Defining novel pathways that arrest genetically unstable tetraploid cells

项目总结/摘要 研究：四倍体细胞是细胞分裂失败的常见副产品，在遗传上不稳定 并且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细胞周期来限制这些细胞的进一步增殖 逮捕了然而，与其他激活p53并促进G1停滞的常见细胞损伤不同，例如， DNA损伤反应，四倍体反应中G1期阻滞的调控机制在很大程度上仍然存在 未开发的这项提案的一个主要目标是揭示细胞如何“感知”与细胞变化相关的细胞变化。 然后将该信息传递到p53通路中以阻止细胞周期进展。解决 为了解决这个基本问题，我开发了一种新的全基因组RNAi筛选试验， 鉴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， 诱导四倍体。该筛选的结果，结合互补的生物信息学和 生物化学方法，将阐明与四倍体相关的应激的性质：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 它们最终被癌细胞所战胜。将检查来自该筛选的强基因命中以确定 是否同样需要它们来激活G1期阻滞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 在体外和体内测试肿瘤抑制活性。总的来说，本提案所述的目标 很有可能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识别新的途径， 可以被化疗药物靶向，以加强异常癌细胞中的G1期阻滞。 候选人职业目标：我的长期职业目标是在一个领先的大学获得终身教职。 学术机构，并建立一个实验室，在解开细胞周期的许多奥秘的前沿 进展特别是，我注意到细胞生物学家使用的方法存在很大分歧， 癌症生物学家了解癌症的原因和后果：在我的实验室里，我计划建立一个桥梁， 通过将我现有的细胞生物学专业知识与新的培训机会相结合， 全基因组筛选、生物信息学和本提案中描述的转化测定。最后， 我的目标是融合这两个领域，以便采取一种独特的多方面实验方法来探索 细胞周期进展的详细机制。K99/R 00奖将提供受保护的 为了实现这个职业目标，我需要时间进行有针对性的高级培训。我希望辅导阶段 该提案的主要内容包括完成全基因组筛选和表征新的途径， 规范p53，需要1-2年，并导致至少一个高质量的出版物。以下独立 这个阶段的奖励将允许我进一步探索这些信号的激活和调节 途径，以及发现新的肿瘤抑制途径。这些数据将用于 证明未来的研究建议在一个RO 1补助金申请，我希望在年初提交的 独立第三年。 环境：Dana-Farber癌症研究所（DFCI）的儿科肿瘤学系， 哈佛医学院拥有国际公认的研究项目， 细胞增殖、肿瘤发生和癌细胞生物学领域的研究人员。我召集了一个 一流的指导和咨询委员会（大卫佩尔曼博士、威廉凯林博士、罗恩迪平霍博士、马特博士 Meyerson和William Hahn博士），这将极大地受益于我的研究和培训经验。我将会晤 根据具体科学或实验建议的需要，与该委员会成员进行非正式接触， 每年两次正式讨论我的项目的总体进展情况，确定未来的主要方向，并计划 我向独立的过渡。此外，哈佛医疗区拥有所有必要的物理设施， 我完成拟议的培训和研究所需的资源，包括 流式细胞分选、质谱、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.