The Notch-2 Gene Is Regulated by Wnt Signaling in Cultured Colorectal Cancer Cells

• Background: Notch 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/beta-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 Findings: In 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 beta-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 beta-catenin/Lef-1. Wild type (wt)-Apc negatively regulates beta-catenin. By induction of wt-Apc or beta-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 Apc(Min/+) 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 beta-catenin. Conclusions: In this paper, we identified Notch-2 as a novel target for beta-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.

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