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、发展和转移直接相关。我们已经证明，一次吸烟、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，我们已经证明BRMS1可以抑制肺癌的转移。这项建议的目的是在表观遗传和翻译后水平上表征和更好地理解BRMS1在肺癌中的调控。鉴于大多数肺癌患者都患有转移性疾病，而且它仍然是世界上头号癌症杀手，了解BRMS1是如何在转录和翻译后修饰中调节的是很重要的。本提案目标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。具体地说，核因子-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重新聚集到染色质，从而转录沉默BRMS1。这一目标的目的是确定RelA/p65调节BRMS1转录的机制，并利用人类肺癌标本扩展这些观察。除了BRMS1的转录调控外，肿瘤坏死因子、尼古丁和NNK还可以促进酪蛋白激酶II介导的BRMS1蛋白的翻译后修饰，包括针对BRMS1的蛋白酶体的磷酸化和泛素化。该提案目标II中的实验将确定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(S)对BRMS1核输出以及随后的泛素化和蛋白酶体介导的降解有什么影响。其他实验将使用质谱学绘制泛素化的特定赖氨酸残基的图谱，以及这些功能相关的赖氨酸残基(S)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翻译后修饰在转移发生中的特异性和单独贡献(S)。总的来说，本提案中概述的实验将阐明BRMS1在染色质水平上和通过翻译后修饰独立调节的机制。我们的观察结果表明，BRMS1受这两种机制的调控，其抑制可以独立于一种机制的选择抑制而发生，具有显著的翻译潜力，并直接说明了该项目与临床癌症的相关性和意义。

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.