Mechanistic or mammalian target of rapamycin (mTOR) may determine robustness in young male mice at the cost of accelerated aging.
ABSTRACT: Males, who are bigger and stronger than females, live shorter in most species from flies to mammals including humans. Cellular mass growth is driven in part by mTOR (Target of Rapamycin). When developmental growth is completed, then, instead of growth, mTOR drives aging, manifested by increased cellular functions, such as hyper-secretion by fibroblasts, thus altering homeostasis, leading to age-related diseases and death. We hypothesize that MTOR activity is elevated in male mice compared with females. Noteworthy, 6 months old males were 28 % heavier than females. Also levels of phosphorylated S6 (pS6) and phospho-AKT (p-AKT, Ser 473), markers of the mTOR activity, were higher in male organs tested. Levels of pS6 were highly variable among mice and correlated with body weight and p-AKT. With age, the difference between levels of pS6 between sexes tended to minimize, albeit males still had hyperactive mTOR. Unlike fasting, the intraperitoneal (i.p.) administration of rapamycin eliminated pS6 in all organs of all females measured by immunoblotting and immunohistochemistry without affecting p-AKT and blood insulin. Although i.p. rapamycin dramatically decreased levels of pS6 in males too, it was still detectable by immunoblotting upon longer exposure. Our study demonstrated that both tissue p-AKT and pS6 were higher in young male mice and were associated with increased body weight and insulin. These data can explain bigger body size and faster aging in males. Our data suggest higher efficacy of rapamycin compared to fasting. Higher sensitivity of females to rapamycin may explain more pronounced life extension by rapamycin observed in females compared to males in several studies.
Project description:Adaptive cardiac remodeling is characterized by enhanced signaling of mTORC2 downstream kinase Akt. In females, 17ß-estradiol (E2), as well as Akt contribute essentially to sex-related premenopausal cardioprotection. Pharmacologic mTOR targeting with rapamycin is increasingly used for various clinical indications, yet burdened with clinical heterogeneity in therapy responses. The drug inhibits mTORC1 and less-so mTORC2. In male rodents, rapamycin decreases maladaptive cardiac hypertrophy whereas it leads to detrimental dilative cardiomyopathy in females. We hypothesized that mTOR inhibition could interfere with 17?-estradiol (E2)-mediated sexual dimorphism and adaptive cell growth and tested responses in murine female hearts and cultured female cardiomyocytes. Under physiological in vivo conditions, rapamycin compromised mTORC2 function only in female, but not in male murine hearts. In cultured female cardiomyocytes, rapamycin impaired simultaneously IGF-1 induced activation of both mTOR signaling branches, mTORC1 and mTORC2 only in presence of E2. Use of specific estrogen receptor (ER)?- and ER?-agonists indicated involvement of both estrogen receptors (ER) in rapamycin effects on mTORC1 and mTORC2. Classical feedback mechanisms common in tumour cells with upregulation of PI3K signaling were not involved. E2 effect on Akt-pS473 downregulation by rapamycin was independent of ERK as shown by sequential mTOR and MEK-inhibition. Furthermore, regulatory mTORC2 complex defining component rictor phosphorylation at Ser1235, known to interfere with Akt-substrate binding to mTORC2, was not altered. Functionally, rapamycin significantly reduced trophic effect of E2 on cell size. In addition, cardiomyocytes with reduced Akt-pS473 under rapamycin treatment displayed decreased SERCA2A mRNA and protein expression suggesting negative functional consequences on cardiomyocyte contractility. Rictor silencing confirmed regulation of SERCA2A expression by mTORC2 in E2-cultured female cardiomyocytes. These data highlight a novel modulatory function of E2 on rapamycin effect on mTORC2 in female cardiomyocytes and regulation of SERCA2A expression by mTORC2. Conceivably, rapamycin abrogates the premenopausal "female advantage".
Project description:Vulnerabilities to fear-related disorders can be enhanced following early life adversity. This study sought to determine whether post-weaning social isolation (PSI), an animal model of early life adversity, alters the development of social fear in an innovative model of conditioned social fear. Male and female Sprague-Dawley rats underwent either social rearing (SR) or PSI for 4 weeks following weaning. Rats were then assigned to groups consisting of either Footshock only, Social conditioned stimulus (CS) only, or Paired footshock with a social CS. Social behavior was assessed the next day. We observed a novel behavioral response in PSI rats, running in circles, that was rarely observed in SR rats; moreover, this behavior was augmented after Paired treatment in PSI rats. Other social behaviors were altered by both PSI and Paired footshock and social CS. The mammalian target of rapamycin (mTOR) pathway was assessed using immunohistochemistry for phosphorylated ribosomal protein S6 (pS6) in subregions of the prefrontal cortex (PFC) and amygdala. Paired treatment produced opposite effects in the PFC and amygdala in males, but no differences were observed in females. Conditioned social fear produced alterations in social behavior and the mTOR pathway that are dependent upon rearing condition and sex.
Project description:The phosphoinositide 3-kinase (PI3K)/Akt pathway is constitutively activated in pancreatic cancer and the mammalian target of rapamycin (mTOR) kinase is an important mediator for its signaling. Our recent in vitro studies suggest that prolonged exposure of pancreatic cancer cells to mTOR inhibitors can promote insulin receptor substrate-PI3K interactions and paradoxically increase Akt phosphorylation and cyclin D1 expression in pancreatic cancer cells (negative feedback loop). The addition of erlotinib to rapamycin can down-regulate rapamycin-stimulated Akt and results in synergistic antitumor activity with erlotinib in preclinical tumor models.Two studies prospectively enrolled adult patients with advanced pancreatic cancer, Eastern Cooperative Oncology Group performance status 0-1, adequate hematologic, hepatic and renal parameters and measurable disease. In Study A, temsirolimus was administered intravenously at 25 mg weekly. In Study B, everolimus was administered orally at 30 mg weekly and erlotinib was administered at 150 mg daily. The primary endpoint in both studies was overall survival at 6 months. Secondary endpoints included time to progression, progression-free survival, overall survival, response rate, safety and toxicity. Pretreatment tumor biopsies were analyzed by immunofluorescence and laser scanning cytometry for the expression of pmTOR/mTOR, pAkt/Akt, pErk/Erk, pS6, p4EBP-1 and PTEN.Five patients enrolled in Study A; Two patients died within a month (rapid disease progression and hemorrhagic stroke, respectively). One patient developed dehydration and another developed asthenia. Sixteen patients enrolled in Study B.: 12 males, all ECOG PS = 1. Median cycles = 1 (range 1-2). Grade 4 toxicity: hyponatremia (n = 1), Grade 3: diarrhea (n = 1), cholangitis (n = 3), hyperglycemia (n = 1), fatigue (n = 1). Grade 2: pneumonia (n = 2), dehydration (n = 2), nausea (n = 2), neutropenia (n = 1), mucositis (n = 2) & rash (n = 2). Four patients were hospitalized. Progressive disease occurred in 15 and 1 was non-evaluable. Pretreatment biopsies revealed a higher pAkt/Akt ratio in tumor specimens that in nonmalignant pancreatic tissue. No such trends were noted for the other biomarkers.Neither study with mTOR inhibitors demonstrated objective responses or disease stability. The negative feedback loop resulting from mTOR inhibition may account for the disease progression and toxicity noted in these studies. Future strategies should aim for a broader targeting of the PI3K pathway in pancreatic cancer.NCT 0075647.NCT00640978.
Project description:Inhibition of the TOR signalling pathway by genetic or pharmacological intervention extends lifespan in invertebrates, including yeast, nematodes and fruitflies; however, whether inhibition of mTOR signalling can extend lifespan in a mammalian species was unknown. Here we report that rapamycin, an inhibitor of the mTOR pathway, extends median and maximal lifespan of both male and female mice when fed beginning at 600 days of age. On the basis of age at 90% mortality, rapamycin led to an increase of 14% for females and 9% for males. The effect was seen at three independent test sites in genetically heterogeneous mice, chosen to avoid genotype-specific effects on disease susceptibility. Disease patterns of rapamycin-treated mice did not differ from those of control mice. In a separate study, rapamycin fed to mice beginning at 270 days of age also increased survival in both males and females, based on an interim analysis conducted near the median survival point. Rapamycin may extend lifespan by postponing death from cancer, by retarding mechanisms of ageing, or both. To our knowledge, these are the first results to demonstrate a role for mTOR signalling in the regulation of mammalian lifespan, as well as pharmacological extension of lifespan in both genders. These findings have implications for further development of interventions targeting mTOR for the treatment and prevention of age-related diseases.
Project description:Improved efficacy of neoadjuvant endocrine-targeting therapies in luminal breast carcinomas could be achieved with optimal use of pathway targeting agents. In a mouse model of ductal breast carcinoma we identify a tumor regressive stromal reaction that is induced by neoadjuvant endocrine therapy. This reparative reaction is characterized by tumor neovascularization accompanied by infiltration of immune cells and carcinoma-associated fibroblasts that stain for phosphorylated ribosomal protein S6 (pS6), downstream the PI3K/Akt/mTOR pathway. While tumor variants with higher PI3K/Akt/mTOR activity respond well to a combination of endocrine and PI3K/Akt/mTOR inhibitors, tumor variants with lower PI3K/Akt/mTOR activity respond more poorly to the combination therapy than to the endocrine therapy alone, associated with inhibition of stromal pS6 and the reparative reaction. In human breast cancer xenografts we confirm that such differential sensitivity to therapy is primarily determined by the level of PI3K/Akt/mTOR in tumor cells. We further show that the clinical response of breast cancer patients undergoing neoadjuvant endocrine therapy is associated with the reparative stromal reaction. We conclude that tumor level and localization of pS6 are associated with therapeutic response in breast cancer and represent biomarkers to distinguish which tumors will benefit from the incorporation of PI3K/Akt/mTOR inhibitors with neoadjuvant endocrine therapy.
Project description:Previous studies support a role for mitogen-activated protein kinase pathway signaling, and more recently Akt/mammalian target of rapamycin (mTOR), in pediatric low-grade glioma (PLGG), including pilocytic astrocytoma (PA). Here we further evaluate the role of the mTORC1/mTORC2 pathway in order to better direct pharmacologic blockade in these common childhood tumors.We studied 177 PLGGs and PAs using immunohistochemistry and tested the effect of mTOR blockade on 2 PLGG cell lines (Res186 and Res259) in vitro.Moderate (2+) to strong (3+) immunostaining was observed for pS6 in 107/177 (59%) PAs and other PLGGs, while p4EBP1 was observed in 35/115 (30%), pElF4G in 66/112 (59%), mTOR (total) in 53/113 (47%), RAPTOR (mTORC1 component) in 64/102 (63%), RICTOR (mTORC2 component) in 48/101 (48%), and pAkt (S473) in 63/103 (61%). Complete phosphatase and tensin homolog protein loss was identified in only 7/101 (7%) of cases. In PA of the optic pathways, compared with other anatomic sites, there was increased immunoreactivity for pS6, pElF4G, mTOR (total), RICTOR, and pAkt (P < .05). We also observed increased pS6 (P = .01), p4EBP1 (P = .029), and RICTOR (P = .05) in neurofibromatosis type 1 compared with sporadic tumors. Treatment of the PLGG cell lines Res186 (PA derived) and Res259 (diffuse astrocytoma derived) with the rapalog MK8669 (ridaforolimus) led to decreased mTOR pathway activation and growth.These findings suggest that the mTOR pathway is active in PLGG but varies by clinicopathologic subtype. Additionally, our data suggest that mTORC2 is differentially active in optic pathway and neurofibromatosis type 1-associated gliomas. MTOR represents a potential therapeutic target in PLGG that merits further investigation.
Project description:The ketogenic diet (KD) is an effective treatment for epilepsy, but its mechanisms of action are poorly understood. We investigated the hypothesis that the KD inhibits mammalian target of rapamycin (mTOR) pathway signaling. The expression of pS6 and pAkt, markers of mTOR pathway activation, was reduced in hippocampus and liver of rats fed KD. In the kainate model of epilepsy, KD blocked the hippocampal pS6 elevation that occurs after status epilepticus. Because mTOR signaling has been implicated in epileptogenesis, these results suggest that the KD may have anticonvulsant or antiepileptogenic actions via mTOR pathway inhibition.
Project description:Rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR), robustly extends the lifespan of model organisms including mice. We recently found that chronic treatment with rapamycin not only inhibits mTOR complex 1 (mTORC1), the canonical target of rapamycin, but also inhibits mTOR complex 2 (mTORC2) in vivo. While genetic evidence strongly suggests that inhibition of mTORC1 is sufficient to promote longevity, the impact of mTORC2 inhibition on mammalian longevity has not been assessed. RICTOR is a protein component of mTORC2 that is essential for its activity. We examined three different mouse models of Rictor loss: mice heterozygous for Rictor, mice lacking hepatic Rictor, and mice in which Rictor was inducibly deleted throughout the body in adult animals. Surprisingly, we find that depletion of RICTOR significantly decreases male, but not female, lifespan. While the mechanism by which RICTOR loss impairs male survival remains obscure, we find that the effect of RICTOR depletion on lifespan is independent of the role of hepatic mTORC2 in promoting glucose tolerance. Our results suggest that inhibition of mTORC2 signaling is detrimental to males, which may explain in part why interventions that decrease mTOR signaling show greater efficacy in females.
Project description:Phorbol ester (PMA or TPA), a tumor promoter, can cause either proliferation or cell cycle arrest, depending on cellular context. For example, in SKBr3 breast cancer cells, PMA hyper-activates the MEK/MAPK pathway, thus inducing p21 and cell cycle arrest. Here we showed that PMA-induced arrest was followed by conversion to cellular senescence (geroconversion). Geroconversion was associated with active mTOR and S6 kinase (S6K). Rapamycin suppressed geroconversion, maintaining quiescence instead. In this model, PMA induced arrest (step one of a senescence program), whereas constitutively active mTOR drove geroconversion (step two). Without affecting Akt phosphorylation, PMA increased phosphorylation of S6K (T389) and S6 (S240/244), and that was completely prevented by rapamycin. Yet, T421/S424 and S235/236 (p-S6K and p-S6, respectively) phosphorylation became rapamycin-insensitive in the presence of PMA. Either MEK or mTOR was sufficient to phosphorylate these PMA-induced rapamycin-resistant sites because co-treatment with U0126 and rapamycin was required to abrogate them. We next tested whether activation of rapamycin-insensitive pathways would shift quiescence towards senescence. In HT-p21 cells, cell cycle arrest was caused by IPTG-inducible p21 and was spontaneously followed by mTOR-dependent geroconversion. Rapamycin suppressed geroconversion, whereas PMA partially counteracted the effect of rapamycin, revealing the involvement of rapamycin-insensitive gerogenic pathways. In normal RPE cells arrested by serum withdrawal, the mTOR/pS6 pathway was inhibited and cells remained quiescent. PMA transiently activated mTOR, enabling partial geroconversion. We conclude that PMA can initiate a senescent program by either inducing arrest or fostering geroconversion or both. Rapamycin can decrease gero-conversion by PMA, without preventing PMA-induced arrest. The tumor promoter PMA is a gero-promoter, which may be useful to study aging in mammals.
Project description:BACKGROUND:Mammalian target of rapamycin (mTOR) is a master regulator of various cellular responses by forming two functional complexes, mTORC1 and mTORC2. mTOR signaling is frequently dysregulated in pancreatic neuroendocrine tumors (PNETs). mTOR inhibitors have been used in attempts to treat these lesions, and prolonged progression free survival has been recorded. If this holds true also for the multiple endocrine neoplasia type 1 (MEN1) associated PNETs is yet unclear. We investigated the relationship between expression of the MEN1 protein menin and mTOR signaling in the presence or absence of the mTOR inhibitor rapamycin. METHODS:In addition to use of menin wild type and menin-null mouse embryonic fibroblasts (MEFs), menin was silenced by siRNA in pancreatic neuroendocrine tumor cell line BON-1. Panels of protein phosphorylation, as activation markers downstream of PI3k-mTOR-Akt pathways, as well as menin expression were evaluated by immunoblotting. The impact of menin expression in the presence and absence of rapamycin was determinate upon Wound healing, migration and proliferation in MEFs and BON1 cells. RESULTS:PDGF-BB markedly increased phosphorylation of mTORC2 substrate Akt, at serine 473 (S473) and threonine 450 (T450) in menin-/- MEFs but did not alter phosphorylation of mTORC1 substrates ribosomal protein S6 or eIF4B. Acute rapamycin treatment by mTORC1-S6 inhibition caused a greater enhancement of Akt phosphorylation on S473 in menin-/- cells as compared to menin+/+ MEFs (116% vs 38%). Chronic rapamycin treatment, which inhibits both mTORC1and 2, reduced Akt phosphorylation of S473 to a lesser extent in menin-/- MEFs than menin+/+ MEFs (25% vs 75%). Silencing of menin expression in human PNET cell line (BON1) also enhanced Akt phosphorylation at S473, but not activation of mTORC1. Interestingly, silencing menin in BON1 cells elevated S473 phosphorylation of Akt in both acute and chronic treatments with rapamycin. Finally, we show that the inhibitory effect of rapamycin on serum mediated wound healing and cell migration is impaired in menin-/- MEFs, as well as in menin-silenced BON1 cells. CONCLUSIONS:Menin is involved in regulatory mechanism between the two mTOR complexes, and its reduced expression is accompanied with increased mTORC2-Akt signaling, which consequently impairs anti-migratory effect of rapamycin.