Genetic approaches to next-generation breast cancer therapy

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 这一过程可以通过实验进行操纵，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：设计和构建针对全人类的RNAi文库和 小鼠基因组和基因条形码策略的发展，以促进全基因组RNAi筛选。 这两个目标都实现了。我们已经建造了20多万个建筑，目标几乎是 整个人类和小鼠基因组，我们已经开发并验证了微阵列条码策略 这使得一次可以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 这种疾病的死亡率。我们的RNAi文库代表着全面和系统的独特机会 大规模的功能研究，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的长期目标是筛选整个基因组 鉴定和表征a)新的假定的肿瘤抑制因子和b)合成致死相互作用 ERBB2激活。我将通过以下目标实现这些主要目标： A1)全基因组RNAi筛选以确定促进抗凋亡和不受控制的基因 正常人乳腺上皮细胞体外增殖的研究。A2)在目标A1中选择的候选对象 一种原位小鼠模型，以确定体内促进乳腺癌的基因。A3)采用蜂窝、 用分子和生物化学方法阐明候选基因的功能和生理相关性 在目标a2中确定。 B1)全基因组RNAi筛选，以确定显示合成致命相互作用的候选基因 体外激活ErbB2。B2)确认ErbB2激活和 来自体内目标b1的候选基因。B3)使用细胞、分子和生化方法来获得 深入到b2中证实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用于乳房综合功能基因组学研究的简化方法 癌症。基本的想法是组织一个系统的管道，从全基因组的RNA功能开始 体外研究和在小鼠体内验证后，得出了分子和生化方面的结论 对新识别的基因进行表征。这个研究核心的安排将为我提供一个 一致和均匀的检测平台，以研究乳腺癌的不同方面。

RNA interference (RNAi) is conserved through evolution as a mechanism to regulated gene expression. This process can be manipulated experimentaly to repress the expression of any specific gene. My postdoctoral research has focused on two goals: the design and construct an RNAi library targeting the whole human and mouse genome 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 oportunity 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 in 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 the 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 the 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 the aim b1 in vivo. b3) Employ cellular, molecular and biochemical approaches to gain inside 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.