Protein Kinase mTOR Research Articles

The mRNA encoding the JUND tumor suppressor detains nuclear RNA-binding proteins to assemble polysomes that are unaffected by mTOR

One long-standing knowledge gap is the role of nuclear proteins in mRNA translation. Nuclear RNA helicase A (DHX9/RHA) is necessary for the translation of the mRNAs of JUND (JunD proto-oncogene AP-1 transcription factor subunit) and HIV-1 genes, and nuclear cap-binding protein 1 (NCBP1)/CBP80 is a component of HIV-1 polysomes. The protein kinase mTOR activates canonical messenger ribonucleoproteins by post-translationally down-regulating the eIF4E inhibitory protein 4E-BP1. We posited here that NCBP1 and DHX9/RHA (RHA) support a translation pathway of JUND RNA that is independent of mTOR. We present evidence from reciprocal immunoprecipitation experiments indicating that NCBP1 and RHA both are components of messenger ribonucleoproteins in several cell types. Moreover, tandem affinity and RT-quantitative PCR results revealed that JUND mRNA is a component of a previously unknown ribonucleoprotein complex. Results from the tandem IP indicated that another component of the JUND-containing ribonucleoprotein complex is NCBP3, a recently identified ortholog of NCBP2/CBP20. We also found that NCBP1, NCBP3, and RHA, but not NCBP2, are components of JUND-containing polysomes. Mutational analysis uncovered two dsRNA-binding domains of RHA that are necessary to tether JUND-NCBP1/NCBP3 to polysomes. We also found that JUND translation is unaffected by inhibition of mTOR, unless RHA was down-regulated by siRNA. These findings uncover a noncanonical cap-binding complex consisting of NCBP1/NCBP3 and RHA substitutes for the eukaryotic translation initiation factors 4E and 4G and activates mTOR-independent translation of the mRNA encoding the tumor suppressor JUND.

Metformin rescues rapamycin-induced mitochondrial dysfunction and attenuates rheumatoid arthritis with metabolic syndrome

BackgroundRapamycin, an inhibitor of the serine/threonine protein kinase mTOR, is an immunosuppressant used to treat renal transplant recipients, but it can cause endothelial and mitochondrial dysfunction. Metformin is used for the treatment of type 2 diabetes and was reported to exert therapeutic effects against rheumatoid arthritis and obesity by improving mitochondrial dysfunction via the activation of fibroblast growth factor 21. We investigated the therapeutic effects of rapamycin–metformin combination therapy in obese mice with collagen-induced arthritis (CIA).MethodsMouse embryonic fibroblasts were treated with rapamycin, metformin, or rapamycin–metformin, and their respiratory level and mitochondrial gene expression were assayed. Mice were fed a high-fat diet, immunized with type II collagen, and subsequently treated with rapamycin–metformin daily for 10 weeks.ResultsRapamycin-treated cells exhibited dysfunction of mitochondrial respiration and decreased mitochondrial gene expression compared with rapamycin–metformin-treated cells. Moreover, rapamycin–metformin reduced the clinical arthritis score and the extent of histological inflammation and improved the metabolic profile in obese mice with CIA. Rapamycin–metformin enhanced the balance between T helper 17 and regulatory T cells in vitro and in vivo.ConclusionsThese results suggest that rapamycin–metformin is a potential therapeutic option for autoimmune arthritis.

Tracking mTORC1 Activity in the Nucleus: Distinct Regulation Revealed by Locationspecific Inhibition of Akt

The mechanistic target of rapamycin complex 1 (mTORC1) is a kinase complex that contains the protein kinase mTOR, the key scaffolding protein Raptor, and several other core components. It is known to sense diverse upstream signals, such as growth factors, nutrient and energy status. When activated, mTORC1 integrates these signals to critically regulate distinct cellular processes, including cell growth, metabolism, and autophagy. An intriguing question is how mTORC1 is spatially regulated to exert widespread controls on downstream effectors. Besides the well studied mTORC1 activation at the lysosome, the regulation of this complex within other subcellular compartments is less clear. Previously, we showed that growth factor induces nuclear mTORC1 activity by utilizing a genetically encoded mTORC1 activity reporter targeted to the nucleus, providing initial evidence for active mTORC1 in the nucleus. Here we probe the regulation of growth factor‐induced nuclear mTORC1 activity by combining location‐selective kinase inhibition, live‐cell imaging and cellular biochemistry. We developed Akt Substrate‐based Tandem Occupancy Peptide Sponge (Akt‐STOPS) that allows location‐specific perturbation of Akt, a critical upstream kinase, at distinct subcellular compartments. Expression of Akt‐STOPS in the nucleus largely suppressed the growth factor stimulated Akt activity without significant inhibition of cytosolic Akt activity. Using Akt‐STOPS, we showed that growth factor‐stimulated nuclear mTORC1 activity requires nuclear Akt activity. Furthermore, we showed that growth factor stimulation induces nuclear translocation of Raptor, which is sensitive to importin inhibition, and that Raptor forms a complex with mTOR in the nucleus . The nuclear translocation of Raptor specifically depends on the nuclear Akt activity, as expression of nuclear Akt‐STOPS significantly lowered the nuclear translocation of Raptor and reduced the phosphorylation of RanBP, a critical regulator of Ran GTPase. Lastly, localizing exogenous Raptor to the nucleus in Raptor knock‐down cells rescues nuclear mTORC1 activity. Taken together, these results reveal a new mode of dynamic regulation of mTORC1 by recruiting a critical scaffolding protein, distinct from the mode of lysosomal mTORC1 activation. Diversity in the regulation of mTORC1 could lead to specificity in functional controls and also provide opportunities for selective targeting.Support or Funding InformationThis work was supported by NIH grants, R35 CA197622 and R01 GM111665 to J.Z., and NS047101 to UCSD Microscopy Core.

MTOR pathway somatic variants and the molecular pathogenesis of hemimegalencephaly.

ObjectivesRecently, defects in the protein kinase mTOR (mammalian target of rapamycin) and its associated pathway have been correlated with hemimegalencephaly (HME). mTOR acts as a central regulator of important physiological cellular functions such as growth and proliferation, metabolism, autophagy, death, and survival. This study was aimed at identifying specific variants in mTOR signaling pathway genes in patients diagnosed with HME.MethodsUsing amplicon and whole exome sequencing (WES) of resected brain and paired blood samples from five HME patients, we were able to identify pathogenic mosaic variants in the mTOR pathway genes MTOR, PIK3CA, and DEPDC5.ResultsThese results strengthen the hypothesis that somatic variants in PI3K‐Akt‐mTOR pathway genes contribute to HME. We also describe one patient presenting with a pathogenic variant on DEPDC5 gene, which reinforces the role of DEPDC5 on cortical structural changes due to mTORC1 hyperactivation. These findings also provide insights into when in brain development these variants occurred. An early developmental variant is expected to affect a larger number of cells and to result in a larger malformation, whereas the same variant occurring later in development would cause a minor malformation.SignificanceIn the future, numerous somatic variants in known or new genes will undoubtedly be revealed in resected brain samples, making it possible to draw correlations between genotypes and phenotypes and allow for a genetic clinical diagnosis that may help to predict a given patient's outcome.

Ca2+ signalling plays a role in celastrol-mediated suppression of synovial fibroblasts of rheumatoid arthritis patients and experimental arthritis in rats.

Background and PurposeCelastrol exhibits anti‐arthritic effects in rheumatoid arthritis (RA), but the role of celastrol‐mediated Ca2+ mobilization in treatment of RA remains undefined. Here, we describe a regulatory role for celastrol‐induced Ca2+ signalling in synovial fibroblasts of RA patients and adjuvant‐induced arthritis (AIA) in rats.Experimental ApproachWe used computational docking, Ca2+ dynamics and functional assays to study the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump (SERCA). In rheumatoid arthritis synovial fibroblasts (RASFs)/rheumatoid arthritis fibroblast‐like synoviocytes (RAFLS), mechanisms of Ca2+‐mediated autophagy were analysed by histological, immunohistochemical and flow cytometric techniques. Anti‐arthritic effects of celastrol, autophagy induction, and growth rate of synovial fibroblasts in AIA rats were monitored by microCT and immunofluorescence staining. mRNA from joint tissues of AIA rats was isolated for transcriptional analysis of inflammatory genes, using siRNA methods to study calmodulin, calpains, and calcineurin.Key ResultsCelastrol inhibited SERCA to induce autophagy‐dependent cytotoxicity in RASFs/RAFLS via Ca2+/calmodulin‐dependent kinase kinase‐β–AMP‐activated protein kinase–mTOR pathway and repressed arthritis symptoms in AIA rats. BAPTA/AM hampered the in vitro and in vivo effectiveness of celastrol. Inflammatory‐ and autoimmunity‐associated genes down‐regulated by celastrol in joint tissues of AIA rat were restored by BAPTA/AM. Knockdown of calmodulin, calpains, and calcineurin in RAFLS confirmed the role of Ca2+ in celastrol‐regulated gene expression.Conclusion and ImplicationsCelastrol triggered Ca2+ signalling to induce autophagic cell death in RASFs/RAFLS and ameliorated arthritis in AIA rats mediated by calcium‐dependent/‐binding proteins facilitating the exploitation of anti‐arthritic drugs based on manipulation of Ca2+ signalling.

Rapamycin-induced autophagy decreases Myf5 and MyoD proteins in C2C12 myoblast cells

Rapamycin is an immunosuppressant that inhibits the mammalian or mechanistic target of rapamycin (mTOR) protein kinase and extends lifespan in organisms including mice. Myf5 and MyoD act as muscle-specific transcriptional factors for skeletal muscle differentiation. In this study, we determined whether rapamycin-induced autophagy causes the decrease of Myf5 and MyoD protein in C2C12 myoblast cells. Rapamycin induced a significant increase in the expression of the microtubule-associated protein 1 light chain 3 (LC3) II protein in a dose-dependent manner for 12 h. Rapamycin treatment also significantly increased p-ERK, p-Akt, and catalase expressions, and decreased Mn-SOD expression in a dose-dependent manner. Bax expression was significantly high compared to Bcl-2 expression in a dose-dependent manner of rapamycin for 12 h. For further study of rapamycin-induced autophagy in C2C12 myoblast cells, we investigated rapamycin treatment for 24, 36, and 48 h. Cell viability did not change with rapamycin treatment for 24, 36, and 48 h. Rapamycin-induced LC3-II, Beclin-1, Bax, and Bcl-2 proteins were significantly increased compared to without rapamycin. p-ERK expression increased with rapamycin treatment for 24 and 36 h compared to that without rapamycin, but decreased for 48 h. p-Akt expression decreased with rapamycin treatment for 36 and 48 h compared to that without rapamycin. In the same conditions, rapamycin-induced autophagy significantly reduced the Myf5 and MyoD proteins. Together, these results suggest that rapamycin-induced autophagy results in the decrease of Myf5 and MyoD proteins in C2C12 myoblast cells.

Curcumin Regulates the Progression of Colorectal Cancer via LncRNA NBR2/AMPK Pathway.

Objective:To identify the effect of curcumin on tumor suppression and the possible molecular pathways involved.Methods:The expression of long noncoding RNA neighbor of BRCA1 lncRNA 2 (NBR2) was quantified using reverse transcription-polymerase chain reaction on cultured colorectal cancer cells. Next, we used Western blot to measure the activation of adenosine monophosphate-activated protein kinase and mechanistic target of rapamycin kinase (mTOR) signaling molecules. Both cell proliferation and viability were measured via MTT assay, and the cell ratio and S phase were detected by BrdU assay. Colorectal cancer cells were pretreated with curcumin or transfected with shNBR2 or adenosine monophosphate-activated protein kinase inhibitor Compound C to examine the molecular pathway involved.Results:Current data showed that glucose deficiency increased the expression of NBR2 in colorectal cancer cells, and NBR2 knockdown affected the progression of colorectal cancer cells under glucose starvation conditions. When NBR2 was silenced in the treated colorectal cancer cells, the proliferation, the clone formation, and the percentage of S-phase cells suppressed by glucose deprivation were compromised. Furthermore, NBR2 knockdown could suppress glucose deprivation-induced adenosine monophosphate-activated protein kinase activation plus mTOR inactivation. Similarly, when colorectal cancer cells were treated with curcumin, the expression of NBR2 was significantly increased. NBR2 knockdown reversed curcumin-suppressed proliferation, clone formation, and the percentage of S-phase colorectal cancer cells. Furthermore, NBR2 knockdown abolished curcumin-induced activation of adenosine monophosphate-activated protein kinase and inactivation of the mTOR signaling pathway.Conclusion:This study revealed a novel mechanism by which long noncoding RNA NBR2 mediates curcumin suppression of colorectal cancer proliferation by activating adenosine monophosphate-activated protein kinase and inactivating the mTOR signaling pathway.

Potential efficacy and prognosis of silencing the CRLF2‑mediated AKT/mTOR pathway in pediatric acute B‑cell lymphoblastic leukemia.

Acute B-cell lymphoblastic leukemia (B-ALL) is a common type of blood cancer, which is associated with aberrant gene expression. Cytokine receptor-like factor 2 (CRLF2) serves a crucial role in the growth and allergic and inflammatory responses of dendritic cells and T cells. The purpose of the present study was to investigate the potential therapeutic and prognostic effect of silencing the CRLF2-mediated RAC-α serine/threonine-protein kinase (AKT)/serine/threonine-protein kinase mTOR (mTOR) pathway in B-ALL. In our study, bone marrow specimens were collected from 128 children with B-ALL and 26 healthy children. The expression of CRLF2 in bone marrow tissue was detected using immunohistochemistry. The survival rates were compared among the children with high and low CRLF2 expression levels. BaF3 leukemia cells were treated with CRLF2 short hairpin RNA knockdown and/or the AKT/mTOR pathway specific inhibitor LY294002. mRNA and protein expression associated with CRLF2 and the AKT/mTOR pathway in each group was detected by reverse transcription-quantitative polymerase chain reaction analysis and western blotting. The viability of BaF3 cells in all the groups was assessed by Cell Counting Kit-8 assay; the migration and invasion of BaF3 cells were determined by wound healing and Transwell invasion assays; and the sensitivity of BaF3 cells to the chemotherapeutic drug imatinib was detected using flow cytometry. The results demonstrated that CRLF2 overexpression is associated with a poor prognosis in B-ALL, and the CRLF2/AKT/mTOR pathway is involved in the migration, invasion and chemotherapeutic agent-induced apoptosis of BaF3 cells.

Lysosome: The metabolic signaling hub

For the past five decades, the lysosome has been characterized as an unglamorous cellular recycling center. This notion has undergone a radical shift in the last 10 years, with new research revealing that this organelle serves as a major hub for metabolic signaling pathways. The discovery that master growth regulators, including the protein kinase mTOR (mechanistic target of rapamycin), make their home at the lysosomal surface has generated intense interest in the lysosome's key role in nutrient sensing and cellular homeostasis. The transcriptional networks required for lysosomal maintenance and function are just being unraveled and their connection to lysosome-based signaling pathways revealed. The catabolic and anabolic pathways that converge on the lysosome connect this organelle with multiple facets of cellular function; when these pathways are deregulated they underlie multiple human diseases, and promote cellular and organismal aging. Thus, understanding how lysosome-based signaling pathways function will not only illuminate the fascinating biology of this organelle but will also be critical in unlocking its therapeutic potentials.

Effect of interaction between leucine and acetate on the milk protein synthesis in bovine mammary epithelial cells.

The interaction between Leucine (Leu) and acetate affecting milk protein synthesis in the bovine mammary epithelial cells (BMECs), and underlying the molecular mechanisms are not well understood. The objectives of this study were to investigate the effect of Leu, acetate, and their interaction on the expression of genes involved in milk protein synthesis, and JACK2/STAT5, mTOR and AMP-activated protein kinase (AMPK) signaling pathway. The study was a 2×6 factorial arrangement with treatments: Leu concentration (0.45 and 1.8mM) and acetate concentration (0, 4, 6, 8, 10, and 12mM). The results showed that 1.8mM Leu or 8-10mM acetate had positive effect on ATP content, the expression of casein genes, JACK2/STAT5 and phosphorylation of mTOR pathway, but reduced AMPK phosphorylation. Leu at 1.8mM had a positive effect on the up-regulation of acetate on ATP content, the expression of CSN1S1, CSN2, CSN3, and JACK2, the expression and phosphorylation of eukaryotic initiation factor 4E, p70 ribosomal protein S6 kinase-1, and mTOR, but reducing AMPK phosphorylation. The results suggest that acetate, Leu, and their interaction have effect on milk protein synthesis through the JACK2/STAT5, mTOR, and AMPK pathway. Acetate addition up-regulated the effect of Leu on milk protein synthesis, and Leu facilitated the up-regulation of acetate on milk protein synthesis through these pathways.

Regnase-1 controls colon epithelial regeneration via regulation of mTOR and purine metabolism

Damage to intestinal epithelial cell (IEC) layers during intestinal inflammation is associated with inflammatory bowel disease. Here we show that the endoribonuclease Regnase-1 controls colon epithelial regeneration by regulating protein kinase mTOR (the mechanistic target of rapamycin kinase) and purine metabolism. During dextran sulfate sodium-induced intestinal epithelial injury and acute colitis, Regnase-1∆IEC mice, which lack Regnase-1 specifically in the intestinal epithelium, were resistant to body weight loss, maintained an intact intestinal barrier, and showed increased cell proliferation and decreased epithelial apoptosis. Chronic colitis and tumor progression were also attenuated in Regnase-1∆IEC mice. Regnase-1 predominantly regulates mTORC1 signaling. Metabolic analysis revealed that Regnase-1 participates in purine metabolism and energy metabolism during inflammation. Furthermore, increased expression of ectonucleotidases contributed to the resolution of acute inflammation in Regnase-1∆IEC mice. These findings provide evidence that Regnase-1 deficiency has beneficial effects on the prevention and/or blocking of intestinal inflammatory disorders.

A LINK BETWEEN AUTOPHAGY REGULATORY PROTEINS M-TOR AND BECLIN-1 AND PARAMETERS OF LYMPHOGENIC METASTASIS IN COLORECTAL CANCER

Currently the impact of autophagy on carcinogenesis remains understudied. On the one hand, autophagy acts as a tumor suppressor, as it activates degradation of oncoproteins, toxic proteins, and damaged cell organelles, that may be aggressive and lead to DNA damage. On the other hand, autophagy may promote tumor cell survival under hypoxia and in the presence of reactive oxygen species, which occurs primarily due to blocking of apoptosis mechanisms, raising the chances for maintaining tumor clone dynamics. Autophagy regulation is a complicated and multi-stage process. The main regulator here is a signaling pathway that activates serine/threonine protein kinase m-TOR (the mammalian target of rapamycin). Data on the impact of autophagic proteins ATG5, LC3A, LC3B, and Beclin-1 on malignant cell survival as well as on tumor growth and progression have been reported in literature. However, studies aimed at seeking possible relationships between autophagy and pathogenetic mechanisms of carcinogenesis are of great interest.Theaimof the study is to investigate a relationship between the expression parameters of autophagy regulatory proteins m-TOR and Beclin-1 and the features of lymphogenic metastasis in colorectal cancer.Materials and methods. The study included 105 patients with T1-4N0-3M0 colorectal cancer treated in the Thoracic and Abdominal Department of Cancer Research Institute of Tomsk Research Medical Center from 2012 to 2015. The average age of patients was 59.7±4.3 years. Morphological verification of the diagnosis was performed on the biopsy samples of primary tumor tissue. Staging of colorectal cancer was determined according to the TNM classification of malignant tumors (2002).Results.Analysis of the frequency of lymphogenic metastasis depending on the presence or absence of m-Tor and Beclin-1 expression in tumor cell cytoplasm revealed a statistically significant link between these variables.Conclusion.The obtained findings clearly exhibit that deceleration or loss of autophagic activity in the tumor is accompanied by implementation of lymphogenic dissemination, which is a predictor of an unfavorable outcome of the disease.

Effect of combined treatment with insulin, metformin, and gliclazide on the expression and activity of Akt, mTOR, and p70S6K protein kinases in lymphocytes of diabetic patients

Effect of combined treatment with insulin, metformin, and gliclazide on the expression and activity of Akt, mTOR, and p70S6K protein kinases in lymphocytes of diabetic patients

Extracellular matrix promotes proliferation, migration and adhesion of airway smooth muscle cells in a rat model of chronic obstructive pulmonary disease via upregulation of the PI3K/AKT signaling pathway.

Extracellular matrix (ECM) creates the tissue microenvironment and serves a role in airway wall remodeling in chronic obstructive pulmonary disease (COPD). However, the biological function of ECM in COPD remains to be elucidated. In the present study, 24 healthy Sprague Dawley rats were randomized to normal and COPD groups. COPD was established by intratracheal injection with lipopolysaccharide over 30 days. Subsequently, airway smooth muscle cells (ASMCs) were isolated from rats and served as a model to assess the effects of three ECM components, including collagen type I, laminin and collagen type III (COL-3). Functional analysis in vitro, using cell counting kit-8, flow cytometry, wound healing and cell adhesion assays indicated that the ECM components could promote cell proliferation, cell cycle progression, migration and adhesion ability, respectively. Furthermore, as demonstrated by ELISA, treatment with ECM components increased levels of C-X-C motif chemokine ligand 1 (CXCL1), CXCL8 and interleukin-6 in ASMCs. Expression of transforming growth factor β1 (TGFβ1), fibroblast growth factor-1 (FGF-1) and tissue inhibitor of metalloproteinase 1 (TIMP1) was increased, and expression of matrix metalloproteinase-9 (MMP-9) was decreased following treatment with ECM components, as demonstrated by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Additionally, specific activation of phosphoinositide 3-kinase (PI3K) signaling, using insulin-like growth factor-1 (IGF-1), promoted cell proliferation and cell cycle progression, increased expression of TGFβ1, FGF-1, PI3K, AKT, phospho-AKT, serine/threonine-protein kinase mTOR (mTOR), phospho-mTOR and TIMP1, promoted cell migration capacity and reduced the expression level of MMP-9 in cells from COPD rats. Consistently, PI3K inhibitor LY294002 exerted the opposite effect to IGF-1. In conclusion, ECM proteins promoted proliferation, migration and adhesion of ASMCs form rat models of COPD through activation of the PI3K/AKT signaling pathway.

Abstract 252: Prostate carcinogenesis inhibition through AKT-PKCα signaling pathway by diosmetin

Abstract Mammalian target of rapamycin (mTOR) integrates nutrient and mitogen signals to regulate cell growth (increased cell mass and cell size) and cell division. mTOR protein kinase have two different complexes; complex-I contains major component raptor, whereas complex II (insensitive to rapamycin), has major component rictor. Rictor plays a critical role in AKT (Ser-473 phosphorylation), which facilitates phosphorylation of AKT on Thr-308 by PDK1 for full activation. When we genetically disrupted Rictor, it decreased AKT phosphorylation (Ser-473) and impaired PKCα (Ser-657) expressions. Growth factors viz., IGF-1 and IL-6 are the major player in prostate cancers, these increases Rictor expression. We need to have an agent that can modulate kinase activity of AKT and PKCα to inhibit prostate cancer cell growth. Diosmetin (5, 7-Trihydroxy-4′-methoxyflavone) could be such an agent. Diosmetin, a natural flavone present in plant legumes, has anti-mutagenic and anti-allergic properties. Diosmetin inhibited IGF-1 and IL-6 induced rictor expression in LNCaP and PC-3 prostate cancer cells. Diosmetin dose response treatment to these cells inhibited cell growth and induced apoptosis with no significant growth inhibition in normal prostate epithelial cells (RWPE1). Diosmetin treatment to prostate cancer cells modulated cell survival machinery by down regulating key molecules viz., c-Myc, Survivin and XIAP (X-Linked Inhibitor of Apoptosis). Similarly diosmetin (20 and 50μg/animal/day) feeding to nude mice models, which were orthotopically implanted with luciferase tag PC-3 cells in ventral prostate represented significant decrease in tumor volume than control mice. Diosmetin fed tumor bearing mice reduced phosphorylation of Rictor, (Thr-1135), AKT (Ser-473) and PKCα (Ser-657) expressions to inhibit tumor growth significantly. Moreover, luciferase activity of implanted tumor decreased after diosmetin dose response feeding than control mice. Additionally bone radiographic images suggested that diosmetin treatment inhibited homing of PC-3 cells in the bone. These effects were associated with decrease in cyclin D1 expression and their activating partner, cyclin-dependent kinase (cdk) 2 and 4 with concomitant upregulation of p27/KIP1. Further diosmetin treatment induced apoptosis which was evident by increased expression of cleaved caspase-3. We are documenting these evidences for the first time in animal model system that diosmetin acts against potential molecular targets to alter cellular events to elicit anticancer effects in prostate cancer. Citation Format: Rebecca Pakradooni, Ahmad Khalifa, Ilaha Isali, Sanjeev Shukla. Prostate carcinogenesis inhibition through AKT-PKCα signaling pathway by diosmetin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 252.

Knockdown of long non‑coding RNA PVT1 reverses multidrug resistance in colorectal cancer cells.

Multidrug resistance (MDR) is one of the primary causes of chemotherapy failure in colorectal cancer (CRC), and extensive biological studies into MDR are required. The non-coding RNA plasmacytoma variant translocation 1 (PVT1) has been demonstrated to be associated with low survival rates in patients with CRC. However, whether PVT1 serves a critical function in the MDR of CRC remains to be determined. To determine the association between PVT1 expression and 5-fluorouracil (5-FU) resistance in CRC, the expression levels of PVT1 mRNA in 5-FU-resistant CRC tissues and cell lines (HCT-8/5-FU and HCT-116/5-FU) were assessed by a reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cytotoxicity was evaluated using a Cell Counting Kit-8 assay and apoptosis rates were assessed via flow cytometry. In the present study, PVT1 mRNA was highly expressed in 5-FU-resistant CRC tissues and cell lines. HCT-8/5-FU and HCT-116/5-FU cells transfected with small interfering RNA PVT1 and treated with 5-FU exhibited higher apoptotic rates and lower survival rates. By contrast, overexpression of PVT1 in HCT-8 and HCT-116 cells transfected with lentiviral vector-PVT1-green fluorescent protein and treated with 5-FU exhibited lower apoptosis rates and higher survival rates. RT-qPCR and western blotting demonstrated that the overexpression of PVT1 increased the mRNA and protein expression levels of multidrug resistance-associated protein 1, P-glycoprotein, serine/threonine-protein kinase mTOR and apoptosis regulator Bcl2. The present study indicates that PVT1 overexpression may promote MDR in CRC cells, and suggested that inhibition of PVT1 expression may be an effective therapeutic strategy for reversing MDR in CRC.

Autophagy induced by cardamonin is associated with mTORC1 inhibition in SKOV3 cells.

The mammalian target of rapamycin (mTOR) integrates energy level to modulate cell proliferation and autophagy. Cardamonin exhibits anti-proliferative activity through inhibiting mTOR. In this study, the effect of cardamonin on autophagy and its mechanism on mTOR inhibition were investigated. Cell viability and proliferation were measured by MTT assay and BrdU incorporation, respectively. Cell apoptosis was assayed by flow cytometry and cell autophagy was detected by electron microscopy and GFP-LC3 fluorescence. The mechanism of cardamonin on mTORC1 inhibition was investigated by Raptor siRNA and Raptor over-expression. The cell viability and proliferation were inhibited by cardamonin. The autophagosomes and the protein level of LC3-II were increased by cardamonin. Cell apoptosis and the levels of cleaved PARP and Caspase-3 were increased by cardamonin. Cardamonin inhibited the phosphorylation of mTOR and ribosome S6 protein kinase 1 (S6K1) as well as the protein level of regulatory associated protein of mTOR (Raptor). However, cardamonin had no effect on the component of mTORC2 and its downstream substrate Akt. The inhibitory effect of cardamonin on the phosphorylation of mTOR and S6K1 was eliminated by Raptor knockdown with siRNA, whereas this effect of cardamonin was stronger than that of rapamycin and AZD8055 in Raptor over-expression cells. Cell viability was inhibited by cardamonin in both Raptor knockdown and Raptor over-expression cells, which was consistent with the inhibitory effect of cardamonin on mTOR. These findings demonstrated that the autophagy induced by cardamonin was associated with mTORC1 inhibition through decreasing the protein level of Raptor in SKOV3 cells.

Galectins Control mTOR in Response to Endomembrane Damage

The Ser/Thr protein kinase mTOR controls metabolic pathways, including the catabolic process of autophagy. Autophagy plays additional, catabolism-independent roles in homeostasis of cytoplasmic endomembranes and whole organelles. How signals from endomembrane damage are transmitted to mTOR to orchestrate autophagic responses is not known. Here we show that mTOR is inhibited by lysosomal damage. Lysosomal damage, recognized by galectins, leads to association of galectin-8 (Gal8) with the mTOR apparatus on the lysosome. Gal8 inhibits mTOR activity through its Ragulator-Rag signaling machinery, whereas galectin-9 activates AMPK in response to lysosomal injury. Both systems converge upon downstream effectors including autophagy and defense against Mycobacterium tuberculosis. Thus, a novel galectin-based signal-transduction system, termed here GALTOR, intersects with the known regulators of mTOR on the lysosome and controls them in response to lysosomal damage. VIDEO ABSTRACT.

JTC-801 exerts anti-proliferative effects in human osteosarcoma cells by inducing apoptosis

Background: The research of G protein-coupled receptors (GPCRs) is a promising strategy for drug discovery. In cancer therapy, there is a need to discover novel agents that can inhibit proliferation and induce apoptosis in cancer cells. JTC-801 is a novel GPCR antagonist with the function of reversing pain and anxiety symptoms. This study aims to investigate the antitumor effects of JTC-801 on human osteosarcoma cells (U2OS) and elucidate the underlying mechanism.Materials and methods: The Cell Counting Kit-8 assay was used to detect the viability of U2OS cells treated with JTC-801 in vitro. The cell apoptosis was evaluated using a flow cytometry assay with Annexin V-FITC/PI double staining. The inhibitory effect of JTC-801 on invasion and migration of U2OS cells were determined by the Transwell assays. Western blot assay was performed to measure the levels of proteins related to cell apoptosis and its mechanism.Results: The JTC-801 significantly decreased the viability of U2OS cells (p < .05) as a result of its anti-proliferative effect through induction of apoptosis associated with activation of BAX, Caspase-3 and down-regulating BCL-2 expression. The invasive and migratory cells were obviously reduced after JTC-801 treatment (p < .05). Further, the phosphorylated AKT, mTOR and active p70 S6 protein kinase in the PI3K/AKT signaling pathway were obviously lessened in the JTC-801 treated U2OS group (p < .05).Conclusions: JTC-801 may exert osteosarcoma cell growth inhibition by promoting cell apoptosis, through PI3K/AKT signaling pathway participation.

Role of the PI3K‑AKT‑mTOR pathway in hepatitis B virus infection and replication.

The replication of hepatitis B virus (HBV) may be modulated by a variety of cell signaling pathways, including the phosphatidylinositol 3‑kinase (PI3K)‑RAC‑α serine/threonine‑protein kinase (AKT)‑serine/threonine‑protein kinase mTOR (mTOR) pathway. The aim of the present study was to determine the regulatory effects of this pathway on the infection and replication of HBV. The results indicated that the HBV entry process may activate the AKT pathway, as demonstrated by AKT phosphorylation in HBV natural infection. However, inhibition of AKT phosphorylation by short‑term treatment with AKT inhibitors was unable to block HBV entry, which suggested that AKT activation induced by HBV infection is not essential for viral entry process. Prolonged treatment with PI3K‑AKT‑mTOR pathway inhibitors markedly promoted HBV replication in HBV replicating and natural infection models. The PI3K‑AKT‑mTOR pathway was therefore identified to be a negative regulator of HBV replication. These inhibitors enhanced the replication and transcription of HBV in an HBx‑dependent way. The results additionally indicated that a PI3K inhibitor, Ly294002, inhibited the secretion of the small surface antigen of HBV in a PI3K‑AKT‑independent manner. The inhibitor Ly294002 may be used as a tool for the drug development of surface antigen secretion inhibitors.