mTOR Activity Research Articles

Dehydroandrographolide ameliorates doxorubicin-mediated cardiotoxicity by regulating autophagy through the mTOR-TFEB pathway

The clinical application of doxorubicin (DOX) was limited by the serious cardiotoxicity. The traditional Chinese medicine Andrographis paniculata and its principal active component (Dehydroandrographolide, DA) have been well known for their diverse cardiovascular protective effects. However, the effects of DA on DOX-induced cardiotoxicity (DIC) were still unknown. In this study, we evaluated the effects and revealed the potential mechanisms of DA on DIC both in vivo and in vitro. The effects of DA on DIC were systematically assessed by echocardiography and histological assays. Western blot and flow cytometry were used to measure apoptosis of cardiomyocytes. Transmission electron microscopy and StubRFP-SensGFP-LC3 lentivirus were further used to assay autophagic flux. Our results showed that DA administration significantly improved cardiac function and attenuated DOX-induced cardiomyocyte apoptosis. Mechanically, DA restored autophagic flux and lysosome functions via inhibiting DOX-induced mTOR signal pathway activation and increasing the translocation of TFEB to the nucleus. However, activation of mTOR or knockdown of TFEB significantly inhibited the protective effects of DA against DIC by impacting lysosomal functions and autophagic flux. In conclusion, our results revealed that DA might be a potential cardioprotective agent against DIC.

Exposure to serum from exclusive heated tobacco product smokers induces mTOR activation and fibrotic features in human cardiac stromal cells

Chronic smokers have increased risk of fibrosis-related atrial fibrillation. The use of heated-tobacco products (HTPs) is increasing exponentially, and their health impact is still uncertain. We aim to investigate the effects of circulating molecules in exclusive HTP chronic smokers on the fibrotic behavior of human atrial cardiac stromal cells (CSCs). CSCs were isolated from atrial tissue of elective cardiac surgery patients, and exposed to serum lots from young healthy subjects, stratified in exclusive HTP smokers, tobacco combustion cigarette (TCC) smokers, or nonsmokers (NS). CSCs treated with TCC serum displayed impaired migration and increased expression of pro-inflammatory cytokines. Cells cultured with HTP serum showed increased levels of pro-fibrotic markers, and reduced expression of connexin-43. Both TCC and HTP sera increased collagen release and reduced secretion of angiogenic protective factors from CSCs, compared to NS serum. Paracrine support to tube-formation by endothelial cells and to viability of cardiomyocytes was significantly impaired. Treatment with sera of both smokers groups impaired H2O2/NO release balance by CSCs and reduced early phosphorylation of several pathways compared to NS serum, leading to mTOR activation. Cotreatment with rapamycin was able to reduce mTOR phosphorylation and differentiation into aSMA-positive myofibroblasts in CSCs exposed to TCC and HTP sera. In conclusion, the circulating molecules in the serum of chronic exclusive HTP smokers induce fibrotic behavior in CSCs through activation of the mTOR pathway, and reduce their beneficial paracrine effects on endothelial cells and cardiomyocytes. These results point to a potential risk for cardiac fibrosis in chronic HTP users.

Therapeutic effects of exendin-4 on spinal cord injury via restoring autophagy function and decreasing necroptosis in neuron.

Necroptosis is one of programmed death that may aggravate spinal cord injury (SCI). We aimed to investigate the effect and mechanism of exendin-4 (EX-4) on the recovery of motor function and necroptosis after SCI. The SD rats with left hemisection in the T10 spinal cord as SCI model were used. The behavior tests were measured within 4 weeks. The effects of EX-4 on necroptosis-associated proteins and autophagy flux were explored. In addition, the SHSY5Y cell model was introduced to explore the direct effect of EX-4 on neurons. The effect of lysosome was explored using mTOR activator and AO staining. EX-4 could improve motor function and limb strength, promote the recovery of autophagy flux, and accelerate the degradation of necroptosis-related protein at 3 d after injury in rats. EX-4 reduced lysosome membrane permeability, promoted the recovery of lysosome function and autophagy flux, and accelerated the degradation of necroptosis-related proteins by inhibiting the phosphorylation level of mTOR in the SHSY5Y cell model. Our results demonstrated that EX-4 may improve motor function after SCI via inhibiting mTOR phosphorylation level and accelerating the degradation of necroptosis-related proteins in neurons. Our findings may provide new therapeutic targets for clinical treatment after SCI.

Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.

Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status.

20(S)-Ginsenoside Rh2 induces apoptosis and autophagy in melanoma cells via suppressing Src/STAT3 signaling

Background20(S)-Ginsenoside Rh2 (GRh2) has been extensively studied for multifaceted health benefits. However, the anti-melanoma effect of GRh2 remains poorly understood. Herein, the anti-melanoma effects and underlying mechanisms of GRh2 were investigated. MethodsMTT assays, the EdU staining assay, flow cytometric analysis, the cellular thermal shift assay (CETSA), confocal microscope analysis, molecular docking, molecular dynamics (MD), immunoblotting, a B16F10 cell bearing mouse model were adopted to examine the anti-melanoma effect of mechanism of action of GRh2. ResultsIn melanoma cells, GRh2 was found to suppress cell proliferation, arrest cell cycle at G0/G1 phase and evoke apoptosis. GRh2 initiated autophagy and inhibited the activity of mTOR, the autophagy negative regulator, in melanoma cells. Repressing autophagy enhanced the anti-melanoma efficacy of GRh2. Molecular docking, MD and CETSA studies revealed that GRh2 stably bound to Src protein (one of the upstream kinases of STAT3). GRh2 suppressed Src and STAT3 activities, thereof prohibiting STAT3 nuclear translocation in melanoma cells. STAT3 over-activation attenuated the cytotoxic, apoptotic and autophagy inductive effects of GRh2. Additionally, GRh2 suppressed B16F10 tumor growth without inducing obvious toxicity in mice. It downregulated phospho-Src, phospho-STAT3, phospho-mTOR and Mcl-1 protein levels, while elevated cleaved-PARP and LC3B-II protein levels in B16F10 tumors. ConclusionGRh2 exerts anti-melanoma effects through suppressing Src/STAT3 signaling. This study advances our understanding on the anti-melanoma mechanism of GRh2 and indicates that the intake of GRh2 has the potential to retard melanoma progression.

Strength and muscle mass development after a resistance-training period at terrestrial and normobaric intermittent hypoxia

This study investigated the effect of a resistance training (RT) period at terrestrial (HH) and normobaric hypoxia (NH) on both muscle hypertrophy and maximal strength development with respect to the same training in normoxia (N). Thirty-three strength-trained males were assigned to N (FiO2 = 20.9%), HH (2,320 m asl) or NH (FiO2 = 15.9%). The participants completed an 8-week RT program (3 sessions/week) of a full body routine. Muscle thickness of the lower limb and 1RM in back squat were assessed before and after the training program. Blood markers of stress, inflammation (IL-6) and muscle growth (% active mTOR, myostatin and miRNA-206) were measured before and after the first and last session of the program. Findings revealed all groups improved 1RM, though this was most enhanced by RT in NH (p = 0.026). According to the moderate to large excess of the exercise-induced stress response (lactate and Ca2+) in HH and N, results only displayed increases in muscle thickness in these two conditions over NH (ES > 1.22). Compared with the rest of the environmental conditions, small to large increments in % active mTOR were only found in HH, and IL-6, myostatin and miR-206 in NH throughout the training period. In conclusion, the results do not support the expected additional benefit of RT under hypoxia compared to N on muscle growth, although it seems to favour gains in strength. The greater muscle growth achieved in HH over NH confirms the impact of the type of hypoxia on the outcomes.

PDCD4 as a marker of mTOR pathway activation and therapeutic target in mycobacterial infections.

This study emphasizes the critical role of the mammalian target of rapamycin (mTOR) pathway in macrophage responses to mycobacterial infections, elucidating how mycobacteria activate mTOR, resulting in PDCD4 degradation. The utilization of the (Tor)-signal-indicator (TOSI) vector for real-time monitoring of mTOR activity represents a significant advancement in understanding mTOR regulation during mycobacterial infection. These findings deepen our comprehension of mycobacteria's innate immune mechanisms and introduce PDCD4 as a novel marker for mTOR activity in infectious diseases. Importantly, this research laid the groundwork for high-throughput screening of mTOR inhibitors using FDA-approved drugs, offering the potential for repurposing treatments against mycobacterial infections. The identification of drugs that inhibit mTOR activation opens new avenues for host-directed therapies, marking a significant step forward in combating tuberculosis and other mycobacterial diseases.

Glutamine maintains the stability of alveolar structure and function after lung transplantation by inhibiting autophagy

Excessive autophagy may lead to degradation and damage of alveolar epithelial cells after lung transplantation, eventually leading to alveolar epithelial cell loss, affecting the structural integrity and function of alveoli. Glutamine (Gln), a nutritional supplement, regulates autophagy through multiple signaling pathways. In this study, we explored the protective role of Gln on alveolar epithelial cells by inhibiting autophagy. In vivo, a rat orthotopic lung transplant model was carried out to evaluate the therapeutic effect of glutamine. Ischemia/reperfusion (I/R) induced alveolar collapse, edema, epithelial cell apoptosis, and inflammation, which led to a reduction of alveolar physiological function, such as an increase in peak airway pressure, and a decrease in lung compliance and oxygenation index. In comparison, Gln preserved alveolar structure and function by reducing alveolar apoptosis, inflammation, and edema. In vitro, a hypoxia/reoxygenation (H/R) cell model was performed to simulate IR injury on mouse lung epithelial (MLE) cells and human lung bronchus epithelial (Beas-2B) cells. H/R impaired the proliferation of epithelial cells and triggered cell apoptosis. In contrast, Gln normalized cell proliferation and suppressed I/R-induced cell apoptosis. The activation of mTOR and the downregulation of autophagy-related proteins (LC3, Atg5, Beclin1) were observed in Gln-treated lung tissues and alveolar epithelial cells. Both in vivo and in vitro, rapamycin, a classical mTOR inhibitor, reversed the beneficial effects of Gln on alveolar structure and function. Taken together, Glnpreserved alveolar structure and function after lung transplantation by inhibiting autophagy.

Pivotal Role of mTOR in Non-Skin Manifestations of Psoriasis.

Psoriasis is a chronic inflammatory condition affecting 2% of the Western population. It includes diverse manifestations influenced by genetic predisposition, environmental factors, and immune status. The sustained activation of mTOR is a key element in psoriasis pathogenesis, leading to an uncontrolled proliferation of cytokines. Furthermore, mTOR activation has been linked with the transition from psoriasis to non-skin manifestations such as psoriatic arthritis and cardiovascular events. While therapies targeting pro-inflammatory cytokines have shown efficacy, additional pathways may offer therapeutic potential. The PI3K/Akt/mTOR pathway, known for its role in cell growth, proliferation, and metabolism, has emerged as a potential therapeutic target in psoriasis. This review explores the relevance of mTOR in psoriasis pathophysiology, focusing on its involvement in cutaneous and atheromatous plaque proliferation, psoriatic arthritis, and cardiovascular disease. The activation of mTOR promotes keratinocyte and synovial cell proliferation, contributing to plaque formation and joint inflammation. Moreover, mTOR activation may exacerbate the cardiovascular risk by promoting pro-inflammatory cytokine production and dysregulation lipid and glucose metabolism. The inhibition of mTOR has shown promise in preclinical studies, reducing skin inflammation and plaque proliferation. Furthermore, mTOR inhibition may mitigate cardiovascular risk by modulating cholesterol metabolism and attenuating atherosclerosis progression. Understanding the role of mTOR in psoriasis, psoriatic arthritis, and cardiovascular disease provides insight into the potential treatment avenues and sheds light on the complex interplay of the immune and metabolic pathways in these conditions.

Resveratrol Induces Myotube Development by Altering Circadian Metabolism via the SIRT1-AMPK-PP2A Axis.

Resveratrol is a polyphenol known to have metabolic as well as circadian effects. However, there is little information regarding the metabolic and circadian effect of resveratrol on muscle cells. We sought to investigate the metabolic impact of resveratrol throughout the circadian cycle to clarify the associated signaling pathways. C2C12 myotubes were incubated with resveratrol in the presence of increasing concentrations of glucose, and metabolic and clock proteins were measured for 24 h. Resveratrol led to SIRT1, AMPK and PP2A activation. Myotubes treated with increasing glucose concentrations showed higher activation of the mTOR signaling pathway. However, resveratrol did not activate the mTOR signaling pathway, except for P70S6K and S6. In accordance with the reduced mTOR activity, resveratrol led to advanced circadian rhythms and reduced levels of pBMAL1 and CRY1. Resveratrol increased myogenin expression and advanced its rhythms. In conclusion, resveratrol activates the SIRT1-AMPK-PP2A axis, advances circadian rhythms and induces muscle development.

Early Postnatal Exposure to Midazolam Causes Lasting Histological and Neurobehavioral Deficits via Activation of the mTOR Pathway.

Exposure to general anesthetics can adversely affect brain development, but there is little study of sedative agents used in intensive care that act via similar pharmacologic mechanisms. Using quantitative immunohistochemistry and neurobehavioral testing and an established protocol for murine sedation, we tested the hypothesis that lengthy, repetitive exposure to midazolam, a commonly used sedative in pediatric intensive care, interferes with neuronal development and subsequent cognitive function via actions on the mechanistic target of rapamycin (mTOR) pathway. We found that mice in the midazolam sedation group exhibited a chronic, significant increase in the expression of mTOR activity pathway markers in comparison to controls. Furthermore, both neurobehavioral outcomes, deficits in Y-maze and fear-conditioning performance, and neuropathologic effects of midazolam sedation exposure, including disrupted dendritic arborization and synaptogenesis, were ameliorated via treatment with rapamycin, a pharmacologic mTOR pathway inhibitor. We conclude that prolonged, repetitive exposure to midazolam sedation interferes with the development of neural circuitry via a pathologic increase in mTOR pathway signaling during brain development that has lasting consequences for both brain structure and function.

Phytic acid-based nanomedicine against mTOR represses lipogenesis and immune response for metabolic dysfunction-associated steatohepatitis therapy

Abstract Metabolic dysfunction-associated steatohepatitis (MASH) is one of the most common chronic liver diseases and is mainly caused by metabolic disorders and systemic inflammatory responses. Recent studies have indicated that the activation of the mammalian (or mechanistic) target of rapamycin (mTOR) signaling participates in MASH progression by facilitating lipogenesis and regulating the immune microenvironment. Although several molecular medicines have been demonstrated to inhibit the phosphorylation or activation of mTOR, their poor specificity and side effects limit their clinical application in MASH treatment. Phytic acid (PA), as an endogenous and natural antioxidant in the liver, presents significant anti-inflammatory and lipid metabolism-inhibiting functions to alleviate MASH. In this study, considering the unique phosphate-rich structure of PA, we developed a cerium-PA (CePA) nanocomplex by combining PA with cerium ions possessing phosphodiesterase activity. CePA intervened in the S2448 phosphorylation of mTOR through the occupation effect of phosphate groups, thereby inhibiting the inflammatory response and mTOR-sterol regulatory element-binding protein 1 (SREBP1) regulation axis. The in vivo experiments suggested that CePA alleviated MASH progression and fat accumulation in high-fat diet-fed mice. Mechanistic studies validated that CePA exerts a liver-targeted mTOR repressive function, making it a promising candidate for MASH and other mTOR-related disease treatments.

GlyT1 Inhibition by NFPS Promotes Neuroprotection in Amyloid-β-Induced Alzheimer's Disease Animal Model.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β, leading to N-methyl-D-aspartate (NMDA) receptor-dependent synaptic depression, spine elimination, and memory deficits. Glycine transporter type 1 (GlyT1) modulates glutamatergic neurotransmission via NMDA receptors (NMDAR), presenting a potential alternative therapeutic approach for AD. This study investigates the neuroprotective potential of GlyT1 inhibition in an amyloid-β-induced AD mouse model. C57BL/6 mice were treated with N-[3-([1,1-Biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine (NFPS), a GlyT1 inhibitor, 24h prior to intrahippocampal injection of amyloid-β. NFPS pretreatment prevented amyloid-β-induced cognitive deficits in short-term and long-term memory, evidenced by novel object recognition and spatial memory tasks. Moreover, NFPS pretreatment curbed microglial activation, astrocytic reactivity, and subsequent neuronal damage from amyloid-β injection. An extensive label-free quantitative UPLC-MSE proteomic analysis was performed on the hippocampi of mice treated with NFPS. In proteomics, KEGG enrichment analysis revealed increased in dopaminergic synapse, purine-containing compound biosynthetic process and long-term potentiation, and a reduction in Glucose catabolic process and glycolytic process pathways. The western blot analysis confirmed that NFPS treatment elevated BDNF levels, correlating with enhanced TRKB phosphorylation and mTOR activation. Moreover, NFPS treatment reduced the GluN2B expression after 6h, which was associated with an increase on CaMKIV and CREB phosphorylation. Collectively, these findings demonstrate that GlyT1 inhibition by NFPS activates diverse neuroprotective pathways, enhancing long-term potentiation signaling and countering amyloid-β-induced hippocampal damage.

Extensive location bias of the GPCR-dependent translatome via site-selective activation of mTOR.

G protein-coupled receptors (GPCRs) modulate various physiological functions by re-wiring cellular gene expression in response to extracellular signals. Control of gene expression by GPCRs has been studied almost exclusively at the transcriptional level, neglecting an extensive amount of regulation that takes place translationally. Hence, little is known about the nature and mechanisms of gene-specific post-transcriptional regulation downstream of receptor activation. Here, we apply an unbiased multiomics approach to delineate an extensive translational regulatory program initiated by the prototypical beta2-adrenergic receptor (β2-AR) and provide mechanistic insights into how these processes are orchestrated. Using ribosome profiling (Ribo-seq), we identify nearly 120 novel gene targets of adrenergic receptor activity which expression is exclusively regulated at the level of translation. We next show that all translational changes are induced selectively by endosomal β2-ARs. We further report that this proceeds through activation of the mammalian target of rapamycin (mTOR) pathway. Specifically, within the set of translational GPCR targets we discover significant enrichment of genes with 5' terminal oligopyrimidine (TOP) motifs, a gene class classically known to be translationally regulated by mTOR. We then demonstrate that endosomal β2-ARs are required for mTOR activation and subsequent mTOR-dependent TOP mRNA translation. Together, this comprehensive analysis of drug-induced translational regulation establishes a critical role for location-biased GPCR signaling in fine-tuning the cellular protein landscape.

Antitumor activity of afatinib in EGFR T790M-negative human oral cancer therapeutically targets mTOR/Mcl-1 signaling axis.

This study investigates the role and effectiveness of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in oral cancer, focusing on the clinical relevance of EGFR and myeloid cell leukemia-1 (Mcl-1) in head and neck cancers (HNCs). It aims to explore the molecular mechanism of afatinib, a TKI, in treating human oral cancer. We conducted an in silico analysis using databases like The Cancer Genome Atlas, Gene Expression Omnibus, and Clinical Proteomic Tumor Analysis Consortium, along with immunohistochemistry staining, to study EGFR and Mcl-1 expression in HNCs. For investigating afatinib's anticancer properties, we performed various in vitro and in vivo analyses, including trypan blue exclusion assay, Western blotting, 4'-6-diamidino-2-phenylindole staining, flow cytometry, quantitative real-time PCR, Mitochondrial membrane potential assay, overexpression vector construction, transient transfection, and a tumor xenograft model. Higher expression levels of EGFR and Mcl-1 were observed in HNC patient tissues compared to normal tissues, with their co-expression significantly linked to poor prognosis. There was a strong correlation between EGFR and Mcl-1 expressions in oral cancer patients. Afatinib treatment induced apoptosis and suppressed Mcl-1 in oral cancer cell lines without the EGFR T790M mutation. The mechanism of afatinib-induced apoptosis involved the EGFR/mTOR/Mcl-1 axis, as shown by the effects of mTOR activator MHY1485 and inhibitor rapamycin. Afatinib also increased Bim expression, mitochondrial membrane permeabilization, and cytochrome c release. It significantly lowered tumor volume without affecting body, liver, and kidney weights. Afatinib, targeting the EGFR/mTOR/Mcl-1 axis, shows promise as a therapeutic strategy for oral cancer, especially in patients with high EGFR and Mcl-1 expressions.

MiR-181a expressed in the dorsal hippocampus regulates the reinstatement of cocaine CPP by targeting PRKAA1

Neuroadaptive changes in the hippocampus underlie addictive-like behaviors in humans or animals chronically exposed to cocaine. miR-181a, which is widely expressed in the hippocampus, acts as a regulator for synaptic plasticity, while its role in drug reinstatement is unclear. In this study, we found that miR-181a regulates the reinstatement of cocaine conditioned place preference(CPP), and altered miR-181a expression changes the complexity of hippocampal neurons and the density and morphology of dendritic spines. By using a luciferase gene reporter, we found that miR-181a targets PRKAA1, an upstream molecule in the mTOR pathway. High miR-181a expression reduced the expression of the PRKAA1 mRNA and promoted mTOR activity and the reinstatement of cocaine CPP. These results indicate that miR-181a is involved in neuronal structural plasticity induced by reinstatement of cocaine CPP, possibly through the activation of the mTOR signaling pathway. This study provides new microRNA targets and a theoretical foundation for the prevention of cocaine-induced reinstatement.

Hesperidin Alleviates Hepatic Injury Caused by Deoxynivalenol Exposure through Activation of mTOR and AKT/GSK3β/TFEB Pathways.

Deoxynivalenol (DON) is a common agricultural mycotoxin that is chemically stable and not easily removed from cereal foods. When organisms consume food made from contaminated crops, it can be hazardous to their health. Numerous studies in recent years have found that hesperidin (HDN) has hepatoprotective effects on a wide range of toxins. However, few scholars have explored the potential of HDN in attenuating DON-induced liver injury. In this study, we established a low-dose DON exposure model and intervened with three doses of HDN, acting on male C57 BL/6 mice and AML12 cells, which served as in vivo and in vitro models, respectively, to investigate the protective mechanism of HDN against DON exposure-induced liver injury. The results suggested that DON disrupted hepatic autophagic fluxes, thereby impairing liver structure and function, and HDN significantly attenuated these changes. Further studies revealed that HDN alleviated DON-induced excessive autophagy through the mTOR pathway and DON-induced lysosomal dysfunction through the AKT/GSK3β/TFEB pathway. Overall, our study suggested that HDN could ameliorate DON-induced autophagy flux disorders via the mTOR pathway and the AKT/GSK3β/TFEB pathway, thereby reducing liver injury.

Abstract PR007: Delineating functional drivers of esophageal adenocarcinoma to identify synthetic lethal interactions

Abstract Application of molecular targeted therapies for esophageal adenocarcinoma (EAC) has been limited by a lack of druggable oncogenic drivers. We propose that synthetic lethal interactions may provide new opportunities for targeted therapies in EAC. We have taken an integrated multi-omics approach incorporating Perturb-Seq (CRISPR editing combined with single cell RNA sequencing) and in vivo tumorigenesis assays to perform high-throughput characterisation of >70 high-confidence EAC driver genes, and genome-wide CRISPR-Cas9 knockout screens in isogenic models of EAC tumorigenesis to identify disease relevant synthetic lethal genetic interactions. MS-based proteomics, reverse phase protein arrays, polysome profiling and bulk RNA-sequencing of isogenic models were utilised to interrogate the biology of bona fide EAC drivers and associated driver specific gene dependencies. The overall goals were to (i) enhance our understanding of EAC tumorigenesis, (ii) identify potential opportunities for therapeutic interventions targeting EAC drivers via synthetic lethal-like approaches, and (iii) reduce the complexity of genetic heterogeneity by categorising EAC drivers with similar phenotypic outcomes. Through our approach we have identified complex crosstalk between the tumor suppressor SMAD4 and regulation of mTOR signaling, with specific downstream effects on translational reprogramming in EAC. Mutation or loss of SMAD4 occurs in up to 20% of EAC, but not pre-malignant tissue (Barrett’s esophagus), and is sufficient to promote transformation of pre-malignant cells in our in vivo tumorigenesis model. In this model, xenotransplanted SMAD4-deficient (via CRISPR-Cas9 knockout or shRNA knockdown) Barrett’s metaplasia cells formed invasive, metastatic tumors after a period of latency. SMAD4 deficient cells had downregulated expression of 4E-BP1, which inhibits EIF4E, the cap-dependent translation initiation factor. This was accompanied by increased mTOR activity, including phosphorylation and inactivation of 4EBP1. Moreover, we found that SMAD4-deficient cells preferentially upregulate cap-dependent translation at the expense of IRES mediated translation. Furthermore, perturbation of additional negative regulators of mTOR signaling in combination with SMAD4 knockout exacerbated these effects and accelerated tumorigenesis in vivo. We have extended these findings to a model of Barrett’s esophagus patient-derived organoids (PDOs) and observed increased proliferative potential of our genetically modified PDOs. Finally, analysing gene ontologies for differentially expressed genes from Perturb-seq revealed that driver-dependent transcriptional changes can be categorized into a smaller number of functional pathways allowing us to potentially consider groups of drivers as functional units. This work advances our understanding of EAC tumorigenesis, provides new mechanistic insights into SMAD4-driven transformation as well as novel potential therapeutic avenues for SMAD4-deficient EAC. Citation Format: Julia V. Milne, Ebtihal Mustafa, Kenji Fujihara, Eric Kusnadi, Anna Trigos, Niko Thio, Maree Pechlivanis, Carlos Cabalag, Twishi Gulati, Kaylene Simpson, Cuong Duong, Luc Furic, Wayne Phillips, Nicholas Clemons. Delineating functional drivers of esophageal adenocarcinoma to identify synthetic lethal interactions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR007.

γ-Mangosteen, an autophagy enhancer, prevents skin-aging via activating KEAP1/NRF2 signaling and downregulating MAPKs/AP-1/NF-κB-mediated MMPs

BackgroundMangosteens, a naturally occurring xanthones, found abundantly in mangosteen fruits. The anti-skin aging potential of γ-mangosteen (GM) remains unexplored; therefore, we investigated the UVB-induced anti-skin aging of GM via activation of autophagy. HypothesisWe hypothesized that GM exerts antioxidant and anti-aging capabilities both in vitro and in vivo through activation of autophagy as well as control of KEAP1/NRF2 signaling and MAPKs/AP-1/NF-κB-mediated MMPs pathways. MethodsThe anti-skin aging effects of GM were studied using HDF cells and a mice model. Various assays, such as DPPH, ABTS, CUPRAC, FRAP, and ROS generation, assessed antioxidant activities. Kits measured antioxidant enzymes, SA-β-gal staining, collagen, MDA content, si-RNA experiments, and promoter assays. Western blotting evaluated protein levels of c-Jun, c-Fos, p-IκBα/β, p-NF-κB, MAPK, MMPs, collagenase, elastin, KEAP1, NRF2, HO-1, and autophagy-related proteins. ResultsGM exhibited strong antioxidant, collagenase and elastase enzyme inhibition activity surpassing α- and β-mangosteen. GM competitively inhibited elastase with a Ki value of 29.04 µM. GM orchestrated the KEAP1-NRF2 pathway, enhancing HO-1 expression, and suppressed UVB-induced ROS in HDF cells. NRF2 knockdown compromised GM's antioxidant efficacy, leading to uncontrolled ROS post-UVB. GM bolstered endogenous antioxidants, curbing lipid peroxidation in UVB-exposed HDF cells and BALB/c mice. GM effectively halted UVB-induced cell senescence, and reduced MMP-1/-9, while elevated TIMP-1 levels, augmented COL1A1, ELN, and HAS-2 expression in vitro and in vivo. Additionally, it suppressed UVB-induced MAPKs, AP-1, NF-κB phosphorylation. Pharmacological inhibitors synergistically enhanced GM's anti-skin aging potential. Moreover, GM inhibited UVB-induced mTOR activation, upregulated LC3-II, Atg5, Beclin 1, and reduced p62 in both UVB induced HDF cells and BALB/c mice, while blocking of autophagy successfully halt the GM effects against the UVB-induced increase of cell senescence, degradation of collagen through upregulation of MMP-1, underscoring GM's substantial anti-skin aging impact via autophagy induction in vitro and in vivo. ConclusionTogether, GM has potent antioxidant and anti-skin aging ingredients that can be used to formulate skin care products for both the nutraceutical and cosmeceutical industries.

Adiponectin rescues synaptic plasticity in the dentate gyrus of a mouse model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and is the leading known single-gene cause of autism spectrum disorder. Patients with FXS display varied behavioural deficits that include mild to severe cognitive impairments in addition to mood disorders. Currently, there is no cure for this condition; however, there is an emerging focus on therapies that inhibit mechanistic target of rapamycin (mTOR)-dependent protein synthesis owing to the clinical effectiveness of metformin for alleviating some behavioural symptoms in FXS. Adiponectin (APN) is a neurohormone that is released by adipocytes and provides an alternative means to inhibit mTOR activation in the brain. In these studies, we show that Fmr1 knockout mice, like patients with FXS, show reduced levels of circulating APN and that both long-term potentiation (LTP) and long-term depression (LTD) in the dentate gyrus (DG) are impaired. Brief (20 min) incubation of hippocampal slices in APN (50 nM) was able to rescue both LTP and LTD in the DG and increased both the surface expression and phosphorylation of GluA1 receptors. These results provide evidence for reduced APN levels in FXS playing a role in decreasing bidirectional synaptic plasticity and show that therapies which enhance APN levels may have therapeutic potential for this and related conditions.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.