【作者】 胡英斌；

【导师】 胡铁辉；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 研究背景及目的：乳腺癌是女性最常见的恶性肿瘤之一,目前其发病率在全世界范围内呈明显上升趋势。而乳腺癌是一种全身性的疾病,单纯的手术治疗不能显著提高其生存率。为了提高其治愈率,乳腺癌的综合治疗显得非常重要。近年来,科学家发现了HER-2基因在20%-30%的乳腺癌中存在明显扩增和过表达,并针对性的研究出来了赫赛汀(Herceptin)这种药物临床应用取得了很好的效果。乳腺癌其发生发展是一个多因素、多基因及多途径改变的复杂过程,其癌变机制仍不是十分明确。本实验从酪氨酸磷酸化修饰入手,研究乳腺肿瘤蛋白质酪氨酸磷酸化的表达差异。为研究乳腺癌发生,发展,治疗,检测及防治研究开辟新的思路。方法：本实验收集了20例乳腺癌恶性肿瘤和12例乳腺癌良性肿瘤的新鲜标本,采用酪氨酸磷酸化免疫富集的方法收集多肽,并用LTQ Orbitrap质谱仪进行鉴定分析,对可能有意义的差异表达酪氨酸磷酸化蛋白质采用免疫印迹技术加以验证。并在四例乳腺癌细胞株进行培养,收集培养后的乳腺癌细胞进行差异酪氨酸磷酸化蛋白研究,比较人乳腺癌和癌株在酪氨酸磷酸化蛋白质表达的差异,为乳腺癌的进一步研究提供模型。结果：本实验对人乳腺肿瘤的研究中共找到了772种酪氨酸磷酸化差异性蛋白质,其中在恶性肿瘤中有581种磷酸化差异蛋白上调,良性肿瘤中有191种酪氨酸磷酸化蛋白上调。根据P<0.05,良恶性肿瘤中鉴别出来了116种酪氨酸磷酸化差异蛋白质。另外在恶性肿瘤中根据P<0.05中的P数值由低到高排列出来了51种差异化酪氨酸磷酸化蛋白质。发现了PKCD、DCP1、AXL在人乳腺癌中表达上调。IGFIR在人乳腺纤维瘤中表达上调。并应用免疫印迹法验证(Western Blotting) PKCD、CDCP1酪氨酸磷酸化等蛋白质。我们还选择了Cal-85-1、hcc-159、du-4475、MCF-7等四株乳腺癌细胞株进行培养,收获乳腺癌细胞进行磷酸化蛋白质组学研究。发现了Cal-85-1、hcc-1599、du-4475这三株乳腺癌株中PKCD、CDCP1表达上调,而MCF-7乳腺癌株中PKCD、CDCP1未见明显表达。并通过免疫印迹法(Western Blotting)对PKCD、CDCP1等加以验证。结论：1.收集了乳腺癌及乳腺纤维瘤组织,并培养了乳腺癌细胞株,裂解并富集了酪氨酸磷酸化蛋白质,为今后的乳腺癌酪氨酸磷酸化蛋白质组学的进一步研究打下基础。2.本次试验首次应用酪氨酸磷酸化免疫沉淀的方法富集了乳腺肿瘤及细胞株的酪氨酸磷酸化蛋白,初步探索了乳腺癌发生、发展和治疗,及预后检测的分子机制,为乳腺癌的诊断提供了新的肿瘤标志物。本实验避免了以往磷酸化蛋白质研究及肿瘤发病机制,进程及治疗等领域研究针对个别蛋白质的局限性。首次把酪氨酸磷酸化定量蛋白应用于乳腺癌发病机制及治疗的研究。克服了普通双向凝胶电泳重复性差,动态范围小的缺点。并且建立新的定量比较蛋白质组学的技术路线,并掌握了蛋白质组学研究的基本方法。3.筛选了多个乳腺肿瘤差异性磷酸化蛋白质,其中PKCD、CDCP1、AXL、IGF1R等在乳腺癌细胞中参与了各项生理过程,其中的一些为乳腺癌的个体化治疗提供潜在靶向药物,另一些能够作为肿瘤标志物对乳腺癌疾病的诊断和术后检测提供信息。4.在Cal-85-1、Hcc-159、Du-4475等细胞株中发现了与乳腺癌中表达量上调一致的PKCD、CDCP1等酪氨酸磷酸化差异蛋白质,这些细胞株能够为研究未来这些蛋白质功能和新药研发提供模型。本实验研究为未来乳腺癌的研究打下了坚实的基础。总之,本实验应用了新的半定量磷酸化蛋白质组学的方法来研究乳腺癌。这种方法是灵敏的和可重复性的,用功能研究的方法去验证蛋白激酶活性和磷酸化底物。而这些蛋白激酶和磷酸化底物在已前的信号传导网络中没有报道过。另外,在本文中,酪氨酸蛋白激酶在人类多种癌症磷酸化组学分析中起了重要的作用,并且提供了功能筛选和酪氨酸激酶活性验证。这些新的发现极大的丰富了酪氨酸激酶的活性和下游信号传导网络的研究。更多还原

【Abstract】 Background and purpose:Breast cancer is one of the most frequent female malignancies. Currently, its incidence is still rising worldwide. Breast cancer is a systematic disease, surgical treatment alone does not significantly improve the survival rate of the patients. In order to improve its cure rate, comprehensive treatment of breast cancer is very important. In recent years, amplification of HER-2 gene and overexpression of HER-2 protein were reported in 20% to 30% of breast cancer patients, targeted therapy against HER-2 kinase with Herceptin (Herceptin) has shown promising results.Oncogenesis of breast cancer is a multi-factorial complex process. The molecular pathogenesis of breast cancer is not clear. Here, we investigated differential protein tyrosine phosphorylation between malignant and benign breast tumors. This study will provide new insights into the molecular pathogenesis and treatment of breast cancer.Methods:In this study, we collected 20 cases of malignant breast cancer and 12 benign cases. Tyrosine phosphorylated peptides were enriched by immunoaffinity purification, and analyzed by LTQ Orbitrap mass spectrometer。Phosphoproteins with statistical difference were further validated by immunoblotting. In addition, we cultured and profiled four breast cancer cell lines by mass spectrometer. Comparison of primary human breast tumors and cancer cell lines suggested that cell lines can be used as models to validate targets identified in primary tumors.Result; In this study, we identified 772 tyrosine phosphorylated proteins.581 of them were enriched in malignant tumors,191 were enriched in benign tumors。There are 116 differentially tyrosine phosphorylated proteins with p<0.05,51 of them are from malignant tumors. Among them, we found that PKCD, CDCP1, AXL upregulated in human breast cancer and IGF1R in human breast fibroma. We validated tyrosine phosphorylation of PKCD and CDCP1 by Western blot.We investigated the phosphorylation and expression of PKCD and CDCP1 in four breast cancer cell lines. Protein lysates from Cal-85-1, hcc-1599, du-4475, and MCF-7 were harvested and phosphoproteomics analysis was performed. In Cal-85-1, hcc-1599, and du-4475, we observed phosphorylation and expression of PKCD and CDCP1, while we did not observe significant expression of PKCD and CDCP1 in MCF-7 breast cancer cell line. These results were confirmed by Western blot.Conclusion:1. In this study, we collected malignant and benign breast cancer tissue, enriched tyrosine phosphorylated proteins, and performed breast cancer cell lines culture. These tyrosine phosphorylated proteomics data will serve as foundation for future research.2.This is a novel application of peptide immunoprecipitation method and LTQ-Orbit Trap for studing tyrosine phosphorylation in breast tumors and cell lines. It provides novel insights into the occurrence, development and treatment of breast cancer. It might identify new biomarkers for the prognosis of the disease as well. It overcame shortcomings of the poor reproducibility, small dynamic range of the ordinary two-dimensional gel electrophoresis. In addition, we applied semi-quantitative phosphoproteomics technology, and acquired basic knowledge of proteomics research.3.This study identified a number of differentially tyrosine phosphorylated proteins, including PKCD, CDCP1, AXL, and IGF1R, which are involved in various physiological processes of the breast cancer. Some of them are potential therapeutic targets for personalized medicine, others could be served as biomarkers to monitor disease progression and provide information for prognosis.4.In malignant breast cancer samples, we identified tyrosine phosphorylation of PKCD and CDCP1. Furthermore, we confirmed these findings in several breast cancer cell lines (Cal-85-1, hcc-1599, du-4475). Thus, these cell lines can be used as models for understanding the function of these proteins and for drug development in the future. This study provides a solid foundation for breast cancer research in the future.In summary, we applied a new method of semi-quantitative phosphoproteomics to study breast cancer. This method is a sensitive and reproducible functional strategy to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks. Furthermore, in the context where protein tyrosine kinases are known to play an important role in many human cancer genes phosphoproteomic analysis provides a functional screening assay to rapidly identify constitutively activated tyrosine kinases regardless of the molecular mechanism of activation. This analysis generated a deep and broad view of tyrosine kinase activity and downstream signaling networks that were not revealed before.更多还原