Genetic approaches to next-generation breast cancer therapy

描述(申请人提供)：RNA干扰(RNAi)作为一种调节基因表达的机制，在进化过程中是保守的。这一过程可以通过实验进行操作，以抑制任何特定基因的表达。我的博士后研究集中在两个目标上：设计和构建一个针对整个人类和小鼠基因组的RNAi文库，以及开发一种遗传条形码策略，以促进全基因组RNAi筛选。这两个目标都实现了。我们已经构建了20多万个几乎针对整个人类和小鼠基因组的结构，我们已经开发并验证了一种微阵列条形码策略，该策略允许同时对数千个基因进行功能丧失研究。 尽管已知乳腺癌的重要调控因素，但这些发现并未显著降低乳腺癌的死亡率。我们的RNAi文库为全面和系统的大规模功能研究提供了一个独特的机会，以揭示与肿瘤发生有关的基因。在这笔赠款中，我建议使用我们的RNAi文库和我们开发的技术，长期目标是筛选整个基因组，以识别和表征a)新的假定的肿瘤抑制因子和b)与ErbB2激活的合成致命相互作用。我将通过以下目标实现这些主要目标： A1)全基因组RNAi筛选，以确定在体外正常人类乳腺上皮细胞中促进抗凋亡和不受控制的增殖的基因。A2)在原位小鼠模型中筛选Aim A1中选择的候选基因，以确定体内促进乳腺癌的基因。A3)使用细胞、分子和生物化学方法来阐明目标a2中确定的候选者的功能和生理相关性。 B1)全基因组RNAi筛选，以确定与激活的ErbB2在体外显示人工致死相互作用的候选基因。B2)证实ErbB2的激活与来自目标b1的候选基因在体内的合成致死作用。B3)使用细胞、分子和生化方法来洞察b2中确认的遗传相互作用的生物学。 我提议的研究的完成将增加我们对肿瘤发生的理解，并将揭示新的潜在治疗靶点。 我的职业目标是开发一种简化的方法，用于乳腺癌的综合功能基因组研究。其基本思想是组织一个系统的管道，从体外全基因组RNA功能研究开始，在小鼠模型中进行体内验证后，得出新识别基因的分子和生化特征。这一研究核心的安排将为我提供一个一致和均一的分析平台，以研究乳腺癌的不同方面。

DESCRIPTION (provided by applicant): RNA interference (RNAi) is conserved through evolution as a mechanism to regulate gene expression. This process can be manipulated experimentally to repress the expression of any specific gene. My postdoctoral research has focused on two goals: the design and construction of an RNAi library targeting the whole human and mouse genomes and the development of a genetic-barcode strategy to facilitate genome-wide RNAi screens. Both aims have been accomplished. We have constructed more than 200,000 constructs that target almost the entire human and mouse genomes and we have developed and validated a microarray barcode strategy that allows the loss of function study of thousands of genes at a time. Although important regulators of breast cancer are known, these findings have not decreased remarkably the mortality of this disease. Our RNAi library represents an unique opportunity for comprehensive and systematic large scale functional studies to uncover relevant genes to tumorigenesis. In this grant I propose to use our RNAi library and the technology we have developed with the long-term goal to screen the entire genome to identify and characterize a) Novel Putative Tumor Suppressors and b) Synthetic Lethal Interactions with ErbB2 Activation. I will approach these main objectives through the following aims: a1) Genome-wide RNAi screens to identify genes that promote resistance to apoptosis and uncontrolled proliferation in normal human breast epithelial cells in vitro. a2) Screen candidates selected in aim a1 in an orthotopic mouse model to identify genes that promote breast cancer in vivo. a3) Employ cellular, molecular and biochemical approaches to elucidate the functions and physiological relevance of candidates identified in aim a2. b1) Genome-wide RNAi screen to identify candidate genes that show synthetic lethal interaction with activated ErbB2 in vitro. b2) Confirm the synthetic lethal interaction between ErbB2 activation and the candidate genes from aim b1 in vivo. b3) Employ cellular, molecular and biochemical approaches to gain insight into the biology of the genetic interaction confirmed in b2. The completion of the research I am proposing will increase our understanding of tumorigenesis and will reveal new potential therapeutic targets. My career goal is to develop a streamlined approach for integrative functional genomics studies in breast cancer. The basic idea is to organize a systematic pipeline that begins with genome-wide RNA functional studies in vitro and after in vivo validation in mouse models it concludes with the molecular and biochemical characterization of newly identify genes. The arrangement of this research core will provide me with a consistent and homogeneous assay platform to investigate different aspects of breast cancer.