Comparative Proteomic Analysis Of Human Breast Cancer Cell Lines With Differently Spontaneous Lung Metastatic Capability And Study On The Regulation Of The Differently Secreted Protein

Objective:To use human breast cancer cell lines with differently spontaneous lung metastatic capability as suitable models for the study of the molecular mechanisms related to breast cancer metastasis and finding out protein markers with metastatic potential.Methods:MDA-MB-231HM was identified as one kind of cell lines derived from parental MDA-MB-231 cells by analyzing the karyotype of the MDA-MB-231HM cells with Giemsa chromosome banding stain.Then metastatic potential of both cell lines was tested in vitro by transwell and in vivo by comparing the weight of orthotopic tumor and the number of metastasis.Using two-dimensional electrophoresis(2-DE),we performed a proteomic comparison of both cell lines. Subsequently differentially expressed protein spots were identified by mass spectrometry.Differential expression was confirmed by Western blotting analysis and immunohistochemistry.Results:All the MDA-MB-231HM cells exhibited X shaped chromosomes, which is completely different fromâ…¤shaped chromosomes of mice.Metastatic potential of MDA-MB-231HM cells was found higher than that of MDA-MB-231cells both in vitro and in vivo.We finally identified 14 unique proteins by proteomic analysis including Macrophage-capping protein(CapG),Galectin-1, Chloride intracellular channel protein 1,Endoplasmic reticulum protein ERp29 precursor,Transgelin-2,Peptidyl-prolyl cis-trans isomerase A(PPIase A),Stathmin-1 (STMN1),Isoform 1 of Uridine-cytidine kinase 2(UCK2),Rho GDP-dissociation inhibitor 2(ARHGDIB),Isocitrate dehydrogenase[NADP]cytoplasmic(IDH1), NDRG1 Protein NDRG1,Sperm protein associated with the nucleus on the X chromosome B/F,Neuron-specific calcium-binding hippocalcin,HSPA5 protein. Transgelin-2 and HSPA5 protein were down-regulated in MDA-MB-231HM cells, whereas Sperm protein associated with the nucleus on the x chromosome B/F and hippocalcin were only detected in MDA-MB-231 cells.CapG,Galectin-1,Chloride intracellular channel protein 1,ERp29,PPIase A,STMN1,UCK2,Rho GDP-dissociation inhibitor 2,Isocitrate dehydrogenase[NADP]cytoplasmic(IDH1) and NDRG1 were up-regulated in MDA-MB-231HM cells.Change of CapG,STMN1 and transgelin-2 was confirmed by western blotting analysis.Immunohistochemical analysis showed transgelin-2 expression was higher correlation in lymph node-negative tissues(19/30) and lower correlation in lymph node-positive tissues (5/30).Conclusions:MDA-MB-231HM,a human breast carcinoma cell line with high capability of spontaneous lung metastasis,was a good tool for the study of breast cancer metastasis.Fourteen unique proteins in our study can both reveal molecular mechanisms related to breast cancer metastasis and be used as biological markers with metastatic potential for clinical diagnosis in breast cancer metastasis. Objective:To compare the secretome of both human breast cancer cell lines with differently spontaneous lung metastatic capability for finding out biological markers with metastatic potential for clinical diagnosis in breast cancer metastasis. Further more,to study the regulation of the protein differently secreted by MDA-MB-231HM cells and MDA-MB-231 cells.Methods:MDA-MB-231HM cells and MDA-MB-231 cells were cultured in serum-free medium for 24 hours,and the culture supernant of cells was desalted and concentrated by ultrafiltration to prepare the total secreted proteins.Two-dimensional gel electrophoresis(2-DE) was used to separate the secreted proteins.The differently secreted protein spots gels were identified by desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) and bioinformatics.Western blot and RT-PCR were used to further confirm differential expression and secretion of matrix metalloproteinase 1(MMP1).The expression vector of MMP1 specific shRNA(short hairpin RNA) and negative shRNA in vitro were constructed and transfected into MDA-MB-231 cells by lipofectamine2000 for the study of the function of MMP1 related to metastasis.The potential of invasion in vitro was tested by Transwell experiment.When MMP1 was immunoprecipitated by anti-MMP1 antibody for the study of the secretory regulation of MMP1,components from immunoprecipitated complex was separated by SDS-polyaerylamide gel electrophoresis and then identified by mass spectrometry.Interaction of MMP1 and GRP75 from immunoprecipitated complex was studied by confocalmicroscopy and co-immunoprecipitation.CXCR4 antagonist AMD3100 was used to interfere T47D cells,MDA-MB-231HM cells and MDA-MB-231 cells to study whether CXCR4 can affect the expressive regulation of MMP1.Results:The secretory protein profiles reveal 22 proteins differently secreted by MDA-MB-231HM cells and MDA-MB-231 cells,whereas only five protein spots were identified by MALDI-TOF-MS including pro-MMP1,mature-MMP1,TIMP-2, TCTP,beta-2 microglobulin.MMP1 and TCTP were up-regulated in MDA-MB-231HM cells,whereas TIMP-2 and beta-2 microglobulin were up-regulated in MDA-MB-231 cells.Up-regulated expression and secretion of MMP1 were confirmed by western blot and RT-PCR,which also revealed secretory difference of both cell lines was more significant than expressive difference of both cell lines.Transwell experiment revealed that the number of MDA-MB-231 cells into which were transfected by the vector of MMP1 specific shRNA for 36 hours to penetrate polycarbonates coated with Matrigel was significantly lower than that of MDA-MB-231 cells into which were transfected by the vector of MMP1 negative shRNA for 36 hours.GRP75 was identified in immunoprecipitated complex.When MMP1 was immunoprecipitated by anti-MMP1 antibody,MMP1 was identified by western blotting with anti-MMP1 antibody from immunoprecipitated complex. Co-localization of GRP75 and MMP1 was determined by confocalmicroscopy, whereas western blot revealed there was no differently expressive amount of GRP75 between MDA-MB-231 cells and MDA-MB-231HM cells.AMD3100 can down-regulated the expression of MMP1 in T47D cells,MDA-MB-231HM cells and MDA-MB-231 cells.Conclusions:Many kinds of secretory proteins were differently secreted from both human breast cancer cell lines with differently spontaneous lung metastatic capability.MMP1 may contribute to breast cancer metastasis and be used as a potential protein marker related to breast cancer metastasis.Contrasted with differently internal expression of MMP1 between the interior of MDA-MB-231 cells and MDA-MB-231HM cells,differently secretion of MMP1 between MDA-MB-231 cells and MDA-MB-231HM cells may play a predominant role in enhancing metastatic potential.GRP75 can interact with MMP1,whereas this kind of interaction may not be the effect of different secretion of MMP1 between MDA-MB-231 cells and MDA-MB-231HM cells.CXCR4 may regulate the expression of MMP1 in breast cancer cells.