Overexpression Of Beta-catenin Research Articles

Baf-mediated transcriptional regulation of teashirt is essential for the development of neural progenitor cell lineages

Accumulating evidence hints heterochromatin anchoring to the inner nuclear membrane as an upstream regulatory process of gene expression. Given that the formation of neural progenitor cell lineages and the subsequent maintenance of postmitotic neuronal cell identity critically rely on transcriptional regulation, it seems possible that the development of neuronal cells is influenced by cell type-specific and/or context-dependent programmed regulation of heterochromatin anchoring. Here, we explored this possibility by genetically disrupting the evolutionarily conserved barrier-to-autointegration factor (Baf) in the Drosophila nervous system. Through single-cell RNA sequencing, we demonstrated that Baf knockdown induces prominent transcriptomic changes, particularly in type I neuroblasts. Among the differentially expressed genes, our genetic analyses identified teashirt (tsh), a transcription factor that interacts with beta-catenin, to be closely associated with Baf knockdown-induced phenotypes that were suppressed by the overexpression of tsh or beta-catenin. We also found that Baf and tsh colocalized in a region adjacent to heterochromatin in type I NBs. Notably, the subnuclear localization pattern remained unchanged when one of these two proteins was knocked down, indicating that both proteins contribute to the anchoring of heterochromatin to the inner nuclear membrane. Overall, this study reveals that the Baf-mediated transcriptional regulation of teashirt is a novel molecular mechanism that regulates the development of neural progenitor cell lineages.

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.

Immunohistochemical appraisal of epithelial mesenchymal transition type III in gall bladder cancer.

Epithelial-mesenchymal transition (EMT) is the heart of invasion. EMT associated with cancer progression and metastasis is known as type III EMT. Beta-catenin, E-cadherin, and MMP9 markers of EMT are routinely employed for diagnostic purposes. We employed these markers to study EMT by immunohistochemistry (IHC) in gall bladder cancer (GBC) with respect to depth of tumor invasion, clinical outcome, and disease-free survival. This was a prospective case-control study. Seventy gall bladders were included (50 GBC and 20 CC). After detailed histology, immunoexpression was studied in terms of percentage and strength of expression. Expression was compared between CC and GBC by Student t test and analysis of variance. Kaplan-Meier was used for survival analysis, and the extent of agreement ("Kappa") was calculated. The age of incidence of GBC was 49.40 (+11.6) years with female predominance (F:M = 4:1). In 88% (44/50) of GBC, the fundus was involved. Moderately differentiated adenocarcinoma was most frequent [54%; 27/50]. Significant downregulation of E-cadherin (P = 0.022) and beta-catenin (P < 0.001) and upregulation in MMP9 (P < 0.001) were seen in GBC with respect to CC with significant association among them. MMP9 expression was significantly associated with higher tumor stage but with chemotherapeutic response. Our results display that epithelial-mesenchymal transition type III plays a role in GBC invasion. MMP9 overexpression and loss of membranous beta-catenin may be considered a marker for poor clinical outcomes and advanced disease.

Overexpression of MicroRNA-340-5p Attenuates Lung Cancer Cell Proliferation and Invasion through Wnt/ Beta-Catenin Signaling Pathway

Numerous studies have screened and dysregulated the microRNA in various cancers via microarray assay. In this dysregulated microRNA, microRNA-340-5p attracted our attention due to its high conservation and fold change. MicroRNA-340-5p is abnormally expressed in several cancers and plays an important role. However, the expression and function of microRNA-340-5p in lung cancer are still unknown. Our aim was to explore the role of microRNA-340-5p in lung cancer and its underlying molecular mechanism. The lung cancer cell lines of human lung squamous cell carcinoma cell line SKMES1, human lung adenocarcinoma cell line A549, lung adenocarcinoma cells H23, human lung cancer cell line SPC-A1 and human squamous cell carcinoma H520 were used in present study. Real time polymerase chain reaction was performed to detect microRNA-340-5p and beta-catenin messenger RNA expression. Transwell assay was conducted to determine the invasion of human squamous cell carcinoma H520 cells under different treatment conditions. Our study showed that the expression of microRNA-340-5p in lung cancer tissues and was significantly lower than normal tissues. Moreover, overexpression of microRNA-340-5p could suppress the invasion and proliferation of lung cancer cells. We also confirmed that microRNA-340-5p and beta-catenin are negatively correlated in lung cancer tissues and the role of microRNA-340-5p in lung cancer can be reversed by overexpression of beta-catenin. Our results demonstrate that up-regulation of microRNA-340-5p attenuates lung cancer cell invasion and proliferation via inhibiting wingless-related integration site/beta-catenin pathway. The data will provide new insights for the treatment of lung cancer and new guidance for clinical treatment of lung cancer.

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.

FOXC1 induces cancer stem cell-like properties through upregulation of beta-catenin in NSCLC

BackgroundAccumulating evidence suggests that cancer stem cells (CSCs) play a critical role in tumor initiation, progression and therapy, and recent studies have indicated that Forkhead box C1 (FOXC1) is strongly associated with CSCs. This study investigates the regulatory effects of FOXC1 on CSC-like properties in non-small cell lung cancer (NSCLC).MethodsWe analyzed FOXC1 expression in NSCLC using the Cancer Genome Atlas (TCGA) database on UALCANC and performed survival analyses of NSCLC patients on Human Protein Atlas. CSC-like properties were analyzed based on CSC marker-positive cell population, self-renewal ability, stemness-related gene expression, tumorigenicity and drug resistance. The percentage of CD133+ cells was analyzed by flow cytometric analysis. Self-renewal ability was detected by sphere-formation analysis. Real-time PCR, western blotting and immunohistochemical staining were employed to detect mRNA and protein levels. Tumorigenicity was determined based on a xenograft formation assay, and effects of FOXC1 on drug resistance were assessed by cell viability and apoptosis assays. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to investigate the binding of FOXC1 to beta-catenin promoter.ResultsFOXC1 expression was found to be elevated in NSCLC tissues and negatively correlated with patient survival. FOXC1 knockdown reduced CD133+ cell percentage, suppressed self-renewal ability, decreased expression of stemness-related genes (Oct4, NANOG, SOX2 and ABCG2) and inhibited NSCLC cell tumorigenicity in vivo. Moreover, FOXC1 knockdown increased cisplatin and docetaxel sensitivity and reduced gefitinib resistance, whereas FOXC1 overexpression enhanced CSC-like properties. Luciferase reporter and ChIP assays showed beta-catenin to be a direct transcriptional target of FOXC1. Furthermore, overexpression of beta-catenin reversed the CSC-like property inhibition induced by FOXC1 knockdown, and knockdown of beta-catenin attenuated the CSC-like properties induced by FOXC1 overexpression.ConclusionsThis study demonstrates that FOXC1 induces CSC-like properties in NSCLC by promoting beta-catenin expression. The findings indicate that FOXC1 is a potential molecular target for anti-CSC-based therapies in NSCLC.

Abstract 5831: Activated Wnt signaling for the treatment of recurrent medulloblastoma

Abstract Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current molecular subgroups of MB recognize distinct disease entities of which activated Wnt signaling (monosomy 6, exon 3 mutations in CTNNB1, and Wnt gene signature) is associated with a distinct subgroup and the best overall outcome. In contrast, only non-Wnt MBs are characterized by metastatic disease, increased rate of recurrence, and poor overall survivorship. Given the excellent clinical outcome in patients with Wnt-driven MB, we aimed to convert treatment-resistant MB subgroups into an ostensibly benign tumor through selective targeting by small molecules and transgenic patient-derived lines containing a stabilized beta-catenin mutant. Activated Wnt signaling by way of Wnt agonists in treatment-refractory MBs resulted in decreased in vitro self-renewal and promoted differentiation. Comparative gene expression profiling of control and transgenic lines containing a stabilized beta-catenin mutant demonstrated a reduction in stem cell self-renewal genes following beta-catenin overexpression, including Sox2 and Bmi1. In order to validate the therapy-sensitive nature of Wnt-activated cells, we developed stable patient-derived lines containing a 7XTOPFlash reporter for endogenous Wnt signaling. Rare subclonal Wnt-active cells demonstrated a reduced self-renewal and tumor-initiating capacity through in vivo limiting dilution assays when compared to bulk Wnt-inactive cells. The therapeutic relevance of these findings were demonstrated with an in vivo survival advantage in mice with orthotopic injections of cells containing a stabilized beta-catenin mutant representative of constitutively active Wnt signaling or endogenous Wnt-active cells. Xenografts generated from Wnt-activated tumors were smaller in size, maintained a lower rate of proliferation, and reduction in MB self-renewal genes. To further illustrate the clinical utility of activated Wnt signaling, we modified the Children’s Oncology Group therapy protocol for childhood MB so that xenografts may receive chemo/radiotherapy. Tumors generated from Wnt-active xenografts were much more radiosensitive and displayed a significant reduction in spinal metastasis when compared to mice receiving standard therapy without Wnt activation. To develop a rationale clinical therapeutic, we developed unique agonist antibodies that target the Wnt co-receptor LRP5. Treatment with LRP5 antibodies showed a significant reduction in tumor burden and increase in survival of patient-derived tumors that were otherwise treatment-resistant. Our work establishes for the first time activated Wnt signaling as a novel treatment paradigm in childhood MB, identifies a rationale therapeutic approach for recurrent MB, and provides evidence for the context-specific tumor suppressive function of the canonical Wnt pathway. Note: This abstract was not presented at the meeting. Citation Format: Branavan Manoranjan, Chitra Venugopal, Zvezdan Pavlovic, David Bakhshinyan, Michelle Kameda-Smith, Minomi Subapanditha, Sujeivan Mahendram, Jason Moffat, Bradley W. Doble, Sheila Singh. Activated Wnt signaling for the treatment of recurrent medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5831. doi:10.1158/1538-7445.AM2017-5831

Relationship Between Self-Renewal and Differentiation Pathways in Stem Cells and Leukemia

Hematopoietic stem cells are capable of perpetual self-renewal and multi-lineage differentiation, properties that are maintained throughout life by minimal cell cycle activity. Our work has focused on deciphering transcriptional driven differentiation versus self-renewal pathways in stem and progenitor cells. To this end, we have studied transcription factors that control the fate of hematopoietic stem cells by combining mouse models of activated self-renewal with models that can report transcription factor expression. We chose to study the Wnt pathway, activated in several types of leukemia, in combination with the ets family PU.1 transcription factor, vital to almost all myeloid and lymphoid lineages. PU.1 regulates a number of important myeloid specific genes that mediate differentiation to a specific cell fate.To understand the interaction of these pathways, we found that over-expression of Wnt signaling or beta-catenin, the downstream signaling component of the Wnt pathway, was able to inhibit PU.1-mediated differentiation in a PU.1-inducible cell line. There was little to no up-regulation of the myeloid markers Mac1 or Gr1 with activation of Wnt signaling upon induction with 4-hydroxy-tamoxifen (4-OHT). Additionally, many genes related to myeloid differentiation were not increased as compared to control-induced cultures. To understand how these interactions might function in vitro, we crossed a Cre-responsive activated beta-catenin (floxed allele Exon3) mouse to a PU.1-GFP knock-in mouse. From this model, we are able to see changes in PU.1 (GFP) expression in specific populations of hematopoietic progenitors upon activation of beta-catenin. Most importantly, in the LT-HSCs (defined by Lin- cKitHi Sca1+ CD150+ CD48-), we observed a significant increase in GFP (PU.1) intensity upon activation of active beta-catenin. Additionally, there was an increase in the total number of LT-HSCs, as defined by surface markers. LT-HSCs with active beta-catenin and GFP (PU.1) were found to be more in cycle and they express lower levels of transcription factors related to differentiation. These results demonstrate that when beta-catenin is activated, PU.1’s role is modified and the self-renewal program is enhanced at the expense of differentiation.Furthermore, activation of beta-catenin in the hematopoietic cells of mice has been shown to lead to impaired differentiation and eventual death. Even though active beta-catenin has been shown to be essential in several subtypes of myeloid leukemias using murine models, its over-expression is not sufficient to lead to leukemic development. However, heterozygous PU.1/GFP knock-in mice were crossed to the beta-catenin overexpression model, they rapidly developed leukemia post Cre induction. This is not observed in the PU.1/GFP knock-in mice in the absence of beta-catenin activation, suggesting that Wnt signaling adds to a block in differentiation needed for leukemic transformation. These mice show splenomegaly and increased myelocytic populations in the peripheral blood. The leukemia was transplantable to secondary mice and expressed high levels of GFP (PU.1) in the spleen, bone marrow and peripheral blood.These findings demonstrate that the interaction and crosstalk between these two pathways regulate hematopoietic stem cell fate. Future studies will focus on understanding how this interaction between transcription factor and self-renewal pathways becomes disrupted in leukemic stem cells. DisclosuresNo relevant conflicts of interest to declare.

Associations of beta-catenin alterations and MSI screening status with expression of key cell cycle regulating proteins and survival from colorectal cancer

BackgroundDespite their pivotal roles in colorectal carcinogenesis, the interrelationship and prognostic significance of beta-catenin alterations and microsatellite instability (MSI) in colorectal cancer (CRC) needs to be further clarified. In this paper, we studied the associations between beta-catenin overexpression and MSI status with survival from CRC, and with expression of p21, p27, cyclin D1 and p53, in a large, prospective cohort study.MethodsImmunohistochemical MSI-screening status and expression of p21, p27 and p53 was assessed in tissue microarrays with tumours from 557 cases of incident CRC in the Malmö Diet and Cancer Study. Chi Square and Spearman’s correlation tests were used to explore the associations between beta-catenin expression, MSI status, clinicopathological characteristics and investigative parameters. Kaplan-Meier analysis and Cox proportional hazards modelling were used to assess the relationship between beta-catenin overexpression, MSI status and cancer specific survival (CSS).ResultsPositive MSI screening status was significantly associated with older age, female sex, proximal tumour location, non-metastatic disease, and poor differentiation, and inversely associated with beta-catenin overexpression. Beta-catenin overexpression was significantly associated with distal tumour location, low T-stage and well-differentiated tumours. Patients with MSI tumours had a significantly prolonged CSS in the whole cohort, and in stage III-IV disease, also in multivariable analysis, but not in stage I-II disease. Beta-catenin overexpression was associated with a favourable prognosis in the full cohort and in patients with stage III-IV disease. Neither MSI nor beta-catenin status were predictive for response to adjuvant chemotherapy in curatively treated stage III patients. P53 and p27 expression was positively associated with beta-catenin overexpression and inversely associated with MSI. Cyclin D1 expression was positively associated with MSI and beta-catenin overexpression, and p21 expression was positively associated with MSI but not beta-catenin overexpression.ConclusionsFindings from this large, prospective cohort study demonstrate that MSI screening status in colorectal cancer is an independent prognostic factor, but not in localized disease, and does not predict response to adjuvant chemotherapy. Beta-catenin overexpression was also associated with favourable outcome but not a treatment predictive factor. Associations of MSI and beta-catenin alterations with other investigative and clinicopathological factors were in line with the expected.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/8778585058652609

Bilateral Sertoli cell tumors of the testis—a likely new extracolonic manifestation of familial adenomatous polyposis

Testicular Sertoli cell tumors are rare and usually sporadic and unifocal. The large cell calcifying Sertoli cell tumor variant is known to be associated with Carney and Peutz-Jeghers syndromes and can be bilateral in these patient populations. There has been no documented association of Sertoli cell tumor with familial adenomatous polyposis (FAP) in the literature. The case presented is a bilateral Sertoli cell tumor occurring in a 34-year-old patient with FAP. The tumor had a conventional Sertoli cell tumor morphology, but with different morphology in the left and right sites. Beta-catenin immunostain showed strong nuclear reactivity in the tumor cells but not the nonneoplastic Sertoli cells. The presence of bilaterality as well as overexpression of beta-catenin by this tumor supports an association of the development of Sertoli cell tumor with the patient's FAP syndrome and adenomatous polyposis coli inactivation.

Molecular correlates and prognostic significance of SATB1 expression in colorectal cancer

BackgroundSpecial AT-rich sequence-binding protein 1 (SATB1) is a global gene regulator that has been reported to confer malignant behavior and associate with poor prognosis in several cancer forms. SATB1 expression has been demonstrated to correlate with unfavourable tumour characteristics in rectal cancer, but its association with clinical outcome in colorectal cancer (CRC) remains unclear. In this study, we examined the prognostic impact of SATB1 expression in CRC, and its association with important molecular characteristics; i.e. beta-catenin overexpression, microsatellite instability (MSI) screening status, and SATB2 expression.MethodsImmunohistochemical expression of SATB1 and beta-catenin was assessed in tissue microarrays with tumours from 529 incident CRC cases in the prospective population-based Malmö Diet and Cancer Study, previously analysed for SATB2 expression and MSI screening status. Spearmans Rho and Chi-Square tests were used to explore correlations between SATB1 expression, clinicopathological and investigative parameters. Kaplan Meier analysis and Cox proportional hazards modelling were used to explore the impact of SATB1 expression on cancer specific survival (CSS) and overall survival (OS).ResultsSATB1 was expressed in 222 (42%) CRC cases and negative, or sparsely expressed, in adjacent colorectal mucosa (n = 16). SATB1 expression was significantly associated with microsatellite stable tumours (p < 0.001), beta-catenin overexpression (p < 0.001) and SATB2 expression (p < 0.001). While not prognostic in the full cohort, SATB1 expression was significantly associated with poor prognosis in SATB2 negative tumours (HR = 2.63; 95% CI 1.46-4.71; pinteraction = 0.011 for CSS and HR = 2.31; 95% CI 1.32-4.04; pinteraction = 0.015 for OS), remaining significant in multivariable analysis.ConclusionsThe results of this study demonstrate that SATB1 expression in CRC is significantly associated with beta-catenin overexpression, microsatellite stability and SATB2 expression. Furthermore, SATB1 expression is a factor of poor prognosis in SATB2 negative tumours. Altogether, these data indicate an important role for SATB1 in colorectal carcinogenesis and suggest prognostically antagonistic effects of SATB1 and SATB2. The mechanistic basis for these observations warrants further study.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1922643082772076

Abstract 692: Prognostic indicators associated with breast cancer molecular subtypes in African-American women

Abstract Background: Overexpression of Fascin, Endoglin (CD105), and Beta-Catenin has been found to be associated with unfavorable prognosis and progression in hormone-receptor negative breast carcinomas. Specifically, fascin promotes cell motility, while Beta-catenin is involved in cancer invasion, cellular transformation, and metastasis. CD105 is selectively upregulated in small, immature tumor vessels in malignant tumors. The objective of this study was to correlate fascin, CD105, and Beta-Catenin protein expression with clinicopathological factors including age, grade, tumor size, stage, regional node status, survival, and with the four major molecular breast cancer subtypes (luminal A, luminal B, HER2 positive, and Triple Negative [TN]). Methods: Tissue microarrays (TMAs) were constructed from optimally-fixed formalin-fixed, paraffin-embedded tumor blocks from primary breast carcinomas in 203 African-American females. Sections were stained with antitibodies against fascin, CD105, and beta-catenin. The sections were evaluated for the intensity of reactivity (0-3) and the percentage of reactive cells. Cases were categorized as having negative/weak (score &amp;lt;10) or moderate/strong (score &amp;gt;10) fascin expression, negative (score=0) or positive (score&amp;gt;0) CD105 (in tumor cells only) and beta-catenin (cytoplasmic and nuclear) expression. Bivariate analysis was done via χ2 analysis and survivability data was calculated via the generation of Kaplan-Meier curves (SPSS v19). Statistical significance was assumed if P &amp;lt; 0.05. Results: On the TMA, there were 67 triple negatives, 88 Luminal As, 29 Luminal Bs, and 18 Her2 overexpressing cancers. The mean age for the patients in the study was 57.65. Fascin expression was significantly linked to the triple negative subtype (p&amp;lt;0.0001), ER negativity (p&amp;lt;0.0001), PR negativity (0.0001), and Grade III differentiation (p=0.03). CD105 cytoplasmic expression significantly correlated with HER2 positive subtype (p&amp;lt;0.0001), Luminal B subtype (0.01), HER2 positivity (p&amp;lt;0.0001), tumor size (p&amp;lt;0.01) and was associated with decreased disease-free survival (p=0.038). Cytoplasmic beta-catenin expression was significantly associated with triple negative subtype (p&amp;lt;0.03), HER2+ subtype (p&amp;lt;0.001), ER negativity (p&amp;lt;0.001), PR negativity (p&amp;lt;0.001), and HER2 negativity (p=0.03). Conclusions: Our results indicate that fascin, CD105, and Beta-catenin may be potential markers of aggressive breast cancer subtypes and predictors of recurrence and survival. These data also support pharmacological targets for these aggressive subtypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 692. doi:1538-7445.AM2012-692

Abstract 825: Wnt/beta-catenin signaling regulates the expression of O6-methylguanine DNA-methyltransferase in cancer cells: a new strategy to overcome resistance for DNA alkylators in cancer therapy

Abstract Background: A number of DNA-damaging agents attack the O6 position on guanine and thereby form the most potent cytotoxic DNA adducts known. The DNA-repair protein O6-alkylguanine (O6-AG) DNA alkyltransferase (AGT) encoded by the gene O6-Methylguanine (O6-MG)-DNA-methyltransferase (MGMT) repair DNA adducts caused by alkylating agents. MGMT has important implications in cancer treatment since its expression correlates inversely with sensitivity to agents that form O6-alkylguanine adducts, such as temozolamide. It is therefore of great interest to find agents that induce MGMT deficiency, increase the sensitivity and possible overcoming resistance to alkylating chemotherapeutic agents. Methods: Cell lines from medulloblastomas, gliomas, colon cancer and neuroblastomas were examined for Wnt/beta-catenin activity and MGMT expression. We used Western blot, Real-Time quantitative PCR (Q-PCR), siRNA knockdown and cDNA overexpression of beta-catenin, MGMT promotor reporter plasmids and cells with inducible siRNAs targeting beta-catenin to study beta-catenin mediated regulation of MGMT. The correlation between Wnt activity and MGMT expression was also investigated in primary medulloblastomas, gliomas and colon cancer using gene expression cohorts. Cell cytotoxicity and clonogenicity of chemotherapeutic drugs in combination with celecoxib were examined in cell lines using fluorometric microculture cytotoxicity assay and clonogenic assay, respectively. Compounds targeting Wnt signaling was investigated in combination with temolzolamide in vitro and in vivo. Results: MGMT expression level was shown be correlated to Wnt signaling activation both in primary tumors and cell lines of different origins. Proinflammatory prostaglandin E2 activates the Wnt/beta-catenin signaling and increase MGMT expression. Transfection experiments and cells with inducible siRNAs revealed that beta-catenin directly regulates MGMT expression via Tcf/LEF binding. Wnt inhibiting drugs and compounds potentiates the cytotoxic effect of the DNA alkylating drug, temozolomide in cells with elevated MGMT expression in vitro and in vivo. Conclusions: Our data demonstrate that MGMT is a direct Wnt/beta-catenin target, and agents that inhibit Wnt signalling reduces the transcription of MGMT through a prostaglandin E2-Wnt/beta-catenin route and thus increases the sensitivity to temozolomide. This provides a rational approach for improved efficacy of chemotherapeutic drugs inducing DNA alkylation in cancer treatment. The data also suggest that Wnt/beta-catenin is an important target for therapeutic interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 825. doi:1538-7445.AM2012-825

Nuclear beta-catenin overexpression in metastatic sentinel lymph node is associated with synchronous liver metastasis in colorectal cancer.

BackgroundBeta-catenin, a component of the Wingless/Wnt signaling pathway, can activate target genes linking with the adenomatous polyposis coli (APC) gene in colorectal cancer. The purpose of this study is to investigate whether nuclear beta-catenin overexpression in metastatic sentinel lymph node(s) [SLN(s)] is associated with synchronous liver metastasis.MethodsClinicopathological data from 355 patients (93 cases with liver metastasis and 262 cases without liver metastasis) were reviewed. Beta-catenin expression in metastatic SLN(s) and liver metastatic lesions was examined by immunohistochemistry. The association of nuclear beta-catenin expression in metastatic SLN(s) and liver metastatic lesions was evaluated, and the relationship between nuclear beta-catenin expression and clinicopathological characteristics was analyzed. Finally, univariate and logistic multivariate regression analyses were adopted to discriminate the risk factors of liver metastasis.ResultsNuclear beta-catenin overexpression in metastatic SLN(s) was observed in 70 patients with liver metastasis and 31 patients without liver metastasis (75.3% vs. 11.8%; P < 0.001). Nuclear beta-catenin expression was noted in all the metastatic lesions. Spearman rank correlation analysis demonstrated that nuclear beta-catenin expression in metastatic SLN(s) had a positive correlation with that in metastatic lesions (r = 0.425, P < 0.001). Univariate and multivariate analyses indicated that nuclear beta-catenin overexpression in metastatic SLN(s) correlated with liver metastasis.ConclusionsNuclear beta-catenin overexpression in metastatic SLN(s) is strongly associated with liver metastasis and may contribute to predict liver metastasis.

Activation of the SKP2 protooncogene is a common effector pathway in hepatocarcinogenesis and contributes to malignant transformation of the liver

We have recently shown that upregulation of SKP2 protooncogene is associated with the development and progression of human hepatocellular carcinoma (HCC). In the present study, we further investigated the functional role of SKP2 by using in vitro and in vivo approaches. Forced overexpression of a number of known liver oncogenes, including AKT, E2F1, N-Ras, ERK2, STAT5b, and mutated beta-catenin in human HCC cell lines led to increased proliferation, which was associated with the rise in the levels and activation of SKP2 (as assessed by SKP2-mediated degradation of p27, p57, and p130 target genes). Although the protooncogene FOXM1 has been shown to transcriptionally activate SKP2, induction of SKP2 was mediated by FOXM1 only in N-Ras- and ERK2-overexpressing cells. On the other hand, the growth promoting effects driven by AKT, ERK2, E2F1, N-Ras, STAT5b, and mutated beta-catenin overexpression in vitro were significantly decreased when their overexpression was paralleled by siRNA mediated silencing of SKP2. Forced induction of SKP2 gene via hydrodynamic gene delivery did not induce significant morphologic alterations in the mouse liver, similar to that occurring in mice transfected with the N-Ras gene. However, when SKP2 and N-Ras genes were co-injected via hydrodynamic transfection, preneoplastic liver lesions developed, which finally evolved to HCC 18 weeks after injection. Furthermore, SKP2 accelerated hepatocarcinogenesis induced by the AKT protooncogene when both genes were co-injected in the mouse liver. In conclusion, the in vitro and in vivo data obtained in this study provide solid evidence that SKP2 functions as an oncogene in liver cancer, acting downstream of many signaling pathways involved in human HCC development. Therapeutic approaches aimed at inhibiting SKP2 might be highly beneficial for the treatment of human HCC.

Anti-Tumor Activity of Novel Small Molecule Wnt Signaling Inhibitor, CWP232291, In Multiple Myeloma

AbstractAbstract 3038Multiple myeloma (MM), one of the most incurable hematological malignancies in adults, is a disorder of plasma cells characterized by accumulation of clonal proliferation of malignant plasma cells in the bone marrow (BM). Overexpression of beta-catenin, the downstream effector of the canonical Wnt signaling pathway, has been reported in both MM cell lines and patient samples. Activated Wnt signaling pathway has also been reported to play a critical role in progression of MM cell proliferation, thus highlighting the need for new therapeutic approaches, particularly those targeting Wnt molecular pathway. Here we report the discovery of a novel inhibitor of Wnt signaling CWP232291, which promotes degradation of beta-catenin. CWP232291 exhibits potent growth inhibitory activity in several MM cell lines (RPMI-8226, OPM-2, NCI-H929, JJN3, and EJM) with IC50 values of 13 – 73 nM. The inhibitory activity of CWP232291 on Wnt signaling is demonstrated by reporter gene assay and by its effect in down-regulation of Wnt target genes. Using HEK293 cells, CWP232291 treatment dose dependently reduces promoter activity of TOPflash induced by Wnt-3a-Conditioned Media, at a calculated IC50 value of 273 nM. Furthermore, MM cells treated with CWP232291 show downregulated expression of Wnt target genes such as survivin by triggering degradation of beta-catenin. Treatment of these cells with CWP232291 results in activation of caspase-3 and PARP cleavage, indicating induction of apoptosis. To investigate the potential in vivo anti-tumor efficacy of CWP232291, we utilized two MM tumor bearing mice models. Daily or intermittent intravenous (i.v.) administration of CWP232291 led to significant tumor growth inhibition (TGI) in OPM-2 (50 mg/kg, qdx5: regression and 100 mg/kg, biw: 95% TGI) and RPMI-8226 (100 mg/kg, qdx5: regression and 100 mg/kg tiw: 80% TGI) xenograft model with no obvious change in body weight. The anti-tumor efficacies of CWP232291 were dose-dependent and had strong correlations with degradation of beta-catenin in the tumor samples. Potent induction of apoptosis through cleavage of Caspase-3 and PARP and significant decrease of plasma M protein levels in OPM-2 tumor bearing mice were detected as early as 2 and up to 24 hours after single i.v. administration of CWP232291. Taken together, these data clearly demonstrate the impressive anti-tumor profile of CWP232291 with a good therapeutic window and suggest a potential therapeutic application of CWP232291 for the treatment of MM. Disclosures:Cha:Choongwae Pharma Corp.: Employment. Jung:Choongwae Pharma Corp.: Employment. Lee:Choongwae Pharma Corp.: Employment. Briaud:Theriac Pharmaceutical Corp.: Employment. Tenzin:Theriac Pharmaceutical Corp.: Employment. Jung:Choongwae Pharma Corp.: Employment. Pyon:Choongwae Pharma Corp.: Employment. Lee:Choongwae Pharma Corp.: Employment. Chung:Choongwae Pharma Corp.: Employment. Lee:Choongwae Pharma Corp.: Employment. Oh:Choongwae Pharma Corp.: Employment. Jung:Choongwae Pharma Corp.: Employment. Pai:Choongwae Pharma Corp.: Employment. Emami:Theriac Pharmaceutical Corp.: Employment.

Human Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) gene is transcriptionally regulated by CREB and Wnt/β‐catenin signaling

The aberrant activation of Wnt signaling is a key process in colorectal tumorigenesis. Canonical Wnt signaling controls transcription of target genes via beta-catenin and T-cell factor/lymphoid enhancer factor family transcription factor complex. Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) is a novel member of the Armadillo family which has two Armadillo repeats as opposed to more than six repeats in the classical Armadillo family members. Here we examine cis-regulatory elements and trans-acting factors involved in the transcriptional regulation of the ALEX1 gene. Site-directed mutations of a cyclic AMP response element (CRE) and an E-box impaired the basal activity of human ALEX1 promoter in colorectal and pancreatic cancer cell lines. Moreover, overexpression of CRE-binding protein (CREB) increased the ALEX1 promoter activity in these cell lines, whereas knockdown of CREB expression decreased the expression level of ALEX1 mRNA. Interestingly, luciferase reporter analysis and quantitative real-time RT-PCR demonstrated that the ALEX1 promoter was up-regulated in a CRE-dependent manner by continuous activation of Wnt/beta-catenin signaling induced by a glycogen synthase kinase-3 inhibitor and overexpression of beta-catenin. These results indicate that the CRE and E-box sites are essential cis-regulatory elements for ALEX1 promoter activity, and ALEX1 expression is regulated by CREB and Wntk/beta-catenin signaling.

Loss of p53, rather than beta-catenin overexpression, induces survivin-mediated resistance to apoptosis in an esophageal cancer cell line

Survivin, an important inhibitor of apoptosis, is overexpressed in esophageal cancer and negatively affects survival. The complex regulation of survivin transcription involves enhancement by beta-catenin and repression by p53. The purpose of this study is to test whether inhibition of beta-catenin or overexpression of p53 can decrease survivin expression and render esophageal cancer cells more susceptible to apoptosis. Studies were performed in normal human esophageal epithelial cells and the human esophageal cancer cell line TE7. Levels of beta-catenin, survivin, and p53 were measured by Western blot. Apoptosis was induced after treatment with camptothecin and measured by release of caspase 3 and morphologic criteria. The roles of survivin and beta-catenin in preventing apoptosis were tested by their silencing with specific small interfering RNA molecules. The effect of p53 overexpression on survivin promoter activity was measured using a survivin promoter-luciferase reporter construct and by real-time polymerase chain reaction measurement of survivin mRNA levels. Both beta-catenin and survivin are overexpressed in TE7 cells, whereas p53 expression is negligible. TE7 cells demonstrate resistance to camptothecin-induced apoptosis (P < .01). This effect is significantly reduced by inhibition of survivin, but not of beta-catenin (P < .01). Overexpression of p53 in TE7 cells reduces survivin transcription and mRNA levels (P < .01), without reducing survivin protein levels. Survivin plays a critical role in TE7 cell resistance to camptothecin-induced apoptosis. This effect is not dependent on beta-catenin expression. Overexpression of p53 decreases survivin transcription but does not decrease levels of survivin protein, suggesting posttranscriptional control of survivin expression.

SplicerAV: a tool for mining microarray expression data for changes in RNA processing

BackgroundOver the past two decades more than fifty thousand unique clinical and biological samples have been assayed using the Affymetrix HG-U133 and HG-U95 GeneChip microarray platforms. This substantial repository has been used extensively to characterize changes in gene expression between biological samples, but has not been previously mined en masse for changes in mRNA processing. We explored the possibility of using HG-U133 microarray data to identify changes in alternative mRNA processing in several available archival datasets.ResultsData from these and other gene expression microarrays can now be mined for changes in transcript isoform abundance using a program described here, SplicerAV. Using in vivo and in vitro breast cancer microarray datasets, SplicerAV was able to perform both gene and isoform specific expression profiling within the same microarray dataset. Our reanalysis of Affymetrix U133 plus 2.0 data generated by in vitro over-expression of HRAS, E2F3, beta-catenin (CTNNB1), SRC, and MYC identified several hundred oncogene-induced mRNA isoform changes, one of which recognized a previously unknown mechanism of EGFR family activation. Using clinical data, SplicerAV predicted 241 isoform changes between low and high grade breast tumors; with changes enriched among genes coding for guanyl-nucleotide exchange factors, metalloprotease inhibitors, and mRNA processing factors. Isoform changes in 15 genes were associated with aggressive cancer across the three breast cancer datasets.ConclusionsUsing SplicerAV, we identified several hundred previously uncharacterized isoform changes induced by in vitro oncogene over-expression and revealed a previously unknown mechanism of EGFR activation in human mammary epithelial cells. We analyzed Affymetrix GeneChip data from over 400 human breast tumors in three independent studies, making this the largest clinical dataset analyzed for en masse changes in alternative mRNA processing. The capacity to detect RNA isoform changes in archival microarray data using SplicerAV allowed us to carry out the first analysis of isoform specific mRNA changes directly associated with cancer survival.

β-Catenin is involved in alterations in mitochondrial activity in non-transformed intestinal epithelial and colon cancer cells

Background:Alteration in respiratory activity and mitochondrial DNA (mtDNA) transcription seems to be an important feature of cancer cells. Leukotriene D4 (LTD4) is a proinflammatory mediator implicated in the pathology of chronic inflammation and cancer. We have shown earlier that LTD4 causes translocation of β-catenin both to the mitochondria, in which it associates with the survival protein Bcl-2 identifying a novel role for β-catenin in cell survival, and to the nucleus in which it activates the TCF/LEF transcription machinery.Methods:Here we have used non-transformed intestinal epithelial Int 407 cells and Caco-2 colon cancer cells, transfected or not with wild type and mutated (S33Y) β-catenin to analyse its effect on mitochondria activity. We have measured the ATP/ADP ratio, and transcription of the mtDNA genes ND2, ND6 and 16 s in these cells stimulated or not with LTD4.Results:We have shown for the first time that LTD4 triggers a cellular increase in NADPH dehydrogenase activity and ATP/ADP ratio. In addition, LTD4 significantly increased the transcription of mtDNA genes. Overexpression of wild-type β-catenin or a constitutively active β-catenin mutant mimicked the effect of LTD4 on ATP/ADP ratio and mtDNA transcription. These elevations in mitochondrial activity resulted in increased reactive oxygen species levels and subsequent activations of the p65 subunit of NF-κB.Conclusions:The present novel data show that LTD4, presumably through β-catenin accumulation in the mitochondria, affects mitochondrial activity, lending further credence to the idea that inflammatory signalling pathways are intrinsically linked with potential oncogenic signals.