Inflammation Induced Cytosine Deamination and the Initiation of Cancer

炎症诱导的胞嘧啶脱氨和癌症的发生

• 批准号: 10206050
• 负责人: Chia Wei Hsu
• 金额: $ 3.56万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10206050
• 关键词: Address; Affect; Age; Base Excision Repairs; Base Pairing; Biological Assay; Cause of Death; Cell Extracts; Cell Line; Cells; Cessation of life; Characteristics; Codon Nucleotides; Colitis associated colorectal cancer; Colorectal Cancer; Cytosine; DNA Damage; DNA Repair; DNA Repair Pathway; Deamination; Development; Diagnosis; Diagnostic Procedure; Dinucleoside Phosphates; Disease; Early Diagnosis; Etiology; Event; Exons; Formalin; Frequencies; Genes; Genomic DNA; Guanine; Histologic; Human Cell Line; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Investigation; Lead; Link; Location; Malignant Neoplasms; Masks; Mass Spectrum Analysis; Measures; Methodology; Methods; Molecular; Mutagenesis; Mutation; Nitrogen; Nucleotide Excision Repair; Oxygen; Paraffin Embedding; Patient-Focused Outcomes; Patients; Population; Positioning Attribute; Production; Prone Position; Public Health; Recording of previous events; Reporting; Research Project Grants; Role; Sampling; Scanning; Silent Mutation; TP53 gene; Techniques; Temperature; Testing; Thymine; Thymine DNA Glycosylase; Time; Tissue Embedding; Tissues; Tracer; Ulcerative Colitis; United States; base; cancer diagnosis; cancer initiation; design; driver mutation; experimental study; human tissue; improved; inflammatory milieu; knock-down; new therapeutic target; next generation sequencing; novel; preference; prognostic; thermostability; tumor; tumorigenesis; tumorigenic; uracil analog

PROJECT SUMMARY/ABSTRACT Cancer remains one of the leading causes of death in the United States and worldwide with more than 8.8 million cancer related deaths each year. To reduce this public health burden, investigation of the most commonly observed substitution mutation, a cytosine to thymine transition, is crucial. An example of this predominance is evident in inflammation driven colitis associated colorectal cancer (CAC). Development of CAC involves an early critical p53 mutation dominated by cytosine to thymine transitions at 5’ cytosine, 3’ guanine (CpG) dinucleotides. These mutations arise from 5-methylcytosine deamination into thymine, forming a T:G mispaired intermediate. Subsequent replication propagates the mutation to the opposing strand, forming a T:A base pair. Although mutations in p53 have been demonstrated at the time of CAC diagnosis, little has been done to detect the exact location of low frequency mutations and mispaired intermediates in noncancerous tissues. It is also unknown how long a mutation is present before cancer diagnosis or how inflammatory conditions can promote cytosine to thymine transitions. Current sequencing methodologies involving standard PCR mask the presence of rare but potentially important mutations because amplification exponentially increases the predominant sequence and reports only that sequence. To solve these unanswered questions, we aim to mechanistically determine how inflammation increases the rate of these critical mutations and develop novel methods to detect low frequency mispaired intermediates and mutations. Given the critical role of inflammation in the etiology of CAC, we hypothesize that inflammation increases the number of mutations by providing substrates for competing DNA repair pathways and that an inflammatory environment will greatly increase the number of mutations and mispaired intermediates which leads to cancer development. We will address this hypothesis by first proving our mechanistic hypothesis of competing DNA repair and then developing methods to determine the number of global mispaired intermediates at a p53 mutation hotspot, described through three Specific Aims: 1) determine a potential mechanism by which inflammatory mediators can induce characteristic cytosine to thymine transitions; 2) design a novel glycosylase-mass spectrometry assay to measure global levels of mispaired intermediates and determine how inflammation affects mispaired intermediate production; and 3) develop a novel method to scan p53 exon 7 for cytosine deamination mutations and mispaired intermediates. By measuring low frequency mispaired intermediates and mutations long before tumor formation, we will be able to estimate the number of cells in a population that contains critical mutation and predict its prior DNA damage history. The mechanistic studies will elucidate the role of inflammation on the beginning stages of tumorigenesis, and together, the results of this proposal could lead to novel therapeutic targets or diagnostic methods for CAC or other inflammatory conditions.

项目概要/摘要 癌症仍然是美国和全世界死亡的主要原因之一，死亡人数超过 8.8 每年有 100 万人因癌症相关死亡。为了减轻这种公共卫生负担，调查了最 常见的取代突变，即胞嘧啶到胸腺嘧啶的转变，是至关重要的。一个这样的例子 炎症驱动的结肠炎相关结直肠癌（CAC）中的优势很明显。发展 CAC 涉及早期关键的 p53 突变，主要是 5' 胞嘧啶、3' 处的胞嘧啶到胸腺嘧啶的转变 鸟嘌呤 (CpG) 二核苷酸。这些突变源自 5-甲基胞嘧啶脱氨成胸腺嘧啶，形成 T:G 错配中间体。随后的复制将突变传播到相反的链，形成 T：碱基对。尽管在 CAC 诊断时已证实 p53 突变，但几乎没有发现任何突变。 已完成检测低频突变和错配中间体的确切位置 非癌组织。目前还不清楚突变在癌症诊断前存在多长时间或如何发生。 炎症条件可以促进胞嘧啶向胸腺嘧啶的转变。目前的测序方法 涉及标准 PCR 掩盖了罕见但可能重要的突变的存在，因为扩增 指数增加主要序列并仅报告该序列。为了解决这些 对于尚未解答的问题，我们的目标是从机制上确定炎症如何增加这些比率 关键突变并开发新方法来检测低频错配中间体和突变。 鉴于炎症在 CAC 病因学中的关键作用，我们假设炎症会增加 通过为竞争性 DNA 修复途径提供底物来减少突变数量，并且炎症 环境会大大增加突变和错配中间体的数量，从而导致癌症 发展。我们将首先证明我们的竞争 DNA 机制假设来解决这个假设 修复，然后开发方法来确定 p53 处全局错配中间体的数量 突变热点，通过三个具体目标进行描述：1）确定一种潜在机制，通过该机制 炎症介质可以诱导特征性胞嘧啶向胸腺嘧啶的转变； 2）设计一部小说 糖基化酶质谱测定法测量错配中间体的总体水平并确定如何 炎症影响错配的中间产物； 3) 开发一种扫描 p53 外显子 7 的新方法 胞嘧啶脱氨基突变和错配中间体。通过测量低频错配 在肿瘤形成之前很久就已经存在中间体和突变，我们将能够估计肿瘤中的细胞数量 包含关键突变的群体并预测其先前的 DNA 损伤历史。机理研究将 阐明炎症在肿瘤发生的起始阶段的作用，并共同得出这一结果 该提案可能会为 CAC 或其他炎症带来新的治疗靶点或诊断方法 状况。