Dissecting the role of TCF7L1 in colorectal cancer

项目总结 去调控的Wnt/β-连环蛋白信号是结直肠癌的一个共同特征，但这一通路是如何 腐败靶基因的表达还不完全清楚。T细胞因子/淋巴增强因子(Tcf/Lef； 此后，转录因子结合Wnt反应元件(WRes)来控制Wnt/β-连接蛋白 靶基因表达。在四个TCF家族成员(TCF7、LEF1、TCF7L1和TCF7L2)中，很少有 已知结直肠癌中的TCF7L1及其直接调控的靶基因集。通过使用shRNA耗尽 在已建立的人结直肠癌细胞系中，我们发现TCF7L1能促进细胞生长。虽然微阵列分析 在TCF7L1基因敲除细胞和对照细胞中差异表达的转录本发现了数百个 基因，令人惊讶的是，我们没有在这个列表中识别出WNT靶基因签名。此外，还出版了 ChIP-Seq数据表明，相当一部分TCF结合的DNA元件缺乏共识的TCF结合 图案。这些发现表明，TCF7L1可能主要在规范的Wnt信号之外发挥作用 促进肿瘤发生的途径。还需要额外的工作来确定直接靶基因的星座 TCF7L1调控，以及是否需要其DNA结合功能来促进结直肠癌生长。在目标1中，我们 将通过进行无偏见的全基因组功能基因组学来鉴定TCF7L1转录组 对照和TCF7L1耗尽的CRC细胞的方法。在TCF7L1缺失的品系中，我们将引入野生型 和DNA结合缺陷的TCF7L1 cDNA来分类其表达独立于 TCF7L1直接与DNA结合。在目标2中，我们将确定TCF7L1的DNA结合能力是否 对于结直肠癌细胞的增殖、细胞周期的进展、在不依赖于锚定的环境中生长是必需的 方式，和体内的肿瘤发生。我们还将评估非规范目标是否具有差异性 在原发的人类结肠组织和肿瘤中表达。绝大多数现有的治疗方法旨在 针对Wnt途径的研究主要集中在核Tcf/β-连环蛋白复合体作为结直肠癌的关键调节因子 转录组。我们的发现将确立TCF7L1是典型的Wnt/β-之外的基因的关键调节因子。 并将开辟一个全新的研究领域，以开发这些靶点用于治疗 干预。

PROJECT SUMMARY Deregulated Wnt/β-catenin signaling is a common feature of colorectal cancer (CRC), but how this pathway corrupts target gene expression is not fully understood. The T-cell factor/Lymphoid enhancer factor (TCF/Lef; hereafter TCF) transcription factors bind Wnt-responsive DNA elements (WREs) to control Wnt/β-catenin target gene expression. Of the four TCF family members (TCF7, LEF1, TCF7L1, and TCF7L2) very little is known about TCF7L1 in CRC and the set of target genes it directly regulates. By using shRNAs to deplete TCF7L1 in established human CRC lines, we found that it promotes cell growth. Although microarray analysis of transcripts differentially expressed in TCF7L1 knockdown cells versus controls uncovered hundreds of genes, surprisingly, we failed to identify a Wnt target gene signature among this list. Moreover, published ChIP-Seq data indicates that a substantial fraction of TCF-bound DNA elements lack consensus TCF binding motifs. These findings suggest that TCF7L1 may primarily function outside the canonical Wnt signaling pathway to promote oncogenesis. Additional work is needed to identify the constellation of direct target genes that TCF7L1 regulates and whether its DNA-binding function is required to promote CRC growth. In Aim 1, we will identify the TCF7L1-transcriptome by conducting unbiased and genome-wide functional genomics approaches in control and TCF7L1-depleted CRC cells. In TCF7L1-depleted lines, we will introduce wild-type and DNA-binding deficient TCF7L1 cDNAs to classify targets whose expression is regulated independently of direct TCF7L1 binding to DNA. In Aim 2, we will determine whether the DNA-binding capacity of TCF7L1 is required for CRC cell proliferation, progression through the cell cycle, growth in an anchorage-independent manner, and tumorigenesis in vivo. We will also assess whether non-canonical targets are differentially expressed in primary human colonic tissues and tumors. The vast majority of existing therapeutics designed to target the Wnt pathway have focused on nuclear TCF/β-catenin complexes as the key regulator of the CRC transcriptome. Our findings will establish TCF7L1 as a critical regulator of genes outside the canonical Wnt/β- catenin pathway and will open a whole new field of research to exploit those targets for therapeutic intervention.