Beta-catenin Transcriptional Activity Research Articles

Intestinal epithelial CAR‐Like Membrane Protein promotes mucosal barrier function and prevents colitis‐associated cancer in mice

CAR‐Like Membrane Protein (CLMP) is a transmembrane glycoprotein structurally related to Coxsackie and Adenovirus Receptor. Mutations in CLMP gene have been correlated with the development of an intestinal pediatric disorder termed congenital short bowel syndrome (CSBS). To date, not only is the pathogenesis of CSBS incompletely understood, the role of CLMP in gut homeostasis and disease has not yet been elucidated. We found that CLMP expression is significantly reduced in colorectal cancer (CRC) biopsies and increased in inflammatory bowel disease (IBD) compared to normal mucosa samples. Given the expression pattern of CLMP, we generated mice harboring a tamoxifen inducible intestinal epithelial cell (IEC)‐specific deletion of CLMP as well as model human IEC in which CLMP expression was silenced or upregulated. We then assessed the role of CLMP in regulating intestinal mucosal barrier function, the development of chronic inflammation and colitis‐associated cancer. In this study we report that CLMP protein expression is abundant in differentiated IEC compared to undifferentiated and highly proliferative IEC. CLMP is associated with tight junction controlling the paracellular permeability to solutes and macromolecules. Epithelial CLMP deficiency resulted in enhanced paracellular permeability in vivo and in vitro.These findings indicate an important role for CLMP in regulating epithelial barrier function. Mechanistically, we found that CLMP is involved in homotypic trans‐interactions that facilitate cell‐cell cohesion and promotes cell‐matrix adhesion through integrin beta 1 activity. Lastly, we observed that loss of CLMP in human and mouse IEC resulted in increased cell proliferation. Conversely, CLMP overexpression in IEC resulted in reduced cell proliferation. These results were corroborated in murine models of cancer that included xenograft experiments with CLMP‐deficient tumor cells versus CLMP overexpressing tumor cells and Azoxymethane/Dextran Sulfate Sodium‐induced colitis‐associated cancer. In these models, CLMP deficiency worsened tumor growth. Biochemical analyses revealed CLMP‐dependent regulation of IEC proliferation to be mediated through effects on Akt signaling and beta‐catenin transcriptional activity. Collectively, our findings provide the first report of a key role for CLMP in the positive regulation of intestinal epithelial barrier function and prevention of tumor development.

Targeting Wnt/beta-catenin signaling in CTNNB1-mutant endometrial cancer

<h3>Objectives:</h3> <i>CTNNB1</i> (gene encoding for beta-catenin) mutations convey increased rates of recurrence in early stage, low grade endometrial cancer (EC). The role of beta-catenin/TCF transcriptional activity in EC recurrence is not well understood. We aim to assess the impact of Wnt/beta-catenin inhibition in <i>in vitro</i> and <i>in vivo</i> EC models. <h3>Methods:</h3> Using The Cancer Genome Atlas (TCGA) of Uterine Corpus Endometrial Carcinoma (PanCancer Atlas), we evaluated <i>CTNNB1</i>-mutant vs -wildtype tumors in a low-risk population. We studied <i>CTNNB1</i>-wildtype (HEC1B, Ishikawa) and CTNNB1-mutant (HEC108, HEC265, HEC1B-S33Y, Ishikawa-S33Y) EC cell lines. <i>CTNNB1</i>-S33Y cell lines were created via retroviral transduction. Dose response curves were determined for 5 <i>Wnt</i>/beta-catenin pathway inhibitors (Wnt-C59, XAV-939, PyrPam, PRI-724, SM04690). Cell viability was assessed with Licor Cell Staining. TCF transcriptional activity was determined via TOP/FOP reporter assay. Apoptosis following treatment with SM04690 was evaluated via Annexin V/propidium iodide (PI). HEC1B, HEC1B-S33Y and HEC265 tumor-bearing athymic nude mice were treated with vehicle or SM04690 25mg/kg. Tumor size was measured using calipers. Tumors were evaluated with immunohistochemistry for proliferation (Ki67) and apoptosis (cl-caspase 3). <h3>Results:</h3> TCGA analysis confirmed that <i>CTNNB1</i> mutations are enriched in recurrent low-risk EC and showed that aberrant Wnt/beta-catenin pathway activation is associated with disease recurrence. <i>In vitro,</i> XAV939, Wnt-C59 and PyrPam inhibited function upstream of beta-catenin transcriptional activity and were ineffective at inhibiting EC cell viability. In contrast, PRI724 and SM04690 indirectly inhibited beta-catenin transcriptional activity and significantly reduced cell viability in <i>CTNNB1</i>-mutant EC cell lines. Treatment with SM04690 reduced cell viability in all EC cell lines, but was significantly lower in HEC108, HEC265 and HEC1B-S33Y compared to HEC1B (24.2%, 32.3%, 44.4%, vs. 71.4%, p<0.01). Compared to control, SM04690 significantly induced apoptosis in HEC265 cells (3.98% vs. 6.91% AnnexinV/PI+, p=0.044) and reduced TCF transcriptional activity in HEC1B-S33Y (-84%, p=0.017) and HEC108 (-74%, p=0.002) cells. <i>In vivo,</i> HEC1B, HEC1B-S33Y and HEC265 tumors treated with SM04690 had smaller mean tumor volumes than those treated with vehicle (HEC1B 146.4 vs 335.4mm3, p<0.001; HEC1B-S33Y 136.4 vs. 243.1mm3, p=0.014; HEC265 105.7 vs. 321.8mm3, p=0.06). In HEC1B-S33Y and HEC265 tumors, SM04690 treatment significantly reduced Ki67 H-scores compared to vehicle (129.8 vs. 146.7, p=0.035 and 87.08 vs.106.4, p=0.024, respectively). <h3>Conclusions:</h3> Targeting the Wnt/beta-catenin pathway in <i>CTNNB1-</i>mutant EC effectively inhibited proliferation and beta-catenin/TCF transcriptional activity. Further, the inhibitor SM04690 blunted tumor progression in <i>in vivo</i> models. These studies suggest beta-catenin transcriptional inhibitors are effective in EC and have a more significant effect in <i>CTNNB1</i>-mutant than -wildtype EC. These findings highlight a potential therapeutic vulnerability for treatment of <i>CTNNB1</i>-mutant EC.

Abstract PO043: Targeting beta-catenin and Wnt signaling in CTNNB1-mutated endometrial cancer

Abstract Background: CTNNB1 mutations convey increased rates of disease recurrence in early stage, low grade endometrial cancer (EC). The role of beta-catenin transcriptional activity in CTNNB1-mutated EC recurrence is not well understood. We aim to assess the impact of Wnt/beta-catenin inhibition in EC models. Methods: Data mining The Cancer Genome Atlas (TCGA) of endometrial carcinoma (PanCancer Atlas), we evaluated CTNNB1-mutated vs -wildtype tumors in a low-risk population. We then studied EC cell lines that are CTNNB1-wildtype (HEC1B, Ishikawa) or CTNNB1-mutated (HEC108, HEC265, HEC1B-S33Y, Ishikawa-S33Y). CTNNB1-S33Y cell lines were created via retroviral transduction. Treatment dose curves were determined for 5 Wnt/beta-catenin pathway inhibitors (Wnt-C59, XAV-939, PyrPam, PRI-724, SM04690). Cell viability was assessed with Licor Cell Staining. TCF transcriptional activity was determined via TOP/FOP reporter assay. Apoptosis following treatment with SM04690 was evaluated via Annexin V/propidium iodide (PI). HEC1B, HEC1B-S33Y and HEC265 tumor-bearing athymic nude mice were treated with vehicle or SM04690 25mg/kg. Tumor size was measured using calipers. Tumor cell proliferation and apoptosis were evaluated with immunohistochemistry for Ki67 and Cleaved Caspase 3. Results: TCGA database analysis parallels previous findings that CTNNB1 mutations are enriched in recurrent low-risk EC. XAV939, Wnt-C59 and PyrPam all inhibit function upstream of beta-catenin transcriptional activity and were ineffective at inhibiting EC cell viability. In contrast, PRI724 and SM04690 indirectly inhibit beta-catenin transcriptional activity and significantly reduce cell viability in CTNNB1-mutated EC cell lines. Treatment with SM04690 reduced cell viability in all EC cell lines, but was significantly lower in HEC108, HEC265 and HEC1B-S33Y compared to HEC1B (24.2%, 32.3%, 44.4%, 71.4%, p&amp;lt;0.01). Compared to control, SM04690 significantly induced apoptosis in HEC265 cells (3.98% vs. 6.91% AnnexinV/PI+, p=0.0443) and reduced TCF transcriptional activity in HEC1B-S33Y (-84%, p=0.017) and HEC108 (-74%, p=0.002) cells. In the mouse model, HEC1B, HEC1B-S33Y and HEC265 tumors treated with SM04690 had smaller mean tumor volumes than those treated with vehicle (146.4 vs 335.4cm3, p&amp;lt;0.001; 136.4 vs. 243.1cm3, p=0.014; 105.7 vs. 321.8cm3, p=0.06). In HEC1B-S33Y tumors, SM04690 treatment significantly reduced Ki67 H-scores compared to vehicle (129.8 vs. 146.7, p=0.0351). Conclusions: Targeting the Wnt/beta-catenin pathway in CTNNB1-mutated EC effectively inhibited proliferation and beta-catenin/TCF transcriptional activity. Further, the inhibitor SM04690 blunted tumor progression in multiple in vivo models. These studies demonstrate beta-catenin transcriptional inhibitors are effective in CTNNB1-mutated EC and have a more significant effect in CTNNB1-mutated EC than CTNNB1-wildtype EC. These findings highlight a potential therapeutic vulnerability for treatment of CTNNB1-mutated EC. Citation Format: Marisa R. Moroney, Elizabeth R. Woodruff, Lubna Qamar, Benjamin G. Bitler, Bradley R. Corr. Targeting beta-catenin and Wnt signaling in CTNNB1-mutated endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO043.

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.

The Vitamin D Receptor as Tumor Suppressor in Skin.

Cutaneous malignancies including melanomas and keratinocyte carcinomas (KC) are the most common types of cancer, occurring at a rate of over one million per year in the United States. KC, which include both basal cell carcinomas and squamous cell carcinomas, are substantially more common than melanomas and form the subject of this chapter. Ultraviolet radiation (UVR), both UVB and UVA, as occurs with sunlight exposure is generally regarded as causal for these malignancies, but UVB is also required for vitamin D synthesis in the skin. Keratinocytes are the major cell in the epidermis. These cells not only produce vitamin D but contain the enzymatic machinery to metabolize vitamin D to its active metabolite, 1,25(OH)2D, and express the receptor for this metabolite, the vitamin D receptor (VDR). This allows the cell to respond to the 1,25(OH)2D that it produces. Based on our own data and that reported in the literature, we conclude that vitamin D signaling in the skin suppresses UVR-induced epidermal tumor formation. In this chapter we focus on four mechanisms by which vitamin D signaling suppresses tumor formation. They are inhibition of proliferation/stimulation of differentiation with discussion of the roles of hedgehog, Wnt/β-catenin, and hyaluronan/CD44 pathways in mediating vitamin D regulation of proliferation/differentiation, regulation of the balance between oncogenic and tumor suppressor long noncoding RNAs, immune regulation, and promotion of DNA damage repair (DDR).

Quercetin as a Novel Treatment for Developmental Abnormalities of the Kidney

Renal dysplasia is the abnormal development of the kidney and is the leading cause of chronic kidney disease and kidney failure in children. Despite this, there is no cure. Invasive symptom management such as dialysis and kidney transplants have significant adverse effects. Normal kidney development involves the interaction between three cell types: ureteric epithelium, metanephric mesenchyme, and renal stroma. The ureteric epithelium undergoes branching morphogenesis to form the collecting system, and the metanephric mesenchyme undergoes nephrogenesis to form the nephrons. The renal stroma provides structural support to the developing kidney and regulates proper branching morphogenesis and nephrogenesis. Beta‐catenin is a transcription factor essential for normal branching morphogenesis and nephrogenesis. In human renal dysplasia, beta‐catenin levels are markedly increased in the nucleus of the stroma, mesenchyme and epithelium. Our findings from transgenic mouse models show that elevated levels of beta‐catenin in the nucleus cause dysregulation of genes leading to abnormal branching morphogenesis and nephrogenesis, resulting in renal dysplasia. Our objective is to inhibit beta‐catenin transcriptional activity in the kidney and determine its effects on normal and dysplastic kidney development. Quercetin is a flavonoid that inhibits beta‐catenin activity and was effective in reducing beta‐catenin levels that resulted to improved outcomes in different cancer types and in kidney fibrosis. We tested if Quercetin alters beta‐catenin activity during normal kidney development. Wild‐type embryonic day 13.5 mouse kidneys were cultured in growth medium supplemented with 0uM (control), 40uM, 80uM, and 160uM Quercetin for 48 hours. A dose‐dependent disruption in branching morphogenesis and nephrogenesis was observed by performing immunofluorescence for cytokeratin, Pax2, and Six2. In addition, quantitative reverse‐transcriptase PCR (qRT‐PCR) showed a disrupted expression of beta‐catenin target genes essential for kidney development (Pax2, Wnt4, and GDNF). Consistent with these findings, immunohistochemistry for beta‐catenin demonstrated that Quercetin reduced nuclear beta‐catenin expression and increased cytoplasmic expression in a dose‐dependent manner. Next, to determine if Quercetin rescues renal dysplasia, we utilized transgenic mice models that over‐express beta‐catenin specifically in the kidney stroma (Foxd1;B‐catGOFs), or in the mesenchyme, stroma, and ureteric epithelium (Rarb2;B‐catGOFsmu). Both of these animal models have severe renal dysplasia characterized by disorganized branching morphogenesis and disrupted nephrogenesis. Immunofluorescence for cytokeratin and Pax2 showed that in‐vitro treatment of embryonic day 13.5 dysplastic kidneys with 40uM Quercetin resulted in a markedly improved kidney with improved branching morphogenesis and nephrogenesis, and closely resembles wild‐type kidney development. Our findings demonstrate that Quercetin reduces beta‐catenin activity in normal and dysplastic kidneys, reduces the transcription of genes that play central roles in kidney development, and resulted to a reduced severity of renal dysplasia.Support or Funding InformationNSERC, CIHR and KFOCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 5897: Single cell-derived analysis of desmoid tumors for studying tumor-stroma interactions

Abstract Cancer associated fibroblasts play an important role in the maintenance and remodeling of the tumor microenvironment, providing the appropriate conditions for neoplastic cell growth and invasion. Desmoid tumors (DT), also called aggressive fibromatosis, are rare, locally invasive soft tissue tumors that consist of fibroblastic cells embedded in extracellular matrix. Identification of the stromal cells to study tumor-stroma interactions is difficult due to both populations displaying a fibroblastic phenotype, and no cell marker exists that reliably differentiates between the two populations. Majority of DT arise sporadically due to somatic activating mutations in beta-catenin (CTNNB1), a major effector molecule of canonical Wnt signaling. We established single cell derived colonies from multiple DT samples and characterized the beta-catenin mutation status of each clone by Sanger sequencing. Indeed, we were able to establish both mutant and non-mutant colonies from DT samples. Quantitative PCR for beta-catenin targets AXIN2 and LEF1 confirmed differential activity between the mutant and non-mutant colonies. The specific CTNNB1 codon mutation had no difference on beta-catenin transcriptional activity. We next performed a high throughput surface antigen screen to identify cell markers that can distinguish between the two subpopulations. Our screen found CD142 to be uniquely expressed by the mutant colonies, while the non-mutant colonies uniquely expressed Podoplanin. Quantitative PCR confirmed the differential expression of these markers. Furthermore, the CD142-positive population in heterogeneous DT samples correlated with their mutation frequency. Importantly, CD142-based cell sorting allowed the isolation of the mutant subpopulation even in samples that appeared as wild-type by Sanger sequencing. We also studied the expression of secreted factors in our mutant and non-mutant populations. We observed that CTHRC1, a ligand related to the Wnt/PCP pathway, is highly elevated exclusively in the mutant subpopulations. Recombinant CTHRC1 increased the proliferation rate of DT primary cultures, as measured by BrdU incorporation, while neutralizing antibodies against CTHRC1 decreased cell proliferation. The importance of tumor-stroma interactions cannot be studied without first identifying and characterizing the two populations. This has been especially difficult in soft tissue sarcomas where both the neoplastic and stromal cells exhibit a mesenchymal phenotype. Our study offers a novel method for identifying the mutant and non-mutant subpopulations within desmoid tumors to study how they may interact. Rapidly quantifying tumor composition will also support efforts to understand the natural progression of disease and how it responds to therapy. Citation Format: Mushriq Al-Jazrawe, Steven Xu, Qingxia Wei, Raymond Poon, Benjamin Alman. Single cell-derived analysis of desmoid tumors for studying tumor-stroma interactions [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 5897. doi:10.1158/1538-7445.AM2017-5897

Bioinformatics Knowledge Map for Analysis of Beta-Catenin Function in Cancer.

Given the wealth of bioinformatics resources and the growing complexity of biological information, it is valuable to integrate data from disparate sources to gain insight into the role of genes/proteins in health and disease. We have developed a bioinformatics framework that combines literature mining with information from biomedical ontologies and curated databases to create knowledge “maps” of genes/proteins of interest. We applied this approach to the study of beta-catenin, a cell adhesion molecule and transcriptional regulator implicated in cancer. The knowledge map includes post-translational modifications (PTMs), protein-protein interactions, disease-associated mutations, and transcription factors co-activated by beta-catenin and their targets and captures the major processes in which beta-catenin is known to participate. Using the map, we generated testable hypotheses about beta-catenin biology in normal and cancer cells. By focusing on proteins participating in multiple relation types, we identified proteins that may participate in feedback loops regulating beta-catenin transcriptional activity. By combining multiple network relations with PTM proteoform-specific functional information, we proposed a mechanism to explain the observation that the cyclin dependent kinase CDK5 positively regulates beta-catenin co-activator activity. Finally, by overlaying cancer-associated mutation data with sequence features, we observed mutation patterns in several beta-catenin PTM sites and PTM enzyme binding sites that varied by tissue type, suggesting multiple mechanisms by which beta-catenin mutations can contribute to cancer. The approach described, which captures rich information for molecular species from genes and proteins to PTM proteoforms, is extensible to other proteins and their involvement in disease.

Abstract A13: A positive feedback loop for transcriptional regulation of beta-catenin that favors sustained colorectal cancer cell invasion

Abstract Wnt/beta-catenin signaling is a branch of a functional network that is involved in a broad range of biological systems and deregulation of components involved in Wnt/beta-catenin signaling has been implicated in a wide spectrum of cancers. We have shown earlier that beta-catenin, is up regulated in the invasion front compared to the inner parts of the tumor in a murine model of colorectal liver metastasis and clinical specimens. This activity is due to the fact that beta-catenin acts as co-transcription factor of its own promoter contributing to the dynamic changes of beta-catenin levels upon the confrontation of tumor cells with the host microenvironment. To examine the putative role of the host cells for transcriptional activation of beta-catenin and its promoter, co-culture experiments and application of recombinant proteins or small molecule inhibitors were performed. To find out which cells play a role in activation of the beta-catenin promoter both hepatocytes and hepatic stellate cells were co-cultured with a colorectal tumor cell line LS174T that was stably transfected with a SEAP reporter gene driven by a fragment of the beta-catenin promoter. We could demonstrate that co-culture of LS174T cells with the hepatocytes (AML12) results in an increase of the SEAP reporter indicating an activation of the beta-catenin promoter. In contrast, co-culturing of the LS174T with hepatic stellate cells (LX-2) caused a decrease of reporter gene expression. Treatment of LS174T beta-catenin cells with FCS showed a 5-fold up-regulation of the beta-catenin promoter as compared to serum-free conditions concluding that fetal calf serum contains factors that activate the beta-catenin promoter. Application of different cytokines and growth factors that are involved in tumor formation and metastasis did not lead to any alteration of the reporter gene expression. Upon application of several small molecule inhibitors, Wortmannin and LY492004 that inhibits the PI3 kinase showed a pronounced up-regulation of the beta-catenin promoter in a concentration dependent manner. PI3K is known to activate Akt that in turn stabilizes beta-catenin and translocates to the nucleus. Inhibition of the above described PI3K-akt- beta-catenin pathway by application of PI3K inhibitors leads to the activation of the beta-catenin-promoter indicating the presence of a feed-back loop that leads to increased beta-catenin transcription in response to decreased beta-catenin protein levels that in turn results in sustained up regulation of the invasiveness of CRC cells. These results warrant for the development of small molecule inhibitors targeting beta-catenin rather than the PI3K, Wnt/beta-catenin signaling pathways. Citation Format: Franziska Ehrmann, Linda Frank, Peter Schirmacher, Karsten Brand, Obul R. Bandapalli. A positive feedback loop for transcriptional regulation of beta-catenin that favors sustained colorectal cancer cell invasion. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A13.

MCC inhibits beta-catenin transcriptional activity by sequestering DBC1 in the cytoplasm.

The mutated in colorectal cancer (MCC) is a multifunctional gene showing loss of expression in colorectal and liver cancers. MCC mutations can drive colon carcinogenesis in the mouse and in vitro experiments suggest that loss of MCC function promotes cancer through several important cellular pathways. In particular, the MCC protein is known to regulate beta-catenin (β-cat) signaling, but the mechanism is poorly understood. Here we show that the β-cat repressor function of MCC is strongly impaired by the presence of a disease-associated mutation. We also identify deleted in breast cancer 1 (DBC1) as a new MCC interacting partner and regulator of β-cat signaling. RNA interference experiments show that DBC1 promotes β-cat transcriptional activity and that the presence of DBC1 is required for MCC-mediated β-cat repression. In contrast to all other DBC1 interacting partners, MCC does not interact through the DBC1 Leucine Zipper domain but with a glutamic-acid rich region located between the Nudix and EF-hand domains. Furthermore, MCC overexpression relocalizes DBC1 from the nucleus to the cytoplasm and reduces β-cat K49 acetylation. Treatment of cells with the SIRT1 inhibitor Nicotinamide reverses MCC-induced deacetylation of β-cat K49. These data suggest that the cytoplasmic MCC-DBC1 interaction sequesters DBC1 away from the nucleus, thereby removing a brake on DBC1 nuclear targets, such as SIRT1. This study provides new mechanistic insights into the DBC1-MCC axis as a new APC independent β-cat inhibitory pathway.

Alterations of Axis Inhibition Protein 1 (AXIN1) in Hepatitis B Virus-Related Hepatocellular Carcinoma and Overexpression of AXIN1 Induces Apoptosis in Hepatocellular Cancer Cells

Axis inhibition protein 1 (AXIN1) is a negative regulator of Wnt/beta-catenin signaling via regulating the level of beta-catenin. However, the role of AXIN1 in the tumorigenesis and progression of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is less clear. PCR sequence analysis, immunohistochemistry, and Western blot were performed on 22 HBV-related HCC samples and corresponding nontumor liver tissues to detect variants in AXIN1 gene and the expression level of AXIN1. Human hepatoma cell lines SNU475 and SNU423 were transfected with pCDNA3.1-AXIN1-myc or AXIN1 G425S-myc mutant. The growth curve and apoptosis rate of cell lines, phosphorylation of beta-catenin, and cell cycle regulatory proteins depending on beta-catenin transcriptional activity were detected. We identified four mutations of AXIN1 in 22 primary HBV-related HCCs and demonstrated a lower expression of AXIN1 in HBV-related HCC tissues than that in paired adjacent nontumor tissues. Overexpression of AXIN1 wild-type but not AXIN1 mutant inhibited the growth of HCC cell lines, accelerated their apoptosis, and negatively regulated beta-catenin-dependent transcriptional activity. Our study revealed that alterations of AXIN1 were involved in HBV-related HCC. Overexpression of AXIN1 but not AXIN1 mutant negatively regulated beta-catenin-dependent transcriptional activity and downregulated the level of cell cycle regulatory proteins, suggesting that AXIN1 may be a potential target for gene therapy of primary HCC.

P2-05-05: Receptor-Like Protein Tyrosine Phosphatase Kappa (PTPRK) and Its Biological Role in Angiogenesis.

Abstract Introduction: Protein Tyrosine Phosphatases (PTPs) are known as signalling molecules which affect cell growth, differentiation and oncogenic transformation. PTPs have been indicated in tumourigenesis and progression of various solid tumours. PTPRK, receptor-type protein tyrosine phosphatase kappa, has been shown to down-regulate transcriptional activity of beta-catenin and impact distribution of beta-catenin/E-cadherin complexes on cancer cell membrane. However, the role played by PTPRK in angiogenesis remains unknown. In present study, the effect of PTPRK knock-down on functions and tubule formation ability of HECV cells was investigated. (or In the present study, the effect of PTPRK on angiogenesis process was investigated.) Methods: anti-PTPRK ribozyme transgenes were constructed to knock-down PTPRK expression in vascular endothelial cells, HECV cells. The subsequent effect upon in vitro cell growth, adhesion, migration and microvascular tubule formation was examined using a variety of functional assays. Result: Knock-down PTPRK in HECV cells (HECVΔPTPRK) resulted in a decrease of cell growth in vitro. The growth rate of HECVΔPTPRK was 276.3±16.4, p=0.01 compared with HECVpEF (314.8±21.9) controls. However, knock-down of PTPRK in HECV cells increased cell motility. The cell migration distance of HECVΔPTPRK was 83.8±19.8μm, p=0.008 compared with HECVpEF (61.8±11.8μm) controls. No effect on cell adhesion by PTPRK knockdown was seen in HECVΔPTPRK (81.2±6.6), compared with HECVpEF control (78.8±15.6). Furthermore, knock-down of PTPRK suppressed tubule formation in HECV cells, the length of total tubules (μm) in HECVΔPTPRK was 2167.4±943.2, p&amp;lt;0.05 compared with HECVpEF (3130.6±386.9) control. Conclusion: Knock-down of PTPRK reduced the growth and tubule formation abilities of vascular endothelial cells. It suggests that PTPRK is pivotal regulator for angiogenic process. Further investigations are required to identify the downstream pathways involved in these impacts. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-05.

Abstract A56: The telomerase inhibitor imetelstat exhibits antitumor and anticancer stem cell effects through perturbation of casein kinase-2 signaling.

Abstract Background: Activation of telomerase is essential for the indefinite replication potential of most cancer cells. Inhibition of telomerase is expected to lead to loss of telomere maintenance resulting in cell cycle arrest and/or cancer cell death, making telomerase inhibition an attractive anti-cancer approach. Cancer stem cells (CSCs) are rare cells in tumors implicated in cancer initiation and potentiation, as well as persistence or recurrence after standard treatment. Unlike normal tissue counterparts, all CSCs tested to date have upregulated telomerase activity. Recent identification of several non-canonical roles of telomerase components may expand the functionality of telomerase-inhibiting drugs. Imetelstat, a potent telomerase inhibitor currently in Phase II clinical trials, has been shown to reduce proliferative potential in multiple cancer models. Several recent studies have demonstrated that imetelstat depletes CSCs in various tumor types. The relatively rapid onset of CSC depletion could probably indicate a mechanism independent of telomere shortening. To understand the contributions of non-canonical pathways in the response of cancer cells to imetelstat treatment, we analyzed modulations of key signaling pathways in imetelstat-treated glioblastoma multiforme cancer cell lines (U87-MG and U118) and their CSC and bulk subsets. Results: Imetelstat inhibited telomerase activity, proliferative capacity and colony-forming potential in the U87 and U118 cell lines. Imetelstat treatment (2 weeks at 3uM) resulted in decreased expression of casein kinase 2 (CK2) subunits alpha and beta, and a reduction in phosphorylation of downstream CK2 substrates such as the DNA repair protein XRCC1. In addition, imetelstat reduced the transcriptional activity of beta-catenin, which is regulated by members of the CK2 family, in U87-MG cells. Cyclin D1, which is regulated by beta-catenin activity, was found to be down regulated by imetelstat treatment. These pathway modulations were not observed in T98G, a cell line resistant to imetelstat-mediated proliferation effects. Reducing CK2-alpha expression had an additive effect on cell growth inhibition in combination with imetelstat in U87-MG cells. Knock-down of the CK2-alpha, but not CK2-beta subunits, with siRNAs rapidly reduced the numbers of CSCs in U87-MG and U118 cell lines. Concomitant with the down-regulation of CK2-alpha signaling, imetelstat treatment depleted the number of CSCs in U78-MG and U118 cell lines. Preliminary data indicate a more marked reduction of CK2-alpha levels in the CSC subset of U87-MG cells compared to the bulk cells upon short term imetelstat treatment (1 week at 3uM). These in-vitro exposure concentrations of imetelstat are comparable to those attainable intratumorally in a xenograft mouse model. Conclusions: Our results suggest that CK2-alpha signaling and dysregulation of its downstream targets may play a key role in survival of CSCs as well as bulk tumor cells in U87 and U118 glioblastoma cell lines. These novel insights may help identify drugs that can act synergistically with imetelstat in treating cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A56.

Abstract LB-281: Enteric bacterial protein AvrA enhances tumorigenesis through activating intestinal STAT/beta-catenin signaling pathways

Abstract Enteric bacteria play an important role in the pathogenesis of colon cancer. More than 1000 species of bacteria coexist in the human colon. The number of microbial cells is about 10 times larger than the number of eukaryotic cells in the human body. Due to the complexity of the gut flora, identification of the specific microbial agents contributing to colon cancer remains challenging. How bacterial products directly contribute to colorectal cancer is still unknown. Many researchers have taken advantage of the colitis induced by Salmonella Typhimurium to study the early phase of inflammation and infection. A few reports have been on chronic infection in vivo. Bacteria can modulate the host by secreting bacterial effector proteins to the host cells. AvrA is a pathogenic gene of Salmonella and E.coli, whose encoded protein is inserted into intestinal mucosa. AvrA influences eukaryotic cell pathways utilizing ubiquitin and acetylation. We hypothesize that the bacterial effector AvrA activates the STAT/beta-catenin pathway to promote colonic tumorigenesis. In the current study, we investigated a chronic bacterial infected cancer model with Salmonella Typhimurium colonization in the mouse intestine for 49 weeks. Mice were colonized with AvrA-sufficient or deficient bacterial strains, then stimulated with a carcinogen azoxymethane (10mg/kg body weight, intraperitoneal injection) and 1% dextran sodium sulfate (induced colitis) in vivo. We found that mice infected with AvrA-expressed bacteria had significantly high incidence of tumor in colon. We found that AvrA expression decreased the phosphorylated-beta-catenin and inhibits the ubiquitination of beta-catenin in mouse colonic epithelial cells in vivo. Additionally, bacterial AvrA expression enhanced the acetylated beta-catenin, which regulates the beta-catenin transcription activity and stabilizes beta-catenin. Moreover, we found the infected colon had higher STAT 1 and 3 expressions. Inflammatory cytokines such as IL-6 and INF-gamma in serum were increased. The STAT pathway was activated by infection and promoted epithelial cancer. STAT and its acute STAT activation promoted inflammation in the infected colon. Overall, AvrA activation of the STAT/beta-catenin pathway promoted colonic tumorigenesis. Our findings provide important insights into how a bacterial effector protein contributes to the development of colon cancer. We believe that determining the responses and underlying mechanisms by which bacteria modulate the STAT/beta-catenin pathway will identify novel approaches to target the specific signal pathway that contributes to tumorigenesis. Our findings can also be applied to the risk assessment and prevention of colon carcinogenesis. This work was supported by NIH DK075386 and ACS RSG-09–075–01-MBC to Jun Sun. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-281. doi:10.1158/1538-7445.AM2011-LB-281

Abstract A105: The extracellular calcium sensing receptor regulates the proliferation of colonic epithelial cells

Abstract The progression of colorectal cancer (CRC) from normal colonic epithelium to the malignant phenotype is associated with diverse genetic and epigenetic changes, including mutations of APC or beta-catenin genes. Extensive experimental and epidemiological evidence also suggest that differences in diet contribute to more variation in CRC incidence than any other factor. For example, several animal experimental studies support the notion that dietary Ca2+ prevents the development of CRC. The precise mechanism(s) mediating the chemopreventive properties of Ca2+ remain incompletely understood. The extracellular Ca2+-sensing receptor (CaR), a GPCRs originally cloned from parathyroid chief cells, plays a major role in the control of extracellular Ca2+ concentration by regulating parathyroid hormone secretion. However, subsequent studies demonstrated that the CaR is also expressed in other tissues and organs, including the entire gastrointestinal (GI) tract. Interestingly, CaR expression in colonic carcinomas is greatly reduced or lost, suggesting that the suppression of its expression may be associated with abnormal differentiation and malignant progression. Here, we assessed the role of the CaR in colonic epithelial cells proliferation. In order to elucidate the mechanisms involved, we examined in vitro changes in beta-catenin phosphorylation and intracellular distribution and cellular proliferation triggered by CaR stimulation. We found that CaR stimulation promotes, in human epithelial cells derived from normal colon and from colorectal adenocarcinoma, a decrease of up to 60% in beta-catenin Ser-552 phosphorylation, a residue involved in beta-catenin nuclear import and transcriptional activity regulation. This change in phosphorylation was accompanied by a redistribution of beta-catenin from the cytoplasm and nuclei to the plasma membrane and by a marked proliferation inhibition. To determine the role of the CaR in the proliferation of colonic cells in vivo, we employed a novel mouse model where the expression of the CaR was specifically knocked out in the GI tract. The results show that the crypts in the distal colon are deeper in knock out (KO) mice (389 ± 7.39 Arbitrary Units, mean ± SEM) than controls littermates (268 ± 6.35 AU) P &amp;lt; 0.0001. Similarly, the crypts in the proximal colon are deeper in KO mice (289 ± 7.02 AU) than controls (232 ± 5.36 AU) P &amp;lt; 0.0001. We also found significant morphological differences in the crypt’ structure of several regions in the proximal colon of CaR KO mice, including the absence of a well-organized surface layer of epithelial cells and the presence of disorganized crypts and numerous enlarged globet cells. Further analysis of the colon of KO mice showed that the number of proliferating cells, as revealed by Ki-67 proliferation marker immunostaining, was significantly larger than control littermates. Specifically, proliferating cells in the KO mice were detected in the base of the crypt and up to 60% of the crypt column. In contrast, in control littermates the proliferating cells were fewer and present mostly at the base of the crypt. We hypothesize that pathways emanating from the CaR lead to proliferation inhibition of colon-derived epithelial cells by a mechanism that involves beta-catenin Ser-552 dephosphorylation and that the genetic ablation of this receptor promotes hyperproliferation of colonic crypts. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A105.

Adenomatous polyposis coli regulates endothelial cell migration independent of roles in beta-catenin signaling and cell-cell adhesion.

Adenomatous polyposis coli (APC), a tumor suppressor commonly mutated in cancer, is a cytoskeletal organizer for cell migration and a scaffold for GSK3 beta/CKI-mediated phosphorylation and degradation of the Wnt effector beta-catenin. It remains unclear whether these different APC functions are coupled, or independently regulated and localized. In primary endothelial cells, we show that GSK3 beta/CKI-phosphorylated APC localizes to microtubule-dependent clusters at the tips of membrane extensions. Loss of GSK3 beta/CKI-phosphorylated APC from these clusters correlates with a decrease in cell migration. GSK3 beta/CKI-phosphorylated APC and beta-catenin at clusters is degraded rapidly by the proteasome, but inhibition of GSK3 beta/CKI does not increase beta-catenin-mediated transcription. GSK3 beta/CKI-phosphorylated and -nonphosphorylated APC also localize along adherens junctions, which requires actin and cell-cell adhesion. Significantly, inhibition of cell-cell adhesion results in loss of lateral membrane APC and a concomitant increase in GSK3 beta/CKI-phosphorylated APC in clusters. These results uncouple different APC functions and show that GSK3 beta/CKI phosphorylation regulates APC clusters and cell migration independently of cell-cell adhesion and beta-catenin transcriptional activity.

β-catenin signaling involves HGF-enhanced HepG2 scattering through activating MMP-7 transcription

It is well accepted that cell scattering (dispersion of clustered cells into single cells) is the initial step of tumor metastasis, and the downregulation of E-cadherin is associated with metastatic potential of tumor cells; however, the molecular mechanisms underlying loss of E-cadherin during tumor development are still poorly understood. Here, we report that hepatocyte growth factor (HGF) induced E-cadherin downregulation and cell scattering are attributed to the activation of Wnt/beta-catenin signaling and transcriptional activation of matrix metalloproteinase MMP-7. Furthermore, the increased MMP-7 is secreted into the medium and cleaves the ectodomain of E-cadherin. Inhibition of HGF signal by siRNA of c-Met, blocking the beta-catenin transcriptional activity through a dominant negative form of TCF4, MMP-7 knockdown by siRNA or suppression of MMP-7 enzymatic activity with a neutralization antibody allowed inhibition of HGF-induced loss of E-cadherin and HepG2 scattering. Our data presented here revealed the intrinsic mechanism of HGF activated Wnt/beta-catenin signaling regulation of HepG2 cell scattering through MMP-7 transcription activation and E-cadherin degradation. The results suggest that the blocking of HGF/c-Met/beta-catenin/MMP-7/E-cadherin signaling pathway might present a practical therapeutic target for interference with hepatocellular carcinoma metastasis.

Sonic Hedgehog Acts as a Negative Regulator of β-Catenin Signaling in the Adult Tongue Epithelium

Wnt/beta-catenin signaling has been implicated in taste papilla development; however, its role in epithelial maintenance and tumor progression in the adult tongue remains elusive. We show Wnt/beta-catenin pathway activation in reporter mice and by nuclear beta-catenin staining in the epithelium and taste papilla of adult mouse and human tongues. beta-Catenin activation in APC(min/+) mice, which carry a mutation in adenomatous poliposis coli (APC), up-regulates Sonic hedgehog (Shh) and Jagged-2 (JAG2) in the tongue epithelium without formation of squamous cell carcinoma (SCC). We demonstrate that Shh suppresses beta-catenin transcriptional activity in a signaling-dependent manner in vitro and in vivo. A similar regulation and function was observed for JAG2, suggesting that both pathways negatively regulate beta-catenin, thereby preventing SCC formation in the tongue. This was supported by reduced nuclear beta-catenin in the tongue epithelium of Patched(+/-) mice, exhibiting dominant active Shh signaling. At the invasive front of human tongue cancer, nuclear beta-catenin and Shh were increased, suggesting their participation in tumor progression. Interestingly, Shh but not JAG2 was able to reduce beta-catenin signaling in SCC cells, arguing for a partial loss of negative feedback on beta-catenin transcription in tongue cancer. We show for the first time that the putative Wnt/beta-catenin targets Shh and JAG2 control beta-catenin signaling in the adult tongue epithelium, a function that is partially lost in lingual SCC.

Abstract 5077: Signaling through the cytoplasmic tail of MUC1 directly activates transcription of the metastasis promoting growth factor CTGF in pancreatic adenocarcinoma

Abstract Transmembrane mucin MUC1 is highly expressed in pancreatic adenocarcinoma and contributes to tumor growth, invasion, and metastasis. Differential phosphorylation of the cytoplasmic tail of MUC1 (MUC1.CT) by growth factor receptor tyrosine kinases is associated with morphogenetic signal transduction, as it enables interactions with signal transducers and leads to nuclear accumulation of MUC1.CT. MUC1.CT contains no known DNA binding motif but localizes to promoters of genes implicated in tumorigenesis in complex with transcription factors, modulating their regulatory activity. Using microarray analysis, we identified genes differentially transcribed in S2013 pancreatic adenocarcinoma cells stably overexpressing MUC1. These data were correlated with transcriptional targets of MUC1.CT identified on a genome-wide scale in pancreatic adenocarcinoma cells using a combination of chromatin immunoprecipitation and promoter array methodologies (ChIP-on-chip). We further investigated the transcriptional regulation of one novel target, connective tissue growth factor (CTGF/CCN2), by MUC1.CT. CTGF is highly expressed in pancreatic cancer and is a known mediator of tumor growth, angiogenesis, and metastasis. Overexpression of MUC1 significantly increased CTGF expression in pancreatic tumor cells in response to EGF, PDGF-BB, or HGF stimulation. The MUC1.CT becomes phosphorylated by EGFR, PDGFR-beta, and c-Met and overexpression of MUC1 constructs harboring mutations within the target phosphorylation motifs for these receptor tyrosine kinases reduced CTGF expression. We determined that MUC1.CT directly regulated the CTGF gene transcription through co-occupancy with transcription factors at several regions within and surrounding the CTGF promoter. MUC1 overexpression promoted localization of mutant p53 (R273H) and beta-catenin to the CTGF promoter, while decreasing occupancy by a repressor of CTGF transcription, c-Jun. We propose that MUC1, when overexpressed in pancreatic adenocarcinoma, enables expression of CTGF in response to growth factor stimulations by promoting mutant p53 and beta-catenin transcriptional activity and alleviating CTGF transcriptional repression by c-Jun. Taken together, these results reveal a novel signaling pathway that selectively induces CTGF expression in MUC1-overexpressing pancreatic tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5077.

Vitamin D receptor ligands, adenomatous polyposis coli, and the vitamin D receptor FokI polymorphism collectively modulate β‐catenin activity in colon cancer cells

The activity of beta-catenin, commonly dysregulated in human colon cancers, is inhibited by the vitamin D receptor (VDR), and this mechanism is postulated to explain the putative anti-cancer activity of vitamin D metabolites in the colon. We investigated the effect of a common FokI restriction site polymorphism (F/f) in the human VDR gene as well as the effect of anti-tumorigenic 1,25-dihydroxyvitamin D(3) (1,25D) and pro-tumorigenic lithocholic acid (LCA) VDR ligands on beta-catenin transcriptional activity. Furthermore, the influence of a major regulatory protein of beta-catenin, the APC tumor suppressor gene, on VDR-dependent inhibition of beta-catenin activity was examined. We report herein that beta-catenin-mediated transcription is most effectively suppressed by the VDR FokI variant F/M4 when 1,25D is limiting. Using Caco-2 colorectal cancer (CRC) cells, it was observed that VDR ligands, 1,25D and LCA, both suppress beta-catenin transcriptional activity, though 1,25D exhibited significantly greater inhibition. Moreover, 1,25D, but not LCA, suppressed endogenous expression of the beta-catenin target gene DKK-4 independent of VDR DNA-binding activity. These results support beta-catenin sequestration away from endogenous gene targets by 1,25D-VDR. This activity is most efficiently mediated by the FokI gene variant F/M4, a VDR allele previously associated with protection against CRC. Interestingly, we found the inhibition of beta-catenin activity by 1,25D-VDR was significantly enhanced by wild-type APC. These results reveal a previously unrecognized role for 1,25D-VDR in APC/beta-catenin cross talk. Collectively, these findings strengthen evidence favoring a direct effect on the Wnt-signaling molecule beta-catenin as one anti-cancer target of 1,25D-VDR action in the colorectum.