Tenofovir disoproxil fumarate directly ameliorates liver fibrosis by inducing hepatic stellate cell apoptosis via downregulation of PI3K/Akt/mTOR signaling pathway

Background Antifibrotic agent for the treatment of liver fibrosis has not been developed so far. Long term treatment of chronic hepatitis B patients with antiviral drugs tenofovir disoproxil fumarate (TDF) and entecavir (ETV) results in the regression of liver fibrosis, but the underlying mechanism has not been clarified. Therefore, we aimed to investigate the direct impact of TDF and ETV on liver fibrosis. Methods Activated hepatic stellate cell (HSC) cell lines were used to evaluate the effects of TDF and ETV. After treatment with each antiviral agent, cell viability, morphology, apoptotic features, autophagy and antifibrosis signalling pathways were examined. Then, collagen deposition, fibrosis markers and activated HSCs were measured in liver tissues of the liver fibrosis model mice. Results After TDF treatment, the viabilities of LX2 and HSC-T6 cells were decreased, and the cells exhibited apoptotic features, but ETV did not induce these effects. Cleavage of PARP and Caspase-3 and the inhibition of the antiapoptotic gene Bcl-xl indicated activated HSC apoptosis following TDF treatment. TDF simultaneously increased autophagy, which also regulated apoptosis through crosstalk. TDF inactivated the PI3K/Akt/mTOR signalling pathway, which was associated with the activation of both apoptosis and autophagy. In the liver fibrosis mouse model, the fibrotic area and activated HSC markers were decreased by TDF but not ETV treatment. Additionally, apoptotic cells were concentrated in the periportal fibrotic area after TDF treatment, which indicated the specific antifibrotic effect of TDF. Conclusions TDF directly ameliorates liver fibrosis by downregulating the PI3K/Akt/mTOR signalling pathway, which results in the apoptosis of activated HSCs. The antifibrotic effects of TDF indicate that it may be a therapeutic agent for the treatment of liver fibrosis.

Negatively Regulated by miR-29c-3p, MTFR1 Promotes the Progression and Glycolysis in Lung Adenocarcinoma via the AMPK/mTOR Signalling Pathway

Background: Lung adenocarcinoma (LUAD) is the major form of lung cancer that presents a major peril to public health. Owing to the high rates of morbidity, mortality and chemoresistance, it is necessary to develop more effective therapeutic targets of LUAD. Mitochondrial fission regulator 1 (MTFR1) affects the occurrence and development of some diseases by regulating mitochondrial dynamics and is dysregulated in LUAD. However, the functions and molecular mechanisms of MTFR1 in LUAD have not been investigated.Methods: Immunohistochemical (IHC) analysis, real-time quantitative polymerase chain reaction (RT-qPCR), bioinformatic analysis and western blot (WB) were performed to assess the expression of MTFR1 at both protein and mRNA levels. The biological functions of MTFR1 in LUAD cells were assessed based on various in vivo and in vitro experiments. The dual-luciferase reporter assay and some rescue experiments were performed to evaluate the underlying mechanism of MTFR1 in LUAD.Results: MTFR1 was upregulated in LUAD cells and tissues and correlated with dismal clinicopathologic features and a worse prognosis of patients with LUAD. Functionally, MTFR1 overexpression stimulated the proliferation, invasion, migration and glycolytic capacity and impeded the apoptosis of LUAD cells; however, opposite results were obtained when MTFR1 expression was knocked down. MTFR1, which was directly targeted by miR-29c-3p, may exert its biological functions through the AMPK/mTOR signalling pathway.Conclusion: MTFR1 promotes the progression of LUAD. Therefore, targeting MTFR1 can offer an effective therapeutic strategy for LUAD treatment.

Leucine supplementation during late gestation globally altered placental metabolism and nutrient transport via modulation of the PI3K/AKT/mTOR signalling pathway

Background Our previous study found that sow dietary leucine supplementation signicicantly improved fetal intrauterine growth and newborn piglet birth weight. But we still have limited knowledge how leucine regulated placental functions to promote the nutrient supply to fetus to support its intrauterine development. Methods 150 sows at day 90 of gestiation were divided into three groups and fed with either control diet (CON), CON + 0.4% Leu or CON + 0.8% Leu separately until parturition. Placental metabolomics, full spectrum amino acids and nutrient transporters were systematically analyzed. Results Leu supplementation lead to an altered placental metabolism with higher amount of metabolites related to glycolysis and fatty acids oxidatation, and more amino acids accumulation in placenta. Besides, nutrient transporters including amino acids transporters, glucose transporters and fatty acids transporters in placenta were globally enhanced and several enzymes related to energy metabolism including hexokinase, succinatede hydrogenase, lactated hydrogenase, glycogen phosphorylase and hydroxyaryl-CoA-dehydrogenase were significantly increased with no change in antioxidative status in the groups with Leu supplementation. Futhermore, Leu supplementation significantly increased phosphalation of PI3K, Akt, and mTOR in placenta compared with control group. Conclusions Leu supplementation during late gestation globally altered placental metabolism, nutrient transport (amino acids, glucose, and fatty acids) via modulation of the PI3K/Akt/mTOR signalling pathway.

Complete response to neoadjuvant chemoradiotherapy in rectal cancer is associated with RAS/AKT mutations and high tumour mutational burden

Background Pathological complete response (pathCR) in rectal cancer is beneficial, as up to 75% of patients do not experience regrowth of the primary tumour, but it is poorly understood. We hypothesised that the changes seen in the pre-treatment biopsies of pathCR but not seen in residual tumour after chemoradiotherapy were the determinants of responsiveness. Methods Two groups of patients with either complete response (pathCR group, N = 24) or no response (poor response group, N = 24) were retrieved. Pre-treatment biopsies of cancers from these patients underwent high read depth amplicon sequencing for a targeted panel, exome sequencing, methylation profiling and immunohistochemistry for DNA repair pathway proteins. Results Twenty four patients who underwent pathCR and twenty-four who underwent poor response underwent molecular characterisation. Patients in the pathCR group had significantly higher tumour mutational burden and neoantigen load, frequent copy number alterations but fewer structural variants and enrichment for driver mutations in the PI3K/AKT/mTOR signalling pathway. There were no significant differences in tumour heterogeneity as measured by MATH score. Methylation analysis demonstrated enrichment for hypomethyation in the PI3K/AKT/mTOR signalling pathway. Discussion The phenomenon of pathCR in rectal cancer may be related to immunovisibility caused by a high tumour mutational burden phenotype. Potential therapy resistance mechanisms involve the PI3K/AKT/mTOR signalling pathway, but tumour heterogeneity does not seem to play a role in resistance.

O-080 Activated AKT/mTOR signalling in peripheral blood of women with premature ovarian insufficiency and its correlation with variable FMR1 expression profiles

Study question How predictive are gene expression levels of AKT/mTOR-signalling-pathway genes in peripheral blood of patients with premature ovary insufficiency (POI) and is there a link to FMR1-expression? Summary answer AKT1, TSC2, mTOR, S6K and FOXO3-expression-levels are significantly upregulated in POI-patients and demonstrate a positive correlation with FMR1-expression-level in case of mTOR-, S6K and FOXO3. What is known already The AKT/mTOR-signalling-pathway is involved in a range of cellular functions. In female germline it regulates early follicular-activation and follicular-pool-maintenance. Over the past few years AKT-activation has been experimentally applied to induce follicular maturation in POI-patients. Additionally, first evidence of a linked FMR1 – AKT/mTOR signaling in female germline have been reported. FMR1 is a major control gene in folliculogenesis. Due to increased (CGG)-triplet-numbers (54 < n < 200) in its 5′-untranslated-region, named premutation, increased FMR1-expression-levels and reduced FMRP-production have been described, associated with POI in 20% of cases. A former study found premutation independent, large transcript-level-variances of FMR1 in leukocyte RNA-samples of POI-patients. Study design, size, duration 74 POI patients and 56 fertile controls were prospectively enrolled in this study. Accordingly, expression levels of genes associated with the AKT/mTOR-signaling pathway and FMR1 were analyzed and correlated on the mRNA level of their leukocytes. Participants/materials, setting, methods All patients provided written informed consent. mRNA was extracted from EDTA blood after lysis; quantitative expression analyses of FMR1, AKT, mTOR, S6K, FOXO3, FOXO1 genes were performed with specific TaqMan-Assays. Statistical analyses was performed with SPSS; statistical significance was set to P < 0.05. Main results and the role of chance Gene expression levels of AKT1, TSC2, mTOR, S6K, FOXO3 are significant higher in POI patients compared to controls (P < 0.009 or less). The rate of FMR1-expression is highly correlated with mTOR-, S6K and FOXO3-expression levels (P < 0.001) in all patients, in addition. When grouped according to ovarian reserve this effect is more pronounced in POI than in control patients. Additionally, the correlation of FMR1 with FOXO3 remained significant only in the POI subgroup. The upregulation of AKT/mTOR-signaling in POI may reflect a compensative mechanism in POI aiming the activation of the last remaining follicles. The linkage of FMR1 with AKT/mTOR-signalling in peripheral blood comparable to prior results from germline, support its putative impact on the pathogenesis of POI and other folliculogenesis related disorders, such as poor ovarian response. Limitations, reasons for caution Results are based on limited patient numbers. More patients, stratified for age and other risks factors, are needed to further elucidate this mechanism. Wider implications of the findings This is the first evidence that FMR1 is linked to an AKT/mTOR activation in POI potentially involved in its pathogenesis. Such a marker in peripheral blood offers a perspective towards its usability as a predictive tool in the diagnostics and prognosis of POI, if results are consistent in further studies. Trial registration number not applicable