Microsatellite instability and cancer gene expression

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。微卫星突变子表型(MMP型)是通过检测DNA复制过程中因链错位滑动引起的重复序列中的缺失/插入突变而发现的。微卫星序列在癌症发生发展中的重要性已经通过基质金属蛋白酶的机制得到了很好的说明：在基质金属蛋白酶阳性的肿瘤中，在其编码区含有微卫星重复序列的癌症相关基因被特异性地突变。非编码微卫星在基质金属蛋白酶癌症发病机制中的作用仅被稀少的实验数据和理论推测所证实。这一建议的工作假设是，基质金属蛋白酶不可避免地导致位于癌症相关基因非编码区的微卫星突变，这些突变可能通过调节基因转录、翻译、RNA剪接或mRNA稳定性来影响其表达。这些负性和正性细胞生长调节因子水平的变化反过来将有助于觉醒和/或增加细胞的肿瘤潜能。这项拟议研究的目标是直接检验这一假说。这一假说的独特之处在于，在基质金属蛋白酶的癌症途径中，这些非编码微卫星突变，尽管它们的致癌潜力可能不大，但对肿瘤的发生可能与其他更有效的编码突变一样重要，因为它们很早就发生了。为了验证这一假说，我们将：分析MMP阳性癌症中EGFR CA重复序列的扩增；通过转基因和小干扰RNA检测，估计不同重复长度的MMP细胞亚克隆中EGFR的表达水平，以及它们的凋亡反应；将EGFR重复序列扩增与K-ras癌基因突变和细胞凋亡相关基因的表达相关联；检查有关EGFR表达和重复序列扩增的肿瘤考古学；以及通过成瘤性试验(特定目标1)测试EGFR CA重复序列扩增在体内的致癌潜力。我们将通过基因转染法和致瘤性分析来确定fl-catenin基因3‘端非编码区缺失对蛋白质表达和肿瘤表型的影响。我们还将测试P53和其他已确定候选基因3‘UTR区缺失对基因表达的影响(特定目标2)。我们还将识别具有调控微卫星重复的其他癌症相关基因，并表征前面概述的EGFR和β-catenin基因的非编码微卫星表达调控。(具体目标3)。这一建议将为肿瘤发生过程中癌症基因表达调控的新途径提供见解。

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.