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

 描述（由申请人提供）：拟议研究的目标是研究CXCL 12驱动的核LASP 1在乳腺癌表观遗传事件调节中的作用。LASP-1介导几种乳腺癌细胞系中的细胞迁移、增殖和存活。LASP-1的沉默抑制增殖和迁移45%。此前，LASP-1被证明与CXC趋化因子受体CXCR 1、CXCR 2、CXCR 3和CXCR 4直接相互作用，这些受体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CXCR 2介导的细胞迁移。乳腺癌细胞中的表观遗传学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的方法，发现UHRF 1是一种新的LASP-1相关蛋白。随后，DNMT 1、G9 a和Snail 1也被观察到与LASP-1相关。特别地，发现Snail 1直接结合LASP-1。这种直接相互作用的生物学意义尚不清楚。首先，我建议映射LASP-1和蜗牛1之间的相互作用位点的突变和生化方法。我进一步建议研究LASP 1-Snail 1复合物调节E-钙粘蛋白启动子的能力。此外，由于Snail 1直接结合 赖氨酸去甲基酶1（LSD 1）的表达，通过生物化学研究分析LSD 1是否与LASP 1-Snail 1复合物结合。在功能上，我建议进行染色质免疫沉淀（ChIP）分析LASP-1，LSD 1，和K4/9组蛋白H3的甲基化，看看LASP-1/LSD 1共定位到区域与去甲基化组蛋白CXCL 12刺激后。或者，将重组LASP-1与纯化的LSD-1混合，观察LASP-1是否增强标记的组蛋白H3或核小体底物的体外去甲基化活性。此外，将在牙本质盘测定中测试非沉默的和LASP 1敲低的基底样乳腺癌细胞促进骨吸收的能力。LASP 1-Snail 1轴可能成为小分子抑制剂的新药物靶点，以延长尤其是三阴性乳腺癌患者的生存率。新的抑制剂可能在对目前化疗药物耐药的乳腺癌病例中更有用。

 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.