O overreaching não funcional (NFOR) induzido pelo exercício excêntrico (EE) em modelo animal está associado com a diminuição de desempenho físico, dano no DNA (amostras de músculo esquelético e soro), estresse oxidativo (amostras de músculo esquelético e soro), inflamação crônica de baixo grau (amostras de músculo esquelético e soro) e prejuízo da via de sinalização da insulina (amostras de músculo esquelético). No entanto, as adaptações do músculo cardíaco em resposta ao estado de NFOR induzido ou não pela predominância do EE ainda não foram investigadas. Além disso, sabe-se que a mTOR (mammalian target of rapamycin) possui um efeito protetor no músculo cardíaco, suprimindo o aumento de citocinas pró-inflamatórias, que estão relacionadas à disfunções cardíacas. Assim, o presente estudo teve como objetivo comparar os efeitos do NFOR em declive com outros dois protocolos de mesmo volume e intensidade, mas realizados sem inclinação e em aclive, no conteúdo das proteínas relacionadas às vias moleculares inflamatória e hipertrófica, no conteúdo de fibrose intersticial e na expresão gênica em músculo cardíaco de camundongos. Os animais foram divididos em 6 grupos: Naíve (N; camundongos sedentários), Controle (C; camundongos sedentários submetidos aos testes físicos), Treinado (TR; camundongos submetidos ao protocolo de treinamento), Overtraining em declive (OTR/down; camundongos submetidos ao protocolo de OT com corrida na descida), Overtraining sem inclinação (OTR; camundongos submetidos ao protocolo de OT com corrida sem inclinação) e Overtraining em aclive (OTR/up; camundongos submetidos ao protocolo de OT com corrida na subida). Em relação aos parâmetros metabólicos, o grupo OTR/down apresentou menor variação de peso corporal na semana 8. Todos os grupos que passaram pelo protocolo de OT demonstraram queda de desempenho ao final da semana 8, aumento no conteúdo de tecido conjuntivo no ventrículo esquerdo e menor ativação da proteína AMPKalfa. O grupo OTR/down apresentou menor conteúdo da proteína mTOR e S6RP, e aumento na expressão do gene beta-MHC. Pode-se concluir que os protocolos de OT provocaram indícios de hipertrofia patológica no ventrículo esquerdo, sendo esse efeito mais pronunciado no grupo OTR/down. / Nonfunctional overreaching (NFOR) induced by eccentric exercise (EE) in animal model is associated with performance decrement, DNA damage (muscle and serum samples), oxidative stress (muscle and serum samples), low grade chronic inflammation (muscle and serum samples) and insulin signaling impairment (muscle and serum samples). However, the adaptations of cardiac muscle in response to NFOR induced or not induced by EE are unknown. In addition, the mammalian target of rapamycin (mTOR) has a protector effect in the cardiac muscle, suppressing the increase of the proinflammatory cytokines that is related to cardiac dysfunctions. Thus, the main aim of present study was to compare the effects of NFOR based on EE (downhill running) with other two protocols with similar intensity and volume, but performed in uphill and without inclination, on the protein contents related to hypertrophic and inflammatory signaling, on the content of interstitial fibrotic tissue and on genes expression in mice cardiac muscle. The animals were divided on six groups: Naïve (N; sedentary mice), Control (C; sedentary mice submitted to physical tests), Trained (TR; mice submitted to training protocol), Overtraining in downhill (OTR/down; mice submitted to the overtraining protocol in downhill), Overtraining without inclination (OTR; mice submitted to the overtraining protocol without inclination), and Overtraining in uphill (OTR/up; mice submitted to the overtraining protocol in uphill). Regarding metabolic parameters, OTR/down group presented reduced body weight variation at week 8. All OT groups presented performance drop at end of week 8, increased connective tissue content in left ventricle and reduced AMPKalpha activation. OTR/down group presented reduced mTOR and S6RP protein content, and increased betaMHC gene expression. The conclusion is that OT protocols provoked signs of pathological hypertrophy in left ventricle, being this effect more pronounced in OTR/down group.

MTor est une protéine centrale de la voie de signalisation PI3K/AKT/mTor et est impliquée dans la croissance, la prolifération et la survie cellulaire. Cette protéine joue un rôle majeur particulièrement dans la prolifération tumorale. Ainsi des inhibiteurs de mTor ont été développés à partir des années 1980, notamment la rapamycine, et deux sont utilisés actuellement en clinique: l’évérolimus et le temsirolimus. Ces deux inhibiteurs font partie des différentes possibilités thérapeutiques dans les cancers du sein et du rein métastatiques. Les patients traités présentent des réponses variées avec des cas de patients très bons répondeurs et des cas de patients résistants d'emblée ou après exposition au traitement. Face à cette notion de résistance, nous avons voulu caractériser les mécanismes d’adaptation de la cellule tumorale après exposition aux anti-mtor. L’analyse de l’expression génomique de huit lignées cellulaires tumorales variées après traitement par la rapamycine a montré une modulation de l’expression de certains gènes dont celle de la pseudokinase TRIB3, diminuée dans toutes les lignées. Cette donnée in vitro est aussi retrouvée chez les patients. En effet après traitement par évérolimus, nous avons pu observer une diminution de l’expression du gène TRIB3 au niveau sanguin. Nous avons donc cherché à comprendre le rôle de cette pseudokinase dans la réponse aux anti-mTor. Nos résultats mettent en évidence, d’une part, que la rapamycine régule l’expression de TRIB3 en agissant sur son promoteur via une interaction avec GCF2. D’autre part, l’hyperexpression de TRIB3 limite les effets anti-tumoraux de la rapamycine dans différentes lignées cellulaires tumorales. Pour étudier plus en détails ce mécanisme, nous avons cherché à déterminer les partenaires de TRIB3. Par des approches de protéomique haut-débit, nous avons mis en évidence un lien avec des protéines impliquées dans l’épissage. Ainsi la rapamycine semble inhiber la machinerie d’épissage via la diminution d’expression de TRIB3. Ce travail relève l’intérêt de TRIB3 dans la réponse aux anti-mTor comme un potentiel biomarqueur et illustre également le mode d’action de la rapamycine. / MTor is a central protein of the PI3K/AKT/mTor signaling pathway and is involved in growth, proliferation and cell survival. This protein plays a major role particularly in tumor proliferation. Thus, mTor inhibitors have been developed since the 1980s, including rapamycin, and two are currently used in daily practice: everolimus and temsirolimus. These two inhibitors are part of the different therapeutic possibilities in metastatic breast and kidney cancers. Patients treated present different responses with cases of very good responders and cases of resistant patients either immediately or after exposure to treatment. According to the concept of resistance, we wanted to characterize the mechanisms of adaptation of the tumor cell after exposure to mTor inhibitors. Analysis of the genomic expression of eight variant tumor cell lines after treatment with rapamycin showed a modulation of the expression of different genes including the pseudokinase TRIB3, decreased in all the lines. This in vitro data is also found in patients. Indeed, after treatment with everolimus, we observed a decreased expression of the TRIB3 gene in blood. We therefore sought to understand the role of this pseudokinase in the response to mTor inhibitors. Our results show, that rapamycin regulates the expression of TRIB3 through a GCF2-dependent inhibition of promoter activity. Moreover, TRIB3 overexpression limits the anti-tumor effects of rapamycin in different tumor cell lines. To investigate this mechanism, we sought to identify TRIB3 partners. By high-throughput proteomic approaches, we have demonstrated a link with proteins involved in splicing. Thus, rapamycin appears to inhibit splicing machinery with the decreased expression of TRIB3. This work highlights the interest of TRIB3 in the response to mTor inhibitors as a potential biomarker and investigates also how rapamycin acts on cells.

No description available.

Introduction: Over the past decades, considerable advances in understanding of cell biology at genetic, epigenetic and proteomic levels led to development of new strategies for better outcome of cancer therapy. One of these new strategies is targeting the class I PI3K/Akt/mTOR signaling pathway, in that this pathway plays a key role in regulation of many cellular functions, including proliferation, survival, metabolism, autophagy and motility. Dysregulation of the class I PI3K/Akt/mTOR pathway has been documented in a variety of human tumours and inhibition of this pathway has been observed to hamper tumour proliferation in vitro and prevent tumour progression in vivo and in clinic. More recently, emerging evidence suggests that the class I PI3K/Akt/mTOR pathway is associated with Cancer Stem Cell (CSC) biology, in light of maintenance, viability and conventional therapy resistance of CSCs. The CSC theory conceptualizes that a subset of tumour cells with Stem Cell-like properties, including self-renewal, multipotency, differentiation, and resistance to chemotherapy and radiotherapy, can recapitulate new tumours and resistance to cancer therapy. Materials and Methods: To explore class I PI3K/Akt/mTOR signaling pathway and CSCs as therapeutic targets in canine oncology, in one series of experiments, smallmolecular inhibitors Wortmannin, ZSTK474, KP372-1 and Rapamycin, which selectively target pan-class I PI3K, pan-class I PI3K, Akt and mTOR, respectively, were utilized to treat canine cancer cell lines using inhibitors alone or in combination with conventional therapeutic drugs. The human acute lymphoblastic leukaemia of T-cell origin cell line (Jurkat T cell line) was used as a comparative control. In another, a stem cell culture system was performed to isolate CSCs from canine glioma J3T cell line. Subsequently, microarray analysis of transcriptional expression profiles of J3T spheres (the putative CSCs) versus J3T parental cells was performed. Results: In this study, small molecules ZSTK474 and KP372-1 were found to significantly decrease cell viability at lower micromolar and nanomolar ranges, respectively. Rapamycin decreased cell viability at lower micromolar concentrations. However, the efficacy of Wortmannin varied from one cell line to another. Dissection of the mechanism of these inhibitors using Western Blot analysis and annexin V staining showed that all inhibitors functioned by decreasing phosphorylation of class I PI3K pathway members. Notably, the efficacy of Wortmannin for this pathway inhibition is confined to certain cell lines. In addition, Wortmannin had shorter drug duration than the other three inhibitors. Annexin V staining showed that KP372-1 was a potent inducer of apoptosis, with decreasing potency in hierarchy order, Rapamycin, Wortmannin and ZSTK474. The data obtained from the combination of pan-class I PI3K inhibitor (Wortmannin or ZSTK474) and mTOR inhibitor (Rapamycin) suggested that additive/synergistic effects were, in part, due to inactivation of Akt. The class I PI3K pathway inhibitors enhanced the efficacy of Doxorubicin in SB cells but not in canine REM, 3132 and J3T cells. The CSC colonies of canine glioma J3T cells were successfully isolated and expanded in the neurosphere formation assay. By microarray analysis, several class I PI3K signaling network-associated genes, particularly IGFBP2 (27-fold), FYN (9.3- fold), and DDIT4 (8.5-fold), were found to be highly up-regulated in the J3T CSCs. However, the genes encoding components, such as Akt1 and eIF4E, of class I PI3K/Akt/mTOR axis signaling were either unchanged or down-regulated in the CSCs. The majority of the genes encoding translation initiation factors were also downregulated in the CSCs. Conclusions: This study demonstrates that class I PI3K/Akt/mTOR signaling pathway is critical for proliferation and survival of cell lines derived from human acute lymphoblastic leukemia of T cell origin (Jurkat T cell line) and a variety of canine tumours. However, it appears that this pathway is dispensible for maintainence and viability of the CSCs isolated from canine gloma J3T cell line. This study suggests that the strategy of dual inhibition of class I PI3K and mTOR kinases may have better outcomes than the combination inhibitors of this pathway (such as ZSTK474 and KP372-1) with Doxorubicin in canine oncology.

Bone is a highly dynamic organ system comprised of various cell types that are constantly working to maintain the health and stability of bone. The main cells involved are the osteoblasts that form bone, the osteoclasts that degrade bone, and the osteocytes that act as sensors of the microenvironment and coordinate a response. An imbalance of the interactions between the cell types can potentially result in pathological states in bone at the microscopic level that can then affect the entire skeleton. Moreover, a number of genetic mutations can also lead to pathogenic changes in bone. An example of such is the development of sclerotic bone lesions in patients with the disease tuberous sclerosis complex. Tuberous sclerosis complex, or TSC, is an autosomal dominant disorder affecting approximately 1.5 million people worldwide. It is caused by a mutation in one of the genes encoding either member of the TSC1-TSC2 complex. Molecularly, TSC1-TSC2 negatively regulate the mechanistic target of rapamycin (mTOR) kinase in the mutli-protein complex mTORC1. Activation of mTORC1 leads to an upregulation of protein synthesis and cell growth. Tuberous sclerosis patients are heterozygous for TSC1 or TSC2, and post-natal loss of the second allele results in the development of multiple, benign, tumor-like hamartomas in various organ systems, most notably affecting the brain, kidneys, lungs, skin, and heart. Additionally, CT scans of patients reveal multiple loci of dense, compact bone termed sclerotic bone lesions. The bone lesions were most commonly seen in the posterior elements of the vertebrae and while they are asymptomatic, a remarkably high frequency of patients express them. To further investigate and better understand the mechanisms of tuberous sclerosis complex in bone, we analyzed a mouse model with heterozygous deletion in Tsc2. Initial examination showed the Tsc2+/- mice recapitulated tumors in various organ systems, most notably the kidney, and presented bone lesions in the pelvis and elements of the vertebrae. To further investigate the mechanism driving the disease state, we used a Cre driver thought to be specific for osteoclast (Cathepsin K-Cre, or Ctsk-Cre) to selectively delete Tsc2. Cathepsin K-Cre; Tsc2fl/fl mice exhibit a remarkably high bone mass. This study examined three specific aspects of this high bone mass phenotype. First, we sought to verify that the increased bone mass caused by Ctsk-Cre driven Tsc2 deletion was dependent on mTORC1 upregulation. This was done by generating Ctsk-Cre;Tsc2fl/fl mice lacking Raptor, a mTORC1 component essential for function. Next, we investigated the cell of origin driving the increase bone density by utilizing additional Cre drivers specific for osteoclasts and osteocytes. Additionally, we used radiation chimeras to assess if donated wild type cells could rescue the observed phenotype. We lastly explored the role of a secreted signaling molecule, CTHRC1, that has been proposed as a candidate to mediate osteoclast-osteoblast interaction, in the high bone mass phenotype of Ctsk-Cre;Tsc2fl/fl mice. Selective deletion of Tsc2 in bone cells provides an excellent model to investigate pathways regulating bone mass and strength and may provide new candidate targets for treating diseases of low bone mass, such as osteoporosis.

Macrophages are key orchestrators of the innate immune response with a dynamic role in the promotion and resolution of inflammation. Macrophage polarization to a pro-inflammatory or anti-inflammatory phenotype must be tightly controlled to maintain appropriate responses to stimuli as well as to maintain tissue homeostasis. The nutrient and energy sensor Mammalian Target of Rapamycin (mTOR) integrates upstream signals from the PI3K/Akt pathway to orchestrate cellular protein, lipid, and glucose metabolism. This key metabolic pathway has been implicated in T-helper cell skewing and in the innate immune regulation. The mechanisms of innate immune regulation by mTOR are currently unclear as most studies use pharmacological inhibitors with potential off target effects. In this study, we use a novel model of TSC1 deficiency in myeloid lineage cells to elucidate a role for mTOR in macrophage polarization. We show, for the first time, that Tsc1-deficiency and constitutive mTORC1 activity in macrophages leads to a marked defect in M2 polarization when stimulated with the Th2 cytokine IL-4. Tsc1-deficient macrophages display attenuated Akt signaling in response to IL-4 consistent with negative feedback of mTORC1 on upstream IRS2/PI3K signaling, and we demonstrate that this parallel signaling pathway is critical for induction of a subset of M2 markers. Tsc1-deficient macrophages fail to upregulate the M2 genes Pgc-1!, Arg-1, Fizz-1, and Mgl1 in addition to other M2 markers despite normal STAT6 signaling in response to IL-4. Consistent with downregulation of Pgc-1!, Tsc1-deficient macrophages also display defects in fatty acid metabolism and mitchochondrial biogenesis. Furthermore, LPS stimulation in Tsc-1 deficient macrophages leads to an enhanced inflammatory response with increased production of pro-inflammatory cytokines. We believe that Tsc1-deficient macrophages are a model of constitutive mTORC1 activity akin to obesity, where chronic nutrient excess leads to increases in mTORC1 activity, attenuation of IRS/PI3K/Akt signaling, and defective M2 polarization of macrophages in metabolic tissues.

Histotroph, secretions from luminal (LE), superficial glandular (sGE) and glandular (GE) epithelia and molecules selectively transported into the uterine lumen, are essential for peri-implantation ovine conceptus development and maternal recognition of pregnancy. Among them, several components of histotroph including nutrients, cell matrix proteins and growth factors may activate mTOR (mammalian target of rapamycin; also known as FRAP1) to stimulate hypertrophy, hyperplasia, and/or migration of conceptus trophectoderm cells, as well as expression of IFNT for pregnancy recognition and critical proteins for conceptus development. Therefore, studies were conducted to examine effects of select nutrients (arginine, leucine, glutamine and glucose), IGF2 and SPP1 on mTOR signal transduction pathways and determine their biological effects on proliferation, migration and/or attachment of ovine trophectoderm (oTr) cells and conceptuses (embryo and it extra-embryonic membranes). The first study defined the expression of IGF2, RPS6K, phosphorylated AKT, RPS6K, P38 and ERK1/2 MAPK by the uterus and conceptus during the periimplantation period. In addition, effects of IGF2 on the PI3K signaling pathway were evaluated using oTr cells isolated from Day 15 conceptuses. IGF2 was most abundant in compact stroma of endometrial caruncles and also present in all cells of the conceptus, but particularly abundant in the endoderm and yolk sac. Phosphorylated AKT1, RPS6K, P38 and ERK1/2 proteins were abundant in nuclei of endometrial LE and conceptus trophectoderm. IGF2 activated multiple cell signaling pathways including PDK/AKT/mTOR/RPS6K and MAPKs that are critical to survival, growth and migration of the ovine trophoblast cells. The second study demonstrated the multifunctional effects of secreted phosphoprotein 1 (SPP1) on oTr cells including cell signaling transduction, migration, and adhesion. Novel results of this study indicated that SPP1 binds ?v?3 and ?5?1 integrins to activate PI3K/mTOR/RPS6K, MAPK as well as crosstalk between mTOR and MAPK pathways that are essential for expansion and elongation of conceptuses and attachment of trophectoderm to uterine LE during implantation. The third study identified effects of arginine (Arg), leucine (Leu), glutamine (Gln) and glucose on oTr cells. Arg, Leu and glucose, but not Gln, activated PI3KAKT1 and mTOR-RPS6K-RPS6 signaling pathways. Arg, Leu and glucose increased abundance of p-RPS6K in nuclei and p-RPS6 in cytoplasm of oTr cells. In addition, results of this study demonstrated that Arg and Leu are remarkably stimulatory to cell proliferation and migration. The fourth study determined effects of Arg on signal transduction pathways and oTr cell proliferation, as well as inhibition of oTr cell proliferation by L-NAME (an inhibitor of NOS) or Nor-NOHA (an inhibitor of arginase) on oTr cells. Arg increased p-mTOR, RPS6K and 4EBP1 protein and also increased protein synthesis and reduced protein degradation in oTr cells. Both NO and polyamines enhanced cell proliferation in a dose-dependent manner. The effects of Arg were partially inhibited by both L-NAME and Nor-NOHA. These results indicate that Arg enhances production of polyamines and NO and activates the mTOR-FRAP1-RPS6K-RPS6 signaling pathway to stimulate proliferation of oTr. The fifth study identified differential effects of Arg, Leu, Gln and glucose on gene expression and protein translation in explants cultures of ovine conceptuses. Expression of mRNAs was not affected by treatments with the select nutrients; however, Arg, Leu, Gln and glucose increased abundance of total and phosphorylated forms of mTOR, RPS6K, 4E-BP1 and RPS6. Arg, Leu, Gln and glucose also increased the amounts of NOS and ODC1, but only Arg stimulated a significant increase in abundance of IFNT. Collectively, these studies indicated that IGF2, SPP1 and select nutrients activate mTOR cell-signaling pathways that converge on AKT1 and that are likely critical to mechanism(s) responsible for survival, elongation an development of conceptuses. A more complete understanding of this mechanism will be important to development of strategies to reduce early embryonic losses in ruminants and and in other species including humans.

The complex PI3K/mTOR pathway regulates tumor progression via effects on cellular proliferation, apoptosis, autophagy, and motility. New drugs that inhibit the catalytic site of both PI3K and mTOR have shown promise in clinical trials. Here, we report the first use of a novel, dual PI3K/mTOR catalytic site inhibitor (PF-04691502, PF1502) in a xenograft model of breast cancer metastasis to bone. Metastatic MDA-MB-1833 cells showed PI3K/mTOR activation relative to parental MDA-MB-231. Low-dose PF1502 significantly impaired tumor cell motility and invasion in vitro without causing cell cycle arrest, apoptosis, or reduced proliferation. Pre-treatment of tumor cells at this dose reduced bone metastatic outgrowth in vivo. The atypical tumor suppressor, p27KIP1, is phosphorylated in its C-terminal region by multiple AGC kinases downstream of PI3K/mTOR. These phosphorylation events promote cytoplasmic mislocalzation of p27 which, in turn, facilitates inhibition of the RhoA cytoskeletal regulatory protein. The resulting turnover of the actin cytoskeleton is thought to underlie the increased cellular motility attributed to cytoplasmic p27. In MDA-MB-1833 cells, PI3K/mTOR inhibition reduced p27 C-terminal phosphorylation at T157 and T198 and reduced cytoplasmic p27 levels. Overexpression of a p27T157D/T198D phospho-mimetic mutant conferred resistance to the anti-motility effects of PF1502 in vitro. MDA-MB-1833 cells demonstrate p27-dependent inhibition of RhoA-ROCK signaling, as well as p27-dependent motility and invasion in vitro, however, RhoA knockdown did not confer resistance to the anti-motility effects of PF1502. p27shRNA dramatically impaired the bone metastatic outgrowth of MDA-MB-1833 in vivo. In an effort to explore potentially novel RhoA-independent mechanisms whereby cytoplasmic p27 might drive tumor cell motility and metastasis, we turned to the process known as epithelial-to-mesenchymal transition (EMT). The EMT program has been implicated as a critical driver of tumor metastasis in a variety of cancer models. PI3K/mTOR inhibition and shRNA p27 treatment both reversed expression of EMT markers in MDA-MB-1833. Thus, PI3K/mTOR appears to drive p27-dependent motility and metastasis at least in part by induction of an EMT-like phenotype, a novel mechanism through which p27 might act to promote tumor progression. These results provide an important new clinical rationale supporting the use of PI3K/mTOR inhibitors as anticancer agents via their inhibition of tumor invasion and metastasis.

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No description available.