The intestine-specific transcription factor Cdx2 inhibits beta-catenin/TCF transcriptional activity by disrupting the beta-catenin-TCF protein complex

Abstract

Cdx2 is an intestine-specific transcription factor known to regulate proliferation and differentiation. We have reported previously that Cdx2 limits the proliferation of human colon cancer cells by inhibiting the transcriptional activity of the beta-catenin-T-cell factor (TCF) bipartite complex. Herein we further elucidate this mechanism. Studies with a classic Cdx2 target gene and a canonical Wnt/beta-catenin/TCF reporter suggest that Cdx2 regulates these promoters by distinctly different processes. Specifically, inhibition of beta-catenin/TCF activity by Cdx2 does not require Cdx2 transcriptional activity. Instead, Cdx2 binds beta-catenin and disrupts its interaction with the DNA-binding TCF factors, thereby silencing beta-catenin/TCF target gene expression. Using Cdx2 mutants, we map the Cdx2 domains required for the inhibition of beta-catenin/TCF activity. We identify a subdomain in the N-terminus that is highly conserved and when mutated significantly reduces Cdx2 inhibition of beta-catenin/TCF transcriptional activity. Mutation of this subdomain also abrogates Cdx2's anti-proliferative effects in colon cancer cells. In summary, we conclude that Cdx2 binds beta-catenin and disrupts the beta-catenin-TCF complex. Considering the pivotal role of beta-catenin/TCF activity in driving proliferation of normal intestinal epithelial and colon cancer cells, our findings suggest a novel mechanism for Cdx2-mediated regulation of Wnt/beta-catenin signaling and cell proliferation.

Fig. 1.

Comparison of TOPFLASH inhibition with SI promoter activation in DLD1 and 293T cells. (A and B) Luciferase activity was determined after Flag-Cdx1 (X1; gray bar) or Flag-Cdx2 (X2; white bar) expression vectors or an empty vector control (E; black bar), were co-transfected with TOPFLASH and SI reporters; 2 μg or 400 ng of the expression vectors were transfected into DLD1 or 293T cells (400 ng of expression vectors). a, Significantly differs from empty control and Cdx1, P < 0.01. b, Significantly differs from empty control, P < 0.05. (C and D) Protein levels of Flag-Cdx1 and Flag-Cdx2 after transfection of DLD1 and 293T cells.

Fig. 2.

The inhibition of TOPFLASH by Cdx2 requires distinct domains in the N-terminus. The Cdx2-responsive reporter SI-Luc or the β-catenin/TCF reporter TOPFLASH were transfected into 293T cells to determine the effect of Cdx2 mutations upon their transcriptional activity. (A) Luciferase activity after co-transfection with Cdx2 wild-type and truncation mutants. (B) Additional Cdx2 truncation mutants were generated and tested for their ability to activate SI-Luc or inhibit TOPFLASH. In Cdx2 diagram, HD: homeodomain; A, B,C: conserved domains. Insets: Western blots showing equal protein levels of new mutants and wild-type Cdx2 after transfection. Blots were probed for Cdx2 with tubulin as a loading control. Cdx2ND and Cdx2^S56,57,58,60 were probed using a polyclonal antibody against the Cdx2 C-terminus. a, significantly differs from Cdx2 wild-type, P < 0.001; b, significantly differs from Cdx2 wild-type and Empty vector, P < 0.05; c, Differs from Empty vector and Cdx2Δ50, P < 0.01. *Significantly differs from empty vector control, P < 0.001.

Fig. 3.

The inhibition of TOPFLASH by Cdx2 correlates with disruption of the β-catenin–TCF complex. (A) Constitutively activated chimeric LEF/TCF proteins VP16-Lef1 (17) and CatCLef (26) or the stabilized β-catenin mutant (S33Y) were transfected into 293T cells along with TOPFLASH and an expression vector for Cdx2 (white bar) or an empty control vector (gray bar). Luciferase activity was determined as before; a, differs from all other transfections, P < 0.001; b, differs from CatCLef and empty vector, P < 0.05; (B) A myc-tagged TCF4 and the stabilized β-catenin mutant were co-transfected into 293T cells along with an empty vector control or increasing amounts of a Cdx2 expression vector. Then myc-TCF4 was immunoprecipitated, and the products were analyzed for the presence of β-catenin. (C) Similar study except it was carried out in DLD1 colon cancer cells without the addition of the β-catenin mutant S33Y. (D) Wild-type Cdx1 and Cdx2 truncation mutants are tested for their ability to disrupt the β-catenin–TCF co-immunoprecipitation complex in 293T cells; 50 ng of each Cdx expression vector was transfected for this study.

Fig. 4.

Cdx2 protein binds β-catenin in vivo but not in vitro. (A) A Flag-tagged Cdx2 or the empty Flag vector were co-transfected along with β-catenin (S33Y) into 293T cells, and FLAG or β-catenin immunoprecipitations were performed. (B) β-catenin co-immunoprecipitates with Cdx2 in human Caco2 cells. (C) ³⁵S-labeled Cdx2 was incubated with a full-length GST–β-catenin, or with the control Cdx2-binding GST–UBC9 protein, and then GST pull-down was performed. As a control, the GST–β-catenin construct was incubated with an ³⁵S-labeled TCF4 protein. (D) Increasing amounts of an in vitro translated Cdx2 protein were pre-incubated with GST–β-catenin before the addition of ³⁵S-TCF4.

Fig. 5.

A subdomain located in the N-terminus is required for Cdx2 transcriptional activity and TOPFLASH inhibition. (A) N-terminal amino acid sequences of five Cdx2 mutants generated for this study. Mutant sequences in bold and underlined. (B) Cdx2 mutants were tested for their ability to transactivate an SI reporter or block β-catenin/TCF-mediated TOPFLASH activation as before. S33Y-β-catenin was co-transfected into 293T cells along with wild-type Cdx2 (white bar), mutant Cdx2s (gray bars), or the empty vector control (black bar); a, significantly different from wild-type Cdx2 and empty vector controls, P < 0.005; b, differs from all other expression vectors, P < 0.001; c, significantly differs from wild-type Cdx2 and empty vector controls, P < 0.05. (C) Alignment of murine Cdx2 with other Cdx sequences using MacVector (Oxford Molecular). Genebank accession numbers indicated. Conserved subdomain indicated by box.

Fig. 6.

Inhibition of cancer cell proliferation by Cdx2 requires a conserved subdomain in the N-terminus. (A) HCT116 cells were transfected with a GFP expression vector as well as wild-type Cdx2, the Cdx2 truncation mutants, with Cdx2Mut4, or the empty vector as control. At 48 h, the cells were stained with propidium iodide and DNA content quantitated in the GFP+ cells. *Significantly differs from empty vector, Cdx2ND, and Cdx2Δ50, P < 0.05 by. (B) Model of Cdx2 inhibition of Wnt/β-catenin transcriptional activity. β-catenin translocates to the nucleus where it partners with a TCF family member, bind DNA and activates target genes. In the presence of Cdx2 expression and an as yet unidentified factor or post-translational modification (X), β-catenin associates with Cdx2. This interaction prevents β-catenin/TCF transcriptional activity.