DNA Methylation Changes During Development and Progression of Lung Adenocarcinoma

描述(申请人提供)：肺癌是美国癌症死亡的主要原因。腺癌是从不吸烟和曾经吸烟的人中最常见的组织亚型，现在是男性和女性最常见的肺癌类型。肺腺癌发病率的增加凸显了了解这种致命疾病的发展和进展的重要性。启动子CpG岛的DMA高甲基化是肿瘤抑制基因失活的关键机制。利用一组独特的侵袭前病变、临床分期IA-IIIA的肺腺癌和对照肺样本，我们建议在全基因组范围内寻找与腺癌的发生和发展相关的DNA甲基化变化。这项研究的具体目的是：1)通过全基因组DNA甲基化分析，确定肺腺癌与正常肺组织相比的新的表观遗传学变化。我们将使用CpG岛和表达微阵列对早期和晚期腺癌和非癌肺进行比较，以确定甲基化和表达的同步变化。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)确定在肺腺癌的发生发展过程中是否存在AIM 1基因座的DNA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、侵袭前病变和非肿瘤肺中各50个档案样本的甲基化情况。3)确定在AIM 2中观察到的甲基化情况与患者存活率之间是否存在相关性。4)通过体外功能实验：强迫基因表达或通过RNAi人为地沉默基因，以检验Aim 2中确定的基因座的生物学意义。这些操作对代表癌前病变和癌病变的细胞系的影响将通过测量增殖率、细胞周期分布、细胞迁移率、非锚定生长和在裸鼠体内形成肿瘤的能力来评估。每年有超过15万美国人死于肺癌。及早发现是预防癌症死亡的最好方法。识别肺腺癌发展过程中发生的序贯表观遗传学改变，可以为早期发现和预测提供新的标志物，也可能为药物开发提供新的靶点，从而挽救或延长成千上万肺癌患者的生命。

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.