DNA Methylation Changes During Development and Progression of Lung Adenocarcinoma

描述（由申请人提供）：肺癌是美国癌症死亡的主要原因。腺癌是从不吸烟者和曾经吸烟者中最常见的组织学亚型，现在是男性和女性中最常见的肺癌类型。肺腺癌发病率的增加强调了了解这种致命疾病的发展和进展的重要性。DNA甲基化是肿瘤抑制基因失活的重要机制。使用一个独特的收集的浸润前病变，肺腺癌的临床阶段IA-IIIA，和对照肺样本，我们建议进行全基因组搜索与腺癌的发展和进展相关的DNA甲基化变化。本研究的具体目的是：1）通过全基因组DNA甲基化谱来鉴定肺腺癌与组织学正常肺相比的新的表观遗传学变化。我们将比较早期和晚期腺癌和非癌肺，以确定甲基化和表达的同时变化，使用CpG岛和表达微阵列。选择的基因座将通过亚硫酸氢盐测序和定量RT-PCR进行验证。2)目的：探讨肺腺癌发生、发展过程中Aim 1基因位点的甲基化改变。使用高通量系统MethyLight，我们将比较IA、IB、IIA、IIB和MIA期肺腺癌、浸润前病变和非肿瘤肺中各50个存档样本的甲基化谱。3)确定目标2中观察到的甲基化谱与患者生存率之间是否存在相关性。4)通过体外功能实验，研究目标2下识别的基因座的生物学意义：强制基因表达或通过RNAi人工沉默基因。这些操作对代表癌前病变和癌性病变的细胞系的影响将通过测量增殖率、细胞周期分布、细胞移动性、非贴壁依赖性生长和在裸鼠中形成肿瘤的能力来评价。每年有超过15万美国人死于肺癌。早期发现是预防癌症死亡的最佳方法。鉴定肺腺癌发展过程中发生的连续表观遗传学改变可以为早期检测和诊断提供新的标志物，以及可能的药物开发新靶点，从而挽救或延长数千名肺癌患者的生命。

DESCRIPTION (provided by applicant): Lung cancer is the leading cause of cancer death in the United States. Adenocarcinoma, the histological subtype most frequently seen in never smokers and former smokers, is now the most common type of lung cancer in men and women. The increasing incidence of lung adenocarcinoma underlines the importance of understanding the development and progression of this lethal disease. DMA hypermethylation at promoter CpG islands is a key mechanism for tumor suppressor gene inactivation in cancer. Using a unique collection of pre-invasive lesions, lung adenocarcinomas of clinical stages IA-IIIA, and control lung samples, we propose to undertake a genome-wide search for DNA methylation changes associated with adenocarcinoma development and progression. The Specific Aims of this study are: 1) To identify new epigenetic changes in lung adenocarcinoma compared to histologically normal lung by genome-wide DNA methylation profiling. We will compare early and late stage adenocarcinoma and non-cancer lung to identify concurrent changes in methylation and expression, using CpG island and expression microarrays. Select loci will be verified by bisulfite sequencing and quantitative RT-PCR. 2) To determine whether DNA methylation changes for loci identified in Aim 1 occur during development and progression of lung adenocarcinoma. Using the high throughput system MethyLight, we will compare methylation profiles in 50 archival samples each of stages IA, IB, IIA, IIB and MIA lung adenocarcinoma, in pre-invasive lesions, and in non-tumor lung. 3) To determine whether correlations exist between the methylation profiles observed in Aim 2 and patient survival. 4) To examine the biological significance of loci identfied under Aim 2 using functional experiments in vitro: forcing expression of genes or artifically silencing them by RNAi. The effects of these manipulations on cell lines representing precancerous and cancerous lesions will be evaluated by measuring proliferation rates, cell cycle distribution, cell mobility, anchorage-independent growth and the ability to form tumors in nude mice. Over 150,000 Americans die every year from lung cancer. Early detection is the best way to prevent cancer deaths. The identification of sequential epigenetic alterations that occur during lung adenocarcinoma development could provide new markers for early detection and prognostication as well as possible new targets for drug development, thereby saving or extending the lives of thousands of lung cancer patients.