Microsatellite Instability and the DNA Mismatch Repair System

描述（由申请人提供）：结直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驱动正常结肠开始和进展为良性腺瘤和恶性腺癌（与组织学变化平行），因为这些突变影响信号通路，使正常结肠干细胞的标志性行为失调。DNA错配修复（MMR）是一种进化保守的系统，可以修复DNA合成后的聚合酶错误，并纠正整个基因组中微卫星序列的插入/缺失（I/D）环，在家族性结肠癌（Lynch综合征）的生殖系中被破坏，以及在高达20%的散发性结直肠肿瘤中由于DNA MMR组分之一的超甲基化而失活。破坏DNA MMR的遗传后果是在DNA异源双链体首先形成后，在非编码以及编码微卫星区域中发生的聚合酶错误和插入/缺失环的不修复，导致肿瘤形成开始，其定义了临床综合征。我们假设人类DNA MMR在I/D环的靶向修复中具有特异性识别保真度，但异源双链DNA如何形成的基本原理不依赖于DNA MMR。我们的初步数据，使用新的结构，其中测量人类DNA MMR突变表明，（a）异源双链DNA形成突变之前，似乎独立的DNA MMR状态，只有在有缺陷的DNA MMR，和（B）微卫星形成I/D环和随后的突变的能力是由侧翼的DNA序列周围的微卫星的影响。此外，越来越多的证据表明，在60%的结肠癌标本中发现的选定四核苷酸重复序列（EMAST）的微卫星不稳定性升高可能与炎症诱导的DNA MMR蛋白hMSH 3减少有关，从而导致这种遗传特征。EMAST如何适应结肠肿瘤的发病机制尚不明确。在这个继续的建议，我们将集中在hMSH 3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hMSH 3阻止四核苷酸移码，这将在具体目标1中进行检查。我们进一步的目的是了解hMSH 3在影响潜在的突变靶基因中的作用（特异性目的2）。我们还将通过hMSH 3表达来定义EMAST，并检查炎症与hMSH 3和EMAST（特异性目的3）的联系。这一建议的影响在于理解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理学或直接干预来预防结直肠癌发生或死亡的潜在干预能力。

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.