Activation Of mTOR Pathway Research Articles

Luteolin prevents cadmium-induced PC12 cell death by suppressing the Akt/mTOR signaling pathway.

Cadmium (Cd) is an environmental pollutant that can cause neurodegenerative disorders. Luteolin (Lut) is a natural flavonoid compound. However, whether Lut protects against Cd-induced nerve cell death remains unclear. In the present study, PC12 cells were used to investigate the neuroprotective effect of Lut against Cd poisoning. Changes in cell viability, apoptosis, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein expression, and protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway activity were analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst 33258 staining, flow cytometry, and western blotting. Lut markedly attenuated the Cd-induced reduction in cell viability, nuclear fragmentation, condensation, and the decrease in the Bcl-2/Bcl-2-associated X protein ratio in PC12 cells. Furthermore, Lut blocked the Cd-mediated activation of the Akt/mTOR signaling pathway. Moreover, inhibition of the Akt/mTOR signaling pathway with LY294002 (a PI3K inhibitor) enhanced the protective effect of Lut against Cd-induced cell death by suppressing Cd-induced activation of Akt, mTOR, and eukaryotic initiation factor 4E binding protein 1. The results showed that Lut prevented Cd-induced cell death partly by blocking the Akt/mTOR signaling pathway. Lut may be a potential agent for preventing Cd-induced nerve cell damage and neurodegenerative diseases.

Novel Driver Genes and Mutations in Lung Pecoma: A Case Report

Introduction: Perivascular epithelioid cell tumors (PEComa) are rare mesenchymal neoplasms characterized by perivascular epithelioid cells. Despite their common occurrence in the uterus, gastrointestinal tract, and retroperitoneum, this study presents an exceptional case of PEComa identified in the lung, warranting unique molecular exploration. Case Report: A 50-year-old man was diagnosed with a 6 cm neoplasm in the lower lobe of the right lung without enlarged lymph nodes during a routine examination. Thoracotomy, extended lower bilobectomy, and D3 lymphadenectomy were performed. Histological and immunohistochemical analysis diagnosed a PEComa. No conventional TSC1 and TSC2 mutations specific to PEComa, which resulted in mTOR pathway activation, were detected by whole-exome sequencing. In contrast, mutations were unveiled in the MTOR, EIF4EBP1, and PRAME genes that could be an alternative mechanism governing mTOR activation. Conclusion: These findings provide novel insights into the molecular intricacies of lung PEComa, showcasing the distinctive roles of MTOR, EIF4EBP1, and PRAME mutations.

SMAC mimetic BV6 acts in synergy with mTOR inhibitor to increase cisplatin sensitivity in ovarian cancer.

The objective of this study is to observe the antitumor efficacy of the second mitochondria-derived activator of caspases (SMAC) mimetic bivalent smac mimetic (BV6) in combination with target of rapamycin (mTOR) inhibitor on DDP (cisplatin) sensitivity. Ovarian cancer cells were exposed to cisplatin, BV6, DDP + BV6, and DDP + BV6 + mTOR inhibitor Rapamycin. Using proteomics and bioinformatics, protein expression profiles in ovarian cancer were determined. Bagg Albino color nude mice were treated with DDP or BV6 alone or in combination, or BV6 + DDP + Rapamycin. The effects of different treatments on ovarian cancer cells and tumor growth were evaluated in vivo and in vitro. Proteomics and bioinformatics analysis revealed significant changes of protein kinase (AKT)/mTOR pathway. Consistently, western blot data indicated that AKT/mTOR axis was gradually activated in BV6-treated ovarian cancer cells and attenuated the cytotoxic effect of BV6. Functional assays showed that DDP or BV6 treatment alone significantly enhanced the sensitivity and inhibited the migration of ovarian cancer cells, but without any synergistic effects. In addition, combination with BV6 and mTOR inhibitor Rapamycin significantly decreased cell viability and inhibited migration of ovarian cancer cells exposed to DDP. Consistently, the xenograft model showed that co-treatment with Rapamycin with BV6 had significantly suppressed tumor growth and metastasis. Our study demonstrated that SMAC analogue BV6 exhibits a strong anticancer effect on ovarian cancer in vitro and in vivo. Combination with Rapamycin overcomes the activation of mTOR pathway by BV6 and increases the chemosensitivity to DDP. These data suggest a potential application of triple combination with DDP + BV6 + Rapamycin in clinical management of ovarian cancer.

Fat body glycolysis defects inhibit mTOR and promote distant muscle disorganization through TNF-α/egr and ImpL2 signaling in Drosophila larvae

The fat body in Drosophila larvae functions as a reserve tissue and participates in the regulation of organismal growth and homeostasis through its endocrine activity. To better understand its role in growth coordination, we induced fat body atrophy by knocking down several key enzymes of the glycolytic pathway in adipose cells. Our results show that impairing the last steps of glycolysis leads to a drastic drop in adipose cell size and lipid droplet content, and downregulation of the mTOR pathway and REPTOR transcriptional activity. Strikingly, fat body atrophy results in the distant disorganization of body wall muscles and the release of muscle-specific proteins in the hemolymph. Furthermore, we showed that REPTOR activity is required for fat body atrophy downstream of glycolysis inhibition, and that the effect of fat body atrophy on muscles depends on the production of TNF-α/egr and of the insulin pathway inhibitor ImpL2.

The mTOR pathway controls phosphorylation of BRAF at T401

BRAF serves as a gatekeeper of the RAS/RAF/MEK/ERK pathway, which plays a crucial role in homeostasis. Since aberrant signalling of this axis contributes to cancer and other diseases, it is tightly regulated by crosstalk with the PI3K/AKT/mTOR pathway and ERK mediated feedback loops. For example, ERK limits BRAF signalling through phosphorylation of multiple residues. One of these, T401, is widely considered as an ERK substrate following acute pathway activation by growth factors. Here, we demonstrate that prominent T401 phosphorylation (pT401) of endogenous BRAF is already observed in the absence of acute stimulation in various cell lines of murine and human origin. Importantly, the BRAF/RAF1 inhibitor naporafenib, the MEK inhibitor trametinib and the ERK inhibitor ulixertinib failed to reduce pT401 levels in these settings, supporting an alternative ERK-independent pathway to T401 phosphorylation. In contrast, the mTOR inhibitor torin1 and the dual-specific PI3K/mTOR inhibitor dactolisib significantly suppressed pT401 levels in all investigated cell types, in both a time and concentration dependent manner. Conversely, genetic mTOR pathway activation by oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increased pT401, an effect that was reverted by dactolisib and torin1 but not by trametinib. We also show that shRNAmir mediated depletion of the mTORC1 complex subunit Raptor significantly enhanced the suppression of T401 phosphorylation by a low torin1 dose, while knockdown of the mTORC2 complex subunit Rictor was less effective. Using mass spectrometry, we provide further evidence that torin1 suppresses the phosphorylation of T401, S405 and S409 but not of other important regulatory phosphorylation sites such as S446, S729 and S750. In summary, our data identify the mTOR axis and its inhibitors of (pre)clinical relevance as novel modulators of BRAF phosphorylation at T401.

Alternative splicing of ALDOA confers tamoxifen resistance in breast cancer.

The cancer-associated alternative splicing (AS) events generate cancer-related transcripts which are involved in uncontrolled cell proliferation and drug resistance. However, the key AS variants implicated in tamoxifen (TAM) resistance in breast cancer remain elusive. In the current study, we investigated the landscape of AS events in nine pairs of primary and relapse breast tumors from patients receiving TAM-based therapy. We unrevealed a notable association between the inclusion of exon 7.2 in the 5'untranslated region (5'UTR) of ALDOA mRNA and TAM resistance. Mechanistically, the inclusion of ALDOA exon 7.2 enhances the translation efficiency of the transcript, resulting in increased ALDOA protein expression, mTOR pathway activity, and the promotion of TAM resistance in breast cancer cells. Moreover, the inclusion of exon 7.2 in ALDOA mRNA is mediated by MSI1 via direct interaction. In addition, elevated inclusion of ALDOA exon 7.2 or expression of MSI1 is associated with an unfavorable prognosis in patients undergoing endocrine therapy. Notably, treatment with Aldometanib, an ALDOA inhibitor, effectively restrains the growth of TAM-resistant breast cancer cells in vitro and in vivo. The present study unveils the pivotal role of an AS event in ALDOA, under the regulation of MSI1, in driving TAM resistance in breast cancer. Therefore, this study provides a promising therapeutic avenue targeting ALDOA to combat TAM resistance.

Rumen-protected methionine supplementation during the transition period under artificially induced heat stress: Effects on cow-calf performance

This study aimed to evaluate the effects of rumen-protected Met on lactation performance, inflammation and immune response, and liver glutathione of lactating dairy cows during a subclinical mastitis challenge (SMC). Thirty-two Holstein cows (145 ± 51 DIM) were enrolled in a randomized complete block design. At -21 d relative to the SMC, cows were assigned to dietary treatments, and data were collected before and during the SMC. Cows were blocked according to parity, DIM, and milk yield and received a basal diet (17.4% CP; Lys 7.01% MP and Met 2.14% MP) plus 100 g/d of ground corn (CON; n = 16) or a basal diet plus 100 g/d of ground corn and rumen-protected Met (SM, Smartamine M at 0.09% of dietary DM; n = 16), fed as a top-dress. At 0 d, the mammary gland's rear right quarter was infused with 100,000 cfu of Streptococcus uberis (O140J). Milk yield was recorded twice daily from 0 until 3 d relative to SMC. Milk samples were collected during each milking from 0 to 3 d relative to SMC, blood samples were collected at 0, 6, 12, 24, 48, and 72 h relative to SMC. The mTOR pathway activation was assessed in immune cells in blood and milk samples by measuring quantity and phosphorylation status of mTOR-related proteins, including AKT, S6RP, and 4EBP1. For the ratio of phosphorylated to total AKT, S6RP, and 4EBP1, blood samples were collected at 0, 12, and 24 h, and milk samples at 24 h relative to SMC. Liver biopsies were performed at -10 d and 24 h relative to SMC for measurement of glutathione. Linear mixed models with repeated measures were used to analyze the results. There was a trend for greater milk yield per milking (+ 0.8 kg) and per day (+1.7 kg) after SMC in SM cows compared with CON. The DMI was not affected by dietary treatments. Reactive oxygen metabolites (ROM) were lower in SM cows than in CON. Milk somatic cell linear score was not affected by dietary treatments, and a score >4 at 24 h confirmed subclinical mastitis. The SM cows had greater milk fat percentage at 24 and 36 h post SMC, resulting in overall greater milk fat. Milk protein tended to be greater in SM cows than in CON. We observed greater liver glutathione in SM cows than in CON. Among inflammation biomarkers, ceruloplasmin was lower for SM cows compared with CON. In milk, greater pAKT:AKT and pS6RP:S6RP ratios were observed in immune cell populations from SM cows compared with CON. Blood neutrophils had a greater p4EBP1:4EBP1 ratio in SM cows compared with CON. Overall, our results show that Met supplementation during an SMC positively affected milk performance, lowered the risk of oxidative stress, and attenuated inflammation partially by increasing liver glutathione and immune cells' protein synthesis via mTOR signaling.

Complement C5a Receptor Signaling in Macrophages Enhances Trained Immunity Through mTOR Pathway Activation.

Complement C5a receptor (C5aR) signaling in immune cells has various functions, inducing inflammatory or anti-inflammatory responses based on the type of ligand present. The Co1 peptide (SFHQLPARSRPLP) has been reported to activate C5aR signaling in dendritic cells. We investigated the effect of C5aR signaling via the Co1 peptide on macrophages. In peritoneal macrophages, the interaction between C5aR and the Co1 peptide activated the mTOR pathway, resulting in the production of pro-inflammatory cytokines. Considering the close associations of mTOR signaling with IL-6 and TNF-α in macrophage training, our findings indicate that the Co1 peptide amplifies β-glucan-induced trained immunity. Overall, this research highlights a previously underappreciated aspect of C5aR signaling in trained immunity, and posits that the Co1 peptide is a potentially effective immunomodulator for enhancing trained immunity.

Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8+ T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

PIM1 is a potential therapeutic target for the leukemogenic effects mediated by JAK/STAT pathway mutations in T-ALL/LBL

Precursor T-cell neoplasms (T-ALL/LBL) are aggressive hematological malignancies that arise from the malignant transformation of immature thymocytes. Despite the JAK/STAT pathway is recurrently altered in these neoplasms, there are not pharmacological inhibitors officially approved for the treatment of T-ALL/LBL patients that present oncogenic JAK/STAT pathway mutations. In the effort to identify potential therapeutic targets for those patients, we followed an alternative approach and focused on their transcriptional profile. We combined the analysis of molecular data from T-ALL/LBL patients with the generation of hematopoietic cellular models to reveal that JAK/STAT pathway mutations are associated with an aberrant transcriptional profile. Specifically, we demonstrate that JAK/STAT pathway mutations induce the overexpression of the PIM1 gene. Moreover, we show that the pan-PIM inhibitor, PIM447, significantly reduces the leukemogenesis, as well as the aberrant activation of c-MYC and mTOR pathways in cells expressing different JAK/STAT pathway mutations, becoming a potential therapeutic opportunity for a relevant subset of T-ALL/LBL patients.

Acute rapamycin treatment reveals novel mechanisms of behavioral, physiological, and functional dysfunction in a maternal inflammation mouse model of autism and sensory over-responsivity.

Maternal inflammatory response (MIR) during early gestation in mice induces a cascade of physiological and behavioral changes that have been associated with autism spectrum disorder (ASD). In a prior study and the current one, we find that mild MIR results in chronic systemic and neuro-inflammation, mTOR pathway activation, mild brain overgrowth followed by regionally specific volumetric changes, sensory processing dysregulation, and social and repetitive behavior abnormalities. Prior studies of rapamycin treatment in autism models have focused on chronic treatments that might be expected to alter or prevent physical brain changes. Here, we have focused on the acute effects of rapamycin to uncover novel mechanisms of dysfunction and related to mTOR pathway signaling. We find that within 2 hours, rapamycin treatment could rapidly rescue neuronal hyper-excitability, seizure susceptibility, functional network connectivity and brain community structure, and repetitive behaviors and sensory over-responsivity in adult offspring with persistent brain overgrowth. These CNS-mediated effects are also associated with alteration of the expression of several ASD-,ion channel-, and epilepsy-associated genes, in the same time frame. Our findings suggest that mTOR dysregulation in MIR offspring is a key contributor to various levels of brain dysfunction, including neuronal excitability, altered gene expression in multiple cell types, sensory functional network connectivity, and modulation of information flow. However, we demonstrate that the adult MIR brain is also amenable to rapid normalization of these functional changes which results in the rescue of both core and comorbid ASD behaviors in adult animals without requiring long-term physical alterations to the brain. Thus, restoring excitatory/inhibitory imbalance and sensory functional network modularity may be important targets for therapeutically addressing both primary sensory and social behavior phenotypes, and compensatory repetitive behavior phenotypes.

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.

Quantitative cellular pathology of the amygdala in temporal lobe epilepsy and correlation with magnetic resonance imaging volumetry, tissue microstructure, and sudden unexpected death in epilepsy risk factors.

Amygdala enlargement can occur in temporal lobe epilepsy, and increased amygdala volume is also reported in sudden unexpected death in epilepsy (SUDEP). Apnea can be induced by amygdala stimulation, and postconvulsive central apnea (PCCA) and generalized seizures are both known SUDEP risk factors. Neurite orientation dispersion and density imaging (NODDI) has recently provided additional information on altered amygdala microstructure in SUDEP. In a series of 24 surgical temporal lobe epilepsy cases, our aim was to quantify amygdala cellular pathology parameters that could predict enlargement, NODDI changes, and ictal respiratory dysfunction. Using whole slide scanning automated quantitative image analysis methods, parallel evaluation of myelin, axons, dendrites, oligodendroglia, microglia, astroglia, neurons, serotonergic networks, mTOR-pathway activation (pS6) and phosphorylated tau (pTau; AT8, AT100, PHF) in amygdala, periamygdala cortex, and white matter regions of interest were compared with preoperative magnetic resonance imaging data on amygdala size, and in 13 cases with NODDI and evidence of ictal-associated apnea. We observed significantly higher glial labeling (Iba1, glial fibrillary acidic protein, Olig2) in amygdala regions compared to cortex and a strong positive correlation between Olig2 and Iba1 in the amygdala. Larger amygdala volumes correlated with lower microtubule-associated protein (MAP2), whereas higher NODDI orientation dispersion index correlated with lower Olig2 cell densities. In the three cases with recorded PCCA, higher MAP2 and pS6-235 expression was noted than in those without. pTau did not correlate with SUDEP risk factors, including seizure frequency. Histological quantitation of amygdala microstructure can shed light on enlargement and diffusion imaging alterations in epilepsy to explore possible mechanisms of amygdala dysfunction, including mTOR pathway activation, that in turn may increase the risk for SUDEP.

N2M2/NOA-20: Phase I/IIa umbrella trial of molecularly matched targeted therapies plus radiotherapy in patients with newly diagnosed glioblastoma without MGMT promoter hypermethylation.

2000 Background: Patients with glioblastoma without MGMT promoter hypermethylation are unlikely to benefit from temozolomide (TMZ). Trials aiming at replacing TMZ with targeted agents in not molecularly selected patient populations have failed. Methods: This phase I/IIa umbrella trial aimed at showing safety, feasibility, and preliminary efficacy of targeted compounds in addition to standard radiotherapy initiated within 42 days postoperatively. Molecular diagnostics and bioinformatic evaluation are performed within 28 days after surgery. Stratification for treatment takes place in five subtrials, including alectinib, idasanutlin, palbociclib, vismogedib and temsirolimus, according to the best matching molecular alteration. Patients without matching alterations are randomized between subtrials without strong biomarkers using atezolizumab and asunercept and TMZ as standard of care. Primary objective of the phase I parts of the trial was dose finding or dose validation. In the phase IIa trials, centrally determined progression-free survival at six months (PFS-6) is used as endpoint for efficacy with interim analyses for futility ( H0: p=0.231). Results: From May 2018 through July 2022, 301 patients were enrolled and 228 treated in 13 German NOA sites. The alectinib and vismodegib subtrials were closed since no molecularly matching patients were accrued; the idasanutlin subtrial was closed prior to the optimal dose at nine patients at discretion of the company providing the drug. The TMZ subtrial showed a PFS-6 of 18.52% (10/54 patients) (p=0.831) and a median overall survival (OS) of 12.1 months. Asunercept: PFS-6 of 15.4% (4/26) (p=0.8825) and OS of 12.8 months. Atezolizumab: PFS-6 of 21.4% (9/42) (p=0.660) and OS of 11.7 months. Palbociclib with patients demonstrating CDK4 amplification or CDKN2A/B codeletion: PFS-6 of 24.4% (10/41) (p=0.4823) and OS of 12.6 months. Temsirolimus with patients demonstrating mTOR activation: PFS-6 of 39.1% (18/46) (p=0.0109) and OS of 15.4 months. The regimen-limiting toxicity (RLT)-rate is 34.8%, which is insignificantly above the predefined unacceptable rate for RLTs of 30%. Most RLTs had severity grade 3, one RLT had severity grade 4. No RLTs resulted in death. Conclusions: N2M2 allows for elaborate molecular testing being integrated into the treatment decision and efficient determination of treatment activity for patients with newly diagnosed glioblastoma. There is clinical activity of temsirolimus in patients with tumors harboring an activated mTOR pathway although this is not positively prognostic without mTOR inhibition; there is no clinical activity for asunercept and atezolizumab in not molecularly selected patients and also palbociclib in molecularly selected patients. Clinical trial information: NCT03158389 .

High glucose enhances fibrosis in human annulus fibrosus cells by activating mTOR, PKCδ, and NF-κB signaling pathways.

Low back pain stands as a significant factor in disability, largely resulting from intervertebral disc degeneration (IVDD). High glucose (HG) levels have been implicated in the pathogenesis of IVDD. However, the detailed mechanism of HG in IVDD is largely unknown. Our clinical results revealed that fibrosis markers such as CTGF, Col1a1, ATF4, and EIF2 are highly expressed in advanced-stage IVDD patients. Stimulation of human annulus fibrosus cells (HAFCs) with HG, but not mannitol, promotes fibrosis protein production. Ingenuity Pathway Analysis in the GSE database found that the mTOR, PKCδ, and NF-κB pathways were significantly changed during IVDD. The mTOR, PKCδ, and NF-κB inhibitors or siRNAs all abolished HG-induced fibrosis protein production. In addition, treatment of HAFCs with HG enhances the activation of mTOR, PKCδ, and NF-κB pathways. Thus, HG facilitates fibrosis in IVDD through mTOR, PKCδ, and NF-κB pathways. These results underscore the critical role of HG as a fibrotic factor in the progression of IVDD.

A role of dentate gyrus mechanistic target of rapamycin activation in epileptogenesis in a mouse model of posttraumatic epilepsy.

The mechanistic target of rapamycin (mTOR) pathway has been implicated in promoting epileptogenesis in animal models of acquired epilepsy, such as posttraumatic epilepsy (PTE) following traumatic brain injury (TBI). However, the specific anatomical regions and neuronal populations mediating mTOR's role in epileptogenesis are not well defined. In this study, we tested the hypothesis that mTOR activation in dentate gyrus granule cells promotes neuronal death, mossy fiber sprouting, and PTE in the controlled cortical impact (CCI) model of TBI. An adeno-associated virus (AAV)-Cre viral vector was injected into the hippocampus of Rptorflox/flox (regulatory-associated protein of mTOR) mutant mice to inhibit mTOR activation in dentate gyrus granule cells. Four weeks after AAV-Cre or AAV-vehicle injection, mice underwent CCI injury and were subsequently assessed for mTOR pathway activation by Western blotting, neuronal death, and mossy fiber sprouting by immunopathological analysis, and posttraumatic seizures by video-electroencephalographic monitoring. AAV-Cre injection primarily affected the dentate gyrus and inhibited hippocampal mTOR activation following CCI injury. AAV-Cre-injected mice had reduced neuronal death in dentate gyrus detected by Fluoro-Jade B staining and decreased mossy fiber sprouting by ZnT3 immunostaining. Finally, AAV-Cre-injected mice exhibited a decrease in incidence of PTE. mTOR pathway activation in dentate gyrus granule cells may at least partly mediate pathological abnormalities and epileptogenesis in models of TBI and PTE. Targeted modulation of mTOR activity in this hippocampal network may represent a focused therapeutic approach for antiepileptogenesis and prevention of PTE.

Lack of p38 activation in T cells increases IL-35 and protects against obesity by promoting thermogenesis

Obesity is characterized by low-grade inflammation, energy imbalance and impaired thermogenesis. The role of regulatory T cells (Treg) in inflammation-mediated maladaptive thermogenesis is not well established. Here, we find that the p38 pathway is a key regulator of T cell-mediated adipose tissue (AT) inflammation and browning. Mice with T cells specifically lacking the p38 activators MKK3/6 are protected against diet-induced obesity, leading to an improved metabolic profile, increased browning, and enhanced thermogenesis. We identify IL-35 as a driver of adipocyte thermogenic program through the ATF2/UCP1/FGF21 pathway. IL-35 limits CD8+ T cell infiltration and inflammation in AT. Interestingly, we find that IL-35 levels are reduced in visceral fat from obese patients. Mechanistically, we demonstrate that p38 controls the expression of IL-35 in human and mouse Treg cells through mTOR pathway activation. Our findings highlight p38 signaling as a molecular orchestrator of AT T cell accumulation and function.

LncRNA FOXD2-AS1 promotes the growth, invasion and migration of OSCC cells by regulating the MiR-185–5p/PLOD1/Akt/mTOR pathway

Although lncRNAs are recognized to contribute to the development of oral squamous-cell carcinoma (OSCC), their exact function in invasion and cell migration is not clear. In this research, we explored the molecular and cellular mechanisms of FOXD2-AS1 in OSCC. Prognostic and bioinformatics analyses were used to test for the differential expression of FOXD2-AS1-PLOD1. Following FOXD2-AS1 suppression or overexpression, changes in cell viability were measured using the CCK-8 test; changes in cell migration and invasion abilities were measured using the migration and the Transwell assay. The expression of associated genes and proteins was found using Western blot and RT-qPCR. Analysis of luciferase reporter genes was done to look for regulatory connections between various molecules. The FOXD2-AS1-PLOD1 pair, which was highly expressed in OSCC, was analyzed and experimentally verified to be closely related to the prognosis of OSCC, and a nomogram model and correction curve were constructed. The inhibition of FOXD2-AS1 resulted in the reduction of cell activity, migration, invasion ability and changes in genes related to invasion and migration. In vivo validation showed that inhibition of FOXD2-AS1 expression slowed tumor growth, and related proteins changed accordingly. The experiments verified that FOXD2-AS1 negatively regulated miR-185–5 p and that miR-185–5 p negatively regulated PLOD1. In addition, it was found that the expression of PLOD1, p-Akt and p-mTOR proteins in OSCC cells was reduced by the inhibition of FOXD2-AS1, and FOXD2-AS1 and PLOD1 were closely related to the Akt/mTOR pathway. Increased expression of FOXD2-AS1 promotes OSCC growth, invasion and migration, which is important in part by targeting miR-185–5 p/PLOD1/Akt/mTOR pathway activity.

Abstract PO5-13-09: Clinical usage of a CLIA accredited protein/phosphoprotein assay for breast cancer: Primary usage cases and significant findings in the HER2- setting

Abstract Background: A CLIA-accredited assay that measures the expression and/or activation levels of 32 clinically actionable protein and phosphoprotein analytes in clinical tissue biopsy samples was launched in January 2021. The current intended use population for the assay is for patients with late/advanced stage breast cancer wherein molecular profiling is being utilized for clinical decision making. Assessment of physician ordering practices, clinical use dynamics as well as protein/phosphoprotein actionability frequencies were determined for clinically actionable biomarkers. Methods: A total of 219 cases have been processed to date, with mean turn-around time of 16.4 days. The assay utilizes laser capture microdissection to enrich tumor epithelium from FFPE core needle biopsy and resection material coupled to reverse phase protein array. Results for each of the 32 analytes are generated by comparing each patient to a reference population and reported both as a relative quartile and as a continuous percentile compared to the reference population (0-100%). For the purposes of this analysis, significant expression/activation cutpoints were set at the 40th percentile threshold. Results: To date, clinicians have requested the test most frequently to identify potential therapeutic targets in patients with HER2-negative disease (84% of all tested patients, 36% were triple-negative). Despite the majority of cases being HER2-negative, we found that phosphorylated (p) HER2 (Y1248) was found to be activated in 17% of all HER2-negative cases and 19% of triple negative breast cancer cases (TNBC). pHER2 was concurrently activated with pHER3 (Y1289) in 15% (12/80) of TNBC cases and 13% (24/187) of all HER2-negative cases. pHER2 presented concurrently with pEGFR (Y1068 or T654) in 14% (11/80) of TNBC cases and 14% (26/187) of all HER2- cases. Expression of TROP2 was observed in 16% of HER2-negative patients including 11% of ER+/HER2- and 23% of TNBC cancers. MHCII expression, a recently described candidate predictive biomarker of anti-PDL1/PD1 response in TNBC, was found to be expressed in 29% of TNBC patients. Activation frequencies of other clinically relevant drug targets in HER2-negative disease such AKT and mTOR pathway activation was also assessed. We found that pAKT (S473 and/or T308) was activated in 7% of ER+/HER2- cases and 14% of TNBC. Concurrent with AKT activation, at least one additional member of the AKT-mTOR pathway (mTORC1 (S2448), 4E-BP1 (S65), p70S6K (T389), or S6RP (S235/S236) was co-activated in 50% and 45% of ER+/HER2- and TNBC cancers respectively. Conclusion: This initial assessment of a CLIA LDT protein/phosphoprotein assay utilized to date found that physicians most often order the test for advanced stage HER2-negative breast cancer. Activation/expression of clinically important protein drug targets with potential actionability in HER2-negative disease was observed in a significant number of cases and demonstrates the power of the assay to elucidate actionable targets for potential therapeutic intervention. Citation Format: Claudius Mueller, Brian Corgiat, Emanuel Petricoin, Kayla Sparks, Joyce O'Shaughnessy, Chelsea Gawryletz, Matthew Schwartz, Justin Davis. Clinical usage of a CLIA accredited protein/phosphoprotein assay for breast cancer: Primary usage cases and significant findings in the HER2- setting [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-09.

Molecular mechanisms of the effects of mulberry (Morus Rubra) extracts on UV-radiation and oxidative stress-induced skin cell damage

Solar ultraviolet (UV) radiation, while beneficial for the homeostasis of human skin at proper doses, causes skin cell damage under extreme conditions, with a result of photoaging and skin cancer. With the depletion of the ozone layer, the prevalence of UV-induced skin cancer including melanoma has increased dramatically worldwide. Unfortunately, that poses a public health challenge for almost all countries. Over the past two decades, we have been working extensively on understanding the cellular and molecular mechanisms through which UV radiation induces skin aging and skin cancer. We have observed that UV radiation activates cell surface receptors, more pronouncedly EGFR, leading to a cascade of activation of MAP kinase pathways, with a result of MMP activation and in turn matrix degradation, and apoptosis. We have also found that UV radiation induces the downregulation of water channel aquaporins and skin cell dehydration. This novel discovery together with other reports has resulted in the emergence of numerous skin care products. Moreover, UV radiation induces the generation of reactive oxygen species, which exacerbates skin cell damage. With this important line of discovery, more skin care products with added antioxidants have been developed. Mulberries ( Morus Rubra) have been known to be rich in antioxidants and applied to cosmetic products in Asian countries with reported effcacy although the cellular and molecular mechanisms of its actions remain elusive. The objective of this study was to investigate these mechanisms from various perspectives. We hypothesize that the mulberry extracts affect cell signaling pathways that lead to protection against UV radiation or oxidative stressed-induced cell damage. First, we extracted the antioxidants with methanol from two types of mulberries, red and white. Human skin keratinocytes (HaCaT cells) were treated with UV radiation and hydrogen peroxide (H2O2), with and without mulberry extracts. Confocal microscopy and Western blot analysis were employed to measure various cell signaling components. The results showed that mulberry extracts protect against UV radiation and H2O2 induced cell death, with red extracts showing better protection than white extracts. Mulberry extracts attenuate H2O2-induced mitochondrial activity as tested by MitoTracker™ Red CMXRos. Our previous studies have shown that UV radiation induces mTOR pathway activation evidenced by elevation of phosphorylated S6. Interestingly, in this study, Western blot analysis data indicated that mulberry extracts potentiate UV-induced S6 phosphorylation and H2O2-induced MEK activation. Furthermore, confocal data demonstrated that mulberry extract pretreatment inhibits IL1β induced NFκB p65 translocation from the cytoplasm to the nucleus. Taken all together, our data suggest that mulberry extracts containing abundant antioxidants affect MAPK kinase and mTOR pathways and modulate cell signaling pathways associated with skin cell survival and inflammation under UV radiation and oxidative stress. Funding from the Committee on Aid to Faculty Research (CAFR) from Providence College. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.