Nucleoporins in Chromosomal Instability and Cancer

摘要 绝大多数人类癌症的染色体数目都不正常，也就是所谓的非整倍体。 然而，非整倍体的分子基础及其在肿瘤发展中的作用仍然知之甚少。 最近的研究导致了一类新的有丝分裂调节器的鉴定，这些有丝分裂调节器由介导 间期核质运输。我们广泛的长期目标是提供对生物学的洞察 这些核运输因子的相关性及其在防止染色体不稳定和 肿瘤发生学。当前提案的具体目标是剖析核孔的有丝分裂功能。 在分子、细胞和生物水平上的复杂蛋白RanBP2，并确定RanBP2如何 下调调控促进了肿瘤的发生。我们已经培育了一系列小鼠，在这些小鼠中，核孔素 RanBP2蛋白通过使用野生型、敲除和 亚形等位基因。缺乏RanBP2的小鼠在胚胎上是致命的，但具有非常低数量的 蛋白质是有活力的，而且明显是正常的。与RanBP2在有丝分裂中的作用一致，我们发现这些小鼠 形成严重的非整倍体。我们观察到的主要有丝分裂缺陷是染色质桥形成。 后期，一种使人联想到受损的拓扑异构酶II功能的表型。在具体目标一中，我们将使用 用遗传和生化方法建立RanBP2调控精确的机制 姐妹染色单体后期分离。此外，使用条件敲除单元格，我们将确定 RanBP2的关键功能域(S)。初步研究表明，RanBP2水平较低的小鼠 增加对自发性和致癌物诱发的肿瘤，特别是肺癌的易感性。重要的是 通过对原发人类肿瘤和人类癌细胞株的定量RT-PCR分析，我们发现 在许多肺腺癌中，RanBP2的表达显著降低，这表明RanBP2具有 对小鼠和人类都有肿瘤抑制作用。在具体目标二中，我们将使用已经确立的和 新设计的RanBP2突变小鼠模型解决RanBP2不足的机制 促进肿瘤的发生。RanBP2突变小鼠对致癌物DMBA的高度敏感性表明 RanBP2基因缺失与其他基因突变在肿瘤发生中具有很强的协同作用。以特定的目标 第三，我们将利用“睡美人”转座子系统来鉴定这些癌症基因突变。在……里面 除了这种无偏见的方法外，我们还将使用候选基因方法来确定K-ras 与RanBP2缺乏症在肺癌发生中具有协同作用。在基础研究层面，完成这些 AIMS将提供对一个重要的核运输因子维持的机制的洞察 染色体稳定，预防癌症。在临床层面上，这些研究可能为 改进对人类癌症的检测、预防和治疗。

ABSTRACT The vast majority of human cancers have abnormal numbers of chromosomes, known as aneuploidy. However, the molecular basis of aneuploidy and its role in tumor development remain poorly understood. Recent studies led to the identification of a new class of mitotic regulators consisting of proteins that mediate nucleocytoplasmic transport in interphase. Our broad long-term goal is to provide insight into the biological relevance of these nuclear transport factors and their possible role in preventing chromosomal instability and tumorigenesis. The specific goal of the current proposal is to dissect the mitotic functions of the nuclear pore complex protein RanBP2 at the molecular, cellular and organismal levels, and to determine how RanBP2 downregulation promotes tumorigenesis. We have generated a series of mice in which the nucleoporin RanBP2 protein is reduced in a graded fashion from normal to zero by the use of wild-type, knockout and hypomorphic alleles. Mice lacking RanBP2 are embryonically lethal, but mice with very low amounts of the protein are viable and overtly normal. Consistent with a role for RanBP2 in mitosis, we find that these mice develop severe aneuploidy. The main mitotic defect that we observe is chromatin-bridge formation in anaphase, a phenotype reminiscent of impaired topoisomerase II¿ function. In specific aim one, we will use both genetic and biochemical approaches to establish the mechanism by which RanBP2 regulates accurate sister chromatid segregation in anaphase. Furthermore, using conditional knockout cells we will determine the critical functional domain(s) of RanBP2. Preliminary studies show that mice with low levels of RanBP2 have increased susceptibility to spontaneous and carcinogen-induced tumors, especially lung tumors. Importantly, using quantitative RT-PCR analysis of primary human tumors and human cancer cell lines we found that RanBP2 expression is dramatically reduced in many lung adenocarcinomas, suggesting that RanBP2 has a tumor suppressive function in both mice and humans. In specific aim two, we will use already established and newly designed RanBP2 mutant mouse models to resolve the mechanism by which RanBP2 insufficiency promotes tumorigenesis. The profound sensitivity of RanBP2 mutant mice to the carcinogen DMBA indicates that RanBP2 insufficiency strongly synergizes with other gene mutations in tumorigenesis. In specific aim three, we will identify these cancer gene mutations by the use of the "Sleeping Beauty" transposon system. In addition to this unbiased approach, we will use a candidate gene approach to determine whether K-ras synergizes with RanBP2 deficiency in lung carcinogenesis. At the basic research level, completion of these aims will provide insight into the mechanism by which a prominent nuclear transport factor maintains chromosomal stability and prevents cancer. At the clinical level, these studies may provide the basis for improved detection, prevention and treatment of cancer in humans.