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到良性腺瘤到恶性腺癌的发生和发展（平行的组织学改变），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（DNA mismatch repair， 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上的插入/缺失（I/D）环，但在家族性结肠癌（Lynch综合征）的种系中，DNA错配修复被破坏，并且在高达20%的散发性结直肠肿瘤中，由于DNA错配修复的一种成分的超甲基化而失活。DNA MMR中断的遗传后果是DNA异双链首先形成后，聚合酶错误和插入/删除环在非编码和编码微卫星区域无法修复，导致肿瘤的开始，这定义了临床综合征。我们假设人类DNA MMR在针对I/D环的修复中具有特定的识别保真度，但异双工DNA形式的基本原理与DNA MMR无关。我们使用新型结构来测量人类DNA MMR突变的初步数据表明：(a)异双工DNA在突变之前形成，并且与DNA MMR状态无关，并且只有在DNA MMR缺陷中才能看到完全突变，并且(b)微卫星形成I/D环和随后突变的能力受到微卫星周围侧翼DNA序列的影响。此外，越来越多的证据表明，在60%的结肠癌标本中，选择性四核苷酸重复序列（EMAST）的微卫星不稳定性升高可能与炎症诱导的DNA MMR蛋白hMSH3的减少有关，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。在本续提案中，我们将重点关注hMSH3及其缺乏如何导致EMAST，并确定EMAST的生物学后果。更具体地说，我们假设hMSH3可以阻止四核苷酸的帧移，这将在Specific Aim 1中进行研究。我们进一步旨在了解hMSH3在影响潜在靶基因突变方面的作用（Specific aim 2）。我们还将通过hMSH3表达来定义EMAST，并研究炎症与hMSH3和EMAST之间的联系（Specific Aim 3）。这一建议的影响在于了解DNA如何改变以驱动常见癌症的基本原理，以及我们最终可能通过药物或直接干预来预防结直肠癌的发生或死亡的能力。

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.