Defining novel pathways that arrest genetically unstable tetraploid cells

描述（由申请人提供）： 研究：四倍体细胞是细胞分裂失败的常见副产品，其遗传不稳定并具有促进肿瘤发生的能力。因此，细胞具有 p53 依赖性肿瘤抑制机制，通过促进持久的 G1 细胞周期停滞来限制这些细胞的进一步增殖。然而，与激活 p53 并促进 G1 停滞的其他常见细胞损伤（例如 DNA 损伤反应）不同，四倍体响应的 G1 停滞的控制机制在很大程度上仍未被探索。该提案的一个主要目标是揭示细胞如何“感知”与四倍体相关的细胞变化，然后将该信息传递到 p53 途径以阻止细胞周期进展。为了解决这个基本问题，我开发了一种新型全基因组 RNAi 筛选方法，以全面鉴定在胞质分裂失败和诱导四倍体后激活或维持 G1 细胞周期停滞所必需的蛋白质。该筛选的结果与互补的生物信息学和生化方法相结合，将阐明与四倍体相关的应激的本质：这包括确定这些应激如何被感知，它们如何进入激活 p53 的信号级联，以及它们最终如何被癌细胞克服。将检查来自该筛选的强基因命中，以确定它们是否同样需要激活 G1 停滞以响应其他常见的细胞缺陷，以及它们是否在人类癌症中通常丢失或突变。最终，最有趣的候选基因将在体外和体内测试肿瘤抑制活性。总体而言，该提案中描述的目标具有识别与四倍体相关的细胞缺陷以及揭示 p53 的新调控机制的巨大潜力。此外，这项工作还可能导致发现新的途径，化疗药物可以针对这些途径加强异常癌细胞中的 G1 期停滞。 候选人的职业目标：我的长期职业目标是在领先的学术机构获得终身教职，并建立一个处于揭开细胞周期进展许多谜团最前沿的实验室。特别是，我注意到细胞生物学家和癌症生物学家在了解癌症的原因和后果时所使用的方法存在巨大分歧：在我的实验室中，我计划通过将我现有的细胞生物学专业知识与本提案中描述的全基因组筛选、生物信息学和转化测定方面的新培训机会相结合来弥补这一差距。最终，我的目标是将这两个领域融合起来，以便采用独特的多方面实验方法来探讨控制细胞周期进展的详细机制。 K99/R00 奖项将为我提供集中高级培训所需的受保护时间，以实现这一职业目标。我预计该提案的指导阶段（包括完成全基因组筛选和表征调节 p53 的新途径）需要 1-2 年时间，并产生至少一篇高质量的出版物。接下来的独立阶段的奖项将使我能够进一步探索这些信号通路的激活和调节，以及发现新的肿瘤抑制通路。这些数据将共同用于证明 R01 拨款申请中提出的未来研究的合理性，我预计在独立阶段的第三年年初提交该申请。 环境：达纳法伯癌症研究所 (DFCI) 和哈佛医学院的儿科肿瘤科拥有国际公认的研究项目，拥有细胞增殖、肿瘤发生和癌细胞生物学领域的许多专家研究人员。我组建了一个出色的指导和咨询委员会（David Pellman 博士、William Kaelin 博士、Ron Depinho 博士、Matt Meyerson 博士和 William Hahn 博士），这将极大地有益于我的研究和培训经验。我将根据需要与该委员会的成员进行非正式会面，以获取具体的科学或实验建议，并每年两次正式会面，讨论我的项目的总体进展，确定未来的关键方向，并为我向独立的过渡制定计划。此外，哈佛医学区拥有我完成拟议的培训和研究所需的所有必要的物理资源，包括 FACS 分选、质谱、微阵列分析和全基因组筛查设施。

DESCRIPTION (provided by applicant): 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 candidate 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 R01 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.