Beta-catenin Activation Research Articles

Ergosterol inhibits the proliferation of breast cancer cells by suppressing AKT/GSK-3beta/beta-catenin pathway

Breast cancer is a prevalent malignancy affecting women globally, necessitating effective treatment strategies. This study explores the potential of ergosterol, a bioactive compound found in edible mushrooms, as a candidate for breast cancer treatment. Breast cancer cell lines (MCF-7 and MDA-MB-231) were treated with ergosterol, revealing its ability to inhibit cell viability, induce cell cycle arrest, and suppress spheroid formation. Mechanistically, ergosterol demonstrated significant inhibitory effects on the Wnt/beta-catenin signaling pathway, a critical regulator of cancer progression, by attenuating beta-catenin translocation in the nucleus. This suppression was attributed to the inhibition of AKT/GSK-3beta phosphorylation, leading to decreased beta-catenin stability and activity. Additionally, ergosterol treatment impacted protein synthesis and ubiquitination, potentially contributing to its anti-cancer effects. Moreover, the study revealed alterations in metabolic pathways upon ergosterol treatment, indicating its influence on metabolic processes critical for cancer development. This research sheds light on the multifaceted mechanisms through which ergosterol exerts anti-tumor effects, mainly focusing on Wnt/beta-catenin pathway modulation and metabolic pathway disruption. These findings provide valuable insights into the potential of ergosterol as a therapeutic candidate for breast cancer treatment, warranting further investigation and clinical application.

#2215 Early molecular and cellular features of aortic remodeling in a mild CKD-MBD model with low bone turnover

Abstract Background and Aims Vascular calcification associated with a maladaptive bone response is common in CKD and is a major cause of mortality (up to 60% depending on CKD stage). Alterations in several intracellular signaling pathways have been reported during the cellular phenotype transition and vascular calcification, however the initial phases of vascular remodeling in early CKD-MBD remain poorly understood. The aim of this study was to investigate the early molecular and cellular features of aortic remodeling prior to calcification in a model of mild CKD-MBD with low bone turnover. Method We induced CKD-MBD by 3/4 nephrectomy (Nx, n = 8) in adult male spontaneously hypertensive rats (SHR) with normal phosphate (Pi) intake (0.6%). Controls were sham-operated Wistar rats (n = 8) and SHR (n = 8). Chronic kidney injury and Pi exchange parameters, serum levels of calciprotein particles (CPPs), intact PTH, intact FGF23, dickkopf-1 and sclerostin, static bone histomorphometry, aortic morphology (H&E, Masson's trichrome, calcein, von Kossa stain), CD31, aSMA, nestin, Col1a1, PDGFR-beta, PiT1, LGR4 and intracellular signaling molecules—phospho-ERK1/2, active (non-phospho) beta-catenin, Hes1, Gli1 were analyzed by IHC with quantitative morphometry and IF after 4 months. Confocal microscopy was performed at the Centre for Collective Use of the Pavlov Institute of Physiology, Russian Academy of Sciences (Zeiss LSM 710). Results The study found that Nx rats with chronic kidney injury comparable to human CKD S2 exhibited lower bone turnover (Fig. 1). The Nx group had higher serum levels of Pi and CPPs vs. control group, but there were no differences in Pi-regulatory factors (Figs 1, 2b). Additionally, the Nx group showed aorta remodeling, which was characterized by an increase in intima thickness, cellularity, and collagen accumulation in the medial layer (Figs 1, 2a). The histological parameters of intimal remodeling and perivascular fibrosis were found to be directly correlated with serum levels of Pi and CPPs. In Nx, there was a decrease in medial αSMA expression accompanied by an increase in phospho-ERK1/2, PiT1, nestin, Gli1, beta-catenin, dickkopf-1, and an decrease in Hes1 immunomorphological staining (Figs 1, 2a, c). αSMA-negative cells demonstrated the expression of osteogenesis-related molecules, such as phospho-ERK1/2 (Fig. 2a, c), and occasionally, RunX2 (Fig. 2a). Phospho-ERK1/2 and PiT1 were found to co-localize in Nx (Fig. 2c). Intimal remodeling was observed, characterized by increased beta-catenin and nestin expression (Fig. 2c). Conclusion In mild CKD-MBD with low bone turnover, early intimal and medial aortic remodeling was associated with higher serum Pi and CPPs levels, and upregulation of PiT1, ERK1/2. In addition, other signals likely related to osteogenesis (RunX2, LGR4) and regulation of vascular progenitor cells (Nestin, Wnt, Notch, Hedgehog) may be implicated.

Abstract LB199: Condensate modulator sequesters beta-catenin into depot condensates and demonstrates competitive anti-tumor activity in animal models ofcolorectal cancer

Abstract Constitutive activation of beta-catenin is a well-known driver of malignancy. However, traditional drug discovery approaches have proven challenging and largely unsuccessful in identifying therapeutic agents to modulate the function of beta-catenin. Biomolecular condensates have recently been demonstrated to play key roles in regulating most cellular processes and biological pathways by compartmentalizing biomolecules in membranelles organelles, thus lending novel opportunities to drug previously “undruggable” targets. Here, we leverage condensate biology to discover small molecules that reverse the hyperactive function of beta-catenin in colorectal cancer by entrapping it into depot condensates. We developed a high-throughput phenotypic assay that identifies beta-catenin condensate-modifying compounds (c-mods). Through condensate screening and functional secondary assays, we have identified c-mods that sequester beta-catenin into depots, induce selective cancer cell killing, and reverse oncogenic beta-catenin specific gene expression programs. C-mods identified in our screen are effective in genetically diverse colorectal cancers and a range of Wnt-associated cancers, providing the opportunity to treat a broad patient population. Furthermore, oral dosing of lead c-mod demonstrates competitive tumor growth inhibition as a single agent in xenograft and PDX models of colorectal cancer. Taken together, these results highlight the promise of condensate biology in drugging previously intractable high-value targets in oncology and developing novel treatments for patients suffering from diseases of high unmet need. Citation Format: Costa Salojin, Douglas Baumann, Adam Talbot, Kip West, Thomas Durand-Reville, Isaac Klein, Ann Boija. Condensate modulator sequesters beta-catenin into depot condensates and demonstrates competitive anti-tumor activity in animal models ofcolorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB199.

A reappraisal of transcriptional regulation by NR5A1 and beta-catenin in adrenocortical carcinoma.

Overexpression of the transcription factor NR5A1 and constitutive activation of canonical Wnt signalling leading to nuclear translocation of beta-catenin are hallmarks of malignancy in adrenocortical carcinoma (ACC). Based on the analysis of genomic profiles in H295R ACC cells, Mohan etal. (Cancer Res. 2023; 83: 2123-2141) recently suggested that a major determinant driving proliferation and differentiation in malignant ACC is the interaction of NR5A1 and beta-catenin on chromatin to regulate gene expression. I reanalyzed the same set of data generated by Mohan etal. and other published data of knockdown-validated NR5A1 and beta-catenin target genes. Beta-catenin is mainly found in association to canonical T cell factor/lymphoid enhancer factor (TCF/LEF) motifs in genomic DNA. NR5A1 and beta-catenin regulate distinct target gene sets in ACC cells. Overall, my analysis suggests a model where NR5A1 overexpression and beta-catenin activation principally act independently, rather than functionally interacting, to drive ACC malignancy.

Localization of beta catenin across the domain of odontogenic lesions: A systematic review.

CTNNB1 gene encodes beta catenin, a transcriptional activator of Wnt pathway involved in the pathogenesis of odontogenic lesions. Though located intramembranously, its translocation into cytoplasm and nucleus could trigger cell proliferation, inhibition of apoptosis, invasion and migration of the tumour cell. Five electronic databases including MEDLINE by PubMed, Google scholar, Scopus, Trip, Cochrane library and EMBASE until 1 January 2023 without period restriction were thoroughly searched. Those articles that identified CTNNB1 mutation and beta catenin in odontogenic lesions were included for review. Risk of bias was analysed for each study using QUADAS 2 tool and Review Manager 5.3 was used to output its result. Thirty four published articles were included for data synthesis. A total of 1092 cases of odontogenic lesions were assessed for both CTNNB1 mutation and beta catenin expression. CTNNB1 mutation was observed in ameloblastoma, calcifying odontogenic cyst, calcifying cystic odontogenic tumour and all malignant odontogenic tumours. The beta catenin expression (nuclear and cytoplasmic) was maximum in odontogenic keratocyst and calcifying odontogenic cyst. The expression was variable in ameloblastomas, membranous in odontomas, calcifying cystic odontogenic tumour and nuclear in all malignant tumours. High recurrence of odontogenic keratocyst and aggressiveness of solid ameloblastoma and malignant odontogenic tumours could be associated with the nuclear translocation of beta catenin. Disparity between CTNNB1 mutation and beta catenin expression within odontogenic lesions suggests alternate routes of beta catenin activation. The review results support the unique localisation of beta catenin as a helpful diagnostic factor in the pathogenesis of odontogenic lesions.

Gain-of-function mutant p53 together with ERG proto-oncogene drive prostate cancer by beta-catenin activation and pyrimidine synthesis

Whether TMPRSS2-ERG fusion and TP53 gene alteration coordinately promote prostate cancer (PCa) remains unclear. Here we demonstrate that TMPRSS2-ERG fusion and TP53 mutation / deletion co-occur in PCa patient specimens and this co-occurrence accelerates prostatic oncogenesis. p53 gain-of-function (GOF) mutants are now shown to bind to a unique DNA sequence in the CTNNB1 gene promoter and transactivate its expression. ERG and β-Catenin co-occupy sites at pyrimidine synthesis gene (PSG) loci and promote PSG expression, pyrimidine synthesis and PCa growth. β-Catenin inhibition by small molecule inhibitors or oligonucleotide-based PROTAC suppresses TMPRSS2-ERG- and p53 mutant-positive PCa cell growth in vitro and in mice. Our study identifies a gene transactivation function of GOF mutant p53 and reveals β-Catenin as a transcriptional target gene of p53 GOF mutants and a driver and therapeutic target of TMPRSS2-ERG- and p53 GOF mutant-positive PCa.

OS-023-YI - Remodeling of myeloid cells during hepatocellular carcinoma driven by beta-catenin activation

OS-023-YI - Remodeling of myeloid cells during hepatocellular carcinoma driven by beta-catenin activation

Human respiratory syncytial virus activates beta-catenin pathway in lung epithelial cells to regulate pro-inflammatory response

Abstract Respiratory Syncytial Virus (RSV) is a non-segmented negative-sense RNA virus belonging to the paramyxovirus family. RSV infects respiratory tract to cause pneumonia and bronchiolitis in infants, elderly, and immunocompromised patients. Effective clinical therapeutic options and vaccine to combat RSV infection are still lacking. Therefore, to develop effective therapeutic interventions, it is imperative to understand virus-host interactions during RSV infection. Canonical Wnt/beta-catenin pathway is a well-established signaling cascade regulating expression of Wnt target genes involved in various biological and physiological functions. Our study shows RSV infection of human lung epithelial A549 cells triggering stabilization of beta-catenin protein and subsequent activation of beta-catenin mediated transcriptional activity. Further studies with beta-catenin inhibitors and beta-catenin lacking cells revealed a role of beta-catenin in positively regulating pro-inflammatory response in RSV infected A549 cells. Activation of beta-catenin during RSV infection was mediated by cell surface receptor complex comprising of frizzled receptors and low-density lipoprotein receptor-related protein 5 and 6 (LRP5 and LRP6). Thus, our studies have revealed an involvement of beta-catenin pathway in inducing pro-inflammatory response during RSV infection. The mechanism(s) and host derived factors regulating RSV-mediated beta-catenin activation will be discussed.

1332 Janus kinase inhibitor increases hair growth by increasing beta-catenin activity in outer root sheath cells

1332 Janus kinase inhibitor increases hair growth by increasing beta-catenin activity in outer root sheath cells

Abstract 6166: TBL1, a multifunctional transcriptional regulator, is highly expressed in osteosarcoma and correlates with activated beta catenin

Abstract Background: Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents. Two-thirds of OS patients are cured through chemotherapy and tumor resection. However, survival rates have plateaued over the past 30 years, especially for patients with relapsed and/or metastatic disease, with overall long-term outcomes of less than 30%. Transducin beta-like protein 1 (TBL1) protects beta-catenin from nuclear degradation and mediates Wnt targeted transcription by forming a TBL1-beta-catenin complex. Overexpression of TBL1 and nuclear beta-catenin are positively correlated to adverse clinicopathological features and poor prognosis for osteosarcoma patients. Functional experiments reveal that down-regulation of TBL1 and nuclear beta-catenin activity inhibit proliferation, migration, and invasion of osteosarcoma cells. The objective of this study was to determine TBL1 levels within a panel of OS patient-derived xenograft (PDX) tumors. Methods: TBL1 and beta-catenin protein levels were measured via western blot and immunohistochemistry in OS samples. We examined protein levels in OS PDX cell lines collected at distinct clinical stages of metastatic OS and OS primary tumor samples. TBL1, total beta-catenin, and activated beta-catenin levels were measured by immunohistochemical (IHC) staining in tissue microarray (TMA) derived from patient derived xenograft (PDX) tumor and clinical samples. Each tumor sample were formalin fixed, paraffin embedded, and spotted in triplicate on the TMA. Results: Beta-catenin and TBL1 protein were detected in OS samples, and we found higher TBL1 protein levels in cells derived from advanced disease stages. Assessment of TBL1 protein was expanded to OS tissue microarray where TBL1, total beta-catenin, and active beta-catenin were independently stained in a TMA. TBL1, total beta-catenin, and activated beta-catenin were present in 100% of OS PDX cores. Finally, a majority of OS cores were shown to have high expression for both TBL1 and active beta-catenin. Conclusion: Elevated TBL1 levels have been associated with metastatic disease and poor survival. We have measured TBL1 and activated beta-catenin protein levels in a variety of osteosarcoma samples and identified a large percentage of osteosarcoma that co-express both proteins. These results indicate that metastatic OS represents a TBL1 enriched patient population and is a candidate for therapeutics that target TBL1. Citation Format: Kimberly R. Holloway, Taku Yamamichi, Bikesh Nirala, Nino Rainusso, Jason T. Yustein, Stephen Horrigan. TBL1, a multifunctional transcriptional regulator, is highly expressed in osteosarcoma and correlates with activated beta catenin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6166.

PSII-5 Endotoxin-Induced Inflammation on Immune Markers and Steroidogenesis in Granulosa Cells

Abstract Reproductive performance is central to profitability in beef cattle operations. Pathogenic stress can result from gram-negative bacteria release of the lipopolysaccharide (LPS) endotoxin which has the potential to accumulate in follicular fluid and impair folliculogenesis and steroidogenesis, leading to reduced fertility. Elevated LPS concentrations observed in acute clinical bacterial infections are recognized to have detrimental effects on female fertility, however, definitive effects of low concentrations of LPS present in subclinical or undetectable infection remains to be fully elucidated. This study investigated the effect of varying concentrations of LPS on mechanisms regulating ovarian function and subsequent steroidogenesis. A granulosa cell line (KGN) was cultured with or without increasing doses of LPS (0.0001 to 10 µg/mL) for 30 min or 48 h. Cells collected 30 min after treatment were analyzed for abundance of phosphorylated proteins and cells collected 48 h after treatment were analyzed for total protein abundance and gene expression. Media were analyzed by radioimmunoassay for steroid hormone concentrations. Quantitative and semi-quantitative PCR was used to evaluate aromatase, beta-catenin, and cytokines IL-6 and IL-8, respectively. No differences were detected among LPS treatment groups compared to vehicle-treated controls for protein abundance of total beta-catenin (P = 0.99) or active beta-catenin lacking phosphorylation at serine 33/37 (P = 0.95) evaluated at 48 h. Similar results were observed in protein abundance of phosphorylated forms of beta-catenin at serine 552 (P = 0.98) and 675 (P = 0.85) evaluated at 30 min. Additionally, estradiol concentrations were not different at 0.0001 to 1.0 µg/mL LPS; however, estradiol was significantly decreased at 10 µg/mL LPS treatment compared to the vehicle-treated control (P = 0.03). Progesterone concentrations were also similar within 0.0001 to 1.0 µg/mL LPS treatment groups with a tendency to decrease at 10 µg/mL LPS treatment (P = 0.08) compared to the vehicle-treated control. Varying concentrations of LPS did not change mRNA expression of beta-catenin (P = 0.28), aromatase (P = 0.32), and IL-6 (P = 0.35). However, a significant increase in cytokine IL-8 mRNA expression was observed at 10 µg/mL LPS compared to the vehicle-treated control (P = 0.04). These findings suggest that inflammatory response against LPS at higher concentrations may influence distinct cytokine expression and steroidogenesis. Conversely, basal expression of key steroidogenic enzymes and ovarian steroidogenic modulators are less affected by low concentrations of LPS. Therefore, immune response of granulosa cells is dependent on concentration of LPS exposure that has the ability to impact fertility by modulating the ovarian hormonal and follicular environment.

High glucose impairs osteogenic differentiation of embryonic stem cells via early diversion of beta-catenin from Forkhead box O to T cell factor interaction.

Diabetes, which is characterized by an increase in blood glucose concentration, is accompanied by low bone turnover, increased fracture risk, and the formation of embryonic skeletal malformations. Yet, there are few studies elucidating the underlying alterations in signaling pathways leading to these osteogenic defects. We hypothesized here that bone formation deficiencies in a high glucose environment result from altered activity of beta-catenin (CTNNB1), a key contributor to osteogenic differentiation, dysregulation of which has also been implicated in the development of diabetes. To test this hypothesis, we used a previously established embryonic stem cell (ESC) model of differentiation that mimics the diabetic environment of the developing embryo. We differentiated murine ESCs within osteogenic-inducing media containing either high (diabetic) or low (physiological) levels of D-glucose and performed time course analyses to study the influence of high glucose on early and late bone cell differentiation. Endpoint measures for osteogenic differentiation were reduced in a glucose-dependent manner and expression of precursor-specific markers altered at multiple time points. Furthermore, transcriptional activity of the lymphoid enhancer factor (LEF)/T cell factor (TCF) transcription factors during precursor formation stages was significantly elevated while levels of CTNNB1 complexed with Forkhead box O 3a (FOXO3a) declined. Modulation of AKT, a known upstream regulator of both LEF/TCF and FOXO3a, as well as CTNNB1 rescued some of the reductions in osteogenic output seen in the high glucose condition. Within our in vitro model, we found a clear involvement of LEF/TCF and FOXO3a signaling pathways in the regulation of osteogenic differentiation, which may account for the skeletal deficiencies found in newborns of diabetic mothers.

Abstract A028: Growth factor signaling and kinase inhibitors regulate oncogenesis in desmoid fibromatosis by modulating activity of the beta-catenin transcription target ABL1

Abstract Introduction: While activation of beta-catenin is associated with desmoid fibromatosis (DF), mechanisms by which this oncogene initiates tumorigenesis are unclear as are factors underlying variable biologic behavior in the disease and vulnerability to targeted therapies. This study sought to define downstream pathways dysregulated by beta-catenin that may be modulated to potentially affect patient outcome. Methods: Multiple primary DF cell lines were developed from surgical specimens, validated by Sanger sequencing, and immortalized by ectopic expression of TERT. Gene expression was assessed in DF tumors (n=45) with U133A arrays and by RNA-seq in cells. Direct targets of beta-catenin were identified by CHIP-seq. Lentiviral systems were used to deliver shRNA (or scramble control) and overexpression constructs. Cell proliferation, protein levels/phosphorylation and gene expression were assessed by CyQuant DNA quantification, immunoblot, and RT-PCR, respectively. Endothelial cell (HUVEC) tube formation was quantitated using light microscopy. Results: Gene set enrichment analysis performed on RNA-seq data comparing DF cells treated with shRNA directed against CTNNB1 showed downregulation of hypoxia-regulated genes, and unsupervised analysis clustered 45 DF tumors separately from normal mesenchymal tissue based on the expression levels of these genes. CTNNB1 knockdown (KD) was associated with reduction in HIF1A and ability of DF cells to induced endothelial tube formation in HUVEC co-cultures (71%, p<0.001); ectopic expression of HIF1A in CTNNB1 KDs rescued this effect. HIF1A KD itself inhibited DF induction of HUVEC tube formation (49%, p<0.001), but did not affect DF cell proliferation. CHIP-seq nominated ABL1, a known regulator of HIF1 translation, as a direct target of beta-catenin. CTNNB1 KD caused 65% (p=0.01) decrease in ABL1 expression, and reduction in levels of c-ABL, its downstream target p-CRKL, and HIF1-alpha. Unlike HIF1A, ABL1 KD also reduced proliferation in multiple DF cell lines (up to 90%) as did direct inhibition of c-ABL with its inhibitor dasatinib (IC50 <50nM). Dasatinib and sorafenib, a PDGFR-beta inhibitor of clinical benefit in DF, both reduced cellular levels of p-ABL, p-CRKL and HIF1a expression in DF cells. Sorafenib also inhibited HUVEC tube-formation (59% at 1uM, p<0.05) induced by DF. Conversely, exogenous PDGF-BB stimulated DF proliferation (53% increase at 20ng/ml, p<0.05), increased p-ABL, p-CRKL and HIF1a in DF and promoted endothelial cell tube formation (2-fold, p<0.05) when added to DF and HUVEC co-cultures but not HUVEC cell cultures alone. Conclusion: ABL1 is a transcriptional target of beta-catenin in DF cells and is necessary for proliferation and maintenance of HIF1-alpha levels. Regulation of c-ABL activity by PDGFR-beta and targeted therapies modulates DF cell proliferation and paracrine signaling, suggesting a reason for variable biologic behavior between tumors and a mechanism for sorafenib activity in DF. This finding may point to markers predictive of outcome in patients. Citation Format: Jia Hu, Anthony Villano, Rachael O'Connor, Yuliy Rozenberg, Alankrta Venkatesh, Mrinal Gounder, Nicholas Socci, Samuel Singer, Meera Hameed, Aimee M. Crago. Growth factor signaling and kinase inhibitors regulate oncogenesis in desmoid fibromatosis by modulating activity of the beta-catenin transcription target ABL1 [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A028.

PGM3 regulates beta-catenin activity to promote colorectal cancer cell progression.

The hexosamine biosynthetic pathway (HBP) is connected to abnormal N- and O-linked protein glycosylation in cancer, which performs critical roles in tumorigenesis. However, the regulation mechanisms of HBP and its role in colorectal cancer (CRC) progression remain unexplained. This study analyzed the expression level of phosphoglucomutase 3 (PGM3), a key enzyme in HBP, and identified its function in CRC cell lines. Analysis of publicly available CRC microarray data determined that PGM3 is upregulated in CRC tumor tissues. Furthermore, functional experiments emphasized the significant roles of PGM3 in facilitating CRC cell proliferation and migration. Mechanistically, we demonstrated that the activity of β-catenin in CRC was maintained by PGM3-mediated O-GlcNAcylation. PGM3 knockdown or inhibition of O-GlcNAc transferase decreased β-catenin activity and the expression levels of its downstream targets. Collectively, our findings indicate that PGM3 exhibits tumor-promoting roles by elevating O-GlcNAcylation level and maintaining β-catenin activity, and might serve as a prognostic biomarker and treatment target in CRC.

Abstract 2528: PDLIM2 expression in triple negative breast cancer stromal macrophages

Abstract PDLIM2 is a PDZ-LIM domain protein expressed in hematopoietic and epithelial cells that regulates the stability of transcription factors including NFΚB, STATs and beta catenin. PDLIM2 function is associated with tumour suppression and also with the acquisition of a metastatic or epithelial mesenchymal transition (EMT) phenotype. We recently showed that PDLIM2 is more highly expressed in Triple Negative Breast Cancer (TNBC) tumours than in other breast cancer types or normal breast tissue, and that this is associated with active beta catenin in TNBC (1). Interestingly, in PDLIM2-positive TNBC tumours, PDLIM2 was also present in the tumour-associated stroma. However, whether stromal PDLIM2 expression contributes to TNBC cancer progression is unknown. To investigate this further we assessed the phenotype of PDLIM2-expressing stromal cells and determined that they most strongly represented a M2-like macrophage population. We then investigated the requirement for PDLIM2 in polarization towards either the M1 and M2-like macrophage phenotype using cultured bone marrow-derived macrophages from PDLIM2 knockout mice compared with wild type. This demonstrated that PDLIM2 is required for full adoption of IL-4-induced M2 macrophage polarization and function, including cell adhesion and migration. M1 polarization was less dependent on PDLIM2, although potential for activation of STATs and NFKB was similar in both macrophage populations.We conclude that high expression of PDLIM2 in TNBC influences the tumour microenvironment through the presence of an M2 macrophage population. Reference: (1) Cox, O. T., Edmunds, S. J., Simon-Keller, K., Li, B., Moran, B., Buckley, N. E., Bustamante-Garrido, M., Healy, N., O'flanagan, C. H., Gallagher, W. M., Kennedy, R. D., Bernards, R., Caldas, C., Chin, S. F., Marx, A. & O'Connor, R. 2019. Pdlim2 Is A Marker Of Adhesion And Beta-Catenin Activity In Triple-Negative Breast Cancer. Cancer Res, 79, 2619-2633. Citation Format: Orla T. Cox, Stephanie Ward, Emilie Tresse, Rosemary O'Connor. PDLIM2 expression in triple negative breast cancer stromal macrophages [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 2528.

Hepatic Adenomas in Patients 60 and Older Are Enriched for HNF1A Inactivation and Malignant Transformation.

Hepatic adenomas occur most commonly in women between the ages of 20 and 40 years, but rarely they occur in older aged persons, including those 60 years of age or older. This group of adenomas, however, has not been systemically examined. Twenty-six hepatic adenomas in persons 60 years of age or older were studied, along with a control group of 50 hepatic adenomas in persons aged 30 to 39. Hepatic adenomas in persons 60 or more years of age were found in 21 women and 5 men, while the control group had 44 women and 6 men. Subtyping the adenomas in persons 60 years or older showed the following results: 18 HNF1A-inactivated adenomas (69%), 4 inflammatory adenomas (15%), and 4 unclassified adenomas (15%). In contrast, the control group showed a significantly different pattern (P=0.003), with a greater percentage of inflammatory adenomas (28, 56%), fewer HNF1A-inactivated adenomas (8, 16%), and more unclassified adenomas (14, 28%). Atypia and malignant transformation within the hepatic adenomas was studied next. Of the hepatic adenomas in persons age 60 or greater, 3 (12%) showed atypical histologic features, and 6 (23%) had a malignant transformation. In contrast, for hepatic adenomas in the control group, only 4 (8%) adenomas showed atypical histologic features, and 3 (6%) had undergone malignant transformation. In addition, the hepatic adenomas that were atypical or showed early malignant transformation were less likely to have beta-catenin activation in patients over 60 (2/9 cases) compared with those between 30 and 39 years (5/7 cases). Myxoid change and heavy lipofuscin deposition were also more common in adenomas in older aged persons. In conclusion, hepatic adenomas in persons 60 years of age or older are enriched for HNF1A-inactivated adenomas and have a higher frequency of malignant transformation. Malignant transformation, however, is less likely to develop through activation of the beta-catenin pathway.

Circular RNA MTCL1 promotes advanced laryngeal squamous cell carcinoma progression by inhibiting C1QBP ubiquitin degradation and mediating beta-catenin activation

BackgroundCircular RNAs (circRNAs) are involved in regulatory processes of ubiquitination and deubiquitination in various tumors at post-transcriptional epigenetic modification level. However, the underlying mechanism and its biological functions of circRNAs in the advanced laryngeal squamous cell carcinoma (LSCC) remain obscure.MethodsRNA sequencing and quantitative real-time PCR (qRT-PCR) assays were applied to screen for circRNAs differentially expressed in LSCC tissues and cell lines. The candidate RNA-binding proteins and target signalling pathway were detected by RNA pull-down and mass spectrometry, in situ hybridization (ISH), immunohistochemistry (IHC), qRT-PCR assays, and bioinformatics analysis. The functional roles of these molecules were investigated using in vitro and in vivo experiments including EdU, transwell, wound healing, western blot assays, and the xenograft mice models. The molecular mechanisms were identified using RNA pull-down assays, RNA immunoprecipitation (RIP), Co-IP, ISH, Ubiquitination assay, bioinformatics analysis, and the rescue experiments.ResultsHere, we unveil that microtubule cross-linking factor 1 circRNA (circMTCL1, circ0000825) exerts its critical oncogenic functions by promoting complement C1q-binding protein (C1QBP)-dependent ubiquitin degradation and subsequently activating Wnt/β-catenin signalling in laryngeal carcinoma initiation and development. Specifically, circMTCL1 was remarkably up-regulated in the paired tissues of patients with LSCC (n = 67), which predicted a worse clinical outcome. Functionally, circMTCL1 exerted oncogenic biological charactersistics by promoting cell proliferative capability and invasive and migrative abilities. Ectopic circMTCL1 augumented cell proliferation, migration, and invasion of LSCC cells, and this effect could be reversed by C1QBP knocking down in vitro and in vivo. Mechanistically, circMTCL1 directly recruited C1QBP protein by harboring the specific recognized sequence (+ 159 − + 210), thereby accelerating the translation of C1QBP expression by inhibiting its ubiquitin–proteasome-mediated degradation. Importantly, the direct interaction of C1QBP with β-catenin protein was enhanced via suppressing the β-catenin phosphorylation and accelerating its accumulation in cytoplasm and nucleus.ConclusionOur findings manifested a novel circMTCL1-C1QBP-β-catenin signaling axis involving in LSCC tumorigenesis and progression, which shed new light on circRNAs-ubiquitous acidic glycoprotein mediated ubiquitin degradation and provided strategies and targets in the therapeutic intervention of LSCC.

Changes in beta-catenin expression and activation during progression of primary sclerosing cholangitis predict disease recurrence

Primary sclerosing cholangitis (PSC) is a rare, chronic, cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts. We have previously demonstrated the importance of Wnt/β-catenin signaling in mouse models of PSC. In this study, we wished to determine the clinical relevance of β-catenin localization in patient samples. In livers explanted from patients diagnosed with PSC, the majority (12/16; 75%) lacked β-catenin protein expression. Biopsies from patients post-transplant were classified as recurrent or non-recurrent based on pathology reports and then scored for β-catenin activation as a function of immunohistochemical localization. Despite lack of statistical significance, patients with recurrent primary disease (n = 11) had a greater percentage of samples with nuclear, transcriptionally active β-catenin (average 58.8%) than those with no recurrence (n = 10; 40.53%), while non-recurrence is correlated with β-catenin staining at the cell surface (average 52.63% for non-recurrent vs. 27.34% for recurrent), as determined by three different methods of analysis. β-catenin score and years-to-endpoint are both strongly associated with recurrence status (p = 0.017 and p = 0.00063, respectively). Finally, there was significant association between higher β-catenin score and increased alkaline phosphatase, a marker of biliary injury and disease progression. Thus, β-catenin expression and activation changes during the progression of PSC, and its localization may be a useful prognostic tool for predicting recurrence of this disease.

PATH-38. APC MUTATION AS A DRIVER ONCOGENE IN NON-CTNNB1 MUTANT ADAMANTINOMATOUS CRANIOPHARYNGIOMAS

Abstract Craniopharyngiomas arise from the embryonic remnants of Rathke’s pouch in the craniopharyngeal duct. Their invasive capacity and geographic proximity to the optic apparatus, pituitary gland and stalk, third ventricle, and the hypothalamus can cause progressive visual, hormonal, and neurological deficits and transform these lesions into behaviorally malignant tumors. The classic subtypes of craniopharyngiomas, adamantinomatous and papillary, have conventionally been approached similarly in regards to therapeutic strategies. However, recent exome sequencing studies have revealed mutually exclusive pathways for their genetic origins. Recurrent mutations in BRAF (V600E), a regulator of MAP kinase/ERK signaling pathway have been observed in 95% of papillary craniopharyngiomas and may be amenable to targeted therapies with BRAF inhibitors. However, CTNNB1 mutations are observed in only 75%–96% of adamantinomatous craniopharyngiomas, revealing a gap in the classic genetic-histological correlation. We describe a 74-year-old male who underwent resection of a craniopharyngioma with suprasellar and third ventricular involvement. The pathology demonstrated adamantinomatous craniopharyngioma with aberrant nuclear beta-catenin activity on immunohistochemistry. Whole-exome sequencing, performed in accordance with an institutional review board-approved protocol on the tumor and blood, revealed wildtype CTNNB1 but notably two somatic stop-codon mutations in APC (rs786201856:c.C793T:p.R265X and rs587779780:c.1159T:p.R387X), considered pathogenic variants in other reported cancer types. Loss of APC leads to constitutive activation of the Wnt signaling pathway, similar to the observed downstream effects of activating CTNNB1 mutations in adamantinomatous craniopharyngioma. Our case suggests that craniopharyngiomas may arise from somatic loss of APC, which previously was only described in a handful of individuals with familial adenomatous polyposis, harboring germline APC mutations. This report adds new insight into the pathogenesis of adamantinomatous craniopharyngiomas, particularly those that are otherwise wildtype for CTNNB1. Based on accumulating evidence, we propose that along with BRAF and CTNNB1, APC should also be routinely checked in cranipharyngiomas, especially if the former two have been negative.

LRP5-Mediated Lipid Uptake Modulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells.

Nutritional microenvironment determines the specification of progenitor cells, and lipid availability was found to modulate osteogenesis in skeletal progenitors. Here, we investigated the implications of lipid scarcity in the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) and the role of low-density lipoprotein receptor-related protein 5 (LRP5), a co-receptor transducing canonical Wnt/beta-catenin signals, in BMSC lipid uptake during osteogenesis. The osteogenic differentiation of murine BMSCs was suppressed by lipid scarcity and partially rescued by additional fatty acid treatment with oleate. The enhancement of osteogenesis by oleate was found to be dosage-dependent, along with the enhanced activation of beta-catenin and Wnt target genes. Conditional knockout (CKO) of Lrp5 gene in murine mesenchymal lineage using Lrp5fl/fl;Prrx1-cre mice led to decreased bone quality and altered fat distribution in vivo. After Lrp5 ablation using adenoviral Cre-recombinase, the accumulation of lipid droplets in BMSC cytoplasm was significantly reduced, and the osteogenesis of BMSCs was suppressed. Moreover, the impaired osteogenesis due to either lipid scarcity or Lrp5 ablation could be rescued by recombinant Wnt3a protein, indicating that the osteogenesis induced by Wnt/beta-catenin signaling was independent of LRP5-mediated lipid uptake. In conclusion, lipid scarcity suppresses BMSC osteogenic differentiation. LRP5 plays a role in the uptake of lipids in BMSCs and therefore mediates osteogenic specification.