Wnt Signaling in Colon Cancer

描述（由申请人提供）：Wnt信号是与肠道癌变相关的最常见途径之一。介导Wnt信号的转录因子是淋巴增强因子/T细胞因子（LEF/TCF）家族的成员。这个四成员家族是复杂的，因为它有全长激活形式，截断的显性阴性形式，以及修改Wnt靶基因识别的选择性剪接同种异构体。TCF-1和TCF-4是在肠上皮隐窝区表达的两个家族成员。通常情况下，TCF-1以显性负异构体表达，它抵消了TCF-4的作用，后者以wnt介导的活性异构体表达。这种模式在癌症中发生变化，因为TCF-1表达转换为全长Wnt介导形式。我们在TCF-1和TCF-4同工异构体中发现了第二个DNA结合域，称为C-clamp。我们已经证明C-clamp对于调节结肠癌细胞的增殖是重要的。我们还发现了在结肠癌细胞中触发TCF-1输出的wnt。TCF-1的输出在细胞核中留下C-clamp形式的TCF-4介导异常的Wnt信号传导。为这个项目提出了三个目标。首先，将定义指导TCF-1核输出的细胞外wnt和细胞内组分（Aim1）。其次，输出的生物学影响将在结肠癌细胞和结肠癌启动3D培养（Aim2）中定义。第三，将通过全基因组结合测定（ChIP-Seq）、高通量蛋白结合微阵列和体外生化分析（Aim3）确定C-clamp形式的TCF-1和TCF-4的DNA结合特异性和靶基因选择。总体目标是确定结肠癌发生中TCF表达变化背后的机制，并确定细胞外wnt和TCF的DNA结合特异性如何影响稳定-catenin的结肠癌细胞中的异常信号传导。

DESCRIPTION (provided by applicant): Wnt signaling is one of the most common pathways linked to carcinogenesis in the intestine. The transcription factors that mediate Wnt signals are members of the Lymphoid Enhancer Factor/T Cell Factor (LEF/TCF) family. This four-member family is complex in that there are full-length activating forms, truncated dominant negative forms, and alternatively spliced isoforms that modify Wnt target gene recognition. TCF-1 and TCF-4 are the two family members expressed in the epithelial crypt compartment of the intestine. Normally, TCF-1 is expressed as a dominant negative isoform and it counteracts the action of TCF-4 which is expressed as a Wnt-mediating, active isoform. This pattern changes in cancer because TCF-1 expression switches to a full-length, Wnt mediating form. We have discovered a second DNA binding domain in TCF-1 and TCF-4 isoforms called the C-clamp. We have shown that the C-clamp is important for regulating cell proliferation of colon cancer cells. We have also identified Wnts that trigger TCF-1 export in colon cancer cells. Export of TCF-1 leaves the C-clamp form of TCF-4 in the nucleus to mediate aberrant Wnt signaling. Three aims are proposed for this project. First, the extracellular Wnts and intracellular components that direct TCF-1 nuclear export will be defined (Aim1). Second, the biological impact of export will be defined in colon cancer cells and colon cancer initiating 3D cultures (Aim2). Third, the DNA binding specificities and target gene selection of C-clamp forms of TCF-1 and TCF-4 will be defined through genome-wide binding assays (ChIP-Seq), high-throughput protein binding microarrays, and in vitro biochemical analysis (Aim3). The overall goal is to define the mechanisms behind changes in TCF expression in colon carcinogenesis, and to define how extracellular Wnts and DNA binding specificities of TCFs influence aberrant signaling in colon cancer cells with stabilized ?-catenin. PUBLIC HEALTH RELEVANCE: In colon cancer, Wnt signaling is an overactive and strong signal for tumor initiation and progression. Wnt signaling is mediated by the TCF transcription factors and we have discovered their activities and expression to be different in colon cancer versus normal colon tissue. This project investigates basic mechanisms of TCF localization and gene targeting and asks how differences in these attributes contribute to the cancer cell phenotype.