Beta-catenin Protein Research Articles

Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes

Oncomarkers play important roles in the detection and management of human malignancies. Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by cancer cells and can be detected in the blood serum. We investigated the effect of CEA production by MIP101 colorectal carcinoma cell lines on E-cadherin adherens junction (AJ) protein complexes. No direct interaction between E-cadherin and CEA was detected; however, the functional relationships between E-cadherin and its AJ partners: α-, β- and p120 catenins were impaired. We discovered a novel interaction between CEA and beta-catenin protein in the CEA producing cells. It is shown in the current study that CEA overexpression alters the splicing of p120 catenin and triggers the release of soluble E-cadherin. The influence of CEA production by colorectal cancer cells on the function of E-cadherin junction complexes may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer.

Use of an Activated Beta-Catenin to Identify Wnt Pathway Target Genes inCaenorhabditis elegans, Including a Subset of Collagen Genes Expressed in Late Larval Development

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin−dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

Combination of a Novel Photosensitizer DTPP with 650 nm Laser Results in Efficient Apoptosis and Cytoskeleton Collapse in Breast Cancer MCF-7 Cells

Luminal A type breast cancer was suitable for Photodynamic therapy (PDT) as its strong adhesion ability, low malignancy and easily being exposed to laser. To examine the novel photosensitizer agent 5-5-(4-N, N-diacetoxylphenyl-10, 15, 20-tetraphenylporphyrin)(DTPP) mediate PDT in breast cancer cell, Luminal A type breast cancer MCF-7 cells were used in this study, various concentrations of DTPP (0, 2, 4, 6, 8, 10, 12, 15, 20, 25, 30 μg/mL) and different time intervals (0, 0.5, 1, 2, 4, 6, 8 min) of laser exposure at 650 nm wavelength (power of 20 mW) were tested in PDT. The survival rates of MCF-7 cells were measured using a sensitive cell proliferation assay (MTT) to establish optimal semilethal dose and optimal time exposure, a further study of effects on cytoskeleton and apoptosis were also performed. Cell cycle and apoptosis variation were assayed by flow cytometry. Microtubule, microfilament, and nuclei were observed using laser scanning confocal microscopy. Oncoproteins Bcl-2, beta-tubulin, and beta-catenin were detected by means of electrophoresis. The novel DTPP showed an efficient growth inhibition of MCF-7 during PDT, effective combinations in MCF-7 cells were shown to be 4 μg mL(-1) PS irradiated for 8 min at least or 15 μg mL(-1) irradiated for 2 min at least. Microtubule, microfilament, and nucleus staining demonstrated that cytoskeletal collapse occurs at 0.5 h after PDT. Bcl-2 and skeleton adhesion proteins beta-catenin were reduced in the level of expression; whereas, skeleton proteins beta-tubulin and actin maintained similar levels of expression 12 h after PDT. These results provided a better understanding of DTPP-PDT in MCF-7 cells.

Beta-Catenin and Epithelial Tumors: A Study Based on 374 Oropharyngeal Cancers

Introduction. Although altered regulation of the Wnt pathway via beta-catenin is a frequent event in several human cancers, its potential implications in oral/oropharyngeal squamous cell carcinomas (OSCC/OPSCC) are largely unexplored. Work purpose was to define association between beta-catenin expression and clinical-pathological parameters in 374 OSCCs/OP-SCCs by immunohistochemistry (IHC). Materials and Methods. Association between IHC detected patterns of protein expression and clinical-pathological parameters was assessed by statistical analysis and survival rates by Kaplan-Meier curves. Beta-catenin expression was also investigated in OSCC cell lines by Real-Time PCR. An additional analysis of the DNA content was performed on 22 representative OSCCs/OPSCCs by DNA-image-cytometric analysis. Results and Discussion. All carcinomas exhibited significant alterations of beta-catenin expression (P < 0.05). Beta-catenin protein was mainly detected in the cytoplasm of cancerous cells and only focal nuclear positivity was observed. Higher cytoplasmic expression correlated significantly with poor histological differentiation, advanced stage, and worst patient outcome (P < 0.05). By Real-Time PCR significant increase of beta-catenin mRNA was detected in OSCC cell lines and in 45% of surgical specimens. DNA ploidy study demonstrated high levels of aneuploidy in beta-catenin overexpressing carcinomas. Conclusions. This is the largest study reporting significant association between beta-catenin expression and clinical-pathological factors in patients with OSCCs/OPSCCs.

Regulated Wnt/Beta-Catenin Signaling Sustains Adult Spermatogenesis in Mice1

The importance of Wnt signaling for postnatal testis function has been previously studied in several mouse models, with chronic pathway disruption addressing its function in Sertoli cells and in postmeiotic germ cells. While chronic beta-catenin deletion in Sertoli cells does not profoundly affect testis development, new data indicate that Wnt signaling is required at multiple stages of spermatogenesis. We used two mouse models that allow acute disruption of Wnt signaling to explore the importance of regulated Wnt pathway activity for normal germ cell development in adult male mice. Short-term induction of mutations in Adenomatous polyposis coli (Apc) and beta-catenin (Ctnnbl), which increase and decrease Wnt signaling levels, were generated in AhCre Apc(fl/fl) and AhCre Ctnnb1(fl/fl) mice, respectively. Each exhibited a distinct phenotype of disrupted spermatogenesis that was evident within 24 h and persisted for up to 4 days. Outcomes included germ cell apoptosis and rapid loss and altered blood-testis barrier protein distribution and morphology. The functional significance of nuclear localized beta-catenin protein in spermatocytes and round spermatids, indicative of active Wnt signaling, was highlighted by the profound loss of postmitotic germ cells in both models. Developmentally regulated Wnt signaling mediators identified through transcriptional profiling of wild-type and AhCre Ctnnb1(fl/fl) mouse testes identified Wnt receptors (e.g., Fzd4) and ligands (e.g., Wnt3, Wnt3a, Wnt5b, Wnt7a, and Wnt8b). This demonstration that Wnt signaling control is essential for adult spermatogenesis supports the growing understanding that its disruption may underpin certain cases of male infertility.

Cancer chemopreventive effect of dietary Zataria multiflora essential oils

Zataria multiflora Boiss., with the common name Avishan-e-Shirazi, is native to Iran. This herb has been found to possess varied pharmacological properties. In the present study, for the first time, colon chemopreventive effects of Z. multiflora essential oils (0.01% and 0.1% in the diet) in rats treated with 1,2-dimethylhydrazine (DMH) were demonstrated. For this purpose, the oxidative stress/antioxidant parameters (lipid peroxidation, glutathione, superoxide dismutase, catalase, and ferric reducing ability of plasma) concomitant with xenobiotic metabolizing enzymes (CYP450 and GST) were considered. Moreover, the colonic Beta-catenin protein was examined in colon tissues followed by histopathological analysis. The results showed that the dietary intervention of Z. multiflora oils in tumor-bearing rats induced with DMH caused significant modulatory effects on DMH-metabolizing enzymes, but with lack of oxidative stress/antioxidant status. In parallel, the elevated protein Beta-catenin induced by DMH decreased significantly in treatment groups. However, the decreased tumor formations in histopathological biopsies in treated groups further confirmed these results. Thus, with reference to histopathological and biochemical data, it can be safely concluded that inhibition of colon premalignant lesions induced by DMH was mediated by the interference of Z. multiflora oils through the modulatory effect of DMH-metabolizing enzymes in association with Beta-catenin and no impact of antioxidant/oxidative stress state.

Recurrent Chromosome 22 Deletions in Osteoblastoma Affect Inhibitors of the Wnt/Beta-Catenin Signaling Pathway

Osteoblastoma is a bone forming tumor with histological features highly similar to osteoid osteoma; the discrimination between the tumor types is based on size and growth pattern. The vast majority of osteoblastomas are benign but there is a group of so-called aggressive osteoblastomas that can be diagnostically challenging at the histopathological level. The genetic aberrations required for osteoblastoma development are not known and no genetic difference between conventional and aggressive osteoblastoma has been reported. In order to identify recurrent genomic aberrations of importance for tumor development we applied cytogenetic and/or SNP array analyses on nine conventional and two aggressive osteoblastomas. The conventional osteoblastomas showed few or no acquired genetic aberrations while the aggressive tumors displayed heavily rearranged genomes. In one of the aggressive osteoblastomas, three neighboring regions in chromosome band 22q12 were homozygously deleted. Hemizygous deletions of these regions were found in two additional cases, one aggressive and one conventional. In total, 10 genes were recurrently and homozygously lost in osteoblastoma. Four of them are functionally involved in regulating osteogenesis and/or tumorigenesis. MN1 and NF2 have previously been implicated in the development of leukemia and solid tumors, and ZNRF3 and KREMEN1 are inhibitors of the Wnt/beta-catenin signaling pathway. In line with deletions of the latter two genes, high beta-catenin protein expression has previously been reported in osteoblastoma and aberrations affecting the Wnt/beta-catenin pathway have been found in other bone lesions, including osteoma and osteosarcoma.

Enhanced beta-catenin expression and inflammation are associated with human ectopic tubal pregnancy

Is there a molecular link between Wnt signaling in fallopian tube inflammation and ectopic tubal implantation? Enhanced beta-catenin expression, reduced E-cadherin expression and glycogen accumulation in the tubal epithelia and hyperplasia in tubal arteries were found in ectopic tubal pregnancy, consistent with the effects induced by Wnt signaling and inflammation. Chronic inflammation caused by infection can alter gene expression in the fallopian tube cells possibly leading to the development of ectopic pregnancy. Knockout mouse models have shown a relationship between Wnt/beta-catenin signaling and predisposition to tubal ectopic pregnancy. Women with ectopic tubal pregnancy (n = 18) were included in the case group, while women with chronic salpingitis (n = 13) and non-pregnant women undergoing sterilization procedures or salpingectomy for benign uterine disease (n = 10) were set as the controls. This study was performed between January 2012 and November 2012. The ampullary segments of fallopian tubes were collected from patients. Tissues of tubal pregnancy were separated into implantation sites and non-implantation sites. Beta-catenin and E-cadherin expression were determined using immunohistological and immunofluorescence staining. Glycogen production was measured with periodic acid Schiff by staining. The diameter and wall thickness of tubal arteries were evaluated by histological analysis method. Immunohistological staining revealed that beta-catenin protein expression was 100% positive in the ectopic pregnant and inflamed tubal tissues, and the staining intensity was significantly higher than in non-pregnant tubal tissues. In contrast, E-cadherin expression was reduced in ectopic pregnant fallopian tubes, possibly as a consequence of increased Wnt signaling. Moreover, glycogen accumulated in the tubal cells, and hyperplasia was observed in the tubal arteries with ectopic pregnancy, which is consistent with the effects induced by Wnt signaling and inflammation. All these changes could create the permissive environment that promotes embryos to ectopically implant into the fallopian tube. This finding requires a further confirmation about what activates Wnt signaling in ectopic tubal pregnancies. Also, it is generally recognized that Chlamydia infection is associated with ectopic pregnancy, and disturbs tubal epithelia via the Wnt signaling. However, the infection type in the samples used was salpingitis. A better understanding of the underlying mechanisms leading to ectopic pregnancies may contribute to our knowledge of the pathogenesis of tubal disorders and infertility and to the prevention of tubal ectopic pregnancy.

Abstract 3132: Beta-catenin regulates c-Myc and CDKN1A expression in breast cancer cells.

Abstract Basal-like breast cancer (BLBC) is associated with a poor prognosis and lacks therapeutic targets. The molecular pathways leading to the development of basal-like tumors are not well understood. We reported recently that nuclear and cytosolic accumulation of beta-catenin was enriched in BLBC, suggesting Wnt pathway activation in this specific subtype. c-Myc has been placed as a downstream effector of Wnt/beta-catenin pathway in colorectal cancer. However, exactly how the Wnt pathway regulates c-Myc in breast cancer and the biological significance of this regulation are poorly understood. In this study, we found that c-Myc is highly expressed in the basal-like subtype in comparison to the other four breast cancer subtypes by IHC staining. In addition, we confirmed the concordant expression of beta-catenin and c-Myc in basal-like breast tumors. Both c-Myc and beta-catenin proteins are highly expressed in the basal-like cell lines in comparison with non-basal-like cell lines. After silencing beta-catenin using siRNA, c-Myc expression was decreased in none-BLBC cells. In contrast, c-Myc mRNA and protein expression was up-regulated in the BLBC cell lines, suggesting that beta-catenin regulates c-Myc expression in a cell type dependent manner in breast cancer. Although we did not detect any TCF promoter activity in all breast cancer cell lines, decreased c-Myc promoter activity was observed after inhibiting beta-catenin by siRNA in non-BLBC cells; however, inhibition of beta-catenin or over-expression of dominant-negative LEF had no effect on c-Myc promoter activity in BLBC cell lines. In contrast, CDKN1A mRNA and p21 protein expression were significantly increased in all breast cancer cell lines upon beta-catenin silencing. Interestingly, inhibiting beta-catenin expression alone does not induce apoptosis in breast cancer cell lines despite c-Myc regulation,, but we observed a modest increase of cells in G1 and decrease of cells in S phase in the cell lines with beta-catenin silencing. Collectively, these findings suggest that the regulation of c-Myc in breast cancer cells is dependent on the molecular subtype, and that beta-catenin-mediated control of c-Myc and p21 may control the balance of cell death and proliferation in breast cancer by both TCF-dependent and TCF-independent mechanisms. Citation Format: Jinhua Xu, Yinghua Chen, Andrey Khramtsov, Dezheng Huo, Kathleen Goss, Olufunmilayo I. Olopade. Beta-catenin regulates c-Myc and CDKN1A expression in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3132. doi:10.1158/1538-7445.AM2013-3132

Differential expression of beta-catenin and dickkopf-1 in the third trimester placentas from normal and preeclamptic pregnancies: a comparative study

BackgroundBeta-catenin is a key nuclear effector of Wnt signaling which could be antagonized by dickkopf-1(DKK1). Beta-catenin and DKK1 are involved in a variety of biological processes; however, their expression in the placenta with severe preeclampsia (PE) has not been elucidated. This study was aimed to detect the localization and compare the expression of beta-catenin and DKK1 in normal and preeclamptic placenta.MethodsSixty pregnant women who underwent cesarean section were enrolled in this study, including 30 healthy pregnant women in the control group and 30 preeclamptic women in the severe PE group. Real-time polymerase chain reaction (real-time-PCR) and western blot were employed to detect the beta-catenin and DKK1 mRNA and protein expression levels, respectively, and their locations were evaluated by immunohistochemistry (IHC).ResultsOur results indicated that beta-catenin and DKK1 were expressed predominantly in the syncytiotrophoblast and the extravillous trophoblast (EVT). The beta-catenin mRNA and protein expressions were significantly decreased, whereas the DKK1 significantly increased in preeclamptic placental tissues compared to normal placental controls.ConclusionsIn conclusion, decreased beta-catenin expression, as well as DKK1 over-expression might be associated with the process of the pathogenesis of PE. Further studies would elucidate their exact roles in the pathogenesis of PE.

Giant solid pseudopapillary neoplasm of the pancreas in a middle-aged man: A case report

M. Iseki , M. Mizuma , H. Yoshida , T. Okada , K. Nakagawa, H. Hayashi , T. Morikawa , S. Ottomo, N. Sakata , H. Ohtsuka , K. Fukase , T. Aoki , F. Motoi , T. Naitoh , Y. Katayose , S. Egawa , M. Unno . Division of Hepato-Biliary-Pancretic Surgery, Department of Surgery, Tohoku University Graduate School of Medicine, Japan Division of Integrated Surgery and Oncology, Department of Surgery, Tohoku University Graduate School of Medicine, Japan Introduction: A solid pseudopapillary neoplasm (SPN) of the pancreas is a rare neoplasm of low malignant potential. It typically afflicts in young women, but uncommon in men. We report giant solid pseudopapillary neoplasm of the pancreas in a middle-aged man. Case report: A 47-year-old manwas admitted to the previous hospital for continuous epigastric pain and bodyweight loss. A giantmasswas palpated in the upper abdominal area. The abdominal CT scan revealed 21 cmmass with central necrosis and peripheral calcification in the pancreas. This tumor showed enhanced solid component in the marginal area. The patient underwent total pancreatectomy with portal vein resection and reconstruction. In the resected specimen, 26 cm tumor of the pancreas, surrounded by a fibrous pseudocapsule, gave rise to central necrosis and intratumoral hemorrhage. Histological examination revealed that the tumor cells with enlarged nucleus and granular eosinophillic cytoplasm were arranged in sheets and nests, forming pseudopapilla formation. They were immunohistologically positive for CD10 and vimentin. Since nuclear accumulation of beta-catenin protein was shown, this tumor was pathologically diagnosed with SPN. This had malignant potential, indicated by venous invasion. Discussion: This case show atypical clinical feature (age, gender) and typical radiographical feature, including the enhancement of solid component and peripheral calcification. Giant SPN with central necrosis and intratumoral hemorrhage is similar to other giant pancreatic tumors, such as neuroendcrine tumor. Giant SPN is difficult to diagnose by imaging modalities preoperatively, especially in men.

Reduced Expression of DKK3 Is Associated With Adverse Clinical Outcomes of Uterine Cervical Squamous Cell Carcinoma

The aim of this study was to assess the expression of DKK3 protein and its target, beta-catenin, in uterine cervical squamous cell carcinoma and to determine potential clinical correlations. Six carcinoma in-situ (CIS) tissues and 88 invasive cervical cancer tissues were included in the study. Twenty-two normal cervical tissues and one gastric cancer tissue were used as controls. The expression of DKK3 and beta-catenin proteins was evaluated by immunohistochemical analysis. Clinical and pathological parameters were obtained from medical records. Survival data were estimated using Kaplan-Meier estimates and compared with a log-rank test. Multivariate analysis was performed using the Cox regression method. DKK3 was predominantly present in the cytoplasm. Beta-catenin was observed only on the cellular membrane of both normal and cancer cells in contrast to earlier reports, in which beta-catenin was localized to the cytoplasm and nucleus of cancer cells. The expressions of beta-catenin and DKK3 were not correlated. Three of 6 CIS (50%) and 57 of 88 invasive cancer specimens (64.8%) had lower DKK3 expression than normal controls. DKK3 expression was decreased in a stage-dependent manner (P = 0.021). The patients with low expression of DKK3 were older than those with high expression of DKK3 (P < 0.01). Moreover, the patients with low DKK3 expression had a significantly lower 5-year disease-free survival rate than those with high DKK3 expression (P = 0.026). A multivariate analysis showed that International Federation of Gynecology and Obstetrics clinical stage and parametrial involvement were independent prognostic factors. Decreased DKK3 expression was associated with advanced International Federation of Gynecology and Obstetrics clinical stages and was predictive of lower disease-free survival in patients with cervical squamous cell carcinoma. DKK3 may be implicated in cervical carcinogenesis through a beta-catenin-independent mechanism.

Expression Profiling of Proliferation and Apoptotic Markers along the Adenoma-Carcinoma Sequence in Familial Adenomatous Polyposis Patients

Introduction. Familial adenomatous polyposis (FAP) patients have a germline mutation in the adenomatous polyposis coli (APC) gene. The APC protein interacts with beta-catenin, resulting in the activation of the Wnt signalling pathway. This results in alterations in cell proliferation and apoptosis. We investigated the expression of beta-catenin and related proliferation and apoptotic factors in FAP patients, exploring the expression along the adenoma-carcinoma sequence. Methods. The expression of beta-catenin, p53, bcl-2, cyclin-D1, caspase-3, CD10, and Ki-67 proteins was studied by immunohistochemistry in samples of colonic nonneoplastic mucosa (n = 71), adenomas (n = 152), and adenocarcinomas (n = 19) from each of the16 FAP patients. Results. The expression of beta-catenin, caspase-3, cyclin-D1, and Ki-67 was increased in both adenomas and carcinomas in FAP patients, compared with normal mucosa. p53 and CD10 expression was only slightly increased in adenomas, but more frequently expressed in carcinomas. Bcl-2 expression was increased in adenomas, but decreased in carcinomas. Conclusion. This is the first study investigating collectively the expression of these molecules together in nonneoplastic mucosa, adenomas, and carcinomas from FAP patients. We find that beta-catenin and related proliferative and apoptotic factors (cyclin-D1, bcl-2, caspase-3, and Ki-67) are expressed early in the sequence, in adenomas. However, p53 and CD10 are often expressed later in the sequence, in carcinomas.

Abstract P5-07-02: Analysis of a novel synergistic relationship between the FK506 binding protein FKBP52 and beta catenin in androgen receptor signaling pathways

Abstract Hormone-dependent cancers depend largely on folding and regulation by a chaperone-steroid receptor complex. The final stage of the receptor-chaperone complex consists of a receptor, a heat shock protein 90 (Hsp90) dimer, p23, and an immunophilin. Hormone-dependent prostate cancer progresses due to key interactions between the androgen receptor complex and the ligand dihydrotestosterone. While current treatments block androgen receptor-ligand interactions, these therapies are no longer effective in advanced stage, hormone independent prostate cancer (HRPC), when the receptor is activated even in miniscule levels of hormone. Therefore, we are interested in targeting other members of the androgen receptor complex and signaling cascade, namely the immunophilin FK506 binding protein (FKBP52) and the Wnt cell signaling protein β-catenin. FKBP52 has the ability to potentiate activity of the androgen, progesterone, and glucocorticoid receptors and is a prostate cancer biomarker. β-catenin is a member of the cellular adhesion complex with E-cadherin in healthy cells, but in aberrant situations, β-catenin actively promotes progression and invasion in a number of cancers. Our data from FKBP52 knockout mouse embryonic fibroblast cell lines suggests that FKBP52 and β-catenin work in tandem to synergize androgen receptor (AR) transcriptional activity with the probasin promoter. When AR, FKBP52, and β-catenin are co-transfected, a synergistic up-regulation of AR signaling is observed, as assessed by luciferase reporter assays. According to pull down assays, FKBP52 and β-catenin can interact in the absence of other proteins. Luciferase assays of mutant FKBP52 proteins suggest that FKBP52's binding to β-catenin involves the FKBP52 proline-rich loop and may act independently of Hsp90 binding. Interestingly, it appears that this synergistic effect is promoter-specific, suggesting that regulation by these proteins occurs at the transcriptional level. We have successfully abrogated the synergism of these proteins using the FKBP52-specific inhibitor MJC13, a small molecule developed for use in our laboratory. According to surface plasmon resonance studies, this molecule binds the androgen receptor at the BF-3 regulatory surface, the interaction surface for FKBP52 regulation of receptor. According to recent findings, AR, Wnt, and Human Epidermal Growth Factor Receptor 2 signaling pathways are linked in molecular apocrine breast cancer progression. We therefore hypothesize that our findings could be relevant to breast cancer as well as prostate cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-07-02.

Fap1 Controls Beta-Catenin Activity in a Gsk3-Beta Dependent Manner in Chronic Myeloid Leukemia.

Abstract 2404Fap1 (Fas-associated phosphatase 1) interacts with the C-terminus of multiple proteins via a PDZ-type protein-protein interaction domain. Interaction of Fap1 with Fas results in Fas-de-phosphorylation and inhibits Fas-induced apoptosis. In previous studies, we determined that PTPN13, the gene encoding Fap1, is a target gene for the interferon consensus sequence binding protein (referred to as Icsbp or Irf8). Icsbp functions as a leukemia suppressor in chronic myeloid leukemia (CML), and we previously demonstrated Icsbp/Fap1-dependent apoptosis resistance in cells expressing the CML-associated oncoprotein, Bcr-abl. Another known interaction partner for Fap1 is the adenomatous polyposis coli protein (Apc). However, the functional significance of this interaction has not been previously explored. In the current stuides, we investigate whether interaction between Fap1 and Apc impacts the pathogenesis of CML. Apc is involved in assembly of a multi-protein complex that includes Apc, Axin, Gsk3-beta and beta-catenin. Association of these proteins results in serine/threonine phosphorylation (pS/T) of beta-catenin by Gsk3-beta. pS/T beta-catenin is released from the complex and degraded by the proteasome. Previous studies demonstrated that pS/T of beta-catenin, and consequent beta-catenin degradation, is impaired in CML, but the mechanism for this impairment is unknown in most cases. We find that Icsbp-dependent increase in Fap1 results in increased interaction between Fap1 and Apc in Bcr-abl+ myeloid progenitor cells. We also find that increased Fap1/Apc interaction is associated with inactivation (de-phosphorylation) of Gsk3-beta and a decrease in inhibitory pS/T of βcatenin. We find that this results in increased beta-catenin protein and activity. We also find that Fap1 co-immuno-precipitates with Apc, Axin, Gsk3-beta and beta-catenin. This result suggests the possibility that Gsk3-beta is a substrate for Fap1 protein tyrosine phosphatase activity. Consistent with this hypothesis, we find that Fap1 is able to dephosphorylate Gsk3-beta in vitro. We tested the functional significance of this observation in experiments using an activated form of Gsk3-beta that cannot be dephosphorylated at a key residue (Y216D-Gsk3-beta). We find that expression of this Gsk3-beta mutant partly reverses the effect of Bcr-abl and Fap1 on beta-catenin protein stability and activity. Increased beta-catenin activity is a characteristic of leukemia stem cells (LSC) in CML, and increasing beta-catenin activity in the bone marrow is associated with poor prognosis in CML. Therefore, these studies identify a novel pathway that regulates events significant to CML pathogenesis. These studies also identify a role for Apc in the pathogenesis of leukemia, and another mechanism for the leukemia suppressor effect of Icsbp. Disclosures:No relevant conflicts of interest to declare.

Variation of medium osmolalities induces changes in cilia related signalling pathways in primary cultured renal inner medullary collecting duct cells

Compared to other organs the cells of the renal inner medulla are challenged with an environment of highly variable osmolality. Cellular adaption to changing osmolality is associated with changes in gene expression. To identify novel genes and pathways that are affected by high osmolality we performed microarray experiments using primary cultured rat inner medullary collecting duct cells cultivated at 300, 600, or 900 mosmol/kg for six days. Compared to 300 mosmol/kg more than 2000 genes showed significant changes in expression at 600 or 900 mosmol/kg. Pathway analysis revealed that the WNT/beta-catenin pathway was also affected. Western blot analysis confirmed the down regulation of beta-catenin and of phosphorylated beta-catenin protein. Immunofluorescence analysis indicated massive changes in intracellular localization of beta-catenin. At 300 mosmol/kg beta-catenin was distributed diffusively within the cells. At higher osmolalities beta-catenin was concentrated at the cell to cell contacts and in the nucleus. Similar effects were observed for phosphorylated beta-catenin. We also observed striking changes in cell morphology. While at 300 mosmol/kg the actin filaments were diffusively distributed the cultivation at 600 mosmol/kg was associated with massive reorganisation and enrichment of the actin filaments at the cell to cell contacts. Staining of the microtubules with beta-tubulin clearly showed the primary cilia in cells cultivated at 300 mosmol/kg. Surprisingly the length of the cilia was drastically reduced in cells cultivated at 600 mosmol/kg. The analysis of the underlying physiological mechanisms and the consequences for cilia related signalling at different osmolalities could help to add new aspects to cilia function.

377 Selective Inhibition of AKT Protein Isoforms Augments Gefitinib Response in EGFR Mutated NSCLC Cells

Introduction: Genetic changes occurring in the triple negative (TN) subset of breast tumors (BT) contribute to different phenotypes in the tumor cells via deregulation of specific signaling-pathways. We identified a differential upregulation of the Wnt-beta-catenin pathway in TNBT (Barwick etal, 2008; Dey etal. 2009), and reported that in TNBT, the poor prognosis and the higher incidence of metastasis are associated with an activation of this pathway (Barwick etal., 2009). We also reported a role of the pathway in the control of migration and invasion of tumor cells (Dey etal. 2009, 2010). Since integrin-directed migration and invasion of the tumor cells are instrumental for the metastasis, and TN tumor cells are highly invasive in nature, we hypothesized that the upregulation of this pathway in TNBT is functionally connected to the control of metastasis-associated phenotypes. This hypothesis is further strengthened by our identification of MMP7 as a selective-biomarker of TNBT (Abramovitz etal, 2009). Purpose: We studied the involvement of the pathway in the control of metastasis-associated phenotypes in TN breast cancer (BC) cells. Methods: We tested the effects of (1) different chemical modulators of the Wnt signaling, and (2) downregulation of beta-catenin on the (a) cell signaling marker(s), (b) cell proliferation, (c) clonogenic growth, (d) migration, (e) invasion, and (f) vascular mimicry (VM) using a panel of six TNBT cells. Results: (1) CHIR99021 (selective GSK3b inhibitor which activates Wnt signaling) enhanced clonogenic growth, and VM while WntC59 (palmitylates Wnt proteins and blocks the Wnt secretion/activity), as well as XAV939 (tankyrase inhibitor that blocks beta-catenin-mediated transcription via axin stabilization) blocked clonogenic growth, cell proliferation, fibronectindirected migration, and VM in TNBT cells. (2) Sulindac sulfide (decreases cellular levels of total and active beta-catenin) inhibited migration, invasion, VM, actin cytoskeleton, podia-parameters, RAC1/Cdc42 activation, and MMP7 in TNBC cells. (3) SiRNA mediated downregulation of betacatenin protein in TNBC cells inhibited cell proliferation, clonogenic growth, migration, invasion, c-MYC, and VM. Conclusion: Wnt-beta-catenin pathway activates cellular signaling for the metastasis-associated phenotypes like proliferation, clonogenic growth, migration, invasion and VM in TNBT cells and this function is directly mediated by via cellular levels of beta-catenin. An inhibition of the Wntbeta-catenin pathway might be an effective combination treatment strategy in TNBC.

Vitamin D3 regulates beta-catenin protein expression in human fibroid cells: implications for MED12 role in fibroid pathogenesis

Uterine fibroids are the most common benign tumors in the reproductive age women and hysterectomy is the main effective treatment for uterine fibroids till now. African American women have four times higher risk of uterine fibroids and they have ten times more hypovitaminosis D than their counterpart white women. We recently found that the mediator complex 12 (Med12) gene exon 2 somatic mutations in more than 70% cases in USA women with uterine fibroids. Med12 gene exon 2 somatic mutations are associated with uterine fibroid tumorigenicity and activation of Med12 gene regulates Wnt/beta-catenin pathways.

Chemoprevention of azoxymethane-initiated colon cancer in rat by using a novel polymeric nanocarrier–curcumin

Curcumin is a potential natural anticancer drug with limited bioavailability due to the lack of solubility in aqueous solvents. The present study is designed to investigate the preventive effects of polymeric nanocarrier–curcumin (PNCC) on colon carcinogenesis in an azoxymethane-induced rat tumor. Forty rats were divided into control, curcumin- and PNCC-treated groups. Animals received azoxymethane (AOM) as a carcinogenic agent (15mg/kg, s.c.) weekly for two consecutive weeks. They were given curcumin 0.2% and PNCC two weeks before till 14 weeks after the last injection of AOM. In the end, post euthanasia, the entire gastrointestinal tract was scrutinized for tumors, and the rest of the body for metastatic deposits. Tumor number, size and location were characterized. The histopathological and immunohistochemistry examinations were also performed on colon tissue. In vivo, curcumin nanoparticles inhibited colon cancer growth in animal model. The tumors incidence and number decreased by nanocurcumin comparison with control. Furthermore, the nuclear/cytoplasmic ratio, epithelial stratification, nuclear dispolarity, goblet depletion, structural abnormality, and the expression of Beta-catenin and Bcl-2 proteins were reduced in PNCC compared to others groups (P<0.05). In addition, Bax protein expression was significantly increased in PNCC in comparison with control and curcumin-treated groups (P<0.001). The present study demonstrated the potential anticancer effects of PNCC in a typical animal model. The results provide evidence that nanopolymeric curcumin exerts a significant chemopreventive effect on AOM-initiated colon cancer through cell proliferation inhibition and apoptosis induction. More investigations are needed to confirm its safety for human use.

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