The function of BCL9 in Wnt/beta-catenin signaling and colorectal cancer cells

Abstract

Background: Most cases of colorectal cancer are initiated by hyperactivation of the Wnt/beta-catenin pathway due to mutations in the APC tumour suppressor, or in beta-catenin itself. A recently discovered component of this pathway is Legless, which is essential for Wnt-induced transcription during Drosophila development. Limited functional information is available for its two mammalian relatives, BCL9 and B9L/BCL9-2: like Legless, these proteins bind to beta-catenin, and RNAi-mediated depletion of B9L/BCL9-2 has revealed that this protein is required for efficient beta-catenin-mediated transcription in mammalian cell lines. No loss-of-function data are available for BCL9.

Methods: We have used overexpression of dominant-negative forms of BCL9, and RNAi-mediated depletion, to study its function in human cell lines with elevated Wnt pathway activity, including colorectal cancer cells.

Results: We found that BCL9 is required for efficient beta-catenin-mediated transcription in Wnt-stimulated HEK 293 cells, and in the SW480 colorectal cancer cell line whose Wnt pathway is active due to APC mutation. Dominant-negative mutants of BCL9 indicated that its function depends not only on its beta-catenin ligand, but also on an unknown ligand of its C-terminus. Finally, we show that BCL9 and B9L are both Wnt-inducible genes, hyperexpressed in colorectal cancer cell lines, indicating that they are part of a positive feedback loop.

Conclusion: BCL9 is required for efficient beta-catenin-mediated transcription in human cell lines whose Wnt pathway is active, including colorectal cancer cells, indicating its potential as a drug target in colorectal cancer.

Figure 1

Mutants of BCL9 proteins that cannot bind to β-catenin. A, Sequence alignments of the HD2 domains of Lgs, BCL9 and BCL9-2/B9L (residue numbers in the latter two refer to mouse or human HD2 sequences, which are identical). Individual conserved residues (boxed) were mutated, and semiquantitative estimates of their in vitro binding affinities (++/+/-) to the β-catenin ARD are given underneath. B, pull-down assays between in vitro translated wt and mutant BCL9 HD2 and GST-ARD, as indicated; input lanes, 5% of the total binding reaction. C and D, co-immunoprecipitations of FLAG-tagged wt and mutant BCL9 or BCL9-2 with HA-β-catenin expressed in HEK 293T cells; bottom panels, protein expression levels in the lysates.

Figure 2

Subcellular distribution and transactivation potential of wt and mutant BCL9 and B9L. A and B, SW480 cells overexpressing FLAG-BCL9 or FLAG-BCL9-2, fixed and stained with antibodies against β-catenin and FLAG, as indicated in panels (the staining pattern of GFP-B9L was similar to that of FLAG-BCL9-2; not shown). C-E, TOPFLASH assays in SW480 and Wnt-stimulated HEK 293 cells (as indicated in panels), expressing wt or mutant FLAG-BCL9 or FLAG-BCL9-2 (see text). Control FOPFLASH assays were also conducted, but the values were less than 5% of TOPFLASH values and did not vary significantly between samples (not shown). Standard deviations are given by bars, and statistical significance relative to values from cells transfected with pCDNA3 (arbitrarily set to 100) is indicated above bars (*, p < 0.05; **, p < 0.001).

Figure 3

BCL9 and B9L are Wnt-inducible genes. A, Transcript levels of BCL9 and B9L in comparison to AXIN2, as measured by RT-qPCR, after induction of Wnt pathway activity in HEK 293 cells by addition of Wnt3A-conditioned medium, or 20 mM LiCl, for 6 hours. Statistical significance (p < 0.01) relative to Wnt-stimulated cells (arbitrarily set to 100) is indicated by asterisks. B and C, Transcript levels of c-myc and AXIN2 in Wnt-stimulated HEK 293 cells, measured by RT-qPCR as in A, after overexpression of wt or mutant FLAG-BCL9, or FLAG-BCL9-2. Statistical significance relative to Wnt-stimulated cells (arbitrarily set to 100) is indicated by asterisks (*, p < 0.05; **, p < 0.005). D, Transcript levels of BCL9, B9L, AXIN2, c-myc and CD44 relative to TBP (as internal control) in HEK 293 (with or without Wnt stimulation), SW480 or HCT116 cells. Statistical significance relative to uninduced HEK 293 cells is indicated by asterisks (*, p < 0.01; **, p < 0.001).

Figure 4

BCL9 is required for the transcription of endogenous Wnt target genes. Transcript levels of BCL9, B9L, c-MYC and AXIN2, as measured by RT-qPCR, in (A) SW480 cells or (B) Wnt-induced HEK 293 cells (as in Fig. 3), treated with siRNA to deplete β-catenin, BCL9 or B9L. Statistical significance (*, p < 0.01 and **, p < 0.001) relative to controls (set arbitrarily to 100%) is indicated above bars.

Figure 5

Requirement of the C-terminus of BCL9 for Wnt-induced transcription. Transcript levels of c-MYC and AXIN2, as measured by RT-qPCR, in Wnt-induced HEK 293 cells (as in Fig. 3) after overexpression of wt or mutant (A) FLAG-BCL9 or (B) FLAG-BCL9-2. Statistical significance (p < 0.01) relative to Wnt-stimulated control cells (set arbitrarily to 100%) is indicated by asterisks above bars.