Genome Instability in Cancer Development

癌症发展中的基因组不稳定性

• 批准号: 6988951
• 负责人: Kyungjae Myung
• 金额: --
• 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/6988951
• 关键词: DNA binding protein; DNA damage; DNA repair; Saccharomyces cerevisiae; cancer risk; cell component structure /function; cell cycle; cell growth regulation; chromosome aberrations; fungal genetics; fungal proteins; gene expression; gene mutation; gene rearrangement; human tissue; intermolecular interaction; molecular oncology; molecular pathology; neoplasm /cancer genetics; neoplastic transformation; protein structure function; telomere

Genome instability is a characteristic of cancer cells. Different types of genome instability such as the accumulation of mutations, genome rearrangements and aneuploidy have been observed in different genetic disorders including cacners. There is growing evidence supporting the idea that acquisition of a mutator phenotype is required to account for the high rate of accumulating genetic changes in cancer cells. Some of the many examples are cancer susceptibility genes such as ATM, NBS, BLM, BRCA1 and BRCA2 whose protein products have been linked to defects in DNA damage responses and/or DNA repair. In some cases, tumors and even normal blood cells from mutation-bearing individuals show an abnormally high frequency of chromosomal aberrations. While there are many observations of genetic cancer syndromes associated with genome instability, there is little work linking these gene defects to the molecular events that cause genome instability. To understand the mechanisms of genome instability, we have studied 1) The role of Ku protein in suppression of GCR through telomere maintenance, 2) The role of mitotic checkpoint to increase the GCRs and 3) Characterization of MRX (Mre11-Rad50-Xrs2) complex for its role of suppression of GCRs. 1) The role of Ku protein in suppression of GCR through telomere maintenance. Telomeres are the terminal structures of linear chromosomes. Telomeres appear to perform at least two functions; a) they allow for the replication of the ends of chromosomes and b) they stabilize chromosomes by keeping them from recombining with one another. Ku86 plays a key role in nonhomologous end joining (NHEJ) in organisms as evolutionarily disparate as bacteria and humans. In eukaryotic cells, Ku86 has also been implicated in the regulation of telomere length although the effect of Ku86 mutations varies considerably between species. Indeed, telomeres either shorten significantly, shorten slightly, remain unchanged, or lengthen significantly in budding yeast, fission yeast, chicken cells or plants, respectively, that are null for Ku86 expression. Thus, it has been unclear which model system is most relevant for humans. We found that the functional inactivation of even a single allele of Ku86 in human somatic cells results in profound telomere loss, which is accompanied by an increase in chromosomal fusions, translocations and genomic instability. Together, these experiments demonstrate that Ku86, separate from its role in nonhomologous end joining, performs that additional function in human somatic cells of suppressing genomic instability through the regulation of telomere length. Furthermore, we investigated the mechanism how Ku proteins suppress genome instability by using yeast GCR assay system. We found that the overexpression of yeast Ku70-Ku86 heterodimer suppressed the GCR formation either spontaneously generated or induced by treatments with different DNA damaging agents, which are sometimes used for radio- and chemotherapies for cancer. The suppression of GCR formation by the yKu70-yKu86 overexpression was disappeared only when the DNA damage checkpoint is despaired suggesting that the GCR suppression by Ku protein is its interaction through the DNA damage checkpoint not through its role in NHEJ. The Ku overexpression caused cell growth delay, which is dependent on intact Okazaki fragment maturation proteins. Furthermore, the inactivation of telomerase inhibitor, Pif1 along with Ku overexpression arrested cell cycle at S phase in DNA damage checkpoint dependent fashion. 2) The role of mitotic checkpoint to increase the GCRs. Checkpoints are surveillance mechanisms designed to ensure correct transmission of genetic information during cell division. There are a number of checkpoints that respond to DNA damage and as well as aberrant DNA structures that occur when DNA replication is blocked. The DNA damage checkpoint arrests the cell cycle eithher in G1 or G2 in response to DNA damage and also results in the slowing of DNA replication when DNA damage occurs during S phase; this latter checkpoint response is sometimes called the intra-S checkpoint. The DNA replication checkpoint arrests cell cycle progression and suppresses the firing of late replication origins in response to blocked DNA replication. The mitotic checkpoints respond to the failure of spindle assembly and arrests the cell cycle at M phase. Genetic defects in various DNA damage and S-phase checkpoints have been demonstrated to result in differing degrees of increased spontaneous GCR rates, increased chromosome loss and increased recombination. However, compared to the significant increases in GCR rates caused by defects in S phase checkpoints including the replication checkpoint and intra-S checkpoints, defects in the mitotic checkpoint did not appear to increase GCR rates. The mitotic checkpoint, also known as the spindle checkpoint, ensures proper chromosome segregation by arresting the cell cycle at mitosis by responding to improper or incomplete spindle assembly. In S. cerevisiae the genes that function in the mitotic checkpoint include MAD1, 2, 3, BUB1, 3 and MPS1 and these in part function through the anaphase-promoting complex (APC). Bub2 functions in the MEN by inhibiting the degradation of mitotic cyclins and other regulators of the exit from mitosis. Mitotic exit is achieved by inactivation of Tem1 by conversion of bound GTP to GDP by the Bub2 and Bfa1 GTPase activating proteins. Mutations in genes encoding mitotic checkpoint and MEN proteins lead to increased missegregation of chromosomes even in the absence of spindle damages and failure of mitotic cell cycle arrest in response to spindle depolymerizing drugs such as nocodazole or benomyl. We found that defects in the mitotic checkpoint and the mitotic exit network (MEN) often result in the suppression of GCRs in strains containing defects that increase the GCR rate. These data strongly suggest that functional mitotic checkpoints can play an important role in the formation of genome rearrangements. 3) Characterization of MRX (Mre11-Rad50-Xrs2) complex for its role of suppression of GCRs. Mutation in any of genes in the MRX complex (MRE11, RAD50 or XRS2) causes sensitivity to alkylating agents and ionizing radiation, defects in mitotic and meiotic recombination and NHEJ and also results in a decrease in telomere length. The fact that the mammalian MRX complex equivalent forms foci at the site of DNA damage suggests that the MRX complex functions in cell cycle checkpoints and DNA repair in mammalian cells. Furthermore, mutations of genes encoding these subunits have been identified in many cancer prone syndromes including Ataxia Telangiectasia Like Disorder (ATLD) and Nijmegen Breakage Syndrome (NBS). Null mutations in any of the MRX genes increased the GCR rate up to 1000 fold. However, because of the multiple functions and biochemical activities of the MRX complex, it is unclear which functions and what biochemical activities of the MRX complex are important for suppression of GCRs. To understand which function of MRX complex is the main function of GCR suppression, we generated different types of point mutations that specifically inactivate different MRX functions. We found that at least three different activities of the MRX complex are important for suppression of GCRs. The nuclease activity of Mre11, an activity related to MRX complex formation and the telomere maintenance function of the MRX complex are important for the suppression of GCRs. An activity related to MRX complex formation is especially important for the suppression of translocation type of GCRs.

基因组不稳定是癌细胞的一个特征。在包括癌症在内的不同遗传性疾病中观察到了不同类型的基因组不稳定性，例如突变的积累、基因组重排和非整倍性。越来越多的证据支持这样一种观点，即需要获得突变表型才能解释癌细胞中积累的高速率遗传变化。许多例子中的一些是癌症易感性基因，例如 ATM、NBS、BLM、BRCA1 和 BRCA2，其蛋白质产物与 DNA 损伤反应和/或 DNA 修复的缺陷有关。在某些情况下，肿瘤甚至来自携带突变个体的正常血细胞都显示出异常高频率的染色体畸变。虽然有许多与基因组不稳定相关的遗传性癌症综合征的观察，但很少有人将这些基因缺陷与导致基因组不稳定的分子事件联系起来。为了了解基因组不稳定的机制，我们研究了 1) Ku 蛋白通过端粒维持抑制 GCR 的作用，2) 有丝分裂检查点增加 GCR 的作用，以及 3) MRX (Mre11-Rad50-Xrs2) 复合物抑制 GCR 作用的表征。 1) Ku蛋白通过端粒维持抑制GCR的作用。 端粒是线性染色体的末端结构。端粒似乎至少执行两种功能： a) 它们允许染色体末端的复制；b) 它们通过防止染色体彼此重组来稳定染色体。 Ku86 在进化上不同的生物体（如细菌和人类）的非同源末端连接 (NHEJ) 中发挥着关键作用。在真核细胞中，Ku86 也参与端粒长度的调节，尽管 Ku86 突变的影响在物种之间存在很大差异。事实上，在对 Ku86 表达无效的芽殖酵母、裂殖酵母、鸡细胞或植物中，端粒分别显着缩短、稍微缩短、保持不变或显着延长。因此，尚不清楚哪种模型系统与人类最相关。我们发现，即使人类体细胞中 Ku86 的单个等位基因的功能失活也会导致端粒严重丧失，并伴随着染色体融合、易位和基因组不稳定性的增加。总之，这些实验表明，Ku86 除了在非同源末端连接中的作用之外，还在人类体细胞中通过调节端粒长度来发挥抑制基因组不稳定性的附加功能。此外，我们利用酵母GCR检测系统研究了Ku蛋白抑制基因组不稳定性的机制。我们发现酵母 Ku70-Ku86 异二聚体的过度表达抑制了 GCR 的形成，GCR 的形成要么是自发产生的，要么是由不同 DNA 损伤剂治疗诱导的，这些药物有时用于癌症的放射和化疗。仅当 DNA 损伤检查点绝望时，yKu70-yKu86 过表达对 GCR 形成的抑制才消失，这表明 Ku 蛋白的 GCR 抑制是其通过 DNA 损伤检查点的相互作用，而不是通过其在 NHEJ 中的作用。 Ku 过度表达导致细胞生长延迟，这依赖于完整的冈崎片段成熟蛋白。此外，端粒酶抑制剂 Pif1 的失活以及 Ku 的过度表达以 DNA 损伤检查点依赖性方式将细胞周期阻滞在 S 期。 2)有丝分裂检查点增加GCR的作用。 检查点是旨在确保细胞分裂过程中遗传信息正确传输的监视机制。有许多检查点可以对 DNA 损伤以及 DNA 复制受阻时出现的异常 DNA 结构做出反应。 DNA损伤检查点将细胞周期停滞在G1或G2以响应DNA损伤，并且当DNA损伤发生在S期时也会导致DNA复制减慢；后一个检查点响应有时称为 S 内检查点。 DNA 复制检查点可阻止细胞周期进程，并抑制晚期复制起点的激发，以应对 DNA 复制受阻。有丝分裂检查点对纺锤体组装失败做出反应，并将细胞周期停滞在 M 期。各种DNA损伤和S期检查点的遗传缺陷已被证明会导致不同程度的自发GCR率增加、染色体丢失增加和重组增加。然而，与S期检查点（包括复制检查点和S内检查点）缺陷引起的GCR率显着增加相比，有丝分裂检查点的缺陷似乎并未增加GCR率。 有丝分裂检查点，也称为纺锤体检查点，通过响应不正确或不完整的纺锤体组装来阻止有丝分裂的细胞周期，从而确保适当的染色体分离。在酿酒酵母中，在有丝分裂检查点发挥作用的基因包括 MAD1、2、3、BUB1、3 和 MPS1，这些基因部分通过后期促进复合体 (APC) 发挥作用。 Bub2 在 MEN 中通过抑制有丝分裂细胞周期蛋白和有丝分裂退出的其他调节因子的降解来发挥作用。有丝分裂退出是通过 Bub2 和 Bfa1 GTPase 激活蛋白将结合的 GTP 转化为 GDP 来灭活 Tem1 来实现的。编码有丝分裂检查点和 MEN 蛋白的基因突变会导致染色体错误分离增加，即使没有纺锤体损伤，也不会因诺考达唑或苯菌灵等纺锤体解聚药物而导致有丝分裂细胞周期停滞。我们发现，有丝分裂检查点和有丝分裂出口网络 (MEN) 中的缺陷通常会导致含有缺陷的菌株中 GCR 受到抑制，从而增加 GCR 率。这些数据强烈表明功能性有丝分裂检查点可以在基因组重排的形成中发挥重要作用。 3) MRX (Mre11-Rad50-Xrs2) 复合物抑制 GCR 作用的表征。 MRX 复合物（MRE11、RAD50 或 XRS2）中任何基因的突变都会导致对烷化剂和电离辐射的敏感性、有丝分裂和减数分裂重组以及 NHEJ 的缺陷，还会导致端粒长度缩短。哺乳动物 MRX 复合物等价物在 DNA 损伤部位形成焦点这一事实表明，MRX 复合物在哺乳动物细胞的细胞周期检查点和 DNA 修复中发挥作用。此外，编码这些亚基的基因突变已在许多癌症易发综合征中被发现，包括共济失调性毛细血管扩张样疾病（ATLD）和奈梅亨断裂综合征（NBS）。任何 MRX 基因的无效突变可使 GCR 率增加高达 1000 倍。然而，由于MRX复合物的多种功能和生化活性，尚不清楚MRX复合物的哪些功能和哪些生化活性对于GCR的抑制很重要。为了了解 MRX 复合物的哪个功能是 GCR 抑制的主要功能，我们生成了不同类型的点突变，这些突变专门使不同的 MRX 功能失活。我们发现 MRX 复合物至少有三种不同的活性对于抑制 GCR 很重要。 Mre11 的核酸酶活性、与 MRX 复合物形成相关的活性以及 MRX 复合物的端粒维持功能对于 GCR 的抑制非常重要。与 MRX 复合物形成相关的活性对于抑制 GCR 的易位类型尤其重要。