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Osteoporosis (OP) is a highly prevalent disorder characterized by low bone mass that severely reduces patient quality of life. Although numerous treatments for OP have been introduced in clinic, many have side effects and high costs. Therefore, there is still an unmet need for optimal solutions. Here, raw signal analysis was used to identify potential high-risk factors for OP, and the biological functions and possible mechanisms of action (MOAs) of these factors were explored via gene set enrichment analysis (GSEA). Subsequently, molecular biological experiments were performed to verify and analyze the discovered risk factors in vitro and in vivo. PMAIP1 was identified as a potential risk factor for OP and significantly suppressed autophagy in osteoblasts via the AMPK/mTOR pathway, thereby inhibiting the proliferation and differentiation of osteoblasts. Furthermore, we constructed an ovariectomy (OVX) model of OP in rats and simultaneously applied si-PMAIP1 for in vivo interference. si-PMAIP1 upregulated the expression of LC3B and p-AMPK and downregulated the expression of p-mTOR, and these effects were reversed by the autophagy inhibitor. Micro-CT revealed that, si-PMAIP1 significantly inhibited the development of osteoporosis in OVX model rats, and this therapeutic effect was attenuated by treatment with an autophagy inhibitor. This study explored the role and mechanism of PMAIP1 in OP and demonstrated that PMAIP1 may serve as a novel target for OP treatment., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

Endothelial-lined valves assure unidirectional flow in the lymphatic system. In this issue, Saygili Demir et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202207049) demonstrate how continuous repair of these valves occur, beginning with mTOR-activated cell replication in valve sinuses, and followed by cell migration to cover the valve surface., (© 2023 Arany.)

Background: Gastric adenocarcinoma (GAC) is a lethal disease with limited therapeutic options. Genetic alterations in chromatin remodelling gene AT-rich interactive domain 1A ( ARID1A ) and mTOR pathway activation occur frequently in GAC. Targeting the mechanistic target of rapamycin (mTOR) pathway in unselected patients has failed to show survival benefit. A deeper understanding of GAC might identify a subset that can benefit from mTOR inhibition., Methods: Genomic alterations in ARID1A were analysed in GAC. Mouse gastric epithelial cells from CK19-Cre-Arid1A fl/fl and wild-type mice were used to determine the activation of oncogenic genes due to loss of Arid1A. Functional studies were performed to determine the significance of loss of ARID1A and the sensitivity of ARID1A-deficient cancer cells to mTOR inhibition in GAC., Results: More than 30% of GAC cases had alterations (mutations or deletions) of ARID1A and ARID1A expression was negatively associated with phosphorylation of S6 and SOX9 in GAC tissues and patient-derived xenografts (PDXs). Activation of mTOR signalling (increased pS6) and SOX9 nuclear expression were strongly increased in Arid1A -/- mouse gastric tissues which could be curtailed by RAD001, an mTOR inhibitor. Knockdown of ARID1A in GAC cell lines increased pS6 and nuclear SOX9 and increased sensitivity to an mTOR inhibitor which was further amplified by its combination with fluorouracil both in vitro and in vivo in PDXs., Conclusions: The loss of ARID1A activates pS6 and SOX9 in GAC, which can be effectively targeted by an mTOR inhibitor. Therefore, our studies suggest a new therapeutic strategy of clinically targeting the mTOR pathway in patients with GAC with ARID1A deficiency., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Hepatocellular carcinoma (HCC) is a substantial health concern worldwide. Increasing studies have suggested that circle RNAs (circRNAs) function as new regulators in HCC progression. The present work explored the role of hsa_circ_0007059 (circ_0007059) in the developing process of hepatocarcinogenesis., Methods: The circ_0007059 level in HCC was determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and northern blot. Its biological role in HCC cells was assessed using 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT), colony formation, flow cytometry, Transwell, sphere formation and western blotting analyses. Bioinformatics analysis, luciferase reporter, and RNA immunoprecipitation (RIP) assays were used to test the regulatory mechanisms of circ_0007059., Results: Our results revealed that circ_0007059 expression was downregulated in HCC samples and cells. Moreover, circ_0007059 overexpression inhibited HCC cell proliferation, migration, invasion, and stem cell-like property, and strengthened cell apoptosis. In mechanism, circ_0007059 suppressed AKT/mTOR pathway by positively regulating phosphatase and tensin homolog (PTEN) expression. Additionally, circ_0007059 acted as a positive regulator of PTEN through controlling the availability of miR-421. Rescue assays demonstrated that PTEN knockdown or SC79 (AKT agonist) eliminated the effect of circ_0007059 on HCC cell phenotypes., Conclusion: Circ_0007059 sponges miR-421 to inhibit oncogenic cellular process in HCC by mediating the PTEN-AKT/mTOR pathway., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Background: Sodium/iodide symporter (NIS) acts as a vital role in regulation of iodide uptake in thyroid cancer. However, the efficient approach to increase NIS expression and the mechanism of NIS-mediated iodide uptake in thyroid cancer remain unclear., Methods: Small activating RNA (saRNA) was used to promote NIS expression. And the cell viability, apoptosis, and autophagy were detected using Cell count-kit 8 (CCK-8), Annexin V-FITC/PI double staining, and GFP-LC3 immunofluorescence assays, respectively. The protein levels of caspase 3, Bax, Bcl-2, ATG5, ATG12, LC3B Ⅱ to LC3B Ⅰ, Beclin 1, P62, AMPK, mTOR, P70S6K, actin, and phosphorylation of AMPK, mTOR, P70S6K were determined by western blotting. Moreover, a nude murine node with transplanted NC-dsRNA or NIS-482-transfected SW579 cells was used to examine the effect of NIS-mediated autophagy in vivo. And the levels of caspase 3 and ki67 were examined by immunohistochemical staining assay., Results: saRNA mediated NIS mRNA and protein upregulated in SW579 cells. saRNA-mediated NIS expression inhibited cell proliferation, induced apoptosis and autophagy, and promoted iodide uptake in SW579 cells. Moreover, the effects of NIS on cells were enhanced by autophagy activator Rapamycin whereas reversed by autophagy inhibitor 3-Methyladenine (3-MA). For mechanism analysis, we found that NIS upregulation exerted the effects on cell proliferation, apoptosis, autophagy, and iodide uptake via regulating AMPK/mTOR pathway. We also demonstrated that saRNA-mediated NIS expression promoted iodide uptake in vivo., Conclusion: saRNA-mediated NIS expression acted as a critical role in increasing iodide uptake via AMPK/mTOR pathway in thyroid cancer., (Copyright © 2021. Published by Elsevier GmbH.)

Background: While stimulator of interferon genes (STING) activation in innate immune cells of the tumor microenvironment can result in CD8 T cell-dependent antitumor immunity, whether STING signaling affects CD4 T-cell responses remains elusive., Methods: Here, we tested whether STING activation modulated the effector functions of CD4 T cells in vivo by analyzing tumor-infiltrating CD4 T cells and evaluating the contribution of the CD4 T cell-derived cytokines in the antitumor activity of the STING ligand 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) in two mouse tumor models. We performed ex vivo experiments to assess the impact of STING activation on CD4 T-cell differentiation and investigate the underlying molecular mechanisms. Finally, we tested whether STING activation enhances T H 9 cell antitumor activity against mouse melanoma upon adoptive transfer., Results: We found that activation of STING signaling cell-intrinsically enhances the differentiation and antitumor functions of T H 1 and T H 9 cells by increasing their respective production of interferon gamma (IFN-γ) and interleukin-9. IRF3 and type I interferon receptors (IFNARs) are required for the STING-driven enhancement of T H 1 cell differentiation. However, STING activation favors T H 9 cell differentiation independently of the IFNARs/IRF3 pathway but through mammalian target of rapamycin (mTOR) signaling, underscoring that STING activation differentially affects the fate of distinct CD4 T-cell subsets. The therapeutic effect of STING activation relies on T H 1 and T H 9-derived cytokines, and STING activation enhances the antitumor activity of T H 9 cells upon adoptive transfer., Conclusion: Our results reveal the STING signaling pathway as a therapeutic target to boost CD4 T-cell effector functions and antitumor immunity., Competing Interests: Competing interests: LA performed consultancy work for Roche, Merck, Bristol-Myers Squibb, and Orega Biotech and was a recipient of a research grant from Sanofi., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Mechanistic target of rapamycin (mTOR) forms two distinct complexes, mTOR complex 1 (mTORC1) and mTORC2. Here we investigated the antitumor effect of dual mTORC1/2 inhibitor AZD2014 on epithelial ovarian cancer (EOC) and its potential effect on immunosuppressive myeloid-derived suppressor cells (MDSCs). Immunohistochemical analysis of mTORC1 and mTORC2 was performed on a human ovarian cancer tissue microarray. High mTORC2 expression level was associated with shorter survival in EOC, whereas mTORC1 was not correlate with patients' prognosis. AZD2014 suppressed mTOR signaling pathway in ovarian cancer cells, inhibited proliferation and induced G1-phase cell cycle arrest and apoptosis. In tumor-bearing mice, AZD2014 treatment limited tumor growth, reduced peritoneal ascites, and prolonged survival. AZD2014 specifically reduced MDSCs migration and accumulation in EOC peritoneal fluid but not in the spleen. Moreover, subsequent AZD2014 treatment after cisplatin chemotherapy delayed EOC recurrence. Collectively, we observed that high mTORC2 expression level in EOC indicated a poor prognosis. Remarkably, in tumor-bearing mice, AZD2014 diminished MDSC accumulation and delayed tumor growth and recurrence., (Copyright © 2021. Published by Elsevier B.V.)

Membrane vesicles, including exosomes and microparticles (MPs), serve to package and transfer the cellular cargo during inter/extracellular communication, which is of great interest in cancer development, especially in the dissemination of signal transduction-associated traits from donor cells to recipient cells. Although increasing evidence suggests that microparticles (MPs) contribute to the development of cancer, their unique characteristics remain to be exploited. Here, we examined the secretion of MPs in tumor tissues from triple-negative breast cancer (TNBC) patients and found that the tumor cells could release MPs loaded with immune checkpoint molecular programmed cell death ligand 1 (PD-L1), especially in patients treated with traditional clinical interventions, such as chemotherapy and radiotherapy. These PD-L1-loading MPs contribute to the suppressive immune microenvironment, eventually resulting in the tumor progression in TNBC. Mechanically, we proved that PD-L1-loading MPs could suppress the activation and function of functional cluster of differentiation CD8 + T cells. Meanwhile, the PD-L1-loading MPs could mediate the differentiation of macrophages toward the immune-suppressive M2 phenotype via the activation of the TANK-binding kinase 1 (TBK1)/signal transducer and activator of transcription 6 (STAT6) signal and suppression of the serine-threonine kinase (AKT)/mammalian target of rapamycin (mTOR) signal. Given the increasing MP production induced by traditional clinical interventions, we further combined chemotherapy with the PD-L1 inhibitor atezolizumab (ATZ) to efficiently abrogate the immunosuppression caused by the PD-L1-loading MPs. Therefore, our study unveils the mechanism by which tumor cells systemically evade immune surveillance by releasing the PD-L1-loading MPs, and provides new insights into clinical TNBC immunotherapy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ + NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yao, Yang, Yang, Li, Peng, Wu, Wang, Zhang, Huang, Wang, Xiong, Luo, Li, Jia, Deng and Deng.)

Synovitis is the primary driving factor for the occurrence and development of knee osteoarthritis (KOA) and fibroblast-like synoviocytes (FLSs) and plays a crucial role during this process. Our previous works revealed that transient receptor potential ankyrin 1 (TRPA1) ion channels mediate the amplification of KOA synovitis. In recent years, essential oils have been proved to have blocking effect on transient receptor potential channels. Meanwhile, the therapeutic effect of Sanse Powder on KOA synovitis has been confirmed in clinical trials and basic studies; although, the mechanism remains unclear. In the present study, Sanse Powder essential oil nanoemulsion (SP-NEs) was prepared, and then chemical composition, physicochemical properties, and stability were investigated. Besides, both in MIA-induced KOA rats and in LPS-stimulated FLSs, we investigated whether SP-NES could alleviate KOA synovitis by interfering with AMP-activated protein kinase- (AMPK-) mammalian target of rapamycin (mTOR), an energy sensing pathway proved to negatively regulate the TRPA1. Our research shows that the top three substances in SP-NEs were tumerone, delta-cadinene, and Ar-tumerone, which accounted for 51.62% of the total, and should be considered as the main pharmacodynamic ingredient. Less inflammatory cell infiltration and type I collagen deposition were found in the synovial tissue of KOA rats treated with SP-NEs, as well as the downregulated expressions of interleukin (IL)-1 β , IL-18, and TRPA1. Besides, SP-NEs increased the phosphorylation level of AMPK and decreased the phosphorylation level of mTOR in the KOA model, and SP-NEs also upregulated expressions of peroxisome proliferator-activated receptor-gamma (PPAR γ ) and PPAR γ coactivator-1 α and downstream signaling molecules of AMPK-mTOR in vivo and in vitro. To conclude, a kind of Chinese herbal medicine for external use which is effective in treating synovitis of KOA was extracted and prepared into essential oil nanoemulsion with stable properties in the present study. It may alleviate synovitis in experimental KOA through the negative regulation of TRPA1 by AMPK-mTOR signaling., Competing Interests: All authors declare that there are no actual or potential conflicts of interest, including any financial, personal, or relationships with other people or organizations that could inappropriately influence or be perceived to influence our work., (Copyright © 2021 Mingchao Li et al.)

Akt signaling has been associated with adult neurogenesis in the hippocampal dentate gyrus (DG). We reported cognitive dysfunction in Akt3 knockout (Akt3-KO) mice with the down-regulation of mTOR activation. However, little is known about the effects of Akt3 signaling on hippocampal neurogenesis. Herein, we show that progenitor cells, neuroblasts, and mature newborn neurons in hippocampal DG expressed Akt3 protein. The Akt3 phosphorylation in hippocampal DG was increased after voluntary wheel running for 7 days in wild-type mice (running WT mice), but not in Akt3-KO mice (running Akt3-KO mice). Subsequently, we observed that the proliferation of progenitor cells was suppressed in Akt3-KO mice and the mTOR inhibitor rapamycin-treated mice, whereas enhanced in running WT mice rather than running Akt3-KO mice. Neurite growth of neuroblasts was impaired in Akt3-KO mice and rapamycin-treated mice. In contrast, neither differentiation of progenitor cells nor migrating of newly generated neurons was altered in Akt3-KO mice or running WT mice. The levels of p70S6K and 4EBP1 phosphorylation were declined in Akt3-KO mice and elevated in running WT mice depending on mTOR activation. Furthermore, telomerase activity, telomere length, and expression of telomerase reverse transcriptase (TERT) were decreased in Akt3-KO mice but increased in running WT mice rather than running Akt3-KO mice, which required the mTOR activation. The study provides in vivo evidence that Akt3-mTOR signaling plays an important role in the proliferation of progenitor cells and neurite growth through positive regulated TERT expression and activation of p70S6K and 4EBP1., (© 2021 International Society for Neurochemistry.)

Slit2 exerts antitumor effects in various cancers; however, the underlying mechanism, especially its role in regulating the immune, especially in the bone marrow niche, system is still unknown. Elucidating the behavior of macrophages in tumor progression can potentially improve immunotherapy. Using a spontaneous mammary tumor virus promoter-polyoma middle T antigen (PyMT) breast cancer mouse model, we observed that Slit2 increased the abundance of antitumor M1 macrophage in the bone marrow upon differentiation in vitro . Moreover, myeloablated PyMT mice injected with Slit2-treated bone marrow allografts showed a marked reduction in tumor growth, with enhanced recruitment of M1 macrophage in their tumor stroma. Mechanistic studies revealed that Slit2 significantly enhanced glycolysis and reduced fatty acid oxidation in bone marrow-derived macrophages (BMDMs). Slit2 treatment also altered mitochondrial respiration metabolites in macrophages isolated from healthy human blood that were treated with plasma from breast cancer patients. Overall, this study, for the first time, shows that Slit2 increases BMDM polarization toward antitumor phenotype by modulating immune-metabolism. Furthermore, this study provides evidence that soluble Slit2 could be developed as novel therapeutic strategy to enhance antitumor immune response., Competing Interests: NKJ serves as a consultant for Capture Collective Inc. RKG serves as consultant for Guidepoint consultation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kaul, Benej, Mishra, Ahirwar, Yadav, Stanford, Jacob, Denko and Ganju.)

Neuroinflammation plays a critical role during sepsis triggered by microglial activation. Mammalian target of rapamycin (mTOR) has gained attraction in neuroinflammation, however, the mechanism remains unclear. Our goal was to assess the effects of mTOR inhibition by rapamycin on inflammation, microglial activation, oxidative stress, and apoptosis associated with the changes in the inhibitor-κB (IκB)-α/nuclear factor-κB (NF-κB)/hypoxia-inducible factor-1α (HIF-1α) pathway activity following a systemic challenge with lipopolysaccharide (LPS). Rats received saline (10 mL/kg), LPS (10 mg/kg), and (or) rapamycin (1 mg/kg) intraperitoneally. Inhibition of mTOR by rapamycin blocked phosphorylated form of ribosomal protein S6, NF-κB p65 activity by increasing degradation of IκB-α in parallel with HIF-1α expression increased by LPS in the kidney, heart, lung, and brain tissues. Rapamycin attenuated the increment in the expression of tumor necrosis factor-α and interleukin-1β, the inducible nitric oxide synthase, gp91 phox , and p47 phox in addition to nitrite levels elicited by LPS in tissues or sera. Concomitantly, rapamycin treatment reduced microglial activation, brain expression of caspase-3, and Bcl-2-associated X protein while it increased expression of B cell lymphoma 2 induced by LPS. Overall, this study supports the hypothesis that mTOR contributes to the detrimental effect of LPS-induced systemic inflammatory response associated with neuroinflammation via IκB-α/NF-κB/HIF-1α signaling pathway.

A critical cell type participating in cardiac outflow tract development is a subpopulation of the neural crest cells, the cardiac neural crest cells (NCCs), whose defect causes a spectrum of cardiovascular abnormalities. Accumulating evidence indicates that mTOR, which belongs to the PI3K-related kinase family and impacts multiple signaling pathways in a variety of contexts, plays a pivotal role for NCC development. Here, we investigated functional roles of mTOR for cardiac neural crest development using several lines of mouse genetic models. We found that disruption of mTOR caused NCC defects and failure of cardiac outflow tract separation, which resulted in a spectrum of cardiac defects including persistent truncus arteriosus, ventricular septal defect and ventricular wall defect. Specifically, mutant neural crest cells showed reduced migration into the cardiac OFT and prematurely exited the cell cycle. A number of critical factors and fundamental signaling pathways, which are important for neural crest and cardiomyocyte development, were impaired. Moreover, actin dynamics was disrupted by mTOR deletion. Finally, by phenotyping the neural crest Rptor and Rictor knockout mice respectively, we demonstrate that mTOR acts principally through the mTORC1 pathway for cardiac neural crest cells. Altogether, these data established essential roles of mTOR for cardiac NCC development and imply that dysregulation of mTOR in NCCs may underline a spectrum of cardiac defects., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The target of rapamycin (TOR) protein kinase is an atypical Ser/Thr protein kinase and evolutionally conserved among yeasts, plants, and mammals. TOR has been established as a central hub for integrating nutrient, energy, hormone, and environmental signals in all the eukaryotes. Despite the conserved functions across eukaryotes, recent research has shed light on the multifaceted roles of TOR signaling in plant-specific functional and mechanistic features. One of the most specific features is the involvement of TOR in plant photosynthesis. The recent development of tools for the functional analysis of plant TOR has helped to uncover the involvement of TOR signaling in several steps preceding photoautotrophy and maintenance of photosynthesis. Here, we present recent novel findings relating to TOR signaling and its roles in regulating plant photosynthesis, including carbon nutrient sense, light absorptions, and leaf and chloroplast development. We also provide some gaps in our understanding of TOR function in photosynthesis that need to be addressed in the future.

We studied the possibilities of inhibition of neurodegeneration in MPTP-induced model of Parkinson's disease (PD) in C57Bl/6J mice and transgenic model of early PD stage (5-monthold B6.Cg-Tg(Prnp-SNCA*A53T)23Mkle/J mice) by autophagy activation through mTOR-dependent and mTOR-independent pathways with rapamycin and trehalose, respectively. Therapy with autophagy inducers in a "postponed" mode (7 days after MPTP intoxication) restored the expression of the dopaminergic neuron marker tyrosine hydroxylase and markedly improved cognitive function in the conditioned passive avoidance response (CPAR; fear memory). The transgenic model also showed an increase in the expression of tyrosine hydroxylase in the nigrostriatal system of the brain. An enhanced therapeutic effect of the combined treatment with the drugs was revealed on the expression of tyrosine hydroxylase, but not in the CPAR test. Thus, activation of both pathways of autophagy regulation in PD models with weakened neuroinflammation can restore the dopaminergic function of neurons and cognitive activity in mice., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: The extent to which routine genomic sequencing can identify relevant secondary genomic alterations among BRAF V600E -mutant papillary thyroid carcinoma (PTC) is unknown. Such markers would prove highly valuable for prognostic purposes., Experimental Design: We reviewed clinicopathologic data of 225 patients with BRAF V600E -mutant PTC and integrated them with genomic data derived from targeted next-generation sequencing (NGS) on tumor specimens. We defined patient subgroups based on bona fide secondary oncogenic events (separate from BRAF V600E ) and compared their clinical features and outcomes with those without additional oncogenic alterations., Results: Additional oncogenic alterations were identified in 16% of tumors. Patients in the " BRAF +additional mutations" group were more likely to be at high American Thyroid Association (ATA) risk of recurrence (48.6% vs. 17.6%; P = 0.0009), had larger baseline tumor (2.7 vs. 1.9 cm; P = 0.0005) and more advanced stage at presentation (14.3% vs. 1.1% stage 4; P < 0.0001). Importantly, over a 65-month follow-up, disease-specific mortality (DSM) was increased when additional mutations were identified (13.8% vs. 1.4% in the BRAF -only group; P = 0.005). Separately, we identified a subcluster of patients harboring oncogenic mutations in key effectors of the PI3K/AKT/mTOR pathway, which were independently associated with DSM (OR = 47.9; 95% confidence interval, 3.5-1,246.5; P = 0.0043)., Conclusions: Identification of additional PIK3/AKT/mTOR alterations in patients with BRAF V600E -mutant PTC provides important and actionable prognostic risk stratification. These data support genomic profiling of PTC tumors to inform prognosis and clinical strategy., (©2021 American Association for Cancer Research.)

Objective: To investigate if PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells. Participants . Eight lean insulin-sensitive nondiabetic participants (4 females and 4 males; age: 21.0 ± 1.0 years; BMI: 22.0 ± 0.7 kg/m 2 ; 2-hour OGTT: 97.0 ± 6.0 mg/dl; HbA1c: 5.3 ± 0.1%; fasting plasma glucose: 87.0 ± 2.0 mg/dl; M value; 11.0 ± 1.0 mg/kgBW/min)., Design: A hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects, and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells (shown to retain metabolic characteristics of donors) were cultured from these muscle biopsies that included 8 lean insulin-sensitive participants. Cultured cells were expanded, differentiated into myotubes, and treated with 50  μ M metformin for 24 hours before harvesting. PP2Ac activity was measured by a phosphatase activity assay kit (Millipore) according to the manufacturer's protocol., Results: The results indicated that metformin significantly increased the activity of PP2A in the myotubes for all 8 lean insulin-sensitive nondiabetic participants, and the average fold increase is 1.54 ± 0.11 ( P < 0.001)., Conclusions: These results provided the first evidence that metformin can activate PP2A in human skeletal muscle cells derived from lean healthy insulin-sensitive participants and may help to understand metformin's action in skeletal muscle in humans., Competing Interests: The authors declare no conflict of interest and have nothing to disclose., (Copyright © 2021 Aktham Mestareehi et al.)

Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy produced by mutations in the CAPN3 gene. It is a rare disease and there is no cure or treatment for the disease while the pathophysiological mechanism by which the absence of calpain 3 provokes the dystrophy in muscles is not clear. However, key proteins implicated in Wnt and mTOR signaling pathways, which regulate muscle homeostasis, showed a considerable reduction in their expression and in their phosphorylation in LGMDR1 patients' muscles. Finally, the administration of tideglusib and VP0.7, ATP non-competitive inhibitors of glycogen synthase kinase 3β (GSK-3β), restore the expression and phosphorylation of these proteins in LGMDR1 cells, opening the possibility of their use as therapeutic options.

Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.

As the world navigates the coronavirus disease 2019 (COVID-19) pandemic, there is a growing need to assess its impact in patients with autoimmune rheumatic diseases, such as systemic lupus erythematosus (SLE). Patients with SLE are a unique population when considering the risk of contracting COVID-19 and infection outcomes. The use of systemic glucocorticoids and immunosuppressants, and underlying organ damage from SLE are potential susceptibility factors. Most patients with SLE have evidence of high type I interferon activity, which may theoretically act as an antiviral line of defense or contribute to the development of a deleterious hyperinflammatory response in COVID-19. Other immunopathogenic mechanisms of SLE may overlap with those described in COVID-19, thus, studies in SLE could provide some insight into immune responses occurring in severe cases of the viral infection. We reviewed the literature to date on COVID-19 in patients with SLE and provide an in-depth review of current research in the area, including immune pathway activation, epidemiology, clinical features, outcomes, and the psychosocial impact of the pandemic in those with autoimmune disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The nuclear envelope component proline-rich protein 14 (PRR14) is involved in the nuclear morphological alteration and activation of the mTOR (mammalian target of rapamycin) signaling pathway, and has been repeatedly shown to be upregulated in patients with Parkinson's disease (PD). The aim of this study was to explore whether PRR14 can be used as a potential biomarker for the diagnosis of PD. We compared PRR14 expression in PD patients and normal controls in gene expression omnibus (GEO) data. Quantitative enzyme-linked immunosorbent assay (ELISA) was used to detect PRR14 expression in PD patients and age- and sex-matched controls. The relationship between serum PRR14 and clinical phenotype was evaluated using correlation analysis and logistic regression. The expression of PRR14 in whole blood, substantia nigra, and medial substantia nigra was significantly higher in PD patients than in the healthy control group. Compared to plasma, serum was more suitable for the detection of PRR14. Furthermore, serum PRR14 level in PD patients was significantly higher than that in age- and sex-matched controls. The area under the curve for serum PRR14 level in the ability to identify PD versus age- and sex-matched controls was 0.786. In addition, serum PRR14 level was found to correlate with constipation in PD patients. Our findings demonstrate for the first time that serum PRR14 is a potential biomarker for PD.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular homeostasis that integrates environmental and nutrient signals to control cell growth and survival. Over the past two decades, extensive studies of mTOR have implicated the importance of this protein complex in regulating a broad range of metabolic functions, as well as its role in the progression of various human diseases. Recently, mTOR has emerged as a key signaling molecule in regulating animal entry into a hypometabolic state as a survival strategy in response to environmental stress. Here, we review current knowledge of the role that mTOR plays in contributing to natural hypometabolic states such as hibernation, estivation, hypoxia/anoxia tolerance, and dauer diapause. Studies across a diverse range of animal species reveal that mTOR exhibits unique regulatory patterns in an environmental stressor-dependent manner. We discuss how key signaling proteins within the mTOR signaling pathways are regulated in different animal models of stress, and describe how each of these regulations uniquely contribute to promoting animal survival in a hypometabolic state.

Scope: Nutritional supplementation of the maternal diet can modify the cancer susceptibility in adult offspring. Therefore, the authors evaluate the effects of a fish-oil diet administered to a long-term, during pre-mating, gestation, and lactation, in reducing cancer-cachexia damages in adult Walker-256 tumor-bearing offspring., Methods and Results: Female rats receive control or fish oil diet during pre-mating, gestation, and lactation. After weaning, male offspring are fed the control diet until adulthood and distributed in (C) control adult-offspring; (W) adult tumor-bearing offspring; (OC) adult-offspring of maternal fish oil diet; (WOC) adult tumor-bearing offspring of maternal fish oil diet groups. Fat body mass is preserved, muscle expression of mechanistic target of rapamicin (mTOR) and eukariotic binding protein of eukariotic factor 4E (4E-BP1) is modified, being associated with lower 20S proteasome protein expression, and the liver alanine aminotransferase (ALT) enzyme content maintained in the WOC group. Also, the OC group shows reduced triglyceridemia., Conclusion: In this experimental model of cachexia, the long-term maternal supplementation is a positive strategy to improve liver function and lipid metabolism, as well as to modify muscle proteins expression in the mTOR pathway and also reduce the 20S muscle proteasome protein, without altering the tumor development and muscle wasting in adult tumor-bearing offspring., (© 2021 Wiley-VCH GmbH.)

Farnesyltransferase inhibitors (FTIs) suppress tumor aggressiveness in several malignancies by inhibiting Ras signaling. However, treatment of cells with a low dose of the FTI tipifarnib suppresses the expression of hypoxia-inducible factor-1α (HIF-1α) and results in antitumor effects without inhibiting the Ras pathway. Although we previously reported that elevated HIF-1α expression is associated with an aggressive phenotype in gastric cancer (GC), little is known about the antitumor effects of FTIs on GC. In this study, we examined the relationship between the antitumor effects of low-dose tipifarnib and HIF-1α expression in GC cells. Under normoxic conditions, HIF-1α was expressed only in MKN45 and KATOIII cells. The inhibitory effect of tipifarnib on HIF-1α was observed in HIF-1α-positive cells. Low-dose tipifarnib had antitumor effects only on HIF-1α-positive cells both in vitro and in vivo. Furthermore, low-dose tipifarnib inactivated ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling and decreased intracellular reactive oxygen species (ROS) levels in HIF-1α-positive GC cells. Our results that the antitumor effects of low-dose tipifarnib are at least partially mediated through suppression of mTOR signaling and HIF-1α expression via inhibition of Rheb farnesylation and reduction in ROS levels. These findings suggest that low-dose tipifarnib may be capable of exerting an antitumor effect that is dependent on HIF-1α expression in GC cells. Tipifarnib may have potential as a novel therapeutic agent for HIF-1α-expressing GC exhibiting an aggressive phenotype., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Rationale: Metastasis, the development of secondary malignant growth at a distance from a primary tumor, is the main cause of cancer-associated death. However, little is known about how metastatic cancer cells adapt to and colonize in the new organ environment. Here we sought to investigate the functional mechanism of cholesterol metabolic aberration in colorectal carcinoma (CRC) liver metastasis. Methods: The expression of cholesterol metabolism-related genes in primary colorectal tumors (PT) and paired liver metastases (LM) were examined by RT-PCR. The role of SREBP2-dependent cholesterol biosynthesis pathway in cell growth and CRC liver metastasis were determined by SREBP2 silencing in CRC cell lines and experimental metastasis models including, intra-splenic injection models and liver orthotropic injection model. Growth factors treatment and co-culture experiment were performed to reveal the mechanism underlying the up-regulation of SREBP2 in CRC liver metastases. The in vivo efficacy of inhibition of cholesterol biosynthesis pathway by betulin or simvastatin were evaluated in experimental metastasis models. Results: In the present study, we identify a colorectal cancer (CRC) liver metastasis-specific cholesterol metabolic pathway involving the activation of SREBP2-dependent cholesterol biosynthesis, which is required for the colonization and growth of metastatic CRC cells in the liver. Inhibiting this cholesterol biosynthesis pathway suppresses CRC liver metastasis. Mechanically, hepatocyte growth factor (HGF) from liver environment activates SREBP2-dependent cholesterol biosynthesis pathway by activating c-Met/PI3K/AKT/mTOR axis in CRC cells. Conclusion: Our findings support the notion that CRC liver metastases show a specific cholesterol metabolic aberration. Targeting this cholesterol biosynthesis pathway could be a promising treatment for CRC liver metastasis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Fluorochloridone (FLC), a selective pyrrolidone herbicide, has been recognized as a potential endocrine disruptor and reported to induce male reproductive toxicity, but the underlying mechanism is unclear. The aim of this study was to investigate the mechanism of FLC-induced reproductive toxicity on male mice with particular emphasis on the role of autophagy in mice' TM4 Sertoli cells., Methods: Adult C57BL/6 mice were divided into one control group (0.5% sodium carboxymethyl cellulose), and four FLC-treated groups (3,15,75,375 mg/kg). The animals (ten mice per group) received gavage for 28 days. After treatment, histological analysis, sperm parameters, the microstructure of autophagy and the expression of autophagy-associated proteins in testis were evaluated. Furthermore, to explore the autophagy mechanism, TM4 Sertoli cells were treated with FLC (0,40,80,160 μM) in vitro for 24 h. Cell activity and cytoskeletal changes were measured by MTT assay and F-actin immunofluorescence staining. The formation of autophagosome, accumulation of reactive oxygen species (ROS), expression of autophagy marker proteins (LC3, Beclin-1 and P62) and AKT-related pathway proteins (AKT, mTOR) were observed. The ROS scavenger N-acetylcysteine (NAC) and AKT agonist (SC79) were used to treat TM4 cells to observe the changes of AKT-mTOR pathway and autophagy., Results: In vivo, it showed that FLC exposure caused testicular injuries, abnormality in epididymal sperm. Moreover, FLC increased the formation of autophagosomes, the accumulation of LC3II/LC3I, Beclin-1 and P62 protein, which is related to the degradation of autophagy. In vitro, FLC triggered TM4 cell autophagy by increasing the formation of autophagosomes and upregulating of LC3II/LC3I, Beclin-1 and P62 levels. In addition, FLC induced ROS production and inhibited the activities of AKT and mTOR kinases. The Inhibition of AKT/mTOR signaling pathways and the activation of autophagy induced by FLC could be efficiently reversed by pretreatment of NAC. Additionally, decreased autophagy and increased cell viability were observed in TM4 cells treated with SC79 and FLC, compared with FLC alone, indicating that FLC-induced autophagy may be pro-death., Conclusion: Taken together, our study provided the evidence that FLC promoted autophagy in TM4 Sertoli cells and that this process may involve ROS-mediated AKT/mTOR signaling pathways.

Background & Aims: Our previous study found that platelet counts were positively associated with body fat percentage in human. In the present study, we conducted a reverse translational study to explore the role of platelets in modulating pre-adipocyte proliferation in mice., Methods: Mouse pre-adipocyte cell line (3T3-L1) and human pre-adipocytes harvested from female subcutaneous fat were used. Pre-adipocytes were co-cultured with platelets or platelet releasate, which were isolated from mice or humans. The cell viability and proliferative ability of the pre-adipocytes were examined by MTT and flow cytometry assays. Western blotting analysis was used to determine the phosphorylation levels of proteins in the mTOR pathway., Results: The number of platelets in the adipose tissues from obese mice was significantly higher than that from lean mice. Platelets and collagen-activated platelet releasate stimulated the proliferation of human pre-adipocytes and 3T3-L1 cells in vitro. Besides, platelets from obese mice were more potent in stimulating pre-adipocyte proliferation than those from lean control mice. Mechanistically, platelets enhanced pre-adipocyte proliferation through the acceleration of cell cycle progression from G0/G1 to S phase cell cycle progression. At the molecular level, platelets promoted pre-adipocyte proliferation through mTOR pathway-mediated upregulation of cyclin D1 expression., Conclusion: In conclusion, platelets and platelet releasate play an important role in the proliferation of pre-adipocytes. Our study may provide new clues and the molecular mechanism of the causal pathways between platelets and body fat to explain the finding we observed in population study., Competing Interests: Conflicts of interest The authors declare no conflict of interest pertinent to this study., (Copyright © 2020 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Obesity and obesogenic comorbidities have been associated with COVID-19 susceptibility and mortality. However, the mechanism of such correlations requires an in-depth understanding. Overnutrition/excess serum amino acid profile during obesity has been linked with inflammation and reprogramming of translational machinery through hyperactivation of amino acid sensor mammalian target of rapamycin (mTOR), which is exploited by SARS-CoV-2 for its replication. Conversely, we have shown that the activation of general control nonderepressible 2 (GCN2)-dependent amino acid starvation sensing pathway suppresses intestinal inflammation by inhibiting the production of reactive oxygen species (ROS) and interleukin-1 beta (IL-1β). While activation of GCN2 has shown to mitigate susceptibility to dengue infection, GCN2 deficiency increases viremia and inflammation-associated pathologies. These findings reveal that the amino acid sensing pathway plays a significant role in controlling inflammation and viral infections. The current fact is that obesity/excess amino acids/mTOR activation aggravates COVID-19, and it might be possible that activation of amino acid starvation sensor GCN2 has an opposite effect. This article focuses on the amino acid sensing pathways through which host cells sense the availability of amino acids and reprogram the host translation machinery to mount an effective antiviral response. Besides, how SARS-CoV-2 hijack and exploit amino acid sensing pathway for its replication and pathogenesis is also discussed., (© 2021 World Obesity Federation.)

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disorder resulting from an aberrant expansion of a polyglutamine stretch in the ataxin-3 protein and subsequent neuronal death. The underlying intracellular signaling pathways are currently unknown. We applied the Reverse-phase Protein MicroArray (RPMA) technology to assess the levels of 50 signaling proteins (in phosphorylated and total forms) using three in vitro and in vivo models expressing expanded ataxin-3: (i) human embryonic kidney (HEK293T) cells stably transfected with human ataxin-3 constructs, (ii) mouse embryonic fibroblasts (MEF) from SCA3 transgenic mice, and (iii) whole brains from SCA3 transgenic mice. All three models demonstrated a high degree of similarity sharing a subset of phosphorylated proteins involved in the PI3K/AKT/GSK3/mTOR pathway. Expanded ataxin-3 strongly interfered (by stimulation or suppression) with normal ataxin-3 signaling consistent with the pathogenic role of the polyglutamine expansion. In comparison with normal ataxin-3, expanded ataxin-3 caused a pro-survival stimulation of the ERK pathway along with reduced pro-apoptotic and transcriptional responses.

The current COVID-19 is one of the deadliest pandemics in recent decades. In the lack of a specific treatment for this novel infection, knowing the role of cell signaling pathways in the pathogenesis of this infection could be useful in finding effective drugs against this disease. The mammalian or mechanistic target of rapamycin (mTOR) is an important cell signaling pathway that has important role in the regulation of cell growth, protein synthesis, and metabolism in reactance to upstream signals in both pathological and normal physiological conditions. Recently, some researchers have suggested the therapeutic potential of mTOR inhibitors such as rapamycin against COVID-19. However, it is important to consider the role of activation of this pathway in controlling immune system response against viral activity in drug repositioning of rapamycin and other mTOR inhibitors in SARS-CoV-2 infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi's sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman's disease). While in patients with late stage of Kaposi's sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. KEY MESSAGES: In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance.

The mechanistic target of rapamycin (mTOR) is a key mediator of energy metabolism, cell growth, and survival. While previous studies using transgenic mice with cardiac-specific overexpression of mTOR (mTOR-Tg) demonstrated the protective effects of cardiac mTOR against ischemia-reperfusion (I/R) injury in both ex vivo and in vivo models, the mechanisms underlying the role of cardiac mTOR in cardiac function following I/R injury are not well-understood. Torin1, a pharmacological inhibitor of mTOR complex (mTORC) 1 and mTORC2, significantly decreased functional recovery of LV developed pressure in ex vivo I/R models (p < 0.05). To confirm the role of mTOR complexes in I/R injury, we generated cardiac-specific mTOR-knockout (CKO) mice. In contrast to the effects of Torin1, CKO hearts recovered better after I/R injury than control hearts (p < 0.05). Interestingly, the CKO hearts had exhibited irregular contractions during the reperfusion phase. Calcium is a major factor in Excitation-Contraction (EC) coupling via Sarcoplasmic Reticulum (SR) calcium release. Calcium is also key in opening the mitochondrial permeability transition pore (mPTP) and cell death following I/R injury. Caffeine-induced SR calcium release in isolated CMs showed that total SR calcium content was lower in CKO than in control CMs. Western blotting showed that a significant amount of mTOR localizes to the SR/mitochondria and that GSK3-β phosphorylation, a key factor in SR calcium mobilization, was decreased. These findings suggest that cardiac mTOR located to the SR/mitochondria plays a vital role in EC coupling and cell survival in I/R injury., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Cells have evolved highly intertwined kinase networks to finely tune cellular homeostasis to the environment. The network converging on the mechanistic target of rapamycin (MTOR) kinase constitutes a central hub that integrates metabolic signals and adapts cellular metabolism and functions to nutritional changes and stress. Feedforward and feedback loops, crosstalks and a plethora of modulators finely balance MTOR-driven anabolic and catabolic processes. This complexity renders it difficult - if not impossible - to intuitively decipher signaling dynamics and network topology. Over the last two decades, systems approaches have emerged as powerful tools to simulate signaling network dynamics and responses. In this review, we discuss the contribution of systems studies to the discovery of novel edges and modulators in the MTOR network in healthy cells and in disease., (© 2021 The Author(s).)

A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982)., (© 2021 by The American Society of Hematology.)

Purpose: There is not much progress in the treatment for lung squamous cell carcinoma LSCC in the past few years. Rapamycin Rapa, an inhibitor of mammalian target of rapamycin mTOR, has exhibited antitumor efficacy in a variety of malignant tumors. It has recently been reported that Rapamycin can induce autophagy signaling pathway in lung cancer and Glypican-3GPC3 can promote the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling pathway. The aim of this study is to investigate the mechanisms of rapamycin's antitumor efficacy in relation to GPC3/Wnt/β-catenin pathway and autophagy in LSCC., Methods: SK-MES-1 cells, a LSCC cell line, were treated with various concentrations of rapamycin with or without Glypican-3 GPC3-targeting siRNA. SK-MES-1 cell proliferation was determined by MTT assay. Protein expression levels of GPC3, β-catenin, Beclin-1 were checked via western blotting. We established the xenograft mice model to investigate the suppression effect of rapamycin on LSCC. In addition, we further testified the metabolism protein of autophagy process using the xenograft tumor tissue., Results: Rapamycin could inhibit the SK-MES-1 cell proliferation in a concentration-dependent manner both in vitro and in vivo by decreasing the GPC3 expression and downregulating the glypican-3/Wnt/β-catenin signaling pathway. In addition, we found that GPC3 silencing can activate the glypican-3/Wnt/β-catenin pathway and autophagy, which contribute to the suppression of tumor growth both in vitro and in vivo., Conclusion: Rapamycin suppresses the growth of lung cancer through down-regulating glypican-3/Wnt/β-catenin signaling, which mediates with activation of autophagy. This study suggests GPC3 is a new promising target for rapamycin in the treatment of lung cancer.

Background: Bronchopulmonary dysplasia remains one of the most common complications of prematurity, despite significant improvements in perinatal care. Functional modeling of human lung development and disease, like BPD, is limited by our ability to access the lung and to maintain relevant progenitor cell populations in culture., Methods: We supplemented Rho/SMAD signaling inhibition with mTOR inhibition to generate epithelial basal cell-like cell lines from tracheal aspirates of neonates., Results: Single-cell RNA-sequencing confirmed the presence of epithelial cells in tracheal aspirates obtained from intubated neonates. Using Rho/SMAD/mTOR triple signaling inhibition, neonatal tracheal aspirate-derived (nTAD) basal cell-like cells can be expanded long term and retain the ability to differentiate into pseudostratified airway epithelium., Conclusions: Our data demonstrate that neonatal tracheal aspirate-derived epithelial cells can provide a novel ex vivo human cellular model to study neonatal lung development and disease., Impact: Airway epithelial basal cell-like cell lines were derived from human neonatal tracheal aspirates. mTOR inhibition significantly extends in vitro proliferation of neonatal tracheal aspirate-derived basal cell-like cells (nTAD BCCs). nTAD BCCs can be differentiated into functional airway epithelium. nTAD BCCs provide a novel model to investigate perinatal lung development and diseases.

The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5'ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Primary liver cancers, including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), are highly lethal tumors, with high worldwide frequency and few effective treatment options. The mammalian target of rapamycin (mTOR) complex is a central regulator of cell growth and metabolism that integrates inputs from amino acids, nutrients, and extracellular signals. The mTOR protein is incorporated into two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Specifically, mTORC1 regulates protein synthesis, glucose and lipid metabolism, and autophagy, whereas mTORC2 promotes liver tumorigenesis through modulating the adenine/cytosine/guanine family of serine/threonine kinases, especially the protein kinase B proteins. In human HCC and iCCA samples, genomics analyses have revealed the frequent deregulation of the mTOR complexes. Both in vitro and in vivo studies have demonstrated the key role of mTORC1 and mTORC2 in liver-tumor development and progression. The first-generation mTOR inhibitors have been evaluated for effectiveness in liver-tumor treatment and have provided unsatisfactory results. Current research efforts are devoted to generating more efficacious mTOR inhibitors and identifying biomarkers for patient selection as well as for combination therapies. Here, we provide a comprehensive review of the mechanisms leading to a deregulated mTOR signaling cascade in liver cancers, the mechanisms whereby the mTOR pathway contributes to HCC and iCCA molecular pathogenesis, the therapeutic strategies, and the challenges to effectively inhibit mTOR in liver-cancer treatment. Conclusion: Deregulated mTOR signaling significantly contributes to HCC and iCCA molecular pathogenesis. mTOR inhibitors, presumably administered in association with other drugs, might be effective against subsets of human liver tumors., (© 2020 by the American Association for the Study of Liver Diseases.)

Leukemias are routinely sub-typed for risk/outcome prediction and therapy choice using acquired mutations and chromosomal rearrangements. Down syndrome acute lymphoblastic leukemia (DS-ALL) is characterized by high frequency of CRLF2-rearrangements, JAK2-mutations, or RAS-pathway mutations. Intriguingly, JAK2 and RAS-mutations are mutually exclusive in leukemic sub-clones, causing dichotomy in therapeutic target choices. We prove in a cell model that elevated CRLF2 in combination with constitutionally active JAK2 is sufficient to activate wtRAS. On primary clinical DS-ALL samples, we show that wtRAS-activation is an obligatory consequence of mutated/hyperphosphorylated JAK2. We further prove that CRLF2-ligand TSLP boosts the direct binding of active PTPN11 to wtRAS, providing the molecular mechanism for the wtRAS activation. Pre-inhibition of RAS or PTPN11, but not of PI3K or JAK-signaling, prevented TSLP-induced RAS-GTP boost. Cytotoxicity assays on primary clinical DS-ALL samples demonstrated that, regardless of mutation status, high-risk leukemic cells could only be killed using RAS-inhibitor or PTPN11-inhibitor, but not PI3K/JAK-inhibitors, suggesting a unified treatment target for up to 80% of DS-ALL. Importantly, protein activities-based principal-component-analysis multivariate clusters analyzed for independent outcome prediction using Cox proportional-hazards model showed that protein-activity (but not mutation-status) was independently predictive of outcome, demanding a paradigm-shift in patient-stratification strategy for precision therapy in high-risk ALL.

This study was designed to investigate the effect of naringin in oxygen-glucose deprivation/reoxygenation (OGD/R) model and its mechanism. The target gene of naringin and the enriched pathways of the gene were searched and identified using bioinformatics analysis. Then OGD/R model was built using PC12 cells, after which the cells were treated with different concentrations of naringin. Subsequently, cell proliferation and apoptosis were evaluated by cell counting kit-8 (CCK-8) and flow cytometry assays, respectively. Meanwhile, the expression of NFKB1 in PC12 cells underwent OGD/R-induced injury was detected by qRT-PCR, while apoptosis-related and pathway-related proteins were checked by Western blot. DCF-DA kit was utilized to measure the level of ROS. Our results revealed that NFKB1, which was upregulated in MACO rats and OGD/R-treated PC12 cells, was a target gene of naringin. Naringin could alleviate OGD/R-induced injury via promoting the proliferation, and repressing the apoptosis of PC12 cells through regulating the expression of NFKB1 and apoptosis-associated proteins and ROS level. Besides, the depletion of NFKB1 was positive to cell proliferation but negative to cell apoptosis. Moreover, the depletion of NFKB1 enhanced the influences of naringin on cell proliferation and apoptosis as well as the expression of apoptosis-related proteins and ROS level. Western blotting indicated that both naringin treatment and depletion of NFKB1 could increase the expression of HIF-1α, p-AKT, and p-mTOR compared with OGD/R group. What's more, treatment by naringin and si-NFKB1 together could significantly increase these effects. Nevertheless, the expression of AKT and mTOR among each group was almost not changed. In conclusion, naringin could prevent the OGD/R-induced injury in PC12 cells in vitro by targeting NFKB1 and regulating HIF-1α/AKT/mTOR-signaling pathway, which might provide novel ideas for the therapy of cerebral ischemia-reperfusion (I/R) injury.

Intrahepatic cholangiocarcinoma (ICC) is a type of cancer that is difficult to cure; chemoresistance of cholangiocarcinoma cells affect the prognosis of patients who cannot be treated with surgery. The mechanism underlying this chemoresistance remains unknown. Mesenchymal stem cells (MSCs) are known to be important components of the tumor microenvironment. In the present study, a large number of MSCs were observed to infiltrate the tumor sites of ICC; thus, MSCs were isolated from ICC tumor tissues. It was revealed that herpesvirus entry mediator (HVEM) was overexpressed in ICC‑MSCs. The present study then investigated the role of HVEM‑overexpressing MSCs in the chemoresistance of cholangiocarcinoma cells. It was demonstrated that HVEM‑overexpressing MSCs could support cell survival of chemotherapeutic cholangiocarcinoma cells and inhibited their apoptosis. Further investigations revealed that HVEM‑overexpressing MSCs could secrete IL‑6 and also activated AMPK/mTOR‑dependent autophagy of cholangiocarcinoma cells. Thus, it was concluded that ICC‑MSC‑induced autophagy is the primary cause of chemoresistance in ICC.

Cellular senescence, cancer and aging are highly interconnected. Among many important molecular machines that lie at the intersection of this triad, the mechanistic (formerly mammalian) target of rapamycin (mTOR) is a central regulator of cell metabolism, proliferation, and survival. The mTOR signaling cascade is essential to maintain cellular homeostasis in normal biological processes or in response to stress, and its dysregulation is implicated in the progression of many disorders, including age-associated diseases. Accordingly, the pharmacological implications of mTOR inhibition using rapamycin or others rapalogs span the treatment of various human diseases from immune disorders to cancer. Importantly, rapamycin is one of the only known pan-species drugs that can extend lifespan. The molecular and cellular mechanisms explaining the phenotypic consequences of mTOR are vast and heavily studied. In this review, we will focus on the potential role of mTOR in the context of cellular senescence, a tumor suppressor mechanism and a pillar of aging. We will explore the link between senescence, autophagy and mTOR and discuss the opportunities to exploit senescence-associated mTOR functions to manipulate senescence phenotypes in age-associated diseases and cancer treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Impaired fear extinction, combined with the likelihood of fear relapse after exposure therapy, contributes to the persistence of many trauma-related disorders such as anxiety and post-traumatic stress disorder. Identifying mechanisms to aid fear extinction and reduce relapse could provide novel strategies for augmentation of exposure therapy. Exercise can enhance learning and memory and augment fear extinction of traumatic memories in humans and rodents. One factor that could contribute to enhanced fear extinction following exercise is the mammalian target of rapamycin (mTOR). mTOR is a translation regulator involved in synaptic plasticity and is sensitive to many exercise signals such as monoamines, growth factors, and cellular metabolism. Further, mTOR signaling is increased after chronic exercise in brain regions involved in learning and emotional behavior. Therefore, mTOR is a compelling potential facilitator of the memory-enhancing and overall beneficial effects of exercise on mental health.The goal of the current study is to test the hypothesis that mTOR signaling is necessary for the enhancement of fear extinction produced by acute, voluntary exercise. We observed that intracerebral-ventricular administration of the mTOR inhibitor rapamycin reduced immunoreactivity of phosphorylated S6, a downstream target of mTOR, in brain regions involved in fear extinction and eliminated the enhancement of fear extinction memory produced by acute exercise, without reducing voluntary exercise behavior or altering fear extinction in sedentary rats. These results suggest that mTOR signaling contributes to exercise-augmentation of fear extinction., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A class of proteins that were discovered to bind the immunosuppressant drug FK506, called FK506-binding proteins (FKBPs), are members of a sub-family of immunophilins. Although they were first identified in human, FKBPs exist in all three domains of life. In this report, a rice FKBP12 homolog was first identified as a biotic stress-related gene through suppression subtractive hybridization screening. By ectopically expressing OsFKBP12 in the heterologous model plant system, Arabidopsis thaliana , for functional characterization, OsFKBP12 was found to increase susceptibility of the plant to the pathogen, Pseudomonas syringae pv. tomato DC3000 ( Pst DC3000). This negative regulatory role of FKBP12 in biotic stress responses was also demonstrated in the AtFKBP12- knockout mutant, which exhibited higher resistance towards Pst DC3000. Furthermore, this higher-plant FKBP12 homolog was also shown to be a negative regulator of salt tolerance. Using yeast two-hybrid tests, an ancient unconventional G-protein, OsYchF1, was identified as an interacting partner of OsFKBP12. OsYchF1 was previously reported as a negative regulator of both biotic and abiotic stresses. Therefore, OsFKBP12 probably also plays negative regulatory roles at the convergence of biotic and abiotic stress response pathways in higher plants.

The target of rapamycin (TOR) is an evolutionarily-conserved serine/threonine kinase that senses and integrates signals from the environment to coordinate developmental and metabolic processes. TOR senses nutrients, hormones, metabolites, and stress signals to promote cell and organ growth when conditions are favorable. However, TOR is inhibited when conditions are unfavorable, promoting catabolic processes such as autophagy. Autophagy is a macromolecular degradation pathway by which cells degrade and recycle cytoplasmic materials. TOR negatively regulates autophagy through phosphorylation of ATG13, preventing activation of the autophagy-initiating ATG1-ATG13 kinase complex. Here we review TOR complex composition and function in photosynthetic and non-photosynthetic organisms. We also review recent developments in the identification of upstream TOR activators and downstream effectors of TOR. Finally, we discuss recent developments in our understanding of the regulation of autophagy by TOR in photosynthetic organisms.

Pancreatic cancer is one of the most lethal solid malignancies, with a poor prognosis and a high mortality rate. Pancreatic cancer cells exhibit enhanced glycolysis to maintain their rapid growth. Canagliflozin (CANA) is a sodium‑glucose co‑transporter 2 inhibitor used for the clinical treatment of diabetes. Recent studies have demonstrated the potential ability of CANA to suppress hepatocellular carcinoma, whereas its therapeutic effects on and mechanisms in pancreatic cancer have rarely been reported. In the present study, the antitumor effects of CANA on pancreatic cancer were investigated. The data obtained indicated that pancreatic cancer growth was effectively suppressed by CANA in a dose‑dependent manner, with peak inhibition rates of 54.3 and 57.6% in cultured Capan‑1 and PANC‑1 cells respectively. The tumor inhibitory rate reached 45.2% in nude mice with PANC‑1‑derived tumors, suggesting its effective antitumor activity against pancreatic cancer in vitro and/or in vivo. In addition, the combined treatment of Capan‑1 and PANC‑1 cells with gemcitabine and CANA exhibited a greater efficacy compared with that of treatment with gemcitabine alone. Moreover, glucose uptake and lactate production were decreased, and the mRNA levels of the glycolysis‑associated genes, including glucose transporter‑1 and lactate dehydrogenase A were decreased, indicating the inhibitory effects caused by the combination treatment on the metabolism of glucose in pancreatic cancer cells. Furthermore, CANA induced apoptosis, notably early apoptosis, and decreased the protein levels of PI3K, p‑AKT, p‑mTOR and HIF‑1α, which indicated that the PI3K/AKT/mTOR signaling pathway was involved in the glycolytic process. These results demonstrated that pancreatic cancer growth was effectively inhibited by CANA via the suppression of glycolysis. This was mediated primarily by the PI3K/AKT/mTOR signaling pathway, revealing the underlying role and potential of this pathway for the clinical treatment of pancreatic cancer. Novel applications for the existing drug CANA can be explored, which could reduce the cost and time required for drug development in the field of drug discovery.

Prostate cancer is the most commonly diagnosed type of cancer in North American men and is typically classified as either androgen receptor positive or negative depending on the expression of the androgen receptor (AR). AR positive prostate cancer can be treated with hormone therapy while AR negative prostate cancer is aggressive and does not respond to hormone therapy. It has been previously reported that rosemary extract (RE) has antioxidant, anti-inflammatory and anti-cancer properties. In the present study, we found that treatment of the androgen-insensitive PC-3 prostate cancer cells with RE resulted in a significant inhibition of proliferation, survival, migration, Akt, and mTOR signaling. In addition, treatment of the androgen-sensitive 22RV1 prostate cancer cells with RE resulted in a significant inhibition of proliferation and survival while RE had no effect on normal prostate epithelial PNT1A cells. These findings suggest that RE has potent effects against prostate cancer and warrants further investigation., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Rhizoma Paris is a popular Chinese medicine in clinics. It contains four main saponins which are its major bioactive compounds. These saponins are Paris saponin I, II, VI and VII (PSI, PSII, PSVI and PSVII, respectively). Up to now, the research using HUVEC cells to evaluate the anti-angiogenic activity of four saponins is blank. The purpose of this study was to evaluate the anti-angiogenic properties (also known as angiotoxicity) of the four saponins in Rhizoma Paris on vascular endothelial cells-HUVEC cells, and to investigate the underlying mechanism, which has not been studied before. In this study, MTT assay, Lactate dehydrogenase (LDH) assay, wound healing experiments, transwell cell invasion assay, tubule formation experiment, DAPI staining, AV-PI double staining, and cell cycle analysis were used to determine the effects of Paris saponins. The results showed that, with increases in concentrations of PSI, PSII, PSVI and PSVII, the viability of HUVEC cells decreased significantly. In addition, four saponins dose-dependent enhanced LDH release and inhibited HUVEC cell migration, invasion, and angiogenesis. In terms of mechanism, PSI significantly inhibited protein expression in multiple signaling pathways. In particular, with the VEGF2 as the target, it activate the downstream PI3K / AKT / mTOR, SRC / eNOS, P38, PLCγ / ERK / MERK and JAK2/STAT3 signaling pathways. In conclusion, PSI, PSII, PSVI and PSVII can inhibit endothelial cell proliferation, migration and invasion, block endothelial cell cycle, induce endothelial cell apoptosis, act on protein expression in several anti-angiogenic signaling pathways, and finally inhibit angiogenesis in vitro. This study provides further data support for the clinical application of Paris saponins as antiangiogenic drugs., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)