Wnt Signaling in intestinal stem cells, homeostasis, and cancer

项目摘要/摘要 Wnt家族分泌蛋白通过转录共激活因子β-连环蛋白(Wnt/β-连环蛋白)传递信号 途径)在肠道干细胞(ISCs)的调节和胃肠道的内稳态中起核心作用 (GI)肠道。R-响应蛋白是一种分泌型分子，可通过增强Wnt/β-连环蛋白信号转导通路 稳定Wnt受体，对ISCs的自我更新和增殖有很强的刺激作用。 Wnt/RSPO信号异常可导致包括结直肠癌(CRC)在内的胃肠道疾病。 Wnt/β-catenin信号通过ISC特异的基因表达程序控制ISCs，该程序由 T细胞因子/淋巴增强因子(T细胞因子/淋巴增强因子)家族转录因子与β- 连锁素。Tcf/β-catenin介导的转录调控是我们理解 WnT/β-连环蛋白通路参与间质细胞癌的调控和结直肠癌的发病。 更好地了解Wnt/RSPO信号转导机制，寻找治疗结直肠癌的新靶点 处理后，我们进行了功能cdna表达筛选，并鉴定出锌指(Znf)转录 因子作为Tcf/β依赖的连环蛋白转录的有效刺激因子。我们的初步数据表明，这 锌是必需的：(I)Wnt/RSPO刺激小鼠肠道器官中ISC的扩张；(Ii)Tcf/β- 连环蛋白介导的Wnt靶基因/干细胞信号基因在人结直肠癌细胞系中的表达；以及(Iii)促进增殖 CRC细胞系。我们的初步数据进一步表明，锌F与Tcf结合，并与Tcf/β共同占据- 染色质中Wnt靶基因增强子/启动子的连环蛋白。我们的初步发现确定了一部小说 在ISC和CRC细胞中的WNT/RSPO信号的关键组件，并揭示了在 Tcf/β-catenin介导的基因激活的机制。 我们在这一应用中提出了三个特定的目标来研究在ISC调节中Wnt/RSPO信号中的znf 以及结直肠癌的发病机制。在目标1中，我们将定义在Tcf/β-连环蛋白驱动的转录中对锌的需求，通过 全基因组RNA-seq和芯片-seq技术，从而解决这一因素是否对所有或 Tcf/β-catenin靶基因的一个子集；在目标2中，我们将研究人类肠道对锌的需求。 有机化合物和初级CRC有机化合物，试图验证其在人类胃肠道和癌症生物学中的关键作用； 在目标3中，我们将产生条件性的zif缺失突变小鼠，从而研究其在肠道组织中的作用。 体内动态平衡与肿瘤的形成。

Project Summary/Abstract Signaling by the Wnt family of secreted proteins through transcription coactivator β-catenin (the Wnt/β-catenin pathway) plays central roles in regulation of intestinal stem cells (ISCs) and homeostasis of the gastrointestinal (GI) tract. R-spondin (Rspo) proteins are secreted molecules that enhance Wnt/β-catenin signaling through stabilizing Wnt receptors, and they exhibit potent stimulation effect on self-renewal and proliferation of ISCs. Anomaly of Wnt/Rspo signaling leads to GI diseases including colorectal cancer (CRC). Wnt/β-catenin signaling controls ISCs through an ISC-specific gene expression program, which is driven by the DNA-bound TCF/LEF (T cell factor/Lymphoid enhancer factor) family of transcription factors in complex with β- catenin. TCF/β-catenin-mediated transcriptional regulation has been a cornerstone for our understanding of the Wnt/β-catenin pathway including in ISC regulation and CRC pathogenesis. To better understand Wnt/Rspo signaling and search for additional potential therapeutic target for CRC treatment, we performed a functional cDNA expression screen and identified a Zinc-finger (Znf) transcription factor as a potent stimulators of TCF/β-catenin-dependent transcription. Our preliminary data suggest that this Znf is required for (i) Wnt/Rspo stimulation of ISC expansion in mouse intestinal organoids; (ii) for TCF/β- catenin-mediated Wnt target genes/stem cell signature genes in human CRC cell lines; and (iii) for proliferation of CRC cell lines. Our preliminary data further suggest that the Znf binds to TCF, and co-occupies with TCF/β- catenin on enhancers/promoters of Wnt target genes in chromatin. Our preliminary findings identify a novel critical component of Wnt/Rspo signaling in ISCs and CRC cells, and reveal an unappreciated complexity in the mechanism by which TCF/β-catenin-mediated gene activation is achieved. We propose three specific aims in this application to investigate the Znf in Wnt/Rspo signaling in ISC regulation and CRC pathogenesis. In Aim 1 we will define the Znf requirement in TCF/β-catenin-driven transcription via genome-wide RNA-seq and CHIP-seq techniques, thereby addressing whether this factor is required for all or a subset of TCF/β-catenin target genes; In Aim 2 we will examine the Znf requirement in human intestinal organoids and primary CRC organoids, attempting to validate its critical role in human GI and cancer biology; and in Aim 3 we will generate conditional Znf deletion mutant mice, thereby studying its role in intestinal tissue homeostasis and tumor formation in vivo.