An epigenetic link from CXCL12-CXCR4 axis through nuclear LASP-1 in breast cancer

 描述(申请人提供)：拟议研究的目标是调查CXCL12驱动的核LASP1在调节乳腺癌表观遗传事件中的作用。LASP-1在几种乳腺癌细胞系中介导细胞迁移、增殖和存活。沉默LASP-1可抑制45%的增殖和迁移。以往研究表明，LASP-1与CXC趋化因子受体CXCR1、CXCR2、CXCR3和CXCR4直接相互作用，CXCR3和CXCR4在促进乳腺癌进展和转移的肿瘤微环境中发挥关键作用。特别是，LASP-1增强了CXCR2介导的细胞迁移。乳腺癌细胞的表观遗传改变将其转化为侵袭性和转移性表型。本研究通过蛋白质组学研究发现，uhrf1是一种新的LASP-1结合蛋白。随后，还观察到DNMT1、G9a和Snail1与LASP-1相关。特别是，Snail1被发现直接与LASP-1结合。这种直接相互作用的生物学含义尚不清楚。首先，我建议通过突变和生化方法定位LASP-1和Snail1之间的相互作用位点。我进一步建议研究LASP1-Snail1复合体对E-钙粘素启动子的调节能力。此外，由于Snail1直接绑定 对于赖氨酸脱甲基酶1(LSD1)，将通过生化研究来分析其是否与LASP1-Snail1复合体有关。在功能上，我建议对组蛋白H3的LASP-1、LSD1和K4/9甲基化进行染色质免疫沉淀(CHIP)分析，以确定在CXCL12刺激下，LASP-1/LSD1是否共定位于带有去甲基化的组蛋白的区域。或者，将重组LASP-1与纯化的LSD-1混合，观察LASP-1是否在标记的组蛋白H3或核小体底物上增强体外去甲基化活性。此外，非沉默和LASP1基因敲除的基底样乳腺癌细胞将在牙本质盘试验中测试它们促进骨吸收的能力。LASP1-Snail1轴有望成为小分子抑制剂的一个新的药物靶点，以延长生存率，特别是对三阴性乳腺癌患者。对于对当前化疗药物具有耐药性的乳腺癌患者，新型抑制剂可能更加有用。

 DESCRIPTION (provided by applicant): The goal of the proposed research is to investigate the role of CXCL12-driven nuclear LASP1 in modulation of epigenetic events in breast cancer. LASP-1 mediates cell migration, proliferation and survival in several breast cancer cell lines. Silencing of LASP-1 inhibits proliferation and migration by 45%. Previously, LASP-1 was demonstrated to directly interact with CXC chemokine receptors, CXCR1, CXCR2, CXCR3 and CXCR4 that play a key role in the tumor microenvironment facilitating breast cancer progression and metastasis. In particular, LASP-1 augmented CXCR2-mediated cell migration. Epigenetic alterations in breast cancer cells convert them into aggressive and metastatic phenotype. In this study, through proteomics, UHRF1 was discovered as a novel LASP-1 associating protein. Subsequently, DNMT1, G9a and Snail1 were also observed to associate with LASP-1. In particular, Snail1 was found to directly bind to LASP-1. The biological implication of this direct interaction is unclear. First, I propose to map the interaction sites between LASP-1 and Snail1 by mutational and biochemical approaches. I further propose to study the LASP1-Snail1 complex for its ability to modulate the E-cadherin promoter. Additionally, as Snail1 directly binds to lysine demethyalse1 (LSD1), LSD1 will be analyzed whether it associates with LASP1-Snail1 complex by biochemical studies. Functionally, I propose to perform a chromatin immunoprecipitation (ChIP) analysis for LASP-1, LSD1, and K4/9 methylation of histone H3 to see if LASP-1/LSD1 co-localize to regions with demethylated histones upon stimulation with CXCL12. Alternatively, recombinant LASP-1 will be mixed with purified LSD-1 and see if LASP-1 enhances in vitro demethylation activity on labeled histone H3 or nucleosome substrates. Additionally, non-silenced and LASP1-knock down basal- like breast cancer cells will be tested for their ability to facilitate bone resorption in a dentine disk assay. LASP1-Snail1 axis would become a novel drug target for small molecule inhibitors with the aim of prolonging the survival rate especially in triple-negative breast cancer patients. Novel inhibitors might be even more useful in cases of breast cancer that are resistant to current chemotherapeutic agents.