51), a cytochrome P450 enzyme involved in the biosynthesis of ergosterol in the parasite. Compound 4 also inhibits mammalian CYP isoforms but is trypanocidal at concentrations below those required to significantly inhibit mammalian CYPs in vitro. A chemical-proteomics approach employing an activity-based probe derived from 1 was used to identify mammalian cathepsin B as a potentially important off-target of 1 and 4. Computational docking studies and the evaluation of truncated analogues of 4 reveal structural determinants for TcCYP51 binding, information that will be useful in further optimization of this new class of inhibitors.

  3. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension.

    PubMed

    Aghamohammadzadeh, Reza; Zhang, Ying-Yi; Stephens, Thomas E; Arons, Elena; Zaman, Paula; Polach, Kevin J; Matar, Majed; Yung, Lai-Ming; Yu, Paul B; Bowman, Frederick P; Opotowsky, Alexander R; Waxman, Aaron B; Loscalzo, Joseph; Leopold, Jane A; Maron, Bradley A

    2016-07-01

    Activation of the mammalian target of rapamycin complex 1 (mTORC1) subunit Raptor induces cell growth and is a downstream target of Akt. Elevated levels of aldosterone activate Akt, and, in pulmonary arterial hypertension (PAH), correlate with pulmonary arteriole thickening, which suggests that mTORC1 regulation by aldosterone may mediate adverse pulmonary vascular remodeling. We hypothesized that aldosterone-Raptor signaling induces abnormal pulmonary artery smooth muscle cell (PASMC) survival patterns to promote PAH. Remodeled pulmonary arterioles from SU-5416/hypoxia-PAH rats and monocrotaline-PAH rats with hyperaldosteronism expressed increased levels of the Raptor target, p70S6K, which provided a basis for investigating aldosterone-Raptor signaling in human PASMCs. Aldosterone (10(-9) to 10(-7) M) increased Akt/mTOR/Raptor to activate p70S6K and increase proliferation, viability, and apoptosis resistance in PASMCs. In PASMCs transfected with Raptor-small interfering RNA or treated with spironolactone/eplerenone, aldosterone or pulmonary arterial plasma from patients with PAH failed to increase p70S6K activation or to induce cell survival in vitro Optimal inhibition of pulmonary arteriole Raptor was achieved by treatment with Staramine-monomethoxy polyethylene glycol that was formulated with Raptor-small interfering RNA plus spironolactone in vivo, which decreased arteriole muscularization and pulmonary hypertension in 2 experimental animal models of PAH in vivo Up-regulation of mTORC1 by aldosterone is a critical pathobiologic mechanism that controls PASMC survival to promote hypertrophic vascular remodeling and PAH.-Aghamohammadzadeh, R., Zhang, Y.-Y., Stephens, T. E., Arons, E., Zaman, P., Polach, K. J., Matar, M., Yung, L.-M., Yu, P. B., Bowman, F. P., Opotowsky, A. R., Waxman, A. B., Loscalzo, J., Leopold, J. A., Maron, B. A. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth

  4. miR-24 represses metastasis of human osteosarcoma cells by targeting Ack1 via AKT/MMPs pathway.

    PubMed

    Liu, Zhendong; Liu, Zhitao; Zhang, Yuanjun; Li, Yan; Liu, Bo; Zhang, Kexiang

    2017-02-08

    The expression levels of the protein tyrosine kinase Ack1 has been reported to be dysregulated in various cancers and involve in oncogenesis and progression. However, the expression and role of Ack1 in osteosarcoma remains unknown. In this study, we found that Ack1 were evidently upregulated in human osteosarcoma tissues and cell lines. In addition, the clinical data showed that high expression level of Ack1 is closely associated with clinical stage and positive distant metastasis, and negatively correlated with overall survival. Then, bioinformatics prediction and luciferase reporter assay indicated Ack1 as a direct target of miR-24, and Ack1 could be downregulated by miR-24 at both the mRNA and protein expression levels. Moreover, Ack1 expression levels were inversely correlated with that of miR-24 in osteosarcoma tissues. Furthermore, functional assay showed that miR-24 significantly suppressed osteosarcoma progression partially mediated by inhibiting Ack1 expression. Finally, western bolt assay revealed that miR-24 regulate AKT/MMPs pathway via Ack1 in osteosarcoma cells. In conclusion, our study demonstrated the suppression of miR-24 on osteosarcoma metastasis by targeting Ack1 via AKT/MMPs pathways, providing a novel strategy for the diagnosis and treatment of osteosarcoma patients.

  5. MicroRNA-302a Functions as a Putative Tumor Suppressor in Colon Cancer by Targeting Akt

    PubMed Central

    Sun, Shengjie; Zhang, Guoqing; Wu, Zhiyong; Shi, Weiwei; Yang, Bo; Li, Ying

    2014-01-01

    Micro RNAs (miRNAs) are important regulators involved in various physical and pathological processes, including cancer. The miRNA-302 family has been documented as playing a critical role in carcinogenesis. In this study, we investigated the role of miRNA-302a in colon cancer. MiRNA-302a expression was detected in 44 colon cancer tissues and 10 normal colon tissues, and their clinicopathological significance was analyzed. Cell proliferation and cell cycle analysis were performed on colon cancer cells that stably expressed miRNA-302a. The target gene of miRNA-302a and the downstream pathway were further investigated. Compared with normal colon tissues, miRNA-302a expression was downregulated in colon cancer tissues. Overexpression of miRNA-302a induced G1/S cell cycle arrest in colon cancer cells, and suppressed colon cancer cell proliferation both in vitro and in vivo. Furthermore, miRNA-302a inhibited AKT expression by directly binding to its 3′ untranslated region, resulting in subsequent alterations of the AKT-GSK3β-cyclin D1 pathway. These results reveal miRNA-302a as a tumor suppressor in colon cancer, suggesting that miRNA-302a may be used as a potential target for therapeutic intervention in colon cancer. PMID:25542007

  6. Synthetic neomycin-kanamycin phosphotransferase, type II coding sequence for gene targeting in mammalian cells.

    PubMed

    Jin, Seung-Gi; Mann, Jeffrey R

    2005-07-01

    The bacterial neomycin-kanamycin phosphotransferase, type II enzyme is encoded by the neo gene and confers resistance to aminoglycoside drugs such as neomycin and kanamycin-bacterial selection and G418-eukaryotic cell selection. Although widely used in gene targeting in mouse embryonic stem cells, the neo coding sequence contains numerous cryptic splice sites and has a high CpG content. At least the former can cause unwanted effects in cis at the targeted locus. We describe a synthetic sequence, sneo, which encodes the same protein as that encoded by neo. This synthetic sequence has no predicted splice sites in either strand, low CpG content, and increased mammalian codon usage. In mouse embryonic stem cells sneo expressability is similar to neo. The use of sneo in gene targeting experiments should substantially reduce the probability of unwanted effects in cis due to splicing, and perhaps CpG methylation, within the coding sequence of the selectable marker.

  7. Recent advances in developing molecular tools for targeted genome engineering of mammalian cells.

    PubMed

    Lim, Kwang-il

    2015-01-01

    Various biological molecules naturally existing in diversified species including fungi, bacteria, and bacteriophage have functionalities for DNA binding and processing. The biological molecules have been recently actively engineered for use in customized genome editing of mammalian cells as the molecule-encoding DNA sequence information and the underlying mechanisms how the molecules work are unveiled. Excitingly, multiple novel methods based on the newly constructed artificial molecular tools have enabled modifications of specific endogenous genetic elements in the genome context at efficiencies that are much higher than that of the conventional homologous recombination based methods. This minireview introduces the most recently spotlighted molecular genome engineering tools with their key features and ongoing modifications for better performance. Such ongoing efforts have mainly focused on the removal of the inherent DNA sequence recognition rigidity from the original molecular platforms, the addition of newly tailored targeting functions into the engineered molecules, and the enhancement of their targeting specificity. Effective targeted genome engineering of mammalian cells will enable not only sophisticated genetic studies in the context of the genome, but also widely-applicable universal therapeutics based on the pinpointing and correction of the disease-causing genetic elements within the genome in the near future.

  8. Multifaceted role of insulin-like growth factors and mammalian target of rapamycin in skeletal muscle.

    PubMed

    Frost, Robert A; Lang, Charles H

    2012-06-01

    This review describes the current literature on the interaction between insulin-like growth factors, endocrine hormones, and branched-chain amino acids on muscle physiology in healthy young individuals and during select pathologic conditions. Emphasis is placed on the mechanism by which physical and hormonal signals are transduced at the cellular level to either grow or atrophy skeletal muscle. The key role of the mammalian target of rapamycin and its ability to respond to hypertrophic and atrophic signals informs our understanding how a combination of physical, nutritional, and pharmacologic therapies may be used in tandem to prevent or ameliorate reductions in muscle mass.

  9. Prediction of human miRNA target genes using computationally reconstructed ancestral mammalian sequences

    PubMed Central

    Leclercq, Mickael; Diallo, Abdoulaye Baniré; Blanchette, Mathieu

    2017-01-01

    MicroRNAs (miRNA) are short single-stranded RNA molecules derived from hairpin-forming precursors that play a crucial role as post-transcriptional regulators in eukaryotes and viruses. In the past years, many microRNA target genes (MTGs) have been identified experimentally. However, because of the high costs of experimental approaches, target genes databases remain incomplete. Although several target prediction programs have been developed in the recent years to identify MTGs in silico, their specificity and sensitivity remain low. Here, we propose a new approach called MirAncesTar, which uses ancestral genome reconstruction to boost the accuracy of existing MTGs prediction tools for human miRNAs. For each miRNA and each putative human target UTR, our algorithm makes uses of existing prediction tools to identify putative target sites in the human UTR, as well as in its mammalian orthologs and inferred ancestral sequences. It then evaluates evidence in support of selective pressure to maintain target site counts (rather than sequences), accounting for the possibility of target site turnover. It finally integrates this measure with several simpler ones using a logistic regression predictor. MirAncesTar improves the accuracy of existing MTG predictors by 26% to 157%. Source code and prediction results for human miRNAs, as well as supporting evolutionary data are available at http://cs.mcgill.ca/∼blanchem/mirancestar. PMID:27899600

  10. Molecular and Enzymatic Profiles of Mammalian DNA Methyltransferases: Structures and Targets for Drugs

    PubMed Central

    Xu, F.; Mao, C.; Ding, Y.; Rui, C.; Wu, L.; Shi, A.; Zhang, H.; Zhang, L.; Xu, Z.

    2010-01-01

    DNA methylation is an epigenetic event involved in a variety array of processes that may be the foundation of genetic phenomena and diseases. DNA methyltransferase is a key enzyme for cytosine methylation in DNA, and can be divided into two functional families (Dnmt1 and Dnmt3) in mammals. All mammalian DNA methyltransferases are encoded by their own single gene, and consisted of catalytic and regulatory regions (except Dnmt2). Via interactions between functional domains in the regulatory or catalytic regions and other adaptors or cofactors, DNA methyltransferases can be localized at selective areas (specific DNA/nucleotide sequence) and linked to specific chromosome status (euchromatin/heterochromatin, various histone modification status). With assistance from UHRF1 and Dnmt3L or other factors in Dnmt1 and Dnmt3a/Dnmt3b, mammalian DNA methyltransferases can be recruited, and then specifically bind to hemimethylated and unmethylated double-stranded DNA sequence to maintain and de novo setup patterns for DNA methylation. Complicated enzymatic steps catalyzed by DNA methyltransferases include methyl group transferred from cofactor Ado-Met to C5 position of the flipped-out cytosine in targeted DNA duplex. In the light of the fact that different DNA methyltransferases are divergent in both structures and functions, and use unique reprogrammed or distorted routines in development of diseases, design of new drugs targeting specific mammalian DNA methyltransferases or their adaptors in the control of key steps in either maintenance or de novo DNA methylation processes will contribute to individually treating diseases related to DNA methyltransferases. PMID:20939822

  11. Transcriptional regulation of gene expression during osmotic stress responses by the mammalian target of rapamycin.

    PubMed

    Ortells, M Carmen; Morancho, Beatriz; Drews-Elger, Katherine; Viollet, Benoit; Laderoute, Keith R; López-Rodríguez, Cristina; Aramburu, Jose

    2012-05-01

    Although stress can suppress growth and proliferation, cells can induce adaptive responses that allow them to maintain these functions under stress. While numerous studies have focused on the inhibitory effects of stress on cell growth, less is known on how growth-promoting pathways influence stress responses. We have approached this question by analyzing the effect of mammalian target of rapamycin (mTOR), a central growth controller, on the osmotic stress response. Our results showed that mammalian cells exposed to moderate hypertonicity maintained active mTOR, which was required to sustain their cell size and proliferative capacity. Moreover, mTOR regulated the induction of diverse osmostress response genes, including targets of the tonicity-responsive transcription factor NFAT5 as well as NFAT5-independent genes. Genes sensitive to mTOR-included regulators of stress responses, growth and proliferation. Among them, we identified REDD1 and REDD2, which had been previously characterized as mTOR inhibitors in other stress contexts. We observed that mTOR facilitated transcription-permissive conditions for several osmoresponsive genes by enhancing histone H4 acetylation and the recruitment of RNA polymerase II. Altogether, these results reveal a previously unappreciated role of mTOR in regulating transcriptional mechanisms that control gene expression during cellular stress responses.

  12. Transcriptional regulation of gene expression during osmotic stress responses by the mammalian target of rapamycin

    PubMed Central

    Ortells, M. Carmen; Morancho, Beatriz; Drews-Elger, Katherine; Viollet, Benoit; Laderoute, Keith R.; López-Rodríguez, Cristina; Aramburu, Jose

    2012-01-01

    Although stress can suppress growth and proliferation, cells can induce adaptive responses that allow them to maintain these functions under stress. While numerous studies have focused on the inhibitory effects of stress on cell growth, less is known on how growth-promoting pathways influence stress responses. We have approached this question by analyzing the effect of mammalian target of rapamycin (mTOR), a central growth controller, on the osmotic stress response. Our results showed that mammalian cells exposed to moderate hypertonicity maintained active mTOR, which was required to sustain their cell size and proliferative capacity. Moreover, mTOR regulated the induction of diverse osmostress response genes, including targets of the tonicity-responsive transcription factor NFAT5 as well as NFAT5-independent genes. Genes sensitive to mTOR-included regulators of stress responses, growth and proliferation. Among them, we identified REDD1 and REDD2, which had been previously characterized as mTOR inhibitors in other stress contexts. We observed that mTOR facilitated transcription-permissive conditions for several osmoresponsive genes by enhancing histone H4 acetylation and the recruitment of RNA polymerase II. Altogether, these results reveal a previously unappreciated role of mTOR in regulating transcriptional mechanisms that control gene expression during cellular stress responses. PMID:22287635

  13. Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo

    PubMed Central

    Li, Wenyan; Shen, Jun

    2016-01-01

    Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction. PMID:28116172

  14. Targeted deletion of Kif18a protects from colitis-associated colorectal (CAC) tumors in mice through impairing Akt phosphorylation

    SciTech Connect

    Zhu, Houbao; Xu, Wangyang; Zhang, Hongxin; Liu, Jianbing; Xu, Haimin; Lu, Shunyuan; Dang, Suying; Kuang, Ying; Jin, Xiaolong; Wang, Zhugang

    2013-08-16

    Highlights: •Kif18A is up-regulated in CAC of mouse model. •Kif18a{sup −/−} mice are protected from CAC. •Tumor cells from Kif18a{sup −/−} mice undergo more apoptosis. •Kif18A deficiency induces poor Atk phosphorylation. -- Abstract: Kinesins are a superfamily of molecular motors involved in cell division or intracellular transport. They are becoming important targets for chemotherapeutic intervention of cancer due to their crucial role in mitosis. Here, we demonstrate that the kinesin-8 Kif18a is overexpressed in murine CAC and is a crucial promoter during early CAC carcinogenesis. Kif18a-deficient mice are evidently protected from AOM–DSS-induced colon carcinogenesis. Kif18A is responsible for proliferation of colonic tumor cells, while Kif18a ablation in mice promotes cell apoptosis. Mechanistically, Kif18a is responsible for induction of Akt phosphorylation, which is known to be associated with cell survival regulation. In conclusion, Kif18a is critical for colorectal carcinogenesis in the setting of inflammation by mechanisms of increased PI3K-AKT signaling. Inhibition of Kif18A activity may be useful in the prevention or chemotherapeutic intervention of CAC.

  15. Mefloquine effectively targets gastric cancer cells through phosphatase-dependent inhibition of PI3K/Akt/mTOR signaling pathway.

    PubMed

    Liu, Yanwei; Chen, Sen; Xue, Rui; Zhao, Juan; Di, Maojun

    2016-02-05

    Deregulation of PI3K/Akt/mTOR pathway has been recently identified to play a crucial role in the progress of human gastric cancer. In this study, we show that mefloquine, a FDA-approved anti-malarial drug, effectively targets human gastric cancer cells. Mefloquine potently inhibits proliferation and induces apoptosis of a panel of human gastric cancer cell lines, with EC50 ∼ 0.5-0.7 μM. In two independent gastric cancer xenograft mouse models, mefloquine significantly inhibits growth of both tumors. The combination of mefloquine with paclitaxel enhances the activity of either drug alone in in vitro and in vivo. In addition, mefloquine potently decreased phosphorylation of PI3K, Akt, mTOR and rS6. Overexpression of constitutively active Akt significantly restored mefloquine-mediated inhibition of mTOR phosphorylation and growth, and induction of apoptosis, suggesting that mefloquine acts on gastric cancer cells via suppressing PI3K/Akt/mTOR pathway. We further show that mefloquine-mediated inhibition of Akt/mTOR singaling is phosphatase-dependent as pretreatment with calyculin A does-dependently reversed mefloquine-mediated inhibition of Akt/mTOR phosphorylation. Since mefloquine is already available for clinic use, these results suggest that it is a useful addition to the treatment armamentarium for gastric cancer.

  16. Targeted detection of mammalian species using carrion fly-derived DNA.

    PubMed

    Schubert, Grit; Stockhausen, Melanie; Hoffmann, Constanze; Merkel, Kevin; Vigilant, Linda; Leendertz, Fabian H; Calvignac-Spencer, Sébastien

    2015-03-01

    DNA analysis from carrion flies (iDNA analysis) has recently been promoted as a powerful tool for cost- and time-efficient monitoring of wildlife. While originally applied to identify any mammalian species present in an area, it should also allow for targeted detection of species and individuals. Using carrion flies captured in the Taï National Park, Côte d'Ivoire, we assessed this possibility by (i) screening carrion fly DNA extracts with nonspecific and species-specific PCR systems, respectively, targeting mitochondrial DNA (mtDNA) fragments of any mammal or of Jentink's duiker (Cephalophus jentinki), three colobine monkeys (subfamily Colobinae) and sooty mangabey (Cercocebus atys); and (ii) genotyping carrion fly extracts containing sooty mangabey mtDNA. In comparison with the nonspecific PCR assay, the use of specific PCRs increased the frequency of detection of target species up to threefold. Detection rates partially reflected relative abundances of target species in the area. Amplification of seven microsatellite loci from carrion flies positive for sooty mangabey mtDNA yielded an average PCR success of 46%, showing that the identification of individuals is, to some extent, possible. Regression analysis of microsatellite PCR success and mtDNA concentration revealed that, among all carrion flies analysed for this study, 1% contained amounts of mammal mtDNA sufficient to attempt genotyping with potentially high success. We conclude that carrion fly-derived DNA analysis represents a promising tool for targeted monitoring of mammals in their natural habitat.

  17. The effect of hydroxyurea and trichostatin a on targeted nucleotide exchange in yeast and Mammalian cells.

    PubMed

    Parekh-Olmedo, Hetal; Engstrom, Julia U; Kmiec, Eric B

    2003-12-01

    Targeted nucleotide exchange (TNE) is a process by which a synthetic DNA oligonucleotide, partially complementary to a site in a chromosomal or an episomal gene directs the reversal of a single nucleotide at a specific site. To protect against nuclease digestion, the oligonucleotide is modified with derivative linkages among the terminal bases. We have termed these molecules modified single-stranded oligonucleotides (MSOs). Current models suggest that the reaction occurs in two steps. The first, DNA pairing, involves the alignment of the MSO with the target site and its assimilation into the target helix forming a D-loop. The second phase centers around the repair of a single base mismatch formed between the MSO and its complementary strand in the D-loop. Nucleotide exchange is promoted in all likelihood by the mismatch repair system. A critical feature of successful TNE is the accessibility of the target site for the MSO and the factors that increase the dynamic nature of the chromatin that will likely increase the frequency. Here, we report that two factors, trichostatin A and hydroxyurea, elevate gene repair of a mutant hygromycin gene in Saccharomyces cerevisiae and a mutant eGFP gene in a mammalian cell line, MCF-10AT1 cells. Trichostatin A (TSA) acts by preventing the deacetylation of histones while hydroxyurea (HU) reduces the rate of replication. Both of these activities, by their very nature, create a more open configuration of the MSO into the target site.

  18. Mammalian Target of Rapamycin (mTOR) Activation Increases Axonal Growth Capacity of Injured Peripheral Nerves*

    PubMed Central

    Abe, Namiko; Borson, Steven H.; Gambello, Michael J.; Wang, Fan; Cavalli, Valeria

    2010-01-01

    Unlike neurons in the central nervous system (CNS), injured neurons in the peripheral nervous system (PNS) can regenerate their axons and reinnervate their targets. However, functional recovery in the PNS often remains suboptimal, especially in cases of severe damage. The lack of regenerative ability of CNS neurons has been linked to down-regulation of the mTOR (mammalian target of rapamycin) pathway. We report here that PNS dorsal root ganglial neurons (DRGs) activate mTOR following damage and that this activity enhances axonal growth capacity. Furthermore, genetic up-regulation of mTOR activity by deletion of tuberous sclerosis complex 2 (TSC2) in DRGs is sufficient to enhance axonal growth capacity in vitro and in vivo. We further show that mTOR activity is linked to the expression of GAP-43, a crucial component of axonal outgrowth. However, although TSC2 deletion in DRGs facilitates axonal regrowth, it leads to defects in target innervation. Thus, whereas manipulation of mTOR activity could provide new strategies to stimulate nerve regeneration in the PNS, fine control of mTOR activity is required for proper target innervation. PMID:20615870

  19. Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma.

    PubMed

    Makinoshima, Hideki; Takita, Masahiro; Saruwatari, Koichi; Umemura, Shigeki; Obata, Yuuki; Ishii, Genichiro; Matsumoto, Shingo; Sugiyama, Eri; Ochiai, Atsushi; Abe, Ryo; Goto, Koichi; Esumi, Hiroyasu; Tsuchihara, Katsuya

    2015-07-10

    Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.

  20. Targeting the AKT/GSK3{beta}/Cyclin D1/Cdk4 Survival Signaling Pathway for Eradication of Tumor Radioresistance Acquired by Fractionated Radiotherapy

    SciTech Connect

    Shimura, Tsutomu; Kakuda, Satoshi; Ochiai, Yasushi; Kuwahara, Yoshikazu; Takai, Yoshihiro; Fukumoto, Manabu

    2011-06-01

    Purpose: Radioresistance is a major cause of treatment failure of radiotherapy (RT) in human cancer. We have recently revealed that acquired radioresistance of tumor cells induced by fractionated radiation is attributable to cyclin D1 overexpression as a consequence of the downregulation of GSK3{beta}-dependent cyclin D1 proteolysis mediated by a constitutively activated serine-threonine kinase, AKT. This prompted us to hypothesize that targeting the AKT/GSK3{beta}/cyclin D1 pathway may improve fractionated RT by suppressing acquired radioresistance of tumor cells. Methods and Materials: Two human tumor cell lines with acquired radioresistance were exposed to X-rays after incubation with either an AKT inhibitor, AKT/PKB signaling inhibitor-2 (API-2), or a Cdk4 inhibitor (Cdk4-I). Cells were then subjected to immunoblotting, clonogenic survival assay, cell growth analysis, and cell death analysis with TUNEL and annexin V staining. In vivo radiosensitivity was assessed by growth of human tumors xenografted into nude mice. Results: Treatment with API-2 resulted in downregulation of cyclin D1 expression in cells with acquired radioresistance. Cellular radioresistance disappeared completely both in vitro and in vivo with accompanying apoptosis when treated with API-2. Furthermore, inhibition of cyclin D1/Cdk4 by Cdk4-I was sufficient for abolishing radioresistance. Treatment with either API-2 or Cdk4-I was also effective in suppressing resistance to cis-platinum (II)-diamine-dichloride in the cells with acquired radioresistance. Interestingly, the radiosensitizing effect of API-2 was canceled by overexpression of cyclin D1 whereas Cdk4-I was still able to sensitize cells with cyclin D1 overexpression. Conclusion: Cyclin D1/Cdk4 is a critical target of the AKT survival signaling pathway responsible for tumor radioresistance. Targeting the AKT/GSK3{beta}/cyclin D1/Cdk4 pathway would provide a novel approach to improve fractionated RT and would have an impact on tumor

  1. Targeting the dysregulated mammalian target of rapamycin pathway in organ transplantation: killing 2 birds with 1 stone.

    PubMed

    Haidinger, Michael; Werzowa, Johannes; Weichhart, Thomas; Säemann, Marcus D

    2011-10-01

    Dysregulation and hyperactivation of the mammalian target of rapamycin (mTOR) pathway define the molecular basis of the hamartoma syndromes, including Cowden syndrome, tuberous sclerosis complex (TSC)/lymphangioleiomyomatosis, and Peutz-Jeghers syndrome. Loss of the tumor suppressors phosphatase and tensin homolog (PTEN), TSC1, TSC2, and LKB1 results in uncontrolled growth of usually benign tumors in various organs that, however, frequently lead to organ failure. Therefore, organ transplantation is a common therapeutic option in distinct patients with hamartoma syndromes, especially those with TSC/lymphangioleiomyomatosis. mTOR inhibitors are currently used in allogeneic transplantation as immunosuppressants and for the treatment of a growing number of cancers with dysregulated mTOR/phosphoinositide 3-kinase pathway. This dual targeting provides the unique opportunity for mTOR inhibitors to affect hamartoma syndromes at the molecular level along with potent immunosuppression in transplanted individuals. Here, we review the molecular mechanisms of hamartoma syndromes and discuss the recent clinical progress in transplant patients with hamartomas. Combining the identification of novel molecular targets of the phosphoinositide 3-kinase/mTOR pathway with insights into the clinical effectiveness of current therapeutic strategies sets the stage for a broader translational potential essential for further progress both in the treatment of cancer and for transplantation.

  2. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development

    PubMed Central

    Guan, Yingjie; Yang, Xu; Yang, Wentian; Charbonneau, Cherie; Chen, Qian

    2014-01-01

    Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and expression of chondrogenic genes, including Indian hedgehog (Ihh), a critical mediator of mechanotransduction. Conversely, cyclic loading (1 Hz, 5% matrix deformation) of embryonic chicken growth plate chondrocytes in 3-dimensional (3D) collagen scaffolding induced sustained activation of mTOR. Mechanical activation of mTOR occurred in serum-free medium, indicating that it is independent of growth factor or nutrients. Treatment of chondrocytes with Rapa abolished mechanical activation of cell proliferation and Ihh gene expression. Cyclic loading of chondroprogenitor cells deficient in SH2-containing protein tyrosine phosphatase 2 (Shp2) further enhanced mechanical activation of mTOR, cell proliferation, and chondrogenic gene expression. This result suggests that Shp2 is an antagonist of mechanotransduction through inhibition of mTOR activity. Our data demonstrate that mechanical activation of mTOR is necessary for cell proliferation, chondrogenesis, and cartilage growth during bone development, and that mTOR is an essential mechanotransduction component modulated by Shp2 in the cytoplasm.—Guan, Y., Yang, X., Yang, W., Charbonneau, C., Chen, Q. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development. PMID:25002119

  3. Live imaging and modeling of inner nuclear membrane targeting reveals its molecular requirements in mammalian cells

    PubMed Central

    Boni, Andrea; Politi, Antonio Z.; Strnad, Petr; Xiang, Wanqing; Hossain, M. Julius

    2015-01-01

    Targeting of inner nuclear membrane (INM) proteins is essential for nuclear architecture and function, yet its mechanism remains poorly understood. Here, we established a new reporter that allows real-time imaging of membrane protein transport from the ER to the INM using Lamin B receptor and Lap2β as model INM proteins. These reporters allowed us to characterize the kinetics of INM targeting and establish a mathematical model of this process and enabled us to probe its molecular requirements in an RNA interference screen of 96 candidate genes. Modeling of the phenotypes of genes involved in transport of these INM proteins predicted that it critically depended on the number and permeability of nuclear pores and the availability of nuclear binding sites, but was unaffected by depletion of most transport receptors. These predictions were confirmed with targeted validation experiments on the functional requirements of nucleoporins and nuclear lamins. Collectively, our data support a diffusion retention model of INM protein transport in mammalian cells. PMID:26056140

  4. Targeted Myostatin Gene Editing in Multiple Mammalian Species Directed by a Single Pair of TALE Nucleases.

    PubMed

    Xu, Li; Zhao, Piming; Mariano, Andrew; Han, Renzhi

    2013-07-30

    Myostatin (MSTN) is a negative regulator of skeletal muscle mass. Strategies to block myostatin signaling pathway have been extensively pursued to increase muscle mass in various disease settings including muscular dystrophy. Here, we report a new class of reagents based on transcription activator-like effector nucleases (TALENs) to disrupt myostatin expression at the genome level. We designed a pair of MSTN TALENs to target a highly conserved sequence in the coding region of the myostatin gene. We demonstrate that codelivery of these MSTN TALENs induce highly specific and efficient gene disruption in a variety of human, cattle, and mouse cells. Based upon sequence analysis, this pair of TALENs is expected to be functional in many other mammalian species. Moreover, we demonstrate that these MSTN TALENs can facilitate targeted integration of a mCherry expression cassette or a larger muscular dystrophy gene (dysferlin) expression cassette into the MSTN locus in mouse or human cells. Therefore, targeted editing of the myostatin gene using our highly specific and efficient TALEN pair would facilitate cell engineering, allowing potential use in translational research for cell-based therapy.Molecular Therapy-Nucleic Acids (2013) 2, e112; doi:10.1038/mtna.2013.39; published online 30 July 2013.

  5. The mammalian target of rapamycin modulates the immunoproteasome system in the heart.

    PubMed

    Zhang, Hong-Mei; Fu, Jianliang; Hamilton, Ryan; Diaz, Vivian; Zhang, Yiqiang

    2015-09-01

    The mammalian target of rapamycin (mTOR) plays an important role in cardiac development and function. Inhibition of mTOR by rapamycin has been shown to attenuate pathological cardiac hypertrophy and improve the function of aging heart, accompanied by an inhibition of the cardiac proteasome activity. The current study aimed to determine the potential mechanism(s) by which mTOR inhibition modulates cardiac proteasome. Inhibition of mTOR by rapamycin was found to reduce primarily the immunoproteasome in both H9c2 cells in vitro and mouse heart in vivo, without significant effect on the constitutive proteasome and protein ubiquitination. Concurrent with the reduction of the immunoproteasome, rapamycin reduced two important inflammatory response pathways, the NF-κB and Stat3 signaling. In addition, rapamycin attenuated the induction of the immunoproteasome in H9c2 cells by inflammatory cytokines, including INFγ and TNFα, by suppressing NF-κB signaling. These data indicate that rapamycin indirectly modulated immunoproteasome through the suppression of inflammatory response pathways. Lastly, the role of the immunoproteasome during the development of cardiac hypertrophy was investigated. Administration of a specific inhibitor of the immunoproteasome ONX 0914 attenuated isoproterenol-induced cardiac hypertrophy, suggesting that the immunoproteasome may be involved in the development of cardiac hypertrophy and therefore could be a therapeutic target. In conclusion, rapamycin inhibits the immunoproteasome through its effect on the inflammatory signaling pathways and the immunoproteasome could be a potential therapeutic target for pathological cardiac hypertrophy.

  6. miR-451a is underexpressed and targets AKT/mTOR pathway in papillary thyroid carcinoma

    PubMed Central

    Minna, Emanuela; Romeo, Paola; Dugo, Matteo; De Cecco, Loris; Todoerti, Katia; Pilotti, Silvana; Perrone, Federica; Seregni, Ettore; Agnelli, Luca; Neri, Antonino; Greco, Angela; Borrello, Maria Grazia

    2016-01-01

    Papillary Thyroid Carcinoma (PTC) is the most frequent thyroid cancer. Although several PTC-specific miRNA profiles have been reported, only few upregulated miRNAs are broadly recognized, while less consistent data are available about downregulated miRNAs. In this study we investigated miRNA deregulation in PTC by miRNA microarray, analysis of a public dataset from The Cancer Genome Atlas (TCGA), literature review and meta-analysis based on a univocal miRNA identifier derived from miRBase v21. A list of 18 miRNAs differentially expressed between PTC and normal thyroid was identified and validated in the TCGA dataset. Furthermore, we compared our signature with miRNA profiles derived from 15 studies selected from literature. Then, to select possibly functionally relevant miRNA, we integrated our miRNA signature with those from two in vitro cell models based on the PTC-driving oncogene RET/PTC1. Through this strategy, we identified commonly deregulated miRNAs, including miR-451a, which emerged also by our meta-analysis as the most frequently reported downregulated miRNA. We showed that lower expression of miR-451a correlates with aggressive clinical-pathological features of PTC as tall cell variant, advanced stage and extrathyroid extension. In addition, we demonstrated that ectopic expression of miR-451a impairs proliferation and migration of two PTC-derived cell lines, reduces the protein levels of its recognized targets MIF, c-MYC and AKT1 and attenuates AKT/mTOR pathway activation. Overall, our study provide both an updated overview of miRNA deregulation in PTC and the first functional evidence that miR-451a exerts tumor suppressor functions in this neoplasia. PMID:26871295

  7. Mammalian target of rapamycin signaling modulates photic entrainment of the suprachiasmatic circadian clock

    PubMed Central

    Cao, Ruifeng; Li, Aiqing; Cho, Hee-yeon; Lee, Boyoung; Obrietan, Karl

    2010-01-01

    Inducible gene expression appears to be an essential event that couples light to entrainment of the master mammalian circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Recently, we reported that light triggers phase-dependent activation of the mammalian target of rapamycin (mTOR) signaling pathway, a major regulator of protein synthesis, in the SCN, thus raising the possibility that mTOR-evoked mRNA translation contributes to clock entrainment. Here, we employed a combination of cellular, molecular and behavioral assays to address this question. To this end, we show that the in vivo infusion of the mTOR inhibitor rapamycin led to a significant attenuation of the phase-delaying effect of early night light. Conversely, disruption of mTOR during the late night augmented the phase-advancing effect of light. To assess the role of mTOR signaling within the context of molecular entrainment, the effects of rapamycin on light-induced expression of PERIOD1 and PERIOD2 were examined. At both the early and late night time points, abrogation of mTOR signaling led to a significant attenuation of light-evoked PERIOD protein expression. Our results also reveal that light-induced mTOR activation leads to translation of mRNAs with a 5′-terminal oligopyrimidine tract such as eukaryotic elongation factor 1 A (eEF1A) and the immediate early gene JunB. Together, these data indicate that the mTOR pathway functions as potent and selective regulator of light-evoked protein translation and SCN clock entrainment. PMID:20445056

  8. Mammalian target of rapamycin signaling modulates photic entrainment of the suprachiasmatic circadian clock.

    PubMed

    Cao, Ruifeng; Li, Aiqing; Cho, Hee-yeon; Lee, Boyoung; Obrietan, Karl

    2010-05-05

    Inducible gene expression appears to be an essential event that couples light to entrainment of the master mammalian circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Recently, we reported that light triggers phase-dependent activation of the mammalian target of rapamycin (mTOR) signaling pathway, a major regulator of protein synthesis, in the SCN, thus raising the possibility that mTOR-evoked mRNA translation contributes to clock entrainment. Here, we used a combination of cellular, molecular, and behavioral assays to address this question. To this end, we show that the in vivo infusion of the mTOR inhibitor rapamycin led to a significant attenuation of the phase-delaying effect of early-night light. Conversely, disruption of mTOR during the late night augmented the phase-advancing effect of light. To assess the role of mTOR signaling within the context of molecular entrainment, the effects of rapamycin on light-induced expression of PERIOD1 and PERIOD2 were examined. At both the early- and late-night time points, abrogation of mTOR signaling led to a significant attenuation of light-evoked PERIOD protein expression. Our results also reveal that light-induced mTOR activation leads to the translation of mRNAs with a 5'-terminal oligopyrimidine tract such as eukaryotic elongation factor 1A and the immediate early gene JunB. Together, these data indicate that the mTOR pathway functions as potent and selective regulator of light-evoked protein translation and SCN clock entrainment.

  9. Effect of supplemental dietary zinc on the mammalian target of rapamycin (mTOR) signaling pathway in skeletal muscle and liver from post-absorptive mice.

    PubMed

    McClung, James P; Tarr, Tyson N; Barnes, Brian R; Scrimgeour, Angus G; Young, Andrew J

    2007-07-01

    Zinc (Zn) is an essential trace element that functions in cellular signaling. The mammalian target of rapamycin (mTOR) regulates the initiation of protein synthesis. The objective of this study was to determine whether Zn could stimulate protein phosphorylation in the mTOR pathway in vivo. Mice (C57BL/6J, n = 30) were fed Zn marginal diets (ZM, 5 mg/kg) for 4 weeks, followed by fasting (F) and/or refeeding with ZM or Zn supplemental (300 mg/kg, ZS) diets for 3 or 6 h. Plasma insulin was greater (P < 0.05) in refed animals as compared to F animals. Protein phosphorylation was detected using multiplex analysis and Western blotting. Multiplex analysis indicated greater (P < 0.05) p70 S6 kinase (p70S6K) and glycogen synthase kinase 3 (GSK-3 alpha/beta) phosphorylation in livers from 6-h refed ZS animals as compared to F animals. Western blots indicated increased (P < 0.05) Akt (Ser 473) phosphorylation in skeletal muscle from animals refed ZS diets for 3 and 6 h as compared to F animals. The ZS diet affected phosphorylation of GSK-3 (alpha/beta) in liver, as 3-h ZS refed animals had greater (P < 0.01) phosphorylation than F animals. These findings indicate that Zn may contribute to the initiation of protein synthesis as a signaling molecule in vivo.

  10. Traumatic Brain Injury Stimulates Neural Stem Cell Proliferation via Mammalian Target of Rapamycin Signaling Pathway Activation

    PubMed Central

    Seekaew, Pich

    2016-01-01

    Abstract Neural stem cells in the adult brain possess the ability to remain quiescent until needed in tissue homeostasis or repair. It was previously shown that traumatic brain injury (TBI) stimulated neural stem cell (NSC) proliferation in the adult hippocampus, indicating an innate repair mechanism, but it is unknown how TBI promotes NSC proliferation. In the present study, we observed dramatic activation of mammalian target of rapamycin complex 1 (mTORC1) in the hippocampus of mice with TBI from controlled cortical impact (CCI). The peak of mTORC1 activation in the hippocampal subgranular zone, where NSCs reside, is 24–48 h after trauma, correlating with the peak of TBI-enhanced NSC proliferation. By use of a Nestin-GFP transgenic mouse, in which GFP is ectopically expressed in the NSCs, we found that TBI activated mTORC1 in NSCs. With 5-bromo-2′-deoxyuridine labeling, we observed that TBI increased mTORC1 activation in proliferating NSCs. Furthermore, administration of rapamycin abolished TBI-promoted NSC proliferation. Taken together, these data indicate that mTORC1 activation is required for NSC proliferation postinjury, and thus might serve as a therapeutic target for interventions to augment neurogenesis for brain repair after TBI. PMID:27822507

  11. Managing stomatitis in patients treated with Mammalian target of rapamycin inhibitors.

    PubMed

    Pilotte, Amy Potter; Hohos, Melissa Beth; Polson, Kathleen M O; Huftalen, Tarsha Marie; Treister, Nathaniel

    2011-10-01

    Mammalian target of rapamycin (mTOR) inhibitors are a class of targeted cancer therapeutic agents with clinical benefit for multiple tumor types. Oral ulcerations are a common side effect of mTOR inhibitors; however, the clinical findings resemble aphthous stomatitis rather than the mucositis seen with chemotherapy. Consequently, the appearance of aphthous-like oral ulcerations has been referred to as mTOR inhibitor-associated stomatitis (mIAS). The severity of mIAS can be minimized by following common preventive steps and initiating treatment at the first sign of mouth discomfort, thereby reducing the likelihood of treatment discontinuation. mIAS can be managed through prophylactic measures, such as patient education in oral hygiene and avoidance of triggers. Patients who develop mIAS may be treated topically using rinses or other local therapies, including corticosteroids. In severe cases, dose modifications may be required. Oncology nurses have an important role in the management of patients with cancer and are well positioned to offer strategies for minimizing the occurrence and impact of mIAS.

  12. Defining the Domain Arrangement of the Mammalian Target of Rapamycin Complex Component Rictor Protein

    PubMed Central

    Zhou, Ping; Zhang, Ning; Nussinov, Ruth

    2015-01-01

    Abstract Mammalian target of rapamycin (mTOR) complexes play a pivotal role in the cell. Raptor and Rictor proteins interact with mTOR to form two distinct complexes, mTORC1 and mTORC2, respectively. While the domain structure of Raptor is known, current bioinformatics tools failed to classify the domains in Rictor. Here we focus on identifying specific domains in Rictor by searching for conserved regions. We scanned the pdb structural database and constructed three protein domain datasets. Next we carried out multiple pairwise sequence alignments of the proteins in the domain dataset. By analyzing the z-scores of Rictor sequence similarity to protein sequences in the dataset, we assigned the structural and functional domains of Rictor. We found that, like Raptor, Rictor also has HEAT and WD40 domains, which could be the common motif binding to mTORC. Rictor may also have pleckstrin homology domains, which mediate cellular localization and transmit signals to downstream targets, as well as a domain that is homologous to 50S protein L17 and human 39S protein L17. This putative ribosome binding domain could mediate mTORC2–ribosome interaction. PMID:26176550

  13. SHEDDING NEW LIGHT ON NEURODEGENERATIVE DISEASES THROUGH THE MAMMALIAN TARGET OF RAPAMYCIN

    PubMed Central

    Chong, Zhao Zhong; Shang, Yan Chen; Wang, Shaohui; Maiese, Kenneth

    2012-01-01

    Neurodegenerative disorders affect a significant portion of the world's population leading to either disability or death for almost 30 million individuals worldwide. One novel therapeutic target that may offer promise for multiple disease entities that involve Alzheimer's disease, Parkinson's disease, epilepsy, trauma, stroke, and tumors of the nervous system is the mammalian target of rapamycin (mTOR). mTOR signaling is dependent upon the mTORC1 and mTORC2 complexes that are composed of mTOR and several regulatory proteins including the tuberous sclerosis complex (TSC1, hamartin/ TSC2, tuberin). Through a number of integrated cell signaling pathways that involve those of mTORC1 and mTORC2 as well as more novel signaling tied to cytokines, Wnt, and forkhead, mTOR can foster stem cellular proliferation, tissue repair and longevity, and synaptic growth by modulating mechanisms that foster both apoptosis and autophagy. Yet, mTOR through its proliferative capacity may sometimes be detrimental to central nervous system recovery and even promote tumorigenesis. Further knowledge of mTOR and the critical pathways governed by this serine/threonine protein kinase can bring new light for neurodegeneration and other related diseases that currently require new and robust treatments. PMID:22980037

  14. Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells

    PubMed Central

    Kato, Yuzuru; Katsuki, Takeo; Kokubo, Hiroki; Masuda, Aki; Saga, Yumiko

    2016-01-01

    Evolutionally conserved Nanos RNA-binding proteins play crucial roles in germ cell development. While a mammalian Nanos family protein, NANOS2, is required for sexual differentiation of male (XY) germ cells in mice, the underlying mechanisms and the identities of its target RNAs in vivo remain elusive. Using comprehensive microarray analysis and a bacterial artificial chromosome transgenic system, here we identify Dazl, a germ cell-specific gene encoding an RNA-binding protein implicated in translation, as a crucial target of NANOS2. Importantly, removal of the Dazl 3′-untranslated region in XY germ cells stabilizes the Dazl mRNA, resulting in elevated meiotic gene expression, abnormal resumption of the cell cycle and impaired processing-body formation, reminiscent of Nanos2-knockout phenotypes. Furthermore, our data suggest that NANOS2 acts as an antagonist of the DAZL protein. We propose a dual system of NANOS2-mediated suppression of Dazl expression as a pivotal molecular mechanism promoting sexual differentiation of XY germ cells. PMID:27072294

  15. Blocking mammalian target of rapamycin alleviates bladder hyperactivity and pain in rats with cystitis

    PubMed Central

    Li, Jie; Gou, Xin; Chen, Daihui

    2016-01-01

    Background Bladder disorders associated with interstitial cystitis are frequently characterized by increased contractility and pain. The purposes of this study were to examine (1) the effects of blocking mammalian target of rapamycin (mTOR) on the exaggerated bladder activity and pain evoked by cystitis and (2) the underlying mechanisms responsible for the role of mTOR in regulating cystic sensory activity. Results The expression of p-mTOR, mTOR-mediated phosphorylation of p70 ribosomal S6 protein kinase 1 (p-S6K1), 4 E–binding protein 4 (p-4 E-BP1), as well as phosphatidylinositide 3-kinase (p-PI3K) pathway were amplified in cyclophosphamide rats as compared with control rats. Blocking mTOR by intrathecal infusion of rapamycin attenuated bladder hyperactivity and pain. In addition, blocking PI3K signal pathway attenuated activities of mTOR, which was accompanied with decreasing bladder hyperactivity and pain. Inhibition of either mTOR or PI3K blunted the enhanced spinal substance P and calcitonin gene-related peptide in cyclophosphamide rats. Conclusions The data for the first time revealed specific signaling pathways leading to cyclophosphamide-induced bladder hyperactivity and pain, including the activation of mTOR and PI3K. Inhibition of these pathways alleviates cystic pain. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of overactive bladder and pain often observed in cystitis. PMID:27780878

  16. ets-2 Is a Target for an Akt (Protein Kinase B)/Jun N-Terminal Kinase Signaling Pathway in Macrophages of motheaten-viable Mutant Mice

    PubMed Central

    Smith, James L.; Schaffner, Alicia E.; Hofmeister, Joseph K.; Hartman, Matthew; Wei, Guo; Forsthoefel, David; Hume, David A.; Ostrowski, Michael C.

    2000-01-01

    The transcription factor ets-2 was phosphorylated at residue threonine 72 in a colony-stimulating factor 1 (CSF-1)- and mitogen-activated protein kinase-independent manner in macrophages isolated from motheaten-viable (me-v) mice. The CSF-1 and ets-2 target genes coding for Bcl-x, urokinase plasminogen activator, and scavenger receptor were also expressed at high levels independent of CSF-1 addition to me-v cells. Akt (protein kinase B) was constitutively active in me-v macrophages, and an Akt immunoprecipitate catalyzed phosphorylation of ets-2 at threonine 72. The p54 isoform of c-jun N-terminal kinase–stress-activated kinase (JNK- SAPK) coimmunoprecipitated with Akt from me-v macrophages, and treatment of me-v cells with the specific phosphatidylinositol 3-kinase inhibitor LY294002 decreased cell survival, Akt and JNK kinase activities, ets-2 phosphorylation, and Bcl-x mRNA expression. Therefore, ets-2 is a target for phosphatidylinositol 3-kinase–Akt–JNK action, and the JNK p54 isoform is an ets-2 kinase in macrophages. Constitutive ets-2 activity may contribute to the pathology of me-v mice by increasing expression of genes like the Bcl-x gene that promote macrophage survival. PMID:11027273

  17. Combined blockade of AKT/mTOR pathway inhibits growth of human hemangioma via downregulation of proliferating cell nuclear antigen.

    PubMed

    Ou, J M; Qui, M-K; Dai, Y-X; Dong, Q; Shen, J; Dong, P; Wang, X-F; Liu, Y-B; Fei, Z-W

    2012-01-01

    Protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway plays a crucial role in the tumorigenesis and progression of multiple tumors, and has been shown to be important therapeutic targets for cancer. The present study aimed to explore the role and molecular mechanisms of AKT/mTOR pathway in human hemangioma (HA). Twenty-five cases of human HA tissues were collected. The expression of AKT, mTOR and proliferating cell nuclear antigen (PCNA) proteins was evaluated using semi-quantitative immunohistochemistry in biopsy samples in different phases of HA. AKT/mTOR pathway was blocked by recombinant small hairpin RNA adenovirus vector rAd5-AKT+mTOR (rAd5-Am), used for infecting proliferating phase HA-derived endothelial cells (HDEC). The expression of AKT, mTOR and PCNA was detected by Real-time PCR and Western blot assays. Cell proliferative activities were determined by MTT assay, and cell cycle distribution and apoptosis were analyzed by flow cytometry. As a consequence, the expression of AKT, mTOR and PCNA was significantly increased in proliferative phase HA, while that was decreased in involutive phase. Combined blockade of AKT/mTOR pathway by rAd5-Am diminished cell proliferative activities, and induced cell apoptosis and cycle arrest with the decreased expression of AKT, mTOR and PCNA in proliferative phase HDEC. In conclusion, the activity of AKT/mTOR pathway was increased in proliferative phase HA, while it was decreased in involutive phase. Combined blockade of AKT/mTOR pathway might suppress cell proliferation via down-regulation of PCNA expression, and induce apoptosis and cycle arrest in proliferative phase HDEC, suggesting that AKT/mTOR pathway might represent the important therapeutic targets for human HA.

  18. ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin: Design and Synthesis of Highly Potent and Selective Pyrazolopyrimidines

    SciTech Connect

    Zask, Arie; Verheijen, Jeroen C.; Curran, Kevin; Kaplan, Joshua; Richard, David J.; Nowak, Pawel; Malwitz, David J.; Brooijmans, Natasja; Bard, Joel; Svenson, Kristine; Lucas, Judy; Toral-Barza, Lourdes; Zhang, Wei-Guo; Hollander, Irwin; Gibbons, James J.; Abraham, Robert T.; Ayral-Kaloustian, Semiramis; Mansour, Tarek S.; Yu, Ker

    2009-09-18

    The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.

  19. Phase I Study of Apitolisib (GDC-0980), Dual Phosphatidylinositol-3-Kinase and Mammalian Target of Rapamycin Kinase Inhibitor, in Patients with Advanced Solid Tumors

    PubMed Central

    Dolly, Saoirse O.; Wagner, Andrew J.; Bendell, Johanna C.; Kindler, Hedy L.; Krug, Lee M.; Seiwert, Tanguy Y.; Zauderer, Marjorie G.; Lolkema, Martijn P.; Apt, Doris; Yeh, Ru-Fang; Fredrickson, Jill O.; Spoerke, Jill M.; Koeppen, Hartmut; Ware, Joseph A.; Lauchle, Jennifer O.; Burris, Howard A.; de Bono, Johann S.

    2016-01-01

    Purpose This first-in-human phase I trial assessed the safety, tolerability, and preliminary anti-tumor activity of apitolisib (GDC-0980), a dual inhibitor of class I phosphatidylinositol-3-(PI3K) and mammalian target of rapamycin (mTOR) kinases. Experimental Design Once-daily (QD) oral apitolisib was administered to patients with solid tumors for days 1-21 or 1-28 of 28-day cycles. Pharmacokinetic and pharmacodynamic parameters were assessed. Results Overall, 120 patients were treated at doses between 2-70 mg. The commonest ≥G3 toxicities related to apitolisib at the recommended phase 2 dose (RP2D) at 40mg QD included hyperglycemia (18%), rash (14%), liver dysfunction (12%), diarrhea (10%), pneumonitis (8%), mucosal inflammation (6%), and fatigue (4%). Dose-limiting toxicities (one patient each) were G4 fasting hyperglycemia at 40 mg (21/28-schedule), and G3 maculopapular rash and G3 fasting hyperglycemia at 70 mg (21/28-schedule). The pharmacokinetic profile was dose-proportional. Phosphorylated serine-473 AKT levels were suppressed by ≥90% in platelet-rich plasma within 4 hours at the maximum tolerated dose (50 mg). Pharmacodynamic decreases in FDG-PET uptake of >25% occurred in 66% (21/32) of patients dosed at 40 mg QD. Evidence of single agent activity included ten RECIST partial responses (confirmed for peritoneal mesothelioma, PIK3CA mutant head- and-neck cancer, and three pleural mesotheliomas). Conclusion Apitolisib exhibited dose-proportional pharmacokinetics with target modulation at doses ≥16 mg. The RP2D was 40 mg QD 28/28-schedule; severe on-target toxicities were apparent at ≥40 mg, particularly pneumonitis. Apitolisib was reasonably tolerated at 30 mg, the selected dose for pleural mesothelioma patients given limited respiratory reserve. Modest but durable anti-tumor activity was demonstrated. PMID:26787751

  20. Synergistic effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway or NUP214-ABL1 fusion protein in human Acute Lymphoblastic Leukemia

    PubMed Central

    Martelli, Alberto M.; Zauli, Giorgio; Milani, Daniela; McCubrey, James A.; Capitani, Silvano; Neri, Luca M.

    2016-01-01

    Philadelphia chromosome-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) accounts for 25–30% of adult ALL and its incidence increases with age in adults >40 years old. Irrespective of age, the ABL1 fusion genes are markers of poor prognosis and amplification of the NUP214-ABL1 oncogene can be detected mainly in patients with T-ALL. T cell malignancies harboring the ABL1 fusion genes are sensitive to many cytotoxic agents, but up to date complete remissions have not been achieved. The PI3K/Akt/mTOR signaling pathway is often activated in leukemias and plays a crucial role in leukemogenesis. We analyzed the effects of three BCR-ABL1 tyrosine kinase inhibitors (TKIs), alone and in combination with a panel of selective PI3K/Akt/mTOR inhibitors, on three NUP214-ABL1 positive T-ALL cell lines that also displayed PI3K/Akt/mTOR activation. Cells were sensitive to anti BCR-ABL1 TKIs Imatinib, Nilotinib and GZD824, that specifically targeted the ABL1 fusion protein, but not the PI3K/Akt/mTOR axis. Four drugs against the PI3K/Akt/mTOR cascade, GSK690693, NVP-BGT226, ZSTK474 and Torin-2, showed marked cytotoxic effects on T-leukemic cells, without affecting the NUP214-ABL1 kinase and related pathway. Dephosphorylation of pAkt and pS6 showed the cytotoxicity of these compounds. Either single or combined administration of drugs against the different targets displayed inhibition of cellular viability associated with a concentration-dependent induction of apoptosis, cell cycle arrest in G0/G1 phase and autophagy, having the combined treatments a significant synergistic cytotoxic effect. Co-targeting NUP214-ABL1 fusion gene and PI3K/Akt/mTOR signaling pathway could represent a new and effective pharmacological strategy to improve the outcome in NUP214-ABL1 positive T-ALL. PMID:27821800

  1. Targeting the mammalian target of rapamycin pathway with everolimus: implications for the management of metastatic breast cancer.

    PubMed

    Ng, Vin Cci; Johnson, Jeremy J; Cuellar, Sandra

    2015-12-01

    The inhibitors of mammalian target of rapamycin (mTOR) have documented antitumor activity via disruption of various signaling pathways leading to impaired cellular growth, proliferation, and survival. In preclinical studies, mTOR inhibitors use in combination with hormonal therapy has shown promising results in overcoming endocrine resistance in breast cancer cells. The role of everolimus in breast cancer was established in the Breast Cancer Trial of Oral Everolimus-2 (BOLERO-2) trial in combination with exemestane for patients with advanced metastatic hormone receptor-positive (HR+) breast cancer, who relapsed after initial hormonal manipulation. The study met its primary endpoint of significant improvement in progression free survival (PFS) with a median time to progression of 6.9 months in the combination group versus 2.8 months in exemestane group. Favorable improvements in PFS were reported across all patient subgroups regardless of age, Eastern Cooperative Oncology Group performance status, number of prior therapies, and presence of visceral metastases. Adverse events were mostly mild to moderate in severity and consistent with the known safety profile of everolimus. Major toxicities reported include stomatitis, non-infectious pneumonitis, and hyperglycemia. The purpose of this review is to discuss the role of everolimus as a valuable component in advanced metastatic breast cancer and delineate current strategies to prevent and manage the most common toxicities associated with this combination regimen.

  2. Inhibition of lung tumor growth by targeting EGFR/VEGFR-Akt/NF-κB pathways with novel theanine derivatives.

    PubMed

    Zhang, Guoying; Ye, Xinshan; Ji, Dexin; Zhang, Huarong; Sun, Fujia; Shang, Chunqing; Zhang, Ying; Wu, Erxi; Wang, Fengfei; Wu, Fei; Tian, Huihui; Liu, Xin; Chen, Linlin; Liu, Kun; Wang, Yishan; Liu, Hanchen; Zhang, Wenhua; Guan, Yukun; Wang, Qinwen; Zhao, Xiaohang; Wan, Xiaochun

    2014-09-30

    The molecularly targeted agents, including anti-VEGF or anti-EGFR monoclonal antibody and some inhibitors of EGFR tyrosine kinase, are effective in the treatment of non-small-cell lung cancer (NSCLC) to a certain extent, but the benefit for a proportion of patients is still limited. Hence, it is necessary and urgent to develop more selective and effective molecular targeted agents against lung cancer. Here, we have synthesized novel theanine derivatives, methyl coumarin-3-carboxylyl L-theanine (TMC), ethyl coumarin-3-carboxylyl L-theanine (TEC), ethyl 6-fluorocoumarin- 3-carboxylyl L-theanine (TFC), and ethyl 6-nitrocoumarin-3-carboxylyl L-theanine (TNC), which are fluorescent small molecules, based on their parental compound theanine and studied their anticancer activities in vitro, ex vivo and in vivo models of human and mouse cancers. Our results show that the four theanine derivatives significantly inhibit the lung cancer cell migration and the growth of lung cancer and leukemia cell lines. TFC and TNC display enhanced effects with anticancer drugs cytarabine vincristine, andmethotrexate on inhibition of lung cancer cell growth and no toxicity to the normal human embryonic lung fibroblast and peripheral blood lymphocytes. TFC and TNC exhibit strong suppression of the highly metastatic Lewis lung cancer (LLC) and A549 tumor growth in tumor-bearing mice without toxicity to mice. TFC and TNC can effectively suppress the growth of lung cancer cells in vitro, ex vivo and in vivo by targeting EGFR/VEGFR-Akt/NF-κB pathways. Our study has suggested that TFC and TNC may have the therapeutic and/or adjuvant therapeutic applications in the treatment of lung cancers and other cancer.

  3. USP39, a direct target of microRNA-133a, promotes progression of pancreatic cancer via the AKT pathway.

    PubMed

    Cai, Jing; Liu, Tiande; Huang, Peng; Yan, Wei; Guo, Changkuo; Xiong, Le; Liu, Anwen

    2017-04-22

    Ubiquitin specific protease 39 (USP39) is one of the deubiquitinating enzymes without ubiquitin protease activity, which has been implicated in the progression of several cancers. However, the role of USP39 in pancreatic cancer (PC) is largely unknown. In present study, we found that USP39 expression was elevated in PC tissues than adjacent non-tumor tissues. Importantly, we demonstrated that overexpression of USP39 is closely correlated with tumor progression and poor survival in PC patients. Furthermore, high USP39 expression was observed in PC cell lines and ectopic expression of USP39 significantly enhanced in vitro cell proliferation and promoted in vivo tumor growth, whereas silencing USP39 suppressed growth of PC cells. Besides, our experimental data revealed that knockdown of USP39 induced cell apoptosis through inhibition of AKT signaling pathway in PC cells. Moreover, USP39 was a direct target of miR-133a, a microRNA that has been reported to be involved in progression of PC. Taken together, our data provide a novel PC regulatory axis that is miR-133a/USP39, the dysfunction of which drives diverse aspects of the progression of PC.

  4. PI3K/AKT-mediated upregulation of WDR5 promotes colorectal cancer metastasis by directly targeting ZNF407.

    PubMed

    Tan, Xin; Chen, Shuai; Wu, Jiangxue; Lin, Jiaxin; Pan, Changchuan; Ying, Xiaofang; Pan, Zhizhong; Qiu, Lin; Liu, Ranyi; Geng, Rong; Huang, Wenlin

    2017-03-16

    Colorectal cancer (CRC) is the third most common cause of cancer deaths, and has a high rate of liver and lung metastasis. Unfortunately, distant metastasis is the main barrier for advanced CRC therapy and leads to a very low survival rate. In this study, we identified WDR5, a vital factor that regulates vertebrate development and cell self-renewal and reprogramming, as a novel prognostic marker and therapeutic target for CRC patients. We demonstrate that WDR5 is upregulated in CRC tissues and promotes CRC metastasis both in vitro and in vivo. In an effort to investigate the impact of WDR5 on CRC cell fate, we treated CRC cells with growth factor and inhibitor. We report that WDR5 is a novel factor in the metastasis of CRC by triggering epithelial-mesenchymal transition (EMT) process in response to the PI3K/AKT signaling pathway. Moreover, WDR5 shows a direct binding to the ZNF407 promoter on regulating cellular EMT process, leading to CRC metastasis. Hence, our findings strongly position WDR5 as a valuable marker for CRC, and inhibiting WDR5 or the associated signaling pathways may be an effective strategy for the future development of anti-CRC therapy.

  5. Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-kappaB and Akt as targets.

    PubMed

    Albini, Adriana; Dell'Eva, Raffaella; Vené, Roberta; Ferrari, Nicoletta; Buhler, Donald R; Noonan, Douglas M; Fassina, Gianfranco

    2006-03-01

    Xanthohumol (XN), the principal flavonoid of the hop plant (Humulus lupulus L.) and a constituent of beer, has been suggested to have potential cancer chemopreventive activities. We have observed that most cancer chemopreventive agents show antiangiogenic properties in vitro and in vivo, a concept we termed "angioprevention." Here we show for the first time that XN can inhibit growth of a vascular tumor in vivo. Histopathology and in vivo angiogenesis assays indicated that tumor angiogenesis inhibition was involved. Further, we show the mechanisms for its inhibition of angiogenesis in vivo and related endothelial cell activities in vitro. XN repressed both the NF-kappaB and Akt pathways in endothelial cells, indicating that components of these pathways are major targets in the molecular mechanism of XN. Moreover, using in vitro analyses, we show that XN interferes with several points in the angiogenic process, including inhibition of endothelial cell invasion and migration, growth, and formation of a network of tubular-like structures. Our results suggest that XN can be added to the expanding list of antiangiogenic chemopreventive drugs whose potential in cancer prevention and therapy should be evaluated.

  6. A central role for the mammalian target of rapamycin in LPS-induced anorexia in mice.

    PubMed

    Yue, Yunshuang; Wang, Yi; Li, Dan; Song, Zhigang; Jiao, Hongchao; Lin, Hai

    2015-01-01

    Bacterial lipopolysaccharide (LPS), also known as endotoxin, induces profound anorexia. However, the LPS-provoked pro-inflammatory signaling cascades and the neural mechanisms underlying the development of anorexia are not clear. Mammalian target of rapamycin (mTOR) is a key regulator of metabolism, cell growth, and protein synthesis. This study aimed to determine whether the mTOR pathway is involved in LPS-induced anorexia. Effects of LPS on hypothalamic gene/protein expression in mice were measured by RT-PCR or western blotting analysis. To determine whether inhibition of mTOR signaling could attenuate LPS-induced anorexia, we administered an i.c.v. injection of rapamycin, an mTOR inhibitor, on LPS-treated male mice. In this study, we showed that LPS stimulates the mTOR signaling pathway through the enhanced phosphorylation of mTOR(Ser2448) and p70S6K(Thr389). We also showed that LPS administration increased the phosphorylation of FOXO1(Ser256), the p65 subunit of nuclear factor kappa B (P<0.05), and FOXO1/3a(Thr) (24) (/) (32) (P<0.01). Blocking the mTOR pathway significantly attenuated the LPS-induced anorexia by decreasing the phosphorylation of p70S6K(Thr389), FOXO1(Ser256), and FOXO1/3a(Thr) (24) (/) (32). These results suggest promising approaches for the prevention and treatment of LPS-induced anorexia.

  7. Regulation of the glutamate transporter EAAT3 by mammalian target of rapamycin mTOR.

    PubMed

    Almilaji, Ahmad; Pakladok, Tatsiana; Guo, Anne; Munoz, Carlos; Föller, Michael; Lang, Florian

    2012-05-04

    The serine/threonine kinase mammalian target of rapamycin (mTOR) is stimulated by insulin, growth factors and nutrients and confers survival of several cell types. The kinase has previously been shown to stimulate amino acid uptake. In neurons, the cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs) decreases excitation and thus confers protection against excitotoxicity. In epithelia, EAAT3 accomplishes transepithelial glutamate and aspartate transport. The present study explored, whether mTOR regulates EAAT3 (SLC1A1). To this end, cRNA encoding EAAT3 was injected into Xenopus oocytes with or without cRNA encoding mTOR and the glutamate induced current (I(glu)), a measure of glutamate transport, determined by dual electrode voltage clamp. Moreover, EAAT3 protein abundance was determined utilizing chemiluminescence. As a result, I(glu) was observed in Xenopus oocytes expressing EAAT3 but not in water injected oocytes. Coexpression of mTOR significantly increased I(glu), an effect reversed by rapamycin (100 nM). mTOR coexpression increased EAAT3 protein abundance in the cell membrane. The decay of I(glu) following inhibition of carrier insertion with brefeldin A in oocytes coexpressing EAAT3 with mTOR was similar in the presence and absence of rapamycin (100 nM). In conclusion, mTOR is a novel powerful regulator of EAAT3 and may thus contribute to protection against neuroexcitotoxicity.

  8. Phospholipase D Mediates Nutrient Input to Mammalian Target of Rapamycin Complex 1 (mTORC1)*

    PubMed Central

    Xu, Limei; Salloum, Darin; Medlin, Phil S.; Saqcena, Mahesh; Yellen, Paige; Perrella, Benjamin; Foster, David A.

    2011-01-01

    The mammalian target of rapamycin (mTOR) is a critical sensor of nutritional sufficiency. Although much is known about the regulation of mTOR in response to growth factors, much less is known about the regulation of mTOR in response to nutrients. Amino acids have no impact on the signals that regulate Rheb, a GTPase required for the activation of mTOR complex 1 (mTORC1). Phospholipase D (PLD) generates a metabolite, phosphatidic acid, that facilitates association between mTOR and the mTORC1 co-factor Raptor. We report here that elevated PLD activity in human cancer cells is dependent on both amino acids and glucose and that amino acid- and glucose-induced increases in mTORC1 activity are dependent on PLD. Amino acid- and glucose-induced PLD and mTORC1 activity were also dependent on the GTPases RalA and ARF6 and the type III phosphatidylinositol-3-kinase hVps34. Thus, a key stimulatory event for mTORC1 activation in response to nutrients is the generation of phosphatidic acid by PLD. PMID:21622984

  9. Mammalian target of rapamycin complex 1 is involved in differentiation of regenerating myofibers in vivo.

    PubMed

    Miyabara, Elen H; Conte, Talita C; Silva, Meiricris T; Baptista, Igor L; Bueno, Carlos; Fiamoncini, Jarlei; Lambertucci, Rafael H; Serra, Carmen S; Brum, Patricia C; Pithon-Curi, Tania; Curi, Rui; Aoki, Marcelo S; Oliveira, Antonio C; Moriscot, Anselmo S

    2010-11-01

    This work was undertaken to provide further insight into the role of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle regeneration, focusing on myofiber size recovery. Rats were treated or not with rapamycin, an mTORC1 inhibitor. Soleus muscles were then subjected to cryolesion and analyzed 1, 10, and 21 days later. A decrease in soleus myofiber cross-section area on post-cryolesion days 10 and 21 was accentuated by rapamycin, which was also effective in reducing protein synthesis in these freeze-injured muscles. The incidence of proliferating satellite cells during regeneration was unaltered by rapamycin, although immunolabeling for neonatal myosin heavy chain (MHC) was weaker in cryolesion+rapamycin muscles than in cryolesion-only muscles. In addition, the decline in tetanic contraction of freeze-injured muscles was accentuated by rapamycin. This study indicates that mTORC1 plays a key role in the recovery of muscle mass and the differentiation of regenerating myofibers, independently of necrosis and satellite cell proliferation mechanisms.

  10. Cross-Talk between NFkB and the PI3-Kinase/AKT Pathway Can Be Targeted in Primary Effusion Lymphoma (PEL) Cell Lines for Efficient Apoptosis

    PubMed Central

    Hussain, Azhar R.; Ahmed, Saeeda O.; Ahmed, Maqbool; Khan, Omar S.; Al AbdulMohsen, Sally; Platanias, Leonidas C.; Al-Kuraya, Khawla S.; Uddin, Shahab

    2012-01-01

    Background A number of constitutively activated signaling pathways play critical roles in the survival and growth of primary effusion lymphoma cells (PELs) including NFkB and PI3/AKT kinase cascades. NFkBis constitutively activated in a number of malignancies, including multiple myeloma, Burkitt’s lymphoma and diffuse large cell B-cell lymphoma. However, its role in primary effusion lymphoma has not been fully explored. Methodology/Principal Findings We used pharmacological inhibition and gene silencing to define the role of NFkB in growth and survival of PEL cells. Inhibition of NFkB activity by Bay11-7085 resulted in decreased expression of p65 in the nuclear compartment as detected by EMSA assays. In addition, Bay11-7085 treatment caused de-phosphorylation of AKT and its downstream targets suggesting a cross-talk between NFkB and the PI3-kinase/AKT pathway. Importantly, treatment of PEL cells with Bay11-7085 led to inhibition of cell viability and induced apoptosis in a dose dependent manner. Similar apoptotic effects were found when p65 was knocked down using specific small interference RNA. Finally, co-treatment of PEL cells with suboptimal doses of Bay11-7085 and LY294002 led to synergistic apoptotic responses in PEL cells. Conclusion/Significance These data support a strong biological-link between NFkB and the PI3-kinase/AKT pathway in the modulation of anti-apoptotic effects in PEL cells. Synergistic targeting of these pathways using NFKB- and PI3-kinase/AKT- inhibitors may have a therapeutic potential for the treatment of PEL and possibly other malignancies with constitutive activation of these pathways. PMID:22768179

  11. Mammalian Target of Rapamycin: Hitting the Bull's-Eye for Neurological Disorders

    PubMed Central

    Chong, Zhao Zhong; Shang, Yan Chen; Zhang, Lijie; Wang, Shaohui; Maiese, Kenneth

    2010-01-01

    The mammalian target of rapamycin (mTOR) and its associated cell signaling pathways have garnered significant attention for their roles in cell biology and oncology. Interestingly,the explosion of information in this field has linked mTOR to neurological diseases with promising initial studies. mTOR, a 289 kDa serine/threonine protein kinase, plays an important role in cell growth and proliferation and is activated through phosphorylation in response to growth factors, mitogens and hormones. Growth factors, amino acids, cellular nutrients and oxygen deficiency can downregulate mTOR activity. The function of mTOR signaling is mediated primarily through two mTOR complexes: mTORC1 and mTORC2. mTORC1 initiates cap-dependent protein translation, a rate-limiting step of protein synthesis, through the phosphorylation of the targets eukaryotic initiation factor 4E-binding protein 1 (4EBP1) and p70 ribosomal S6 kinase (p70S6K). In contrast, mTORC2 regulates development of the cytoskeleton and also controls cell survival. Although closely tied to tumorigenesis, mTOR and the downstream signaling pathways are significantly involved in the central nervous system (CNS) with synaptic plasticity, memory retention, neuroendocrine regulation associated with food intake and puberty and modulation of neuronal repair following injury. The signaling pathways of mTOR also are believed to be a significant component in a number of neurological diseases, such as Alzheimer disease, Parkinson disease and Huntington disease, tuberous sclerosis, neurofibromatosis, fragile X syndrome, epilepsy, traumatic brain injury and ischemic stroke. Here we describe the role of mTOR in the CNS and illustrate the potential for new strategies directed against neurological disorders. PMID:21307646

  12. Circadian regulation of mammalian target of rapamycin signaling in the mouse suprachiasmatic nucleus.

    PubMed

    Cao, R; Anderson, F E; Jung, Y-J; Dziema, H; Obrietan, K

    2011-05-05

    Circadian (24-h) rhythms influence virtually every aspect of mammalian physiology. The main rhythm generation center is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, and work over the past several years has revealed that rhythmic gene transcription and post-translational processes are central to clock timing. In addition, rhythmic translation control has also been implicated in clock timing; however the precise cell signaling pathways that drive this process are not well known. Here we report that a key translation activation cascade, the mammalian target of rapamycin (mTOR) pathway, is under control of the circadian clock in the SCN. Using phosphorylated S6 ribosomal protein (pS6) as a marker of mTOR activity, we show that the mTOR cascade exhibits maximal activity during the subjective day, and minimal activity during the late subjective night. Importantly, expression of S6 was not altered as a function of circadian time. Rhythmic S6 phosphorylation was detected throughout the dorsoventral axis of the SCN, thus suggesting that rhythmic mTOR activity was not restricted to a subset of SCN neurons. Rather, rhythmic pS6 expression appeared to parallel the expression pattern of the clock gene period1 (per1). Using a transgenic per1 reporter gene mouse strain, we found a statistically significant cellular level correlation between pS6 and per1 gene expression over the circadian cycle. Further, photic stimulation triggered a coordinate upregulation of per1 and mTOR activation in a subset of SCN cells. Interestingly, this cellular level correlation between mTOR activity and per1 expression appears to be specific, since a similar expression profile for pS6 and per2 or c-FOS was not detected. Finally, we show that mTOR activity is downstream of the ERK/MAPK signal transduction pathway. Together these data reveal that mTOR pathway activity is under the control of the SCN clock, and suggests that mTOR signaling may contribute to distinct aspects of the

  13. microRNA-133a regulates the cell cycle and proliferation of breast cancer cells by targeting epidermal growth factor receptor through the EGFR/Akt signaling pathway.

    PubMed

    Cui, Wenjing; Zhang, Shuai; Shan, Changliang; Zhou, Li; Zhou, Zhemin

    2013-08-01

    microRNAs are small, highly conserved, non-coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition, and are widely involved in carcinogenesis and cancer development. In this study, the expression profile of microRNA-133a (miR-133a) was examined in breast cancer cells and breast cancer tissues. The results showed that expression of miR-133a in both breast cancer cells and breast cancer tissues was significantly down-regulated. Over-expression of miR-133a in tumor cells arrested the cell cycle by drastically decreasing the G2 /S phase and retarded the newly synthesized DNA, suggesting a regulatory role for miR-133a in proliferation of breast cancer cells. Bioinformatics prediction showed that epidermal growth factor receptor (EGFR) is a potential target for miR-133a. A dual luciferase reporter gene assay showed that miR-133a bound to the 3' UTR of EGFR but not a mutated 3' UTR, thereby down-regulating the protein expression level. Accordingly, we found that expression of EGFR protein decreased with increased expression of miR-133a in MCF-7 and MDA-MB-231 cells. Over-expression of miR-133a in breast cancer cells resulted in suppression of the level of phosphorylated Akt protein (p-Akt) and inhibition of p-Akt nuclear translocation. These results demonstrate that miR-133a, which may act as a tumor suppressor in breast cancer, regulates the cell cycle and proliferation in tumorigenesis by targeting EGFR through the downstream signal molecule Akt. Overall, these results show that miR-133a may be used as biomarker and/or therapeutic target for diagnosis and therapy of breast cancer.

  14. Rictor/mammalian target of rapamycin 2 regulates the development of Notch1 induced murine T-cell acute lymphoblastic leukemia via forkhead box O3.

    PubMed

    Hua, Chunlan; Guo, Huidong; Bu, Jiachen; Zhou, Mi; Cheng, Hui; He, Fuhong; Wang, Jinhong; Wang, Xiaomin; Zhang, Yinchi; Wang, Qianfei; Zhou, Jianfeng; Cheng, Tao; Xu, Mingjiang; Yuan, Weiping

    2014-12-01

    Mammalian target of rapamycin (mTOR) is composed of two distinct biochemical complexes, mTORC1 and mTORC2. In response to nutrients and growth factors, mTORC1 is known to control cellular growth by regulating the translational regulators S6 kinase 1 and 4E binding protein 1, whereas mTORC2 mediates cell proliferation and survival by activating Akt through phosphorylation at Ser473. Studies have shown that the deregulation of mTORC2 leads to the development of myeloproliferative disorder and leukemia in the phosphatase and tensin homolog deleted on chromosome ten (PTEN)-deleted mouse model. However, the mechanism by which mTORC2 specifically affects leukemogenesis is still not fully understood. Here, we investigated the role of mTORC2 in NOTCH1-driven T-cell acute lymphoblastic leukemia (T-ALL) in a Rictor-deficient mouse model. We found that, by deleting Rictor, an essential component of mTORC2, leukemia progression was significantly suppressed by arresting a greater proportion of Rictor(△/△) leukemic cells at the G0 phase of the cell cycle. Furthermore, the absence of Rictor led to the overexpression of chemotaxis-related genes, such as CCR2, CCR4 and CXCR4, which contributed to the homing and migration of Rictor-deficient T-ALL cells to the spleen but not the bone marrow. In addition, we demonstrated that inactivation of mTORC2 caused the overexpression of forkhead box O3 and its downstream effectors and eased the progression of leukemia in T-ALL mice. Our study thus indicates that forkhead box O3 could be a potential drug target for the treatment of T-ALL leukemia.

  15. The novel orally bioavailable inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin, NVP-BEZ235, inhibits growth and proliferation in multiple myeloma

    SciTech Connect

    Baumann, Philipp Mandl-Weber, Sonja; Oduncu, Fuat; Schmidmaier, Ralf

    2009-02-01

    NVP-BEZ235 is a new inhibitor of phosphoinositol-3-kinase (PI3 kinase) and mammalian target of rapamycin (mTOR) whose efficacy in advanced solid tumours is currently being evaluated in a phase I/II clinical trial. Here we show that NVP-BEZ235 inhibits growth in common myeloma cell lines as well as primary myeloma cells at nanomolar concentrations in a time and dose dependent fashion. Further experiments revealed induction of apoptosis in three of four cell lines. Inhibition of cell growth was mainly due to inhibition of myeloma cell proliferation, as shown by the BrdU assay. Cell cycle analysis revealed induction of cell cycle arrest in the G1 phase, which was due to downregulation of cyclin D1, pRb and cdc25a. NVP-BEZ235 inhibited phosphorylation of protein kinase B (Akt), P70S6k and 4E-BP-1. Furthermore we show that the stimulatory effect of CD40-ligand (CD40L), insulin-like growth factor 1 (IGF-1), interleukin-6 (IL-6) and conditioned medium of HS-5 stromal cells on myeloma cell growth is completely abrogated by NVP-BEZ235. In addition, synergism studies revealed synergistic and additive activity of NVP-BEZ235 together with melphalan, doxorubicin and bortezomib. Taken together, inhibition of PI3 kinase/mTOR by NVP-BEZ235 is highly effective and NVP-BEZ235 represents a potential new candidate for targeted therapy in multiple myeloma.

  16. Contribution of mammalian target of rapamycin in the pathophysiology of cirrhotic cardiomyopathy

    PubMed Central

    Saeedi Saravi, Seyed Soheil; Ghazi-Khansari, Mahmoud; Ejtemaei Mehr, Shahram; Nobakht, Maliheh; Mousavi, Seyyedeh Elaheh; Dehpour, Ahmad Reza

    2016-01-01

    AIM: To explore the role of mammalian target of rapamycin (mTOR) in the pathogenesis of cirrhotic cardiomyopathy and the potential of rapamycin to improve this pathologic condition. METHODS: Male albino Wistar rats weighing 100-120 g were treated with tetrachloride carbon (CCl4) for 8 wk to induce cirrhosis. Subsequently, animals were administered rapamycin (2 mg/kg per day). The QTc intervals were calculated in a 5-min electrocardiogram. Then, the left ventricular papillary muscles were isolated to examine inotropic responsiveness to β-adrenergic stimulation using a standard organ bath equipped by Powerlab system. Phosphorylated-mTOR localization in left ventricles was immunohistochemically assessed, and ventricular tumor necrosis factor (TNF)-α was measured. Western blot was used to measure levels of ventricular phosphorylated-mTOR protein. RESULTS: Cirrhosis was confirmed by hematoxylin and eosin staining of liver tissues, visual observation of lethargy, weight loss, jaundice, brown urine, ascites, liver stiffness, and a significant increase of spleen weight (P < 0.001). A significant prolongation in QTc intervals occurred in cirrhotic rats exposed to CCl4 (P < 0.001), while this prolongation was decreased with rapamycin treatment (P < 0.01). CCl4-induced cirrhosis caused a significant decrease of contractile responsiveness to isoproterenol stimulation and a significant increase in cardiac TNF-α. These findings were correlated with data from western blot and immunohistochemical studies on phosphorylated-mTOR expression in left ventricles. Phosphorylated-mTOR was significantly enhanced in cirrhotic rats, especially in the endothelium, compared to controls. Rapamycin treatment significantly increased contractile force and myocardial localization of phosphorylated-mTOR and decreased cardiac TNF-α concentration compared to cirrhotic rats with no treatment. CONCLUSION: In this study, we demonstrated a potential role for cardiac mTOR in the pathophysiology of

  17. Inhibition of the mammalian target of rapamycin complex 1 signaling pathway reduces itch behaviour in mice.

    PubMed

    Obara, Ilona; Medrano, Maria C; Signoret-Genest, Jérémy; Jiménez-Díaz, Lydia; Géranton, Sandrine M; Hunt, Stephen P

    2015-08-01

    Activated mammalian target of rapamycin (P-mTOR) has been shown to maintain the sensitivity of subsets of small-diameter primary afferent A-nociceptors. Local or systemic inhibition of the mTOR complex 1 (mTORC1) pathway reduced punctate mechanical and cold sensitivity in neuropathic pain and therefore offered a new approach to chronic pain control. In this study, we have investigated the effects of the rapamycin analog temsirolimus (CCI-779) on itch. Bouts of scratching induced by the histamine-dependent pruritogenic compound 48/80 and histamine-independent pruritogens, chloroquine and SLIGRL-NH2, injected intradermally were significantly reduced by local (intradermal) or systemic (intraperitoneal, i.p.) pretreatment with CCI-779. We also investigated the action of metformin, a drug taken to control type 2 diabetes and recently shown to inhibit mTORC1 in vivo. Although the response to nonhistaminergic stimuli was reduced at all of the time points tested, scratching to compound 48/80 was modified by metformin only when the drug was injected 24 hours before this pruritogen. We also examined the colocalization of P-mTOR with gastrin-releasing peptide, a putative marker for some itch-sensitive primary afferents, and found that P-mTOR was coexpressed in less than 5% of gastrin-releasing peptide-positive fibers in the mouse skin. Taken together, the data highlight the role that P-mTOR-positive A-fibers play in itch signaling and underline the importance of the mTORC1 pathway in the regulation of homeostatic primary afferent functions such as pain and itch. The actions of the antidiabetic drug metformin in ameliorating nonhistamine-mediated itch also suggest a new therapeutic route for the control of this category of pruritus.

  18. Is proteinuria reversible, after withdrawal of mammalian target of rapamycin inhibitors?

    PubMed

    Arnau, A; Ruiz, J C; Rodrigo, E; Quintanar, J A; Arias, M

    2011-01-01

    Conversion to mammalian target of rapamycin inhibitors (mTORi) is an ever more frequent practice in renal transplant recipients, even if it is not always satisfactory, needing to be suspended for various reasons in certain patients. We analyzed the evolution of proteinuria as a marker of kidney damage after withdrawal of mTORi for any reason in order to assess conversion failure risk. Among 1633 renal transplant patients with 185 converted to mTORi, we considered the 52 (28%) who withdrew as result of intolerance or a bad evolution after at least 3 months use (median: 142 days after conversion). Four groups were defined according to the evolution of proteinuria: group 1 (G1), stable after conversion; group 2 (G2), increased with complete recovery (<1 g); group 3 (G3), increased with partial recovery (>1 g); or group 4 (G4), increased without recovery. The evolution according to the groups was: G1 (57.1%), G2 (17.2%), G3 (5.7%), and G4 (20%). There were no differences between the good (G1 and G2) and the bad evolution groups (G3 and G4) in proteinuria at the time of conversion (838 ± 641 vs 532 ± 404 mg/d) or renal function (1.95 ± 0.47 vs 1.90 ± 0.4 mg/dL). Six months after withdrawal, proteinuria was stable in G1 and G2 but worse in G3 and G4 (781 ± 643 vs 4479 ± 3235 mg/d); the same observation was noted for renal failure (2.1 ± 0.71 vs 2.8 ± 1.57 mg/dL). Among about 75% of patients in whom mTORi was withdrawn, no injury remained in the medium term whereas among the other 25%, there was a residual injury.

  19. AKT-STAT3 Pathway as a Downstream Target of EGFR Signaling to Regulate PD-L1 Expression on NSCLC cells.

    PubMed

    Abdelhamed, Sherif; Ogura, Keisuke; Yokoyama, Satoru; Saiki, Ikuo; Hayakawa, Yoshihiro

    2016-01-01

    While cancer development and progression can be controlled by cytotoxic T cells, it is also known that tumor-specific CD8(+)T cells become functionally impaired by acquiring a group of inhibitory receptors known as immune checkpoints. Amongst those, programmed death-1 (PD-1) is one of the most recognized negative regulators of T cell function. In non-small lung cancers (NSCLCs), the aberrant activation of epidermal growth factor receptor (EGFR) is known to induce PD-L1 expression and further the treatment with gefitinib, a tyrosine kinase inhibitor (TKI) for EGFR, decrease the expression of PD-L1 on NSCLC. Given the acquired resistance to gefitinib treatment frequently observed by developing secondary-site mutations limiting its efficacy, it is important to understand the downstream mechanism of activated-EGFR signaling for regulating PD-L1 in NSCLC. In this study, we demonstrated that AKT-STAT3 pathway could be a potential target for regulating the surface expression of PD-L1 on NSCLCs with aberrant EGFR activity and, further, the inhibition of AKT or STAT3 activity could down-regulate the expression of PD-L1 even in gefitinib-resistant NSCLCs. These results highlight an importance of AKT-STAT3 pathway as a promising target for potentiating anti-tumor immune responses by regulating PD-L1 expression on cancer cells with aberrant EGFR activity.

  20. miR-222 attenuates cisplatin-induced cell death by targeting the PPP2R2A/Akt/mTOR Axis in bladder cancer cells.

    PubMed

    Zeng, Li-Ping; Hu, Zheng-Mao; Li, Kai; Xia, Kun

    2016-03-01

    Increased miR-222 levels are associated with a poor prognosis in patients with bladder cancer. However, the role of miR-222 remains unclear. In the present study, we found that miR-222 enhanced the proliferation of both the T24 and the 5637 bladder cancer cell lines. Overexpression of miR-222 attenuated cisplatin-induced cell death in bladder cancer cells. miR-222 activated the Akt/mTOR pathway and inhibited cisplatin-induced autophagy in bladder cancer cells by directly targeting protein phosphatase 2A subunit B (PPP2R2A). Blocking the activation of Akt with LY294002 or mTOR with rapamycin significantly prevented miR-222-induced proliferation and restored the sensitivity of bladder cancer cells to cisplatin. These findings demonstrate that miR-222 modulates the PPP2R2A/Akt/mTOR axis and thus plays a critical role in regulating proliferation and chemotherapeutic drug resistance. Therefore, miR-222 may be a novel therapeutic target for bladder cancer.

  1. Akt- or MEK-mediated mTOR inhibition suppresses Nf1 optic glioma growth

    PubMed Central

    Kaul, Aparna; Toonen, Joseph A.; Cimino, Patrick J.; Gianino, Scott M.; Gutmann, David H.

    2015-01-01

    Background Children with neurofibromatosis type 1 (NF1) develop optic pathway gliomas, which result from impaired NF1 protein regulation of Ras activity. One obstacle to the implementation of biologically targeted therapies is an incomplete understanding of the individual contributions of the downstream Ras effectors (mitogen-activated protein kinase kinase [MEK], Akt) to optic glioma maintenance. This study was designed to address the importance of MEK and Akt signaling to Nf1 optic glioma growth. Methods Primary neonatal mouse astrocyte cultures were employed to determine the consequence of phosphatidylinositol-3 kinase (PI3K)/Akt and MEK inhibition on Nf1-deficient astrocyte growth. Nf1 optic glioma–bearing mice were used to assess the effect of Akt and MEK inhibition on tumor volume, proliferation, and retinal ganglion cell dysfunction. Results Both MEK and Akt were hyperactivated in Nf1-deficient astrocytes in vitro and in Nf1 murine optic gliomas in vivo. Pharmacologic PI3K or Akt inhibition reduced Nf1-deficient astrocyte proliferation to wild-type levels, while PI3K inhibition decreased Nf1 optic glioma volume and proliferation. Akt inhibition of Nf1-deficient astrocyte and optic glioma growth reflected Akt-dependent activation of mammalian target of rapamycin (mTOR). Sustained MEK pharmacologic blockade also attenuated Nf1-deficient astrocytes as well as Nf1 optic glioma volume and proliferation. Importantly, these MEK inhibitory effects resulted from p90RSK-mediated, Akt-independent mTOR activation. Finally, both PI3K and MEK inhibition reduced optic glioma–associated retinal ganglion cell loss and nerve fiber layer thinning. Conclusion These findings establish that the convergence of 2 distinct Ras effector pathways on mTOR signaling maintains Nf1 mouse optic glioma growth, supporting the evaluation of pharmacologic inhibitors that target mTOR function in future human NF1–optic pathway glioma clinical trials. PMID:25534823

  2. The Effects of Glucagon-like Peptide-2 on the Tight Junction and Barrier Function in IPEC-J2 Cells through Phosphatidylinositol 3-kinase–Protein Kinase B–Mammalian Target of Rapamycin Signaling Pathway

    PubMed Central

    Yu, Changsong; Jia, Gang; Deng, Qiuhong; Zhao, Hua; Chen, Xiaoling; Liu, Guangmang; Wang, Kangning

    2016-01-01

    Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that 100 μg/mL LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ’s expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway. PMID:26954146

  3. Structure-Activity Analysis of Niclosamide Reveals Potential Role for Cytoplasmic pH in Control of Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling*

    PubMed Central

    Fonseca, Bruno D.; Diering, Graham H.; Bidinosti, Michael A.; Dalal, Kush; Alain, Tommy; Balgi, Aruna D.; Forestieri, Roberto; Nodwell, Matt; Rajadurai, Charles V.; Gunaratnam, Cynthia; Tee, Andrew R.; Duong, Franck; Andersen, Raymond J.; Orlowski, John; Numata, Masayuki; Sonenberg, Nahum; Roberge, Michel

    2012-01-01

    Mammalian target of rapamycin complex 1 (mTORC1) signaling is frequently dysregulated in cancer. Inhibition of mTORC1 is thus regarded as a promising strategy in the treatment of tumors with elevated mTORC1 activity. We have recently identified niclosamide (a Food and Drug Administration-approved antihelminthic drug) as an inhibitor of mTORC1 signaling. In the present study, we explored possible mechanisms by which niclosamide may inhibit mTORC1 signaling. We tested whether niclosamide interferes with signaling cascades upstream of mTORC1, the catalytic activity of mTOR, or mTORC1 assembly. We found that niclosamide does not impair PI3K/Akt signaling, nor does it inhibit mTORC1 kinase activity. We also found that niclosamide does not interfere with mTORC1 assembly. Previous studies in helminths suggest that niclosamide disrupts pH homeostasis of the parasite. This prompted us to investigate whether niclosamide affects the pH balance of cancer cells. Experiments in both breast cancer cells and cell-free systems demonstrated that niclosamide possesses protonophoric activity in cells and in vitro. In cells, niclosamide dissipated protons (down their concentration gradient) from lysosomes to the cytosol, effectively lowering cytoplasmic pH. Notably, analysis of five niclosamide analogs revealed that the structural features of niclosamide required for protonophoric activity are also essential for mTORC1 inhibition. Furthermore, lowering cytoplasmic pH by means other than niclosamide treatment (e.g. incubation with propionic acid or bicarbonate withdrawal) recapitulated the inhibitory effects of niclosamide on mTORC1 signaling, lending support to a possible role for cytoplasmic pH in the control of mTORC1. Our data illustrate a potential mechanism for chemical inhibition of mTORC1 signaling involving modulation of cytoplasmic pH. PMID:22474287

  4. PI3K/Akt responses to oxytocin stimulation in Caco2BB gut cells.

    PubMed

    Klein, Benjamin Y; Tamir, Hadassah; Welch, Martha G

    2011-11-01

    Recently, we discovered oxytocin receptor (OTR) expression in the developing gut villus epithelium that emerges in villus-crypt junctions after weaning. Oxytocin (OT) and OTR regulate many physiological functions in various tissues; however, their function in gut epithelium is unknown. We explored responses of PI3K and Akt phosphoisoforms to OT stimuli in the Caco2BB human gut cell line. In Caco2BB cells, PI3K and pAkt levels peaked at 62.5  nM OT. At higher concentrations, PI3K decreased more gradually than pAkt(S473) suggesting that the pAkt(S473) response is separate from PI3K. At ≤7.8  nM OT, pAkt(T308) increased while pAkt(S473) decreased. Using a specific OTR antagonist, we demonstrated that responses of pAkt(T308) to OT depend on OTR in contrast to the partial OTR-dependence of the pAkt(S473) response. Differential pAkt phosphoisoform responses included pAkt phosphoserine 473 persistently free of phosphothreonine 308. The reduction in PI3K after 62.5  nM OT for 30  min coincided with OTR internalization. The PI3K/Akt activation profile was somewhat different in other cell lines (MCF-7 breast cancer cells, HT29 gut cells), which have PI3K activating mutations, that were examined to establish experimental parameters. In Caco2BB cells, the divergent effects of OT upon pAkt phosphoisoforms suggests separate sub-pathways; pAkt (T308) activation depends on OTR via the PI3K pathway and pAkt(S473) presumably results from its specific kinase mTORC2 (mammalian target of rapamycin complex 2). Thus, OT may modulate gut cell functions downstream of mTOR complexes (e.g., translation control as suggested by others in uterine cells). We will next explore OT-stimulated kinase activities downstream of mTOR related to pAkt phosphoisoforms.

  5. Angiographic and volumetric effects of mammalian target of rapamycin inhibitors on angiomyolipomas in tuberous sclerosis

    PubMed Central

    Sheth, Rahul A; Feldman, Adam S; Paul, Elahna; Thiele, Elizabeth A; Walker, T Gregory

    2016-01-01

    AIM: To investigate the angiographic and volumetric effects of mammalian target of rapamycin (mTOR) inhibitors on angiomyolipomas (AMLs) in a case series of patients with tuberous sclerosis complex. METHODS: All patients who underwent catheter angiography prior to and following mTOR inhibitor therapy (n = 3) were evaluated. All cross-sectional imaging studies were analyzed with three-dimensional volumetrics, and tumor volume curves for all three tissue compartments (soft tissue, vascular, and fat) were generated. Segmentation analysis tools were used to automatically create a region of interest (ROI) circumscribing the AML. On magnetic resonance images, the “fat only” map calculated from the in- and opposed-phase gradient recalled echo sequences was used to quantify fat volume within tumors. Tumor vascularity was measured by applying a thresholding tool within the ROI on post-contrast subtraction images. On computed tomography images, volume histogram analysis of Hounsfield unit was performed to quantify tumor tissue composition. The angiography procedures were also reviewed, and tumor vascularity based on pre-embolization angiography was characterized in a semi-quantitative manner. RESULTS: Patient 1 presented at the age of 15 with a 6.8 cm right lower pole AML and a 4.0 cm right upper pole AML. Embolization was performed of both tumors, and after a few years of size control, the tumors began to grow, and the patient was initiated on mTOR inhibitor therapy. There was an immediate reduction in the size of both lesions. The patient then underwent repeat embolization and discontinuation of mTOR inhibition, after which point there was a substantial regrowth in both tumors across all tissue compartments. Patient 2 presented at the age of 18 with a right renal AML. Following a brief period of tumor reduction after embolization, she was initiated on mTOR inhibitor therapy, with successful reduction in tumor size across all tissue compartments. As with patient 1

  6. GRP1 PH Domain, Like AKT1 PH Domain, Possesses a Sentry Glutamate Residue Essential for Specific Targeting to Plasma Membrane PI(3,4,5)P3

    PubMed Central

    Pilling, Carissa; Landgraf, Kyle E.; Falke, Joseph J.

    2011-01-01

    During the appearance of the signaling lipid PI(3,4,5)P3, an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P3-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P2 and bind the rare PI(3,4,5)P3 target lipid with sufficiently high affinity. Our previous study of the E17K mutant of protein kinase B (AKT1) PH domain, together with evidence from Carpten et al (1), revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P2, thereby playing an essential role in specific PI(3,4,5)P3 targeting (2). The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P3-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P2 affinity and constitutive plasma membrane targeting. To test this hypothesis the present study investigates the E345 residue, a putative sentry glutamate, of General Receptor for Phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into GRP1 PH domain enhances PI(4,5)P2 affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P2 releases E345K GRP1 PH domain into the cytoplasm and the efficiency of this release increases when target Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K (1, 3). Analysis of available PH domain structures suggests that a lone glutamate residue (or, in some cases an aspartate) is a common, perhaps ubiquitous, feature of PI(3,4,5)P3-specific binding

  7. Metabonomics applied in exploring the antitumour mechanism of physapubenolide on hepatocellular carcinoma cells by targeting glycolysis through the Akt-p53 pathway

    PubMed Central

    Ma, Ting; Fan, Bo-Yi; Zhang, Chao; Zhao, Hui-Jun; Han, Chao; Gao, Cai-Yun; Luo, Jian-Guang; Kong, Ling-Yi

    2016-01-01

    Metabolomics can be used to identify potential markers and discover new targets for future therapeutic interventions. Here, we developed a novel application of the metabonomics method based on gas chromatography-mass spectrometry (GC/MS) analysis and principal component analysis (PCA) for rapidly exploring the anticancer mechanism of physapubenolide (PB), a cytotoxic withanolide isolated from Physalis species. PB inhibited the proliferation of hepatocellular carcinoma cells in vitro and in vivo, accompanied by apoptosis-related biochemical events, including the cleavage of caspase-3/7/9 and PARP. Metabolic profiling analysis revealed that PB disturbed the metabolic pattern and significantly decreased lactate production. This suggests that the suppression of glycolysis plays an important role in the anti-tumour effects induced by PB, which is further supported by the decreased expression of glycolysis-related genes and proteins. Furthermore, the increased level of p53 and decreased expression of p-Akt were observed, and the attenuated glycolysis and enhanced apoptosis were reversed in the presence of Akt cDNA or p53 siRNA. These results confirm that PB exhibits anti-cancer activities through the Akt-p53 pathway. Our study not only reports for the first time the anti-tumour mechanism of PB, but also suggests that PB is a promising therapeutic agent for use in cancer treatments and that metabolomic approaches provide a new strategy to effectively explore the molecular mechanisms of promising anticancer compounds. PMID:27416811

  8. A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

    NASA Astrophysics Data System (ADS)

    Deyle, Kaycie M.; Farrow, Blake; Qiao Hee, Ying; Work, Jeremy; Wong, Michelle; Lai, Bert; Umeda, Aiko; Millward, Steven W.; Nag, Arundhati; Das, Samir; Heath, James R.

    2015-05-01

    Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate.

  9. Activation of mammalian target of rapamycin signaling in skeletal muscle of neonatal chicks: effects of dietary leucine and age.

    PubMed

    Deng, Huiling; Zheng, Aijuan; Liu, Guohua; Chang, Wenhuan; Zhang, Shu; Cai, Huiyi

    2014-01-01

    The mammalian target of rapamycin (mTOR) signaling pathway is necessary for cellular protein synthesis regulation. Leucine was reported to stimulate muscle protein synthesis in mammalian embryos and neonates, but in higher animals (chickens) the effect of dietary leucine on mTOR signaling is unknown. Thus, we investigated the effects of dietary leucine and age on mRNA expression and phosphorylation of mTOR as well as its downstream targets, ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in chick pectoral muscles. One hundred eighty newly hatched male chicks were randomly assigned to 1 of 3 dietary leucine treatment groups (1.43, 1.73, and 2.03% leucine) for 14 d, respectively. Each treatment group consisted of 6 cages with 10 chicks each. On d 3, 7, and 14, plasma insulin and leucine were measured and target gene expression and phosphorylation was assessed. Dietary leucine influenced plasma leucine but not insulin, and plasma leucine and insulin declined with chick age. The mTOR, S6K1, and 4E-BP1 mRNA expression and phosphorylation within chick pectoral muscles were upregulated with increased dietary leucine but downregulated with increased chick age. Thus, high dietary leucine activates target of rapamycin signaling pathways in skeletal muscle of neonatal chicks to stimulate muscle protein synthesis, and this pathway is attenuated with aging.

  10. Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells

    PubMed Central

    Bressan, Raul Bardini; Dewari, Pooran Singh; Kalantzaki, Maria; Gangoso, Ester; Matjusaitis, Mantas; Garcia-Diaz, Claudia; Blin, Carla; Grant, Vivien; Bulstrode, Harry; Gogolok, Sabine; Skarnes, William C.

    2017-01-01

    Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable – experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis. PMID:28096221

  11. Role of inhibitors of mammalian target of rapamycin in the treatment of luminal breast cancer.

    PubMed

    Ciruelos, Eva; Cortes-Funes, Hernán; Ghanem, Ismael; Manso, Luis; Arteaga, Carlos

    2013-09-01

    Approximately 75% of patients with breast cancer present hormone receptor-positive tumors. This subtype of breast cancer initially shows a high overall response rate to hormonal treatments. However, resistance eventually develops, resulting in tumor progression. The PI3K/Akt/mTOR pathway regulates several cellular functions in cancer such as cell growth, survival, and proliferation. In addition, a high activation level of the PI3K/Akt/mTOR pathway is related to resistance to conventional chemotherapy and hormone therapy. The mTOR inhibitor everolimus, in combination with hormonal treatments, has led to excellent results in progression-free survival in patients with metastatic breast cancer resistant to hormone therapies. Therefore, everolimus has entered the National Comprehensive Cancer Network (NCCN) guidelines 2012 and its combination with exemestane was approved recently by the US Food and Drug Administration and the European Medicines Agency. This is the first time that a drug will have been approved for the restoration of hormone sensitivity in breast cancer.

  12. Akt Regulates TNFα Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

    PubMed Central

    McNamara, Colleen R.; Ahuja, Ruchita; Osafo-Addo, Awo D.; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D.; Gerber, Scott; Degterev, Alexei

    2013-01-01

    Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation. PMID:23469174

  13. Akt Regulates TNFα synthesis downstream of RIP1 kinase activation during necroptosis.

    PubMed

    McNamara, Colleen R; Ahuja, Ruchita; Osafo-Addo, Awo D; Barrows, Douglas; Kettenbach, Arminja; Skidan, Igor; Teng, Xin; Cuny, Gregory D; Gerber, Scott; Degterev, Alexei

    2013-01-01

    Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

  14. Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

    SciTech Connect

    Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

    2008-04-01

    Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3{beta} (GSK-3{beta}) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-{alpha} (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.

  15. MicroRNA-21 plays an oncogenic role by targeting FOXO1 and activating the PI3K/AKT pathway in diffuse large B-cell lymphoma

    PubMed Central

    Kim, Pil-Jong; Kim, Young-Goo; Nam, Soo Jeong; Paik, Jin Ho; Kim, Tae Min; Heo, Dae Seog; Kim, Chul-Woo; Jeon, Yoon Kyung

    2015-01-01

    The prognostic implications of miR-21, miR-17-92 and miR-155 were evaluated in diffuse large B-cell lymphoma (DLBCL) patients, and novel mechanism by which miR-21 contributes to the oncogenesis of DLBCL by regulating FOXO1 and PI3K/AKT/mTOR pathway was investigated. The expressions of miR-21, miR-17-92 and miR-155 measured by quantitative reverse-transcription-PCR were significantly up-regulated in DLBCL tissues (n=200) compared to control tonsils (P=0.012, P=0.001 and P<0.0001). Overexpression of miR-21 and miR-17-92 was significantly associated with shorter progression-free survival (P=0.003 and P=0.014) and overall survival (P=0.004 and P=0.012). High miR-21 was an independent prognostic factor in DLBCL patients treated with rituximab-combined chemotherapy. MiR-21 level was inversely correlated with the levels of FOXO1 and PTEN in DLBCL cell lines. Reporter-gene assay showed that miR-21 directly targeted and suppressed the FOXO1 expression, and subsequently inhibited Bim transcription in DLBCL cells. MiR-21 also down-regulated PTEN expression and consequently activated the PI3K/AKT/mTOR pathway, which further decreased FOXO1 expression. Moreover, miR-21 inhibitor suppressed the expression and activity of MDR1, thereby sensitizing DLBCL cells to doxorubicin. These data demonstrated that miR-21 plays an important oncogenic role in DLBCL by modulating the PI3K/AKT/mTOR/FOXO1 pathway at multiple levels resulting in strong prognostic implication. Therefore, targeting miR-21 may have therapeutic relevance in DLBCL. PMID:25909227

  16. MiR-148a Functions as a Tumor Suppressor by Targeting CCK-BR via Inactivating STAT3 and Akt in Human Gastric Cancer

    PubMed Central

    Su, Liping; Li, Jianfang; Yu, Yingyan; Gu, Qinlong; Yan, Min; Zhu, Zhenggang; Liu, Bingya

    2016-01-01

    MicroRNAs (miRNAs) have been widely accepted as a class of gene expression regulators which post-translationally regulate protein expression. These small noncoding RNAs have been proved closely involved in the modulation of various pathobiological processes in cancer. In this research, we demonstrated that miR-148a expression was significantly down-regulated in gastric cancer tissues in comparison with the matched normal mucosal tissues, and its expression was statistically associated with lymph node metastasis. Ectopic expression of miR-148a inhibited tumor cell proliferation and migration in vitro, and inhibited tumor formation in vivo. Subsequently, we identified cholecystokinin B receptor (CCK-BR) as a direct target of miR-148a using western blot and luciferase activity assay. More importantly, siRNA-induced knockdown of CCK-BR elicited similar anti-oncogenic effects (decreased proliferation and migration) as those induced by enforced miR-148a expression. We also found that miR-148a-mediated anti-cancer effects are dependent on the inhibition of STAT3 and Akt activation, which subsequently regulates the pathways involved in cell proliferation and migration. Taken together, our results suggest that miR-148a serves as a tumor suppressor in human gastric carcinogenesis by targeting CCK-BR via inactivating STAT3 and Akt. PMID:27518872

  17. MicroRNA-130b targets PTEN to mediate drug resistance and proliferation of breast cancer cells via the PI3K/Akt signaling pathway

    PubMed Central

    Miao, Yuan; Zheng, Wei; Li, Nana; Su, Zhen; Zhao, Lifen; Zhou, Huimin; Jia, Li

    2017-01-01

    Multidrug resistance (MDR) correlates with treatment failure and poor prognosis among breast cancer patients. This study was aimed to investigate the possible mechanism by which microRNA-130b-3p (miR-130b) mediates the chemoresistance and proliferation of breast cancer. MiR-130b was found to be up-regulated in tumor tissues versus adjacent tissues of breast cancer, as well as in adriamycin (ADR) resistant breast cancer cell line (MCF-7/ADR) versus its parental line (MCF-7) and the non-malignant breast epithelial cell line (MCF-10A), demonstrating its crucial relevance for breast cancer biology. We identified that PTEN was a direct target of miR-130b and inversely correlated with miR-130b expression in breast cancer. Moreover, over-expression of miR-130b promoted drug resistance, proliferation and decreased apoptosis of MCF-7 cells, while suppression of miR-130b enhanced drug cytotoxicity and apoptosis, as well as reduced proliferation of MCF-7/ADR cells in vitro and in vivo. Particularly, miR-130b mediated the activity of phosphoinositide-3 kinase (PI3K)/Akt signaling pathway as well as the chemoresistance and proliferation of breast cancer cell lines, which was partially blocked following knockdown of PTEN. Altogether, miR-130b targets PTEN to induce MDR, proliferation, and apoptosis via PI3K/Akt signaling pathway. This provides a novel promising candidate for breast cancer therapy. PMID:28165066

  18. EGFR-PI3K-AKT-mTOR Signaling in Head and Neck Squamous Cell Carcinomas - Attractive Targets for Molecular-Oriented Therapy

    PubMed Central

    Freudlsperger, Christian; Burnett, Jeffrey R.; Friedman, Jay A.; Kannabiran, Vishnu R.; Chen, Zhong; Van Waes, Carter

    2012-01-01

    Importance of the field Recent advances in understanding the oncogenesis of head and neck squamous cell carcinomas (HNSCC) have revealed multiple dysregulated signaling pathways. One frequently altered axis is the EGFR/PI3K/Akt/mTOR pathway. This pathway plays a central role in numerous cellular processes including metabolism, cell growth, apoptosis, survival and differentiation, which ultimately contributes to HNSCC progression. What the reader will gain This article reviews the current understanding of EGFR/PI3K/Akt/mTOR signaling in HNSCC, including the impact of both genetic and epigenetic alterations. This review further highlights the potential of targeting this signaling cascade as a promising therapeutic approach in the treatment of HNSCC. Areas covered in this review Books, journals, databases and websites have been searched to provide a current review on the subject. Take home message Genetic alterations of several nodes within this pathway, including both genetic and epigenetic changes, leading to either oncogene activation or inactivation of tumor suppressors, have frequently been implicated in HNSCC. Consequently, drugs that target the central nodes of this pathway have become attractive for molecular oriented cancer therapies. Numerous preclinical and clinical studies are being performed in HNSCC, however, more studies are still needed to better understand the biology of this pathway. PMID:21110697

  19. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

    NASA Astrophysics Data System (ADS)

    Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

    2015-07-01

    Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

  20. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

    PubMed Central

    Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

    2015-01-01

    Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein–DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells. PMID:26151127

  1. MicroRNA-126 affects rheumatoid arthritis synovial fibroblast proliferation and apoptosis by targeting PIK3R2 and regulating PI3K-AKT signal pathway

    PubMed Central

    Deng, Jia-Xin; Zhang, Yu-Ping; Liang, Wan-Yi; Jiang, Zhen-Lan; Yu, Qing-Hong; Li, Juan

    2016-01-01

    Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes inflammation and destruction of the joints as well as an increased risk of cardiovascular disease. RA synovial fibroblasts (RASFs) are involved in the progression of RA and release pro-inflammatory cytokines. On the other hand, microRNAs (miRs) may help control the inflammatory response of immune and non-immune cells. Therefore, our study used lentiviral expression vectors to test the effects of miR-126 overexpression on RASF proliferation and apoptosis. Luciferase experiments verified the targeting relationship between miR-126 and PIK3R2 gene. The co-transfection of anti-miR-126 and PIK3R2 siRNA to RASFs were used to identify whether PIK3R2 was directly involved in proliferation and apoptosis of miR-126-induced RASFs. Real-time polymerase chain reaction (PCR) was used to detect miR-126 and PIK3R2 expressions. MTT assay was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis and cell cycle. Western blotting was used to detect PIK3R2, PI3K, AKT and p-AKT proteins. After Lv-miR-126 infected RASFs, the relative expression of miR-126 was significantly enhanced. MiR-126 promoted RASF proliferation and inhibited apoptosis. Levels of PIK3R2 decreased while total PI3K and p-AKT levels increased in RASFs overexpressing miR-126. Co-transfection of anti-miR-126 and PIK3R2 siRNA also increased PI3K and p-AKT levels as well as RASF proliferation and reduced apoptosis, as compared to anti-miR-126 treatment alone. Finally, luciferase reporter assays showed that miR-126 targeted PIK3R2. Our data indicate that miR-126 overexpression in RASFs inhibits PIK3R2 expression and promotes proliferation while inhibiting apoptosis. This suggests inhibiting miR-126 may yield therapeutic benefits in the treatment of RA. PMID:27729613

  2. MiR-34a targets GAS1 to promote cell proliferation and inhibit apoptosis in papillary thyroid carcinoma via PI3K/Akt/Bad pathway

    SciTech Connect

    Ma, Yanfei; Qin, Huadong; Cui, Yunfu

    2013-11-29

    Highlights: •MiR-34a is up- and GAS1 is down-regulated in papillary thyroid carcinoma. •GAS1 is a direct target for miR-34a. •MiR-34a promotes PTC cells proliferation and inhibits apoptosis through PI3K/Akt/Bad pathway. -- Abstract: MicroRNAs (miRNAs) are fundamental regulators of cell proliferation, differentiation, and apoptosis, and are implicated in tumorigenesis of many cancers. MiR-34a is best known as a tumor suppressor through repression of growth factors and oncogenes. Growth arrest specific1 (GAS1) protein is a tumor suppressor that inhibits cancer cell proliferation and induces apoptosis through inhibition of RET receptor tyrosine kinase. Both miR-34a and GAS1 are frequently down-regulated in various tumors. However, it has been reported that while GAS1 is down-regulated in papillary thyroid carcinoma (PTC), miR-34a is up-regulated in this specific type of cancer, although their potential roles in PTC tumorigenesis have not been examined to date. A computational search revealed that miR-34a putatively binds to the 3′-UTR of GAS1 gene. In the present study, we confirmed previous findings that miR-34a is up-regulated and GAS1 down-regulated in PTC tissues. Further studies indicated that GAS1 is directly targeted by miR-34a. Overexpression of miR-34a promoted PTC cell proliferation and colony formation and inhibited apoptosis, whereas knockdown of miR-34a showed the opposite effects. Silencing of GAS1 had similar growth-promoting effects as overexpression of miR-34a. Furthermore, miR-34a overexpression led to activation of PI3K/Akt/Bad signaling pathway in PTC cells, and depletion of Akt reversed the pro-growth, anti-apoptotic effects of miR-34a. Taken together, our results demonstrate that miR-34a regulates GAS1 expression to promote proliferation and suppress apoptosis in PTC cells via PI3K/Akt/Bad pathway. MiR-34a functions as an oncogene in PTC.

  3. Localization and Targeting of Voltage-Gated Ion Channels in Mammalian Central Neurons

    PubMed Central

    Vacher, Helene; Mohapatra, Durga P.; Trimmer, James S.

    2008-01-01

    The intrinsic electrical properties and the synaptic input-output relationships of neurons are governed by the action of voltage-dependent ion channels. The localization of specific population of ion channels with distinct functional properties at discrete sites in neurons dramatically impacts excitability and synaptic transmission. Molecular cloning studies have revealed a large family of genes encoding voltage-dependent ion channel principal and auxiliary subunits, most of which are expressed in mammalian central neurons. Much recent effort has focused on determining which of these subunits co-assemble into native neuronal channel complexes, and the cellular and subcellular distributions of these complexes, as a crucial step in understanding the contribution of these channels to specific aspects of neuronal function. Here we review progress made on recent studies aimed at determining the cellular and subcellular distribution of specific ion channel subunits in mammalian brain neurons using in situ hybridization, and immunohistochemistry. We also discuss the repertoire of ion channel subunits in specific neuronal compartments and implications for neuronal physiology. Finally, we discuss the emerging mechanisms for determining the discrete subcellular distributions observed for many neuronal ion channels. PMID:18923186

  4. CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet {beta}-cells mediated by hepatocyte growth factor

    SciTech Connect

    Li, Xin-Yu; Zhan, Xiao-Rong; Liu, Xiao-Min; Wang, Xiao-Chen

    2011-01-14

    Research highlights: {yields} CREB is a regulatory target for the protein kinase Akt/PKB in pancreatic duct cells. {yields} Activation of the PI3K/AKT/CREB pathway plays a critical role in the HGF-mediated differentiation of pancreatic duct cells in vivo. {yields} CREB was causally linked to the expression of transcription factors during PDEC differentiation induced by HGF. -- Abstract: We have previously reported that the PI3K/Akt signaling pathway is involved in hepatocyte growth factor (HGF)-induced differentiation of adult rat pancreatic ductal epithelial cells (PDECs) into islet {beta}-cells in vitro. The transcription factor CREB is one of the downstream key effectors of the PI3K/Akt signaling pathway. Recent studies showing that CREB is required for the survival of certain cell types prompted us to examine whether CREB is a nuclear target for activation via the HGF-dependent Ser/Thr kinase Akt/PKB in the differentiation of pancreatic duct cell into islet {beta}-cells. In this study, we first attempted to examine whether HGF modulates the Akt-dependent activation of target gene CREB and then investigated whether CREB activity affects the differentiation of HGF-induced PDECs. Finally, we studied the role of CREB in modulating the expression of transcription factors in PDECs during the differentiation of HGF-induced PDECs. Our results demonstrated that CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet {beta}-cells mediated by HGF.

  5. Akt activation enhances ribosomal RNA synthesis through casein kinase II and TIF-IA.

    PubMed

    Nguyen, Le Xuan Truong; Mitchell, Beverly S

    2013-12-17

    Transcription initiation factor I (TIF-IA) plays an essential role in regulating ribosomal RNA (rRNA) synthesis by tethering RNA polymerase I (Pol I) to the rDNA promoter. We have found that activated Akt enhances rRNA synthesis through the phosphorylation of casein kinase IIα (CK2α) on a threonine residue near its N terminus. CK2 in turn phosphorylates TIF-IA, thereby increasing rDNA transcription. Activated Akt also stabilizes TIF-IA, induces its translocation to the nucleolus, and enhances its interaction with Pol I. Treatment with AZD8055, an inhibitor of both Akt and mammalian target of rapamycin phosphorylation, but not with rapamycin, disrupts Akt-mediated TIF-IA stability, translocation, and activity. These data support a model in which activated Akt enhances rRNA synthesis both by preventing TIF-IA degradation and phosphorylating CK2α, which in turn phosphorylates TIF-IA. This model provides an explanation for the ability of activated Akt to promote cell proliferation and, potentially, transformation.

  6. A monoclonal antibody (Mc178-Ab) targeted to the ecto-ATP synthase β-subunit-induced cell apoptosis via a mechanism involving the MAPKase and Akt pathways.

    PubMed

    Wang, Wen-Juan; Ma, Zhan; Liu, Yi-Wen; He, Yi-Qing; Wang, Ying-Zhi; Yang, Cui-Xia; Du, Yan; Zhou, Mu-Qing; Gao, Feng

    2012-03-01

    Ecto-ATP synthase has been considered to be an effective target for cancer recently. As inhibitors of ecto-ATP synthase were found to be cytotoxic for tumor cells, a monoclonal antibody (Mc178-Ab) against ecto-ATP synthase was generated in our previous study that exhibited both anti-angiogenic and anti-tumorigenic effects. However, the mechanism of action of Mc178-Ab and its downstream pathways for anti-tumor effects remain unclear. In this research, we intended to investigate the mechanism of the anti-tumor action of Mc178-Ab. The expressions of cell surface ATP synthase on A549 and CHO cells were confirmed by flow cytometry and confocal microscope. Proliferation and apoptosis were examined after the treatment with Mc178-Ab. In order to examine the activity of ecto-ATP synthase changed by Mc178-Ab, extracellular ATP generation and intracellular pH levels were assessed. The phosphorylation of the signaling molecules, MAPKase and Akt, was analyzed by western blot. Cell proliferation was blocked, and apoptosis was induced in A549 cells treated with Mc178-Ab, as determined by MTT assay and flow cytometry analysis of Annexin-V/PI staining separately. The intracellular pH level and extracellular ATP generation were also decreased after Mc178-Ab treatment. Finally, western blot data revealed that the phosphorylation of JNK and p38 was increased, while the phosphorylation of ERK and Akt was decreased in A549 cells treated with Mc178-Ab. Compared with A549 cells, Mc178-Ab had less effect on CHO cells. The decreased intracellular pH levels and the altered concentration of extracellular ATP may contribute to the mechanisms of the effect of Mc178-Ab on A549 and CHO cells. The results also suggested that the anti-tumor effect of Mc178-Ab was associated with MAPKase and Akt pathways.

  7. AKT/PKB Signaling: Navigating Downstream

    PubMed Central

    Manning, Brendan D.; Cantley, Lewis C.

    2009-01-01

    The serine/threonine kinase Akt, also known as protein kinase B (PKB), is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Aberrant loss or gain of Akt activation underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes and cancer. Here, we review the molecular properties of Akt and the approaches used to characterize its true cellular targets. In addition, we discuss those Akt substrates that are most likely to contribute to the diverse cellular roles of Akt, which include cell survival, growth, proliferation, angiogenesis, metabolism, and migration. PMID:17604717

  8. The natural compound sulforaphene, as a novel anticancer reagent, targeting PI3K-AKT signaling pathway in lung cancer

    PubMed Central

    Liu, Weilin; Kuang, Pengqun; Liang, Hao; Yuan, Qipeng

    2016-01-01

    Lung cancer is one of the leading causes of cancer death worldwide. Isothiocyanates from cruciferous vegetables been shown to possess anticarcinogenic activities in lung malignances. We previously found sulforaphene (4-methylsufinyl-3-butenyl isothiocyanate, SFE), one new kind of isothiocyanates, existing in a relative high abundance in radish seeds. An efficient methodology based on macroporous resin and preparative high-performance liquid chromatography was developed to isolate SFE in reasonably large quantities, high purity and low cost. However, it is still largely unclear whether SFE could function as an antineoplastic compound, especially in lung cancer. In this study, we systematically investigated the anti-cancer effects of SFE in vitro as well as its possible underling molecular mechanisms in lung cancer. The acute toxicity tests and pharmacokinetics tests for SFE were performed to evaluate its drugability in mice. Also, we evaluated the in vivo anti-cancer effects of SFE using nude Balb/C mice with lung cancer xenograft. SFE can induce apoptosis of multiple lung cancer celllines and, thus, inhibited cancer cell proliferation. Lung cancer cells treated with SFE exhibit significant inhibition of the PI3K-AKT signaling pathway, including depressed PTEN expression and inhibition of AKT phosphoralation. At well-tolerated doses, administration of SFE to mice bearing lung cancer xenografts leads to significant inhibitions of tumor growth. In summary, our work identifies SFE as a novel natural broad-spectrum small molecule inhibitor for lung cancer. PMID:27765931

  9. Black raspberry extracts inhibit benzo(a)pyrene diol-epoxide-induced activator protein 1 activation and VEGF transcription by targeting the phosphotidylinositol 3-kinase/Akt pathway.

    PubMed

    Huang, Chuanshu; Li, Jingxia; Song, Lun; Zhang, Dongyun; Tong, Qiangsong; Ding, Min; Bowman, Linda; Aziz, Robeena; Stoner, Gary D

    2006-01-01

    Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway.

  10. A new functional role for mechanistic/mammalian target of rapamycin complex 1 (mTORC1) in the circadian regulation of L-type voltage-gated calcium channels in avian cone photoreceptors.

    PubMed

    Huang, Cathy Chia-Yu; Ko, Michael Lee; Ko, Gladys Yi-Ping

    2013-01-01

    In the retina, the L-type voltage-gated calcium channels (L-VGCCs) are responsible for neurotransmitter release from photoreceptors and are under circadian regulation. Both the current densities and protein expression of L-VGCCs are significantly higher at night than during the day. However, the underlying mechanisms of circadian regulation of L-VGCCs in the retina are not completely understood. In this study, we demonstrated that the mechanistic/mammalian target of rapamycin complex (mTORC) signaling pathway participated in the circadian phase-dependent modulation of L-VGCCs. The activities of the mTOR cascade, from mTORC1 to its downstream targets, displayed circadian oscillations throughout the course of a day. Disruption of mTORC1 signaling dampened the L-VGCC current densities, as well as the protein expression of L-VGCCs at night. The decrease of L-VGCCs at night by mTORC1 inhibition was in part due to a reduction of L-VGCCα1 subunit translocation from the cytosol to the plasma membrane. Finally, we showed that mTORC1 was downstream of the phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT) signaling pathway. Taken together, mTORC1 signaling played a role in the circadian regulation of L-VGCCs, in part through regulation of ion channel trafficking and translocation, which brings to light a new functional role for mTORC1: the modulation of ion channel activities.

  11. Targeting the PI3K/AKT pathway via GLI1 inhibition enhanced the drug sensitivity of acute myeloid leukemia cells

    PubMed Central

    Liang, Hui; Zheng, Qi-Li; Fang, Peng; Zhang, Jian; Zhang, Tuo; Liu, Wei; Guo, Min; Robinson, Christopher L.; Chen, Shui-bing; Chen, Xiao-Ping; Chen, Fang-Ping; Zeng, Hui

    2017-01-01

    Combination targeted therapy is commonly used to treat acute myeloid leukemia (AML) patients, particularly in refractory/relapse (RR) population. However, concerns have been raised regarding the safety and patient tolerance of combination chemotherapy. It is critical to choose the appropriate treatment for precision therapy. We performed genome-wide RNA profiling using RNA-Seq to compare the RR group and the complete remission (CR) group (a total of 42 adult AML patients). The Hedgehog (Hh) and PI3K/AKT pathways were upregulated in the RR population, which was further confirmed by western blot and/or qPCR. Overexpression of GLI1 in AML cells led to increased AKT phosphorylation and decreased drug sensitivity, which was attenuated by GLI1 inhibition. By contrast, neither the expression of GLI1 nor apoptosis in response to Ara-C treatment of AML cells was significantly affected by PI3K inhibition. Furthermore, co-inhibition of GLI1 and PI3K induced apoptosis of hematopoietic stem/progenitor cells (HSPCs), which raised serious concerns about the side effects of this treatment. These results indicated that GLI1 inhibition alone, but not combined inhibition, is sufficient to enhance AML drug sensitivity, which provides a novel therapeutic strategy for AML treatment. PMID:28098170

  12. Diosmetin inhibits cell proliferation and induces apoptosis by regulating autophagy via the mammalian target of rapamycin pathway in hepatocellular carcinoma HepG2 cells

    PubMed Central

    Liu, Jie; Ren, Hao; Liu, Bin; Zhang, Qingyu; Li, Mingyi; Zhu, Runzhi

    2016-01-01

    Hepatocellular carcinoma (HCC), which is a type of malignant tumor, is the fifth most common cancer in men and ninth in women worldwide. The aim of the present study was to investigate the antitumor effect of diosmetin (DIOS) in hepatocellular carcinoma HepG2 cells. The proliferation, apoptosis and autophagy rates of HepG2 cells were measured following treatment with DIOS. The effects of DIOS treatment on HepG2 cell proliferation and apoptosis rates were analyzed using MTT assays and Annexin V staining, respectively. The effect of DIOS treatment on autophagy levels was assessed using transmission electron microscopy, green fluorescent protein (GFP)-microtubule-associated protein 1 light chain (LC3) transfection and LysoTracker Red staining. Furthermore, bafilomycin A1 (BA1), an autophagy inhibitor, was used to assess the association between DIOS and cell autophagy, proliferation and apoptosis. In addition, the expression of autophagy-related proteins [mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase, P70S6K, phosphoinositide-dependent kinase-1, extracellular signal-regulated kinase, 5′-AMP-activated protein kinase and Akt] and apoptosis-related proteins [B-cell lymphoma (Bcl)-2-associated X protein, Bak, p53, Bcl-2 and caspase-3] were analyzed by western blotting. The results revealed that DIOS significantly inhibited proliferation (P<0.01) and induced apoptosis (P<0.001) in HepG2 cells. It was also demonstrated that DIOS triggered autophagy by regulating the mTOR pathway in HepG2 cells. Notably, following treatment of HepG2 cells with the autophagy inhibitor, BA1, the expression of apoptosis-related proteins, including Bax, Bak and p53, were significantly decreased (P<0.05), and cell viability was recovered to a certain extent. In conclusion, DIOS inhibits cell proliferation and induces apoptosis in HepG2 cells via regulation of the mTOR pathway. Thus, the results of the current study indicate that DIOS may present a potential therapeutic

  13. Reversal of the glycolytic phenotype of primary effusion lymphoma cells by combined targeting of cellular metabolism and PI3K/Akt/ mTOR signaling

    PubMed Central

    Bertacchini, Jessika; Frasson, Chiara; Bosco, Raffaella; Accordi, Benedetta; Basso, Giuseppe; Bonora, Massimo; Calabrò, Maria Luisa; Mattiolo, Adriana; Sgarbi, Gianluca; Baracca, Alessandra; Pinton, Paolo; Riva, Giovanni; Rampazzo, Enrico; Petrizza, Luca; Prodi, Luca; Milani, Daniela; Luppi, Mario; Potenza, Leonardo; De Pol, Anto; Cocco, Lucio; Capitani, Silvano; Marmiroli, Sandra

    2016-01-01

    PEL is a B-cell non-Hodgkin lymphoma, occurring predominantly as a lymphomatous effusion in body cavities, characterized by aggressive clinical course, with no standard therapy. Based on previous reports that PEL cells display a Warburg phenotype, we hypothesized that the highly hypoxic environment in which they grow in vivo makes them more reliant on glycolysis, and more vulnerable to drugs targeting this pathway. We established here that indeed PEL cells in hypoxia are more sensitive to glycolysis inhibition. Furthermore, since PI3K/Akt/mTOR has been proposed as a drug target in PEL, we ascertained that pathway-specific inhibitors, namely the dual PI3K and mTOR inhibitor, PF-04691502, and the Akt inhibitor, Akti 1/2, display improved cytotoxicity to PEL cells in hypoxic conditions. Unexpectedly, we found that these drugs reduce lactate production/extracellular acidification rate, and, in combination with the glycolysis inhibitor 2-deoxyglucose (2-DG), they shift PEL cells metabolism from aerobic glycolysis towards oxidative respiration. Moreover, the associations possess strong synergistic cytotoxicity towards PEL cells, and thus may reduce adverse reaction in vivo, while displaying very low toxicity to normal lymphocytes. Finally, we showed that the association of 2-DG and PF-04691502 maintains its cytotoxic and proapoptotic effect also in PEL cells co-cultured with human primary mesothelial cells, a condition known to mimic the in vivo environment and to exert a protective and pro-survival action. All together, these results provide a compelling rationale for the clinical development of new therapies for the treatment of PEL, based on combined targeting of glycolytic metabolism and constitutively activated signaling pathways. PMID:26575168

  14. PI3K/Akt/mTOR signaling & its regulator tumour suppressor genes PTEN & LKB1 in human uterine leiomyomas

    PubMed Central

    Makker, Annu; Goel, Madhu Mati; Mahdi, Abbas Ali; Bhatia, Vikram; Das, Vinita; Agarwal, Anjoo; Pandey, Amita

    2016-01-01

    Background & objectives: Despite their high occurrence and associated significant level of morbidity manifesting as spectrum of clinical symptoms, the pathogenesis of uterine leiomyomas (ULs) remains unclear. We investigated expression profile of tumour suppressor genes PTEN (phosphatase and tensin homolog deleted on chromosome ten) and LKB1 (liver kinase B1), and key signaling components of P13K (phosphatidylinositol 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) pathway in leiomyomas and adjacent normal myometrium in women of reproductive age, to explore the possibility of targeting this pathway for future therapeutic implications. Methods: Real time PCR (qPCR) was used to quantify relative gene expression levels of PTEN, Akt1, Akt2, mTOR, LKB1 and VEGFA (vascular endothelial growth factor A) in leiomyoma as compared to adjacent normal myometrium. Immunohistochemistry was subsequently performed to analyze expression of PTEN, phospho-Akt, phospho-mTOR, phospho-S6, LKB1 and VEGFA in leiomyoma and adjacent normal myometrium. Results: Significant upregulation of PTEN (2.52 fold; P=0.03) and LKB1 (3.93 fold; P=0.01), and downregulation of VEGFA (2.95 fold; P=0.01) genes were observed in leiomyoma as compared to normal myometrium. Transcript levels of Akt1, Akt2 and mTOR did not vary significantly between leiomyoma and myometrium. An increased immunoexpression of PTEN (P=0.015) and LKB1 (P<0.001) and decreased expression of VEGFA (P=0.01) was observed in leiomyoma as compared to myometrium. Immunostaining for activated (phosphorylated) Akt, mTOR and S6 was absent or low in majority of leiomyoma and myometrium. Interpretation & conclusions: Upregulation of PTEN and LKB1 in concert with negative or low levels of activated Akt, mTOR and S6 indicates that PI3K/Akt/mTOR pathway may not play a significant role in pathogenesis of leiomyoma. PMID:27748285

  15. Targeted Deletion of a Plasmodium Site-2 Protease Impairs Life Cycle Progression in the Mammalian Host

    PubMed Central

    Goulielmaki, Evi; Chalari, Anna; Withers-Martinez, Chrislaine; Siden-Kiamos, Inga; Matuschewski, Kai

    2017-01-01

    Site-2 proteases (S2P) belong to the M50 family of metalloproteases, which typically perform essential roles by mediating activation of membrane–bound transcription factors through regulated intramembrane proteolysis (RIP). Protease-dependent liberation of dormant transcription factors triggers diverse cellular responses, such as sterol regulation, Notch signalling and the unfolded protein response. Plasmodium parasites rely on regulated proteolysis for controlling essential pathways throughout the life cycle. In this study we examine the Plasmodium-encoded S2P in a murine malaria model and show that it is expressed in all stages of Plasmodium development. Localisation studies by endogenous gene tagging revealed that in all invasive stages the protein is in close proximity to the nucleus. Ablation of PbS2P by reverse genetics leads to reduced growth rates during liver and blood infection and, hence, virulence attenuation. Strikingly, absence of PbS2P was compatible with parasite life cycle progression in the mosquito and mammalian hosts under physiological conditions, suggesting redundant or dispensable roles in vivo. PMID:28107409

  16. Mammalian Target of Rapamycin: Its Role in Early Neural Development and in Adult and Aged Brain Function

    PubMed Central

    Garza-Lombó, Carla; Gonsebatt, María E.

    2016-01-01

    The kinase mammalian target of rapamycin (mTOR) integrates signals triggered by energy, stress, oxygen levels, and growth factors. It regulates ribosome biogenesis, mRNA translation, nutrient metabolism, and autophagy. mTOR participates in various functions of the brain, such as synaptic plasticity, adult neurogenesis, memory, and learning. mTOR is present during early neural development and participates in axon and dendrite development, neuron differentiation, and gliogenesis, among other processes. Furthermore, mTOR has been shown to modulate lifespan in multiple organisms. This protein is an important energy sensor that is present throughout our lifetime its role must be precisely described in order to develop therapeutic strategies and prevent diseases of the central nervous system. The aim of this review is to present our current understanding of the functions of mTOR in neural development, the adult brain and aging. PMID:27378854

  17. Mechanistic or mammalian target of rapamycin (mTOR) may determine robustness in young male mice at the cost of accelerated aging

    PubMed Central

    Leontieva, Olga V.; Paszkiewicz, Geraldine M.; Blagosklonny, Mikhail V.

    2012-01-01

    Males, who are bigger and stronger than females, die younger in most species from flies to mammals including humans. Cellular mass growth is driven in part by mTOR (Target of Rapamycin). When developmental growth is completed, then, instead of growth, mTOR drives aging, manifested by increased cellular functions, such as hyper-secretion by fibroblasts, thus altering homeostasis, leading to age-related diseases and death. We hypothesize that MTOR activity is elevated in male mice compared with females. Noteworthy, 6 months old males were 28 % heavier than females. Also levels of phosphorylated S6 (pS6) and phospho-AKT (p-AKT, Ser 473), markers of the mTOR activity, were higher in male organs tested. Levels of pS6 were highly variable among mice and correlated with body weight and p-AKT. With age, the difference between levels of pS6 between sexes tended to minimize, albeit males still had hyperactive mTOR. Unlike fasting, the intraperitoneal (i.p.) administration of rapamycin eliminated pS6 in all organs of all females measured by immunoblotting and immunohistochemistry without affecting p-AKT and blood insulin. Although i.p. rapamycin dramatically decreased levels of pS6 in males too, it was still detectable by immunoblotting upon longer exposure. Our study demonstrated that both tissue p-AKT and pS6 were higher in young males than young females and were associated with increased body weight and insulin. These data can explain larger body size and faster aging in males. Our data suggest higher efficacy of rapamycin compared to fasting. Higher sensitivity of females to rapamycin may explain more pronounced life extension by rapamycin observed in females compared to males in several studies. PMID:23443503

  18. Synthesis of Fluorophores that Target Small Molecules to the Endoplasmic Reticulum of Living Mammalian Cells**

    PubMed Central

    Matthew Meinig, J.; Fu, Liqiang; Peterson, Blake R.

    2015-01-01

    The endoplasmic reticulum (ER) plays critical roles in the processing of secreted and transmembrane proteins. To deliver small molecules to this organelle, we synthesized fluorinated hydrophobic analogues of the fluorophore rhodol. These cell-permeable fluorophores are exceptionally bright, with quantum yields of ~ 0.8, and specifically accumulate in the ER of living HeLa cells, as imaged by confocal laser scanning microscopy. To target a biological pathway controlled by the ER, we linked a fluorinated hydrophobic rhodol to 5-nitrofuran-2-acrylaldehyde. In contrast to an untargeted nitrofuran warhead, delivery of this electrophilic nitrofuran to the ER by the rhodol resulted in cytotoxicity comparable to the ER-targeted cytotoxin eeyarestatin I, and specifically inhibited protein processing by the ubiquitin-proteasome system. Fluorinated hydrophobic rhodols represent outstanding fluorophores that enable delivery of small molecules for targeting of ER-associated proteins and pathways. PMID:26118368

  19. Targeted killing of a mammalian cell based upon its specialized metabolic state.

    PubMed

    Alexander, Peter B; Wang, Jian; McKnight, Steven L

    2011-09-20

    Mouse ES cells use a mitochondrial threonine dehydrogenase (TDH) enzyme to catabolize threonine into glycine and acetyl-CoA. Measurements of mRNA abundance have given evidence that ES cells express upwards of 1,000-fold higher levels of TDH mRNA than any of seven other mouse tissues tested. When cell culture medium is deprived of threonine, ES cells rapidly discontinue DNA synthesis, arrest cell division, and eventually die. Such studies led to the conclusion that mouse ES cells exist in a threonine-dependent metabolic state. Proceeding with the assumption that the active TDH enzyme should be essential for the growth and viability of mouse ES cells, we performed a drug screen in search of specific inhibitors of the purified TDH enzyme. Such efforts led to the discovery of a class of quinazolinecarboxamide (Qc) compounds that inhibit the ability of the TDH enzyme to catabolize threonine into glycine and acetyl-CoA. Administration of Qc inhibitors of TDH to mouse ES cells impeded cell growth and resulted in the induction of autophagy. By contrast, the same chemicals failed to affect the growth of HeLa cells at concentrations 300-fold higher than that required to kill mouse ES cells. It was likewise observed that the Qc class of TDH inhibitors failed to affect the growth or viability of ES cell-derived embryoid body cells known to have extinguished TDH expression. These studies demonstrate how it is possible to kill a specific mammalian cell type on the basis of its specialized metabolic state.

  20. MicroRNA-107 inhibits tumor growth and metastasis by targeting the BDNF-mediated PI3K/AKT pathway in human non-small lung cancer.

    PubMed

    Xia, Huan; Li, Yang; Lv, Xiaohong

    2016-10-01

    Abnormal expression of microRNA-107 (miR-107) was found in non-small cell lung cancer (NSCLC). However, little is known about its role and molecular mechanism in NSCLC progression and metastasis. Therefore, the aims of this study were to clarify the potential role of miR-107 and molecular mechanism in NSCLC progression and metastasis. Quantitative real-time polymerase chain reaction assay showed that miR-107 expression levels were significantly decreased in NSCLC tissue and cell lines. Low miR-107 levels in tumor tissue correlated with advanced TNM stage and lymph node metastasis. Function assays showed that overexpression of miR-107 suppressed cell proliferation, migration and invasion in A549 cells in vitro, and inhibited NSCLC tumor growth in vivo. Further mechanism assays suggested the brain-derived neurotrophic factor (BDNF) was identified as a target gene of miR-107 in NSCLC cells. In addition, BDNF expression was upregulated, and inversely correlated with miR-107 in NSCLC tissues. Enforced overexpression of BDNF effectively reversed the tumor suppressive functions of miR-107 on NSCLC proliferation, migration and invasion. miR-107 overexpression or downregulation of BDNF was able to inhibit activation of PI3K/AKT signaling pathway. Taken together, our findings present the first evidence that miR-107 could suppress NSCLC metastasis by targeting BDNF and indirectly regulating PI3K/AKT signaling pathway, which might lead to a potential therapeutic strategy focusing on miR-107 and BDNF for human NSCLC.

  1. MicroRNA-320 inhibits invasion and induces apoptosis by targeting CRKL and inhibiting ERK and AKT signaling in gastric cancer cells

    PubMed Central

    Zhao, Yue; Dong, Qianze; Wang, Enhua

    2017-01-01

    MicroRNA-320 (miR-320) downregulation has been reported in several human cancers. Until now, its expression pattern and biological roles in human cancer remain unknown. This study aims to clarify its clinical expression pattern and biological function in gastric cancers. We found miR-320 level was downregulated in gastric cancer tissues. miR-320 mimic was transfected in SGC-7901 cells with low endogenous expression. miR-320 inhibitor was used in BGC-823 cells with high endogenous expression. We found that miR-320 inhibited SGC-7901 proliferation and invasion, with decreased expression of cyclin D1 and MMP9 at both mRNA and protein levels. We also found that miR-320 mimic downregulated chemoresistance and cell survival of gastric cancer cells when treated with 5-fluorouracil. miR-320 inhibitor displayed the opposite effects in BGC-823 cell line. In addition, we discovered that miR-320 mimic could inhibit AKT and ERK activity. By using luciferase reporter assay, we found that CRKL serves as the target of miR-320. miR-320 mimic downregulated CRKL expression, whereas miR-320 inhibitor upregulated CRKL expression. miR-320 suppressed CRKL-3′-untranslated region reporter intensity in SGC-7901 cells. Furthermore, CRKL depletion abrogated the effects of miR-320. In gastric cancer tissues, we observed a negative correlation between CRKL and miR-320. In conclusion, our study demonstrated that downregulation of miR-320 was closely related with malignant progression of gastric cancer. miR-320 inhibits proliferation, invasion, and chemoresistance through ERK and AKT signaling by targeting CRKL. PMID:28255248

  2. MicroRNA-143-3p inhibits hyperplastic scar formation by targeting connective tissue growth factor CTGF/CCN2 via the Akt/mTOR pathway.

    PubMed

    Mu, Shengzhi; Kang, Bei; Zeng, Weihui; Sun, Yaowen; Yang, Fan

    2016-05-01

    Post-traumatic hypertrophic scar (HS) is a fibrotic disease with excessive extracellular matrix (ECM) production, which is a response to tissue injury by fibroblasts. Although emerging evidence has indicated that miRNA contributes to hypertrophic scarring, the role of miRNA in HS formation remains unclear. In this study, we found that miR-143-3p was markedly downregulated in HS tissues and fibroblasts (HSFs) using qRT-PCR. The expression of connective tissue growth factor (CTGF/CCN2) was upregulated both in HS tissues and HSFs, which is proposed to play a key role in ECM deposition in HS. The protein expression of collagen I (Col I), collagen III (Col III), and α-smooth muscle actin (α-SMA) was obviously inhibited after treatment with miR-143-3p in HSFs. The CCK-8 assay showed that miR-143-3p transfection reduced the proliferation ability of HSFs, and flow cytometry showed that either early or late apoptosis of HSFs was upregulated by miR-143-3p. In addition, the activity of caspase 3 and caspase 9 was increased after miR-143-3p transfection. On the contrary, the miR-143-3p inhibitor was demonstrated to increase cell proliferation and inhibit apoptosis of HSFs. Moreover, miR-143-3p targeted the 3'-UTR of CTGF and caused a significant decrease of CTGF. Western blot demonstrated that Akt/mTOR phosphorylation and the expression of CTGF, Col I, Col III, and α-SMA were inhibited by miR-143-3p, but increased by CTGF overexpression. In conclusion, we found that miR-143-3p inhibits hypertrophic scarring by regulating the proliferation and apoptosis of human HSFs, inhibiting ECM production-associated protein expression by targeting CTGF, and restraining the Akt/mTOR pathway.

  3. MicroRNA-101 suppresses progression of lung cancer through the PTEN/AKT signaling pathway by targeting DNA methyltransferase 3A

    PubMed Central

    Wang, Lumin; Yao, Jiayi; Sun, Hongfei; He, Kang; Tong, Dongdong; Song, Tusheng; Huang, Chen

    2017-01-01

    It is well established that transcriptional silencing of critical tumor suppressor genes by DNA methylation is a fundamental process in the initiation of lung cancer. However, the involvement of microRNAs (miRNAs) in restoring abnormal DNA methylation patterns in lung cancer is not well understood. Therefore, and since miRNA-101 is complementary to the 3′-untranslated region of DNA methyltransferase 3A (DNMT3A), we investigated whether miRNA-101 could restore normal DNA methylation patterns in lung cancer cell lines. Bioinformatics has indicated that DNMT3A is a major target of miR-101. In addition, the overexpression of miR-101 downregulates DNMT3A. Using a methylation-specific polymerase chain reaction assay, we demonstrated that methylation of the phosphatase and tensin homolog (PTEN) promoter was reduced in A549 cells transfected with miR-101, but not in the transfected control. Furthermore, overexpression of miR-101 and silencing of DNMT3A suppressed lung cell proliferation and S/G2 transition, and increased apoptosis through the PTEN/AKT pathway in vitro. Furthermore, we observed the opposite phenomenon in A549 cells transfected with a miR-101 inhibitor. Subsequent investigation revealed that overexpression of miR-101 significantly inhibited the tumorigenicity of A549 cells in a nude mouse xenograft model. These results demonstrate that miR-101 affects lung cancer progression through the PTEN/AKT signaling pathway by targeting DNMT3A in lung cells, suggesting that miR-101 may be a novel potential therapeutic strategy in lung cancer treatment. PMID:28123563

  4. Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tunicamycins, streptovirudins, and corynetoxins are natural products that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg**2+ complex established as a transition state analog for hexosamine-1-phosphate:pren...

  5. The Complexity of Targeting PI3K-Akt-mTOR Signalling in Human Acute Myeloid Leukaemia: The Importance of Leukemic Cell Heterogeneity, Neighbouring Mesenchymal Stem Cells and Immunocompetent Cells.

    PubMed

    Brenner, Annette K; Andersson Tvedt, Tor Henrik; Bruserud, Øystein

    2016-11-11

    Therapeutic targeting of PI3K-Akt-mTOR is considered a possible strategy in human acute myeloid leukaemia (AML); the most important rationale being the proapoptotic and antiproliferative effects of direct PI3K/mTOR inhibition observed in experimental studies of human AML cells. However, AML is a heterogeneous disease and these effects caused by direct pathway inhibition in the leukemic cells are observed only for a subset of patients. Furthermore, the final effect of PI3K-Akt-mTOR inhibition is modulated by indirect effects, i.e., treatment effects on AML-supporting non-leukemic bone marrow cells. In this article we focus on the effects of this treatment on mesenchymal stem cells (MSCs) and monocytes/macrophages; both these cell types are parts of the haematopoietic stem cell niches in the bone marrow. MSCs have unique membrane molecule and constitutive cytokine release profiles, and mediate their support through bidirectional crosstalk involving both cell-cell contact and the local cytokine network. It is not known how various forms of PI3K-Akt-mTOR targeting alter the molecular mechanisms of this crosstalk. The effect on monocytes/macrophages is also difficult to predict and depends on the targeted molecule. Thus, further development of PI3K-Akt-mTOR targeting into a clinical strategy requires detailed molecular studies in well-characterized experimental models combined with careful clinical studies, to identify patient subsets that are likely to respond to this treatment.

  6. Targeted Cytoplasmic Irradiation with Alpha Particles Induces Mutations in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Wu, Li-Jun; Randers-Pehrson, Gerhard; Xu, An; Waldren, Charles A.; Geard, Charles R.; Yu, Zengliang; Hei, Tom K.

    1999-04-01

    Ever since x-rays were shown to induce mutation in Drosophila more than 70 years ago, prevailing dogma considered the genotoxic effects of ionizing radiation, such as mutations and carcinogenesis, as being due mostly to direct damage to the nucleus. Although there was indication that alpha particle traversal through cellular cytoplasm was innocuous, the full impact remained unknown. The availability of the microbeam at the Radiological Research Accelerator Facility of Columbia University made it possible to target and irradiate the cytoplasm of individual cells in a highly localized spatial region. By using dual fluorochrome dyes (Hoechst and Nile Red) to locate nucleus and cellular cytoplasm, respectively, thereby avoiding inadvertent traversal of nuclei, we show here that cytoplasmic irradiation is mutagenic at the CD59 (S1) locus of human-hamster hybrid (AL) cells, while inflicting minimal cytotoxicity. The principal class of mutations induced are similar to those of spontaneous origin and are entirely different from those of nuclear irradiation. Furthermore, experiments with radical scavenger and inhibitor of intracellular glutathione indicated that the mutagenicity of cytoplasmic irradiation depends on generation of reactive oxygen species. These findings suggest that cytoplasm is an important target for genotoxic effects of ionizing radiation, particularly radon, the second leading cause of lung cancer in the United States. In addition, cytoplasmic traversal by alpha particles may be more dangerous than nuclear traversal, because the mutagenicity is accomplished by little or no killing of the target cells.

  7. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter

    PubMed Central

    Ide, Satoru; Dejardin, Jerome

    2015-01-01

    The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals. PMID:25812914

  8. Target-specific variants of Flp recombinase mediate genome engineering reactions in mammalian cells.

    PubMed

    Shah, Riddhi; Li, Feng; Voziyanova, Eugenia; Voziyanov, Yuri

    2015-09-01

    Genome engineering relies on DNA-modifying enzymes that are able to locate a DNA sequence of interest and initiate a desired genome rearrangement. Currently, the field predominantly utilizes site-specific DNA nucleases that depend on the host DNA repair machinery to complete a genome modification task. We show here that genome engineering approaches that employ target-specific variants of the self-sufficient, versatile site-specific DNA recombinase Flp can be developed into promising alternatives. We demonstrate that the Flp variant evolved to recombine an FRT-like sequence, FL-IL10A, which is located upstream of the human interleukin-10 gene, and can target this sequence in the model setting of Chinese hamster ovary and human embryonic kidney 293 cells. This target-specific Flp variant is able to perform the integration reaction and, when paired with another recombinase, the dual recombinase-mediated cassette exchange reaction. The efficiency of the integration reaction in human cells can be enhanced by 'humanizing' the Flp variant gene and by adding the nuclear localization sequence to the recombinase.

  9. Dynamics of tRNA fragments and their targets in aging mammalian brain

    PubMed Central

    Karaiskos, Spyros; Grigoriev, Andrey

    2016-01-01

    Background: The progress of next-generation sequencing technologies has unveiled various non-coding RNAs that have previously been considered products of random degradation and attracted only minimal interest. Among small RNA families, microRNA (miRNAs) have traditionally been considered key post-transcriptional regulators. However, recent studies have reported evidence for widespread presence of fragments of tRNA molecules (tRFs) across a range of organisms and tissues, and of tRF involvement in Argonaute complexes.  Methods:To elucidate potential tRF functionality, we compared available RNA sequencing datasets derived from the brains of young, mid-aged and old rats. Using sliding 7-mer windows along a tRF, we searched for putative seed sequences with high numbers of conserved complementary sites within 3' UTRs of 23 vertebrate genomes. We analyzed Gene Ontology term enrichment of predicted tRF targets and compared their transcript levels with targets of miRNAs in the context of age.  Results and Discussion: We detected tRFs originating from 3’- and 5’-ends of tRNAs in rat brains at significant levels. These fragments showed dynamic changes: 3’ tRFs monotonously increased with age, while 5’ tRFs displayed less consistent patterns. Furthermore, 3’ tRFs showed a narrow size range compared to 5’ tRFs, suggesting a difference in their biogenesis mechanisms. Similar to our earlier results in Drosophila and compatible with other experimental findings, we found “seed” sequence locations on both ends of different tRFs. Putative targets of these fragments were found to be enriched in neuronal and developmental functions. Comparison of tRFs and miRNAs increasing in abundance with age revealed small, but distinct changes in brain target transcript levels for these two types of small RNA, with the higher proportion of tRF targets decreasing with age. We also illustrated the utility of tRF analysis for annotating tRNA genes in sequenced genomes. PMID

  10. Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets.

    PubMed

    Turanov, Anton A; Kehr, Sebastian; Marino, Stefano M; Yoo, Min-Hyuk; Carlson, Bradley A; Hatfield, Dolph L; Gladyshev, Vadim N

    2010-09-01

    The classical Trx (thioredoxin) system, composed of TR (Trx reductase), Trx and NADPH, defines a major pathway of cellular thiol-based redox regulation. Three TRs have been identified in mammals: (i) cytosolic TR1, (ii) mitochondrial TR3 and (iii) testes-specific TGR (Trx-glutathione reductase). All three are selenocysteine-containing enzymes with broad substrate specificity in in vitro assays, but which protein substrates are targeted by TRs in vivo is not well understood. In the present study, we used a mechanism-based approach to characterize the molecular targets of TR1. Cytosolic Trx1 was the major target identified in rat and mouse liver, as well as in rat brain and mouse serum. The results suggest that the main function of TR1 is to reduce Trx1. We also found that TR1-based affinity resins provide a convenient tool for specific isolation of Trxs from a variety of biological samples. To better assess the role of TRs in redox homoeostasis, we comparatively analysed TR1- and TR3-knockdown cells. Although cells deficient in TR1 were particularly sensitive to diamide, TR3-knockdown cells were more sensitive to hydrogen peroxide. To further examine the TR1-Trx1 redox pair, we used mice with a liver-specific knockout of selenocysteine tRNA. In this model, selenocysteine insertion into TR1 was blocked, but the truncated form of this protein was not detected. Instead, TR1 and TR3 levels were decreased in the knockout samples. Diminished hepatic TR1 function was associated with elevated Trx1 levels, but this protein was mostly in the oxidized state. Overall, this study provides evidence for the key role of the TR1-Trx1 pair in redox homoeostasis.

  11. The N-methyl-D-aspartate neurotransmitter receptor is a mammalian brain target for the dinoflagellate Pfiesteria piscicida toxin.

    PubMed

    El-Nabawi, A; Quesenberry, M; Saito, K; Silbergeld, E; Vasta, G; Eldefrawi, A

    2000-11-15

    Blooms of Pfiesteria piscicida, a dinoflagellate in eastern U.S. coastal rivers, are believed to secrete toxins that kill fish and produce short-term memory loss in humans. Only one or two of Pfiesteria's multiple stages secrete the toxin, and only under certain environmental conditions. Thus, neither the presence of Pfiesteria nor fish kill alone can be indicative of toxin presence. The objective of this study was to identify the mammalian molecular brain target for the toxin that is associated with decrements in memory. Seven rat brain neurotransmitter receptors were selected to study because of their reported roles in cognitive function: receptors for nicotine, muscarine, AMPA/kainate, N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid, and dopamine 1 and 2. The effects of 17 environmental and laboratory samples on radioactive ligand binding to these receptors were studied. Of the seven receptors, binding only to the NMDA receptor was inhibited by only the two Pfiesteria-containing waters (identified by PCR) that also killed fish, and not by any of the other 15 samples tested. It is suggested that inhibition of NMDA-receptor binding is the cause of memory loss in exposed humans. Thus, it could be a useful biomarker for the toxin's presence in rivers for decisions on closures and for identification of the fractions containing the toxin during its purification. Knowledge of the toxin's molecular target, and how it affects its function, also leads to suggestions for therapeutics to use in animal models.

  12. Coupling nutrient sensing to metabolic homoeostasis: the role of the mammalian target of rapamycin complex 1 pathway.

    PubMed

    André, Caroline; Cota, Daniela

    2012-11-01

    The mammalian target of rapamycin complex 1 (mTORC1) pathway is known to couple different environmental cues to the regulation of several energy-demanding functions within the cell, spanning from protein translation to mitochondrial activity. As a result, at the organism level, mTORC1 activity affects energy balance and general metabolic homoeostasis by modulating both the activity of neuronal populations that play key roles in the control of food intake and body weight, as well as by determining storage and use of fuel substrates in peripheral tissues. This review focuses on recent advances made in understanding the role of the mTORC1 pathway in the regulation of energy balance. More particularly, it aims at providing an overview of the status of knowledge regarding the mechanisms underlying the ability of certain amino acids, glucose and fatty acids, to affect mTORC1 activity and in turn illustrates how the mTORC1 pathway couples nutrient sensing to the hypothalamic regulation of the organisms' energy homoeostasis and to the control of intracellular metabolic processes, such as glucose uptake, protein and lipid biosynthesis. The evidence reviewed pinpoints the mTORC1 pathway as an integrator of the actions of nutrients on metabolic health and provides insight into the relevance of this intracellular pathway as a potential target for the therapy of metabolic diseases such as obesity and type-2 diabetes.

  13. Ubiquilin-mediated Small Molecule Inhibition of Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling*

    PubMed Central

    Coffey, Rory T.; Shi, Yuntao; Long, Marcus J. C.; Marr, Michael T.; Hedstrom, Lizbeth

    2016-01-01

    Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism, growth, and proliferation. mTORC1 has been implicated in many diseases such as cancer, diabetes, and neurodegeneration, and is a target to prolong lifespan. Here we report a small molecule inhibitor (Cbz-B3A) of mTORC1 signaling. Cbz-B3A inhibits the phosphorylation of eIF4E-binding protein 1 (4EBP1) and blocks 68% of translation. In contrast, rapamycin preferentially inhibits the phosphorylation of p70S6k and blocks 35% of translation. Cbz-B3A does not appear to bind directly to mTORC1, but instead binds to ubiquilins 1, 2, and 4. Knockdown of ubiquilin 2, but not ubiquilins 1 and 4, decreases the phosphorylation of 4EBP1, suggesting that ubiquilin 2 activates mTORC1. The knockdown of ubiquilins 2 and 4 decreases the effect of Cbz-B3A on 4EBP1 phosphorylation. Cbz-B3A slows cellular growth of some human leukemia cell lines, but is not cytotoxic. Thus Cbz-B3A exemplifies a novel strategy to inhibit mTORC1 signaling that might be exploited for treating many human diseases. We propose that Cbz-B3A reveals a previously unappreciated regulatory pathway coordinating cytosolic protein quality control and mTORC1 signaling. PMID:26740621

  14. MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines

    PubMed Central

    Kulkarni, Varun; Naqvi, Afsar Raza; Uttamani, Juhi Raju; Nares, Salvador

    2016-01-01

    MicroRNAs are 18–22 nucleotides long, non-coding RNAs that bind transcripts with complementary sequences leading to either mRNA degradation or translational suppression. However, the inherent differences in preferred mode of miRNA regulation among cells of different origin have not been examined. In our previous transcriptome profiling studies, we observed that post-transcriptional regulation can differ substantially depending on the cell in context. Here we examined mechanistic differences in the regulation of a let-7a targeted (wild type) or resistant (mutant) engineered renilla transcript across various mammalian cell lines of diverse origin. Dual luciferase assays show that compared to mutant (mut), the reporter gene containing wild type (wt) let-7a binding sites was efficiently suppressed upon transfection in various cell lines. Importantly, the strength of miRNA regulation varied across the cell lines. Total RNA analysis demonstrates that wt renilla mRNA was expressed to similar or higher levels compared to mut suggesting that translation repression is a predominant mode of miRNA regulation. Nonetheless, transcript degradation was observed in some cell lines. Ago-2 immunoprecipitation show that miRNA repressed renilla mRNA are associated with functional mi-RISC (miRNA-RNA induced silencing complex). Given the immense potential of miRNA as a therapeutic option, these findings highlight the necessity to thoroughly examine the mode of mRNA regulation in order to achieve the beneficial effects in targeting cells. PMID:26761000

  15. Phosphoinositide 3-kinase targeting by the β galactoside binding protein cytokine negates akt gene expression and leads aggressive breast cancer cells to apoptotic death

    PubMed Central

    Wells, Valerie; Mallucci, Livio

    2009-01-01

    Introduction Phosphoinositide 3-kinase (PI3K)-activated signalling has a critical role in the evolution of aggressive tumourigenesis and is therefore a prime target for anticancer therapy. Previously we have shown that the β galactoside binding protein (βGBP) cytokine, an antiproliferative molecule, induces functional inhibition of class 1A and class 1B PI3K. Here, we have investigated whether, by targeting PI3K, βGBP has therapeutic efficacy in aggressive breast cancer cells where strong mitogenic input is fuelled by overexpression of the ErbB2 (also known as HER/neu, for human epidermal growth factor receptor 2) oncoprotein receptor and have used immortalised ductal cells and non-aggressive mammary cancer cells, which express ErbB2 at low levels, as controls. Methods Aggressive BT474 and SKBR3 cancer cells where ErbB2 is overexpressed, MCF10A immortalised ductal cells and non-invasive MCF-7 cancer cells which express low levels of ErbB2, both in their naive state and when forced to mimic aggressive behaviour, were used. Class IA PI3K was immunoprecipitated and the conversion of phosphatidylinositol (4,5)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) assessed by ELISA. The consequences of PI3K inhibition by βGBP were analysed at proliferation level, by extracellular signal-regulated kinase (ERK) activation, by akt gene expression and by apoptosis. Apoptosis was documented by changes in mitochondrial membrane potential, alteration of the plasma membrane, caspase 3 activation and DNA fragmentation. Phosphorylated and total ERK were measured by Western blot analysis and akt mRNA levels by Northern blot analysis. The results obtained with the BT474 and SKBR3 cells were validated in the MCF10A ductal cells and in non-invasive MCF-7 breast cancer cells forced into mimicking the in vitro behaviour of the BT474 and SKBR3 cells. Results In aggressive breast cancer cells, where mitogenic signalling is enforced by the ErbB2 oncoprotein receptor

  16. eIF4B is a convergent target and critical effector of oncogenic Pim and PI3K/Akt/mTOR signaling pathways in Abl transformants

    PubMed Central

    Chen, Ke; Yang, Jianling; Li, Jianning; Wang, Xuefei; Chen, Yuhai; Huang, Shile; Chen, Ji-Long

    2016-01-01

    Activation of eIF4B correlates with Abl-mediated cellular transformation, but the precise mechanisms are largely unknown. Here we show that eIF4B is a convergent substrate of JAK/STAT/Pim and PI3K/Akt/mTOR pathways in Abl transformants. Both pathways phosphorylated eIF4B in Abl-transformed cells, and such redundant regulation was responsible for the limited effect of single inhibitor on Abl oncogenicity. Persistent inhibition of one signaling pathway induced the activation of the other pathway and thereby restored the phosphorylation levels of eIF4B. Simultaneous inhibition of the two pathways impaired eIF4B phosphorylation more effectively, and synergistically induced apoptosis in Abl transformed cells and inhibited the growth of engrafted tumors in nude mice. Similarly, the survival of Abl transformants exhibited a higher sensitivity to the pharmacological inhibition, when combined with the shRNA-based silence of the other pathway. Interestingly, such synergy was dependent on the phosphorylation status of eIF4B on Ser422, as overexpression of eIF4B phosphomimetic mutant S422E in the transformants greatly attenuated the synergistic effects of these inhibitors on Abl oncogenicity. In contrast, eIF4B knockdown sensitized Abl transformants to undergo apoptosis induced by the combined blockage. Collectively, the results indicate that eIF4B integrates the signals from Pim and PI3K/Akt/mTOR pathways in Abl-expressing leukemic cells, and is a promising therapeutic target for such cancers. PMID:26848623

  17. miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6

    PubMed Central

    Xu, Junfen; Wan, Xiaoyun; Chen, Xiaojing; Fang, Yifeng; Cheng, Xiaodong; Xie, Xing; Lu, Weiguo

    2016-01-01

    Persistent infection with oncogenic human papillomavirus viruses (HPVs) is a casual factor for cervical cancer and its precursors, and the abnormal constitutive expression of viral oncoprotein E6 is a key event during the malignant transformation. Here, we performed miRNA microarray to identify changes of miRNAs following ectopic HPV16 E6 overexpression in HEK293T cells and found miR-2861 was greatly decreased in both HEK293T and HaCaT cells expressing HPV16 E6 compared to vector control. Further, we demonstrated a biological link among HPV16 E6, miR-2861, EGFR, AKT2, and CCND1 in cervical cancer cells. We showed that miR-2861 was downregulated in cervical cancer tissues and negatively correlated with advanced tumor stage and lymph node metastasis. Overexpression of miR-2861 suppressed cervical cancer cell proliferation and invasion and enhanced apoptosis. Subsequent investigation revealed that EGFR, AKT2, and CCND1 were all the direct targets of miR-2861. Importantly, silencing EGFR, AKT2, and/or CCND1 recapitulated the cellular effects seen upon miR-2861 overexpression. Restoration of EGFR, AKT2, and/or CCND1 counteracted the effects of miR-2861 expression. Thus, we identified a new pathway employing miR-2861, EGFR, AKT2, and CCND1 that may mediate HPV16 E6 induced initiation and progression of cervical cancer. PMID:27364926

  18. miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6.

    PubMed

    Xu, Junfen; Wan, Xiaoyun; Chen, Xiaojing; Fang, Yifeng; Cheng, Xiaodong; Xie, Xing; Lu, Weiguo

    2016-07-01

    Persistent infection with oncogenic human papillomavirus viruses (HPVs) is a casual factor for cervical cancer and its precursors, and the abnormal constitutive expression of viral oncoprotein E6 is a key event during the malignant transformation. Here, we performed miRNA microarray to identify changes of miRNAs following ectopic HPV16 E6 overexpression in HEK293T cells and found miR-2861 was greatly decreased in both HEK293T and HaCaT cells expressing HPV16 E6 compared to vector control. Further, we demonstrated a biological link among HPV16 E6, miR-2861, EGFR, AKT2, and CCND1 in cervical cancer cells. We showed that miR-2861 was downregulated in cervical cancer tissues and negatively correlated with advanced tumor stage and lymph node metastasis. Overexpression of miR-2861 suppressed cervical cancer cell proliferation and invasion and enhanced apoptosis. Subsequent investigation revealed that EGFR, AKT2, and CCND1 were all the direct targets of miR-2861. Importantly, silencing EGFR, AKT2, and/or CCND1 recapitulated the cellular effects seen upon miR-2861 overexpression. Restoration of EGFR, AKT2, and/or CCND1 counteracted the effects of miR-2861 expression. Thus, we identified a new pathway employing miR-2861, EGFR, AKT2, and CCND1 that may mediate HPV16 E6 induced initiation and progression of cervical cancer.

  19. Mammalian polyamine catabolism: a therapeutic target, a pathological problem, or both?

    PubMed

    Wang, Yanlin; Casero, Robert A

    2006-01-01

    With the recent discovery of the polyamine catabolic enzyme spermine oxidase (SMO/PAOh1), the apparent complexity of the polyamine metabolic pathway has increased considerably. Alone or in combination with the two other known members of human polyamine catabolism, spermidine/spermine N(1)-acetyltransferase, and N(1)-acetylpolyamine oxidase (PAO), SMO/PAOh1 expression has the potential to alter polyamine homeostasis in response to normal cellular signals, drug treatment and environmental and/or cellular stressors. The activity of the oxidases producing toxic aldehydes and the reactive oxygen species (ROS) H(2)O(2), suggest a mechanism by which these oxidases can be exploited as an antineoplastic drug target. However, inappropriate activation of the pathways may also lead to pathological outcomes, including DNA damage that can lead to cellular transformation. The most recent data suggest that the two polyamine catabolic pathways exhibit distinct properties and understanding these properties should aid in their exploitation for therapeutic and/or chemopreventive strategies.

  20. The Akt-mTOR axis is a pivotal regulator of eccentric hypertrophy during volume overload

    PubMed Central

    Ikeda, Masataka; Ide, Tomomi; Fujino, Takeo; Matsuo, Yuka; Arai, Shinobu; Saku, Keita; Kakino, Takamori; Oga, Yasuhiro; Nishizaki, Akiko; Sunagawa, Kenji

    2015-01-01

    The heart has two major modalities of hypertrophy in response to hemodynamic loads: concentric and eccentric hypertrophy caused by pressure and volume overload (VO), respectively. However, the molecular mechanism of eccentric hypertrophy remains poorly understood. Here we demonstrate that the Akt-mammalian target of rapamycin (mTOR) axis is a pivotal regulator of eccentric hypertrophy during VO. While mTOR in the heart was activated in a left ventricular end-diastolic pressure (LVEDP)-dependent manner, mTOR inhibition suppressed eccentric hypertrophy and induced cardiac atrophy even under VO. Notably, Akt was ubiquitinated and phosphorylated in response to VO, and blocking the recruitment of Akt to the membrane completely abolished mTOR activation. Various growth factors were upregulated during VO, suggesting that these might be involved in Akt-mTOR activation. Furthermore, the rate of eccentric hypertrophy progression was proportional to mTOR activity, which allowed accurate estimation of eccentric hypertrophy by time-integration of mTOR activity. These results suggested that the Akt-mTOR axis plays a pivotal role in eccentric hypertrophy, and mTOR activity quantitatively determines the rate of eccentric hypertrophy progression. As eccentric hypertrophy is an inherent system of the heart for regulating cardiac output and LVEDP, our findings provide a new mechanistic insight into the adaptive mechanism of the heart. PMID:26515499

  1. Akt-dependent enhanced migratory capacity of Th17 cells from children with lupus nephritis.

    PubMed

    Kshirsagar, Sudhir; Riedl, Magdalena; Billing, Heiko; Tönshoff, Burkhard; Thangavadivel, Shanmugapriya; Steuber, Christian; Staude, Hagen; Wechselberger, Gottfried; Edelbauer, Monika

    2014-11-15

    Th17 cells infiltrate the kidneys of patients with lupus nephritis (LN) and are critical for the pathogenesis of this disease. In this study, we show that enhanced activity of Stat3 in CD4(+)CD45RA(-)Foxp3(-) and Foxp3(low) effector T cells from children with LN correlates with increased frequencies of IL-17-producing cells within these T cell populations. The levels of retinoic acid-related orphan receptor c and IL-17 mRNA are significantly higher in PBMCs from children with LN than in those from controls. Mammalian target of rapamycin inhibition by rapamycin reduces both Stat3 activation in effector T cells and the frequency of IL-17-producing T cells in lupus patients. Complement factor C5a slightly increases the expression of IL-17 and induces activation of Akt in anti-CD3-activated lupus effector T cells. Th17 cells from children with LN exhibit high Akt activity and enhanced migratory capacity. Inhibition of the Akt signaling pathway significantly decreases Th17 cell migration. These findings indicate that the Akt signaling pathway plays a significant role in the migratory activity of Th17 cells from children with LN and suggest that therapeutic modulation of the Akt activity may inhibit Th17 cell trafficking to sites of inflammation and thus suppress chronic inflammatory processes in children with LN.

  2. Akt Pathway Activation by Human T-cell Leukemia Virus Type 1 Tax Oncoprotein.

    PubMed

    Cherian, Mathew A; Baydoun, Hicham H; Al-Saleem, Jacob; Shkriabai, Nikoloz; Kvaratskhelia, Mamuka; Green, Patrick; Ratner, Lee

    2015-10-23

    Human T-cell leukemia virus (HTLV) type 1, the etiological agent of adult T-cell leukemia, expresses the viral oncoprotein Tax1. In contrast, HTLV-2, which expresses Tax2, is non-leukemogenic. One difference between these homologous proteins is the presence of a C-terminal PDZ domain-binding motif (PBM) in Tax1, previously reported to be important for non-canonical NFκB activation. In contrast, this study finds no defect in non-canonical NFκB activity by deletion of the Tax1 PBM. Instead, Tax1 PBM was found to be important for Akt activation. Tax1 attenuates the effects of negative regulators of the PI3K-Akt-mammalian target of rapamycin pathway, phosphatase and tensin homologue (PTEN), and PHLPP. Tax1 competes with PTEN for binding to DLG-1, unlike a PBM deletion mutant of Tax1. Forced membrane expression of PTEN or PHLPP overcame the effects of Tax1, as measured by levels of Akt phosphorylation, and rates of Akt dephosphorylation. The current findings suggest that Akt activation may explain the differences in transforming activity of HTLV-1 and -2.

  3. The Akt-mTOR axis is a pivotal regulator of eccentric hypertrophy during volume overload.

    PubMed

    Ikeda, Masataka; Ide, Tomomi; Fujino, Takeo; Matsuo, Yuka; Arai, Shinobu; Saku, Keita; Kakino, Takamori; Oga, Yasuhiro; Nishizaki, Akiko; Sunagawa, Kenji

    2015-10-30

    The heart has two major modalities of hypertrophy in response to hemodynamic loads: concentric and eccentric hypertrophy caused by pressure and volume overload (VO), respectively. However, the molecular mechanism of eccentric hypertrophy remains poorly understood. Here we demonstrate that the Akt-mammalian target of rapamycin (mTOR) axis is a pivotal regulator of eccentric hypertrophy during VO. While mTOR in the heart was activated in a left ventricular end-diastolic pressure (LVEDP)-dependent manner, mTOR inhibition suppressed eccentric hypertrophy and induced cardiac atrophy even under VO. Notably, Akt was ubiquitinated and phosphorylated in response to VO, and blocking the recruitment of Akt to the membrane completely abolished mTOR activation. Various growth factors were upregulated during VO, suggesting that these might be involved in Akt-mTOR activation. Furthermore, the rate of eccentric hypertrophy progression was proportional to mTOR activity, which allowed accurate estimation of eccentric hypertrophy by time-integration of mTOR activity. These results suggested that the Akt-mTOR axis plays a pivotal role in eccentric hypertrophy, and mTOR activity quantitatively determines the rate of eccentric hypertrophy progression. As eccentric hypertrophy is an inherent system of the heart for regulating cardiac output and LVEDP, our findings provide a new mechanistic insight into the adaptive mechanism of the heart.

  4. Overexpression of Akt1 Enhances Adipogenesis and Leads to Lipoma Formation in Zebrafish

    PubMed Central

    Rajendran, R. Samuel; Shen, Chia-Ning; Chen, Te-Hao; Yen, Chueh-Chuan; Chuang, Chih-Kuang; Lin, Dar-Shong; Hsiao, Chung-Der

    2012-01-01

    Background Obesity is a complex, multifactorial disorder influenced by the interaction of genetic, epigenetic, and environmental factors. Obesity increases the risk of contracting many chronic diseases or metabolic syndrome. Researchers have established several mammalian models of obesity to study its underlying mechanism. However, a lower vertebrate model for conveniently performing drug screening against obesity remains elusive. The specific aim of this study was to create a zebrafish obesity model by over expressing the insulin signaling hub of the Akt1 gene. Methodology/Principal Findings Skin oncogenic transformation screening shows that a stable zebrafish transgenic of Tg(krt4Hsa.myrAkt1)cy18 displays severely obese phenotypes at the adult stage. In Tg(krt4:Hsa.myrAkt1)cy18, the expression of exogenous human constitutively active Akt1 (myrAkt1) can activate endogenous downstream targets of mTOR, GSK-3α/β, and 70S6K. During the embryonic to larval transitory phase, the specific over expression of myrAkt1 in skin can promote hypertrophic and hyperplastic growth. From 21 hour post-fertilization (hpf) onwards, myrAkt1 transgene was ectopically expressed in several mesenchymal derived tissues. This may be the result of the integration position effect. Tg(krt4:Hsa.myrAkt1)cy18 caused a rapid increase of body weight, hyperplastic growth of adipocytes, abnormal accumulation of fat tissues, and blood glucose intolerance at the adult stage. Real-time RT-PCR analysis showed the majority of key genes on regulating adipogenesis, adipocytokine, and inflammation are highly upregulated in Tg(krt4:Hsa.myrAkt1)cy18. In contrast, the myogenesis- and skeletogenesis-related gene transcripts are significantly downregulated in Tg(krt4:Hsa.myrAkt1)cy18, suggesting that excess adipocyte differentiation occurs at the expense of other mesenchymal derived tissues. Conclusion/Significance Collectively, the findings of this study provide direct evidence that Akt1 signaling plays an

  5. Inhibiting PLK1 induces autophagy of acute myeloid leukemia cells via mammalian target of rapamycin pathway dephosphorylation.

    PubMed

    Tao, Yan-Fang; Li, Zhi-Heng; Du, Wei-Wei; Xu, Li-Xiao; Ren, Jun-Li; Li, Xiao-Lu; Fang, Fang; Xie, Yi; Li, Mei; Qian, Guang-Hui; Li, Yan-Hong; Li, Yi-Ping; Li, Gang; Wu, Yi; Feng, Xing; Wang, Jian; He, Wei-Qi; Hu, Shao-Yan; Lu, Jun; Pan, Jian

    2017-03-01

    Decreased autophagy is accompanied by the development of a myeloproliferative state or acute myeloid leukemia (AML). AML cells are often sensitive to autophagy‑inducing stimuli, prompting the idea that targeting autophagy can be useful in AML cytotoxic therapy. AML NB4 cells overexpressing microtubule-associated protein 1 light chain 3-green fluorescent protein were screened with 69 inhibitors to analyze autophagy activity. AML cells were treated with the polo-like kinase 1 (PLK1) inhibitors RO3280 and BI2536 before autophagy analysis. Cleaved LC3 (LC3-II) and the phosphorylation of mammalian target of rapamycin (mTOR), adenosine monophosphate-activated protein kinase, and Unc-51-like kinase 1 during autophagy was detected with western blotting. Autophagosomes were detected using transmission electron microscopy. Several inhibitors had promising autophagy inducer effects: BI2536, MLN0905, SK1-I, SBE13 HCL and RO3280. Moreover, these inhibitors all targeted PLK1. Autophagy activity was increased in the NB4 cells treated with RO3280 and BI2536. Inhibition of PLK1 expression in NB4, K562 and HL-60 leukemia cells with RNA interference increased LC3-II and autophagy activity. The phosphorylation of mTOR was reduced significantly in NB4 cells treated with RO3280 and BI2536, and was also reduced significantly when PLK1 expression was downregulated in the NB4, K562 and HL-60 cells. We demonstrate that PLK1 inhibition induces AML cell autophagy and that it results in mTOR dephosphorylation. These results may provide new insights into the molecular mechanism of PLK1 in regulating autophagy.

  6. Inhibiting PLK1 induces autophagy of acute myeloid leukemia cells via mammalian target of rapamycin pathway dephosphorylation

    PubMed Central

    Tao, Yan-Fang; Li, Zhi-Heng; Du, Wei-Wei; Xu, Li-Xiao; Ren, Jun-Li; Li, Xiao-Lu; Fang, Fang; Xie, Yi; Li, Mei; Qian, Guang-Hui; Li, Yan-Hong; Li, Yi-Ping; Li, Gang; Wu, Yi; Feng, Xing; Wang, Jian; He, Wei-Qi; Hu, Shao-Yan; Lu, Jun; Pan, Jian

    2017-01-01

    Decreased autophagy is accompanied by the development of a myeloproliferative state or acute myeloid leukemia (AML). AML cells are often sensitive to autophagy-inducing stimuli, prompting the idea that targeting autophagy can be useful in AML cytotoxic therapy. AML NB4 cells overexpressing microtubule-associated protein 1 light chain 3-green fluorescent protein were screened with 69 inhibitors to analyze autophagy activity. AML cells were treated with the polo-like kinase 1 (PLK1) inhibitors RO3280 and BI2536 before autophagy analysis. Cleaved LC3 (LC3-II) and the phosphorylation of mammalian target of rapamycin (mTOR), adenosine monophosphate-activated protein kinase, and Unc-51-like kinase 1 during autophagy was detected with western blotting. Autophagosomes were detected using transmission electron microscopy. Several inhibitors had promising autophagy inducer effects: BI2536, MLN0905, SK1-I, SBE13 HCL and RO3280. Moreover, these inhibitors all targeted PLK1. Autophagy activity was increased in the NB4 cells treated with RO3280 and BI2536. Inhibition of PLK1 expression in NB4, K562 and HL-60 leukemia cells with RNA interference increased LC3-II and autophagy activity. The phosphorylation of mTOR was reduced significantly in NB4 cells treated with RO3280 and BI2536, and was also reduced significantly when PLK1 expression was downregulated in the NB4, K562 and HL-60 cells. We demonstrate that PLK1 inhibition induces AML cell autophagy and that it results in mTOR dephosphorylation. These results may provide new insights into the molecular mechanism of PLK1 in regulating autophagy. PMID:28184925

  7. The critical role of Akt in cardiovascular function.

    PubMed

    Abeyrathna, Prasanna; Su, Yunchao

    2015-11-01

    Akt kinase, a member of AGC kinases, is important in many cellular functions including proliferation, migration, cell growth and metabolism. There are three known Akt isoforms which play critical and diverse roles in the cardiovascular system. Akt activity is regulated by its upstream regulatory pathways at transcriptional and post-translational levels. Beta-catenin/Tcf-4, GLI1 and Stat-3 are some of few known transcriptional regulators of AKT gene. Threonine 308 and serine 473 are the two critical phosphorylation sites of Akt1. Translocation of Akt to the cell membrane facilitates PDK1 phosphorylation of the threonine site. The serine site is phosphorylated by mTORC2. Ack1, Src, PTK6, TBK1, IKBKE and IKKε are some of the non-canonical pathways which affect the Akt activity. Protein-protein interactions of Akt to actin and Hsp90 increase the Akt activity while Akt binding to other proteins such as CTMP and TRB3 reduces the Akt activity. The action of Akt on its downstream targets determines its function in cardiovascular processes such as cell survival, growth, proliferation, angiogenesis, vasorelaxation, and cell metabolism. Akt promotes cell survival via caspase-9, YAP, Bcl-2, and Bcl-x activities. Inhibition of FoxO proteins by Akt also increases cell survival by transcriptional mechanisms. Akt stimulates cell growth and proliferation through mTORC1. Akt also increases VEGF secretion and mediates eNOS phosphorylation, vasorelaxation and angiogenesis. Akt can increase cellular metabolism through its downstream targets GSK3 and GLUT4. The alterations of Akt signaling play an important role in many cardiovascular pathological processes such as atherosclerosis, cardiac hypertrophy, and vascular remodeling. Several Akt inhibitors have been developed and tested as anti-tumor agents. They could be potential novel therapeutics for the cardiovascular diseases.

  8. Glutathione-s-transferase A 4 (GSTA4) suppresses tumor growth and metastasis of human hepatocellular carcinoma by targeting AKT pathway

    PubMed Central

    Liu, Chang Jun; Yang, Jin Hui; Huang, Fei Zhou; Nie, Wan Pin; Liu, Chu Ping; Mao, Xian Hai; Yin, Xin Min; Shen, Xian Bo; Peng, Chuang; Chen, Mei Fu; Jiang, Bo; Liu, Xun Yang; Wu, Jin Shu

    2017-01-01

    Hepatocellular carcinoma (HCC) is one of the most lethal malignancies of cancers and its prognosis remains dismal due to the paucity of effective therapeutic targets. Up-regulation of glutathione-s-transferase A 4 (GSTA4) is associated with poor prognosis of HCC, but its functional mechanism in HCC remains unclear. In this study, we investigated the roles of GSTA4 in tumor growth and metastasis of HCC and found that GSTA4 was frequently up-regulated in HCC tissues. Through gain- and loss-of-function studies, GSTA4 was demonstrated to significantly regulate cell proliferation, migration, and invasion in vitro. Furthermore, GSTA4 overexpressing significantly promoted the tumorigenicity and metastasis of HCC cells in nude mice models bearing human HCC, whereas silencing endogenous GSTA4 caused an opposite outcome. Moreover, we demonstrated that GSTA4 enhanced HCC aggressiveness by activating protein kinase B (AKT) signaling. In multivariate analysis, our results GSTA4 overexpression promotes the progression of hepatocellular carcinoma and might represent a novel therapeutic target for its treatment.

  9. MiRNA-21 mediates the antiangiogenic activity of metformin through targeting PTEN and SMAD7 expression and PI3K/AKT pathway

    PubMed Central

    Luo, Mao; Tan, Xiaoyong; Mu, Lin; Luo, Yulin; Li, Rong; Deng, Xin; Chen, Ni; Ren, Meiping; Li, Yongjie; Wang, Liqun; Wu, Jianbo; Wan, Qin

    2017-01-01

    Metformin, an anti-diabetic drug commonly used for type 2 diabetes therapy, is associated with anti-angiogenic effects in conditions beyond diabetes. miR-21 has been reported to be involved in the process of angiogenesis. However, the precise regulatory mechanisms by which the metformin-induced endothelial suppression and its effects on miR-21-dependent pathways are still unclear. Bioinformatic analysis and identification of miR-21 and its targets and their effects on metformin-induced antiangiogenic activity were assessed using luciferase assays, quantitative real-time PCR, western blots, scratch assays, CCK-8 assays and tubule formation assays. In this study, miR-21 was strikingly downregulated by metformin in a time- and dose-dependent manner. miR-21 directly targeted the 3′-UTR of PTEN and SMAD7, and negatively regulated their expression. Overexpression of miR-21 abrogated the metformin-mediated inhibition of endothelial cells proliferation, migration, tubule formation and the TGF-β-induced AKT, SMAD- and ERK-dependent phosphorylations, and conversely, down-regulation of miR-21 aggravated metformin’s action and revealed significant promotion effects. Our study broadens our understanding of the regulatory mechanism of miR-21 mediating metformin-induced anti-angiogenic effects, providing important implications regarding the design of novel miRNA-based therapeutic strategies against angiogenesis. PMID:28230206

  10. Inactivation of fatty acid synthase impairs hepatocarcinogenesis driven by AKT in mice and humans

    PubMed Central

    Li, Lei; Pilo, Giulia M.; Li, Xiaolei; Cigliano, Antonio; Latte, Gavinella; Che, Li; Joseph, Christy; Mela, Marta; Wang, Chunmei; Jiang, Lijie; Ribback, Silvia; Simile, Maria M.; Pascale, Rosa M.; Dombrowski, Frank; Evert, Matthias; Semenkovich, Clay F.; Chen, Xin; Calvisi, Diego F.

    2015-01-01

    Background & Aims Cumulating evidence underlines the crucial role of aberrant lipogenesis in human hepatocellular carcinoma (HCC). Here, we investigated the oncogenic potential of fatty acid synthase (FASN), the master regulator of de novo lipogenesis, in the mouse liver. Methods FASN was overexpressed in the mouse liver, either alone or in combination with activated N-Ras, c-Met, or SCD1, via hydrodynamic injection. Activated AKT was overexpressed via hydrodynamic injection in livers of conditional FASN or Rictor knockout mice. FASN was suppressed in human hepatoma cell lines via specific small interfering RNA. Results Overexpression of FASN, either alone or in combination with other genes associated with hepatocarcinogenesis, did not induce histological liver alterations. In contrast, genetic ablation of FASN resulted in the complete inhibition of hepatocarcinogenesis in AKT-overexpressing mice. In human HCC cell lines, FASN inactivation led to a decline in cell proliferation and a rise in apoptosis, which were paralleled by a decrease in the levels of phosphorylated/activated AKT, an event controlled by the mammalian target of rapamycin complex 2 (mTORC2). Downregulation of AKT phosphorylation/activation following FASN inactivation was associated with strong inhibition of rapamycin-insensitive companion of mTOR (Rictor), the major component of mTORC2, at post-transcriptional level. Finally, genetic ablation of Rictor impaired AKT-driven hepatocarcinogenesis in mice. Conclusions FASN is not oncogenic per se in the mouse liver, but is necessary for AKT-driven hepatocarcinogenesis. Pharmacological blockade of FASN might be highly useful in the treatment of human HCC characterized by activation of the AKT pathway. PMID:26476289

  11. Distinct domains of the spinal muscular atrophy protein SMN are required for targeting to Cajal bodies in mammalian cells.

    PubMed

    Renvoisé, Benoît; Khoobarry, Kevinee; Gendron, Marie-Claude; Cibert, Christian; Viollet, Louis; Lefebvre, Suzie

    2006-02-15

    Mutations of the survival motor neuron gene SMN1 cause the inherited disease spinal muscular atrophy (SMA). The ubiquitous SMN protein facilitates the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs). The protein is detected in the cytoplasm, nucleoplasm and enriched with snRNPs in nuclear Cajal bodies. It is structurally divided into at least an amino-terminal region rich in basic amino acid residues, a central Tudor domain, a self-association tyrosine-glycine-box and an exon7-encoded C-terminus. To examine the domains required for the intranuclear localization of SMN, we have used fluorescently tagged protein mutants transiently overexpressed in mammalian cells. The basic amino acid residues direct nucleolar localization of SMN mutants. The Tudor domain promotes localization of proteins in the nucleus and it cooperates with the basic amino acid residues and the tyrosine-glycine-box for protein localization in Cajal bodies. Moreover, the most frequent disease-linked mutant SMNDeltaex7 reduces accumulation of snRNPs in Cajal bodies, suggesting that the C-terminus of SMN participates in targeting to Cajal bodies. A reduced number of Cajal bodies in patient fibroblasts associates with the absence of snRNPs in Cajal bodies, revealing that intranuclear snRNA organization is modified in disease. These results indicate that direct and indirect mechanisms regulate localization of SMN in Cajal bodies.

  12. Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery.

    PubMed

    Panavas, Tadas; Lu, Jin; Liu, Xuesong; Winkis, Ann-Marie; Powers, Gordon; Naso, Michael F; Amegadzie, Bernard

    2011-09-01

    Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity.

  13. Antisense Oligonucleotides Targeting Parasite Inositol 1,4,5-Trisphosphate Receptor Inhibits Mammalian Host Cell Invasion by Trypanosoma cruzi

    NASA Astrophysics Data System (ADS)

    Hashimoto, Muneaki; Nara, Takeshi; Hirawake, Hiroko; Morales, Jorge; Enomoto, Masahiro; Mikoshiba, Katsuhiko

    2014-02-01

    Chagas disease is caused by an intracellular parasitic protist, Trypanosoma cruzi. As there are no highly effective drugs against this agent that also demonstrate low toxicity, there is an urgent need for development of new drugs to treat Chagas disease. We have previously demonstrated that the parasite inositol 1,4,5-trisphosphate receptor (TcIP3R) is crucial for invasion of the mammalian host cell by T. cruzi. Here, we report that TcIP3R is a short-lived protein and that its expression is significantly suppressed in trypomastigotes. Treatment of trypomastigotes, an infective stage of T. cruzi, with antisense oligonucleotides specific to TcIP3R deceased TcIP3R protein levels and impaired trypomastigote invasion of host cells. Due to the resulting instability and very low expression level of TcIP3R in trypomastigotes indicates that TcIP3R is a promising target for antisense therapy in Chagas disease.

  14. Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain.

    PubMed

    Vieux-Rochas, Maxence; Fabre, Pierre J; Leleu, Marion; Duboule, Denis; Noordermeer, Daan

    2015-04-14

    Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters centered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics, and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra- and interchromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral lamina-associated domains. Interactions are independent of nearby H3K27me3-marked loci and determined by chromosomal distance and cell-type-specific scaling factors, thus inducing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Polycomb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active, nuclear environments.

  15. Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA

    PubMed Central

    Zhang, Lin; Hou, Dongxia; Chen, Xi; Li, Donghai; Zhu, Lingyun; Zhang, Yujing; Li, Jing; Bian, Zhen; Liang, Xiangying; Cai, Xing; Yin, Yuan; Wang, Cheng; Zhang, Tianfu; Zhu, Dihan; Zhang, Dianmu; Xu, Jie; Chen, Qun; Ba, Yi; Liu, Jing; Wang, Qiang; Chen, Jianqun; Wang, Jin; Wang, Meng; Zhang, Qipeng; Zhang, Junfeng; Zen, Ke; Zhang, Chen-Yu

    2012-01-01

    Our previous studies have demonstrated that stable microRNAs (miRNAs) in mammalian serum and plasma are actively secreted from tissues and cells and can serve as a novel class of biomarkers for diseases, and act as signaling molecules in intercellular communication. Here, we report the surprising finding that exogenous plant miRNAs are present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired orally, through food intake. MIR168a is abundant in rice and is one of the most highly enriched exogenous plant miRNAs in the sera of Chinese subjects. Functional studies in vitro and in vivo demonstrated that MIR168a could bind to the human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA, inhibit LDLRAP1 expression in liver, and consequently decrease LDL removal from mouse plasma. These findings demonstrate that exogenous plant miRNAs in food can regulate the expression of target genes in mammals. PMID:21931358

  16. Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA.

    PubMed

    Zhang, Lin; Hou, Dongxia; Chen, Xi; Li, Donghai; Zhu, Lingyun; Zhang, Yujing; Li, Jing; Bian, Zhen; Liang, Xiangying; Cai, Xing; Yin, Yuan; Wang, Cheng; Zhang, Tianfu; Zhu, Dihan; Zhang, Dianmu; Xu, Jie; Chen, Qun; Ba, Yi; Liu, Jing; Wang, Qiang; Chen, Jianqun; Wang, Jin; Wang, Meng; Zhang, Qipeng; Zhang, Junfeng; Zen, Ke; Zhang, Chen-Yu

    2012-01-01

    Our previous studies have demonstrated that stable microRNAs (miRNAs) in mammalian serum and plasma are actively secreted from tissues and cells and can serve as a novel class of biomarkers for diseases, and act as signaling molecules in intercellular communication. Here, we report the surprising finding that exogenous plant miRNAs are present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired orally, through food intake. MIR168a is abundant in rice and is one of the most highly enriched exogenous plant miRNAs in the sera of Chinese subjects. Functional studies in vitro and in vivo demonstrated that MIR168a could bind to the human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA, inhibit LDLRAP1 expression in liver, and consequently decrease LDL removal from mouse plasma. These findings demonstrate that exogenous plant miRNAs in food can regulate the expression of target genes in mammals.

  17. Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity.

    PubMed

    Wang, Yuji; Li, Pingxin; Wang, Shu; Hu, Jing; Chen, Xiangyun Amy; Wu, Jianhui; Fisher, Megan; Oshaben, Kira; Zhao, Na; Gu, Ying; Wang, Dong; Chen, Gong; Wang, Yanming

    2012-07-27

    Tumor suppressor genes are frequently silenced in cancer cells by enzymes catalyzing epigenetic histone modifications. The peptidylarginine deiminase family member PAD4 (also called PADI4) is markedly overexpressed in a majority of human cancers, suggesting that PAD4 is a putative target for cancer treatment. Here, we have generated novel PAD inhibitors with low micromolar IC(50) in PAD activity and cancer cell growth inhibition. The lead compound YW3-56 alters the expression of genes controlling the cell cycle and cell death, including SESN2 that encodes an upstream inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Guided by the gene expression profile analyses with YW3-56, we found that PAD4 functions as a corepressor of p53 to regulate SESN2 expression by histone citrullination in cancer cells. Consistent with the mTORC1 inhibition by SESN2, the phosphorylation of its substrates including p70S6 kinase (p70S6K) and 4E-BP1 was decreased. Furthermore, macroautophagy is perturbed after YW3-56 treatment in cancer cells. In a mouse xenograft model, YW3-56 demonstrates cancer growth inhibition activity with little if any detectable adverse effect to vital organs, whereas a combination of PAD4 and histone deacetylase inhibitors further decreases tumor growth. Taken together, our work found that PAD4 regulates the mTORC1 signaling pathway and that PAD inhibitors are potential anticancer reagents that activate tumor suppressor gene expression alone or in combination with histone deacetylase inhibitors.

  18. MicroRNA-432 targeting E2F3 and P55PIK inhibits myogenesis through PI3K/AKT/mTOR signaling pathway.

    PubMed

    Ma, Meilin; Wang, Xiangming; Chen, Xiaochang; Cai, Rui; Chen, Fenfen; Dong, Wuzi; Yang, Gongshe; Pang, Weijun

    2017-03-04

    Skeletal muscle is the dominant executant in locomotion and regulator in energy metabolism. Embryonic myogenesis and postnatal muscle growth are controlled by a cascade of transcription factors and epigenetic regulatory mechanisms. MicroRNAs (miRNAs), a family of non-coding RNA of 22 nucleotides in length, post-transcriptionally regulates expression of mRNA by pairing the seed sequence to 3' UTR of target mRNA. Increasing evidence has demonstrated that miRNAs are important regulators in diverse myogenic processes. The profiling of miRNA expression revealed that miR-432 is more enriched in the longissimus dorsi of 35-day-old piglets than that of adult pigs. Our gain of function study showed that miR-432 can negatively regulate both myoblast proliferation and differentiation. Mechanically, we found that miR-432 is able to down-regulate E2F transcription factor 3 (E2F3) to inactivate the expression of cell cycle and myogenic genes. We also identified that phosphatidylinositol 3-kinase regulatory subunit (P55PIK) is another target gene of miR-432 in muscle cells. downregulation of P55PIK by miR-432 leads to inhibition of P55PIK-mediated PI3K/AKT/mTOR signaling pathway during differentiation. The blocking effect of miR-432 on this pathway can be rescued by insulin treatment. Taken together, our findings identified microRNA-432 as a potent inhibitor of myogenesis which functions by targeting E2F3 and P55PIK in muscle cells.

  19. Phosphorylated Mammalian Target of Rapamycin p-mTOR Is a Favorable Prognostic Factor than mTOR in Gastric Cancer

    PubMed Central

    Zhang, Jia-wei; Chen, Bo; Xiong, Mao-ming

    2016-01-01

    Aims The mammalian target of rapamycin (mTOR) and phosphorylated mTOR (p-mTOR) occurring downstream in the PI3K/Akt/mTOR pathway, are regarded as potential prognostic markers for gastric cancer (GC). However, the prognostic value of mTOR/p-mTOR expression remains controversial. In this study, we determined the expression of mTOR, p-mTOR, p70S6k, and p-p70S6K in GC, and investigated the correlation between their overexpression, clinicopathological parameters, and overall survival (OS). Methods The expression of mTOR, p-mTOR, p70S6k, and p-p70S6K was examined in 120 GC patients by immunohistochemistry (IHC). The association of protein expression with clinicopathological features and OS was explored. The p-mTOR expression was detected in normal, adjacent, and GC tissues using Western blot. Eligible studies retrieved from PubMed, Ovid, Web of Science and Cochrane databases, were reviewed in this meta-analysis. Results IHC showed that the rates of expression of the signal transduction molecules mTOR, p-mTOR, p70S6k and p-p70S6K in GC were 60.8%, 54.2%, 53.3% and 53.3%, respectively. Overexpression of mTOR and p70S6K showed no significant association with clinical variables. Expression of p-mTOR was significantly associated with differentiation (P < 0.01), depth of invasion (P < 0.01), lymph node metastasis (P = 0.04) and TNM stage (P = 0.02). Expression of p-p70S6K was associated with differentiation (P = 0.006), depth of invasion (P < 0.001), and TNM stage (P = 0.02). In survival analysis, differentiation, depth of invasion, lymph node metastasis and TNM stage were not related to OS (all P > 0.05). Furthermore, p-mTOR and p-p70S6K expression, but not mTOR and p70S6K, were tightly associated with OS of GC patients (P = 0.006 and P < 0.001, respectively). In Western blot, p-mTOR was significantly higher in GC tissues than in normal and adjacent tissues. In the present meta-analysis, mTOR overexpression showed no relationship with any clinicopathological variables

  20. A New Functional Role for Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) in the Circadian Regulation of L-Type Voltage-Gated Calcium Channels in Avian Cone Photoreceptors

    PubMed Central

    Huang, Cathy Chia-Yu; Ko, Michael Lee; Ko, Gladys Yi-Ping

    2013-01-01

    In the retina, the L-type voltage-gated calcium channels (L-VGCCs) are responsible for neurotransmitter release from photoreceptors and are under circadian regulation. Both the current densities and protein expression of L-VGCCs are significantly higher at night than during the day. However, the underlying mechanisms of circadian regulation of L-VGCCs in the retina are not completely understood. In this study, we demonstrated that the mechanistic/mammalian target of rapamycin complex (mTORC) signaling pathway participated in the circadian phase-dependent modulation of L-VGCCs. The activities of the mTOR cascade, from mTORC1 to its downstream targets, displayed circadian oscillations throughout the course of a day. Disruption of mTORC1 signaling dampened the L-VGCC current densities, as well as the protein expression of L-VGCCs at night. The decrease of L-VGCCs at night by mTORC1 inhibition was in part due to a reduction of L-VGCCα1 subunit translocation from the cytosol to the plasma membrane. Finally, we showed that mTORC1 was downstream of the phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT) signaling pathway. Taken together, mTORC1 signaling played a role in the circadian regulation of L-VGCCs, in part through regulation of ion channel trafficking and translocation, which brings to light a new functional role for mTORC1: the modulation of ion channel activities. PMID:23977383

  1. AKT1 and AKT2 isoforms play distinct roles during breast cancer progression through the regulation of specific downstream proteins

    PubMed Central

    Riggio, Marina; Perrone, María C.; Polo, María L.; Rodriguez, María J.; May, María; Abba, Martín; Lanari, Claudia; Novaro, Virginia

    2017-01-01

    The purpose of this study was to elucidate the mechanisms associated with the specific effects of AKT1 and AKT2 isoforms in breast cancer progression. We modulated the abundance of specific AKT isoforms in IBH-6 and T47D human breast cancer cell lines and showed that AKT1 promoted cell proliferation, through S6 and cyclin D1 upregulation, but it inhibited cell migration and invasion through β1-integrin and focal adhesion kinase (FAK) downregulation. In contrast, AKT2 promoted cell migration and invasion through F-actin and vimentin induction. Thus, while overexpression of AKT1 promoted local tumor growth, downregulation of AKT1 or overexpression of AKT2 promoted peritumoral invasion and lung metastasis. Furthermore, we evaluated The Cancer Genome Atlas (TCGA) dataset for invasive breast carcinomas and found that increased AKT2 but not AKT1 mRNA levels correlated with a worse clinical outcome. We conclude that AKT isoforms play specific roles in different steps of breast cancer progression, with AKT1 involved in the local tumor growth and AKT2 involved in the distant tumor dissemination, having AKT2 a poorer prognostic value and consequently being a worthwhile target for therapy. PMID:28287129

  2. Complex regulation of mammalian target of rapamycin complex 1 in the basomedial hypothalamus by leptin and nutritional status.

    PubMed

    Villanueva, Eneida C; Münzberg, Heike; Cota, Daniela; Leshan, Rebecca L; Kopp, Keely; Ishida-Takahashi, Ryoko; Jones, Justin C; Fingar, Diane C; Seeley, Randy J; Myers, Martin G

    2009-10-01

    The medial basal hypothalamus, including the arcuate nucleus (ARC) and the ventromedial hypothalamic nucleus (VMH), integrates signals of energy status to modulate metabolism and energy balance. Leptin and feeding regulate the mammalian target of rapamycin complex 1 (mTORC1) in the hypothalamus, and hypothalamic mTORC1 contributes to the control of feeding and energy balance. To determine the mechanisms by which leptin modulates mTORC1 in specific hypothalamic neurons, we immunohistochemically assessed the mTORC1-dependent phosphorylation of ribosomal protein S6 (pS6). In addition to confirming the modulation of ARC mTORC1 activity by acute leptin treatment, this analysis revealed the robust activation of mTORC1-dependent ARC pS6 in response to fasting and leptin deficiency in leptin receptor-expressing Agouti-related protein neurons. In contrast, fasting and leptin deficiency suppress VMH mTORC1 signaling. The appropriate regulation of ARC mTORC1 by mutant leptin receptor isoforms correlated with their ability to suppress the activity of Agouti-related protein neurons, suggesting the potential stimulation of mTORC1 by the neuronal activity. Indeed, fasting- and leptin deficiency-induced pS6-immunoreactivity (IR) extensively colocalized with c-Fos-IR in ARC and VMH neurons. Furthermore, ghrelin, which activates orexigenic ARC neurons, increased ARC mTORC1 activity and induced colocalized pS6- and c-Fos-IR. Thus, neuronal activity promotes mTORC1/pS6 in response to signals of energy deficit. In contrast, insulin, which activates mTORC1 via the phosphatidylinositol 3-kinase pathway, increased ARC and VMH pS6-IR in the absence of neuronal activation. The regulation of mTORC1 in the basomedial hypothalamus thus varies by cell and stimulus type, as opposed to responding in a uniform manner to nutritional and hormonal perturbations.

  3. Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

    SciTech Connect

    Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun; Song, Sang Heon; Shin, Hwa Kyoung; Bae, Sun Sik

    2015-08-14

    Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs.

  4. The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions.

    PubMed

    O'Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A

    2009-07-15

    Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K-PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K-PKB is a transient event (<15 min), while the activation of mTOR is sustained for a long duration (>12 h). Furthermore, inhibition of PI3K-PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K-PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K-PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K-PKB-independent mechanism that requires PLD and PA.

  5. MicroRNA-21 Regulates PI3K/Akt/mTOR Signaling by Targeting TGFβI during Skeletal Muscle Development in Pigs.

    PubMed

    Bai, Lijing; Liang, Ruyi; Yang, Yalan; Hou, Xinhua; Wang, Zishuai; Zhu, Shiyun; Wang, Chuduan; Tang, Zhonglin; Li, Kui

    2015-01-01

    MicroRNAs (miRNAs), which are short (22-24 base pairs), non-coding RNAs, play critical roles in myogenesis. Using Solexa deep sequencing, we detected the expression levels of 229 and 209 miRNAs in swine skeletal muscle at 90 days post-coitus (E90) and 100 days postnatal (D100), respectively. A total of 138 miRNAs were up-regulated on E90, and 31 were up-regulated on D100. Of these, 9 miRNAs were selected for the validation of the small RNA libraries by quantitative RT-PCR (RT-qPCR). We found that miRNA-21 was down-regulated by 17-fold on D100 (P<0.001). Bioinformatics analysis suggested that the transforming growth factor beta-induced (TGFβI) gene was a potential target of miRNA-21. Both dual luciferase reporter assays and western blotting demonstrated that the TGFβI gene was regulated by miRNA-21. Co-expression analysis revealed that the mRNA expression levels of miRNA-21 and TGFβI were negatively correlated (r = -0.421, P = 0.026) in skeletal muscle during the 28 developmental stages. Our results revealed that more miRNAs are expressed in prenatal than in postnatal skeletal muscle. The miRNA-21 is a novel myogenic miRNA that is involved in skeletal muscle development and regulates PI3K/Akt/mTOR signaling by targeting the TGFβI gene.

  6. Multi-drug loaded micelles delivering chemotherapy and targeted therapies directed against HSP90 and the PI3K/AKT/mTOR pathway in prostate cancer

    PubMed Central

    Le, Bao; Powers, Ginny L.; Tam, Yu Tong; Schumacher, Nicholas; Malinowski, Rita L.; Steinke, Laura; Kwon, Glen; Marker, Paul C.

    2017-01-01

    Background Advanced prostate cancers that are resistant to all current therapies create a need for new therapeutic strategies. One recent innovative approach to cancer therapy is the simultaneous use of multiple FDA-approved drugs to target multiple pathways. A challenge for this approach is caused by the different solubility requirements of each individual drug, resulting in the need for a drug vehicle that is non-toxic and capable of carrying multiple water-insoluble antitumor drugs. Micelles have recently been shown to be new candidate drug solubilizers for anti cancer therapy. Methods This study set out to examine the potential use of multi-drug loaded micelles for prostate cancer treatment in preclinical models including cell line and mouse models for prostate cancers with Pten deletions. Specifically antimitotic agent docetaxel, mTOR inhibitor rapamycin, and HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin were incorporated into the micelle system (DR17) and tested for antitumor efficacy. Results In vitro growth inhibition of prostate cancer cells was greater when all three drugs were used in combination compared to each individual drug, and packaging the drugs into micelles enhanced the cytotoxic effects. At the molecular level DR17 targeted simultaneously several molecular signaling axes important in prostate cancer including androgen receptor, mTOR, and PI3K/AKT. In a mouse genetic model of prostate cancer, DR17 treatment decreased prostate weight, which was achieved by both increasing caspase-dependent cell death and decreasing cell proliferation. Similar effects were also observed when DR17 was administered to nude mice bearing prostate cancer cells xenografts. Conclusion These results suggest that combining these three cancer drugs in multi-drug loaded micelles may be a promising strategy for prostate cancer therapy. PMID:28350865

  7. Analysis of AKT and ERK1/2 protein kinases in extracellular vesicles isolated from blood of patients with cancer

    PubMed Central

    van der Mijn, Johannes C.; Sol, Nik; Mellema, Wouter; Jimenez, Connie R.; Piersma, Sander R.; Dekker, Henk; Schutte, Lisette M.; Smit, Egbert F.; Broxterman, Henk J.; Skog, Johan; Tannous, Bakhos A.; Wurdinger, Thomas; Verheul, Henk M. W.

    2014-01-01

    Background Extracellular vesicles (EVs) are small nanometre-sized vesicles that are circulating in blood. They are released by multiple cells, including tumour cells. We hypothesized that circulating EVs contain protein kinases that may be assessed as biomarkers during treatment with tyrosine kinase inhibitors. Methods EVs released by U87 glioma cells, H3255 and H1650 non-small-cell lung cancer (NSCLC) cells were profiled by tandem mass spectrometry. Total AKT/protein kinase B and extracellular signal regulated kinase 1/2 (ERK1/2) levels as well as their relative phosphorylation were measured by western blot in isogenic U87 cells with or without mutant epidermal growth factor receptor (EGFRvIII) and their corresponding EVs. To assess biomarker potential, plasma samples from 24 healthy volunteers and 42 patients with cancer were used. Results In total, 130 different protein kinases were found to be released in EVs including multiple drug targets, such as mammalian target of rapamycin (mTOR), AKT, ERK1/2, AXL and EGFR. Overexpression of EGFRvIII in U87 cells results in increased phosphorylation of EGFR, AKT and ERK1/2 in cells and EVs, whereas a decreased phosphorylation was noted upon treatment with the EGFR inhibitor erlotinib. EV samples derived from patients with cancer contained significantly more protein (p=0.0067) compared to healthy donors. Phosphorylation of AKT and ERK1/2 in plasma EVs from both healthy donors and patients with cancer was relatively low compared to levels in cancer cells. Preliminary analysis of total AKT and ERK1/2 levels in plasma EVs from patients with NSCLC before and after sorafenib/metformin treatment (n=12) shows a significant decrease in AKT levels among patients with a favourable treatment response (p<0.005). Conclusion Phosphorylation of protein kinases in EVs reflects their phosphorylation in tumour cells. Total AKT protein levels may allow monitoring of kinase inhibitor responses in patients with cancer. PMID:25491250

  8. Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress

    PubMed Central

    Thompson, Scott M.; Callstrom, Matthew R.; Jondal, Danielle E.; Butters, Kim A.; Knudsen, Bruce E.; Anderson, Jill L.; Lien, Karen R.; Sutor, Shari L.; Lee, Ju-Seog; Thorgeirsson, Snorri S.; Grande, Joseph P.; Roberts, Lewis R.; Woodrum, David A.

    2016-01-01

    Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2–3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin. PMID:27611696

  9. A novel AKT inhibitor, AZD5363, inhibits phosphorylation of AKT downstream molecules, and activates phosphorylation of mTOR and SMG-1 dependent on the liver cancer cell type

    PubMed Central

    ZHANG, YUNCHENG; ZHENG, YUANWEN; FAHEEM, ALI; SUN, TIANTONG; LI, CHUNYOU; LI, ZHE; ZHAO, DIANTANG; WU, CHAO; LIU, JUN

    2016-01-01

    Due to frequent phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway dysregulation, AKT is typically accepted as a promising anticancer therapeutic target. mTOR, in particular, represents a suitable therapeutic target for hepatocellular carcinoma, whilst suppressor with morphogenetic effect on genitalia family member-1 (SMG-1) is believed to serve a potential tumor suppressor role in human cancer. Despite SMG-1 and mTOR belonging to the same PI3K-related kinase family, the interactions between them are not yet fully understood. In the present study, a novel pyrrolopyrimidine-derived compound, AZD5363, was observed to suppress proliferation in liver cancer Hep-G2 and Huh-7 cells by inhibiting the phosphorylation of downstream molecules in the AKT signal pathway, in a dose- and time-dependent manner. AZD5363 activated the phosphorylation of mTOR, dependent on the liver cancer cell type, as it may have differing effects in various liver cancer cell lines. Additionally, AZD5363 also activated SMG-1 within the same liver cancer cells types, which subsequently activated the phosphorylation of mTOR. In conclusion, the present study indicates that AZD5363 inhibited phosphorylation of AKT downstream molecules, and activated phosphorylation of mTOR and SMG-1, dependent on the liver cancer type. PMID:26998062

  10. Obesity Increases Vascular Senescence and Susceptibility to Ischemic Injury Through Chronic Activation of Akt and mTOR

    PubMed Central

    Wang, Chao-Yung; Kim, Hyung-Hwan; Hiroi, Yukio; Sawada, Naoki; Salomone, Salvatore; Benjamin, Laura E.; Walsh, Kenneth; Moskowitz, Michael A.; Liao, James K.

    2009-01-01

    Obesity and age are important risk factors for cardiovascular disease. However, the signaling mechanism linking obesity with age-related vascular senescence is unknown. Here we show that mice fed a high-fat diet show increased vascular senescence and vascular dysfunction compared to mice fed standard chow and are more prone to peripheral and cerebral ischemia. All of these changes involve long-term activation of the protein kinase Akt. In contrast, mice with diet-induced obesity that lack Akt1 are resistant to vascular senescence. Rapamycin treatment of diet-induced obese mice or of transgenic mice with long-term activation of endothelial Akt inhibits activation of mammalian target of rapamycin (mTOR)–rictor complex 2 and Akt, prevents vascular senescence without altering body weight, and reduces the severity of limb necrosis and ischemic stroke. These findings indicate that long-term activation of Akt-mTOR signaling links diet-induced obesity with vascular senescence and cardiovascular disease. PMID:19293429

  11. Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics.

    PubMed

    Costa, Céu; Pereira, Sofia; Lima, Luís; Peixoto, Andreia; Fernandes, Elisabete; Neves, Diogo; Neves, Manuel; Gaiteiro, Cristiana; Tavares, Ana; Gil da Costa, Rui M; Cruz, Ricardo; Amaro, Teresina; Oliveira, Paula A; Ferreira, José Alexandre; Santos, Lúcio L

    2015-01-01

    Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin

  12. Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics

    PubMed Central

    Lima, Luís; Peixoto, Andreia; Fernandes, Elisabete; Neves, Diogo; Neves, Manuel; Gaiteiro, Cristiana; Tavares, Ana; Gil da Costa, Rui M.; Cruz, Ricardo; Amaro, Teresina; Oliveira, Paula A.; Ferreira, José Alexandre; Santos, Lúcio L.

    2015-01-01

    Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin

  13. Per2 participates in AKT-mediated drug resistance in A549/DDP lung adenocarcinoma cells.

    PubMed

    Chen, Bo; Tan, Yaoxi; Liang, Yan; Li, Yan; Chen, Lei; Wu, Shuangshuang; Xu, Wei; Wang, Yan; Zhao, Weihong; Wu, Jianqing

    2017-01-01

    Period2 (Per2) is a key mammalian circadian clock protein, and additionally has a tumor suppressive function. The present study aimed to investigate its role in drug resistance in A549/cisplatin (DDP) lung adenocarcinoma cells. Per2 knockdown and overexpression in A549/DDP cells were used to compare cell proliferation (by MTT assay), apoptosis (active-caspase 3 western blot) and clone forming assay. The activation of AKT/mechanistic target of rapamycin (mTOR) was investigated by a western blot assay. The Per2 expression level was decreased in A549/DDP cells compared with A549 cells. Per2 knockdown by short hairpin RNA protects A549/DDP cells from apoptosis, and promotes proliferation and migration. Per2 knockdown results in increased activation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway. Overexpression of Per2 in A549/DDP cells may reduce the activity of the PI3K/AKT/mTOR signaling pathway, and promote apoptosis of A549 cells. The results of the present study suggest that Per2 participates in AKT-mediated drug resistance in A549/DDP lung adenocarcinoma cells.

  14. Per2 participates in AKT-mediated drug resistance in A549/DDP lung adenocarcinoma cells

    PubMed Central

    Chen, Bo; Tan, Yaoxi; Liang, Yan; Li, Yan; Chen, Lei; Wu, Shuangshuang; Xu, Wei; Wang, Yan; Zhao, Weihong; Wu, Jianqing

    2017-01-01

    Period2 (Per2) is a key mammalian circadian clock protein, and additionally has a tumor suppressive function. The present study aimed to investigate its role in drug resistance in A549/cisplatin (DDP) lung adenocarcinoma cells. Per2 knockdown and overexpression in A549/DDP cells were used to compare cell proliferation (by MTT assay), apoptosis (active-caspase 3 western blot) and clone forming assay. The activation of AKT/mechanistic target of rapamycin (mTOR) was investigated by a western blot assay. The Per2 expression level was decreased in A549/DDP cells compared with A549 cells. Per2 knockdown by short hairpin RNA protects A549/DDP cells from apoptosis, and promotes proliferation and migration. Per2 knockdown results in increased activation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway. Overexpression of Per2 in A549/DDP cells may reduce the activity of the PI3K/AKT/mTOR signaling pathway, and promote apoptosis of A549 cells. The results of the present study suggest that Per2 participates in AKT-mediated drug resistance in A549/DDP lung adenocarcinoma cells. PMID:28123577

  15. MicroRNA-145 inhibits the activation of the mTOR signaling pathway to suppress the proliferation and invasion of invasive pituitary adenoma cells by targeting AKT3 in vivo and in vitro

    PubMed Central

    Zhou, Kai; Fan, Yan-Dong; Wu, Peng-Fei; Duysenbi, Serick; Feng, Zhao-Hai; Du, Guo-Jia; Zhang, Ting-Rong

    2017-01-01

    Purpose This study was designed to explore how miR-145 regulates the mTOR signaling pathway in invasive pituitary adenoma (IPA) by targeting AKT3. Methods A total of 71 cases of IPA tissues and 66 cases of non-IPA tissues were obtained in this study. In vitro, the IPA cells were assigned into blank control, empty plasmid, miR-145 mimic, miR-145 inhibitor, miR-145 mimic + rapamycin, miR-145 inhibitor + rapamycin and rapamycin groups. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to detect the protein expressions of PI3K, AKT3, mTOR mRNA and the mRNA expression of miR-145 both in vivo and in vitro. Additionally, the S6K and RPS6 mRNA and protein expressions as well as the relative phosphorylation levels were determined in vitro. MTT assay, flow cytometry and transwell assay were used to testify the cell proliferation, apoptosis and invasion ability, respectively. Results The IPA tissues exhibited significantly lower expression of miR-145 but higher PI3K, AKT3 and mTOR mRNA and protein expressions when compared with the non-IPA tissues. Compared with the blank control and empty plasmid groups, the miR-145 mimic group showed significantly decreased PI3K, AKT3, mTOR, S6K and RPS6 mRNA and protein expressions as well as phosphorylation levels; besides, the IPA cell proliferation, migration and invasion ability were strongly inhibited, accompanied with the increased number of apoptotic cells. In the miR-145 inhibitor group, the PI3K, AKT3, mTOR, S6K and RPS6 mRNA and protein expressions as well as the phosphorylation levels were significantly increased; cell proliferation, migration and invasion ability were remarkably elevated, accompanied with reduced apoptotic cell number. Conclusion The study demonstrates that miR-145 inhibits the mTOR signaling pathway to suppress the IPA cell proliferation and invasion and promotes its apoptosis by targeting AKT3. PMID:28352194

  16. Medium-chain triacylglycerol suppresses the decrease of plasma albumin level through the insulin-Akt-mTOR pathway in the livers of malnourished rats.

    PubMed

    Sekine, Seiji; Terada, Shin; Aoyama, Toshiaki

    2013-01-01

    Recent studies have shown that medium-chain triacylglycerol (MCT) improved serum albumin concentration in elderly people with protein-energy malnutrition (PEM) and in malnourished rats. However, the mechanism for this effect has not been clarified. Dietary MCT promotes insulin secretion from the pancreas, and insulin activates mammalian target of rapamycin (mTOR) complex 1 (mTORC1) via the activation of phosphoinositide 3-kinase (PI3K) and its downstream effecter, Akt. mTORC1 promotes mRNA translation through S6K and 4E-BP1. Therefore, we hypothesized that dietary MCT elevates albumin synthesis through promotion of insulin-Akt-mTOR transduction in the liver. To test this hypothesis, we measured phosphorylated Akt, mTOR and albumin in the livers of malnourished rats. In the present study we examined rats fed low-protein diets containing either MCT or long-chain triacylglycerol (LCT) with energy restriction. The plasma and liver albumin levels were significantly higher in the MCT-fed group than in the LCT-fed group. In addition, plasma insulin concentration, liver phosphorylated Akt/Akt and phosphorylated mTOR/mTOR levels were significantly higher in the MCT-fed group than in the LCT-fed group. These results suggest that one of the mechanisms for the albumin improvement effect of dietary MCT is the promotion of albumin synthesis through the insulin-Akt-mTOR signaling pathway of the liver.

  17. Power of PTEN/AKT: Molecular switch between tumor suppressors and oncogenes

    PubMed Central

    XIE, YINGQIU; NAIZABEKOV, SANZHAR; CHEN, ZHANLIN; TOKAY, TURSONJAN

    2016-01-01

    An increasing amount of evidence has shown that tumor suppressors can become oncogenes, or vice versa, but the mechanism behind this is unclear. Recent findings have suggested that phosphatase and tensin homolog (PTEN) is one of the powerful switches for the conversion between tumor suppressors and oncogenes. PTEN regulates a number of cellular processes, including cell death and proliferation, through the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Furthermore, a number of studies have suggested that PTEN deletions may alter various functions of certain tumor suppressor and oncogenic proteins. The aim of the present review was to analyze specific cases driven by PTEN loss/AKT activation, including aberrant signaling pathways and novel drug targets for clinical application in personalized medicine. The findings illustrate how PTEN loss and/or AKT activation switches MDM2-dependent p53 downregulation, and induces conversion between oncogene and tumor suppressor in enhancer of zeste homolog 2, BTB domain-containing 7A, alternative reading frame 2, p27 and breast cancer 1, early onset, through multiple mechanisms. This review highlights the genetic basis of complex drug targets and provides insights into the rationale of precision cancer therapy. PMID:27347153

  18. Phosphatidylserine is a critical modulator for Akt activation

    PubMed Central

    Huang, Bill X.; Akbar, Mohammed; Kevala, Karl

    2011-01-01

    Akt activation relies on the binding of Akt to phosphatidylinositol-3,4,5-trisphosphate (PIP3) in the membrane. Here, we demonstrate that Akt activation requires not only PIP3 but also membrane phosphatidylserine (PS). The extent of insulin-like growth factor–induced Akt activation and downstream signaling as well as cell survival under serum starvation conditions positively correlates with plasma membrane PS levels in living cells. PS promotes Akt-PIP3 binding, participates in PIP3-induced Akt interdomain conformational changes for T308 phosphorylation, and causes an open conformation that allows for S473 phosphorylation by mTORC2. PS interacts with specific residues in the pleckstrin homology (PH) and regulatory (RD) domains of Akt. Disruption of PS–Akt interaction by mutation impairs Akt signaling and increases susceptibility to cell death. These data identify a critical function of PS for Akt activation and cell survival, particularly in conditions with limited PIP3 availability. The novel molecular interaction mechanism for Akt activation suggests potential new targets for controlling Akt-dependent cell survival and proliferation. PMID:21402788

  19. Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains

    PubMed Central

    Gao, Xinxin

    2008-01-01

    As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane. PMID:18701703

  20. Myostatin signaling regulates Akt activity via the regulation of miR-486 expression.

    PubMed

    Hitachi, Keisuke; Nakatani, Masashi; Tsuchida, Kunihiro

    2014-02-01

    Myostatin, also known as growth and differentiation factor-8, is a pivotal negative regulator of skeletal muscle mass and reduces muscle protein synthesis by inhibiting the insulin-like growth factor-1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. However, the precise mechanism by which myostatin inhibits the IGF-1/Akt/mTOR pathway remains unclear. In this study, we investigated the global microRNA expression profile in myostatin knockout mice and identified miR-486, a positive regulator of the IGF-1/Akt pathway, as a novel target of myostatin signaling. In myostatin knockout mice, the expression level of miR-486 in skeletal muscle was significantly increased. In addition, we observed increased expression of the primary transcript of miR-486 (pri-miR-486) and Ankyrin 1.5 (Ank1.5), the host gene of miR-486, in myostatin knockout mice. In C2C12 cells, myostatin negatively regulated the expression of Ank1.5. Moreover, canonical myostatin signaling repressed the skeletal muscle-specific promoter activity of miR-486/Ank1.5. This repression was partially mediated by the E-box elements in the proximal region of the promoter. We also show that overexpression of miR-486 induced myotube hypertrophy in vitro and that miR-486 was essential to maintain skeletal muscle size both in vitro and in vivo. In addition, inhibition of miR-486 led to a decrease in Akt activity in C2C12 myotubes. Our findings indicate that miR-486 is one of the intermediary molecules connecting myostatin signaling and the IGF-1/Akt/mTOR pathway in the regulation of skeletal muscle size.

  1. Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin increases chemosensitivity of CaSki cells to paclitaxel.

    PubMed

    Faried, L S; Faried, A; Kanuma, T; Nakazato, T; Tamura, T; Kuwano, H; Minegishi, T

    2006-05-01

    Paclitaxel, a potent anti-neoplastic agent, has been found to be effective against several tumours, including cervical cancer. However, the exact mechanism underlying the cytotoxic effects of pacitaxel, especially in the survival-signalling pathway, is poorly understood. The aim of this study was to investigate the molecular pathway of the cytotoxic effect of paclitaxel in human cervical cancer cell lines. Four human cervical cancer cell lines were treated for 24 h with various concentration of paclitaxel, and the sensitivity was analysed by an MTT assay. The cell cycle progression and sub-G1 population were analysed by flow cytometry. Apoptosis was further measured by DNA fragmentation and microscope examination. The protein expression was determined by Western blot analysis. Our results showed that HeLa cells demonstrated the highest sensitivity to paclitaxel, whereas CaSki cells showed the lowest. In cervical cancer cells, paclitaxel induced apoptosis through an intrinsic pathway with prior G2/M arrest. In addition, we showed that paclitaxel downregulated the phosphorylation of Akt in both HeLa and CaSki cells. Interestingly, in CaSki cells, which were more suggestive of a resistant phenotype, paclitaxel induced the activation of mTOR as a downstream target of Akt. Pre-treatment with rapamycin inhibited activation of mTOR signalling and significantly enhanced the sensitivity of CaSki cells to paclitaxel by increasing apoptotic cell death. This effect was mediated, at least partly, through caspase activation. Overall, paclitaxel exerts its anti-tumour effects on cervical cancer cells by inducing apoptosis through intrinsic pathway, and rapamycin targeted to mTOR can sensitise paclitaxel-resistant cervical cancer cells.

  2. GIV/girdin links vascular endothelial growth factor signaling to Akt survival signaling in podocytes independent of nephrin.

    PubMed

    Wang, Honghui; Misaki, Taro; Taupin, Vanessa; Eguchi, Akiko; Ghosh, Pradipta; Farquhar, Marilyn G

    2015-02-01

    Podocytes are critically involved in the maintenance of the glomerular filtration barrier and are key targets of injury in many glomerular diseases. Chronic injury leads to progressive loss of podocytes, glomerulosclerosis, and renal failure. Thus, it is essential to maintain podocyte survival and avoid apoptosis after acute glomerular injury. In normal glomeruli, podocyte survival is mediated via nephrin-dependent Akt signaling. In several glomerular diseases, nephrin expression decreases and podocyte survival correlates with increased vascular endothelial growth factor (VEGF) signaling. How VEGF signaling contributes to podocyte survival and prevents apoptosis remains unknown. We show here that Gα-interacting, vesicle-associated protein (GIV)/girdin mediates VEGF receptor 2 (VEGFR2) signaling and compensates for nephrin loss. In puromycin aminonucleoside nephrosis (PAN), GIV expression increased, GIV was phosphorylated by VEGFR2, and p-GIV bound and activated Gαi3 and enhanced downstream Akt2, mammalian target of rapamycin complex 1 (mTORC1), and mammalian target of rapamycin complex-2 (mTORC2) signaling. In GIV-depleted podocytes, VEGF-induced Akt activation was abolished, apoptosis was triggered, and cell migration was impaired. These effects were reversed by introducing GIV but not a GIV mutant that cannot activate Gαi3. Our data indicate that after PAN injury, VEGF promotes podocyte survival by triggering assembly of an activated VEGFR2/GIV/Gαi3 signaling complex and enhancing downstream PI3K/Akt survival signaling. Because of its important role in promoting podocyte survival, GIV may represent a novel target for therapeutic intervention in the nephrotic syndrome and other proteinuric diseases.

  3. Maximising the potential of AKT inhibitors as anti-cancer treatments.

    PubMed

    Brown, Jessica S; Banerji, Udai

    2017-04-01

    PI3K/AKT signalling is commonly disrupted in human cancers, with AKT being a central component of the pathway, influencing multiple processes that are directly involved in tumourigenesis. Targeting AKT is therefore a highly attractive anti-cancer strategy with multiple AKT inhibitors now in various stages of clinical development. In this review, we summarise the role and regulation of AKT signalling in normal cellular physiology. We highlight the mechanisms by which AKT signalling can be hyperactivated in cancers and discuss the past, present and future clinical strategies for AKT inhibition in oncology.

  4. The GRP1 PH domain, like the AKT1 PH domain, possesses a sentry glutamate residue essential for specific targeting to plasma membrane PI(3,4,5)P(3).

    PubMed

    Pilling, Carissa; Landgraf, Kyle E; Falke, Joseph J

    2011-11-15

    During the appearance of the signaling lipid PI(3,4,5)P(3), an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P(3)-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P(2) and bind the rare PI(3,4,5)P(3) target lipid with sufficiently high affinity. Our previous study of the E17K mutant of the protein kinase B (AKT1) PH domain, together with evidence from Carpten et al. [Carpten, J. D., et al. (2007) Nature 448, 439-444], revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P(2), thereby playing an essential role in specific PI(3,4,5)P(3) targeting [Landgraf, K. E., et al. (2008) Biochemistry 47, 12260-12269]. The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P(3)-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P(2) affinity and constitutive plasma membrane targeting. To test this hypothesis, we investigated the E345 residue, a putative sentry glutamate, of the general receptor for phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into the GRP1 PH domain enhances PI(4,5)P(2) affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in the AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P(2) releases the E345K GRP1 PH domain into the cytoplasm, and the efficiency of this release increases when Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K [Carpten, J. D., et al. (2007) Nature 448, 439-444; Lindhurst, M. J., et al

  5. Combined inhibition of AKT/mTOR and MDM2 enhances Glioblastoma Multiforme cell apoptosis and differentiation of cancer stem cells

    PubMed Central

    Daniele, Simona; Costa, Barbara; Zappelli, Elisa; Da Pozzo, Eleonora; Sestito, Simona; Nesi, Giulia; Campiglia, Pietro; Marinelli, Luciana; Novellino, Ettore; Rapposelli, Simona; Martini, Claudia

    2015-01-01

    The poor prognosis of Glioblastoma Multiforme (GBM) is due to a high resistance to conventional treatments and to the presence of a subpopulation of glioma stem cells (GSCs). Combination therapies targeting survival/self-renewal signals of GBM and GSCs are emerging as useful tools to improve GBM treatment. In this context, the hyperactivated AKT/mammalian target of the rapamycin (AKT/mTOR) and the inhibited wild-type p53 appear to be good candidates. Herein, the interaction between these pathways was investigated, using the novel AKT/mTOR inhibitor FC85 and ISA27, which re-activates p53 functionality by blocking its endogenous inhibitor murine double minute 2 homologue (MDM2). In GBM cells, FC85 efficiently inhibited AKT/mTOR signalling and reactivated p53 functionality, triggering cellular apoptosis. The combined therapy with ISA27 produced a synergic effect on the inhibition of cell viability and on the reactivation of p53 pathway. Most importantly, FC85 and ISA27 blocked proliferation and promoted the differentiation of GSCs. The simultaneous use of these compounds significantly enhanced GSC differentiation/apoptosis. These findings suggest that FC85 actively enhances the downstream p53 signalling and that a combination strategy aimed at inhibiting the AKT/mTOR pathway and re-activating p53 signalling is potentially effective in GBM and in GSCs. PMID:25898313

  6. Mammalian target of rapamycin complex (mTOR) pathway modulates blood-testis barrier (BTB) function through F-actin organization and gap junction.

    PubMed

    Li, Nan; Cheng, C Yan

    2016-09-01

    mTOR (mammalian target of rapamycin) is one of the most important signaling molecules in mammalian cells which regulates an array of cellular events, ranging from cell metabolism to cell proliferation. Based on the association of mTOR with the core component proteins, such as Raptor or Rictor, mTOR can become the mTORC1 (mammalian target of rapamycin complex 1) or mTORC2, respectively. Studies have shown that during the epithelial cycle of spermatogenesis, mTORC1 promotes remodeling and restructuring of the blood-testis barrier (BTB) in vitro and in vivo, making the Sertoli cell tight junction (TJ)-permeability barrier "leaky"; whereas mTORC2 promotes BTB integrity, making the Sertoli cell TJ-barrier "tighter". These contrasting effects, coupled with the spatiotemporal expression of the core signaling proteins at the BTB that confer the respective functions of mTORC1 vs. mTORC2 thus provide a unique mechanism to modulate BTB dynamics, allowing or disallowing the transport of biomolecules and also preleptotene spermatocytes across the immunological barrier. More importantly, studies have shown that these changes to BTB dynamics conferred by mTORC1 and mTORC2 are mediated by changes in the organization of the actin microfilament networks at the BTB, and involve gap junction (GJ) intercellular communication. Since GJ has recently been shown to be crucial to reboot spermatogenesis and meiosis following toxicant-induced aspermatogenesis, these findings thus provide new insightful information regarding the integration of mTOR and GJ to regulate spermatogenesis.

  7. Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

    SciTech Connect

    Truong Le Xuan Nguyen; Choi, Joung Woo; Lee, Sang Bae; Ye, Keqiang; Woo, Soo-Dong; Lee, Kyung-Hoon; Ahn, Jee-Yin . E-mail: jyahn@med.skku.ac.kr

    2006-10-20

    Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions.

  8. Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway

    PubMed Central

    Varghese, Binny V.; Koohestani, Faezeh; McWilliams, Michelle; Colvin, Arlene; Gunewardena, Sumedha; Kinsey, William H.; Nowak, Romana A.; Nothnick, Warren B.; Chennathukuzhi, Vargheese M.

    2013-01-01

    Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase–protein kinase B/AKT–mammalian target of rapamycin (PI3K/AKT–mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K–AKT–mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids. PMID:23284171

  9. Genome-scale RNA interference screen identifies antizyme 1(OAZ1) as a target for improvement of recombinant protein production in mammalian cells†

    PubMed Central

    Xiao, Su; Chen, Yu Chi; Buehler, Eugene; Mandal, Swati; Mandal, Ajeet; Betenbaugh, Michael; HeePark, Myung; Martin, Scott; Shiloach, Joseph

    2017-01-01

    For the purpose of improving recombinant protein production from mammalian cells, an unbiased, high-throughput whole-genome RNA interference screen was conducted using human embryonic kidney 293 (HEK 293) cells expressing firefly luciferase. 21,585 human genes were individually silenced with three different siRNAs for each gene. The screen identified 56 genes that led to the greatest improvement in luciferase expression. These genes were found to be included in several pathways involved in spliceosome formation and mRNA processing, transcription, metabolic processes, transport and protein folding. The 10 genes that most enhanced protein expression when down regulated, were further confirmed by measuring the effect of their silencing on the expression of three additional recombinant proteins. Among the confirmed genes, OAZ1- the gene encoding the ornithine decarboxylase antizyme1- was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of the ornithine decarboxylase enzyme and the cellular levels of putrescine and spermidine; an indication that increased cellular polyamines enhances luciferase expression without affecting its transcription. The study shows that OAZ1 is a novel target for improving expression of recombinant proteins. The genome-scale screening performed in this work can establish the foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications. PMID:27215166

  10. Targeted Regulation of PI3K/Akt/mTOR/NF-κB Signaling by Indole Compounds and their Derivatives: Mechanistic Details and Biological Implications for Cancer Therapy

    PubMed Central

    Ahmad, Aamir; Biersack, Bernhard; Li, Yiwei; Kong, Dejuan; Bao, Bin; Schobert, Rainer; Padhye, Subhash B.; Sarkar, Fazlul H.

    2014-01-01

    Indole compounds, found in cruciferous vegetables, are potent anti-cancer agents. Studies with indole-3-carbinol (I3C) and its dimeric product, 3,3’-diindolylmethane (DIM) suggest that these compounds have the ability to deregulate multiple cellular signaling pathways, including PI3K/Akt/mTOR signaling pathway. These natural compounds are also effective modulators of downstream transcription factor NF-κB signaling which might help explain their ability to inhibit invasion and angiogenesis, and the reversal of epithelial-to-mesenchymal transition (EMT) phenotype and drug resistance. Signaling through PI3K/Akt/mTOR and NF-κB pathway is increasingly being realized to play important role in EMT through the regulation of novel miRNAs which further validates the importance of this signaling network and its regulations by indole compounds. Here we will review the available literature on the modulation of PI3K/Akt/mTOR/NF-κB signaling by both parental I3C and DIM, as well as their analogs/derivatives, in an attempt to catalog their anticancer activity. PMID:23272910

  11. Degradation of Akt Using Protein Catalyzed Capture Agents

    PubMed Central

    Das, Samir; Nag, Arundhati; Tang, Grace; Tang, Kevin; Sutherland, Alexander M.; Heath, James R.

    2016-01-01

    Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry we recently developed multiple protein catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein based assay. Here we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare Proteolysis Targeting Chimeric molecules (PROTACs) that are shown to promote the rapid degradation of Akt in live cancer cells. These novel PROTACs demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets. PMID:26880702

  12. Redox-Sensitive Oxidation and Phosphorylation of PTEN Contribute to Enhanced Activation of PI3K/Akt Signaling in Rostral Ventrolateral Medulla and Neurogenic Hypertension in Spontaneously Hypertensive Rats

    PubMed Central

    Wu, Kay L.H.; Wu, Chiung-Ai; Wu, Chih-Wei; Chan, Samuel H.H.; Chang, Alice Y.W.

    2013-01-01

    Abstract Aims: The activity of phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (Akt) is enhanced under hypertension. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of PI3K signaling, and its activity is redox-sensitive. In the rostral ventrolateral medulla (RVLM), which is responsible for the maintenance of blood pressure, oxidative stress plays a pivotal role in neurogenic hypertension. The present study evaluated the hypothesis that redox-sensitive inactivation of PTEN results in enhanced PI3K/Akt signaling in RVLM, leading to neurogenic hypertension. Results: Compared to age-matched normotensive Wistar-Kyoto (WKY) rats, PTEN inactivation in the form of oxidation and phosphorylation were greater in RVLM of spontaneously hypertensive rats (SHR). PTEN inactivation was accompanied by augmented PI3K activity and PI3K/Akt signaling, as reflected by the increase in phosphorylation of Akt and mammalian target of rapamycin. Intracisternal infusion of tempol or microinjection into the bilateral RVLM of adenovirus encoding superoxide dismutase significantly antagonized the PTEN inactivation and blunted the enhanced PI3K/Akt signaling in SHR. Gene transfer of PTEN to RVLM in SHR also abrogated the enhanced Akt activation and promoted antihypertension. Silencing PTEN expression in RVLM with small-interfering RNA, on the other hand, augmented PI3K/Akt signaling and promoted long-term pressor response in normotensive WKY rats. Innovation: The present study demonstrated for the first time that the redox-sensitive check-and-balance process between PTEN and PI3K/Akt signaling is engaged in the pathogenesis of hypertension. Conclusion: We conclude that an aberrant interplay between the redox-sensitive PTEN and PI3k/Akt signaling in RVLM underpins neural mechanism of hypertension. Antioxid. Redox Signal. 18, 36–50. PMID:22746319

  13. MicroRNA-130a alleviates human coronary artery endothelial cell injury and inflammatory responses by targeting PTEN via activating PI3K/Akt/eNOS signaling pathway

    PubMed Central

    Song, Chun-Li; Liu, Bin; Shi, Yong-Feng; Liu, Ning; Yan, You-You; Zhang, Ji-Chang; Xue, Xin; Wang, Jin-Peng; Zhao, Zhuo; Liu, Jian-Gen; Li, Yang-Xue; Zhang, Xiao-Hao; Wu, Jun-Duo

    2016-01-01

    Our study aims to investigate the roles of microRNA-130a (miR-130a) in human coronary artery endothelial cells (HCAECs) injury and inflammatory responses by targeting PTEN through the PI3K/Akt/eNOS signaling pathway. HCAECs were treated with 1.0 mmol/L homocysteine (HCY) and assigned into eight groups: the blank group, the negative control (NC) group, the miR-130a mimics group, the miR-130a inhibitors group, the si-PTEN group, the Wortmannin group, the miR-130a inhibitors + si-PTEN group and the miR-130a mimics + Wortmannin group. Luciferase reporter gene assay was used to validate the relationship between miR-130a and PTEN. The expressions of miR-130a, PTEN and PI3K/Akt/eNOS signaling pathway-related proteins were detected by qRT-PCR assay and Western blotting. MTT assay and Hoechst 33258 staining were adopted to testify cell growth and apoptosis. The NO kit assay was used to detect the NO release. ELISA was conducted to measure serum cytokine levels. Luciferase reporter gene assay confirmed the target relationship between miR-130a and PTEN. Compared with the blank and NC groups, the miR-130a mimics and si-PTEN groups showed significant increases in the expressions of PI3K/Akt/eNOS signaling pathway-related proteins, cell viability and the NO release, while serum cytokine levels and cell apoptosis were decreased; by contrast, an opposite trend was observed in miR-130a inhibitors and Wortmannin groups. However, no significant difference was found in the miR-130a inhibitors + si-PTEN and miR-130a mimics + Wortmannin groups when compared with the blank group. These results indicate that miR-130a could alleviate HCAECs injury and inflammatory responses by down-regulating PTEN and activating PI3K/Akt/eNOS signaling pathway. PMID:27713121

  14. Isorhamnetin inhibits cell proliferation and induces apoptosis in breast cancer via Akt and mitogen-activated protein kinase kinase signaling pathways

    PubMed Central

    HU, SHAN; HUANG, LIMING; MENG, LIWEI; SUN, HE; ZHANG, WEI; XU, YINGCHUN

    2015-01-01

    Breast cancer is the most common cause of female cancer-associated mortality. Although treatment options, including chemotherapy, radiotherapy and surgery have led to a decline in the mortality rates associated with breast cancer, drug resistance remains one of the predominant causes for poor prognosis and high recurrence rates. The present study investigated the potential effects of the natural product, isorhamnetin on breast cancer, and examined the effects of isorhamnetin on the Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK) signaling cascades, which are two important signaling pathways for endocrine therapy resistance in breast cancer. The results of the present study indicate that isorhamnetin inhibits cell proliferation and induces cell apoptosis. In addition, isorhamnetin was observed to inhibit the Akt/mTOR and the MEK/extracellular signal-regulated kinase phosphorylation cascades. The inhibition of these two signaling pathways was attenuated by the two Akt and MEK1 inhibitors, but not by the nuclear factor-κB inhibitor. Furthermore, epidermal growth factor inhibited the effects of isorhamnetin via activation of the Akt and MEK signaling pathways. These results indicate that isorhamnetin exhibits antitumor effects in breast cancer, which are mediated by the Akt and MEK signaling pathways. PMID:26502751

  15. Apocynin inhibits Toll-like receptor-4-mediated activation of NF-κB by suppressing the Akt and mTOR pathways.

    PubMed

    Nam, Yoon Jeong; Kim, Arum; Sohn, Dong Suep; Lee, Chung Soo

    2016-12-01

    Microbial product lipopolysaccharide has been shown to be involved in the pathogenesis of inflammatory skin diseases. Apocynin has demonstrated to have an anti-inflammatory effect. However, the effect of apocynin on the Toll-like receptor-4-dependent activation of Akt, mammalian target of rapamycin (mTOR), and nuclear factor (NF)-κB pathway, which is involved in productions of inflammatory mediators in keratinocytes, has not been studied. Using human keratinocytes, we investigated the effect of apocynin on the inflammatory mediator production in relation to the Toll-like receptor-4-mediated-Akt/mTOR and NF-κB pathways, which regulates the transcription genes involved in immune and inflammatory responses. Apocynin, Akt inhibitor SH-5, Bay 11-7085 and N-acetylcysteine each attenuated the lipopolysaccharide-induced production of cytokines, PGE2, and chemokines, changes in the levels of Toll-like receptor-4, p-Akt, mTOR, and NF-κB, and production of reactive oxygen species in keratinocytes. The results show that apocynin appears to attenuate the lipopolysaccharide-stimulated production of inflammatory mediators in keratinocytes by suppressing the Toll-like receptor-4-mediated activation of the Akt, mTOR, and NF-κB pathways. The effect of apocynin appears to be attributed to its inhibitory effect on the production of reactive oxygen species. Apocynin appears to attenuate the microbial product-mediated inflammatory skin diseases.

  16. Isorhamnetin inhibits cell proliferation and induces apoptosis in breast cancer via Akt and mitogen‑activated protein kinase kinase signaling pathways.

    PubMed

    Hu, Shan; Huang, Liming; Meng, Liwei; Sun, He; Zhang, Wei; Xu, Yingchun

    2015-11-01

    Breast cancer is the most common cause of female cancer-associated mortality. Although treatment options, including chemotherapy, radiotherapy and surgery have led to a decline in the mortality rates associated with breast cancer, drug resistance remains one of the predominant causes for poor prognosis and high recurrence rates. The present study investigated the potential effects of the natural product, isorhamnetin on breast cancer, and examined the effects of isorhamnetin on the Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK) signaling cascades, which are two important signaling pathways for endocrine therapy resistance in breast cancer. The results of the present study indicate that isorhamnetin inhibits cell proliferation and induces cell apoptosis. In addition, isorhamnetin was observed to inhibit the Akt/mTOR and the MEK/extracellular signal-regulated kinase phosphorylation cascades. The inhibition of these two signaling pathways was attenuated by the two Akt and MEK1 inhibitors, but not by the nuclear factor-κB inhibitor. Furthermore, epidermal growth factor inhibited the effects of isorhamnetin via activation of the Akt and MEK signaling pathways. These results indicate that isorhamnetin exhibits antitumor effects in breast cancer, which are mediated by the Akt and MEK signaling pathways.

  17. STC2 promotes head and neck squamous cell carcinoma metastasis through modulating the PI3K/AKT/Snail signaling

    PubMed Central

    Yang, Shuwen; Ji, Qinghai; Chang, Bin; Wang, Yan; Zhu, Yongxue; Li, Duanshu; Huang, Caiping; Wang, Yulong; Sun, Guohua; Zhang, Ling; Guan, Qing; Xiang, Jun; Wei, Wenjun; Lu, Zhongwu; Liao, Tian; Meng, Jiao; Wang, Ziliang; Ma, Ben; Zhou, Li; Wang, Yu; Yang, Gong

    2017-01-01

    The mammalian peptide hormone stanniocalcin 2 (STC2) plays an oncogenic role in many human cancers. However, the exact function of STC2 in human head and neck squamous cell carcinoma (HNSCC) is unclear. We aimed to examine the function and clinical significance of STC2 in HNSCC. Using in vitro and in vivo assays, we show that overexpression of STC2 suppressed cell apoptosis, promoted cell proliferation, migration, invasion, and cell cycle arrest at the G1/S transition. By contrast, silencing of STC2 inhibited these activities. We further show that STC2 upregulated the phosphorylation of AKT and enhanced HNSCC metastasis via Snail-mediated increase of vimentin and decrease of E-cadherin. These responses were blocked by silencing of STC2/Snail expression or inhibition of pAKT activity. Furthermore, clinical data indicate that high STC2 expression was associated with high levels of pAKT and Snail in tumor samples from HNSCC patients with regional lymph node metastasis (P < 0.01). Thus, we conclude that STC2 controls HNSCC metastasis via the PI3K/AKT/Snail signaling axis and that targeted therapy against STC2 may be a novel strategy to effectively treat patients with metastatic HNSCC. PMID:27863406

  18. Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted G.C-->T.A transversions in simian kidney cells.

    PubMed Central

    Moriya, M

    1993-01-01

    A single-stranded shuttle vector has been developed for the purpose of investigating translesional events in mammalian cells. The vector is designed to permit site-specific introduction of defined DNA lesions between a gene for neomycin resistance and its promoter. Efficiencies of translesional synthesis in simian kidney cells (COS) and Escherichia coli are established by determining the number of neomycin- and ampicillin-resistant colonies recovered, respectively, after introduction of a modified vector. Fidelity of translesional synthesis is evaluated by analyzing the nucleotide sequence of progeny phagemid DNA in the region corresponding to the lesion site. This experimental system, capable of detecting mutagenic and nonmutagenic events at and adjacent to the lesion site, was used to establish the mutagenic potential of a single 8-oxoguanine residue in DNA. This modified base, produced by attack of reactive oxygen species on cellular DNA, did not cause a decrease in the number of transformants when single-stranded DNA containing the lesion replicated in COS cells or E. coli. The predominant mutations observed (> 78%) were G-->T transversions targeted to the site of the lesion. The mutation frequencies for this event were 2.5-4.8% in COS cells and 1.8% in E. coli. It is concluded that a single-stranded shuttle vector, utilized in conjunction with a site-specific approach, can be used to investigate translesional events in mammalian cells and in bacteria. Images PMID:8430083

  19. Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted G.C-->T.A transversions in simian kidney cells.

    PubMed

    Moriya, M

    1993-02-01

    A single-stranded shuttle vector has been developed for the purpose of investigating translesional events in mammalian cells. The vector is designed to permit site-specific introduction of defined DNA lesions between a gene for neomycin resistance and its promoter. Efficiencies of translesional synthesis in simian kidney cells (COS) and Escherichia coli are established by determining the number of neomycin- and ampicillin-resistant colonies recovered, respectively, after introduction of a modified vector. Fidelity of translesional synthesis is evaluated by analyzing the nucleotide sequence of progeny phagemid DNA in the region corresponding to the lesion site. This experimental system, capable of detecting mutagenic and nonmutagenic events at and adjacent to the lesion site, was used to establish the mutagenic potential of a single 8-oxoguanine residue in DNA. This modified base, produced by attack of reactive oxygen species on cellular DNA, did not cause a decrease in the number of transformants when single-stranded DNA containing the lesion replicated in COS cells or E. coli. The predominant mutations observed (> 78%) were G-->T transversions targeted to the site of the lesion. The mutation frequencies for this event were 2.5-4.8% in COS cells and 1.8% in E. coli. It is concluded that a single-stranded shuttle vector, utilized in conjunction with a site-specific approach, can be used to investigate translesional events in mammalian cells and in bacteria.

  20. Salvianolic acid A reverses paclitaxel resistance in human breast cancer MCF-7 cells via targeting the expression of transgelin 2 and attenuating PI3 K/Akt pathway.

    PubMed

    Cai, Jiangxia; Chen, Siying; Zhang, Weipeng; Zheng, Xiaowei; Hu, Sasa; Pang, Chengsen; Lu, Jun; Xing, Jianfeng; Dong, Yalin

    2014-10-15

    Chemotherapy resistance represents a major problem for the treatment of patients with breast cancer and greatly restricts the use of first-line chemotherapeutics paclitaxel. The purpose of this study was to investigate the role of transgelin 2 in human breast cancer paclitaxel resistance cell line (MCF-7/PTX) and the reversal mechanism of salvianolic acid A (SAA), a phenolic active compound extracted from Salvia miltiorrhiza. Western blotting and real-time quantitative polymerase chain reaction (qRT-PCR) indicated that transgelin 2 may mediate paclitaxel resistance by activating the phosphatidylinositol 3-kinase (PI3 K)/Akt signaling pathway to suppress MCF-7/PTX cells apoptosis. The reversal ability of SAA was confirmed by MTT assay and flow cytometry, with a superior 9.1-fold reversal index and enhancement of the apoptotic cytotoxicity induced by paclitaxel. In addition, SAA effectively prevented transgelin 2 and adenosine-triphosphate binding cassette transporter (ABC transporter) including P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1), and breast cancer resistance protein (BCRP) up-regulation and exhibited inhibitory effect on PI3 K/Akt signaling pathway in MCF-7/PTX cells. Taken together, SAA can reverse paclitaxel resistance through suppressing transgelin 2 expression by mechanisms involving attenuation of PI3 K/Akt pathway activation and ABC transporter up-regulation. These results not only provide insight into the potential application of SAA in reversing paclitaxel resistance, thus facilitating the sensitivity of breast cancer chemotherapy, but also highlight a potential role of transgelin 2 in the development of paclitaxel resistance in breast cancer.

  1. Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection

    PubMed Central

    Cottle, Renee N.; Lee, Ciaran M.; Archer, David; Bao, Gang

    2015-01-01

    Tal-effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are genome editing tools with unprecedented potential. However, the ability to deliver optimal amounts of these nucleases into mammalian cells with minimal toxicity poses a major challenge. Common delivery approaches are transfection- and viral-based methods; each associated with significant drawbacks. An alternative method for directly delivering genome-editing reagents into single living cells with high efficiency and controlled volume is microinjection. Here, we characterize a glass microcapillary-based injection system and demonstrate controlled co-injection of TALENs or CRISPR/Cas9 together with donor template into single K562 cells for targeting the human β-globin gene. We quantified nuclease induced insertions and deletions (indels) and found that, with β-globin-targeting TALENs, similar levels of on- and off-target activity in cells could be achieved by microinjection compared with nucleofection. Furthermore, we observed 11% and 2% homology directed repair in single K562 cells co-injected with a donor template along with CRISPR/Cas9 and TALENs respectively. These results demonstrate that a high level of targeted gene modification can be achieved in human cells using glass-needle microinjection of genome editing reagents. PMID:26558999

  2. Ischemic post-conditioning facilitates brain recovery after stroke by promoting Akt/mTOR activity in nude rats.

    PubMed

    Xie, Rong; Wang, Peng; Ji, Xunming; Zhao, Heng

    2013-12-01

    While pre-conditioning is induced before stroke onset, ischemic post-conditioning (IPostC) is performed after reperfusion, which typically refers to a series of mechanical interruption of blood reperfusion after stroke. IPostC is known to reduce infarction in wild-type animals. We investigated if IPostC protects against brain injury induced by focal ischemia in Tcell-deficient nude rats and to examine its effects on Akt and the mammalian target of rapamycin (mTOR) pathway. Although IPostC reduced infarct size at 2 days post-stroke in wild-type rats, it did not attenuate infarction in nude rats. Despite the unaltered infarct size in nude rats, IPostC increased levels of phosphorylated Akt (p-Akt) and Akt isoforms (Akt1, Akt2, Akt3), and p-mTOR, p-S6K and p-4EBP1 in the mTOR pathway, as well as growth associated Protein 43 (GAP43), both in the peri-infarct area and core, 24 h after stroke. IPostC improved neurological function in nude rats 1-30 days after stroke and reduced the extent of brain damage 30 days after stroke. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC. We determined that IPostC did not inhibit acute infarction in nude rats but did provide long-term protection by enhancing Akt and mTOR activity during the acute post-stroke phase. Post-conditioning did not attenuate infarction in nude rats measured 2 days post-stroke, but improved neurological function in nude rats and reduced brain damage 30 days after stroke. It resulted in increased-activities of Akt and mTOR, S6K and p-4EBP1. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC.

  3. c-Src activation promotes nasopharyngeal carcinoma metastasis by inducing the epithelial-mesenchymal transition via PI3K/Akt signaling pathway: a new and promising target for NPC

    PubMed Central

    Lu, Jinping; Xia, Weixiong; Yu, Yahui; Peng, Yongjian; Wang, Li; Wang, Gang; Ye, Yanfang; Yang, Jing; Liang, Hu; Kang, Tiebang; Lv, Xing

    2016-01-01

    Aberrant activation of cellular Src (c-Src), a non-receptor tyrosine kinase, could promote cancer progression through activating its downstream signaling pathways. However, the roles of c-Src and phosphorylated-Src (p-Src) in nasopharyngeal carcinoma (NPC) progression are rarely investigated. Herein, we have identified high c-Src concentrations in the serum of NPC patients with distant metastasis using high-throughput protein microarrays. Levels of c-Src in serum and p-Src in human primary NPC samples were unfavorable independent prognostic factors for cancer-specific survival, disease-free survival, and distant metastasis-free survival. Depletion or inactivation of c-Src in NPC cells using sgRNA with CRISPR/Cas9 system or PP2 decreased cell viability, colony formation, migration and invasion in vitro and metastasis in vivo. In contrast, these malignancies could be up-regulated by overexpressed c-Src in a NPC cell line with low-metastasis potential. Furthermore, p-Src was involved in promoting NPC cell metastasis by inducing the epithelial-mesenchymal transition (EMT) process via activating the PI3K/Akt pathway and cytoskeleton remodeling. The p-Src-induced EMT process could be retarded by PP2, which mediated by down-regulating the PI3K/Akt pathway. In conclusion, elevated levels of c-Src in serum and p-Src in primary NPC tissue correlated with poor outcomes of NPC patients. And aberrant activation of c-Src facilitated NPC cells with malignant potential, especially metastasis ability, which mediated by the PI3K/Akt pathway activation and sequentially induced the EMT process. These findings unveiled a promising approach for targeted therapy of advanced NPC. PMID:27078847

  4. EGFR-AKT-mTOR activation mediates epiregulin-induced pleiotropic functions in cultured osteoblasts.

    PubMed

    Fan, Jian-Bo; Liu, Wei; Zhu, Xin-Hui; Yuan, Kun; Xu, Da-Wei; Chen, Jia-Jia; Cui, Zhi-Ming

    2015-01-01

    Epidermal growth factor (EGF) receptor (EGFR) emerges as an essential molecule for the regulating of osteoblast cellular functions. In the current study, we explored the effect of epiregulin, a new EGFR ligand, on osteoblast functions in vitro, and studied the underlying mechanisms. We found that epiregulin-induced EGFR activation in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, epiregulin activated AKT-mammalian target of rapamycin (mTOR) and Erk-mitogen-activated protein kinase (MAPK) signalings in cultured osteoblasts, which were blocked by EGFR inhibitor AG1478 or monoclonal antibody against EGFR (anti-EGFR). Further, in primary and MC3T3-E1 osteoblasts, epiregulin promoted cell proliferation and increased alkaline phosphatase activity, while inhibiting dexamethasone (Dex)-induced cell death. Such effects by epiregulin were largely inhibited by AG1478 or anti-EGFR. Notably, AKT-mTOR inhibitors, but not Erk inhibitors, alleviated epiregulin-induced above pleiotropic functions in osteoblasts. Meanwhile, siRNA depletion of Sin1, a key component of mTOR complex 2 (mTORC2), also suppressed epiregulin-exerted effects in MC3T3-E1 cells. Together, these results suggest that epiregulin-induced pleiotropic functions in cultured osteoblasts are mediated through EGFR-AKT-mTOR signalings.

  5. MFN2 suppresses cancer progression through inhibition of mTORC2/Akt signaling

    PubMed Central

    Xu, Ke; Chen, Guo; Li, Xiaobo; Wu, Xiaoqin; Chang, Zhijie; Xu, Jianhua; Zhu, Yu; Yin, Peihao; Liang, Xin; Dong, Lei

    2017-01-01

    The mitochondrial GTPase mitofusin-2 (MFN2) has previously been reported to play a role in regulating cell proliferation, apoptosis and differentiation in a number of cell types. Here, we report that breast cancer patients with low MFN2 expression are associated with poor prognosis as compared to patients with high MFN2 expression. We find that MFN2 knockout from MCF7 and A549 cells via Crispr/Cas9 greatly promotes cell viability, colony formation, and invasion of cancer cells in vitro and in vivo, which were confirmed by colony formation assay, transwell invasion assay, and tumor xenograft model. Signaling analyses suggest the mammalian target of rapamycin complex 2 (mTORC2)/Akt signaling pathway is highly elevated in MFN2 knockout cancer cells. The elevated mTORC2 promotes cancer cell growth and metastasis via AktS437 phosphorylation mediated signaling pathway. Mechanistic studies reveal that MFN2 suppresses mTORC2 through direct interaction by binding its domain HR1. Inhibition of mTORC2 significantly suppresses MFN2 deficient tumor growth. Collectively, this study provides novel insights into the tumor progression associated with MFN2 deficiency and suggests that the importance of mTORC2 inhibitor in the treatment of MFN2 downregulated cancer patients. PMID:28176801

  6. Dynamic Akt/mTOR Signaling in Children with Autism Spectrum Disorder

    PubMed Central

    Onore, Charity; Yang, Houa; Van de Water, Judy; Ashwood, Paul

    2017-01-01

    Autism spectrum disorder (ASD) is a behaviorally defined disorder affecting 1 in 68 children. Currently, there is no known cause for the majority of ASD cases nor are there physiological diagnostic tools or biomarkers to aid behavioral diagnosis. Whole-genome linkage studies, genome-wide association studies, copy number variation screening, and SNP analyses have identified several ASD candidate genes, but which vary greatly among individuals and family clusters, suggesting that a variety of genetic mutations may result in a common pathology or alter a common mechanistic pathway. The Akt/mammalian target of rapamycin (mTOR) pathway is involved in many cellular processes including synaptic plasticity and immune function that can alter neurodevelopment. In this study, we examined the activity of the Akt/mTOR pathway in cells isolated from children with ASD and typically developing controls. We observed higher activity of mTOR, extracellular receptor kinase, and p70S6 kinase and lower activity of glycogen synthase kinase 3 (GSK3)α and tuberin (TSC2) in cells from children with ASD. These data suggest a phosphorylation pattern indicative of higher activity in the Akt/mTOR pathway in children with general/idiopathic ASD and may suggest a common pathological pathway of interest for ASD. PMID:28361047

  7. Neuroprotective role of Sirt1 in mammalian models of Huntington's disease through activation of multiple Sirt1 targets.

    PubMed

    Jiang, Mali; Wang, Jiawei; Fu, Jinrong; Du, Lin; Jeong, Hyunkyung; West, Tim; Xiang, Lan; Peng, Qi; Hou, Zhipeng; Cai, Huan; Seredenina, Tamara; Arbez, Nicolas; Zhu, Shanshan; Sommers, Katherine; Qian, Jennifer; Zhang, Jiangyang; Mori, Susumu; Yang, X William; Tamashiro, Kellie L K; Aja, Susan; Moran, Timothy H; Luthi-Carter, Ruth; Martin, Bronwen; Maudsley, Stuart; Mattson, Mark P; Cichewicz, Robert H; Ross, Christopher A; Holtzman, David M; Krainc, Dimitri; Duan, Wenzhen

    2011-12-18

    Huntington's disease is a fatal neurodegenerative disorder caused by an expanded polyglutamine repeat in huntingtin (HTT) protein. We previously showed that calorie restriction ameliorated Huntington's disease pathogenesis and slowed disease progression in mice that model Huntington's disease (Huntington's disease mice). We now report that overexpression of sirtuin 1 (Sirt1), a mediator of the beneficial metabolic effects of calorie restriction, protects neurons against mutant HTT toxicity, whereas reduction of Sirt1 exacerbates mutant HTT toxicity. Overexpression of Sirt1 improves motor function, reduces brain atrophy and attenuates mutant-HTT-mediated metabolic abnormalities in Huntington's disease mice. Further mechanistic studies suggested that Sirt1 prevents the mutant-HTT-induced decline in brain-derived neurotrophic factor (BDNF) concentrations and the signaling of its receptor, TrkB, and restores dopamine- and cAMP-regulated phosphoprotein, 32 kDa (DARPP32) concentrations in the striatum. Sirt1 deacetylase activity is required for Sirt1-mediated neuroprotection in Huntington's disease cell models. Notably, we show that mutant HTT interacts with Sirt1 and inhibits Sirt1 deacetylase activity, which results in hyperacetylation of Sirt1 substrates such as forkhead box O3A (Foxo3a), thereby inhibiting its pro-survival function. Overexpression of Sirt1 counteracts the mutant-HTT-induced deacetylase deficit, enhances the deacetylation of Foxo3a and facilitates cell survival. These findings show a neuroprotective role for Sirt1 in mammalian Huntington's disease models and open new avenues for the development of neuroprotective strategies in Huntington's disease.

  8. Construction of simple and efficient siRNA validation systems for screening and identification of effective RNAi-targeted sequences from mammalian genes.

    PubMed

    Tsai, Wen-Hui; Chang, Wen-Tsan

    2014-01-01

    RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing induced by double-stranded RNAs (dsRNAs). Among the widely used dsRNAs, small interfering RNAs (siRNAs) and short hairpin RNAs have evolved as extremely powerful and the most popular gene silencing reagents. The key challenge to achieving efficient gene silencing especially for the purpose of therapeutics is mainly dependent on the effectiveness and specificity of the selected RNAi-targeted sequences. Practically, only a small number of dsRNAs are capable of inducing highly effective and sequence-specific gene silencing via RNAi mechanism. In addition, the efficiency of gene silencing induced by dsRNAs can only be experimentally examined based on inhibition of the target gene expression. Therefore, it is essential to develop a fully robust and comparative validation system for measuring the efficacy of designed dsRNAs. In this chapter, we focus our discussion on a reliable and quantitative reporter-based siRNA validation system that has been previously established in our laboratory. The system consists of a short synthetic DNA fragment containing an RNAi-targeted sequence of interest and two expression vectors for targeting reporter and triggering siRNA expressions. The efficiency of siRNAs is determined by their abilities to inhibit expression of the targeting reporters with easily quantified readouts including enhanced green fluorescence protein and firefly luciferase. Since only a readily available short synthetic DNA fragment is needed for constructing this reliable and efficient reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective RNAi-targeted sequences from mammalian genes but also implicates the use of RNAi-based dsRNA reagents for reverse functional genomics and molecular therapeutics.

  9. Synthesis and evaluation of indazole based analog sensitive Akt inhibitors.

    PubMed

    Okuzumi, Tatsuya; Ducker, Gregory S; Zhang, Chao; Aizenstein, Brian; Hoffman, Randy; Shokat, Kevan M

    2010-08-01

    The kinase Akt is a key signaling node in regulating cellular growth and survival. It is implicated in cancer by mutation and its role in the downstream transmission of aberrant PI3K signaling. For these reasons, Akt has become an increasingly important target of drug development efforts and several inhibitors are now reaching clinical trials. Paradoxically it has been observed that active site kinase inhibitors of Akt lead to hyperphosphorylation of Akt itself. To investigate this phenomenon we here describe the application of a chemical genetics strategy that replaces native Akt with a mutant version containing an active site substitution that allows for the binding of an engineered inhibitor. This analog sensitive strategy allows for the selective inhibition of a single kinase. In order to create the inhibitor selective for the analog sensitive kinase, a diversity of synthetic approaches was required, finally resulting in the compound PrINZ, a 7-substituted version of the Abbott Labs Akt inhibitor A-443654.

  10. Position based nucleotide analysis of miR168 family in higher plants and its targets in mammalian transcripts.

    PubMed

    Javed, Mohammed; Solanki, Manish; Sinha, Anshika; Shukla, Lata Israni

    2017-02-15

    The conserved miR168 family is evaluated for position based nucleotide preference in higher plants. The mature miRNA sequences include miR168-5p and miR168-3p, were obtained from miRBase (v21, June 2014) for 15 families (28 plants) containing a-c subfamilies. The preferred position based nucleotide sequences were obtained for miR168-5p and miR168-3p using Data Analysis in Molecular Biology and Evolution (DAMBE). miR168-5p shows same nucleotides at positions 1-6, 8-9, 11-12, 15-17 and 19. Also, miR168-3p is present in 3 families (10 plants) shows the same nucleotide at position 1-11, 13-15 and 17-21. Our work suggests that miR168 family has conserved sequence in higher plants. The miR168-5p was subjected to cross kingdom analysis using psRNATarget. The seed region position 2-8 shows 70-95% pairing and cleavage site at position 10-14 were analysed for the base preference, in which pairing with the targets showed 80-96% Watson Crick pairing. The 123 targets in human transcriptome were identified showing 58% cleavage and 41% translation repression. Earlier reported Low density lipoprotein receptor adaptor protein 1(LDLRAP1) target validated for miR168a obtained from rice origin, could also be targeted from miR168 from any other plant sources. The randomly selected 10 targets include some important genes like RPL34, ATXN1, AKAPI3 and ALS2 and is involved in transcription, cell trafficking, cell metabolism and neurodegenerative disorder. This paper provides DAMBE analysis for miR168 across the plant kingdom and identification of new cross kingdom targets for miR168 using psRNATarget.

  11. Neuroprotective effect of miR-665 against sevoflurane anesthesia-induced cognitive dysfunction in rats through PI3K/Akt signaling pathway by targeting insulin-like growth factor 2

    PubMed Central

    Lu, Xihua; Lv, Shuaiguo; Mi, Yan; Wang, Lei; Wang, Gensheng

    2017-01-01

    The aim of this study was to investigate the in vivo and in vitro effects of miR-665 on sevoflurane anesthesia-induced cognitive dysfunction. SH-SY5Y cells and male SD rats were treated with sevoflurane to simulate anesthesia-induced cognitive dysfunction. The cells and rats both were transfected with a miR-665 mimic, inhibitor, scramble, IGF-2 siRNA, or treated with P13K/Akt inhibitor LY294002. The cell apoptosis, autophagy, growth related proteins, and mRNA levels were measured using different methods. The motor performance was assessed using the Morris water maze (MWM) test. Finally, the differences were statistically analyzed. It was noted that sevoflurane-induced miR-665 downregulation accompanied with the upregulation of IGF-2 in vivo and motor deficits in vitro. Moreover, sevoflurane also induced hippocampal neuroapoptosis; reduced regular autophagy; increased Bax/Bcl-2 ratio; decreased the expression of Beclin 1, PSD95, and p-CREB; and activated P13K/Akt signaling pathway. However, the treatment by miR-665 mimics significantly reversed all the molecular changes and improved motor performance. Our data demonstrate the neuroprotective effect of miR-665 against sevoflurane anesthesia-induced cognitive impairment. This study suggests that miR-665 might be explored as a potential target of therapy for sevoflurane-induced cognitive impairment. PMID:28386360

  12. Gefitinib induces lung cancer cell autophagy and apoptosis via blockade of the PI3K/AKT/mTOR pathway

    PubMed Central

    ZHAO, ZHONG-QUAN; YU, ZHONG-YANG; LI, JIE; OUYANG, XUE-NONG

    2016-01-01

    Gefitinib is a selective inhibitor of the tyrosine kinase epidermal growth factor receptor, which inhibits tumor pathogenesis, metastasis and angiogenesis, as well as promoting apoptosis. Therefore, gefitinib presents an effective drug for the targeted therapy of lung cancer. However, the underlying mechanisms by which gefitinib induces lung cancer cell death remain unclear. To investigate the effects of gefitinib on lung cancer cells and the mechanism of such, the present study analyzed the effect of gefitinib on the autophagy, apoptosis and proliferation of the A549 and A549-gefitinib-resistant (GR) cell lines GR. The regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway was also investigated. Acridine orange staining revealed that gefitinib induced autophagy of A549 cells but not A549-GR cells. In addition, gefitinib promoted apoptosis and inhibited proliferation of A549 cells but not A549-GR cells. Furthermore, western blot analysis demonstrated that gefitinib treatment led to the downregulation of PI3K, AKT, pAKT, mTOR and phosphorylated-mTOR protein expression in A549 cells but not A549-GR cells. LY294002 blocked the PI3K/AKT/mTOR pathway and induced autophagy and apoptosis of A549 cells, however, no synergistic effect was observed following combined treatment with gefitinib and LY294002. In conclusion, the results of the present study indicate that gefitinib promotes autophagy and apoptosis of lung cancer cells via blockade of the PI3K/AKT/mTOR pathway, which leads to lung cancer cell death. PMID:27347100

  13. NFκB up-regulation of glucose transporter 3 is essential for hyperactive mammalian target of rapamycin-induced aerobic glycolysis and tumor growth.

    PubMed

    Zha, Xiaojun; Hu, Zhongdong; Ji, Shuang; Jin, Fuquan; Jiang, Keguo; Li, Chunjia; Zhao, Pan; Tu, Zhenzhen; Chen, Xianguo; Di, Lijun; Zhou, Haisheng; Zhang, Hongbing

    2015-04-01

    Accumulating evidence indicates that mammalian target of rapamycin (mTOR) exerts a crucial role in aerobic glycolysis and tumorigenesis, but the underlying mechanisms remain largely obscure. Results from Tsc1- or Tsc2-null mouse embryonic fibroblasts (MEFs) and human cancer cell lines consistently indicate that the expression of glucose transporter 3 (Glut3) is dramatically up-regulated by mTOR. The rapamycin-sensitive mTOR complex 1 (mTORC1), but not the rapamycin-insensitive mTOR complex 2 (mTORC2), was involved in the regulation of Glut3 expression. Moreover, mTORC1 enhances Glut3 expression through the activation of the IKK/NFκB pathway. Depletion of Glut3 led to the suppression of aerobic glycolysis, the inhibition of cell proliferation and colony formation, and the attenuation of the tumorigenic potential of the cells with aberrantly hyper-activated mTORC1 signaling in nude mice. We conclude that Glut3 is a downstream target of mTORC1, and it is critical for oncogenic mTORC1-mediated aerobic glycolysis and tumorigenesis. Hence Glut3 may be a potential target for therapy against cancers caused by the aberrantly activated mTORC1 signaling.

  14. Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins. The potential for selective inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tunicamycins are a heterologous family of nucleoside antibiotics that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg2+ complex established as a transition state analog for hexosamine-1-phosphate: prenol pho...

  15. Akt Regulates TPP1 Homodimerization and Telomere Protection

    PubMed Central

    Han, Xin; Liu, Dan; Zhang, Yi; Li, Yujing; Lu, Weisi; Chen, Junjie; Songyang, Zhou

    2014-01-01

    Summary Telomeres are specialized structures at the ends of eukaryotic chromosomes that are important for maintaining genome stability and integrity. Telomere dysfunction has been linked to aging and cancer development. In mammalian cells, extensive studies have been carried out to illustrate how core telomeric proteins assemble on telomeres to recruit the telomerase and additional factors for telomere maintenance and protection. In comparison, how changes in growth signaling pathways impact telomeres and telomere-binding proteins remains largely unexplored. The phosphatidylinositol 3-kinase (PI3-K)/Akt (also known as PKB) pathway, one of the best characterized growth signaling cascades, regulates a variety of cellular function including cell proliferation, survival, metabolism, and DNA repair, and dysregulation of PI3-K/Akt signaling has been linked to aging and diseases such as cancer and diabetes. In this study, we provide evidence that the Akt signaling pathway plays an important role in telomere protection. Akt inhibition either by chemical inhibitors or small interfering RNAs induced telomere dysfunction. Furthermore, we found that TPP1 could homodimerize through its OB fold, a process that was dependent on the Akt kinase. Telomere damage and reduced TPP1 dimerization as a result of Akt inhibition was also accompanied by diminished recruitment of TPP1 and POT1 to the telomeres. Our findings highlight a previously unknown link between Akt signaling and telomere protection. PMID:23862686

  16. CWF-145, a novel synthetic quinolone derivative exerts potent antimitotic activity against human prostate cancer: Rapamycin enhances antimitotic drug-induced apoptosis through the inhibition of Akt/mTOR pathway.

    PubMed

    Hung, Chao-Ming; Lin, Ying-Chao; Liu, Liang-Chih; Kuo, Sheng-Chu; Ho, Chi-Tang; Way, Tzong-Der

    2016-12-25

    CWF-145, a synthetic 2-phenyl-4-quinolone derivative exerted potent cytotoxicity against prostate cancer. CWF-145 inhibited prostate cancer cell lines PC-3, DU-145 and LNCap. It had a very low IC50 about 200 nM against castrate-resistant prostate cancer (CRPC) PC-3. We found that CWF-145 had a similar effect to clinical trial antimitotic agents in cancer cells and normal cells. CWF-145 arrested cell cycle at G2/M phase by binding to the β-tubulin at the colchicine-binding site then disrupted microtubule polymerization. Furthermore, the damaged microtubule affected the Akt/mammalian target of rapamycin (mTOR) signaling pathway. Our data showed that CWF-145 activated Akt and mTOR expression to increase emi1 accumulation and inhibit APC. The increased cyclin B1 and securin arrested cell cycle at G2/M phase. Moreover, we showed that Akt activation markedly increased resistance to microtubule-directed agents, including CWF-145, colchicine, and paclitaxel. Interestingly, rapamycin inhibited Akt-mediated therapeutic resistance, indicating that these effects were dependent on mTOR. Taken together, these observations suggest that activation of the Akt/mTOR signaling pathway can promote resistance to chemotherapeutic agents that do not directly target metabolic regulation. These data may provide insight into potentially synergistic combinations of anticancer therapies.

  17. Mango polyphenolics suppressed tumor growth in breast cancer xenografts in mice: role of the PI3K/AKT pathway and associated microRNAs.

    PubMed

    Banerjee, Nivedita; Kim, Hyemee; Krenek, Kimberly; Talcott, Stephen T; Mertens-Talcott, Susanne U

    2015-08-01

    The cytotoxic and anti-inflammatory properties of mango polyphenolics including gallic acid and gallotannins have been demonstrated in numerous types of cancers. We hypothesized that the phosphoinositide 3-kinase (PI3K)/AKT pathway and the expression of related miRNAs are involved in the chemotherapeutic activities of mango polyphenolics in a mouse xenograft model for breast cancer. The objectives of this research were to determine the tumor-cytotoxic activities of mango polyphenolics and the underlying molecular mechanisms involving posttranscriptional targets in BT474 breast cancer cells and xenografts in mice. In vitro findings showed cytotoxic effects of mango polyphenolics in BT474 breast cancer cells within a concentration range of 2.5 to 20 mg/L gallic acid equivalents. Mango polyphenolics suppressed the expression of PI3K, AKT, hypoxia inducible factor-1α, and vascular endothelial growth factor (VEGF) mRNA, and pAKT, AKT, pPI3K (p85), VEGF and nuclear factor-kappa B protein levels. The involvement of miR-126 was verified by using antagomiR for miR-126, where mango reversed the effect of the antagomiR of miR-126. In vivo, the intake of mango polyphenolics decreased the tumor volume by 73% in BT474 xenograft-bearing mice compared with the control group. In addition, mango reduced the expression of nuclear factor-kappa B (p65), pAKT, pPI3K, mammalian target of rapamycin, hypoxia inducible factor-1α, and VEGF protein in athymic nude mice. A screening for miRNA expression changes confirmed that mango polyphenolics modulated the expression of cancer-associated miRNAs including miR-126 in the xenografted tumors. In summary, mango polyphenolics have a chemotherapeutic potential against breast cancer that at least in part is mediated through the PI3K/AKT pathway and miR-126.

  18. Mitochondrial import of human and yeast fumarase in live mammalian cells: Retrograde translocation of the yeast enzyme is mainly caused by its poor targeting sequence

    SciTech Connect

    Singh, Bhag; Gupta, Radhey S. . E-mail: gupta@mcmaster.ca

    2006-08-04

    Studies on yeast fumarase provide the main evidence for dual localization of a protein in mitochondria and cytosol by means of retrograde translocation. We have examined the subcellular targeting of yeast and human fumarase in live cells to identify factors responsible for this. The cDNAs for mature yeast or human fumarase were fused to the gene for enhanced green fluorescent protein (eGFP) and they contained, at their N-terminus, a mitochondrial targeting sequence (MTS) derived from either yeast fumarase, human fumarase, or cytochrome c oxidase subunit VIII (COX) protein. Two nuclear localization sequences (2x NLS) were also added to these constructs to facilitate detection of any cytosolic protein by its targeting to nucleus. In Cos-1 cells transfected with these constructs, human fumarase with either the native or COX MTSs was detected exclusively in mitochondria in >98% of the cells, while the remainder 1-2% of the cells showed varying amounts of nuclear labeling. In contrast, when human fumarase was fused to the yeast MTS, >50% of the cells showed nuclear labeling. Similar studies with yeast fumarase showed that with its native MTS, nuclear labeling was seen in 80-85% of the cells, but upon fusion to either human or COX MTS, nuclear labeling was observed in only 10-15% of the cells. These results provide evidence that extramitochondrial presence of yeast fumarase is mainly caused by the poor mitochondrial targeting characteristics of its MTS (but also affected by its primary sequence), and that the retrograde translocation mechanism does not play a significant role in the extramitochondrial presence of mammalian fumarase.

  19. Mammalian DNA Repair. Final Report

    SciTech Connect

    2003-01-24

    The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  20. PIK3R1 targeting by miR-21 suppresses tumor cell migration and invasion by reducing PI3K/AKT signaling and reversing EMT, and predicts clinical outcome of breast cancer.

    PubMed

    Yan, Li-Xu; Liu, Yan-Hui; Xiang, Jian-Wen; Wu, Qi-Nian; Xu, Lei-Bo; Luo, Xin-Lan; Zhu, Xiao-Lan; Liu, Chao; Xu, Fang-Ping; Luo, Dong-Lan; Mei, Ping; Xu, Jie; Zhang, Ke-Ping; Chen, Jie

    2016-02-01

    We have previously shown that dysregulation of miR-21 functioned as an oncomiR in breast cancer. The aim of the present study was to elucidate the mechanisms by which miR-21 regulate breast tumor migration and invasion. We applied pathway analysis on genome microarray data and target-predicting algorithms for miR-21 target screening, and used luciferase reporting assay to confirm the direct target. Thereafter, we investigated the function of the target gene phosphoinositide-3-kinase, regulatory subunit 1 (α) (PIK3R1), and detected PIK3R1 coding protein (p85α) by immunohistochemistry and miR-21 by RT-qPCR on 320 archival paraffin-embedded tissues of breast cancer to evaluate the correlation of their expression with prognosis. First, we found that PIK3R1 suppressed growth, invasiveness, and metastatic properties of breast cancer cells. Next, we identified the PIK3R1 as a direct target of miR-21 and showed that it was negatively regulated by miR-21. Furthermore, we demonstrated that p85α overexpression phenocopied the suppression effects of antimiR-21 on breast cancer cell growth, migration and invasion, indicating its tumor suppressor role in breast cancer. On the contrary, PIK3R1 knockdown abrogated antimiR‑21-induced effect on breast cancer cells. Notably, antimiR-21 induction increased p85α, accompanied by decreased p-AKT level. Besides, antimiR-21/PIK3R1-induced suppression of invasiveness in breast cancer cells was mediated by reversing epithelial-mesenchymal transition (EMT). p85α downregulation was found in 25 (7.8%) of the 320 breast cancer patients, and was associated with inferior 5-year disease-free survival (DFS) and overall survival (OS). Taken together, we provide novel evidence that miR-21 knockdown suppresses cell growth, migration and invasion partly by inhibiting PI3K/AKT activation via direct targeting PIK3R1 and reversing EMT in breast cancer. p85α downregulation defined a specific subgroup of breast cancer with shorter 5-year DFS and OS

  1. Quantitative Persulfide Site Identification (qPerS-SID) Reveals Protein Targets of H2S Releasing Donors in Mammalian Cells

    PubMed Central

    Longen, Sebastian; Richter, Florian; Köhler, Yvette; Wittig, Ilka; Beck, Karl-Friedrich; Pfeilschifter, Josef

    2016-01-01

    H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents. PMID:27411966

  2. Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells

    SciTech Connect

    Li, Kuiqing; Chen, Xu; Liu, Cheng; Gu, Peng; Li, Zhuohang; Wu, Shaoxu; Xu, Kewei; Lin, Tianxin; Huang, Jian

    2015-05-01

    Pirarubicin is widely used in intravesical chemotherapy for bladder cancer, but its efficacy is limited due to drug resistance; the mechanism has not been well studied. Emerging evidence shows that autophagy can be a novel target for cancer therapy. This study aimed to investigate the role of autophagy in pirarubicin-treated bladder cancer cells. Bladder cancer cells EJ and J82 were treated with pirarubicin, siRNA, 3-methyladenine or hydroxychloroquine. Cell proliferation and apoptosis were tested by cell survival assay and flow cytometric analysis, respectively. Autophagy was evaluated by immunoblotting before and after the treatments. The phosphorylated mammalian target of rapamycin, serine/threonine kinase p70 S6 kinase, and eukaryotic translation initiation factor 4E binding protein 1 were also investigated by immunoblotting. We found that pirarubicin could induce autophagy in bladder cancer cells. Inhibition of autophagy by 3-methyladenine, hydroxychloroquine or knockdown of autophagy related gene 3 significantly increased apoptosis in pirarubicin-treated bladder cancer cells. Pirarubicin-induced autophagy was mediated via the mTOR/p70S6K/4E-BP1 signaling pathway. In conclusion, autophagy induced by pirarubicin plays a cytoprotective role in bladder cancer cells, suggesting that inhibition of autophagy may improve efficacy over traditional pirarubicin chemotherapy in bladder cancer patients. - Highlights: • Pirarubicin induced autophagy in bladder cancer cells. • Inhibition of autophagy enhanced pirarubicin-induced apoptosis. • Pirarubicin induced autophagy through inhibition of mTOR signaling pathway.

  3. Device-based local delivery of siRNA against mammalian target of rapamycin (mTOR) in a murine subcutaneous implant model to inhibit fibrous encapsulation

    PubMed Central

    Takahashi, Hironobu; Wang, Yuwei; Grainger, David W.

    2010-01-01

    Fibrous encapsulation of surgically implant devices is associated with elevated proliferation and activation of fibroblasts in tissues surrounding these implants, frequently causing foreign body complications. Here we test the hypothesis that inhibition of the expression of mammalian target of rapamycin (mTOR) in fibroblasts can mitigate the soft tissue implant foreign body response by suppressing fibrotic responses around implants. In this study, mTOR was knocked down using small interfering RNA conjugated with branched cationic polyethylenimine (bPEI) in fibroblastic lineage cells in serum-based cell culture as shown by both gene and protein analysis. This mTOR knockdown led to an inhibition in fibroblast proliferation by 70% and simultaneous down-regulation in the expression of type I collagen in fibroblasts in vitro. These siRNA/bPEI complexes were released from poly(ethylene glycol) (PEG)-based hydrogel coatings surrounding model polymer implants in a subcutaneous rodent model in vivo. No significant reduction in fibrous capsule thickness and mTOR expression in the foreign body capsules was observed. Observed siRNA inefficacy in this in vivo implant model was attributed to siRNA dosing limitations in the gel delivery system, and lack of targeting ability of the siRNA complex specifically to fibroblasts. While in vitro data supported mTOR knock-down in fibroblast cultures, in vivo siRNA delivery must be further improved to produce clinically relevant effects on fibrotic encapsulation around implants. PMID:20727922

  4. Antagonism of microRNA-99a promotes cell invasion and down-regulates E-cadherin expression in pancreatic cancer cells by regulating mammalian target of rapamycin.

    PubMed

    Li, Dan; Li, Xiaohan; Cao, Wei; Qi, Yafei; Yang, Xianghong

    2014-06-01

    MicroRNA-99a (miRNA-99a), a potential tumor suppressor, has been implicated in tumorigenesis of many human malignancies. However, the role of miRNA-99a in pancreatic cancer remains unclear. In the present study, we transfected miRNA-99a antagonism into human pancreatic cancer AsPC-1 cells to inhibit miRNA-99a expression and investigated its influence on cell migration and invasion as well as the underlying possible mechanisms. We found that miRNA-99a antagonism significantly increased proliferation, migration and invasion abilities of AsPC-1 cells, which was accompanied by increased expression of mesenchymal phenotype cell biomarkers (N-cadherin, Vimentin, and α-SMA), and decreased expression of epithelial phenotype cell biomarker (E-cadherin). Interestingly, small interfering RNA (siRNA)-mediated knockdown of mammalian target of rapamycin (mTOR) remarkably restored miRNA-99a antagonism-induced down-regulation of E-cadherin. In conclusion, our data suggest that miRNA-99a is involved in pancreatic cancer migration and invasion by regulating mTOR, and may provide a target for effective therapies against pancreatic cancer.

  5. Inhibition of Mammalian Target of Rapamycin (mTOR) Signaling in the Insular Cortex Alleviates Neuropathic Pain after Peripheral Nerve Injury

    PubMed Central

    Kwon, Minjee; Han, Jeongsoo; Kim, Un Jeng; Cha, Myeounghoon; Um, Sun Woo; Bai, Sun Joon; Hong, Seong-Karp; Lee, Bae Hwan

    2017-01-01

    Injury of peripheral nerves can trigger neuropathic pain, producing allodynia and hyperalgesia via peripheral and central sensitization. Recent studies have focused on the role of the insular cortex (IC) in neuropathic pain. Because the IC is thought to store pain-related memories, translational regulation in this structure may reveal novel targets for controlling chronic pain. Signaling via mammalian target of rapamycin (mTOR), which is known to control mRNA translation and influence synaptic plasticity, has been studied at the spinal level in neuropathic pain, but its role in the IC under these conditions remains elusive. Therefore, this study was conducted to determine the role of mTOR signaling in neuropathic pain and to assess the potential therapeutic effects of rapamycin, an inhibitor of mTORC1, in the IC of rats with neuropathic pain. Mechanical allodynia was assessed in adult male Sprague-Dawley rats after neuropathic surgery and following microinjections of rapamycin into the IC on postoperative days (PODs) 3 and 7. Optical recording was conducted to observe the neural responses of the IC to peripheral stimulation. Rapamycin reduced mechanical allodynia and downregulated the expression of postsynaptic density protein 95 (PSD95), decreased neural excitability in the IC, thereby inhibiting neuropathic pain-induced synaptic plasticity. These findings suggest that mTOR signaling in the IC may be a critical molecular mechanism modulating neuropathic pain. PMID:28377693

  6. Reduced signaling of PI3K-Akt and RAS-MAPK pathways is the key target for weight-loss-induced cancer prevention by dietary calorie restriction and/or physical activity.

    PubMed

    Standard, Joseph; Jiang, Yu; Yu, Miao; Su, Xiaoyu; Zhao, Zhihui; Xu, Jianteng; Chen, Jie; King, Brenee; Lu, Lizhi; Tomich, John; Baybutt, Richard; Wang, Weiqun

    2014-12-01

    Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics and lipidomics was employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into four groups for 10 weeks: ad-libitum-fed sedentary control, ad-libitum-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE) and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67 and 110 genes were significantly changed in DCR, PE and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including up-regulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears that the reduction of both Ras-MAPK and PI3K-Akt signaling pathways is a cancer preventive target that has been consistently demonstrated by three bioinformatics approaches.

  7. Reduced signaling of PI3K-Akt and RAS-MAPK pathways are the key targets for weight loss-induced cancer prevention by dietary calorie restriction and/or physical activity

    PubMed Central

    Standard, Joseph; Jiang, Yu; Yu, Miao; Su, Xiaoyu; Zhao, Zhihui; Xu, Jianteng; Chen, Jie; King, Brenee; Lu, Lizhi; Tomich, John; Baybutt, Richard; Wang, Weiqun

    2014-01-01

    Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics, and lipidomics were employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into 4 groups for 10 weeks: ad lib-fed sedentary control, ad lib-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE), and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics, and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67, and 110 genes were significantly changed in DCR, PE, and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including upregulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears the reduction of both Ras-MAPK and PI3K-Akt signaling pathways are cancer preventive targets that have been consistently demonstrated by three bioinformatics approaches. PMID:25283328

  8. Recruitment of the Mammalian Histone-modifying EMSY Complex to Target Genes Is Regulated by ZNF131.

    PubMed

    Varier, Radhika A; Carrillo de Santa Pau, Enrique; van der Groep, Petra; Lindeboom, Rik G H; Matarese, Filomena; Mensinga, Anneloes; Smits, Arne H; Edupuganti, Raghu Ram; Baltissen, Marijke P; Jansen, Pascal W T C; Ter Hoeve, Natalie; van Weely, Danny R; Poser, Ina; van Diest, Paul J; Stunnenberg, Hendrik G; Vermeulen, Michiel

    2016-04-01

    Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1, and EMSY. Here, we characterize the EMSY/KDM5A/SIN3B complex in detail by quantitative interaction proteomics and ChIP-sequencing. We identify a novel substoichiometric interactor of the complex, transcription factor ZNF131, which recruits EMSY to a large number of active, H3K4me3 marked promoters. Interestingly, using an EMSY knock-out line and subsequent rescue experiments, we show that EMSY is in most cases positively correlated with transcriptional activity of its target genes and stimulates cell proliferation. Finally, by immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner. Taken together, these data open venues for exploring the possibility that sporadic breast cancer patients with EMSY amplification might benefit from epigenetic combination therapy targeting both the KDM5A demethylase and histone deacetylases.

  9. Effect of lithium on ventricular remodelling in infarcted rats via the Akt/mTOR signalling pathways.

    PubMed

    Lee, Tsung-Ming; Lin, Shinn-Zong; Chang, Nen-Chung

    2017-04-28

    Activation of phosphoinositide 3-kinase (PI3K)/Akt signalling is the molecular pathway driving physiological hypertrophy. As lithium, a PI3K agonist, is highly toxic at regular doses, we assessed the effect of lithium at a lower dose on ventricular hypertrophy after myocardial infarction (MI). Male Wistar rats after induction of MI were randomized to either vehicle or lithium (1 mmol/kg per day) for 4 weeks. The dose of lithium led to a mean serum level of 0.39 mM, substantially lower than the therapeutic concentrations (0.8-1.2 mM). Infarction in the vehicle was characterized by pathological hypertrophy in the remote zone; histologically, by increased cardiomyocyte sizes, interstitial fibrosis and left ventricular dilatation; functionally, by impaired cardiac contractility; and molecularly, by an increase of p-extracellular-signal-regulated kinase (ERK) levels, nuclear factor of activated T cells (NFAT) activity, GATA4 expression and foetal gene expressions. Lithium administration mitigated pathological remodelling. Furthermore, lithium caused increased phosphorylation of eukaryotic initiation factor 4E binding protein 1 (p-4E-BP1), the downstream target of mammalian target of rapamycin (mTOR). Blockade of the Akt and mTOR signalling pathway with deguelin and rapamycin resulted in markedly diminished levels of p-4E-BP1, but not ERK. The present study demonstrated that chronic lithium treatment at low doses mitigates pathological hypertrophy through an Akt/mTOR dependent pathway.

  10. Mitochondrial Akt Regulation of Hypoxic Tumor Reprogramming.

    PubMed

    Chae, Young Chan; Vaira, Valentina; Caino, M Cecilia; Tang, Hsin-Yao; Seo, Jae Ho; Kossenkov, Andrew V; Ottobrini, Luisa; Martelli, Cristina; Lucignani, Giovanni; Bertolini, Irene; Locatelli, Marco; Bryant, Kelly G; Ghosh, Jagadish C; Lisanti, Sofia; Ku, Bonsu; Bosari, Silvano; Languino, Lucia R; Speicher, David W; Altieri, Dario C

    2016-08-08

    Hypoxia is a universal driver of aggressive tumor behavior, but the underlying mechanisms are not completely understood. Using a phosphoproteomics screen, we now show that active Akt accumulates in the mitochondria during hypoxia and phosphorylates pyruvate dehydrogenase kinase 1 (PDK1) on Thr346 to inactivate the pyruvate dehydrogenase complex. In turn, this pathway switches tumor metabolism toward glycolysis, antagonizes apoptosis and autophagy, dampens oxidative stress, and maintains tumor cell proliferation in the face of severe hypoxia. Mitochondrial Akt-PDK1 signaling correlates with unfavorable prognostic markers and shorter survival in glioma patients and may provide an "actionable" therapeutic target in cancer.

  11. Control of fibroblast fibronectin expression and alternative splicing via the PI3K/Akt/mTOR pathway

    SciTech Connect

    White, Eric S.; Sagana, Rommel L.; Booth, Adam J.; Yan, Mei; Cornett, Ashley M.; Bloomheart, Christopher A.; Tsui, Jessica L.; Wilke, Carol A.; Moore, Bethany B.; Ritzenthaler, Jeffrey D.; Roman, Jesse; Muro, Andres F.

    2010-10-01

    Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-{beta}, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten{sup -/-} fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten{sup -/-} cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten{sup -/-} cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.

  12. Curcumin Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells through the Inhibition of Akt/mTOR Pathway.

    PubMed

    Zhu, Fang-Qiang; Chen, Min-Jia; Zhu, Ming; Zhao, Rong-Seng; Qiu, Wei; Xu, Xiang; Liu, Hong; Zhao, Hong-Wen; Yu, Rong-Jie; Wu, Xiong-Fei; Zhang, Keqin; Huang, Hong

    2017-01-01

    Curcumin has exhibited a protective effect against development of renal fibrosis in animal models, however, its underlying molecular mechanisms are largely unclear. Therefore, we investigated the anti-fibrosis effects of curcumin in transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT), and the mechanism by which it mediates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Human kidney tubular epithelial cells (HKCs) were treated with TGF-β1 or curcumin alone, or TGF-β1 in combination with curcumin. The effect of curcumin on cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of E-cadherin, cytokeratin, vimentin, alpha smooth muscle actin (α-SMA), fibroblast-specific protein 1 (FSP1) and key proteins of Akt/mammalian target of rapamycin (mTOR) pathway were analyzed by immunocytochemistry, real-time PCR and Western blot. Low dose curcumin (3.125 and 25 µmol/L) effectively promoted HKC proliferation. When HKCs were co-incubated with TGF-β1 and curcumin for 72 h, curcumin maintained the epithelial morphology in a dose-dependent manner, decreased expression of vimentin, α-SMA and FSP1 normally induced by TGF-β1, and increased expression of E-cadherin, cytokeratin. Importantly, we found that curcumin reduced Akt, mTOR and P70S6K phosphorylation, effectively suppressing the activity of the Akt/mTOR pathway in HKCs. Curcumin also promoted HKC proliferation, and antagonized TGF-β1-driven EMT through the inhibition of Akt/mTOR pathway activity, which may suggest an alternative therapy for renal fibrosis.

  13. RY-2f, an isoflavone analog, overcomes cisplatin resistance to inhibit ovarian tumorigenesis via targeting the PI3K/AKT/mTOR signaling pathway

    PubMed Central

    Ren, Yi; Li, Hanbin; Yang, Gong; Zhang, Qian

    2015-01-01

    Ovarian cancer remains the leading cause of death in gynecologic malignancies partially because of resistance to chemotherapy. In the present study, we show that RY-2f, a chemically synthesized isoflavone analog, inhibited ovarian cancer cell proliferation, blocked cell cycle in G2/M phase and induced cellular apoptosis through up-regulation of p21, cyclin B1, Bax, Bad and cleaved-PARP, and suppression of cyclin A, CDK2 and Bcl-2. We also show that RY-2f could increase the chemotherapeutic efficacy of cisplatin as tested by cell proliferation and colony formation assays, indicating a synergistic effect of RY-2f and cisplatin. Mechanistic study revealed that RY-2f exerted the anti-tumor activities mainly through suppression of the PI3K/AKT/mTOR signaling. Finally, in vivo studies showed that RY-2f blocked the A2780-induced xenograft tumor growth without detectable toxicity in the animals at the therapeutic doses, and whereas RY-2f re-sensitized the cisplatin resistant cell line A2780/CDDP induced xenograft tumor to cisplatin treatment. Thus, RY-2f may be developed as a potential therapeutic agent to treat ovarian cancer. PMID:26325371

  14. BCL-3 expression promotes colorectal tumorigenesis through activation of AKT signalling

    PubMed Central

    Urban, Bettina C; Collard, Tracey J; Eagle, Catherine J; Southern, Samantha L; Greenhough, Alexander; Hamdollah-Zadeh, Maryam; Ghosh, Anil; Paraskeva, Christos; Silver, Andrew; Williams, Ann C

    2016-01-01

    Objective Colorectal cancer remains the fourth most common cause of cancer-related mortality worldwide. Here we investigate the role of nuclear factor-κB (NF-κB) co-factor B-cell CLL/lymphoma 3 (BCL-3) in promoting colorectal tumour cell survival. Design Immunohistochemistry was carried out on 47 tumour samples and normal tissue from resection margins. The role of BCL-3/NF-κB complexes on cell growth was studied in vivo and in vitro using an siRNA approach and exogenous BCL-3 expression in colorectal adenoma and carcinoma cells. The question whether BCL-3 activated the AKT/protein kinase B (PKB) pathway in colorectal tumour cells was addressed by western blotting and confocal microscopy, and the ability of 5-aminosalicylic acid (5-ASA) to suppress BCL-3 expression was also investigated. Results We report increased BCL-3 expression in human colorectal cancers and demonstrate that BCL-3 expression promotes tumour cell survival in vitro and tumour growth in mouse xenografts in vivo, dependent on interaction with NF-κB p50 or p52 homodimers. We show that BCL-3 promotes cell survival under conditions relevant to the tumour microenvironment, protecting both colorectal adenoma and carcinoma cells from apoptosis via activation of the AKT survival pathway: AKT activation is mediated via both PI3K and mammalian target of rapamycin (mTOR) pathways, leading to phosphorylation of downstream targets GSK-3β and FoxO1/3a. Treatment with 5-ASA suppressed BCL-3 expression in colorectal cancer cells. Conclusions Our study helps to unravel the mechanism by which BCL-3 is linked to poor prognosis in colorectal cancer; we suggest that targeting BCL-3 activity represents an exciting therapeutic opportunity potentially increasing the sensitivity of tumour cells to conventional therapy. PMID:26033966

  15. Metastasis and AKT activation.

    PubMed

    Qiao, Meng; Sheng, Shijie; Pardee, Arthur B

    2008-10-01

    Metastasis is responsible for 90% of cancer patient deaths. More information is needed about the molecular basis for its potential detection and treatment. The activated AKT kinase is necessary for many events of the metastatic pathway including escape of cells from the tumor's environment, into and then out of the circulation, activation of proliferation, blockage of apoptosis, and activation of angiogenesis. A series of steps leading to metastatic properties can be initiated upon activation of AKT by phosphorylation on Ser-473. These findings lead to the question of how this activation is connected to metastasis. Activated AKT phosphorylates GSK-3beta causing its proteolytic removal. This increases stability of the negative transcription factor SNAIL, thereby decreasing transcription of the transmembrane protein E-cadherin that forms adhesions between adjacent cells, thereby permitting their detachment. How is AKT hyperactivated in metastatic cells? Increased PI3K or TORC2 kinase activity- or decreased PHLPP phosphatase could be responsible. Furthermore, a positive feedback mechanism is that the decrease of E-cadherin lowers PTEN and thereby increases PIP3, further activating AKT and metastasis.

  16. Dissecting signalling by individual Akt/PKB isoforms, three steps at once.

    PubMed

    Osorio-Fuentealba, Cesar; Klip, Amira

    2015-09-01

    The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1(W80A) or Akt2(W80A)). Akt1(W80A) or Akt2(W80A) are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBβ can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBβ assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase.

  17. Cross-communication between histone H3 and H4 acetylation and Akt-mTOR signalling in prostate cancer cells.

    PubMed

    Makarević, Jasmina; Tawanaie, Nassim; Juengel, Eva; Reiter, Michael; Mani, Jens; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A

    2014-07-01

    Molecular tumour targeting has significantly improved anti-cancer protocols. Still, the addition of molecular targeting to the treatment regime has not led to a curative breakthrough. Combined mammalian target of Rapamycin (mTOR) and histone deacetylase (HDAC) inhibition has been shown not only to enhance anti-tumour potential, but also to prevent resistance development seen under mono-drug therapy. This investigation was designed to evaluate whether cross-communication exists between mTOR signalling and epigenetic events regulated by HDAC. DU-145 prostate cancer cells were treated with insulin-like growth factor (IGF) to activate the Akt-mTOR cascade or with the HDAC-inhibitor valproic acid (VPA) to induce histone H3 and H4 acetylation (aH3, aH4). Subsequently, mTOR, Rictor, Raptor, p70s6k, Akt (all: total and phosphorylated), H3 and H4 (total and acetylated) were analysed by western blotting. Both techniques revealed a link between mTOR and the epigenetic machinery. IGF activated mTOR, Rictor, Raptor, p70s6k and Akt, but also enhanced aH3 and aH4. Inversely, IGFr blockade and knock-down blocked the Akt-mTOR axis, but simultaneously diminished aH3 and aH4. VPA treatment up-regulated histone acetylation, but also activated mTOR-Akt signalling. HDAC1 and 2 knock-down revealed that the interaction with the mTOR system is initiated by histone H3 acetylation. HDAC-mTOR communication, therefore, is apparent whereby tumour-promoting (Akt/mTOR(high), aH3/aH4(low)) and tumour-suppressing signals (Akt/mTOR(low) , aH3/aH4(high)) are activated in parallel. Combined use of an HDAC- and mTOR inhibitor might then diminish pro-tumour effects triggered by the HDAC- (Akt/mTOR(high)) or mTOR inhibitor (aH3/aH4(low)) alone.

  18. Cross-communication between histone H3 and H4 acetylation and Akt-mTOR signalling in prostate cancer cells

    PubMed Central

    Makarević, Jasmina; Tawanaie, Nassim; Juengel, Eva; Reiter, Michael; Mani, Jens; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A

    2014-01-01

    Molecular tumour targeting has significantly improved anti-cancer protocols. Still, the addition of molecular targeting to the treatment regime has not led to a curative breakthrough. Combined mammalian target of Rapamycin (mTOR) and histone deacetylase (HDAC) inhibition has been shown not only to enhance anti-tumour potential, but also to prevent resistance development seen under mono-drug therapy. This investigation was designed to evaluate whether cross-communication exists between mTOR signalling and epigenetic events regulated by HDAC. DU-145 prostate cancer cells were treated with insulin-like growth factor (IGF) to activate the Akt-mTOR cascade or with the HDAC-inhibitor valproic acid (VPA) to induce histone H3 and H4 acetylation (aH3, aH4). Subsequently, mTOR, Rictor, Raptor, p70s6k, Akt (all: total and phosphorylated), H3 and H4 (total and acetylated) were analysed by western blotting. Both techniques revealed a link between mTOR and the epigenetic machinery. IGF activated mTOR, Rictor, Raptor, p70s6k and Akt, but also enhanced aH3 and aH4. Inversely, IGFr blockade and knock-down blocked the Akt-mTOR axis, but simultaneously diminished aH3 and aH4. VPA treatment up-regulated histone acetylation, but also activated mTOR-Akt signalling. HDAC1 and 2 knock-down revealed that the interaction with the mTOR system is initiated by histone H3 acetylation. HDAC-mTOR communication, therefore, is apparent whereby tumour-promoting (Akt/mTORhigh, aH3/aH4low) and tumour-suppressing signals (Akt/mTORlow, aH3/aH4high) are activated in parallel. Combined use of an HDAC- and mTOR inhibitor might then diminish pro-tumour effects triggered by the HDAC- (Akt/mTORhigh) or mTOR inhibitor (aH3/aH4low) alone. PMID:24779401

  19. Novel agents and associated toxicities of inhibitors of the pi3k/Akt/mtor pathway for the treatment of breast cancer

    PubMed Central

    Chia, S.; Gandhi, S.; Joy, A.A.; Edwards, S.; Gorr, M.; Hopkins, S.; Kondejewski, J.; Ayoub, J.P.; Califaretti, N.; Rayson, D.; Dent, S.F.

    2015-01-01

    The pi3k/Akt/mtor (phosphatidylinositol 3 kinase/ Akt/mammalian target of rapamycin) signalling pathway is an established driver of oncogenic activity in human malignancies. Therapeutic targeting of this pathway holds significant promise as a treatment strategy. Everolimus, an mtor inhibitor, is the first of this class of agents approved for the treatment of hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer. Everolimus has been associated with significant improvements in progression-free survival; however, it is also associated with increased toxicity related to its specific mechanism of action. Methods A comprehensive review of the literature conducted using a focused medline search was combined with a search of current trials at http://ClinicalTrials.gov/. Summary tables of the toxicities of the various classes of pi3k/Akt/mtor inhibitors were created. A broad group of Canadian health care professionals was assembled to review the data and to produce expert opinion and summary recommendations for possible best practices in managing the adverse events associated with these pathway inhibitors. Results Differing toxicities are associated with the various classes of pi3k/Akt/mtor pathway inhibitors. The most common unique adverse events observed in everolimus clinical trials in breast cancer include stomatitis (all grades: approximately 60%), noninfectious pneumonitis (15%), rash (40%), hyperglycemia (15%), and immunosuppression (40%). To minimize grades 3 and 4 toxicities and to attempt to attain optimal outcomes, effective management of those adverse events is critical. Management should be interdisciplinary and should use approaches that include education, early recognition, active intervention, and potentially prophylactic strategies. Discussion Everolimus likely represents the first of many complex oral targeted therapies for the treatment of breast cancer. Using this agent as a template, it is essential to

  20. A novel mouse model of inflammatory bowel disease links mammalian target of rapamycin-dependent hyperproliferation of colonic epithelium to inflammation-associated tumorigenesis.

    PubMed

    Deng, Lin; Zhou, Jin-Feng; Sellers, Rani S; Li, Jiu-Feng; Nguyen, Andrew V; Wang, Yubao; Orlofsky, Amos; Liu, Qiang; Hume, David A; Pollard, Jeffrey W; Augenlicht, Leonard; Lin, Elaine Y

    2010-02-01

    Inflammatory bowel disease (IBD) is a high-risk condition for human colorectal cancer. However, our mechanistic understanding of the link between inflammation and tumorigenesis in the colon is limited. Here we established a novel mouse model of colitis-associated cancer by genetically inactivating signal transducer and activator of transcription 3 (Stat3) in macrophages, with partial deletion in other myeloid and lymphoid cells. Inflammation developed in the colon of mutant mice spontaneously, and tumor lesions, including invasive carcinoma, arose in the inflamed region of the intestine with a frequency similar to that observed in human IBD patients. The development of both inflammation and tumors in the mutant mice required the presence of microflora. Indeed, inflammation was associated with disruption of colonic homeostasis, fulminant epithelial/tumor cell proliferation, and activation of the mammalian target of rapamycin (mTOR)-Stat3 pathway in epithelial and tumor cells. The activation of this pathway was essential for both the excess proliferation of epithelial/tumor cells and the disruption of colonic homeostasis in the mutant mice. Notably, a similar abnormal up-regulation of mTOR-Stat3 signaling was consistently observed in the colonic epithelial cells of human IBD patients with active disease. These studies demonstrate a novel mouse model of IBD-colorectal cancer progression in which disrupted immune regulation, mTOR-Stat3 signaling, and epithelial hyperproliferation are integrated and simultaneously linked to the development of malignancy.

  1. The Mammalian “Obesogen” Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish

    PubMed Central

    Lyssimachou, Angeliki; Santos, Joana G.; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C. Marisa R.; Teixeira, Catarina; Castro, L. Filipe C.; Santos, Miguel M.

    2015-01-01

    Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an “obesogenic” phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound. PMID:26633012

  2. 8-Methoxypsoralen induced mutations are highly targeted at crosslinkable sites of photoaddition on the non-transcribed strand of a mammalian chromosomal gene.

    PubMed Central

    Sage, E; Drobetsky, E A; Moustacchi, E

    1993-01-01

    We have determined the mutational specificity of 8-methoxypsoralen photoaddition at the endogenous adenine phosphoribosyltransferase gene of Chinese hamster ovary cells hemizygous for this locus. In addition, the distribution of 8-methoxypsoralen photo-adducts was resolved in vitro at the DNA sequence level, and compared with the observed site specificity for mutation. Among 27 mutants characterized, all were single base changes at AT base pairs: 16 A:T-->T:A, six A:T-->C:G, four A:T-->G:C and one -T frameshift. All these vents were targeted to potential sites of photoaddition. The vast majority of these sites were also detectable in vitro, suggesting that 8-methoxypsoralen plus UVA-induced mutational hotspots may be damage hotspots. Furthermore 26/27 mutations occurred at crosslinkable 5'TpA sites, supporting the notion that 8-methoxypsoralen biadducts rather than monoadducts are major premutagenic lesions in mammalian cells. Since 90% of our mutation collection could have resulted from damage on the non-transcribed strand, it appears that photoadducted thymine residues on the transcribed strand of the adenine phosphoribosyltransferase gene may be preferentially repaired. We therefore suggest a model for mutagenesis, induced by psoralen biadducts, based on the preferential incision of biadducts followed by translesion synthesis past modified T bases persisting on the non-transcribed strand. Images PMID:8440233

  3. Screening of mammalian target of rapamycin inhibitors in natural product extracts by capillary electrophoresis in combination with high performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Zhang, Yanmei; Li, Feng; Li, Mingxia; Kang, Jingwu

    2015-04-03

    In this study, capillary electrophoresis (CE) combined with HPLC-MS/MS were used as a powerful platform for screening of inhibitors of mammalian target of rapamycin (mTOR) in natural product extracts. The screening system has been established by using 5-carboxyfluorescein labeled substrate peptide F-4EBP1, a known mTOR inhibitor AZD8055, and a small chemical library consisted of 18 natural product extracts. Biochemical screening of natural product extracts was performed by CE with laser induced fluorescence detection. The CE separation allowed a quantitative measurement of the phosphorylated product, hence the quantitation of enzymatic inhibition as well as inhibition kinetics. The hits are readily identified as long as the peak area of the phosphorylated product is reduced in comparison with the negative control. Subsequent assay-guided isolation of the active natural product extract was performed with HPLC-MS/MS to track the particular active components. The structures of the identified active components were elucidated by the molecular ions and fragmentation information provided by MS/MS analysis. The CE-based assay method only requires minute pure compounds, which can be readily purified by HPLC. Therefore, the combination of CE and HPLC-MS/MS provides a high-throughput platform for screening bioactive compounds from the crude nature extracts. By taking the advantage of the screening system, salvianolic acid A and C in extract of Salvia miltiorrhiza were discovered as the new mTOR inhibitors.

  4. Cellular senescence or EGFR signaling induces Interleukin 6 (IL-6) receptor expression controlled by mammalian target of rapamycin (mTOR)

    PubMed Central

    Garbers, Christoph; Kuck, Fabian; Aparicio-Siegmund, Samadhi; Konzak, Kirstin; Kessenbrock, Mareike; Sommerfeld, Annika; Häussinger, Dieter; Lang, Philipp A; Brenner, Dirk; Mak, Tak W.; Rose-John, Stefan; Essmann, Frank; Schulze-Osthoff, Klaus; Piekorz, Roland P; Scheller, Jürgen

    2013-01-01

    Interleukin 6 (IL-6) signaling plays a role in inflammation, cancer, and senescence. Here, we identified soluble IL-6 receptor (sIL-6R) as a member of the senescence-associated secretory phenotype (SASP). Senescence-associated sIL-6R upregulation was mediated by mammalian target of rapamycin (mTOR). sIL-6R was mainly generated by a disintegrin and metalloprotease 10 (ADAM10)-dependent ectodomain shedding to enable IL-6 trans-signaling. In vivo, heterozygous PTEN-knockout mice exhibited higher mTOR activity and increased sIL-6R levels. Moreover, aberrant EGF receptor (EGFR) activation triggered IL-6 synthesis. In analogy to senescence, EGFR-induced activation of mTOR also induced IL-6R expression and sIL-6R generation. Hence, mTOR activation reprograms IL-6 non-responder cells into IL-6 responder cells. Our data suggest that mTOR serves as a central molecular switch to facilitate cellular IL-6 classic and trans-signaling via IL-6R upregulation with direct implications for cellular senescence and tumor development. PMID:24047696

  5. The Mammalian "Obesogen" Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish.

    PubMed

    Lyssimachou, Angeliki; Santos, Joana G; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C Marisa R; Teixeira, Catarina; Castro, L Filipe C; Santos, Miguel M

    2015-01-01

    Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an "obesogenic" phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound.

  6. A Novel Mouse Model of Inflammatory Bowel Disease Links Mammalian Target of Rapamycin-Dependent Hyperproliferation of Colonic Epithelium to Inflammation-Associated Tumorigenesis

    PubMed Central

    Deng, Lin; Zhou, Jin-Feng; Sellers, Rani S.; Li, Jiu-Feng; Nguyen, Andrew V.; Wang, Yubao; Orlofsky, Amos; Liu, Qiang; Hume, David A.; Pollard, Jeffrey W.; Augenlicht, Leonard; Lin, Elaine Y.

    2010-01-01

    Inflammatory bowel disease (IBD) is a high-risk condition for human colorectal cancer. However, our mechanistic understanding of the link between inflammation and tumorigenesis in the colon is limited. Here we established a novel mouse model of colitis-associated cancer by genetically inactivating signal transducer and activator of transcription 3 (Stat3) in macrophages, with partial deletion in other myeloid and lymphoid cells. Inflammation developed in the colon of mutant mice spontaneously, and tumor lesions, including invasive carcinoma, arose in the inflamed region of the intestine with a frequency similar to that observed in human IBD patients. The development of both inflammation and tumors in the mutant mice required the presence of microflora. Indeed, inflammation was associated with disruption of colonic homeostasis, fulminant epithelial/tumor cell proliferation, and activation of the mammalian target of rapamycin (mTOR)-Stat3 pathway in epithelial and tumor cells. The activation of this pathway was essential for both the excess proliferation of epithelial/tumor cells and the disruption of colonic homeostasis in the mutant mice. Notably, a similar abnormal up-regulation of mTOR-Stat3 signaling was consistently observed in the colonic epithelial cells of human IBD patients with active disease. These studies demonstrate a novel mouse model of IBD-colorectal cancer progression in which disrupted immune regulation, mTOR-Stat3 signaling, and epithelial hyperproliferation are integrated and simultaneously linked to the development of malignancy. PMID:20042677

  7. Temporal changes in mammalian target of rapamycin (mTOR) and phosphorylated-mTOR expressions in the hippocampal CA1 region of rat with vascular dementia

    PubMed Central

    Park, Jin-A

    2017-01-01

    Mammalian target of rapamycin (mTOR) has an important role in various biological processes in cells. In the present study, we investigated temporal changes in mTOR and phosphorylated-mTOR (p-mTOR) expressions in the rat hippocampal CA1 region following chronic cerebral hypoperfusion (CCH) induced by permanent bilateral common carotid arteries occlusion (2VO). The mTOR immunoreactivity in the pyramidal neurons and mTOR protein level in the hippocampal CA1 region were markedly decreased at 21 and 28 days after 2VO surgery. However, p-mTOR protein expression was significantly increased at 7 days following CCH but then decreased with time. The results indicate that mTOR and p-mTOR expressions change in the hippocampal CA1 region after 2VO surgery and that reduced expressions of mTOR and p-mTOR may be closely related to the CCH-induced neuronal damage in the hippocampal CA1 region. PMID:27297423

  8. Activation of mammalian target of rapamycin contributes to pain nociception induced in rats by BmK I, a sodium channel-specific modulator.

    PubMed

    Jiang, Feng; Hua, Li-Ming; Jiao, Yun-Lu; Ye, Pin; Fu, Jin; Cheng, Zhi-Jun; Ding, Gang; Ji, Yong-Hua

    2014-02-01

    The mammalian target of rapamycin (mTOR) pathway is essential for maintenance of the sensitivity of certain adult sensory neurons. Here, we investigated whether the mTOR cascade is involved in scorpion envenomation-induced pain hypersensitivity in rats. The results showed that intraplantar injection of a neurotoxin from Buthus martensii Karsch, BmK I (10 μg), induced the activation of mTOR, as well as its downstream molecules p70 ribosomal S6 protein kinase (p70 S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), in lumbar 5-6 dorsal root ganglia neurons on both sides in rats. The activation peaked at 2 h and recovered 1 day after injection. Compared with the control group, the ratios of p-mTOR/p-p70 S6K/p-4EBP1 in three types of neurons changed significantly. The cell typology of p-mTOR/p-p70 S6K/p-4E-BP1 immuno-reactive neurons also changed. Intrathecal administration of deforolimus, a specific inhibitor of mTOR, attenuated BmK I-induced pain responses (spontaneous flinching, paroxysmal pain-like behavior, and mechanical hypersensitivity). Together, these results imply that the mTOR signaling pathway is mobilized by and contributes to experimental scorpion sting-induced pain.

  9. Novel PI3K/AKT targeting anti-angiogenic activities of 4-vinylphenol, a new therapeutic potential of a well-known styrene metabolite

    PubMed Central

    Yue, Grace Gar-Lee; Lee, Julia Kin-Ming; Kwok, Hin-Fai; Cheng, Ling; Wong, Eric Chun-Wai; Jiang, Lei; Yu, Hua; Leung, Hoi-Wing; Wong, Yuk-Lau; Leung, Ping-Chung; Fung, Kwok-Pui; Lau, Clara Bik-San

    2015-01-01

    The pneumo- and hepato-toxicity of 4-vinylphenol (4VP), a styrene metabolite, has been previously reported. Nevertheless, the present study reported the novel anti-angiogenic activities of 4VP which was firstly isolated from the aqueous extract of a Chinese medicinal herb Hedyotis diffusa. Our results showed that 4VP at non-toxic dose effectively suppressed migration, tube formation, adhesion to extracellular matrix proteins, as well as protein and mRNA expressions of metalloproteinase-2 of human endothelial cells (HUVEC and HMEC-1). Investigation of the signal transduction revealed that 4VP down-regulated PI3K/AKT and p38 MAPK. Besides, 4VP interfered with the phosphorylation of ERK1/2, the translocation and expression of NFkappaB. In zebrafish embryo model, the new blood vessel growth was significantly blocked by 4VP (6.25–12.5 μg/mL medium). The VEGF-induced blood vessel formation in Matrigel plugs in C57BL/6 mice was suppressed by 4VP (20–100 μg/mL matrigel). In addition, the blood vessel number and tumor size were reduced by intraperitoneal 4VP (0.2–2 mg/kg) in 4T1 breast tumor-bearing BALB/c mice, with doxorubicin as positive control. Together, the in vitro and in vivo anti-angiogenic activities of 4VP were demonstrated for the first time. These findings suggest that 4VP has great potential to be further developed as an anti-angiogenic agent. PMID:26053458

  10. Mammalian pheromones.

    PubMed

    Liberles, Stephen D

    2014-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors.

  11. Mammalian Pheromones

    PubMed Central

    Liberles, Stephen D.

    2015-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d ) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors. PMID:23988175

  12. Allosteric Small-Molecule Inhibitors of the AKT Kinase

    NASA Astrophysics Data System (ADS)

    Dalafave, D. S.

    This research addresses computational design of small druglike molecules for possible anticancer applications. AKT and SGK are kinases that control important cellular functions. They are highly homologous, having similar activators and targets. Cancers with increased SGK activity may develop resistance to AKT-specific inhibitors. Our goal was to design new molecules that would bind both AKT and SGK, thus preventing the development of drug resistance. Most kinase inhibitors target the kinase ATP-binding site. However, the high similarity in this site among kinases makes it difficult to target specifically. Furthermore, mutations in this site can cause resistance to ATP-competitive kinase inhibitors. We used existing AKT inhibitors as initial templates to design molecules that could potentially bind the allosteric sites of both AKT and SGK. Molecules with no implicit toxicities and optimal drug-like properties were used for docking studies. Binding energies of the stable complexes that the designed molecules formed with AKT and SGK were calculated. Possible applications of the designed putative inhibitors against cancers with overexpressed AKT/SGK is discussed.

  13. AKT as locus of fragility in robust cancer system.

    PubMed

    Radisavljevic, Ziv

    2008-08-15

    Metastatic cancer is a complex positive feedback loop system. Such as system has a tendency to acquire extreme robustness. Signaling pathways controlling that robustness can fail completely if an essential element from the signaling is removed. That element is a locus of fragility. Targeting that locus represents the best way to target the cancer robustness. This prospect presents another locus of fragility in signaling complex system network, controlling the cell cycle progression through the PI3K/AKT/mTOR/RAN pathway and cell migration and angiogenesis through the VEGF/PI3K/AKT/NO/ICAM-1 pathway. The locus of fragility of these pathways is AKT, which is regulated by a balance of catalase/H2O2 or by AKT inhibitor. Tiny and trivial perturbations such as change in redox state in the cells by antioxidant enzyme catalase, scavenging H2O2 signaling molecule, regulates robust signaling molecule AKT, abolishing its phosporilation and inducing cascading failure of robust signaling pathways for cell growth, proliferation, migration, and angiogenesis. An anticancer effect of the antioxidant is achieved through the AKT locus, by abolishing signals from growth factors VEGF, HGF, HIF-1alpha and H2O2. Previously reported locus of fragility nitric oxide (NO) and locus AKT are close in the complex signaling interactome network, but they regulate distinct signaling modules. Simultaneously targeted loci represents new principles in cancer robustness chemotherapy by blocking cell proliferation, migration, angiogenesis and inducing rather slow then fast apoptosis leading to slow eradication of cancer.

  14. Oral Adverse Events Associated with Tyrosine Kinase and Mammalian Target of Rapamycin Inhibitors in Renal Cell Carcinoma: A Structured Literature Review

    PubMed Central

    Epstein, Joel B.; Raber-Durlacher, Judith E.; Ouwerkerk, Jan; Logan, Richard M.; Brakenhoff, Jan A.; Lacouture, Mario E.; Gelderblom, Hans

    2012-01-01

    Background. Oral adverse events (OAEs) associated with multitargeted tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin inhibitors (mTORIs) are underestimated but frequent and novel presentations of mucosal manifestations. Because optimal antitumor activity requires maintaining the optimal dose, it is essential to avoid unintended treatment delays or interruptions. Methods. We review the reported prevalence and appearance of OAEs with TKIs and mTORIs and the current oral assessment tools commonly used in clinical trials. We discuss the correlations between OAEs and hand–foot skin reaction (HFSR) and rash. Results. The reported prevalence of oral mucositis/stomatitis of any grade is 4% for pazopanib, 28% for sorafenib, 38% for sunitinib, 41% for temsirolimus, and 44% for everolimus. Oral lesions associated with these agents have been reported to more closely resemble aphthous stomatitis than OM caused by conventional agents. In addition, these agents may result in symptoms such as oral mucosal pain, dysgeusia, and dysphagia, in the absence of clinical lesions. Because of these factors, OAEs secondary to targeted agents may be underreported. In addition, a correlation between OAEs and HFSR was identified. Conclusions. OAEs caused by TKIs and mTORIs may represent dose-limiting toxicities, especially considering the fact that even low grades of OAEs may be troubling to the patient. We discuss how these novel AEs can be assessed because current mucositis assessment tools have limitations. Prospective studies investigating the pathogenesis, risk factors, and management of OAEs are needed in order to minimize the impact on patient's health-related quality of life. PMID:22207567

  15. Mammalian target of rapamycin complex 1 activation is required for the stimulation of human skeletal muscle protein synthesis by essential amino acids.

    PubMed

    Dickinson, Jared M; Fry, Christopher S; Drummond, Micah J; Gundermann, David M; Walker, Dillon K; Glynn, Erin L; Timmerman, Kyle L; Dhanani, Shaheen; Volpi, Elena; Rasmussen, Blake B

    2011-05-01

    The relationship between mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis during instances of amino acid surplus in humans is based solely on correlational data. Therefore, the goal of this study was to use a mechanistic approach specifically designed to determine whether increased mTORC1 activation is requisite for the stimulation of muscle protein synthesis following L-essential amino acid (EAA) ingestion in humans. Examination of muscle protein synthesis and signaling were performed on vastus lateralis muscle biopsies obtained from 8 young (25 ± 2 y) individuals who were studied prior to and following ingestion of 10 g of EAA during 2 separate trials in a randomized, counterbalanced design. The trials were identical except during 1 trial, participants were administered a single oral dose of a potent mTORC1 inhibitor (rapamycin) prior to EAA ingestion. In response to EAA ingestion, an ~60% increase in muscle protein synthesis was observed during the control trial, concomitant with increased phosphorylation of mTOR (Ser(2448)), ribosomal S6 kinase 1 (Thr(389)), and eukaryotic initiation factor 4E binding protein 1 (Thr(37/46)). In contrast, prior administration of rapamycin completely blocked the increase in muscle protein synthesis and blocked or attenuated activation of mTORC1-signaling proteins. The inhibition of muscle protein synthesis and signaling was not due to differences in either extracellular or intracellular amino acid availability, because these variables were similar between trials. These data support a fundamental role for mTORC1 activation as a key regulator of human muscle protein synthesis in response to increased EAA availability. This information will be useful in the development of evidence-based nutritional therapies targeting mTORC1 to counteract muscle wasting associated with numerous clinical conditions.

  16. The role of the PI3K/Akt/mTOR signalling pathway in human cancers induced by infection with human papillomaviruses.

    PubMed

    Zhang, Lifang; Wu, Jianhong; Ling, Ming Tat; Zhao, Liang; Zhao, Kong-Nan

    2015-04-17

    Infection with Human papillomaviruses (HPVs) leads to the development of a wide-range of cancers, accounting for 5% of all human cancers. A prominent example is cervical cancer, one of the leading causes of cancer death in women worldwide. It has been well established that tumor development and progression induced by HPV infection is driven by the sustained expression of two oncogenes E6 and E7. The expression of E6 and E7 not only inhibits the tumor suppressors p53 and Rb, but also alters additional signalling pathways that may be equally important for transformation. Among these pathways, the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signalling cascade plays a very important role in HPV-induced carcinogenesis by acting through multiple cellular and molecular events. In this review, we summarize the frequent amplification of PI3K/Akt/mTOR signals in HPV-induced cancers and discuss how HPV oncogenes E6/E7/E5 activate the PI3K/Akt/mTOR signalling pathway to modulate tumor initiation and progression and affect patient outcome. Improvement of our understanding of the mechanism by which the PI3K/Akt/mTOR signalling pathway contributes to the immortalization and carcinogenesis of HPV-transduced cells will assist in devising novel strategies for preventing and treating HPV-induced cancers.

  17. The Prolyl Peptidases PRCP/PREP Regulate IRS-1 Stability Critical for Rapamycin-induced Feedback Activation of PI3K and AKT*

    PubMed Central

    Duan, Lei; Ying, Guoguang; Danzer, Brian; Perez, Ricardo E.; Shariat-Madar, Zia; Levenson, Victor V.; Maki, Carl G.

    2014-01-01

    The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/mammalian target of rapamycin (mTOR) pathway conveys signals from receptor tyrosine kinases (RTKs) to regulate cell metabolism, proliferation, survival, and motility. Previously we found that prolylcarboxypeptidase (PRCP) regulate proliferation and survival in breast cancer cells. In this study, we found that PRCP and the related family member prolylendopeptidase (PREP) are essential for proliferation and survival of pancreatic cancer cells. Depletion/inhibition of PRCP and PREP-induced serine phosphorylation and degradation of IRS-1, leading to inactivation of the cellular PI3K and AKT. Notably, depletion/inhibition of PRCP/PREP destabilized IRS-1 in the cells treated with rapamycin, blocking the feedback activation PI3K/AKT. Consequently, inhibition of PRCP/PREP enhanced rapamycin-induced cytotoxicity. Thus, we have identified PRCP and PREP as a stabilizer of IRS-1 which is critical for PI3K/AKT/mTOR signaling in pancreatic cancer cells. PMID:24936056

  18. Fibroblast growth factor 2 induces E-cadherin down-regulation via PI3K/Akt/mTOR and MAPK/ERK signaling in ovarian cancer cells.

    PubMed

    Lau, Man-Tat; So, Wai-Kin; Leung, Peter C K

    2013-01-01

    Fibroblast growth factor 2 (FGF2) is produced by ovarian cancer cells and it has been suggested to play an important role in tumor progression. In this study, we report that FGF2 treatment down-regulated E-cadherin by up-regulating its transcriptional repressors, Slug and ZEB1, in human ovarian cancer cells. The pharmacological inhibition of phosphatidylinositol-3-kinase (PI3K), mammalian target of rapamycin (mTOR), and MEK suggests that both PI3K/Akt/mTOR and MAPK/ERK signaling are required for FGF2-induced E-cadherin down-regulation. Moreover, FGF2 up-regulated Slug and ZEB1 expression via the PI3K/Akt/mTOR and MAPK/ERK signaling pathways, respectively. Finally, FGF2-induced cell invasion was abolished by the inhibition of the PI3K/Akt/mTOR and MAPK/ERK pathways, and the forced expression of E-cadherin diminished the intrinsic invasiveness of ovarian cancer cells as well as the FGF2-induced cell invasion. This study demonstrates a novel mechanism in which FGF2 down-regulates E-cadherin expression through the activation of PI3K/Akt/mTOR and MAPK/ERK signaling, and the up-regulation of Slug and ZEB1 in human ovarian cancer cells.

  19. Clinical potential of novel therapeutic targets in breast cancer: CDK4/6, Src, JAK/STAT, PARP, HDAC, and PI3K/AKT/mTOR pathways

    PubMed Central

    Hosford, Sarah R; Miller, Todd W

    2014-01-01

    Breast cancers expressing estrogen receptor α, progesterone receptor, or the human epidermal growth factor receptor 2 (HER2) proto-oncogene account for approximately 90% of cases, and treatment with antiestrogens and HER2-targeted agents has resulted in drastically improved survival in many of these patients. However, de novo or acquired resistance to antiestrogen and HER2-targeted therapies is common, and many tumors will recur or progress despite these treatments. Additionally, the remaining 10% of breast tumors are negative for estrogen receptor α, progesterone receptor, and HER2 (“triple-negative”), and a clinically proven tumor-specific drug target for this group has not yet been identified. Therefore, the identification of new therapeutic targets in breast cancer is of vital clinical importance. Preclinical studies elucidating the mechanisms driving resistance to standard therapies have identified promising targets including cyclin-dependent kinase 4/6, phosphoinositide 3-kinase, poly adenosine diphosphate–ribose polymerase, Src, and histone deacetylase. Herein, we discuss the clinical potential and status of new therapeutic targets in breast cancer. PMID:25206307

  20. Akt stimulates hepatic SREBP1c and lipogenesis through parallel mTORC1-dependent and independent pathways

    PubMed Central

    Yecies, Jessica L.; Zhang, Hui H.; Menon, Suchithra; Liu, Sihao; Yecies, Derek; Lipovsky, Alex I.; Gorgun, Cem; Kwiatkowski, David J.; Hotamisligil, Gökhan S.; Lee, Chih-Hao; Manning, Brendan D.

    2011-01-01

    Through unknown mechanisms, insulin activates the sterol regulatory element-binding protein (SREBP1c) transcription factor to promote hepatic lipogenesis. We find that this induction is dependent on the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). To further define the role of mTORC1 in the regulation of SREBP1c in the liver, we generated mice with liver-specific deletion of TSC1 (LTsc1KO), which results in insulin-independent activation of mTORC1. Surprisingly, the LTsc1KO mice are protected from age- and diet-induced hepatic steatosis and display hepatocyte-intrinsic defects in SREBP1c activation and de novo lipogenesis. These phenotypes result from attenuation of Akt signaling driven by mTORC1-dependent insulin resistance. Therefore, mTORC1 activation is not sufficient to stimulate hepatic SREBP1c in the absence of Akt signaling, revealing the existence of an additional downstream pathway also required for this induction. We provide evidence that this mTORC1-independent pathway involves Akt-mediated suppression of Insig2a, a liver-specific transcript encoding the SREBP1c inhibitor INSIG2. PMID:21723501

  1. The Transcription Factor p8 Regulates Autophagy in Response to Palmitic Acid Stress via a Mammalian Target of Rapamycin (mTOR)-independent Signaling Pathway.

    PubMed

    Jia, Sheng-Nan; Lin, Cheng; Chen, Dian-Fu; Li, An-Qi; Dai, Li; Zhang, Li; Zhao, Ling-Ling; Yang, Jin-Shu; Yang, Fan; Yang, Wei-Jun

    2016-02-26

    Autophagy is an evolutionarily conserved degradative process that allows cells to maintain homoeostasis in numerous physiological situations. This process also functions as an essential protective response to endoplasmic reticulum (ER) stress, which promotes the removal and degradation of unfolded proteins. However, little is known regarding the mechanism by which autophagy is initiated and regulated in response to ER stress. In this study, different types of autophagy were identified in human gastric cancer MKN45 cells in response to the stress induced by nutrient starvation or lipotoxicity in which the regulation of these pathways is mammalian target of rapamycin (mTOR)-dependent or -independent, respectively. Interestingly, we found that p8, a stress-inducible transcription factor, was enhanced in MKN45 cells treated with palmitic acid to induce lipotoxicity. Furthermore, an increase in autophagy was observed in MKN45 cells stably overexpressing p8 using a lentivirus system, and autophagy induced by palmitic acid was blocked by p8 RNAi compared with the control. Western blotting analyses showed that autophagy was regulated by p8 or mTOR in response to the protein kinase-like endoplasmic reticulum kinase/activating transcription factor 6-mediated ER stress of lipotoxicity or the parkin-mediated mitochondrial stress of nutrient starvation, respectively. Furthermore, our results indicated that autophagy induced by palmitic acid is mTOR-independent, but this autophagy pathway was regulated by p8 via p53- and PKCα-mediated signaling in MKN45 cells. Our findings provide insights into the role of p8 in regulating autophagy induced by the lipotoxic effects of excess fat accumulation in cells.

  2. Mammalian target of rapamycin/p70 ribosomal S6 protein kinase signaling is altered by sevoflurane and/or surgery in aged rats.

    PubMed

    Liu, Yongzhe; Ma, Li; Jiao, Linbo; Gao, Minglong; Guo, Wenzhi; Chen, Lin; Pan, Ningling; Ma, Yaqun

    2015-12-01

    The mammalian target of rapamycin (mTOR)/p70 ribosomal S6 protein kinase (p70S6k) pathway exerts anti‑apoptotic effects that may contribute to disease pathogenesis. The memory impairment in patients with Alzheimer's disease (AD) has been suggested to be contributed to by abnormal mTOR signaling. The aim of the current study was to investigate the association between sevoflurane and/or surgery and AD through the mTOR/p70S6K signaling pathway. Sprague‑Dawley rats were randomly assigned to the sevoflurane, surgery or control groups. The animals in the surgery group received a partial hepatectomy under sevoflurane anesthesia. The hippocampal levels of phosphorylated (p)‑mTOR, p‑p70S6K, caspase‑3 and p‑tau/total (t)‑tau were analyzed. The Morris water maze (MWM) was used to evaluate cognitive function following treatment. The levels of p‑mTOR and p‑p70S6K were reduced, whereas caspase‑3 levels were increased in the surgery group compared with the sevoflurane group. The p‑tau/t‑tau levels were increased, however, tau mRNA was unaffected by sevoflurane and/or surgery. The rats in the surgery group required a significantly longer time to locate the platform in the MWM test compared with the control and sevoflurane groups. Sevoflurane treatment and/or surgery reduced anti‑apoptotic activity, and the postoperative cognitive dysfunction following surgery may be due to mTOR signaling pathway inhibition in aged rats. Increased neuronal apoptosis and tau phosphorylation are suggested to be involved in the association between anesthesia and AD occurrence.

  3. Peroxisome proliferator-activated receptor-γ agonist inhibits the mammalian target of rapamycin signaling pathway and has a protective effect in a rat model of status epilepticus

    PubMed Central

    SAN, YONG-ZHI; LIU, YU; ZHANG, YU; SHI, PING-PING; ZHU, YU-LAN

    2015-01-01

    Peroxisome proliferator-activated receptor γ (PPAR-γ) has a protective role in several neurological diseases. The present study investigated the effect of the PPAR-γ agonist, pioglitazone, on the mammalian target of rapamycin (mTOR) signaling pathway in a rat model of pentylenetetrazol (PTZ)-induced status epilepticus (SE). The investigation proceeded in two stages. First, the course of activation of the mTOR signaling pathway in PTZ-induced SE was examined to determine the time-point of peak activity, as reflected by phopshorylated (p)-mTOR/mTOR and p-S6/S6 ratios. Subsequently, pioglitazone was administrated intragastrically to investigate its effect on the mTOR signaling pathway, through western blot and immunochemical analyses. The levels of the interleukin (IL)-1β and IL-6 inflammatory cytokines were detected using ELISA, and neuronal loss was observed via Nissl staining. In the first stage of experimentation, the mTOR signaling pathway was activated, and the p-mTOR/mTOR and p-S6/S6 ratios peaked on the third day. Compared with the vehicle treated-SE group, pretreatment with pioglitazone was associated with the loss of fewer neurons, lower levels of IL-1β and IL-6, and inhibition of the activation of the mTOR signaling pathway. Therefore, the mTOR signaling pathway was activated in the PTZ-induced SE rat model, and the PPAR-γ agonist, pioglitazone, had a neuroprotective effect, by inhibiting activation of the mTOR pathway and preventing the increase in the levels of IL-1β and IL-6. PMID:25891824

  4. Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways*

    PubMed Central

    Agarwal, Swati; Tiwari, Shashi Kant; Seth, Brashket; Yadav, Anuradha; Singh, Anshuman; Mudawal, Anubha; Chauhan, Lalit Kumar Singh; Gupta, Shailendra Kumar; Choubey, Vinay; Tripathi, Anurag; Kumar, Amit; Ray, Ratan Singh; Shukla, Shubha; Parmar, Devendra; Chaturvedi, Rajnish Kumar

    2015-01-01

    The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A1 increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A1) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell's compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be

  5. Mammalian target of rapamycin/eukaryotic initiation factor 4F pathway regulates follicle growth and development of theca cells in mice.

    PubMed

    Zhang, Chao; Liu, Xiao-Ran; Cao, Yong-Chun; Tian, Jin-Ling; Zhen, Di; Luo, Xiao-Fei; Wang, Xin-Mei; Tian, Jian-Hui; Gao, Jian-Ming

    2016-01-11

    The aim of the present study was to clarify the roles of the mammalian target of rapamycin (mTOR) signalling pathway in follicular growth and development of thecal cells. Using in vivo-grown and in vitro-cultured ovaries, histological changes were evaluated using haematoxylin and eosin (HE) staining. Differentially expressed genes (DEGs) from 0 day post partum (d.p.p.) to 8 d.p.p. ovaries were screened by microarray and verified by quantitative real-time polymerase chain reaction. Forty-two DEGs related to cell proliferation and differentiation were screened out, with most DEGs being related to the to mTOR signalling pathway. Then, 3 d.p.p. ovaries were retrieved and used to verify the role of mTOR signalling in follicle and thecal cell development using its activators (Ras homologue enriched in brain (Rheb) and GTP) and inhibitor (rapamycin). The development of follicles and thecal cells was significantly impaired in ovaries cultured in vitro Day 3 to Day 8. In in vitro-cultured ovaries, Rheb and GTP (is 100 ng mL-1 Rheb and 500 ng mL-1 GTP for 48 h) significantly increased follicle diameter, the percentage of primary and secondary follicles and the umber of thecal cells, and upregulated expression of mTOR, phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), eukaryotic initiation factor (eIF) 4F and cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1). Rapamycin (10 nM rapamycin for 24 h) had opposite effects to those of Rheb and GTP, and partly abrogated (significant) the effects of Rheb and GTP when added to the culture in combination with these drugs. Thus, mTOR signalling plays an important role in follicle growth and thecal cell development.

  6. The role of diacylglycerol kinase ζ and phosphatidic acid in the mechanical activation of mammalian target of rapamycin (mTOR) signaling and skeletal muscle hypertrophy.

    PubMed

    You, Jae-Sung; Lincoln, Hannah C; Kim, Chan-Ran; Frey, John W; Goodman, Craig A; Zhong, Xiao-Ping; Hornberger, Troy A

    2014-01-17

    The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.

  7. Ketamine Exhibits Different Neuroanatomical Profile After Mammalian Target of Rapamycin Inhibition in the Prefrontal Cortex: the Role of Inflammation and Oxidative Stress.

    PubMed

    Abelaira, Helena M; Réus, Gislaine Z; Ignácio, Zuleide M; Dos Santos, Maria Augusta B; de Moura, Airam B; Matos, Danyela; Demo, Júlia P; da Silva, Júlia B I; Danielski, Lucineia G; Petronilho, Fabricia; Carvalho, André F; Quevedo, João

    2016-09-02

    Studies indicated that mammalian target of rapamycin (mTOR), oxidative stress, and inflammation are involved in the pathophysiology of major depressive disorder (MDD). Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been identified as a novel MDD therapy; however, the antidepressant mechanism is not fully understood. In addition, the effects of ketamine after mTOR inhibition have not been fully investigated. In the present study, we examined the behavioral and biochemical effects of ketamine in the prefrontal cortex (PFC), hippocampus, amygdala, and nucleus accumbens after inhibition of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2 nmol) or vehicle into the PFC and then a single dose of ketamine (15 mg/kg, i.p.). Immobility was assessed in forced swimming tests, and then oxidative stress parameters and inflammatory markers were evaluated in the brain and periphery. mTOR activation in the PFC was essential to ketamine's antidepressant-like effects. Ketamine increased lipid damage in the PFC, hippocampus, and amygdala. Protein carbonyl was elevated in the PFC, amygdala, and NAc after ketamine administration. Ketamine also increased nitrite/nitrate in the PFC, hippocampus, amygdala, and NAc. Myeloperoxidase activity increased in the hippocampus and NAc after ketamine administration. The activities of superoxide dismutase and catalase were reduced after ketamine administration in all brain areas studied. Inhibition of mTOR signaling pathways by rapamycin in the PFC was required to protect against oxidative stress by reducing damage and increasing antioxidant enzymes. Finally, the TNF-α level was increased in serum by ketamine; however, the rapamycin plus treatment group was not able to block this increase. Activation of mTOR in the PFC is involved in the antidepressant-like effects of ketamine; however, the inhibition of this pathway was able to protect certain brain areas against

  8. Mammalian target of rapamycin inhibitor-associated stomatitis in hematopoietic stem cell transplantation patients receiving sirolimus prophylaxis for graft-versus-host disease.

    PubMed

    Villa, Alessandro; Aboalela, Ali; Luskin, Katharine A; Cutler, Corey S; Sonis, Stephen T; Woo, Sook Bin; Peterson, Douglas E; Treister, Nathaniel S

    2015-03-01

    The mammalian target of rapamycin (mTOR) inhibitor sirolimus is effective in reducing incidence of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Agents that inhibit the mTOR pathway are known to be associated with significant and potentially dose-limiting toxicities, including stomatitis. The objective of this study was to report the clinical features and management outcomes of sirolimus-associated oral ulcers in the context of post-HSCT prophylaxis of GVHD. Seventeen patients, from a study cohort of 967, who were treated with sirolimus as prophylaxis for GVHD after allogeneic HSCT at the Dana-Farber/Brigham and Women's Cancer Center developed oral ulcers and were referred to the oral medicine clinic for evaluation and treatment over a period of 6 years. Clinical characteristics (appearance, anatomic site, size) and therapeutic outcomes (time to complete resolution) were documented. Median time to onset of oral ulceration was 55 days after allogeneic HSCT (range, 6 to 387 days); 92.9% of ulcers were located on nonkeratinized mucosa, with the ventrolateral tongue the most common site of involvement. Thirteen patients were treated with topical corticosteroid therapy; 12 of these patients also required intralesional corticosteroid injections. Clinical improvement (resolution of the lesions and improvement of symptoms) was noted in all cases, with no reported adverse events. Median time to complete resolution after onset of therapy was 14 days (range, 2 to 70 days). Patients receiving sirolimus for GVHD prophylaxis may develop painful oral ulcerations, which can be effectively managed with topical steroid treatment. Further prospective studies are needed to better elucidate the incidence of this complication, identify risk factors, and evaluate the effectiveness of interventions.

  9. A treadmill exercise reactivates the signaling of the mammalian target of rapamycin (mTor) in the skeletal muscles of starved mice.

    PubMed

    Zheng, Dong-Mei; Bian, Zehua; Furuya, Norihiko; Oliva Trejo, Juan Alejandro; Takeda-Ezaki, Mitsue; Takahashi, Katsuyuki; Hiraoka, Yuka; Mineki, Reiko; Taka, Hikari; Ikeda, Shin-Ichi; Komatsu, Masaaki; Fujimura, Tsutomu; Ueno, Takashi; Ezaki, Junji

    2015-01-02

    It has been well established that a starvation-induced decrease in insulin/IGF-I and serum amino acids effectively suppresses the mammalian target of rapamycin (mTor) signaling to induce autophagy, which is a major degradative cellular pathway in skeletal muscles. In this study, we investigated the systematic effects of exercise on the mTor signaling of skeletal muscles. Wild type C57BL/6J mice were starved for 24h under synchronous autophagy induction conditions. Under these conditions, endogenous LC3-II increased, while both S6-kinse and S6 ribosomal protein were dephosphorylated in the skeletal muscles, which indicated mTor inactivation. Using GFP-LC3 transgenic mice, it was also confirmed that fluorescent GFP-LC3 dots in the skeletal muscles increased, including soleus, plantaris, and gastrocnemius, which clearly showed autophagosomal induction. These starved mice were then subjected to a single bout of running on a treadmill (12m/min, 2h, with a lean of 10 degrees). Surprisingly, biochemical analyses revealed that the exercise elicited a decrease in the LC3-II/LC3-I ratio as well as an inversion from the dephosphorylated state to the rephosphorylated state of S6-kinase and ribosomal S6 in these skeletal muscles. Consistently, the GFP-LC3 dots of the skeletal muscles were diminished immediately after the exercise. These results indicated that exercise suppressed starvation-induced autophagy through a reactivation of mTor signaling in the skeletal muscles of these starved mice.

  10. Targeting glutamine metabolism and the focal adhesion kinase additively inhibits the mammalian target of the rapamycin pathway in spheroid cancer stem-like properties of ovarian clear cell carcinoma in vitro.

    PubMed

    Sato, Masakazu; Kawana, Kei; Adachi, Katsuyuki; Fujimoto, Asaha; Yoshida, Mitsuyo; Nakamura, Hiroe; Nishida, Haruka; Inoue, Tomoko; Taguchi, Ayumi; Ogishima, Juri; Eguchi, Satoko; Yamashita, Aki; Tomio, Kensuke; Wada-Hiraike, Osamu; Oda, Katsutoshi; Nagamatsu, Takeshi; Osuga, Yutaka; Fujii, Tomoyuki

    2017-04-01

    Ovarian cancer is one of the leading causes of death in the world, which is linked to its resistance to chemotherapy. Strategies to overcome chemoresistance have been keenly investigated. Culturing cancer cells in suspension, which results in formation of spheroids, is a more accurate reflection of clinical cancer behavior in vitro than conventional adherent cultures. By performing RNA-seq analysis, we found that the focal adhesion pathway was essential in spheroids. The phosphorylation of focal adhesion kinase (FAK) was increased in spheroids compared to adherent cells, and inhibition of FAK in spheroids resulted in inhibition of the downstream mammalian target of the rapamycin (mTOR) pathway in ovarian clear cell carcinomas. This result also suggested that only using a FAK inhibitor might have limitations because the phosphorylation level of FAK could not be reduced to the level in adherent cells, and it appeared that some combination therapies might be necessary. We previously reported that glutamine and glutamate concentrations were higher in spheroids than adherent cells, and we investigated a synergistic effect targeting glutamine metabolism with FAK inhibition on the mTOR pathway. The combination of AOA, a pan-transaminase inhibitor, and PF 573228, a FAK inhibitor, additively inhibited the mTOR pathway in spheroids from ovarian clear cell carcinomas. Our in vitro study proposed a rationale for the positive and negative effects of using FAK inhibitors in ovarian clear cell carcinomas and suggested that targeting glutamine metabolism could overcome the limitation of FAK inhibitors by additively inhibiting the mTOR pathway.

  11. Notoginsenoside R1 attenuates glucose-induced podocyte injury via the inhibition of apoptosis and the activation of autophagy through the PI3K/Akt/mTOR signaling pathway

    PubMed Central

    Huang, Guodong; Zou, Bingyu; Lv, Jianzhen; Li, Tongyu; Huai, Guoli; Xiang, Shaowei; Lu, Shilong; Luo, Huan; Zhang, Yaping; Jin, Yi; Wang, Yi

    2017-01-01

    Injury to terminally differentiated podocytes contributes ignificantly to proteinuria and glomerulosclerosis. The aim of this study was to examine the protective effects of notoginsenoside R1 (NR1) on the maintenance of podocyte number and foot process architecture via the inhibition of apoptosis, the induction of autophagy and the maintenance pf podocyte biology in target cells. The effects of NR1 on conditionally immortalized human podocytes under high glucose conditions were evaluated by determining the percentage apoptosis, the percentage autophagy and the expression levels of slit diaphragm proteins. Our results revealed that NR1 protected the podocytes against high glucose-induced injury by decreasing apoptosis, increasing autophagy and by promoting cytoskeletal recovery. The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was further investigated in order to elucidate the mechanisms responsible for the protective effects of NR1 on podocytes. Our data indicated that treatment with NR increased the phosphorylation levels of PI3K, Akt and mTOR, leading to the activation of the PI3K/Akt/mTOR signaling pathway in podocytes. To the best of our knowledge, this is the first in vitro study to demonstrate that NR1 protects podocytes by activating the PI3K/Akt/mTOR pathway. PMID:28112381

  12. Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

    SciTech Connect

    Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.; Burgos, Patricia V.; Villanueva, Carolina I.; Figueroa, Carlos D.; González, Carlos B.

    2013-11-29

    Highlights: •AVP induces mTOR phosphorylation in A-10 cells cultured in high glucose concentration. •The mTOR phosphorylation is mediated by the PI3K/Akt pathway activation. •The AVP-induced mTOR phosphorylation inhibited autophagy and stimulated cell proliferation. -- Abstract: Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. The AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.

  13. Essential role of AKT in tumor cells addicted to FGFR.

    PubMed

    Hu, Yi; Lu, Huiru; Zhang, Jinchao; Chen, Jun; Chai, Zhifang; Zhang, Jingxin

    2014-02-01

    Tumor cells with genetic amplifications or mutations in the fibroblast growth factor receptor (FGFR) family are often addicted to FGFR and heavily dependent on its signaling to survive. Although it is critical to understand which signaling pathway downstream of FGFR plays an essential role to guide the research and development of FGFR inhibitors, it has remained unclear partly because the tool compounds used in the literature also hit many other kinases, making the results difficult to interpret. With the development of a potent FGFR-specific inhibitor, BGJ398, we are now able to dissect various pathways with low drug concentrations to minimize multiple-target effects. Importantly, here, we show that inhibition of FGFR signaling by BGJ398 leads to only transient inhibition of ERK1/2 phosphorylation, whereas the inhibitory effect on AKT phosphorylation is sustainable, indicating that AKT, not ERK as commonly believed, serves as an appropriate pharmacodynamic biomarker for BGJ398. Although AKT inhibition by a pan-PI3K inhibitor alone has almost no effect on cell growth, heterologous expression of myr-AKT, an active form of AKT, rescues BGJ398-mediated suppression of tumor cell proliferation. These results indicate that AKT is an essential component downstream of FGFR. Finally, combination of the FGFR inhibitor BGJ398 with rapamycin significantly inhibits AKT phosphorylation and enhances their antiproliferative effects in FGFR-addicted cells, suggesting an effective combination strategy for clinical development of FGFR inhibitors.

  14. Gremlin promotes retinal pigmentation epithelial (RPE) cell proliferation, migration and VEGF production via activating VEGFR2-Akt-mTORC2 signaling

    PubMed Central

    Liu, Yuan; Chen, Zhijun; Cheng, Haixia; Chen, Juan; Qian, Jing

    2017-01-01

    Retinopathy of prematurity (ROP) is characterized by late-phase pathologic retinal vasoproliferation. Gremlin is a novel vascular endothelial growth factors (VEGF) receptor 2 (VEGFR2) agonist and promotes angiogenic response. We demonstrated that gremlin expression was significantly increased in retinas of ROP model mice, which was correlated with VEGF upregulation. In retinal pigmentation epithelial (RPE) cells, gremlin activated VEGFR2-Akt-mTORC2 (mammalian target of rapamycin complex 2) signaling, and promoted cell proliferation, migration and VEGF production. VEGFR inhibition (by SU5416) or shRNA knockdown almost abolished gremlin-mediated pleiotropic functions in RPE cells. Further, pharmacological inhibition of Akt-mTOR, or shRNA knockdown of key mTORC2 component (Rictor or Sin1) also attenuated gremlin-exerted activities in RPE cells. We conclude that gremlin promotes RPE cell proliferation, migration and VEGF production possibly via activating VEGFR2-Akt-mTORC2 signaling. Gremlin could be a novel therapeutic target of ROP or other retinal vasoproliferation diseases. PMID:27894090

  15. Histone Deacetylase Inhibitors Inhibit the Proliferation of Gallbladder Carcinoma Cells by Suppressing AKT/mTOR Signaling.

    PubMed

    Zhang, Peng; Guo, Zhiyong; Wu, Ying; Hu, Ronglin; Du, Jun; He, Xiaoshun; Jiao, Xingyuan; Zhu, Xiaofeng

    2015-01-01

    Gallbladder carcinoma is an aggressive malignancy with high mortality mainly due to the limited potential for curative resection and its resistance to chemotherapeutic agents. Here, we show that the histone deacetylase inhibitors (HDACIs) trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA) reduce the proliferation and induce apoptosis of gallbladder carcinoma cells by suppressing the AKT/mammalian target of rapamycin (mTOR) signaling. Gallbladder carcinoma SGC-996 cells were treated with different concentrations of TSA and SAHA for different lengths of time. Cell proliferation and morphology were assessed with MTT assay and microscopy, respectively. Cell cycle distribution and cell apoptosis were analyzed with flow cytometry. Western blotting was used to detect the proteins related to apoptosis, cell cycle, and the AKT/mTOR signaling pathway. Our data showed that TSA and SAHA reduced SGC-996 cell viability and arrested cell cycle at the G1 phase in a dose- and time-dependent manner. TSA and SAHA promoted apoptosis of SGC-996 cells, down-regulated the expression of cyclin D1, c-Myc and Bmi1, and decreased the phosphorylation of AKT, mTOR p70S6K1, S6 and 4E-BP1. Additionally, the mTOR inhibitor rapamycin further reduced the cell viability of TSA- and SAHA-treated SGC-996 cells and the phosphorylation of mTOR, whereas the mTOR activator 1,2-dioctanoyl-sn-glycero-3-phosphate (C8-PA) exerted the opposite influence. Our results demonstrate that histone deacetylase inhibitors (HDACIs) suppress the proliferation of gallbladder carcinoma cell via inhibition of AKT/mTOR signaling. These findings offer a mechanistic rationale for the application of HDACIs in gallbladder carcinoma treatment.

  16. Melatonin prevents hemorrhagic shock-induced liver injury in rats through an Akt-dependent HO-1 pathway.

    PubMed

    Hsu, Jun-Te; Kuo, Chia-Jung; Chen, Tsung-Hsing; Wang, Frank; Lin, Chun-Jun; Yeh, Ta-Sen; Hwang, Tsann-Long; Jan, Yi-Yin

    2012-11-01

    Although melatonin treatment following trauma-hemorrhage or ischemic reperfusion prevents organs from dysfunction and injury, the precise mechanism remains unknown. This study tested whether melatonin prevents liver injury following trauma-hemorrhage involved the protein kinase B (Akt)-dependent heme oxygenase (HO)-1 pathway. After a 5-cm midline laparotomy, male rats underwent hemorrhagic shock (mean blood pressure approximately 40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, melatonin (2 mg/kg), or melatonin plus phosphoinositide 3-kinase (PI3K) inhibitor wortmannin (1 mg/kg). At 2 hr after trauma-hemorrhage, the liver tissue myeloperoxidase activity, malondialdehyde, adenosine triphosphate, serum alanine aminotransferase, and aspartate aminotransferase levels were significantly increased compared with sham-operated control. Trauma-hemorrhage resulted in a significant decrease in the Akt activation in comparison with the shams (relative density, 0.526 ± 0.031 versus 1.012 ± 0.066). Administration of melatonin following trauma-hemorrhage normalized liver Akt phosphorylation (0.993 ± 0.061), further increased mammalian target of rapamycin (mTOR) activation (5.263 ± 0.338 versus 2.556 ± 0.225) and HO-1 expression (5.285 ± 0.325 versus 2.546 ± 0.262), and reduced cleaved caspase-3 levels (2.155 ± 0.297 versus 5.166 ± 0.309). Coadministration of wortmannin abolished the melatonin-mediated attenuation of the shock-induced liver injury markers. Our results collectively suggest that melatonin prevents hemorrhagic shock-induced liver injury in rats through an Akt-dependent HO-1 pathway.

  17. SMYD3-mediated lysine methylation in the PH domain is critical for activation of AKT1

    PubMed Central

    Yoshioka, Yuichiro; Suzuki, Takehiro; Matsuo, Yo; Nakakido, Makoto; Tsurita, Giichiro; Simone, Cristiano; Watanabe, Toshiaki; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-01-01

    AKT1 is a cytosolic serine/threonine kinase that is overexpressed in various types of cancer and has a central role in human tumorigenesis. Although it is known that AKT1 is post-translationally modified in various ways including phosphorylation and ubiquitination, methylation has not been reported so far. Here we demonstrate that the protein lysine methyltransferase SMYD3 methylates lysine 14 in the PH domain of AKT1 both in vitro and in vivo. Lysine 14-substituted AKT1 shows significantly lower levels of phosphorylation at threonine 308 than wild-type AKT1, and knockdown of SMYD3 as well as treatment with a SMYD3 inhibitor significantly attenuates this phosphorylation in cancer cells. Furthermore, substitution of lysine 14 diminishes the plasma membrane accumulation of AKT1, and cancer cells overexpressing lysine 14-substiuted AKT1 shows lower growth rate than those overexpressing wild-type AKT1. These results imply that SMYD3-mediated methylation of AKT1 at lysine 14 is essential for AKT1 activation and that SMYD3-mediated AKT1 methylation appears to be a good target for development of anti-cancer therapy. PMID:27626683

  18. Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity.

    PubMed

    Oeck, S; Al-Refae, K; Riffkin, H; Wiel, G; Handrick, R; Klein, D; Iliakis, G; Jendrossek, V

    2017-02-17

    The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition.

  19. Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity

    PubMed Central

    Oeck, S.; Al-Refae, K.; Riffkin, H.; Wiel, G.; Handrick, R.; Klein, D.; Iliakis, G.; Jendrossek, V.

    2017-01-01

    The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition. PMID:28209968

  20. PDGF inactivates forkhead family transcription factor by activation of Akt in glomerular mesangial cells.

    PubMed

    Ghosh Choudhury, Goutam; Lenin, Mahimainathan; Calhaun, Cheresa; Zhang, Jian-Hua; Abboud, Hanna E

    2003-02-01

    Regulation of the forkhead domain transcription factors by PDGF has not been studied. In this report, we investigated the role of PDGF-induced Akt in regulating forkhead domain protein FKHRL1 in glomerular mesangial cells. PDGF increased phosphorylation of FKHRL1 in a time- and PI 3 kinase-dependent manner. Expression of dominant negative Akt by adenovirus-mediated gene transfer blocked PDGF-induced FKHRL1 phosphorylation. PDGF inhibited transcription of a forkhead DNA binding element-driven reporter gene. This inhibition was mimicked by constitutively active myristoylated Akt. Moreover, FKHR1-mediated transcription of the reporter gene was completely attenuated by both PDGF and Myr-Akt. One of the targets of forkhead transcription factors is the proapoptotic Fas ligand (FasL) gene. PDGF, as well as Myr-Akt, inhibited transcription of FasL. In contrast, inhibition of PI 3 kinase and dominant negative Akt increased FasL gene transcription, suggesting that suppression of PI 3 kinase/Akt signalling may induce apoptosis in mesangial cells via upregulation of FasL expression. However, expression of dominant negative Akt by adenovirus did not induce apoptosis in mesangial cells, suggesting that Akt-independent antiapoptotic mechanisms also exist. Together, our data demonstrate for the first time that PDGF inactivates forkhead domain transcription factor by Akt-dependent phosphorylation and that suppression of Akt signalling is not sufficient to induce apoptosis in mesangial cells.

  1. The underlying mechanism of proinflammatory NF-κB activation by the mTORC2/Akt/IKKα pathway during skin aging

    PubMed Central

    Choi, Yeon Ja; Moon, Kyoung Mi; Chung, Ki Wung; Jeong, Ji Won; Park, Daeui; Kim, Dae Hyun; Yu, Byung Pal; Chung, Hae Young

    2016-01-01

    Mammalian target of rapamycin complex 2 (mTORC2), one of two different enzymatic complexes of mTOR, regulates a diverse set of substrates including Akt. mTOR pathway is one of well-known mediators of aging process, however, its role in skin aging has not been determined. Skin aging can be induced by physical age and ultraviolet (UV) irradiation which are intrinsic and extrinsic factors, respectively. Here, we report increased mTORC2 pathway in intrinsic and photo-induced skin aging, which is implicated in the activation of nuclear factor-κB (NF-κB). UVB-irradiated or aged mice skin revealed that mTORC2 activity and its component, rictor were significantly upregulated which in turn increased Akt activation and Akt-dependent IκB kinase α (IKKα) phosphorylation at Thr23 in vivo. We also confirmed that UVB induced the mTORC2/Akt/IKKα signaling pathway with HaCaT human normal keratinocytes. The increased mTORC2 signaling pathway during skin aging were associated to NF-κB activation. Suppression of mTORC2 activity by the treatment of a mTOR small inhibitor or knockdown of RICTOR partially rescued UVB-induced NF-κB activation through the downregulation of Akt/IKKα activity. Our data demonstrated the upregulation of mTORC2 pathway in intrinsic and photo-induced skin aging and its role in IKKα/NF-κB activation. These data not only expanded the functions of mTOR to skin aging but also revealed the therapeutic potential of inhibiting mTORC2 in ameliorating both intrinsic skin aging and photoaging. PMID:27486771

  2. Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

    PubMed

    Bakker, Astrid D; Gakes, Tom; Hogervorst, Jolanda M A; de Wit, Gerard M J; Klein-Nulend, Jenneke; Jaspers, Richard T

    2016-06-01

    Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts.

  3. Hematopoietic Akt2 deficiency attenuates the progression of atherosclerosis

    PubMed Central

    Rotllan, Noemi; Chamorro-Jorganes, Aránzazu; Araldi, Elisa; Wanschel, Amarylis C.; Aryal, Binod; Aranda, Juan F.; Goedeke, Leigh; Salerno, Alessandro G.; Ramírez, Cristina M.; Sessa, William C.; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-01-01

    Atherosclerosis is the major cause of death and disability in diabetic and obese subjects with insulin resistance. Akt2, a phosphoinositide-dependent serine-threonine protein kinase, is highly express in insulin-responsive tissues; however, its role during the progression of atherosclerosis remains unknown. Thus, we aimed to investigate the contribution of Akt2 during the progression of atherosclerosis. We found that germ-line Akt2-deficient mice develop similar atherosclerotic plaques as wild-type mice despite higher plasma lipids and glucose levels. It is noteworthy that transplantation of bone marrow cells isolated from Akt2−/− mice to Ldlr−/− mice results in marked reduction of the progression of atherosclerosis compared with Ldlr−/− mice transplanted with wild-type bone marrow cells. In vitro studies indicate that Akt2 is required for macrophage migration in response to proatherogenic cytokines (monocyte chemotactic protein-1 and macrophage colony-stimulating factor). Moreover, Akt2−/− macrophages accumulate less cholesterol and have an alternative activated or M2-type phenotype when stimulated with proinflammatory cytokines. Together, these results provide evidence that macrophage Akt2 regulates migration, the inflammatory response and cholesterol metabolism and suggest that targeting Akt2 in macrophages might be beneficial for treating atherosclerosis.—Rotllan, N., Chamorro-Jorganes, A., Araldi, E., Wanschel, A. C., Aryal, B., Aranda, J. F., Goedeke, L., Salerno, A. G., Ramírez, C. M., Sessa,W. C., Suárez, Y., Fernández-Hernando, C. Hematopoietic Akt2 deficiency attenuates the progression of atherosclerosis. PMID:25392271

  4. Mammalian sleep

    NASA Astrophysics Data System (ADS)

    Staunton, Hugh

    2005-05-01

    This review examines the biological background to the development of ideas on rapid eye movement sleep (REM sleep), so-called paradoxical sleep (PS), and its relation to dreaming. Aspects of the phenomenon which are discussed include physiological changes and their anatomical location, the effects of total and selective sleep deprivation in the human and animal, and REM sleep behavior disorder, the latter with its clinical manifestations in the human. Although dreaming also occurs in other sleep phases (non-REM or NREM sleep), in the human, there is a contingent relation between REM sleep and dreaming. Thus, REM is taken as a marker for dreaming and as REM is distributed ubiquitously throughout the mammalian class, it is suggested that other mammals also dream. It is suggested that the overall function of REM sleep/dreaming is more important than the content of the individual dream; its function is to place the dreamer protagonist/observer on the topographical world. This has importance for the developing infant who needs to develop a sense of self and separateness from the world which it requires to navigate and from which it is separated for long periods in sleep. Dreaming may also serve to maintain a sense of ‘I’ness or “self” in the adult, in whom a fragility of this faculty is revealed in neurological disorders.

  5. TGF-β effects on prostate cancer cell migration and invasion are mediated by PGE2 through activation of PI3K/AKT/mTOR pathway.

    PubMed

    Vo, Baohan T; Morton, Derrick; Komaragiri, Shravan; Millena, Ana C; Leath, Chelesie; Khan, Shafiq A

    2013-05-01

    TGF-β plays an important role in the progression of prostate cancer. It exhibits both tumor suppressor and tumor-promoting activities. Correlations between cyclooxygenase (COX)-2 overexpression and enhanced production of prostaglandin (PG)E2 have been implicated in cancer progression; however, there are no studies indicating that TGF-β effects in prostate cancer cells involve PGE2 synthesis. In this study, we investigated TGF-β regulation of COX-1 and COX-2 expression in prostate cancer cells and whether the effects of TGF-β on cell proliferation and migration are mediated by PGE2. COX-1 protein was ubiquitously expressed in prostate cells; however, COX-2 protein levels were detected only in prostate cancer cells. TGF-β treatment increased COX-2 protein levels and PGE2 secretion in PC3 cells. Exogenous PGE2 and PGF2α had no effects on cell proliferation in LNCaP, DU145, and PC3 cells whereas PGE2 and TGF-β induced migration and invasive behavior in PC3 cells. Only EP2 and EP4 receptors were detected at mRNA levels in prostate cells. The EP4-targeting small interfering RNA inhibited PGE2 and TGF-β-induced migration of PC3 cells. TGF-β and PGE2 induce activation of PI3K/AKT/mammalian target of rapamycin pathway as indicated by increased AKT, p70S6K, and S6 phosphorylation. Rapamycin completely blocked the effects of TGF-β and PGE2 on phosphorylation of p70S6K and S6 but not on AKT phosphorylation. PGE2 and TGF-β induced phosphorylation of AKT, which was blocked by antagonists of PGE2 (EP4) receptors (L161982, AH23848) and PI3K inhibitor (LY294002) in PC3 cells. Pretreatment with L161982 or AH23848 blocked the stimulatory effects of PGE2 and TGF-β on cell migration, whereas LY294002 or rapamycin completely eliminated PGE2, TGF-β, and epidermal growth factor-induced migration in PC3 cells. We conclude that TGF-β increases COX-2 levels and PGE2 secretion in prostate cancer cells which, in turn, mediate TGF-β effects on cell migration and invasion through

  6. miR-7 reverses the resistance to BRAFi in melanoma by targeting EGFR/IGF-1R/CRAF and inhibiting the MAPK and PI3K/AKT signaling pathways.

    PubMed

    Sun, Xiaoyan; Li, Jun; Sun, Yanhong; Zhang, Yi; Dong, Liyun; Shen, Chen; Yang, Liu; Yang, Ming; Li, Yan; Shen, Guanxin; Tu, Yating; Tao, Juan

    2016-08-16

    MicroRNAs (miRNAs) are attractive therapeutic targets for various therapy-resistant tumors. However, the association between miRNA and BRAF inhibitor resistance in melanoma remains to be elucidated. We used microarray analysis to comprehensively study the miRNA expression profiling of vemurafenib resistant (VemR) A375 melanoma cells in relation to parental A375 melanoma cells. MicroRNA-7 (miR-7) was identified to be the most significantly down-regulated miRNA in VemR A375 melanoma cells. We also found that miR-7 was down-regulated in Mel-CVR cells (vemurafenib resistant Mel-CV melanoma cells). Reestablishment of miR-7 expression could reverse the resistance of both cells to vemurafenib. We showed that epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R) and CRAF were over-expressed in VemR A375 melanoma cells. Introduction of miR-7 mimics could markedly decrease the expressions of EGFR, IGF-1R and CRAF and further suppressed the activation of MAPK and PI3K/AKT pathway in VemR A375 melanoma cells. Furthermore, tumor growth was inhibited in an in vivo murine VemR A375 melanoma tumor model transfected with miR-7 mimics. Collectively, our study demonstrated that miR-7 could reverse the resistance to BRAF inhibitors in certain vemurafenib resistant melanoma cell lines. It could advance the field and provide the basis for further studies in BRAF inhibitor resistance in melanoma.

  7. Inhibition of Adipogenesis by Oligonol through Akt-mTOR Inhibition in 3T3-L1 Adipocytes

    PubMed Central

    Park, Jae-Yeo; Kim, Younghwa; Im, Jee Ae; You, Seungkwon

    2014-01-01

    Polyphenols have recently become an important focus of study in obesity research. Oligonol is an oligomerized polyphenol, typically comprised of catechin-type polyphenols from a variety of fruits, which has been found to exhibit better bioavailability and bioreactivity than natural polyphenol compounds. Here, we demonstrated that Oligonol inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression. During adipogenesis, Oligonol downregulated the mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and δ (C/EBPδ) in a dose-dependent manner and the expression of genes involved in lipid biosynthesis. The antiadipogenic effect of Oligonol appears to originate from its ability to inhibit the Akt and mammalian target of rapamycin (mTOR) signaling pathway by diminishing the phosphorylation of ribosomal protein S6 kinase (p70S6K), a downstream target of mTOR and forkhead box protein O1 (Foxo1). These results suggest that Oligonol may be a potent regulator of obesity by repressing major adipogenic genes through inhibition of the Akt signaling pathway, which induces the inhibition of lipid accumulation, ultimately inhibiting adipogenesis. PMID:25295069

  8. The PI3K/Akt/mTOR signaling pathway mediates insulin-like growth factor 1-induced E-cadherin down-regulation and cell proliferation in ovarian cancer cells.

    PubMed

    Lau, Man-Tat; Leung, Peter C K

    2012-12-30

    Insulin-like growth factor 1 (IGF1) is produced by ovarian cancer cells and it has been suggested that it plays an important role in tumor progression. In this study, we report that IGF1 treatment down-regulated E-cadherin by up-regulating E-cadherin transcriptional repressors, Snail and Slug, in human ovarian cancer cells. The pharmacological inhibition of phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) suggests that PI3K/Akt/mTOR signaling is required for IGF1-induced E-cadherin down-regulation. Moreover, IGF1 up-regulated Snail and Slug expression via the PI3K/Akt/mTOR signaling pathway. Finally, IGF1-induced cell proliferation was abolished by inhibiting PI3K/Akt/mTOR signaling. This study demonstrates a novel mechanism in which IGF1 down-regulates E-cadherin expression through the activation of PI3K/Akt/mTOR signaling and the up-regulation of Snail and Slug in human ovarian cancer cells.

  9. Mammalian Ste20-like kinase 4 promotes pituitary cell proliferation and survival under hypoxia.

    PubMed

    Xiong, Weipeng; Knox, Aaron J; Xu, Mei; Kiseljak-Vassiliades, Katja; Colgan, Sean P; Brodsky, Kelley S; Kleinschmidt-Demasters, Bette K; Lillehei, Kevin O; Wierman, Margaret E

    2015-03-01

    The genetic and molecular mechanisms that initiate and maintain pituitary tumorigenesis are poorly understood. Nonfunctioning tumors of the gonadotrope lineage represent 35% of all tumors; are usually macroadenomas, often resulting in hypopituitarism; and have no medical treatments. Using expression microarrays combined with whole-genome copy number screens on individual human tumors, we identified the mammalian sterile-20-like kinase (MST4) transcript, which was amplified within chromosome Xq26.2 in one tumor and up-regulated in all gonadotrope tumor samples. MST4 mRNA and protein were consistently overexpressed in human tumors compared with normal pituitaries. To mimic the pituitary tumor microenvironment, a hypoxia model using LβT2 murine gonadotrope cells was created to examine the functional role of the kinase. During long-term hypoxia, MST4 expression increased colony formation in a soft agar assay and rates of cell proliferation by activating p38 MAPK and AKT. Under short-term severe hypoxic stress, MST4 decreased the rates of apoptosis via p38 MAPK, AKT, hypoxia-inducible factor-1, and its cell-specific downstream targets. Analysis of MST4 mutants confirmed the importance of the kinase sequence but not the regulatory C terminus for its functional effects. Together these data identify the MST4 kinase as a novel candidate to mediate human pituitary tumorigenesis in a hypoxic environment and position it as a potential therapeutic target.

  10. Mammalian Ste20-Like Kinase 4 Promotes Pituitary Cell Proliferation and Survival Under Hypoxia

    PubMed Central

    Xiong, Weipeng; Knox, Aaron J.; Xu, Mei; Kiseljak-Vassiliades, Katja; Colgan, Sean P.; Brodsky, Kelley S.; Kleinschmidt-Demasters, Bette K.; Lillehei, Kevin O.

    2015-01-01

    The genetic and molecular mechanisms that initiate and maintain pituitary tumorigenesis are poorly understood. Nonfunctioning tumors of the gonadotrope lineage represent 35% of all tumors; are usually macroadenomas, often resulting in hypopituitarism; and have no medical treatments. Using expression microarrays combined with whole-genome copy number screens on individual human tumors, we identified the mammalian sterile-20-like kinase (MST4) transcript, which was amplified within chromosome Xq26.2 in one tumor and up-regulated in all gonadotrope tumor samples. MST4 mRNA and protein were consistently overexpressed in human tumors compared with normal pituitaries. To mimic the pituitary tumor microenvironment, a hypoxia model using LβT2 murine gonadotrope cells was created to examine the functional role of the kinase. During long-term hypoxia, MST4 expression increased colony formation in a soft agar assay and rates of cell proliferation by activating p38 MAPK and AKT. Under short-term severe hypoxic stress, MST4 decreased the rates of apoptosis via p38 MAPK, AKT, hypoxia-inducible factor-1, and its cell-specific downstream targets. Analysis of MST4 mutants confirmed the importance of the kinase sequence but not the regulatory C terminus for its functional effects. Together these data identify the MST4 kinase as a novel candidate to mediate human pituitary tumorigenesis in a hypoxic environment and position it as a potential therapeutic target. PMID:25650755

  11. Novel B55α-PP2A mutations in AML promote AKT T308 phosphorylation and sensitivity to AKT inhibitor-induced growth arrest

    PubMed Central

    Shouse, Geoffrey; de Necochea-Campion, Rosalia; Mirshahidi, Saied; Liu, Xuan; Chen, Chien-Shing

    2016-01-01

    Activation of the Protein Kinase B (PKB), or AKT pathway has been shown to correlate with acute myeloid leukemia (AML) prognosis. B55α-Protein Phosphatase 2A (PP2A) has been shown to dephosphorylate AKT at Thr-308 rendering it inactive. In fact, low expression of the PP2A regulatory subunit B55α was associated with activated phospho-AKT and correlated with inferior outcomes in AML. Despite this fact, no studies have specifically demonstrated a mechanism whereby B55α expression is regulated in AML. In this study, we demonstrate novel loss of function mutations in the PPP2R2A gene identified in leukemic blasts from three AML patients. These mutations eliminate B55α protein expression thereby allowing constitutive AKT activation. In addition, leukemic blasts with PPP2R2A gene mutation were more sensitive to treatment with the AKT inhibitor MK2206, but less responsive to the PP2A activator FTY720. Using leukemia cell lines, we further demonstrate that B55α expression correlates with AKT Thr-308 phosphorylation and predicts responsiveness to AKT inhibition and PP2A activation. Together our data illustrate the importance of the B55α-PP2A-AKT pathway in leukemogenesis. Screening for disruptions in this pathway at initial AML diagnosis may predict response to targeted therapies against AKT and PP2A. PMID:27531894

  12. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  13. The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleated tetraploid liver cells in rodents

    PubMed Central

    Celton-Morizur, Séverine; Merlen, Grégory; Couton, Dominique; Margall-Ducos, Germain; Desdouets, Chantal

    2009-01-01

    The formation of polyploid cells is part of the developmental program of several tissues. During postnatal development, binucleated tetraploid cells arise in the liver, caused by failure in cytokinesis. In this report, we have shown that the initiation of cytokinesis failure events and the subsequent appearance of binucleated tetraploid cells are strictly controlled by the suckling-to-weaning transition in rodents. We found that daily light/dark rhythms and carbohydrate intake did not affect liver tetraploidy. In contrast, impairment of insulin signaling drastically reduced the formation of binucleated tetraploid cells, whereas repeated insulin injections promoted the generation of these liver cells. Furthermore, inhibition of Akt activity decreased the number of cytokinesis failure events, possibly through the mammalian target of rapamycin signaling complex 2 (mTORC2), which indicates that the PI3K/Akt pathway lies downstream of the insulin signal to regulate the tetraploidization process. To our knowledge, these results are the first demonstration in a physiological context that insulin signaling through Akt controls a specific cell division program and leads to the physiologic generation of binucleated tetraploid liver cells. PMID:19603546

  14. Licochalcone A induces autophagy through PI3K/Akt/mTOR inactivation and autophagy suppression enhances Licochalcone A-induced apoptosis of human cervical cancer cells.

    PubMed

    Tsai, Jen-Pi; Lee, Chien-Hsing; Ying, Tsung-Ho; Lin, Chu-Liang; Lin, Chia-Liang; Hsueh, Jung-Tsung; Hsieh, Yi-Hsien

    2015-10-06

    The use of dietary bioactive compounds in chemoprevention can potentially reverse, suppress, or even prevent cancer progression. However, the effects of licochalcone A (LicA) on apoptosis and autophagy in cervical cancer cells have not yet been clearly elucidated. In this study, LicA treatment was found to significantly induce the apoptotic and autophagic capacities of cervical cancer cells in vitro and in vivo. MTT assay results showed dose- and time-dependent cytotoxicity in four cervical cancer cell lines treated with LicA. We found that LicA induced mitochondria-dependent apoptosis in SiHa cells, with decreasing Bcl-2 expression. LicA also induced autophagy effects were examined by identifying accumulation of Atg5, Atg7, Atg12 and microtubule-associated protein 1 light chain 3 (LC3)-II. Treatment with autophagy-specific inhibitors (3-methyladenine and bafilomycin A1) enhanced LicA-induced apoptosis. In addition, we suggested the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of mTOR pathway by LicA. Furthermore, the inhibition of PI3K/Akt by LY294002/si-Akt or of mTOR by rapamycin augmented LicA-induced apoptosis and autophagy. Finally, the in vivo mice bearing a SiHa xenograft, LicA dosed at 10 or 20 mg/kg significantly inhibited tumor growth. Our findings demonstrate the chemotherapeutic potential of LicA for treatment of human cervical cancer.

  15. Modulatory effect of curcumin on survival of irradiated human intestinal microvascular endothelial cells: role of Akt/mTOR and NF-κB

    PubMed Central

    Binion, David G.; Wellner, Michael; Behmaram, Behnaz; Floer, Martin; Mitton, Elizabeth; Nie, Linghui; Zhang, Zhihong; Otterson, Mary F.

    2010-01-01

    Radiation therapy is an essential modality in the treatment of colorectal cancers. Radiation exerts an antiangiogenic effect on tumors, inhibiting endothelial proliferation and survival in the tumor microvasculature. However, damage from low levels of irradiation can induce a paradoxical effect, stimulating survival in endothelial cells. We used human intestinal microvascular endothelial cells (HIMEC) to define effects of radiation on these gut-specific endothelial cells. Low-level irradiation (1–5 Gy) activates NF-κB and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is involved in cell cycle reentry and cell survival in HIMEC. A downstream target of PI3K/Akt is mammalian target of rapamycin (mTOR), which contributes to endothelial proliferation and angiogenesis. The aim of this study was to investigate the signaling molecules involved in the radiosensitizing effects of curcumin on HIMEC subjected to low levels of irradiation. We have demonstrated that exposure of HIMEC to low levels of irradiation induced Akt and mTOR phosphorylation, which was attenuated by curcumin, rapamycin, LY294002, and mTOR small interference RNA (siRNA). Activation of NF-κB by low levels of irradiation was inhibited by curcumin, SN-50, and mTOR siRNA. Curcumin also induced apoptosis by induction of caspase-3 cleavage in irradiated HIMEC. In conclusion, curcumin significantly inhibited NF-κB and attenuated the effect of irradiation-induced prosurvival signaling through the PI3K/Akt/mTOR and NF-κB pathways in these gut-specific endothelial cells. Curcumin may be a potential radiosensitizing agent for enhanced antiangiogenic effect in colorectal cancer radiation therapy. PMID:20299603

  16. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    NASA Astrophysics Data System (ADS)

    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

    2016-04-01

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  17. Phospholipase D2 mediates survival signaling through direct regulation of Akt in glioblastoma cells.

    PubMed

    Bruntz, Ronald C; Taylor, Harry E; Lindsley, Craig W; Brown, H Alex

    2014-01-10

    The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipaseD(PLD) is a novel regulator of Akt inGBM.Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase.

  18. Phospholipase D2 Mediates Survival Signaling through Direct Regulation of Akt in Glioblastoma Cells*♦

    PubMed Central

    Bruntz, Ronald C.; Taylor, Harry E.; Lindsley, Craig W.; Brown, H. Alex

    2014-01-01

    The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipase D (PLD) is a novel regulator of Akt in GBM. Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase. PMID:24257753

  19. MYOCARDIAL AKT: THE OMNIPRESENT NEXUS

    PubMed Central

    Sussman, Mark A.; Völkers, Mirko; Fischer, Kimberlee; Bailey, Brandi; Cottage, Christopher T.; Din, Shabana; Gude, Natalie; Avitabile, Daniele; Alvarez, Roberto; Sundararaman, Balaji; Quijada, Pearl; Mason, Matt; Konstandin, Mathias H.; Malhowski, Amy; Cheng, Zhaokang; Khan, Mohsin; McGregor, Michael

    2013-01-01

    One of the greatest examples of integrated signal transduction is revealed by examination of effects mediated by AKT kinase in myocardial biology. Positioned at the intersection of multiple afferent and efferent signals, AKT exemplifies a molecular sensing node that coordinates dynamic responses of the cell in literally every aspect of biological responses. The balanced and nuanced nature of homeostatic signaling is particularly essential within the myocardial context, where regulation of survival, energy production, contractility, and response to pathological stress all flow through the nexus of AKT activation or repression. Equally important, the loss of regulated AKT activity is primarily the cause or consequence of pathological conditions leading to remodeling of the heart and eventual decompensation. This review presents an overview compendium of the complex world of myocardial AKT biology gleaned from more than a decade of research. Summarization of the widespread influence that AKT exerts upon myocardial responses leaves no doubt that the participation of AKT in molecular signaling will need to be reckoned with as a seemingly omnipresent regulator of myocardial molecular biological responses. PMID:21742795

  20. DMH1 increases glucose metabolism through activating Akt in L6 rat skeletal muscle cells.

    PubMed

    Xie, Xin; Xu, Xiao-Ming; Li, Na; Zhang, Yong-Hui; Zhao, Yu; Ma, Chun-Yan; Dong, De-Li

    2014-01-01

    DMH1(4-[6-(4-Isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl] quinoline) is a compound C analogue with the structural modifications at the 3- and 6-positions in pyrazolo[1,5-a]pyrimidine backbone. Compound C was reported to inhibit both AMPK and Akt. Our preliminary work found that DMH1 activated Akt. Since Akt was involved in glucose metabolism, we aimed to identify the effects of DMH1 on glucose metabolism in L6 rat muscle cells and the potential mechanism. Results showed that DMH1 increased lactic acid release and glucose consumption in L6 rat muscle cells in a dose-dependent manner. DMH1 activated Akt in L6 cells. Akt inhibitor inhibited DMH1-induced Akt activation and DMH1-induced increases of glucose uptake and consumption. DMH1 had no cytotoxicity in L6 cells, but inhibited mitochondrial function and reduced ATP production. DMH1 showed no effect on AMPK, but in the presence of Akt inhibitor, DMH1 significantly activated AMPK. Compound C inhibited DMH1-induced Akt activation in L6 cells. Compound C inhibited DMH1-induced increase of glucose uptake, consumption and lactic acid release in L6 cells. DMH1 inhibited PP2A activity, and PP2A activator forskolin reversed DMH1-induced Akt activation. We concluded that DMH1 increased glucose metabolism through activating Akt and DMH1 activated Akt through inhibiting PP2A activity in L6 rat muscle cells. In view of the analogue structure of DMH1 and compound C and the contrasting effects of DMH1 and compound C on Akt, the present study provides a novel leading chemical structure targeting Akt with potential use for regulating glucose metabolism.

  1. Targeting isoprenylcysteine methylation ameliorates disease in a mouse model of progeria.

    PubMed

    Ibrahim, Mohamed X; Sayin, Volkan I; Akula, Murali K; Liu, Meng; Fong, Loren G; Young, Stephen G; Bergo, Martin O

    2013-06-14

    Several progeroid disorders, including Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (ZMPSTE24 deficiency), arise when a farnesylated and methylated form of prelamin A accumulates at the nuclear envelope. Here, we found that a hypomorphic allele of isoprenylcysteine carboxyl methyltransferase (ICMT) increased body weight, normalized grip strength, and prevented bone fractures and death in Zmpste24-deficient mice. The reduced ICMT activity caused prelamin A mislocalization within the nucleus and triggered prelamin A-dependent activation of AKT-mammalian target of rapamycin (mTOR) signaling, which abolished the premature senescence of Zmpste24-deficient fibroblasts. ICMT inhibition increased AKT-mTOR signaling and proliferation and delayed senescence in human HGPS fibroblasts but did not reduce the levels of misshapen nuclei in mouse and human cells. Thus, targeting ICMT might be useful for treating prelamin A-associated progeroid disorders.

  2. Electroporation into Cultured Mammalian Embryos

    NASA Astrophysics Data System (ADS)

    Nomura, Tadashi; Takahashi, Masanori; Osumi, Noriko

    Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms under lying complex developmental processes (Mak, 2007). However, our understanding of cell—cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, which limit their use.

  3. Akt inhibitors in cancer treatment: The long journey from drug discovery to clinical use (Review)

    PubMed Central

    NITULESCU, GEORGE MIHAI; MARGINA, DENISA; JUZENAS, PETRAS; PENG, QIAN; OLARU, OCTAVIAN TUDOREL; SALOUSTROS, EMMANOUIL; FENGA, CONCETTINA; SPANDIDOS, DEMETRIOS A.; LIBRA, MASSIMO; TSATSAKIS, ARISTIDIS M.

    2016-01-01

    Targeted cancer therapies are used to inhibit the growth, progression, and metastasis of the tumor by interfering with specific molecular targets and are currently the focus of anticancer drug development. Protein kinase B, also known as Akt, plays a central role in many types of cancer and has been validated as a therapeutic target nearly two decades ago. This review summarizes the intracellular functions of Akt as a pivotal point of converging signaling pathways involved in cell growth, proliferation, apoptotis and neo-angiogenesis, and focuses on the drug design strategies to develop potent anticancer agents targeting Akt. The discovery process of Akt inhibitors has evolved from adenosine triphosphate (ATP)-competitive agents to alternative approaches employing allosteric sites in order to overcome the high degree of structural similarity between Akt isoforms in the catalytic domain, and considerable structural analogy to the AGC kinase family. This process has led to the discovery of inhibitors with greater specificity, reduced side-effects and lower toxicity. A second generation of Akt has inhibitors emerged by incorporating a chemically reactive Michael acceptor template to target the nucleophile cysteines in the catalytic activation loop. The review outlines the development of several promising drug candidates emphasizing the importance of each chemical scaffold. We explore the pipeline of Akt inhibitors and their preclinical and clinical examination status, presenting the potential clinical application of these agents as a monotherapy or in combination with ionizing radiation, other targeted therapies, or chemotherapy. PMID:26698230

  4. Pancreatic Endocrine Tumors: Expression Profiling Evidences a Role for AKT-mTOR Pathway

    PubMed Central

    Missiaglia, Edoardo; Dalai, Irene; Barbi, Stefano; Beghelli, Stefania; Falconi, Massimo; della Peruta, Marco; Piemonti, Lorenzo; Capurso, Gabriele; Di Florio, Alessia; delle Fave, Gianfranco; Pederzoli, Paolo; Croce, Carlo M.; Scarpa, Aldo

    2010-01-01

    Purpose We investigated the global gene expression in a large panel of pancreatic endocrine tumors (PETs) aimed at identifying new potential targets for therapy and biomarkers to predict patient outcome. Patients and Methods Using a custom microarray, we analyzed 72 primary PETs, seven matched metastases, and 10 normal pancreatic samples. Relevant differentially expressed genes were validated by either quantitative real-time polymerase chain reaction or immunohistochemistry on tissue microarrays. Results Our data showed that: tuberous sclerosis 2 (TSC2) and phosphatase and tensin homolog (PTEN) were downregulated in most of the primary tumors, and their low expression was significantly associated with shorter disease-free and overall survival; somatostatin receptor 2 (SSTR2) was absent or very low in insulinomas compared with nonfunctioning tumors; and expression of fibroblast growth factor 13 (FGF13) gene was significantly associated with the occurrence of liver metastasis and shorter disease-free survival. TSC2 and PTEN are two key inhibitors of the Akt/mammalian target of rapamycin (mTOR) pathway and the specific inhibition of mTOR with rapamycin or RAD001 inhibited cell proliferation of PET cell lines. Conclusion Our results strongly support a role for PI3K/Akt/mTOR pathway in PET, which ties in with the fact that mTOR inhibitors have reached phase III trials in neuroendocrine tumors. The finding of differential SSTR expression raises the potential for SSTR expression to be evaluated as a marker of response to somatostatin analogs. Finally, we identified FGF13 as a new prognostic marker that predicted poorer outcome in patients who were clinically considered free from disease. PMID:19917848

  5. Threonine-4 of mammalian RNA polymerase II CTD is targeted by Polo-like kinase 3 and required for transcriptional elongation

    PubMed Central

    Hintermair, Corinna; Heidemann, Martin; Koch, Frederic; Descostes, Nicolas; Gut, Marta; Gut, Ivo; Fenouil, Romain; Ferrier, Pierre; Flatley, Andrew; Kremmer, Elisabeth; Chapman, Rob D; Andrau, Jean-Christophe; Eick, Dirk

    2012-01-01

    Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5′ and 3′ regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo-like kinase 3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4-P levels in the 3′ region of genes occurring subsequently to an increase of Ser2-P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells. PMID:22549466

  6. In search of AKT kinase inhibitors as anticancer agents: structure-based design, docking and molecular dynamics studies of 2,4,6-trisubstituted pyridines.

    PubMed

    Trejo-Soto, Pedro Josué; Hernández-Campos, Alicia; Romo-Mancillas, Antonio; Medina-Franco, José L; Castillo, Rafael

    2017-02-02

    The AKT isoforms are a group of key kinases that play a critical role in tumorigenesis. These enzymes are overexpressed in different types of cancers, such as breast, colon, prostate, ovarian and lung. Because of its relevance the AKT isoforms are attractive targets for the design of anticancer molecules. However, it has been found that AKT1 and AKT3 isoforms have a main role in tumor progression and metastasis; thus, the identification of AKT isoforms specific inhibitors seems to be a challenge. Previously, we identified an ATP binding pocket pan-AKT inhibitor, this compound is a 2,4,6-trisubstituted pyridine (compound 11), which represents a new interesting scaffold for the developing of AKT inhibitors. Starting from the 2,4,6-trisubstituted pyridine scaffold, and guided by structure-based design technique, 42 new inhibitors were designed and further evaluated in the three AKT isoforms by multiple docking approach and molecular dynamics. Results showed that seven compounds presented binding selectivity for AKT1 and AKT3, better than for AKT2. The binding affinities of these seven compounds on AKT1 and AKT3 isoforms were mainly determined by hydrophobic contributions between the aromatic portion at position 4 of the pyridine ring with residues Phe236/234, Phe237/235, Phe438/435 and Phe442/439 in the ATP binding pocket. Results presented in this work provide an addition knowledge leading to promising selective AKT inhibitors.

  7. Nuclear Akt2 opposes limbal keratinocyte stem cell self-renewal by repressing a FOXO-mTORC1 signaling pathway.

    PubMed

    Saoncella, Stefania; Tassone, Beatrice; Deklic, Erika; Avolio, Fabio; Jon, Cristina; Tornillo, Giusy; De Luca, Elisa; Di Iorio, Enzo; Piva, Roberto; Cabodi, Sara; Turco, Emilia; Pandolfi, Pier Paolo; Calautti, Enzo

    2014-03-01

    Signals downstream of Akt can either favor or oppose stem cell (SC) maintenance, but how this dual role can be achieved is still undefined. Using human limbal keratinocyte stem cells (LKSCs), a SC type used in transplantation therapies for corneal regeneration, we show that Akt signaling is prominent in SC populations both in vivo and in vitro, and that Akt1 promotes while Akt2 opposes SC self-renewal. Noteworthy, loss of Akt2 signaling enhances LKSC maintenance ex vivo, whereas Akt1 depletion anticipates SC exhaustion. Mechanistically, the antagonistic functions of Akt1 and Akt2 in SC control are mainly dictated by their differential subcellular distribution, being nuclear Akt2 selectively implicated in FOXO inhibition. Akt2 downregulation favors LKSC maintenance as a result of a gain of FOXO functions, which attenuates the mechanistic target of rapamycin complex one signaling via tuberous sclerosis one gene induction, and promotes growth factor signaling through Akt1. Consistently, Akt2 deficiency also enhances limbal SCs in vivo. Thus, our findings reveal distinct roles for nuclear versus cytosolic Akt signaling in normal epithelial SC control and suggest that the selective Akt2 inhibition may provide novel pharmacological strategies for human LKSC expansion in therapeutic settings and mechanistic research.

  8. [IL-12 induces autophagy via AKT/mTOR/STAT3 signaling pathway in human hepatoma cells].

    PubMed

    Liu, Cuiying; Xie, Changli; Lin, Yan; Wu, Bitao; Wang, Qin; Li, Ziwei; Tu, Zhiguang

    2016-07-01

    Objective To investigate the effect of IL-12 on autophagy and the relative possible mechanism in HepG2 and SMMC-7721 human hepatoma cells. Methods The hepatoma cells were treated with IL-12 (10 ng/mL) for 6 hours. Western blotting was applied to detect the expressions of microtubule-associated protein 1 light chain 3 (LC-3), Beclin 1 and the phosphorylated levels of protein kinase B (AKT), mammalian target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3); immunofluorescence assay (IFA) and transmission electron microscopy (TEM) were used to observe the formation of autophagosome. After STAT3 was inhibited by STATTIC or siSTAT3 and AKT was activated by insulin-like growth factor (IGF-1), Western blotting and IFA were performed again to analyze the change of IL-12-induced autophagy. After the cells were treated with IL-12 (10 ng/mL) for 1, 2, 3, 4, 5 days, CCK-8 assay was used to determine the growth ability. After the hepatoma cells were treated with IL-12 (10 ng/mL) for 48 hours, trypan blue staining was used to detect the death rate of the cells. After cell autophagy was inhibit by siBeclin 1, CCK-8 assay and trypan blue staining were performed again to study the effect of IL-12 on the proliferation and death of human hepatoma cells. Results IL-12 induced autophagy and inhibited cell growth in the hepatoma cells. Silencing Beclin 1 gene enhanced IL-12-mediated growth inhibition and cell death. Furthermore, IL-12 treatment also decreased the expressions of p-AKT, p-mTOR and p-STAT3. The pretreatment of siSTAT3 or STATTIC inhibited STAT3-enhanced IL-12-induced autophagy. Accordingly, activation of AKT with IGF-1 decreased IL-12-induced autophagy. Conclusion IL-12 could induce autophagy through AKT/mTOR/STAT3 signaling pathways and the induction of autophagy attenuates the growth-inhibitory effect of IL-12 on hepatoma cells.

  9. Curcumin activates autophagy and attenuates oxidative damage in EA.hy926 cells via the Akt/mTOR pathway.

    PubMed

    Guo, Shouyu; Long, Mingzhi; Li, Xiuzhen; Zhu, Shushu; Zhang, Min; Yang, Zhijian

    2016-03-01

    Curcumin, which is the effective component of turmeric (Curcuma longa), has previously been shown to exert potent antioxidant, antitumor and anti‑inflammatory activities in vitro and in vivo. However, the mechanism underlying the protective effects of curcumin against oxidative damage in endothelial cells remains unclear. The present study aimed to examine the effects of curcumin on hydrogen peroxide (H2O2)‑induced apoptosis and autophagy in EA.hy926 cells, and to determine the underlying molecular mechanism. Cultured EA.hy926 cells were treated with curcumin (5‑20 µmol/l) 4 h prior to and for 4 h during exposure to H2O2 (200 µmol/l). Oxidative stress resulted in a significant increase in the rate of cell apoptosis, which was accompanied by an increase in the expression levels of caspase‑3 and B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax), and a decrease in the expression levels of Bcl‑2. Treatment with curcumin (5 or 20 µmol/l) significantly inhibited apoptosis, and reversed the alterations in caspase‑3, Bcl‑2 and Bax expression. Furthermore, curcumin induced autophagy and microtubule‑associated protein 1A/1B‑light chain 3‑Ⅱ expression, and suppressed the phosphorylation of Akt and mammalian target of rapamycin (mTOR). These results indicated that curcumin may protect cells against oxidative stress‑induced damage through inhibiting apoptosis and inducing autophagy via the Akt/mTOR pathway.

  10. De novo somatic mutations in components of the PI3K-AKT3-mTOR pathway cause hemimegalencephaly

    PubMed Central

    Lee, Jeong Ho; Huynh, My; Silhavy, Jennifer L; Kim, Sangwoo; Dixon-Salazar, Tracy; Heiberg, Andrew; Scott, Eric; Bafna, Vineet; Hill, Kiley J; Collazo, Adrienne; Funari, Vincent; Russ, Carsten; Gabriel, Stacey B; Mathern, Gary W; Gleeson, Joseph G

    2015-01-01

    De novo somatic mutations in focal areas are well documented in diseases such as neoplasia but are rarely reported in malformation of the developing brain. Hemimegalencephaly (HME) is characterized by overgrowth of either one of the two cerebral hemispheres. The molecular etiology of HME remains a mystery. The intractable epilepsy that is associated with HME can be relieved by the surgical treatment hemispherectomy, allowing sampling of diseased tissue. Exome sequencing and mass spectrometry analysis in paired brain-blood samples from individuals with HME (n = 20 cases) identified de novo somatic mutations in 30% of affected individuals in the PIK3CA, AKT3 and MTOR genes. A recurrent PIK3CA c.1633G>A mutation was found in four separate cases. Identified mutations were present in 8–40% of sequenced alleles in various brain regions and were associated with increased neuronal S6 protein phosphorylation in the brains of affected individuals, indicating aberrant activation of mammalian target of rapamycin (mTOR) signaling. Thus HME is probably a genetically mosaic disease caused by gain of function in phosphatidylinositol 3-kinase (PI3K)-AKT3-mTOR signaling. PMID:22729223

  11. A Positive Feedback Loop between Akt and mTORC2 via SIN1 Phosphorylation.

    PubMed

    Yang, Guang; Murashige, Danielle S; Humphrey, Sean J; James, David E

    2015-08-11

    The mechanistic target of rapamycin complex 2 (mTORC2) regulates cell survival and cytoskeletal organization by phosphorylating its AGC kinase substrates; however, little is known about the regulation of mTORC2 itself. It was previously reported that Akt phosphorylates the mTORC2 subunit SIN1 at T86, activating mTORC2 through a positive feedback loop, though another study reported that S6K phosphorylates SIN1 at the same site, inhibiting mTORC2 activity. We performed extensive analysis of SIN1 phosphorylation upon inhibition of Akt, S6K, and mTOR under diverse cellular contexts, and we found that, in all cell lines and conditions studied, Akt is the major kinase responsible for SIN1 phosphorylation. These findings refine the activation mechanism of the Akt-mTORC2 signaling branch as follows: PDK1 phosphorylates Akt at T308, increasing Akt kinase activity. Akt phosphorylates SIN1 at T86, enhancing mTORC2 kinase activity, which leads to phosphorylation of Akt S473 by mTORC2, thereby catalyzing full activation of Akt.

  12. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    SciTech Connect

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa; Shin, Incheol

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  13. Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-gamma1-driven activation of mTOR/p70S6-kinase pathway.

    PubMed

    Markova, B; Albers, C; Breitenbuecher, F; Melo, J V; Brümmendorf, T H; Heidel, F; Lipka, D; Duyster, J; Huber, C; Fischer, T

    2010-02-04

    In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-gamma1 (PLC-gamma1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-gamma1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-gamma1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-gamma1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-gamma1 and Bcr-Abl inhibition. The pivotal role of PLC-gamma1 was further confirmed in a mouse leukemogenesis model.

  14. Rosemary extract reduces Akt/mTOR/p70S6K activation and inhibits proliferation and survival of A549 human lung cancer cells.

    PubMed

    Moore, Jessy; Megaly, Mark; MacNeil, Adam J; Klentrou, Panagiota; Tsiani, Evangelia

    2016-10-01

    Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Rosemary extract contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt, mammalian target of rapamycin (mTOR) and p70S6K, and the apoptotic protein poly ADP ribose polymerase (PARP) are key modulators of cancer cell growth and survival. In this study, we examined the effects of rosemary extract on proliferation, survival and apoptosis of human non-small cell lung cancer (NSCLC) cells and its influence on signaling events. Human NSCLC adenocarcinoma A549 cells were used. Cell proliferation and clonogenic survival were assessed using specific assays. Immunoblotting was used to examine total and phosphorylated levels of Akt, mTOR and p70S6K, and cleavage of PARP. Rosemary extract dose-dependently inhibited cell proliferation and reduced clonogenic survival of A549 cells, while PARP cleavage, an indicator of apoptosis, was enhanced. Rosemary extract significantly reduced total and phosphorylated/activated Akt, mTOR and p70S6K levels. In conclusion, rosemary extract inhibited proliferation, blocked clonogenic survival, and enhanced apoptosis of A549 lung cancer cells. These effects were associated with inhibition of Akt and downstream mTOR and p70S6K activity. Our data suggest that rosemary extract may have considerable anti-tumor and chemoprevention properties in lung cancer and deserves further systematic investigation in animal models of lung cancer.

  15. Effects of orexin A on glucose metabolism in human hepatocellular carcinoma in vitro via PI3K/Akt/mTOR-dependent and -independent mechanism.

    PubMed

    Liu, Yuanyuan; Zhao, Yuyan; Guo, Lei

    2016-01-15

    Orexins are hypothalamic neuropeptides that regulate food intake, energy homeostasis, reward system and sleep/wakefulness states. The purpose of this study was to investigate the effects of orexin A on glucose metabolism in human hepatocellular carcinoma cell line, Hep3B, and determine the possible mechanisms. Hep3B cells were incubated with different concentrations of orexin A (10(-9)-10(-7) M) in vitro in the presence or absence of the orexin receptor 1 (OX1R) inhibitor (SB334867), Akt inhibitor (PF-04691502) and mammalian target of rapamycin (mTOR) inhibitor (temsirolimus). Subsequently, OX1R protein expression, glucose transporter 1 (GLUT1) expression, glucose uptake, the mRNA expression of lactate dehydrogenase (LDHA), pyruvate dehydrogenase kinase 1 (PDK1) and pyruvate dehydrogenase B (PDHB), lactate generation and mitochondrial pyruvate dehydrogenase (PDH) enzyme activity were measured. The activity of phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling was also determined. OX1R was expressed in hepatoma tissues and Hep3B cells. Stimulation of the Hep3B cells with orexin A resulted in a dose-dependent increase of GLUT1 expression and glucose uptake, which was associated with the activation of PI3K/Akt/mTOR pathway. Further, orexin A increased PDHB expression and PDH enzyme activity, decreased LDHA, PDK1 mRNA levels and lactate generation independent of PI3K/Akt/mTOR pathway. Our results demonstrated that orexin A directed the cellular metabolism towards mitochondrial glucose oxidation rather than glycolysis. These findings provide functional evidence of the metabolic actions of orexin A in hepatocellular carcinoma cells.

  16. Gastrin induces sodium-hydrogen exchanger 3 phosphorylation and mTOR activation via a phosphoinositide 3-kinase-/protein kinase C-dependent but AKT-independent pathway in renal proximal tubule cells derived from a normotensive male human.

    PubMed

    Liu, Tianbing; Jose, Pedro A

    2013-02-01

    Gastrin is natriuretic, but its renal molecular targets and signal transduction pathways are not fully known. In this study, we confirmed the existence of CCKBR (a gastrin receptor) in male human renal proximal tubule cells and discovered that gastrin induced S6 phosphorylation, a downstream component of the phosphatidylinositol 3 kinase (PI3 kinase)-mammalian target of rapamycin pathway. Gastrin also increased the phosphorylation of sodium-hydrogen exchanger 3 (NHE3) at serine 552, caused its internalization, and decreased its expression at the cell surface and NHE activity. The phosphorylation of NHE3 and S6 was dependent on PI3 kinases because it was blocked by 2 different PI3-kinase inhibitors, wortmannin and LY294,002. The phosphorylation of NHE3 and S6 was not affected by the protein kinase A inhibitor H-89 but was blocked by a pan-PKC (chelerythrine) and a conventional PKC (cPKC) inhibitor (Gö6976) (10 μM) and an intracellular calcium chelator, 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The cPKC involved was probably PKCα because it was phosphorylated by gastrin. The gastrin-mediated phosphorylation of NHE3, S6, and PKCα was via phospholipase C because it was blocked by a phospholipase C inhibitor, U73122 (10 μM). The phosphorylation (activation) of AKT, which is usually upstream of mammalian target of rapamycin in the classic PI3 kinase-AKT-p70S6K signaling pathway, was not affected, suggesting that the gastrin-induced phosphorylation of NHE3 and S6 is dependent on both PI3 kinase and PKCα but not AKT.

  17. AKT Regulates BRCA1 Stability in Response to Hormone Signaling

    PubMed Central

    Nelson, Andrew C.; Lyons, Traci R.; Young, Christian D.; Hansen, Kirk C.; Anderson, Steven M.; Holt, Jeffrey T.

    2015-01-01

    BRCA1, with its binding partner BARD1, regulates the cellular response to DNA damage in multiple tissues, yet inherited mutations within BRCA1 result specifically in breast and ovarian cancers. This observation, along with several other lines of evidence, suggests a functional relationship may exist between hormone signaling and BRCA1 function. Our data demonstrates that AKT activation promotes the expression of BRCA1 in response to estrogen and IGF-1 receptor signaling. Further, we have identified a novel AKT phosphorylation site in BRCA1 at S694 which is responsive to activation of these signaling pathways. This rapid increase in BRCA1 protein levels appears to occur independently of new protein synthesis and treatment with the clinically utilized proteasome inhibitor bortezomib similarly leads to a rapid increase in BRCA1 protein levels. Together, these data suggest that AKT phosphorylation of BRCA1 increases total protein expression by preventing proteasomal degradation. AKT activation also appears to support nuclear localization of BRCA1, and co-expression of activated AKT with BRCA1 decreases radiation sensitivity, suggesting this interaction has functional consequences for BRCA1's role in DNA repair. We conclude that AKT regulates BRCA1 protein stability and function through direct phosphorylation of BRCA1. Further, the responsiveness of the AKT-BRCA1 regulatory pathway to hormone signaling may, in part, underlie the tissue specificity of BRCA1 mutant cancers. Pharmacological targets within this pathway could provide strategies for modulation of BRCA1 protein, which may prove therapeutically beneficial for the treatment of breast and ovarian cancers. PMID:20085797

  18. Discovery of a novel class of AKT pleckstrin homology domain inhibitors.

    PubMed

    Mahadevan, Daruka; Powis, Garth; Mash, Eugene A; George, Benjamin; Gokhale, Vijay M; Zhang, Shuxing; Shakalya, Kishore; Du-Cuny, Lei; Berggren, Margareta; Ali, M Ahad; Jana, Umasish; Ihle, Nathan; Moses, Sylvestor; Franklin, Chloe; Narayan, Satya; Shirahatti, Nikhil; Meuillet, Emmanuelle J

    2008-09-01

    AKT, a phospholipid-binding serine/threonine kinase, is a key component of the phosphoinositide 3-kinase cell survival signaling pathway that is aberrantly activated in many human cancers. Many attempts have been made to inhibit AKT; however, selectivity remains to be achieved. We have developed a novel strategy to inhibit AKT by targeting the pleckstrin homology (PH) domain. Using in silico library screening and interactive molecular docking, we have identified a novel class of non-lipid-based compounds that bind selectively to the PH domain of AKT, with "in silico" calculated K(D) values ranging from 0.8 to 3.0 micromol/L. In order to determine the selectivity of these compounds for AKT, we used surface plasmon resonance to measure the binding characteristics of the compounds to the PH domains of AKT1, insulin receptor substrate-1, and 3-phosphoinositide-dependent protein kinase 1. There was excellent correlation between predicted in silico and measured in vitro K(D)s for binding to the PH domain of AKT, which were in the range 0.4 to 3.6 micromol/L. Some of the compounds exhibited PH domain-binding selectivity for AKT compared with insulin receptor substrate-1 and 3-phosphoinositide-dependent protein kinase 1. The compounds also inhibited AKT in cells, induced apoptosis, and inhibited cancer cell proliferation. In vivo, the lead compound failed to achieve the blood concentrations required to inhibit AKT in cells, most likely due to rapid metabolism and elimination, and did not show antitumor activity. These results show that these compounds are the first small molecules selectively targeting the PH domain of AKT.

  19. AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1.

    PubMed

    Corum, Daniel G; Tsichlis, Philip N; Muise-Helmericks, Robin C

    2014-01-01

    Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1α and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1α nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1α target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (~5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ~1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.

  20. Notch-1 inhibition by Withaferin-A: a therapeutic target against colon carcinogenesis.

    PubMed

    Koduru, Srinivas; Kumar, Raj; Srinivasan, Sowmyalakshmi; Evers, Mark B; Damodaran, Chendil

    2010-01-01

    Notch signaling plays a crucial role in the development of colon cancer; targeting the Notch pathway may sensitize colon cancers to various adjuvant agents. The focus of our current study is to identify natural compounds that target Notch signaling and that might be beneficial for the prevention and treatment of colon cancer. Withaferin-A (WA) is a bioactive compound derived from Withania somnifera, which inhibits Notch-1 signaling and downregulates prosurvival pathways, such as Akt/NF-kappaB/Bcl-2, in three colon cancer cell lines (HCT-116, SW-480, and SW-620). In addition, WA downregulated the expression of mammalian target of rapamycin signaling components, pS6K and p4E-BP1, and activated c-Jun-NH(2)-kinase-mediated apoptosis in colon cancer cells. We also established the molecular link between Notch/Akt/mammalian target of rapamycin signaling by complementary approaches (i.e., overexpression of Notch-1 or inhibition of Notch-1 by small interfering RNA). Our results suggest that WA inhibits Notch-mediated prosurvival signaling, which facilitates c-Jun-NH(2)-kinase-mediated apoptosis in colon cancer cell lines. These results underscore the anticancer activity of WA, which exhibits potential for further development for targeted chemotherapy and/or chemoprevention strategies in the context of colon cancer.

  1. Photodynamic inactivation of mammalian viruses and bacteriophages.

    PubMed

    Costa, Liliana; Faustino, Maria Amparo F; Neves, Maria Graça P M S; Cunha, Angela; Almeida, Adelaide

    2012-07-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process.

  2. Recent advances in mammalian protein production

    PubMed Central

    Bandaranayake, Ashok D.; Almo, Steven C.

    2014-01-01

    Mammalian protein production platforms have had a profound impact in many areas of basic and applied research, and an increasing number of blockbuster drugs are recombinant mammalian proteins. With global sales of these drugs exceeding US$120 billion per year, both industry and academic research groups continue to develop cost effective methods for producing mammalian proteins to support preclinical and clinical evaluations of potential therapeutics. While a wide range of platforms have been successfully exploited for laboratory use, the bulk of recent biologics have been produced in mammalian cell lines due to the requirement for post translational modification and the biosynthetic complexity of the target proteins. In this review we highlight the range of mammalian expression platforms available for recombinant protein production, as well as advances in technologies for the rapid and efficient selection of highly productive clones. PMID:24316512

  3. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    PubMed Central

    Costa, Liliana; Faustino, Maria Amparo F.; Neves, Maria Graça P. M. S.; Cunha, Ângela; Almeida, Adelaide

    2012-01-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process. PMID:22852040

  4. Mammalian Polyamine Metabolism and Function

    PubMed Central

    Pegg, Anthony E.

    2009-01-01

    Summary Polyamines are ubiquitous small basic molecules that play multiple essential roles in mammalian physiology. Their cellular content is highly regulated and there is convincing evidence that altered metabolism is involvement in many disease states. Drugs altering polyamine levels may therefore have a variety of important targets. This review will summarize the current state of understanding of polyamine metabolism and function, the regulation of polyamine content, and heritable pathological conditions that may be derived from altered polyamine metabolism. PMID:19603518

  5. Differential Phosphatidylinositol-3-Kinase-Akt-mTOR Activation by Semliki Forest and Chikungunya Viruses Is Dependent on nsP3 and Connected to Replication Complex Internalization

    PubMed Central

    Biasiotto, Roberta; Eng, Kai; Neuvonen, Maarit; Götte, Benjamin; Rheinemann, Lara; Mutso, Margit; Utt, Age; Varghese, Finny; Balistreri, Giuseppe; Merits, Andres; Ahola, Tero; McInerney, Gerald M.

    2015-01-01

    ABSTRACT Many viruses affect or exploit the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, a crucial prosurvival signaling cascade. We report that this pathway was strongly activated in cells upon infection with the Old World alphavirus Semliki Forest virus (SFV), even under conditions of complete nutrient starvation. We mapped this activation to the hyperphosphorylated/acidic domain in the C-terminal tail of SFV nonstructural protein nsP3. Viruses with a deletion of this domain (SFV-Δ50) but not of other regions in nsP3 displayed a clearly delayed and reduced capacity of Akt stimulation. Ectopic expression of the nsP3 of SFV wild type (nsP3-wt), but not nsP3-Δ50, equipped with a membrane anchor was sufficient to activate Akt. We linked PI3K-Akt-mTOR stimulation to the intracellular dynamics of viral replication complexes, which are formed at the plasma membrane and subsequently internalized in a process blocked by the PI3K inhibitor wortmannin. Replication complex internalization was observed upon infection of cells with SFV-wt and SFV mutants with deletions in nsP3 but not with SFV-Δ50, where replication complexes were typically accumulated at the cell periphery. In cells infected with the closely related chikungunya virus (CHIKV), the PI3K-Akt-mTOR pathway was only moderately activated. Replication complexes of CHIKV were predominantly located at the cell periphery. Exchanging the hypervariable C-terminal tail of nsP3 between SFV and CHIKV induced the phenotype of strong PI3K-Akt-mTOR activation and replication complex internalization in CHIKV. In conclusion, infection with SFV but not CHIKV boosts PI3K-Akt-mTOR through the hyperphosphorylated/acidic domain of nsP3 to drive replication complex internalization. IMPORTANCE SFV and CHIKV are very similar in terms of molecular and cell biology, e.g., regarding replication and molecular interactions, but are strikingly different regarding pathology: CHIKV is a relevant human

  6. mRNA Targeting to Endoplasmic Reticulum Precedes Ago Protein Interaction and MicroRNA (miRNA)-mediated Translation Repression in Mammalian Cells.

    PubMed

    Barman, Bahnisikha; Bhattacharyya, Suvendra N

    2015-10-09

    MicroRNA (miRNA) binds to the 3'-UTR of its target mRNAs to repress protein synthesis. Extensive research was done to understand the mechanism of miRNA-mediated repression in animal cells. Considering the progress in understanding the mechanism, information about the subcellular sites of miRNA-mediated repression is surprisingly limited. In this study, using an inducible expression system for an miRNA target message, we have delineated how a target mRNA passes through polysome association and Ago2 interaction steps on rough endoplasmic reticulum (ER) before the miRNA-mediated repression sets in. From this study, de novo formed target mRNA localization to the ER-bound polysomes manifested as the earliest event, which is followed by Ago2 micro-ribonucleoprotein binding, and translation repression of target message. Compartmentalization of this process to rough ER membrane ensures enrichment of miRNA-targeted messages and micro-ribonucleoprotein components on ER upon reaching a steady state.

  7. Loss of Akt activity increases circulating soluble endoglin release in preeclampsia: identification of inter-dependency between Akt-1 and heme oxygenase-1

    PubMed Central

    Cudmore, Melissa J.; Ahmad, Shakil; Sissaoui, Samir; Ramma, Wenda; Ma, Bin; Fujisawa, Takeshi; Al-Ani, Bahjat; Wang, Keqing; Cai, Meng; Crispi, Fatima; Hewett, Peter W.; Gratacós, Eduard; Egginton, Stuart; Ahmed, Asif

    2012-01-01

    Aims Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an antagonist of transforming growth factor-β (TGF-β) signalling, which is known to be elevated in preeclampsia. Methods and results Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin, which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt (Aktdn) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, overexpression of a constitutively active Akt (Aktmyr) inhibited PTEN and cytokine-induced sEng release. Systemic delivery of Aktmyr to mice significantly reduced circulating sEng, whereas Aktdn promoted sEng release. Phosphorylation of Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Furthermore, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Aktmyr failed to suppress the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated inhibition of sEng. Conclusion The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers a strategy to circumvent endothelial dysfunction. PMID:21411816

  8. Context-dependent antagonism between Akt inhibitors and topoisomerase poisons.

    PubMed

    Gálvez-Peralta, Marina; Flatten, Karen S; Loegering, David A; Peterson, Kevin L; Schneider, Paula A; Erlichman, Charles; Kaufmann, Scott H

    2014-05-01

    Signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is aberrantly activated in >50% of carcinomas, inhibits apoptosis and contributes to drug resistance. Accordingly, several Akt inhibitors are currently undergoing preclinical or early clinical testing. To examine the effect of Akt inhibition on the activity of multiple widely used classes of antineoplastic agents, human cancer cell lines were treated with the Akt inhibitor A-443654 [(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine; ATP-competitive] or MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride; allosteric inhibitor) or with small interfering RNA (siRNA) targeting phosphoinositide-dependent kinase 1 (PDK1) along with cisplatin, melphalan, camptothecin, or etoposide and assayed for colony formation. Surprisingly different results were observed when Akt inhibitors were combined with different drugs. Synergistic effects were observed in multiple cell lines independent of PI3K pathway status when A-443654 or MK-2206 was combined with the DNA cross-linking agents cisplatin or melphalan. In contrast, effects of the Akt inhibitors in combination with camptothecin or etoposide were more complicated. In HCT116 and DLD1 cells, which harbor activating PI3KCA mutations, A-443654 over a broad concentration range enhanced the effects of camptothecin or etoposide. In contrast, in cell lines lacking activating PI3KCA mutations, partial inhibition of Akt signaling synergized with camptothecin or etoposide, but higher A-443654 or MK-2206 concentrations (>80% inhibition of Akt signaling) or PDK1 siRNA antagonized the topoisomerase poisons by diminishing DNA synthesis, a process that contributes to effective DNA damage and killing by these agents. These results indicate that the effects of combining inhibitors of the PI3K/Akt pathway with certain classes of chemotherapeutic agents might be more

  9. Dynamin1 Is a Novel Target for IRSp53 Protein and Works with Mammalian Enabled (Mena) Protein and Eps8 to Regulate Filopodial Dynamics*

    PubMed Central

    Chou, Ai Mei; Sem, Kai Ping; Wright, Graham Daniel; Sudhaharan, Thankiah; Ahmed, Sohail

    2014-01-01

    Filopodia are dynamic actin-based structures that play roles in processes such as cell migration, wound healing, and axonal guidance. Cdc42 induces filopodial formation through IRSp53, an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain protein. Previous work from a number of laboratories has shown that IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through its Src homology 3 domain binding partners. Here, we show that dynamin1 (Dyn1), the large guanosine triphosphatase, is an interacting partner of IRSp53 through pulldown and Förster resonance energy transfer analysis, and we explore its role in filopodial formation. In neuroblastoma cells, Dyn1 localizes to filopodia, associated tip complexes, and the leading edge just behind the anti-capping protein mammalian enabled (Mena). Dyn1 knockdown reduces filopodial formation, which can be rescued by overexpressing wild-type Dyn1 but not the GTPase mutant Dyn1-K44A and the loss-of-function actin binding domain mutant Dyn1-K/E. Interestingly, dynasore, an inhibitor of Dyn GTPase, also reduced filopodial number and increased their lifetime. Using rapid time-lapse total internal reflection fluorescence microscopy, we show that Dyn1 and Mena localize to filopodia only during initiation and assembly. Dyn1 actin binding domain mutant inhibits filopodial formation, suggesting a role in actin elongation. In contrast, Eps8, an actin capping protein, is seen most strongly at filopodial tips during disassembly. Taken together, the results suggest IRSp53 partners with Dyn1, Mena, and Eps8 to regulate filopodial dynamics. PMID:25031323

  10. Dynamin1 is a novel target for IRSp53 protein and works with mammalian enabled (Mena) protein and Eps8 to regulate filopodial dynamics.

    PubMed

    Chou, Ai Mei; Sem, Kai Ping; Wright, Graham Daniel; Sudhaharan, Thankiah; Ahmed, Sohail

    2014-08-29

    Filopodia are dynamic actin-based structures that play roles in processes such as cell migration, wound healing, and axonal guidance. Cdc42 induces filopodial formation through IRSp53, an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain protein. Previous work from a number of laboratories has shown that IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through its Src homology 3 domain binding partners. Here, we show that dynamin1 (Dyn1), the large guanosine triphosphatase, is an interacting partner of IRSp53 through pulldown and Förster resonance energy transfer analysis, and we explore its role in filopodial formation. In neuroblastoma cells, Dyn1 localizes to filopodia, associated tip complexes, and the leading edge just behind the anti-capping protein mammalian enabled (Mena). Dyn1 knockdown reduces filopodial formation, which can be rescued by overexpressing wild-type Dyn1 but not the GTPase mutant Dyn1-K44A and the loss-of-function actin binding domain mutant Dyn1-K/E. Interestingly, dynasore, an inhibitor of Dyn GTPase, also reduced filopodial number and increased their lifetime. Using rapid time-lapse total internal reflection fluorescence microscopy, we show that Dyn1 and Mena localize to filopodia only during initiation and assembly. Dyn1 actin binding domain mutant inhibits filopodial formation, suggesting a role in actin elongation. In contrast, Eps8, an actin capping protein, is seen most strongly at filopodial tips during disassembly. Taken together, the results suggest IRSp53 partners with Dyn1, Mena, and Eps8 to regulate filopodial dynamics.

  11. AKT capture by feline leukemia virus.

    PubMed

    Kawamura, Maki; Umehara, Daigo; Odahara, Yuka; Miyake, Ariko; Ngo, Minh Ha; Ohsato, Yoshiharu; Hisasue, Masaharu; Nakaya, Masa-Aki; Watanabe, Shinya; Nishigaki, Kazuo

    2016-12-22

    Oncogene-containing retroviruses are generated by recombination events between viral and cellular sequences, a phenomenon called "oncogene capture". The captured cellular genes, referred to as "v-onc" genes, then acquire new oncogenic properties. We report a novel feline leukemia virus (FeLV), designated "FeLV-AKT", that has captured feline c-AKT1 in feline lymphoma. FeLV-AKT contains a gag-AKT fusion gene that encodes the myristoylated Gag matrix protein and the kinase domain of feline c-AKT1, but not its pleckstrin homology domain. Therefore, it differs structurally from the v-Akt gene of murine retrovirus AKT8. AKT may be involved in the mechanisms underlying malignant diseases in cats.

  12. Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue*

    PubMed Central

    Schrötter, Sandra; Leondaritis, George; Eickholt, Britta J.

    2016-01-01

    The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1–3 kinases are specifically activated by two phosphorylation events on residues Thr308 and Ser473 upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser473 and Thr308 phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser473 and Thr308 phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser473-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons. PMID:26945062

  13. Optogenetic activation reveals distinct roles of PIP3 and Akt in adipocyte insulin action.

    PubMed

    Xu, Yingke; Nan, Di; Fan, Jiannan; Bogan, Jonathan S; Toomre, Derek

    2016-05-15

    Glucose transporter 4 (GLUT4; also known as SLC2A4) resides on intracellular vesicles in muscle and adipose cells, and translocates to the plasma membrane in response to insulin. The phosphoinositide 3-kinase (PI3K)-Akt signaling pathway plays a major role in GLUT4 translocation; however, a challenge has been to unravel the potentially distinct contributions of PI3K and Akt (of which there are three isoforms, Akt1-Akt3) to overall insulin action. Here, we describe new optogenetic tools based on CRY2 and the N-terminus of CIB1 (CIBN). We used these 'Opto' modules to activate PI3K and Akt selectively in time and space in 3T3-L1 adipocytes. We validated these tools using biochemical assays and performed live-cell kinetic analyses of IRAP-pHluorin translocation (IRAP is also known as LNPEP and acts as a surrogate marker for GLUT4 here). Strikingly, Opto-PIP3 largely mimicked the maximal effects of insulin stimulation, whereas Opto-Akt only partially triggered translocation. Conversely, drug-mediated inhibition of Akt only partially dampened the translocation response of Opto-PIP3 In spatial optogenetic studies, focal targeting of Akt to a region of the cell marked the sites where IRAP-pHluorin vesicles fused, supporting the idea that local Akt-mediated signaling regulates exocytosis. Taken together, these results indicate that PI3K and Akt play distinct roles, and that PI3K stimulates Akt-independent pathways that are important for GLUT4 translocation.

  14. Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue.

    PubMed

    Schrötter, Sandra; Leondaritis, George; Eickholt, Britta J

    2016-05-06

    The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1-3 kinases are specifically activated by two phosphorylation events on residues Thr(308) and Ser(473) upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser(473) and Thr(308) phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser(473) and Thr(308) phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser(473)-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons.

  15. Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated β-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine.

    PubMed

    Zhang, Feng-Ying; Hu, Yi; Que, Zhong-You; Wang, Ping; Liu, Yun-Hui; Wang, Zhen-Hua; Xue, Yi-Xue

    2015-10-09

    Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 μmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the expression of phosphorylated β-catenin (p-β-catenin) and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-β-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1).

  16. Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated β-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine

    PubMed Central

    Zhang, Feng-Ying; Hu, Yi; Que, Zhong-You; Wang, Ping; Liu, Yun-Hui; Wang, Zhen-Hua; Xue, Yi-Xue

    2015-01-01

    Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 μmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the expression of phosphorylated β-catenin (p-β-catenin) and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-β-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1). PMID:26473829

  17. Activation of p53/p21/PUMA alliance and disruption of PI-3/Akt in multimodal targeting of apoptotic signaling cascades in cervical cancer cells by a pentacyclic triterpenediol from Boswellia serrata.

    PubMed

    Bhushan, Shashi; Malik, Fayaz; Kumar, Ajay; Isher, Harpreet Kaur; Kaur, Indu Pal; Taneja, Subhash Chandra; Singh, Jaswant

    2009-12-01

    Cervical carcinoma is a growing menace to women health worldwide. This study reports the apoptotic cell death in human cervical cancer HeLa and SiHa cells by a pentacyclic triterpenediol (TPD) from Boswellia serrata by a mechanism different from reported in HL-60 cells. It caused oxidative stress by early generation of nitric oxide and reactive oxygen species that robustly up regulated time-dependent expression of p53/p21/PUMA while conversely abrogating phosphatidylinositol-3-kinase (PI3K)/Akt pathways in parallel. TPD also decreased the expression of PI3K/pAkt, ERK1/2, NF-kappaB/Akt signaling cascades which coordinately contribute to cancer cell survival through these distinct pathways. The tumor suppressor p53 pathway predominantly activated by TPD further up-regulated PUMA, which concomitantly decreased the Bcl-2 level, caused mitochondrial membrane potential loss with attendant translocation of Bax and drp1 to mitochondria and release of pro-apoptotic factors such as cytochrome c and Smac/Diablo to cytosol leading to caspases-3 and -9 activation. In addition both the phospho-p53 and p21 were found to accumulate heavily in the nuclear fraction with attendant decrease in topoisomarase II and survivin levels. On the contrary, TPD did not affect the extrinsic signaling transduction pathway effectively through apical death receptors. Interestingly, N-acetyl cysteine, ascorbate and s-methylisothiourea (sMIT) rescued cells significantly from TPD induced DNA damage and caspases activation. TPD may thus find usefulness in managing and treating cervical cancer.

  18. Factors Influencing the Central Nervous System Distribution of a Novel Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor GSK2126458: Implications for Overcoming Resistance with Combination Therapy for Melanoma Brain Metastases

    PubMed Central

    Vaidhyanathan, Shruthi; Wilken-Resman, Brynna; Ma, Daniel J.; Parrish, Karen E.; Mittapalli, Rajendar K.; Carlson, Brett L.; Sarkaria, Jann N.

    2016-01-01

    Small molecule inhibitors targeting the mitogen-activated protein kinase pathway (Braf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) have had success in extending survival for patients with metastatic melanoma. Unfortunately, resistance may occur via cross-activation of alternate signaling pathways. One approach to overcome resistance is to simultaneously target the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway. Recent reports have shown that GSK2126458 [2,4-difluoro-N-(2-methoxy-5-(4-(pyridazin-4-yl)quinolin-6-yl)pyridin-3-yl) benzenesulfonamide], a dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor, can overcome acquired resistance to Braf and mitogen-activated protein kinase kinase inhibitors in vitro. These resistance mechanisms may be especially important in melanoma brain metastases because of limited drug delivery across the blood–brain barrier. The purpose of this study was to investigate factors that influence the brain distribution of GSK2126458 and to examine the efficacy of GSK2126458 in a novel patient-derived melanoma xenograft (PDX) model. Both in vitro and in vivo studies indicate that GSK2126458 is a substrate for P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), two dominant active efflux transporters in the blood–brain barrier. The steady-state brain distribution of GSK2126458 was 8-fold higher in the P-gp/Bcrp knockout mice compared with the wild type. We also observed that when simultaneously infused to steady state, GSK212658, dabrafenib, and trametinib, a rational combination to overcome mitogen-activated protein kinase inhibitor resistance, all had limited brain distribution. Coadministration of elacridar, a P-gp/Bcrp inhibitor, increased the brain distribution of GSK2126458 by approximately 7-fold in wild-type mice. In the PDX model, GSK2126458 showed efficacy in flank tumors but was ineffective in intracranial melanoma. These results show

  19. The Akt1/IL-6/STAT3 pathway regulates growth of lung tumor initiating cells

    PubMed Central

    Malanga, Donatella; De Marco, Carmela; Guerriero, Ilaria; Colelli, Fabiana; Rinaldo, Nicola; Scrima, Marianna; Mirante, Teresa; De Vitis, Claudia; Zoppoli, Pietro; Ceccarelli, Michele; Riccardi, Miriam; Ravo, Maria; Weisz, Alessandro; Federico, Antonella; Franco, Renato; Rocco, Gaetano; Mancini, Rita; Rizzuto, Antonia; Gulletta, Elio; Ciliberto, Gennaro; Viglietto, Giuseppe

    2015-01-01

    Here we report that the PI3K/Akt1/IL-6/STAT3 signalling pathway regulates generation and stem cell-like properties of Non-Small Cell Lung Cancer (NSCLC) tumor initiating cells (TICs). Mutant Akt1, mutant PIK3CA or PTEN loss enhances formation of lung cancer spheroids (LCS), self-renewal, expression of stemness markers and tumorigenic potential of human immortalized bronchial cells (BEAS-2B) whereas Akt inhibition suppresses these activities in established (NCI-H460) and primary NSCLC cells. Matched microarray analysis of Akt1-interfered cells and LCSs identified IL-6 as a critical target of Akt signalling in NSCLC TICs. Accordingly, suppression of Akt in NSCLC cells decreases IL-6 levels, phosphorylation of IkK and IkB, NF-kB transcriptional activity, phosphorylation and transcriptional activity of STAT3 whereas active Akt1 up-regulates them. Exposure of LCSs isolated from NSCLC cells to blocking anti-IL-6 mAbs, shRNA to IL-6 receptor or to STAT3 markedly reduces the capability to generate LCSs, to self-renew and to form tumors, whereas administration of IL-6 to Akt-interfered cells restores the capability to generate LCSs. Finally, immunohistochemical studies in NSCLC patients demonstrated a positive correlative trend between activated Akt, IL-6 expression and STAT3 phosphorylation (n = 94; p < 0.05). In conclusion, our data indicate that aberrant Akt signalling contributes to maintaining stemness in lung cancer TICs through a NF-kB/IL-6/STAT3 pathway and provide novel potential therapeutic targets for eliminating these malignant cells in NSCLC. PMID:26486080

  20. The Akt1/IL-6/STAT3 pathway regulates growth of lung tumor initiating cells.

    PubMed

    Malanga, Donatella; De Marco, Carmela; Guerriero, Ilaria; Colelli, Fabiana; Rinaldo, Nicola; Scrima, Marianna; Mirante, Teresa; De Vitis, Claudia; Zoppoli, Pietro; Ceccarelli, Michele; Riccardi, Miriam; Ravo, Maria; Weisz, Alessandro; Federico, Antonella; Franco, Renato; Rocco, Gaetano; Mancini, Rita; Rizzuto, Antonia; Gulletta, Elio; Ciliberto, Gennaro; Viglietto, Giuseppe

    2015-12-15

    Here we report that the PI3K/Akt1/IL-6/STAT3 signalling pathway regulates generation and stem cell-like properties of Non-Small Cell Lung Cancer (NSCLC) tumor initiating cells (TICs). Mutant Akt1, mutant PIK3CA or PTEN loss enhances formation of lung cancer spheroids (LCS), self-renewal, expression of stemness markers and tumorigenic potential of human immortalized bronchial cells (BEAS-2B) whereas Akt inhibition suppresses these activities in established (NCI-H460) and primary NSCLC cells. Matched microarray analysis of Akt1-interfered cells and LCSs identified IL-6 as a critical target of Akt signalling in NSCLC TICs. Accordingly, suppression of Akt in NSCLC cells decreases IL-6 levels, phosphorylation of IkK and IkB, NF-kB transcriptional activity, phosphorylation and transcriptional activity of STAT3 whereas active Akt1 up-regulates them. Exposure of LCSs isolated from NSCLC cells to blocking anti-IL-6 mAbs, shRNA to IL-6 receptor or to STAT3 markedly reduces the capability to generate LCSs, to self-renew and to form tumors, whereas administration of IL-6 to Akt-interfered cells restores the capability to generate LCSs. Finally, immunohistochemical studies in NSCLC patients demonstrated a positive correlative trend between activated Akt, IL-6 expression and STAT3 phosphorylation (n = 94; p < 0.05). In conclusion, our data indicate that aberrant Akt signalling contributes to maintaining stemness in lung cancer TICs through a NF-kB/IL-6/STAT3 pathway and provide novel potential therapeutic targets for eliminating these malignant cells in NSCLC.

  1. HER2-induced metastasis is mediated by AKT/JNK/EMT signaling pathway in gastric cancer

    PubMed Central

    Choi, Yiseul; Ko, Young San; Park, Jinju; Choi, Youngsun; Kim, Younghoon; Pyo, Jung-Soo; Jang, Bo Gun; Hwang, Douk Ho; Kim, Woo Ho; Lee, Byung Lan

    2016-01-01

    AIM To investigated the relationships between HER2, c-Jun N-terminal kinase (JNK) and protein kinase B (AKT) with respect to metastatic potential of HER2-positive gastric cancer (GC) cells. METHODS Immunohistochemistry was performed on tissue array slides containing 423 human GC specimens. Using HER2-positve GC cell lines SNU-216 and NCI-N87, HER2 expression was silenced by RNA interference, and the activations of JNK and AKT were suppressed by SP600125 and LY294002, respectively. Transwell assay, Western blot, semi-quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining were used in cell culture experiments. RESULTS In GC specimens, HER2, JNK, and AKT activations were positively correlated with each other. In vitro analysis revealed a positive regulatory feedback loop between HER2 and JNK in GC cell lines and the role of JNK as a downstream effector of AKT in the HER2/AKT signaling pathway. JNK inhibition suppressed migratory