乳腺癌耐药新靶点核仁磷酸蛋白的分子机制及其靶向逆转剂的作用研究

Chemotherapy is one of important means in the comprehensive treatment of breast cancer, which is an effective therapeutic method for breast cancer throughout the entire course of treatment. However, with continuous application of chemotherapeutic drugs, the occurrence of multidrug resistance (MDR) greatly hindered the clinical efficacy of chemotherapeutics. Therefore, screening molecular markers which could predict drug resistance in breast cancer, clarifying their roles in drug resistance, and screening reversal agents that would effectively reverse the drug resistance according to the function of resistant targets, these would be becoming the focus issues. In previous study, we successfully established the methotrexate-resistant breast cancer cell model, and found that nucleophosmin (NPM) could be a potential novel target of breast cancer resistance, but its role pathway and molecular mechanism are not clear, and its corresponding reversal agent is also difficult to find. This study aimed to explore the molecular mechanism of NPM inducing breast cancer MDR from cellular level, nude mice model and clinical tissue samples using various analysis techniques, screen effective histone deacetylase inhibitors as reversal agent of breast cancer resistance based on the feature that NPM is a chaperone of histone, and investigate their reversal effect of breast cancer resistance both in vitro and in vivo. These will provide new theoretical foundation and scientific basis for solving the problem of breast cancer MDR in clinical.

化学治疗（化疗）是乳腺癌综合治疗的重要手段之一，它是贯穿乳腺癌病程治疗的有效方法。然而，化疗药物不断应用出现的多药耐药现象，却极大地限制了其临床疗效。因此，筛选能够预测乳腺癌耐药的分子标志物，阐明其在耐药产生过程中的作用，并根据作用靶点筛选有效的耐药逆转剂，研究其作用效果是目前研究的热点问题。在我们的前期研究中，成功建立了甲氨蝶呤耐药的乳腺癌细胞模型，并发现核仁磷酸蛋白（Nucleophosmin, NPM）是潜在的乳腺癌耐药新靶标，但其作用途径和分子机制并未明确，相应的靶向逆转剂也难以发现。本研究通过多种分析手段从细胞水平、裸鼠模型及组织样本三方面探讨NPM诱导乳腺癌多药耐药的分子机制，并针对NPM是组蛋白分子伴侣这一功能，筛选有效的组蛋白去乙酰化酶抑制剂，以期作为乳腺癌耐药的逆转剂，从体内、体外研究其逆转乳腺癌多药耐药的作用，为临床解决乳腺癌多药耐药问题提供了新的理论基础和科学依据。

在青年科学基金的资助下，本课题组基于前期研究鉴定的潜在乳腺癌耐药分子标志物—核仁磷酸蛋白（Nucleophosmin, NPM），探究了其在乳腺癌多药耐药产生过程中的作用机制。同时，以NPM为作用靶点，筛选了具有逆转乳腺癌耐药作用的抗肿瘤化合物—新型组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitor, HDACi），以期作为乳腺癌多药耐药的逆转剂，通过分子生物学方法和裸鼠移植瘤模型从体外、体内揭示其逆转乳腺癌多药耐药的作用机制。结果显示，抑制乳腺癌耐药细胞中NPM的表达，可增强该细胞对化疗药物的敏感性并抑制多药耐药相关因子的异常表达，降低细胞的耐药性。此外，抑制NPM可增强PTEN的表达，使磷脂酰肌醇3-激酶/蛋白激酶B（phosphatidylinositol 3 kinase/serine-threonine kinase, PI3K/Akt）信号通路失活，激活下游线粒体凋亡途径，从而阻滞乳腺癌多药耐药的产生。另一方面，Akt抑制剂MK-2206可减弱NPM的表达，明确NPM在乳腺癌耐药形成过程中的重要作用，阐明它与PI3K/Akt信号通路及 caspase家族凋亡途径之间的相互作用关系，为我们解决临床患者的耐药问题提供了理论基础。随后针对NPM的功能，筛选出可显著性抑制乳腺癌细胞生长且具有较强逆转活性的新型HDACi，体内外实验阐明HDACi可降低细胞中耐药分子靶标NPM的表达，使PI3K/Akt通路失活，从而促进线粒体凋亡途径活化，最终诱导耐药细胞凋亡。结果表明新型HDACi 为逆转乳腺癌多药耐药提供了有效的候选化合物，并对逆转药物的开发具有重要的意义。