Harnessing Functional Genomics to Reveal Cancer Specific Determinants of Mitosis

广泛的证据表明，支配癌症分子病理学的一个统一原则是驱动增殖和抑制凋亡的核心机制相互依赖的异常调节。 参与支持致癌调控环境的异常蛋白可能是异种癌细胞群体中的最佳干预靶点。基于RNAi的功能基因组学的出现为在机械关系的先入为主的概念框架外获得支持关键生物系统的有效基因靶标的无偏见的全面收集提供了机会。在这里，我结合了一个高通量的基于细胞的一孔/一基因筛选平台和一个排列的全基因组合成siRNA文库，用于系统地询问癌细胞化学反应的分子基础。NCI-H1155是一种人类非小细胞肺癌细胞株，用于紫杉醇依赖的合成致死筛选，旨在识别在紫杉醇浓度不佳的情况下特定降低细胞活力的基因靶点。使用严格的客观统计算法将错误发现率降低到5%以下，我们分离了一组87个基因，这些基因代表了癌细胞对微管去除的自主反应的主要支点 动力学。重要的是，许多这些靶点使肺癌细胞对紫杉醇浓度的敏感度低于显著反应所需浓度的1000倍，并揭示了癌症相关异常基因表达程序与强劲有丝分裂进程所需的基本细胞机制之间的新的机制关系。在这里提出的研究中，我计划表征这些靶点的作用机制，并确定它们是否是体内肿瘤细胞存活和化疗耐药所必需的。我的具体目标是：1：确定新型紫杉醇增敏剂对癌细胞纺锤体组装和功能的贡献；2：验证肺癌原位异种移植模型候选治疗靶点的有效性。 在公共卫生方面，这些研究将有助于确定新的化疗药物以及现有化疗药物的新组合，以更好地治疗癌症。

Widespread evidence suggests that a unifying principle governing the molecular pathology of cancer is the co-dependent aberrant regulation of core machinery driving proliferation and suppressing apoptosis. Anomalous proteins engaged to support a tumorigenic regulatory environment likely represent optimal intervention targets in a heterogenous population of cancer cells. The advent of RNAi-based functional genomics provides the opportunity to derive unbiased comprehensive collections of validated gene targets supporting critical biological systems outside the framework of preconceived notions of mechanistic relationships. Here, I have combined a high throughput cell-based one-well/one-gene screening platform with an arrayed genome-wide synthetic siRNA library for systematic interrogation of the molecular underpinnings of cancer cell chemoresponsiveness. NCI-H1155, a human non small cell lung cancer line, was employed for a paclitaxel-dependent synthetic lethal screen designed to identify gene targets that specifically reduce cell viability in the presence of otherwise suboptimal pactlitaxel concentrations. Using a stringent objective statistical algorithm to reduce false discovery rates below 5%, we isolated a panel of 87 genes that represent major fulcrums of the cancer cell autonomous response to abrogation of microtubule dynamics. Importantly, a number of these targets sensitize lung cancer cells to paclitaxel concentrations 1000-fold lower than otherwise required for a significant response, and reveal novel mechanistic relationships between cancer-associated aberrant gene expression programs and the basic cellular machinery required for robust mitotic progression. In the studies proposed herein, I plan to characterize the mechanism of action of these targets and determine if they are required for tumor cell survival and chemoresistance in vivo. My specific aims are to 1: Defining the contribution of novel paclitaxel sensitizers to cancer cell spindle assembly and function and 2: Validate effectiveness of candidate therapeutic targets with orthotopic xenograft models of lung cancer. With respect to public health, these studies will aid in identifying both novel chemotherapeutics as well as novel combinations of existing chemotherapeutics to better treat cancer.