TGF-β Activation Research Articles

Integration of histone modification-based risk signature with drug sensitivity analysis reveals novel therapeutic strategies for lower-grade glioma

BackgroundLower-grade glioma (LGG) exhibits significant heterogeneity in clinical outcomes, and current prognostic markers have limited predictive value. Despite the growing recognition of histone modifications in tumor progression, their role in LGG remains poorly understood. This study aimed to develop a histone modification-based risk signature and investigate its relationship with drug sensitivity to guide personalized treatment strategies.MethodsWe performed single-cell RNA sequencing analysis on LGG samples (n = 4) to characterize histone modification patterns. Through integrative analysis of TCGA-LGG (n = 513) and CGGA datasets (n = 693 and n = 325), we constructed a histone modification-related risk signature (HMRS) using machine learning approaches. The model's performance was validated in multiple independent cohorts. We further conducted comprehensive analyses of molecular mechanisms, immune microenvironment, and drug sensitivity associated with the risk stratification.ResultsWe identified distinct histone modification patterns across five major cell populations in LGG and developed a robust 20-gene HMRS from 129 candidate genes that effectively stratified patients into high- and low-risk groups with significantly different survival outcomes (training set: AUC = 0.77, 0.73, and 0.71 for 1-, 3-, and 5-year survival; P < 0.001). Integration of HMRS with clinical features further improved prognostic accuracy (C-index >0.70). High-risk tumors showed activation of TGF-β and IL6-JAK-STAT3 signaling pathways, and distinct mutation profiles including TP53 (63% vs 28%), IDH1 (68% vs 85%), and ATRX (46% vs 20%) mutations. The high-risk group demonstrated significantly elevated immune and stromal scores (P < 0.001), with distinct patterns of immune cell infiltration, particularly in memory CD4+ T cells (P < 0.001) and CD8+ T cells (P = 0.001). Drug sensitivity analysis revealed significant differential responses to six therapeutic agents including Temozolomide and targeted drugs (P < 0.05).ConclusionOur study establishes a novel histone modification-based prognostic model that not only accurately predicts LGG patient outcomes but also reveals potential therapeutic targets. The identified associations between risk stratification and drug sensitivity provide valuable insights for personalized treatment strategies. This integrated approach offers a promising framework for improving LGG patient care through molecular-based risk assessment and treatment selection.

Critical role for Transglutaminase 2 in scleroderma skin fibrosis and in the development of dermal sclerosis in a mouse model of scleroderma.

Scleroderma is a life-threatening autoimmune disease characterized by inflammation, tissue remodelling, and fibrosis. This study aimed to investigate the expression and function of transglutaminase 2 (TGM2) in scleroderma skin and experimentally-induced dermal fibrosis to determine its potential role and therapeutic implications. We performed immunohistochemistry on skin sections to assess TGM2 expression and localisation, and protein biochemistry of both SSc-derived and healthy control dermal fibroblasts to assess TGM2 expression, function and ECM deposition in the presence of a TGM2 and TGFβ neutralizing antibodies and a small molecule inhibitor of the TGFβRI kinase. Mice with a complete deficiency of TGM2 (Tgm2-/-) were investigated in the bleomycin-induced model of skin fibrosis. TGM2 was found to be widely expressed in both control and scleroderma skin samples, as well as in cultured fibroblasts. Scleroderma fibroblasts exhibited elevated TGM2 expression, which correlated with increased expression of fibrosis markers (collagen type 1, αSMA and CCN2). Inhibition of TGM2 using a neutralizing antibody reduced the expression of key markers of fibrosis. The effects of TGM2 inhibition were similar to those observed with TGFβ neutralization, suggesting a potential cross-talk between TGM2 and TGFβ signalling. Moreover, TGM2 knock-out mice showed significantly reduced dermal fibrosis compared to wild-type mice. In vitro experiments with TGM2-deleted fibroblasts demonstrated impaired cell migration and collagen matrix contraction, which could be partially restored by exogenous TGFβ. TGM2 can regulate several key pro-fibrotic activities of TGFβ suggesting that attenuating TGM2 function may be of benefit in severe forms of connective tissue disease with skin fibrosis.

Targeting circFOXO3 to Modulate Integrin β6 Expression in Periodontitis: A Potential Therapeutic Approach.

Circular RNA forkhead box O3 (circFOXO3) is crucial in regulating inflammation in lung and heart injuries. However, its role in periodontitis remains unclear. We sought to elucidate the effects of circFOXO3 on periodontitis progression and related molecular mechanisms. Reverse-transcription quantitative polymerase chain reaction and fluorescence insitu hybridization were used toquantify and localize circFOXO3 expression. The mechanism by which circFOXO3 promotes inflammation in periodontitiswas investigated using epithelial cells, human gingival epithelium and a rat model of ligature-induced periodontitis. circFOXO3 expression was abnormally high in the gingival epithelial tissues of patients with periodontitis. Elevated circFOXO3 levels down-regulated microRNA (miR)-141-3p, leading to increased FOXO3 expression. FOXO3 interacted with JunB to form a transcriptional-repression complex that inhibited the integrin β6 (ITGβ6)-mediated activation of transforming growth factor β (TGF-β) in epithelial cells. Through the miR-141-3p/FOXO3/JunB axis, circFOXO3 suppressed TGF-β signalling, thereby exacerbating periodontal inflammation. Finally, circFOXO3 inhibition hindered disease progression and restored TGF-β activity invivo via the FOXO3/JunB/ITGβ6 pathway. Our study identified a novel mechanism by which circFOXO3 contributes to periodontal inflammation through a complex transcriptional regulatory network involving miR-141-3p, FOXO3, JunB and ITGβ6. These findings highlight potential therapeutic targets for the development of effective treatments for this debilitating disease.

Targeting BMP-1 enhances anti-tumoral effects of doxorubicin in metastatic mammary cancer: common and distinct features of TGF-β inhibition.

Mammary carcinoma is comprised heterogeneous groups of cells with different metastatic potential. 4T1 mammary carcinoma cells metastasized to heart (4THM), liver (4TLM) and brain (4TBM) and demonstrate cancer-stem cell phenotype. Using these cancer cells we found thatTGF-β is the top upstream regulator of metastatic process. In addition, secretion of bone morphogenetic protein 1 (BMP-1), which is crucial for the proteolytic release of TGF-β, was markedly high in metastatic mammary cancer cells compared to non-metastatic cells. Although TGF-β inhibitors are in clinical trials, systemic inhibition of TGF-β may produce heavy side effects. We here hypothesize that inhibition of BMP-1 proteolytic activity inhibits TGF-β activity and induces anti-tumoral effects. Effects of specific BMP-1 inhibitor on liver and brain metastatic murine mammary cancer cells (4TLM and 4TBM), as well as on human mammary cancer MDA-MB-231 and MCF-7 cells, were examined and compared with the results of TGF-β inhibition. Inhibition of BMP-1 activity markedly suppressed proliferation of cancer cells and enhanced anti-tumoral effects of doxorubicin. Inhibition of BMP-1 activity but not of TGF-β activity decreased colony and spheroid formation. Differential effects of BMP-1 and TGF-β inhibitors on TGF-β secretion was also observed. These results demonstrated for the first time that the inhibition of BMP-1 activity has therapeutic potential for treatment of metastatic mammary cancer and enhances the anti-tumoral effects of doxorubicin.

An eCIRP inhibitor attenuates fibrosis and ferroptosis in ischemia and reperfusion induced chronic kidney disease

BackgroundChronic kidney disease (CKD) is a leading cause of death in the United States, and renal fibrosis represents a pathologic hallmark of CKD. Extracellular cold-inducible RNA-binding protein (eCIRP) is a stress response protein involved in acute inflammation, tissue injury and regulated cell death. However, the role of eCIRP in chronic inflammation and tissue injury has not been elucidated. We hypothesize that eCIRP is involved in renal ischemia/reperfusion (RIR)-induced CKD and that C23, an antagonist to eCIRP, is beneficial in attenuating renal fibrosis and ferroptosis in RIR-induced CKD.MethodsC57BL/6 (WT) or CIRP−/− mice underwent renal injury with total blockage of blood perfusion by clamping bilateral renal pedicles for 28 min. In the WT mice at the time of reperfusion, they were treated with C23 (8 mg/kg) or vehicle. Blood and kidneys were harvested for further analysis at 21 days thereafter. In a separate cohort, mice underwent bilateral RIR and treatment with C23 or vehicle and were then subjected to left nephrectomy 72 h thereafter. Mice were then monitored for additional 19 days, and glomerular filtration rate (GFR) was assessed using a noninvasive transcutaneous method.ResultsIn the RIR-induced CKD, CIRP−/− mice showed decreased collagen deposition, fibronectin staining, and renal injury as compared to the WT mice. Administration of C23 ameliorated renal fibrosis by decreasing the expression of active TGF-β1, α-SMA, collagen deposition, fibronectin and macrophage infiltration to the kidneys. Furthermore, intervention with C23 significantly decreased renal ferroptosis by reducing iron accumulation, increasing the expression of glutathione peroxidase 4 (GPX4) and lipid peroxidation in the kidneys of RIR-induced CKD mice. Treatment with C23 also attenuated BUN and creatinine. Finally, GFR was significantly decreased in RIR mice with left nephrectomy and C23 treatment partially prevented their decrease.ConclusionOur data show that eCIRP plays an important role in RIR-induced CKD. Treatment with C23 decreased renal inflammation, alleviated chronic renal injury and fibrosis, and inhibited ferroptosis in the RIR-induced CKD mice.

Mechanism of THBS1 Regulation of MDCK Cell Proliferation and Apoptosis Through TGF-β/Smad Signalling.

Madin-Darby Canine Kidney (MDCK) cells are a key cell line for influenza vaccine production, due to their high viral yield and low mutation resistance. In our laboratory, we established a tertiary cell bank (called M60) using a standard MDCK cell line imported from American Type Culture Collection (ATCC) in the USA. Due to their controversial tumourigenicity, we domesticated non-tumourigenic MDCK cells (named CL23) for influenza vaccine production via monoclonal screening in the early stage of this study, and the screened CL23 cells were characterised based on their low proliferative capacity, which had certain limitations in terms of expanding their production during cell resuscitation. It was thus our objective to enhance the proliferation efficiency of MDCK cells for influenza vaccine production following cell resuscitation, with a view to improving the production of non-tumourigenic MDCK cells for vaccines and enhancing the production of influenza virus lysate vaccines from MDCK cells through genetic intervention. We concentrated on the protein thrombosponin-1 (THBS1), which was markedly differentiated in the proteomics data of the two MDCK cells. By integrating this finding with related studies, we were able to ascertain that THBS1 exerts a significant influence on the level of cell proliferation and apoptosis. Consequently, our objective was to investigate the impact of THBS1 expression on MDCK cell apoptosis by verifying the differences in THBS1 expression between the two MDCK cells and by interfering with THBS1 expression in the MDCK cells. We found that the knockdown of THBS1 significantly increased the proliferation and apoptosis of CL23 cells without causing significant changes in cell migration and invasion, and its overexpression significantly decreased the proliferation of M60 cells and increased cell migration, invasion, and apoptosis. In addition, the TGF-β/Smad pathway target genes transforming growth factor-β1 (TGF-β1), mothers against decapentaplegic homolog 2 (Smad2), and mothers against decapentaplegic homolog 3 (Smad3), were significantly down-regulated in CL23 cells after THBS1 knockdown and up-regulated in M60 cells after overexpression, with consistent expression identified at both the mRNA and protein levels. The treatment of cells with TGF-β activators and inhibitors further demonstrated that THBS1 regulated MDCK cell proliferation and apoptosis through the TGF-β/Smad signalling pathway. Finally, we found that THBS1 also regulated H1N1 influenza virus replication. These findings enable a comprehensive understanding of the regulatory mechanisms of THBS1 regarding MDCK cell proliferation and apoptosis functions and the effects of influenza virus replication.

Arketamine alleviates cognitive impairments and demyelination in mice with postoperative cognitive dysfunction via TGF-β1 activation.

Postoperative cognitive dysfunction (POCD) is characterized by a decline in cognitive functions, including memory, attention, and executive abilities, following surgery, with no effective therapeutic drugs currently available. Arketamine, the (R)-enantiomer of ketamine, has shown promise in mitigating cognitive deficits in animal models. In this study, we investigated whether arketamine could ameliorate cognitive deficits in a mouse model of POCD, with a focus on the role of transforming growth factor (TGF)-β1 in its effects. POCD mice displayed cognitive impairments and demyelination in the corpus callosum. A single arketamine injection (10mg/kg) significantly improved both cognitive function and demyelination in the corpus callosum of POCD mice. Notably, pretreatment with RepSox (10mg/kg), a TGF-β receptor 1 inhibitor, significantly blocked the beneficial effects of arketamine on cognitive deficits and demyelination. Moreover, intranasal administration of TGF-β1 (3.0μg/kg) markedly alleviated cognitive impairments and demyelination in POCD mice. These findings suggest that arketamine exerts its effects through a TGF-β1-dependent mechanism, positioning it as a potential therapeutic option for POCD.

Hydrogen decreases susceptibility to AngII-induced atrial fibrillation and atrial fibrosis via the NOX4/ROS/NLRP3 and TGF-β1/Smad2/3 signaling pathways.

Atrial fibrillation (AF) represents the commonly occurring cardiac arrhythmia and the main factor leading to stroke and heart failure. Hydrogen (H2) is a gaseous signaling molecule that has the effects of anti-inflammation and antioxidation. Our study provides evidence that hydrogen decreases susceptibility to AngII-mediated AF together with atrial fibrosis. Following continuous AngII administration for a 28-day period, AngII+H2 treated rats showed decreased susceptibility to AF, a decrease in atrial fibrosis, a decrease in ROS in atrial myocytes, an inhibition of NLRP3 inflammasome activation, an improvement in electrical remodeling, and an inhibition of proliferation and migration of cardiac fibroblasts. We further found that hydrogen regulates the activation of inflammasome and thus improves Ca2+ handling and IKAch and IKur by inhibiting the activity of NOX4 in vivo. In addition, hydrogen was involved in AngII-mediated atrial fibrosis through inhibiting TGF-β1/Smad2/3 pathway through suppressing TGF-β1 activation and secretion in vivo. Our findings suggest that hydrogen is important for preventing and treating AngII-mediated AF and atrial fibrosis, suggesting that hydrogen could be used as the candidate way to prevent and treat AF.

Epigenetic Inhibitors Differentially Impact TGF-β1 Signaling Cascades in COPD Airway Smooth Muscle Cells.

Background: Chronic obstructive pulmonary disease (COPD) is characterized by progressive and incurable airflow obstruction and chronic inflammation. Both TGF-β1 and CXCL8 have been well described as fundamental to COPD progression. DNA methylation and histone acetylation, which are well-understood epigenetic mechanisms regulating gene expression, are associated with COPD progression. However, a deeper understanding of the complex mechanisms associated with DNA methylation, histone post-translational changes and RNA methylation in the context of regulatory pathways remains to be elucidated. We here report on how DNA methylation and histone acetylation inhibition differentially affect CXCL8 signaling in primary human non-COPD and COPD airway cells. Methods: Airway smooth muscle (ASM) cells, a pivotal cell type in COPD, were isolated from the small airways of heavy smokers with and without COPD. Histone acetylation and DNA methylation were inhibited before the TGF-β1 stimulation of cells. Subsequently, CXCL8 production and the abundance and activation of pertinent transcription regulatory proteins (NF-κB, p38 MAPK and JNK) were analyzed. Results: TGF-β1-stimulated CXCL8 release from ASM cells from 'healthy' smoker subjects was significantly modulated by DNA methylation (56.32 pg/mL and 56.60 pg/mL) and acetylation inhibitors (27.50 pg/mL and 48.85 pg/mL) at 24 and 48 h, respectively. However, modulation via the inhibition of DNA methylation (34.06 pg/mL and 43.18 pg/mL) and acetylation (23.14 pg/mL and 27.18 pg/mL) was observed to a lesser extent in COPD ASM cells. These changes were associated with differences in the TGF-β1 activation of NF-κB and MAPK pathways at 10 and 20 min. Conclusions: Our findings offer insight into differential epigenetics in controlling COPD ASM cells and provide a foundation warranting future studies on epigenetic differences associated with COPD diagnosis. This would provide a scope for developing therapeutic interventions targeting signaling and epigenetic pathways to improve patient outcomes.

Evaluating the role of active TGF-β1 as inflammatory biomarker in Kashmiri (North-Indian) patients with systemic sclerosis: a case-control study

Abstract Background As a master immune system regulator, transforming growth factor β1 (TGF-β1) is closely linked to the complicated pathophysiology and development of systemic sclerosis (SSc), a multisystem fibrotic disease. Objective We aim to evaluate the transcriptional levels of TGF-β1 mRNA in PBMCs, assess the TGF-β1 serum levels of SSc patients, and compare them with those of healthy subjects. Methods PBMCs were isolated from whole blood of 50 SSc patients and in 30 healthy controls. After total RNA was extracted from isolated PBMCs, complementary DNA (cDNA) synthesis was performed. Afterward, the expression of TGF-β1 mRNA was assessed using quantitative real-time PCR using the SYBR Green, GAPDH, and TGF-β1 specific primers. The serum levels of TGF-β1 were determined using a commercially available ELISA kit. Results There was a significant upregulation of TGF-β1 relative expression (p < 0.0001), when SSc patients were compared to the control group. The diffuse subgroup was more common in patients with elevated TGF-β1 mRNA expression (p < 0.0001). However, an insignificant difference was observed between the disease subsets of SSc. Serum TGF- β1 levels were upregulated in SSc patients (78.35 ± 23.16) compared to healthy subjects (61.06 ± 15.90), and were considerably higher in SSc patients with ILD (p < 0.01) and positive anti-topo-Isomerase antibody (p < 0.0001). Conclusion In patients with SSc, elevated levels of TGF-β1 in serum and their correlation with clinical symptoms imply that this cytokine may serve as a marker for fibrotic and vascular involvement in SSc.

A hybrid epithelial-mesenchymal transition program enables basal epithelial cells to bypass stress-induced stasis and contributes to a metaplastic breast cancer progenitor state

BackgroundHuman mammary epithelial cell (HMEC) cultures encounter a stress-associated barrier termed stasis, during which most cells adopt a senescence-like phenotype. From these cultures, rare variants emerge from the basal epithelial population, re-initiating growth. Variants exhibit pre-malignant properties, including an aberrant epigenetic program that enables continued proliferation and acquisition of genetic changes. Following oncogenic transformation, variants produce tumors that recapitulate the histopathological characteristics of metaplastic breast cancer (MBC), a rare and aggressive subtype marked by the differentiation of neoplastic epithelium into squamous and mesenchymal elements.MethodsUsing a serum-free HMEC culture system, we probed the capacity for phenotypic plasticity inherent to basal epithelial cell populations from human breast tissue as they navigated stasis and emerged as variant populations.ResultsWe observed robust activation of a TGF-β-dependent epithelial-mesenchymal transition (EMT) program in basal epithelial cells during stasis, followed by subsequent attenuation of this program in emerging variants. Inhibition of the TGF-β pathway or depleting the EMT regulators Snail or Slug allowed basal epithelial cells to collectively bypass stasis, demonstrating that cellular dysfunction and arrest resulting from TGF-β and EMT activation are central to this in vitro barrier. The spontaneous emergence of variants from stasis cultures was associated with a restricted EMT trajectory, characterized by the stabilization of hybrid EMT states associated with greater proliferative capacity, rather than progressing to a complete mesenchymal state characterized by irreversible growth arrest. Epigenetic mechanisms, which contributed to the dysregulated growth control characteristic of the variant phenotype, also contributed to the stability of the hybrid EMT program in variants. By overcoming the cellular dysfunction and growth arrest resulting from TGF-β and complete EMT, variants exhibited a higher oncogenic transformation efficiency compared to pre-stasis basal epithelial cells. Inhibiting the TGF-β pathway prior to stasis significantly reduced EMT in the basal epithelial population, alleviated selective pressure driving variant emergence, and also enhanced oncogenic transformation efficiency, resulting in tumors with markedly diminished metaplastic differentiation.ConclusionsThis study reveals how an epigenetic program governs basal epithelial cell fate decisions and contributes to the development of MBC progenitors by restricting access to terminal mesenchymal states that induce growth arrest and, instead, favoring hybrid EMT states with enhanced tumorigenic potential.

RNA-seq analysis of the biological process and regulatory signal of TGF-β1-mediated changes in ovarian granulosa cells in small-tail Han sheep

Transforming growth factor beta-1 (TGF-β1) regulates the proliferation of ovarian granulosa cells and participates in follicular development in small-tail Han sheep via the SMAD pathway. However, which additional biological processes and regulatory mechanisms are involved in TGF-β1-mediated regulation of granulosa cell changes remains unknown. In this study, TGF-β1-treated (10 ng/mL) ovarian granulosa cells of small-tail Han sheep were used as the model, RNA-Seq was employed to screen differentially expressed genes (DEGs), and rescue experiments were used to verify selected key pathways. In total, 1179 upregulated and 873 downregulated DEGs were screened using RNA-Seq. Gene Ontology (GO) enrichment analysis showed that the DEGs were mainly involved in the biological processes of cell adhesion, cell migration, cell cycle, cell proliferation and apoptosis, and endocrine regulation. Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the DEGs were primarily associated with pathways relating to ECM-receptor interaction, PI3K-AKT, focal adhesion, MAPK, TNF, and FOXO signaling, among others. The addition of doramapimod confirmed that the p38 pathway participates in the TGF-β1-induced proliferation and apoptosis of ovarian granulosa cells as well as the regulation of steroid hormone secretion. These results revealed a novel TGF-β1/p38 pathway-mediated mechanism that induces both the proliferation and apoptosis of ovarian granulosa cells. Our findings provide a basis for better understanding the genetic mechanism of TGF-β1 action in follicle development.

MiR-629-3p inhibits fine particulate matter exposure-induced lung function decline: results from the two-stage population study and in vitro study.

MiRNAs were reported to play crucial roles in the pathogenesis of health damage caused by environmental pollutants. However, its potential role in fine particulate matter (PM2.5) exposure-induced lung function decline has rarely been elucidated. The present study was developed to profile specific miRNAs that were related to both PM2.5 exposure and lung function decline, and to investigate the regulating role in PM2.5 exposure-induced lung injury. Based on the Wuhan-Zhuhai cohort, in the discovery stage, plasma miRNA profiling for PM2.5 exposure was conducted through next-generation sequencing among 60 participants with 120 observations in a repeated-measures design. Plasma miRNA profiling for lung function decline was conducted among 10 pairs of lung function decline incident cases and matched healthy controls. In the validating stage, miR-629-3p was selected from miRNAs that were related to both PM2.5 exposure and lung function decline, and was measured by quantitative real-time PCR among 475 residents to validate its association with PM2.5 exposure as well as lung function. In vitro, PM2.5-treated A549 and BEAS-2B cell models and miR-629-3p mimic/inhibitor models were used to explore the role and underlying mechanism of miR-629-3p on epithelial-mesenchymal transition (EMT) induced by PM2.5 exposure. The two-stage population study found a negative association between personal PM2.5 exposure and plasma miR-629-3p, while a positive association between miR-629-3p and lung function. In vitro, PM2.5 treatment stimulated the expressions of EMT-related factors, accompanied by the activation of TGF-β1/TGF-βR1 signal pathway. Overexpression of miR-629-3p could inhibit PM2.5-induced TGF-βR1 expression and alleviate EMT process. And inhibition of miR-629-3p could promote TGF-βR1 expression and aggravate EMT process. In conclusion, miR-629-3p may alleviate the lung injury induced by PM2.5 exposure through inhibiting TGF-β1/TGF-βR1 pathway.

Exploring the relation between TGF-β pathway activity and response to checkpoint inhibition in patients with metastatic melanoma.

Immune checkpoint inhibition (ICI) is highly effective for the treatment of melanoma, but intrinsic resistance is present in a subgroup of patients. TGF-β pathway activity may play a role in this resistance by preventing T-cells from entering the tumor microenvironment, causing immune escape. We investigated the association of TGF-β signal transduction pathway activity with resistance to ICI treatment in advanced melanoma. Furthermore, other pathway activities were analyzed to better understand their potential role in ICI resistance. The activity of 8 signaling pathways (TGF-β, Hedgehog, MAPK, AR, NOTCH, PI3K, JAK/STAT1-2, and NFkB) was analyzed from tumor tissue from patients with advanced melanoma. Pathway activity scores (PAS) were explored for associations with survival and response to ICI in 34 patients (19 non-responders and 15 responders). A second, independent method to investigate the predictive value of TGF-β pathway activation was conducted by determining levels of phosphorylated SMAD2. The mean TGF-β PAS of responders vs non-responders was 53.9 vs 56.8 (P = 0.265). No significant relation with progression-free survival was detected for TGF-β activity (P = 0.078). No association between pSMAD2 staining and treatment response or survival was identified. In contrast, Hedgehog scores of responders versus non-responders were 35.7 vs 41.6 (P = 0.038). High Hedgehog PAS was the sole significant predictor of resistance to ICI (OR 0.88, P = 0.033) and worse progression-free survival (HR 1-1.1, P = 0.012). TGF-β pathway activation showed no significant relation with treatment response to ICI or survival in patients with advanced melanoma. Hedgehog PAS was identified as a possible biomarker associated with both treatment response and survival.

The Correlation between BRAFV600E Expression and Integrin β3 in Melanoma Cells

This study investigated the relationship between ITGB3 and BRAFV600E expression in melanoma. It specifically focused on how the overexpression of BRAFV600E and Integrin $\beta $3 impacts wound healing and the TGF-β pathway. The findings provide valuable insights into melanoma progression and highlight potential therapeutic targets. Homozygous BRAFV600E-carrying A375 and heterozygous BRAFV600E-carrying M14 melanoma cell lines were utilized. ITGB3 mRNA levels were measured in both A375 and M14 cells, while Integrin β3 protein levels were analyzed in BRAFV600E-overexpressing A375 cells. The impact of increased BRAFV600E and Integrin β3 on cell migration was assessed using an in vitro wound healing assay. Additionally, the influence of BRAFV600E and Integrin β3 overexpression on the TGF-β pathway was evaluated through luciferase reporter assays and real-time PCR. ITGB3 levels were significantly higher in A375 cells compared to M14 cells. Overexpression of BRAFV600E in A375 cells resulted in a marked increase in Integrin β3 levels. Cells overexpressing both BRAFV600E and Integrin β3 demonstrated enhanced wound healing rates and elevated TGF-β activity. These findings suggest a strong correlation between BRAFV600E expression and Integrin β3 levels in melanoma cells. This study uncovers a significant relationship between BRAFV600E expression and Integrin β3 levels in melanoma cells. The overexpression of both BRAFV600E and Integrin β3 enhances wound healing and promotes TGF-β signalling. These findings suggest that Integrin β3 may contribute to melanoma progression and poor prognosis by influencing cell migration and the TGF-β pathway. Targeting integrins, the TGF-β pathway, and BRAFV600E present potential therapeutic strategies for melanoma treatment.

PIEZO1 attenuates Marfan syndrome aneurysm development through TGF-β signaling pathway inhibition via TGFBR2.

Marfan syndrome (MFS) is a hereditary disorder primarily caused by mutations in the FBN1 gene. Its critical cardiovascular manifestation is thoracic aortic aneurysm (TAA), which poses life-threatening risks. Owing to the lack of effective pharmacological therapies, surgical intervention continues to be the current definitive treatment. In this study, the role of Piezo-type mechanosensitive ion channel component 1 (Piezo1) in MFS was investigated and the activation of PIEZO1 was identified as a potential treatment for MFS. PIEZO1 expression was detected in MFS mice (Fbn1C1041G/+) and patients. Piezo1 conditional knockout mice in vascular smooth muscle cells of MFS mice (MFS × CKO) was generated, and bioinformatics analysis and experiments in vitro and in vivo were performed to investigate the role of Piezo1 in MFS. PIEZO1 expression decreased in the aortas of MFS mice; MFS × CKO mice showed aggravated TAA, inflammation, extracellular matrix remodelling, and TGF-β pathway activation compared to MFS mice. Mechanistically, PIEZO1 knockout exacerbated the activation of the TGF-β signalling pathway by inhibiting the endocytosis and autophagy of TGF-β receptor 2 mediated by Rab GTPase 3C. Additionally, the pharmacological activation PIEZO1 through Yoda1 prevented TGF-β signalling pathway activation and reversed TAA in MFS mice. Piezo1 deficiency aggravates MFS aneurysms by promoting TGF-β signalling pathway activation via TGF-β receptor 2 endocytosis and a decrease in autophagy. These data suggest that PIEZO1 may be a potential therapeutic target for MFS treatment.

HUVECs-derived exosomes increase neovascularization and decrease limb necrosis in hindlimb ischemia

Chronic limb-threatening ischemia (CLTI) is the most severe manifestation of peripheral arterial disease (PAD) and imposes a significantly high burden due to its high risk of mortality and amputation. Revascularization is the first-line treatment for CLTI; however, the amputation rate remains high, and approximately one-third of patients are not eligible for this treatment. Therefore, there is an urgent need for more effective therapeutic strategies. The aim of this study was to investigate the effects and mechanisms of human umbilical vein endothelial cells (HUVECs)-derived exosomes on neovascularization and the degree of necrosis in a hindlimb ischemia model and to study the biological processes underlying their mechanisms. This is an in vivo experimental study with a post-test-only control group design. Forty BALB/c mice were randomized to receive injections of exosomes, conditioned media, and phosphate-buffered saline (PBS) one day after unilateral double ligation. A sham-operated group was also included as a control. Capillary density, arteriole lumen diameter, and histopathological necrosis were measured after seven days, while clinical necrosis was observed daily. MicroRNA profiling, in silico analysis, and transcriptomic analysis of vascular endothelial growth factor (VEGF) mRNA expression were performed to determine the possible biological processes. No amputation was found in the exosome group, as well as in the conditioned media and sham-operated groups, compared to three out of seven mice (43%) in the PBS group. The capillary density was higher in the exosome than in the PBS group (p=0.026). The arteriole lumen diameter in the exosome group was larger than in the PBS (p=0.033) and sham-operated (p=0.034) groups. The scores of clinical necrosis and histopathological necrosis in the exosome group were lower than the PBS group (p=0.005), while the histopathological necrosis scores were also lower but statistically insignificant. In silico analysis showed improvement in neovascularization and necrosis, possibly through energy regulation, PI3K/AKT and TGF-β activation, the ubiquitin-proteasome system, and tyrosine kinases receptors. HUVEC exosomes were associated with lower VEGF mRNA expression, which may indicate a more effective compensatory mechanism under ischemic conditions. The exosome group had the lowest VEGF mRNA expression compared to other groups, although the difference was not statistically significant. This study highlights that HUVECs-derived exosomes improve neovascularization and decrease necrosis in a hindlimb ischemia mice model, potentially by modulating several possible mechanisms.

Discovery and preclinical evaluation of BPB-101: a novel triple functional bispecific antibody targeting GARP-TGF-β complex/SLC, free TGF-β and PD-L1.

In the tumor microenvironment (TME), the transforming growth factor-β (TGF-β) and programmed cell death receptor 1 (PD-1)/programmed death ligand 1 (PD-L1) signaling axes are complementary, nonredundant immunosuppressive signaling pathways. Studies have revealed that active TGF-β is mainly released from the glycoprotein A repetitions predominant (GARP)-TGF-β complex on the surface of activated regulatory T cells (Tregs), B cells, natural killer (NK) cells, and tumor cells. The currently available antibodies or fusion proteins that target TGF-β are limited in their abilities to simultaneously block TGF-β release and neutralize active TGF-β in the TME, thus limiting their antitumor effects. We designed and constructed a bispecific, trifunctional antibody, namely, BPB-101, that specifically targets the GARP-TGF-β complex and/or small latent complex (SLC), active TGF-β, and PD-L1. The binding ability of BPB-101 to the different antigens was determined by ELISA, FACS, and biolayer interferometry (BLI). The blocking ability of BPB-101 to the TGF-β and PD-1/PD-L1 signaling axes was determined by reporter gene assay (RGA). The antitumor effect and biosafety of BPB-101 were determined in a transgenic mouse tumor model and cynomolgus monkeys, respectively. Stability assessments, including stability in serum, after exposure to light, after repeated freeze-thaw cycles, and after high-temperature stress tests had been completed to evaluate the stability of BPB-101. BPB-101 bound efficiently to different antigenic proteins: the GARP-TGF-β complex and/or SLC, active TGF-β, and PD-L1. Data showed that BPB-101 not only effectively inhibited the release of TGF-β from human Tregs, but also blocked both the TGF-β and PD-1/PD-L1 signaling pathways. In an MC38-hPD-L1 tumor-bearing C57BL/6-hGARP mouse model, BPB-101 at a dose of 5 mg/kg significantly inhibited tumor growth, with a complete elimination rate of 50%. Stability assessments confirmed the robustness of BPB-101. Furthermore, BPB-101 showed a favorable safety profile in nonhuman primate (NHP) toxicity studies. BPB-101 is a potentially promising therapeutic candidate that may address unmet clinical needs in cancer immunotherapy, thus, BPB-101 warrants further clinical investigation.

A patient stratification signature mirrors the immunogenic potential of high grade serous ovarian cancers

BackgroundWhile high-grade serous ovarian cancer (HGSC) has proven largely resistant to immunotherapy, sporadic incidents of partial and complete response have been observed in clinical trials and case reports. These observations suggest that a molecular basis for effective immunity may exist within a subpopulation of HGSC. Herein, we developed an algorithm, CONSTRU (Computing Prognostic Marker Dependencies by Successive Testing of Gene-Stratified Subgroups), to facilitate the discovery and characterization of molecular backgrounds of HGSC that confer resistance or susceptibility to protective anti-tumor immunity.MethodsWe used CONSTRU to identify genes from tumor expression profiles that influence the prognostic power of an established immune cytolytic activity signature (CYTscore). From the identified genes, we developed a stratification signature (STRATsig) that partitioned patient populations into tertiles that varied markedly by CYTscore prognostic power. The tertile groups were then analyzed for distinguishing biological, clinical and immunological properties using integrative bioinformatics approaches.ResultsPatient survival and molecular measures of immune suppression, evasion and dysfunction varied significantly across STRATsig tertiles in validation cohorts. Tumors comprising STRATsig tertile 1 (S-T1) showed no immune-survival benefit and displayed a hyper-immune suppressed state marked by activation of TGF-β, Wnt/β-catenin and adenosine-mediated immunosuppressive pathways, with concurrent T cell dysfunction, reduced potential for antigen presentation, and enrichment of cancer-associated fibroblasts. By contrast, S-T3 tumors exhibited diminished immunosuppressive signaling, heightened antigen presentation machinery, lowered T cell dysfunction, and a significant CYTscore-survival benefit that correlated with mutational burden in a manner consistent with anti-tumor immunoediting. These tumors also showed elevated activity of DNA damage/repair, cell cycle/proliferation and oxidative phosphorylation, and displayed greater proportions of Th1 CD4 + T cells. In these patients, but not those of S-T1 or S-T2, validated predictors of immunotherapy response were prognostic of longer patient survival. Further analyses showed that STRATsig tertile properties were not explained by known HGSC molecular or clinical subtypes or singular immune mechanisms.ConclusionsSTRATsig is a composite of parallel immunoregulatory pathways that mirrors tumor immunogenic potential. Approximately one-third of HGSC cases classify as S-T3 and display a hypo-immunosuppressed and antigenic molecular composition that favors immunologic tumor control. These patients may show heightened responsiveness to current immunotherapies.

Tankyrase inhibitor IWR-1 modulates HIPPO and Transforming Growth Factor β signaling in primed bovine embryonic stem cells cultured on mouse embryonic fibroblasts

The use of tankyrase inhibitors is essential for capturing livestock embryonic stem cells (ESC), yet their mechanisms of action remain largely uncharacterized. Previous studies indicate that their roles extend beyond the suppression of canonical WNT signaling. This study investigates the effects of the tankyrase inhibitor IWR-1 on maintaining the pluripotency of bovine embryonic stem cells (bESC) cultured on mitotically inactivated mouse embryonic fibroblasts (MEF). Notably, bESC exhibited significant differentiation after one month of IWR-1 withdrawal, without a clear bias toward any specific germ layer. IWR-1 effectively inhibited TNKS2 activity in bESC, whereas it suppressed TNKS1 protein level in growth-arrested MEF. Early differentiation upon IWR-1 removal induced more substantial transcriptomic changes in MEF than in bESC. Furthermore, cell communication analysis predicted that IWR-1 influenced several paracrine and autocrine signals within the culture system. We also observed that IWR-1 repressed protein abundance of the HIPPO pathway components including TEAD4 and YAP1 in bESC and decreased transcription of HIPPO targeted genes CYR61. Protein analysis in growth-arrested MEF suggested that IWR-1 modulated MEF function by impeding TGF-β1 activation and activin A secretion which mitigated nuclear localization of SMAD2/3 in the bESC. This study underscores the role of tankyrase inhibitors in ESC self-renewal by modulating key signaling pathways and orchestrating cell-cell interactions, which may be meaningful in understanding the delicate signaling control of pluripotency in livestock and improving the culture system.