Development of ESC-derived Intestinal Cells for Colon Cancer Research

用于结肠癌研究的 ESC 衍生肠细胞的开发

• 批准号: 8302854
• 负责人: CHARLES A. GIARDINA
• 金额: $ 18.39万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8302854
• 关键词: Address; Affect; BRAF gene; Biological Assay; Biological Factors; CDKN2A gene; Cell Count; Cell Culture System; Cell Cycle; Cell Cycle Regulation; Cell Differentiation process; Cell Proliferation; Cells; Chemoprevention; Chemopreventive Agent; Colon; Colon Carcinoma; Colonoscopy; Colorectal Cancer; Complement; Complex; CpG Island Methylator Phenotype; DNA Repair; Defect; Development; Endoderm; Engineering; Epigenetic Process; Epithelial Cells; Event; Eye; Future; Gene Silencing; Generations; Genes; Genetic; Genetic Polymorphism; Goals; Growth; Human; In Vitro; Individual; Inhibition of Apoptosis; Intestinal Mucosa; Intestines; KRAS2 gene; Libraries; MLH1 gene; Malignant Neoplasms; Methods; Modeling; Molecular; Mucous Membrane; Mus; Mutate; Mutation; Nutrient; Oncogene Activation; Oncogenes; Organoids; Pathway interactions; Phenotype; Polypoid Lesion; Prevention; Prevention strategy; Proteins; Regulator Genes; Relative (related person); Reporting; Risk; Role; Screening procedure; Signal Pathway; Signal Transduction; Small Interfering RNA; Staging; Stem cells; Structure; System; Testing; Tissues; Transgenic Animals; Tumor Suppressor Genes; Work; adenoma; anticancer research; base; cancer chemoprevention; cancer prevention; cancer risk; cell growth; cell transformation; clinically significant; colon carcinogenesis; combinatorial; design; embryonic stem cell; flexibility; high throughput screening; human embryonic stem cell; in vivo; in vivo Model; innovation; interest; knockout animal; mouse model; notch protein; novel; pre-clinical; prevent; promoter; research study; response; small molecule; stem cell differentiation; villin

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.

描述（由申请人提供）：我们建议开发一种用于研究结肠癌发展的非常早期阶段的新颖和创新的系统。这种体外培养系统涉及通过定形内胚层中间体促进胚胎干细胞（ESC）分化为肠类器官，然后通过癌基因表达或肿瘤抑制基因敲低启动细胞转化。我们一直在用小鼠和人类胚胎干细胞开发的这个实验系统，预计将是强大的和高度灵活的，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。该系统的优点包括与营养素、小分子、siRNA或其他生物制剂的快速分析相容的体外形式。此外，肠类器官可以从人类ESC中产生，从而可以详细研究具有不同结肠癌风险的个体的肠细胞。在拟议的研究中，我们将评估肠道类器官用于研究Braf癌基因激活的影响的用途。我们对BRAF突变细胞的兴趣源于这样一个事实，即BRAF突变的癌症经常在人类近端结肠中发展，在那里它们很难被检测和去除，这使得有效的化学预防这些癌症成为一个重要的目标。肠类器官将从小鼠ESC制备，其中Braf癌基因在肠谱系定型期间被激活（使用Villin-Cre/LSL-BrafV 600 E小鼠），并且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组织的类器官。这项工作将集中在癌基因对细胞周转动力学的影响， 肠干细胞扩增和细胞分化。由于Braf激活与表观遗传改变相关，我们还将确定癌基因激活对表观遗传修饰因子表达和生长调节基因沉默的影响。我们还将确定细胞生长调节化学预防剂纠正表达癌基因的肠类器官中的分子和增殖缺陷的能力。最后，我们将开发高通量检测系统，以筛选天然化合物和药理学试剂库，寻找选择性抑制表达Braf的肠细胞生长的新型化学预防剂。我们预计识别新的化学预防剂（或药物组合），在随后的建议进一步发展。最后，建立用于癌症研究的类器官系统可以促进研究影响癌症促进和预防的许多因素和变量。 公共卫生相关性：KRAS和BRAF癌基因可以在结肠癌发生的早期被激活，并刺激促生长和抗生长信号通路的复杂组合，最终决定细胞是否可以进展为结肠癌。在这个提议中，我们将从胚胎干细胞中获得肠细胞，以建立一个简单的系统来研究KRAS和BRAF癌基因激活后的复杂事件。我们的重点将是确定表观遗传学在促进癌基因的生长效应中的作用，并着眼于了解促生长信号如何被抑制以预防结肠癌。