Stabilization of beta-catenin in the mouse zygote leads to premature epithelial-mesenchymal transition in the epiblast

Many components of the Wnt/beta-catenin signaling pathway are expressed during mouse pre-implantation embryo development, suggesting that this pathway may control cell proliferation and differentiation at this time. We find no evidence for a functional activity of this pathway in cleavage-stage embryos using the Wnt-reporter line, BAT-gal. To further probe the activity of this pathway, we activated beta-catenin signaling by mating a zona pellucida3-cre (Zp3-cre) transgenic mouse line with a mouse line containing an exon3-floxed beta-catenin allele. The result is expression of a stabilized form of beta-catenin, resistant to degradation by the GSK3beta-mediated proteasome pathway, expressed in the developing oocyte and in each cell of the resulting embryos. Nuclear localization and signaling function of beta-catenin were not observed in cleavage-stage embryos derived from these oocytes. These results indicate that in pre-implantation embryos, molecular mechanisms independent of the GSK3beta-mediated ubiquitination and proteasome degradation pathway inhibit the nuclear function of beta-catenin. Although the mutant blastocysts initially developed normally, they then exhibited a specific phenotype in the embryonic ectoderm layer of early post-implantation embryos. We show a nuclear function of beta-catenin in the mutant epiblast that leads to activation of Wnt/beta-catenin target genes. As a consequence, cells of the embryonic ectoderm change their fate, resulting in a premature epithelial-mesenchymal transition.     

Nuclear beta-catenin localization is not sufficient for canonical Wnt signaling activation in human meianoma cell lines

In most cases, advanced stages of melanoma are practically incurable due to high metastatic potential of tumor cells. Multiple observations support the idea that aberrations in Wnt signaling pathway play a significant role in melanoma development and progression. Canonical Wnt signaling activation results in stabilization and accumulation of the major effector molecule called beta-catenin. Mutations promoting beta-catenin stabilization and, thereby, activation of canonical Wnt signaling pathway are frequently found in different cancers, but rarely observed in melanomas. Nevertheless, beta-catenin nuclear and cytoplasmic accumulation is the feature of many human melanoma cell lines and original tumors. That is why, the aim of the investigation was to elucidate the relation between beta-catenin intracellular localization and activity status of Wnt signaling pathway in human melanoma cell lines. Ten human melanoma cell lines were characterized on the basis of the following parameters: canonical Wnt ligand expression, intracellular beta-catenin localization, and activity status of canonical Wnt signaling pathway. Here, it has been demonstrated that nuclear localization of beta-catenin does not always correspond to active status canonical Wnt signaling pathway. Moreover, in the majority of cell lines with nuclear beta-catenin canonical Wnt signaling can't be activated by exogenous expression of an appropriate ligand. Human melanoma cell lines differ in activity of canonical Wnt signaling pathway as well as in mechanisms of its regulation. Therefore, the pathway-targeted potential antineoplastic therapy requires the formation of a "molecular pattern of cancer" for localization of the defect in Wnt signaling cascade in the each case.     

Casein kinase Iepsilon modulates the signaling specificities of dishevelled

Wnt signaling is critical to many aspects of development, and aberrant activation of the Wnt signaling pathway can cause cancer. Dishevelled (Dvl) protein plays a central role in this pathway by transducing the signal from the Wnt receptor complex to the beta-catenin destruction complex. Dvl also plays a pivotal role in the planar cell polarity pathway that involves the c-Jun N-terminal kinase (JNK). How functions of Dvl are regulated in these two distinct pathways is not clear. We show that deleting the C-terminal two-thirds of Dvl, which includes the PDZ and DEP domains and is essential for Dvl-induced JNK activation, rendered the molecule a much more potent activator of the beta-catenin pathway. We also found that casein kinase Iepsilon (CKIepsilon), a previously identified positive regulator of Wnt signaling, stimulated Dvl activity in the Wnt pathway, but dramatically inhibited Dvl activity in the JNK pathway. Consistent with this, overexpression of CKIepsilon in Drosophila melanogaster stimulated Wnt signaling and disrupted planar cell polarity. We also observed a correlation between the localization and the signaling activity of Dvl in the beta-catenin pathway and the JNK pathway. Furthermore, by using RNA interference, we demonstrate that the Drosophila CKIepsilon homologue Double time positively regulates the beta-catenin pathway through Dvl and negatively regulates the Dvl-induced JNK pathway. We suggest that CKIepsilon functions as a molecular switch to direct Dvl from the JNK pathway to the beta-catenin pathway, possibly by altering the conformation of the C terminus of Dvl.     

Dapper1 is a nucleocytoplasmic shuttling protein that negatively modulates Wnt signaling in the nucleus

Wnt signaling, via the activation of the canonical beta-catenin and lymphoid enhancer factor (LEF)/T-cell factor pathway, plays an important role in embryogenesis and cancer development by regulating the expression of genes involved in cell proliferation, differentiation, and survival. Dapper (Dpr), as a Dishevelled interactor, has been suggested to modulate Wnt signaling by promoting Dishevelled degradation. Here, we provide evidence that Dpr1 shuttles between the cytoplasm and the nucleus. Although overexpressed Dpr1 was mainly found in the cytoplasm, endogenous Dpr1 was localized over the cell, and Wnt1 induced its nuclear export. Treatment with leptomycin B induced nuclear accumulation of both endogenous and overexpressed Dpr1. We further identified the nuclear localization signal and the nuclear export signal within Dpr1. Using reporter assay and in vivo zebrafish embryo assay, we demonstrated that the forced nuclearly localized Dpr1 possessed the ability to antagonize Wnt signaling. Dpr1 interacted with beta-catenin and LEF1 and disrupted their complex formation. Furthermore, Dpr1 could associate with histone deacetylase 1 (HDAC1) and enhance the LEF1-HDAC1 interaction. Together, our findings suggest that Dpr1 negatively modulates the basal activity of Wnt/beta-catenin signaling in the nucleus by keeping LEF1 in the repressive state. Thus, Dpr1 controls Wnt/beta-catenin signaling in both the cytoplasm and the nucleus.     

Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm

The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Here, defective axis conversion due to Otx2 deficiency can be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the beta-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of beta-catenin localization is impaired in the Otx2-deficient and Wnt-misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. We propose that Wnt/beta-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo.     

Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling

Canonical Wnt signaling critically regulates cell fate and proliferation in development and disease. Nuclear localization of beta-catenin is indispensable for canonical Wnt signaling; however, the mechanisms governing beta-catenin nuclear localization are not well understood. Here we demonstrate that nuclear accumulation of beta-catenin in response to Wnt requires Rac1 activation. The role of Rac1 depends on phosphorylation of beta-catenin at Ser191 and Ser605, which is mediated by JNK2 kinase. Mutations of these residues significantly affect Wnt-induced beta-catenin nuclear accumulation. Genetic ablation of Rac1 in the mouse embryonic limb bud ectoderm disrupts canonical Wnt signaling and phenocopies deletion of beta-catenin in causing severe truncations of the limb. Finally, Rac1 interacts genetically with beta-catenin and Dkk1 in controlling limb outgrowth. Together these results uncover Rac1 activation and subsequent beta-catenin phosphorylation as a hitherto uncharacterized mechanism controlling canonical Wnt signaling and may provide additional targets for therapeutic intervention of this important pathway.     

Epigenetic alterations of CDH1 and APC genes: relationship with activation of Wnt/beta-catenin pathway in invasive ductal carcinoma of breast

Activation of canonical Wnt/beta-catenin pathway in Invasive Ductal Carcinoma of Breast (IDCs) was recently reported from our laboratory. Herein, we analyzed promoter methylation status of CDH1 and Adenomatous polyposis coli (APC) genes in 50 IDCs and correlated with expression of E-cadherin (E-CD) and APC proteins and with activation of oncogenic Wnt/beta-catenin signaling pathway components, Dvl, beta-catenin and CyclinD1. Further, Wnt/beta-catenin driven epithelial mesenchymal transition (EMT) was investigated by correlating the expression of Dvl, beta-catenin and CyclinD1 with vimentin expression in these IDCs. Promoter hypermethylation was observed in 25/50 (50%) IDCs for CDH1 and in 11/50 (22%) tumors for APC, associated with loss of expression of E-CD and APC proteins; concordant hypermethylation of these genes was observed in paired patients' sera. Further, 57% of tumors harboring CDH1 methylation and 50% tumors harboring the methylated APC gene showed nuclear localization of beta-catenin, suggesting activation of the canonical Wnt/beta-catenin pathway. Our study demonstrates significant association between vimentin expression and nuclear beta-catenin (p=0.001; Odds ratio (OR)=25.6) and Dvl (p=0.023; OR=8.0), suggesting that EMT may be driven by Wnt/beta-catenin activation in IDCs. In conclusion, we demonstrate correlation of CDH1 and APC promoter methylation with loss of E-CD and APC proteins and with activation of Wnt/beta-catenin signaling pathway. Association of nuclear Dvl and beta-catenin with vimentin expression suggests the importance of Wnt/beta-catenin pathway driven EMT in IDCs. The concordance between CDH1 and APC methylation in IDCs and paired circulating DNA underscores the utility of serum DNA as a non-invasive tool for methylation analysis in IDC patients.     

Nuclear localization of beta-catenin in vegetal pole cells during early embryogenesis of the starfish Asterina pectinifera

Recently, beta-catenin has been reported to control the expression of morphogenetic genes through the Wnt signaling pathway in invertebrate embryogenesis. In this study, the distribution pattern of beta-catenin during starfish embryogenesis was investigated using immunohistochemistry. In 16-cell stage embryos, beta-catenin began to accumulate in some nuclei at the vegetal pole. During the early cleavage stage, the cells expressing nuclear beta-catenin increased in number in the vegetal pole region of the embryos, and the beta-catenin signal increased in intensity in each nucleus. At the blastula stage, signal for beta-catenin was also found in the cytoplasm of the cells with nuclear beta-catenin. At the vegetal plate stage, almost all vegetal plate cells expressed beta-catenin in both the nucleus and cytoplasm. When the embryos developed to early gastrulae, cells with nuclear beta-catenin were restricted to the archenteron tip, and the signal gradually faded in later stages. The localization and temporal change of beta-catenin expression suggests that beta-catenin has a pivotal role in archenteron formation in starfish embryos.     

A transgenic Lef1/beta-catenin-dependent reporter is expressed in spatially restricted domains throughout zebrafish development.

The Wnt/beta-catenin signaling pathway plays multiple roles during embryonic development, only a few of which have been extensively characterized. Although domains of Wnt expression have been identified throughout embryogenesis, anatomical and molecular characterization of responding cells has been mostly unexplored. We have generated a transgenic zebrafish line that expresses a destabilized green fluorescent protein (GFP) variant under the control of a beta-catenin responsive promoter. Early zygotic expression of this transgene (TOPdGFP) mirrors known domains of Wnt signaling in the embryo. Loss of Lef1 activity results in decreased reporter expression and posterior defects, while loss of Tcf3 (Headless, Hdl) activity does not alter reporter expression, even though it results in loss of forebrain structures. In addition, ectopic Wnt1 expression can activate the reporter. In older embryos, we identify a number of transgene-expressing cell populations as novel sites of beta-catenin signaling. We conclude that our TOP-dGFP reporter line faithfully illustrates domains of beta-catenin activity and enables the identification of responsive cell populations.     

HDAC6 is required for epidermal growth factor-induced beta-catenin nuclear localization

Nuclear translocation of beta-catenin is a hallmark of Wnt signaling and is associated with various cancers. In addition to the canonical Wnt pathway activated by Wnt ligands, growth factors such as epidermal growth factor (EGF) also induce beta-catenin dissociation from the adherens junction complex, translocation into the nucleus, and activation of target genes such as c-myc. Here we report that EGF-induced beta-catenin nuclear localization and activation of c-myc are dependent on the deacetylase HDAC6. We show that EGF induces HDAC6 translocation to the caveolae membrane and association with beta-catenin. HDAC6 deacetylates beta-catenin at lysine 49, a site frequently mutated in anaplastic thyroid cancer, and inhibits beta-catenin phosphorylation at serine 45. HDAC6 inactivation blocks EGF-induced beta-catenin nuclear localization and decreases c-Myc expression, leading to inhibition of tumor cell proliferation. These results suggest that EGF-induced nuclear localization of beta-catenin is regulated by HDAC6-dependent deacetylation and provide a new mechanism by which HDAC inhibitors prevent tumor growth.