Mechanisms of error-free cell division after whole-genome doubling

项目摘要 二倍体细胞非整倍体的一个普遍途径是发生全基因组加倍（WGD） 随后染色体丢失。WGD缓冲剂对在生长期间必需基因变化的有害影响 肿瘤细胞进化然而，WGD也导致中心体含量加倍，这可能是因为 在有丝分裂期间有害。这是因为每个中心体都使微管成核，从而导致微管的分裂。 多极纺锤体当这种情况发生时，有两种潜在的结果：细胞可以启动多极分裂， 这是致命的，或者它们可以通过聚集额外的 中心体后一种结果允许双极细胞分裂和存活，但增加了细胞分裂的速率。 染色体分离错误在WGD后，正常的未转化的人细胞经历细胞分裂和细胞分裂。 频率大致相等。相比之下，癌细胞系通常更擅长于 聚集额外的中心体，但这是如何发生的还不清楚。这项工作的长期目标是， 定义允许细胞在WGD后在有丝分裂中存活的机制。本建议的总体目标是 定义蛋白磷酸酶2A（PP 2A）中的癌症相关突变和缺失如何改变有丝分裂， WGD。中心假设是PP 2A失活通过以下途径增加WGD后细胞的活力： 增强中心体聚类和减少染色体分离错误。这个假设是基于 关于PP 2A-A中普遍存在的错义突变的研究，PP 2A-A是异源三聚体PP 2A复合物的核心成分。 这项研究的基本原理是，了解癌细胞如何减轻与癌症相关的压力， 更具体地说，PP 2A-A突变如何改变有丝分裂， 来治疗癌细胞中的这些通路。我们的目标将通过以下两个方面来实现 目的：（1）研究PP 2A对中心体成簇的调控作用。NuMA和TPX 2的磷酸化，两个 纺锤体组装的调节因子，通过PP 2A-A突变而改变。互补的体内和体外方法 将用于确定这如何影响WGD后的中心体聚类。(2)了解PP 2A如何控制 染色体分离的精确性。高分辨率活细胞成像，FRET磷酸化 生物传感器和内源性动粒底物的定量免疫荧光将用于测试 PP 2A-A突变通过改变磷酸化水平来减少染色体分离错误的假设， 在着丝粒上发出信号PP 2A-A突变对细胞基因组稳定性的长期影响， 还将确定WGD的经验。这项工作的成功完成将建立人类癌症如何 “微调”磷酸化以确保WGD后的增殖。这些知识可以为战略提供信息， 靶向具有倍性改变或额外中心体的肿瘤细胞。 ! !

PROJECT SUMMARY A prevalent route to aneuploidy for diploid cells is the occurrence of a Whole-Genome Doubling (WGD) followed by chromosome loss. WGD buffers against deleterious effects of changes in essential genes during tumor cell evolution. However, WGD also results in a doubling of centrosome content, which can be deleterious during mitosis. This is because each of the centrosomes nucleates microtubules, resulting in a multipolar spindle. There are two potential outcomes when this happens: cells can initiate a multipolar division, which is lethal, or they can re-organize the multipolar spindle into a bipolar spindle by clustering the extra centrosomes. The latter outcome allows for bipolar cell division and survival, but with an increased rate of chromosome segregation errors. After WGD, normal untransformed human cells experience both cell division outcomes with approximately equal frequency. Cancer cell lines, by contrast, are generally more proficient at clustering extra centrosomes, but how this occurs is not known. The long-term goal of this work, therefore, is to define mechanisms that allow cells to survive mitosis after WGD. The overall objective of this proposal is to define how cancer-associated mutations and deletions in protein phosphatase 2A (PP2A) alter mitosis after WGD. The central hypothesis is that PP2A inactivation increases the viability of cells after WGD through enhanced centrosome clustering and reduction in chromosome segregation errors. This hypothesis is based on studies of a prevalent mis-sense mutation in PP2A-A, a core component of heterotrimeric PP2A complexes. The rationale for the proposed research is that knowledge of how cancer cells mitigate the stress associated with WGD, and more specifically, how recurrent PP2A-A mutations change mitosis, could potentially be used to therapeutically target those pathways in cancer cells. Our objective will be realized by the following two aims: (1) Identify how PP2A controls centrosome clustering. Phosphorylation of NuMA and TPX2, two regulators of spindle assembly, is altered by PP2A-A mutation. Complementary in vivo and in vitro approaches will be used to determine how this impacts centrosome clustering after WGD. (2) Identify how PP2A controls the fidelity of chromosome segregation. High-resolution live-cell imaging, FRET-based phosphorylation biosensors, and quantitative immunofluorescence of endogenous kinetochore substrates will be used to test the hypothesis that PP2A-A mutation reduces chromosome segregation errors through altered phospho- signaling at kinetochores. The long-term impact of PP2A-A mutation on genome stability in cells that experience WGD will also be determined. Successful completion of this work will establish how human cancers `fine-tune' phosphorylation to ensure proliferation after WGD. This knowledge could inform on strategies to target tumor cells with ploidy changes or supernumerary centrosomes. ! !