mTOR Research Articles

A novel acquired resistance mechanism to 5-aminolevulinic acid-mediated photodynamic therapy with ABCG2 inhibition.

We report the occurrence of acquired tumor cell resistance to 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) in combination with ABCG2 inhibition. ALA-PDT in combination with either an ABCG2 tool inhibitor Ko143 or a repurposed clinically-relevant ABCG2 inhibitor lapatinib was highly effective in eradicating the H4 human glioma cells, resulting in minimal cell survival after treatment. However, after seven rounds of repeated treatments with light dose escalation, the resultant tumor cells became resistant to the combination therapy. The resistant sublines and the parental cell line showed similar ABCG2 activities and protein levels, indicating that it was not ABCG2 that caused the resistance. They also exhibited similar responses to PpIX-PDT and mTOR inhibitor AZD2014, suggesting that alterations in PDT sensitivity and mTOR pathway had little contribution to the development of resistance phenotype. By determining the intracellular and extracellular PpIX levels, the activities and protein levels of heme biosynthesis enzymes, we found that porphobilinogen deaminase (PBGD) activity and protein level were significantly reduced in the resistant sublines, causing resistance to PDT by substantially reducing PpIX biosynthesis. A novel acquired resistance mechanism to ALA-PDT with ABCG2 inhibition has been uncovered.

Increased oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in mice with surgically induced osteoarthritis.

Chondrocytes can shift their metabolism to oxidative phosphorylation (OxPhos) in the early stages of osteoarthritis (OA), but as the disease progresses, this metabolic adaptation becomes limited and eventually fails, leading to mitochondrial dysfunction and oxidative stress. Here we investigated whether enhancing OxPhos through the inhibition of pyruvate dehydrogenase kinase (PDK) 2 affects the metabolic flexibility of chondrocytes and cartilage degeneration in a surgical model of OA. Among the PDK isoforms, PDK2 expression was increased by IL-1β in vitro and in the articular cartilage of the DMM model in vivo, accompanied by an increase in phosphorylated PDH. Mice lacking PDK2 showed significant resistance to cartilage damage and reduced pain behaviors in the DMM model. PDK2 deficiency partially restored OxPhos in IL-1β-treated chondrocytes, leading to increases in APT and the NAD+/NADH ratio. These metabolic changes were accompanied by a decrease in reactive oxygen species and senescence in chondrocytes, as well as an increase in the expression of antioxidant proteins such as NRF2 and HO-1 after IL-1β treatment. At the signaling level, PDK2 deficiency reduced p38 signaling and maintained AMPK activation without affecting the JNK, mTOR, AKT and NF-κB pathways. p38 MAPK signaling was critically involved in reactive oxygen species production under glycolysis-dominant conditions in chondrocytes. Our study provides a proof of concept for PDK2-mediated metabolic reprogramming toward OxPhos as a new therapeutic strategy for OA.

Proteomic analysis and effects on osteogenic differentiation of exosomes from patients with ossification of the spinal ligament

Abstract Ossification of the spinal ligament (OSL), including ossification of the posterior longitudinal ligament (OPLL) and ossification of the ligamentum flavum (OLF), is a multifactorial disease that includes genetic predisposition. The association between the rate of ossification in the spinal canal and the severity of myelopathy symptoms is well known, but the degree of progression varies widely among patients. Although many candidate genes and biomarkers have been reported, there are no definitive and quantitative conclusions to date, probably because of low reproducibility due to individual differences. In this study, we focused on exosomes secreted by ossified spinal ligament cells. Exosomes are crucial for intercellular communication during development and progression of disease. In a co-culture study of non-OLF cells with OLF cells, there was increased osteogenic differentiation, including Runx2 and Wnt3a expression, with use of exosome-penetrating filters (1.2 μm) compared to exosome-non-penetrating filters (0.03 μm). Dose-dependent increases in alkaline phosphatase activity and mineral deposition were observed in non-OLF cells treated with OLF-derived exosomes. These results support the hypothesis that OLF-derived exosomes are involved in regulation of osteogenic differentiation. In comparative proteomics analysis, 32 factors were increased and 40 were decreased in OLF-derived exosomes compared to non-OLF-derived exosomes. Molecular network analysis of these 72 factors indicated 10 significant pathways, including the MMP signaling, mTOR signaling, Wnt signaling and VDR-associated pathways. Among the upregulated exosomal membrane proteins in OLF samples, COL IV, FMNL3, mTORC2, and PIP4K showed increased expression with greater ossification, suggesting they may serve as biomarkers of disease activity and therapeutic targets. These factors are involved in the PI3K/Akt/mTOR signaling pathway, and particularly mTOR is known to regulate osteogenic and chondrogenic differentiation. In contrast, FABP5, several KRT family proteins, S100A8, SERPINB3, and TGM, were significantly downregulated in OLF-derived exosomes. These findings provide novel insights into the molecular mechanisms underlying OSL pathogenesis.

Cabergoline-induced NDFIP1 upregulation in pituitary neuroendocrine tumor cells activates mTOR signaling and contributes to cabergoline resistance.

To investigate the molecular mechanisms underlying resistance to dopamine agonists (DA). LC-MS/MS analysis was performed on rat pituitary neuroendocrine tumors (PitNET) cell line GH3 to identify differentially expressed proteins induced by cabergoline (CAB) treatment. A total of 180 human PITNET samples were subjected to transcriptome analysis. Immunohistochemistry (IHC) was conducted on 29 tumor samples to validate NDFIP1 alteration. A xenograft mouse model was established by subcutaneously injecting GH3 cells, with or without NDFIP1 overexpression, into nude mice to investigate tumor growth. PitNET cell lines were treated with CAB. Cell proliferation was assessed using the CCK-8, and protein expression levels were examined through Western blot analysis. CAB treatment upregulated FDFT1 and NDFIP1 protein expression in GH3 cells, with NDFIP1 showing a significant positive correlation with tumor size, as confirmed by IHC results. MMQ and GH3 cells overexpressing NDFIP1 exhibited enhanced viability and reduced sensitivity to CAB. In vivo experiments demonstrated that subcutaneous injection of NDFIP1-overexpressing GH3 cells led to enhanced tumor growth compared to parental GH3 cells. Although the total levels of PTEN remained unaltered, NDFIP1 overexpression induced PTEN nuclear translocation, potentially activating the mTOR pathway. This was supported by increased phosphorylation of key mTOR pathway components, including p-AKT and p-4EBP1, in cells overexpressing NDFIP1. CAB treatment induces the upregulation of NDFIP1 in PitNET cells, which correlates with tumor size and contributes to reduced CAB sensitivity, potentially through activation of the mTOR pathway. NDFIP1 as a potential therapeutic target for overcoming DA resistance in PitNET patients.

CN7:1h Alleviates Inflammation, Apoptosis and Extracellular Matrix Degradation in Osteoarthritis by Modulating the NF-κB and mTOR Pathways.

Osteoarthritis (OA) is a degenerative joint disease with a complex aetiology, which includes inflammation, cellular growth dysregulation and extracellular matrix (ECM) degradation. This study investigated the therapeutic potential of a small-molecule compound, 2-amino-4-(3,4,5-trimethoxyphenyl)-4H-benzo[h]chromene-3-carbonitrile (CN7:1h) in modulating these critical biochemical pathways in OA. Cellular models and rat models of OA were used to explore the impact of CN7:1h on the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and mechanistic target of rapamycin (mTOR) signalling pathways. Parameters such as autophagy, apoptosis and ECM preservation were evaluated. CN7:1h demonstrated a non-cytotoxic profile at a concentration as high as 140 μM as confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. At a concentration of 5 μM, CN7:1h was shown to inhibit the activation of NF-κB and mTOR pathways. CN7:1h was also shown to promote autophagy and reduce apoptosis in cellular models. In rat models, CN7:1h facilitated cartilage repair and demonstrating the therapeutic efficacy of this compound. In conclusion, CN7:1h is a promising bioactive compound for the modulation of key biochemical pathways with therapeutic benefits in degenerative conditions, such as OA. Its high bioavailability and lack of cytotoxicity make CN7:1h an excellent candidate for further research aimed at clinical applications.

Engagement of peroxisome proliferator-activated receptor gamma (PPARγ) and mammalian target of rapamycin (mTOR) in the triclosan-induced disruption of Cyp450 enzyme activity in an in vitro model of mouse embryo fibroblasts (3T3-L1).

Triclosan (TCS) is commonly used worldwide due to its bactericidal and antifungal properties. There are data suggesting the involvement of aryl hydrocarbon receptors (AhR) and peroxisome proliferator-activated receptors (PPARγ). Since the effect of TCS on mouse fibroblasts has not been described so far, we decided to investigate the mechanism of action of this compound in the mouse embryonic fibroblast cell line (3T3-L1). Our results showed that high µM concentrations of TCS increased caspase-3 activity and decreased cell viability after 24-h exposure. The molecular analysis confirmed that 1 µM TCS decreased Ki67 mRNA expression and PCNA protein expression with a similar tendency to that of AhR. The analyses of mRNA levels after treatment with αNF or βNF alone and αNF in combination with TCS showed an increase in Ki67 mRNA expression. TCS alone increased AhR mRNA but had different effects on Cyp1a1 and Cyp1b1 expression. These results suggest the involvement of the PPARγ pathway in the inhibition of Cyp1b1 by TCS. After the TCS exposure, we observed a decrease in PPARγ, and this effect was enhanced in the presence of an AhR agonist and antagonist. These results support the theory about the interaction between the AhR and PPARγ pathways. In the experiments, the strongest increase in PI3K protein expression was observed in the group treated simultaneously with TCS and βNF. Changes in the PI3K level were reflected in changes in the examined mTOR protein. TCS caused a decrease in both mTOR and Cyp1b1 after 24 hours, while opposite effects were observed after 48 hours. Given the crucial role of Cyp1b1, PPARγ, and mTOR in cellular metabolism, we can conclude that TCS is able to disrupt a number of cellular processes. Our data suggest that TCS reduces the metabolism of this xenobiotic in mouse preadipocytes.

Metabolic Reprogramming in Epstein-Barr Virus Associated Diseases.

Epstein-Barr virus (EBV) is the first human cancer-causing viral pathogen to be discovered; it has been epidemiologically associated with a wide range of diseases, including cancers, autoimmunity, and hyperinflammatory disorders. Its evolutionary success is underpinned by coordinated expression of viral transcription factors (EBV nuclear antigens), signaling proteins (EBV latent membrane proteins), and noncoding RNAs, which orchestrate cell transformation, immune evasion, and dissemination. Each of those activities entails significant metabolic rewiring, which is achieved by viral subversion of key host metabolic regulators such as the mammalian target of rapamycin (mTOR), MYC, and hypoxia-inducible factor (HIF). In this review, we systemically discuss how EBV-encoded factors regulate metabolism to achieve viral persistence and propagation, as well as potential research questions and directions in EBV-driven metabolism.

Targeting SIRT1 by Scopoletin to Inhibit XBB.1.5 COVID-19 Life Cycle.

Natural products have historically driven pharmaceutical discovery, but their reliance has diminished with synthetic drugs. Approximately 35% of medicines originate from natural products. Scopoletin, a natural coumarin compound found in herbs, exhibits antioxidant, hepatoprotective, antiviral, and antimicrobial properties through diverse intracellular signaling mechanisms. Furthermore, it also enhances the activity of antioxidants. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes viral pneumonia through cytokine storms and systemic inflammation. Cellular autophagy pathways play a role in coronavirus replication and inflammation. The Silent Information Regulator 1 (SIRT1) pathway, linked to autophagy, protects cells via FOXO3, inhibits apoptosis, and modulates SIRT1 in type-II epithelial cells. SIRT1 activation by adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) enhances the autophagy cascade. This pathway holds therapeutic potential for alveolar and pulmonary diseases and is crucial in lung inflammation. Angiotensin-converting enzyme 2 (ACE-2) activation, inhibited by reduced expression, prevents COVID-19 virus entry into type-II epithelial cells. The coronavirus disease 2019 (COVID-19) virus binds ACE-2 to enter into the host cells, and XBB.1.5 COVID-19 displays high ACE-2-binding affinity. ACE-2 expression in pneumocytes is regulated by signal transducers and activators of transcription-3 (STAT3), which can increase COVID-19 virus replication. SIRT1 regulates STAT3, and the SIRT1/STAT3 pathway is involved in lung diseases. Therapeutic regulation of SIRT1 protects the lungs from inflammation caused by viral-mediated oxidative stress. Scopoletin, as a modulator of the SIRT1 cascade, can regulate autophagy and inhibit the entry and life cycle of XBB.1.5 COVID-19 in host cells.

Synergistic Effect of HAD-B1 and Osimertinib Against Gefitinib Resistant HCC827 Non-Small Cell Lung Cancer Cells.

In this study, we investigated the synergistic effect of co-administration of osimertinib and HAD-B1 using gefitinib-resistant non-small cell lung cancer cells, HCC827-GR. HAD-B1 is composed of 4 natural drugs, Panax Notoginseng Radix, Panax ginseng C. A. Meyer, Cordyceps militaris, and Boswellia carterii Birdwood, and has been reported to have therapeutic effects on patients with advanced non-small cell lung cancer in several studies. Resistance to gefitinib in HCC827 cells was acquired through MET activity. Co-treatment with osimertinib and HAD-B1 reduced the cell viability of HCC827-GR cells. In addition, phosphorylation of MET and ERK were effectively suppressed for HCC827-GR cells. And, compared to when osimertinib and HAD-B1 were administered alone, cell proliferation was significantly inhibited and apoptosis was effectively induced when osimertinib and HAD-B1 were co-administered to HCC827-GR cells. We found that the synergistic effect of osimertinib and HAD-B1 combination therapy resulted in cancer cell death and cell cycle arrest by targeting the ERK and mTOR signaling pathways. In conclusion, this study confirmed that the combination of osimertinib, a third-generation anticancer drug, and HAD-B1, a natural anticancer drug, had a potentially synergistic effect on non-small cell lung cancer resistant to EGFR-targeted anticancer drugs.

Exploring the antiproliferative effect of PI3K/Akt/mTOR pathway and CDK4/6 inhibitors in human papillomavirus‑positive and ‑negative head and neck squamous cell carcinoma cell lines.

Human papillomavirus (HPV)‑positive and -negative head and neck squamous cell carcinoma (HNSCC) are often associated with activation of the phosphatidylinositol 3‑kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway due to mutations or amplifications in PI3KCA, loss of PTEN or activation of receptor tyrosine kinases. In HPV‑negative tumors, CDKN2A (encoding p16 protein) inactivation or CCND1 (encoding Cyclin D1 protein) amplification frequently results in sustained cyclin‑dependent kinase (CDK) 4/6 activation. The present study aimed to investigate the efficacy of the CDK4/6 inhibitors (CDKi) palbociclib and ribociclib, and the PI3K/Akt/mTOR pathway inhibitors (PI3Ki) gedatolisib, buparlisib and alpelisib, in suppressing cell viability of HPV‑positive and ‑negative HNSCC cell lines. Inhibitor efficacy was assessed in vitro using MTT assay and western blotting analysis. Cell cycle analysis was performed using flow cytometry and apoptosis was assessed using annexin V staining. Metabolic changes in terms of glycolysis and oxidative metabolism were measured by Seahorse XF96 extracellular Flux analysis. The results of the present study showed that both HPV‑positive and ‑negative HNSCC cell lines were sensitive to PI3Ki. In general, PI3Ki decreased PI3K/Akt/mTOR pathway activity, resulting in apoptosis, and decreased oxidative and glycolytic metabolism. The CDKi were particularly effective in blocking HPV‑negative cell line viability, showing decreased retinoblastoma expression and G1‑phase cell cycle arrest, whereas apoptosis was not induced. Thus, PI3Ki and CDKi efficiently inhibited their respective pathways and HNSCC cell viability in vitro, with the latter occurring only in HPV‑negative cell lines. Whereas PI3Ki induced apoptosis and attenuated cellular metabolism, CDKi led to cell cycle arrest. Further research should be performed to elucidate whether (a combination of) these inhibitors may be effective therapeutic agents for patients with HNSCC.

Copper excess induces autophagy dysfunction and mitochondrial ROS-ferroptosis progression, inhibits cellular biosynthesis of milk protein and lipid in bovine mammary epithelial cells.

Excessive copper (Cu) has the potential risk to ecosystems and organism health, with its impact on dairy cow mammary glands being not well-defined. This study used a bovine mammary epithelial cell (MAC-T) model to explore how copper excess affects cellular oxidative stress, autophagy, ferroptosis, and protein and lipid biosynthesis in milk. Results showed the increased intracellular ROS, MDA, and CAT (P < 0.05), alongside decreased T-SOD and GSH in CuSO4-treated cells (P < 0.05). Transmission electron microscopy and Ad-mCherry-GFP-LC3B assays revealed significant autophagosome accumulation in CuSO4 exposed cells (P < 0.05). Additionally, CuSO4 exposure modulated autophagy markers, evidenced by upregulation of genes such as LC3, ATG5, JNK1, and Beclin1, and downregulation of genes such as ATG4B, and p62 (P < 0.05). CuSO4 also led to notable mitochondrial changes, including size reduction, membrane rupture, and cristae loss, and reduced expression of the ferroptosis inhibitor GPX4 (P < 0.05). The expression of mTOR, HIF-1α and β-catenin signaling pathway were inhibited in differentiated MAC-T cells by CuSO4 exposure (P < 0.05), activated autophagy through activation of the AMPK-mTOR pathway which in turn affected downstream levels of genes related to milk protein and lipid. In conclusion, excessive copper induces oxidative stress in MAC-T cells, promoting autophagy through JNK-Bcl2, Beclin1-Vps34 and AMPK-mTOR pathways, leading to cell ferroptosis, as well as inhibits the cellular biosynthesis of milk protein and lipid.

Role of Cathepsin K in bone invasion of pituitary adenomas: A dual mechanism involving cell proliferation and osteoclastogenesis.

This study aimed to investigate the regulation and underlying mechanism of Cathepsin K (CTSK) in bone-invasive pituitary adenomas (BIPAs). A total of 1437 patients with pituitary adenomas were included and followed up. RNA sequencing, immunohistochemistry, and qRT-PCR were used to analyze CTSK expression. The effects of CTSK on cellular proliferation, bone matrix degradation, and osteoclast differentiation were determined by gain/loss of function experiments in vitro and in vivo. The exploration of signaling pathways was determined through molecular biology experiments. Here, we reported a significant fraction (∼10%) of pituitary adenoma patients developed bone invasion, which was correlated with tumor recurrence. Patients with BIPAs had shorter recurrence-free survival. CTSK expression was increased in BIPA patients and was strongly associated with a worse prognosis. Increased CTSK expression enhanced pituitary adenoma cell proliferation through the activation of the mammalian target of rapamycin (mTOR) signaling pathway and promoted bone invasion by increasing osteoclast differentiation both in vitro and in vivo. Treatment with the CTSK inhibitor odanacatib effectively inhibited pituitary adenoma cell proliferation and bone invasion in these models. Additionally, CTSK facilitated osteoclast differentiation by promoting RANKL expression in MC3T3-E1 cells via interaction with TLR4. Based on these findings, we conclude that CTSK has the potential to become a novel predictive biomarker and therapeutic target for BIPAs.

Repressing cytokine storm-like response in macrophages by targeting the eIF2α-integrated stress response pathway.

Cytokine storm is a life-threatening systemic hyper-inflammatory state caused by different etiologies, in which the bulk production of pro-inflammatory cytokines from activated macrophages has a central role. Integrated stress response (ISR) comprises several protective signaling pathways, leading to phosphorylation of eukaryotic initiation factor 2α (eIF2α) and repression of protein translation. Emerging evidence suggests that ISR induction may elicit anti-inflammatory effects. Currently, however, it is unclear whether targeting eIF2α phosphorylation is sufficient to inhibit the cytokine storm-like response in macrophages. Here we carried out a proof-of-concept study, employing two approaches: (1) ectopic expression of the eIF2α-S51D mutant (mimicking the phosphorylated eIF2α); (2) treatment with salubrinal, a small molecule inhibitor of eIF2α dephosphorylation. Experiments were performed in lipopolysaccharides (LPS)-stimulated macrophages and in murine models with LPS-induced acute endotoxemia. We demonstrated that in macrophages, ectopic expression of eIF2α-S51D, treatment with salubrinal, and gene silencing of PP1/GADD34 (the phosphatase holoenzyme mediating eIF2α dephosphorylation) significantly inhibited LPS-induced cytokine productions without changing their mRNA levels. Polysome PCR and puromycin incorporation assays confirmed that salubrinal suppressed de novo protein translation of the cytokines. In vivo, salubrinal pre-treatment mitigated LPS-induced acute lung injury and significantly reduced the concentration of circulating TNF-α. Salubrinal did not exhibit any effects on the Toll-like receptor 4-mediated signaling or the activation of mammalian target of rapamycin (mTOR). Our data suggest that direct manipulation of eIF2α phosphorylation, thereby bypassing all associated upstream signaling events, may suppress the cytokine storm-like response in activated macrophages, likely by decoupling the gene transcription and protein translation. Inhibiting eIF2α dephosphorylation with small molecule inhibitors may be a viable therapeutic strategy to treat disorders involving cytokine storm-like responses.

Agmatine suppresses glycolysis via the PI3K/Akt/mTOR/HIF-1α signaling pathway and improves mitochondrial function in microglia exposed to lipopolysaccharide.

Modulating metabolic pathways in activated microglia can alter their phenotype, which is relevant in uncontrolled neuroinflammation as a component of various neurodegenerative diseases. Here, we investigated how pretreatment with agmatine, an endogenous polyamine, affects metabolic changes in an in vitro model of neuroinflammation, a murine microglial BV-2 cell line exposed to lipopolysaccharide (LPS). Hence, we analyzed gene expression using qPCR and protein levels using Western blot and ELISA. Microglial metabolic status was assessed by measuring lactate release and cellular ATP by enzymatic and luminescence spectrophotometry. Mitochondrial functionality was analyzed by fluorescent probes detecting mitochondrial membrane potential (mtMP) and superoxide production. Our findings suggest that kinase pathways associated with hypoxia-inducible factor-1α (HIF-1α) regulate energy metabolism in pro-inflammatory activated microglia. We have shown that LPS induces HIF-1α and genes for glucose transporter and glycolytic rate, increases lactate production and causes mitochondrial dysfunction, suggesting a metabolic shift towards glycolysis. Agmatine inhibits the PI3K/Akt pathway and negatively regulates mammalian target of rapamycin (mTOR) phosphorylation and HIF-1α levels, reducing lactate and tumor necrosis factor (TNF) production, which is supported by pharmacological blockade of PI3K. Pretreatment with agmatine also rescues mitochondrial function by counteracting the LPS-induced decline in mtMP and increase in mitochondrial superoxide, resulting in an anti-apoptotic effect. Agmatine alone increases intracellular ATP levels and maintains this effect even under pro-inflammatory conditions. Our study emphasizes the ability of agmatine to engage in metabolic reprogramming of pro-inflammatory microglia through increased ATP production and modulation of signaling pathway involved in promoting glycolysis and cytokine release.

Vasoactive intestinal peptide induces metabolic rewiring of human-derived cytotrophoblast cells to promote cell migration.

The placenta has an extraordinary metabolic rate with high oxygen consumption. Extravillous cytotrophoblast cells (EVT) metabolism and function are critical to sustain their invasive phenotype supporting fetal development. Deficient EVT function underlies pregnancy complications as preeclampsia (PE) and fetal growth restriction (FGR). The vasoactive intestinal peptide (VIP) promotes human cytotrophoblast cell migration and invasion through mTOR signaling pathways suggesting its crucial role during placentation. Here we explored fatty acid uptake as well as lipid and glucose metabolism in human-derived cytotrophoblast cell function upon VIP stimulation. We found that VIP induced long chain fatty acid (LCFAs) uptake along with the expression of FATP2 transporter, CPT1 fatty acid oxidation (FAO)-rate limiting step importer, and lipid droplet accumulation. VIP induced the expression of glucose 6-P-dehydrogenase, a rate-limiting enzyme of the pentose phosphate pathway (PPP) and pyruvate dehydrogenase complex enzyme DLAT E2, without altering lactate secretion. This metabolic rewiring of trophoblast cells induced by VIP takes place without compromising mitochondrial function or reactive oxygen species (ROS) production. Moreover, cytotrophoblast cell migration induced by VIP required the three glycolysis, oxidative phosphorylation (OXPHOS) and FAO pathways. Our results provide evidence supporting VIP as a metabolic regulatory peptide in cytotrophoblast cells sustaining proper placentation and fetal growth.

Phosphatidic acid as a cofactor of mTORC1 in platinum-based chemoresistance: Mechanisms and therapeutic potential.

Platinum-based chemotherapeutics, such as cisplatin and carboplatin, are widely used to treat various malignancies. However, the development of chemoresistance remains a significant challenge, limiting their efficacy. This review explores the multifaceted mechanisms of platinum-based chemoresistance, with a particular focus on the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, which plays a critical role in promoting tumor survival and resistance to platinum compounds. Additionally, we examined the role of phosphatidic acid (PA) and its synthesizing enzymes, phospholipase D (PLD) and lysophosphatidic acid acyltransferase (LPAAT), in the regulation of mTORC1 activity. Given the involvement of mTORC1 in chemoresistance, we evaluated the potential of mTOR inhibitors as a therapeutic strategy to overcome platinum resistance. Finally, we discuss combination therapies targeting the mTOR pathway alongside conventional chemotherapy to improve treatment outcomes. This review highlights the potential of targeting mTORC1 and related pathways to improve therapeutic strategies for chemoresistant cancers.

A study of the homologous recombination deficiency signature (HRDSig) status of advanced esophageal squamous cell carcinoma (AESCC) by utilizing comprehensive genomic profiling (CGP).

485 Background: The role of poly-ADP-ribose polymerase (PARP) inhibitors is currently under investigation as a potential therapeutic option for AESCC. Homologous recombination deficiency signature status (HRDSig) positivity has been shown to predict biallelic loss of BRCA1/2 which may simultaneously portend a worse prognosis and a sensitivity to poly-ADP-ribose polymerase (PARP) inhibitors. In this study, we attempt to characterize the genomic alterations present in AESCC based on HRDSig status utilizing comprehensive genomic profiling (CGP) techniques. Methods: 2,029 cases of AESCC underwent comprehensive genomic profiling with an examination of all genomic alterations (GA). MSI high status, tumor mutation burden (TMB) levels, genomic ancestry and genomic trinucleotide signatures were determined from the sequencing data. HRDSig status was calculated using a broad set of genome-wide copy number features (PMID 37769224). Results were compared using the Fisher exact system with the Benjamini-Hochberg adjustment to correct for false discovery. Results: 154 (7.6%) of the 2029 AESCC cases featured a positive HRDSig status (HRDSig+). The age (66-67) and gender (58%-63% male) distribution were similar in the HRDSig+ and HRDSig- AECSS cases as was the frequency of GA/tumor (9 for both). The median TMB was higher in the HRDSig+ (6.3 vs 3.8; P&lt;.0001) as was frequency of TMB &gt; 10 mutations/Mb (19.5% vs 10.0%; P=.004). An APOBEC trinucleotide signature was also more frequent in the HRDSig+ AESCC (11.0% vs 5.4%; P=.05). As anticipated, GA in genes associated with HRD including BRCA1 (10.4% vs 1.4%; P&lt;.0001) and BRCA2 (14.3% vs 2.0%; P&lt;.0001) was present. MTOR pathway activating mutations were also more frequent in the HRDSig+ AESCC group including PTEN (13.0% vs 7.3%; P=.06). Conclusions: With a 7.6% frequency, HRDSig+ status is a relatively rare event in AESCC. CGP with determination of HRDSig status in AESCC may prove useful in tailoring PARP inhibitor-based treatment regimens and may potentially uncover other genomic alterations that can aid in designing targeted therapy combinations in future. Characteristics of comprehensive genomic profiling of clinically advanced esophageal squamous cell carcinoma by HRDSig status. AESCC HRDSig- (n=1875) AESCC HRDSig+ (n=154) P-value Pathogenic genomic alterations BRCA1 1.4% (26) 10.4% (16) 0.00 BRCA2 2.0% (36) 14.3% (22) 0.00 PTEN 7.3% (137) 13.0% (20) 0.06 COSMIC trinucleotide signature APOBEC 5.4% (101) 11.0% (17) 0.05 Tumor mutational burden (TMB) Median TMB (range) (IQR) 3.8 (0-85) (2.5-6.3) 6.3 (0-61) (3.8-8.8) 0.00 TMB≥10 mut/Mb 10.0% (188) 19.5% (30) 0.00

Positive homologous recombination signature (HRDsig+) and the genomic landscape of intrahepatic cholangiocarcinoma (iCCA).

627 Background: Defective DNA repair has not been a major focus in iCCA clinical research. In this comprehensive genomic profiling (CGP) study, we queried whether the presence of a scar-based HRDsig biomarker could identify a subset of patients with unique genomic alterations (GA) and potential for responsiveness to PARP inhibitor-based treatment regimens. Methods: 6,271 cases of clinically advanced iCCA underwent hybrid capture based CGP to evaluate all classes of GA. Microsatellite instability (MSI) status, tumor mutation burden (TMB) levels, genomic ancestry and genomic trinucleotide signatures were determined from the sequencing data. HRDsig status was calculated using a broad set of genome-wide copy number features (PMID 37769224). PD-L1 was determined by IHC using the Dako 22C3 tumor proportional score (TPS). Results were compared using the Fisher exact system with the Benjamini-Hochberg procedure. Results: 288 (4.6%) of the sequenced iCCA featured an HRDsig+ status. Gender distribution (46-49% male) and median age (67 years) were similar in both HRDsig+ and HRDsig- iCCA. More pathogenic GA per tumor were found in HRDsig+ vs HRDsig- iCCA (5 vs 4; P&lt;0.0001). Genomic ancestry distribution was similar; European ancestry ranged from 73% to 76% in the two cohorts. The APOBEC signature was uncommon but slightly more frequent in the HRDSig+ cases (1.7% vs 0.4%; P=.034). MSI High status was slightly more frequent in the HRDsig- group (1.8% vs 0.0%; P=0.034). Median TMB was higher in the HRDsig+ cases (3.6 vs 1.2 mut/Mb; P&lt;0.0001) as was the frequency of TMB &gt; 10 mut/Mb (9.1% vs 3.5%; P&lt;0.0001). PD-L1 expression was similar with low level (1-49% TPS) ranging from 19% to 23% in the 2 groups. As anticipated, GA in genes associated with HRD including BRCA1 (6.6% vs 0.8%; P&lt;0.0001), BRCA2 (25.0% vs 1.3%; P&lt;0.0001), PALB2 (8.7% vs 0.3%; P&lt;0.0001), and ATM (6.3% vs 3.4%; P=0.033) were all more frequent in the HRDsig+ iCCA. In contrast, GA in genes associated with iCCA targeted therapies were less frequent in the HRDsig+ cases including FGFR2 (8.0% vs 12.5%; P=0.033), IDH1 (3.5% vs 14.0%; P&lt;0.0001) and ERBB2 (3.5% vs 5.7%; NS). In the HRDsig- group, 83.3%/74.1% of BRCA1 / BRCA2 mutated iCCA were mono-allelic likely non-driver GA respectively. MTOR pathway activating GA were more frequent in the HRDsig+ cases including PTEN (7.3% vs 3.3%; P=0.003) and NF1 (8.0% vs 2.8%; P&lt;0.0001). MTAP deletion, an emerging iCCA target, was more frequent in the HRDsig+ iCCA (23.3% vs 16.9%; P=0.024). Conclusions: Nearly 5% of advanced iCCA feature HRDsig positive status which is associated with both significant differences in the frequencies of therapy associated genomic targets and the potential for introducing PARP inhibitors for these patients. HRDsig- iCCAN = 5983 HRDsig+ iCCAN = 288 P-value ATM 3.4% 6.3% 0.033 BRCA1 0.8% (83.3% mono-allelic) 6.6% &lt;.0001 BRCA2 1.3% (74.1% mono-allelic) 25.0% &lt;.0001 PALB2 0.3% 8.7% &lt;.0001

Role of autophagy in plant growth and adaptation to salt stress.

Under salt stress, autophagy regulates ionic balance, scavenges ROS, and supports nutrient remobilization, thereby alleviating osmotic and oxidative damage. Salt stress is a major environmental challenge that significantly impacts plant growth and agricultural productivity by disrupting nutrient balance, inducing osmotic stress, and causing the accumulation of toxic ions like Na+. Autophagy, a key cellular degradation and recycling pathway, plays a critical role in enhancing plant salt tolerance by maintaining cellular homeostasis and mitigating stress-induced damage. While autophagy has traditionally been viewed as a response to nutrient starvation, recent research has highlighted its importance under various environmental stresses, particularly salt stress. Under such conditions, plants activate autophagy through distinct signaling pathways involving autophagy-related genes (ATGs), Target of Rapamycin (TOR) proteins, and reactive oxygen species (ROS). Salt stress induces the expression of ATG genes and promotes the formation of autophagosomes, which facilitate the degradation of damaged organelles, denatured proteins, and the sequestration of Na+ into vacuoles, thereby improving stress tolerance. Recent studies have also suggested that autophagy may play a direct role in salt stress signaling, linking it to the regulation of metabolic processes. This review discusses the molecular mechanisms underlying autophagy induction in plants under salt stress, including the roles of ATGs and TOR, as well as the physiological significance of autophagy in mitigating oxidative damage, maintaining ion balance, and enhancing overall salt tolerance. In addition, we discussed the metabolic changes related to autophagy in stressed plants and examined the broader implications for managing plant stress and improving crops.

Temporal dynamics of the interstitial fluid proteome in human skeletal muscle following exhaustive exercise.

The skeletal muscle interstitial space is the extracellular region around myofibers and mediates cross-talk between resident cell types. We applied a proteomic workflow to characterize the human skeletal muscle interstitial fluid proteome at rest and in response to exercise. Following exhaustive exercise, markers of skeletal muscle damage accumulate in the interstitial space followed by the appearance of immune cell-derived proteins. Among the proteins up-regulated after exercise, we identified cathelicidin-related antimicrobial peptide (CAMP) as a bioactive molecule regulating muscle fiber development. Treatment with the bioactive peptide derivative of CAMP (LL-37) resulted in the growth of larger C2C12 skeletal muscle myotubes. Phosphoproteomics revealed that LL-37 activated pathways central to muscle growth and proliferation, including phosphatidylinositol 3-kinase, AKT serine/threonine kinase 1, mitogen-activated protein kinases, and mammalian target of rapamycin. Our findings provide a proof of concept that the interstitial fluid proteome is quantifiable via microdialysis sampling in vivo. These data highlight the importance of cellular communication in the adaptive response to exercise.