Beta-catenin Expression Research Articles

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

SummaryEstrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

Wingless ligands and beta-catenin expression in the rat endometrium: The role of Wnt3 and Wnt7a/beta-catenin pathway at the embryo-uterine interface.

Wnt/beta-catenin signaling may play an essential role in endometrial decidualization, placentation, and the establishment of pregnancy. We investigate here the possible roles, immunolocalizations, and synthesis of the Wnt3, Wnt7a, and beta-catenin proteins in the rat endometrium during the estrous cycle and early postimplantation period. Wnt3 and Wnt7a had a similar localization and dynamic expression relative to the endometrial stages. Wnt7a immunostaining was not limited only to the luminal epithelial cells, but also to strong stainings in the stromal and endothelial cells. Wnt3, Wnt7a, and beta-catenin were highly synthesized and colocalized at the trophoblast-decidual interface; and were more obvious in the primary decidual zone, the GTCs, and the ectoplacental cone. Beta-catenin was strongly localized at the borders of the mature decidual cells; however, Wnt3 and Wnt7a immunolocalizations were decreased in those cells. As such, the immunolocalization of Wnt3, Wnt7a, and beta-catenin shifted with decidualization and placentation. The expression level of Wnt3, Wnt7a, and beta-catenin messenger RNAs increased in early pregnancy, and especially between Days 8.5 and 9.5. The dramatic changes in the expression of Wnt3, Wnt7a, and beta-catenin observed during the early days of pregnancy and the estrous cycle may indicate their roles in decidualization, stromal cell proliferation, and trophoblast invasion.

Nuclear Beta-Catenin Expression in Endometrioid Intraepithelial Neoplasia (Atypical Hyperplasia) Does Not Predict Carcinoma on Subsequent Hysterectomy

Beta-catenin (BC) mutations are associated with a high risk of recurrence in otherwise low-grade, early-stage uterine endometrioid adenocarcinomas. Recent literature suggests nuclear BC expression by immunohistochemistry is highly sensitive and specific for BC mutations. The significance of BC expression in endometrioid intraepithelial neoplasia (EIN/atypical hyperplasia) and its relationship to altered differentiation patterns in EIN has yet to be fully explored. Cases meeting current diagnostic criteria for EIN based on H&E examination were obtained from 2 institutions (years 1999-2014). Patterns of altered differentiation (eg, tubal, squamous morular metaplasia, mucinous, secretory) were noted. Representative blocks were stained for BC, and expression patterns recorded. Follow-up and demographic data was obtained from the electronic medical record. Ninety-six cases were included (84 biopsies, 12 hysterectomies). BC nuclear expression was identified in 41 cases (42.7%), with 33 of 41 demonstrating foci of nonmorular BC staining. BC staining in any component of EIN was not significantly associated with the presence of carcinoma on subsequent hysterectomy (P=0.79). When restricting to nonmorular BC, the results were the same (P=0.56). Cases with tubal differentiation were significantly less likely to demonstrate nonmorular BC than cases with no specific pattern of differentiation (P<0.01). EIN frequently demonstrates BC nuclear positivity, especially in cases without tubal differentiation. BC nuclear expression in EIN does not appear to be associated with an increased likelihood of carcinoma on subsequent hysterectomy. Our results do not support routine use of BC immunohistochemistry as a prognostic biomarker in cases of EIN.

Abstract LB-203: The mRNA binding protein Human Antigen R (HuR, ELAVL1) is a novel regulator in pancreatic ductal adenocarcinoma metastatic progression and persistence

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death with a five-year survival rate of 10%. Yet, for the majority of patients who present with metastatic burden (50-60%), this rate drops to a mere 3%. Systemic chemotherapeutic regimens such as FOLFIRINOX or gemcitabine abraxane, have only shown marginal efficacy in delaying tumor growth and metastatic progression. Thus, there is a dire need for more robust treatment options to effectively target metastatic PDAC. Work by Iacobuzio-Donahue et al. and others have interrogated genetic drivers of patient metastases. They have demonstrated through genomic sequencing of matched primary and metastatic tumors that no single genetic event can account for driving metastasis. Therefore, metastatic progression is most likely driven by mechanisms beyond genomic mutations. Our lab has characterized one such alternative, the RNA binding protein Human Antigen R (HuR). HuR is a major regulator of pleiotropic gene regulation in response to demands from the pancreatic tumor microenvironment (e.g. nutrient deprivation, hypoxia). HuR generally stabilizes and enhances the translation of pro-survival mRNA transcripts (e.g., PIM1, IDH1, WEE1, DCK, PARG, etc.). Outside of this role, published data has implicated HuR to regulate epithelial-to-mesenchymal transition diving genes (e.g. SNAI1, PLAU, CTTNB1, MMP9) necessary for migration and invasion. We have been able to demonstrate that HuR is important for the migration and invasion of PDAC cells, in vitro. Through use of short hairpin RNA and CRISPR technology, inhibition of HuR decreased the ability of PDAC cells to migrate and invade in a transwell assay. Additionally, both knockdown and knockout of HuR slowed wound healing and colony formation. To evaluate the role of HuR in metastasis in vivo, we utilized a luciferase-expressing humanized PDAC orthotopic model to monitor tumor growth and spread. Either parental or HuR knockout luciferase-expressing PDAC cells were injected into the tail of the pancreas of NRG mice. A month after tumor engraftment, the majority of mice bearing parental tumors harbored liver and lung metastases (liver-5/5, lung 4/5) while HuR knockout tumor-bearing mice showed little to no evidence of metastasis (liver 2/4, lung 0/4). Employing ribonucleoprotein immunoprecipitation assays identified metastatic-associated mRNA transcripts bound to HuR (i.e., CTNNB1 (beta-catenin) and YAP1 mRNAs). Validation of these findings demonstrated that silencing or knocking out HuR induces a robust loss of beta-catenin and YAP1 expression (p&amp;lt;0.001). Ongoing work will further uncover known and novel pathways that HuR is modulating for the pro-metastatic phenotype often seen in PDAC. Taken together, these data supports our continuing work of developing novel therapeutic strategies to target HuR in PDAC. Citation Format: Grace A. McCarthy, Aditi Jain, Samantha Z. Brown, Charles J. Yeo, Jonathan Brody. The mRNA binding protein Human Antigen R (HuR, ELAVL1) is a novel regulator in pancreatic ductal adenocarcinoma metastatic progression and persistence [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-203.

Abstract 4023: Elevating the reactive oxygen species threshold in prostate cancer stem cells using VK3-OCH3 as a strategy to target prostate cancer

Abstract Prostate cancer (PCa) is the second most leading cause of cancer related deaths in American men. Although several cancer treatments are available, these have a number of drawbacks and can leave the patient with cardiotoxicity, loss of libido and infertility. Androgen deprivation therapy (ADT) is a standard promising therapy for metastatic PCa patients. However, patients within 18-24 months suffer from relapse and progress into castration resistant prostate cancer (CRPC). Experimental evidences suggest that, cancer stem cells (CSC) which constitute as a subpopulation of cancer cells, play a pivotal role in carcinogenesis, metastasis and chemoresistance to therapies. Therefore, targeting CSC can decrease the PCa recurrence rates and can help to offer better treatment options. Elevating the reactive oxygen species (ROS) threshold has been shown to be an effective strategy to eliminate CSC. Hence, in the present study, we hypothesized that VK3-OCH3, a menadione derivative, as a potential ROS enhancer in prostate cancer stem cell line (PCSC). After conducting a cell viability assay with different vitamin K (VK) analogs, our data revealed that VK3-OCH3 was more effective against PCSC compared to other VK analogs. Subsequently, colony formation assay and Trans-well migration assays revealed that VK3-OCH3 exhibits anti-tumorigenic and anti-metastatic properties against PCSC. Further studies showed that antioxidants such as NAC and GSH antagonized the effects of VK3-OCH3 on PCSC survival inhibition, suggesting the activation of oxidative stress by VK3-OCH3 in PCSC. In addition, our data revealed that VK3-OCH3 imparts oxidative stress by increasing the ROS levels which were determined using a ROS probe (DCFDA) by fluorescence analysis. VK3-OCH3 treatment also induced marked elevations in mitochondrial ROS generation in PCSC by FACS analysis which correlated with significant apoptotic cell death in these cells. Furthermore, we tested the effect of VK3-OCH3 on cell cycle progression using FACS and observed a significant G0 cell cycle arrest. By using an oncogene array, it was found that survivin, an apoptotic inhibitor, was downregulated by VK3-OCH3, which was further confirmed by Western blot analysis. Our subsequent confocal studies showed that, VK3-OCH3 downregulates the expression levels of EMT markers such as vimentin and TCF-8. Real time data also revealed that there is a modulation in gene expression of beta-catenin upon VK3-OCH3 treatment in PCSC. Of note, VK3-OCH3 treated PCSCs had a significant reduction in stem cell markers such as CD44 and ALDH1. In conclusion, our results suggest that VK3-OCH3 could be used as a potential ROS targeted anti-CSC strategy in treating PCa. Citation Format: Mona Vudutha, Somaiah Chinnapaka, Pranav Volety, Gnanasekar Munirathinam. Elevating the reactive oxygen species threshold in prostate cancer stem cells using VK3-OCH3 as a strategy to target prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4023.

The symbiotic supplementation modulate gut microbiota, regulation beta-catenin expression and prevents weight gain in ob/ob mice with non-alcoholic fatty liver disease (NAFLD)

The symbiotic supplementation modulate gut microbiota, regulation beta-catenin expression and prevents weight gain in ob/ob mice with non-alcoholic fatty liver disease (NAFLD)

Abstract B40: EWS-FLI1 orchestrates Ewing sarcoma plasticity through a post-translational modification cascade regulating FOXM1 stability

Abstract Oscillations between proliferative and migratory/invasive cell states are at the core of the metastatic process. Transition between these states involves reversible shifts in transcriptional programs that orchestrate cytoskeletal remodeling. Ewing sarcoma pathogenesis depends on EWS-FLI1, an aberrant ETS transcription factor that drives cellular transformation and proliferation of presumably mesenchymal progenitor cells. Recently, it has been reported that transient modulation of EWS-FLI1 expression results in an epithelial-to-mesenchymal transition-like phenotype of Ewing sarcoma cells and in increased metastasis (Chaturvedi et al., 2014; Franzetti et al., 2016). It has been proposed that EWS-FLI1 low cells exist in small amounts in primary Ewing sarcoma tumors and thus provide a potential source for tumor dissemination, leading to adverse prognosis. In our studies, we investigate the mechanistic basis of EWS-FLI1 dose-dependent Ewing sarcoma plasticity and recently reported on the activation of an MRTFB/TEAD transcriptional module promoting cytoskeletal reprograming in response to low EWS-FLI1 levels (Katschnig et al., 2016). Here, we show that the proliferative state depends on the activity of nuclear beta-catenin complexed to FOXM1. We find that EWS-FLI1 not only drives FOXM1 expression on the RNA level, but also sustains nuclear FOXM1 stability by a mechanism involving FOXM1 protein demethylation by lysine-specific demethylase 1 (LSD1/KDM1A). Experimental evidence suggests that EWS-FLI1 regulates LSD1 protein stability in Ewing sarcoma cells by directly activating a ubiquitin-specific protease. Modulation of EWS-FLI1 led to loss of expression of the deubiquitinting enzyme (DUB), and consequently to rapid depletion of LSD1 and FOXM1 proteins and subcellular redistribution of beta-catenin to the cytoplasm. Genetic (siRNA) and pharmacologic inhibition of the DUB or LSD1 recapitulated the destabilizing effect on FOXM1 in EWS-FLI1-high Ewing sarcoma cells, blocking tumor cell proliferation. In addition, they led to complete loss of beta-catenin expression by a mechanism that is currently under investigation. Therefore, our study identifies targetable enzymatic activities downstream of EWS-FLI1 that are required to sustain Ewing sarcoma proliferation. Citation Format: Jozef Ban, Dave Aryee, Lisa Bierbaumer, Heinrich Kovar. EWS-FLI1 orchestrates Ewing sarcoma plasticity through a post-translational modification cascade regulating FOXM1 stability [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B40.

Potential Anticancer Activity of Crude Ethanol, Ethyl Acetate, and Water Extracts of Ephedra foeminea on Human Osteosarcoma U2OS Cell Viability and Migration.

Medicinal plants are potential sources for a wide range of complex compounds with probable anticancer activity. Ephedra foeminea Forssk. (E. foeminea), a medicinal plant found in the Eastern Mediterranean, has recently been gaining popularity as a cancer remedy; there is, however, a paucity of empirical evidence supporting this claim. In this study, the effect of E. foeminea ethyl acetate, ethanol, and water crude extracts on viability, migratory ability, and the steady-state mRNA levels of genes involved in these processes was, respectively, examined using MTT assay, wound healing assay, and reverse transcriptase PCR (RT-PCR). The study concludes that all extracts significantly reduce human osteosarcoma U2OS percentage viability in a dose- and time-dependent manner, with varying potencies. The least half-maximal inhibitory concentration (IC50) was observed in the water extract after 48 h incubation (30.761 ± 1.4 μg/mL) followed by the ethyl acetate extract after 72 h incubation (80.35 ± 1.233 μg/mL) and finally the ethanol extract after 48 h incubation (97.499 ± 1.188 μg/mL). Ethanol extract significantly reduced U2OS percentage wound closure. On the other hand, both ethanol and water extracts considerably reduced the steady-state mRNA expression of beta-catenin, promoting both cell proliferation and migration in osteosarcoma by regulating target genes. Additionally, E. foeminea showed no hemolytic activity. These effects suggest that E. foeminea decreases U2OS cell viability and migratory ability by modulating the expression of critical genes involved in regulating these processes and is likely cytocompatible with human erythrocytes.

Overexpression of EIF5A2 Predicts Poor Prognosis in Patients with Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common epithelial malignancy affecting the oral cavity, and it is especially significant in Asian countries. Patients diagnosed with OSCC have an unfavorable prognosis and additional prognostic markers would help improve therapeutic strategies. We sought to investigate the association between eukaryotic translation initiation factor 5A2 (EIF5A2) and epithelial–mesenchymal transition (EMT) markers as well as the prognostic significance of EIF5A2 in OSCC. The expression of EIF5A2 and EMT markers was measured through the immunohistochemical staining of specimens from 272 patients with OSCC. In addition, the correlation between different clinicopathological factors and EIF5A2 expression was analyzed. The prognostic role of EIF5A2 was then analyzed via Kaplan–Meier analysis and Cox proportional hazard models. Among the 272 patients, high EIF5A2 expression was significantly associated with an advanced N value (p = 0.008). High tumor expression of EIF5A2 was prone to the expression of low E-cadherin and high beta-catenin (p = 0.046 and p = 0.020, respectively). Patients with high EIF5A2 expression had unfavorable five-year survival rates as compared with those with low expression (49.7% and 67.3%, respectively). The prognostic role of EIF5A2 was further confirmed through multivariate analysis (hazard ratio = 1.714, 95% confidence interval: 1.134–2.590, p = 0.011). High EIF5A2 expression is associated with an advanced N value and EMT markers and may serve as a marker for an unfavorable prognosis in patients with OSCC.

Quercetin treatment reduces the severity of renal dysplasia in a beta-catenin dependent manner.

Renal dysplasia, the major cause of childhood renal failure, is characterized by defective branching morphogenesis and nephrogenesis. Beta-catenin, a transcription factor and cell adhesion molecule, is markedly increased in the nucleus of kidney cells in human renal dysplasia and contributes to its pathogenesis by altering target genes that are essential for kidney development. Quercetin, a naturally occurring flavonoid, reduces nuclear beta-catenin levels and reduces beta-catenin transcriptional activity. In this study, we utilized wild type and dysplastic mouse kidney organ explants to determine if quercetin reduces beta-catenin activity during kidney development and whether it improves the severity of renal dysplasia. In wild type kidney explants, quercetin treatment resulted in abnormal branching morphogenesis and nephrogenesis in a dose dependent manner. In wild type embryonic kidneys, quercetin reduced nuclear beta-catenin expression and decreased expression of beta-catenin target genes Pax2, Six2, and Gdnf, which are essential for kidney development. Our RDB mouse model of renal dysplasia recapitulates the overexpression of beta-catenin and histopathological changes observed in human renal dysplasia. RDB kidneys treated with quercetin resulted in improvements in the overall histopathology, tissue organization, ureteric branching morphogenesis, and nephrogenesis. Quercetin treatment also resulted in reduced nuclear beta-catenin and reduced Pax2 expression. These improvements were associated with the proper organization of vimentin, NCAM, and E-cadherin, and a 45% increase in the number of developing and maturing nephrons. Further, our results show that in human renal dysplasia, beta-catenin, vimentin, and e-cadherin also have abnormal expression patterns. Taken together, these data demonstrate that quercetin treatment reduces nuclear beta-catenin and this is associated with improved epithelial organization of developing nephrons, resulting in increased developing nephrons and a partial rescue of renal dysplasia.

4530 Elucidating the Influence of Chemotherapy (melphalan) and /or C. difficile toxin B Exposure on Beta-catenin Protein Expression in Caco-2 Monolayers

OBJECTIVES/GOALS: We previously reported that genetic polymorphisms in the beta-catenin gene (CTNNB) are associated with the development of Clostridiodes difficile colitis during autologous stem cell transplantation (https://www-ncbi-nlm-nih-gov.proxy.libraries.uc.edu/pubmed/29594489). To biological validate these findings, we sought to evaluate the development of chemotherapy-associated Clostridiodes difficile infections by assessing the effect of C.difficile toxin B (TcdB) and of using melphalan in beta-catenin protein expression in Caco2 cells. METHODS/STUDY POPULATION: To determine the effect of melphalan and/or C.difficile toxin B on expression of Beta-catenin from human gut epithelial cells: Adenocarcinoma cells (Caco-2) cells were seeded and allowed to grow into monolayersMonolayers were treated with PBS, TcdB, melphalan and/or TcdB + melphalan for 24 hours and then washed with PBSImmunofluorescence was measured on the monolayers to visualize three markers -DAPI-Nuclear Stain (blue),Actin-ccytoskeletal stain (red), B-Catenin (green)Analysis of images with ImageJ (NIH). Statistical analysis of the effect of TcdB and/or melphalan on β-catenin protein levels was determined by One-way ANOVA Cells stained with a primary anti-β catenin antibody and an Alexa-488 secondary antibody were evaluated by flow cytometry to quantify the effect of melphalan and/or C. difficile toxin B on Caco2 cells. RESULTS/ANTICIPATED RESULTS: Immunofluorescent intensity was higher in the control (PSS exposed) cells when compared to melphalan, TcdB and mephalan+TcdB exposed cells (p = 0.026, 0.004 and 0.049 respectively) DISCUSSION/SIGNIFICANCE OF IMPACT: A significant difference was seen in β catenin expression in Caco-2 monolayers exposed to TcdB and/or melphalan. These data support the a role of β-catenin in the pathophysiology of CDI during chemotherapy and support GWAS findings reporting a difference in CDI susceptibility based on β-catenin genotype.

Intracellular localization of beta-catenin expression plays a possible prognostic role on the outcome of hepatoblastoma patients.

Wnt/Beta-catenin pathway plays an essential role in liver development and regeneration. Abnormal activation in this pathway leads to development of hepatoblastoma (HB). Although its importance has invoked attention, its prognostic role is debatable. We aimed to evaluate the significance of intracellular localization of beta-catenin (BC) expression in the outcome of hepatoblastoma patients. Medical records of HB patients between 2004 and 2018 were reviewed. Patients were grouped according to intracellular localization of BC expression by immunohistochemistry as being cytoplasmic or nuclear. Demographics, radiological images, PRETEXT classifications, vascular involvement, risk groups, chemotherapy responses, and survival rates were analyzed and compared between groups. There were 41 patients. Thirteen patients were excluded for unavailability of records in four, negative/unclear BC expressions in seven. Cytoplasmic expression of BC was observed in 17 patients whereas 13 patients displayed nuclear expression. Demographics were similar in both groups. Cytoplasmic BC expression was associated with poor chemotherapy response (p = 0.001) and increased vascular involvement (p = 0.0162) requiring more extensive surgeries (p = 0.039). Although the numbers are limited in our series, the intracellular localization of BC expression has been found to be a promising determining factor for hepatoblastoma prognosis. With larger patient series, more reliable results can be achieved.

Downregulation of beta-catenin in chemo-tolerant TNBC through changes in receptor and antagonist profiles of the WNT pathway: Clinical and prognostic implications.

In approximately 30% of triple-negative breast cancer (TNBC) patients a complete pathological response is achieved. However, after neo-adjuvant chemotherapy treatment (NACT) residual tumour cells can be intrinsically resistant to chemotherapy. In this study, associations of the WNT/beta-catenin pathway with chemo-tolerance of NACT treated TNBC patients were compared to that of pre-treatment TNBC patients. Expression analyses were performed in both pre-treatment and NACT treated TNBC samples using immunohistochemistry and qRT-PCR, along with DNA copy number variation (CNV) and promoter methylation analyses to elucidate the mechanism(s) underlying chemo-tolerance. In addition, in vitro validation experiments were performed in TNBC cells followed by in vivo clinicopathological correlation analyses. A reduced expression (41.1%) of nuclear beta-catenin together with a low proliferation index was observed in NACT samples, whereas a high expression (59.0%) was observed in pre-treatment samples. The reduced nuclear expression of beta-catenin in the NACT samples showed concordance with reduced expression levels (47-52.9%) of its associated receptors (FZD7 and LRP6) and increased expression levels (35.2-41.1%) of its antagonists (SFRP1, SFRP2, DKK1) compared to those in the pre-treatment samples. The expression levels of the receptors showed no concordance with its respective gene copy number/mRNA expression statuses, regardless treatment. Interestingly, however, significant increases in promoter hypomethylation of the antagonists were observed in the NACT samples compared to the pre-treatment samples. Similar expression patterns of the antagonists, receptors and beta-catenin were observed in the TNBC-derived cell line MDA-MB-231 using the anthracyclines doxorubicin and nogalamycin, suggesting the importance of promoter hypomethylation in chemotolerance. NACT patients showing reduced receptor and/or beta-catenin expression levels and high antagonist expression levels exhibited a comparatively better prognosis than the pre-treatment patients. Our data suggest that reduced nuclear expression of beta-catenin in NACT TNBC samples, due to downregulation of its receptors and upregulation of its antagonists through promoter hypomethylation of the WNT pathway, plays an important role in chemo-tolerance.

Identifying Best Target Pocket for Small Molecule Inhibition of Beta Catenin/E‐Cadherin Binding

The Wnt/beta catenin signaling pathway has been linked to the growth of certain types of cancer cells secondary to an uncontrolled increased expression of beta catenin. Finding a molecule that can inhibit the activity of beta catenin and reduce its concentration in cells could treat cancers involving this pathway. E‐cadherin is a protein involved in maintaining the integrity and adhesiveness of a cell. Disrupting the interaction between E‐cadherin and beta catenin may inhibit beta catenin expression, which may reduce the survivability of the cancer cell. In this study, we aimed to find the best target pocket for beta catenin/E‐cadherin binding based on large volume and high “druggability” score. The “druggability” score measures the binding potential of small molecules to the pocket of interest. We analyze the pocket of interest in both the monomer (beta catenin) and the complex (beta catenin with E‐cadherin bound) in order to identify the best target for small molecules. We hypothesize that the pocket volume and “druggability” score will increase when comparing the pocket of interest in the monomer versus the complex. First, we used the software PyMol and PocketQuery to search for key residues in the E‐cadherin and beta catenin interaction. The residues chosen were the amino acids Leucine 661 and Aspartate 665. Then, we used the website Pharmit to add these residues as features to the beta catenin receptor to screen for potential molecules that may bind using these residues. The pocket that these molecules were bound to was the pocket we chose to focus on. In order to eventually obtain more potential molecules, we ran a pocket volume analysis using the software MDPocket to see if the pocket volume fluctuates over time in both the monomer and the complex. We also used the software Fpocket to calculate a “druggability score” for both the monomer and the complex as the more “druggable” a pocket is, the better target it is. The results showed that the volume fluctuates over time in the monomer and the complex, with the complex having overall larger volumes. Furthermore, the highest “druggability” scores from a scale of −1.0 (“undruggable”) to +1.0 (most “druggable”) in the monomer and complex were 0.601 and 0.984, respectively. With these results, we conclude that the complex may be a better target for future analysis as the properties of having both a larger pocket volume and a higher “druggability” score correlates with an increased chance for a small molecule to bind.

O30 Long non-coding RNA HOTAIR induces beta catenin expression in systemic sclerosis through EZH2 dependent repression of Axin-2

Abstract Background Fibroblasts explanted from affected tissues in systemic sclerosis (SSc) maintain their pro-fibrotic phenotype in vitro. This includes increased secretion of collagens and other extracellular matrix proteins and increased proportion of α-Smooth Muscle Actin (α-SMA) positive cells (myofibroblasts). It has been previously shown that among their profibrotic features, myofibroblasts display activation of WNT signalling, which is linked to a decreased basal expression of AXIN2. Here we aimed to determine whether specific long non-coding RNA (lncRNAs) expressed in myofibroblasts could drive the epigenetically stable decreased expression of Axin 2 in vitro. Methods Full thickness skin biopsies were surgically obtained from the forearms of twelve adult patients with SSc of recent onset. Fibroblasts were isolated and cultured in monolayers and protein and RNA extracted from the fibroblast cultures. Laser capture was performed to isolate cells expressing or not α-SMA as a marker of myofibroblast differentiation. LncRNA HOTAIR was overexpressed in healthy dermal fibroblasts by lentiviral induction. EZH2 was blocked in cultured fibroblasts with the specific inhibitor GSK126. Results HOTAIR expression was increased in SSc patients’ skin (100 fold) and in SSc explanted fibroblasts (5 fold; p &amp;lt; 0.001 for both). Further, laser captured α-SMA expressing fibroblasts expressed in average 2.5 fold higher HOTAIR RNA levels compared to α -SMA negative cells from the same donors (P &amp;lt; 0.05). In vitro, lentiviral induced stable overexpression of HOTAIR in healthy dermal fibroblasts led to their profibrotic activation, including significantly increased expression of Col1A1 and α-SMA both at mRNA and protein levels (2.8 and 1.8 fold respectively, p &amp;lt; 0.05). We further showed that HOTAIR-induced profibrotic activation was due to EZH2 dependent spread of H3k27me3 methylation marker, as demonstrated by complete inhibition by treatment with GSK126. HOTAIR driven EZH2 histone methylation suppressed the expression of Axin 2 in SSc fibroblasts. The reduced Axin 2 levels led to stabilisation of beta catenin and WNT signalling pathway activation. Conclusion Here we show that the epigenetically stable activation of SSc dermal fibroblasts is due to HOTAIR. We also identified a major downstream target of HOTAIR is Axin-2. The results of these studies identify a new venue to modulate fibroblasts biology which could inform clinical research to resolve chronic fibrosis and re-establish tissue homeostasis in SSc. Disclosures C.W. Wasson None. G. Abignano None. R. Ross None. F. Del Galdo Consultancies; AstraZeneca, GSK, Boehringer-Ingelheim, Actelion, Capella Biosciences, Chemomab.

Combined hepatoblastoma and yolk sac tumor of the liver

A 6-year-old patient was admitted to the Petrovsky National Research Center of Surgery in Moscow, diagnosed with hepatoblastoma (HB) of the right lobe of the liver and after undergoing 4 cycles of SIOPEL (super PLADO) pre-operative chemotherapy. An immunohistochemistry test, performed after liver resection, revealed a rare combination of mixed epithelial and mesenchymal hepatoblastoma and yolk sac tumor. The epithelial component of HB consisted of highly differentiated fetal cells, while the mesenchymal component consisted of osteoid. The morphological structure of the second lesion corresponded to yolk sac tumor. Both tumors had regressive changes conditioned by chemotherapy: these changes were most pronounced in HB. In the highly differentiated fetal component of HB it was possible to detect a weak focal perinuclear alpha-fetoprotein expression, HepPar expression, membranous expression of beta-catenin and diffuse glutamine synthetase expression. Mitotic figures were not determined. The node of the yolk sac tumor had the characteristic histological structure of an endodermal sinus tumor with a hepatoid component. In the main tumor node and its nodular elements in the fibrovascular stroma it was possible to identify alpha-fetoprotein expression, CD34, nuclear and cytoplasmic expression of beta-catenin, CDX2, as well as a rare expression of PLAP and multi-cytokeratin, and mitotic activity was high (21 in 10 high power fields, 400x magnification). In the existing literature available to us, there is only one observation of combined hepatoblastoma and yolk sac tumor. Parents patients agreed to use personal data, including the fotos, in research and publications.

Evidence for a Novel Endometrioid Carcinogenic Sequence in the Fallopian Tube With Unique Beta-Catenin Expression.

Epithelial proliferations in the fallopian tube have been characterized by some as stem cell outgrowths (SCOUTs) and divided into type I and type II. Type II SCOUTs exhibit diffuse cellular beta-catenin nuclear staining (β-catenin), implying a CTNNB1 mutation. SCOUTs are more common in perimenopausal and postmenopausal women and are associated with ovarian cancer but have not been linked directly to malignancy. We analyzed type II SCOUTs in various gynecologic conditions, and searched for endometrioid atypical hyperplasias (tubal endometrioid intraepithelial neoplasia) or adenocarcinomas in the tube. β-catenin SCOUT frequency in cases of neoplasia was 66.7% per case and 30.7% per nonfimbrial cross-section for uterine endometrioid carcinomas versus 25% and 13.3% for controls, respectively (P=0.02 and 0.09). Multiple (3 or more) β-catenin SCOUTs in a single section were uncommon; 6 of 9 were associated with a carcinoma or proliferative lesion in the endometrium. Tubal endometrioid intraepithelial neoplasia/atypical hyperplasia displayed complex growth, including focal cribriform growth patterns and squamous morules. Two cases of type II SCOUTs associated with tubal endometrioid intraepithelial neoplasia/atypical hyperplasia and/or adenocarcinomas in the fallopian tube were identified, both of which coexisted with a separate endometrioid adenocarcinoma, one with bilateral ovarian endometrioid adenocarcinomas. Both benign and neoplastic tubal lesions were β-catenin. This report is the first to link components of a unique β-catenin endometrioid carcinogenic sequence in the fallopian tube. It further emphasizes the multifocal nature of endometrioid neoplasia in the female genital tract and poses questions regarding the frequency and biologic underpinnings of β-catenin proliferations in the oviduct.

Bone Morphogenetic Protein 1 Targeting COL1A1 and COL1A2 to Regulate the Epithelial-Mesenchymal Transition Process of Colon Cancer SW620 Cells.

Epithelial-mesenchymal transition (EMT) is an important factor in promoting the metastasis of colon cancer, which leads to clinical incurability. It has been found that bone morphogenetic proteins (BMPs) are closely related to EMT and the prognoses of most malignant tumors, including colon cancer tumors. However, the effects and mechanisms of BMP1 on the EMT of colon cancer are not yet clear. To explore the effects and mechanisms of BMP1 on the EMT of colon cancer, a BMP1 overexpression plasmid vector was used to interfere with SW620 cells and real-time fluorescence quantitative RNA and western blotting were used to detect the effects of BMP1 on the transcription and translation of COL1A1 and COL1A2 genes, as well as EMT-related genes, including beta-catenin, vimentin, and E-cadherin (E-Cad) genes in SW620 cells. MTT assay and Transwell techniques were used to detect the effects of BMP1 on the proliferation and migration of SW620 cells. The results demonstrate that BMP1 expression in SW620 cells is significantly lower than that in HCoEpiC cells, which promotes the expression of COL1A1 and COL1A2. Additionally, the expression of genes related to EMT, including beta-catenin and vimentin, increased, whereas E-Cad expression decreased. This difference was significant, which led to an increase in cell viability and the number of migrating cells in SW620 cells. Based on the overexpression of BMP1, the expression of COL1A1 and COL1A2 in SW620 was inhibited, which inhibited the process of EMT. Specifically, vimentin expression decreased, and E-Cad and beta-catenin expression increased. Additionally, SW620 cell viability decreased and migration ability decreased. Therefore, it can be concluded that the absence of BMP1 promotes the expression of COL1A and COL1A2 in colon cancer and promotes the process of EMT. Increasing the expression of BMP1 can inhibit the process of EMT in colon cancer, thereby inhibiting the migration of tumors.

USP9X promotes the progression of hepatocellular carcinoma by regulating beta-catenin

Hepatocellular carcinoma (HCC) is among the malignant tumors with highest mortality. The role of USP9X in the carcinogenesis of HCC has not yet been determined. In this study, USP9X was found significantly highly expressed in the intratumor tissues. Expression of intratumor USP9X was associated with tumor size and microvascular invasion while USP9X is independent risk factor of HCC disease-free survival and overall survival. In vitro studies revealed that knockdown of USP9X significantly inhibited the proliferation of HCC cells. Mechanically, USP9X promotes HCC cell proliferation by regulating the expression of beta-catenin. The results of the present study demonstrated that high expression of USP9X in intratumoral cells is associated with poor HCC prognosis, which may serve as a potential target for an adjuvant therapy.

Association of different patterns of expression of beta-catenin and cyclin D1 with pathogenesis of breast carcinoma.

Beta-catenin and cyclin D1 have attracted considerable attention in recent studies as potential proto-oncogenes in many human cancers especially colonic cancer. Beta-catenin plays multiple roles within the cell such as canonical Wnt signaling where cyclin D1 has been identified as one of its target genes. The role of beta-catenin and cyclin D1 in breast cancer has been evaluated in many studies but not established yet. The expression of beta-catenin and cyclin D1 was evaluated in 82 cases of breast carcinoma (BCa) and 32 cases of ductal carcinoma in situ(DCIS) by immunohistochemistry (IHC). Their relationship with clinicopathological features was also investigated. Statistical analysis was done to establish an association. Abnormal expression of beta-catenin (ABE) was seen in 80.2% cases of invasive ductal carcinoma (IDC) and 47% cases of DCIS, while the cyclin D1 positive expression rate was 60.9% and 50%, respectively. In the cases showing ABE, cyclin D1 positivity was 88.1%. ABE showed significant association with high-grade BCa. The most common pattern of ABE was loss of membrane with nuclear positivity which is associated with worst prognosis. In addition, ABE in cases of BCa and DCIS showed concordant patterns. Therefore, an association exists between ABE and cyclin D1 in BCa and its precursor lesions implying that Wnt/beta-catenin oncogenic pathway may have a definite role in breast carcinogenesis and can be used for targeted therapy. Also, different patterns of beta-catenin expression may have prognostic and predictive value.