Loss Of Β-catenin Research Articles

Evaluation of epithelial-to-mesenchymal transition and Ki-67 index in aggressive papillary thyroid cancer

ObjectivesThe present study aims to characterize immunohistochemical features of markers associated with Epithelial–Mesenchymal Transition (EMT) and proliferative activity that could lead to death in Papillary Thyroid Cancer (PTC). MethodsClinical data and tumor material were retrospectively collected. The patients were separated into death from PTC (Group 1), metastatic cases with indolent behavior (Group 2) and non-metastatic indolent PTC (Group 3). Immunohistochemical assessment of E-cadherin, β-catenin, Vimentin, ZEB-1 and Ki-67 was performed in each tumor and a semiquantitative estimation of the percentage of expression was fulfilled on the best marking area at high of the tumor invasion front. Results31 patients were included, 15 that died from PTC (Group 1), 6 in Group 2 and 10 in Group 3. The proliferative marker Ki-67 showed a significant difference in its expression in the tumor invasion front between the groups, specifically between groups 1 and 3 (p = 0.006). On the other hand, EMT-related immunohistochemical markers did not show significant difference in their percentage of expression, since loss of E-cadherin, β-catenin and Vimentin was observed in most cases at the invasion front. ConclusionPatients that died from PTC had a significantly higher Ki-67 labelling index compared to patients with indolent disease (cutoff of 11%). Ki-67 may have a role as a prognostic marker and could be considered for routine use in PTC. Level of evidence4.

Loss of β-catenin reveals a role for glutathione in regulating oxidative stress during cholestatic liver disease.

Cholestasis is an intractable liver disorder that results from impaired bile flow. We have previously shown that the Wnt/β-catenin signaling pathway regulates the progression of cholestatic liver disease through multiple mechanisms, including bile acid metabolism and hepatocyte proliferation. To further explore the impact of these functions during intrahepatic cholestasis, we exposed mice to a xenobiotic that causes selective biliary injury. α-naphthylisothiocyanate (ANIT) was administered to liver-specific knockout (KO) of β-catenin and wild-type mice in the diet. Mice were killed at 6 or 14 days to assess the severity of cholestatic liver disease, measure the expression of target genes, and perform biochemical analyses. We found that the presence of β-catenin was protective against ANIT, as KO mice had a significantly lower survival rate than wild-type mice. Although serum markers of liver damage and total bile acid levels were similar between KO and wild-type mice, the KO had minor histological abnormalities, such as sinusoidal dilatation, concentric fibrosis around ducts, and decreased inflammation. Notably, both total glutathione levels and expression of glutathione-S-transferases, which catalyze the conjugation of ANIT to glutathione, were significantly decreased in KO after ANIT. Nuclear factor erythroid-derived 2-like 2, a master regulator of the antioxidant response, was activated in KO after ANIT as well as in a subset of patients with primary sclerosing cholangitis lacking activated β-catenin. Despite the activation of nuclear factor erythroid-derived 2-like 2, KO livers had increased lipid peroxidation and cell death, which likely contributed to mortality. Loss of β-catenin leads to increased cellular injury and cell death during cholestasis through failure to neutralize oxidative stress, which may contribute to the pathology of this disease.

Targeting HER2-positive breast cancer cells by a combination of dasatinib and BMS-202: Insight into the molecular pathways

BackgroundRecent investigations have reported the benefits of using a tyrosine kinase inhibitor, dasatinib (DA), as well as programmed death-ligand 1 (PD-L1) inhibitors in the management of several solid tumors, including breast cancer. Nevertheless, the outcome of the combination of these inhibitors on HER2-positive breast cancer is not explored yet.MethodsHerein, we investigated the impact of DA and PD-L1 inhibitor (BMS-202) combination on HER2-positive breast cancer cell lines, SKBR3 and ZR75.ResultsOur data reveal that the combination significantly inhibits cell viability of both cancer cell lines as compared to monotreatment. Moreover, the combination inhibits epithelial-mesenchymal transition (EMT) progression and reduces cancer cell invasion by restoring E-cadherin and β-catenin expressions and loss of vimentin, major biomarkers of EMT. Additionally, the combination reduces the colony formation of both cell lines in comparison with their matched control. Also, the combination considerably inhibits the angiogenesis of the chorioallantoic membrane model compared with monotreatment. Molecular pathway analysis of treated cells shows that this combination blocks HER2, AKT, β-catenin, and JNK1/2/3 activities.ConclusionOur findings implicate that a combination of DA and BMS-202 could have a significant impact on the management of HER2-positive breast cancer.

Modulation of canonical Wnt signaling regulates peribiliary mesenchymal identity during homeostasis and injury.

The matrix and associated mesenchyme of the extrahepatic bile ducts are distinct, which could drive diseases with a predilection for these ducts, such as primary sclerosing cholangitis. We aimed to understand the molecular drivers of peribiliary mesenchymal cell (PMC) identity in the extrahepatic bile ducts and dissect how this changed in the context of injury using an entirely in vivo approach with transcriptomic analysis. Single-cell sequencing with a receptor-ligand analysis showed that PMCs had the most interactions with surrounding cells. Wnt4, Wnt5a, and Wnt7b were identified as the major ligands secreted from PMCs and cholangiocytes that interacted in both paracrine and autocrine fashion. Bile duct ligation caused an increase in all 3 Wingless/Integrated ligands and Axin2 with an associated increase in the transcription factors T-box transcription factor (Tbx)2 and Tbx3. Conversely, Indian hedgehog secretion decreased without an associated decrease in hedgehog signaling effectors. Loss of smoothened within PMCs did not impact hedgehog signaling effectors or cellular identity, whereas smoothened gain of function led to myofibroblast transdifferentiation with upregulation of Tbx2 and Tbx3 without injury. Loss of β-catenin caused a decrease in expression of all 3 Gli transcription factors and associated mesenchymal gene expression, which was phenocopied with compound Gli2 and Gli3 loss in uninjured PMCs. With injury, loss of β-catenin resulted in decreased myofibroblast transdifferentiation with reduced Tbx2 and Tbx3 expression. Our results show how modulation of canonical Wingless/Integrated signaling in PMCs is important for regulating basal mesenchymal gene expression and initiating a myogenic gene transcriptional program during injury. They also highlight reciprocating interactions between the hedgehog and Wingless/Integrated signaling pathways within PMCs.

β-Catenin regulates endocardial cushion growth by suppressing p21.

Endocardial cushion formation is essential for heart valve development and heart chamber separation. Abnormal endocardial cushion formation often causes congenital heart defects. β-Catenin is known to be essential for endocardial cushion formation; however, the underlying cellular and molecular mechanisms remain incompletely understood. Here, we show that endothelial-specific deletion of β-catenin in mice resulted in formation of hypoplastic endocardial cushions due to reduced cell proliferation and impaired cell migration. By using a β-catenin DM allele in which the transcriptional function of β-catenin is selectively disrupted, we further reveal that β-catenin regulated cell proliferation and migration through its transcriptional and non-transcriptional function, respectively. At the molecular level, loss of β-catenin resulted in increased expression of cell cycle inhibitor p21 in cushion endocardial and mesenchymal cells in vivo. In vitro rescue experiments with HUVECs and pig aortic valve interstitial cells confirmed that β-catenin promoted cell proliferation by suppressing p21. In addition, one savvy negative observation is that β-catenin was dispensable for endocardial-to-mesenchymal fate change. Taken together, our findings demonstrate that β-catenin is essential for cell proliferation and migration but dispensable for endocardial cells to gain mesenchymal fate during endocardial cushion formation. Mechanistically, β-catenin promotes cell proliferation by suppressing p21. These findings inform the potential role of β-catenin in the etiology of congenital heart defects.

Hepatocyte β-catenin loss is compensated by Insulin-mTORC1 activation to promote liver regeneration.

Liver regeneration (LR) following partial hepatectomy (PH) occurs via activation of various signaling pathways. Disruption of a single pathway can be compensated by activation of another pathway to continue LR. The Wnt-β-catenin pathway is activated early during LR and conditional hepatocyte loss of β-catenin delays LR. Here, we study mechanism of LR in the absence of hepatocyte-β-catenin. Eight-week-old hepatocyte-specific Ctnnb1 knockout mice (β-catenin ΔHC ) were subjected to PH. These animals exhibited decreased hepatocyte proliferation at 40-120 h and decreased cumulative 14-day BrdU labeling of <40%, but all mice survived, suggesting compensation. Insulin-mediated mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) activation was uniquely identified in the β-catenin ΔHC mice at 72-96 h after PH. Deletion of hepatocyte regulatory-associated protein of mTOR (Raptor), a critical mTORC1 partner, in the β-catenin ΔHC mice led to progressive hepatic injury and mortality by 30 dys. PH on early stage nonmorbid Raptor ΔHC -β-catenin ΔHC mice led to lethality by 12 h. Raptor ΔHC mice showed progressive hepatic injury and spontaneous LR with β-catenin activation but died by 40 days. PH on early stage nonmorbid Raptor ΔHC mice was lethal by 48 h. Temporal inhibition of insulin receptor and mTORC1 in β-catenin ΔHC or controls after PH was achieved by administration of linsitinib at 48 h or rapamycin at 60 h post-PH and completely prevented LR leading to lethality by 12-14 days. Insulin-mTORC1 activation compensates for β-catenin loss to enable LR after PH. mTORC1 signaling in hepatocytes itself is critical to both homeostasis and LR and is only partially compensated by β-catenin activation. Dual inhibition of β-catenin and mTOR may have notable untoward hepatotoxic side effects.

Loss of β-catenin causes cementum hypoplasia by hampering cementogenic differentiation of Axin2-expressing cells.

Although cementum plays an essential role in tooth attachment and adaptation to occlusal force, the regulatory mechanisms of cementogenesis remain largely unknown. We have previously reported that Axin2-expressing (Axin2+ ) mesenchymal cells in periodontal ligament (PDL) are the main cell source for cementum growth, and constitutive activation of Wnt/β-catenin signaling in Axin2+ cells results in hypercementosis. Therefore, the aim of the present study was to further evaluate the effects of β-catenin deletion in Axin2+ cells on cementogenesis. We generated triple transgenic mice to conditionally delete β-catenin in Axin2-lineage cells by crossing Axin2CreERT2/+ ; R26RtdTomato/+ mice with β-cateninflox/flox mice. Multiple approaches, including X-ray analysis, micro-CT, histological stainings, and immunostaining assays, were used to analyze cementum phenotypes and molecular mechanisms. Our data revealed that loss of β-catenin in Axin2+ cells led to a cementum hypoplasia phenotype characterized by a sharp reduction in the formation of both acellular and cellular cementum. Mechanistically, we found that conditional removal of β-catenin in Axin2+ cells severely impaired the secretion of cementum matrix proteins, for example, bone sialoprotein (BSP), dentin matrix protein 1 (DMP1) and osteopontin (OPN), and markedly inhibited the differentiation of Axin2+ mesenchymal cells into osterix+ cementoblasts. Our findings confirm the vital role of Axin2+ mesenchymal PDL cells in cementum growth and demonstrate that Wnt/β-catenin signaling shows a positive correlation with cementogenic differentiation of Axin2+ cells.

GATA3 mediates nonclassical β-catenin signaling in skeletal cell fate determination and ectopic chondrogenesis.

Skeletal precursors are mesenchymal in origin and can give rise to distinct sublineages. Their lineage commitment is modulated by various signaling pathways. The importance of Wnt signaling in skeletal lineage commitment has been implicated by the study of β-catenin-deficient mouse models. Ectopic chondrogenesis caused by the loss of β-catenin leads to a long-standing belief in canonical Wnt signaling that determines skeletal cell fate. As β-catenin has other functions, it remains unclear whether skeletogenic lineage commitment is solely orchestrated by canonical Wnt signaling. The study of the Wnt secretion regulator Gpr177/Wntless also raises concerns about current knowledge. Here, we show that skeletal cell fate is determined by β-catenin but independent of LEF/TCF transcription. Genomic and bioinformatic analyses further identify GATA3 as a mediator for the alternative signaling effects. GATA3 alone is sufficient to promote ectopic cartilage formation, demonstrating its essential role in mediating nonclassical β-catenin signaling in skeletogenic lineage specification.

Abstract C064: Prognostic impact of an immunomorphological signature integrating immune, fibroblast and tumor markers in a series of 217 patients operated on for pancreatic adenocarcinoma

Abstract An integrative approach taking into consideration the tumor microenvironment, in particular cancer-associated fibroblasts (CAFs) and tumor-infiltrating lymphocytes (TILs) and its interaction with tumor cells offers a more precise reflect of pancreatic ductal adenocarcinomas (PDACs) biology. In this setting, an immunomorphological signature established from bioinformatics data could thus represent an attractive approach. Moreover to molecular signatures, on which there is still no consensus. This study aimed to evaluate the prognostic value of a panel of morphological and immunohistochemical (IHC) markers on the immune, fibroblastic and epithelial contingents of PDACs for overall (OS) and relapse-free (RFS) survival. Two hundred and seventeen patients operated for PDAC between 2000 and 2017 were included for a tissue microarray (TMA) analysis. A panel of antibodies and special stains was selected based on a previous bioinformatics study (under review) literature data. Slides were digitized and lymphocytes expressing CD3, CD8 and CD20 were quantified. A pixel classifier was used to assess the percentage of collagen and fibroblasts expressing anti-LRRC15, HSP47, α-SMA and FAP antibodies involved in the stroma reaction. Due to the co-marking of stromal and epithelial cells of PTK7 and β-catenin, respectively implicated in embryogenesis and the canonical Wnt pathway, these markers were interpreted semi-quantitatively in a double-blind fashion. After dichotomization using the maximized log-rank method, the association of these parameters with OS and RFS was assessed by univariate and multivariate Cox models. Five years after surgical resection, 26 patients (12.0%) were free of recurrence and 56 (25.8%) were alive. In multivariate analysis, the following parameters were associated with reduced OS and RFS: poor differentiation, lymph node ratio greater than 0.2, loss of tumor β-catenin on tumor cells, a low density of tumor-infiltrating CD3 T lymphocytes and a high percentage of HSP47 fibroblasts. Reduced OS was also associated with an age greater than 65 years at the time of surgery and the absence of adjuvant treatment. In order to evaluate the joint effects of the three tumor compartments markers independentely related to OS and RFS (β-catenin, HSP47, CD3), we grouped them into 3 classes (0/1+, 2+, 3+) according to the number of good prognosis criteria (class 3+ with the greatest number of good prognosis criteria). The multivariate analysis shows an additive effect of these parameters in OS and RFS compared to class 3+ patients. These data underline the interest of simultaneously characterizing the three epithelial, immune and fibroblastic compartments in PDAC. Furthermore, we highlight for the first time the prognostic impact of HSP47 by immunohistochemistry and confirm the β-catenin one. This tissue approach with an IHC panel studied on an operating specimen could be a first step towards applicability on biopsies and lead to a better patient stratification in a therapeutic setting. Citation Format: Franck Monnien, Chloé Molimard, Marine Abad, Marie-Paule Algros, Sophie Félix, Nikolaus Zirganos, Bruno Heyd, Angélique Vienot, Christophe Borg, Frédéric Bibeau. Prognostic impact of an immunomorphological signature integrating immune, fibroblast and tumor markers in a series of 217 patients operated on for pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C064.

RF34 | PMON254 Why Does Androgens Fail to Promote the Survival of the Wolffian Duct in the Absence of Wnt9b?

Abstract The male reproductive tract development has been predominantly dependent upon androgen action, which is mainly mediated by the androgen receptor (AR). Under the influence of androgen actions, the primitive male reproductive tract, also known as the Wolffian duct, is first stabilized then differentiates into the epididymis, vas deference and seminal vesicle. Impairment of androgen signaling during fetal programming causes a spectrum of male reproductive disorders that manifest in adulthood. Despite the dominant roles of androgen action in the male reproductive tract development, it has been noticed that androgen's capability in promoting Wolffian duct survival and development is completely lost in the absence of Wnt9b. Wnt9b belongs to the WNT family of secreted proteins. It is specifically expressed in the Wolffian duct epithelium to regulate the surrounding mesenchyme. This project aims to investigate why the androgen action fails to maintain the male reproductive tract in the absence of Wnt9b. We first examined the expression pattern of Wnt9b during the window of sexual differentiation, which was not investigated in the previous publication. We found that during sexual differentiation, Wnt9b is still specifically expressed in the Wolffian duct epithelium in both XX and XY embryos. Androgen action exists in XY but not in the XX embryos during sexual differentiation. These observations indicate that Wnt9b does not lie downstream of the androgen signaling. We are in process of collecting Wnt9b+/+ and Wnt9b-/- male embryos to examine whether expression of AR and AR downstream genes is altered in the absence of Wnt9b. In addition, WNT9B can activate two intracellular pathways under the context of target cells: β-catenin dependent and independent pathways. Since Wolffian duct mesenchyme is the major target of epithelium-derived WNT9B, we next determined which pathways WNT9B acts through in the mesenchyme by specific deletion of β-catenin in the Wolffian duct mesenchyme. Interestingly, the loss of β-catenin in the mesenchyme led to the partial degeneration of Wolffian ducts at the caudal region and cystic formations at the cranial region, the similar phenotypes observed in mesenchymal AR knockout male embryos. Taken together, our study demonstrates that the local WNT9B/β-catenin pathway provides the permissive signaling for androgen actions in Wolffian duct development. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 1:12 p.m. - 1:17 p.m.

Clinicopathological spectrums and prognosis of primary appendiceal adenocarcinoma, goblet cell adenocarcinoma, and low-grade appendiceal mucinous neoplasms

Primary appendiceal adenocarcinoma (APCA), goblet cell adenocarcinoma (GCA), and low/high-grade appendiceal mucinous neoplasms (LAMN/HAMN) are distinct entities with overlapping clinical presentation and histomorphology, leading to diagnostic challenges. We retrospectively reviewed our archived cases between 2010 and 2018 for diagnosis reappraisal and comparative analysis using updated terminology and modern parameters. A total of 87 cases (22 APCA, 40 GCA, and 25 LAMN pT≥3) were included. The entire cohort had 49 women and 38 men with a median age of 59.9 (range 26-88) years. There were no statistically significant differences in age and sex among the three groups. Clinically, patients with GCA were more likely to present with acute appendicitis (65%) and more likely to have appendectomy as initial surgery (68%). Both APCA and GCA were more likely to involve the proximal appendix while LAMN was more likely to involve the distal appendix (p<0.05). All APCAs were associated with mucosal precursor lesions, most commonly tubular, tubulovillous, or villous adenoma, flat LAMN/HAMN-pTis mucinous epithelium, or mixed, which correlated with distinct histomorphology, tumour differentiation, and stage. Although polypoid precursor lesions were rare in GCA, a significant proportion of GCA showed crypt atypia associated with neoplastic cells. Immunohistochemically, APCA had more frequent β-catenin nuclear positivity and loss of SATB2 expression (p<0.05). KRAS mutation was more common in APCA than in GCA (8/11 vs 1/7, p<0.01). We further validated the three-tiered grading system (G1, G2, G3) in GCA, which correlated well with tumour stage and patient survival. APCA had worse progression-free and disease-specific survivals than GCA and LAMN (pT≥3) with the latter being relatively indolent even when perforated with peritoneal spread. Our study is the first comprehensive comparison between all three appendiceal neoplasms. We also describe a spectrum of previously under-recognised crypt atypia in GCA, which should trigger a diligent search for GCA if present.

NMR-based metabolomic analysis identifies RON-DEK-β-catenin dependent metabolic pathways and a gene signature that stratifies breast cancer patient survival.

BackgroundAdvances in detection techniques and treatment have increased the diagnosis of breast cancer at early stages; however, recurrence occurs in all breast cancer subtypes, and both recurrent and de novo metastasis are typically treatment resistant. A growing body of evidence supports the notion that metabolic plasticity drives cancer recurrence. RON and DEK are proteins that promote cancer metastasis and synergize mechanistically to activate β-catenin, but the metabolic consequences are unknown.MethodsTo ascertain RON-DEK-β-catenin dependent metabolic pathways, we utilized an NMR-based metabolomics approach to determine steady state levels of metabolites. We also interrogated altered metabolic pathway gene expression for prognostic capacity in breast cancer patient relapse-free and distant metastasis-free survival and discover a metabolic signature that is likely associated with recurrence.ResultsRON-DEK-β-catenin loss showed a consistent metabolite regulation of succinate and phosphocreatine. Consistent metabolite alterations between RON and DEK loss (but not β-catenin) were found in media glucose consumption, lactate secretion, acetate secretion, and intracellular glutamine and glutathione levels. Consistent metabolite alterations between RON and β-catenin loss (and not DEK) were found only in intracellular lactate levels. Further pathway hits include β-catenin include glycolysis, glycosylation, TCA cycle/anaplerosis, NAD+ production, and creatine dynamics. Genes in these pathways epistatic to RON-DEK-β-catenin were used to define a gene signature that prognosticates breast cancer patient survival and response to chemotherapy.ConclusionsThe RON-DEK-β-catenin axis regulates the numerous metabolic pathways with significant associations to breast cancer patient outcomes.

Abstract LB045: β-catenin antagonist peptide, ST316, attenuates Wnt-dependent oncogenic activity

Abstract Constitutive activation of the Wnt/β-catenin signaling pathway underlines many breast and colorectal cancers, promoting cancer cell survival, proliferation and migration. B-cell CLL/lymphoma 9 protein (BCL9), a co-activator of β-catenin transcriptional activity, is overexpressed in many tumors and facilitates β-catenin nuclear localization. Disruption of the β-catenin and BCL9 interaction has been shown genetically to suppress oncogenic Wnt/β-catenin transcription, without impacting β-catenin homeostatic functions, and represents a powerful approach to inhibit this previously ‘undruggable’ target. Here, we investigate the impact of ST316, a peptide antagonist of β-catenin that inhibits its interaction with BCL9, on cells displaying hyperactivation of the Wnt/β-catenin pathway. Administration of ST316 to HCT116 human colorectal carcinoma cells, which carry a degradation-escape, gain-of-function mutation in β-catenin, dramatically reduced nuclear β-catenin localization. Furthermore, ST316-mediated loss of nuclear β-catenin was associated with a significant decrease in the expression of Wnt/β-catenin target genes in HCT116 cells stimulated with Wnt3a, including Axin2, hTCF7, Fox01 and CDK4. ST316 reduced β-catenin target gene expression by up to ∼70%, whereas no impact was observed following administration of a negative control peptide. A significant anti-proliferative and dose-dependent cytotoxic effect was observed on HCT116 cells 48 hours post ST316 exposure, with an EC50 of 4.4 µM, and on patient-derived, APC-mutant colorectal adenocarcinoma and breast carcinoma cells 72 hours post ST316 exposure, with EC50 values ranging from 1.5 to 2.6 µM. To extend these findings, we investigated the impact of ST316 on HCT116 tumors in vivo. ST316 (5 mg/kg) administered three times weekly by subcutaneous injection resulted in 99.6% tumor growth inhibition vs. vehicle controls (p&amp;lt;0.05), while a negative control peptide had no significant impact on tumor growth. These data demonstrate the potential of ST316 as a potent therapeutic candidate for Wnt/β-catenin-dependent cancers. Citation Format: Lila Ghamsari, Erin Gallagher, Emad Darvishi, Siok Leong, Mark Koester, Rick Ramirez, Jerel Gonzales, Gene Merutka, Barry Kappel, Jim A. Rotolo. β-catenin antagonist peptide, ST316, attenuates Wnt-dependent oncogenic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB045.

Butyrate Inhibits Colorectal Cancer Cell Proliferation through Autophagy Degradation of β-Catenin Regardless of APC and β-Catenin Mutational Status

Colorectal cancer (CRC) pathogenesis is mainly driven by alterations in WNT signaling, which results in altered transcriptional activity of β-Catenin. Mutations in APC (Adenomatous Polyposis Coli) are reflected in β-Catenin hyperactivation and loss of proliferation control. Certain intestinal bacteria metabolites have shown the ability to limit CRC cell proliferation and CRC pathogenesis. Here, we investigated the molecular mechanism underlying the anti-proliferative activity of butyrate, a microbiota-derived short chain fatty acid, in two CRC cell lines, namely HCT116 and SW620, which bear a mutation in β-Catenin and APC, respectively. In particular, we focused on autophagy, a lysosome-dependent degradation pathway, which was shown to control intestinal tissue homeostasis. Butyrate reduced CRC cell proliferation, as witnessed by the downregulation of proliferation markers. TCGA bioinformatic transcriptomic analysis of CTNNB1 (β-Catenin) gene correlation in CRC patients showed that β-Catenin negatively correlates with the autophagy gene ATG4D. In CRC cells, regardless of the mutational state of APC or β-Catenin genes, butyrate caused the autophagy-mediated degradation of β-Catenin; thus, preventing its transcriptional activity. Autophagy gene silencing restored β-Catenin levels, allowing it to translocate into the nucleus to promote the expression of downstream genes associated with cancer cell proliferation. CRC-affected patients show driver mutations in the WNT pathway; thus, targeting its crucial effector may be a promising therapeutic strategy in CRC treatment; for instance, by using ad hoc probiotics that stimulate autophagy.

BRAF Inhibitors Reprogram Cancer-Associated Fibroblasts to Drive Matrix Remodeling and Therapeutic Escape in Melanoma.

The tumor stroma and its cellular components are known to play an important role in tumor response to treatment. Here, we report a novel resistance mechanism in melanoma that is elicited by BRAF inhibitor (BRAFi)-induced noncanonical activation of nuclear β-catenin signaling in cancer-associated fibroblasts (CAF). Treatment with BRAFi leads to an expanded CAF population with increased β-catenin nuclear accumulation and enhanced biological properties. This CAF subpopulation is essential for melanoma cells to proliferate and acquire resistance to BRAFi/MEK inhibitors (MEKi). Mechanistically, BRAFi induces BRAF-CRAF heterodimerization and subsequent activation of ERK signaling in CAFs, leading to inactivation of the β-catenin destruction complex. RNA-seq identified periostin (POSTN) as a major downstream effector of β-catenin in CAFs. POSTN compensates for the loss of β-catenin in CAFs and mediates melanoma cell BRAFi/MEKi resistance. In melanoma cells, POSTN activates phosphoinositide 3-kinase (PI3K)/AKT signaling and subsequently reactivates the ERK pathway that was inhibited by BRAFi/MEKi. Collectively, these data underscore the role of BRAFi-induced CAF reprogramming in matrix remodeling and therapeutic escape of melanoma cells. SIGNIFICANCE: β-Catenin activation in cancer-associated fibroblasts in response to BRAF inhibitors stimulates POSTN secretion to promote resistance in cancer cells, revealing POSTN as a potential matrix target in cancer therapy.

Prognostic and Clinicopathological Significance of the Aberrant Expression of β-Catenin in Oral Squamous Cell Carcinoma: A Systematic Review and Meta-Analysis.

This systematic review and meta-analysis aims to evaluate the prognostic and clinicopathological significance of the aberrant expression of β-catenin (assessed through the immunohistochemical loss of membrane expression, cytoplasmic and nuclear expression) in oral squamous cell carcinoma (OSCC). We searched for primary-level studies published before October-2021 through PubMed, Embase, Web of Science, Scopus, and Google Scholar, with no limitation in regard to their publication date or language. We evaluated the methodological quality and risk of bias of the studies included using the QUIPS tool, carried out meta-analyses, explored heterogeneity and their sources across subgroups and meta-regression, and conducted sensitivity and small-study effects analyses. Forty-one studies (2746 patients) met inclusion criteria. The aberrant immunohistochemical expression of β-catenin was statistically associated with poor overall survival (HR = 1.77, 95% CI = 1.20-2.60, p = 0.004), disease-free survival (HR = 2.44, 95% CI = 1.10-5.50, p = 0.03), N+ status (OR = 2.39, 95% CI = 1.68-3.40, p < 0.001), higher clinical stage (OR = 2.40, 95% CI = 1.58-3.63, p < 0.001), higher tumour size (OR = 1.76, 95% CI = 1.23-2.53, p = 0.004), and moderately-poorly differentiated OSCC (OR = 1.57, 95% CI = 1.09-2.25, p = 0.02). The loss of β-catenin in the cell membrane showed the largest effect size in most of meta-analyses (singularly for poor overall survival [HR = 2.37, 95% CI = 1.55-3.62, p < 0.001], N+ status [OR = 3.44, 95% CI = 2.40-4.93, p < 0.001] and higher clinical stage [OR = 2.51, 95% CI = 1.17-5.35, p = 0.02]). In conclusion, our findings indicate that immunohistochemical assessment of the aberrant expression of β-catenin could be incorporated as an additional and complementary routine prognostic biomarker for the assessment of patients with OSCC.

β-Catenin-NF-κB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction.

Expansion of biliary epithelial cells (BECs) during ductular reaction (DR) is observed in liver diseases including cystic fibrosis (CF), and associated with inflammation and fibrosis, albeit without complete understanding of underlying mechanism. Using two different genetic mouse knockouts of β-catenin, one with β-catenin loss is hepatocytes and BECs (KO1), and another with loss in only hepatocytes (KO2), we demonstrate disparate long-term repair after an initial injury by 2-week choline-deficient ethionine-supplemented diet. KO2 show gradual liver repopulation with BEC-derived β-catenin-positive hepatocytes and resolution of injury. KO1 showed persistent loss of β-catenin, NF-κB activation in BECs, progressive DR and fibrosis, reminiscent of CF histology. We identify interactions of β-catenin, NFκB, and CF transmembranous conductance regulator (CFTR) in BECs. Loss of CFTR or β-catenin led to NF-κB activation, DR, and inflammation. Thus, we report a novel β-catenin-NFκB-CFTR interactome in BECs, and its disruption may contribute to hepatic pathology of CF.

The missing 'link'? β-Catenin's role in skeletal muscle glucose uptake during exercise and contraction.

The missing 'link'? β-Catenin's role in skeletal muscle glucose uptake during exercise and contraction.

Bmi1 Regulates Wnt Signaling in Hematopoietic Stem and Progenitor Cells.

Polycomb group protein Bmi1 is essential for hematopoietic stem cell (HSC) self-renewal and terminal differentiation. However, its target genes in hematopoietic stem and progenitor cells are largely unknown. We performed gene expression profiling assays and found that genes of the Wnt signaling pathway are significantly elevated in Bmi1 null hematopoietic stem and progenitor cells (HSPCs). Bmi1 is associated with several genes of the Wnt signaling pathway in hematopoietic cells. Further, we found that Bmi1 represses Wnt gene expression in HSPCs. Importantly, loss of β-catenin, which reduces Wnt activation, partially rescues the HSC self-renewal and differentiation defects seen in the Bmi1 null mice. Thus, we have identified Bmi1 as a novel regulator of Wnt signaling pathway in HSPCs. Given that Wnt signaling pathway plays an important role in hematopoiesis, our studies suggest that modulating Wnt signaling may hold potential for enhancing HSC self-renewal, thereby improving the outcomes of HSC transplantation.

Abstract P169: β-catenin C-terminal Domain/Sphingosine-1-Phosphate Receptor 1 Axis Drives Neointima Formation After Carotid Injury

Introduction: The β-catenin C-terminal domain (CTD) is required for artery formation during embryogenesis, but its role in vascular remodeling in adulthood is unknown. Inhibitors targeting specifically the CTD of β-catenin have been developed, but their use in obstructive vascular disease is not yet justified because of this gap of knowledge. Loss of β-catenin in mouse aortic smooth muscle cells (MASMCs) decreases the transcript levels of sphingosine-1-phosphate receptor 1 (S1PR1). However, no studies have evaluated the crosstalk between these proteins in vascular remodeling. Hypothesis: We hypothesized that the CTD of β-catenin promotes neointima formation after vascular injury, and that it acts by inducing S1PR1 expression. Methods: We studied mice bearing a knockin mutant β-catenin allele (ΔC) that lacks β-catenin CTD transcriptional activity. Mice expressing the SMC-specific SMA-CreERT2 and bearing one floxed and one mutant β-catenin allele were treated with tamoxifen to inactivate the floxed allele, leaving the mutant allele as the only source of β-catenin in SMC (SMβCΔC mice). Carotid artery ligation was performed to induce vascular injury. MASMCs were isolated from SMβCΔC mice (βCΔC cells). Results: We found reduced neointima formation after injury in SMβCΔC male (SMβCWT/-: 0.52±0.09 vs SMβCΔC: 0.16±0.05; n=10; P&lt;0.05) and female (SMβCWT/-: 0.56±0.11 vs SMβCΔC: 0.17±0.04; n=10; P&lt;0.05) mice. These findings were associated with reduced SMC proliferation, increased SMC apoptosis, and prevention of loss of SMC markers. In vitro, βCΔC MASMCs showed reduced growth compared to control cells (n=6; P&lt;0.05). βCΔC MASMCs also showed reduced S1PR1 expression and decreased S1pr1 promoter activity, which were both rescued by the transfection of a constitutively active form of β-catenin. Interestingly, overexpression of S1PR1 restored βCΔC MASMC growth towards control levels (n=6; P&lt;0.05). In vivo, we found reduced S1PR1 expression in SMCs of injured arteries from SMβCΔC mice. Conclusion: Our results define an essential SMC β-catenin CTD-S1PR1 axis that drives neointima formation after arterial injury, and point to multiple points for potential therapeutic intervention to control vascular remodeling in disease.