Genome Instability in Cancer Development

癌症发展中的基因组不稳定性

• 批准号: 8349992
• 负责人: Kyungjae Myung
• 金额: $ 130.06万
• 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/8349992
• 关键词: Aftercare; Aneuploidy; Aphidicolin; Ataxia Telangiectasia; BRCA1 gene; BRCA2 gene; Biochemical; Bioinformatics; Biological Assay; Bypass; Cell Culture Techniques; Cell Cycle Checkpoint; Cell Death; Cells; Chemicals; Chromosomal Rearrangement; Chromosome Arm; Chromosomes; Collaborations; Collection; Complement; Complex; Congenital Abnormality; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA repair protein; DNA replication fork; DNA-Directed DNA Polymerase; Daphne plant; Development; Disease; ES Cell Line; Embryo; Endometrial Neoplasms; Event; Fanconi' s Anemia; Gene Activation; Generations; Genes; Genetic; Genetic Recombination; Genomic Instability; Genomics; Genotoxic Stress; Goals; Hereditary Disease; Histones; Homologous Gene; Homologous Protein; Human; Human Cloning; Human Genome; Incidence; Inherited; Knockout Mice; Lead; Libraries; Link; Malignant Neoplasms; Mammals; Methods; Modeling; Modification; Molecular; Molecular Biology; Mus; Mutagens; Mutate; Mutation; National Human Genome Research Institute; Open Reading Frames; Orthologous Gene; Pancytopenia; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Play; Polymerase; Predisposition; Prevention strategy; Proliferating Cell Nuclear Antigen; Proteins; Proto-Oncogenes; Regulation; Regulation of Proteolysis; Role; S Phase; SMARCA3 gene; Screening procedure; Sequence Homology; Signal Transduction; Sister Chromatid; Somatic Mutation; Source; Staging; Stress; Syndrome; System; Tumor Suppressor Proteins; Ubiquitin; Ubiquitination; Work; Yeasts; Zebrafish; activator 1 protein; austin; base; cancer cell; cancer therapy; chemotherapeutic agent; genome database; genome wide association study; homologous recombination; human disease; hydroxyurea; interstitial; irradiation; mouse model; novel; null mutation; overexpression; protein complex; protein function; repaired; response; small hairpin RNA; tumor; tumorigenesis; yeast genetics

Transmitting genetic information without creating deleterious genetic alterations is one of the cell's 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. The recent identification of strong correlations between genes responsible for genetic diseases including cancers and GCRs started to pinpoint the importance of GCRs. However, the mechanisms that are responsible for GCR formation were not studied in depth. One of the major reasons for this is that many genes that suppress and enhance GCR formation have not yet been discovered. 1. Determine the role of RAD5 orthologs in mammalian GCR and further dissect the RAD5 pathway upstream signals and additional factors. Persistent stalled replication forks collapse and cause genomic instability that can lead to cell death if unrepaired. In yeast, stalled replication forks are resolved either by bypassing DNA damage with translesion synthesis (TLS) polymerases or by TS to the nascent strand of the sister chromatid. Different modifications of Proliferating Cell Nuclear Antigen (PCNA) determine the bypass mechanisms. PCNA functions to load different DNA polymerases or DNA repair machinery on DNA. PCNA is monoubiquitinated by RAD18 for damage bypass by TLS, and further poly-ubiquitinated by RAD5 on the monoubiquitinated PCNA for currently uncharacterized TS pathways. We found that yeast Rad18 and Rad5 suppress GCR through the poly-ubiquitination of PCNA. We recently demonstrated that mammals have RAD5-dependent TS pathway for suppression of genomic instability. There are two genes, SHPRH and HLTF, as RAD5 orthologs. We next hypothesized that mice deficient in SHPRH would show a high incidence of tumorigenesis. We have recently generated shprh-/- and hltf-/- and double knockout mice but did not observe tumorigenesis. In collaboration with Dr. Heinz Jacobs, we found that there is a redundant pathway that can complement the lack of SHPRH/HLTF pathway. We are currently searching for this complementary pathway. 2. ELG1: determine whether alternative Replication Factor C (RRFC) complex protein directs DNA repair pathways and communicates with cell cycle checkpoints. To investigate whether the role of ELG1 in GCR suppression is conserved in mammals, we cloned the human ELG1 gene by conducting a sequence homology search in the human genome database with help from the NHGRI Bioinformatics Core. When the expression of the human ELG1 gene was reduced by shRNA, an increase in DNA damage resulted as evidenced by an increase of phosphorylated histone H2Ax and ATM foci. The ELG1 protein was localized at the stalled replication fork after hydroxyurea treatment. We also demonstrated an increase of human ELG1 expression at S-phase and after treatment of cells with various DNA-damaging agents, including MMS, hydroxyurea, aphidicolin, and gamma-irradiation. We found that ELG1 interacts with PCNA and USP1 that removes ubiquitin from PCNA after TLS DNA damage bypass pathway. Based on these observations, we decided to investigate whether mice deficient in ELG1 would show a high incidence of tumorigenesis. In an attempt to create homozygous mice by using a retroviral insertion BayGenomics embryonic stem cell line, we found that the null mutation of mouse ELG1 is lethal at an early developmental stage. We confirmed this embryonic lethality in zebrafish model, too. To overcome this lethal event, we are continuing the last years effort to make a conditional knock out mouse model. Interestingly, we found that haploinsufficiency of ELG1 in mouse generated high incidence of tumors. In addition, in collaboration with Dr. Daphne Bell, we found human somatic mutations of ELG1 gene in many endometrial tumors. Lastly, since ELG1 protein level is increased in response to genotoxic stresses, we developed a robust assay to detect genotoxins. With this assay in collaboration with Drs. Christopher Austin, Menghang Xia, Raymond Tice, we screened 300,000 compounds collections in National Chemical Genomics Center to identify potential chemotherapeutic agents. We got 500 hit compounds. We are currently investigating potency of these compounds as chemotherapeutic agents. 3. Determine the role of Mph1, the yeast homolog of FANCM, in DNA repair By screening genes that enhance GCR formation when overexpressed, we identified MPH1 as the strongest GCR enhancing gene. MPH1 has been implicated in a homologous recombination (HR)-dependent DNA repair pathway. Recently, the human homolog of MPH1 was discovered as the gene mutated in FA complementation group M (FANCM) patients. FA is a genomic instability disorder clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. The FA core complex consists of twelve proteins participating in a DNA damage response network with BRCA1 and BRCA2. FANCM is a recently identified component of the FA complex that is hypothesized to function at an early step of the FA pathway. MPH1 enhanced GCR formation when overexpressed. We hypothesized yeast has a FA like pathway. The first effort to identify more proteins functioning in this pathway, in collaboration with Dr. Weidong Wang, we found two proteins, MHF1 and MHF2 function in FA pathway in yeast as well as mammals. To make a yeast as an attractive model to study intercrosslink repair pathway that is a major pathway controlled by FA pathway, we studied genetic interactions of MPH1, MHF1, MHF2 and CHL1 with other DNA repair pathway. In this effort, we found yeast FA like pathway is regulated by RAD5-dependent TS pathway. We are currently investigating biochemical regulation of the yeast FA pathway by the RAD5 pathway.

传输遗传信息而不创造有害的遗传改变是该细胞最重要的任务之一。细胞已经进化了系统，可以检查和修复潜在的致命DNA损伤。但是，当这些系统无法正常工作时，DNA损伤会积累并导致遗传变化或细胞死亡。在包括癌症在内的各种遗传疾病中经常观察到遗传变化的积累，被定义为基因组不稳定性。基因组不稳定性已被记录为肿瘤抑制基因的多种失活和原始癌基因激活的前一个步骤。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，反转，放大，染色体端到端融合和非整倍性。尽管对在癌细胞中观察到的GCR的起源和机制知之甚少，但最近对遗传性癌症易感综合征突变的基因的研究开始证明，在DNA损伤反应，DNA修复和DNA重新组合中起作用的蛋白质在抑制自发性和/或DNA损害损害的GCR中起着至关重要的作用。最近鉴定了负责遗传疾病（包括癌症和GCR）的基因之间的牢固相关性，开始指出GCR的重要性。但是，未深入研究负责GCR形成的机制。这样做的主要原因之一是，尚未发现许多抑制和增强GCR形成的基因。 1。确定RAD5直系同源物在哺乳动物GCR中的作用，并进一步剖析RAD5途径上游信号和其他因素。 持续停滞的复制叉塌陷并导致基因组不稳定性，如果未经修复，可能导致细胞死亡。在酵母中，通过translesy合成（TLS）聚合酶或TS到姐妹染色单体的新生链中绕过DNA损伤来解决停滞的复制叉。增殖细胞核抗原（PCNA）的不同修饰决定了旁路机制。 PCNA功能可在DNA上加载不同的DNA聚合酶或DNA修复机械。 PCNA由Rad18单泛素化，用于TLS损坏旁路，并在单液化的PCNA上进一步由RAD5进行多聚泛素化，以实现目前未表征的TS途径。我们发现酵母Rad18和Rad5通过PCNA的多泛素化抑制GCR。我们最近证明，哺乳动物具有Rad5依赖性TS途径来抑制基因组不稳定性。有两个基因，SHPRH和HLTF为RAD5直系同源物。接下来，我们假设缺乏SHPRH的小鼠会显示出较高的肿瘤发生率。我们最近产生了SHPRH - / - 和HLTF - / - 和双基因敲除小鼠，但没有观察到肿瘤发生。与Heinz Jacobs博士合作，我们发现有一条多余的途径可以补充SHPRH/HLTF途径的缺乏。我们目前正在搜索此互补途径。 2。ELG1：确定替代复制因子C（RRFC）复合蛋白是否指导DNA修复途径并与细胞周期检查点进行通信。 为了研究ELG1在GCR抑制中的作用是否在哺乳动物中保守，我们在NHGRI生物信息学核心的帮助下通过在人基因组数据库中进行序列同源性搜索来克隆人类ELG1基因。当人类ELG1基因的表达通过SHRNA降低时，DNA损伤的增加导致了磷酸化组蛋白H2AX和ATM灶的增加所见。羟基脲处理后，ELG1蛋白位于停滞的复制叉中。我们还证明了人类ELG1在S期和用各种DNA受损剂（包括MMS，羟基脲，蚜虫和γ-辐照）处理后的ELG1表达增加。我们发现ELG1与PCNA和USP1相互作用，这些PCNA和USP1在TLS DNA损伤旁路途径后从PCNA中去除泛素。基于这些观察结果，我们决定研究缺乏ELG1的小鼠是否显示出肿瘤发生的高发生率。为了通过使用逆转录病毒插入Baygenomics胚胎干细胞系创建纯合小鼠，我们发现小鼠ELG1的无效突变在早期发育阶段是致命的。我们在斑马鱼模型中也证实了这种胚胎致死性。为了克服这一致命事件，我们正在继续努力制作有条件的敲除鼠标模型。有趣的是，我们发现ELG1在小鼠中的单倍不足会产生肿瘤的高发病率。此外，与达芙妮·贝尔（Daphne Bell）博士合作，我们发现了许多子宫内膜肿瘤中ELG1基因的人类体细胞突变。最后，由于ELG1蛋白水平因遗传毒性应激而增加，因此我们开发了一种可检测遗传毒素的强大测定。与Drs合作的测定法。克里斯托弗·奥斯汀（Christopher Austin），孟期夏（Menghang Xia），雷蒙德·蒂斯（Raymond Tice），我们在国家化学基因组学中心筛选了300,000种化合物收集，以鉴定潜在的化学治疗剂。我们有500个命中化合物。我们目前正在研究这些化合物作为化学治疗剂的效力。 3。确定FANCM的酵母同源物MPH1在DNA修复中的作用 通过筛选过表达时增强GCR形成的基因，我们将MPH1确定​​为最强的GCR增强基因。 MPH1与同源重组（HR）依赖性DNA修复途径有关。最近，在FA互补组M（FANCM）患者中发现了MPH1的人类同源物。 FA是一种基因组不稳定性疾病，其临床特征是先天性异常，进行性骨髓衰竭和恶性肿瘤的倾向。 FA核心复合物由与BRCA1和BRCA2一起参与DNA损伤反应网络的十二种蛋白质组成。 FANCM是FA复合物的最近确定的组成部分，假设在FA途径的早期步骤中起作用。 MPH1过表达时增强了GCR的形成。我们假设酵母具有类似FA的途径。与Weidong Wang博士合作，在该途径中识别更多蛋白质的首次努力，我们在酵母中的FA途径以及哺乳动物中发现了两个蛋白质，MHF1和MHF2功能。为了使酵母作为一个有吸引力的模型来研究由FA途径控制的主要途径，我们研究了MPH1，MHF1，MHF2和CHL1与其他DNA修复途径的遗传相互作用。在这项工作中，我们发现像Rad5依赖性TS途径调节酵母FA一样。我们目前正在研究RAD5途径对酵母FA途径的生化调节。