Epigenetic and post-translational regulation of the metastasis suppressor BRMS1

描述（由申请人提供）：吸烟和促炎细胞因子与肺癌的发生、进展和转移直接相关。我们已经证明，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BRMS1，而BRMS1可以抑制肺癌的转移。本提案的目的是在肺癌的表观遗传和翻译后水平上表征和更好地理解BRMS1的调控。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第一大癌症杀手，了解BRMS1如何通过转录和翻译后修饰进行调节是很重要的。本提案Aim I中描述的实验将研究转录因子NF-kB如何调节BRMS1启动子的甲基化和转录沉默。具体来说，NF-kB的转录活性亚基RelA/65与BRMS1 DNA结合，促进DNA和组蛋白甲基转移酶向染色质募集，从而转录沉默BRMS1。目的是确定RelA/p65调控BRMS1转录的机制，并将这些观察结果扩展到人类肺癌标本中。除了BRMS1的转录调控外，TNF、尼古丁和NNK还增强酪蛋白激酶II介导的BRMS1蛋白翻译后修饰，包括将BRMS1靶向到蛋白酶体的磷酸化和泛素化。本提案第二部分的实验将确定BRMS1中特定的酪蛋白激酶II磷酸化残基，以及这些位点的突变对BRMS1核输出、随后的泛素化和蛋白酶体介导的降解有何影响。其他实验将使用质谱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BRMS1蛋白表达的影响。Aim III中提出的实验使用可诱导的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BRMS1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BRMS1在染色质水平上通过翻译后修饰独立调控的机制。我们观察到BRMS1受这两种机制的调控，并且其抑制可以独立于其中一种机制的选择抑制而发生，这具有重要的转化潜力，并直接说明了假定的临床癌症相关性和该项目的意义。公共卫生相关性：肺癌是美国的头号癌症杀手，大多数人患有晚期或转移性疾病。我们的研究重点是了解一种名为BRMS1的特定蛋白质如何帮助调节肺癌转移。这项研究的总体目标是提高我们对肺癌如何扩散的理解，这最终是为这种致命疾病设计新的靶向治疗方法所必需的。

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.