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.     

The mitogen-activated protein kinase kinase kinase kinase GCKR positively regulates canonical and noncanonical Wnt signaling in B lymphocytes

Wnt ligands bind receptors of the Frizzled (Fz) family to control cell fate, proliferation, and polarity. Canonical Wnt/Fz signaling stabilizes beta-catenin by inactivating GSK3beta, leading to the translocation of beta-catenin to the nucleus and the activation of Wnt target genes. Noncanonical Wnt/Fz signaling activates RhoA and Rac, and the latter triggers the activation of c-Jun N-terminal kinase (JNK). Here, we show that exposure of B-lymphocytes to Wnt3a-conditioned media activates JNK and raises cytosolic beta-catenin levels. Both the Rac guanine nucleotide exchange factor Asef and the mitogen-activated protein kinase kinase kinase kinase germinal center kinase-related enzyme (GCKR) are required for Wnt-mediated JNK activation in B cells. In addition, we show that GCKR positively affects the beta-catenin pathway in B cells. Reduction of GCKR expression inhibits Wnt3a-induced phosphorylation of GSK3beta at serine 9 and decreases the accumulation of cytosolic beta-catenin. Furthermore, Wnt signaling induces an interaction between GCKR and GSK3beta. Our findings demonstrate that GCKR facilitates both canonical and noncanonical Wnt signaling in B lymphocytes.     

Cortical beta-catenin and APC regulate asymmetric nuclear beta-catenin localization during asymmetric cell division in C. elegans

In C. elegans, Wnt signaling regulates a number of asymmetric cell divisions. During telophase, WRM-1/beta-catenin localizes asymmetrically to the anterior cortex and the posterior daughter's nucleus. However, cortical WRM-1's functions are not known. Here, we use a membrane-targeted form of WRM-1 to show that cortical WRM-1 inhibits Wnt signaling and the nuclear localization of WRM-1. These functions are mediated by APR-1/APC, which regulates WRM-1 nuclear export. We also show that APR-1 as well as PRY-1/Axin and Dishevelled homologs localize asymmetrically to the cortex. Our results suggest a model in which cortical WRM-1 recruits APR-1 to the anterior cortex before and during division, and the cortical APR-1 stimulates WRM-1 export from the anterior nucleus at telophase. Because beta-catenin and APC are localized to the cortex in many cell types in different species, our results suggest that these cortical proteins may regulate asymmetric divisions or Wnt signaling in other organisms as well.     

Alterations of frizzled (FzE3) and secreted frizzled related protein (hsFRP) expression in gastric cancer.

Wnt signaling pathway is important for development and carcinogenesis. Alterations of this pathway, such as mutations in adenomatous polyposis coli (APC) gene and activation mutations of beta-catenin, would result in stabilization of beta-catenin and subsequent translocation to nucleus where genes are transcribed. Recently, a receptor of Wnt, FzE3 was found to be up-regulated in esophageal carcinoma while a non-receptor antagonist of Wnt, secreted frizzled related protein (hsFRP) was found to be down-regulated in some cancer. These findings suggested that FzE3 is a potential oncogene while hsFRP is a potential tumor suppressor gene. We aimed to investigate whether FzE3 and hsFRP were altered in gastric cancer. Twelve cases of gastric cancer, including 7 cases of intestinal type, 4 cases of diffuse type and I case of mixed type, were studied. FzE3 and hsFRP mRNAs were expressed in most of the paired normal gastric tissues. FzE3 was over-expressed in 9 cases (75%) of gastric carcinoma tissues while hsFRP was down-regulated in 2 cases (16%). Beta-catenin nuclear staining was identified in 3 cases (27%) and cyclin D1 was expressed in 5 cases (41%) of cancer samples. All these cases were associated with either up-regulation of FzE3 or down-regulation of hsFRP. Our results suggested that alterations of FzE3 or hsFRP were frequent in gastric cancer. These provide alternative mechanisms leading to activation of Wnt signaling pathway in gastric carcinogenesis.     

Wnt/β-Catenin Signaling Protects Mouse Liver against Oxidative Stress-induced Apoptosis through the Inhibition of Forkhead Transcription Factor FoxO3

Numerous liver diseases are associated with extensive oxidative tissue damage. It is well established that Wnt/β-catenin signaling directs multiple hepatocellular processes, including development, proliferation, regeneration, nutrient homeostasis, and carcinogenesis. It remains unexplored whether Wnt/β-catenin signaling provides hepatocyte protection against hepatotoxin-induced apoptosis. Conditional, liver-specific β-catenin knockdown (KD) mice and their wild-type littermates were challenged by feeding with a hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet to induce chronic oxidative liver injury. Following the DDC diet, mice with β-catenin-deficient hepatocytes demonstrate increased liver injury, indicating an important role of β-catenin signaling for liver protection against oxidative stress. This finding was further confirmed in AML12 hepatocytes with β-catenin signaling manipulation in vitro using paraquat, a known oxidative stress inducer. Immunofluorescence staining revealed an intense nuclear FoxO3 staining in β-catenin-deficient livers, suggesting active FoxO3 signaling in response to DDC-induced liver injury when compared with wild-type controls. Consistently, FoxO3 target genes p27 and Bim were significantly induced in β-catenin KD livers. Conversely, SGK1, a β-catenin target gene, was significantly impaired in β-catenin KD hepatocytes that failed to inactivate FoxO3. Furthermore, shRNA-mediated deletion of FoxO3 increased hepatocyte resistance to oxidative stress-induced apoptosis, confirming a proapoptotic role of FoxO3 in the stressed liver. Our findings suggest that Wnt/β-catenin signaling is required for hepatocyte protection against oxidative stress-induced apoptosis. The inhibition of FoxO through its phosphorylation by β-catenin-induced SGK1 expression reduces the apoptotic function of FoxO3, resulting in increased hepatocyte survival. These findings have relevance for future therapies directed at hepatocyte protection, regeneration, and anti-cancer treatment.     

Adenomatous polyposis coli is down-regulated by the ubiquitin-proteasome pathway in a process facilitated by Axin

Adenomatous polyposis coli (APC) protein and Axin form a complex that mediates the down-regulation of beta-catenin, a key effector of Wnt signaling. Truncation mutations in APC are responsible for familial and sporadic colorectal tumors due to failure in the down-regulation of beta-catenin. While the regulation of beta-catenin by APC has been extensively studied, the regulation of APC itself has received little attention. Here we show that the level of APC is down-regulated by the ubiquitin-proteasome pathway and that Wnt signaling inhibits the process. The domain responsible for the down-regulation and direct ubiquitination was identified. We also show an unexpected role for Axin in facilitating the ubiquitination-proteasome-mediated down-regulation of APC through the oligomerization of Axin. Our results suggest a new mechanism for the regulation of APC by Axin and Wnt signaling.     

Structure-based discovery of a novel inhibitor targeting the β-catenin/Tcf4 interaction

Overactivation or overexpression of β-catenin in the Wnt (wingless) signaling pathway plays an important role in tumorigenesis. Interaction of β-catenin with T-cell factor (Tcf) DNA binding proteins is a key step in the activation of the proliferative genes in response to upstream signals of this Wnt/β-catenin pathway. Recently, we identified a new small molecule inhibitor, named BC21 (C(32)H(36)Cl(2)Cu(2)N(2)O(2)), which effectively inhibits the binding of β-catenin with Tcf4-derived peptide and suppresses β-catenin/Tcf4 driven reporter gene activity. This inhibitor decreases the viability of β-catenin overexpressing HCT116 colon cancer cells that harbor the β-catenin mutation, and more significantly, it inhibits the clonogenic activity of these cells. Down-regulation of c-Myc and cyclin D1 expression, the two important effectors of the Wnt/β-catenin signaling, is confirmed by treating HCT116 cells with BC21. This compound represents a new and modifiable potential anticancer candidate that targets β-catenin/Tcf-4 interaction.     

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.     

Wnt proteins induce dishevelled phosphorylation via an LRP5/6- independent mechanism, irrespective of their ability to stabilize beta-catenin

Wnt glycoproteins play essential roles in the development of metazoan organisms. Many Wnt proteins, such as Wnt1, activate the well-conserved canonical Wnt signaling pathway, which results in accumulation of beta-catenin in the cytosol and nucleus. Other Wnts, such as Wnt5a, activate signaling mechanisms which do not involve beta-catenin and are less well characterized. Dishevelled (Dvl) is a key component of Wnt/beta-catenin signaling and becomes phosphorylated upon activation of this pathway. In addition to Wnt1, we show that several Wnt proteins, including Wnt5a, trigger phosphorylation of mammalian Dvl proteins and that this occurs within 20 to 30 min. Unlike the effects of Wnt1, phosphorylation of Dvl in response to Wnt5a is not concomitant with beta-catenin stabilization, indicating that Dvl phosphorylation is not sufficient to activate canonical Wnt/beta-catenin signaling. Moreover, neither Dickkopf1, which inhibits Wnt/beta-catenin signaling by binding the Wnt coreceptors LRP5 and -6, nor dominant-negative LRP5/6 constructs could block Wnt-mediated Dvl phosphorylation. We conclude that Wnt-induced phosphorylation of Dvl is independent of LRP5/6 receptors and that canonical Wnts can elicit both LRP-dependent (to beta-catenin) and LRP-independent (to Dvl) signals. Our data also present Dvl phosphorylation as a general biochemical assay for Wnt protein function, including those Wnts that do not activate the Wnt/beta-catenin pathway.     

Regulating the balance between peroxisome proliferator-activated receptor gamma and beta-catenin signaling during adipogenesis. A glycogen synthase kinase 3beta phosphorylation-defective mutant of beta-catenin inhibits expression of a subset of adipogenic genes

The differentiation of preadipocytes into adipocytes requires the suppression of canonical Wnt signaling, which appears to involve a peroxisome proliferator-activated receptor gamma (PPARgamma)-associated targeting of beta-catenin to the proteasome. In fact, sustained activation of beta-catenin by expression of Wnt1 or Wnt 10b in preadipocytes blocks adipogenesis by inhibiting PPARgamma-associated gene expression. In this report, we investigated the mechanisms regulating the balance between beta-catenin and PPARgamma signaling that determines whether mouse fibroblasts differentiate into adipocytes. Specifically, we show that activation of PPARgamma by exposure of Swiss mouse fibroblasts to troglitazone stimulates the degradation of beta-catenin, which depends on glycogen synthase kinase (GSK) 3beta activity. Mutation of serine 37 (a target of GSK3beta) to an alanine renders beta-catenin resistant to the degradatory action of PPARgamma. Ectopic expression of the GSK3beta phosphorylation-defective S37A-beta-catenin in Swiss mouse fibroblasts expressing PPARgamma stimulates the canonical Wnt signaling pathway without blocking their troglitazone-dependent differentiation into lipid-laden cells. Analysis of protein expression in these cells, however, shows that S37A-beta-catenin inhibits a select set of adipogenic genes because adiponectin expression is completely blocked, but FABP4/aP2 expression is unaffected. Furthermore, the mutant beta-catenin appears to have no affect on the ability of PPARgamma to bind to or transactivate a PPAR response element. The S37A-beta-catenin-associated inhibition of adiponectin expression coincides with an extensive decrease in the abundance of C/EBPalpha in the nuclei of the differentiated mouse fibroblasts. Taken together, these data suggest that GSKbeta is a key regulator of the balance between beta-catenin and PPARgamma activity and that activation of canonical Wnt signaling downstream of PPARgamma blocks expression of a select subset of adipogenic genes.     

Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism

Wnt regulation of beta-catenin degradation is essential for development and carcinogenesis. beta-catenin degradation is initiated upon amino-terminal serine/threonine phosphorylation, which is believed to be performed by glycogen synthase kinase-3 (GSK-3) in complex with tumor suppressor proteins Axin and adnomatous polyposis coli (APC). Here we describe another Axin-associated kinase, whose phosphorylation of beta-catenin precedes and is required for subsequent GSK-3 phosphorylation of beta-catenin. This "priming" kinase is casein kinase Ialpha (CKIalpha). Depletion of CKIalpha inhibits beta-catenin phosphorylation and degradation and causes abnormal embryogenesis associated with excessive Wnt/beta-catenin signaling. Our study uncovers distinct roles and steps of beta-catenin phosphorylation, identifies CKIalpha as a component in Wnt/beta-catenin signaling, and has implications to pathogenesis/therapeutics of human cancers and diabetes.     

Metalloprotease ADAM9 cleaves ephrin-B ligands and differentially regulates Wnt and mTOR signaling downstream of Akt kinase in colorectal cancer cells

Ephrin-B signaling has been implicated in many normal and pathological processes, including neural crest development and tumor metastasis. We showed previously that proteolysis of ephrin-B ligands by the disintegrin metalloprotease ADAM13 is necessary for canonical Wnt signal activation and neural crest induction in Xenopus, but it was unclear if these mechanisms are conserved in mammals. Here, we report that mammalian ADAM9 cleaves ephrin-B1 and ephrin-B2 and can substitute for Xenopus ADAM13 to induce the neural crest. We found that ADAM9 expression is elevated in human colorectal cancer (CRC) tissues and that knockdown (KD) of ADAM9 inhibits the migration and invasion of SW620 and HCT116 CRC cells by reducing the activity of Akt kinase, which is antagonized by ephrin-Bs. Akt is a signaling node that activates multiple downstream pathways, including the Wnt and mTOR pathways, both of which can promote CRC cell migration/invasion. Surprisingly, we also found that KD of ADAM9 downregulates Wnt signaling but has negligible effects on mTOR signaling in SW620 cells; in contrast, mTOR activity is suppressed while Wnt signaling remains unaffected by ADAM9 KD in HCT116 cells. These results suggest that mammalian ADAM9 cleaves ephrin-Bs to derepress Akt and promote CRC migration and invasion; however, the signaling pathways downstream of Akt are differentially regulated by ADAM9 in different CRC cell lines, reflecting the heterogeneity of CRC cells in responding to manipulations of upstream Akt regulators.     

Glucagon-like peptide-1 activation of TCF7L2-dependent Wnt signaling enhances pancreatic beta cell proliferation

The insulinotropic hormone GLP-1 (glucagon-like peptide-1) is a new therapeutic agent that preserves or restores pancreatic beta cell mass. We report that GLP-1 and its agonist, exendin-4 (Exd4), induce Wnt signaling in pancreatic beta cells, both isolated islets, and in INS-1 cells. Basal and GLP-1 agonist-induced proliferation of beta cells requires active Wnt signaling. Cyclin D1 and c-Myc, determinants of cell proliferation, are up-regulated by Exd4. Basal endogenous Wnt signaling activity depends on Wnt frizzled receptors and the protein kinases Akt and GSK3beta but not cAMP-dependent protein kinase. In contrast, GLP-1 agonists enhance Wnt signaling via GLP-1 receptor-mediated activation of Akt and beta cell independent of GSK3beta. Inhibition of Wnt signaling by small interfering RNAs to beta-catenin or a dominant-negative TCF7L2 decreases both basal and Exd4-induced beta cell proliferation. Wnt signaling appears to mediate GLP-1-induced beta cell proliferation raising possibilities for novel treatments of diabetes.