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

Stress-induced immunosuppression inhibits immune response to infectious bursal disease virus vaccine partially by miR-27b-3p/SOCS3 regulatory gene network in chicken

ABSTRACT: Stress-induced immunosuppression (SIIS) is one of the common problems in intensive poultry production, which often reduces the prevention and control effects of various vaccines, including infectious bursal disease virus (IBDV) vaccine, and brings enormous economic losses to the poultry industry. However, the molecular mechanisms of SIIS inhibiting immune response to IBDV vaccine remain unclear. In this study, suppressor of cytokine signaling 3 (SOCS3) gene was selected and stress-induced immunosuppressed chickens were simulated using dexamethasone (Dex). Quantitative real-time PCR (qRT-PCR) was conducted to analyze its expression characteristics and game relationships between SOCS3 gene and miR-27b-3p (it could target SOCS3 gene) in the process of SIIS inhibiting immune response to IBDV vaccine in chicken, and the potential application value of circulating miR-27b-3p as a biomarker was also identified. The results showed that SOCS3 gene and miR-27b-3p were significantly differentially expressed in the candidate tissues during SIIS inhibiting the immune response to IBDV (P < 0.05), respectively, which were key factors involved in the process. Moreover, miR-27b-3p and SOCS3 gene showed game regulation relationships in several tissues during the process, so the miR-27b-3p/SOCS3 regulatory network was one of the key mechanisms of SOCS3 gene participating in the process. Circulating miR-27b-3p was differentially expressed in serum at 10 time points (1, 2, 3, 4, 5, 7, 14, 21, 28, and 35 days postimmunization (dpi)) in the process (P < 0.05), showing that circulating miR-27b-3p was a valid candidate target as a molecular marker for detecting SIIS inhibiting the IBDV immune response. This study can provide references for further studying molecular mechanisms of stress affecting immune response

<i>DACT1</i> is highly expressed in human colon cancer cells.

<p>(A) <i>DACT1</i> mRNA levels were assayed using quantitative real-time RT-PCR analysis. Samples of colon adenocarcinoma (T, white bars) and normal-appearing control mucosa (N, black bars) were analyzed in six cases of colon cancer. (B) Expression of <i>DACT1</i> protein in human colon adenocarcinoma (T) and normal-appearing control mucosa (N) in six cases, as determined by Western immunoblotting analysis. The expression of GAPDH was used as the loading control.</p

Correlation between <i>DACT1</i> levels and membrane-associated β-catenin expression in colon cell lines and colon cancer tissue.

<p>(A) Semiquantitative RT-PCR analysis of <i>DACT1</i> expression in HCT116, HT29 and SW480 cells. GAPDH was used as the control. (B) Immunofluorescence analysis of <i>DACT1</i> and β-catenin expression in HCT116, HT29 and SW480 cells. Original magnification, 40×. (C, D) HE staining of samples of normal human colon mucosa (N) and adenocarcinoma (T) tissues by an anti-β-catenin antibody. Original magnification, 40×. (E, F) immunofluorescence (IF) staining of samples of normal human colon mucosa (N) and adenocarcinoma (T) tissues by an anti-β-catenin antibody. Original magnification, 40×. (G, H) Immunohistochemical (IHC) staining of samples of normal human colon mucosa (N) and adenocarcinoma (T) tissues by an anti-β-catenin antibody. Original magnification, 40×.</p

<i>DACT1</i> enhances invasion of colon cancer cells in vitro or in vivo.

<p>(A) Representative images are shown for the overexpression of <i>DACT1</i>-transfected SW480 cells. Cells were seeded in an invasive chamber and allowed to invade the chamber toward cell-specific conditioned medium for 24 h. Photomicrographs of stained invading cells were taken under brightfield illumination (20×). (B, C, D) Representative images are shown for <i>DACT1</i> siRNA-transfected HCT116, LoVo and HT29 cells. Cells were seeded in an invasion chamber and allowed to invade the chamber toward cell-specific conditioned medium for 24 h. Photomicrographs of the stained invading cells were taken under brightfield illumination (20×). (E) Quantification of the migration assay. Results were obtained from three separate experiments each performed in triplicate. Invasion was determined by counting cells in six random microscopic fields per well (mean ± SEM; *p<0.05 versus control group cells).</p

<i>DACT1</i> enhances the migratory and invasive potential of colon cancer cells through changing the subcellular location of β-catenin.

<p>(A) Photomicrographs of empty vector and <i>DACT1</i>-overexpressing SW480 cells immunostained with an anti-β-catenin antibody (red). (B, C, D) Photomicrographs of control siRNA and <i>DACT1</i> siRNA in HCT116, LoVo and HT29 cells immunostained with an anti-β-catenin antibody (red). (E) Representative blots of β-catenin levels in membrane (Mem), nuclear (Nuc), and cytoplasmic (Cyto) fractions and total lysates (Lys) in SW480 cells. Laminin B (nuclear expression) and GAPDH (cytoplasmic expression) were used as the loading controls. “+” represents the overexpression <i>DACT1</i>. “−” is empty vector. (F) Representative blots of β-catenin levels in membrane (Mem), nuclear (Nuc), and cytoplasmic (Cyto) fractions and total lysates (Lys) in HCT116 cells. Laminin B (nuclear expression) and GAPDH (cytoplasmic expression) were used as the loading controls. “+” represents control siRNA. “−” is <i>DACT1</i> siRNA.</p

<i>DACT1</i> affects the subcellular localization of β-catenin through interacting with GSK-3β.

<p>(A) Photomicrographs of control siRNA and <i>DACT1</i> siRNA expressing HCT116 cells immunostained with an anti-phospho-GSK-3β(Ser9) antibody (red). Silencing of <i>DACT1</i> in HCT116 cells decreases levels of phospho-GSK-3β(Ser9). (B) Representative Western blots of HCT116 cells expressing control siRNA and <i>DACT1</i> siRNA. Silencing of <i>DACT1</i> in HCT116 cells decreases levels of phospho-GSK-3β(Ser9). GAPDH was used as the loading control. (C) HCT116 cells expressing control siRNA and <i>DACT1</i> siRNA were treated for 1 h with GSK-3β inhibitory drugs (40 mM LiCl). Cells were stained and analyzed by microscopy to detect the expression of β-catenin. LiCl treatment of HCT116 cells increases the levels of β-catenin at the plasma membrane. (D) Representative Western blots of LiCl-treated HCT116 cells, which express either control siRNA or <i>DACT1</i> siRNA. LiCl treatment of HCT116 cells increases levels of β-catenin at the plasma membrane. KDEL was used as a loading control. 1: HCT116 cells expressing control siRNA that had been treated with 40 mM LiCl for 1 h. 2: HCT116 cells expressing control siRNA that had not been treated with LiCl. 3: HCT116 cells expressing <i>DACT1</i> siRNA that were treated by 40 mM LiCl for 1 h; 4: HCT116 cells expressing <i>DACT1</i> siRNA that had not been treated with LiCl. (E) Photomicrographs of control siRNA and <i>DACT1</i> siRNA expressing HCT116 cells immunostained with an anti-phospho-GSK-3β(Ser9) antibody (red). Silencing of <i>DACT1</i> in HCT116 cells decreases levels of phospho-GSK-3β(Ser9). (F) Representative Western blots of HCT116 cells expressing control siRNA and <i>DACT1</i> siRNA. Silencing of <i>DACT1</i> in HCT116 cells decreases levels of phospho-GSK-3β(Ser9). GAPDH was used as a loading control.</p

Putative model of how <i>DACT1</i> mediates the activation of β-catenin signaling in colon cancer cells.

<p>Left: In unstimulated normal colon cells lacking <i>DACT1</i>, the axin/GSK-3β/APC phosphorylate β-catenin and target it for ubiquitin-mediated degradation. Right: In colon cancer cells that express high levels of <i>DACT1</i>, <i>DACT1</i> binds to β-catenin. <i>DACT1</i> then binds and inhibits GSK-3β, which inhibits the function of the destruction complex, resulting in the release of β-catenin. This leads to increased nuclear and cytoplasmic fractions of β-catenin, and increased levels of membrane-bound β-catenin. Subsequently, the migration and invasion capacities of the colon cancer cells are enhanced and downstream target genes are activated.</p