Epigenetic and post-translational regulation of the metastasis suppressor BRMS1

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DESCRIPTION (provided by applicant): Cigarette smoke and pro-inflammatory cytokines are directly linked to the initiation, progression, and metastasis of lung cancer. We have shown that primary cigarette smoke, nicotine, and the tobacco carcinogen NNK as well as the cytokine TNF suppress the metastasis suppressor gene BRMS1 which we have shown to inhibit metastasis in lung cancer. The purpose of this proposal is to characterize and better understand the regulation of BRMS1 at an epigenetic and post-translational level in lung cancer. Given that the majority of lung cancer patients present with metastatic disease and that it remains the number one cancer killer in the world, understanding how BRMS1 is regulated both transcriptionally and through post-translational modifications is important. Experiments described in Aim I of this proposal will investigate how the transcription factor NF-kB regulates methylation and transcriptional silencing of the BRMS1 promoter. Specifically, RelA/65, the transcriptionally active subunit of NF-kB, binds to BRMS1 DNA and promotes DNA and histone methyltransferases recruitment to chromatin which transcriptionally silences BRMS1. The goal of this aim is determine the mechanisms through which RelA/p65 regulates BRMS1 transcription and to extend these observations using human lung cancer specimens. In addition to transcriptional regulation of BRMS1, TNF, nicotine and NNK enhance casein kinase II mediated post-translational modifications of BRMS1 protein, including phosphorylation and ubiquitination which target BRMS1 to the proteasome. Experiments in Aim II of the proposal will identify specific casein kinase II phosphorylation residues in BRMS1 and what effect mutation(s) of these sites have on BRMS1 nuclear exportation and subsequent ubiquitination and proteasome- mediated degradation. Other experiments will map specific lysine residues that are ubiquitinated using mass spectroscopy and what effect mutations of these functionally relevant lysine residue(s) have on BRMS1 protein expression. Experiments proposed in Aim III use an inducible Cre/lox-P system lung cancer metastasis mouse model to explore the specific and separate contribution(s) of transcriptional and casein-kinase II mediated post- translational modifications of BRMS1 to the development of metastases. Collectively, experiments outlined in this proposal will elucidate the mechanisms through which BRMS1 is independently regulated at the chromatin level and through post-translational modifications. Our observations that BRMS1 is regulated by these two mechanisms and that its suppression can occur independent of selected inhibition of one mechanism has significant translational potential and speaks directly to the putative clinical cancer relevance and the significance of the project. PUBLIC HEALTH RELEVANCE: Lung cancer is the number one cancer killer in the United States and most people present with advanced stage or metastatic disease. Our research is focused on understanding how a specific protein called BRMS1 helps regulate lung cancer metastases. The overall goal of this research is to improve our understanding of how lung cancer spreads, which ultimately is necessary in order to design novel targeted therapies for this deadly disease.