Wnt Signaling in intestinal stem cells, homeostasis, and cancer

项目概要/摘要 分泌蛋白的Wnt家族通过转录辅激活因子β-连环蛋白（Wnt/β-连环蛋白）的信号传导 在调节肠干细胞（ISCs）和胃肠道的稳态中起着重要作用 (GI)道。R-spondin（Rspo）蛋白是分泌的分子，其通过增强Wnt/β-连环蛋白信号传导而增强Wnt/β-连环蛋白信号传导。 稳定Wnt受体，并且它们对ISCs的自我更新和增殖表现出有效的刺激作用。 Wnt/Rspo信号传导异常会导致包括结直肠癌（CRC）在内的胃肠道疾病。 Wnt/β-catenin信号通过ISC特异性基因表达程序控制ISC，该程序由 DNA结合的TCF/LEF（T细胞因子/类磷脂增强因子）转录因子家族与β- 连环蛋白。TCF/β-catenin介导的转录调控一直是我们理解TCF/β-catenin基因的基础。 Wnt/β-连环蛋白途径包括ISC调节和CRC发病机制。 为了更好地了解Wnt/Rspo信号通路，并寻找CRC的其他潜在治疗靶点 治疗后，我们进行了功能性cDNA表达筛选，并确定了锌指（Znf）转录 因子作为TCF/β-连环蛋白依赖性转录的有效刺激物。我们的初步数据表明， Znf是（i）小鼠肠类器官中ISC扩增的Wnt/Rspo刺激;（ii）TCF/β-CD刺激所必需的。 连环蛋白介导的Wnt靶基因/人CRC细胞系中的干细胞标记基因;和（iii）用于增殖 CRC细胞系。我们的初步数据进一步表明Znf与TCF结合，并与TCF/β- 连环蛋白对染色质中Wnt靶基因的增强子/启动子的作用。我们的初步发现确定了一种新的 在ISC和CRC细胞中Wnt/Rspo信号传导的关键组成部分，并揭示了 TCF/β-catenin介导的基因激活的机制。 我们在本申请中提出了三个具体目标来研究ISC调节中Wnt/Rspo信号传导中的Znf 和CRC发病机制。在目标1中，我们将通过以下方式定义TCF/β-连环蛋白驱动的转录中的Znf要求： 全基因组RNA-seq和CHIP-seq技术，从而解决这个因素是否需要所有或 TCF/β-连环蛋白靶基因的一个子集;在目标2中，我们将研究人类肠道中Znf的需求。 类器官和原发性CRC类器官，试图验证其在人类GI和癌症生物学中的关键作用; 目的3我们将建立条件性Znf缺失突变小鼠，从而研究其在肠道组织中的作用 体内稳态和肿瘤形成。

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.