Genetic approaches to next-generation breast cancer therapy

RNA干扰（RNAi）作为一种调控基因表达的机制在进化过程中是保守的。这 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上：设计和构建靶向整个人类的RNAi文库， 小鼠基因组和开发基因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20万个结构， 整个人类和小鼠基因组，我们已经开发并验证了微阵列条形码策略， 它可以一次研究数千个基因的功能丧失。 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著降低乳腺癌的发病率。 这种疾病的死亡率。我们的RNAi文库代表了一个独特的机会， 大规模的功能研究，以揭示相关基因的肿瘤发生。在这份赠款中，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）新推定肿瘤抑制剂和B）与 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筛选以鉴定促进对细胞凋亡和不受控制的细胞凋亡的抗性的基因。 体外正常人乳腺上皮细胞增殖。a2）筛选目标a1中选择的候选人， 原位小鼠模型，以确定基因，促进乳腺癌在体内。a3）采用蜂窝， 分子和生物化学方法来阐明候选物的功能和生理相关性 在目标a2中确定。 b1）全基因组RNAi筛选，以鉴定显示与以下的合成致死相互作用的候选基因： 体外激活ErbB2。b2）确认ErbB2活化和ErbB2活化之间的合成致死相互作用。 来自体内aim b1的候选基因。b3）采用细胞、分子和生物化学方法， b2中证实的遗传相互作用的生物学。 我所建议的研究的完成将增加我们对肿瘤发生的理解， 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。 癌基本的想法是组织一个系统的管道，从全基因组RNA功能开始， 体外研究和小鼠模型体内验证后，其结论是分子和生物化学 表征新鉴定基因。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。

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.