Β-catenin Protein Research Articles

Deubiquitinase PSMD14 promotes tumorigenicity of glioblastoma by deubiquitinating and stabilizing β-catenin.

The deubiquitinating enzyme 26S proteasome non-ATPase regulatory subunit 14 (PSMD14), a member of the JAB1/MPN/Mov34 metalloenzyme (JAMM) family, has been shown to function as an oncogene in various human cancers. However, the function of PSMD14 in glioma and the underlying mechanism remain unclear. In this study, our findings reveal a dramatic upregulation of PSMD14 in GBMs, which is associated with poor survival outcomes. Knocking down PSMD14 is associated with decreased proliferation and invasion of GBM cells in vitro and inhibited tumor growth in a xenograft mouse model. Mechanistically, PSMD14 directly interacts with β-catenin, leading to a decrease in the K48-linked ubiquitination of β-catenin and subsequent β-catenin stabilization. Increased β-catenin expression significantly reverses the inhibitory effects of PSMD14 knockdown on the migration, invasion, and tumor growth of GBM cells. Moreover, we observed a significant correlation between PSMD14 and β-catenin expression in human GBM samples. In summary, our results reveal that PSMD14 is a crucial deubiquitinase that is responsible for stabilizing the β-catenin protein, highlighting its potential for use as a therapeutic target for GBM.

F. Nucleatum enhances oral squamous cell carcinoma proliferation via E-cadherin/β-Catenin pathway

BackgroundFusobacterium nucleatum (F. nucleatum) is a microbial risk factor whose presence increases the risk of oral squamous cell carcinoma (OSCC) progression. However, whether it can promote the proliferation of OSCC cells remains unknown.MethodsIn this study, we investigated F. nucleatum effect on OSCC cell proliferation using in vitro and in vivo experiments.ResultsOur results showed that F. nucleatum promoted OSCC cell proliferation, doubling the cell count after 72 h (CCK-8 assay). Cell cycle analysis revealed G2/M phase arrest. F. nucleatum interaction with CDH1 triggered phosphorylation, upregulating downstream protein β-catenin and activating cyclinD1 and Myc. Notably, F. nucleatum did not affect noncancerous cells, unrelated to CDH1 expression levels in CAL27 cells. Overexpression of phosphorylated CDH1 in 293T cells did not upregulate β-catenin and cycle-related genes. In vivo BALB/c nude experiments showed increased tumor volume and Ki-67 proliferation index after F. nucleatum intervention.ConclusionOur study suggests that F. nucleatum promotes OSCC cell proliferation through the CDH1/β-catenin pathway, advancing our understanding of its role in OSCC progression and highlighting its potential as a therapeutic target.

TM7SF2-induced lipid reprogramming promotes cell proliferation and migration via CPT1A/Wnt/β-Catenin axis in cervical cancer cells

Cervical cancer poses a serious threat to women’s health globally. Our previous studies found that upregulation of TM7SF2, which works as an enzyme involved in the process of cholesterol biosynthesis expression, was highly correlated with cervical cancer. However, the mechanistic basis of TM7SF2 promoting cervical cancer progression via lipid metabolism remains poorly understood. Therefore, quantification of fatty acids and lipid droplets were performed in vitro and in vivo. The protein-protein interaction was verified by Co-IP technique. The mechanism and underlying signaling pathway of TM7SF2 via CPT1A associated lipid metabolism in cervical cancer development were explored using Western blotting, IHC, colony formation, transwell assay, and wound healing assay. This study reported that overexpression of TM7SF2 increased fatty acids content and lipid droplets both in vivo and in vitro experiments. While knockout of TM7SF2 obviously attenuated this process. Moreover, TM7SF2 directly bonded with CPT1A, a key enzyme in fatty acid oxidation, and regulated CPT1A protein expression in cervical cancer cells. Notably, the proliferation and metastasis of cervical cancer cells were elevated when their CPT1A expression was upregulated. Then, rescue assay identified that CPT1A overexpressed could enhance the cell viability and migration in TM7SF2-knockout cells. Furthermore, depletion of TM7SF2 significantly inhibited WNT and β-catenin proteins expression, which was enhanced by CPT1A-overexpressed. The proliferation and migration of cervical cancer cells were reversed in CPT1A-overexpressed cells with the treatment of MSAB, an inhibitor of Wnt/β-Catenin pathway. This study put forward an idea that TM7SF2-induced lipid reprogramming promotes proliferation and migration via CPT1A/Wnt/β-Catenin axis in cervical cancer, underlying the progression of cervical cancer.

LncRNA DLG5-AS1 facilitates breast cancer cell proliferation and invasion by promoting EZH2-mediated transcriptional silencing of SFRP1

Rapid proliferation and metastasis of breast cancer contributed to poor clinical prognosis. Accumulating evidence revealed that the dysregulation of long noncoding RNAs (lncRNAs) was associated with breast cancer progression. However, the role of lncRNA DLG5-AS1 in breast cancer has not been established. Here, we investigated the mechanisms of DLG5-AS1 in the development of breast cancer. We found that the expression of DLG5-AS1 was significantly upregulated in breast cancer tissues and cell lines. DLG5-AS1 interference markedly restrained AU565 cell proliferation, invasion, the expression of apoptosis related (caspase3 and caspase8) and Wnt/β-catenin pathway related proteins (wnt5a, β-Catenin and c-Myc), as well as promoted cell apoptosis, whereas DLG5-AS1 overexpression showed an opposite effects. In addition, DLG5-AS1 could directly bind with miR-519 b-3p. We also found that enhancer of zeste homolog 2 (EZH2) is a direct target of miR-519 b-3p, and DLG5-AS1 upregulated EZH2 expression by inhibiting the expression of miR-519 b-3p. EZH2 restrained secreted frizzled related protein 1 (SFRP1) expression through inducing H3 histone methylation in its promoter. MiR-519 b-3p overexpression or SFRP1 knockdown memorably reversed the effects of DLG5-AS1 overexpression on cell functions and Wnt/β-Catenin pathway related protein expression. Finally, in vivo experiments demonstrated that silencing of DLG5-AS1 inhibited xenograft tumor development in mice. Taken together, these findings demonstrated that DLG5-AS1 facilitated cell proliferation and invasion by promoting EZH2-mediated transcriptional silencing of SFRP1 in breast cancer.

MiR-199a-5p modulates choroidal neovascularization by regulating Wnt7b/Wnt/β-catenin signaling pathway.

Choroidal neovascularization (CNV) can be seen in many fundus diseases, and lead to fundus exudation, bleeding, or vision loss. miRNAs are vital regulator in CNV. miR-199a-5p has been proved to be involved in regulating vascular formation of endothelial cells, but its role in CNV remains unclear. This study aims to study the role of miR-199a-5p in CNV. Laser irradiation was used to induce CNV model. The lesion area of CNV was calculated by high-resolution angiography with fluorescein isothiocyanate-dextran. Wnt family member 7b (Wnt7b), β-catenin, and Wnt pathway proteins was measured by western blot. Immunofluorescence was performed to test Wnt7b, β-catenin, CD31, and p-p65. miR-199a-5p and Wnt7b mRNA were tested by reverse transcription real-time polymerase chain reaction. Cell count kit-8, wound healing, Transwell, tube formation, and flow cytometry were used to detect the function of miR-199a-5p and Wnt7b on human retinal microvascular endothelial cells (HRMEC). TargetScan database and dual-luciferase reporter assay verified the interaction between miR-199a-5p and Wnt7b. The results revealed that Wnt7b increased in CNV rats. Knocking down Wnt7b repressed cell proliferation, migration, invasion, and angiogenesis, and accelerated cell apoptosis of HRMEC. Dual-luciferase reporter assay verified that miR-199a-5p targeted Wnt7b. Overexpression of miR-199a-5p inhibited the angiogenesis of HRMEC and promoted cell apoptosis by inhibiting Wbt7b. In vivo experiment found that Wnt7b rescued the promotion of miR-199a-5p inhibition on CNV lesion of rats. In addition, Wnt7b positively regulated Wnt/β-catenin signaling pathway and promoted the angiogenesis of HRMEC. In conclusion, overexpression of miR-199a-5p inhibited the angiogenesis of HRMEC by regulating Wnt7b/Wnt/β-catenin signaling pathway, which may serve as a promising therapy target of CNV.

Identification of potential hub genes and biological mechanism in rheumatoid arthritis and non-small cell lung cancer via integrated bioinformatics analysis

BackgroundAlthough there is evidence of the association between RA and NSCLC, little is known about their interaction mechanisms. The aim of this study is to identify potential hub genes and biological mechanism in RA and NSCLC via integrated bioinformatics analysis. MethodsThe gene expression datasets of RA and NSCLC were downloaded to discover and validate hub genes. After identifying DEGs, we performed enrichment analysis, PPI network construction and module analysis, selection and validation of hub genes. Moreover, we selected the hub gene PTPRC for expression and prognosis analysis, immune analysis, mutation and methylation analysis in NSCLC. Finally, we performed real-time PCR, colony formation assay, wound healing assay, transwell invasion assay, sphere formation assay and western blotting to validate the role of PTPRC in A549 cells. ResultsWe obtained 320 DEGs for subsequent analysis. Enrichment results showed that the DEGs were mainly involved in Th1, Th2 and Th17 cell differentiation. In addition, four hub genes, BIRC5, PTPRC, PLEK, and FYN, were identified after selection and validation. These hub genes were subsequently shown to be closely associated with immune cells and related pathways. In NSCLC, PTPRC was downregulated, positively correlated with immune infiltration and immune cells. Experiments showed that PTPRC could promote the proliferation, migration and invasion, and the ability to form spheroids of A549 cells. In addition, PTPRC could regulate the increased expression of CD45, β-catenin, c-Myc and LEF1 proteins. ConclusionsThis study explored the hub genes and related mechanisms of RA and NSCLC, demonstrated the central role of the inflammatory response and the adaptive immune system, and identified PTPRC as an immune-related biomarker and potential therapeutic target for RA and NSCLC patients. In addition, PTPRC can significantly promote the proliferation, migration and invasion of A549 cells, and its mechanism may be to promote the EMT process by regulating the Wnt signaling pathway and promote cell stemness, which in turn has a promoting effect on A549 cells.

Stereo-selective cardiac toxicity induced by metconazole via oxidative stress and the wnt/β-catenin signaling pathway in zebrafish embryos

Metconazole (MEZ), a chiral triazole fungicide, produces enantioselective adverse effects in non-target organisms. Among MEZ's isomers, cis-MEZ displays robust antimicrobial properties. Evaluating MEZ and cis-MEZ's toxicity may mitigate fungicide usage and safeguard non-target organisms. Our study evaluated the toxicity of MEZ and its cis-isomers at concentrations of 0.02, 0.2, 2, and 4 mg L−1. We report stereoselectivity and severe cardiovascular defects in zebrafish, including pericardial oedema, decreased heart rate, increased sinus venous and bulbous arteries distances, intersegmental vessel defects, and altered cardiovascular development genes (hand2, gata4, nkx2.5, tbx5, vmhc, amhc, dll4, vegfaa, and vegfc). Further, MEZ significantly increased oxidative stress and apoptosis in zebrafish, primarily in the cardiac region. Isoquercetin, an antioxidant found in plants, partially mitigates MEZ-induced cardiac defects. Furthermore, MEZ upregulated the Wnt/β-catenin pathway genes (wnt3, β-catenin, axin2, and gsk-3β) and β-catenin protein expression. Inhibitor of Wnt Response-1 (IWR-1) rescued MEZ-induced cardiotoxicity. Our findings highlight oxidative stress, altered cardiovascular development genes, and upregulated Wnt/β-catenin signaling as contributors to cardiovascular toxicity in response to MEZ and cis-MEZ treatments. Importantly, 1R,5S-MEZ exhibited greater cardiotoxicity than 1S,5R-MEZ. Thus, our study provides a comprehensive understanding of cis-MEZ's cardiovascular toxicity in aquatic life.

BMP Stimulation Differentially Affects Phosphorylation and Protein Stability of β-Catenin in Breast Cancer Cell Lines

Increased expression and nuclear translocation of β-CATENIN is frequently observed in breast cancer, and it correlates with poor prognosis. Current treatment strategies targeting β-CATENIN are not as efficient as desired. Therefore, detailed understanding of β-CATENIN regulation is crucial. Bone morphogenetic proteins (BMP) and Wingless/Integrated (WNT) pathway crosstalk is well-studied for many cancer types including colorectal cancer, whereas it is still poorly understood for breast cancer. Analysis of breast cancer patient data revealed that BMP2 and BMP6 were significantly downregulated in tumors. Since mutation frequency in genes enhancing β-CATENIN protein stability is relatively low in breast cancer, we aimed to investigate whether decreased BMP ligand expression could contribute to a high protein level of β-CATENIN in breast cancer cells. We demonstrated that downstream of BMP stimulation, SMAD4 is required to reduce β-CATENIN protein stability through the phosphorylation in MCF7 and T47D cells. Consequently, BMP stimulation reduces β-CATENIN levels and prevents its nuclear translocation and target gene expression in MCF7 cells. Conversely, BMP stimulation has no effect on β-CATENIN phosphorylation or stability in MDA-MB-231 and MDA-MB-468 cells. Likewise, SMAD4 modulation does not alter the response of those cells, indicating that SMAD4 alone is insufficient for BMP-induced β-CATENIN phosphorylation. While our data suggest that considering BMP activity may serve as a prognostic marker for understanding β-CATENIN accumulation risk, further investigation is needed to elucidate the differential responsiveness of breast cancer cell lines.

Endothelial deubiquinatase YOD1 mediates Ang II-induced vascular endothelial-mesenchymal transition and remodeling by regulating β-catenin.

Hypertension is a prominent contributor to vascular injury. Deubiquinatase has been implicated in the regulation of hypertension-induced vascular injury. In the present study we investigated the specific role of deubiquinatase YOD1 in hypertension-induced vascular injury. Vascular endothelial endothelial-mesenchymal transition (EndMT) was induced in male WT and YOD1-/- mice by administration of Ang II (1 μg/kg per minute) via osmotic pump for four weeks. We showed a significantly increased expression of YOD1 in mouse vascular endothelial cells upon Ang II stimulation. Knockout of YOD1 resulted in a notable reduction in EndMT in vascular endothelial cells of Ang II-treated mouse; a similar result was observed in Ang II-treated human umbilical vein endothelial cells (HUVECs). We then conducted LC-MS/MS and co-immunoprecipitation (Co-IP) analyses to verify the binding between YOD1 and EndMT-related proteins, and found that YOD1 directly bound to β-catenin in HUVECs via its ovarian tumor-associated protease (OTU) domain, and histidine at 262 performing deubiquitination to maintain β-catenin protein stability by removing the K48 ubiquitin chain from β-catenin and preventing its proteasome degradation, thereby promoting EndMT of vascular endothelial cells. Oral administration of β-catenin inhibitor MSAB (20 mg/kg, every other day for four weeks) eliminated the protective effect of YOD1 deletion on vascular endothelial injury. In conclusion, we demonstrate a new YOD1-β-catenin axis in regulating Ang II-induced vascular endothelial injury and reveal YOD1 as a deubiquitinating enzyme for β-catenin, suggesting that targeting YOD1 holds promise as a potential therapeutic strategy for treating β-catenin-mediated vascular diseases.

Wnt signaling in cranial bones as a proponent of TH-induced craniosynostosis.

Craniosynostosis (CS) is the premature fusion of the sutures within the skull during embryonic development. Recently, thyrotoxicosis in pregnant women has been shown to increase the risk of craniosynostosis in the developing fetus, but the specific mechanism relating the accelerated bone development in CS to increased thyroid hormone (TH) is unknown. Different cell signaling pathways, such as Wnt, Ihh, BMP, and IGF-1, have been identified in TH regulated bone development. However, the Wnt pathway has been chosen in this study for its extensive involvement in bone development. It is known that Wnt-signaling increases levels of β-catenin which then drives the transcription of Wnt/β-catenin target genes such as Twist1 and Runx2. When upregulated, these genes have been linked to increased osteoblast differentiation and bone formation. In this study, we aimed to determine the level of Wnt signaling activation, in cranial bones of differing embryonic origins (frontal and parietal bones) from an avian model of thyrotoxicosis. Collected bones were processed for assessing β-catenin protein expression by western blotting and Twist1 and Runx2 gene expression by qRT-PCR. Analysis is currently in progress. We predict that differences will be observed between the parietal and frontal bones as the parietal bones derive from the mesoderm whereas the frontal bones derive from the neural crest.

Wnt signaling regulates chemokine production and cell migration of circulating human monocytes

The β-catenin dependent canonical Wnt signaling pathway plays a crucial role in maintaining normal homeostasis. However, when dysregulated, Wnt signaling is closely associated with various pathological conditions, including inflammation and different types of cancer.Here, we show a new connection between the leukocyte inflammatory response and the Wnt signaling pathway. Specifically, we demonstrate that circulating human primary monocytes express distinct Wnt signaling components and are susceptible to stimulation by the classical Wnt ligand—Wnt-3a. Although this stimulation increased the levels of β-catenin protein, the expression of the classical Wnt-target genes was not affected. Intriguingly, treating circulating human monocytes with Wnt-3a induces the secretion of cytokines and chemokines, enhancing monocyte migration. Mechanistically, the enhanced monocyte migration in response to Wnt stimuli is mediated through CCL2, a strong monocyte-chemoattractant.To further explore the physiological relevance of these findings, we conducted ex-vivo experiments using blood samples of patients with rheumatic joint diseases (RJD) – conditions where monocytes are known to be dysfunctional. Wnt-3a generated a unique cytokine expression profile, which was significantly distinct from that observed in monocytes obtained from healthy donors.Thus, our results provide the first evidence that Wnt-3a may serve as a potent stimulator of monocyte-driven immune processes. These findings contribute to our understanding of inflammatory diseases and, more importantly, shed light on the role of a core signaling pathway in the circulation.

Melatonin inhibits tongue squamous cell carcinoma: Interplay of ER stress-induced apoptosis and autophagy with cell migration

Tongue squamous cell carcinoma (TSCC) occupies a high proportion of oral squamous cell carcinoma. TSCC features high lymph node metastasis rates and chemotherapy resistance with a poor prognosis. Therefore, an effective therapy strategy is needed to improve patient prognosis. Melatonin (MT) is a natural indole compound shown to have anti-tumor effects in several cancers. This study focused on the role and mechanism of MT in TSCC cells. The results of the study suggest that MT could inhibit cell proliferation in CRL-1623 cells. Western blot analysis showed the down-regulate of cyclin B1 and the up-regulate P21 protein by MT. MT was also shown to down-regulate the expression of Zeb1, Wnt5A/B, and β-catenin protein and up-regulate E-cadherin to inhibit the migration of CRL-1623 cells. MT also promoted the expression of ATF4, ATF6, Bip, BAP31 and CHOP in CRL-1623 cells leading to endoplasmic reticulum stress, and induced autophagy and apoptosis in CRL-1623 cells. Western blots showed that MT could promote the expression of Bax, LC3, and Beclin1 proteins and inhibit the expression of p62. We screened differentially expressed long non-coding RNAs (lncRNAs) in MT-treated cells and found that the expression of MALAT1 and H19 decreased. Moreover, MT inhibited tumor growth in nude mice inoculated with CRL-1623 cells. These results suggest that MT could induce autophagy, promote apoptosis, and provide a potential natural compound for the treatment of TSCC.

Dietary copper improves intestinal structural integrity in juvenile grass carp (Ctenopharyngodon idella) probably related to its increased intestinal antioxidant capacity and apical junction complex

This research evaluated the effects of copper (Cu) on intestinal antioxidant capacity and apical junctional complex (AJC) in juvenile grass carp. A total of 1080 healthy juvenile grass carp (11.16 ± 0.01 g) were fed six diets including different dosages of Cu, namely 0, 2, 4, 6, 8 (Cu citrate [CuCit] as Cu source) and 3 mg/kg (CuSO4·5H2O as Cu source), lasting for 9 weeks. The findings revealed that dietary optimal Cu supplementation (2.2 to 4.1 mg/kg) promoted intestinal growth, including intestinal length, intestinal length index, intestinal weight, and intestinal somatic index (P < 0.05). Furthermore, optimal Cu boosted the intestinal mucosal barrier in juvenile grass carp. On the one hand, optimal Cu reduced diamine oxidase and D-lactate levels in serum (P < 0.05), reduced levels of the oxidative damage indicators malondialdehyde, reactive oxygen species (ROS), protein carbonyl, and also elevated superoxide dismutase and catalase mRNA levels (P < 0.05), thus boosting intestinal antioxidant capacity, in which the binding protein Keap1a/1b/Nrf2 signaling pathway might be involved. Optimal Cu had no impact on glutathione peroxidase 1b (GPx1b) gene expression (P > 0.05). On the other hand, optimal Cu increased intestinal tight junction (TJ) proteins (except for claudin 15b) and adherens junction (AJ) proteins (E-cadherin, α-catenin, β-catenin, nectin and afadin) mRNA levels (P < 0.05), which could be connected to the signaling pathway formed by the Ras homolog gene family, member A (RhoA), Rho-associated protein kinase (ROCK), and myosin light chain kinase (MLCK). Finally, based on serum indicator D-lactate and intestinal oxidative damage index (ROS), Cu requirement (CuCit as Cu source) for juvenile grass carp from initial weight to final weight (from 11 to 173 g) was determined to be 4.14 and 4.12 mg/kg diet, respectively. This work may provide a theoretical foundation for identifying putative Cu regulation pathways on fish intestinal health.

"Simmer pus and grow flesh" method promotes chronic wound healing in rats via bFGF-Wnt β-catenin signaling pathway.

The purpose of this study was to explore the mechanism of "simmer pus and grow meat" method based on bFGF regulating WNT / β-Catenin signaling pathway. Of 100 SPF rats, 25 were randomly selected as blank group, and 75 rats were established chronic infectious wound model and divided into blank group, model group (normal saline treatment, n = 25), experimental group (purple and white ointment treatment, n = 25), and wet burn ointment group (wet burn treatment, n = 25). The wound healing rate of rats was compared. The protein expressions of PCAN, VEGF, bFGF, β-Catenin, GSK-3β and C-Myc in granulation tissues were detected. On the 7th day, the wound healing rate of the model group was lower than that of the other 3 groups (P<0.05), and the wound healing rate of the positive control group was higher than that of the experimental group and the control group (P<0.05). The expressions of bFGF, GSK-3β and C-MyC in model group were higher than those in control group (P<0.05). The β-catenin protein expression in the model group was lower than that in the control group (P<0.05), and the β-catenin protein expression in the experimental group and the positive control group was higher than that in the model group (P<0.05). The expressions of PCAN and VEGF in model group were lower than those in model group (P<0.05). We found that Zibai ointment promotes chronic wound healing by modulating the bFGF/Wnt/β-Catenin signaling pathway.

17β-Estradiol promotes metastasis in triple-negative breast cancer through the Calpain/YAP/β-catenin signaling axis.

β-catenin is an important regulator of malignant progression. 17β-Estradiol (E2), an important sex hormone in women, promotes the growth and metastasis of triple-negative breast cancer (TNBC). However, whether β-catenin is involved in E2-induced metastasis of TNBC remains unknown. In this study, we show that E2 induces the proliferation, migration, invasion, and metastasis of TNBC cells. E2 induces β-catenin protein expression and nuclear translocation, thereby regulating the expression of target genes such as Cyclin D1 and MMP-9. The inhibition of β-catenin reversed the E2-induced cell malignant behaviors. Additionally, E2 activated Calpain by increasing intracellular Ca2+ levels and reducing calpastatin levels. When Calpain was inhibited, E2 did not induce the proliferation, migration, invasion, or metastasis of TNBC cells. In addition, E2 promoted translocation of YAP into the nucleus by inhibiting its phosphorylation. Calpain inhibition reversed the E2-induced YAP dephosphorylation. Inhibition of YAP transcriptional activity reversed the effects of E2 on the proliferation, migration, invasion, and β-catenin of TNBC cells. In conclusion, we demonstrated that E2 induced metastasis-related behaviors in TNBC cells and this effect was mediated through the Calpain/YAP/β-catenin signaling pathway.

The Role of Heat-Induced Stress Granules in the Blood-Testis Barrier of Mice.

Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4-8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood-testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, β-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, β-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies.

Glutamine Promotes Porcine Intestinal Epithelial Cell Proliferation through the Wnt/β-Catenin Pathway.

Glutamine (Gln) is a critical nutrient required by neonatal mammals for intestinal growth, especially for newborn piglets. However, the mechanisms underlying the role of Gln in porcine intestinal epithelium development are not fully understood. The objective of the current study was to explore the possible signaling pathway involved in the promotion of porcine intestinal epithelial cell (IPEC-J2) proliferation by Gln. The results showed that 1 mM Gln promoted IPEC-J2 cell proliferation, and tandem mass tag proteomics revealed 973 differentially expressed proteins in Gln-treated IPEC-J2 cells, 824 of which were upregulated and 149 of which were downregulated. Moreover, gene set enrichment analysis indicated that the Wnt signaling pathway is activated by Gln treatment. Western blotting analysis further confirmed that Gln activated the Wnt/β-catenin signaling pathway. In addition, Gln increased not only cytosolic β-catenin but also nuclear β-catenin protein expression. LF3 (a β-catenin/TCF4 interaction inhibitor) assay and β-catenin knockdown demonstrated that Gln-mediated promotion of Wnt/β-catenin signaling and cell proliferation were blocked. Furthermore, the inhibition of TCF4 expression suppressed Gln-induced cell proliferation. These findings further confirmed that Wnt/β-catenin signaling is involved in the promotion of IPEC-J2 cell proliferation by Gln. Collectively, these findings demonstrated that Gln positively regulated IPEC-J2 cell proliferation through the Wnt/β-catenin pathway. These data greatly enhance the current understanding of the mechanism by which Gln regulates intestinal development.

Abstract 2026: Single-cell RNA-sequencing analysis of tumor endothelial cells reveals suppression of specific endothelial subtypes by the combination treatment of Wnt/β-catenin signaling modulator E7386 plus lenvatinib

Abstract Background: Aberrant activation of Wnt/β-catenin signaling plays an important role in both carcinogenesis and modulation of the tumor microenvironment. E7386, a novel orally active antitumor agent, inhibits the interaction between β-catenin and CREB-binding protein (CBP) and thereby modulates Wnt/β-catenin signaling. We previously reported that E7386 in combination with lenvatinib (LEN), a multiple receptor tyrosine kinase inhibitor mainly targeting VEGFRs and FGFRs, showed a greater antitumor activity and reduction of tumor microvessels than each treatment alone in preclinical tumor models. Multiple clinical studies are in progress including the combination of E7386 plus LEN (NCT04008797). Here, we further tried to elucidate the mechanism underlying the antitumor and antiangiogenic effect of the combination of E7386 plus LEN by using single-cell RNA sequencing (scRNA-seq) of tumor endothelial cells (ECs). Methods: Mice bearing subcutaneous RAG (mouse renal cell carcinoma cell line) tumors were treated with E7386 at 25 mg/kg and/or with LEN at 10 mg/kg by daily oral gavage for 8 days. Tumor ECs (CD31+/CD45−) were isolated by magnetic cell separation and flow cytometry and were subjected to scRNA-seq using 10x Genomics pipeline. Bioinformatics data analysis was conducted by Clarivate Analytics. After quality control and filtering, ECs were gated by their expression of pan-EC markers and were further classified into 9 subtypes (tip, capillary, arterial, venous, proliferating, immature, interferon, activated artery, and lymphatic) according to previously reported markers for each subtype. Results: Among classified subtypes, proliferating and immature subtypes showed clear reduction in the percentage of total ECs with combination of E7386 plus LEN versus LEN monotherapy. In both proliferating and immature subtypes, a subset of Wnt/β-catenin downstream genes showed increased expression with LEN monotreatment. Conversely, these Wnt/β-catenin downstream genes were downregulated with the combination of E7386 plus LEN, suggesting that Wnt/β-catenin signaling may be associated with a drug resistance mechanism against LEN in these subtypes. Pathway analysis indicated that genes involved in cell proliferation and survival were downregulated by the E7386 plus LEN combination versus LEN monotreatment in both proliferating and immature subtypes. Conclusion: Taken together, these results suggest a role for E7386 in suppressing the activation of Wnt/β-catenin signaling induced by LEN monotreatment in specific EC subtypes; this activity leads to enhanced antiangiogenic and antitumor activity with E7386 plus LEN combination therapy in a preclinical tumor model. Citation Format: Masahiko Kume, Ryoga Ishida, Yoichi Ozawa, Jialing Shen, Hiroto Konishi, Megumi Kuronishi, Takayuki Kimura, Yusuke Adachi, Yu Kato. Single-cell RNA-sequencing analysis of tumor endothelial cells reveals suppression of specific endothelial subtypes by the combination treatment of Wnt/β-catenin signaling modulator E7386 plus lenvatinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2026.

Abstract 3076: Unveiling the therapeutic potential of β-catenin inhibition in triple negative breast cancers: Targeting the Warburg effect

Abstract Introduction: Triple Negative Breast Cancer (TNBC) exhibits increased glycolysis, known as the Warburg effect. Recent research, including our own Nanostring nCounter studies, identify β-catenin as an oncoprotein linked to elevated glycolysis in TNBC tumors. Our current research explores the impact of β-catenin inhibitors on glycolysis in TNBC cell lines and an in vivo CDX model. Method: We employed immunohistochemistry to assess 98 TNBCs for the presence of β-catenin, PFKP, LDH-A, and MCT1 proteins, exploring their correlation with clinicopathological factors. In in-vitro experiments using MDA-MB-231, MDA-MB-468, and 4T1 cell lines, we investigated how β-catenin inhibitors (XAV939 &amp; Axitinib) affected aerobic glycolysis components, and TNBC cell characteristics (proliferation, migration, invasion, and stemness). For in-vivo validation, 4T1 TNBC xenografts were employed to confirm our findings. Results: Among the 98 TNBC patients, 63 (64%) displayed cytoplasmic/or nuclear β-catenin staining. Additionally, cytoplasmic accumulation of PFKP was observed in 52/98 (53%), increased cytoplasmic LDH-A expression in 57/98 (58%), and membranous MCT-1 expression in 50/98 (51%) TNBC patients. Notably, significant positive correlations were found between nuclear/cytoplasmic β-catenin and cytoplasmic PFKP (p = 0.001) as well as membranous MCT1 (p = 0.014). Furthermore, β-catenin, PFKP, LDHA, and MCT1 proteins were overexpressed in all TNBC cell lines thereby supporting their glycolytic nature. Comparative analyses between XAV939 and Axitinib revealed that Axitinib significantly reduced various TNBC cell parameters, including migration, invasion, lactate production, colony-forming ability, and expression of aerobic glycolytic pathway proteins. In the in-vivo studies, 30mg/kg Axitinib in a female Balb/c-4T1 xenograft model resulted in a substantial reduction in tumor volume and weight. Conclusion: Our findings show that increased levels of β-catenin play an important role in regulating aerobic glycolysis in TNBCs. Furthermore, our findings also highlight Axitinib's potential as an effective therapeutic agent for the treatment of β-catenin-positive TNBCs. Citation Format: Sheikh Mohammad Umar, Shruti kahol, Arundhathi Dev JR, Akanksha Kashyap, Sandeep R. Mathur, Ajay Gogia, S.V.S Deo, Chandra Prasad. Unveiling the therapeutic potential of β-catenin inhibition in triple negative breast cancers: Targeting the Warburg effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3076.

Abstract 7579: Dual inhibition of enhancer of zeste homolog 1 2 induces pyroptosis, modulates oncogenic signaling pathways &amp; sensitizes hepatocellular carcinoma to valemetostat &amp; docetaxel treatments

Abstract HCC is a global health concern, with chemotherapy resistance and patient recurrence posing significant challenges to liver cancer treatment. EZH2 is a crucial part of the polycomb repressive complex 2 (PRC2). It, along with its homolog EZH1, is a focus for cancer therapeutic targets. Dysregulation of EZH1/2 can cause cancer development due to significant changes in geneexpression. The current study aims to investigate the interaction between EZH1/2 and oncogenic signals responsible for HCC progression and pyroptosis induction. Two cell lines, HepG2 and SK-Hep1, were subjected to two FDA-approved drugs, Valemetostat (Val) and Docetaxel (Doc), to determine the impact of EZH1/2 on tumorigenicity and drug resistance. The WST-1 assay was utilized to gauge cell proliferation. The results indisputablydemonstrated a synergistic effect of the drugs after 48 h of treatment. The inhibition of viability was attributed to apoptosis induction in both tested cell lines, and the maximum effect was observed in the combined treatment as compared to either the control or each drug alone. Furthermore, our datashowed activation of caspase-3, upregulation of BAX, indicating apoptotic induction, and downregulation of Bcl-2, Bcl-XL, AKT, S6k, oncogenic β-catenin, and Stat3 protein expression levels, suggesting a modulation of oncogenic signaling pathways at the protein level. After 24 h of Val/Doc treatment, pyroptosis was observed, characterized by cell swelling and bubbles on themembrane and a significant release of lactate dehydrogenase, indicating pyroptotic cell cytotoxicity. The antitumor effects and pyroptosis were enhanced by using a dual inhibitor, UNC1999, for EZH1/2, resulting in a marked increase in the sensitivity of tested cells to the Val/Doc treatment invitro. The present study unequivocally demonstrated that dual inhibition of EZH1/2 acts as a potent sensitizer of HCC cells to Val/Doc treatment, which might be a promising therapeutic approach for inducing cellular pyroptosis in HCC cell lines. Citation Format: Asmaa Elkhder Harira, Adham Maher, Ahmed Sultan. Dual inhibition of enhancer of zeste homolog 1 2 induces pyroptosis, modulates oncogenic signaling pathways &amp; sensitizes hepatocellular carcinoma to valemetostat &amp; docetaxel treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7579.