Microsatellite instability and cancer gene expression

微卫星不稳定性与癌症基因表达

• 批准号: 6680627
• 负责人: MANUEL PERUCHO
• 金额: $ 42.72万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6680627
• 关键词: DNA replication; apoptosis; cadherins; cell line; enzyme linked immunosorbent assay; flow cytometry; gene expression; gene mutation; genetic transcription; genetic translation; human tissue; immunocytochemistry; messenger RNA; microarray technology; neoplasm /cancer genetics; neoplastic growth; neoplastic process; phenotype; polymerase chain reaction; protooncogene; transfection

DESCRIPTION (provided by applicant): Genetic or epigenetic inactivation of DNA mismatch repair in tumor precursor cells causes a profound mutator phenotype. The microsatellite mutator phenotype (MMP) was discovered by the detection of deletion/insertion mutations in repeated sequences due to slippage by strand misalignment during DNA replication. The importance of microsatellite sequences for the development of cancer has been well illustrated through the MMP mechanism: cancerrelated genes that contain microsatellite repeats in their coding regions are specifically mutated in MMP-positive tumors. The role of non-coding microsatellites in MMP cancer pathogenesis has only been suggested by scarce experimental data and theoretical speculations. The working hypothesis of this proposal is that the MMP inevitably causes mutations in microsatellites located in non-coding regions of cancer-related genes, and that these mutations may affect their expression through modulation of gene transcription, translation, RNA splicing or mRNA stability. These changes in the levels of negative and positive cell growth regulators in turn, will contribute to awake and/or increase the cell neoplastic potential. The goal of the proposed research is to directly test this hypothesis. The unique feature of this hypothesis is that in the MMP cancer pathway these non-coding microsatellite mutations, despite their presumable modest oncogenic potency, may be equally important for tumorigenesis than other more potent coding mutations, because they occur well before. To test this hypothesis, we will: analyze EGFR CA repeat expansion in MMP-positive cancers; estimate the levels of EGFR expression in subclones of MMP cell lines with different repeat lengths, and their apoptotic response by transfection and small interference RNA assays; correlate EGFR repeat expansion with K-ras oncogene mutations and expression of apoptosis-related genes; examine tumor archeology regarding EGFR expression and repeat expansion; and test the oncogenic potential in vivo of EGFR CA repeat expansion by tumorigenicity assays (Specific aim 1). We will determine the effect of deletions in the 3'UTR of the fl-catenin gene in protein expression and in tumor phenotype by transfection and tumorigenicity assays. We will also test the effect in gene expression of deletions in the 3'UTR of the p53 and other identified candidate genes (Specific aim 2). We will also identify other cancer-related genes with regulatory microsatellite repeats and characterize the non-coding microsatellite expression modulation as outlined before for the EGFR and beta-catenin genes. (Specific aim 3). This proposal will provide insights on a novel pathway of cancer gene expression modulation in tumorigenesis.

描述（由申请人提供）：肿瘤前体细胞中DNA错配修复的遗传或表观遗传失活导致深刻的突变表型。微卫星突变表型（microsatellite mutator phenotype， MMP）是通过检测DNA复制过程中由于链错位引起的滑动而导致的重复序列中的缺失/插入突变而发现的。微卫星序列对癌症发展的重要性已经通过MMP机制得到了很好的说明：在其编码区含有微卫星重复序列的癌症相关基因在MMP阳性肿瘤中特异性突变。非编码微卫星在MMP癌症发病机制中的作用仅通过缺乏实验数据和理论推测得到证实。本研究的工作假设是，MMP不可避免地导致位于癌症相关基因非编码区的微卫星发生突变，这些突变可能通过调节基因转录、翻译、RNA剪接或mRNA稳定性等方式影响其表达。这些细胞生长调节因子的阳性和阴性水平的变化反过来，将有助于唤醒和/或增加细胞的肿瘤潜能。本研究的目的是直接验证这一假设。这一假说的独特之处在于，在MMP癌症通路中，这些非编码微卫星突变，尽管可能具有适度的致癌效力，但与其他更有效的编码突变相比，可能对肿瘤发生同样重要，因为它们发生得早。为了验证这一假设，我们将：分析EGFR CA重复扩增在mmp阳性癌症中；用转染法和小干扰RNA法测定不同重复长度的MMP细胞系亚克隆中EGFR的表达水平及其凋亡反应；EGFR重复扩增与K-ras癌基因突变和凋亡相关基因表达的相关性；检查肿瘤的EGFR表达和重复扩增的考古学；并通过致瘤性试验测试EGFR CA重复扩增在体内的致癌潜力（特异性目的1）。我们将通过转染和致瘤性实验确定fl-catenin基因3'UTR缺失对蛋白质表达和肿瘤表型的影响。我们还将测试p53和其他已确定的候选基因的3'UTR缺失对基因表达的影响（Specific aim 2）。我们还将鉴定其他具有调控微卫星重复序列的癌症相关基因，并描述EGFR和β -连环蛋白基因的非编码微卫星表达调制。（具体目标3）。这一建议将为肿瘤发生中癌症基因表达调节的新途径提供见解。