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.

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。最近对包括癌症在内的遗传性疾病的基因与GCRs之间的强相关性的鉴定开始确定GCRs的重要性。然而，对GCR形成的机制尚未深入研究。其中一个主要原因是许多抑制和增强GCR形成的基因尚未被发现。确定RAD5同源基因在哺乳动物GCR中的作用，并进一步剖析RAD5通路上游信号和其他因素。持续停滞的复制分叉会崩溃，导致基因组不稳定，如果不进行修复，可能导致细胞死亡。在酵母中，停滞的复制分叉要么通过翻译合成（TLS）聚合酶绕过DNA损伤，要么通过TS到姐妹染色单体的新生链上来解决。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上装载不同的DNA聚合酶或DNA修复机制。对于TLS的损伤旁路，PCNA被RAD18单泛素化，而对于目前尚未表征的TS通路，单泛素化的PCNA被RAD5进一步多泛素化。我们发现酵母Rad18和Rad5通过PCNA的多泛素化抑制GCR。虽然rad18依赖的TLS通路在酵母和哺乳动物中得到了广泛的研究，但哺乳动物中RAD5和PCNA多泛素化通路的存在尚未得到研究，主要是因为没有发现哺乳动物RAD5的同源物。我们假设哺乳动物中存在TS的RAD5通路并抑制GCR。虽然我们无法使用传统的序列同源性搜索找到RAD5同源物，但在NHGRI生物信息学核心的帮助下，我们使用了SMART搜索（http://smart.embl-heidelberg.de/），该搜索基于域结构找到同源物。我们发现两个基因，SHPRH和HLTF，作为推定的RAD5同源基因。我们证实了SHPRH是酵母RAD5的同源物：1)SHPRH和HLTF都促进DNA损伤诱导的PCNA赖氨酸164的多泛素化；2) SHPRH和HLTF与人PCNA、RAD18和泛素偶联酶UBC13结合；3) shRNA使SHPRH或HLTF失活，增加了人类细胞对DNA损伤剂的敏感性，增强了细胞的诱变和染色体断裂以及染色体结构异常。
&#10；我们接下来假设SHPRH缺失的小鼠会表现出高的肿瘤发生发生率。利用BayGenomics的胚胎干细胞系，在小鼠SHPRH位点插入逆转录病毒，我们最近产生了SHPRH -/-小鼠，我们目前正在监测肿瘤发生的情况。此外，我们与丁浩博士合作，引进了HLTF基因被敲除的小鼠模型。我们将shprh和hltf小鼠进行交配，创造出两种基因都有缺陷的小鼠，目前正在监测肿瘤的发生。为了研究ELG1在哺乳动物GCR抑制中的作用是否保守，我们在NHGRI Bioinformatics Core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，导致DNA损伤增加，磷酸化组蛋白H2Ax和ATM灶增加。羟基脲处理后，ELG1蛋白定位在停滞的复制叉上。我们还证明了在s期和用各种dna损伤剂（包括MMS、羟基脲、阿希霉素和γ辐射）处理细胞后，人ELG1的表达增加。我们还发现，ELG1蛋白水平的诱导是由atr依赖性的蛋白降解抑制引起的。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的缺陷是否会导致各种基因组不稳定。基于这些观察，我们决定研究缺乏ELG1的小鼠是否会表现出高发生率的肿瘤发生。在使用逆转录病毒插入BayGenomics胚胎干细胞系创建纯合子小鼠的尝试中，我们发现小鼠ELG1的零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造一种条件敲除小鼠模型。有趣的是，我们发现小鼠ELG1的单倍不足会导致肿瘤的高发。详细分析发现，这是由于TGF β信号的激活。最后，我们发现ELG1在小鼠体内的单倍体不足可诱导多种肿瘤。确定FANCM的酵母同源物Mph1在DNA修复中的作用。通过筛选过表达时促进GCR形成的基因，我们发现MPH1是最强的GCR增强基因。MPH1参与同源重组（HR）依赖的DNA修复途径。最近，在FA互补组M （FANCM）患者中发现了MPH1的人类同源基因突变。FA是一种基因组不稳定性疾病，临床特征为先天性异常、进行性骨髓衰竭和易患恶性肿瘤。FA核心复合体由12个蛋白质组成，参与与BRCA1和BRCA2的DNA损伤反应网络。FANCM是最近发现的FA复合物的组成部分，假设在FA通路的早期阶段起作用。MPH1在过表达时增强GCR的形成，并且在TLS、TS和HR通路上的额外突变进一步提高GCR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。对携带不同ATPase或解旋酶结构域突变的MPH1蛋白的分析表明，MPH1过表达对GCR的增强不是通过ATPase或解旋酶活性实现的，而是通过其与rad52依赖性HR通路的相互作用实现的。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。通过蛋白质组学方法，我们发现了几种与MPH1相互作用的候选物质。