| Breast cancer is one of the most malignant cancers and the leading cause of cancer deaths of women in the world. The majority of patient deaths are due to the growth of disseminated tumor cells in distant organs, namely metastasis. Performing advanced proteomic techniques on breast cancer metastasis research, one would systematically identify and compare the protein composition during the metastasis process to find the novel therapy targets. In this thesis, focusing on breast cancer metastasis process, we performed LC‐MS/MS proteomic strategy to search for key factors which contribute significantly to the metastasis process.We upgraded the hardware systems and improved the software systems based on existing micro‐proteomic platform in the laboratory, and set up a nano‐proteomic research platform for complex protein detection. The standard sample test showed that the proteomic platform could separate peptide mixture stably with the flowing speed of nanoliter, and the detective sensitivity is up to femtomole. Establishing a successful and effective proteomic platform laid the foundation of proteomic research in our laboratory, and also served as a basis for this thesis.Firstly, we utilized2D‐LC‐MS/MS strategy on breast cancer serum proteome to identify potential biomarkers for early metastatic diagnosis. We selected three groups of21human sera including lymph node‐negative/positive breast cancer patients and benigh breast disease controls. After sample pre‐treatment, proteomic identification and data analysis, a fruitful list of serum proteins with significant differences in abundance accompanying the progression of breast cancer were found. Among the selected proteins, TNXB was further validated by the ELISA method in131serum samples as a potential biomarker for early metastasis of breast cancer.Then, we applied GeLC‐MS/MS on comparing secretomes of cells with varied bone‐tropic capacities to screen the key factors of breast cancer bone metastasis, and did functional validation in animal model. We preformed a label‐free proteomic analysis to identify the secretomes of four MDA231derivative cell lines (SCP2, SCP4, SCP6, SCP46). By quantification with label‐free spectral counting, a total of128proteins were found to be consistently up‐/down‐regulated in the CM of bone‐tropic cancer cells, only10proteins were reported by transcriptomics research previously. After molecular function enrichment analysis, we found that the function of peptidase inhibition was significantly enriched including four members of cystatin protein family. We further verified the function of CST6in bone metastasis of breast cancer in vivo. The results showed that overexpression of CST6rescued mice from overt osteolytic metastasis and deaths in the animal study, while CST6knockdown markedly enhanced cancer cell bone metastasis and shortened animal survival. Overall, our study revealed that secreted CST6played an important inhibited function during breast cancer bone metastasis.At last, we screened the key factors promoting immune suppression in breast cancer metastasis process via comparing the4T1series cell lines and performed functional validation. Myeloid‐derived suppressor cells (MDSCs) are a population of cells that have a remarkable ability to suppress anti‐tumor immunology. We selected4T1series cell lines (67NR,168FARN,4TO7,4T1) from the same spontaneous mouse breast cancer with different metastatic capacities as research model. With tumor‐bearing mouse model, FACS and IHC results showed that the ability of MDSCs induction was positively correlated with the increasing metastatic potential of4T1series tumor, and secreted proteins played a major role in the induction of MDSCs. We used the proteomic strategy of GeLC‐MS/MS to efficiently identify and compare the secretomes of4T1series cell lines. After a rigorous data analysis, we obtained26secreted proteins significantly related to breast cancer metastasis, including a variety of cytokines, immune regulated proteins and enzyme activity regulators. We further validated the ability of MDSCs induction by G‐CSF, uPA and LIF. The results showed that G‐CSF highly expressed by malignant cell lines strongly promoted the expansion and recruitment of activated MDSCs. Our work demonstrated that G‐CSF is a key factor mediated immune suppression via induction of MDSC during breast cancer metastasis.In summary, we have established a nano proteomic platform for sensitive detection of complex protein samples, and performed the study in breast cancer metastasis research. This platform was applied for clinical serum proteomic analysis to screen biomarkers for breast cancer early metastatic diagnosis. By comparing the secretomes of isogenetic cell lines with different metastatic capacities, combining the metastatic characteristics of immune regulation and organ tropic, we also obtained a list of key factors associated with breast cancer metastasis. The functions of some of these factors were further studied. The results of the thesis will provide valuable information for the design of therapeutic drug targets specifically against breast cancer metastasis. |
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