Downstream Effector Of mTOR Research Articles

Manuka Honey Inhibits Human Breast Cancer Progression in Preclinical Models.

Manuka honey (MH) exhibits potential antitumor activity in preclinical models of a number of human cancers. Treatment in vitro with MH at concentrations ranging from 0.3 to 5.0% (w/v) led to significant dose-dependent inhibition of proliferation of human breast cancer MCF-7 cells, but anti-proliferative effects of MH were less pronounced in MDA-MB-231 breast cancer cells. Effects of MH were also tested on non-malignant human mammary epithelial cells (HMECs) at 2.5% w/v, and it was found that MH reduced the proliferation of MCF-7 cells but not that of HMECs. Notably, the antitumor activity of MH was in the range of that exerted by treatment of MCF-7 cells with the antiestrogen tamoxifen. Further, MH treatment stimulated apoptosis of MCF-7 cells in vitro, with most cells exhibiting acute and significant levels of apoptosis that correlated with PARP activation. Additionally, the effects of MH induced the activation of AMPK and inhibition of AKT/mTOR downstream signaling. Treatment of MCF7 cells with increased concentrations of MH induced AMPK phosphorylation in a dose-dependent manner that was accompanied by inhibition of phosphorylation of AKT and mTOR downstream effector protein S6. In addition, MH reduced phosphorylated STAT3 levels in vitro, which may correlate with MH and AMPK-mediated anti-inflammatory properties. Further, in vivo, MH administered alone significantly inhibited the growth of established MCF-7 tumors in nude mice by 84%, resulting in an observable reduction in tumor volume. Our findings highlight the need for further research into the use of natural compounds, such as MH, for antitumor efficacy and potential chemoprevention and investigation of molecular pathways underlying these actions.

Abstract PO4-23-09: Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells

Abstract In HER2-enriched (HER2E) breast cancer (BC), the dual HER2/EGFR inhibitor Lapatinib is a standard of care for advanced staged disease. Anti-HER2 therapies disrupt tumor progression through inhibition of the PI3K/AKT pathway. Some treated tumors will override inhibition by shifting to MAPK/ERK signaling to fuel cell growth, thus fostering resistance to anti-HER2 therapy. The tyrosine receptor kinase FGFR4 is included in the profile for a HER2E subtype. FGFR4 is overexpressed in ~30% of BC and can signal through PI3K/AKT and MAPK/ERK. Inhibitors for FGFR4 such as BLU554 are being investigated in phase I/II clinical trials for hepatocellular carcinoma, showing success in tumors that are positive for FGFR4’s preferred ligand FGF19. According to cBioPortal patient data, FGF19 is amplified in ~20% of BC, and has been shown to trigger an autocrine loop in triple negative BC through FGFR4. We examined BLU554 as a dual therapeutic with Lapatinib and dissected intracellular signaling stemming from the combination in HER2E BC. MDA-MB-453 BC cells (HER2+FGFR4+) were used for a dual strategy with BLU554 and Lapatinib. Cell viability assays were performed at matched doses, then as a dose response matrix, ranging from 0.1-20 µM. Findings were used for synergy analysis using synergyfinder.fimm.fi. Western blots determined protein activity in response to treatments. Ridaforolimus (mTOR inhibitor) was used in combination with either BLU554 or Lapatinib to assess MAPK/ERK and PI3K/AKT pathway crosstalk in MB-453 cells. Additionally, FGF19 was added to cells after a 2-hour pre-treatment to challenge BLU554 + Lapatinib and compare changes in signaling with FGF19 present. MB-453 cells treated with BLU554 decreased cell viability similarly to Lapatinib at 10 µM (65% [SD ± 3.3] and 63% [SD ± 2.1], respectively). Dual treatment decreased viability compared to single agents at all concentrations but the greatest difference was observed at 20 µM (38.5% in dual compared to Lapatinib [p-value = 0.00006]; 45.1% in dual compared to BLU554 [p-value = 0.00016]). Dose-response matrix of dual treatment yielded a ZIP synergy score of 16.852 ± 2.97 (>10 is synergistic), with the greatest synergy and inhibition at 10 µM and 20 µM of both drugs. BLU554 caused a therapeutic potency shift of Lapatinib, such that lower doses of Lapatinib (0.1 µM, 1 µM) are effective at higher doses of BLU554 (10 µM, 20 µM). Westerns of MB-453 with one dose of BLU554 increased phospho-ERK1/2 (pERK1/2) at 16- or 48-hr time points versus control, but when BLU554 was re-dosed then pERK1/2 was successfully inhibited. Both pAKT and pS6RP were increased by re-dosing BLU554. Cells treated for 2 hrs with Lapatinib decreased pAKT and pS6RP and did not change pERK, whereas a 2-hr treatment of BLU554 increased pAKT and decreased pERK. Dual treatment exhibited inhibition of ERK, AKT, 4EBP1, and S6RP phosphorylation all to a greater extent than single agents in combination with Ridaforolimus. Challenge by ectopic FGF19 of BLU554 + Lapatinib was unable to restore downstream signaling of AKT and ERK. BLU554 synergistically interacts with Lapatinib to decrease cell viability while increasing its therapeutic potency. Lapatinib primarily inhibits AKT signaling, whereas BLU554 targets ERK. Dual treatment simultaneously blocks MAPK/ERK and PI3K/AKT signaling and yields decreases of mTOR downstream effectors pS6RP and p4EBP1. These effects are maintained even when challenged with FGF19, suggesting that dual inhibition of FGFR4 and HER2 could prevent pathway activation from external stimuli. Citation Format: Jordan Beardsley, Joseph Goode, Deborah Altomare. Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-09.

P4HA2 activates mTOR via hydroxylation and targeting P4HA2-mTOR inhibits lung adenocarcinoma cell growth

,Mammalian target of rapamycin (mTOR) kinase functions as a central regulator of cell growth and metabolism, and its complexes mTORC1 and mTORC2 phosphorylate distinct substrates. Dysregulation of mTOR signaling is commonly implicated in human diseases, including cancer. Despite three decades of active research in mTOR, much remains to be determined. Here, we demonstrate that prolyl 4-hydroxylase alpha-2 (P4HA2) binds directly to mTOR and hydroxylates one highly conserved proline 2341 (P2341) within a kinase domain of mTOR, thereby activating mTOR kinase and downstream effector proteins (e.g. S6K and AKT). Moreover, the hydroxylation of P2341 strengthens mTOR stability and allows mTOR to accurately recognize its substrates such as S6K and AKT. The growth of lung adenocarcinoma cells overexpressing mTORP2341A is significantly reduced when compared with that of cells overexpressing mTORWT. Interestingly, in vivo cell growth assays show that targeting P4HA2-mTOR significantly suppresses lung adenocarcinoma cell growth. In summary, our study reveals an undiscovered hydroxylation-regulatory mechanism by which P4HA2 directly activates mTOR kinase, providing insights for therapeutically targeting mTOR kinase-driven cancers.

Embryonic methionine triggers post-natal developmental programming in Japanese quail

Embryonic development is one of the most sensitive and critical stages when maternal effects may influence the offspring’s phenotype. In birds and other oviparous species, embryonic development is confined to the eggs, therefore females must deposit resources into the eggs to prepare the offspring for the prevailing post-natal conditions. However, the mechanisms of such phenotypic adjustments remain poorly understood. We simulated a maternal nutritional transfer by injecting 1 mg of l-methionine solution into Japanese quail eggs before the onset of incubation. The increase in early methionine concentration in eggs activated the insulin/insulin-like signalling and mechanistic target of rapamycin (IIS/mTOR) signalling pathways and affected post-natal developmental trajectories. Chicks from methionine-supplemented eggs had higher expression of liver IGF1 and mTOR genes at hatching but were similar in size, and the phenotypic effects of increased growth became apparent only a week later and remained up to three weeks. Circulating levels of insulin-like growth factor-1 (IGF-1) and expression of ribosomal protein serine 6 kinase 1 (RPS6K1), the mTOR downstream effector, were elevated only three weeks after hatching. These results show that specific nutritional cues may have phenotypic programming effects by sequentially activating specific nutrient-sensing pathways and achieving transgenerational phenotypic plasticity.

Abstract 6509: GT0486, a novel mTORC1/2 dual inhibitor, exhibits synergistic antitumor efficacy in combination with BTK inhibitors

Abstract Mammalian target of rapamycin (mTOR), a serine/threonine kinase, is the central signal molecule in regulating cell growth, metabolism, angiogenesis, and survival. The first generation of mTOR inhibitors specifically inhibit mTORC1 but are insensitive to mTORC2 and cannot eliminate the downstream signaling cascade in cancer. GT0486, a second-generation mTORC1/2 dual inhibitor, can selectively suppress mTOR kinase activity while has no inhibitory effect on other kinase receptors. We investigate the anti-tumor efficacy of GT0486 in a variety of tumor cells and xenograft animal models in vitro and in vivo. The combination effects of GT0486 and BTKi (BTK inhibitor) were also evaluated. Compared with other mTOR inhibitors (GDC-0349, AZD-2014, Rapamycin, GDC-0941, and CC-223), GT0486 exhibits the stronger inhibitory activity on human tumor cells, such as U87(glioma), PC-3(prostate cancer), MDA-MB-468(breast cancer) and Huh-7(liver cancer). In B-cell lymphoma, compared with the CC-223, GT0486 shows greater inhibitory potency against TMD8, DoHH2 and WSU-DLCL2, with IC50 values of 85nM, 96nM and 83nM, respectively. Moreover, in the U87 and the PC-3 xenograft animal models, GT0486 strongly reduced tumor growth in dose dependent manner. High synergistic effects were observed with GT0486 in combination with BTK inhibitors, including Acalabrutinib, Ibrutinib, Zanubrutinib and Orelabrutinib in the BTKi sensitive TMD8 cells. In BTKi resistant DoHH2 cells, GT0486 also enhanced the drug sensitivity of BTKi. The possible biomarkers on the synergistic mechanism were further studied. GT0486 improves the role of BTKi on cell cycle arrest in phase G1. At the same time, the phosphorylation levels of mTOR downstream effectors AKT, 4EBP1 and S6 were dose-dependently downregulated after GT0486 treatment, and a significant reduction were observed when combined with BTKi. These results demonstrate that GT0486 is a promising dual mTOR1/2 inhibitor, which is currently undergoing clinical phase I study in China for the treatment of solid tumors. Strong synergistic effects observed with GT0486 in combination with BTKi in this study might open a way for a novel treatment strategy in clinical trials. Citation Format: Xiaodan Hou, Honghua Yan, Xin Zhang, Weidong Qian, Dong Chen, Zhihua Ren, Youzhi Tong. GT0486, a novel mTORC1/2 dual inhibitor, exhibits synergistic antitumor efficacy in combination with BTK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6509.

Potential inhibitors of RPS6KB2 and NRF2 in head and neck squamous cell carcinoma

Among the major altered pathways in head and neck squamous cell carcinoma, AKT/mTORC1/S6K and NRF2/KEAP1 pathway are quite significant. The overexpression and overstimulation of proteins from both these pathways makes them the promising candidates in cancer therapeutics. Inhibiting mTOR has been in research from past several decades but the tumour heterogeneity, and upregulation of several compensatory feed-back mechanisms, encourages to explore other downstream targets for inhibiting the pathway. One such downstream effectors of mTOR is S6K2. It is reported to be overexpressed in cancers such as head and neck cancer, breast cancer and prostate cancer. In case of NRF2/KEAP1 pathway, nuclear factor erythroid 2-related factor 2 (NFE2L2 or NRF2) is overexpressed in ∼90% of head and neck squamous cell carcinoma (HNSCC) cases. It associates with poor survival rate and therapeutic resistance in HNSCC treatment. NRF2 pathway is the primary antioxidant pathway in the cell which also serves pro-tumorigenic functions, such as repression of apoptosis, cell proliferation support and chemoresistance. The aim of this work was to explore S6K2 and NRF2 and identify novel and potential inhibitors against them for treating head and neck squamous cell carcinoma. Since the crystal structure of S6K2 was not available at the time of this study, we modelled its structure using homology modelling and performed high throughput screening, molecular dynamics simulations, free energy calculations and protein-ligand interaction studies to identify the inhibitors. We identified natural compounds Crocin and Gypenoside XVII against S6K2 and Chebulinic acid and Sennoside A against NRF2. This study provides a significant in-depth understanding of the two studied pathways and therefore can be used in the development of potential therapeutics against HNSCC. Communicated by Ramaswamy H. Sarma

Phosphorylated S6K1 and 4E-BP1 play different roles in constitutively active Rheb-mediated retinal ganglion cell survival and axon regeneration after optic nerve injury.

Ras homolog enriched in brain (Rheb) is a small GTPase that activates mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that constitutively active Rheb can enhance the regeneration of sensory axons after spinal cord injury by activating downstream effectors of mTOR. S6K1 and 4E-BP1 are important downstream effectors of mTORC1. In this study, we investigated the role of Rheb/mTOR and its downstream effectors S6K1 and 4E-BP1 in the protection of retinal ganglion cells. We transfected an optic nerve crush mouse model with adeno-associated viral 2-mediated constitutively active Rheb and observed the effects on retinal ganglion cell survival and axon regeneration. We found that overexpression of constitutively active Rheb promoted survival of retinal ganglion cells in the acute (14 days) and chronic (21 and 42 days) stages of injury. We also found that either co-expression of the dominant-negative S6K1 mutant or the constitutively active 4E-BP1 mutant together with constitutively active Rheb markedly inhibited axon regeneration of retinal ganglion cells. This suggests that mTORC1-mediated S6K1 activation and 4E-BP1 inhibition were necessary components for constitutively active Rheb-induced axon regeneration. However, only S6K1 activation, but not 4E-BP1 knockdown, induced axon regeneration when applied alone. Furthermore, S6K1 activation promoted the survival of retinal ganglion cells at 14 days post-injury, whereas 4E-BP1 knockdown unexpectedly slightly decreased the survival of retinal ganglion cells at 14 days post-injury. Overexpression of constitutively active 4E-BP1 increased the survival of retinal ganglion cells at 14 days post-injury. Likewise, co-expressing constitutively active Rheb and constitutively active 4E-BP1 markedly increased the survival of retinal ganglion cells compared with overexpression of constitutively active Rheb alone at 14 days post-injury. These findings indicate that functional 4E-BP1 and S6K1 are neuroprotective and that 4E-BP1 may exert protective effects through a pathway at least partially independent of Rheb/mTOR. Together, our results show that constitutively active Rheb promotes the survival of retinal ganglion cells and axon regeneration through modulating S6K1 and 4E-BP1 activity. Phosphorylated S6K1 and 4E-BP1 promote axon regeneration but play an antagonistic role in the survival of retinal ganglion cells.

Dysregulated lipid metabolism in lymphangioleiomyomatosis pathogenesis as a paradigm of chronic lung diseases.

A chronic inflammatory condition characterizes various lung diseases. Interestingly, a great contribution to inflammation is made by altered lipids metabolism, that can be caused by the deregulation of the mammalian target of rapamycin complex-1 (mTORC1) activity. There is evidence that one of mTOR downstream effectors, the sterol regulatory element-binding protein (SREBP), regulates the transcription of enzymes involved in the de novo fatty acid synthesis. Given its central role in cell metabolism, mTOR is involved in several biological processes. Among those, mTOR is a driver of senescence, a process that might contribute to the establishment of chronic lung disease because the characteristic irreversible inhibition of cell proliferation, associated to the acquisition of a pro-inflammatory senescence-associated secretory phenotype (SASP) supports the loss of lung parenchyma. The deregulation of mTORC1 is a hallmark of lymphangioleiomyomatosis (LAM), a rare pulmonary disease predominantly affecting women which causes cystic remodeling of the lung and progressive loss of lung function. LAM cells have senescent features and secrete SASP components, such as growth factors and pro-inflammatory molecules, like cancer cells. Using LAM as a paradigm of chronic and metastatic lung disease, here we review the published data that point out the role of dysregulated lipid metabolism in LAM pathogenesis. We will discuss lipids' role in the development and progression of the disease, to hypothesize novel LAM biomarkers and to propose the pharmacological regulation of lipids metabolism as an innovative approach for the treatment of the disease.

Coordinated control of adiposity and growth by anti-anabolic kinase ERK7.

Energy storage and growth are coordinated in response to nutrient status of animals. How nutrient‐regulated signaling pathways control these processes in vivo remains insufficiently understood. Here, we establish an atypical MAP kinase, ERK7, as an inhibitor of adiposity and growth in Drosophila. ERK7 mutant larvae display elevated triacylglycerol (TAG) stores and accelerated growth rate, while overexpressed ERK7 is sufficient to inhibit lipid storage and growth. ERK7 expression is elevated upon fasting and ERK7 mutant larvae display impaired survival during nutrient deprivation. ERK7 acts in the fat body, the insect counterpart of liver and adipose tissue, where it controls the subcellular localization of chromatin‐binding protein PWP1, a growth‐promoting downstream effector of mTOR. PWP1 maintains the expression of sugarbabe, encoding a lipogenic Gli‐similar family transcription factor. Both PWP1 and Sugarbabe are necessary for the increased growth and adiposity phenotypes of ERK7 loss‐of‐function animals. In conclusion, ERK7 is an anti‐anabolic kinase that inhibits lipid storage and growth while promoting survival on fasting conditions.

Research Progress of 70 kDa Ribosomal Protein S6 Kinase (P70S6K) Inhibitors as Effective Therapeutic Tools for Obesity, Type II Diabetes and Cancer.

At present, diseases such as obesity, type Ⅱ diabetes and cancer have brought serious health problems, which are closely related to mTOR pathway. 70 kDa ribosomal protein S6 kinase (p70S6K), as a significant downstream effector of mTOR, mediates protein synthesis, RNA processing, glucose homeostasis, cell growth and apoptosis. Inhibiting the function of p70S6K can reduce the risk of obesity which helps to treat dyslipidemia, enhance insulin sensitivity, and extend the life span of mammals. Therefore, p70S6K has become a potential target for the treatment of these diseases. So far, except for the first p70S6K specific inhibitor PF-4708671 developed by Pfizer and LY2584702 developed by Lilai, all of them are in preclinical research. This paper briefly introduces the general situation of p70S6K and reviews their inhibitors in recent years, which are mainly classified into two categories: natural compounds and synthetic compounds. In particular, their inhibitory activities, structure-activity relationships (SARs) and mechanisms are highlighted.

Gomisin N Exerts Anti-liver Cancer Effects and Regulates PI3K-Akt and mTOR-ULK1 Pathways in Vitro.

Primary liver cancer is a lethal cancer. The phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway has been implicated in the pathogenesis of liver cancer. Gomisin N (GN), a lignan isolated from the dried fruits of Schisandra chinensis (Turca.) Baill., has been reported to reduce viability of, and induce apoptosis in, HepG2 liver cancer cells. In preadipocytes, GN was found to inhibit Akt activity. In the present study, Akt signaling-related anti-liver cancer mechanisms of GN were investigated. We confirmed that GN reduces cell viability of, and triggers apoptosis in, more liver cancer cell lines. Mechanistic studies revealed that GN lowers protein levels of phospho-PI3K (p85 tyrosine (Tyr)458), phospho-Akt (serine (Ser)473), and Akt downstream molecules Mcl-1 in HepG2 and HCCLM3 cells. Meanwhile, GN activates mTOR and inhibits ULK1 (a negative downstream effector of mTOR) activities. Activation of mTOR has been reported to suppress ULK1 activity and repress autophagy. Indeed, we observed that GN inhibits autophagy in liver cancer cells. In summary, we for the first time demonstrated that GN inhibits the PI3K-Akt pathway and regulates the mTOR-ULK1 pathway in liver cancer cells.

Maf1 regulates dendritic morphogenesis and influences learning and memory

Maf1, a general transcriptional regulator and mTOR downstream effector, is highly expressed in the hippocampus and cortex, but the function of Maf1 in neurons is not well elucidated. Here, we first demonstrate that Maf1 plays a central role in the inhibition of dendritic morphogenesis and the growth of dendritic spines both in vitro and in vivo. Furthermore, Maf1 downregulation paradoxically leads to activation of AKT-mTOR signaling, which is mediated by decreased PTEN expression. Moreover, we confirmed that Maf1 could regulate the activity of PTEN promoter by luciferase reporter assay, and proved that Maf1 could bind to the promoter of PTEN by ChIP-PCR experiment. We also demonstrate that expression of Maf1 in the hippocampus affects learning and memory in mice. Taken together, we show for the first time that Maf1 inhibits dendritic morphogenesis and the growth of dendritic spines through AKT-mTOR signaling by increasing PTEN expression.

MTOR Pathway Expression as Potential Predictive Biomarker in Patients with Advanced Neuroendocrine Tumors Treated with Everolimus.

Background. Everolimus (Eve), which is a mammalian target of Rapamicin (mTOR) inhibitor, is part of the therapeutic armamentarium of neuroendocrine tumors (NETs). Currently, there are no validated biomarkers predicting Eve efficacy in NETs. In this study, we explore whether the expression of phosphorilated (p)-mTOR and p70S6-kinase (S6K), a downstream effector of mTOR, correlates with the outcome of patients with NET that were treated with Eve. Methods. Tissue specimens that were derived from NETs treated with Eve at our Institution were examined for the expression levels of p-mTOR and p-S6K by immunohistochemistry. Response rate (RR), progression-free survival (PFS), and overall survival (OS) were analyzed in two groups: p-mTOR/p-S6K positive (group 1) and p-mTOR/p-S6K negative (group 2). Univariate and multivariate Cox regression analysis were performed. Results. Twenty-four patients with advanced NETs that were treated with Eve were included in the analysis. Eight out 24 (33.3%) patients were both p-mTOR and p-S6K positive. A better median PFS and OS in group 1 (18.2 and 39.9 months) as compared to group 2 (13 and 32.4 months) was depicted, with a toxicity profile that was comparable with data literature. Conclusions. Our study suggests that the activation of mTOR pathway can predict better outcomes in patients with NET treated with Eve. However, these results warrant further confirmation in a prospective setting.

Ribosomal Protein S6 Hypofunction in Postmortem Human Brain Links mTORC1-Dependent Signaling and Schizophrenia.

The mechanistic target of rapamycin (also known as mammalian target of rapamycin) (mTOR)-dependent signaling pathway plays an important role in protein synthesis, cell growth, and proliferation, and has been linked to the development of the central nervous system. Recent studies suggest that mTOR signaling pathway dysfunction could be involved in the etiopathogenesis of schizophrenia. The main goal of this study was to evaluate the status of mTOR signaling pathway in postmortem prefrontal cortex (PFC) samples of subjects with schizophrenia. For this purpose, we quantified the protein expression and phosphorylation status of the mTOR downstream effector ribosomal protein S6 as well as other pathway interactors such as Akt and GSK3β. Furthermore, we quantified the status of these proteins in the brain cortex of rats chronically treated with the antipsychotics haloperidol, clozapine, or risperidone. We found a striking decrease in the expression of total S6 and in its active phosphorylated form phospho-S6 (Ser235/236) in the brain of subjects with schizophrenia compared to matched controls. The chronic treatment with the antipsychotics haloperidol and clozapine affected both the expression of GSK3β and the activation of Akt [phospho-Akt (Ser473)] in rat brain cortex, while no changes were observed in S6 and phospho-S6 (Ser235/236) protein expression with any antipsychotic treatment. These findings provide further evidence for the involvement of the mTOR-dependent signaling pathway in schizophrenia and suggest that a hypofunctional S6 may have a role in the etiopathogenesis of this disorder.

Polycystin-1 Inhibits Cell Proliferation through Phosphatase PP2A/B56α.

Autosomal dominant polycystic kidney disease (ADPKD) is associated with a number of cellular defects such as hyperproliferation, apoptosis, and dedifferentiation. Mutations in polycystin-1 (PC1) account for ∼85% of ADPKD. Here, we showed that wild-type (WT) or mutant PC1 composed of the last five transmembrane (TM) domains and the C-terminus (termed PC1-5TMC) inhibits cell proliferation and protein translation, as well as the downstream effectors of mTOR, consistent with previous reports. Knockdown of B56α, a subunit of the protein phosphatase 2A (PP2A) complex, or application of PP2A inhibitor okadaic acid or calyculin A, abolished the inhibitory effect of PC1 and PC1-5TMC on proliferation, indicating that PP2A/B56α mediates the regulation of cell proliferation by PC1. In addition to the phosphorylated S6 and 4EBP1, B56α was also downregulated by PC1 and PC1-5TMC. Furthermore, the downregulation of B56α, which may be mediated by mTOR but not AKT, can account for the dependence of PC1-inhibited proliferation on PP2A.

Brain somatic mutations in MTOR reveal translational dysregulations underlying intractable focal epilepsy.

Brain somatic mutations confer genomic diversity in the human brain and cause neurodevelopmental disorders. Recently, brain somatic activating mutations in MTOR have been identified as a major etiology of intractable epilepsy in patients with cortical malformations. However, the molecular genetic mechanism of how brain somatic mutations in MTOR cause intractable epilepsy has remained elusive. In this study, translational profiling of intractable epilepsy mouse models with brain somatic mutations and genome-edited cells revealed a novel translational dysregulation mechanism and mTOR activation-sensitive targets mediated by human MTOR mutations that lead to intractable epilepsy with cortical malformation. These mTOR targets were found to be regulated by novel mTOR-responsive 5'-UTR motifs, distinct from known mTOR inhibition-sensitive targets regulated by 5' terminal oligopyrimidine motifs. Novel mTOR target genes were validated in patient brain tissues, and the mTOR downstream effector eIF4E was identified as a new therapeutic target in intractable epilepsy via pharmacological or genetic inhibition. We show that metformin, an FDA-approved eIF4E inhibitor, suppresses intractable epilepsy. Altogether, the present study describes translational dysregulation resulting from brain somatic mutations in MTOR, as well as the pathogenesis and potential therapeutic targets of intractable epilepsy.

Distinct role of 4E-BP1 and S6K1 in regulating autophagy and hepatitis B virus (HBV) replication

AimsTo investigate the role and underlying mechanism of 4E-BP1 and S6K1 in regulating autophagy and hepatitis B virus (HBV) replication. Main methodsThe mRNA relative expression of HBx and its DNA level were detected by real-time PCR. The relative levels of hepatitis B surface antigen (HBsAg) were measured by enzyme-linked immunosorbent assay (ELISA). HBx DNA level of HepG2 cells transfected with pcDNA3.1(+)-HBV1.3 plasmids were detected by Southern blot. Moreover, we determined autophagy through electron microscopy, confocal microscopy and Western blot. Key findingsRapamycin promoted autophagy and the X protein synthesis concomitantly with elevation in Akt phosphorylation and Beclin1 expression. Either Beclin1 or Akt depletion suppresses the Rapa-enhanced HBV replication, whereas mTOR silencing inhibited HBV replication concurring with a decreased in both S6K1 and 4E-BP1 phosphorylation. Unexpectedly, Akt inhibitor suppressed Rapa-dependent autophagic flux and increased the level of p62/SQSTM1. While S6K1 ablation impaired autophagy and decreased X protein expression, 4E-BP1 silencing slightly influenced autophagy and increased X protein level. SignificanceThe underlying mechanism of 4E-BP1 and S6K1, two main downstream effectors of mTOR, in mediating HBV replication and HBV-induced autophagy remains largely unknown. Here, we propose that Akt is required for both HBV replication and Rapa-induced autophagy, and 4E-BP1 and S6K1 play a distinct role in the virus replication and autophagic process.

Ribosomal protein S6 kinase 1 promotes the survival of photoreceptors in retinitis pigmentosa

Retinitis pigmentosa (RP) is a heterogeneous group of inherited disorders caused by mutations in genes that are mostly expressed by rod photoreceptors, which results in initial death of rods followed by cone photoreceptors. The molecular mechanisms that lead to both rod and cone degeneration are not yet fully understood. The mTOR pathway is implicated in RP. However, it remains unclear whether S6K1 plays an essential role downstream of the mTOR pathway in mediating photoreceptor survival in RP. Our in vitro studies demonstrated that PTEN (phosphatase and tensin homolog) overexpression deactivated mTOR activity and induced 661W cone cell apoptosis. In addition, we identified that S6K1 but not 4EBP1 was the downstream effector of PTEN neurotoxicity using gain- and loss-of-function approaches. Moreover, our in vivo data corroborated the results of our in vitro studies. S6K1 overexpression either in rods or cones promoted these cell survival and function and improved visual performance in the rd10 mouse model of RP. Our data demonstrated that S6K1 was the downstream effector of mTOR and that S6K1 was critical for both rod and cone survival in RP. Our findings make a strong case for targeting S6K1 as a promising therapeutic strategy for promoting the survival of photoreceptors in RP.

MTOR signaling in the arcuate nucleus of the hypothalamus mediates the anorectic action of estradiol.

Current evidence suggests that estradiol (E2), the main ovarian steroid, modulates energy balance by regulating both feeding and energy expenditure at the central level, through the energy sensor AMP-activated protein kinase (AMPK). We hypothesized that the hypothalamic mechanistic target of rapamycin (mTOR) pathway, a well-established nutrient sensor and modulator of appetite and puberty, could also mediate the anorectic effect of E2. Our data showed that ovariectomy (OVX) elicited a marked downregulation of the mTOR signaling in the arcuate nucleus of the hypothalamus (ARC), an effect that was reversed by either E2 replacement or central estrogen receptor alpha (ERα) agonism. The significance of this molecular signaling was given by the genetic inactivation of S6 kinase B1 (S6K1, a key downstream mTOR effector) in the ARC, which prevented the E2-induced hypophagia and weight loss. Overall, these data indicate that E2 induces hypophagia through modulation of mTOR pathway in the ARC.

The Ketogenic Diet Attenuates Both Hyperactivity in mTOR pathway and Astrogliosis Through Regulation of AMPK signaling in the Epileptic Brain

BackgroundThe high‐fat, low carbohydrate ketogenic diet (KD) is a proven therapy for patients with medically refractory epilepsy. The metabolic changes induced by the KD mimics the physiological alteration of fasting and calorie restriction – wherein ketone levels are elevated (ketosis). Both the KD and its metabolic substrates are involved in modulating anti‐seizure effects, but the molecular target of these beneficial effects have yet to be fully elucidated. One potential molecular explanation links the mammalian target of rapamycin (mTOR) signaling pathway. Disruptions in the mTOR pathway may explain in part susceptibility to and pathogenesis of epilepsy. The primary purpose of this study was to investigate the role of metabolic changes in mTOR signaling pathway in the Kcna1‐null mouse model of epilepsy and whether its mechanistic effects are linearly correlated with ketosis.MethodsSpontaneously epileptic Kcna1‐null (KO) mice were generated using heterozygous breeding pairs. At P21‐ 23, mice were treated with the KD and either standard diet (SD) or SD with subcutaneous administration of BHB through osmotic mini‐pumps for 2–3 weeks. Blood from tail clippings were used to measure BHB and glucose levels. EEG electrodes were implanted through parasaggital burr holes in either wild type (WT) mice or KO mice around P28. Following a 3‐day recovery period, seizure activity was assessed over 72 continuous hours. After over 2 weeks of treatment, hippocampal lysates were collected from: (1) SD‐fed WT; (2) SD‐fed KO; (3) KD‐fed WT; (4) KD‐fed KO; (5) saline‐treated WT; (6) saline‐treated KO; (7) BHB‐treated WT; and (8) BHB–treated KO mice. Samples were probed with antibodies against upstream modulators of mTOR, either phosphorylated ‐ or total –AMPK, and downstream effectors of mTOR, phospho‐ or total ‐ S6, as well as a downstream effectors of AMPK, either glial fibrillary acidic protein (GFAP) or Tubulin. Total RNA obtained from hippocampus or whole brain using TRIzol was reverse transcribed into cDNA by RT‐PCR and then the single‐stranded cDNA was amplified by PCR with primers for GFAP and β‐actin.ResultsUsing western blot analysis, mTOR signaling was significantly increased in KO mice accompanied with spontaneous epileptic discharge, and levels of upstream pAMPK were decreased in these animals compared to control and KD‐fed KO mice. KO mice showed a significant enhancement in the downstream markers pS6 and p4EBP1, whereas this effect was strongly suppressed by the KD treatment. KD treatment led to a reduction in pS6 in kainic acid (KA)‐treated control mice. KA resulted in a gradual increase of GFAP. In contrast, at 24 hours post KA injection, KD‐fed mice exhibited a significant decrease in expression level of GFAP.ConclusionCollectively, either the KD or ketone not only reduces the severity of seizures in two different mouse models, but also attenuates mTOR signaling pathways. Further, our finding that phospho‐S6 is reduced by KD treatment suggests a mechanistic link between mTOR inhibition and decreases in astrogliosis, both mechanisms that could contribute to the anti‐seizure and neuroprotective effects of the KD. The results provide evidence for the functional protective link between the KD and its primary metabolic substrate, β‐hydroxybutyrate, in spontaneously epileptic Kcna1‐null mice. Finally, the therapeutic effects of BHB and the KD may be in part inhibitory action of glial cell activation through modulation of mTOR signaling pathway Effects of the ketogenic diet on seizure‐evoked upregulation of mTOR signaling pathways in the hippocampus of Kcna1‐null mice. (A & D)