基于亲和纯化-定量质谱技术研究HER2阳性乳腺癌细胞耐药性生物标记物

Trastuzumab is widely used in the first-line treatment of epidermal growth factor receptor 2 (HER2) positive breast cancers as a monoclonal antibody, which could significantly improve the survival rate of patients. However, the trastuzumab resistance developed in patients is the leading cause of treatment failure. At present, many resistance mechanisms have been discovered, most of which are associated with abnormal activation of downstream signaling pathway of HER2 signaling complex. But there are still no methods to differentiate accurately the different resistance mechanisms produced in patients, which requires urgently the development of more effective biomarkers for the clinical diagnosis and treatment of breast cancers. Affinity purification-quantitative mass spectrometry involves isolating protein complexes directly from cell lysates through affinity interaction between the antibody and the epitope tag in bait protein to eliminate the background proteins for the enrichment of targeted protein complex, and then identifying and quantifying sample by using mass spectrometry. This technique has the properties of simple sample preparation, high sensitivity, high throughput, wide linear range, high reproducibility, therefore, greatly promote the research of protein complexes in recent years. In this project, an approach based on affinity purification-quantitative mass spectrometry for the determination of protein complex in cancer cells will be established to analyze quantitatively the differential assembly of HER2 signaling complex among breast cancer cell lines with different sensitivities to drug. The potential biomarkers related to drug resistance would be discovered. This project would not only be useful for the prediction of breast cancer response to drug, but also promote the further investigation of drug resistance mechanisms. Therefore, it is of great academic significance and clinical application values.

曲妥珠单抗广泛应用于表皮生长因子受体2（HER2）阳性乳腺癌的一线治疗，能够显著提升患者存活率，但耐药性的产生是治疗失败的主要原因。目前已发现多种耐药机制，多与HER2信号复合体下游信号途径异常激活有关，但缺乏对不同耐药机制准确鉴别的方法，亟待开发更有效的生物标记物作为乳腺癌临床诊治指标。亲和纯化-定量质谱技术通过抗体对诱饵蛋白表位标签的亲和作用，从细胞裂解液中直接纯化蛋白复合体，去除背景蛋白实现富集，并进行质谱鉴定和定量分析。该技术样品准备简单、灵敏度高、高通量、线形范围宽、重复性好，近年来极大促进了蛋白复合体研究。本项目拟建立对癌细胞中蛋白复合体进行分析的亲和纯化-定量质谱技术，定量分析对药物有不同敏感性乳腺癌细胞系中HER2信号复合体的差异性组装，发现与药物敏感性相关潜在生物标记物。该研究成果将不仅有利于乳腺癌药物反应预测，还能促进耐药机制的深入研究，具有重要学术意义和临床应用价值。

乳腺癌是世界范围内常见的恶性肿瘤，是造成女性癌症死亡的首要原因，20-30%乳腺癌患者HER2阳性。原发或继发性耐药性普遍存在，是造成治疗失败的主要原因。目前研究主要集中在关键信号因子表达水平的测定，但是难以准确反映蛋白质功能的缺陷，而蛋白相互作用是蛋白功能的直接反应。该项目从蛋白相互作用层面研究乳腺癌耐药性标志物。主要完成工作如下：（1）建立了对细胞内ERBB复合体定量分析的亲和纯化-定量质谱技术体系，该方法具高灵敏度，高通量，定量范围宽等优点；（2）应用该方法定量分析20株癌细胞系中的ERBB复合体的动态组装，发现ERBB复合体的组装具有细胞特异性，和ERBB受体相关，组成成分之间呈功能模块化关联；（3）把癌细胞的ERBB复合体和359种靶向药物的药物敏感性进行相关性分析，发现ERBB受体的相互作用强度，ERBB占比和药物敏感性相关；（4）应用数据库中临床数据，在基因表达水平验证了EGFR, ERBB3和HER2阳性乳腺癌病人的生存率相关。同时功能模块中基因组合EGFR、LRRK1和HER2阳性乳腺癌病人的生存率关联性更大，ERBB3、PIK3CA、PIK3CB和ARHGEF5组合不仅和HER2阳性病人生存率相关，而且和Lumina A型病人的生存率相关；（5）应用所建立方法，定量分析了在7种配体刺激下的ERBB复合体的差异性组装，发现BTC, ERBB4会造成HER2阳性乳腺癌细胞对靶向ERBB2药物的耐药性；（6）结合密度梯度离心，定量分析了ERBB复合体在在信号传导中的时间空间动态变化；（7）结合甲醛交联，定量分析了参与ERBB复合体在信号过程中的激酶，主要涉及弱/瞬时相互作用，并经行了生物验证。（8）对质谱采集到的空间数据进行处理过程中，通过分析和优化数据滤波算法的种类及其在管道中的位置，提出了一种改进的空间分割管道。本项目所建立的信号复合体亲和纯化-质谱定量方法可应用于其他目的研究及生物标志物的验证。耐药性潜在标志物的发现，对药物研发和乳腺癌治疗方案的改进具有重要意义。本项目执行期间与本基金项目相关的有3篇在投、审稿、修稿SCI文章，另外2篇在整理文章中，已培养硕士3名。