Oncogenic function of DACT1 in colon cancer through the regulation of β-catenin

The Wnt/β-catenin signaling pathway plays important roles in the progression of colon cancer. DACT1 has been identified as a modulator of Wnt signaling through its interaction with Dishevelled (Dvl), a central mediator of both the canonical and noncanonical Wnt pathways. However, the functions of DACT1 in the WNT/β-catenin signaling pathway remain unclear. Here, we present evidence that DACT1 is an important positive regulator in colon cancer through regulating the stability and sublocation of β-catenin. We have shown that DACT1 promotes cancer cell proliferation in vitro and tumor growth in vivo and enhances the migratory and invasive potential of colon cancer cells. Furthermore, the higher expression of DACT1 not only increases the nuclear and cytoplasmic fractions of β-catenin, but also increases its membrane-associated fraction. The overexpression of DACT1 leads to the increased accumulation of nonphosphorylated β-catenin in the cytoplasm and particularly in the nuclei. We have demonstrated that DACT1 interacts with GSK-3β and β-catenin. DACT1 stabilizes β-catenin via DACT1-induced effects on GSK-3β and directly interacts with β-catenin proteins. The level of phosphorylated GSK-3β at Ser9 is significantly increased following the elevated expression of DACT1. DACT1 mediates the subcellular localization of β-catenin via increasing the level of phosphorylated GSK-3β at Ser9 to inhibit the activity of GSK-3β. Taken together, our study identifies DACT1 as an important positive regulator in colon cancer and suggests a potential strategy for the therapeutic control of the β-catenin-dependent pathway.     

Regulation of E-cadherin/Catenin association by tyrosine phosphorylation

Alteration of cadherin-mediated cell-cell adhesion is frequently associated to tyrosine phosphorylation of p120- and beta-catenins. We have examined the role of this modification in these proteins in the control of beta-catenin/E-cadherin binding using in vitro assays with recombinant proteins. Recombinant pp60(c-src) efficiently phosphorylated both catenins in vitro, with stoichiometries of 1.5 and 2.0 mol of phosphate/mol of protein for beta-catenin and p120-catenin, respectively. pp60(c-src) phosphorylation had opposing effects on the affinities of beta-catenin and p120 for the cytosolic domain of E-cadherin; it decreased (in the case of beta-catenin) or increased (for p120) catenin/E-cadherin binding. However, a role for p120-catenin in the modulation of beta-catenin/E-cadherin binding was not observed, since addition of phosphorylated p120-catenin did not modify the affinity of phosphorylated (or unphosphorylated) beta-catenin for E-cadherin. The phosphorylated Tyr residues were identified as Tyr-86 and Tyr-654. Experiments using point mutants in these two residues indicated that, although Tyr-86 was a better substrate for pp60(c-src), only modification of Tyr-654 was relevant for the interaction with E-cadherin. Transient transfections of different mutants demonstrated that Tyr-654 is phosphorylated in conditions in which adherens junctions are disrupted and evidenced that binding of beta-catenin to E-cadherin in vivo is controlled by phosphorylation of beta-catenin Tyr-654.     

KHC-4 inhibits β-catenin expression in prostate cancer cells

ABSTRACT

KHC-4 is a 2-phenyl-4-quinolone analogue that exhibits anticancer activity. Aberrant activation of β-catenin signaling contributes to prostate cancer development and progression. Therefore, targeting β-catenin expression could be a useful approach to treating prostate cancer. We found that KHC-4 can inhibit β-catenin expression and its signaling pathway in DU145 prostate cancer cells. Treatment with KHC-4 decreased total β-catenin expression and concomitantly decreased β-catenin levels in both the cytoplasm and nucleus of cells. KHC-4 treatment also inhibited β-catenin expression and that of its target proteins, PI3K, AKT, GSK3β and TBX3. We monitored the stability of β-catenin with the proteasomal inhibitor, MG132, in DU145 cells and found that MG132 reversed KHC-4-induced proteasomal β-catenin degradation. We verified CDK1/β-catenin expression in KHC-4 treated DU145 cells. We found that roscovitine treatment reversed cell proliferation by arresting the cell cycle at the G2/M phase and β-catenin expression caused by KHC-4 treatment. We suggest that KHC-4 inhibits β-catenin signaling in DU145 prostate cancer cells.     

Dual alterations in casein kinase I-epsilon and GSK-3beta modulate beta-catenin stability in hyperproliferating colonic epithelia

Casein kinase I (CKI)-epsilon and GSK-3beta phosphorylate beta-catenin at Ser(45) (beta-cat(45)) and Thr(41)/Ser(37,33) (beta-cat(33,37,41)) residues, thereby facilitating its ubiquitination and proteasomal degradation. We used a Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model to determine Ser/Thr phosphorylation and biological function of beta-catenin during crypt hyperproliferation. TMCH was associated with 3-fold and 3.3-fold increases in CKI-epsilon cellular abundance and 2-fold and 1.8-fold increase in its activity at 6 and 12 days after infection, respectively. beta-Catenin coimmunoprecipitated with both cellular and nuclear CKI-epsilon and cellular axin at these time points. Cellular beta-catenin was constitutively phosphorylated at Ser(45) and underwent subcellular redistribution to cytoskeletal and nuclear fractions at days 6 and 12 of TMCH, respectively. beta-cat(33,37,41), however, exhibited only subtle changes in either phosphorylation status or subcellular distribution even after blocking proteasomal degradation in vivo. Interestingly, GSK-3beta underwent increased phosphorylation at Ser(9), leading to 40% and 70% decreases in its activity at these time points, respectively. Coimmunoprecipitation studies exhibited strong association of GSK-3beta with PKC-zeta at either time point. Cellular beta-cat(45) stabilized and, along with unphosphorylated beta-catenin, underwent nuclear translocation, associated with nuclear accumulated Tcf-4 and cAMP response element binding protein binding protein, and was significantly acetylated, leading to increases in DNA binding. Priming of beta-catenin at Ser(45) exists in vivo. However, beta-cat(45) does not necessarily enter the degradation pathway. Impairment in linking beta-cat(45) to subsequent GSK-3beta-mediated phosphorylation and degradation may account for increased steady-state levels of both unphosphorylated as well as Ser(45)-phosphorylated beta-catenin, which may be causally linked to increases in cell census during TMCH.     

Regulation of β-catenin nuclear dynamics by GSK-3β involves a LEF-1 positive feedback loop

Nuclear localization of β-catenin is integral to its role in Wnt signaling and cancer. Cellular stimulation by Wnt or lithium chloride (LiCl) inactivates glycogen synthase kinase-3β (GSK-3β), causing nuclear accumulation of β-catenin and transactivation of genes that transform cells. β-catenin is a shuttling protein; however, the mechanism by which GSK-3β regulates β-catenin nuclear dynamics is poorly understood. Here, fluorescence recovery after photobleaching assays were used to measure the β-catenin-green fluorescent protein dynamics in NIH 3T3 cells before and after GSK-3β inhibition. We show for the first time that LiCl and Wnt3a cause a specific increase in β-catenin nuclear retention in live cells and in fixed cells after detergent extraction. Moreover, LiCl reduced the rate of nuclear export but did not affect import, hence biasing β-catenin transport toward the nucleus. Interestingly, the S45A mutation, which blocks β-catenin phosphorylation by GSK-3β, did not alter nuclear retention or transport, implying that GSK-3β acts through an independent regulator. We compared five nuclear binding partners and identified LEF-1 as the key mediator of Wnt3a and LiCl-induced nuclear retention of β-catenin. Thus, Wnt stimulation triggered a LEF-1 positive feedback loop to enhance the nuclear chromatin-retained pool of β-catenin by 100-300%. These findings shed new light on regulation of β-catenin nuclear dynamics.