The Wnt signaling pathway in hepatocellular carcinoma: Regulatory mechanisms and therapeutic prospects.

Hepatocellular carcinoma (HCC) is a malignant tumor that arises from hepatocytes. Multiple signaling pathways play a regulatory role in the occurrence and development of HCC, with the Wnt signaling pathway being one of the primary regulatory pathways. In normal hepatocytes, the Wnt signaling pathway maintains cell regeneration and organ development. However, when aberrant activated, the Wnt pathway is closely associated with invasion, cancer stem cells(CSCs), drug resistance, and immune evasion in HCC. Among these factors, the development of drug resistance is one of the most important factors affecting the efficacy of HCC treatment. These mechanisms form the basis for tumor cell adaptation and evolution within the body, enabling continuous changes in tumor cells, resistance to drugs and immune system attacks, leading to metastasis and recurrence. In recent years, there have been numerous new discoveries regarding these mechanisms. An increasing number of drugs targeting the Wnt signaling pathway have been developed, with some already entering clinical trials. Therefore, this review encompasses the latest research on the role of the Wnt signaling pathway in the onset and progression of HCC, as well as advancements in its therapeutic strategies.

The overexpression of R-spondin 3 affects hair morphogenesis and hair development along with the formation and maturation of the hair follicle stem cells.

Mice hair follicles (HFs) are a valuable model for studying various aspects of hair biology, including morphogenesis, development, and regeneration due to their easily observable phenotype and genetic manipulability. The initiation and progression of hair follicle morphogenesis, as well as the hair follicle cycle, are regulated by various signaling pathways, of which the main role is played by the Wingless-type MMTV integration site family (Wnt) and the Bone Morphogenic Protein (BMP). During the hair follicle cycle, the BMP pathway maintains hair follicle stem cells (HFSCs) in a dormant state while the Wnt pathway activates them for hair growth. Given the pivotal role of the Wnt pathway in hair biology and HFSCs regulation, we investigated the influence of the Wnt modulator - R-spondin 3 (Rspo3), in these processes. For this purpose, we developed a transgenic mice model with the overexpression of Rspo3 (Rspo3GOF) in the whole ectoderm and its derivatives, starting from early morphogenesis. Rspo3GOF mice exhibited a distinct phenotype with sparse hair and visible bald areas, caused by reduced proliferation and increased apoptosis of hair matrix progenitor cells, which resulted in a premature anagen-to-catagen transition with a shortened growth phase and decreased overall length of all hair types. In addition, Rspo3GOF promoted induction of auchene and awl, canonical Wnt-dependent hair type during morphogenesis, but the overall hair amount remained reduced. We also discovered a delay in the pre-bulge formation during morphogenesis and prolonged immaturity of the HFSC population in the bulge region postnatally, which further impaired proper hair regeneration throughout the mice's lifespan. Our data supported that Rspo3 function observed in our model works in HFSCs' formation of pre-bulge during morphogenesis via enhancing activation of the canonical Wnt pathway, whereas in contrast, in the postnatal immature bulge, activation of canonical Wnt signaling was attenuated. In vitro studies on keratinocytes revealed changes in proliferation, migration, and colony formation, highlighting the inhibitory effect of constitutive overexpression of Rspo3 on these cellular processes. Our research provides novel insights into the role of Rspo3 in the regulation of hair morphogenesis and development, along with the formation and maturation of the HFSCs, which affect hair regeneration.

Preliminary investigation on the mechanism of baicalein regulating the effects of Nischarin on invasion and apoptosis of human breast cancer cells MCF-7 through Wnt3α/ß-catenin pathway.

BACKGROUND: Breast cancer (BC) remains the leading cause of cancer-related mortality in women. Here, we investigate the anti-tumor effects of baicalein on human BC cells (MCF-7 cells) and explore if it regulates the Nischarin protein via Wnt3α/ß-catenin signaling pathway. METHODS: We employed Wnt3α and DKK-1 to activate and inhibit the Wnt/ß-catenin signaling pathway, respectively. We used CCK-8 cell viability, flow cytometry apoptosis, wound-healing and transwell migration/invasion assays. Further, using western blotting and real-time quantitative PCR (q-PCR) we analyzed expression levels of Nischarin, MMP-9, Wnt/ß-catenin pathway (ß-catenin, Axin 1), and apoptotic pathway (Bax, Bcl-2) proteins and their mRNAs. RESULTS: We found that baicalein inhibits MCF-7 cell viability and promotes apoptosis (evidenced by increased Bax and decreased Bcl-2 expressions) in a concentration-dependent manner. It also inhibits TPA-induced migration and invasion, and downregulates MMP-9 expression. Baicalein reverses the increase in cell viability caused by Wnt3α-induced Wnt/ß-catenin pathway activation. Conversely, baicalein counteracts the increase in apoptosis caused by DKK-1 mediated inhibition of the Wnt/ß-catenin pathway. Additionally, baicalein upregulates Nischarin expression via modulating the Wnt/ß-catenin pathway as indicated by the antagonistic effects of Wnt3α and DKK-1 on this effect of baicalein. CONCLUSION: Baicalein exerts anti-tumor effects on MCF-7 cells through the Wnt3α/ß-catenin signaling pathway, and promotes apoptosis and inhibits migration and invasion. The upregulation of Nischarin by baicalein further suggests a potential therapeutic target for BC treatment.

SLAMF8 regulates osteogenesis and adipogenesis of bone marrow mesenchymal stem cells via S100A6/Wnt/ß-catenin signaling pathway.

BACKGROUND: The inflammatory microenvironment plays an essential role in bone healing after fracture. The signaling lymphocytic activation molecule family (SLAMF) members deeply participate in inflammatory response and make a vast difference. METHODS: We identified SLAMF8 in GEO datasets (GSE129165 and GSE176086) and co-expression analyses were performed to define the relationships between SLAMF8 and osteogenesis relative genes (RUNX2 and COL1A1). In vitro, we established SLAMF8 knockdown and overexpression mouse bone marrow mesenchymal stem cells (mBMSCs) lines. qPCR, Western blot, ALP staining, ARS staining, Oil Red O staining and Immunofluorescence analyses were performed to investigate the effect of SLAMF8 in mBMSCs osteogenesis and adipogenesis. In vivo, mice femoral fracture model was performed to explore the function of SLAMF8. RESULTS: SLAMF8 knockdown significantly suppressed the expression of osteogenesis relative genes (RUNX2, SP7 and COL1A1), ALP activity and mineral deposition, but increased the expression of adipogenesis relative genes (PPARγ and C/EBPα). Additionally, SLAMF8 overexpression had the opposite effects. The role SLAMF8 played in mBMSCs osteogenic and adipogenic differentiation were through S100A6 and Wnt/ß-Catenin signaling pathway. Moreover, SLAMF8 overexpression mBMSCs promoted the healing of femoral fracture. CONCLUSIONS: SLAMF8 promotes osteogenesis and inhibits adipogenesis of mBMSCs via S100A6 and Wnt/ß-Catenin signaling pathway. SLAMF8 overexpression mBMSCs effectively accelerate the healing of femoral fracture in mice.

USP7 facilitates deubiquitination of LRRC42 in colorectal cancer to accelerate tumorigenesis and augment Wnt/ß-catenin signaling.

Colorectal cancer is a prevalent malignancy with an increasing incidence worldwide. Leucine-rich repeat-containing protein 42 (LRRC42) is known to be dysregulated in tumor tissues, yet its role in colorectal cancer remains largely unexplored. Herein, the function of LRRC42 in colorectal cancer was investigated using clinical samples, cellular experiments, animal models, and multiple omics techniques. The results demonstrated that LRRC42 was highly expressed in colorectal cancer tissues and was associated with poor clinical outcomes. Silencing LRRC42 suppressed cell proliferation, induced G0/G1 phase arrest, and promoted apoptosis by reducing Bcl2 expression while elevating the expression of Bax, cleaved PARP and cleaved caspase 3. Conversely, LRRC42 overexpression exhibited the opposite effects. Consistent findings were observed in vivo. Additionally, ubiquitin specific peptidase 7 was identified as a potential LRRC42-interacting protein through immunoprecipitation-mass spectrometry, with ubiquitin specific peptidase 7 stabilizing LRRC42 expression by promoting its deubiquitination. Notably, LRRC42 overexpression partially reversed the effects of ubiquitin specific peptidase 7 silencing on tumor cell proliferation and apoptosis. mRNA sequencing analysis revealed that differentially expressed genes in LRRC42 overexpressing cells were linked to Wnt signaling pathway, suggesting that LRRC42 overexpression may activate this pathway. Furthermore, LRRC42 was proved to elevate the levels of ki67, cyclin D1 and WNT3, while reducing the level of p-ß-catenin. These findings suggest that LRRC42 perhaps serve as a potential oncogenic factor in colorectal cancer, regulated by ubiquitin specific peptidase 7 and capable of activating Wnt/ß-catenin signaling pathway.

Adaptor protein Abelson interactor 1 in homeostasis and disease.

Dysregulation of Abelson interactor 1 (ABI1) is associated with various states of disease including developmental defects, pathogen infections, and cancer. ABI1 is an adaptor protein predominantly known to regulate actin cytoskeleton organization processes such as those involved in cell adhesion, migration, and shape determination. Linked to cytoskeleton via vasodilator-stimulated phosphoprotein (VASP), Wiskott-Aldrich syndrome protein family (WAVE), and neural-Wiskott-Aldrich syndrome protein (N-WASP)-associated protein complexes, ABI1 coordinates regulation of various cytoplasmic protein signaling complexes dysregulated in disease states. The roles of ABI1 beyond actin cytoskeleton regulation are much less understood. This comprehensive, protein-centric review describes molecular roles of ABI1 as an adaptor molecule in the context of its dysregulation and associated disease outcomes to better understand disease state-specific protein signaling and affected interconnected biological processes.

Exploring circulating MiRNA signature for osteosarcoma detection: Bioinformatics-based analyzing and validation.

Early detection followed by efficient treatment still remain a considerable challenge for osteosarcoma (OS), indicating the importance of emerging innovative diagnostic methods. Circulating miRNAs offer a promising and non-invasive approach to assess the OS molecular landscapes. This study utilized RNAseq data from OS plasma miRNA expression profiles (PRJEB30542) and PCR Array data (GSE65071) from GEO and ENA databases. In total, 43 miRNAs demonstrated significant differential expression in OS samples of training dataset. A diagnostic model, including hsa-miR-30a-5p, hsa-miR-556-3p, hsa-miR-200a-3p, and hsa-miR-582-5p was identified through multivariate logistic regression analysis and demonstrated significant efficacy in differentiating OS patients from healthy controls in the validation group (AUC: 0.917, sensitivity: 1, specificity: 0.85). The result of target gene prediction and functional enrichment analyses revealed significant associations with terms such as epithelial morphogenesis, P53 and Wnt signaling pathways, and neoplasm metastasis. Further bioinformatics-based evaluations showed that the down-regulation of these miRNAs significantly correlates with poor prognosis and lower survival rate in OS patients and propose their tumor suppressor function in pathogenesis of OS. Furthermore, the study developed a miRNA-mRNA subnetwork that connects these miRNAs to the P53 and Wnt signaling pathways, which are critical pathways with oncogenic effects on OS progression. This comprehensive approach not only presents a promising diagnostic model but also proposes potential molecular markers for OS early diagnosis, making prognosis, and targeted therapy. The identified miRNA-mRNA functional axis holds promise as a valuable resource for further research in understanding OS pathogenesis and establishing therapeutic modalities.

Targeting Canonical Wnt-signaling Through GSK-3ß in Arrhythmogenic Cardiomyopathy: Conservative or Progressive?

Arrhythmogenic cardiomyopathy is a primary myocardial disease and a major cause of sudden death in all populations of the world. Canonical Wnt signalling is a critical pathway controlling numerous processes including cellular differentiation, hypertrophy and development. GSK3ß is a ubiquitous serine/threonine kinase, which acts downstream of Wnt to promote protein ubiquitination and proteasomal degradation. Several studies now suggest that inhibiting GSK3ß can prevent and reverse key pathognomonic features of ACM in a range of experimental models. However, varying concerns are reported throughout the literature including the risk of paradoxical arrhythmias, cancer and off-target effects in upstream or downstream pathways. CLINICAL RELEVANCE: In light of the start of the phase 2 TaRGET clinical trial, designed to evaluate the potential therapeutic efficacy of GSK3ß inhibition in patients with arrhythmogenic cardiomyopathy, this report aims to review the advantages and disadvantages of this strategy.

TMEM132A regulates Wnt/ß-catenin signaling through stabilizing LRP6 during mouse embryonic development.

The Wnt/ß-catenin signaling pathway is crucial for embryonic development and adult tissue homeostasis. Dysregulation of Wnt signaling is linked to various developmental anomalies and diseases, notably cancer. Although numerous regulators of the Wnt signaling pathway have been identified, their precise function during mouse embryo development remains unclear. Here, we revealed that TMEM132A is a crucial regulator of canonical Wnt/ß-catenin signaling in mouse development. Mouse embryos lacking Tmem132a displayed a range of malformations, including open spina bifida, caudal truncation, syndactyly, and renal defects, similar to the phenotypes of Wnt/ß-catenin mutants. Tmem132a knockdown in cultured cells suppressed canonical Wnt/ß-catenin signaling. In developing mice, loss of Tmem132a also led to diminished Wnt/ß-catenin signaling. Mechanistically, we showed that TMEM132A interacts with the Wnt co-receptor LRP6, thereby stabilizing it and preventing its lysosomal degradation. These findings shed light on a novel role for TMEM132A in regulating LRP6 stability and canonical Wnt/ß-catenin signaling during mouse embryo development. This study provides valuable insights into the molecular intricacies of the Wnt signaling pathway. Further research may deepen our understanding of Wnt pathway regulation and offer its potential therapeutic applications.

2-Ethylhexyl diphenyl phosphate aggravates colitis-induced neuroinflammation and behavioral abnormalities by inhibiting the PI3K-AKT-NF-κB and Wnt/GSK3ß signaling pathways.

2-Ethylhexyl diphenyl phosphate (EHDPHP), a widely used organophosphorus flame retardant (OPFR), is ubiquitous in daily life because of its extensive application in plastic production. EHDPHPs, which are only superficially applied and not chemically bonded to products, are released into the environment, posing potential health risks. With increasing environmental concentrations, EHDPHP is a growing threat, particularly to individuals with preexisting health conditions who are more susceptible to environmental pollutants. This study examined the effects of EHDPHP exposure in a colitis model, reflecting a rising chronic health issue, by assessing changes in neuroinflammation and neurobehavioral abnormalities. Healthy and dextran sulfate sodium (DSS)-induced colitis C57BL/6 J mice were treated with either 0.2 % Tween or EHDPHP solution (10 mg/kg body weight/day) for 28 days. The study revealed significant increases in the serum and expression levels of TNFα and IL-1ß, accompanied by depressive and anxiety-like behaviors. Coexposure to EHDPHP and DSS exacerbated these neurobehavioral impairments. RNA sequencing confirmed that EHDPHP triggered inflammation via the PI3K-Akt-NF-κB and Wnt/GSK3ß signaling pathways, as confirmed by Western blot analysis. These findings suggest that EHDPHP aggravates colitis-induced neuroinflammation and neurobehavioral abnormalities, highlighting the harmful impact of EHDPHP, particularly in individuals with preexisting inflammatory conditions.

Dietary titanium dioxide nanoparticles impair intestinal epithelial regeneration by perturbating the function of intestinal stem cells.

Intestinal health is closely linked to intestinal stem cells (ISCs), which are highly sensitive to the harmful substances in the lumen. However, there is limited knowledge regarding the effects of food additives on ISCs. This study aims to investigate the impact of dietary titanium dioxide nanoparticles (TiO2 NPs) compared with titanium dioxide microparticles (TiO2 MPs) on intestinal health associated with ISCs in response to dextran sodium sulfate (DSS)-induced enteritis in mice, as well as the related mechanism. We found that exposure to 1% (w/w) TiO2 NPs aggravated DSS-induced enteritis in mice, while this effect could not be observed under exposure to TiO2 MPs. Additionally, 1% (w/w) TiO2 NPs exposure under DSS-induced enteritis worsened the ISC-mediated regeneration of intestinal epithelium by decreasing the epithelial cell proliferation and epithelial turnover rate while increasing epithelial cell death. Meanwhile, using a 3D intestinal organoid model, we discovered that 20 µg/mL TiO2 NPs impaired ISC function and disrupted ISC fate specification both ex vivo and in vitro. Furthermore, TiO2 NPs hindered the nuclear translocation of ß-catenin, reducing the overall output of Wnt signaling. Together, TiO2 NPs deteriorated the intestinal epithelial regeneration of mice with DSS-induced enteritis by perturbating ISC function and fate specification through a mechanism involving Wnt signaling. These findings highlight the adverse effect of dietary TiO2 NPs on ISCs and shed light on the particle size optimization of TiO2 food additive.

Exploring DIX-DIX Homo- and Hetero-Oligomers in Wnt Signaling with AlphaFold2.

Wnt signaling is involved in embryo development and cancer. The binding between the DIX domains of Axin1/2, Dishevelled1/2/3, and Coiled-coil-DIX1 is essential for Wnt/ß-catenin signaling. Structural and biological studies have revealed that DIX domains are polymerized through head-to-tail interface interactions, which are indispensable for activating ß-catenin Wnt signaling. Although different isoforms of Dvl and Axin proteins display both redundant and specific functions in Wnt signaling, the specificity of DIX-mediated interactions remains unclear due to technical challenges. Using AlphaFold2(AF2), we predict the structures of 6 homodimers and 22 heterodimers of DIX domains without templates and compare them with the reported X-ray complex structures. PRODIGY is used to calculate the binding affinities of these DIX complexes. Our results show that the Axin2 DIX homodimer has a stronger binding affinity than the Axin1 DIX homodimer. Among Dishevelled (Dvl) proteins, the binding affinity of the Dvl1 DIX homodimer is stronger than that of Dvl2 and Dvl3. The Coiled-coil-DIX1(Ccd1) DIX homodimer shows weaker binding than the Axin1 DIX homodimer. Generally, heterodimer interactions tend to be stronger than those of homodimers. Our findings provide insights into the mechanism of the Wnt signaling pathway and highlight the potential of AF2 and PRODIGY for studying protein-protein interactions in signaling pathways.

Zonated Wnt/ß-catenin signal-activated cardiomyocytes at the atrioventricular canal promote coronary vessel formation in zebrafish.

Cells functioning at a specific zone by clustering according to gene expression are recognized as zonated cells. Here, we demonstrate anatomical and functional zones in the zebrafish heart. The cardiomyocytes (CMs) at the atrioventricular canal between the atrium and ventricle could be grouped into three zones according to the localization of signal-activated CMs: Wnt/ß-catenin signal+, Bmp signal+, and Tbx2b+ zones. Endocardial endothelial cells (ECs) changed their characteristics, penetrated the Wnt/ß-catenin signal+ CM zone, and became coronary ECs covering the heart. Coronary vessel length was reduced when the Wnt/ß-catenin signal+ CMs were depleted. Collectively, we demonstrate the importance of anatomical and functional zonation of CMs in the zebrafish heart.

Variational Approaches for Drug-Disease-Gene Links in Periodontal Inflammation.

INTRODUCTION AND OBJECTIVES: Oral diseases, including gingivitis and periodontitis, are linked to the Wnt signaling pathway, vital for bone metabolism, cementum homeostasis, and mesenchymal stem cell differentiation. Advances in generative AI techniques, such as variational autoencoders (VAEs) and quantum variational classifiers (QVCs), offer promising tools for predicting gene associations between drugs and diseases. This study aims to compare the predictive performance of VAEs and QVCs in modeling drug-disease gene networks within the Wnt signaling pathway in periodontal inflammation. METHODS: Genes associated with Wnt-related periodontal inflammation were identified through comprehensive literature reviews and genomic databases. Their roles in various biological processes were evaluated using gene set enrichment analysis, employing tools like Enrichr, which integrates diverse gene sets from sources such as DSigDB, DisGeNET, and Lincs_l1000.drug. The study then applied VAEs and QVCs to predict gene-disease associations related to the Wnt signaling pathway. RESULTS: The analysis revealed an extensive network comprising 1738 nodes and 1498 edges, averaging 1.992 neighbors per node. The network exhibited a diameter of 2, a radius of 1, and a characteristic path length of 1.992, indicating limited interconnectivity. The VQA model demonstrated a high accuracy rate of 97.5%, although it only detected 50% of anomalies. The VQC model achieved a precision of 78%, with Class 1 samples showing improved recall and a balanced F1 score. CONCLUSION: VQC and VAE models exhibit strong potential for discovering FDA-approved drugs by predicting gene-drug associations in periodontitis based on the Wnt signaling pathway. CLINICAL RELEVANCE: This study highlights the potential of VAEs and QVCs in predicting gene-drug associations for periodontal inflammation. This could lead to more targeted therapies for oral diseases like periodontitis, improving patient outcomes and advancing personalized treatment strategies in clinical practice.

Combinatorial Effects of 5-Fluorouracil and Menadione on Wnt/ß-Catenin Pathway in Human Colorectal Cancer Cells.

The incidence and mortality rates of colorectal cancer (CRC) are alarmingly high, and the scientific community is consistently engaged in developing newer therapeutic options for cancer cure or prevention. The fluoropyrimidine drug, 5-fluorouracil (5FU), remains the first line of treatment against CRC; nevertheless, relapses frequently occur since the cells gain resistance over time through various mechanisms. Studies have highlighted the significance of combinatorial treatment of a Wnt signaling inhibitor and 5FU as a better treatment strategy to overcome 5FU resistance. Small molecules that specifically target and disrupt ß-catenin-TCF interaction, a crucial step of the Wnt signaling, are promising in CRC treatment. In this study, we investigated the synergistic cytotoxic activity of menadione with 5FU as the former has previously been shown to downregulate Wnt signaling in CRC cells. Docking and experimental results suggest that the drug combination interfered with key protein-protein interactions in the ß-catenin-TCF complex, exerted synergistic anti-cancerous effects in CRC cells, and downregulated the expression of Wnt signaling proteins. Taken together, our data suggest that the simultaneous binding of 5FU and menadione to ß-catenin can block Wnt signaling by disrupting ß-catenin-TCF interaction and inhibit the proliferation of CRC cells.

Facilitation of diabetic wound healing by far upstream element binding protein 1 through augmentation of dermal fibroblast activity.

AIMS: Diabetes mellitus (DM) often leads to wound healing complications, partly attributed to the accumulation of advanced glycosylation end products (AGEs) that impair fibroblast function. Far Upstream Element Binding Protein 1 (FUBP1) regulates cell proliferation, migration, and collagen synthesis. However, the impact of FUBP1 on diabetic wound healing remains unknown. This study is designed to explore the function and mechanisms of FUBP1 in diabetic wound healing. METHODS: Eighteen Sprague-Dawley rats (weighing 220-240 g) were randomly assigned to three groups (n = 6): a control group (NC) of healthy rats, a model group (DM) of untreated diabetic rats, and a treatment group (DM + FUBP1) of diabetic rats accepting FUBP1 treatment. A 10 mm diameter circular full-thickness skin defect was created on the back of each rat. On days 1 and 7, rats in the treatment group received local injections of 5 µg FUBP1 protein at the wound site, whereas the control group and model group were administered saline. Wound healing was documented on days 0, 3, 7, 10, and 14, with tissue samples from the wound areas collected on day 14 for histological analysis, including H&E staining, Masson's trichrome staining, and immunohistochemistry. Western blot analysis was utilized to assess the expression of GSK-3ß, Wnt3a, and ß-catenin. In vitro, the effects of various concentrations of AGEs on cell viability and FUBP1 expression were examined in human dermal fibroblasts (HDF). Cells were genetically modified to overexpress FUBP1 using lentiviral vectors and were cultured for 48 h in media with or without AGEs. The impacts on fibroblast proliferation, migration, and Wnt/ß-catenin signaling were evaluated using CCK-8, scratch assays, and Western blot analysis. RESULTS: Animal investigation revealed that from day 7 onwards, the wound healing rate of the treatment group was higher than that of the model group but lower than the control group. On day 14, the wound healing rates were as follows: control group (0.97 ± 0.01), model group (0.84 ± 0.03), and treatment group (0.93 ± 0.01). These differences were statistically significant. Histological analysis indicates that FUBP1 promotes granulation tissue formation, re-epithelialization, and collagen deposition in treatment group. Additionally, FUBP1 protein expression decreased in dermal fibroblasts when exposed to AGEs. Overexpression of FUBP1 significantly enhanced fibroblast proliferation and migration, activating the Wnt/ß-catenin pathway and mitigating the inhibitory effects of AGEs. CONCLUSIONS: Our results suggest that FUBP1 can be a promising therapeutic target for diabetic wound healing, potentially counteracting the detrimental effects of AGEs on dermal fibroblasts through the Wnt/ß-catenin pathway.

Downregulating LKB1 in bone marrow mesenchymal stem cells could inhibit CD4+ T cell proliferation via the PD-1/PD-L1 signaling pathway.

BACKGROUND: Our previous research has shown that LKB1 in amniotic mesenchymal stem cells (MSCs) serves as a vital regulator of regulatory T cell differentiation and T cell proliferation, which may have a similar role in bone marrow MSCs (BMMSCs). Therefore, we investigated the role of LKB1 in BMMSCs for regulating CD4+ T cell proliferation in the bone micro-environment of AML. METHODS: RT-PCR was used to assessed LKB1 expression in BMMSCs derived from AML patients and healthy controls. Subsequently, LKB1 was knocked down in the BMMSCs line HS-5 (HS-5-LKB1KD). Co-cultures in vitro were established to analyze the effect of HS-5-LKB1KD on CD4+ T cell. Flow cytometry was employed to measure PD-L1 and CD4+ T cell proliferation levels. Western blot was utilized to detect related proteins. RESULTS: The expression of LKB1 in BMMSCs derived from AML patients was decreased. Knockdown of LKB1 in HS-5 resulted in upregulation of PD-L1 expression. Co-culture of peripheral blood CD4+ T cell with HS-5-LKB1KD exhibited reduced CD4+ T cell proliferation compared to co-culture with HS-5-LKB1con. Furthermore, blocking PD-L1 in the co-culture conditions could restore the reduced CD4+ T cell proliferation. Additionally, it was found that upregulation of the Wnt signaling pathway-related proteins following LKB1 knockdown in HS-5, indicating that downregulating LKB1 could promote PD-L1 expression through activation of the Wnt signaling pathway. CONCLUSIONS: The decreased expression of LKB1 in BMMSCs may activate the Wnt signaling pathway, leading to increased PD-L1 expression. This inhibited CD4+ T cell proliferation, which might lead to impaired anti-tumor immunity in AML patients and promote AML progression.

Cuproptosis in microsatellite stable colon cancer cells affects the cytotoxicity of CD8+T through the WNT signaling pathway.

The microsatellite stable (MSS) colon cancer (CC) has long been considered resistant to immunotherapy. Cuproptosis, as a novel form of cell death, may interact with tumor immunity. This project focused on the impact of cuproptosis on the cytotoxicity of CD8+T in MSS CC, aiming to provide effective clues for improving the treatment strategy of MSS CC. The study developed an MSS CC cuproptosis model using 50 nM elesclomol and 1 µM CuCl2. Cuproptotic SW480 cells were directly co-cultured with CD8+ T cells. Cuproptosis levels were assessed via intracellular copper ion detection, Western blot, and confocal laser scanning microscopy. CCK-8, Hochest/PI staining, CFSE cell proliferation assay, LDH cytotoxicity detection, and ELISA were used to evaluate CD8+ T cell immune activity and cytotoxicity. Transcriptome sequencing and bioinformatics analysis identified regulated signals in cuproptotic SW480 cells. A rescue experiment utilized a WNT pathway activator (BML-284). PD-L1 expression in cells/membranes was analyzed using qRT-PCR, Western blot, and flow cytometry. NSG mice were immunoreconstituted, and the effects of cuproptosis on immune infiltration and cancer progression in MSS CC mice were assessed using ELISA and immunohistochemistry (IHC). Treatment with 50 nM elesclomol and 1 µM CuCl2 significantly increased cuproptosis in SW480 cells. Co-culture with CD8+ T cells enhanced their cytotoxicity. Sequencing revealed cuproptosis-mediated modulation of immune and inflammatory pathways, including WNT signaling. Rescue experiments showed downregulation of WNT signaling in cuproptotic SW480 cells. Indirectly, CD8+ T cell immune function was enhanced by reducing PD-L1 expression. In mice, cuproptosis resulted in increased infiltration of CD8+ T cells in tumor tissue, leading to delayed cancer progression compared to the control group. Cuproptosis in MSS CC cells enhances the cytotoxicity of CD8+ T cells, which may be achieved through downregulation of the WNT signaling pathway and decreased expression of PD-L1. In the future, drugs that can induce cuproptosis may be a promising approach to improve MSS CC immunotherapy.

Natural small molecule compounds targeting Wnt signaling pathway inhibit HPV infection.

BACKGROUND: High-risk human papillomavirus (HPV) infection is a major risk factor of HPV-related tumors, especially cervical cancer. To date, there is no specific drug for the treatment of HPV infection. PURPOSE: To explore the role of canonical Wnt signaling pathway in HPV16 infection and to screen inhibitors against HPV16 infection from natural small molecule compounds targeting the canonicalWnt pathway. METHODS: Wnt pathway inhibitor IWP-2 and FH535 were used to inhibit Wnt/ß-catenin signaling pathway. HPV16-GFP pseudovirus infectivity were analyzed by fluorescence microscopy and fluorescence activated cell sorting. A small molecule screening of a total of CFDA-approved 29 natural compounds targeting the Wnt pathway was performed. RESULTS: Wnt signaling pathway inhibitor suppressed HPV16-GFP pseudovirus infection in HaCat cells. Natural small molecule compounds screening identified 6-Gingerol, gossypol, tanshinone II2A, and EGCG as inhibitors of HPV16-GFP pseudovirus infection. CONCLUSION: Wnt signaling pathway is involved in the process of HPV infection of host cells. 6-Gingerol, gossypol, tanshinone II2A, and EGCG inhibited HPV16-GFP pseudovirus infection and suppressed Wnt/ß-catenin pathway in HaCat cells.

Cellular localization of a variant RAPGEF5 protein associated with idiopathic epilepsy risk in the Belgian shepherd.

The Wnt signaling pathway is critical for normal embryonic development. Disruptions in the Wnt signaling pathway have been linked to neurological disorders. The RAPGEF5 protein is a partner in Wnt signaling and a RAPGEF5 3-bp insertion is associated with increased risk for idiopathic epilepsy in the Belgian shepherd dog. The 3-bp insertion risk variant introduces an alanine residue predicted to disrupt the protein. Wildtype and the risk variant RAPGEF5 cDNAs were cloned into green fluorescent protein (GFP) expression vectors and transfected into canine kidney cells. The cellular localization of each GFP-labeled RAPGEF5 protein was assessed. Variant RAPGEF5 protein was altered in its localization from that of the wildtype protein and rather than localized to the nucleus and cytoplasm as seen for the wildtype, it was predominantly found in the cytoplasm. Belgian shepherds with the risk variant for RAPGEF5 may have altered Wnt signaling due to modified intracellular localization which in turn could thereby contribute to the expression of idiopathic epilepsy.

A small insertion in the RAPGEF5 gene is shown to be associated with an increased risk for idiopathic epilepsy in Belgian Shepherds. The insertion introduces an additional amino acid that is predicted to disrupt the RAPGEF5 protein. This protein is involved in the Wnt signaling pathway which is critical for normal embryonic development and disturbances in the Wnt pathway have been linked to neurological disorders. To determine the impact of the insertion on RAPGEF5 function, coding DNA sequences for both the normal and risk variant RAPGEF5 were cloned into a fluorescent expression vector and transfected into cultured canine kidney cells. The cellular location of the labeled RAPGEF5 proteins was then visually assessed. In contrast to the localization of the normal protein to the nucleus, the risk variant protein was located predominantly in the cytoplasm. With this altered location within the cell, the risk variant protein may alter the Wnt signaling pathway and contribute to the idiopathic epilepsy observed in Belgian Shepherds.