Normal cellular behaviour in multicellular organisms is achieved by tight control of signaling pathway networks. The mitogen-activated protein kinase (MAPK) signaling cascade is one of these signaling networks, that when deregulated can lead to cellular transformation. Activation of the RAS-RAF-MEK-MAPK (ERK) signal transduction pathway or the SAPK2/p38 pathway results in the activation of mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2). Subsequently, MSKs go on to phosphorylate histone H3 at Ser10 and Ser28.Here, we demonstrate that the activities of ERK and MSK1, but not p38, are elevated in Hras-transformed cells (Ciras-3) relative to these activities in the parental 10T1⁄2 cells. Analyses of the subcellular distribution of MSK1 showed that the H3 kinase was similarly distributed in Ciras-3 and 10T1/2 cells, with most MSK1 being present in the nucleus. In contrast to many other chromatin modifying enzymes, MSK1 was loosely bound in the nucleus and was not a component of the nuclear matrix. Our results provide evidence that oncogene-mediated activation of the RAS-MAPK signal transduction pathway elevates the activity of MSK1, resulting in the increased steady-state levels of phosphorylated H3, which may contribute to the chromatin decondensation and aberrant gene expression observed in oncogene-transformed cells. Furthermore, upon activation of the ERK and p38 MAPK pathways, the MSK1/2- mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2, Ciras-3 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 (the ATPase subunit of the SWI/SNF remodeler) is recruited to the promoter of target genes by transcription factors such as ELK-1 or NFκB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of immediate-early genes enabling the binding of transcription factors like JUN and the onset of transcription. Since RAS-MAPK activated MSKs mediate H3 phosphorylation that is required for expression of various immediate-early gene products involved in cellular transformation, inhibition of MSK activity may be a therapeutic target that could be exploited in cancers with upregulated RAS-MAPK signaling.

The RAS-mitogen-activated protein kinase (MAPK) pathway is an essential signaling mechanism that regulates cellular processes and culminates in the activation of specific gene expression programs. Alterations in the RAS-MAPK signaling cascade can modify epigenetic programs and confer advantages in cell growth and transformation. In fact, deregulation of the cascade is a key event in tumour development with 30% of human cancers harbouring RAS mutations. In breast and pancreatic epithelial cancers, characterization of an aberrant RAS-MAPK pathway has focused on upstream mediators such as receptors and oncogenic RAS molecules but the impact of downstream targets remain poorly defined. Stimulation of the RAS-RAF-MEK-MAPK pathway leads to 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. We postulate that deregulation of the RAS-MAPK pathway produced by constitutive activation and/ or over-expression of upstream components or mitogen stimulation 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 is associated with regulatory regions upon gene transcription. In mouse fibroblasts, 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. We then examined if activation of RAS-MAPK signaling in breast cancer cells elicits similar molecular events. We demonstrate that the RAS-MAPK pathway is induced and enhances the association of MSK1 and H3 phosphorylation on the IEG Trefoil Factor 1 resulting in transcriptional activation. We further observed that mutated K-RAS expression did not correlate with genomic instability or altered signaling in pancreatic cancer cell lines while overexpressed HER2 and EGFR breast cancer cell lines generally exhibit upregulated ERK1/2 and H3 phosphorylation levels. Taken together, our studies contribute to the further understanding of MSK-mediated transcriptional activation in response to RAS-MAPK signaling in oncogene-transformed and cancer cell lines. Inhibition of MSK activity may be an unexplored avenue for combination cancer therapy with abnormal RAS-MAPK signaling pathways.

Normal cellular behaviour in multicellular organisms is achieved by tight control of signaling pathway networks. The mitogen-activated protein kinase (MAPK) signaling cascade is one of these signaling networks, that when deregulated can lead to cellular transformation. Activation of the RAS-RAF-MEK-MAPK (ERK) signal transduction pathway or the SAPK2/p38 pathway results in the activation of mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2). Subsequently, MSKs go on to phosphorylate histone H3 at Ser10 and Ser28.Here, we demonstrate that the activities of ERK and MSK1, but not p38, are elevated in Hras-transformed cells (Ciras-3) relative to these activities in the parental 10T1⁄2 cells. Analyses of the subcellular distribution of MSK1 showed that the H3 kinase was similarly distributed in Ciras-3 and 10T1/2 cells, with most MSK1 being present in the nucleus. In contrast to many other chromatin modifying enzymes, MSK1 was loosely bound in the nucleus and was not a component of the nuclear matrix. Our results provide evidence that oncogene-mediated activation of the RAS-MAPK signal transduction pathway elevates the activity of MSK1, resulting in the increased steady-state levels of phosphorylated H3, which may contribute to the chromatin decondensation and aberrant gene expression observed in oncogene-transformed cells. Furthermore, upon activation of the ERK and p38 MAPK pathways, the MSK1/2- mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2, Ciras-3 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 (the ATPase subunit of the SWI/SNF remodeler) is recruited to the promoter of target genes by transcription factors such as ELK-1 or NFκB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of immediate-early genes enabling the binding of transcription factors like JUN and the onset of transcription. Since RAS-MAPK activated MSKs mediate H3 phosphorylation that is required for expression of various immediate-early gene products involved in cellular transformation, inhibition of MSK activity may be a therapeutic target that could be exploited in cancers with upregulated RAS-MAPK signaling.

The RAS-mitogen-activated protein kinase (MAPK) pathway is an essential signaling mechanism that regulates cellular processes and culminates in the activation of specific gene expression programs. Alterations in the RAS-MAPK signaling cascade can modify epigenetic programs and confer advantages in cell growth and transformation. In fact, deregulation of the cascade is a key event in tumour development with 30% of human cancers harbouring RAS mutations. In breast and pancreatic epithelial cancers, characterization of an aberrant RAS-MAPK pathway has focused on upstream mediators such as receptors and oncogenic RAS molecules but the impact of downstream targets remain poorly defined. Stimulation of the RAS-RAF-MEK-MAPK pathway leads to 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. We postulate that deregulation of the RAS-MAPK pathway produced by constitutive activation and/ or over-expression of upstream components or mitogen stimulation 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 is associated with regulatory regions upon gene transcription. In mouse fibroblasts, 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. We then examined if activation of RAS-MAPK signaling in breast cancer cells elicits similar molecular events. We demonstrate that the RAS-MAPK pathway is induced and enhances the association of MSK1 and H3 phosphorylation on the IEG Trefoil Factor 1 resulting in transcriptional activation. We further observed that mutated K-RAS expression did not correlate with genomic instability or altered signaling in pancreatic cancer cell lines while overexpressed HER2 and EGFR breast cancer cell lines generally exhibit upregulated ERK1/2 and H3 phosphorylation levels. Taken together, our studies contribute to the further understanding of MSK-mediated transcriptional activation in response to RAS-MAPK signaling in oncogene-transformed and cancer cell lines. Inhibition of MSK activity may be an unexplored avenue for combination cancer therapy with abnormal RAS-MAPK signaling pathways.

Nous étudions une nouvelles familles d'inhibiteurs de kinase: les benzopyridoindole. Ces molécules ont des effets antiprolifératifs sur des lignées cancéreuses et représentent les têtes de série de possibles agents anti-cancéreux. We study on a new family of kinase inhibitors: benzopyridoindole. These molecules have antiproliferative effects on cancer cell lines and represent the lead of potential anti-cancer products. / Benzo[e]pyridoindoles are novel potent inhibitors of aurora kinases. We performed a SAR study to improve their activity and water solubility. Amino-benzo[e]pyridoindolones were found to be potent hydrosoluble anti-proliferative molecules. They induced a massive arrest in mitosis, prevented histone H3 phosphorylation as well as disorganizing the mitotic spindles. Upon a delay, cells underwent binucleated and finally died. Taking into account their interesting preclinal characteristics, their efficiency towards xenografts in nude mice and their apparent safety in animals, these molecules are promising new anti-cancer drugs. They probably target a metabolic signaling pathway, besides aurora B inhibition. In addition to their possible applications, these inhibitors are tools for cell biology studies. C4, a low ATP affinity inhibitor of aurora B kinase, revealed that the basal activity of the kinase is required for histone H3 phosphorylation in prophase and for chromosome compaction in anaphase. These waves of activation/deactivation of the kinase, during mitosis, corresponded to different conformations of the passenger chromosomal complex.