Epigenetic and post-translational regulation of the metastasis suppressor BRMS1

描述（由申请人提供）：香烟烟雾和促炎细胞因子与肺癌的发生、进展和转移直接相关。我们已经表明，主要的香烟烟雾，尼古丁，烟草致癌物NNK以及细胞因子TNF抑制转移抑制基因BRMS 1，我们已经表明，抑制肺癌的转移。该提案的目的是表征和更好地理解BRMS 1在肺癌中的表观遗传和翻译后水平的调节。鉴于大多数肺癌患者存在转移性疾病，并且它仍然是世界上头号癌症杀手，因此了解BRMS 1如何通过转录和翻译后修饰进行调控是非常重要的。本提案的目的I中描述的实验将研究转录因子NF-κ B如何调节BRMS 1启动子的甲基化和转录沉默。具体地，NF-κ B的转录活性亚基RelA/65与BRMS 1 DNA结合，并促进DNA和组蛋白甲基转移酶募集到染色质，从而使BRMS 1转录沉默。本研究的目的是确定RelA/p65调节BRMS 1转录的机制，并使用人类肺癌标本扩展这些观察结果。除了BRMS 1的转录调节外，TNF、尼古丁和NNK还增强酪蛋白激酶II介导的BRMS 1蛋白的翻译后修饰，包括磷酸化和泛素化，其将BRMS 1靶向蛋白酶体。该提案的目的II中的实验将鉴定BRMS 1中的特定酪蛋白激酶II磷酸化残基以及这些位点的突变对BRMS 1核输出和随后的泛素化和蛋白酶体介导的降解的影响。其他实验将使用质谱法绘制泛素化的特定赖氨酸残基，以及这些功能相关赖氨酸残基的突变对BRMS 1蛋白表达的影响。目的III中提出的实验使用诱导型Cre/lox-P系统肺癌转移小鼠模型来探索转录和酪蛋白激酶II介导的BRMS 1的翻译后修饰对转移发展的特异性和单独贡献。总的来说，本提案中概述的实验将阐明BRMS 1在染色质水平和翻译后修饰中独立调控的机制。我们观察到BRMS 1受这两种机制调节，并且其抑制可以独立于一种机制的选择性抑制而发生，这具有显著的翻译潜力，并直接说明了推定的临床癌症相关性和该项目的意义。

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.