Bub 1 in Chromosomal Instability and Tumorigenesis

描述(申请人提供)：大多数人类癌症具有异常的染色体内容，也称为非整倍体。然而，非整倍体形成的分子缺陷及其在肿瘤发生中的作用在很大程度上仍不清楚。纺锤体组装检查点是一种监测机制，它通过推迟有丝分裂后期的开始，直到每个着丝粒都正确地附着在有丝分裂纺锤体上，来确保有丝分裂染色体的准确分离。在某些非整倍体癌症中，已发现有丝分裂检查点基因Bub1、BubR1和MAD2发生突变。在非整倍体肿瘤中也观察到了Bub1和BubR1表达的表观遗传下调。为了研究Bub1在有丝分裂、发育和肿瘤发生中的作用，我们培育了一系列小鼠，在这些小鼠中，Bub1的表达通过使用野生型、基因敲除和亚型等位基因来逐步降低。对这些小鼠的初步研究表明，Bub1以剂量依赖的方式防止非整倍体和自发的肿瘤发展。来自相对较低Bub1含量的动物的细胞表现出有丝分裂检查点活性缺陷，染色体错误分离频繁，染色体错误分离后存活率增加，以及大量非整倍体。该提案的总体目标是在分子、细胞和组织水平上剖析Bub1的有丝分裂功能，并确定Bub1功能障碍促进肿瘤发生的机制。在特定的目标一，我们将利用突变的小鼠品系来建立Bub1的生理功能和关键功能结构域。具体目的二是研究BUB1和Skp1之间的一种新的相互作用，Skp1是SCF型E3连接酶的核心成分。我们将通过使用表达不能结合Skp1的Bub1突变形式的敲门小鼠模型以及体外泛素化试验来检验这种相互作用的功能意义。在特定的目标三中，我们将使用我们的等位基因系列的Bub1小鼠来建立Bub1促进肿瘤发生的机制，并识别在肿瘤发展中与Bub1协同的癌症基因。从这些研究中获得的信息将提高我们对这种关键的有丝分裂调节因子维持染色体稳定和预防癌症的机制的理解。我们相信，这些信息最终将改善人类癌症的检测、预防和治疗。

DESCRIPTION (provided by applicant): Most human cancers have an abnormal chromosome content, also known as aneuploidy. However, the molecular defects underlying the development of aneuploidy and its role in tumorigenesis remain largely unclear. The spindle assembly checkpoint is a surveillance mechanism that ensures accurate segregation of mitotic chromosomes by delaying anaphase onset until each kinetochore has properly attached to the mitotic spindle. In certain cancers with aneuploidy, mutations have been identified in the mitotic checkpoint genes Bub1, BubR1 and Mad2. Epigenetic downregulation of Bub1 and BubR1 expression has also been observed in aneuploid tumors. To study the role of Bub1 in mitosis, development and tumorigenesis, we produced a series of mice in which expression of Bub1 is reduced in a graded fashion by the use of wild-type, knockout and hypomorphic alleles. Preliminary studies of these mice show that Bub1 prevents aneuploidy and spontaneous tumor development in a dose dependent fashion. Cells from animals with relatively low amounts of Bub1 exhibit defective mitotic checkpoint activity, frequent chromosome missegregation, increased survival after chromosome missegregation and massive aneuploidy. The overall goal of this proposal is to dissect the mitotic functions of Bub1 at the molecular, cellular and organismal levels, and to determine the mechanisms by which Bub1 dysfunction promotes tumorigenesis. In specific aim one, we will use mutant mouse strains to establish the physiological functions and critical functional domains of Bub1. Specific aim two focuses on a novel interaction between Bub1 and Skp1, a core component of SCF-type E3 ligases. We will examine the functional significance of this interaction by using a knockin mouse model expressing a mutant form of Bub1 that cannot bind Skp1 as well as in vitro ubiquitination assays. In specific aim three, we will use our allelic series of Bub1 mice to establish the mechanism by which Bub1-promotes tumorigenesis and to identify cancer genes that cooperate with Bub1 in tumor development. The information gained from these studies will improve our understanding of the mechanisms by which this key mitotic regulator maintains chromosomal stability and prevents cancer. We believe this information will ultimately lead to improved detection, prevention and treatment of cancer in humans.