LEF/TCF Expression in Colon Cancer

描述(申请人提供)：结肠癌的分子遗传学分析已经证实Wnt信号通路参与肿瘤的早期发展，特别是在结肠癌中。将信号转化为靶基因表达变化的转录因子是淋巴增强因子/T细胞因子(Lef/Tcf)家族的成员。该家族的两个成员TCF1和LEF1的基因产生截短的显性负值形式，限制Wnt信号，并可能作为生长抑制因子发挥作用。我们发现LEF1基因在结肠癌中是异位诱导的，但只有全长激活形式表达-显性阴性形式不表达。LEF1和TCF1在结肠癌进展过程中的表达模式尚不清楚。TCF1在人类结肠中正常表达，但在正常结肠和癌症中全长和显性阴性形式的相对数量也尚不清楚。根据我们的初步研究结果，我们认为LEF/TCFs的全长亚型在结肠癌中占主导地位。此外，我们认为Wnt途径本身在促进全长异构体的表达方面发挥了作用。为了验证这些假设，我们将测定LEF1和TCF1亚型在正常、息肉和结肠癌中的表达(特定目标1)。为了探讨LEF1异常表达的机制，我们将在结肠癌细胞系中评估LEF1基因的遗传完整性及其对染色质结构表观遗传变化的敏感性(特定目标2)。在瞬时转染实验中，TCF1/a-catenin和TCF4/a-catenin复合体能够激活LEF1启动子，而其他LEF/TCFs则不能。我们认为LEF1是肿瘤中特异性TCF1/4/a-连环蛋白复合体的靶点。建议进行实验以探索这一调节，并测试其与结肠癌细胞内源性LEF1基因的相关性(特定目标3)。基于TCFI/4对LEF1启动子的选择性作用，我们假设每个LEF/TCF家族成员都携带独特的靶基因特异性，并且它们在癌症中的表达具有不同的作用。为了验证这一假设，将使用两种策略来扰乱结肠癌细胞中的LEF/TCF功能。效果将通过全球基因表达谱以及细胞生长和活力分析(特定目标4)进行评估。

DESCRIPTION (provided by applicant): Molecular genetic analysis of colon cancers has established that the Wnt signaling pathway is involved in early tumor development, particularly in colon cancer. The transcription factors that commute the signal into changes in target gene expression are members of the Lymphoid Enhancer Factor/T Cell Factor (LEF/TCF) family. The genes for two members of this family, TCF1 and LEF1, produce truncated dominant negative forms that limit Wnt signals and may function as growth suppressors. We have found that the LEF1 gene is ectopically induced in colon cancer, but only the full-length activating form is expressed - the dominant negative form is not. The patterns of LEF1 and TCF1 expression during colon cancer progression are not known. TCF1 is normally expressed in human colon, but the relative amounts of full-length and dominant negative forms in normal colon and cancer are also not known. Based on results from our preliminary studies, we propose that full-length isoforms of LEF/TCFs predominate in colon cancer. Furthermore, we propose that the Wnt pathway itself plays a role in promoting expression of full-length isoforms. To test these hypotheses we will determine the expression of LEF1 and TCF1 isoforms in normal, polyp and colon carcinoma (Specific Aim 1). To probe the mechanism of aberrant LEF1 expression, the genetic integrity of the LEF1 gene and its sensitivity to epigenetic changes in chromatin structure will be assessed in colon cancer cell lines (Specific Aim 2). TCFl/a-catenin and TCF4/ a-catenin complexes can activate the LEF1 promoter in transient transfection assays, other LEF/TCFs cannot. We propose that LEF1 is a target of specific TCFl/4/ a-catenin complexes in cancer. Experiments are proposed to explore this regulation and test for its relevance to the endogenous LEF1 gene in colon cancer cells (Specific Aim 3). Based on the selective effect of TCFI/4 on the LEF1 promoter, we hypothesize that each LEF/TCF family member carries unique target gene specificities and that their expression in cancer imparts distinct effects. To test this hypothesis, two strategies will be used to disrupt LEF/TCF function in colon cancer cells. Effects will be assessed by global gene expression profiles and cell growth and viability assays (Specific Aim 4).