Comprehensive analysis of β-catenin target genes in colorectal carcinoma cell lines with deregulated Wnt/β-catenin signaling

Abstract

Background: Deregulation of Wnt/β-catenin signaling is a hallmark of the majority of sporadic forms of colorectal cancer and results in increased stability of the protein β-catenin. β-catenin is then shuttled into the nucleus where it activates the transcription of its target genes, including the proto-oncogenes MYC and CCND1 as well as the genes encoding the basic helix-loop-helix (bHLH) proteins ASCL2 and ITF-2B. To identify genes commonly regulated by β-catenin in colorectal cancer cell lines, we analyzed β-catenin target gene expression in two non-isogenic cell lines, DLD1 and SW480, using DNA microarrays and compared these genes to β-catenin target genes published in the PubMed database and DNA microarray data presented in the Gene Expression Omnibus (GEO) database.

Results: Treatment of DLD1 and SW480 cells with β-catenin siRNA resulted in differential expression of 1501 and 2389 genes, respectively. 335 of these genes were regulated in the same direction in both cell lines. Comparison of these data with published β-catenin target genes for the colon carcinoma cell line LS174T revealed 193 genes that are regulated similarly in all three cell lines. The overlapping gene set includes confirmed β-catenin target genes like AXIN2, MYC, and ASCL2. We also identified 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are regulated similarly in DLD1 and SW480 cells and one pathway - the steroid biosynthesis pathway - was regulated in all three cell lines.

Conclusions: Based on the large number of potential β-catenin target genes found to be similarly regulated in DLD1, SW480 and LS174T cells as well as the large overlap with confirmed β-catenin target genes, we conclude that DLD1 and SW480 colon carcinoma cell lines are suitable model systems to study Wnt/β-catenin signaling and associated colorectal carcinogenesis. Furthermore, the confirmed and the newly identified potential β-catenin target genes are useful starting points for further studies.

Figure 1

Validation of β-catenin siRNA. DLD1 and SW480 cells were treated with β-catenin siRNA (siβcat) or β-galactosidase siRNA (siβgal) and the protein expression levels of β-catenin were determined by immuno detection using an anti-β-catenin antibody. Actin was used as a loading control (A). The activity of the β-catenin dependent reporter gene 8×TOPflash was determined in DLD1 and SW480 cells after treatment of these cells with β-catenin siRNA or β-galactosidase siRNA. The experiments were performed in duplicates with error bars representing standard deviation (B).

Figure 2

Identification of β-catenin target genes. DLD1 and SW480 cells were treated with β-catenin siRNA and differentially expressed genes were identified using a DNA microarray. The corresponding data for LS174T cells was published earlier and used for comparison. The Venn-like diagrams display the number of differentially expressed genes when analyzing all differentially expressed genes (A), previously described β-catenin target genes listed in Table 1(B), or 96 genes encoding basic helix-loop-helix (bHLH) proteins (PFAM family PF00010) (C). The names of the genes corresponding to the seven sections of each Venn-like diagram are listed in separate tabs in the Additional files 1, 2 and 3.

Figure 3

Identification of known interactions between differentially expressed β-catenin target genes in DLD1, SW480, and LS174T cells. Using the software package “Cytoscape” in combination with the “MiMI” plugin, we identified known interactions between the 193 differentially expressed β-catenin target genes in DLD1, SW480, and LS174T cells (A) and the 335 differentially expressed β-catenin target genes in DLD1 and SW480 cells (B). Only gene networks are displayed that contain three or more nodes. The genes highlighted in pink color have been previously described as β-catenin target genes (see Table 1).