The selective advantage of mismatch repair loss in colonic stem cells

结肠干细胞错配修复缺失的选择性优势

• 批准号: 10375493
• 负责人: Christopher D. Heinen
• 金额: $ 47.16万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10375493
• 关键词: Address; Adult; Affect; Alleles; Apoptosis; Biological Models; CRISPR/Cas technology; Cancer cell line; Cell Cycle Arrest; Cell Cycle Checkpoint; Cell Death; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Colon; Colon Carcinoma; Colorectal; Colorectal Cancer; DNA; DNA Damage; DNA Repair; DNA-Directed DNA Polymerase; Defect; Development; Diagnosis; Disease; Endometrial; Environment; Epithelial Cells; Event; Experimental Models; Frequencies; Genes; Goals; Growth; Hereditary Disease; Hereditary Nonpolyposis Colorectal Neoplasms; High Fat Diet; Human; Inflammation; Inherited; Intestines; Knock-out; Knockout Mice; Laboratories; MLH1 gene; MSH2 gene; MSH6 gene; Malignant Neoplasms; Mediating; Mismatch Repair; Modeling; Mosaicism; Mus; Mutation; Oncogenic; Organoids; Ovarian; PMS2 gene; Pathway interactions; Patients; Penetrance; Phenotype; Play; Prevention; Prevention strategy; Process; Production; Regimen; Risk; Role; Sampling; Stress; System; Testing; Time; Tissue Sample; Tumor Suppressor Genes; cancer therapy; cell growth; colonic crypt; gene repair; human embryonic stem cell; human stem cells; in vivo; insight; intestinal crypt; knockout gene; lifetime risk; mouse model; mutant; novel; novel strategies; oxidative damage; pressure; prevent; repair function; response; stem cell model; stem cell niche; stem cells; treatment strategy; tumor; tumorigenesis; tumorigenic

Project Summary Lynch syndrome (LS) is a hereditary disease that predisposes patients to colorectal, endometrial, ovarian and other cancers. LS is caused by inherited mutations in the DNA mismatch repair (MMR) genes, defects in which also underlie 15-30% of sporadic colorectal cancers. Loss of MMR function is associated with a 1,000-fold increase in mutation rate likely increasing the risk of mutation to important oncogenes and tumor suppressors. The MMR pathway also activates cell cycle checkpoints and cell death in response to exogenous DNA damage, however, the role of this damage response in preventing tumorigenesis is not known. We hypothesize that colonic stem cells (CSCs) that lose this MMR-dependent damage response will gain a selective advantage over neighboring MMR-proficient cells, particularly in a mutagenic environment such as may be found in the colon. We predict that loss of MMR will enhance survival under conditions of increased DNA damage, favoring these cells in a competition for stem cell niche occupancy. Ultimately, this will lead to the production of more hypermutable intestinal cells, increasing the penetrance of the cancer phenotype. Testing this hypothesis has been problematic previously due to lack of a suitable model system. However, the recent development of human colonic organoid and enteroid models now allow us to study the effects of MMR loss on CSC dynamics via the following aims: 1) Determine whether loss of MMR function in human embryonic stem cells (hESCs) leads to an immediate advantage in the absence or presence of exogenous DNA damaging agents. We will use CRISPR/Cas9-mediated gene editing to knock out the MMR genes in hESCs and examine their growth and damage response as well as determine the mechanism underlying those responses. 2) Determine whether MMR-deficient CSCs have a selective advantage in colonic organoids and colonic enteroids. For this purpose, we will differentiate MMR-proficient and deficient hESCs into colonic organoids. As a complementary model, we will also create MMR knock out enteroids from human adult colon tissue samples. Using both systems, we will compare the response to exogenous DNA damaging agents or oncogenic stress. We will also create mixed organoids containing MMR-proficient and deficient cells and test whether the MMR-deficient cells have a growth or survival advantage over time in the presence or absence of DNA damage. 3) Determine whether MMR loss leads to a selective advantage for CSCs in vivo. We will use an inducible, stem cell specific knockout mouse model of Msh2 to create mosaic intestinal crypts and test whether MMR-deficient CSCs outcompete wild-type CSCs. Together, these aims utilize novel approaches to study the mechanism by which loss of MMR function contributes to tumorigenesis providing information that may help explain disease penetrance while guiding the diagnosis, prevention and treatment of LS-associated cancers.

项目概要 林奇综合征 (LS) 是一种遗传性疾病，使患者易患结直肠癌、子宫内膜癌、卵巢癌和 其他癌症。 LS 是由 DNA 错配修复 (MMR) 基因的遗传性突变引起的，其中的缺陷 也是 15-30% 散发性结直肠癌的基础。 MMR 功能丧失与 1,000 倍相关 突变率的增加可能会增加重要癌基因和肿瘤抑制基因突变的风险。 MMR 途径还激活细胞周期检查点和细胞死亡以响应外源 DNA 损伤， 然而，这种损伤反应在预防肿瘤发生中的作用尚不清楚。我们假设 失去这种 MMR 依赖性损伤反应的结肠干细胞 (CSC) 将获得选择性优势 邻近的 MMR 熟练细胞，特别是在诱变环境中，例如结肠中可能存在的细胞。 我们预测，MMR 的丧失将在 DNA 损伤增加的情况下提高生存率，有利于这些 细胞竞争干细胞生态位占据。最终，这将导致生产更多 肠道细胞高度可变，增加癌症表型的外显率。检验这个假设有 由于缺乏合适的模型系统，以前一直存在问题。然而，人类最近的发展 结肠类器官和肠类模型现在使我们能够通过以下方式研究 MMR 损失对 CSC 动态的影响 以下目标： 1) 确定人胚胎干细胞 (hESC) 中 MMR 功能的丧失是否会导致 在不存在或存在外源 DNA 损伤剂的情况下具有直接的优势。我们将使用 CRISPR/Cas9 介导的基因编辑可敲除 hESC 中的 MMR 基因并检查其生长和发育 损伤反应并确定这些反应背后的机制。 2）判断是否 MMR 缺陷的 CSC 在结肠类器官和结肠肠类中具有选择性优势。为此， 我们将把 MMR 丰富和缺陷的 hESC 分化为结肠类器官。作为补充模型，我们 还将从人类成人结肠组织样本中创建 MMR 敲除肠类。使用这两个系统，我们将 比较对外源 DNA 损伤剂或致癌应激的反应。我们还将打造混合 含有 MMR 丰富细胞和缺陷细胞的类器官，并测试 MMR 缺陷细胞是否有生长 或在存在或不存在 DNA 损伤的情况下随时间的生存优势。 3）判断MMR是否丢失 导致 CSC 在体内具有选择性优势。我们将使用可诱导的干细胞特异性敲除小鼠 Msh2 模型创建镶嵌肠隐窝并测试 MMR 缺陷的 CSC 是否能在竞争中胜过野生型 CSC。这些目标共同利用新方法来研究 MMR 功能丧失的机制 有助于肿瘤发生，提供可能有助于解释疾病外显率的信息，同时指导 LS 相关癌症的诊断、预防和治疗。