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. Overall the study contains 32 samples. We studied the influence of seven transcription factors (Fosl1, Gfi1, Hmga2, Junb, Klf6, Otx1, Rela) being knocked down by means of RNA interference. Two independent siRNA duplexes (N1, N2) against the same gene were used. All experiments were done with Cy3/Cy5 dye-swaps. As a negative control, we used scrambled siRNAs. Off-target effect was estimated by comparison of the scrambled siRNA treatment and an unterated cell line ROSEA 25.