The Experts below are selected from a list of 3231 Experts worldwide ranked by ideXlab platform
Joseph Gera - One of the best experts on this subject based on the ideXlab platform.
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mtorc2 akt hsf1 hur constitute a feed forward loop regulating RICTOR expression and tumor growth in glioblastoma
Oncogene, 2018Co-Authors: Brent Holmes, Angelica Benavidesserrato, Ryan S Freeman, Kenna A Landon, Tariq Bashir, Robert N Nishimura, Joseph GeraAbstract:Overexpression of RICTOR has been demonstrated to result in increased mechanistic target of rapamycin C2 (mTORC2) nucleation and activity leading to tumor growth and increased invasive characteristics in glioblastoma multiforme (GBM). However, the mechanisms regulating RICTOR expression in these tumors is not clearly understood. In this report, we demonstrate that RICTOR is regulated at the level of mRNA translation via heat-shock transcription factor 1 (HSF1)-induced HuR activity. HuR is shown to directly bind the 3' untranslated region of the RICTOR transcript and enhance translational efficiency. Moreover, we demonstrate that mTORC2/AKT signaling activates HSF1 resulting in a feed-forward cascade in which continued mTORC2 activity is able to drive RICTOR expression. RNAi-mediated blockade of AKT, HSF1 or HuR is sufficient to downregulate RICTOR and inhibit GBM growth and invasive characteristics in vitro and suppress xenograft growth in mice. Modulation of AKT or HSF1 activity via the ectopic expression of mutant alleles support the ability of AKT to activate HSF1 and demonstrate continued HSF1/HuR/RICTOR signaling in the context of AKT knockdown. We further show that constitutive overexpression of HuR is able to maintain RICTOR expression under conditions of AKT or HSF1 loss. The expression of these components is also examined in patient GBM samples and correlative associations between the relative expression of these factors support the presence of these signaling relationships in GBM. These data support a role for a feed-forward loop mechanism by which mTORC2 activity stimulates RICTOR translational efficiency via an AKT/HSF1/HuR signaling cascade resulting in enhanced mTORC2 activity in these tumors.
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mtorc2 akt hsf1 hur constitute a feed forward loop regulating RICTOR expression and tumor growth in glioblastoma
bioRxiv, 2017Co-Authors: Brent Holmes, Angelica Benavidesserrato, Ryan S Freeman, Kenna A Landon, Tariq Bashir, Robert N Nishimura, Joseph GeraAbstract:Overexpression of RICTOR has been demonstrated to result in increased mTORC2 nucleation and activity leading to tumor growth and increased invasive characteristics in glioblastoma multiforme (GBM). However the mechanisms regulating RICTOR expression in these tumors is not clearly understood. In this report, we demonstrate that RICTOR is regulated at the level of mRNA translation via HSF1-induced HuR activity. HuR is shown to directly bind the 3′ UTR of the RICTOR transcript and enhance translational efficiency. Moreover, we demonstrate that mTORC2/AKT signaling activates HSF1 resulting in a feed-forward cascade in which continued mTORC2 activity is able to drive RICTOR expression. RNAi-mediated blockade of AKT, HSF1 or HuR is sufficient to downregulate RICTOR and inhibit GBM growth and invasive characteristics in vitro and suppresses xenograft growth in mice. We further demonstrate that constitutive overexpression of HuR is able to maintain RICTOR expression under conditions of AKT or HSF1 loss. In an additional level of regulation, miR-218 , a known RICTOR targeting miRNA is shown to be subject to mTORC2/STAT3-mediated repression. The expression of these components is also examined in patient GBM samples and correlative associations between the relative expression of these factors support the presence of these signaling relationships in GBM. These data support a role for a feed-forward loop mechanism by which mTORC2 activity stimulates RICTOR translational efficiency and suppresses miR-218 resulting in enhanced mTORC2 activity in these tumors.
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JR-AB-000 and JR-AB-011 bind to RICTOR and prevent RICTOR-mTOR association.
2017Co-Authors: Angelica Benavides-serrato, Brent Holmes, Kenna A Landon, Tariq Bashir, Jihye Lee, Michael E. Jung, Alan Lichtenstein, Joseph GeraAbstract:(A) Surface plasmon resonance analysis of JR-AB-000 binding to immobilized RICTOR, mSIN1 or mTOR as indicated (left panel). Binding sensorgrams of immobilized mTOR with RICTOR over the indicated concentration range (right panel). The Kon, Koff and Kd were calculated by simultaneous non-linear regression using a 1:1 binding model and BIAevaluation 3.1 software. (B) Competitive binding curves of RICTOR-mTOR association in the absence or presence of JR-AB2-011 or JR-AB2-000 as indicated (left panel). Analysis of selectivity of JR-AB2-011 (middle panel) or JR-AB2-000 (right panel) binding to RICTOR, Raptor, mLST8 or Deptor as shown. Samples were preincubated with inhibitors and RICTOR, Raptor, mLST8 or Deptor proteins as indicated and run over sensor chip containing immobilized mTOR. The IC50 values were calculated using the response units at the dissociation phase. (C) mTOR-Flag coupled beads binding to myc-RICTOR in the presence of increasing JR-AB2-011 (top panels) or JR-AB2-000 (bottom panels). myc-RICTOR was incubated with inhibitor for 1 h followed by incubation with FLAG agarose beads coupled to mTOR-Flag (mTOR-Flag beads). Binding of myc-RICTOR to mTOR-Flag beads (RICTOR-mTOR-Flag beads) was detected by immunoblotting with an anti-myc mAb. The amount of protein bound to FLAG agarose beads was detected with an anti-Flag mAb (loading control). Immunoblots were quantified via densitometric analyses and graphs are shown to the right of the blots. Three independent experiments were performed and one representative result is shown.
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mtorc2 activity is elevated in gliomas and promotes growth and cell motility via overexpression of RICTOR
Cancer Research, 2007Co-Authors: Janine Masri, Andrew Bernath, Jheralyn Martin, Raffi Vartanian, Alexander Funk, Joseph GeraAbstract:mTORC2 is a multimeric kinase composed of the mammalian target of rapamycin kinase (mTOR), mLST8, mSin1, and RICTOR. The complex is insensitive to acute rapamycin exposure and has shown functions in controlling cell growth and actin cytoskeletal assembly. mTORC2 has recently been shown to phosphorylate and activate Akt. Because approximately 70% of gliomas harbor high levels of activated Akt, we investigated whether mTORC2 activity was elevated in gliomas. In this study, we found that mTORC2 activity was elevated in glioma cell lines as well as in primary tumor cells as compared with normal brain tissue (P < 0.05). Moreover, we found that RICTOR protein and mRNA levels were also elevated and correlated with increased mTORC2 activity. Overexpression of RICTOR in cell lines led to increased mTORC2 assembly and activity. These lines exhibited increased anchorage-independent growth in soft agar, increased S-phase cell cycle distribution, increased motility, and elevated integrin beta(1) and beta(3) expression. In contrast, small interfering RNA-mediated knockdown of RICTOR inhibited these oncogenic activities. Protein kinase C alpha (PKC alpha) activity was shown to be elevated in RICTOR-overexpressing lines but reduced in RICTOR-knockdown clones, consistent with the known regulation of actin organization by mTORC2 via PKC alpha. Xenograft studies using these cell lines also supported a role for increased mTORC2 activity in tumorigenesis and enhanced tumor growth. In summary, these data suggest that mTORC2 is hyperactivated in gliomas and functions in promoting tumor cell proliferation and invasive potential due to increased complex formation as a result of the overexpression of RICTOR.
Mark A Magnuson - One of the best experts on this subject based on the ideXlab platform.
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RICTOR mtorc2 deficiency enhances keratinocyte stress tolerance via mitohormesis
Cell Death & Differentiation, 2017Co-Authors: Beatrice Tassone, Mark A Magnuson, Stefania Saoncella, Francesco Neri, Ugo Ala, Davide Brusa, Paolo Provero, Salvatore Oliviero, Chiara RigantiAbstract:How metabolic pathways required for epidermal tissue growth and remodeling influence the ability of keratinocytes to survive stressful conditions is still largely unknown. The mechanistic target of rapamycin complex 2 (mTORC2) regulates growth and metabolism of several tissues, but its functions in epidermal cells are poorly defined. RICTOR is an adaptor protein essential for mTORC2 activity. To explore the roles of mTORC2 in the epidermis, we have conditionally deleted RICTOR in mice via K14-Cre-mediated homologous recombination and found that its deficiency causes moderate tissue hypoplasia, reduced keratinocyte proliferation and attenuated hyperplastic response to TPA. Noteworthy, RICTOR-deficient keratinocytes displayed increased lifespan, protection from senescence, and enhanced tolerance to cellular stressors such as growth factors deprivation, epirubicin and X-ray in vitro and radioresistance in vivo. RICTOR-deficient keratinocytes exhibited changes in global gene expression profiles consistent with metabolic alterations and enhanced stress tolerance, a shift in cell catabolic processes from glycids and lipids to glutamine consumption and increased production of mitochondrial reactive oxygen species (ROS). Mechanistically, the resiliency of RICTOR-deficient epidermal cells relies on these ROS increases, indicating stress resistance via mitohormesis. Thus, our findings reveal a new link between metabolic changes and stress adaptation of keratinocytes centered on mTORC2 activity, with potential implications in skin aging and therapeutic resistance of epithelial tumors.
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fat cell specific ablation of RICTOR in mice impairs insulin regulated fat cell and whole body glucose and lipid metabolism
Diabetes, 2010Co-Authors: Anil Kumar, Mark A Magnuson, Susanna R Keller, John C Lawrence, Dae Young Jung, Jason K Kim, Thurl E HarrisAbstract:Abstract Objective - RICTOR is an essential component of mTOR complex (mTORC) 2, a kinase that phosphorylates and activates Akt, an insulin signaling intermediary that regulates glucose and lipid metabolism in adipose tissue, skeletal muscle and liver. To determine the physiological role of RICTOR/mTORC2 in insulin signaling and action in fat cells, we developed fat cell-specific RICTOR knockout (FRic -/- ) mice. Research Design and Methods - Insulin signaling and glucose and lipid metabolism were studied in FRic -/- fat cells. In vivo glucose metabolism was evaluated by hyperinsulinemic-euglycemic clamp. Results - Loss of RICTOR in fat cells prevents insulin-stimulated phosphorylation of Akt at S473 which, in turn, impairs the phosphorylation of downstream targets such as FoxO3a at T32 and AS160 at T642. However, GSK3β phosphorylation at S9 is not affected. The signaling defects in FRic -/- fat cells lead to impaired insulin-stimulated GLUT4 translocation to the plasma membrane and decreased glucose transport. Furthermore, RICTOR null fat cells are unable to suppress lipolysis in response to insulin leading to elevated circulating free fatty acids and glycerol. These metabolic perturbations are likely to account for defects observed at the whole body level of FRic -/- mice including glucose intolerance, marked hyperinsulinemia, insulin resistance in skeletal muscle and liver and hepatic steatosis. Conclusions - RICTOR/mTORC2 in fat cells plays an important role in whole body energy homeostasis by mediating signaling necessary for the regulation of glucose and lipid metabolism in fat cells.
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RICTOR phosphorylation on the thr 1135 site does not require mammalian target of rapamycin complex 2
PMC, 2010Co-Authors: Delphine R Boulbes, Mark A Magnuson, Chien Hung Chen, Tattym Shaikenov, Nitin K Agarwal, Timothy R Peterson, Terri Addona, Hasmik Keshishian, Steven A Carr, David M SabatiniAbstract:In animal cells, growth factors coordinate cell proliferation and survival by regulating the phosphoinositide 3-kinase/Akt signaling pathway. Deregulation of this signaling pathway is common in a variety of human cancers. The PI3K-dependent signaling kinase complex defined as mammalian target of rapamycin complex 2 (mTORC2) functions as a regulatory Ser-473 kinase of Akt. We find that activation of mTORC2 by growth factor signaling is linked to the specific phosphorylation of its component RICTOR on Thr-1135. The phosphorylation of this site is induced by the growth factor stimulation and expression of the oncogenic forms of ras or PI3K. RICTOR phosphorylation is sensitive to the inhibition of PI3K, mTOR, or expression of integrin-linked kinase. The substitution of wild-type RICTOR with its specific phospho-mutants in RICTOR null mouse embryonic fibroblasts did not alter the growth factor–dependent phosphorylation of Akt, indicating that the RICTOR Thr-1135 phosphorylation is not critical in the regulation of the mTORC2 kinase activity. We found that this RICTOR phosphorylation takes place in the mTORC2-deficient cells, suggesting that this modification might play a role in the regulation of not only mTORC2 but also the mTORC2-independent function of RICTOR. Mol Cancer Res; 8(6); 896–906. ©2010 AACR.
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a RICTOR myo1c complex participates in dynamic cortical actin events in 3t3 l1 adipocytes
Molecular and Cellular Biology, 2008Co-Authors: Nana G Hagan, Mark A Magnuson, Yenshou Lin, Joseph Avruch, Michael P CzechAbstract:Insulin signaling through phosphatidylinositol 3-kinase (PI 3-kinase) activates the protein kinase Akt through phosphorylation of its threonine 308 and serine 473 residues by the PDK1 protein kinase and the RICTOR-mammalian target of rapamycin complex (mTORC2), respectively. Remarkably, we show here that the RICTOR protein is also present in cultured adipocytes in complexes containing Myo1c, a molecular motor that promotes cortical actin remodeling. Interestingly, the RICTOR-Myo1c complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Furthermore, while RNA interference-directed silencing of RICTOR results in the expected attenuation of Akt phosphorylation at serine 473, depletion of Myo1c is without effect. In contrast, loss of either RICTOR or Myo1c inhibits phosphorylation of the actin filament regulatory protein paxillin at tyrosine 118. Furthermore, Myo1c-induced membrane ruffling of 3T3-L1 adipocytes is also compromised following RICTOR knockdown. Interestingly, neither the mTORC2 inhibitor rapamycin nor the PI 3-kinase inhibitor wortmannin affects paxillin tyrosine 118 phosphorylation. Taken together, our findings suggest that the RICTOR-Myo1c complex is distinct from mTORC2 and that Myo1c, in conjunction with RICTOR, participates in cortical actin remodeling events.
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muscle specific deletion of RICTOR impairs insulin stimulated glucose transport and enhances basal glycogen synthase activity
Molecular and Cellular Biology, 2008Co-Authors: Anil Kumar, Thurl E Harris, Mark A Magnuson, Susanna R Keller, Kin M Choi, John C LawrenceAbstract:RICTOR is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either RICTOR or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific RICTOR knockout mice. Muscle from male RICTOR knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking RICTOR, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in RICTOR knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking RICTOR was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in RICTOR knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, RICTOR in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.
Haiying Cheng - One of the best experts on this subject based on the ideXlab platform.
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RICTOR amplification defines a novel subset of patients with lung cancer who may benefit from treatment with mtorc1 2 inhibitors
Cancer Discovery, 2015Co-Authors: Haiying Cheng, Yiyu Zou, Balazs Halmos, Jeffrey S Ross, Kai Wang, Xuewen Liu, Siraj M Ali, Huijie Liu, Amit Verma, Cristina MontagnaAbstract:We identified amplification of RICTOR, a key component of the mTORC2, as the sole actionable genomic alteration in an 18-year-old never smoker with lung adenocarcinoma. It occurs in 13% of lung cancers (1016 cases) in TCGA and at a similar frequency in an independent cohort of 1,070 patients identified by genomic profiling. In the latter series, 11% of cases harbored RICTOR amplification as the only relevant genomic alteration. Its oncogenic roles were suggested by decreased lung cancer cell growth both in vitro and in vivo with RICTOR ablation, and the transforming capacity of RICTOR in a Ba/F3-cell system. The mTOR1/2 inhibitors were significantly more active against RICTOR-amplified lung cancer cells as compared to other agents targeting the PI3K/AKT/mTOR pathway. Moreover, an association between RICTOR amplification and sensitivities to mTOR1/2 inhibitors was observed. The index patient has been treated with mTOR1/2 inhibitors that led to tumor stabilization for over 18 months. SIGNIFICANCE: RICTOR amplification may define a novel and unique molecular subset of lung cancer patients who may benefit from treatment with mTOR1/2 inhibitors.
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abstract 3526 the mtorc2 component RICTOR plays a key role in lung cancer cell growth
Cancer Research, 2014Co-Authors: Haiying Cheng, Yiyu Zou, Alain C Borczuk, Wanglong Qiu, Bilal Piperdi, Mimi Kim, Balazs Halmos, Roman PerezsolerAbstract:RICTOR (Rapamycin-insensitive companion of mTOR protein) is a key component of mTORC2 (the mammalian target of rapamycin complex 2). One of the most-recognized targets for RICTOR-mTORC2 is AKT (Ser473). RICTOR also carries functions independent of mTORC2. RICTOR signaling has been suggested to play key roles in regulating cancer cell migration, invasion and metastasis in breast, ovarian, colorectal cancers and gliomas. The potential roles of RICTOR in lung cancer remain to be elucidated. We first examined the expression profile of RICTOR in primary lung tumor specimens and in lung cancer cell lines by immunohistochemistry. Among 125 FFPE patients9 specimen, ninety nine were stained positive for RICTOR (intensity 1+ to 3+). More interestingly, the RICTOR IHC expression is stronger in squamous cell lung cancer in comparison to adenocarcinoma. Furthermore, twenty four out of 36 lung cancer cell lines showed positive RICTOR IHC staining. Thus, RICTOR is expressed in most lung tumors. To investigate the role of RICTOR in lung cancer cell growth, we found that RICTOR knockdown by siRNA reduced colony formation in A549, HCC827 and Calu3 cells, regardless of KRAS or EGFR mutational status (A549 is KRAS mutated whereas HCC827 is EGFR mutated). Western blot confirmed RICTOR knockdown and also decreased level of its downstream pAKT S473. We further blocked RICTOR signaling by utilizing inducible shRNAs of RICTOR. Similar inhibition of lung cancer cell growth was observed. Moreover, to test the in vivo role of RICTOR, we preformed xenograft mouse experiments. When RICTOR was inducibly knockdown by the presence of doxycycline, the growth of A549 lung tumor xenografts was markedly reduced by 60% (P Citation Format: Haiying Cheng, Yiyu Zou, Alain Borczuk, Wanglong Qiu, Bilal Piperdi, Mimi Kim, Balazs Halmos, Roman Perez-Soler. The mTORC2 component RICTOR plays a key role in lung cancer cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3526. doi:10.1158/1538-7445.AM2014-3526
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RICTOR amplification to define a novel and unique subset of lung cancer patients
Journal of Clinical Oncology, 2014Co-Authors: Haiying Cheng, Balazs Halmos, Vincent A Miller, Jeffrey S Ross, Kai Wang, Xuewen Liu, Abraham Chachoua, Philip J Stephens, Roman Yelensky, Doron LipsonAbstract:8027 Background: Recent advances in molecularly tailored therapy have shifted the treatment paradigm in lung cancer. We recently identified amplification of RICTOR, a key component of the mTORC2 complex, as a new and sole genomic alteration in an 18-year-old never smoker with lung adenocarcinoma. We hypothesizethat RICTOR amplification, unlike previously reported molecular changes in the PI3K/AKT/mTOR pathways, may identify a new subgroup of lung cancer patients amenable to molecularly targeted therapy. Methods: The index patient had RICTOR amplification identified by next generation sequencing (NGS)- genomic profiling of 182 cancer-related genes performed with extracted DNA from FFPE specimens. We then reviewed the database of 1128 lung cancer patients who had profiling performed by Foundation Medicine. Additionally, we examined the in vitro cytotoxicity of 6 classes of drugs against different components of the PI3K/AKT/mTOR pathways in RICTOR-amplified H23 lung cancer cells. Results: RICTOR amplificatio...
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abstract 2033 identification of a novel genetic abnormality the amplification of RICTOR rapamycin insensitive companion of mtor in a patient with non small cell lung cancer
Cancer Research, 2013Co-Authors: Haiying Cheng, Bilal Piperdi, Balazs Halmos, Edward L Schwartz, Changcheng Zhu, Zhenfeng Zhang, Vincent A Miller, Roman PerezsolerAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Despite recent advances, clinically-relevant oncogenic drivers have not been identified in about half of lung adenocarcinormas. Here, we report RICTOR (rapamycin-insensitive companion of mTOR) amplification as a novel genetic abnormality in a young patient with non-small cell lung cancer. This male patient was a never smoker and was diagnosed with lung adenocarcinorma at the age of 18. He initially presented with refractory cough and persistent left lung lesions. Pathology was consistent with lung adenocarcinorma, poorly differentiated. Extensive molecular testing was negative for common genetic alterations in K-RAS, EGFR, ALK, HER2, RET, AKT, PIK3CA, BRAF and ROS1. His tumor was subsequently subjected to next generation DNA sequencing (FoundationOne, Foundation Medicine, Cambridge, MA) targeting a cohort of more than 180 cancer-related genes. This analysis identified RICTOR amplification of at least 6 copies as the sole DNA abnormality. Immunohistochemistry further documented RICTOR overexpression in the patient's lung tumor. The neoplastic lung tissue exhibited diffuse and strong cytoplasmic staining for RICTOR, compared to surrounding non-neoplastic tissues. Furthermore, the tumor tissue showed diffuse overexpression of phospho-S473 Akt, an essential downstream target of RICTOR. RICTOR is a key component of mTORC2 complex, where it modifies actin cytoskeleton organization, cell proliferation and survival. Activation of RICTOR signaling has been suggested to play critical roles in regulating cancer cell migration, invasion and metastasis in breast, ovarian, and colorectal cancers and gliomas. To our knowledge, this is the first report of RICTOR amplification in lung cancer. Moreover, we found that blockade of the RICTOR-mTORC2 pathway by siRNA-mediated RICTOR knockdown or with pharmacological inhibitors led to decreased phosphorylation of S473-AKT and growth inhibition in lung cancer cell lines. Thus, RICTOR amplification may represent a potential novel target in lung cancer. We will perform further studies to determine whether RICTOR amplification is a unique molecular subtype of lung cancer. Further, we hope to identify which targeted drugs are active against tumors with RICTOR amplification and overexpression. Citation Format: Haiying Cheng, Bilal Piperdi, Edward L. Schwartz, Changcheng Zhu, Zhenfeng Zhang, Balazs Halmos, Vincent Miller, Roman Perez-Soler. Identification of a novel genetic abnormality, the amplification of RICTOR (rapamycin-insensitive companion of mTOR), in a patient with non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2033. doi:10.1158/1538-7445.AM2013-2033
Chunsun Dai - One of the best experts on this subject based on the ideXlab platform.
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RICTOR mtorc2 signaling mediates tgfβ1 induced fibroblast activation and kidney fibrosis
Kidney International, 2015Co-Authors: Jiafa Ren, Xin Liu, Lei Jiang, Weiping Yuan, Junwei Yang, Chunsun DaiAbstract:The mammalian target of rapamycin (mTOR) was recently identified in two structurally distinct multiprotein complexes: mTORC1 and mTORC2. Previously, we found that RICTOR/mTORC2 protects against cisplatin-induced acute kidney injury, but the role and mechanisms for RICTOR/mTORC2 in TGFβ1-induced fibroblast activation and kidney fibrosis remains unknown. To study this, we initially treated NRK-49F cells with TGFβ1 and found that TGFβ1 could activate RICTOR/mTORC2 signaling in cultured cells. Blocking RICTOR/mTORC2 signaling with RICTOR or Akt1 small interfering RNAs markedly inhibited TGFβ1-induced fibronection and α-smooth muscle actin expression. Ensuing western blotting or immunostaining results showed that RICTOR/mTORC2 signaling was activated in kidney interstitial myofibroblasts from mice with unilateral ureteral obstruction. Next, a mouse model with fibroblast-specific deletion of RICTOR was generated. These knockout mice were normal at birth and had no obvious kidney dysfunction or kidney morphological abnormality within 2 months of birth. Compared with control littermates, the kidneys of RICTOR knockout mice developed less interstitial extracellular matrix deposition and inflammatory cell infiltration at 1 or 2 weeks after ureteral obstruction. Thus our study suggests that RICTOR/mTORC2 signaling activation mediates TGFβ1-induced fibroblast activation and contributes to the development of kidney fibrosis. This may provide a therapeutic target for chronic kidney diseases.
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RICTOR mtorc2 protects against cisplatin induced tubular cell death and acute kidney injury
Kidney International, 2014Co-Authors: Lei Jiang, Weiping Yuan, Junwei Yang, Junhua Mao, Zhifeng Zeng, Li Fang, Chunsun DaiAbstract:The mammalian target of rapamycin (mTOR) plays a critical role for cell growth and survival in many cell types. While substantial progress has been made in understanding the abnormal activation of mTORC1 in the pathogenesis of kidney disease, little is known about mTORC2 in kidney disease such as acute kidney injury (AKI). To study this, we generated a mouse model with tubule-specific deletion of RICTOR (Tubule-RICTOR-/-). The knockouts were born normal and no obvious kidney dysfunction or kidney morphologic abnormality was found within 2 months of birth. However, ablation of RICTOR in the tubular cells exacerbated cisplatin-induced AKI compared to that in the control littermates. As expected, tubular cell apoptosis, Akt phosphorylation (Ser473), and autophagy were induced in the kidneys from the control littermates by cisplatin treatment. Less cell autophagy or Akt phosphorylation and more cell apoptosis in the kidneys of the knockout mice were identified compared with those in the control littermates. In NRK-52E cells in vitro , RICTOR siRNA transfection sensitized cell apoptosis to cisplatin but with reduced cisplatin-induced autophagy. Metformin, an inducer of autophagy, abolished cell death induced by RICTOR siRNA and cisplatin. Thus, endogenous RICTOR/mTORC2 protects against cisplatin-induced AKI, probably mediated by promoting cell survival through Akt signaling activation and induction of autophagy.
Shiyong Sun - One of the best experts on this subject based on the ideXlab platform.
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RICTOR an essential component of mtor complex 2 undergoes caspase mediated cleavage during apoptosis induced by multiple stimuli
Apoptosis, 2021Co-Authors: Liqun Zhao, Lei Zhu, Guoqing Qian, Z Chen, Shiyong SunAbstract:Caspase-mediated cleavage of proteins ensures the irreversible commitment of cells to undergo apoptosis, and is thus a hallmark of apoptosis. Rapamycin-insensitive companion of mTOR (RICTOR) functions primarily as a core and essential component of mTOR complex 2 (mTORC2) to critically regulate cellular homeostasis. However, its role in the regulation of apoptosis is largely unknown. In the current study, we found that RICTOR was cleaved to generate two small fragments at ~ 50 kD and ~ 130 kD in cells undergoing apoptosis upon treatment with different stimuli such as the death ligand, TRAIL, and the small molecule, AZD9291. This cleavage was abolished when caspases were inhibited and could be reproduced when directly incubating RICTOR protein and caspase-3 in vitro. Furthermore, the cleavage site of caspase-3 on RICTOR was mapped at D1244 (VGVD). These findings together robustly demonstrate that RICTOR is a substrate of caspase-3 and undergoes cleavage during apoptosis. These results add new information for understanding the biology of RICTOR in the regulation of cell survival and growth.
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RICTOR undergoes glycogen synthase kinase 3 gsk3 dependent fbxw7 mediated ubiquitination and proteasomal degradation
Journal of Biological Chemistry, 2015Co-Authors: Junghui Koo, Zixu Mao, Fadlo R Khuri, Shiyong SunAbstract:RICTOR, an essential component of mTOR complex 2 (mTORC2), plays a pivotal role in regulating mTOR signaling and other biological functions. Posttranslational regulation of RICTOR (e.g. via degradation) and its underlying mechanism are largely undefined and thus are the focus of this study. Chemical inhibition of the proteasome increased RICTOR ubiquitination and levels. Consistently, inhibition of FBXW7 with various genetic means including knockdown, knock-out, and enforced expression of a dominant-negative mutant inhibited RICTOR ubiquitination and increased RICTOR levels, whereas enforced expression of FBXW7 decreased RICTOR stability and levels. Moreover, we detected an interaction between FBXW7 and RICTOR. Hence, RICTOR is degraded through an FBXW7-mediated ubiquitination/proteasome mechanism. We show that this process is dependent on glycogen synthase kinase 3 (GSK3): GSK3 was associated with RICTOR and directly phosphorylated the Thr-1695 site in a putative CDC4 phospho-degron motif of RICTOR; mutation of this site impaired the interaction between RICTOR and FBXW7, decreased RICTOR ubiquitination, and increased RICTOR stability. Finally, enforced activation of Akt enhanced RICTOR levels and increased mTORC2 activity as evidenced by increased formation of mTORC2 and elevated phosphorylation of Akt, SGK1, and PKCα. Hence we suggest that PI3K/Akt signaling may positively regulate mTORC2 signaling, likely through suppressing GSK3-dependent RICTOR degradation.
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enhancing mammalian target of rapamycin mtor targeted cancer therapy by preventing mtor raptor inhibition initiated mtor RICTOR independent akt activation
Cancer Research, 2008Co-Authors: Xuerong Wang, Fadlo R Khuri, Ping Yue, Young Ae Kim, Shiyong SunAbstract:It has been shown that mammalian target of rapamycin (mTOR) inhibitors activate Akt while inhibiting mTOR signaling. However, the underlying mechanisms and the effect of the Akt activation on mTOR-targeted cancer therapy are unclear. The present work focused on addressing the role of mTOR/RICTOR in mTOR inhibitor-induced Akt activation and the effect of sustained Akt activation on mTOR-targeted cancer therapy. Thus, we have shown that mTOR inhibitors increase Akt phosphorylation through a mechanism independent of mTOR/RICTOR because the assembly of mTOR/RICTOR was inhibited by mTOR inhibitors and the silencing of RICTOR did not abrogate mTOR inhibitor-induced Akt activation. Moreover, Akt activation during mTOR inhibition is tightly associated with development of cell resistance to mTOR inhibitors. Accordingly, cotargeting mTOR and phosphatidylinositol 3-kinase/Akt signaling prevents mTOR inhibition-initiated Akt activation and enhances antitumor effects both in cell cultures and in animal xenograft models, suggesting an effective cancer therapeutic strategy. Collectively, we conclude that inhibition of the mTOR/raptor complex initiates Akt activation independent of mTOR/RICTOR. Consequently, the sustained Akt activation during mTOR inhibition will counteract the anticancer efficacy of the mTOR inhibitors.
