Genetic approaches to next-generation breast cancer therapy

描述（由申请人提供）：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。这个过程可以通过实验来控制，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：设计和构建针对整个人类和小鼠基因组的RNAi文库，以及开发遗传条形码策略以促进全基因组RNAi筛选。这两个目标都已经实现。我们已经构建了超过200，000个构建体，这些构建体几乎针对整个人类和小鼠基因组，我们已经开发并验证了一种微阵列条形码策略，该策略允许一次对数千个基因进行功能丧失研究。 虽然乳腺癌的重要调节因子是已知的，但这些发现并没有显着降低这种疾病的死亡率。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，以发现与肿瘤发生相关的基因。在这项授权中，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）新型推定肿瘤抑制因子和B）与ErbB 2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筛选以鉴定促进体外正常人乳腺上皮细胞中对凋亡和不受控制的增殖的抗性的基因。a2）在原位小鼠模型中筛选在目的al中选择的候选物以鉴定体内促进乳腺癌的基因。a3）采用细胞、分子和生物化学方法阐明目标a2中鉴定的候选物的功能和生理相关性。 b1）全基因组RNAi筛选以鉴定在体外显示与活化的ErbB2的合成致死相互作用的候选基因。b2）确认ErbB2活化与来自目标bl的候选基因之间的体内合成致死相互作用。b3）采用细胞，分子和生物化学方法来深入了解b2中证实的遗传相互作用的生物学。 我所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。 我的职业目标是为乳腺癌的综合功能基因组学研究开发一种简化的方法。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，以新鉴定基因的分子和生物化学表征结束。这个研究核心的安排将为我提供一个一致和同质的检测平台，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.