Novel Effectors of Colon Tumorigenesis

描述(申请人提供)：这项研究建议的中心假设是，通过高通量筛选模仿RAS 1(KSR1)的分子支架激酶抑制因子(KSR1)的基因和小分子，可以协调揭示癌症治疗的新靶点和化合物。KSR1-/-小鼠及其来源的细胞生长发育正常，但对致癌RAS的肿瘤形成和转化具有抵抗力。这些观察结果表明，KSR1、KSR1下游的基因产物或具有类似KSR1功能特征的基因产物可能成为治疗携带致癌RAS的肿瘤的靶点，但对正常组织的毒性降低或最小。我们开发了一种基因表达高通量筛选(GE-HTS)，它可以协调识别模拟KSR1功能的效应物和这些靶点的小分子抑制物，这些靶点选择性地抑制肿瘤细胞的生长，但不能抑制正常结肠上皮的生长。随后的分析表明，表达突变的、激活的RAS的人结肠肿瘤细胞系表现出KSR1依赖的转录调节因子的表达，这是维持转化表型所必需的。永生化的正常人结肠上皮细胞不表达这些调控因子。这些数据确定了一个以前未被识别的信号网络，该网络促进了结肠肿瘤细胞的存活。基于RNAi的GE-HTS将被用于定义该网络，并通过表征转录调节因子的作用和识别调节KSR1依赖的肿瘤形成潜力的额外网络效应来测试其作为治疗靶点的潜力。使用天然产物库的GE-HTS将用于识别网络效应器的小分子抑制剂。这些效应器和小分子将在体外和体内原位肿瘤模型中进行测试，以验证它们调节肿瘤发展和维持的能力。

DESCRIPTION (provided by applicant): The central hypothesis of this research proposal is that new targets and compounds for cancer therapy can be coordinately revealed by a high throughput screen for genes and small molecules that mimic the effects of the molecular scaffold Kinase Suppressor of Ras 1 (KSR1). KSR1-/- mice and cells derived from those mice grow and develop normally but are resistant to tumorigenesis and transformation by oncogenic Ras. These observations suggest that KSR1, gene products downstream of KSR1, or gene products with functional characteristics similar to KSR1, may serve as targets for therapeutic manipulation in tumors bearing oncogenic Ras, but with reduced or minimal toxicity to normal tissue. We developed a Gene Expression High Throughput Screen (GE-HTS) that can coordinately identify effectors mimicking KSR1 function and small molecule inhibitors of those targets that selectively inhibit tumor cell growth, but not growth of normal colonic epithelium. Subsequent analysis demonstrated that human colon tumor cell lines expressing mutated, activated Ras exhibit KSR1-dependent expression of transcriptional regulators essential for maintenance of the transformed phenotype. Immortalized normal human colonic epithelial cells did not express these regulators. These data identify a previously unrecognized signaling network that promotes colon tumor cell survival. RNAi based GE- HTS will be used to define that network and test its potential as a therapeutic target by characterizing the role of transcriptional regulators and the identification of additional network effectors regulating KSR1-dependent tumorigenic potential. GE-HTS using a natural products library will be used to identify small molecule inhibitors of network effectors. These effectors and small molecules will be tested in vitro and in in vivo orthotopic tumor models to validate their ability to regulate tumor development and maintenance.