Genome Instability in Cancer Development

癌症发展中的基因组不稳定性

• 批准号: 8149429
• 负责人: Kyungjae Myung
• 金额: $ 154.58万
• 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/8149429
• 关键词: Development; Genomic Instability; Malignant Neoplasms

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.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. 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. Although the RAD18-dependent TLS pathway was studied extensively in yeast and mammals, the existence of RAD5 and PCNA polyubiquitination pathways in mammals has not been investigated, mainly because no mammalian RAD5 homolog has been identified. We hypothesized that the RAD5 pathway for TS existed in mammals and suppressed GCR. Although we could not find a RAD5 homolog using conventional sequence homology searches, with the help of the NHGRI Bioinformatics Core, we used the SMART search (http://smart.embl-heidelberg.de/) that finds orthologs based on domain structures. We found two genes, SHPRH and HLTF, as putative RAD5 orthologs. We confirmed that SHPRH is an ortholog of yeast RAD5 by demonstrating: 1) SHPRH and HLTF both promote DNA damage-induced PCNA polyubiquitination at lysine 164; 2) SHPRH and HLTF associate with human PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13; and 3) the inactivation of SHPRH or HLTF by shRNA increase sensitivity to DNA damaging agents and enhance mutagenesis and chromosome breaks and abnormal chromosome structures in human cells. 
We next hypothesized that mice deficient in SHPRH would show a high incidence of tumorigenesis. Using a BayGenomics embryonic stem cell line, which contains a retroviral insertion at the mouse SHPRH locus, we have recently generated shprh-/-mice that we are currently monitoring for occurrence of tumorigenesis. In addition, in collaboration with Dr. Hao Ding, we imported a mouse model having HLTF gene is knocked out. We mated shprh and hltf mice and created mice having defects in both genes and currently monitoring tumorigenesis. 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 also found that the induction of ELG1 protein levels was caused by ATR-dependent inhibition of protein degradation. Lastly, we found that ELG1 interacts with PCNA and USP1 that removes ubiquitin from PCNA. We are currently working on whether defects in ELG1 could cause various genomic instability.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. To overcome this lethal event, we are currently trying to make a conditional knock out mice model. Interestingly, we found that haploinsufficiency of ELG1 in mouse generated high incidence of tumors. Detail analysis found that it was due to the activation of TGF beta signaling. Lastly, we found that haploinsufficiency of ELG1 in mice induces various tumors. 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, and the GCR rate was further elevated by additional mutations in the TLS, TS, and HR pathways. We demonstrated that the GCR enhancement by MPH1 overexpression was caused by the partial inactivation of HR. Analysis of MPH1 proteins carrying a different mutation in ATPase or helicase domains demonstrated that the GCR enhancement by MPH1 overexpression was not accomplished through ATPase or helicase activity but through its interaction with the RAD52-dependent HR pathway. We found that the interaction between MPH1 and single strand binding protein RPA is the key function to promote genomic instability. We found several candidates interacting with MPH1 by using proteomics approach.

传输遗传信息而不创造有害的遗传改变是最重要的任务之一。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性综合征突变基因的研究开始表明，在DNA损伤反应，DNA修复和DNA重新组合中起作用，在抑制遗传中抑制gcr的conteries的蛋白质在DNA损伤反应，DNA修复和DNA重新组合中起着至关重要的作用。癌症和GCR开始确定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。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。尽管在酵母和哺乳动物中广泛研究了Rad18依赖性TLS途径，但尚未研究Rad5和PCNA多泛素化途径的存在，主要是因为尚未鉴定出哺乳动物RAD5同源物。我们假设TS的RAD5途径存在于哺乳动物中并抑制GCR。尽管我们无法使用常规序列同源性搜索找到RAD5同源物，但借助NHGRI BioInformitics核心，我们使用了基于域结构的智能搜索（http://smart.embl-heidelberg.de/）。我们发现两个基因SHPRH和HLTF是推定的Rad5直系同源物。我们通过证明：1）SHPRH和HLTF均促进DNA损伤诱导的PCNA诱导的PCNA多泛素化。 2）SHPRH和HLTF与人PCNA，RAD18和泛素偶联酶UBC13相关； 3）SHRNA通过SHRH或HLTF的失活增加了对DNA破坏剂的敏感性，并增强了人类细胞中诱变和染色体断裂和异常的染色体结构。 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。使用Baygenomics胚胎干细胞系，该干细胞系在小鼠SHPRH基因座上包含逆转录病毒插入，我们最近生成了我们目前正在监测肿瘤发生的SHPRH - / - 小鼠。此外，与Hao ding博士合作，我们进口了一个具有HLTF基因的鼠标模型。我们交配了SHPRH和HLTF小鼠，并创建了在基因上存在缺陷和目前监测肿瘤发生的小鼠。 ELG1：确定替代复制因子C（RRFC）复杂蛋白是否指导DNA修复途径并与细胞周期检查点进行通信，以调查ELG1在哺乳动物中是否保留ELG1在GCR抑制中的作用，我们将人类ELG1基因的克隆通过在人类基因组数据库中与NHGRI BioInefics一起进行人类基因组数据库中的序列同源性搜索来帮助。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。我们还发现，ELG1蛋白水平的诱导是由ATR依赖性抑制蛋白降解引起的。最后，我们发现ELG1与PCNA和USP1相互作用，从PCNA中去除泛素。我们目前正在研究ELG1缺陷是否会引起各种基因组不稳定性。基于这些观察结果，我们决定调查缺乏ELG1的小鼠是否会显示出较高的肿瘤发生率。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。为了克服这一致命事件，我们目前正在尝试制作有条件的敲除小鼠模型。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。细节分析发现，这是由于TGFβ信号的激活。最后，我们发现小鼠中ELG1的单倍不足会诱导各种肿瘤。确定FANCM的酵母同源物在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途径的早期工作。MPH1过表达时增强了GCR的形成，并且通过TLS，TS和HR途径中的其他突变进一步提高了GCR速率。我们证明了MPH1过表达的GCR增强是由HR的部分失活引起的。对ATPase或Helicase结构域中携带不同突变的MPH1蛋白的分析表明，MPH1过表达的GCR增强不是通过ATPase或Helicase活性来实现的，而是通过与Rad52依赖性HR途径的相互作用来实现的。我们发现，MPH1与单链结合蛋白RPA之间的相互作用是促进基因组不稳定性的关键功能。我们发现使用蛋白质组学方法与MPH1相互作用的几个候选人。