Conclusion
Overexpression of MMP-9 increases the malignancy of breast cancer cell lines, largely via activation of the TGF-β/SMAD signalling.
Methods
The distributions of MMP-9 and TGF-β in the tissues of canine breast cancers were screened by immunohistochemical assays. A recombinant plasmid expressing mouse MMP-9 was generated and transiently transfected into three different breast cancer cell lines. Cell Counting Kit-8 and colony formation assay were used to study cell viability. Migration and invasion ability were analysed by wound assay and transwell filters. Western blot and quantitative real-time PCR were used to determine the protein and mRNA expression. Result: Remarkable strong MMP-9 and TGF-β signals were observed in the malignant tissues of canine breast cancers. In the cultured three cell lines receiving recombinant plasmid expressing mouse MMP-9, the cell malignancy was markedly increased, including the cell colony formation, migration and epithelial-mesenchymal transition. The levels of activated TGF-β, as well as SMAD4, SMAD2/3 and phosphorylation of SMAD2, were increased, reflecting an activation of TGF-β/SMAD signalling. We also demonstrated that the inhibitors specific for MMP-9 and TGF-β sufficiently blocked the overexpressing MMP-9 induced the activation of SMAD signalling and enhancement on invasion in the tested breast cancer cell lines.