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The ubiquitin-conjugating enzyme (E2) is a critical component of the ubiquitin-proteasome system and regulates hepatocarcinogenesis by controlling protein degradation. Ubiquitin-conjugating enzyme E2 O (UBE2O), a member of the E2 family, functions as an oncogene in human cancers. Nevertheless, the role of UBE2O in hepatocellular carcinoma (HCC) remains unknown yet. Here, we demonstrated that the UBE2O level was markedly upregulated in HCC compared with adjacent noncancerous tissues. UBE2O overexpression was also confirmed in HCC cell lines. UBE2O overexpression was prominently associated with advanced tumor stage, high tumor grade, venous infiltration, and reduced HCC patients' survivals. UBE2O knockdown inhibited the migration, invasion, and proliferation of HCCLM3 cells. UBE2O overexpression enhanced the proliferation and mobility of Huh7 cells. Mechanistically, UBE2O mediated the ubiquitination and degradation of AMP-activated protein kinase α2 (AMPKα2) in HCC cells. UBE2O silencing prominently increased AMPKα2 level and reduced phosphorylated mechanistic target of rapamycin kinase (p-mTOR), MYC, Cyclin D1, HIF1α, and SREBP1 levels in HCCLM3 cells. UBE2O depletion markedly activated the AMPKα2/mTOR pathway in Huh7 cells. Moreover, AMPKα2 silencing reversed UBE2O downregulation-induced mTOR pathway inactivation. Rapamycin, an inhibitor of mTOR, remarkably abolished UBE2O-induced mTOR phosphorylation and HCC cell proliferation and mobility. To conclude, UBE2O was highly expressed in HCC and its overexpression conferred to the poor clinical outcomes of patients. UBE2O contributed to the malignant behaviors of HCC cells, including cell proliferation, migration, and invasion, by reducing AMPKα2 stability and activating the mTOR pathway.

Background Lymphatic malformations (LMs) are benign congenital malformations that stem from the abnormal development of the lymphatic vessels during early embryogenesis. Somatic PIK3CA gene mutations are conventional cause leading to LMs. Both macrocystic and microcystic LMs arise due to lymphatic endothelial cell-autonomous defects, depending on the time in development at which PIK3CA gene mutation occurs. Recent study finds a PIK3CA mutation in 79% of LMs. However, discovering new genetic events in this disease is crucial to identify the molecular mechanism of the pathogenesis and further develop new targeted therapies. Results Here, we initially performed whole-exome sequencing in six children with LMs to find a new causal gene. Somatic mutations in PIK3CA (c.1633G > A [p. E545K] and PIK3CD (c.1997T > C [p.L666P]) were discovered in two different individuals. In vitro functional studies were conducted to demonstrate the pathogenicity of the novel mutation c.1997T > C in PIK3CD. We found that L666P promoted the cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) and induced hyperactivation of the mTOR pathway. These findings indicate that the PIK3CD mutation affects downstream signalling in endothelial cells, which may impair normal lymphangiogenesis. Conclusions This study reveals a novel candidate gene associated with the development of LMs, which is consistent with previous researches. These findings in our study may offer a novel gene target for developing therapies, which acts in tight interaction with the previously known PIK3CA.

The traditional classification of lung cancer into small cell lung cancer and non-small cell lung cancer (NSCLC) has been transformed with the increased understanding of the molecular alterations and genomic biomarkers that drive the development of lung cancer. Increased activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway leads to numerous hallmarks of cancer and this pathway represents an attractive target for novel anticancer therapies. In NSCLC, the PI3K/Akt/mTOR pathway has been heavily implicated in both tumorigenesis and the progression of disease. A number of specific inhibitors of PI3K, Akt and mTOR are currently under development and in various stages of preclinical investigation and in early phase clinical trials for NSCLC. Early evidence has yielded disappointing results. Clinical trials, however, have been performed on predominantly molecularly unselected populations, and patient enrichment strategies using high-precision predictive biomarkers in future trials will increase the likelihood of success. A greater understanding of the underlying molecular biology including epigenetic alterations is also crucial to allow for the detection of appropriate biomarkers and guide combination approaches.

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E74-like E26 transformation-specific (ETS) transcription factor 3 (ELF3), is a member of the ETS transcription factor family, and has been characterized as an epithelial cell-specific transcription factor. The role of ELF3 in tumor progression remains to be elucidated. Previous studies have indicated that loss of ELF3 mRNA and protein expression was associated with poor outcomes in ovarian cancer (OC). By contrast, the present study demonstrated that ELF3 was upregulated in OC, using data from The Cancer Genome Atlas, and elevated expression levels of ELF3 were associated with a poor prognosis. ELF3 promoted OC cell proliferation in vitro and in vivo. The present study revealed that ELF3 inhibited apoptosis and reduced the cisplatin sensitivity of OC cells. Furthermore, the mTOR pathway was found to be activated by ELF3. Collectively, the results of the present study indicated the role of ELF3 in the development and pathogenesis of OC.

Adipocytes constitute a major component of the tumour microenvironment. Numerous studies have shown that adipocytes promote aggressiveness and invasion by stimulating cancer cells proliferation and modulating their metabolism. Herein, we reported that Notch3 promotes mouse 3T3‐L1 pre‐adipocytes differentiation by performing the integrative transcriptome and TMT‐based proteomic analyses. The results revealed that aminoacyl‐tRNA_biosynthesis pathway was significantly influenced with Nocth3 change during 3T3‐L1 pre‐adipocytes differentiation, and the expression of LARS in this pathway was positively correlated with Notch3. Published studies have shown that LARS is a sensor of leucine that regulates the mTOR pathway activity, and the latter involves in adipogenesis. We therefore supposed that Notch3 might promote 3T3‐L1 pre‐adipocytes differentiation by up‐regulating LARS expression and activating mTOR pathway. CHIP and luciferase activity assay uncovered that Notch3 could transcriptionally regulate the expression of LARS gene. Oil Red staining identified a positive correlation between Notch3 expression and adipocytic differentiation. The activation of mTOR pathway caused by Notch3 overexpression could be attenuated by knocking down LARS expression. Altogether, our study revealed that Notch3 promotes adipocytic differentiation of 3T3‐L1 pre‐adipocytes cells by up‐regulating LARS expression and activating the mTOR pathway, which might be an emerging target for obesity treatment.

Ex vivo limbal stem cell transplantation is the main therapeutic approach to address a complete and functional re‐epithelialization in corneal blindness, the second most common eye disorder. Although important key points were defined, the molecular mechanisms involved in the epithelial phenotype determination are unclear. Our previous studies have demonstrated the pluripotency and immune‐modulatory of fibroblast limbal stem cells (f‐LSCs), isolated from the corneal limbus. We defined a proteomic profile especially enriched in wound healing and cytoskeleton‐remodelling proteins, including Profilin‐1 (PFN1). In this study we postulate that pfn‐1 knock down promotes epithelial lineage by inhibiting the integrin‐β1(CD29)/mTOR pathway and subsequent NANOG down‐expression. We showed that it is possible modulate pfn1 expression levels by treating f‐LSCs with Resveratrol (RSV), a natural compound: pfn1 decline is accompanied with up‐regulation of the specific differentiation epithelial genes pax6 (paired‐box 6), sox17 (sex determining region Y‐box 17) and ΔNp63‐α (p63 splice variant), consistent with drop‐down of the principle stem gene levels. These results contribute to understand the molecular biology of corneal epithelium development and suggest that pfn1 is a potential molecular target for the treatment of corneal blindness based on epithelial cell dysfunction.

Background: Abnormally activated mechanistic target of rapamycin (mTOR) pathway has been reported in several model animals with inherited metabolic myopathies (IMMs). However, the profiles of mTOR pathway in skeletal muscles from patients are still unknown. This study aimed to analyze the activity of mTOR pathway in IMMs muscles. Methods: We collected muscle samples from 25 patients with mitochondrial myopathy (MM), lipid storage disease (LSD) or Pompe disease (PD). To evaluate the activity of mTOR pathway in muscle specimens, phosphorylation of S6 ribosomal protein (p-S6) and p70S6 kinase (p-p70S6K) were analyzed by Western blotting and immunohistochemistry. Results: Western blotting results showed that p-p70S6K/p70S6K in muscles from LSD and MM was up-regulated when compared with normal controls (NC) (NC vs. LSD, U= 2.000, P= 0.024; NC vs. MM: U= 6.000, P= 0.043). Likewise, p-S6/S6 was also upregulated in muscles from all three subgroups of IMMs (NC vs. LSD, U= 0.000, P= 0.006; NC vs. PD, U= 0.000, P= 0.006; NC vs. MM, U= 1.000, P = 0.007). Immunohistochemical study revealed that p-S6 was mainly expressed in fibers with metabolic defect. In MM muscles, most p-S6 positive fibers showed cytochrome C oxidase (COX) deficiency (U= 5.000, P= 0.001). In LSD and PD muscles, p-S6 was mainly overexpressed in fibers with intramuscular vacuoles containing lipid droplets (U= 0.000, P = 0.002) or basophilic materials (U= 0.000, P= 0.002). Conclusion: The mTOR pathway might be activated in myofibers with various metabolic defects, which might provide evidence for mTOR inhibition therapy in human IMMs. Key words: mTOR pathway; Mitochondrial myopathy; Lipid storage disease; Pompe disease

Macroautophagy/autophagy dysfunction is associated with many neurodegenerative diseases. TFEB (transcription factor EB), an important molecule that regulates lysosomal and autophagy function, is regarded as a potential target for treating some neurodegenerative diseases. However, the relationship between autophagy dysfunction and spiral ganglion neuron (SGN) degeneration and the role of TFEB in SGN degeneration has not yet been established. Here, we showed that in degenerated SGNs, induced by sensory epithelial cell loss in the cochlea of mice following kanamycin and furosemide administration, the lipofuscin area and oxidative stress level were increased, the nuclear-to-cytoplasmic TFEB ratio was decreased, and the late stage of autophagic flux was impaired. After autophagy dysfunction was partially ameliorated with an MTOR inhibitor, which promoted TFEB translocation into the nucleus from the cytoplasm, we found that the lysosomal deficits were significantly relieved, the oxidative stress level was reduced, and the density of surviving SGNs and auditory nerve fibers was increased. The results in the present study reveal that autophagy dysfunction is an important component of SGN degeneration, and TFEB may be a potential target for attenuating SGN degeneration following sensory epithelial cell loss in the cochlea of mice. Abbreviations: 3-NT: 3-nitrotyrosine; 4-HNE: 4-hydroxynonenal; 8-OHdG: 8-hydroxy-2ʹ-deoxyguanosine; ABR: auditory brainstem response; APP: amyloid beta (A4) precursor protein; CLEAR: coordinated lysosomal expression and regulation; CTSB: cathespin B; CTSD: cathespin D; SAMR1: senescence-accelerated mouse/resistance 1; SAMP8: senescence-accelerated mouse/prone 8; MAPK1/ERK2: mitogen-activated protein kinase 1; MTOR: mechanistic target of rapamycin kinase; SGN: spiral ganglion neuron; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscope; TFEB: transcription factor EB

Made available in DSpace on 2019-10-06T16:06:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-02-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The mTOR/4E-BP1/eIF4E pathway plays important roles in the neoplastic transformation process and in tumour growth. In men, the mTOR/4E-BP1/eIF4E pathway was described as altered in different tumours, including prostate cancer (PC). Apart from humans, the dog is the only species that develops PC with high frequency and is considered a good model for comparative oncology initiatives. Due to limited information on this pathway in canine tumours, this study aimed to investigate mTOR, 4E-BP1 and eIF4E gene and protein expression in canine PC, as well as in metastatic and normal prostatic tissues, and to evaluate the correlations between gene/protein expression and Gleason score (GS) in PC. A total of 35 formalin-fixed paraffin-embedded (FFPE) samples, including 13 of normal prostatic tissue, 17 PC samples and 5 metastasis samples, were evaluated by immunohistochemistry and qPCR. mTOR gene mutation in the kinase domain was also investigated. We identified higher p-mTOR and eIF4E protein levels in canine PC with higher GS values (≥ 8) and a significant positive correlation in expression between these proteins. eIF4E overexpression was observed in metastasis relative to expression in normal samples. Our data suggest that p-mTOR and eIF4E expression is positively correlated with GS in canine PC, similar to the pattern in humans. More studies of the mTOR/4EBP1/eIF4E pathway should be performed to identify possible correlations of the proteins involved with clinical and pathologic findings in canine PC and the roles of these proteins as therapeutic targets for the treatment of canine PC. Department of Veterinary Pathology School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) Department of Veterinary Pathology School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) Department of Veterinary Clinic School of Veterinary Medicine and Animal Science São Paulo State University (Unesp) CNPq: 443884/2014-5

Objectives Focal cortical dysplasia (FCD) is a major cause of drug‐resistant focal epilepsy in children, and the clinicopathological classification remains a challenging issue in daily practice. With the recent progress in DNA methylation–based classification of human brain tumors we examined whether genomic DNA methylation and gene expression analysis can be used to also distinguish human FCD subtypes. Methods DNA methylomes and transcriptomes were generated from massive parallel sequencing in 15 surgical FCD specimens, matched with 5 epilepsy and 6 nonepilepsy controls. Results Differential hierarchical cluster analysis of DNA methylation distinguished major FCD subtypes (ie, Ia, IIa, and IIb) from patients with temporal lobe epilepsy patients and nonepileptic controls. Targeted panel sequencing identified a novel likely pathogenic variant in DEPDC5 in a patient with FCD type IIa. However, no enrichment of differential DNA methylation or gene expression was observed in mechanistic target of rapamycin (mTOR) pathway–related genes. Significance Our studies extend the evidence for disease‐specific methylation signatures toward focal epilepsies in favor of an integrated clinicopathologic and molecular classification system of FCD subtypes incorporating genomic methylation.

With the advent of the aging society, how to grow old healthily has become an important issue for the whole of society. Effective intervention strategies for healthy aging are most desired, due to the complexity and diversity of genetic information, it is a pressing concern to find a single drug or treatment to improve longevity. In this study, long-term administration of triterpenoids of Ganoderma lucidum (TGL) can mitigate brain physiological decline in normal aging mice. In addition, the age-associated pathological features, including cataract formation, hair loss, and skin relaxation, brown adipose tissue accumulation, the β-galactosidase staining degree of kidney, the iron death of spleen, and liver functions exhibit improvement. We used the APP/PS1 mice and 3 × Tg-AD mice model of Alzheimer’s Disease (AD) to further verify the improvement of brain function by TGL and found that Ganoderic acid A might be the effective constituent of TGL for anti-aging of the brain in the 3 × Tg-AD mice. A potential mechanism of action may involve the regulation of sphingolipid metabolism, prolonging of telomere length, and enhance autophagy, which allows for the removal of pathological metabolites.

Objectives Cutaneous wound healing is one of the major medical problems worldwide. Epigenetic modifiers have been identified as important players in skin development, homeostasis and wound repair. SET domain–containing 2 (SETD2) is the only known histone H3K36 tri‐methylase; however, its role in skin wound healing remains unclear. Materials and Methods To elucidate the biological role of SETD2 in wound healing, conditional gene targeting was used to generate epidermis‐specific Setd2‐deficient mice. Wound‐healing experiments were performed on the backs of mice, and injured skin tissues were collected and analysed by haematoxylin and eosin (H&E) and immunohistochemical staining. In vitro, CCK8 and scratch wound‐healing assays were performed on Setd2‐knockdown and Setd2‐overexpression human immortalized keratinocyte cell line (HaCaT). In addition, RNA‐seq and H3K36me3 ChIP‐seq analyses were performed to identify the dysregulated genes modulated by SETD2. Finally, the results were validated in functional rescue experiments using AKT and mTOR inhibitors (MK2206 and rapamycin). Results Epidermis‐specific Setd2‐deficient mice were successfully established, and SETD2 deficiency resulted in accelerated re‐epithelialization during cutaneous wound healing by promoting keratinocyte proliferation and migration. Furthermore, the loss of SETD2 enhanced the scratch closure and proliferation of keratinocytes in vitro. Mechanistically, the deletion of Setd2 resulted in the activation of AKT/mTOR signalling pathway, while the pharmacological inhibition of AKT and mTOR with MK2206 and rapamycin, respectively, delayed wound closure. Conclusions Our results showed that SETD2 loss promoted cutaneous wound healing via the activation of AKT/mTOR signalling., The epidermis‐specific Setd2‐deficient mice showed accelerated re‐epithelialization during cutaneous wound healing by promoting keratinocytes proliferation and migration. Mechanistically, deletion of Setd2 resulted in the activation the AKT/mTOR signalling pathway and pharmacological inhibitions of AKT and mTOR with MK2206 and rapamycin delayed wound closure, respectively.

Stressful conditions induce the cell to save energy and activate a rescue program modulated by mammalian target of rapamycin (mTOR). Along with transcriptional and translational regulation, the cell relies also on post-transcriptional modulation to quickly adapt the translation of essential proteins. MicroRNAs play an important role in the regulation of protein translation, and their availability is tightly regulated by RNA competing mechanisms often mediated by long noncoding RNAs (lncRNAs). In our paper, we simulated the response to growth adverse condition by bimiralisib, a dual PI3K/mTOR inhibitor, in diffuse large B cell lymphoma cell lines, and we studied post-transcriptional regulation by the differential analysis of exonic and intronic RNA expression. In particular, we observed the upregulation of a lncRNA, lncTNK2-2:1, which correlated with the stabilization of transcripts involved in the regulation of translation and DNA damage after bimiralisib treatment. We identified miR-21-3p as miRNA likely sponged by lncTNK2-2:1, with consequent stabilization of the mRNA of p53, which is a master regulator of cell growth in response to DNA damage.

Simple Summary Our understanding of renal tumors has increased in the last years with the description of several novel entities. The expanding morphological spectrum complicates the pathologist’s diagnosis, often requiring immunohistochemical analysis. The role of cathepsin K immunoexpression is widened as a diagnostic tool in several renal tumors. This review describes the usefulness of cathepsin K in the differential diagnosis of renal neoplasms, highlighting the biological knowledge underpinning its expression. Moreover, cathepsin K seems to be a downstream marker of different genetic alterations, with a possible role as a predictive marker that may prospectively guide the development of therapeutic approaches as a molecular target. Abstract Cathepsin K is a papain-like cysteine protease with high matrix-degrading activity. Among several cathepsins, cathepsin K is the most potent mammalian collagenase, mainly expressed by osteoclasts. This review summarizes most of the recent findings of cathepsin K expression, highlighting its role in renal tumors for diagnostic purposes and as a potential molecular target. Indeed, cathepsin K is a recognized diagnostic tool for the identification of TFE3/TFEB-rearranged renal cell carcinoma, TFEB-amplified renal cell carcinoma, and pure epithelioid PEComa/epithelioid angiomyolipoma. More recently, its expression has been observed in a subgroup of eosinophilic renal neoplasms molecularly characterized by TSC/mTOR gene mutations. Interestingly, both TSC mutations or TFE3 rearrangement have been reported in pure epithelioid PEComa/epithelioid angiomyolipoma. Therefore, cathepsin K seems to be a downstream marker of TFE3/TFEB rearrangement, TFEB amplification, and mTOR pathway activation. Given the established role of mTOR inhibitors as a pharmacological option in renal cancers, cathepsin K could be of use as a predictive marker of therapy response and as a potential target. In the future, uropathologists may implement the use of cathepsin K to establish a diagnosis among renal tumors with clear cells, papillary architecture, and oncocytic features.

Introduction: Focal cortical dysplasias (FCDs) are a group of malformations of cortical development that constitute a common cause of drug-resistant epilepsy, often subjected to neurosurgery, with a suboptimal long-term outcome. The past few years have witnessed a dramatic leap in our understanding of the molecular basis of FCD. This study aimed to provide an updated review on the genomic and epigenetic advances underlying FCD etiology, to understand a genotype–phenotype correlation and identify priorities to lead future translational research.Methods: A scoping review of the literature was conducted, according to previously described methods. A comprehensive search strategy was applied in PubMed, Embase, and Web of Science from inception to 07 May 2020. References were screened based on title and abstract, and posteriorly full-text articles were assessed for inclusion according to eligibility criteria. Studies with novel gene variants or epigenetic regulatory mechanisms in patients that underwent epilepsy surgery, with histopathological diagnosis of FCD type I or II according to Palmini's or the ILAE classification system, were included. Data were extracted and summarized for an overview of evidence.Results: Of 1,156 candidate papers, 39 met the study criteria and were included in this review. The advent of next-generation sequencing enabled the detection in resected FCD tissue of low-level brain somatic mutations that occurred during embryonic corticogenesis. The mammalian target of rapamycin (mTOR) signaling pathway, involved in neuronal growth and migration, is the key player in the pathogenesis of FCD II. Somatic gain-of-function variants in MTOR and its activators as well as germline, somatic, and second-hit mosaic loss-of-function variants in its related repressors have been reported. However, the genetic background of FCD type I remains elusive, with a pleomorphic repertoire of genes affected. DNA methylation and microRNAs were the two epigenetic mechanisms that proved to have a functional role in FCD and may represent molecular biomarkers.Conclusion: Further research into the possible pathogenic causes of both FCD subtypes is required, incorporating single-cell DNA/RNA sequencing as well as methylome and proteomic analysis. The collected data call for an integrated clinicopathologic and molecular genetic diagnosis in current practice not only to improve diagnostic accuracy but also to guide the development of future targeted treatments.

Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, a high mortality disease. The development of such disease depends on the interaction of fungal cells with macrophages, in which they can reside and replicate. In order to dissect the molecular mechanisms by which cryptococcal cells modulate the activity of macrophages, a genome-scale comparative analysis of transcriptional changes in macrophages exposed to Cryptococcus spp. was conducted. Altered expression of nearly 40 genes was detected in macrophages exposed to cryptococcal cells. The major processes were associated with the mTOR pathway, whose associated genes exhibited decreased expression in macrophages incubated with cryptococcal cells. Phosphorylation of p70S6K and GSK-3β was also decreased in macrophages incubated with fungal cells. In this way, Cryptococci presence could drive the modulation of mTOR pathway in macrophages possibly to increase the survival of the pathogen.

Background Hyperactivation of mechanistic target of rapamycin (mTOR) signaling pathway is involved in the regulation of cellular growth, proliferation, and more in general, is a common phenomenon in most types of cancers. Thus, natural substances targeting this pathway can be of great therapeutic potential in supporting the treatment of tumor patients. Rhus tripartita (Ucria) Grande is a plant growing in desertic areas which is traditionally used for the treatment of several diseases in Tunisia. In the present work, the biochemical profile of the main compounds present in the plant leaf extract was determined and the anti-leukemic potential of the plant extracts against acute monocytic leukaemia (AML) THP-1 cells was investigated. Methods After HPLC identification of some phenolic compounds present in the plant extract and the quantification of saponin content, the cytotoxic effect of Rhus tripartita extracts on THP-1 cell culture was evaluated using the colorimetric MTT assay for cell viability. THP-1 cells were incubated with medium containing the relative IC50 concentrations of total plant extract, saponin extract and some standard compounds (rutin (R); kaempferol (K); mixture of catechin, epicatechin, and epicatechin-gallate (CEEG); ellagic acid (EA). Finally, qRT-PCR and western blotting analysis were used to evaluate the effect of some flavonoids present in a crude extract of polyphenols and the total extract of saponins on cell survival and apoptosis. Results Analysis of expression level of some gene (PIK3CA, PTEN, AKT1, mTOR, EIF4E, RPS6KB1, and TSC1) involved in the mTOR pathway and the phosphorylation of S6 and AKT proteins allowed to observe that a total Rhus tripartita extract and some of the compounds found in the extract controls THP-1 cell proliferation and apoptosis via regulation of the PI3K-Akt-mTOR signaling pathway. Conclusion Rhus tripartita-induced inhibition of cell cycle and induction of apoptosis may involve the mTOR pathway. Therefore, Rhus tripartita extract may be a useful candidate as a natural anti-cancer drug to support the treatment of AML.

Background Glioblastoma multiforme (GBM) is one of the most common malignant brain tumors and its average survival time is less than 1 year after diagnosis. Results Firstly, this study aims to develop the novel survival analysis algorithms to explore the key genes and proteins related to GBM. Then, we explore the significant correlation between AEBP1 upregulation and increased EGFR expression in primary glioma, and employ a glioma cell line LN229 to identify relevant proteins and molecular pathways through protein network analysis. Finally, we identify that AEBP1 exerts its tumor-promoting effects by mainly activating mTOR pathway in Glioma. Conclusions We summarize the whole process of the experiment and discuss how to expand our experiment in the future.

International audience; A major challenge in medical research resides in controlling the molecular processes of tissue regeneration, as organ and structure damage are central to several human diseases. A survey of the literature reveals that mTOR (mechanistic/mammalian target of rapamycin) is involved in a wide range of regeneration mechanisms in the animal kingdom. More particularly, cellular processes such as growth, proliferation, and differentiation are controlled by mTOR. In addition, autophagy, stem cell maintenance or the newly described intermediate quiescence state, G alert , imply upstream monitoring by the mTOR pathway. In this review, we report the role of mTOR signaling in reparative regenerations in different tissues and body parts (e.g., axon, skeletal muscle, liver, epithelia, appendages, kidney, and whole-body), and highlight how the mTOR kinase can be viewed as a therapeutic target to boost organ repair. Studies in this area have focused on modulating the mTOR pathway in various animal models to elucidate its contribution to regeneration. The diversity of metazoan species used to identify the implication of this pathway might then serve applied medicine (in better understanding what is required for efficient treatments in human diseases) but also evolutionary biology. Indeed, species-specific differences in mTOR modulation can contain the keys to appreciate why certain regeneration processes have been lost or conserved in the animal kingdom.

International audience; DEP-domain containing 5 (DEPDC5) is part of the GATOR1 complex that functions as key inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1). Loss-of-function mutations in DEPDC5 leading to mTOR hyperactivation have been identified as the most common cause of either lesional or non-lesional focal epilepsy. However, the precise mechanisms by which DEPDC5 loss-of-function triggers neuronal and network hyperexcitability are still unclear. In this study, we investigated the cellular mechanisms of hyperexcitability by comparing the constitutive heterozygous Depdc5 knockout mouse versus different levels of acute Depdc5 deletion (≈40% and ≈80% neuronal knockdown of Depdc5 protein) by RNA interference in primary cortical cultures. While heterozygous Depdc5+/- neurons have only a subtle phenotype, acutely knocked-down neurons exhibit a strong dose-dependent phenotype characterized by mTOR hyperactivation, increased soma size, dendritic arborization, excitatory synaptic transmission and intrinsic excitability. The robust synaptic phenotype resulting from the acute knockdown Depdc5 deficiency highlights the importance of the temporal dynamics of Depdc5 knockdown in triggering the phenotypic changes, reminiscent of the somatic second-hit mechanism in patients with focal cortical dysplasia. These findings uncover a novel synaptic phenotype that is causally linked to Depdc5 knockdown, highlighting the developmental role of Depdc5. Interestingly, the synaptic defect appears to affect only excitatory synapses, while inhibitory synapses develop normally. The increased frequency and amplitude of mEPSCs, paralleled by increased density of excitatory synapses and expression of glutamate receptors, may generate an excitation/inhibition imbalance that triggers epileptogenesis.

Introduction This study assessed whether mesenchymal stem cell (MSC)-derived extracellular vesicles influenced ageing and pluripotency markers in cell cultures where they are added. Methods MSC-derived extracellular vesicles from old and young rat bone marrows were isolated by ultracentrifugation and were characterised by western blotting, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). They were added to young and old MSC cultures. Real-time quantitative reverse transcription polymerase chain reactions and western blot analysis were performed to check the markers of ageing (vinculin and lamin A), pluripotency markers (Nanog and Oct4) and components of the mTOR signalling pathway (Rictor, Raptor, AKT and mTOR) in these cell populations. Subsequently, microRNA (miR)-188-3p expression was transiently inhibited in young MSCs to demonstrate the influence of mTOR2 on MSC ageing. Results Incubation with young MSC-derived extracellular vesicles decreased the levels of ageing markers and components of the mTOR pathway and increased the pluripotency markers from old MSC populations. By contrast, incubation of young MSCs with old MSC-derived extracellular vesicles generated the reverse effects. Inhibition of miR-188-3p expression in young MSCs produced extracellular vesicles that when incubated with old MSCs produced an increase in the levels of Rictor, as well as a decrease of phosphor-AKT, as indicated by a significant decrease in beta-galactosidase staining. Conclusions MSC-derived extracellular vesicles affected the behaviour of MSC cultures, based on their composition, which could be modified in vitro. These experiments represented the basis for the development of new therapies against ageing-associated diseases using MSC-derived extracellular vesicles.

Summary The present study was designed to examine the potential cellular antiseizure mechanisms of everolimus, a mechanistic target of rapamycin (mTOR) pathway blocker, in pediatric epilepsy cases. Cortical tissue samples obtained from pediatric patients (n = 11, ages 0.67–6.75 years) undergoing surgical resections for the treatment of their pharmacoresistant epilepsy were examined electrophysiologically in ex vivo slices. The cohort included mTOR‐mediated pathologies (tuberous sclerosis complex [TSC] and severe cortical dysplasia [CD]) as well as non–mTOR‐mediated pathologies (tumor and perinatal infarct). Bath application of everolimus (2 μm) had practically no effect on spontaneous inhibitory postsynaptic activity. In contrast, long‐term application of everolimus reduced spontaneous excitatory postsynaptic activity, burst discharges induced by blockade of γ‐aminobutyric acid A (GABAA) receptors, and epileptiform activity generated by 4‐aminopyridine, a K+ channel blocker. The antiseizure effects were more pronounced in TSC and CD cases, whereas in non–mTOR‐mediated pathologies, the effects were subtle at best. These results support further clinical trials of everolimus in mTOR pathway–mediated pathologies and emphasize that the effects require sustained exposure over time.

Background/Aims: Ligustrazine (LSZ) has been identified as an antitumor agent against some types of cancers. Nevertheless, its ability to inhibit growth, migration and invasion of medulloblastoma cells is still unclear. This study aimed to explore the effect of LSZ on Daoy cells. Methods: The effects of LSZ on viability, proliferation, apoptosis, migration, and invasion of Daoy cells were analyzed by CCK-8, BrdU, flow cytometry and Transwell assays, respectively. The effect of LSZ on miR-211 expression was analyzed by qRT-PCR. miR-211 inhibitor transfection was performed to suppress miR-211 expression. The effects of LSZ on apoptosis-related factors, MMP-2, MMP-9, and Vimentin (Vim), as well as main factors of PI3K/AKT and mTOR pathways were analyzed by Western blot. Results: LSZ inhibited viability but promoted apoptosis of Daoy cells. Additionally, the proliferative, migratory and invasive abilities of Daoy cells were decreased by LSZ. Meanwhile, LSZ promoted the activations of Caspase-3 and Caspase-9, increased Bax level, decreased Bcl-2 level, as well as inhibited the expressions of MMP-2, MMP-9 and Vim. Additionally, we found that LSZ enhanced miR-211 expression and exerted its anti-medulloblastoma effect by up-regulation of miR-211. Furthermore, LSZ inhibited PI3K/AKT and mTOR signaling pathways by up-regulating miR-211. Conclusion: LSZ suppressed medulloblastoma Daoy cells by up-regulating miR-211 and further modulating the activations of PI3K/AKT and mTOR signaling pathways.

Recent studies have shown the effect of microRNAs on HSC activation and transformation, which is essential for the pathogenesis of liver fibrosis. In our study, we explored the role of miR-185 in liver fibrosis. Plasma miR-185 was detected in hepatitis B virus-related liver fibrosis patients (S2/3, n = 10) by Illumina HiSeq sequencing, and healthy volunteers were selected (n = 8) as the control group. We found that the plasma miR-185 level in fibrosis patients was significantly downregulated. CCl4-induced fibrosis tissues in mouse livers and TGF-β1-activated HSCs also presented downregulated miR-185 concomitant with an increased expression of RHEB and RICTOR. To explore the correlations, LX-2 cells were transiently transfected with miR-185 mimics. The expression levels of α-SMA, collagen I, and collagen III were decreased as well as RHEB and RICTOR. Inhibition of endogenous miR-185 increased fibrogenic activity. Furthermore, dual-luciferase reporter assays indicated that miR-185 inhibited the expression of RHEB and RICTOR by directly targeting their 3′ UTRs. Moreover, silencing RHEB and RICTOR suppressed α-SMA and collagen expression levels. In conclusion, miR-185 prevents liver fibrogenesis by inhibiting HSC activation via inhibition of RHEB and RICTOR. These results provide new insights into the mechanisms behind the anti-fibrotic effect of miR-185.

International audience; Over the last decade, there has been increasing evidence that hyperactivation of the mechanistic target of rapamycin (mTOR) pathway is a hallmark of malformations of cortical development such as focal cortical dysplasia (FCD) or hemimegalencephaly. The mTOR pathway governs protein and lipid synthesis, cell growth and proliferation as well as metabolism and autophagy. The molecular genetic aetiology of mTOR hyperactivation has only been recently clarified. This article will review the current and still evolving genetic advances in the elucidation of the molecular basis of FCD. Activating somatic mutations in the MTOR gene are to date the most frequent mutations found in FCD brain specimens.

Clinical and molecular studies have implicated epidermal growth factor receptor (EGFR), insulin-like growth factor (IGF) and target of rapamycin (mTOR) signaling pathways in the regulation of pancreatic neuroendocrine tumor (PanNET) growth. Interpretation and comparison of these studies is complex due to clinical and molecular tumor heterogeneity. We therefore focused in this study on insulinomas, which we examined for mRNA and protein expression of EGFR, IGF and mTOR signaling pathway components by quantitative real-time PCR (n = 48) and immunohistochemistry (n = 86). Findings were compared with normal pancreatic islets and correlated with histopathological data and clinical outcome. Insulinomas showed low EGFR and high IGF2 expression. IGFBP2, IGFBP3 and IGFBP6 mRNA levels were 2- to 4-folds higher than those in islets. High protein expression of IGF2, IGF1R and INSR (in 51–92% of the tumors) and low-to-moderate expression of mTORC1 pathway proteins p-S6k and p-4EBP1 (7–28% of the tumors) were observed. Correlations were found between (1) ERK1 mRNA expression and that of numerous IGF pathway genes, (2) p-ERK and IGF1R protein expression and (3) decrease of IGF pathway components and both metastatic disease and shorter 10-year disease-free survival. In conclusion, our observations suggest that high expression of IGF signaling pathway components is a hallmark of insulinomas, but does not necessarily lead to increased mTOR signaling. Reduced expression of IGF pathway components may be an adverse prognostic factor in insulinomas.

A 23-year-old New Zealand Māori male with tuberous sclerosis (TSC) and associated neurocognitive abnormalities presented with altered behavior and increasing seizure frequency. Endogenous hyperinsulinemia from an underlying insulinoma was confirmed and this was managed surgically. This case represents only the sixth description of insulinoma in TSC to date. The role of the hamartin–tuberin complex in regulation of the mechanistic target of rapamycin pathway provides a plausible pathogenetic mechanism between insulinoma and TSC. This rare disease association should be considered in TSC patients who present with otherwise unexplained worsening neurocognitive symptoms., We report a sixth case of insulinoma in tuberous sclerosis to date. The role of the hamartin–tuberin complex in mTOR pathway could explain this rare disease association.

Although translational research into autosomal dominant polycystic kidney disease (ADPKD) and its pathogenesis has made considerable progress, there is presently lack of standardized animal model for preclinical trials. In this study, we developed an orthologous mouse model of human ADPKD by cross‐mating Pkd2 conditional‐knockout mice (Pkd2 f3) to Cre transgenic mice in which Cre is driven by a spectrum of kidney‐related promoters. By systematically characterizing the mouse model, we found that Pkd2 f3/f3 mice with a Cre transgene driven by the mouse villin‐1 promoter (Vil‐Cre;Pkd2 f3/f3) develop overt cysts in the kidney, liver and pancreas and die of end‐stage renal disease (ESRD) at 4–6 months of age. To determine whether these Vil‐Cre;Pkd2 f3/f3 mice were suitable for preclinical trials, we treated the mice with the high‐dose mammalian target of rapamycin (mTOR) inhibitor rapamycin. High‐dose rapamycin significantly increased the lifespan, lowered the cystic index and kidney/body weight ratio and improved renal function in Vil‐Cre;Pkd2 f3/f3 mice in a time‐ and dose‐dependent manner. In addition, we further found that rapamycin arrested aberrant epithelial‐cell proliferation in the ADPKD kidney by down‐regulating the cell‐cycle‐associated cyclin‐dependent kinase 1 (CDK1) and cyclins, namely cyclin A, cyclin B, cyclin D1 and cyclin E, demonstrating a direct link between mTOR signalling changes and the polycystin‐2 dysfunction in cystogenesis. Our newly developed ADPKD model provides a practical platform for translating in vivo preclinical results into ADPKD therapies. The newly defined molecular mechanism by which rapamycin suppresses proliferation via inhibiting abnormally elevated CDK1 and cyclins offers clues to new molecular targets for ADPKD treatment.

Background: Depression is a highly prevalent disorder that is one of the leading causes of disability worldwide. Despite an unknown aetiology, evidence suggests that the innate and adaptive immune systems play a significant role in the development and maintenance of major depressive disorder (MDD). The non-competitive glutamatergic N-methyl-D-aspartate receptor (NMDAR) antagonist, (R,S)-ketamine (ketamine), has demonstrated rapid and robust efficacy as an antidepressant when administered at sub-anaesthetic doses. Methods: Our goal was to characterize the pro-inflammatory profile of patients with MDD by measuring proinflammatory cytokines in plasma and circulating monocyte subsets and to understand how ketamine induces an anti-inflammatory program in monocyte and macrophages in vitro and vivo. Finding: Our results show that patients with MDD without other comorbidities (N= 33) exhibited significantly higher levels of pro-inflammatory IL-12 and IL-6 in plasma and that these cytokines were associated with increased numbers of non-classical (CD11b+ CD16brightCD14neg) monocytes and increased activation state (CD40+ CD86+ ) of classical monocytes in circulation. Remarkably, we have demonstrated that sub-anaesthetic doses of ketamine programs human monocytes into M2c-like macrophages by inducing high levels of CD163 and MERTK with intermediate levels of CD64 and stimulating mTOR-associated gene expression in vitro. The NMDAR antagonist MK-801, but not the a-amino-3‑hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonist, NBQX, also polarizes macrophages to an M2c-like phenotype, but this phenotype disappears upon mTOR pathway inhibition. Subanaesthetic doses (10 mg/kg) of ketamine administration in mice both promote reduction of circulating classical pro-inflammatory monocytes and increase of alternative M2 macrophage subtypes in the spleen and CNS. Interpretation: Our results suggest an anti-inflammatory property of ketamine that can skew macrophages to an M2-like phenotype, highlighting potential therapeutic implications not only for patients with MDD but also other inflammatory-based diseases. Fil: Nowak, Wanda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia.; Argentina Fil: Grendas, Leandro Nicolás. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia.; Argentina Fil: Sanmarco, Liliana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Estecho, Ivana Gisele. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia.; Argentina Fil: Arena, Ángeles Romina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia.; Argentina Fil: Eberhardt, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Rodante, Demián Emanuel. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia.; Argentina Fil: Aoki, María Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Daray, Federico Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia.; Argentina Fil: Carrera Silva, Eugenio Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Errasti, Andrea Emilse. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia.; Argentina

The transcript factor LHX2 is dysregulated in many cancers but its role in osteosarcoma (OS) remains unclear. In this study, we confirm that LHX2 is up-regulated in osteosarcoma, and that its silencing inhibits OS malignancy and induces autophagy via mTOR signaling. We further demonstrate that miR-129-5p negatively regulates LHX2 and suppresses the malignant phenotypes of OS. LHX2 overexpression could restore the malignant phenotypes. In conclusion, LHX2 regulates tumorigenesis and autophagy via mTOR in OS and is negatively regulated by miR-129-5p. Targeting the miR-129-5p/LHX2/mTOR axis therefore represents a novel therapeutic strategy for OS treatment.

Osteonecrosis of the femoral head (ONFH) is usually caused by chronic and excessive alcohol dependency, and this condition largely suppresses the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). As a trimethyl derivative of glycine, betaine is an important human nutrient that regulates a series of vital biological processes, including oxidative stress, inflammatory responses, osteoblast differentiation and cellular apoptosis. However, no study has investigated the role of betaine in alcohol-induced ONFH. In this study, we hypothesized that betaine might have protective effects on ethanol-treated BMSCs and decrease the morbidity of alcohol-induced ONFH in a rat model. In vitro, we found that ethanol significantly downregulated the expression of osteocalcin (OCN), collagen 1 (COL1) and RUNX2 via activating the mammalian target of rapamycin (mTOR) signaling cascade. However, the inhibitory effects were rescued by betaine co-treatment at concentrations of 1 mM and 10 mM. In vivo, the typical ONFH pathological changes in a rat model of alcohol-induced ONFH were investigated by using multiple methods, including hematoxylin-eosin staining, micro-CT scans, TdT-mediated dUTP nick end labeling (TUNEL) assays and immunohistochemical staining for OCN and COL1. Osteonecrotic lesions of the femoral head could be alleviated by betaine as evidenced by significant histological and radiological improvements. Collectively, betaine plays a protective role against ethanol-induced suppression of osteogenesis and mineralization of hBMSCs and is thus a potential pharmacotherapy for alcohol-induced ONFH in vivo.

Dendritic cells (DCs) are important antigen-presenting cells (APCs) that play essential roles in bridging innate and adaptive immune responses. Differentiation stages of DC subsets from bone marrow progenitor cells have been well-defined during the past decades. Features that distinguish DC progenitor cells from each differentiation stages, related signaling pathways and transcription factors that are crucial for DC lineage commitment have been well-elucidated in numerous studies. Recently, growing evidence are showing that cellular metabolism, as one of the most fundamental process of cells, has essential role in the modulation of immune system. There have been multiple reports and reviews that focus on the metabolic modulations on DC functions, however little attention had been paid to the metabolic regulation of DC development and differentiation. In recent years, increasing evidence suggests that metabolic regulations also exert significant impact on DC differentiation, as well as on the homeostasis of tissue resident DCs. The focus of this review is to summarize the findings from recent studies on the metabolic regulation of DC differentiation and to discuss the impacts of the three major aspects of metabolism on the processes of DC development and differentiation, namely the changes in metabolic pathways, the molecular signaling pathways that modulate cell metabolism, and the effects of metabolites and nutrients. The aim of this review is to draw attentions to this important and exciting research field where the effects of metabolic process and their regulation in DC differentiation need to be further explored.

International audience; Sea urchin early development is a powerful model to study translational regulation under physiological conditions. Fertilization triggers an activation of the translation machinery responsible for the increase of protein synthesis necessary for the completion of the first embryonic cell cycles. The cap-binding protein eIF4E, the helicase eIF4A and the large scaffolding protein eIF4G are assembled upon fertilization to form an initiation complex on mRNAs involved in cap-dependent translation initiation. The presence of these proteins in unfertilized and fertilized eggs has already been demonstrated, however data concerning the translational status of translation factors are still scarce. Using polysome fractionation, we analyzed the impact of fertilization on the recruitment of mRNAs encoding initiation factors. Strikingly, whereas the mRNAs coding eIF4E, eIF4A, and eIF4G were not recruited into polysomes at 1 h post-fertilization, mRNAs for eIF4B and for non-canonical initiation factors such as DAP5, eIF4E2, eIF4E3, or hnRNP Q, are recruited and are differentially sensitive to the activation state of the mechanistic target of rapamycin (mTOR) pathway. We discuss our results suggesting alternative translation initiation in the context of the early development of sea urchins.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder associated with epilepsy, intellectual disabilities, and autistic behaviors. These neurological symptoms result from synaptic dysregulations, which shift a balance between excitation and inhibition. To decipher the synaptic substrate of hyperexcitability, we examined pan-neuronal Tsc1 knockout mouse and found a reduction in surface expression of a GABA receptor (GABAR) subunit but not AMPA receptor (AMPAR) subunit. Using electrophysiological recordings, we found a significant reduction in the frequency of GABAR-mediated miniature inhibitory postsynaptic currents (GABAR-mIPSCs) but not AMPAR-mediated miniature excitatory postsynaptic currents (AMPAR-mEPSCs) in layer 2/3 pyramidal neurons. To determine a subpopulation of interneurons that are especially vulnerable to the absence of TSC1 function, we also analyzed two strains of conditional knockout mice targeting two of the prominent interneuron subtypes that express parvalbumin (PV) or somatostatin (SST). Unlike pan-neuronal knockout mice, both interneuron-specific Tsc-1 knockout mice did not develop spontaneous seizures and grew into adults. Further, the properties of AMPAR-mEPSCs and GABAR-mIPSCs were normal in both Pv-Cre and Sst-Cre x Tsc1 fl/fl knockout mice. These results indicate that removal of TSC1 from all neurons in a local cortical circuit results in hyperexcitability while connections between pyramidal neurons and interneurons expressing PV and SST are preserved in the layer 2/3 visual cortex. Our study suggests that another inhibitory cell type or a combination of multiple subtypes may be accountable for hyperexcitability in TSC.

Summary Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal disease associated with aging. However, the molecular mechanisms of the aging process that contribute to the pathogenesis of IPF have not been elucidated. IPF is characterized by abundant foci of highly active fibroblasts and myofibroblasts resistant to apoptosis. Remarkably, the role of aging in the autophagy activity of lung fibroblasts and its relationship with apoptosis, as adaptive responses, has not been evaluated previously in this disease. In the present study, we analyzed the dynamics of autophagy in primary lung fibroblasts from IPF compared to young and age-matched normal lung fibroblasts. Our results showed that aging contributes for a lower induction of autophagy on basal conditions and under starvation which is mediated by mTOR pathway activation. Treatment with rapamycin and PP242, that target the PI3K/AKT/mTOR signaling pathway, modified starvation-induced autophagy and apoptosis in IPF fibroblasts. Interestingly, we found a persistent activation of this pathway under starvation that contributes to the apoptosis resistance in IPF fibroblasts. These findings indicate that aging affects adaptive responses to stress decreasing autophagy through activation of mTORC1 in lung fibroblasts. The activation of this pathway also contributes to the resistance to cell death in IPF lung fibroblasts.

Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. To overcome these hurdles, we developed a new experimental model of clinical dormancy employing patient-isolated Circulating Tumor Cells (de novo CTCs) and their injection in xenografts with subsequent tumor monitoring and CTC characterization (ex vivo CTCs). We hypothesized that significant distinctions exist between signaling pathways of bone marrow-homing vs metastasis-competent CTCs upon transplantation in xenografts. Comparative transcriptomic analyses of ex vivo vs de novo CTCs identified increased mTOR signaling&mdash, a critical pathway frequently dysregulated in breast cancer and implicated in cell survival and dormancy&mdash, with contrasting actions by its two complementary arms (mTORC2/mTORC1). Heightened mTORC2 downstream targets augmented quiescent CTCs (Ki67&minus, /RBL2+ cells) in paired breast cancer tissues, along with high mTORC2 activity in solitary BMRCs and tissue-resident CTCs. Further, shRNA mediated the knockdown of RICTOR, an essential component of mTORC2, and augmented Ki67/PCNA biomarker expression and proliferation. Collectively, these findings suggest that the balance between mTORC1 vs mTORC2 signaling regulates CTC-associated mitotic and/or dormancy characteristics.

Respiratory exposure to marine phycotoxins is of increasing concern. Inhalation of sea spray aerosols (SSAs), during harmful Karenia brevis and Ostreopsis ovata blooms induces respiratory distress among others. The biogenics hypothesis, however, suggests that regular airborne exposure to natural products is health promoting via a downregulation of the mechanistic target of rapamycin (mTOR) pathway. Until now, little scientific evidence supported this hypothesis. The current explorative in vitro study investigated both health-affecting and potential health-promoting mechanisms of airborne phycotoxin exposure, by analyzing cell viability effects via cytotoxicity assays and effects on the mTOR pathway via western blotting. To that end, A549 and BEAS-2B lung cells were exposed to increasing concentrations (ng&middot, L&minus, 1 &ndash, mg&middot, 1) of (1) pure phycotoxins and (2) an extract of experimental aerosolized homoyessotoxin (hYTX). The lowest cell viability effect concentrations were found for the examined yessotoxins (YTXs). Contradictory to the other phycotoxins, these YTXs only induced a partial cell viability decrease at the highest test concentrations. Growth inhibition and apoptosis, both linked to mTOR pathway activity, may explain these effects, as both YTXs were shown to downregulate this pathway. This proof-of-principle study supports the biogenics hypothesis, as specific aerosolizable marine products (e.g., YTXs) can downregulate the mTOR pathway.

Sirtuin 1 (SIRT1) is a critical suppressor of T cell immunity. However, whether SIRT1 is involved in the progression of acute graft-vs.-host disease (aGVHD) has still remained unclear. PI3K/Akt/mTOR pathway is a crucial element involved in the activation and functions of T cells. Over-activation of PI3K/Akt/mTOR signaling may be related to the occurrence of aGVHD. STAT3 activation requires phosphorylation and acetylation. A recent study showed that STAT3 hyperphosphorylation in CD4+ T cells may be a trigger of aGVHD. The role of the STAT3 acetylation in aGVHD pathogenesis is still unclear. The present study revealed that SIRT1 deficiency as a critical factor is involved in the excessive activation of mTOR pathway and upregulation of STAT3 acetylation and phosphorylation in CD4+ T cells from patients with aGVHD. Exorbitant activation of IL-1β signaling is the main reason for TAK1-dependent SIRT1 insufficiency. The findings of the present study might provide a new therapeutic target for treating aGVHD.

Antibody production and function represent an essential part of the immune response, particularly in fighting bacterial and viral infections. Multiple immunological phenotypes can result in dysregulation of the immune system humoral compartment, including class-switch recombination (CSR) defects associated with hyper-IgM (HIGM) syndromes. The CSR/HIGM syndromes are defined by the presence of normal or elevated plasma IgM levels in the context of low levels of switched IgG, IgA, and IgE isotypes. Recently described autosomal dominant gain-of-function (GOF) mutations in PIK3CD and PIK3R1 cause combined immunodeficiencies that can also present as CSR/HIGM defects. These defects, their pathophysiology and derived clinical manifestations are described in depth. Previously reported forms of CSR/HIGM syndromes are briefly reviewed and compared to the phosphoinositide 3-kinase (PI3K) pathway defects. Diseases involving the PI3K pathway represent a distinctive subset of CSR/HIGM syndromes, presenting with their own characteristic clinical and laboratory attributes as well as individual therapeutic approaches.

It is well established that estrogens regulate female reproduction through estrogen receptors (ERs) in the ovary. However, the precise physiological role of estrogen/ER signaling in reproduction processes remains poorly defined in zebrafish. In this study, we successfully generated an ERα (esr1) mutant line in zebrafish via transcription activator-like effectors nucleases (TALENs). It was found in the mutant females that the fertility was enhanced and the ovarian histology was normal at 90 days post-fertilization (dpf). However, the number of fertile females decreased with age. By 180 dpf, esr1 mutant females were infertile with degenerated ovaries, while the age-matched wild-type females were still fertile. Additionally, few large vitellogenic granules can be found in full grown (FG) follicles at 90 dpf and the expression of vtg genes were down-regulated at both 90 and 180 dpf in esr1 mutant zebrafish. Moreover, steroidogenesis pathway and mTOR signaling pathway were over-activated at 90 dpf, but declined prematurely in esr1 mutant zebrafish by 180 dpf. Collectively, the present study provides evidences that esr1 is fundamental for ovarian maintenance in zebrafish.

Acyl-CoA synthetase-4 (ACSL4) is an enzyme implicated in estrogen receptor α (ERα) negative regulation and hormone therapy resistance in breast cancer. In addition, ACSL4 has been associated to certain types of hormone resistance in prostate cancer. Chemotherapeutic treatment of disseminated breast cancer is usually faced with therapy resistance associated to ATP-binding cassette (ABC) transporter expression, which detect and eject anti-cancer drugs from cells. In this context, the aim of the present work was to study the role of ACSL4 in anti-cancer drug resistance and the involvement of ABC transporters in the underlying mechanisms. To this end, we used MCF-7 Tet-Off/ACSL4 and MDA-MB-231 mock cells, which overexpress ACSL4, and control line MCF-7 Tet-Off empty vector, MDA-MB-231 shRNA ACSL4 and MDA-MB-231 wild type cells. Assays were conducted on cell viability (MTT), cell proliferation (BrdU), drug efflux (flow cytometry), ACSL4-responsive drug resistance ABC transporter genes (RNA-Seq), transporter mRNA expression, protein levels and signaling pathway participation (real-time PCR and Western blot). Higher survival rates upon chemotherapeutic treatment were obtained in MCF-7 Tet-Off/ACSL4 and MDA-MB-231 mock cells, an effect counteracted by doxycycline- or shRNA-induced ACSL4 inhibition, respectively. A synergic effect of ACSL4 inhibitor triacsin C and chemotherapeutic drugs was observed on the inhibition of MDA-MB-231 wild type cell proliferation. MCF-7 Tet-Off/ACSL4 cells showed greater doxorubicin, Hoechst 33342 and calcein AM efflux. In contrast, MDA-MB-231 shRNA ACSL4 cells evidenced inhibition of chemotherapeutic drug efflux. ABCG2, ABCC4, and ABCC8 were identified as ACSL4-responsive drug resistance genes whose expression was increased in MCF-7 Tet-Off/ACSL4 cells but inhibited in MDA-MB-231 shRNA ACSL4 cells. Further cell survival assays in the presence of Ko 143 and Ceefourin 1, inhibitors of ABCG2 and ABCC4, respectively, upon chemotherapeutic treatment showed greater participation of ABCG2 in anti-cancer drug resistance in cells overexpressing ACSL4. In addition, ACSL4 inhibition and chemotherapeutic treatment combined with rapamycin-induced mTOR inhibition synergically inhibited proliferation and reduced ABCG2 expression in cells overexpressing ACSL4. In sum, ACSL4 may be regarded as a novel therapeutic target regulating the expression of transporters involved in anticancer drug resistance through the mTOR pathway to restore drug sensitivity in tumors with poor prognosis for disease-free and overall survival. Fil: Orlando, Ulises Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Castillo, Ana Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Ríos Medrano, Mayra Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Solano, Angela Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Maloberti, Paula Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Podesta, Ernesto Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina

Background To examine the activity of the mammalian target of rapamycin (mTOR) pathway and its regulators, transforming growth factor (TGF)-β1 and phosphatase and tensin homolog (PTEN), in minor salivary gland biopsies of Sjogren’s syndrome (SS) and systemic sclerosis (SSc) patients. Methods We retrospectively evaluated SS, SSc, and SS-SSc overlap patients admitted to our outpatient rheumatology clinic between January 2007 and December 2015 who underwent a minor salivary gland biopsy. Patient demographics and some clinical features were obtained from hospital records. Immunohistochemistry was used to analyze total mTOR, total PTEN, and TGF-β1 expression in the biopsied tissues. The biopsy specimens were also examined for the presence and degree of fibrosis. Results Minor salivary gland biopsies of 58 SS, 14 SSc, and 23 SS-SSc overlap patients were included in the study. There was no significant difference in mTOR expression between these groups (P = 0.622). PTEN protein was expressed in 87.2% of patients with SS, 57.9% with overlap syndrome, and 100% of the SSC patients, and these differences were statistically different (P = 0.023). Although ductal epithelial TGF-β1 expression was similar between the groups (P = 0.345), acinar cell expression was found to be more frequent in the SSc (72.7%) and overlap patients (85.7%) in comparison with the SS cases (58.2%; P = 0.004). Conclusion mTOR may be one of the common pathways in the pathology of both SS and SSc. Hence, there may be a role for mTOR inhibitors in the treatment of both diseases. Additionally, PTEN and TGF-β1 expression may be a distinctive feature of SSc.

TOR is a serine/threonine protein kinase that assembles into distinct TOR Complexes 1 and 2 (TORC1 or TORC2) to regulate cell growth. In mammalian cells, a single mTOR incorporates stably into mTORC1 and mTORC2. By contrast, in Saccharomyces cerevisiae, two highly similar Tor1 and Tor2 proteins exist, where Tor1 assembles exclusively into TORC1 and Tor2 assembles preferentially into TORC2. To gain insight into TOR complex assembly, we used this bifurcation in yeast to identify structural elements within Tor1 and Tor2 that govern their complex specificity. We have identified a concise region of ~500 amino acids within the N-terminus of Tor2, which we term the Major Assembly Specificity (MAS) domain, that is sufficient to confer significant TORC2 activity when placed into an otherwise Tor1 protein. Consistently, introduction of the corresponding MAS domain from Tor1 into an otherwise Tor2 is sufficient to confer stable association with TORC1-specific components. Remarkably, much like mTOR, this latter chimera also retains stable interactions with TORC2 components, indicating that determinants throughout Tor1/Tor2 contribute to complex specificity. Our findings are in excellent agreement with recent ultrastructural studies of TORC1 and TORC2, where the MAS domain is involved in quaternary interactions important for complex formation and/or stability.

During the last decade the interest for the mesenchymal cells is growing due to their possible uses in therapies to treat certain degenerative pathologies. Mesenchymal stem cells have been found in the bone marrow and they have been shown to be responsible for bone repair and fat cells production. Mesenchymal stromal cells can be obtained from a wide variety of tissues in addition to bone marrow and can differentiate into many other cell types. The study of cell differentiation and programming provides new models for drug discovery and cell therapy that now overcomes gene therapy. Senescence, cancer development and degenerative diseases depend on mesenchymal cells contribution to tissue homeostasis. On the other hand, diet and life style are included among risk factors, which can contribute to the success of pharmacological treatments. This review focuses on nutrients from Mediterranean diet and supplements, which have been shown to influence mesenchymal stem cells and cells derived from them. Dietary intake of nutrients impairs both in vitro and in vivo observations, this review aims to gather the results about the effects of food compounds on mesenchymal cells from which adipocytes and osteoblasts derive. Amino acids and proteins, carbohydrates, lipids, fatty acids and vegetable secondary metabolites, differently act on mesenchymal cells bearing on modulation of gene expression and controlling the fate of cell lineages. Remarkable, the analysis of literature shows that the main effect of nutrients on mesenchymal cells is the stimulation of transcription factors which address the cells toward proliferation or differentiation. For instance, carbohydrates, simple or complex, and lipids appear to stimulate the PPAR receptors, whereas proteins and amino acids result to act on the mTOR system and they can also stimulate the MyoD-1 transcription factor and cooperating proteins. In conclusion, nutrients can promote cell growth and differentiation of mesenchymal cells.

Hypothalamic proopiomelanocortin (POMC) neurons are important players in the regulation of energy homeostasis; we previously demonstrated that environmental stimulation excites arcuate nucleus circuits to undergo plastic remodeling, leading to altered ratio between excitatory and inhibitory synaptic contacts on these neurons. The widely used selective serotonin reuptake inhibitor fluoxetine (FLX) is known to affect body weight. On the other hand, FLX administration mimics the effects of environmental stimulation on synaptic plasticity in the hippocampus and cortex. The mammalian target of rapamycin (mTOR) pathway is instrumental in these phenomena. Thus, we aimed at investigating whether and how FLX affects POMC neurons activity and hypothalamic mTOR function. Adult mice expressing green fluorescent protein (GFP) under the POMC promoter were treated with FLX for 3 weeks resulting in diminished body weight. Patch clamp recordings performed on POMC neurons indicate that FLX increases their firing rate and the excitatory AMPA-mediated transmission, and reduces the inhibitory GABAergic currents at presynaptic level. Immunofluorescence studies indicate that FLX increases the ratio between excitatory and inhibitory synaptic contacts on POMC neurons. These changes are associated with an increased activity of the hypothalamic mTOR pathway. Use of the mTOR inhibitor rapamycin blunts the effects of FLX on body weight and on functional and structural plasticity of POMC neurons. Our findings indicate that FLX is able to remodel POMC neurons, and that this may be partly mediated by the mTOR signaling pathway.

This experiment was to investigate the effects of dietary leucine supplementation on the gene expression of mammalian target of rapamycin (mTOR) signaling pathway and intestinal development of broilers. A total of 384 one-day-old broilers were randomly assigned into 4 treatments with 6 replicates (16 broilers per replicate). Broilers in these treatment groups were offered the following diets with 1.37, 1.77, 2.17 and 2.57% of leucine. These diet treatments were named 1.37TM, 1.77TM, 2.17TM, and 2.57TM. The experiment lasted 21 days and all birds had free access to feed and water. Results indicated that there was no significant difference in body weight, average daily gain and average feed intake among all treatments (P > 0.05). The broiler duodenal villus height in 2.57TM was the lowest, but the highest occurred in 1.37TM on d 7 and 14 (P

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer. Pathologic activation of PI3K/mTOR pathway and elevated expression of c-Myc are frequently detected in MCC. Yet, there is no targeted therapy presently available for this lethal disease. Recently, MLN0128, a second-generation dual TORC1/2 inhibitor is shown to have therapeutic efficacy in preclinical studies. MLN0128 is currently in clinical trials as a potential therapy for advanced cancers. Here we characterize the therapeutic efficacy of MLN0128 in the preclinical setting of MCC and delineate downstream targets of mTORC1/2 in MCC cellular systems. MLN0128 significantly attenuates xenograft MCC tumor growth independent of Merkel cell polyomavirus. Moreover, MLN0128 markedly diminishes MCC cell proliferation and induces apoptosis. Further investigations indicate that senescence does not contribute to MLN0128-mediated repression of xenograft MCC tumor growth. Finally, we also observe robust antitumor effects of MLN0128 when administered as a dual therapy with JQ1, a bromodomain protein BRD4 inhibitor. These results suggest dual blockade of PI3K/mTOR pathway and c-Myc axis is effective in the control of MCC tumor growth. Our results demonstrate that MLN0128 is potent as monotherapy or as a member of combination therapy with JQ1 for advanced MCC.

mTOR is a protein kinase that plays a central role in regulating critical cellular processes. We evaluated the activation and cellular localization of the mTOR pathway in multiple myeloma (MM) and analyzed the role of pomalidomide in regulating mTOR. By immunohistochemistry cytoplasmic p-mTOR stained positive in 57 out 101 (57.6%) cases with a nuclear p-mTOR localization in 14 out 101 samples (13.8%). In the 70 MM samples analyzed for the entire pathway, p-mTOR expression significantly correlated with p-AKT, p-P70S6K, and p-4E-BP1 suggesting that the AKT/mTOR pathway is activated in a subset of MM patients. Immunofluorescence assays demonstrated that mTOR protein is distributed throughout the cytoplasm and the nucleus at baseline in MM cell lines and in plasma cells of 13 MM patients and that pomalidomide facilitated the shift of the mTOR protein in the nucleus. By western blotting, treatment with pomalidomide increased nuclear mTOR and p-mTOR expression levels in the nucleus with a concomitant decrease of the cytoplasmic fractions while does not seem to affect significantly AKT phosphorylation status. In MM cells the anti-myeloma activity of pomalidomide may be mediated by the regulation of the mTOR pathway.

Somatic Mutations Activating the mTOR Pathway in Dorsal Telencephalic Progenitors Cause a Continuum of Cortical Dysplasias D'Gama AM, Woodworth MB, Hossain AA, Bizzotto S, Hatem NE, LaCoursiere CM, Najm I, Ying Z, Yang E, Barkovich AJ, Kwiatkowski DJ, Vinters HV, Madsen JR, Mathern GW, Blümcke I, Poduri A, Walsh CA. Cell Rep. 2017;21:3754-3766. Focal cortical dysplasia (FCD) and hemimegalencephaly (HME) are epileptogenic neurodevelopmental malformations caused by mutations in mTOR pathway genes. Deep sequencing of these genes in FCD/HME brain tissue identified an etiology in 27 (41%) of 66 cases. Radiographically indistinguishable lesions are caused by somatic activating mutations in AKT3, MTOR, and PIK3CA and germline loss-of-function mutations in DEPDC5, NPRL2, and TSC1/2, including TSC2 mutations in isolated HME demonstrating a "two-hit" model. Mutations in the same gene cause a disease continuum from FCD to HME to bilateral brain overgrowth, reflecting the progenitor cell and developmental time when the mutation occurred. Single-cell sequencing demonstrated mTOR activation in neurons in all lesions. Conditional Pik3ca activation in the mouse cortex showed that mTOR activation in excitatory neurons and glia, but not interneurons, is sufficient for abnormal cortical overgrowth. These data suggest that mTOR activation in dorsal telencephalic progenitors, in some cases specifically the excitatory neuron lineage, causes cortical dysplasia.