中国科学院昆明动物研究所机构知识库

乳腺癌，是全球女性发病率第一的乳腺上皮来源恶性肿瘤，严重危害女性身心健康。近年我国乳腺癌死亡率和发病率均不断攀升，且呈年轻化趋势。乳腺癌是一种高度异质性疾病，根据分子分型可将其分为Luminal型（雌激素受体ER和孕激素受体PR均为阳性）、HER2（人表皮生长因子受体-2）过表达型及三阴性乳腺癌（TNBC:PR,ERα,HER2表达均为阴性）。不同亚型治疗策略不同，因此找到其有效的靶向治疗药物，提高患者生存率，是所有乳腺癌研究者的共同目标。蛋白质翻译后的泛素化修饰可被去泛素化酶（DUBs: deubiquitinating enzymes）逆转。通常，蛋白被泛素化修饰后降解，而去泛素化酶（DUBs）则使蛋白去泛素化增强其稳定性，在泛素蛋白酶体调控中发挥重要作用。 大量研究发现，去泛素化酶与肿瘤发生紧密联系，且能被开发为有效的肿瘤靶向治疗药物。丝裂原活化蛋白激酶磷酸酶1（mitogen-activated protein kinase phosphatase1，MKP-1）是一种络氨酸磷酸酶，主要通过直接调控丝裂原活化蛋白激酶系统（mitogen-activated protein kinase system，MAPKs）发挥功能, 具双特异性磷酸酶活性。MAPK体系与细胞生长、分化、增殖、凋亡及炎症等反应密切相关。有文献报道，一方面，MKP-1在乳腺癌病程中一直处于高表达状态，我课题组前期研究也证明，MKP-1受KLF5 调控在乳腺癌中高表达，促进癌细胞的增殖和存活；另一方面，MKP-1过表达是多种癌症在化疗过程中产生耐药的重要原因。虽然MKP-1通过泛素-蛋白酶体途径降解已有报道－SCFSkp2能作为 MKP-1的E3泛素连接酶促进其降解，但关于使之稳定的的去泛素化调控目前还未知。于是，我们提出科学猜想：MKP-1的去泛素化酶通过使MKP-1去泛素化稳定其表达，从而促进乳腺癌细胞的增殖、生存与化疗耐药，具有癌蛋白功能。因此，找寻MKP-1的DUBs，可使之成为一个有效的抗癌药物靶点。首先，我们对去泛素化酶文库约90个去泛素化酶siRNA进行筛选，查找MKP-1的潜在去泛素化酶。经过多轮多遍筛查和验证，最后确定STAMBPL1是MKP-1的候选去泛素化酶，同时发现USP31也是MKP-1的候选DUB之一。为进一步确定STAMBPL1可增加MKP-1的蛋白稳定性，我们在多个细胞系包括SUM149pt、MDA-MB-231、HCC1937和MCF10A中转染靶向STAMBPL1不同位置的siRNA。敲低STAMBPL1后，内源MKP-1蛋白水平在不同细胞系中均显示下调，且MKP-1蛋白半衰期被明显缩短。我们还构建了STAMBBPL1去泛素化酶活突变体STAMBPL1E292A，将二者同时在细胞中过表达，STAMBPL1能够明显提高MKP-1蛋白水平，而突变体则无法实现这一功能。接下来，在体内外泛素化实验中证明了敲低STAMBPL1后，内源MKP-1的泛素化水平明显上调；过表达STAMBPL1，外源MKP-1的泛素化水平下降，而STAMBPL1E292A没有此生理功能。免疫共沉淀实验表明，在内源或外源情况下，STAMBPL1与MKP-1之间均存在相互作用。这一结果为更进一步探索STAMBPL1稳定MKP-1的作用机制奠定了基础。综上所述，这些结果表明STAMBPL1是MKP-1的去泛素化酶，同时为下一步研究STAMBPL1通过MKP-1在乳腺癌中发挥的功能奠定了良好的基础并提供了可靠的依据，并使STAMBPL1可能成为一个新的用于乳腺癌诊断预后和治疗的分子靶标。

Breast cancer is the most common lethal disease of women. According to China cancer registry annual report, breast cancer incidence rate gradually increased within female over the age of 25. 54 to 50 years old group breast cancer rate reached the peak and the mortality rate of breast cancer account for about 14% in all kinds of woman cancer in China. Mitogen activated protein kinase phosphatase-1 (MKP-1) has severed as an important protein in mediating breast cancer oncogenesis and chemoresistance to cancer chemotherapies. So, it is important to research the stabilization mechanism of MKP-1. Here we show that, in breast cancer cells, MKP-1 is stabilized by the deubiquitinase (DUB) STAMBPL1. Previous studies indicated that STAMBPL1 is a Critical Regulator of Human T-Cell Leukemia Virus Type 1 Tax nuclear export and NF-KB activation. With a genome-wide siRNA library screen of DUBs and two rounds of small scale screening including twenty-two DUBs in MDA-MB-231, MCF10A, BT474, SUM149pt and T47D cell lines, we identified STAMBPL1 as a candidate DUB of MKP-1. We found that when knockdown STAMBPL1, the protein level of MKP-1 was downregulated in different breast cancer cell lines and the half-life of MKP-1 was shrunken. Moreover, we also found that overexpressed STAMBPL1 apparently up-regulated the protein level of MKP-1 but not STAMBPL1 E292A mutation. Furthermore, we proved that knockdown STAMBPL1 obviously increased the endogenous ubiquitin-level of MKP-1, and overexpressed STAMBPL1 decreased the exogenous ubiquitin-level of MKP-1 but not STAMBPL1 E292A mutation. Finally, we, using Co-immunoprecipitation assays, proved that STAMBPL1 and MKP-1 were interacted with each other in vitro or in vivo. These results laid foundation for the further study of the stabilizing mechanism of STAMBPL1 to MKP-1.Taking all these data together, we concluded that STAMBPL1 was the deubiquitinase of MKP-1. Meanwhile, our studies laid foundation for the further exploration of the function of STAMBPL1 in breast cancer and may provide a new potential therapeutic target -STAMBPL1- for breast cancer or other cancers.