Gene expression, immunohistochemistry and gel zimography of matrix metalloproteinases and related tissue inhibitors in canine mammary tumours: preliminary results

Introduction: Matrix metalloproteinases (MMPs) are a family of enzymes involved in the degradation and remodelling of extracellular matrix. Previous studies on matrix metalloproteinases (MMPs) have indicated that they are implicated in cancer invasion and metastases. Only few studies, where the entire gene-protein-enzyme machinery expression and activity have been evaluated in dog. Therefore, in the present study MMP2, 9, 14, TIMP1 mRNA and protein pattern expression, and MMP2 and 9 activity (zymography) were measured in canine mammary tumours (CMTs). Materials and Methods: Eleven CMTs, 5 benign and 6 malignant, were considered in the present study. The expression and activities of MMP2, MMP9, MMP14 and TIMP1 were evaluated by immunohistochemistry and gelatin zymography. Gelatinolytic activities of secreted MMP2 and MMP9 were also analysed in pre and post-surgery plasma. Real Time RT-PCR assays were set up by using UPL probes, validated for relative quantification, and gene expression profiles measured. Results: MMP2 and MMP9 activities were significantly higher in malignant neoplasia than in benign tumours. MMP2, 9, 14 and TIMP1 immunolabelling was observed in both benign and malignant tumour tissues, but was stronger in the latter. At the mRNA level, an increase of MMP9, MMP14 and TIMP1 (15.5, 2.1, 35.1 mean –fold changes, respectively) was generally observed in tumour specimens. Conclusions: The immunohistochemical data indicate that MMP2, MMP9 and TIMP1 are highly expressed in CMTs and in association with gel zymography it suggests that the activation of proMMP2 may be an indicator of malignancy. MMP9, MMP14 and TIMP1 induction was also confirmed pre-transcriptionally.