Harnessing Functional Genomics to Reveal Cancer Specific Determinants of Mitosis

描述（由申请人提供）：广泛的证据表明，控制癌症分子病理的统一原则是驱动增殖和抑制凋亡的核心机制的共同依赖异常调节。参与支持致瘤性调节环境的异常蛋白可能代表了异质癌细胞群体的最佳干预目标。基于rnai的功能基因组学的出现提供了一个机会，可以在机械关系的先入为主的概念框架之外，获得支持关键生物系统的经过验证的基因靶点的公正全面的集合。在这里，我将基于细胞的高通量单孔/单基因筛选平台与全基因组合成siRNA库相结合，用于系统地询问癌细胞化学反应性的分子基础。NCI-H1155是一种人类非小细胞肺癌系，用于紫杉醇依赖性合成致死筛选，旨在识别在紫杉醇浓度不理想的情况下特异性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动力学废除的自主反应的主要支点。重要的是，许多这些靶点使肺癌细胞对紫杉醇浓度敏感，而紫杉醇浓度比有丝分裂过程所需的浓度低1000倍，并揭示了癌症相关的异常基因表达程序和强大的有丝分裂过程所需的基本细胞机制之间的新机制关系。在本文提出的研究中，我计划描述这些靶点的作用机制，并确定它们是否为肿瘤细胞存活和体内化疗耐药所必需。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：用肺癌原位异种移植模型验证候选治疗靶点的有效性。3：从有丝分裂突变检查点中分离出癌细胞网络的核心成分，将化疗耐药肿瘤解放出来。

DESCRIPTION (provided by applicant): 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. 2: Validate effectiveness of candidate therapeutic targets with orthotopic xenograft models of lung cancer. 3: Isolate the core components of the cancer cell network that liberate chemoresistant tumors from mitotic catastrophe checkpoints. 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.