The Wnt connection to tumorigenesis

Wnt signaling has been identified as one of the key signaling pathways in cancer, regulating cell growth, motility and differentiation. Because of its widespread activation in diverse human tumor diseases, the Wnt pathway has gained considerable and growing interest in tumor research over recent years. Evidence that altered Wnt signaling is important for human tumor development came from three major findings: (i) the tumor suppressor adenomatous polyposis coli (APC) binds to the Wnt pathway component beta-catenin and is involved in its degradation, (ii) mutations of APC in colon tumors lead to stabilization of the beta-catenin protein and (iii) tumor-associated mutations of beta-catenin in colorectal cancer as well as in other tumor types lead to its stabilisation, qualifying beta-catenin as a proto-oncogene. Here we will describe the biochemical interactions which shape the Wnt pathway and focus on its role in tumorigenesis.     

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.     

H-Ras activation promotes cytoplasmic accumulation and phosphoinositide 3-OH kinase association of beta-catenin in epidermal keratinocytes.

The mechanisms underlying downregulation of the cadherin/catenin complexes and beta-catenin signaling during tumor progression are not fully understood. We have analyzed the effect of oncogenic H-Ras on E-cadherin/catenin complex formation/stabilization and beta-catenin distribution in epidermal keratinocytes. Microinjection or stable expression of V12Ras into keratinocytes promotes the loss of E-cadherin and alpha-catenin and relocalization of beta-catenin to the cytoplasm and nucleus. Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity. Interestingly, a strong association of p85alpha and p110alpha subunits of PI3K with beta-catenin is induced in V12Ras-expressing keratinocytes, and in vitro binding assays show a direct interaction between beta-catenin and p85alpha. Overexpression of either V12Ras or constitutively active p110alpha induces metabolic stabilization of beta-catenin and promotes its accumulation in cytoplasmic and nuclear pools. In addition, the interaction of beta-catenin with the adenomatous polyposis coli protein is blocked in V12Ras and p110alpha transformants though no changes in glycogen synthase kinase 3 beta activity could be detected. Nevertheless, in V12Ras transformants the in vivo phosphorylation of beta-catenin in Ser residues is strongly decreased. These results indicate that H-Ras activation induces the relocalization and cytoplasmic stabilization of beta-catenin by a mechanism involving its interaction with PI3K.     

Transcriptional activation of interleukin-8 by beta-catenin-Tcf4

Nuclear translocation of beta-catenin and its association with Tcf/Lef factors are key steps in transduction of the Wnt signal, which is aberrantly activated in a variety of human cancers. In a search for new beta-catenin-Tcf target genes, we analyzed beta-catenin-induced alterations of gene expression in primary human hepatocytes, after transduction of either dominant stable beta-catenin or its truncated, transactivation-deficient counterpart by means of a lentiviral vector. cDNA microarray analysis revealed a limited set of up-regulated genes, including known Wnt targets such as matrilysin and keratin-1. In this screen, we identified the CXC chemokine interleukin 8 (IL-8) as a direct target of beta-catenin-Tcf4. IL-8 is constitutively expressed in various cancers, and it has been implicated in tumor progression through its mitogenic, motogenic, and angiogenic activities. The IL-8 promoter contains a unique consensus Tcf/Lef site that is critical for IL-8 activation by beta-catenin. We show here that the p300 coactivator was required for efficient transactivation of beta-catenin on this promoter. Ectopic expression of beta-catenin in hepatoma cells promoted IL-8 secretion, which stimulated endothelial cell migration. These data define IL-8 as a Wnt target and suggest that IL-8 induction by beta-catenin might be implicated in developmental and tumorigenic processes.     

Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate beta-catenin

The current view of canonical Wnt signalling is that following Wnt binding to its receptors (Frizzled-Lrp5/6), dishevelled (Dvl) becomes hyperphosphorylated, and the signal is transduced to the APC-GSK3beta-axin-beta-catenin multiprotein complex, which subsequently dissociates. As a result beta-catenin is not phosphorylated, escapes proteosomal degradation and activates its target genes after translocation to the nucleus. Here, we analyzed the importance of the Wnt-3a-induced phosphorylation and shift in electrophoretic migration of Dvl (PS-Dvl) for the activation of beta-catenin. Analysis of Wnt-3a time- and dose-responses in a dopaminergic cell line showed that beta-catenin is activated rapidly (within minutes) and at a low dose of Wnt-3a (1 ng/ml). Surprisingly, PS-Dvl appeared only after 30 min and at greater doses (> or =20 ng/ml) of Wnt-3a. Moreover, we found that a casein kinase 1 inhibitor (D4476) or siRNA for casein kinase 1 delta/epsilon (CK1delta/epsilon) blocked the Wnt-3a-induced PS-Dvl. Interestingly, CK1 inhibition or siRNA for CK1delta/epsilon did not ablate the activation of beta-catenin by Wnt-3a, indicating that there is a PS-Dvl-independent path to activate beta-catenin. The increase in beta-catenin activation by Wnt-3a (PS-Dvl-dependent or -independent) were blocked by Dickkopf1 (Dkk1), suggesting that the effect of Wnt-3a is in both cases mediated by Lrp5/6 receptors. Thus, our results show that Wnt-3a rapidly induce a partial activation of beta-catenin in the absence of PS-Dvl at low doses, while at high doses induce a full activation of beta-catenin in a PS-Dvl-dependent manner.     

Functional interaction between peroxisome proliferator-activated receptor gamma and beta-catenin

Studies have demonstrated cross talk between beta-catenin and peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways. Specifically, activation of PPARgamma induces the proteasomal degradation of beta-catenin in cells that express an adenomatous polyposis coli-containing destruction complex. In contrast, oncogenic beta-catenin is resistant to such degradation and inhibits the expression of PPARgamma target genes. In the present studies, we demonstrate a functional interaction between beta-catenin and PPARgamma that involves the T-cell factor (TCF)/lymphocyte enhancer factor (LEF) binding domain of beta-catenin and a catenin binding domain (CBD) within PPARgamma. Mutation of K312 and K435 in the TCF/LEF binding domain of an oncogenic beta-catenin (S37A) significantly reduces its ability to interact with and inhibit the activity of PPARgamma. Furthermore, these mutations render S37A beta-catenin susceptible to proteasomal degradation in response to activation of PPARgamma. Mutation of F372 within the CBD (helices 7 and 8) of PPARgamma disrupts its binding to beta-catenin and significantly reduces the ability of PPARgamma to induce the proteasomal degradation of beta-catenin. We suggest that in normal cells, PPARgamma can function to suppress tumorigenesis and/or Wnt signaling by targeting phosphorylated beta-catenin to the proteasome through a process involving its CBD. In contrast, oncogenic beta-catenin resists proteasomal degradation by inhibiting PPARgamma activity, which requires its TCF/LEF binding domain.