PTEN Deficiency and Tumor Development

描述(申请人提供)：PTEN缺失与肿瘤发展PTEN是人类癌症中最常见的突变基因之一。我们以前已经证明了PTEN在维持基因组稳定性中起着重要作用，并且PTEN通过多种机制控制基因组的完整性。有丝分裂检查点是确保细胞分裂过程中染色体准确分离的最重要机制。我们的初步数据显示，PTEN基因敲除会导致严重的有丝分裂错位，并增加单极和多极纺锤体的频率，这表明PTEN缺乏会损害动粒聚集和纺锤体两极。此外，破坏Pten可以消除对纺锤体损伤的有丝分裂检查点反应。值得注意的是，我们发现在Pten空细胞中，两个关键的有丝分裂检查点调节因子Bub1和Auora B的水平降低。在这项授权申请中，我们认为PTEN在有丝分裂检查点控制和纺锤体组装中起着关键作用。我们的第一个具体目标是研究PTEN缺乏如何影响着丝粒功能、纺锤体组装和有丝分裂检查点活动。为此，我们将确定在有丝分裂过程中与PTEN相关的潜在动粒或纺锤体因子。我们还将确定野生型PTEN是否能够纠正有丝分裂错误并恢复有丝分裂检查点。我们的第二个特定目的是确定PTEN的磷酸酶活性是否是其有丝分裂功能所必需的，并进一步确定PTEN负责双极纺锤体组装和有丝分裂检查点的功能结构域。各种带有和不带有N-末端磷酸酶结构域的PTEN突变体将被测试其建立纺锤体双极性、维持检查点蛋白的表达水平以及维持功能有丝分裂检查点的能力。我们的第三个具体目标是探索PTEN如何与E2F-1协同参与Bub1和aurora B的调节机制。我们的初步染色质免疫沉淀分析表明，PTEN和E2F-1都位于有丝分裂基因启动子上，这表明PTEN和E2F-1在染色质上存在功能上的协同作用。因此，我们将确定PTEN如何作用于染色质，以调控E2F-1对有丝分裂基因的转录调控。我们最终的具体目标将是进一步评估PTEN在控制有丝分裂检查点和染色体稳定性中的作用，使用PTEN固有突变的考登综合征模型。我们将检测带有突变PTEN的人淋巴细胞中有丝分裂检查点的活性。我们将表征PTEN突变体的功能获得，并确定这些显性负性PTEN突变体是否扰乱有丝分裂检查点并诱导染色体不稳定。最后，我们将使用PTEN189突变敲入小鼠模型来确定PTEN189突变是否导致基因组不稳定和导致肿瘤发生。这个项目的成功完成将把PTEN定义为纺锤体检查点的控制器和基因组的守护者。该项目的新发现可能为PTEN缺陷和随之而来的有丝分裂障碍导致肿瘤发生的机制提供深入的见解。公共卫生相关性：PTEN缺乏与肿瘤发展叙述PTEN肿瘤抑制因子在多种人类癌症中经常发生突变。PTEN的缺失导致了小鼠肿瘤的发生。有丝分裂检查点是确保染色体遗传和预防恶性肿瘤的主要机制。这个项目将通过揭示核PTEN在纺锤体组装和染色体分离中的关键作用来探索核PTEN在维持基因组稳定方面的新功能。这项研究的新发现将回答PTEN缺乏如何损害有丝分裂监测机制，导致肿瘤发展的根本问题。PTEN-有丝分裂通路的识别可能为制定抗肿瘤的治疗策略提供深刻的启示。

DESCRIPTION (provided by applicant): PTEN Deficiency and Tumor Development PTEN is one of the most frequently mutated genes in human cancer. We have previously demonstrated that PTEN plays an essential role in the maintenance of genomic stability and that PTEN controls genome integrity through multiple mechanisms. The mitotic checkpoint is the most important mechanism for ensuring accurate chromosome segregation during cell division. Our preliminary data show that knockdown of PTEN causes severe mitotic misalignment and increases the frequency of monopolar and multipolar spindles, suggesting that PTEN deficiency impairs kinetochore congression and spindle bipolarity. Moreover, disruption of Pten eliminates the mitotic checkpoint response to spindle damage. Of significant interest was our finding that the levels of Bub1 and aurora B which are two critical mitotic checkpoint regulators are reduced in Pten null cells. In this grant application, we propose that PTEN plays a critical role in mitotic checkpoint control and spindle assembly. Our first specific aim is to characterize how PTEN deficiency affects kinetochore functions, spindle assembly and mitotic checkpoint activity. To do so, we will identify potential kinetochore or spindle factors physically associated with PTEN during mitosis. We will also determine whether wild-type PTEN can correct mitotic errors and restore the mitotic checkpoint. Our second specific aim is to determine whether the phosphatase activity of PTEN is necessary for its mitotic function and further define the functional domain of PTEN responsible for bipolar spindle assembly and the mitotic checkpoint. A variety of PTEN mutants with and without the N-terminal phosphatase domain will be tested for their ability to establish spindle bipolarity, sustain the expression levels of checkpoint proteins, and maintain a functional mitotic checkpoint. Our third specific aim is to explore the mechanism of how PTEN participates in the regulation of Bub1 and aurora B in synergy with E2F-1. Our preliminary chromatin immunoprecipitation assays identified both PTEN and E2F-1 on the mitotic gene promoter, which suggests there is functional cooperation of PTEN and E2F-1 on chromatin. We will therefore determine how PTEN acts on chromatin to modulate the transcriptional regulation of mitotic genes by E2F-1. Our final specific aim will be to further evaluate the role of PTEN in controlling the mitotic checkpoint and chromosomal stability using a Cowden syndrome model where PTEN is inherently mutated. We will examine the mitotic checkpoint activity in human lymphocytes with mutant PTEN. We will characterize the gain of function of PTEN mutants and determine whether these dominant-negative PTEN mutants disrupt the mitotic checkpoint and induce chromosome instability. Finally, we will use a PTEN189 mutant knock-in mouse model to determine whether the PTEN189 mutation causes genomic instability and results in tumorigenesis. Successful completion of this project will define PTEN as a controller of the spindle checkpoint and a guardian of the genome. New findings from this project may provide insights into the mechanism whereby PTEN deficiency and consequent mitotic dysfunction lead to tumorigenesis. PUBLIC HEALTH RELEVANCE: PTEN Deficiency and Tumor Development Narrative The PTEN tumor suppressor is frequently mutated in a variety of human cancers. Loss of PTEN leads to tumorigenesis in mouse models. The mitotic checkpoint is a major mechanism for ensuring chromosome inheritance and preventing malignancy. This project will explore novel functions of nuclear PTEN in maintaining genomic stability by revealing its critical role in spindle assembly and chromosome segregation. New findings from this study will answer the fundamental question of how PTEN deficiency impairs the mitotic surveillance machinery, leading to tumor development. Identification of the PTEN-mitotic pathway may offer a profound implication for development of therapeutic strategies against tumorigenesis.