Nuclear Β-catenin Research Articles

MiR-575/RIPK4 axis modulates cell cycle progression and proliferation by inactivating the Wnt/β-catenin signaling pathway through inhibiting RUNX1 in colon cancer.

Receptor interacting protein serine/threonine kinase 4 (RIPK4) is widely involved in human cancer development. Nevertheless, its role in colon cancer (COAD) has not been elucidated till now. Our research aimed at exploring the function and underlying molecular mechanism of RIPK4 in COAD progression. Through bioinformatic analyses and RT-qPCR, RIPK4 was discovered to be increased in COAD cells and tissues, and its high level predicted poor prognosis. Loss-of-function assays revealed that RIPK4 silencing suppressed COAD cell growth, induced cell cycle arrest, and enhanced cell apoptosis. In vivo experiments also proved that tumor growth was inhibited by silencing of RIPK4. Luciferase reporter assay validated that RIPK4 was targeted and negatively regulated by miR-575. Western blotting demonstrated that Wnt3a, phosphorylated (p)-GSK-3β, and cytoplasmic and nuclear β-catenin protein levels, β-catenin nuclear translocation, and Cyclin D1, CDK4, Cyclin E, and c-Myc protein levels were reduced by RIPK4 knockdown, which however was reversed by treatment with LiCl, the Wnt/β-catenin pathway activator. LiCl also offset the influence of RIPK4 knockdown on COAD cell growth, cell cycle process, and apoptosis. Finally, RIPK4 downregulation reduced RUNX1 level, which was upregulated in COAD and its high level predicted poor prognosis. RIPK4 is positively associated with RUNX1 in COAD. Overexpressing RUNX1 antagonized the suppression of RIPK4 knockdown on RUNX1, Wnt3a, p-GSK-3β, cytoplasmic β-catenin, nuclear β-catenin, Cyclin D1, CDK4, Cyclin E, and c-Myc levels. Collectively, miR-575/RIPK4 axis repressed COAD progression via inactivating the Wnt/β-catenin pathway through downregulating RUNX1.

ILK inhibition reduces osteophyte formation through suppression of osteogenesis in BMSCs via Akt/GSK-3β/β-catenin pathway.

Osteophyte development is a common characteristic of inflammatory skeletal diseases. Elevated osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) participates in pathological osteogenesis. Integrin-linked kinase (ILK) positively regulates the osteoblastic differentiation of osteoprogenitors, but whether the ILK blockage prevents osteophytes and its potential mechanism is still unknown. Furthermore, the low-dose tumor necrosis factor-α (TNF-α) promotes osteogenic differentiation, but a lack of study reports on the relationship between this cytokine and ILK. OSU-T315 is a small ILK inhibitor, which was used to determine the effect of ILK inhibition on osteogenesis and osteophyte formation. The osteogenesis of BMSCs was evaluated using Alizarin red S staining, alkaline phosphatase, collagen type I alpha 2 chain, and bone gamma-carboxyglutamate protein. The expression and phosphorylation of protein were assessed through western blot. Immunofluorescence was employed to display the distribution of β-catenin. microCT, hematoxylin-eosin, and safranin O/fast green staining were utilized to observe the osteophyte formation in collagen antibody-induced arthritis mice. We found that ILK blockage significantly declined calcium deposition and osteoblastic markers in a dose- and time-dependent manner. Furthermore, it lowered osteogenesis in the TNF-α-induced inflammatory microenvironment by diminishing the effect of ILK and inactivating the Akt/ GSK-3β/ β-catenin pathway. Nuclear β-catenin was descended by OSU-T315 as well. Finally, the ILK suppression restrained osteophyte formation but not inflammation in vivo. ILK inhibition lowered osteogenesis in TNF-α-related inflammatory conditions by deactivating the Akt/ GSK-3β/ β-catenin pathway. This may be a potential strategy to alleviate osteophyte development in addition to anti-inflammatory treatment.

YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation

Prostate cancer is a leading cause of cancer-related mortality in males. Dysregulation of RNA Adenine N-6 methylation (m6A) contributes to cancer malignancy. m6A on mRNA may affect mRNA splicing, turnover, transportation and translation. m6A exerts these effects, at least partly, through dedicated m6A reader proteins, including YTHDF2. YTHDF2 is necessary for development while its dysregulation is seen in various cancers including prostate cancer. However, the mechanism underlying the dysregulation and function of YTHDF2 in cancer remains elusive. Here, we find that the deubiquitinase OTUB1 increases YTHDF2 protein stability by inhibiting its ubiquitination. With in vivo and in vitro ubiquitination assays, OTUB1 is shown to block ubiquitin transfer to YTHDF2 independent of its deubiquitinase activity. Furthermore, analysis of functional transcriptomic data and m6A-sequencing data identifies PRSS8 as a potential tumor suppressor gene. OTUB1 and YTHDF2 decrease mRNA and protein levels of PRSS8, which is a trypsin-like serine protease. Mechanistically, YTHDF2 binds PRSS8 mRNA and promotes its degradation in an m6A-dependent manner. Further functional study on cellular and mouse models reveals PRSS8 is a critical downstream effector of the OTUB1-YTHDF2 axis in prostate cancer. We find in prostate cancer cells, PRSS8 decreases nuclear β-catenin level through E-cadherin, which is independent of its protease activity. Collectively, our study uncovers a key regulator of YTHDF2 protein stability and establishes a functional OTUB1-YTHDF2-PRSS8 axis in prostate cancer.

Abstract PR008: Location, location, location: Why cellular localization of mutant β-catenin matters in endometrial cancer

Abstract The present study aims to understand the mechanisms of mutant β-catenin in endometrial cancer (EC) patients with exon 3 CTNNB1 mutations. Endometrial cancer patients with exon 3 CTNNB1 mutations tend to be younger in age and have low cancer grades, but they exhibit worse recurrence-free survival than patients with similar clinical characteristics. It is unknown why EC patients with CTNNB1 mutations have worse survival. CTNNB1 encodes the protein β-catenin, which functions primarily at the cell membrane in the adherens junction complex and in the Wnt signaling pathway, where it localizes in the cytoplasm or nucleus. Exon 3 CTNNB1 mutations in EC are classically thought to promote nuclear localization of β-catenin. In this study, we conducted immunohistochemistry for β-catenin localization on 53 EC cases with an exon 3 CTNNB1 mutation from the UNCseq project. Surprisingly, 32 cases had primarily non-nuclear, either cytoplasmic or membranous, localization and 21 cases had primarily nuclear localization. Additionally, we identified 16 cases with intratumoral heterogeneity containing both nuclear and non-nuclear β-catenin localization. To investigate the functional consequences of this differential localization, we performed spatial transcriptomics using the Nanostring GeoMx Whole Transcriptome Analysis. Nuclear and non-nuclear regions of interest (ROI) were selected from 16 CTNNB1 mutant EC cases exhibiting heterogenous β-catenin localization within the same tissue (n=95 total ROIs). We hypothesize that β-catenin nuclear and non-nuclear ROIs will have differing transcriptional outputs in CTNNB1 mutant EC. Differential expression analysis of all ROIs yielded distinct transcriptional profiles based on localization of mutant β-catenin. Gene Set Enrichment Analysis revealed enrichment for Wnt-signaling and the epithelial-to-mesenchymal transition pathways in nuclear ROIs and enrichment for estrogen and androgen signaling pathways in non-nuclear ROIs. Hierarchical clustering of all ROIs yielded two genetically distinct clusters comprised of entirely nuclear or non-nuclear ROIs, with the exception of four ROIs. Higher expression of TACSTD2, encoding the protein Trop2, was enriched in the nuclear ROI cluster. Trop2 is the target of Sacituzumab Govitecan (SG), a drug conjugate with recent success in metastatic triple negative breast cancer patients. Additionally, studies have shown Trop2 to induce nuclear localization of β-catenin in other cancer types. Therefore, we hypothesize that CTNNB1 mutant EC patients with nuclear localization of β-catenin will respond better to SG treatment. To conclude, we established gene expression profiles for areas of nuclear and non-nuclear β-catenin in CTNNB1 mutant EC tissues and identified a novel gene with implications in mechanisms of β-catenin localization which may allow therapeutic targeting of advanced disease. Citation Format: Molly L. Parrish, Andrew B. Gladden, Russell R. Broaddus. Location, location, location: Why cellular localization of mutant β-catenin matters in endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR008.

Ovarian endometrioid carcinoma with a sex cord-like pattern: a morphological, immunohistochemical, and molecular analysis.

Sex cord-like endometrioid carcinoma (SCLEC) is an uncommon entity which may constitute a diagnostic challenge. This study aimed to perform a clinicopathological, immunohistochemical, and molecular reappraisal of ovarian SCLEC. Consecutive ovarian SCLECs cases from a single institution were reviewed during a 13-year period. Twenty-three immunohistochemical markers were tested; 10 genes were analyzed by next-generation sequencing. Nine cases of ovarian SCLEC were identified. Mean patient age was 65.7years; three cases showed extraovarian extension. Architectural pattern included sertoliform (n = 2), granulosa-like (n = 2), and mixed granulosa-like/sertoliform (n = 5). Eosinophilic changes accompanied by increased nuclear atypia were observed in four tumors. Endometrioid features (glands, squamous/morular differentiation) were observed in six cases. Most tumors were positive for cytokeratin-7 (8/9), EMA (9/9), estrogen and progesterone receptor (9/9), CD10 (7/9, including a luminal pattern reminiscent of mesonephric neoplasms), nuclear β-catenin (8/9), and CDX2 (8/9). A minority of cases showed block-type p16 pattern (2/9), PAX8-positivity (3/9), and non-diffuse positivity for WT1 (1/9), inhibin (1/9), chromogranin (1/9), and synaptophysin (2/9). All cases were negative for GATA3, TTF1, calretinin, and SF1. Ki67 range was 15-90%. Six cases showed CTNNB1 exon 3 mutation. Eight cases were of "no specific molecular profile" (NSMP) and one was p53-abnormal. In conclusion, SCLECs frequently exhibit a mixed sertoliform/granulosa-like architecture and express epithelial markers, hormone receptors, nuclear β-catenin, and CDX2, with luminal CD10 positivity and CTNNB1 mutations. PAX8 expression is often lost, while other mesonephric, sex cord, and neuroendocrine markers are negative.

Immunohistochemical expression of beta-catenin, BMP4 and TGF-beta in odontomas.

Changes in the expression of nuclear β-catenin are responsible for tumorigenesis. Beta-catenin acts synergistically with the TGF-β/BMPs pathway. This interaction leads to greater dentin deposition and may explain the differences between distinct tooth morphologies and hamartomas. The aim of this study was to investigate the role of β-catenin, BMP4 and TGF-β in the development of odontomas. This cross-sectional, retrospective, immunohistochemical study evaluated 30 compound odontomas, 30 complex odontomas and 17 tooth germs. The results showed that BMP4 and TGF-β were more immunoexpressed in the ectomesenchyme of complex odontomas (median = 33.7, p < 0.001; median = 76.4, p = 0.002, respectively). Higher immunoexpression of BMP4 and TGF-β was also observed in the epithelium of tooth germs (median = 2.0, p < 0.001; median = 120.3, p < 0.001, respectively). TGF-β and BMP4 showed a positive and significant correlation (p < 0.001). Both TGF-β and BMP4 were positively correlated with nuclear β-catenin in ectomesenchyme (p = 0.047 and p = 0.023, respectively). Developing teeth exhibited higher concentrations of the proteins studied in odontogenic epithelium, especially during the bud and cap stages. Higher immunoexpression in odontomas occurred mainly in the ectomesenchyme. We therefore suggest that changes in the ectomesenchyme can lead to the development of odontomas.

Cytosolic Cadherin 4 promotes angiogenesis and metastasis in papillary thyroid cancer by suppressing the ubiquitination/degradation of β-catenin

BackgroundAlthough the long-term prognosis of papillary thyroid cancer (PTC) is favorable, distant metastasis significantly compromises the prognosis and quality of life for patients with PTC. The Cadherin family plays a pivotal role in tumor metastasis; however, the involvement of Cadherin 4 (CDH4) in the metastatic cascade remains elusive.MethodsThe expression and subcellular localization of CDH4 were determined through immunohistochemistry, immunofluorescence, and western blot analyses. The impact of CDH4 on cell migration, invasion, angiogenesis, and metastasis was assessed using transwell assays, tube formation assays, and animal experiments. Immunoprecipitation assay and mass spectrometry were employed to examine protein associations. The influence of CDH4 on the subcellular expression of β-catenin and active β-catenin was investigated via western blotting and immunofluorescence. Protein stability and ubiquitination assay were employed to verify the impact of CDH4 on β-catenin degradation. Rescue experiments were performed to ensure the significance of CDH4 in regulating nuclear β-catenin signaling.ResultsCDH4 was found to be significantly overexpressed in PTC tissues and predominantly localized in the cytoplasm. Furthermore, the overexpression of CDH4 in tumor tissues is associated with lymph node metastasis in PTC patients. Cytosolic CDH4 promoted the migration, invasion, and lung metastasis of PTC cells and stimulated the angiogenesis and tumorigenesis of PTC; however, this effect could be reversed by Tegavivint, an antagonist of β-catenin. Mechanistically, cytosolic CDH4 disrupted the interaction between β-catenin and β-TrCP1, consequently impeding the ubiquitination process of β-catenin and activating the nuclear β-catenin signaling.ConclusionsCDH4 induces PTC angiogenesis and metastasis via the inhibition of β-TrCP1-dependent ubiquitination of β-Catenin.

PFKFB3 facilitates cell proliferation and migration in anaplastic thyroid carcinoma via the WNT/β-catenin signaling pathway.

Despite the involvement of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFKFB3) in the proliferation and metastasis of diverse tumor types, its biological functions and related molecular mechanisms in anaplastic thyroid carcinoma (ATC) remain largely unclear. Datasets from the Gene Expression Omnibus, the Cancer Genome Atlas and immunohistochemistry (IHC) analyses were employed to measure the expression level of PFKFB3 in ATC. A series of assays were performed to analyze the role of PFKFB3 and its inhibitor KAN0438757 in ATC cell proliferation and migration. Furthermore, Western blotting (WB), IHC and luciferase reporter assay were conducted to investigate the potential mechanisms underlying the involvement of PFKFB3 and KAN0438757 in ATC. Additionally, we established a subcutaneous xenograft tumor model in nude mice to evaluate the in vivo tumor growth. PFKFB3 exhibited a significant increase in its expression level in ATC tissues. The overexpression of PFKFB3 resulted in the stimulation of ATC cell proliferation and migration. Furthermore, this overexpression was associated with the elevated expression levels of p-AKT (ser473), p-GSK3α/β (ser21/9), nuclear β-catenin, fibronectin1 (FN1), matrix metallopeptidase 9 (MMP-9) and cyclin D1. It also promoted the nuclear translocation of β-catenin and the transcription of downstream molecules. Conversely, contrasting results were observed with the downregulation or KAN0438757-mediated inhibition of PFKFB3 in ATC cells. The selective AKT inhibitor MK2206 was noted to reverse the increased expression of p-AKT (ser473) and p-GSK3α/β (ser21/9) induced by PFKFB3 overexpression. The level of lactate was increased in PFKFB3-overexpressing ATC cells, while the presence of KAN0438757 inhibited lactate production. Moreover, the simultaneous use of PFKFB3 downregulation and KAN0438757 was found to suppress subcutaneous tumor growth in vivo. PFKFB3 can enhance ATC cell proliferation and migration via the WNT/β-catenin signaling pathway and plays a crucial role in the regulation of aerobic glycolysis in ATC cells.

Postnatal Exposure to the Endocrine Disruptor Dichlorodiphenyltrichloroethane Affects Adrenomedullary Chromaffin Cell Physiology and Alters the Balance of Mechanisms Underlying Cell Renewal.

Dichlorodiphenyltrichloroethane (DDT) is a wide-spread systemic pollutant with endocrine disrupting properties. Prenatal exposure to low doses of DDT has been shown to affect adrenal medulla growth and function. The role of postnatal exposure to DDT in developmental disorders remains unclear. The aim of the present investigation is to assess growth parameters and the expression of factors mediating the function and renewal of chromaffin cells in the adult adrenal medulla of male Wistar rats exposed to the endocrine disruptor o,p'-DDT since birth until sexual maturation. The DDT-exposed rats exhibited normal growth of the adrenal medulla but significantly decreased tyrosine hydroxylase production by chromaffin cells during postnatal period. Unlike the control, the exposed rats showed enhanced proliferation and reduced expression of nuclear β-catenin, transcription factor Oct4, and ligand of Sonic hedgehog after termination of the adrenal growth period. No expression of pluripotency marker Sox2 and absence of Ascl 1-positive progenitors were found in the adrenal medulla during postnatal ontogeny of the exposed and the control rats. The present findings indicate that an increase in proliferative activity and inhibition of the formation of reserve for chromaffin cell renewal, two main mechanisms for cell maintenance in adrenal medulla, in the adult DDT-exposed rats may reflect a compensatory reaction aimed at the restoration of catecholamine production levels. The increased proliferation of chromaffin cells in adults suggests excessive growth of the adrenal medulla. Thus, postnatal exposure to DDT alters cell physiology and increases the risk of functional insufficiency and hyperplasia of the adrenal medulla.

HECW1 restrains cervical cancer cell growth by promoting DVL1 ubiquitination and downregulating the activation of Wnt/β-catenin signaling

HECW1 belongs to ubiquitin ligase (E3) HECT family, and is found to be involved in tumorigenesis and tumor progression. However, the function of HECW1 in cervical cancer (CC) remains unknown. Clinical analysis showed that HECW1 is significantly decreased in CC tumor tissues. Ectopic expression of HECW1 suppressed cell growth, promoting cell cycle arrest and apoptosis in CC cells, while downregulation of HECW1 reversed these trends, impeded proliferation and accelerated cell cycle progression of CC cells. Overexpressing of HECW1 reduced mitochondrial membrane potential and the protein expression of voltage-dependent anion channel 1 (VDAC1). In addition, upregulation of HECW1 inhibited nuclear β-catenin accumulation, downregulated β-catenin/TCF/LEF-mediated transcriptional activity and the expression of downstream gene c-Myc, whereas inhibition of HECW1 received opposite results. Further results confirmed HECW1 affects the protein expression of dishevelled-1 (DVL1), a potent activator of Wnt/β‐catenin, and inhibition of HECW1 inhibited the ubiquitination of DVL1, upregulating its expression. Inhibition of DVL1 restrained the promotion effect of HECW1 suppression on cell proliferation. In vivo experiments also verified that HECW1 suppression promoted the tumor formation of CC cells. Summary, we demonstrated that HECW1 inhibits CC cell proliferation and tumor formation by downregulating DVL1 induced Wnt/β‐catenin signaling pathway activation.

Analysis of tumor biomarkers in patients (pts) with advanced hepatocellular carcinoma (HCC) from a phase 1b study of E7386, a CREB-binding protein/β-catenin interaction inhibitor, in combination with lenvatinib.

535 Background: E7386, a novel oral anticancer agent, inhibits β-catenin binding to its transcriptional co-activator CREB-binding protein, thus modulating Wnt/β-catenin signaling. Lenvatinib is a multikinase inhibitor that targets VEGFR 1–3, FGFR 1–4, PDGFRα, RET, and KIT. We previously reported the safety/tolerability, pharmacokinetics, biomarkers, and preliminary efficacy of E7386 plus lenvatinib in 25 pts with HCC in the dose escalation part of a phase 1b study1 (NCT04008797). Here, we present the results of exploratory tumor biomarker analyses from this study. Methods: Pts with HCC were treated with E7386 10–80 mg QD and 60–120 mg BID plus lenvatinib (bodyweight &lt; 60 kg: 8 mg QD; ≥ 60 kg: 12 mg QD). Archival or fresh tumor samples were collected at baseline and post-treatment; tumor tissue was assessed for genetic alteration, immunohistochemistry (IHC), and gene expression. Gene signature scores of interest were calculated using obtained gene expression data. The preliminary association between biomarker status and efficacy was investigated. Results: Of the 25 pts enrolled, 22 had baseline tumor samples available for the gene mutation analysis. TERT (6 pts: 27%), CTNNB1 (5 pts: 23%), and TP53 (4 pts: 18%) mutations were detected. CTNNB1 mutation was confirmed in 3 of 7 responders and 2 of 13 non-responders. Nuclear β-catenin expression was confirmed by IHC analysis in 5 of 16 pts; it was expressed in 3 of 5 pts with CTNNB1 MUT and 2 of 11 pts with CTNNB1 WT. Pts with expression of nuclear β-catenin had 2 partial responses (PRs) among 5 pts; those without β-catenin expression had 2 PRs among 11 pts. Additionally, IHC analysis of tumor microvessel density (MVD; number of CD31+ vessels) at baseline suggested that greater maximum tumor shrinkage (MTS) tended to be observed in the MVD-high population compared to the MVD-low population. 7 Pts had evaluable baseline and post-baseline tumor samples and were included in the gene signature analysis. Despite the small sample size, preliminary results indicated that MTS may be associated with a decrease in Wnt signaling and angiogenesis gene signature scores (spearman r [r]=0.61 for both) and attenuation of hypoxic induction and glycolysis gene signature scores (r=0.79 and 0.68, respectively). Conclusions: These exploratory tumor biomarker analyses suggest that E7386 in combination with lenvatinib may affect the tumor microenvironment in HCC pts through effects on Wnt signaling, angiogenesis, hypoxic response, and glycolysis pathways. This concept is the basis for treatment with this combination. However, given the small sample size, further investigation is needed to confirm these effects on the tumor microenvironment. Enrollment of pts with HCC to the expansion part of Study 102 is ongoing. 1. Ikeda et al. ASCO 2023. Clinical trial information: NCT04008797 .

Ring Finger 43 Hot-spot Frameshift Mutation G659V in Colorectal Cancer Patients: Report from a Tertiary Cancer Care Hospital in North India.

The Ring Finger 43 (RNF43) is a tumor suppressor gene that negatively regulates the Wnt/β-catenin signaling. The p.G659fs is a recurrent RNF43 C-terminal truncating variant frequent in colorectal cancer (CRC) patients. We aimed to identify this hotspot variant in CRC patients and assessed the relationship between the mutation, clinical characteristics, and tumor β-catenin localization. Formalin-fixed, paraffin-embedded tissue samples of upfront, surgically resected, sporadic colorectal adenocarcinoma cases were selected. The p.G659fs mutation was determined by capillary sequencing with sequence-specific primers. Tissue microarray and immunohistochemistry were employed to analyze nuclear β-catenin expression and the expression of mismatch repair (MMR) proteins, respectively. In addition, clinical details were retrieved from the hospital medical records and data were analyzed. The RNF43 p.G659fs mutation was observed in 8% of CRC patients. In total, 25% of tumors showed a loss of immunostaining for one or more MMR proteins and 14.6% of tumors showed positive nuclear β-catenin staining. The p.G659fs variant was significantly enriched in MMR-deficient tumors (P = 0.04). Importantly, no correlation was observed between the variant and nuclear β-catenin localization (P = 0.48), indicating a Wnt-independent role of this variant in CRC tumors. To the best of our knowledge, this is the first study from North India to show the involvement of RNF43 p.G659fs variant in CRC patients. The mutation correlated with MMR protein deficiency and seems to be conferring tumorigenicity independent of the Wnt pathway.

In vitro β-catenin attenuation by a mefloquine-loaded core-shell nano emulsion strategy to suppress liver cancer cells.

Liver cancer, with its robust metastatic propensity, imposes a substantial global health burden of around 800 000 new cases annually. Mutations in the Wnt/β-catenin pathway genes are common in liver cancer, driving over 80% of cases. Targeting this pathway could potentially lead to better treatments. The aim of the present study was to develop a novel strategy for targeting the Wnt/β-catenin pathway while blocking the growth and division, of liver cancer cells and downregulating gene expression. This was achieved by formulating a repurposed drug (mefloquine)-loaded garlic nano-emulsion (GNE) with gold nanoparticles (GNPs) as a core-shell nano-emulsion (MQ/GNE-GNP). The biocompatible core-shell nano-emulsion (MQ/GNE-GNP) exhibited a size distribution in the range of 50-100 nm, high stability, excellent hydrophilicity, good biosafety, and sustained release. Human liver cancer cells were exposed to MQ/GNE, GNPs, and MQ/GNE-GNP at varying concentrations, and the effects were assessed through analysis of the cytotoxicity, reactive oxygen species, cell death, cell cycle analysis, and gene expression studies. It was found that MQ/GNE-GNP arrested HepG2 cells in the sub G0/G1phase and induced apoptosis. The anticancer efficacy of the core-shell nano-emulsion (MQ/GNE-GNP) resulted in higher cell death in the AO/PI staining studies, demonstrating its greater anticancer efficacy. The administration of MQ/GNE-GNP downregulated the overall expression of nuclear β-catenin, thereby suppressing the Wnt/β-catenin pathway. The protein expression level of Wnt 1 was upregulated, while β-catenin expression was significantly decreased. The core-shell nano-emulsion, incorporating a repurposed drug, could disrupt the β-catenin connections in the Wnt/β-catenin pathway. In conclusion, MQ/GNE-GNP could be a promising core-shell nano emulsion for the effective treatment of liver cancer by targeting the Wnt/β-catenin pathway.

Target deubiquitinase OTUB1 as a therapeatic strategy for BLCA via β-catenin/necroptosis signal pathway.

Ubiquitination, a prevalent and highly dynamic reversible post-translational modification, is tightly regulated by the deubiquitinating enzymes (DUBs) superfamily. Among them, OTU Domain-Containing Ubiquitin Aldehyde-Binding Protein 1 (OTUB1) stands out as a critical member of the OTU deubiquitinating family, playing a pivotal role as a tumor regulator across various cancers. However, its specific involvement in BLCA (BLCA) and its clinical significance have remained ambiguous. This study aimed to elucidate the biofunctions of OTUB1 in BLCA and its implications for clinical prognosis. Our investigation revealed heightened OTUB1 expression in BLCA, correlating with unfavorable clinical outcomes. Through in vivo and in vitro experiments, we demonstrated that increased OTUB1 levels promote BLCA tumorigenesis and progression, along with conferring resistance to cisplatin treatment. Notably, we established a comprehensive network involving OTUB1, β-catenin, necroptosis, and BLCA, delineating their regulatory interplay. Mechanistically, we uncovered that OTUB1 exerts its influence by deubiquitinating and stabilizing β-catenin, leading to its nuclear translocation. Subsequently, nuclear β-catenin enhances the transcriptional activity of c-myc and cyclin D1 while suppressing the expression of RIPK3 and MLKL, thereby fostering BLCA progression and cisplatin resistance. Importantly, our clinical data suggest that the OTUB1/β-catenin/RIPK3/MLKL axis holds promise as a potential biomarker for BLCA.

Activation of the Wnt/β-catenin signaling pathway and CTNNB1 mutations in canine intestinal epithelial proliferative lesions.

Nuclear expression of β-catenin has been reported in canine intestinal epithelial tumors (IETs) and colorectal inflammatory polyps (CIPs) with dysplastic epithelia. However, the role of the Wnt/β-catenin signaling pathway in these lesions remains unclear. To investigate the association between the nuclear β-catenin expression and the activation of the Wnt/β-catenin signaling pathway, immunohistochemistry and mutation analyses were conducted on 64 IETs and 20 CIPs. IETs and CIPs with β-catenin nuclear and/or cytoplasm immunolabeling were classified as β-catenin (+). The immunostaining scores of c-Myc and Cyclin D1 and Ki-67 index were significantly higher in β-catenin (+) cases than in β-catenin (-) cases. Identical APC mutations (p.E154D and p.K155X) were detected in 6/41 β-catenin (+) IETs; all 6 of IETs with APC mutations were Jack Russell Terriers. CTNNB1 mutations were detected in 29/42 β-catenin (+) IETs, 3/11 β-catenin (+) CIPs, and 2/22 β-catenin (-) IETs, most of which were hotspots associated with human colorectal carcinoma. In one Miniature Dachshund diagnosed with a CIP that subsequently developed into an IET, the same CTNNB1 mutation was detected in both lesions. The immunohistochemical results suggest that cell proliferative activity in β-catenin (+) cases may be associated with activation of the Wnt/β-catenin signaling pathway. The mutation analysis results suggest that CTNNB1 mutations may be associated with cytoplasmic β-catenin accumulation in IET and CIP. Furthermore, the dysplastic epithelium in CIP may progress to IET through the activation of the Wnt/β-catenin signaling pathway by the CTNNB1 mutation.

BATF2 inhibits the stem cell-like properties and chemoresistance of gastric cancer cells through PTEN/AKT/β-catenin pathway.

Background: Gastric cancer (GC) ranks as the fifth leading cause of cancer mortality, with cancer stem cells (CSCs) playing a critical role in tumor progression and resistance to chemotherapy. Conventional chemotherapy often fails to effectively target these stem cells. BATF2, a tumor suppressor, is known for its role in gastric cancer, but its influence on cancer stem cell-like properties and chemotherapy response remains unclear. Methods: Single-cell RNA sequencing (scRNA-seq) analysis was performed on 9 gastric cancer samples to evaluate the expression and regulatory function of BATF2. In vitro experiments involving cell cultures, tumor cell spheroids, and organoids were conducted to assess BATF2's impact on 5-Fu sensitivity and its interaction with drug transporters and signaling pathways. In vivo studies, including subcutaneous tumor formation assays, immunohistochemistry, and immunoblotting, were used to validate findings. Results: BATF2 was confirmed as a tumor suppressor in gastric cancer through scRNA-seq analysis. Elevated BATF2 expression correlated with improved outcomes from postoperative chemotherapy in GC patients and increased sensitivity to 5-Fu. BATF2 enhanced 5-Fu responsiveness by inhibiting the ABCG2 drug transporter and promoting PTEN stability, which suppressed AKT phosphorylation. This led to reduced nuclear β-catenin levels and decreased expression of stem cell markers CD44, SOX2, and NANOG, ultimately reducing chemoresistance and stem-like properties in GC cells. Conclusions: BATF2 plays a pivotal role in regulating stem-like characteristics and chemoresistance in gastric cancer through the BATF2/PTEN/AKT/ABCG2 pathway. These findings suggest a novel therapeutic strategy targeting BATF2 to enhance chemotherapy effectiveness in gastric cancer treatment.

EBP50 Depletion and Nuclear β-Catenin Accumulation Engender Aggressive Behavior of Colorectal Carcinoma through Induction of Tumor Budding.

Ezin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a scaffold protein that interacts with several partner molecules including β-catenin. Here, we examined the crosstalk between EBP50 and nuclear catenin during colorectal carcinoma (CRC) progression. In clinical samples, there were no correlations between the subcellular location of EBP50 and any clinicopathological factors. However, EBP50 expression was significantly lower specifically in the outer areas of tumor lesions, in regions where tumor budding (BD) was observed. Low EBP50 expression was also significantly associated with several unfavorable prognostic factors, suggesting that EBP50 depletion rather than its overexpression or subcellular distribution plays an important role in CRC progression. In CRC cell lines, knockout of EBP50 induced epithelial-mesenchymal transition (EMT)-like features, decreased proliferation, accelerated migration capability, and stabilized nuclear β-catenin due to disruption of the interaction between EBP50 and β-catenin at the plasma membrane. In addition, Slug expression was significantly higher in outer lesions, particularly in BD areas, and was positively correlated with nuclear β-catenin status, consistent with β-catenin-driven transactivation of the Slug promoter. Together, our data suggest that EBP50 depletion releases β-catenin from the plasma membrane in outer tumor lesions, allowing β-catenin to accumulate and translocate to the nucleus, where it transactivates the Slug gene to promote EMT. This in turn triggers tumor budding and contributes to the progression of CRC to a more aggressive phase.

Primary mucinous adenocarcinoma of the urethra: Aclinicopathological analysis of 35 cases.

Primary mucinous adenocarcinoma of the urethra represents an extremely rare entity. We sought to characterise further these tumours' clinicopathological, immunohistochemical and molecular features. Thirty-five cases were identified, occurring in 18 males and 17 females. The mean age at diagnosis was 65 years (28-89 years). The main presentation symptoms were haematuria and urinary outlet obstruction. Microscopic analysis revealed that all 35 tumours have stromal dissection by mucin. Ten tumours showed villoglandular dysplasia, nine showed mucinous metaplasia, two showed adenocarcinoma in situ and four showed signet ring cell features. All tumours were immunopositive for CEA, while immunonegative for nuclear β-catenin; 19 of 23 (83%) expressed high molecular weight cytokeratin; 19 of 33 (58%) CK7; 28 of 34 (82%) CK20; 32 of 35 (91%) CDX2; 22 of 27 (81%) cadherin-17 (CDH-17); 26 of 29 (90%) SATB2; and one of 31 (3%) GATA3. Mismatch repair gene products, including MLH1, PMS2, MSH2 and MSH6, were immunopositive, suggesting the MSI-low genotype of mucinous adenocarcinoma of the urethra. BRAF V600E and ALK rearrangements were not detected. During the mean follow-up of 20 months, nine patients either developed distant metastasis or succumbed to the illness. Our study, encompassing the most extensive series of 35 cases of primary mucinous adenocarcinoma of the urethra, provides crucial insights into its precise diagnosis, management and potential targeted treatments. We found a greater CDX2, SATB2 and CDH17 sensitivity in these urethral tumours for the first time, to our knowledge. We identified characteristics such as an MSI-low profile, non-V600E BRAF mutations and an absence of ALK rearrangements.

A Complex Interplay between Notch Effectors and β-Catenin Signaling in Morular Differentiation of Endometrial Carcinoma Cells

Notch signaling contributes to tissue development and homeostasis, but little is known about its roles in morular differentiation of endometrial carcinoma (Em Ca) cells. The current study focused on crosstalk between Notch and β-catenin signaling in Em Ca with morules. Promoters of hairy and enhancer of split 1 (Hes1) and mastermind-like 2 (MAML2) were activated by Notch intracellular domain 1 (NICD1) but not β-catenin; we also observed a positive feedback loop between Hes1 and MAML2. Immunoreactivities for nuclear β-catenin, Hes1, and MAML2, as well as the interaction between β-catenin and Hes1 or MAML2, were significantly higher in morular lesions as compared to surrounding carcinoma (Sur Ca) in Em Ca. Inhibition of glycogen synthase kinase-3β (GSK-3β) increased expression of total nuclear and cytoplasmic GSK-3β and its phosphorylated forms, as well as NICD1, Hes1, and active β-catenin. GSK-3β inhibition also decreased proliferation and migration, consistent with the response of cells stably overexpressing Hes1. Finally, the nuclear/cytoplasmic GSK-3β score was significantly higher in morules compared to Sur Ca in Em Ca, and was positively correlated with nuclear β-catenin, Hes1, and MAML2 scores. This complex interplay between Notch effectors and β-catenin signaling through GSK-3β inhibition contributes to the establishment and maintenance of β-catenin-mediated morular differentiation, which is in turn associated with reduced proliferation and inhibition of migration in Em Ca.

Preclinical efficacy of targeting epigenetic mechanisms in AML with 3q26 lesions and EVI1 overexpression.

AML with chromosomal alterations involving 3q26 overexpresses the transcription factor (TF) EVI1, associated with therapy refractoriness and inferior overall survival in AML. Consistent with a CRISPR screen highlighting BRD4 dependency, treatment with BET inhibitor (BETi) repressed EVI1, LEF1, c-Myc, c-Myb, CDK4/6, and MCL1, and induced apoptosis of AML cells with 3q26 lesions. Tegavivint (TV, BC-2059), known to disrupt the binding of nuclear β-catenin and TCF7L2/LEF1 with TBL1, also inhibited co-localization of EVI1 with TBL1 and dose-dependently induced apoptosis in AML cell lines and patient-derived (PD) AML cells with 3q26.2 lesions. TV treatment repressed EVI1, attenuated enhancer activity at ERG, TCF7L2, GATA2 and MECOM loci, abolished interactions between MYC enhancers, repressing AML stemness while upregulating mRNA gene-sets of interferon/inflammatory response, TGF-β signaling and apoptosis-regulation. Co-treatment with TV and BETi or venetoclax induced synergistic in vitro lethality and reduced AML burden, improving survival of NSG mice harboring xenografts of AML with 3q26.2 lesions.