Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas

Abstract

Mutations in the adenomatous polyposis coli or beta-catenin gene lead to cytosolic accumulation of beta-catenin and, subsequently, to increased transcriptional activity of the beta-catenin-T cell-factor/lymphoid-enhancer-factor complex. This process seems to play an essential role in the development of most colorectal carcinomas. To identify genes activated by beta-catenin overexpression, we used colorectal cell lines for transfection with the beta-catenin gene and searched for genes differentially expressed in the transfectants. There are four genes affected by beta-catenin overexpression; three overexpressed genes code for two components of the AP-1 transcription complex, c-jun and fra-1, and for the urokinase-type plasminogen activator receptor (uPAR), whose transcription is activated by AP-1. The direct interaction of the beta-catenin-T cell-factor/lymphoid-enhancer-factor complex with the promoter region of c-jun and fra-1 was shown in a gel shift assay. The concomitant increase in beta-catenin expression and the amount of uPAR was confirmed in primary colon carcinomas and their liver metastases at both the mRNA and the protein levels. High expression of beta-catenin in transfectants, as well as in additionally analyzed colorectal cell lines, was associated with decreased expression of ZO-1, which is involved in epithelial polarization. Thus, accumulation of beta-catenin indirectly affects the expression of uPAR in vitro and in vivo. Together with the other alterations, beta-catenin accumulation may contribute to the development and progression of colon carcinoma both by dedifferentiation and through proteolytic activity.

Figure 1

Analysis of β-catenin expression, APC status, and Tcf4 expression in the cell lines used. (a) Variable expression of β-catenin. Whole cell lysates were separated on a 6% polyacrylamide gel, blotted, and developed with anti-β-catenin mAb 7D11 or, for control of the amount of protein per lane, with anti-β-actin mAb 5441. (b) Cell line CD-NCM425 and LS 174T have APC with the expected molecular mass, whereas, in CSC-1 and SW480, the APC is truncated. Immunoblots from whole cell lysates were developed with FE9 mAb directed against the NH₂-terminal region of APC. The upper part of the membrane was exposed 10-fold longer than the lower part. Cell lines SW480 and LS 174T were blotted in parallel as controls for truncated and intact APC, respectively. (c) Tcf4 is expresed strongly in all cell lines except CSC-1; 25 μg of total RNA per lane was hybridized with probes for Tcf4 or for β-actin for control of amounts of RNA.

Figure 2

Effect of β-catenin transfection on target gene expression. (a) Total RNA (25 μg per lane) from CD-NCM425 and CCO7 cells transfected with pcDNAβ-415 (+) or from CD-NCM425 and CCO7 cells that were mock transfected (−) was hybridized with probes for the genes that were found to be expressed differentially by the Atlas Array. Hybridization with the β-actin probe was performed to control the amounts of RNA applied per lane. (b) Expression of the uPAR and ZO-1 protein in the transfected cell lines. Immunoblots from whole cell lysates were developed with anti-β-catenin mAb 7D11, anti-uPAR rabbit IgG 399R, anti-ZO-1 rabbit IgG 61–7300, or, for control of the amount of protein per lane, anti-β-actin mAb 5441. (Expression differences in a and b are indicated as the percentage of expression in transfected cells related to mock transfected cells after normalization to the β-actin expression on the same blot.)

Figure 3

Promoter regions of fra-1 and c-jun are direct targets of the β-catenin–Tcf/Lef complex. Gel retardation assays were performed on nuclear extracts from SW480 cells with radioactively labeled oligonucleotides from the indicated promoter regions containing Tcf/Lef binding motifs. Anti-β-catenin mAb (0.75 μg) was added to the samples in lanes 3 and 6. No nuclear extracts were added in lanes 1 and 4.

Figure 4

Suppression of ZO-1 protein expression in cell lines with high endogenous β-catenin expression. (a) Immunoblots from whole cell lysates developed with anti-ZO-1 rabbit IgG 61–7300, anti-β-catenin mAb 7D11, or, for control of the amount of protein per lane, anti-β-actin mAb 5441. (b) Expression of ZO-1 and β-catenin protein in the analyzed cell lines, after normalization to β-actin expression (a.u., arbitrary units). The expression of ZO-1 was inversely related to β-catenin expression.

Figure 5

uPAR and β-catenin expression levels are increased concomitantly in primary carcinomas and their liver metastases. (a) Total RNA (25 μg per lane) from tissue samples of normal colonic mucosa (N), primary tumor (T), and metastases (M) were hybridized with probes for uPAR, β-catenin, or β-actin. (b) Immunoblot of whole cell lysates, developed with anti-uPAR rabbit polyclonal antibody 399R or with anti-β-actin mAb 5441. Representative blots from three of six analyzed patients are shown in a and b. (c) Expression of β-catenin mRNA, uPAR mRNA, and uPAR protein in tissue samples from N, T, and M of six analyzed patients after normalization to β-actin expression on the same blots. Bars indicate two standard deviations.

Figure 6

Genes targeted by the β-catenin–Tcf/Lef complex and the related upstream and downstream cellular processes.