Requirement of cell cycle and apoptosis regulator 1 for target gene activation by Wnt and beta-catenin and for anchorage-independent growth of human colon carcinoma cells - PubMed (original) (raw)

Requirement of cell cycle and apoptosis regulator 1 for target gene activation by Wnt and beta-catenin and for anchorage-independent growth of human colon carcinoma cells

Chen-Yin Ou et al. J Biol Chem. 2009.

Abstract

Aberrant Wnt signaling promotes oncogenesis by increasing cellular levels of beta-catenin, which associates with DNA-bound transcription factors and activates Wnt target genes. However, the molecular mechanism by which beta-catenin mediates gene expression is still poorly understood. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1), which was recently shown to function as a transcriptional coactivator for nuclear receptors, also interacts with beta-catenin and enhances the ability of beta-catenin to activate expression of transiently transfected reporter genes. Furthermore, association of CCAR1 with the promoter of an endogenous Wnt/beta-catenin target gene in a colon cancer cell line depends on the presence of beta-catenin. Depletion of CCAR1 inhibits expression of several Wnt/beta-catenin target genes and suppresses anchorage-independent growth of the colon cancer cell line. Thus, CCAR1 is a novel component of Wnt/beta-catenin signaling that plays an important role in transcriptional regulation by beta-catenin and that, therefore, may represent a novel target for therapeutic intervention in cancers involving aberrantly activated Wnt/beta-catenin signaling.

PubMed Disclaimer

Figures

FIGURE 1.

FIGURE 1.

Binding of CCAR1 to β-catenin and LEF1. A, GST pulldown assays were performed as described under “Experimental Procedures,” using bacterially produced GST fusion proteins bound to glutathione-Sepharose beads and HA-tagged proteins translated in vitro. Bound proteins were detected by immunoblot analysis using antibodies against an HA tag. B, HA-tagged β-catenin and CCAR1 were expressed in COS-7 cells by transient transfection, and immunoprecipitation (IP) was performed on cell extracts, using the indicated antibodies against β-catenin or CCAR1, or normal IgG. Precipitated proteins were detected by immunoblot with antibodies against HA-tag. Uncropped images of the blots are shown in the supplemental materials (supplemental Fig.

S1

). C, HA-tagged fragments of β-catenin synthesized in vitro were incubated with GST-CCAR1 in GST pulldown assays, and bound proteins were detected by immunoblot using antibodies against the HA tag, as described in A. The diagram shows the 12 armadillo repeats of β-catenin flanked by N-terminal and C-terminal domains. D, GST pulldown assays were performed as in C using GST-β-catenin and HA-tagged fragments of CCAR1. The diagram shows the domains of CCAR1, including regions with a high content of specific amino acids, an SAF-Acinus-PIAS (SAP) domain, and a poly-A-binding protein (PABP) homology domain. GST pulldown assays were repeated at least twice, with results equivalent to those shown.

FIGURE 2.

FIGURE 2.

Cooperation of CCAR1 and β-catenin as coactivators for transcriptional activation by LEF1. A, CV-1 cells were transfected in 12-well plates with luciferase reporter plasmid GK1-Luc (300 ng) controlled by Gal4 response elements, pM plasmids encoding Gal4-DBD alone or fused to LEF1 or β-catenin (100 ng), and pSG5.HA-CCAR1 (300, 600, or 900 ng). Luciferase assays were conducted on cell extracts as described under “Experimental Procedures.” Results shown are from a single experiment and are representative of three independent experiments. B, CV1 cells were transfected with luciferase reporter plasmids pGL3OT (200 ng) containing LEF1-responsive elements, along with pSG5.HA-LEF1 (10 ng), pSG5.HA-β-catenin (200 ng), and pSG5.HA-CCAR1 (300, 600, or 900 ng). Results are from a single experiment, which is representative of three independent experiments. C and D, plasmids used for reporter gene assay were pGL3OT (200 ng) along with pSG5.HA-LEF1 (0.005 ng in C and 5 ng in D), pSG5.HA-β-catenin (200 ng), pSG5.HA-CARM1 (200 ng), pCMV.p300 (200 ng), pSG5.HA-CoCoA (200 ng), and pSG5.HA-CCAR1 (200 ng), as indicated. Luciferase activity was determined as in A.

FIGURE 3.

FIGURE 3.

Recruitment of CCAR1 to a Wnt target gene depends on β-catenin. A, ChIP assays were performed with HT29 cell chromatin, using antibodies against β-catenin or CCAR1 (CCAR_1 antibody, Bethyl 435A; CCAR_2 antibody, Bethyl 270A), or with normal IgG (IgG_1, Santa Cruz Biotechnology normal rabbit; IgG_2, Bethyl normal rabbit). Immunoprecipitated DNA was analyzed by qPCR with primers for the upstream WRE of the Axin2 c-myc, and DKK1 genes or with primers for the 3′-untranslated region (3′UTR) of the Axin2 gene, an upstream negative control (UNC) site near the DKK1 gene, and the open reading frame (ORF) of the c-myc gene. Relative recruitment was calculated by dividing specific antibody signal by the signal for IgG_2 (Bethyl Laboratories). B, sequential ChIP assays for hAxin2-WRE were performed with the indicated antibodies, and results are expressed relative to input DNA from the unfractionated chromatin. C, HT29 cells were infected with lentivirus encoding a puromycin resistance gene, and shRNAs against a nonspecific sequence (NS), β-catenin, or CCAR1, and puromycin-resistant cells were selected. At 7 days after the infection, cell extracts were analyzed by immunoblot using antibodies against CCAR1, β-catenin, or α-tubulin. D, the infected cells from C were analyzed by ChIP assay as in A. In A, B, and D, the mean and range of variation from duplicate PCR reactions are shown. ChIP results shown are from a single experiment, which is representative of two or more independent experiments.

FIGURE 4.

FIGURE 4.

Requirement of β-catenin and CCAR1 for expression of Wnt target genes. A, HT29 cells infected with lentivirus encoding shRNA against β-catenin, CCAR1, or a nonspecific sequence (NS) were analyzed by immunoblot as in Fig. 3_C. B_, total RNA from the lentivirus-infected cells in A was examined by quantitative reverse transcriptase-PCR analysis, using primers specific for the indicated Wnt target genes. Results shown are normalized to the level of α-tubulin mRNA, are the mean and range of variation for duplicate PCR reactions from a single experiment, and are representative of at least three independent experiments.

FIGURE 5.

FIGURE 5.

Role of CCAR1 in anchorage-independent colony formation. A, HT29 cells were plated in standard tissue culture dishes, and cell proliferation was monitored by MTS assay. B, diluted HT29 cell suspensions containing the indicated number of cells were plated in soft agar, and colony formation was examined by staining after 2–4 weeks (right panels). Microscope images are shown with a scale bar representing 200 μm. Colonies in each dish were counted by Molecular Imager Gel Doc XR System (Bio-Rad). Results shown are mean ± S.D. from triplicate cultures from a single experiment and are representative of three independent experiments. The images from the triplicate plates used for the data shown in Fig. 5_B_ are presented in the supplemental materials (supplemental Fig.

S2

). Results from an independent experiment are also presented in supplemental Fig.

S3

.

Similar articles

Cited by

References

    1. Nusse R. (2008) Cell Res. 18, 523–527 - PubMed
    1. McDonald S. A., Preston S. L., Lovell M. J., Wright N. A., Jankowski J. A. (2006) Nat. Clin. Pract. Gastroenterol. Hepatol. 3, 267–274 - PubMed
    1. Reya T., Clevers H. (2005) Nature 434, 843–850 - PubMed
    1. Hayward P., Kalmar T., Arias A. M. (2008) Development 135, 411–424 - PubMed
    1. Fuerer C., Nusse R., Ten Berge D. (2008) EMBO Rep. 9, 134–138 - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources