Cellular Epigenetic Heterogeneity as a Predeterminant of Malignant Transformation Potential

细胞表观遗传异质性作为恶性转化潜力的决定因素

基本信息

批准号：
10307617
负责人：
PETER W LAIRD
金额：
$ 72.24万
依托单位：
VAN ANDEL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-12-03 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10307617
关键词：
Address Affect Alleles Area Atlases Back Biological Models Carcinogenicity Tests Cell Lineage Cells Characteristics Clonal Expansion Clonality Colon Complex CpG Islands Cryopreservation DNA DNA Methylation DNA Modification Methylases Engraftment Epigenetic Process Event Experimental Designs Frequencies Gatekeeping Gene Combinations Gene Expression Genes Genetic Heterogeneity Histology Human Hypermethylation Individual Intestinal Neoplasms Intestines KRASG12D LGR5 gene Light LoxP-flanked allele Malignant - descriptor Malignant Neoplasms Maps Methods Minor Mus Mutation Normal Cell Normal tissue morphology Oncogene Activation Oncogenes Organoids Outcome Outcome Study Pattern Polycomb Predisposition Prevention Protocols documentation Recovery Research Resistance Risk Assessment Role Screening for cancer Series Site System Tamoxifen Testing Time Tissues Transcriptional Regulation Transplantation Tumor Suppressor Genes Tumor-Suppressor Gene Inactivation Tumorigenicity Ursidae Family Variant adult stem cell bisulfite sequencing cancer cell cancer initiation cancer predisposition cancer prevention cancer risk enema administration epigenetic variation experimental study improved in vivo innovation insight methylation pattern methylome mutant novel premalignant promoter remote control stem cells transcriptome sequencing tumor tumorigenesis whole genome

项目摘要

PROJECT SUMMARY / ABSTRACT Clonal expansion is a pivotal characteristic of cancer, and is thought to be initiated by a genetic alteration in a key gatekeeper driver gene. However, not all normal cells appear to be susceptible to malignant transformation following such an event. Multiple lines of evidence suggest that epigenetic heterogeneity among normal cells may affect their cancer-initiating potential. This is a particularly challenging concept to investigate, since there is no straightforward way a priori to identify which cells may be susceptible or resistant to transformation prior to clonal expansion. We propose an innovative series of experiments to test the hypothesis that pre-existing epigenetic heterogeneity among normal cells affects the likelihood of malignant transformation and further progression upon genetic alteration of a cancer driver gene. In Specific Aim 1, we will use an improved protocol for single-cell whole genome bisulfite sequencing to document the degree of DNA methylation heterogeneity among flow-sorted mouse colon stem cells. For the first time, this will provide insight into whether low-level DNA methylation abnormalities observed in normal tissues are stochastically distributed, or whether subsets of stem cells bear multiple concerted epigenetic abnormalities. In Specific Aim 2, we will first generate large numbers of individual clonally expanded mouse colon organoids harboring conditional alleles for Apc, Trp53, Kras and Braf. Each organoid will be cryopreserved and concurrently subjected to whole genome bisulfite sequencing to delineate each methylome. Organoids representing diverse methylomes will be thawed, the cancer drivers activated, and then tested for tumorigenicity by colon enema engraftment. This innovative experiment aspires to provide the first direct evidence for the contribution of pre-existing epigenetic heterogeneity to cancer predisposition. In Specific Aim 3, we will provide empirical evidence for this concept by using a novel in vivo transcriptional control system to transiently up- or down-regulate DNA methyltransferase activity in mice, prior to activation of conditional key cancer drivers. This aim should provide a proof of concept for a causal role of pre-existing epigenetic variation affecting cancer propensity. This proposal addresses a concept for which there is considerable indirect evidence, but which remains a poorly studied area because of the technical and conceptual challenges presented by the premise. Our proposed experiments are intricate and complex, but we have considerable expertise in all areas of the proposal, and have developed cutting-edge solutions to many of the technical hurdles. We may not be able to conclusively delineate in detail all epigenetic variations that predispose to malignancy, but this exploratory project should provide a proof of principle for the importance of pre-existing epigenetic heterogeneity in cancer susceptibility. The outcome of this study should have a substantial impact on our understanding of lifetime accumulation of cancer risk, with implications for cancer screening and prevention.

项目摘要 /摘要克隆扩张是癌症的关键特征，被认为是由遗传改变引发的关键的网守驱动器基因。但是，并非所有正常细胞似乎都容易受到恶性影响这种事件后的转换。多种证据表明表观遗传异质性正常细胞可能会影响其癌症引发潜力。这是一个特别具有挑战性的概念，由于没有直接的方法可以识别哪些单元可能易感或抗性克隆膨胀之前的转换。我们提出了一系列创新的实验，以测试正常细胞中现有表观遗传异质性的假设会影响恶性肿瘤的可能性癌症驱动基因遗传改变后转化和进一步发展。在特定的目标1中，我们将使用改进的协议将整个基因组Bisulfite测序进行记录流动小鼠结肠干细胞中DNA甲基化异质性的程度。为了第一次，这将提供有关是否在正常情况下观察到的低水平DNA甲基化异常组织随机分布，或者干细胞的亚集是否具有多个协同表观遗传异常。在特定的目标2中，我们将首先生成大量的单个克隆扩展的鼠标结肠机可以带有APC，TRP53，KRAS和BRAF的条件等位基因。每个类型器机器人将是冷冻保存并同时进行整个基因组亚硫酸盐测序，以描绘每个甲基甲基。将解冻代表多种甲基组的器官，激活癌症驱动因素，然后测试结肠灌肠植入的肿瘤性。这种创新的实验渴望提供第一个直接现有表观遗传异质性对癌症易感性的贡献的证据。在特定目标中 3，我们将通过使用新颖的体内转录控制系统来为此概念提供经验证据在激活条件键之前，小鼠中的DNA甲基转移酶活性瞬时或下调癌症司机。这个目标应为预先存在表观遗传变异的因果作用提供概念证明影响癌症倾向。该提议探讨了有大量间接证据的概念，但仍然是由于前提提出的技术和概念挑战，研究领域的研究不足。我们的提出的实验是复杂且复杂的，但是我们在该领域的所有领域都有相当大的专业知识提案，并为许多技术障碍开发了尖端解决方案。我们可能无法最终详细描述所有易于恶性的表观遗传变异，但这种探索性项目应提供原则证明，以证明癌症中现有表观遗传异质性的重要性敏感性。这项研究的结果应该对我们对终生的理解产生重大影响癌症风险的积累，对癌症筛查和预防产生影响。