Development of ESC-derived Intestinal Cells for Colon Cancer Research

描述（由申请人提供）：我们建议开发一种新颖的创新系统来研究非常早期的结肠癌发展。该体外培养系统通过最终的内胚层中间体促进胚胎干细胞（ESC）分化为肠道类器官，然后通过表达癌基因或敲除肿瘤抑制基因启动细胞转化。我们一直在用小鼠和人类ESCs开发这个实验系统，预计它将是强大和高度灵活的，并使癌症生物学家能够解决与结肠癌发生的早期事件有关的基本问题。该系统的优点包括体外格式兼容快速分析营养物质，小分子，sirna或其他生物制剂。此外，肠道类器官可以从人类ESCs中生成，从而可以对具有不同结肠癌风险的个体的肠道细胞进行详细研究。在拟议的研究中，我们将评估肠道类器官在研究Braf致癌基因激活效应方面的作用。我们对Braf突变细胞的兴趣源于这样一个事实，即Braf突变的癌症经常发生在人类近端结肠，在那里它们很难被发现和移除，因此有效的化学预防这些癌症是一个重要的目标。肠道类器官将由小鼠ESCs制备，其中Braf癌基因在肠道谱系承诺过程中被激活（使用Villin- Cre/LSL-BrafV600E小鼠），并由其天然启动子调节。然后，我们将评估癌基因表达如何影响体内内源性肠道组织的类器官。这项工作将集中于癌基因对细胞更新动力学的影响，

DESCRIPTION (provided by applicant): We propose to develop a novel and innovative system for studying very early stages of colon cancer development. This in vitro culture system involves promoting embryonic stem cell (ESC) differentiation into intestinal organoids through a definitive endoderm intermediate, and then initiating cell transformation through oncogene expression or tumor suppressor gene knockdown. This experimental system, which we have been developing with mouse and human ESCs, is anticipated to be robust and highly flexible and enable cancer biologists to address fundamental questions relating to very early events in colon carcinogenesis. Advantages of this system include an in vitro format compatible with the rapid analysis of nutrients, small molecules, siRNAs or other biologics. Furthermore, intestinal organoids can be generated from human ESCs, allowing the detailed study of intestinal cells from individuals with distinct colon cancer risks. In the proposed studies we will assess the utiliy of intestinal organoids for studying the effects of Braf oncogene activation. Our interest in Braf mutated cells stems from the fact that BRAF-mutated cancers frequently develop in the human proximal colon where they can be difficult to detect and remove, making effective chemoprevention of these cancers an important goal. Intestinal organoids will be prepared from mouse ESCs in which Braf oncogene become activated during intestinal lineage commitment (using Villin- Cre/LSL-BrafV600E mice) and are regulated by their native promoters. We will then evaluate how oncogene expression influences the organoids in relationship to endogenous intestinal tissues in vivo. This work will focus on oncogene effects on cellular turnover dynamics, intestinal stem cell expansion and cellular differentiation. Since Braf activation is associated wih epigenetic alterations, we will also determine the effect of oncogene activation on the expression of epigenetic modifying factors and the silencing of growth regulatory genes. We will also determine the ability of cell growth modulating chemopreventive agents to correct molecular and proliferation defects in oncogene-expressing intestinal organoids. Finally, we will develop high throughput assay systems to screen natural compound and pharmacological agent libraries for novel chemopreventive agents that selectively inhibit the growth of Braf-expressing intestinal cells. We anticipate indentifying novel chemopreventive agents (or agent combinations) for further development in subsequent proposals. Finally, establishing the organoid system for cancer research could facilitate the study many factors and variables that impact cancer promotion and prevention. PUBLIC HEALTH RELEVANCE: The KRAS and BRAF oncogenes can be activated early in colon carcinogenesis and stimulate a complex combination of pro-growth and anti-growth signaling pathways that ultimately determine whether the cells can progress to colon cancer or not. In this proposal we will derive intestinal cells from embryonic stem cells to establish a simple system to study the complex events that follow KRAS and BRAF oncogene activation. Our focus will be to determine the role of epigenetics in promoting the growth effects of the oncogenes with an eye towards understanding how the pro-growth signaling might be suppressed for colon cancer prevention.