Microsatellite Instability and the DNA Mismatch Repair System

描述(申请人提供)：大肠肿瘤是由基因突变和表观遗传改变的渐进过程引起的，这些基因突变和表观遗传改变推动了正常结肠到良性腺瘤再到恶性腺癌的起始和进展(与组织学变化平行)，因为这些突变影响了信号通路，从而放松了对正常结肠干细胞标志性行为的调控。DNA错配修复(MMR)是一种进化保守的系统，用于修复DNA合成后的聚合酶错误，并纠正基因组中微卫星序列上的插入/缺失(I/D)环。在家族性结肠癌(林奇综合征)的胚系中，由于DNA错配修复的一种成分的高甲基化，多达20%的散发性结直肠肿瘤中的DNA错配修复失活。DNA MMR中断的遗传后果是在DNA异源双链形成后，发生在非编码和编码微卫星区域的聚合酶错误和插入/缺失环无法修复，导致肿瘤开始形成，从而定义临床症状。我们假设人类DNA MMR在靶向修复I/D环方面具有特定的识别保真度，但异源双链DNA形式独立于DNA MMR的基本原理。我们使用新构建的结构来测量人类DNA MMR突变的初步数据表明：(A)异源双链DNA在突变之前形成，似乎与DNA MMR状态无关，完全突变只有在DNA MMR缺陷的情况下才能看到，以及(B)微卫星形成I/D环和后续突变的能力受到微卫星两侧DNA序列的影响。此外，越来越多的证据表明，在选定的四核苷酸重复序列(EMAST)上，60%的结肠癌样本的微卫星不稳定性增加，可能与炎症诱导的DNA MMR蛋白hMSH3减少有关，导致这种遗传特征。EMAST如何与结肠肿瘤的发病机制相适应还没有明确的定义。在这个继续的提案中，我们将重点关注hMSH3和缺乏如何导致EMAST，并确定EMAST的生物学后果。更具体地说，我们假设hMSH3防止四核苷酸移码，这将在特定的目标1中进行检验。我们进一步的目标是了解hMSH3在影响潜在的突变靶基因上的作用(特定的目标2)。我们还将通过hMSH3的表达来定义EMAST，并研究炎症与hMSH3和EMAST(特异性目标3)的联系。这一提议的影响在于理解DNA如何改变以驱动常见癌症的基本原理，以及我们最终通过药物干预或直接干预以防止结直肠癌发生或死亡的能力。

DESCRIPTION (provided by applicant): Colorectal neoplasia results from a progressive process of gene mutation and epigenetic alterations that drive the initiation and progression of normal colon to benign adenomas to malignant adenocarcinomas (paralleling histological changes) because these mutations affect signaling pathways that deregulate hallmark behaviors of normal colon stem cells. DNA mismatch repair (MMR), an evolutionary-conserved system that repairs polymerase mistakes after DNA synthesis and corrects insertion/deletion (I/D) loops at microsatellite sequences throughout the genome, is disrupted in the germline of familial colon cancer (Lynch syndrome) as well as inactivated in up to 20% of sporadic colorectal neoplasms due to hypermethylation of one of the components of DNA MMR. The genetic consequences of disrupted DNA MMR is non-repair of the polymerase mistakes and insertion/deletion loops that occur in non-coding as well as coding microsatellite regions after DNA heteroduplexes form first, causing neoplasia to commence that defines the clinical syndromes. We hypothesize that human DNA MMR has specific recognition fidelity in targeting repair of I/D loops, but the fundamentals of how heteroduplex DNA forms are independent of DNA MMR. Our preliminary data using novel constructs in which to measure human DNA MMR mutation indicate that (a) heteroduplex DNA forms prior to mutation and appears independent of the DNA MMR status, and that full mutation is only seen with defective DNA MMR, and (b) the ability of microsatellites to form I/D loops and subsequent mutation is influenced by flanking DNA sequences that surround the microsatellite. Additionally, there is growing evidence that elevated microsatellite instability at selected tetranucleotide repeats (EMAST), seen is 60% of colon cancer specimens, may be associated inflammation-induced reduction of the DNA MMR protein hMSH3, causing this genetic signature. How EMAST fits into the pathogenesis of colon neoplasia is not defined. In this continuation proposal, we will focus on hMSH3 and how deficiency causes EMAST, and determine the biological consequences of EMAST. More specifically, we hypothesize that hMSH3 prevents tetranucleotide frameshifts, and this will be examined in Specific Aim 1. We further aim to understand the role of hMSH3 on influencing potential target genes for mutation (Specific Aim 2). We will also define EMAST by hMSH3 expression, and examine the link of inflammation with hMSH3 and EMAST (Specific Aim 3). The impact of this proposal lies in understanding the fundamentals of how DNA is altered to drive a common cancer, and our ability to potentially intervene ultimately by pharmacologic or direct intervention to prevent occurrence or death from colorectal cancer.