Dissection and manipulation of RB function

RB 函数的剖析和操作

• 批准号: 8439189
• 负责人: NICHOLAS J DYSON
• 金额: $ 36.12万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-26 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8439189
• 关键词: Address; Affect; Binding Proteins; Biological Assay; Cancer cell line; Cell Cycle; Cell Cycle Arrest; Cell Line; Cell Proliferation; Cells; Collection; Complement; Consensus; Data; Development; Dissection; Face; Future; Gene Targeting; Genes; Genetic Transcription; Homologous Gene; Human; Individual; Investigation; Libraries; Link; Literature; Maintenance; Malignant Neoplasms; Mediating; Medicine; Molecular; Mutate; Mutation; Normal Cell; Outcome; Pathway interactions; Phenotype; Phosphotransferases; Play; Process; Proliferating; Property; Proteins; RB1 gene; Regulation; Relative (related person); Reporting; Research; Research Personnel; Retinoblastoma Protein; Role; Scientist; Screening procedure; Signal Pathway; Technology; Testing; Therapeutic; Time; Tumor Suppressor Proteins; United States; Work; base; cancer cell; improved; inhibitor/antagonist; knock-down; mortality; neoplastic cell; pancreatic cancer cells; pancreatic neoplasm; protein protein interaction; research study; senescence; small hairpin RNA; tool; tumor

DESCRIPTION (provided by applicant): pRB is one of the best-known tumor suppressors. It plays a central role in the processes that enable cells to stop dividing and to permanently withdraw from the cell cycle. Consistent with its role as a key negative regulator of cell proliferation, pRB's ability to function is thought to be compromised in most, if not all, human cancer cells. The majority of human tumors do not mutate the RB1 gene but acquire changes, such as mutations in the locus encoding the p16 cdk inhibitor that reduce pRB's activity. Despite extensive investigation, the molecular details of pRB's mechanism of action are vague. More than 150 different proteins have been reported to physically interact with pRB and have been proposed to be relevant for pRB function. However, most studies of have investigated just pRB-associated protein, or a small group of pRB-interactors, at any one time. Different groups have championed the importance of individual interactions but because the literature on pRB-associated proteins has accumulated in a piecemeal fashion, there is no information about the relative significance of these interactions, and no consensus over which pRB- associated proteins are genuinely important. We propose to take advantage of the rapid developments in shRNA technologies to systematically test the effect of knocking-down each of the reported pRB-associated protein on a panel of pRB-induced phenotypes. This will enable us to assess the relative importance of each pRB-binding protein for various activities of pRB. To identify pathways that impinge on these protein/protein interactions we will complement this analysis by screening the same set of functional assays with a collection of shRNAs that target the kinome. Preliminary data shows that these function-based screens not only identify proteins that are needed for pRB to act, but also identify pathways that can be targeted to enhance specific outcomes, including the ability of pRB to induce senescence in human tumor cells. Using pancreatic cancer cells as an example of cancers that retain an intact RB gene, we will identify the interacting proteins that are essential for pRB-mediated suppression of cell proliferation, and we will test whether targeting co-operative pathways can enhance the activity of endogenous pRB and improve its ability to induce a permanent cell cycle arrest. Together, these experiments will provide a framework for future analysis of pRB function and will uncover ways to enhance the activity of endogenous pRB in human cancer cells. PUBLIC HEALTH RELEVANCE: Cancer is one of the leading causes of mortality in the United States. This proposal investigates the mechanism of action of pRB, one of the best known tumor suppressors. The experiments address a long- standing issue in the field of pRB research, and will provide much-needed clarity to a large and confusing body of literature on pRB-associated proteins. The experiments will identify the mechanism of pRB-function in pancreatic cancer cells, and will seek to identify new ways to enhance the activity of pRB, potentially providing new strategies for the suppression of tumor cell proliferation.

描述（由申请人提供）：pRB 是最著名的肿瘤抑制因子之一。它在使细胞停止分裂并永久退出细胞周期的过程中发挥着核心作用。与其作为细胞增殖的关键负调节因子的作用一致，pRB 的功能被认为在大多数（如果不是全部）人类癌细胞中受到损害。大多数人类肿瘤不会使 RB1 基因发生突变，但会发生变化，例如编码 p16 cdk 抑制剂的基因座发生突变，从而降低 pRB 的活性。 尽管进行了广泛的研究，pRB 作用机制的分子细节仍不清楚。据报道，超过 150 种不同的蛋白质与 pRB 发生物理相互作用，并被认为与 pRB 功能相关。然而，大多数研究在任何时候都只研究了 pRB 相关蛋白或一小群 pRB 相互作用蛋白。不同的团体都主张个体相互作用的重要性，但由于有关 pRB 相关蛋白的文献以零碎的方式积累，因此没有关于这些相互作用的相对重要性的信息，并且对于哪些 pRB 相关蛋白真正重要也没有达成共识。 我们建议利用 shRNA 技术的快速发展来系统地测试敲低每个已报道的 pRB 相关蛋白对一系列 pRB 诱导表型的影响。这将使我们能够评估每种 pRB 结合蛋白对于 pRB 各种活性的相对重要性。为了确定影响这些蛋白质/蛋白质相互作用的途径，我们将通过使用一系列针对激酶组的 shRNA 筛选相同的功能分析集来补充这一分析。初步数据表明，这些基于功能的筛选不仅可以识别 pRB 发挥作用所需的蛋白质，还可以识别可增强特定结果的靶向途径，包括 pRB 诱导人类肿瘤细胞衰老的能力。以胰腺癌细胞作为保留完整 RB 基因的癌症的例子，我们将鉴定对 pRB 介导的细胞增殖抑制至关重要的相互作用蛋白，以及 我们将测试靶向合作途径是否可以增强内源性 pRB 的活性并提高其诱导永久性细胞周期停滞的能力。 总之，这些实验将为未来分析 pRB 功能提供一个框架，并将揭示增强人类癌细胞内源性 pRB 活性的方法。 公共卫生相关性：癌症是美国的主要原因之一。该提案研究了 pRB（最著名的肿瘤抑制剂之一）的作用机制。这些实验解决了 pRB 研究领域中一个长期存在的问题，并将为大量令人困惑的 pRB 相关蛋白文献提供急需的清晰度。这些实验将确定胰腺癌细胞中 pRB 功能的机制，并寻求增强 pRB 活性的新方法，从而可能为抑制肿瘤细胞增殖提供新策略。