beta-catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer

Abstract

Most colorectal cancers have loss of function mutations in the adenomatosis polyposis coli (APC) tumor suppressor gene. This leads to accumulation of beta-catenin, which together with the DNA binding protein TCF-4 functions as a transcriptional activator. Recently defined target genes are c-myc and cyclin D1, linking the APC gene defect to the capacity for autonomous proliferation of colon tumors. Here we report the identification of the matrix metalloproteinase MMP-7 as another target gene of beta-catenin/TCF-4. MMP-7 is overexpressed in 80% of human colorectal cancers and known to be an important factor for early tumor growth, with a potential function also for later progression steps, like invasion and metastasis. Our results explain the high percentage of MMP-7 overexpression in colon tumors. Moreover they indicate that defects in the APC tumor suppressor gene may also have an influence on later steps of colon tumor progression.

Figure 1.

Identification of two TCF binding sites in the human MMP-7 promoter. a: Localization of the two identified TCF binding sites in the human MMP-7 promoter, TCF(1.) and TCF(2.) (▪). Also indicated are the previously defined TGF-β inhibitory element-like sequences (TIE), the ets (PEA3) and the AP-1 binding sites (□). Numbers indicate nucleotide positions relative to the transcription initiation site (+1). The diagram is not to scale. Also shown are the nucleotide sequences of both sites and the mutated sequences used in the following experiments (TCF(1.m) and TCF(2.m)). b: TCF-4 binds specifically to both TCF sites. EMSA with TCF(1.) and TCF(2.) sequences (wt) or their mutants (mt) as probes, incubated with GST-TCF-4 (TCF-4) or GST alone. For specific competition the unlabeled oligonucleotides used as probes or TBE2 (TCF binding site of the c-myc promoter) were used.

Figure 2.

β-catenin/TCF activate the human MMP-7 promoter. a: Reporter constructs used in transient transfection assays. The phMMP-7prLuc (WT) was mutated (crossed boxes) at only one of the two TCF binding sites (TCF(1.m), and TCF(2.m)), leaving the other intact, or at both sites (TCF1.m/2.m). For both TCF sites four copies of the wild-type (4xTCF) or mutated (4xTCFm) sequences were cloned upstream of a thymidine kinase minimal promoter. All constructs contain luciferase (Luc) cDNA as reporter. b: TCF4 and β-catenin activate the human MMP-7 promoter. Hela cells were cotransfected with WT reporter construct and one μg of the indicated expression vectors (act.) or stimulated with TPA (100 ng/ml). c: Mutations in the TCF binding sites reduce activation of the MMP-7 promoter. HeLa cells were cotransfected with the indicated reporter constructs (rep.) and expression constructs for TCF4 and β-catenin. d: β-catenin/TCF4 activate via isolated TCF sites. HeLa cells were cotransfected with the indicated reporter constructs (rep.) and expression constructs for TCF4 and β-catenin. Mutations in the TCF elements abolished stimulation. In all diagrams columns show activation values compared to cotransfection with the empty expression vector. Data are presented as the mean with standard deviation from three separate experiments. e: Dominant negative TCF-4 (DNTCF4) reduces activity of the human MMP-7 promoter in colon carcinoma cells. HT29 colon carcinoma cells were cotransfected with the indicated reporters (phMMP-7prLuc (WT) or TCF(1.m/2.m)) and expression constructs (vector control, β-catenin +TCF, dNTCF). Note that DNTCF4 leads to a significant reduction only of the WT MMP-7 promoter activity. Shown is the percentage related to the basal activity (100%) of the reporter constructs in HT29 cells.

Figure 3.

Correlation of β-catenin and MMP-7 expression in sporadic colorectal carcinomas (red staining). Immunohistochemistry of two representative colorectal carcinomas with strong (a–d) or very weak (e–h) expression of β-catenin. Serial sections were stained with the indicated antibodies. Note the correlation of strong (a, b) and weak (e, f) expression of β-catenin and MMP-7. The staining efficiencies of the tumor marker CEA and the proliferation marker mib-1 are comparable in both tumors (only few cells are mib-1-positive in the β-catenin strong expressing tumor) (d). Magnification, ×400.