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.

Our understanding of oncogenic signaling pathways has strongly fostered current concepts for targeted therapies in metastatic colorectal cancer. The RALA pathway is novel candidate due to its independent role in controlling expression of genes downstream of RAS.We compared RALA GTPase activities in three colorectal cancer cell lines by GTPase pull-down assay and analyzed the transcriptional and phenotypic effects of transient RALA silencing. Knocking-down RALA expression strongly diminished the active GTP-bound form of the protein. Proliferation of KRAS mutated cell lines was significantly reduced, while BRAF mutated cells were mostly unaffected. By microarray analysis we identified common genes showing altered expression upon RALA silencing in all cell lines. None of these genes were affected when the RAF/MAPK or PI3K pathways were blocked.To investigate the potential clinical relevance of the RALA pathway and its associated transcriptome, we performed a meta-analysis interrogating progression-free survival of colorectal cancer patients of five independent data sets using Cox regression. In each dataset, the RALA-responsive signature correlated with worse outcome.In summary, we uncovered the impact of the RAL signal transduction on genetic program and growth control in KRAS- and BRAF-mutated colorectal cells and demonstrated prognostic potential of the pathway-responsive gene signature in cancer patients.

The EGFR/RAS signaling system plays an imperative role in tumor pathogenesis and therapy response. Downstream effects of the EGFR/RAS signaling axis are mediated by three major pathway branches: the RAF/MAPK, PI3K and RALA pathways. While the RAF/MAPK and PI3K pathways have been extensively characterized in mechanistic, preclinical and clinical studies, the RAL pathway is less well understood. Here we compared RALGTPase activities in three colorectal cancer cell lines by active GTPase pull-down assay and analyzed the effects of transient silencing of RALA expression by siRNA. RALA activity was highest in SW480 [KRAS codon 12 mutation] and HCT116 cells [KRAS codon 13 mutation] and also detectable in HT29 colorectal cancer cells [KRAS wild-type, BRAF V600E mutation]. Silencing of RALA expression strongly diminished the active GTP-bound form of the protein in all cell lines. The proliferation of the two KRAS mutated cell lines was significantly reduced. BRAF mutated cells showed a slight increase in cell death only. Next we interrogated microarrays using RNA prepared from the cell lines treated with siRNA and controls. We identified 550 common genes the expression of which was up-regulated or down-regulated after RALA knock-down (“RALA pathway-responsive genes”). None of them were affected when the RAF/MAPK or PI3K pathways were blocked. To investigate the potential clinical relevance of transcriptional targets regulated by the RALA pathway, we performed a meta-analysis utilizing expression profiles of 1,424 colorectal cancers documented in 8 independent publicly available data sets comprising information on patient survival. Of 19 RALA pathway-responsive genes correlated with progression-free survival, IQGAP1 (IQ-motif containing GTPase-activating protein 1), TOP1 (topoisomerase 1), LGALS1 (lectin galactoside-binding soluble 1), FILIP1L (filamin A-interacting protein 1-like) and TCF4 (transcription factor 4) were identified as the most important RALA pathway targets capable of predicting survival. In conclusion, the RALA pathway impinges on the transcription of a distinct subset of target genes in colorectal cancer cells independent of the KRAS and BRAF mutational status. RALA pathway-responsive genes were unaffected by RAF/MAPK and PI3K signaling. These findings support the concept of a pathway-sensitive modular organization of the transcriptome. In view of the correlation of RAL pathway-responsive genes and patient survival, further investigations of the diagnostic impact in prospective trials and exploitation of therapeutic approaches are warranted. This abstract is also presented as Poster B32. Citation Format: Balazs Gyorffy, Iwona Stelniec-Klotz, Christian Sigler, Attila Szijarto, Yu Qian, Reinhold Schäfer. Impact of RAL signal transduction on genetic program and growth control in KRAS- and BRAF-mutated colorectal cells and prognostic potential of pathway-responsive genes in cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR06. doi: 10.1158/1557-3125.RASONC14-PR06