Nucleoporins in Chromosomal Instability and Cancer

绝大多数人类癌症都有异常数量的染色体，称为非整倍体。然而，非整倍体的分子基础及其在肿瘤发生中的作用仍然知之甚少。最近的研究发现了一类新的有丝分裂调节因子，由介导间期核质转运的蛋白质组成。我们广泛的长期目标是深入了解这些核转运因子的生物学相关性及其在预防染色体不稳定和肿瘤发生中的可能作用。目前建议的具体目标是在分子，细胞和生物体水平上剖析核孔复合物蛋白RanBP 2的有丝分裂功能，并确定RanBP 2下调如何促进肿瘤发生。我们已经产生了一系列的小鼠，其中的核孔蛋白RanBP 2蛋白减少，从正常到零，通过使用野生型，敲除和亚纯型等位基因的分级方式。缺乏RanBP 2的小鼠是胚胎致死的，但具有非常少量蛋白质的小鼠是可行的并且明显正常。与RanBP 2在有丝分裂中的作用一致，我们发现这些小鼠发生严重的非整倍性。主要的有丝分裂缺陷，我们观察到的是染色质桥形成后期，一个表型让人想起受损的拓扑异构酶II？功能在具体的目标之一，我们将使用遗传和生物化学的方法来建立机制，RanBP 2调节准确的姐妹染色单体分离在后期。此外，使用条件性敲除细胞，我们将确定RanBP 2的关键功能结构域。初步研究表明，RanBP 2水平低的小鼠对自发性和致癌物诱导的肿瘤，特别是肺肿瘤的易感性增加。重要的是，使用原发性人类肿瘤和人类癌细胞系的定量RT-PCR分析，我们发现RanBP 2表达在许多肺腺癌中显著降低，表明RanBP 2在小鼠和人类中都具有肿瘤抑制功能。在具体目标二中，我们将使用已经建立和新设计的RanBP 2突变小鼠模型来解决RanBP 2不足促进肿瘤发生的机制。RanBP 2突变小鼠对致癌物DMBA的高度敏感性表明RanBP 2不足与肿瘤发生中的其他基因突变强烈协同作用。在具体目标三中，我们将通过使用“睡美人”转座子系统来识别这些癌症基因突变。除了这种无偏的方法，我们将使用候选基因的方法来确定是否K-ras协同与RanBP 2缺陷在肺癌的发生。在基础研究水平上，这些目标的完成将使人们深入了解一种重要的核转运因子维持染色体稳定性和预防癌症的机制。在临床层面，这些研究可能为改善人类癌症的检测、预防和治疗提供基础。

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.