Genetic analysis of genomic instability and cancer in zebrafish

描述(申请人提供)：癌症是由癌症基因的遗传和表观遗传变化积累而成，包括肿瘤抑制基因；这些变化导致正常体细胞进化为癌细胞。在克努森的两次打击模式中，肿瘤抑制基因功能的丧失分两个步骤进行。这些步骤中的第二步可以通过多种机制发生，包括染色体丢失、重组、插入、缺失、点突变或表观遗传修饰。为了找到参与这些步骤的脊椎动物基因，从而维持基因组的稳定性，我们使用斑马鱼(Danio Rerio)对模拟第二次撞击的基因组不稳定突变进行了筛选。这一筛查使用了马赛克眼球试验，苍白的细胞出现在视网膜色素上皮(RPE)细胞的黑色背景中，这些细胞是隐性色素突变Golddenb1的杂合子。在此筛选中分离到12个ENU诱导的基因组不稳定(GIN)或体细胞突变子突变，这表明可能存在200个这样的基因。大多数GIN突变在杂合子中表现为弱显性，而在纯合子中均表现为较强的表型。最强的突变，GIN-10，在母系中与其他GIN突变一起遗传时，表现出比纯合子表型显著(~10倍)的增强(“GIN-10相互作用组”)；携带GIN-10的人自发癌症的易感性增加9.6倍。这些发现是我们发展斑马鱼作为脊椎动物模型的长期目标的第一步，目的是寻找参与控制脊椎动物基因组稳定性的基因。既然我们已经证明斑马鱼可以用来寻找对癌症敏感的体细胞突变体，我们建议阐明组成GIN-10相互作用组的基因之间惊人相互作用的本质，并更好地表征相关的肿瘤敏感性。这将通过三个具体目标来实现。为了达到特定的目的1，我们将通过对GIN-10和GIN-12进行定位克隆，并使用带有新型转座子载体的敏化插入屏幕来克隆GIN-10相互作用组的成员。具体目的2是通过微卫星、间期FISH分析和外显子测序来表征基因组不稳定的机制，包括镶嵌眼金色RPE细胞中的黄金基因以及与GIN-10相关的肿瘤中P53的类似分析。目的3确定强嵌合型GIN-10/GIN-12反式杂合子的肿瘤易感性。这些拟议的研究将有助于我们理解与基因功能丧失有关的基因相互作用，并阐明它们是如何导致癌症易感性的。我们希望，这样的理解将导致预防或治疗人类癌症的新方法。与公共卫生相关：癌症是一种疾病，在这种疾病中，正常细胞在进化为癌细胞的过程中会积累DNA的变化。我们现在知道，控制我们DNA稳定性的基因在癌症中起着关键作用。我们正在利用斑马鱼作为脊椎动物模型系统的独特力量来寻找和研究对遗传稳定性和癌症至关重要的基因，希望我们越来越多的了解将导致治疗和预防人类癌症的措施。

DESCRIPTION (provided by applicant): Cancer results from an accumulation of genetic and epigenetic changes in cancer genes, including tumor suppressor genes; these changes cause normal somatic cells to evolve into cancer cells. In Knudson's two-hit paradigm, loss of tumor suppressor gene function occurs in two steps. The second of those steps can occur by multiple mechanisms including chromosome loss, recombination, insertion, deletion, point mutation, or epigenetic modification. In order to find vertebrate genes involved in those steps, which serve to maintain genome stability, we have used the zebrafish (Danio rerio) to perform a screen for genomic instability mutants that models this second hit. This screen used the mosaic eye assay, in which pale cells appear in a black background of retinal pigmented epithelial (RPE) cells that are heterozygous for a recessive pigment mutation, goldenb1. Twelve ENU- induced genomic instability (gin), or somatic mutator mutations were isolated in this screen at a rate suggesting the potential existence of 200 such genes. Most of the gin mutations showed weak dominance in heterozygotes, and all showed a stronger phenotype in homozygotes. The strongest mutant, gin-10, showed a striking (~10-fold) phenotypic enhancement over homozygous phenotype when inherited maternally in trans with other gin mutations (the "gin-10 interacting group"); gin-10 carriers show a 9.6-fold increase in susceptibility to spontaneous cancer. These findings represent first steps in our long-term goal of developing the zebrafish as a vertebrate model for finding genes involved in the control of genomic stability in vertebrates. Now that we have shown that the zebrafish can be used to find somatic mutators that are susceptible to cancer, we propose to elucidate the nature of the striking interactions between the genes comprising the gin-10 interacting group, and to better-characterize the associated tumor susceptibility. This will be accomplished in three Specific Aims. For Specific Aim 1, we will clone members of the gin-10 interacting group using positional cloning for gin-10 and gin-12, and using a sensitized insertional screen with a novel "gene breaking" To12 transposon-based vector. Specific Aim 2 is to characterize the mechanism of genomic instability using microsatellite, interphase FISH analysis, and exon sequencing of the golden gene in golden RPE cells from mosaic eyes and by similar analysis of p53 in gin-10-associated tumors. Specific Aim 3 is to determine the tumor susceptibility of the strongly mosaic gin-10/gin-12 trans-heterozygotes. The proposed studies will contribute to our understanding of gene interactions involved in somatic loss of gene function, and to clarify how they contribute to cancer susceptibility. It is our hope that such understanding will lead to novel ways to prevent or treat human cancer. PUBLIC HEALTH RELEVANCE: Cancer is a disease in which normal cells accumulate changes in their DNA as they evolve into cancer cells. We now know that the genes controlling the stability of our DNA play a key role in cancer. We are using the unique power of the zebrafish as a vertebrate model system to find and study genes that are important in genetic stability and cancer, in the hope that our increased understanding will lead to measures to treat and prevent human cancer.