Investigation of Inducible Mitogen and Stress Activated Protein Kinase 1 (MSK1) and Histone H3 Phosphorylation by the RAS-MAPK Pathway in Cancer Cells

The RAS-mitogen-activated protein kinase (MAPK) pathway is an essential signaling mechanism that regulates numerous cellular processes and culminates in the activation of specific gene expression programs. Alterations in the RAS-MAPK pathway by receptor over-expression or gain of function gene mutations can modify epigenetic programs and confer cell growth and/or transformation advantages. In fact, deregulation of the RAS-MAPK signaling cascade is a key event in tumour development and about 30% of human cancers contain RAS mutations. In breast and pancreatic epithelial cancers, characterization of an aberrant RAS-MAPK signaling pathway has focused on upstream mediators such as receptors and oncogenic RAS molecules. However, the impact of downstream targets has not been fully explored.;Our studies focused on MSK-mediated H3 phosphorylation under the frame of inducible and constitutively activated RAS-MAPK signaling. As model cell systems, we used Ras-transformed mouse fibroblasts, human breast cancer cell lines with over-expressed receptors and human pancreatic cancer cell lines with activating K-RAS mutations. We present evidence for the critical involvement of MSK1 and H3 phosphorylation as mediators that bridge the aberrant signals driven by the RAS-MAPK pathway with nucleosomal modifications, chromatin remodeling, IEG expression and malignant transformation in mouse fibroblasts. In order to verify our observations from murine cell model systems, we examined if activation of RAS-MAPK signaling in breast cancer cells elicits similar molecular events. We firstly demonstrate that the RAS-MAPK pathway is induced and consequently enhances the association of MSK1 and H3 phosphorylation on the IEG Trefoil Factor 1 resulting in transcriptional activation. Secondly, we observed that mutated K-RAS expression did not correlate with genomic instability or altered signaling in pancreatic cancer cell lines in comparison to overexpressed HER2 and EGFR breast cancer cell lines which generally exhibit upregulated ERK1/2 and H3 phosphorylation levels.;Taken together, MSK-regulated gene expression may potentially be exploited by epithelial cancerous cells with abnormal MAPK signaling to acquire further growth/transformation advantages. Our studies also contribute to the further understanding of MSK-mediated transcriptional activation in response to RAS-MAPK signaling in oncogenetransformed and cancer cell lines. As such, inhibition of MSK activity may be an unexplored avenue for combination cancer therapy with abnormal RAS-MAPK signaling pathways.;Stimulation of the RAS-MAPK pathway leads to downstream activation of mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2) which are responsible for the phosphorylation of histone H3 on S10 and S28. Herein, we postulate that the deregulation of the RAS-MAPK pathway produced by mitogen stimulation, or that the constitutive activation and/or over-expression of upstream components consequently leads to enhanced MSK1 activity and elevated histone H3 phosphorylation levels. We further hypothesize that MSK1-mediated H3 phosphorylation is critical for immediate early gene (IEG) expression, Ras-driven transformation and associates with regulatory regions upon gene transcription.