mTOR Ser2448 Phosphorylation Research Articles

Glycosphingolipids are mediators of cancer plasticity through independent signaling pathways.

The molecular repertoire promoting cancer cell plasticity is not fully elucidated. Here, we propose that glycosphingolipids (GSLs), specifically the globo and ganglio series, correlate and promote the transition between epithelial and mesenchymal cells. The epithelial character of ovarian cancer remains stable throughout disease progression, and spatial glycosphingolipidomics reveals elevated globosides in the tumor compartment compared with the ganglioside-rich stroma. CRISPR-Cas9 knockin mediated truncation of endogenous E-cadherin induces epithelial-to-mesenchymal transition (EMT) and decreases globosides. The transcriptomics analysis identifies the ganglioside-synthesizing enzyme ST8SIA1 to be consistently elevated in mesenchymal-like samples, predicting poor outcome. Subsequent deletion of ST8SIA1 induces epithelial cell features through mTORS2448 phosphorylation, whereas loss of globosides in ΔA4GALT cells, resulting in EMT, is accompanied by increased ERKY202/T204 and AKTS124. The GSL composition dynamics corroborate cancer cell plasticity, and further evidence suggests that mesenchymal cells are maintained through ganglioside-dependent, calcium-mediated mechanisms.

Canonical and Non-canonical TGFβ Signaling Activate Autophagy in an ULK1-Dependent Manner.

The mechanism(s) in which transforming growth factor beta 1 (TGFβ) modulates autophagy in cancer remain unclear. Here, we characterized the TGFβ signaling pathways that induce autophagy in non-small cell lung cancer cells, using cells lines stably expressing GFP-LC3-RFP-LC3ΔG constructs that measure autophagic flux. We demonstrated that TGFβ1 increases Unc 51-like kinase 1 (ULK1) protein levels, 5′ adenosine monophosphate-activated protein kinase (AMPK)-dependent ULK1 phosphorylation at serine (S) 555 and ULK1 complex formation but decreases mechanistic target of rapamycin (mTOR) activity on ULK1. Further analysis revealed that the canonical Smad4 pathway and the non-canonical TGFβ activated kinase 1/tumor necrosis factor receptor-associated factor 6/P38 mitogen activated protein kinase (TAK1-TRAF6-P38 MAPK) pathway are important for TGFβ1-induced autophagy. The TAK1-TRAF6-P38 MAPK pathway was essential for downregulating mTOR S2448 phosphorylation, ULK1 S555 phosphorylation and autophagosome formation. Furthermore, although siRNA-mediated Smad4 silencing did not alter mTOR-dependent ULK1 S757 phosphorylation, it did reduce AMPK-dependent ULK1 S555 phosphorylation and autophagosome formation. Additionally, Smad4 silencing and inhibiting the TAK1-TRAF6-P38 MAPK pathway decreased autophagosome-lysosome co-localization in the presence of TGFβ. Our results suggest that the Smad4 and TAK1-TRAF6-P38 MAPK signaling pathways are essential for TGFβ-induced autophagy and provide specific targets for the inhibition of TGFβ in tumor cells that utilize autophagy in their epithelial-mesenchymal transition program.

The Effects of an Acute "Train-Low" Nutritional Protocol on Markers of Recovery Optimization in Endurance-Trained Male Athletes.

This study aimed to determine the effects of an acute "train-low" nutritional protocol on markers of recovery optimization compared to standard recovery nutrition protocol. After completing a 2-hour high-intensity interval running protocol, 8 male endurance athletes consumed a standard dairy milk recovery beverage (CHO; 1.2g/kg body mass [BM]of carbohydrate and 0.4g/kg BM of protein) and a low-carbohydrate (L-CHO; isovolumetric with 0.35g/kg BM of carbohydrate and 0.5g/kg BM of protein) dairy milk beverage in a double-blind randomized crossover design. Venous blood and breath samples, nude BM, body water, and gastrointestinal symptom measurements were collected preexercise and during recovery. Muscle biopsy was performed at 0hour and 2hours of recovery. Participants returned to the laboratory the following morning to measure energy substrate oxidation and perform a 1-hour distance test. The exercise protocol resulted in depletion of muscle glycogen stores (250mmol/kg dry weight) and mild body-water losses (BM loss = 1.8%). Neither recovery beverage replenished muscle glycogen stores (279mmol/kg dry weight) or prevented a decrease in bacterially stimulated neutrophil function (-21%). Both recovery beverages increased phosphorylation of mTORSer2448 (main effect of time = P < .001) and returned hydration status to baseline. A greater fold increase in p-GSK-3βSer9/total-GSK-3β occurred on CHO (P = .012). Blood glucose (P = .005) and insulin (P = .012) responses were significantly greater on CHO (618mmol/L per 2h and 3507μIU/mL per 2h, respectively) compared to L-CHO (559mmol/L per 2h and 1147μIU/mL per 2h, respectively). Rates of total fat oxidation were greater on CHO, but performance was not affected. A lower-carbohydrate recovery beverage consumed after exercise in a "train-low" nutritional protocol does not negatively impact recovery optimization outcomes.

The Effect of Leucine-Enriched Essential Amino Acid Supplementation on Anabolic and Catabolic Signaling in Human Skeletal Muscle after Acute Resistance Exercise: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Comparison Trial.

Resistance exercise transiently activates anabolic and catabolic systems in skeletal muscle. Leucine-enriched essential amino acids (LEAAs) are reported to stimulate the muscle anabolic response at a lower dose than whey protein. However, little is known regarding the effect of LEAA supplementation on the resistance exercise-induced responses of the anabolic and catabolic systems. Here, we conducted a randomized, double-blind, placebo-controlled, parallel-group comparison trial to investigate the effect of LEAA supplementation on mechanistic target of rapamycin complex 1 (mTORC1), the ubiquitin–proteasome system and inflammatory cytokines after a single bout of resistance exercise in young men. A total of 20 healthy young male subjects were supplemented with either 5 g of LEAA or placebo, and then they performed 10 reps in three sets of leg extensions and leg curls (70% one-repetition maximum). LEAA supplementation augmented the phosphorylation of mTORSer2448 (+77.1%, p < 0.05), p70S6KThr389 (+1067.4%, p < 0.05), rpS6Ser240/244 (+171.3%, p < 0.05) and 4EBP1Thr37/46 (+33.4%, p < 0.05) after resistance exercise. However, LEAA supplementation did not change the response of the ubiquitinated proteins, MuRF-1 and Atrogin-1 expression. Additionally, the mRNA expression of IL-1β and IL-6 did not change. These data indicated that LEAA supplementation augments the effect of resistance exercise by enhancing mTORC1 signal activation after exercise.

Acute Signaling Responses To Resistance Exercise In Previously Trained And Untrained Skeletal Muscle

It has been indicated that human skeletal muscle has a heightened sensitivity to exercise stimulus when it has been previously trained i.e. possess a “muscle memory”. While previous studies have been directed to the aspects of myonuclear content and epigenetic modifications, no previous study have explored the potential of a muscle memory concerning signaling responses related to acute resistance exercise. PURPOSE: The aim here was to study whether basal and acute resistance exercise induced cell signaling is influenced by previous strength training history in human skeletal muscle. METHODS: 19 training naïve women and men completed 10 weeks of hypertrophy inducing unilateral strength training followed by 20 weeks of detraining. Subsequently, an acute resistance exercise session involving leg press and knee extensions was performed alternated with both legs. Vastus lateralis biopsies taken at rest and 1 h post exercise in both the Control- and Memory-leg. Immunoblotting was used to assess total content and phosphorylation status of proteins in the mTORC1- and related pathways. RESULTS: Following detraining leg muscle hypertrophy had been reversed, but the Memory-leg was on average 19% stronger than the Control-leg. There were no differences between legs with regard to total protein content of all the signaling proteins analyzed. The phosphorylation of AMPKThr172 and eEF2Thr56 was 16%, respectively 21%, higher in the Memory-leg compared to the Control-leg at both time points. The effect on AMPKThr172 was attributed to changes in the women only, whereas the effect on eEF2Thr56 was present in both sexes. Exercise induced an increased phosphorylation of mTORSer2448 (26-36%), S6K1Thr389 (6- to 7-fold) and S6Ser235/236 (13- to 18-fold), that did not differ between the Control- and Memory-leg. In contrast, post exercise phosphorylation of 4E-BP1Thr46 and 4E-BP1Ser65 was 18%, respectively 31%, higher in the Memory-leg compared to the Control-leg. For 4E-BP1Ser65 the effects were attributed to changes in the male subjects only. CONCLUSION: In summary, we illustrate that both basal- and exercise induced cell signaling important for muscle adaptations to strength training can be altered by previous training history, and that some of the changes seem to be sex dependent.

Krill Oil Addition to 8‐weeks of High‐fat Diet Enhances mTORSer2448 Phosphorylation in Rat Heart

Krill oil (KO) derived from shrimp‐like crustaceans, has been marketed as a substantial source of dietary long chain omega‐3 (LC n‐3) polyunsaturated fatty acids, particularly rich in eicosapentaenoic acid (EPA; 20:5 n‐3) and docosahexaenoic acid (DHA; 22:6 n‐3). Consumption of EPA and DHA has been linked to the reduction of cardiovascular disorders including coronary heart disease and myocardial infarction, however the cellular mechanism for these cardioprotective benefits is unclear. The mechanistic target of rapamycin (mTOR) is a Ser/Thr kinase that acts a nutrient sensor, subsequently controlling various key cellular processes including cell growth, cell survival, and protein synthesis. Diets high in saturated fat content have been associated with myocardial dysregulation through alteration of the mTOR signaling pathway, suggesting that interventions that ameliorate mTOR signaling may counter these deleterious effects in the heart. The goal of the current study is to determine the effect of krill oil supplementation to a high saturated fat diet on mTOR signaling in cardiac muscle. Male Sprague‐Dawley rats (approximately 12 months‐old), were assigned by mass to one of three isocaloric dietary groups for 8 weeks: control diet group (CON; 17% saturated fat, 54% carbohydrate, 29% protein; n=8), a high saturated fat group (HF; 42% saturated, 43% carbohydrate, 15% protein; n=12), and a high fat group with krill oil supplementation (KO; 34% saturated fat, 8% krill oil, 43% carbohydrate, 15% protein; n=12). All rats were given ad libitum access to food and water. Body weights and food consumption was recorded weekly. After 8 weeks of dietary intervention and an overnight fast, blood and hearts were collected from anesthetized rats during a terminal surgery. Western immunoblot analysis was used to determine phosphorylation levels of key mTOR signaling proteins including mTORSer2448, p70S6KThr389, 4E‐BP1Thr37/46, eIF4ESer209, and eIF2αSer51. There was no difference in body mass between dietary groups after 8 weeks of treatment. There was a significant increase (p&lt;0.05) in phosphorylation of mTORSer2448 in the KO group when compared to the HF and CON groups. Phosphorylation of downstream targets of mTOR was not different between groups. Eight weeks of KO supplementation can enhance phosphorylation of mTORSer2448, however the cardioprotective impact of this remains unknown. Further research is needed to examine how KO consumption can affect other key downstream mTOR‐related targets associated with improved cardiovascular health.Support or Funding InformationResearch was supported by intramural grants from the College of Health Professions and the Office of Research and Graduate Studies at Central Michigan University.

Beta2‐adrenergic agonist clenbuterol increases energy expenditure and fat oxidation, and induces mTOR phosphorylation in skeletal muscle of young healthy men

Clenbuterol is a beta2 -adrenoceptor agonist marketed as an asthma reliever but is not approved for human use in most countries due to concerns of adverse cardiac effects. Given its demonstrated hypertrophic and lipolytic actions in rodents, clenbuterol is one of the most widely abused doping substances amongt athletes and recreational body-builders seeking leanness. Herein, we examined the effect of clenbuterol ingestion on metabolic rate as well as skeletal muscle mammalian target of rapamycin (mTOR) phosphorylation and protein kinase A (PKA)-signaling in six young men. Before and 140 min after ingestion of 80 μg clenbuterol, resting metabolic rate and contractile function of the quadriceps muscle were measured, and blood samples as well as vastus lateralis muscle biopsies were collected. Clenbuterol increased resting energy expenditure by 21% (P < 0.001), and fat oxidation by 39% (P = 0.006), whereas carbohydrate oxidation was unchanged. Phosphorylation of mTORSer2448 and PKA substrates increased by 121% (P = 0.004) and 35% (P = 0.006), respectively, with clenbuterol. Maximal voluntary contraction torque decreased by 4% (P = 0.026) and the half-relaxation time shortened by 9% (P = 0.046), while voluntary activation, time to peak twitch, and peak twitch torque did not change significantly with clenbuterol. Glycogen content of the vastus lateralis muscle did not change with clenbuterol. Clenbuterol increased circulating levels of glucose (+30%; P < 0.001), lactate (+90%; P = 0.004), insulin (+130%; P = 0.009), and fatty acids (+180%; P = 0.001). Collectively, these findings indicate that clenbuterol is an efficient thermogenic substance that possibly also exerts muscle hypertrophic actions in humans. For these reasons, the restrictions imposed against clenbuterol in competitive sports seem warranted.

Mammalian target of rapamycin and p70S6K mediate thrombin-induced nuclear factor-κB activation and IL-8/CXCL8 release in human lung epithelial cells

Thrombin plays a crucial role in lung inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Thrombin induces the release of interleukin-8 (IL-8)/CXCL8 by lung epithelial cells, and this phenomenon plays a vital role in lung inflammation. Our previous studies have indicated that thrombin stimulates IL-8/CXCL8 expression through PI3K/Akt/IκB kinase (IKK)α/β/nuclear factor-κB (NF-κB) and p300 pathways in human lung epithelial cells. In the present study, we explored the roles of mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K) in thrombin-induced NF-κB activation and IL-8/CXCL8 release in human lung epithelial cells. In this study, we found that rapamycin (an mTOR inhibitor) and p70S6K siRNA diminished thrombin-induced IL-8/CXCL8 release. Thrombin induced mTOR Ser2448 phosphorylation and p70S6K Thr389 phosphorylation in a time-dependent manner. Moreover, rapamycin attenuated thrombin-stimulated p70S6K phosphorylation. We also found that transfection of cells with the dominant negative mutant of Akt (Akt DN) reduced the thrombin-induced increase in mTOR phosphorylation and p70S6K phosphorylation. Moreover, thrombin-stimulated p300 phosphorylation was attenuated by Akt DN, rapamycin, and p70S6K siRNA. Thrombin triggered p70S6K translocation from the cytosol to the nucleus in a time-dependent manner. Thrombin induced the complex formation of p70S6K, p300, and p65; acetylation of p65 Lys310, and recruitment of p70S6K, p300, and p65 to the κB-binding site of the IL-8/CXCL8 promoter region. In conclusion, these results indicate that thrombin initiates the Akt-dependent mTOR/p70S6K signaling pathway to promote p300 phosphorylation and NF-κB activation and finally induces IL-8/CXCL8 release in human lung epithelial cells.

Rapid antidepressant actions of imipramine potentiated by zinc through PKA-dependented regulation of mTOR and CREB signaling

The slow onset of traditional antidepressants has become an urgent clinical issue, researchers are constantly exploring new antidepressants with prompt action. Previous studies have found that zinc levels were decreased in serum and brain of depressed patients or animal models. Zinc treatment can improve depressive symptoms and enhance the antidepressant effects of monoamine antidepressants. However, its mechanism of action is still unclear. This present study aims to investigate whether the zinc can enhance the rapid action of traditional antidepressant imipramine and to explore the potential mechanisms of action through the rapid antidepressant targets CREB (cAMP-response element binding protein) and mTOR (mammalian target of the rapamycin). Drug treatment included intraperitoneal injection of imipramine or zinc alone and imipramine plus zinc. Zinc had a rapid enhanced antidepressive effect on the imipramine and achieved a rapid antidepressant effect similar to ketamine. Combination of zinc with imipramine rapidly enhanced the phosphorylation of mTOR Ser2448 and CREB Ser133, and increased the expression of mTOR and CREB, which were dependent on the activation of PKA. In conclusion, combination therapy with zinc and monoamine antidepressants may overcome the problem of slow-onset action of traditional antidepressants in clinical uses.

Effect of mycophenolate and rapamycin on renal fibrosis in lupus nephritis.

Lupus nephritis (LN) leads to chronic kidney disease (CKD) through progressive fibrosis. Mycophenolate inhibits inosine monophosphate dehydrogenase and is a standard treatment for LN. The mammalian or mechanistic target of rapamycin (mTOR) pathway is activated in LN. Rapamycin inhibits mTOR and is effective in preventing kidney transplant rejection, with the additional merits of reduced incidence of malignancies and viral infections. The effect of mycophenolate or rapamycin on kidney fibrosis in LN has not been investigated. We investigated the effects of mycophenolate and rapamycin in New Zealand Black and White first generation (NZB/W F1) murine LN and human mesangial cells (HMCs), focusing on mechanisms leading to kidney fibrosis. Treatment of mice with mycophenolate or rapamycin improved nephritis manifestations, decreased anti-double stranded (ds) DNA antibody titer and reduced immunoglobulin G (IgG) deposition in the kidney. Both mycophenolate and rapamycin, especially the latter, decreased glomerular mTOR Ser2448 phosphorylation. Renal histology in untreated mice showed mesangial proliferation and progressive glomerulosclerosis with tubular atrophy, and increased expression of transforming growth factor β1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1), α-smooth muscle actin (α-SMA), fibronectin (FN) and collagen. Both mycophenolate and rapamycin ameliorated the histopathological changes. Results from in vitro experiments showed that both mycophenolate and rapamycin decreased mesangial cell proliferation and their binding with anti-dsDNA antibodies. Mycophenolate and rapamycin also down-regulated mTOR and extracellular signal-regulated kinase (ERK) phosphorylation and inhibited fibrotic responses in mesangial cells that were induced by anti-dsDNA antibodies or TGF-β1. Our findings suggest that, in addition to immunosuppression, mycophenolate and rapamycin may reduce fibrosis in LN, which has important implications in preventing CKD in patients with LN.

Mechanisms of insulin resistance by simvastatin in C2C12 myotubes and in mouse skeletal muscle

Statins inhibit cholesterol biosynthesis and lower serum LDL-cholesterol levels. They are generally well tolerated, but can cause insulin resistance in patients. Therefore, we investigated the mechanisms underlying the statin-induced insulin resistance.We used mice and C2C12 myotubes (murine cell line): mice (n = 10) were treated with oral simvastatin (5 mg/kg/day) or water (control) for 21 days and C2C12 cells were exposed to 10 μM simvastatin for 24 h.After intraperitoneal glucose application (2 g/kg), simvastatin-treated mice had higher glucose but equal insulin plasma concentrations than controls and lower glucose transport into skeletal muscle. Similarly, glucose uptake by C2C12 myotubes exposed to 10 μM simvastatin for 24 h was impaired compared to control cells. In simvastatin-treated C2C12 myotubes, mRNA and protein expression of the insulin receptor (IR) β-chain was increased, but the phosphorylation (Tyr1361) was impaired. Simvastatin decreased numerically Akt/PKB Thr308 phosphorylation (via insulin signaling pathway) and significantly Akt/PKB Ser473 phosphorylation (via mTORC2), which was explained by impaired phosphorylation of mTOR Ser2448. Reduced phosphorylation of Akt/PKB impaired downstream phosphorylation of GSK3β, leading to impaired translocation of GLUT4 into plasma membranes of C2C12 myotubes. In contrast, reduced phosphorylation of AS160 could be excluded as a reason for impaired GLUT4 translocation.In conclusion, simvastatin caused insulin resistance in mice and impaired glucose uptake in C2C12 myotubes. The findings in myotubes can be explained by diminished activation of Akt/PKB by mTORC2 and downstream effects on GSK3β, impairing the translocation of GLUT4 and the uptake of glucose.

Deletion of the glucocorticoid receptor chaperone FKBP51 prevents glucocorticoid-induced skin atrophy.

FKBP51 (FK506-binding protein 51) is a known co-chaperone and regulator of the glucocorticoid receptor (GR), which usually attenuates its activity. FKBP51 is one of the major GR target genes in skin, but its role in clinical effects of glucocorticoids is not known. Here, we used FKBP51 knockout (KO) mice to determine FKBP51's role in the major adverse effect of topical glucocorticoids, skin atrophy. Unexpectedly, we found that all skin compartments (epidermis, dermis, dermal adipose and CD34+ stem cells) in FKBP51 KO animals were much more resistant to glucocorticoid-induced hypoplasia. Furthermore, despite the absence of inhibitory FKBP51, the basal level of expression and glucocorticoid activation of GR target genes were not increased in FKBP51 KO skin or CRISPR/Cas9-edited FKBP51 KO HaCaT human keratinocytes. FKBP51 is known to negatively regulate Akt and mTOR. We found a significant increase in AktSer473 and mTORSer2448 phosphorylation and downstream pro-growth signaling in FKBP51-deficient keratinocytes in vivo and in vitro. As Akt/mTOR-GR crosstalk is usually negative in skin, our results suggest that Akt/mTOR activation could be responsible for the lack of increased GR function and resistance of FKBP51 KO mice to the steroid-induced skin atrophy.

Protein translation, proteolysis and autophagy in human skeletal muscle atrophy after spinal cord injury.

Spinal cord injury-induced loss of skeletal muscle mass does not progress linearly. In humans, peak muscle loss occurs during the first 6 weeks postinjury, and gradually continues thereafter. The aim of this study was to delineate the regulatory events underlying skeletal muscle atrophy during the first year following spinal cord injury. Key translational, autophagic and proteolytic proteins were analysed by immunoblotting of human vastus lateralis muscle obtained 1, 3 and 12 months following spinal cord injury. Age-matched able-bodied control subjects were also studied. Several downstream targets of Akt signalling decreased after spinal cord injury in skeletal muscle, without changes in resting Akt Ser473 and Akt Thr308 phosphorylation or total Akt protein. Abundance of mTOR protein and mTOR Ser2448 phosphorylation, as well as FOXO1 Ser256 phosphorylation and FOXO3 protein, decreased in response to spinal cord injury, coincident with attenuated protein abundance of E3 ubiquitin ligases, MuRF1 and MAFbx. S6 protein and Ser235/236 phosphorylation, as well as 4E-BP1 Thr37/46 phosphorylation, increased transiently after spinal cord injury, indicating higher levels of protein translation early after injury. Protein abundance of LC3-I and LC3-II decreased 3 months postinjury as compared with 1 month postinjury, but not compared to able-bodied control subjects, indicating lower levels of autophagy. Proteins regulating proteasomal degradation were stably increased in response to spinal cord injury. Together, these data provide indirect evidence suggesting that protein translation and autophagy transiently increase, while whole proteolysis remains stably higher in skeletal muscle within the first year after spinal cord injury.

Striatal Transcriptome and Interactome Analysis of Shank3-overexpressing Mice Reveals the Connectivity between Shank3 and mTORC1 Signaling.

Mania causes symptoms of hyperactivity, impulsivity, elevated mood, reduced anxiety and decreased need for sleep, which suggests that the dysfunction of the striatum, a critical component of the brain motor and reward system, can be causally associated with mania. However, detailed molecular pathophysiology underlying the striatal dysfunction in mania remains largely unknown. In this study, we aimed to identify the molecular pathways showing alterations in the striatum of SH3 and multiple ankyrin repeat domains 3 (Shank3)-overexpressing transgenic (TG) mice that display manic-like behaviors. The results of transcriptome analysis suggested that mammalian target of rapamycin complex 1 (mTORC1) signaling may be the primary molecular signature altered in the Shank3 TG striatum. Indeed, we found that striatal mTORC1 activity, as measured by mTOR S2448 phosphorylation, was significantly decreased in the Shank3 TG mice compared to wild-type (WT) mice. To elucidate the potential underlying mechanism, we re-analyzed previously reported protein interactomes, and detected a high connectivity between Shank3 and several upstream regulators of mTORC1, such as tuberous sclerosis 1 (TSC1), TSC2 and Ras homolog enriched in striatum (Rhes), via 94 common interactors that we denominated “Shank3-mTORC1 interactome”. We noticed that, among the 94 common interactors, 11 proteins were related to actin filaments, the level of which was increased in the dorsal striatum of Shank3 TG mice. Furthermore, we could co-immunoprecipitate Shank3, Rhes and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) proteins from the striatal lysate of Shank3 TG mice. By comparing with the gene sets of psychiatric disorders, we also observed that the 94 proteins of Shank3-mTORC1 interactome were significantly associated with bipolar disorder (BD). Altogether, our results suggest a protein interaction-mediated connectivity between Shank3 and certain upstream regulators of mTORC1 that might contribute to the abnormal striatal mTORC1 activity and to the manic-like behaviors of Shank3 TG mice.

Whole Eggs and Egg Whites Ingestion Induce Similar Increases in Muscle Anabolic Signaling Phosphorylation after Resistance Exercise in Trained Young Men

The effect of nutrient‐dense food ingestion immediately after resistance exercise as a nutritional strategy to promote muscle anabolism has not been clearly established. Given that a coordinated anabolic response is necessary for the regulation of muscle protein synthesis with exercise and protein ingestion, our aim was to determine whether egg whites or whole eggs ingestion influenced post‐exercise mechanistic target of rapamycin complex 1 (mTORC1) and mitogen‐activated protein kinase (MAPK) signaling phosphorylation in human skeletal muscle. In crossover trials, 10 healthy resistance‐trained men (21±1 y; 88±3 kg; bodyfat: 16±1%; means±SD) completed lower body resistance exercise (4 sets of 10 repetitions at 80% of 10‐RM for both leg press and leg extensions) before ingesting whole eggs (18 g protein, 17 g fat) or egg whites (18 g protein, 0 g fat) in scrambled form. Muscle biopsies were obtained before exercise and at 2 and 5H after protein ingestion to assess the phosphorylation of candidate anabolic signaling molecules by Western blotting. The phosphorylation of mTORser2448 increased ~34% above fasted at 5H following whole egg and egg white consumption (time effect; P&lt;0.01) with no difference between conditions (P&gt;0.66). Downstream of mTORC1, 4E‐BP1Thr37/46 was elevated above fasted at 2H (~22–44%; P&lt;0.01) and was sustained above fasted at 5H (P&lt;0.01) with no differences between conditions (P&gt;0.14). There was no effect of food ingestion on S6K1Thr389 and eEF2Thr56. The phosphorylation of the MAPK ERK1/2Thr202/Tyr204 was similarly suppressed at 2 and 5H following feeding for the whole egg and egg white conditions (~430–780%; P&lt;0.001). Ingestion of isonitrogenous amounts of whole egg and egg white similarly stimulates anabolic signaling in young trained males. These findings would suggest a similar increase in mRNA translation initiation during recovery from resistance exercise in healthy young men. Identification of the intracellular localization of mTORC1 and its colocalization with positive (e.g. Rheb) and negative (e.g. TSC2) regulators via immunofluorescence is ongoing.

Phosphorylation of mTOR Ser2481 is a key target limiting the efficacy of rapalogs for treating hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Although recent studies facilitate the identification of crucial genes and relevant regulatory pathways, therapeutic approaches against advanced HCC are insufficiently effective. Therefore, we aimed here to develop potent therapeutics to provide a reliable biomarker for the therapeutic efficacy in patients with HCC. To this end, we first compared the cytotoxic effects of various anti-cancer drugs between well differentiated (HAK-1A) and poorly differentiated (HAK-1B) cell lines established from a single HCC tumor. Of various drug screened, HAK-1B cells were more sensitive by a factor of 2,000 to the mTORC1 inhibitors (rapalogs), rapamycin and everolimus, than HAK-1A cells. Although rapalogs inhibited phosphorylation of mTOR Ser2448 in HAK-1A and HAK-1B cells, phosphorylation of mTOR Ser2481 was specifically inhibited only in HAK-1B cells. Silencing of Raptor induced apoptosis and inhibited the growth of only HAK-1B cells. Further, three other cell lines established independently from the tumors of three patients with HCC were also approximately 2,000-fold times more sensitive to rapamycin, which correlated closely with the inhibition of mTOR Ser2481 phosphorylation by rapamycin. Treatment with everolimus markedly inhibited the growth of tumors induced by poorly differentiated HAK-1B and KYN-2 cells and phosphorylation of mTOR Ser2481 in vivo. To our knowledge, this is the first study showing that the phosphorylation of mTOR Ser2481 is selectively inhibited by rapalogs in mTORC1-addicted HCC cells and may be a potential reliable biomarker for the therapeutic efficacy of rapalogs for treating HCC patients.

MTOR pathway activation is a favorable prognostic factor in human prostate adenocarcinoma.

Prostate cancer patients with localized disease are treated with curative intent. However, the disease will recur in approximately 30% of patients with a high incidence of morbidity and mortality. Prognostic biomarkers are needed to identify patients with high risk of relapse. mTOR pathway activation is reported in prostate cancer, but clinical trials testing efficacy of mTOR inhibitors were unsuccessful. To explain this clinical observation, we studied the expression and prognostic impact of mTOR-S2448 phosphorylation in localized prostate carcinomas. mTOR-S2448 phosphorylation is indicative for an activated mTOR pathway in prostate cancer. Surprisingly, the mTOR signaling pathway is activated specifically in prostate cancer patients with a favorable outcome. Since tumors from poor-outcome patients have low levels of mTOR-S2448 phosphorylation, this may explain why mTOR inhibitors proved unsuccessful in prostate cancer trials.

The afatinib resistance of in vivo generated H1975 lung cancer cell clones is mediated by SRC/ERBB3/c-KIT/c-MET compensatory survival signaling.

We generated afatinib resistant clones of H1975 lung cancer cells by transient exposure of established tumors to the drug and collected the re-grown tumors. Afatinib resistant H1975 clones did not exhibit any additional mutations in proto-oncogenes when compared to control clones. Afatinib resistant H1975 tumor clones expressed less PTEN than control clones and in afatinib resistant clones this correlated with increased basal SRC Y416, ERBB3 Y1289, AKT T308 and mTOR S2448 phosphorylation, decreased expression of ERBB1, ERBB2 and ERBB3 and increased total expression of c-MET, c-KIT and PDGFRβ. Afatinib resistant clones were selectively killed by knock down of [ERBB3 + c-MET + c-KIT] but not by the individual or doublet knock down combinations. The combination of the ERBB1/2/4 inhibitor afatinib with the SRC family inhibitor dasatinib killed afatinib resistant H1975 cells in a greater than additive fashion; other drugs used in combination with dasatinib such as sunitinib, crizotinib and amufatinib were less effective. [Afatinib + dasatinib] treatment profoundly inactivated ERBB3, AKT and mTOR in the H1975 afatinib resistant clones and increased ATG13 S318 phosphorylation. Knock down of ATG13, Beclin1 or eIF2α strong suppressed killing by [ERBB3 + c-MET + c-KIT] knock down, but were only modestly protective against [afatinib + dasatinib] lethality. Thus afatinib resistant H1975 NSCLC cells rely on ERBB1- and SRC-dependent hyper-activation of residual ERBB3 and elevated signaling, due to elevated protein expression, from wild type c-MET and c-KIT to remain alive. Inhibition of ERBB3 signaling via both blockade of SRC and ERBB1 results in tumor cell death.

Ghrelin induces colon cancer cell proliferation through the GHS-R, Ras, PI3K, Akt, and mTOR signaling pathways

Colon cancer is the third most common malignancy worldwide. Recently, some interesting associations between ghrelin and cancer were reported, and it may participate in colon cancer development. In the present report, we explored the role of the growth hormone secretagogue receptor (GHS-R), Ras, phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) pathways in the ghrelin-induced proliferation of human colon cancer cells. Ghrelin-caused HT-29 proliferation was reduced by [D-Lys3]-GHRP-6 (a GHS-R inhibitor). We also found that a dominant negative mutant of Ras (Ras DN), a PI3K inhibitor (LY 294002), an Akt DN, and an mTOR inhibitor (rapamycin) attenuated ghrelin-caused colon cancer cell proliferation. We found that ghrelin induced time-dependent increases in Ras activity. Moreover, ghrelin-mediated Akt Ser473 phosphorylation was attenuated by a Ras DN and LY 294002. Furthermore, a Ras DN, LY 294002, and an Akt DN all inhibited ghrelin-caused mTOR Ser2448 phosphorylation. These results indicate that the Ras/PI3K/Akt/mTOR cascade plays a critical role in ghrelin-induced colon cancer cell proliferation.

Activity of PIM Kinases in Chronic Lymphocytic Leukemia Modulates Tumor Cell Survival and Stromal Interactions through a Pleiotropic Mechanism Involving Modulation of CXCR4 - mTOR Pathway

The survival of chronic lymphocytic leukemia (CLL) cells depends on their interactions with microenvironment components, such as stromal and T cells. Lymph node microenvironment provides protective signals that enable the formation of proliferation centers and favor resistance to conventional chemotherapeutics. One of the key molecules engaged in the communication of CLL cells with their microenvironment is C-X-C chemokine receptor type 4 (CXCR4). The surface expression of CXCR4 is regulated by PIM1 (provirus integration site for Moloney murine leukemia virus) kinase. PIM 1-3 kinases are overexpressed in CLL cells and recent data suggest that targeting PIMs might be a rational therapeutic approach in this type of leukemia. Herein, we assessed associations of PIM kinase expression with clinical characteristics of CLL patients and investigated the consequences of PIM kinase inhibition for cell survival and CXCR4 - dependent signal transduction and migration of primary CLL cells.In primary CLL cells from peripheral blood, PIM2 transcript abundance was higher than PIM1 and PIM3 (p<0.001). PIM2 expression was higher in patients with advanced disease (Rai 3-4, p=0.004). Higher PIM2 expression was also observed in patients who relapsed after first line treatment (p=0.005). Expression of PIM2 and PIM3 kinases in lymph nodes was significantly higher than in peripheral blood (p=0.024 and p=0.0059, respectively; Herishanu et al., 2010), suggesting a relationship between PIM kinase expression/activity and CLL cell microenvironment. To further explore the role of PIM kinases in these processes, we assessed whether PIM inhibition interferes with CXCR4 - mediated signaling and migration. We incubated primary CLL cells with a novel pan-PIM inhibitor SEL24-B489 and found decreased phospho-CXCR4 (Ser339) level and decreased CXCR4 surface expression. Primary CLL cells incubated with with SDF1 (500 ng/ml, 15-60 min) exhibited highly increased phosphorylations of mTOR (Ser2448) and Akt (Ser437). Since PIM kinases modulate mTOR signaling, we further investigated whether inhibition of PIM kinases with SEL24-B489 interferes with CXCR4/mTOR pathway. SEL24-B489 blocked baseline phosphorylation level of mTOR negative regulator p-TSC2 (Ser1798). Since TSC2 Ser1798 phosphorylation relieves its suppression on RHEB and promotes mTOR activity, we next assessed PIM inhibitor - induced changes in p-mTOR (Ser2448). Upon SEL24-B489 treatment, mTOR Ser2448 phosphorylation and activity of mTOR downstream substrates (p-Akt Ser473 and p-4EBP1 Thr37/46) markedly decreased. Pre-incubation of CLL cells with 10 uM SEL24-B489 or 10 uM mTOR inhibitor OSI-027 before SDF1 treatment restrained the increase of p-mTOR (Ser2448), p-Akt (Ser473), p-4EBP1 (Thr37/46) and p-TSC2 (Ser1798), and consequently impaired CLL cells migration in the SDF1 gradient. In 4 out of 7 analyzed patients the effect of SEL24-B489 on CLL migration was stronger than the effect of OSI-027 and in the remaining 3 patients, the effects of both inhibitors were similar. Since interactions of CLL cells with their microenvironment block the cytotoxicity of chemotherapeutic agents, we next compared the apoptotic response to SEL24-B489 in CLL cells cultured in the absence or presence of human bone marrow HS5 cells. CLL cells were seeded on HS5 monolayers and treated with SEL24-B489 (5 uM and 10 uM) for 48 hours. In 6 out of 7 cases SEL24-B489 overcame the protective signals from HS5 cells and induced apoptosis, although the cytotoxic effect of PIM inhibitor was stronger in the absence of stromal cells.Taken together, our data demonstrate that CXCR4/SDF1 signal in chronic lymphocytic leukemia cells is transduced through mTOR pathway and that CXCR4 - triggered mTOR activity is modulated by PIM kinases. Pan-PIM inhibitor SEL24-B489 decreased CXCR4 surface expression and SDF-1 - triggered mTOR activity. Finally, SEL24-B489 decreased protective effects of tumor microenvironment and induced CLL cells apoptosis even in the presence of stromal cells. Since overexpression of PIM kinases might be associated with adverse clinical characteristics at diagnosis, PIM inhibition might be a rational therapeutic strategy in CLL. DisclosuresCzardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Other: member of Selvita Scientific Advisory Board.