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Definition of the Ras/ERK signal induced chromatin modification at target gene promoters [RNA-seq & ChIP-seq]

ABSTRACT: Ras plays roles across the spectrum of proliferation, invasion and metastasis. The activity of Ras genes, usually K-Ras, is altered by mutations in around 20% of human cancers, and activation of the Ras-ERK pathway is a critical event in melanomas, colorectal and renal cancers but the degree to which Ras- or Raf-driven transformation is dependent on the activity of the SRF network remains largely obscure. Oncogenic active Ras is known to mediate the cellular response to multiple growth factors leading to the activation of immediate-early genes (IE). A key player in IE gene response is the SRF transcriptional network that we identified being crucial to mediate allegedly the entire transcriptional response downstream of Ras/ERK. The Serum response factor (SRF) is a transcription factor with low intrinsic transcriptional activity and requires the recruitment of one of two families of co-activators: the MRTFs (myocardin-related transcription factors) and the TCFs (ternary complex factors, Elk-1, SAP-1, Net). In particular the TCF, member of the wide family of ETS transcription factors, are specifically activated by MAPK signalling downstream of Ras. We characterised the transcriptional program downstream of Ras/ERK signalling using short TPA stimulations (12-O-tetradecanoyl phorbol-13-acetate, compunt known to induced Ras/ERK activation) by RNA-seq. The investigation of the transcriptional response was performed using Mouse Embryonic Fibroblasts (MEFs) derived from different animals: (i) wild type, (ii) TKO (MEFs derived from mice lacking all three TCFs), (iiI) TKO recon pMY Empty (TKO reconstituted with empty vectors by retrovirus infection), (iv) TKO recon Elk-1 wt (TKO reconstituted with retrovirus vectors expressing Elk-1 wild type by retrovirus infection), (v) TKO recon Elk-1 FW (TKO reconstituted with retrovirus vectors expressing Elk-1 mutant lacking the FW residues in its activation domain), (vi) TKO recon Elk-1 nonA (TKO reconstituted with retrovirus vectors expressing Elk-1 mutant where all phosphor-acceptor residues in its activation domain have been mutagenized to alanines). In order to map SRF and Elk-1 binding sites we performed ChIP-seq of SRF in wild type MEFs and TKO and ChIP-seq of Elk-1 in TKO reconstituted cell lines.

ORGANISM(S): Mus musculus

PROVIDER: GSE75667 | GEO | 2016/11/28

SECONDARY ACCESSION(S): PRJNA305011

REPOSITORIES: GEO

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Project description:About 50% of human malignancies exhibit unregulated signalling through the Ras-ERK1/2 (ERK) pathway, as a consequence of activating mutations in members of Ras and Raf families. However, the quest for alternative Ras-ERK pathway-directed therapies is desirable. Upon phosphorylation ERK dimerize. We had previously demonstrated that dimerization is essential for ERK extranuclear but not nuclear signaling. Furthermore, by molecular biology approaches, we showed that specifically inhibiting ERK extranuclear component, by impeding ERK dimerization, is sufficient for curtailing tumor progression. Here, we have identified a small molecule inhibitor for ERK dimerization in vitro and in vivo that, without affecting ERK phosphorylation, prevents tumorigenesis driven by Ras-ERK pathway oncogenes, both in cellular and animal models. Importantly, this compound is unaffected by resistance-acquisition processes that hamper “classical” Ras-ERK pathway inhibitors. Thus, ERK dimerization inhibitors provide the proof of principle for two novel concepts in cancer therapy: 1) The blockade of sublocalization-specific sub-signals, rather than total signals, as a means of effectively counteracting oncogenic Ras-ERK signaling. 2) Targeting regulatory protein-protein interactions such as dimerization, rather than catalytic activities, within a signaling route, as an approach for producing effective anti-tumoral agents. Strategies aimed at preventing aberrant flux through this route remain an attractive option for therapeutic intervention in cancer. In this respect, drugs inhibiting the kinase activities of BRaf and MEK have yielded promising results. A375p cells treated with10 μM of either DEL22379, SCH772984 or DMSO as a control for two hours. mRNA from A375p cells was extrated using RNeasy mini kit (Qiagen, Germany) according to the manufacturer's instructions. Cells were previously treated with10 μM of either DEL22379, SCH772984 or DMSO as a control for two hours.

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Definition of the Ras/ERK signal induced chromatin modification at target gene promoters [RNA-seq & ChIP-seq]

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