Defining novel pathways that arrest genetically unstable tetraploid cells

项目摘要/摘要 研究：四倍体细胞是细胞分裂失败的常见副产品，遗传不稳定 并具有促进肿瘤发生的能力。因此，细胞具有依赖于P53的肿瘤 通过促进持久的G1细胞周期来限制这些细胞进一步增殖的抑制机制 逮捕。然而，与其他常见的激活P53并促进G1期停滞的细胞侮辱不同，例如 DNA损伤反应，控制四倍体反应中G1期停滞的机制在很大程度上仍然存在 未被开发的。这项提议的一个主要目标是揭示细胞如何“感觉”与 四倍体，然后将该信息传递到P53途径，以防止细胞周期进展。致信地址 在这个根本性的问题上，我开发了一种新的全基因组RNAi筛选方法来全面 确定在胞质分裂失败后激活或维持G1细胞周期停滞所必需的蛋白质以及 四倍体的诱导。来自这一筛查的结果，结合互补的生物信息学和 生化方法，将阐明与四倍体相关的胁迫的性质：这包括 确定这些压力是如何被感知的，它们如何进入激活P53的信号级联，以及如何 它们最终会被癌细胞战胜。来自此屏幕的强大基因命中率将被检查以确定 如果它们同样被要求激活G1期以响应其他常见的细胞缺陷，以及是否 它们通常在人类癌症中丢失或突变。最终，最有趣的候选基因将 在体外和体内进行肿瘤抑制活性测试。总体而言，这项提案中描述的目标有 在鉴定与四倍体相关的细胞缺陷以及发现新的 P53的调控机制。此外，这项工作还可能导致确定新的途径， 可以被化疗药物靶向，以加强异常癌细胞的G1期停滞。 求职者的职业目标：我的长期职业目标是在一所领先的大学获得终身教职 建立一个学术机构，并建立一个处于揭开细胞周期许多奥秘的前沿的实验室 进步。特别是，我注意到细胞生物学家和 癌症生物学家了解癌症的原因和后果：在我的实验室里，我计划在 通过将我先前存在的细胞生物学专业知识与 本提案中描述的全基因组筛选、生物信息学和转化分析。最终， 我的目标是将这两个领域融合在一起，以一种独特的、多方面的实验方法来探索 调控细胞周期进程的详细机制。K99/R00奖将提供受保护的 为了实现这个职业目标，我需要时间进行集中的高级培训。我期待着指导阶段 包括完成全基因组筛选和描述新的途径， 调整P53，需要1-2年的时间，并至少产生一份高质量的出版物。以下是独立的 获奖阶段将允许我进一步探索这些信号的激活和调节 以及发现新的肿瘤抑制途径。这些数据加在一起将用于 证明在RO1拨款申请中提出的未来研究是合理的，我预计将在 独立阶段的第三年。 环境：达纳-法伯癌症研究所(DFCI)儿科肿瘤学系和 哈佛医学院有一个国际公认的研究项目，其中包括许多专家 细胞增殖、肿瘤发生和癌细胞生物学领域的研究人员。我已经组装了一个 明星指导和咨询委员会(David Pellman博士、William Kaelin博士、Ron DePinho博士、Matt博士 迈耶森和威廉·哈恩博士)，这将极大地有益于我的研究和培训经验。我会和你见面 根据需要与该委员会成员非正式地就具体的科学或实验建议进行磋商，以及 每年正式两次讨论我的项目的总体进度，确定关键的未来方向，并制定计划 为我向独立的过渡。此外，哈佛医学区拥有所有必要的体检 我完成拟议的培训和研究所需的资源，包括 流式细胞仪分类、质谱学、微阵列分析和全基因组筛选。

Project Summary/Abstract Research: Tetraploid cells, which are a common byproduct of cell division failures, are genetically unstable and have the capacity to facilitate tumorigenesis. Consequently, cells possess p53-dependent tumor suppression mechanisms that limit the further proliferation of these cells by promoting a durable G1 cell cycle arrest. However, unlike other common cellular insults that activate p53 and promote G1 arrest, such as the DNA damage response, the mechanisms governing G1 arrest in response to tetraploidy remain largely unexplored. A major goal of this proposal is to uncover how cells 'sense' cellular changes associated with tetraploidy and then relay that information into the p53 pathway to prevent cell cycle progression. To address this fundamental question, I have developed a novel genome-wide RNAi screening assay to comprehensively identify proteins that are necessary to activate or maintain G1 cell cycle arrest after cytokinesis failure and the induction of tetraploidy. Results from this screen, in combination with complementary bioinformatic and biochemical approaches, will illuminate the nature of the stresses associated with tetraploidy: this includes identifying how these stresses are sensed, how they feed into signaling cascades that activate p53, and how they are ultimately overcome by cancer cells. Strong gene hits from this screen will be examined to determine if they are similarly required to activate G1 arrest in response to other common cellular defects, and whether they are commonly lost or mutated in human cancers. Ultimately, the most interesting candidates genes will be tested for tumor suppressive activities in vitro and in vivo. Overall, the aims described in this proposal have strong potential to identify the cellular defects associated with tetraploidy, as well as to uncover novel regulatory mechanisms of p53. Moreover, this work may also lead to the identification of new pathways that can be targeted by chemotherapeutics to reinforce G1 arrest in abnormal cancer cells. Candidate Career Goals: My long-term career goal is to obtain a tenure-track faculty position at a leading academic institution and establish a lab that is at the forefront of unraveling the many mysteries of cell cycle progression. In particular, I have noticed a great divide in the approaches used by cell biologists and cancer biologists to understand both the causes and consequences of cancer: in my lab, I plan on bridging this gap by combining my pre-existing cell biological expertise with the new training opportunities in genome-wide screening, bioinformatics, and transformation assays described in this proposal. Ultimately, my aim is to blend these two fields in order to take a unique multifaceted experimental approach to probe the detailed mechanisms governing cell cycle progression. The K99/R00 award will provide the protected time I need for focused advanced training in order to achieve this career goal. I expect the mentored phase of this proposal, which includes completing the genome-wide screen and characterizing novel pathways that regulate p53, to take 1-2 years and result in at least one high quality publication. The following independent phase of the award will then permit me to further explore the activation and regulation of these signaling pathways, as well as to uncover new tumor suppressor pathways. Together, these data will be used to justify future studies proposed in an RO1 grant application that I expect to submit at the beginning of the third year of the independent phase. Environment: The Department of Pediatric Oncology at the Dana-Farber Cancer Institute (DFCI) and Harvard Medial School has an internationally recognized research program that houses a number of expert researchers in the areas of cell proliferation, tumorigenesis, and cancer cell biology. I have assembled a stellar mentoring and advisory committee (Dr. David Pellman, Dr. William Kaelin, Dr. Ron Depinho, Dr. Matt Meyerson, and Dr. William Hahn) that will greatly benefit my research and training experience. I will meet with members of this committee informally as needed for specific scientific or experimental advice, and formally twice a year to discuss the general progress of my project, identify key future directions, and plan for my transition to independence. In addition, the Harvard Medical Area has all of the necessary physical resources required to for me to complete the proposed training and research studies, including facilities for FACS sorting, mass spectrometry, microarray analysis, and genome-wide screening.