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.

项目摘要 林奇综合征（LS）是一种遗传性疾病，使患者易于结直肠癌，子宫内膜，卵巢和 其他癌症。 LS是由DNA不匹配修复（MMR）基因中的遗传突变引起的，其中缺陷在其中 同样是15-30％的零星结直肠癌。 MMR功能的损失与1,000倍有关 突变率的增加可能会增加重要的癌基因和抑制肿瘤的突变风险。 MMR途径还激活细胞周期检查点和细胞死亡，以应对外源DNA损伤， 但是，这种损伤反应在预防肿瘤发生中的作用尚不清楚。我们假设这一点 失去MMR依赖性损伤反应的结肠干细胞（CSC）将获得选择性优势 在结肠中可以发现的相邻MMR富含MMR的细胞，特别是在诱变环境中。 我们预测，在DNA损伤增加的条件下，MMR的损失将提高生存率，从而有利于这些 在干细胞小众占用竞争中的细胞。最终，这将导致更多 高过度肠细胞，增加了癌症表型的渗透率。检验该假设具有 由于缺乏合适的模型系统，以前是有问题的。但是，人类的最新发展 现在，结肠器官和肠模型使我们能够研究MMR丢失对CSC动力学的影响 以下目的：1）确定人类胚胎干细胞（HESC）中MMR功能的丧失是否导致 在不存在或存在外源DNA损伤剂的情况下立即优势。我们将使用 CRISPR/CAS9介导的基因编辑，以淘汰hESC中的MMR基因并检查其增长和 损坏响应以及确定这些反应的基础机制。 2）确定是否 MMR缺陷型CSC在结肠机身和结肠肠to中具有选择性的优势。为此， 我们将将MMR的hESC分化为结肠器官。作为互补模型，我们 还将创建MMR从人类成人结肠组织样品中淘汰肠to。使用两个系统，我们将 比较对外源性DNA损害剂或致癌应激的反应。我们还将创造混合 含有MMR的细胞和缺乏细胞的类器官，并测试MMR缺陷型细胞是否具有生长 在存在或不存在DNA损伤的情况下，随着时间的推移而生存优势。 3）确定MMR是否丢失 导致体内CSC的选择性优势。我们将使用可诱导的干细胞特异性基因敲除鼠标 MSH2的模型以创建镶嵌肠道隐窝并测试MMR缺陷的CSC是否超过野生型 CSC。这些目的共同利用新颖的方法来研究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渗透率的信息。 LS相关癌症的诊断，预防和治疗。