Beta-catenin Transcriptional Activity Research Articles

A second protein kinase CK1-mediated step negatively regulates Wnt signalling by disrupting the lymphocyte enhancer factor-1/beta-catenin complex.

Deregulated activation of the canonical Wnt signalling pathway leads to stabilization of beta-catenin and is critically involved in carcinogenesis by an inappropriate induction of lymphocyte enhancer factor (LEF-1)/beta-catenin-dependent transcription of Wnt target genes. Phosphorylation of the pathway components beta-catenin, Dishevelled, Axin and APC (adenomatous polyposis coli) by glycogen synthase kinase-3beta, CK1 and CK2 is of central importance in the regulation of the beta-catenin destruction complex. Here, we identify CK1 and CK2 as major kinases that directly bind to and phosphorylate LEF-1 inducing distinct, kinase-specific changes in the LEF-1/DNA complex. Moreover, CK1-dependent phosphorylation in contrast to CK2 disrupts the association of beta-catenin and LEF-1 but does not impair DNA binding of LEF-1. Sequential phosphorylation assays revealed that for efficient disruption of the LEF-1/beta-catenin complex, beta-catenin also has to be phosphorylated. Consistent with these observations, CK1-dependent phosphorylation inhibits, whereas CK2 activates LEF-1/beta-catenin transcriptional activity in reporter gene assays. These data are in line with a negative regulatory function of CK1 in the Wnt signalling pathway, where CK1 in addition to the beta-catenin destruction complex at a second level acts as a negative regulator of the LEF-1/beta-catenin transcription complex, thereby protecting cells from development of cancer.

Down-regulation of beta catenin inhibits the growth of esophageal carcinoma cells

Introduction Esophageal cancer remains a highly lethal malignancy, with therapeutic options of limited efficacy in the majority of patients. Understanding the molecular events involved in the pathogenesis of esophageal cancer offers insight into potential targets for treatment. Beta catenin and Wnt signaling abnormalities are involved in the development of both adenocarcinoma and squamous carcinoma of the esophagus. We hypothesized that down-regulation of beta catenin would inhibit the growth of human esophageal cancer. Methods A human esophageal squamous cell carcinoma cell line (TE10) was treated with phosphorothioate antisense oligonucleotides to beta catenin. The cells were subsequently assayed for beta catenin mRNA and protein by real-time polymerase chain reaction and Western blot. Beta catenin transcriptional activity was determined by TOPFlash assay. Cell viability and growth was assessed by methyl-thiazol-diphenyl-tetrazolium assay and trypan blue exclusion. A colorimetric assay was employed to assess caspase 3 activity, and flow cytometry was done to determine percentage of cells in a given phase of the cell cycle. Results Following antisense treatment, beta catenin mRNA and protein concentration were decreased. There was corresponding decrease in beta catenin–transcription factor–dependent transcription. Treatment with beta catenin antisense resulted in significantly decreased cell viability and proliferation. The mechanism appears to be increased induction of apoptosis. Conclusions These data suggest a potential role for the targeting of beta catenin in the treatment of esophageal cancer.

Beta-catenin signaling in prostate cancer: an early perspective.

Further understanding of the molecular mechanisms responsible for prostate cancer (CaP) development and progression is paramount for overcoming the current diagnostic and therapeutic hurdles presented by this urologic disease. The beta-catenin nuclear signaling molecule has been widely implicated as an oncogene in human cancer, including CaP. Pooling together knowledge gathered on the contributions of beta-catenin and other factors to human neoplasia may assist in the development of better strategies for management and treatment of prostate tumors of all stages (early, advanced/androgen-dependent, advanced/androgen-independent). Although there is considerable lack of comprehension regarding the function of beta-catenin transcriptional activity in prostate tumors in vivo, recent evidence indicates the probability that beta-catenin contributes to multiple signaling pathways for which a causative role in CaP is already known. In this review, we will approach such pathway interactions, perhaps the most notable being androgen receptor (AR) signaling, in order to highlight those avenues through which beta-catenin may exert its cancer-related function. To the same end, we will draw attention to normal beta-catenin signaling in the prostate; however, as only very limited knowledge exists on this topic, much of the discussion will be correlative. Our final topic will concentrate on how, given realistic scenarios of androgen stimulation or absence in both normal and neoplastic prostate cells, nuclear beta-catenin may ultimately potentiate wnt cell-cell signaling and AR activities. Heightening our comprehension of beta-catenin signaling mechanisms and its phenotypic consequences in CaP - and in normal prostate - may contribute to that body of knowledge which will eventually prove useful for devising more effective therapies.

The PDZ Protein Tax-interacting Protein-1 Inhibits β-Catenin Transcriptional Activity and Growth of Colorectal Cancer Cells

Wnt signaling is essential during development while deregulation of this pathway frequently leads to the formation of various tumors including colorectal carcinomas. A key component of the pathway is beta-catenin that, in association with TCF-4, directly regulates the expression of Wnt-responsive genes. To identify novel binding partners of beta-catenin that may control its transcriptional activity, we performed a mammalian two-hybrid screen and isolated the Tax-interacting protein (TIP-1). The in vivo complex formation between beta-catenin and TIP-1 was verified by coimmunoprecipitation, and a direct physical association was revealed by glutathione S-transferase pull-down experiments in vitro. By using a panel of deletion mutants of both proteins, we demonstrate that the interaction is mediated by the PDZ (PSD-95/DLG/ZO-1 homology) domain of TIP-1 and requires primarily the last four amino acids of beta-catenin. TIP-1 overexpression resulted in a dose-dependent decrease in the transcriptional activity of beta-catenin when tested on the TOP/FOPFLASH reporter system. Conversely, siRNA-mediated knock-down of endogenous TIP-1 slightly increased endogenous beta-catenin transactivation function. Moreover, we show that overexpression of TIP-1 reduced the proliferation and anchorage-independent growth of colorectal cancer cells. These data suggest that TIP-1 may represent a novel regulatory element in the Wnt/beta-catenin signaling pathway.

E-Cadherin Mediates MMP Down-Regulation in Highly Invasive Bronchial Tumor Cells

The disorganization of E-cadherin/catenin complexes and the overexpression of matrix metalloproteinases (MMPs) are frequently involved in the capacity of epithelial cells to acquire an invasive phenotype. The functional link between E-cadherin and MMPs was studied by transfecting invasive bronchial BZR tumor cells with human E-cadherin cDNA. Using different in vitro (cell dispersion, modified Boyden chamber) and in vivo assays (human airway epithelial xenograft), we showed that E-cadherin-positive clones displayed a decrease of invasive abilities. As shown by immunoprecipitation, the re-expressed E-cadherin was able to sequestrate one part of free cytoplasmic beta-catenin in BZR cells. The decrease of beta-catenin transcriptional activity in E-cadherin-transfected clones was demonstrated using the TOP-FLASH reporter construct. Finally, we observed a decrease of MMP-1, MMP-3, MMP-9, and MT1-MMP, both at the mRNA and at the protein levels, in E-cadherin-positive clones whereas no changes in MMP-2, TIMP-1, or TIMP-2 were observed when compared with control clones. Moreover, zymography analysis revealed a loss of MMP-2 activation ability in E-cadherin-positive clones treated with the concanavalin A lectin. These data demonstrate a direct role of E-cadherin/catenin complex organization in the regulation of MMPs and suggest an implication of this regulation in the expression of an invasive phenotype by bronchial tumor cells.

Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors.

Wntbeta-catenin signaling plays key roles in several developmental and pathological processes. Domains of Wnt expression have been extensively investigated in the mouse, but the tissues receiving the signal remain largely unidentified. To define which cells respond to activated beta-catenin during mammalian development, we generated the beta-catenin-activated transgene driving expression of nuclear beta-galactosidase reporter (BAT-gal) transgenic mice, expressing the lacZ gene under the control of beta-cateninT cell factor responsive elements. Reporter gene activity is found in known organizing centers, such as the midhindbrain border and the limb apical ectodermal ridge. Moreover, BAT-gal expression identifies novel sites of Wnt signaling, like notochord, endothelia, and areas of the adult brain, revealing an unsuspected dynamic pattern of beta-catenin transcriptional activity. Expression of the transgene was analyzed in mutant backgrounds. In lipoprotein receptor-related protein 6-null homozygous mice, which lack a Wnt coreceptor, BAT-gal staining is absent in mutant tissues, indicating that BAT-gal mice are bona fide in vivo indicators of Wntbeta-catenin signaling. Analyses of BAT-gal expression in the adenomatous polyposis coli (multiple intestinal neoplasia+) background revealed betacatenin transcriptional activity in intestinal adenomas but surprisingly not in normal crypt cells. In summary, BAT-gal mice unveil the entire complexity of Wntbeta-catenin signaling in mammals and have broad application potentials for the identification of Wnt-responsive cell populations in development and disease.

Regulation of the chondrocyte phenotype by beta-catenin

beta-Catenin regulates important biological processes, including embryonic development and tumorigenesis. We have investigated the role of beta-catenin in the regulation of the chondrocyte phenotype. Expression of beta-catenin was high in prechondrogenic mesenchymal cells, but significantly decreased in differentiated chondrocytes both in vivo and in vitro. Accumulation of beta-catenin by the inhibition of glycogen synthase kinase-3beta with LiCl inhibited chondrogenesis by stabilizing cell-cell adhesion. Conversely, the low level of beta-catenin in differentiated articular chondrocytes was increased by post-translational stabilization during phenotypic loss caused by a serial monolayer culture or exposure to retinoic acid or interleukin-1beta. Ectopic expression of beta-catenin or inhibition of beta-catenin degradation with LiCl or proteasome inhibitor caused de-differentiation of chondrocytes. Transcriptional activation of beta-catenin by its nuclear translocation was sufficient to cause phenotypic loss of differentiated chondrocytes. Expression pattern of Jun, a known target gene of beta-catenin, is essentially the same as that of beta-catenin both in vivo and in vitro suggesting that Jun and possibly activator protein 1 is involved in the beta-catenin regulation of the chondrocyte phenotype.

Wnt signaling in the ovary: identification and compartmentalized expression of wnt-2, wnt-2b, and frizzled-4 mRNAs.

Ovarian cadherins, in addition to acting as structural (adhesion) molecules, also function as modulators of gene activity. The dual role of beta-catenin as an intracellular component of the cadherin adhesion complex and as a transcription factor provides a possible explanation for these cadherin effects. Because the transcriptional activity of beta-catenin is dependent on activation by the wnt signaling cascade, we examined whether components of this cascade are expressed in the rat ovary. Using RT-PCR with degenerate primers on RNA from ovaries of hormone-stimulated immature rats, we identified transcripts for wnt-2 and wnt-2b. RT-PCR and in situ hybridization (ISH) demonstrated that granulosa cells express wnt-2 mRNA. Because the sequence for rat wnt-2b has not been reported, we obtained additional sequence by screening a rat ovarian cDNA library. RT-PCR analysis, using primers designed from this wnt-2b cDNA sequence, failed to detect transcripts in the ovarian follicular compartment (granulosa and oocyte). ISH revealed that the ovarian surface epithelium expresses wnt-2b mRNA. Using a similar degenerate RT-PCR approach, we detected expression of a putative wnt receptor, frizzled-4 (fzd-4), and a cytoplasmic component of the wnt signaling cascade, disheveled-2 (dsh-2), in the rat ovary. Further analyses using both RT-PCR and ISH indicated that granulosa cells express fzd-4 mRNA. The expression of wnt-2b transcripts in rat ovarian surface epithelium prompted us to examine whether the homologous gene is expressed in human ovarian cancer cell lines. RT-PCR, using degenerate and specific primers for wnts, on RNA from five ovarian cancer cell lines confirmed the expression of transcripts for wnt-2b. Two additional wnt transcripts (wnt-5a and wnt-11) were detected in the cancer cell lines and in the rat ovary. These results demonstrate that transcripts corresponding to components of the wnt signaling cascade are expressed in the immature rat ovary. The localization of these transcripts in specific ovarian compartments suggests that this signal transduction pathway may be involved in follicular development and ovarian function. Furthermore, because wnts have been implicated in the oncogenic transformation of epithelial cells, our results raise the possibility that aberrant wnt expression may be involved in ovarian tumorigenesis in humans.

Presenilin 1 Independently Regulates β-Catenin Stability and Transcriptional Activity

Presenilin 1 (PS1) regulates beta-catenin stability; however, published data regarding the direction of the effect are contradictory. We examined the effects of wild-type and mutant forms of PS1 on the membrane, cytoplasmic, nuclear, and signaling pools of endogenous and exogenous beta-catenin by immunofluorescence microscopy, subcellular fractionation, and in a transcription assay. We found that PS1 destabilizes the cytoplasmic and nuclear pools of beta-catenin when stabilized by Wnt or Dvl but not when stabilized at lower levels of the Wnt pathway. The PS1 mutants examined were less able to reduce the stability of beta-catenin. PS1 also inhibited the transcriptional activity of endogenous beta-catenin, and the PS1 mutants were again less inhibitory at the level of Dvl but showed a different pattern of inhibition toward transcription below Dvl. The transcriptional activity of exogenously expressed wild-type beta-catenin and two mutants, DeltaN89beta-catenin and DeltaSTbeta-catenin, were also inhibited by wild-type and mutant PS1. We conclude that PS1 negatively regulates the stability and transcriptional activity of beta-catenin at different levels in the Wnt pathway, that the effect on transcriptional activity appears to be independent of the GSK-3beta mediated degradation of beta-catenin, and that mutations in PS1 differentially affect the stability and transcriptional activity of beta-catenin.

Patterning and Nuclear β-Catenin Expression in the Colonic Adenoma-Carcinoma Sequence: Analogies with Embryonic Gastrulation

Patterning is a process by which ordered arrangements of cells and tissue structure are attained. The term derived from developmental biology is also useful for the study of colonic carcinogenesis, in which the patterning of neoplastic tubules is necessary for properties of growth, invasion, and metastasis. Interestingly the nuclear expression and transcriptional activity of beta-catenin, a major oncoprotein in colonic carcinogenesis, is decisive for the first patterning of a tubule in embryogenesis, which creates the primitive gut and is called the gastrulation. Thus, basic patternings of embryogenesis and carcinogenesis might be linked. To test this hypothesis we compared morphological patterns and immunohistochemical beta-catenin stainings in colonic adenomas and adenocarcinomas with the gastrulation steps. Two analogies were found: 1) the patterning of invasion with reconstruction in adenocarcinomas corresponded to the epithelio-mesenchymal transition, ingression, and rearrangement of cells during the first phase of gastrulation; and 2) the patterning of tubular branching in adenomas and adenocarcinomas resembled the endodermal invagination during the second phase. The intratumorous distribution and intensity of nuclear beta-catenin expression was significantly correlated with the two patternings, similar to the findings in gastrulation. The results indicate microenvironmental regulations of nuclear beta-catenin expression and a return of neoplastic cells to embryonic transcriptional susceptibilities during colonic carcinogenesis.

Restricted High Level Expression of Tcf-4 Protein in Intestinal and Mammary Gland Epithelium

Tcf-4 is a member of the Tcf/Lef family of transcription factors that interact functionally with beta-catenin to mediate Wnt signaling in vertebrates. We have previously demonstrated that the tumor suppressor function of APC in the small intestine is mediated via regulation of Tcf-4/beta-catenin transcriptional activity. To gain further insight into the role of Tcf-4 in development and carcinogenesis we have generated several mouse monoclonal antibodies, one of which is specific for Tcf-4 and another of which recognizes both Tcf-3 and Tcf-4. Immunohistochemistry performed with the Tcf 4- specific monoclonal antibody revealed high levels of expression in normal intestinal and mammary epithelium and carcinomas derived therefrom. Additional sites of Tcf-3 expression, as revealed by staining with the Tcf-3/-4 antibody, occurred only within the stomach epithelium, hair follicles, and keratinocytes of the skin. A temporal Tcf-4 expression gradient was observed along the crypt-villus axis of human small intestinal epithelium: strong Tcf-4 expression was present within the crypts of early (week 16) human fetal small intestine, with the villi showing barely detectable Tcf-4 protein levels. Tcf-4 expression levels increased dramatically on the villi of more highly developed (week 22) fetal small intestine. We conclude that Tcf-4 exhibits a highly restricted expression pattern related to the developmental stage of the intestinal epithelium. The high levels of Tcf-4 expression in mammary epithelium and mammary carcinomas may also indicate a role in the development of this tissue and breast carcinoma.