Duvall-Noelle N, Karwandyar A, Richmond A, Raman D
Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN, USA.
Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN, USA.
Oncogene. 2016 Mar 3;35(9):1122-33. doi: 10.1038/onc.2015.166. Epub 2015 May 18.
Nuclear LASP-1 (LIM and SH3 protein-1) has a direct correlation with overall survival of breast cancer patients. In this study, immunohistochemical analysis of a human breast TMA showed that LASP-1 is absent in normal human breast epithelium but the expression increases with malignancy and is highly nuclear in aggressive breast cancer. We investigated whether the chemokines and growth factors present in the tumor microenvironment could trigger nuclear translocation of LASP-1.Treatment of human breast cancer cells with CXCL12, EGF and HRG, and HMEC-CXCR2 cells with CXCL8 facilitated nuclear shuttling of LASP-1. Data from the biochemical analysis of the nuclear and cytosolic fractions further confirmed the nuclear translocation of LASP-1 upon chemokine and growth factor treatment. CXCL12-dependent nuclear import of LASP-1 could be blocked by CXCR4 antagonist, AMD-3100. Knock down of LASP-1 resulted in alterations in gene expression leading to an increased level of cell-junction and extracellular matrix proteins and an altered cytokine secretory profile. Three-dimensional cultures of human breast cancer cells on Matrigel revealed an altered colony growth, morphology and arborization pattern in LASP-1 knockdown cells. Functional analysis of the LASP-1 knockdown cells revealed increased adhesion to collagen IV and decreased invasion through the Matrigel. Proteomic analysis of immunoprecipitates of LASP-1 and subsequent validation approaches revealed that LASP-1 associated with the epigenetic machinery especially UHRF1, DNMT1, G9a and the transcription factor Snail1. Interestingly, LASP-1 associated with UHRF1, G9a, Snail1 and di- and tri-methylated histoneH3 in a CXCL12-dependent manner based on immunoprecipitation and proximity ligation assays. LASP-1 also directly bound to Snail1 which may stabilize Snail1. Thus, nuclear LASP-1 appears to functionally serve as a hub for the epigenetic machinery.
细胞核内的LASP-1（LIM和SH3结构域蛋白-1）与乳腺癌患者的总生存期直接相关。在本研究中，对人乳腺组织微阵列进行免疫组化分析显示，LASP-1在正常乳腺上皮中不存在，但随着恶性程度增加其表达升高，在侵袭性乳腺癌中高度定位于细胞核。我们研究了肿瘤微环境中存在的趋化因子和生长因子是否能触发LASP-1的核转位。用CXCL12、表皮生长因子（EGF）和肝素结合表皮生长因子（HRG）处理人乳腺癌细胞，用CXCL8处理人微血管内皮细胞系-CXCR2细胞，均促进了LASP-1的核穿梭。细胞核和细胞质组分的生化分析数据进一步证实了趋化因子和生长因子处理后LASP-1发生了核转位。CXCR4拮抗剂AMD-3100可阻断CXCL12依赖的LASP-1核输入。敲低LASP-1导致基因表达改变，导致细胞连接蛋白和细胞外基质蛋白水平升高，细胞因子分泌谱改变。在基质胶上对人乳腺癌细胞进行三维培养，结果显示LASP-1敲低细胞的集落生长、形态和分支模式发生改变。对LASP-1敲低细胞的功能分析显示，其对IV型胶原的黏附增加，通过基质胶的侵袭减少。对LASP-1免疫沉淀产物的蛋白质组学分析及后续验证方法表明，LASP-1与表观遗传机制相关蛋白特别是泛素样含PHD和指环结构域蛋白1（UHRF1）、DNA甲基转移酶1（DNMT1）、组蛋白甲基转移酶G9a和转录因子Snail1相互作用。有趣的是，基于免疫沉淀和邻近连接分析，LASP-1以CXCL12依赖的方式与UHRF1、G9a、Snail1以及二甲基化和三甲基化的组蛋白H3相互作用。LASP-1还直接与Snail1结合，这可能使Snail1稳定。因此，细胞核内的LASP-1似乎在功能上作为表观遗传机制的一个枢纽。
