The <em>Notch-2</em> Gene Is Regulated by Wnt Signaling in Cultured Colorectal Cancer Cells

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.背景：Notch与Wnt信号通路是肠道稳态的关键调控因子，这两条通路的异常改变可促使结直肠癌（colorectal cancer, CRC）的发生发展。在结直肠癌中，Wnt信号通路内的Apc/β-连环蛋白基因常发生突变，而激活的Notch信号通路可通过维持上皮细胞的增殖状态促进肿瘤发生。这两条通路在增殖性腺瘤细胞中同时激活，且既往研究已提出二者在正常发育及癌症进程中存在串扰。 主要研究结果：本研究通过生物信息学分析（in silico）对编码Notch信号通路蛋白的基因进行转录调控潜在启动子的分析，发现多个潜在的LEF-1/TCF结合位点，可作为β-连环蛋白及经典Wnt信号通路的潜在靶点。后续竞争性电泳迁移率变动分析（electrophoretic mobility-shift assay, EMSA）结果显示，Notch通路基因启动子上的多个潜在位点可与体外翻译的β-连环蛋白/Lef-1结合。野生型（wild type, wt）Apc蛋白负向调控β-连环蛋白。通过在HT29细胞中诱导野生型Apc表达或沉默β-连环蛋白，我们观察到包括Notch-2在内的多个Notch通路基因表达下调。最后，在ApcMin/+小鼠模型中验证了激活的Notch信号通路：与正常肠黏膜相比，该模型小鼠肠道肿瘤组织中Hes-1 mRNA的表达水平显著上调。荧光素酶报告基因实验（luciferase assay）显示，当用高表达重组Tcf-4、Lef-1或β-连环蛋白的质粒共转染HCT116细胞时，Notch-2启动子核心区域及近端区域的活性显著升高。 结论：本研究鉴定出Notch-2是依赖β-连环蛋白的Wnt信号通路的全新靶点。此外，本研究数据支持以下观点：结直肠癌中Notch通路的其他基因或许也可被Wnt信号通路实施转录调控。