Abstract
Abstract 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
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