Novel Effectors of Colon Tumorigenesis

描述（由申请人提供）：本研究提案的中心假设是，通过高通量筛选模拟Ras 1（KSR 1）分子支架激酶抑制剂作用的基因和小分子，可以协同揭示癌症治疗的新靶点和化合物。KSR 1-/-小鼠和来自这些小鼠的细胞正常生长和发育，但对致癌Ras的肿瘤发生和转化具有抗性。这些观察结果表明，KSR 1，KSR 1下游的基因产物，或具有类似于KSR 1的功能特征的基因产物，可以作为在携带致癌Ras的肿瘤中进行治疗操作的靶点，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），可以协同识别模拟KSR 1功能的效应物和选择性抑制肿瘤细胞生长但不抑制正常结肠上皮生长的靶点的小分子抑制剂。随后的分析表明，人结肠肿瘤细胞系表达突变，激活Ras表现出KSR 1依赖性表达的转录调节因子的转化表型的维持是必不可少的。永生化的正常人结肠上皮细胞不表达这些调节因子。这些数据确定了一个以前未被认识到的促进结肠肿瘤细胞存活的信号网络。基于RNAi的GE-HTS将用于定义该网络，并通过表征转录调节因子的作用和鉴定调节KSR 1依赖性致瘤潜力的其他网络效应子来测试其作为治疗靶点的潜力。使用天然产物库的GE-HTS将用于鉴定网络效应物的小分子抑制剂。这些效应物和小分子将在体外和体内原位肿瘤模型中进行测试，以验证其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.