Hierarchical regulation in a KRAS-dependent transcriptional network revealed by a reverse-engineering approach

Mutations in RAS proteins occur in 30% of human tumours and have a high relevance in tumor progression. Despite the importance of the underlying genetic network that governs the effects of oncogenic RAS, it is still poorly understood. We developed and applied a reverse-engineering approach in order to reconstruct the network structure of the signaling and gene-regulatory network downstream of RAS from perturbation experiments. We performed microarray, RT-PCR and Western Blot analysis to detect mRNA and protein levels of cytoplasmatic and nuclear targets downstream of RAS after systematic perturbation of the signaling pathways and knock-down of selected transcription factors in KRAS-transformed ovarian surface epithelium cell lines. The reconstructed model shows that the investigated components are connected through a complex network. The transcription factors decomposed into two hierarchically arranged groups. While knock-down of all investigated transcription factors showed a partial reversion of the malignant phenotype, different growth assays show that these two groups of transcription factors control different functions in the malignant anchorage-independent growth and cell cycle regulation of the ROSE cells. Furthermore, the model showed strong regulatory interplay of inhibitory and activating interactions between the RAS-dependent trancriptional network and cytoplasmatic signaling components.RAS蛋白（RAS）突变发生于30%的人类肿瘤中，与肿瘤进展具有高度相关性。尽管调控致癌RAS效应的遗传网络至关重要，但目前对其认知仍较为匮乏。我们开发并应用了反向工程方法，基于扰动实验数据重建RAS下游信号转导与基因调控网络的结构。我们在KRAS转化的卵巢表面上皮细胞系中，对信号通路进行系统性扰动并敲低选定的转录因子，随后通过微阵列（microarray）、逆转录聚合酶链反应（RT-PCR）及蛋白质印迹（Western Blot）分析，检测RAS下游细胞质与细胞核靶标的信使RNA（mRNA）及蛋白表达水平。重建的模型显示，所研究的组分通过复杂网络相互连接；转录因子可被划分为两个层级排布的功能组。尽管对所有所研究转录因子的敲低均可部分逆转恶性表型，但不同生长实验结果表明，这两组转录因子分别调控ROSE细胞的恶性锚定非依赖性生长及细胞周期进程。此外，该模型还揭示了RAS依赖性转录网络与细胞质信号组分之间存在显著的激活与抑制类调控互作。