Activating mTOR Signaling Pathway Research Articles

Low-folate stress reprograms cancer stem cell-like potentials and bioenergetics metabolism through activation of mTOR signaling pathway to promote in vitro invasion and in vivo tumorigenicity of lung cancers

Low-folate (LF) nutritional status is associated with increased risks of lung cancer. It has unexplored effects on lung cancer malignancy, a cancer stem cell (CSC) disease. We hypothesized that LF may reprogram CSC-like potential and bioenergetics metabolism to increase metastasis potential of lung cancers. Cultivation of human non-small-cell lung cancer cells (H23) in an LF medium enhanced CSC-like properties signified by increased expressions of the CSC surface marker CD44 and pluripotency markers Sox2, Oct4 and ALDH1A1, and promoted self-renewal ability of anchorage-independent oncospheroid formation. The CSC-like phenotype of LF-treated H23 cells coupled with the metabolic reprogramming to aerobic glycolysis evident by elevated lactate release and medium acidification suppressed expressions of pyruvate dehydrogenase E1-α, and elevated redox status of the NADH/NAD+ and NADPH/NADP+ ratios. The LF-induced metabostemness phenotype of H23 cells was modified by DNA methylation inhibitor 5-AdC and histone acetylation inhibitor EX. Treatment of H23 cells with mTOR siRNA or the mTOR inhibitor rapamycin abrogated LF-activated Akt-mTOR-Hif1-Foxo signaling and stemness-associated sonic hedgehog pathway, reversed Warburg metabolic switch and diminished invasion of H23 cells. Intrapleural injection of LF-induced lung oncospheres into the LF recipient mice, but not the control recipient mice, caused metastasis xenograft lung tumors. The in vitro and in vivo data corroboratively demonstrate that LF stress reprograms metabostemness signatures through activated mTOR signaling pathway to promote metastasis tumorigenicity of lung cancers.

MiR-181a Participates in Contextual Fear Memory Formation Via Activating mTOR Signaling Pathway.

Long-term memory formation has been proven to require gene expression and new protein synthesis. MicroRNAs (miRNAs), as an endogenous small non-coding RNAs, inhibit the expression of their mRNA targets, through which involve in new memory formation. In this study, elevated miR-181a levels were found to be responsible for hippocampal contextual fear memory consolidation. Using a luciferase reporter assay, we indicated that miR-181a targets 2 upstream molecules of mTOR pathway, namely, PRKAA1 and REDD1. Upregulated miR-181a can downregulate the PRKAA1 and REDD1 protein levels and promote mTOR activity to facilitate hippocampal fear memory consolidation. These results indicate that miR-181a is involved in hippocampal contextual fear memory by activating the mTOR signaling pathway. This work provides a novel evidence for the role of miRNAs in memory formation and demonstrates the implication of mTOR signaling pathway in miRNA processing in the adult brain.

Dietary interventions that reduce mTOR activity rescue autistic-like behavioral deficits in mice

Enhanced mammalian target of rapamycin (mTOR) signaling in the brain has been implicated in the pathogenesis of autism spectrum disorder (ASD). Inhibition of the mTOR pathway improves behavior and neuropathology in mouse models of ASD containing mTOR-associated single gene mutations. The current study demonstrated that the amino acids histidine, lysine, threonine inhibited mTOR signaling and IgE-mediated mast cell activation, while the amino acids leucine, isoleucine, valine had no effect on mTOR signaling in BMMCs. Based on these results, we designed an mTOR-targeting amino acid diet (Active 1 diet) and assessed the effects of dietary interventions with the amino acid diet or a multi-nutrient supplementation diet (Active 2 diet) on autistic-like behavior and mTOR signaling in food allergic mice and in inbred BTBR T+Itpr3tf/J mice. Cow’s milk allergic (CMA) or BTBR male mice were fed a Control, Active 1, or Active 2 diet for 7 consecutive weeks. CMA mice showed reduced social interaction and increased self-grooming behavior. Both diets reversed behavioral impairments and inhibited the mTOR activity in the prefrontal cortex and amygdala of CMA mice. In BTBR mice, only Active 1 diet reduced repetitive self-grooming behavior and attenuated the mTOR activity in the prefrontal and somatosensory cortices. The current results suggest that activated mTOR signaling pathway in the brain may be a convergent pathway in the pathogenesis of ASD bridging genetic background and environmental triggers (food allergy) and that mTOR over-activation could serve as a potential therapeutic target for the treatment of ASD.

Antidepressant-like effects of long-term sarcosine treatment in rats with or without chronic unpredictable stress

Sarcosine, an N-methyl-d-aspartate receptor enhancer, can improve depression-like behavior in rodent models and depression in humans. We found that a single dose of sarcosine exerted antidepressant-like effects with rapid concomitant increases in the mammalian target of rapamycin (mTOR) signaling pathway activation and enhancement of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR) membrane insertion. Sarcosine may play a crucial role in developing novel therapy for depression. For a detailed understanding of sarcosine, this study examined the effects of long-term sarcosine treatment on the forced swim test (FST), mTOR signaling, and AMPAR membrane insertion in rats. The effects of long-term sarcosine treatment were examined in naive rats and rats exposed to chronic unpredictable stress (CUS). Long-term sarcosine treatment (560mg/kg/d for 21 d) significantly ameliorated the increased immobility induced by CUS in the FST, reaffirming the potential role of sarcosine as an antidepressant for depressed patients. The same long-term treatment exhibited no such effect in naive rats despite increased mTOR activation and AMPAR membrane insertion in both groups. Our findings clearly show CUS-exposed rats are sensitive to long-term sarcosine treatment in FST and the response at the same dose is absent in naïve rats. Nevertheless, the distinct sensitivity to long-term sarcosine treatment in rats with or without CUS is not associated with the activated mTOR signaling pathway or increased AMPAR membrane insertion. Additionally, understanding the behavioral and molecular basis of distinct responses is vital important for developing personalized treatment programs to increase the probability of success when treating depression.

Targeted Disruption of miR-17-92 Impairs Mouse Spermatogenesis by Activating mTOR Signaling Pathway.

The miR-17-92 cluster and its 6 different mature microRNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a, play important roles in embryo development, immune system, kidney and heart development, adipose differentiation, aging, and tumorigenicity. Currently, increasing evidence indicates that some members of miR-17-92 cluster may be critical players in spermatogenesis, including miR-17, miR-18a, and miR-20a. However, the roles and underlying mechanisms of miR-17-92 in spermatogenesis remain largely unknown. Our results showed that the targeted disruption of miR-17-92 in the testes of adult mice resulted in severe testicular atrophy, empty seminiferous tubules, and depressed sperm production. This phenotype is partly because of the reduced number of spermatogonia and spermatogonial stem cells, and the significantly increased germ cell apoptosis in the testes of miR-17-92-deficient mice. In addition, overactivation of the mammalian target of rapamycin signaling pathway and upregulation of the pro-apoptotic protein Bim, Stat3, c-Kit, and Socs3 were also observed in miR-17-92-deficient mouse testes, which might be, at least partially if not all, responsible for the aforementioned phenotypic changes in mutant testes. Taken together, these findings suggest that miR-17-92 is essential for normal spermatogenesis in mice.

Leukemia inhibitory factor (LIF) withdrawal activates mTOR signaling pathway in mouse embryonic stem cells through the MEK/ERK/TSC2 pathway.

Leukemia inhibitory factor (LIF) is indispensable to maintain the pluripotent state of mouse embryonic stem cells (ESCs), but the mechanisms underlying the role of LIF/STAT3 pathway are yet poorly understood. Here we first showed that the LIF/STAT3-regulated signaling pathway contributes to the maintenance of self-renewal and pluripotency of mouse ESCs by suppressing mTOR (mammalian target of rapamycin), which is necessary for early differentiation. When LIF is withdrawn from culture medium, the mTOR activity rapidly increases as detected by phosphorylation of its targets – ribosomal protein S6 and translation factor 4EBP1. In turn, suppression of STAT3 phosphorylation on Tyr-705 by a specific small molecule WP1066 also activates phosphorylation of the mTOR target S6 ribosomal protein. LIF removal strongly activates ERK activity indicating that ERK can be involved in either direct phosphorylation of mTOR or phosphorylation of an upstream negative regulator of mTOR – TSC1/TSC2 proteins. According to western blotting data, LIF withdrawal leads to phosphorylation of TSC2 protein thereby relieving its negative effect on mTOR activity. mTOR activation is accompanied by a decrease of pluripotent gene expression Oct-4, Nanog, Sox2 and by an augmentation of fgf5 gene expression – a marker of post-implantation epiblast. Together, these data indicate that LIF-depleted mouse ESCs undergo a transition from the LIF/STAT3-supported pluripotent state to the FGFR/ERK-committed primed-like state with expression of early differentiation markers mediated through activation of mTOR signaling.

Brain-expressed X-linked 2 Is Pivotal for Hyperactive Mechanistic Target of Rapamycin (mTOR)-mediated Tumorigenesis

Frequent alteration of upstream proto-oncogenes and tumor suppressor genes activates mechanistic target of rapamycin (mTOR) and causes cancer. However, the downstream effectors of mTOR remain largely elusive. Here we report that brain-expressed X-linked 2 (BEX2) is a novel downstream effector of mTOR. Elevated BEX2 in Tsc2(-/-) mouse embryonic fibroblasts, Pten(-/-) mouse embryonic fibroblasts, Tsc2-deficient rat uterine leiomyoma cells, and brains of neuronal specific Tsc1 knock-out mice were abolished by mTOR inhibitor rapamycin. Furthermore, BEX2 was also increased in the liver of a hepatic specific Pten knock-out mouse and the kidneys of Tsc2 heterozygous deletion mice, and a patient with tuberous sclerosis complex (TSC). mTOR up-regulation of BEX2 was mediated in parallel by both STAT3 and NF-κB. BEX2 was involved in mTOR up-regulation of VEGF production and angiogenesis. Depletion of BEX2 blunted the tumorigenesis of cells with activated mTOR. Therefore, enhanced STAT3/NF-κB-BEX2-VEGF signaling pathway contributes to hyperactive mTOR-induced tumorigenesis. BEX2 may be targeted for the treatment of the cancers with aberrantly activated mTOR signaling pathway.

Abstract T P85: Mammalian Target of Rapamycin (mTOR) Signaling Pathway Mediates Cerebral Ischemic Brain Damage

BACKGROUND: Mammalian target of rapamycin (mTOR) signaling regulates cell energy metabolism and cell proliferation. Recent studies have demonstrated that mTOR activation is associated with increased cardiac infarct volume and necroptosis. The role of mTOR in cerebral ischemia has not been studied. The aim of this study is to explore how mTOR pathway behavior after cerebral ischemia and whether inhibition of mTOR reduces or increases ischemic brain damage. METHODS: Ten minutes of global ischemia was induced in Wistar rats (2-3 months old). Animals were either treatment with mTOR inhibitor rapamycin (6 mg/kg) or its vehicle. Animal brains were harvested after 3 hrs, 16 hrs and 7 days for histopathology and protein analyses. Sham-operated non-ischemic rats were used as control. Rapamycin was i.p. injected 1 hr after reperfusion in 7-day subgroups and injected 7 days prior to ischemia in 3 and 16 hrs subgroups. RESULTS: 10 minutes of global ischemia caused neuronal damage to the striatum, hippocampal CA1, and neocortical regions at 7 days of reperfusion. Levels of p-mTOR, p-S6K were increased significantly after 3 and 16 hrs in both the hippocampus and cortex. In addition, p-Akt and cytochrome c were elevated in the hippocampi after 3 hrs, peaked at 16 hrs and remained elevated after 7 days of reperfusion. Post-ischemic treatment with rapamycin 1 hrs after reperfusion significantly reduced brain damage in the CA1 and cortical areas at 7 days of reperfusion. Levels of p-mTOR, p-S6K, p-Akt and cytochrome c were significantly suppressed by rapamycin in non-ischemic and ischemic animals. CONCLUSION: Cerebral ischemia activates mTOR signaling pathway and inhibition of mTOR activativity reduces ischemic brain damage.

Laser phototherapy accelerates oral keratinocyte migration through the modulation of the mammalian target of rapamycin signaling pathway

Keratinocytes play a central role in wound healing by responding to tissue injury through the activation of cellular proliferation and migration. Current clinical evidence suggests that the laser phototherapy (LPT) accelerates wound healing in a variety of oral diseases; however, the molecular mechanisms involved in response to LPT are not fully understood. Oral keratinocytes (NOK-SI) maintained under nutritional-deficit culture medium (2% fetal bovine serum) were irradiated with InGaAlP laser (660 nm; 40 mW; 0.04 cm2 spot size) in punctual and contact modes. The energy densities used were 4 and 20 J/cm2 corresponding to 4 and 20 s of exposure times and 0.16 and 0.8 J of energy per point, respectively. Three sessions of irradiations were applied with 6-h intervals. Further, the impact of LPT over cellular migration, proliferation, and activation of the mammalian target of rapamycin (mTOR) pathway, known to play a major role in epithelial migration and wound healing, was analyzed. Compared with control cells, the LPT-treated cells showed accelerated cellular migration without any changes in proliferation. Furthermore, LPT resulted in an increase in the phospho-S6 ribosomal protein, indicating activation of the mTOR signaling pathway. Collectively, these findings suggest that the LPT activates mTOR signaling pathway, promotes epithelial cell migration, and accelerates healing of oral mucosa.

Acute Exposure to Essential Amino Acids (EAA) Activates MTOR/p70 Signaling in Soleus Muscle of Chronically EAA-Treated Aged Rats

Using an in vitro assay we assessed whether the acute exposure of soleus muscle of adult and aged rats to essential amino acid enriched mixture (EAAem) activates mTOR signaling pathway (mTOR and p70S6K) even after prolonged supplementation with the same mixture. A total of 20 adult (9 months of age at the end of treatment) and 20 aged (18 months of age at the end of treatment) male Wistar rats were used. Ten of each group were treated with EAAem (1.5 gr/kg/day in tap water) for 6 months. At the end of treatment the rats were grouped (n = 5 each group) as follows: adult (AD) and aged (AG) untreated controls; adult (AD_EAAem) and aged (AG_EAAem) chronically supplemented with EAAem; adult (AD+EAAem) and aged (AG+EAAem) acutely incubated with EAAem (soleus in 1 percent EAAem for 30 min); AD_EAAem+ and AG_EAAem+ acutely incubated with EAAem. Following treatment the activation level of mTOR and p70S6K was measured by Western blot. The basal level of mTOR and p70S6K activation appeared to be higher in AD compared with AG. In AG+EAAem a significant change in the level of p70S6K activation, unlike mTOR, was observed whereas no change was observed in AD+EAAem. In AD_EAAem muscles the basal level of p70S6K activation, unlike mTOR, was significantly lower than in AD and the acute exposure to EAAem produced a significant reduction of mTOR activation. Contrarily to AG, in AG_EAAem+ the acute exposure to EAAem produced a significant activation of mTOR, unlike p70S6K. Results in the adults indicated a higher basal level of activation and a lower responsiveness of the pathway to acute and chronic exposure to EAA-enriched mixture. On the contrary, in the aged, a lower basal level of activation was associated with a higher responsiveness to EAAem. In particular, although with a different timing, acute exposure to EAAem activated mTOR signaling even following prolonged supplementation.

Hepatitis C Virus Upregulates Beclin1 for Induction of Autophagy and Activates mTOR Signaling

Hepatitis C virus (HCV) induces autophagosome formation in infected human hepatocytes. We have previously reported that HCV exploits autophagic machinery in favor of virus growth and survival in host cells (S. Shrivastava et al., Hepatology 53:406-414, 2011); however, the mechanisms for autophagy induction is poorly understood. In the present study, we observed that HCV infection transcriptionally upregulates Beclin1, which forms complex with Vps34, the class III phosphatidylinositol 3-kinase, as a first step for autophagy initiation. Although Bcl-2 has an anti-autophagy effect by its association with Beclin1 in nutrient-deprived cells, our studies revealed that HCV-mediated autophagy occurs independent of Beclin1-Bcl-2 dissociation. Mammalian target of rapamycin (mTOR) is a positive regulator of cell growth and is recognized as an inhibitor of autophagy induction. Our results demonstrated that HCV infection enhances phospho-mTOR expression and its downstream target 4EBP1 activation, suggesting that mTOR is not a negative regulator of HCV-induced autophagy. On the other hand, HCV infection in autophagy-impaired cells reduced phospho-mTOR, mTOR, and phospho-4EBP1 expression. Together, these results suggested that HCV induces autophagy by upregulating Beclin1 and activates mTOR signaling pathway, which in turn may promote hepatocyte growth.