Abstract
BackgroundNotch and Wnt pathways are key regulators of intestinal homeostasis and alterations in these pathways may lead to the development of colorectal cancer (CRC). In CRC the Apc/β-catenin genes in the Wnt signaling pathway are frequently mutated and active Notch signaling contributes to tumorigenesis by keeping the epithelial cells in a proliferative state. These pathways are simultaneously active in proliferative adenoma cells and a crosstalk between them has previously been suggested in normal development as well as in cancer.Principal FindingsIn this study, in silico analysis of putative promoters involved in transcriptional regulation of genes coding for proteins in the Notch signaling pathway revealed several putative LEF-1/TCF sites as potential targets for β-catenin and canonical Wnt signaling. Further results from competitive electrophoretic mobility-shift assay (EMSA) studies suggest binding of several putative sites in Notch pathway gene promoters to in vitro translated β-catenin/Lef-1. Wild type (wt)-Apc negatively regulates β-catenin. By induction of wt-Apc or β-catenin silencing in HT29 cells, we observed that several genes in the Notch pathway, including Notch-2, were downregulated. Finally, active Notch signaling was verified in the ApcMin/ + mouse model where Hes-1 mRNA levels were found significantly upregulated in intestinal tumors compared to normal intestinal mucosa. Luciferase assays showed an increased activity for the core and proximal Notch-2 promoter upon co-transfection of HCT116 cells with high expression recombinant Tcf-4, Lef-1 or β-catenin.ConclusionsIn this paper, we identified Notch-2 as a novel target for β-catenin-dependent Wnt signaling. Furthermore our data supports the notion that additional genes in the Notch pathway might be transcriptionally regulated by Wnt signaling in colorectal cancer.
Highlights
The epithelium of the gastrointestinal tract is continually replaced with a turn-over rate of two to seven days
Twenty-four of the investigated genes in the Notch pathway were found to contain found in the Jagged-1 promoter at 21933 and 21635 relative to the translation start site, which is in accordance with earlier studies where Jagged-1 has been shown to be a b-catenin/Tcf-4 regulated gene in human colorectal cancer (CRC) [32] as well as in mouse hair follicles [36]
Hes-1 has recently been identified as a direct transcriptional target of b-catenin/Tcf-4-dependent Wnt signaling in CRC [31] and our electrophoretic mobility-shift assay (EMSA) data supports this notion by clearly identifying Hes-1 -528 as a binding site for the b-catenin/Lef-1 complex (Fig. S1)
Summary
The epithelium of the gastrointestinal tract is continually replaced with a turn-over rate of two to seven days. One of the critical components of the b-catenin destruction complex is the adenomatous polyposis coli (Apc) protein [11] Mutational inactivation of this gene causes stabilization of bcatenin [13] and increased cell proliferation and represents one of the most common genetic alterations in colorectal cancer (CRC). In CRC the Apc/b-catenin genes in the Wnt signaling pathway are frequently mutated and active Notch signaling contributes to tumorigenesis by keeping the epithelial cells in a proliferative state. These pathways are simultaneously active in proliferative adenoma cells and a crosstalk between them has previously been suggested in normal development as well as in cancer
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