Genome Instability in Cancer Development

癌症发展中的基因组不稳定性

• 批准号: 8948363
• 负责人: Kyungjae Myung
• 金额: $ 68.42万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8948363
• 关键词: Affect; Alleles; Aneuploidy; Bypass; Cell Culture Techniques; Cell Death; Cell Nucleolus; Cells; Chromosomal Rearrangement; Chromosome Arm; Chromosomes; Collaborations; Core Facility; DNA Damage; DNA Repair; DNA Repair Pathway; DNA repair protein; DNA replication fork; DNA-dependent protein kinase; Daphne plant; Databases; Development; Ectopic Expression; Embryo; Endometrial Neoplasms; Fertilization; Gene Activation; Gene Silencing; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Genetic Transcription; Genome; Genomic Instability; Goals; Hereditary Disease; Histones; Homologous Gene; Human; Incidence; Inherited; International; Knockout Mice; Lead; Malignant Neoplasms; Mammals; Modeling; Modification; Molecular; Mus; Mutate; Mutation; National Human Genome Research Institute; Orthologous Gene; Pathogenesis; Pathway interactions; Phenotype; Phosphorylation; Play; Predisposition; Prevention strategy; Proliferating Cell Nuclear Antigen; Proteins; Proto-Oncogenes; Regulation; Reporting; Ribosomal RNA; Role; SMARCA3 gene; Serine; Sister Chromatid; Site-Directed Mutagenesis; Somatic Mutation; Source; Syndrome; System; Testing; The Cancer Genome Atlas; Tumor Suppressor Proteins; Ubiquitination; Variant; Work; Zebrafish; activator 1 protein; cancer cell; cancer genome; cancer therapy; carcinogenesis; hatching; human FRAP1 protein; human disease; in vivo; interstitial; novel; null mutation; overexpression; prevent; protein complex; protein function; repaired; response; screening; tissue culture; tumor; tumorigenesis; yeast genome; zygote

Transmitting genetic information without creating deleterious genetic alterations is one of the most important tasks. Cells have evolved systems that check for and repair potentially lethal DNA damage. However, when these systems do not work properly, DNA damage accumulates and causes genetic changes or cell death. Accumulation of genetic changes, which is defined as a genomic instability is frequently observed in various types of genetic disorders including cancers. Genomic instability has been documented as a preceding step for multiple inactivations of tumor suppressor genes and activations of proto-oncogenes. One type of genomic instability observed frequently in many cancers is gross chromosomal rearrangement (GCR). GCR includes translocations, deletions of chromosome arm, interstitial deletions, inversions, amplifications, chromosome end-to-end fusion and aneuploidy. Although little is known about the origin and mechanisms of GCRs observed in cancer cells, recent studies on genes mutated in inherited cancer predisposition syndromes have started to demonstrate that proteins that function in DNA damage responses, DNA repair, and DNA recombination, play crucial roles in the suppression of spontaneous and/or DNA damage-induced GCRs. To understand mechanisms how GCRs are generated and how such GCR formation can lead tumorigenesis, we screened the entire yeast genome for mutations or overexpression that increase the rate of GCR formation. RAD5 and ELG1 from mutation screening were selected for further studies of molecular mechanisms of these proteins to protect genome from deleterious GCR formation. 1. Determine the role of RAD5 orthologs, SHPRH. Previously, we identified two RAD5 orthologs in mammals and demonstrated that RAD5 orthologs, SHPRH and HLTF function to prevent collapse of persistent stalled replication forks by assisting template switching DNA damage bypass mechanism that uses the nascent strand of the sister chromatid for recombination mechanism for damage bypass. Among different modifications of Proliferating Cell Nuclear Antigen (PCNA) that determine the bypass mechanisms, we demonstrated that PCNA is poly-ubiquitinated by SHPRH and HLTF. Although we tested for association of SHPRH and HLTF with tumorigenesis in vivo, the inactivation of these genes did not result in an increase in tumorigenesis. SHPRH has a unique histone interaction domain called PHD domain. We recently found that this domain is important for SHPRH localization in the nucleolus. In our characterization of the general DNA damage response regulating PCNA ubiquitination, we unexpectedly found that phosphorylation of Serine 4 and 8 of RPA32, which we used as a DNA damage response control, depends on DNA dependent protein kinase. We found a novel function of SHPRH in the nucleolus that drove the project into a novel molecular mechanism of SHPRH in rRNA transcription in the nucleolus. We found the function of SHPRH for rRNA transcription is mTOR-dependent. 2. ATAD5 (mammalian ELG1 homolog): determine whether alternative Replication Factor C (RFC) complex protein directs DNA repair pathways and replication. We reported that mice haploinsufficient in Atad5 showed a high incidence of tumorigenesis. We recently confirmed that embryonic day 7.5 to 8.5 as embryonic lethality caused by homozygous null mutation of ATAD5. In addition, in collaboration with Dr. Daphne Bells group in NHGRI, we found human somatic mutations of ATAD5 gene in many endometrial tumors. We also found several rare polymorphisms as well as cancer mutations in other tumor types. We recovered a zebrafish atad5 null allele from the TILLING project in the NHGRI zebrafish core facility. Unlike the atad5 null mouse, which dies at embryonic day 8.5, an atad5 null zebrafish survives until seven days post fertilization (dpf). The lethal phenotype of the atad5 null zebrafish became obvious right after hatching when fertilized eggs were treated with MMS. Therefore, we believe that in vivo functional activity of ATAD5 variants identified can be tested with the atad5 null zebrafish model. We have generated various ectopic expression constructs with a single sequence variant found in somatic cancer mutations as well as database search in the International Cancer Genome Consortium for the Cancer Genome Atlas using site-directed mutagenesis. We are currently testing whether these rare mutations found in affect ATAD5s molecular function and cause phenotypes observed in mice and zebrafish as well as in tissue culture system.

在不造成有害基因改变的情况下传递遗传信息是最重要的任务之一。细胞已经进化出了检测和修复潜在致命DNA损伤的系统。然而，当这些系统不能正常工作时，DNA损伤就会累积并导致基因变化或细胞死亡。遗传变化的积累被定义为基因组的不稳定性，在包括癌症在内的各种类型的遗传疾病中经常观察到。基因组不稳定已被证明是肿瘤抑制基因多次失活和原癌基因激活的前一步。在许多癌症中经常观察到的一种基因组不稳定性是染色体重排（GCR）。GCR包括易位、染色体臂缺失、间质缺失、倒位、扩增、染色体端到端融合和非整倍体。尽管对癌细胞中观察到的GCRs的起源和机制知之甚少，但最近对遗传性癌症易感综合征中突变基因的研究已经开始表明，在DNA损伤反应、DNA修复和DNA重组中起作用的蛋白质在抑制自发和/或DNA损伤诱导的GCRs中起着至关重要的作用。为了了解GCR的产生机制以及GCR的形成如何导致肿瘤发生，我们筛选了整个酵母基因组中增加GCR形成率的突变或过表达。从突变筛选中选择RAD5和ELG1，进一步研究这些蛋白保护基因组免受有害GCR形成的分子机制。