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Systematic RNA interference perturbations within ovarian cancer cells reveal a hierarchically organized transcription factor network downstream of the oncogenic RAS pathway. Modules within the network are shown to control distinct aspects of cell growth and migration., Cellular transformation by KRAS oncogenes results in the upregulation of a multitude of transcription factors and a general deregulation of the transcriptome To exploit the network organization of selected transcriptional regulators responding to chronic RAS pathway activation, we used an integrated strategy combining experimental perturbation of transcription factor and signalling kinase expression with a reverse-engineering approach based on modular response analysis (MRA). The network shows strong modularity, high connectivity and hierarchical organization. The network hierarchy is reflected in distinct phenotypic consequences of perturbation within modules that separately control cellular proliferation and anchorage independence., RAS mutations are highly relevant for progression and therapy response of human tumours, but the genetic network that ultimately executes the oncogenic effects is poorly understood. Here, we used a reverse-engineering approach in an ovarian cancer model to reconstruct KRAS oncogene-dependent cytoplasmic and transcriptional networks from perturbation experiments based on gene silencing and pathway inhibitor treatments. We measured mRNA and protein levels in manipulated cells by microarray, RT–PCR and western blot analysis, respectively. The reconstructed model revealed complex interactions among the transcriptional and cytoplasmic components, some of which were confirmed by double pertubation experiments. Interestingly, the transcription factors decomposed into two hierarchically arranged groups. To validate the model predictions, we analysed growth parameters and transcriptional deregulation in the KRAS-transformed epithelial cells. As predicted by the model, we found two functional groups among the selected transcription factors. The experiments thus confirmed the predicted hierarchical transcription factor regulation and showed that the hierarchy manifests itself in downstream gene expression patterns and phenotype.

This study shows that MAPK signalling is robust against protein level changes due to a strong negative feedback from Erk to Raf. Surprisingly, robustness is provided through a fast post-translational mechanism although variation of Erk levels occurs on a timescale of days., MAPK signalling is robust against variation in protein level. Robustness is mediated by a negative feedback to Raf. Loss of negative feedback due to mutation in B-Raf opens the door for targeted intervention., Protein levels within signal transduction pathways vary strongly from cell to cell. For example, it has been reported that concentrations of the last kinase within the MAPK signalling module, Erk, varies about four-fold between clonal cells under the same conditions. In the present study, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity in protein levels. Mathematical analysis of isolated phosphorylation–dephosphorylation cycles predicts that phosphorylation of a signalling molecule is proportional to the protein concentration. We systematically perturbed the protein levels of Erk in human cell lines by siRNA. We found that the steady-state phosphorylation of Erk is very robust against perturbations of Erk protein level, suggesting that there are mechanisms that provide robustness to the pathway against protein fluctuations. Using mathematical modelling, we identified three potential mechanisms that may provide robustness against fluctuating protein levels: 1. Kinetic effects (saturation of the activating kinase Mek), 2. Transcriptional negative feedbacks, 3. Negative feedbacks on the post-translational level. By experimental analysis of the systems, which included analysis of Erk phosphorylation under Mek overexpression, measuring transcript levels of negative feedback regulators, and application of generic inhibitors of transcription and translation, we could exclude kinetic effects and transcriptional negative feedback as mechanisms of robustness. By analysing a panel of cell lines, we found that cells are robust as long as the signal passes through Raf-1. In contrast, cells where the pathway is activated by a mutation in B-Raf lose robustness. Detailed molecular analysis of the system shows that a single post-translational feedback to Raf mediates robustness. Thus, robustness is provided through a fast post-translational mechanism although variation of Erk levels occurs on a timescale of days., Protein levels within signal transduction pathways vary strongly from cell to cell. Here, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity in protein levels. We systematically perturbed the protein levels of Erk, the terminal kinase in the MAPK signalling pathway in a panel of human cell lines. We found that the steady-state phosphorylation of Erk is very robust against perturbations of Erk protein level. Although a multitude of mechanisms exist that may provide robustness against fluctuating protein levels, we found that one single feedback from Erk to Raf-1 accounts for the observed robustness. Surprisingly, robustness is provided through a fast post-translational mechanism although variation of Erk levels occurs on a timescale of days.