Abstract
BackgroundTriple-negative breast cancer (TNBC) is the most difficult cancer to be treated. TNBC expresses high level of matricellular cysteine-rich protein CYR61/CCN1 that plays a key role in producing cancer metastases and is an important target for metastasis chemoprevention. Nitric oxide (NO) can covalently bind to the thiol group of cysteines (termed S-nitrosylation) resulting in regulation of the targeted protein functions. MethodsProtein S-nitrosylation were detected by biotin-switch assay and western blotting assay. CYR61 protein S-nitrosylated sites and 3D structure were determined by mass spectrometry and MODELLER software. Adhesion assay, cell morphology assay, wound healing assay and transwell invasion assay were used to evaluate effects of CYR61 S-nitrosylation on the cell metastatic ability. In vivo metastasis activity of CYR61 S-nitrosylation were tested by intravenous injection and mammary xenograft implantation mouse metastatic models.ResultsS-nitrosylation by GSNO of CYR61 reached a plateau quickly and was confirmed by spectroscopic analysis and biotin-switch assay. Mass-spectrometry proteomic analysis revealed that S-nitrosylation predominantly occurred at Cys100, Cys117, Cys229 and Cys239, resulting in CYR61 structure relaxed and unstable evidenced by protein structure modeling. S-nitrosylation of MDA-MB-231 cells, their CYR61-overexpressed and CYR61–silenced counterparts significantly attenuated the metastatic ability of these cells, including their ability of adhesion, mobility, invasion, and interplay with platelets, and made the adhered cells unattached. The attenuation in metastatic ability proportionally increased with the degree of S-nitrosylation to CYR61 naturally-expressed or genetically-manipulated cells, and was demonstrated in mice, where, S-nitrosylation of these cell lines not only inhibited their acute seeding to lungs after an intravenous injection, but also inhibited the late development of these cells into the metastatic nodes after mammary xenograft implantation. Furthermore, orthotopically-implanted MDA-MB-231 developed mammary tumors and later lung metastasis; whereas, the same cells with S-nitrosylation developed no tumor and metastasis at all. Conclusionwe present the first evidence that S-nitrosylation of CYR61 can significantly inhibit metastatic aggressiveness of the TNBC MDA-MB-231 cells. This conceptual creative study opens a new avenue to prevent the most aggressive TNBC from metastases by S-nitrosylation to CYR61.
Proteomic investigation on bio-corona of Au, Ag and Fe nanoparticles for the discovery of triple negative breast cancer serum protein biomarkers
