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.

传递遗传信息而不产生有害的遗传改变是细胞最重要的任务之一。细胞已经进化出了检查和修复潜在致命 DNA 损伤的系统。然而，当这些系统无法正常工作时，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（定义为基因组不稳定性）经常在包括癌症在内的各种类型的遗传性疾病中观察到。基因组不稳定性已被证明是肿瘤抑制基因多次失活和原癌基因激活的先决条件。在许多癌症中经常观察到的一种基因组不稳定性是总染色体重排（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。尽管人们对癌细胞中观察到的 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中突变基因的研究已开始证明，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 GCR 中发挥着至关重要的作用。最近发现导致包括癌症在内的遗传疾病的基因与 GCR 之间存在很强的相关性，这开始明确 GCR 的重要性。然而，GCR 形成的机制尚未得到深入研究。造成这种情况的主要原因之一是许多抑制和增强GCR形成的基因尚未被发现。 1. 确定 RAD5 直向同源物在哺乳动物 GCR 中的作用，并进一步剖析 RAD5 通路上游信号和其他因素。 持续停滞的复制叉崩溃并导致基因组不稳定，如果不修复，可能导致细胞死亡。在酵母中，停滞的复制叉可以通过使用跨损伤合成 (TLS) 聚合酶绕过 DNA 损伤或通过 TS 到姐妹染色单体的新生链来解决。增殖细胞核抗原（PCNA）的不同修饰决定了旁路机制。 PCNA 的功能是在 DNA 上加载不同的 DNA 聚合酶或 DNA 修复机制。 PCNA 被 RAD18 单泛素化，以通过 TLS 绕过损伤，并进一步被 RAD5 对单泛素化的 PCNA 进行多泛素化，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，作为 RAD5 直向同源物。接下来我们假设 SHPRH 缺陷的小鼠会表现出高肿瘤发生率。我们最近生成了 shprh-/- 和 hltf-/- 以及双敲除小鼠，但没有观察到肿瘤发生。我们与 Heinz Jacobs 博士合作，发现存在一条冗余通路可以补充 SHPRH/HLTF 通路的缺失。我们目前正在寻找这种互补途径。 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通讯。 为了研究 ELG1 在 GCR 抑制中的作用在哺乳动物中是否保守，我们在 NHGRI 生物信息学核心的帮助下，通过在人类基因组数据库中进行序列同源性搜索，克隆了人类 ELG1 基因。当 shRNA 降低人 ELG1 基因的表达时，磷酸化组蛋白 H2Ax 和 ATM 焦点的增加证明了 DNA 损伤的增加。羟基脲处理后，ELG1 蛋白定位于停滞的复制叉。我们还证明，在 S 期以及用各种 DNA 损伤剂（包括 MMS、羟基脲、阿非迪霉素和伽玛射线照射）处理细胞后，人类 ELG1 表达有所增加。我们发现 ELG1 与 PCNA 和 USP1 相互作用，在 TLS DNA 损伤旁路途径后从 PCNA 中去除泛素。基于这些观察，我们决定研究 ELG1 缺陷的小鼠是否会表现出高肿瘤发生率。在尝试使用逆转录病毒插入 BayGenomics 胚胎干细胞系来创建纯合小鼠时，我们发现小鼠 ELG1 的无效突变在早期发育阶段是致命的。我们也在斑马鱼模型中证实了这种胚胎致死性。为了克服这一致命事件，我们正在继续去年的努力，制作条件性基因敲除小鼠模型。有趣的是，我们发现小鼠中 ELG1 的单倍体不足会导致高肿瘤发生率。此外，我们与Daphne Bell博士合作，在许多子宫内膜肿瘤中发现了ELG1基因的人类体细胞突变。最后，由于 ELG1 蛋白水平因基因毒性应激而增加，因此我们开发了一种强大的检测方法来检测基因毒素。与博士合作进行这项测定。 Christopher Austin、Menghang Xia、Raymond Tice，我们筛选了国家化学基因组中心的 300,000 个化合物集合，以确定潜在的化疗药物。我们得到了 500 个命中化合物。我们目前正在研究这些化合物作为化疗药物的效力。 3.确定FANCM的酵母同源物Mph1在DNA修复中的作用 通过筛选过度表达时增强 GCR 形成的基因，我们确定 MPH1 是最强的 GCR 增强基因。 MPH1 与同源重组 (HR) 依赖性 DNA 修复途径有关。最近，MPH1 的人类同源物被发现为 FA 互补组 M (FANCM) 患者的突变基因。 FA 是一种基因组不稳定疾病，临床特征为先天性异常、进行性骨髓衰竭和恶性肿瘤倾向。 FA 核心复合物由 12 种蛋白质组成，它们与 BRCA1 和 BRCA2 一起参与 DNA 损伤反应网络。 FANCM 是最近发现的 FA 复合物的一个组成部分，假设它在 FA 途径的早期步骤发挥作用。 MPH1 过表达时会增强 GCR 形成。我们假设酵母具有类似 FA 的途径。我们与 Weidong Wang 博士合作，首次努力鉴定更多在该途径中发挥作用的蛋白质，在酵母和哺乳动物的 FA 途径中发现了两种蛋白质：MHF1 和 MHF2。为了使酵母成为研究交联修复途径（FA 途径控制的主要途径）的有吸引力的模型，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，我们发现酵母 FA 样途径受到 RAD5 依赖性 TS 途径的调节。我们目前正在研究 RAD5 通路对酵母 FA 通路的生化调节。