Investigation Of Tyrosine Phosphorylation Proteomics In Human Breast Cancer

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.