Dissection and manipulation of RB function

RB 函数的剖析和操作

• 批准号: 8699509
• 负责人: NICHOLAS J DYSON
• 金额: $ 35.02万
• 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/8699509
• 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; 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; screening; 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.

描述（由申请人提供）：pRB是最知名的肿瘤抑制剂之一。它在使细胞停止分裂并永久退出细胞周期的过程中起着核心作用。与其作为细胞增殖的关键负调节剂的作用一致，pRB的功能能力被认为在大多数（如果不是全部）人类癌细胞中受到损害。大多数人类肿瘤不突变RB 1基因，但获得的变化，如突变的基因编码的p16 cdk抑制剂，降低pRB的活性。 尽管广泛的调查，pRB的作用机制的分子细节是模糊的。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相互作用物。不同的研究小组都支持个体相互作用的重要性，但由于关于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，也没有关于哪些pRB相关蛋白真正重要的共识。 我们建议利用shRNA技术的快速发展，系统地测试敲低每个报告的pRB相关蛋白对pRB诱导的表型的影响。这将使我们能够评估每种pRB结合蛋白对pRB的各种活性的相对重要性。为了鉴定影响这些蛋白质/蛋白质相互作用的途径，我们将通过用靶向激酶组的shRNA集合筛选相同的功能测定集来补充该分析。初步数据显示，这些基于功能的筛选不仅鉴定了pRB发挥作用所需的蛋白质，而且还鉴定了可以靶向增强特定结果的途径，包括pRB诱导人类肿瘤细胞衰老的能力。使用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， 我们将测试靶向协同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pRB功能的未来分析提供一个框架，并将揭示增强人类癌细胞中内源性pRB活性的方法。