Epigenetic Alterations in Homeotic Genes

该提案的主要目的是研究由DNA甲基化变化引起的同源异型基因表达改变在乳腺癌的发生和发展中的作用和意义。DNA甲基化的改变是人类肿瘤中常见的表观遗传学异常。我们使用一种名为甲基化敏感扩增片段长度多态性(MS-AFLP)的技术对整个基因组上的DNA甲基化改变进行了公正的评估。在这项研究中，我们分析了乳腺癌、前列腺癌和结肠癌患者正常和肿瘤组织的MS-AFLP甲基化指纹图谱。我们发现，几条显示肿瘤特异性强度变化的指纹条带代表了不同同源基因表达的DNA片段。与MS-AFLP指纹分析方法的不偏不倚的本质一致，我们认为我们的观察只是冰山一角，同源异型基因的DNA甲基化变化可能是癌症发生中极其频繁的事件。同源蛋白是指导胚胎发育和细胞分化的转录因子。因此，同源异型基因的表观遗传甲基化改变可能引发许多基因表达的级联变化，如果这些甲基化改变伴随着同源异型基因表达的变化，则导致细胞分化较少，位置限制较少。我们认为DNA甲基化和同源异型基因在乳腺癌的发生中都很重要。我们将通过提出两个具体目标来验证这一假设：首先，我们将分析乳腺癌组织中各种同源基因的DNA甲基化和基因表达变化，并确定常见的乳腺癌特异性变化。癌症特异性改变和细胞类型特异性改变将通过分析显微镜下选择的正常细胞和癌细胞的统一群体的变化来区分。接下来，我们将研究同源异型基因表达改变在乳腺癌发生中的作用。将为那些高甲基化和表达下调的同源异型基因准备表达载体，并将其引入非表达的乳腺癌细胞。同源异型基因的表达对其他基因的表达以及对细胞表型(锚定依赖/独立生长和运动)的影响将被研究。构造性和诱导性表达系统将被用来检测这些同源基因的肿瘤抑制功能。

The main goal of this proposal is to examine the role and significance of altered expression of homeotic genes, caused by DNA methylation changes, in the pathogenesis and progression of breast cancer. Changes in DNA methylation are frequently detectable epigenetic abnormalities in human neoplasia. We performed an unbiased evaluation of DNA methylation alterations over an entire genome using a technique named Methylation Sensitive-Amplified Fragment Length Polymorphism (MS-AFLP). In that study, we analyzed normal and tumor tissue MS-AFLP methylation fingerprints of breast, prostate, and colon cancer patients. We found that several of the fingerprint bands exhibiting tumor specific intensity changes represented DNA fragments from diverse homeotic gene expression. In concert with the unbiased nature of the MS-AFLP fingerprinting approach, we believe that our observation is the tip of the iceberg, and that DNA methylation changes in homeotic genes might be extremely frequent events in carcinogenesis. Homeoproteins are transcription factors that direct embryogenesis and cell differentiation. Therefore, epigenetic methylation alterations of homeotic genes may trigger a cascade of changes in expression of many genes, resulting in a cell with a less differentiated and less positionally restricted phenotype if those methylation alterations are accompanied by the alterations in homeotic gene expression. We believe that both DNA methylation and homeotic genes are important in breast carcinogenesis. We will test this hypothesis by proposing two specific aims: First, we will analyze DNA methylation and gene expression alterations in various homeotic genes in breast tumor tissues and identify common breast cancer-specific alterations. Cancer-specific alterations and cell type- specific alterations will be discriminated by analyzing the changes in microscopically selected uniform populations of normal and cancer cells. We will next examine the functionality of altered expression of homeotic genes in breast carcinogenesis. Expression constructs will be prepared for those homeotic genes that are hypermethylated and down-regulated in expression and introduced into non-expressor breast cancer cells. Effects of the homeotic gene expression on the expression of other genes as well as on cellular phenotypes (anchorage dependent/independent growth and motility) will be investigated. Both the constitutive and inducible expression systems will be used to examine the tumor suppressor function of these homeotic genes.