Rictor Protein Levels Research Articles

GCN5L1-mediated acetylation prevents Rictor degradation in cardiac cells after hypoxic stress

Cardiomyocyte apoptosis and cardiac fibrosis are the leading causes of mortality in patients with ischemic heart disease. As such, these processes represent potential therapeutic targets to treat heart failure resulting from ischemic insult. We previously demonstrated that the mitochondrial acetyltransferase protein GCN5L1 regulates cardiomyocyte cytoprotective signaling in ischemia-reperfusion injury in vivo and hypoxia-reoxygenation injury in vitro. The current study investigated the mechanism underlying GCN5L1-mediated regulation of the Akt/mTORC2 cardioprotective signaling pathway. Rictor protein levels in cardiac tissues from human ischemic heart disease patients were significantly decreased relative to non-ischemic controls. Rictor protein levels were similarly decreased in cardiac AC16 cells following hypoxic stress, while mRNA levels remained unchanged. The reduction in Rictor protein levels after hypoxia was enhanced by the knockdown of GCN5L1, and was blocked by GCN5L1 overexpression. These findings correlated with changes in Rictor lysine acetylation, which were mediated by GCN5L1 acetyltransferase activity. Rictor degradation was regulated by proteasomal activity, which was antagonized by increased Rictor acetylation. Finally, we found that GCN5L1 knockdown restricted cytoprotective Akt signaling, in conjunction with decreased mTOR abundance and activity. In summary, these studies suggest that GCN5L1 promotes cardioprotective Akt/mTORC2 signaling by maintaining Rictor protein levels through enhanced lysine acetylation.

Circulating exosome-like vesicles of humans with nondiabetic obesity impaired islet β-cell proliferation, which was associated with decreased Omentin-1 protein cargo

The regulation of β-cell mass in the status of nondiabetic obesity remains not well understood. We aimed to investigate the role of circulating exosome-like vesicles (ELVs) isolated from humans with simple obesity in the regulation of islet β-cell mass. Between June 2017 and July 2019, 81 subjects with simple obesity and 102 healthy volunteers with normal weight were recruited. ELVs were isolated by ultra-centrifugation. The proliferations of β-cells and islets were measured by 5-ethynl-2′-deoxyuridine (EdU). Protein components in ELVs were identified by Quantitative Proteomic Analysis and verified by Western blot and ELISA. The role of specific exosomal protein was analyzed by gain-of-function approach in ELVs released by 3T3-L1 preadipocytes. Circulating ELVs from subjects with simple obesity inhibited β-cell proliferation in vitro without affecting its apoptosis, secretion, and inflammation. The protein levels of Rictor and Omentin-1 were downregulated in circulating ELVs from subjects with simple obesity and associated with the obesity-linked pathologic conditions. The ELV-carried Omentin-1 and Omentin-1 protein per se were validated to increase β-cell proliferation and activate Akt signaling pathway. Moreover, Omentin-1 in ELVs was downregulated by insulin. The circulating ELVs may act as a negative regulator for β-cell mass in nondiabetic obesity through inhibiting β-cell proliferation. This effect was associated with downregulated Omentin-1 protein in ELVs. This newly identified ELV-carried protein could be a mediator linking insulin resistance to impaired β-cell proliferation and a new potential target for increasing β-cell mass in obesity and T2DM.

MTORC1/2 Inhibitor Served as a More Ideal Agent Against the Growth of Mouse Lymphocytic Leukemia Both In Vitro and In Vivo.

Chronic lymphocytic leukemia (CLL) still remains an incurable disease as the cells evade apoptosis, which is an obstacle for current therapeutic approaches. Therefore, our aim was to identify an ideal target of leukemic cell growth for developing inhibitors. Mouse lymphocytic leukemia cell line L1210, human Toledo cells and a DBA/2 mouse graft model were used to analyze the activity of dual mTORC1/2 inhibitor AZD2014s. Western blotting and flow cytometry were performed to determine the mechanism. AZD2014 inhibited L1210 and human Toledo cell proliferation. Treatment with AZD2014 reduced the phosphorylation levels of S6K1 and 4EBP1 and the protein levels of Rictor, a component of the mTORC2 pathway. AZD2014 induced cell cycle arrest at the G0-G1 phase by reducing the expression of cyclin D1 and CDK4. Oral administration of AZD2014 significantly inhibited the growth of L1210 cell grafts in DBA/2 mice. The mTORC1/2 inhibitor may be a better therapeutic agent compared to PI3K/mTORC1 inhibitors for treating patients with CLL.

29P - Anticancer activity of the mTOR inhibitor (everolimus) and dual mTORC1/mTORC2 Inhibitor (AZD2014) on mouse lymphocytic leukemia both in vitro and in vivo

The mammalian target of rapamycin (mTOR) controls cell growth and enlargement and has been found to be aberrant in a wide variety of malignancies. Allosteric mTOR inhibitors, which inhibit mTORC1 but not mTORC2, result in feedback activation of AKT signaling, which can attenuate their antitumor activity. AZD2014 is a second generation mTOR inhibitor that blocks activation of both mTORC1 and mTORC2, and activates apoptosis in cancer cells without activation of AKT signaling. Here, we investigated the therapeutic efficacy of everolimus and AZD2014 in lymphocytic leukemia cell line (L1210) and mouse xenograft model. Cytotoxicity effect of AZD2014 and everolimus in L1210 cells was assessed after 24 and 48 h by using cell viability assays, clonogenic survival assays, and cell cycle analyses. Cell cycle, mTOR signal transduction pathway and the relative regulatory molecules were examined in mTOR inhibitor-treated L1210 cells by western blotting to detect protein expression. Then, the in vivo anti-leukemic effect of AZD2014 was assessed in L1210 cell-transplanted DBA/2 mice. AZD2014 significantly inhibited L1210 cell proliferation with an IC50 of 100nM. In contrast, everolimus was a poor growth inhibitor of L1210 cells (IC50 > 200nM). Treatment with AZD2014 was more effective than RAD001 to down-regulate the levels of mTORC1 downstream effectors, including S6K1, 4EBP1,eIF4E and a significant decrease in protein levels of rictor, a component of mTORC2 protein.We also observed inhibition of mTORincreased G1 arrest by reducing cyclin D1 and CDK4 levels, which augmented growth-inhibitory effect of L1210 cells. In vivo, AZD2014 oral administration significantly inhibited the growth of L1210 cell xenograft in DBA/2 mice, and the mice survival was dramatically improved. These data indicate that AZD2014 may be a better therapeutic agent than mTORC1 inhibitors to enhance the antitumor activity of lymphocytic leukemia both in vitro and under xenograft model in vivo conditions. Antitumor activity of AZD2014 was inhibited of both mTORC1 and mTORC2 activity and cell cycle arrest, leading to lymphocytic leukemia growth inhibition.

Cardiac mTOR complex 2 preserves ventricular function in pressure-overload hypertrophy.

Mammalian target of rapamycin (mTOR), a central regulator of growth and metabolism, has tissue-specific functions depending on whether it is part of mTOR complex 1 (mTORC1) or mTORC2. We have previously shown that mTORC1 is required for adaptive cardiac hypertrophy and maintenance of function under basal and pressure-overload conditions. In the present study, we aimed to identify functions of mTORC2 in the heart. Using tamoxifen-inducible cardiomyocyte-specific gene deletion, we generated mice deficient for cardiac rapamycin-insensitive companion of mTOR (rictor), an essential and specific component of mTORC2. Under basal conditions, rictor deficiency did not affect cardiac growth and function in young mice and also had no effects in adult mice. However, transverse aortic constriction caused dysfunction in the rictor-deficient hearts, whereas function was maintained in controls after 1 week of pressure overload. Adaptive increases in cardiac weight and cardiomyocyte cross-sectional area, fibrosis, and hypertrophic and metabolic gene expression were not different between the rictor-deficient and control mice. In control mice, maintained function was associated with increased protein levels of rictor, protein kinase C (PKC)βII, and PKCδ, whereas rictor ablation abolished these increases. Rictor deletion also significantly decreased PKCε at baseline and after pressure overload. Our data suggest that reduced PKCε and the inability to increase PKCβII and PKCδ abundance are, in accordance with their known function, responsible for decreased contractile performance of the rictor-deficient hearts. Our study demonstrates that mTORC2 is implicated in maintaining contractile function of the pressure-overloaded male mouse heart.

Abstract 511: Rictor/mTORC2 drives formation, progression and therapeutic resistance of HER2-amplified breast cancers

Abstract The phosphatidyl inositol-3-kinase (PI3K)/Akt signaling pathway is aberrantly activated in nearly 60% of breast cancers, through HER2 amplification, PIK3CA mutation, PTEN inactivation, and other alterations. The mTOR complexes mTORC1 and mTORC2 operate as activators (mTORC2) or effectors (mTORC1) of Akt. We found that mRNA upregulation in the mTORC2 cofactors RICTOR and MAPKAP1 correlated with poor survival in breast cancer patients, while upregulation in the mTORC1 cofactor RPTOR did not. Genomic gains of RICTOR and MAPKAP1 occurred more frequently in breast cancers with PI3K pathway alterations than genomic RPTOR gains. Importantly, Rictor protein levels were increased in invasive over non-invasive breast tumors, while Raptor levels were not. RICTOR gene ablation in a transgenic mouse model of HER2-amplified breast cancer delayed tumorigenesis and decreased lung metastasis, Akt-S473 phosphorylation, cell growth and survival. In human HER2-amplified breast cancer cells, Rictor loss, but not Raptor loss, decreased Akt-S473 phosphorylation, reducing cell survival and motility/invasion. Interestingly, Rictor/mTORC2 loss or treatment with a dual mTORC1/2 inhibitor improved lapatinib-induced cell killing in parental and lapatinib-resistant tumor cells to a greater extent than mTORC1 inhibition. Akt re-activation rescued cell survival, but not motility/invasion, in Rictor-depleted cells. However, Rictor loss caused accumulation of the Rac inhibitor RhoGDI2, thus impairing Rac1-dependent invasion. We conclude that HER2-amplified breast cancers use Rictor/mTORC2 to drive Akt-mediated cell survival and Rac1-mediated cellular invasion. These studies support additional studies into mTORC2-specific inhibitors. Citation Format: Meghan M. Morrison, Bayley Jones, Violeta Sanchez, Monica V. Estrada, Donna Hicks, Michelle Williams, Dana Brantley-Sieders, Rebecca Cook. Rictor/mTORC2 drives formation, progression and therapeutic resistance of HER2-amplified breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 511. doi:10.1158/1538-7445.AM2015-511

Abstract B28: Rictor/mTORC2 drives formation, progression, and therapeutic resistance of HER2-amplified breast cancers

Abstract Although HER2-targeted therapies substantially improve the outcome for patients with HER2-amplified breast cancer, resistance to HER2 targeting is frequently observed. HER2-amplified tumors depend on the HER2-mediated activation of the phosphatidylinositol-3kinase (PI3K)/Akt pathway, which becomes reactivated in many therapeutically resistant HER2-amplified tumors. We investigated the mTOR complexes mTORC1 and mTORC2 as potential therapeutic targets in HER2-amplified breast cancers, based on their roles as activators (mTORC2) or effectors (mTORC1) of Akt. We found that genomic gains in Rictor, encoding a necessary mTORC2 cofactor, correlated with poor survival in breast cancer patients, while genomic gains in Raptor, an mTORC1 cofactor, did not. Rictor protein levels were increased in invasive breast cancers as compared to ductal carcinoma in situ (DCIS). Conditional Rictor gene ablation decreased growth, cell survival and invasion of human HER2-amplified breast cancer cells by blocking Akt signaling. Treatment of HER2 positive cells with the HER2/EGFR inhibitor Lapatinib caused Rictor upregulation. However, Rictor knockdown improved cell killing in Lapatinib-treated cells and impaired Akt signaling and cell survival in Lapatinib-resistant HER2 positive breast cancer cells. Together, these results demonstrate the pivotal role of mTORC2 in HER2-amplified breast cancers and support efforts to develop mTORC2-targeted therapies. Citation Format: Meghan M. Morrison, Bayley A. Jones, Donna J. Hicks, Violeta Sanchez, Valeria M. Estrada, Michelle M. Williams, Dana Brantley-Sieders, Rebecca S. Cook. Rictor/mTORC2 drives formation, progression, and therapeutic resistance of HER2-amplified breast cancers. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B28.

MTORC1 and mTORC2 Regulate EMT, Motility, and Metastasis of Colorectal Cancer via RhoA and Rac1 Signaling Pathways

Activation of phosphoinositide 3-kinase (PI3K)/Akt signaling is associated with growth and progression of colorectal cancer (CRC). We have previously shown that the mTOR kinase, a downstream effector of PI3K/Akt signaling, regulates tumorigenesis of CRC. However, the contribution of mTOR and its interaction partners toward regulating CRC progression and metastasis remains poorly understood. We found that increased expression of mTOR, Raptor, and Rictor mRNA was noted with advanced stages of CRC, suggesting that mTOR signaling may be associated with CRC progression and metastasis. mTOR, Raptor, and Rictor protein levels were also significantly elevated in primary CRCs (stage IV) and their matched distant metastases compared with normal colon. Inhibition of mTOR signaling, using rapamycin or stable inhibition of mTORC1 (Raptor) and mTORC2 (Rictor), attenuated migration and invasion of CRCs. Furthermore, knockdown of mTORC1 and mTORC2 induced a mesenchymal-epithelial transition (MET) and enhanced chemosensitivity of CRCs to oxaliplatin. We observed increased cell-cell contact and decreased actin cytoskeletal remodeling concomitant with decreased activation of the small GTPases, RhoA and Rac1, upon inhibition of both mTORC1 and mTORC2. Finally, establishment of CRC metastasis in vivo was completely abolished with targeted inhibition of mTORC1 and mTORC2 irrespective of the site of colonization. Our findings support a role for elevated mTORC1 and mTORC2 activity in regulating epithelial-mesenchymal transition (EMT), motility, and metastasis of CRCs via RhoA and Rac1 signaling. These findings provide the rationale for including mTOR kinase inhibitors, which inhibit both mTORC1 and mTORC2, as part of the therapeutic regimen for CRC patients.

Abstract LB-238: Posttranscriptional regulation of rictor by protein kinase D in human colon cancer cells

Abstract Background: mTORC2 phosphorylates Akt at Ser473 thus resulting in Akt activation; knockdown of rictor, a component of mTORC2, blocks Ser473 phosphorylation. Knockdown of rictor leads to growth inhibition and induces apoptosis in colorectal cancer (CRC) cells. However, the molecular mechanism(s) involved in the regulation of rictor expression remains unclear. Protein kinase D (PKD), a serine/threonine kinase family that includes PKD1, PKD2 and PKD3, has been implicated in the regulation of cell proliferation and apoptosis. Recently, PKD3 has been shown to increase Akt phosphorylation at Ser473. The purpose of this study was to investigate the regulation of rictor protein levels by protein kinase D (PKD) in CRC cells. Methods: HT29 and HCT116 CRC cells were treated with MG132, a proteasome inhibitor; cycloheximide (CHX), a protein synthesis inhibitor; Gö6976, a PKC and PKD inhibitor; Gö6983, a PKC inhibitor; or CID755673, a selective PKD inhibitor. Cells were transfected with plasmids encoding PKD1 or PKD2, or infected with an adenovirus construct expressing PKD3. Whole cell lysates were extracted and Western blot analysis performed for rictor protein expression. Total RNA was extracted and real-time RT-PCR was performed for rictor mRNA expression. To investigate whether PKD and rictor are co-localized, PKD and rictor expression was analyzed by immunohistochemistry in human CRCs, liver metastases and corresponding normal mucosa (AccuMax tissue array). Results: MG132 treatment increased rictor protein levels although raptor and mTOR expression was only slightly increased. Moreover, increased Akt Ser473 phosphorylation was observed. Treatment with CHX decreased rictor protein levels in a time-dependent fashion suggesting that rictor is a target of proteasomal degradation in CRC cells. Treatment of HT29 and HCT116 cells with Gö6976, but not Gö6983, decreased rictor protein expression. Consistently, treatment with the selective PKD inhibitor, CID755673, decreased rictor protein expression and inhibited CRC cell growth. Overexpression of PKD1, PKD2 or PKD3 increased rictor protein levels without affecting rictor mRNA expression. In addition, overexpression of PKD3 inhibited rictor degradation suggesting that PKDs are critical for rictor protein expression. Rictor as well as PKD1, PKD2 and PKD3 were up-regulated in primary CRCs and liver metastases compared with normal mucosa, implying a potential role of PKDs upstream of the rictor signaling pathway in the pathogenesis of CRC. Conclusions: Our results demonstrate that PKD post-transcriptionally regulates rictor protein levels in CRC cells. Importantly, these data suggest that PKD-mediated CRC growth is due, at least in part, to an mTORC2-dependent signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-238.