Investigation of the Functional Significance of MMP-8 in Breast Myoepithelial Cells.
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The Matrix Metalloproteinase (MMP) family are conventionally considered as key enzymes contributing to cancer-cell invasion through remodelling of the extracellular matrix (ECM). In contrast, MMP-8 has been shown to exert an anti-cancer role. In normal breast, MMP-8 expression is restricted to tumour suppressor myoepithelial cells (MECs), which form the interface between glandular epithelium and the ECM. In ductal carcinoma in situ (DCIS), MECs are altered; a consistent change is up-regulation of αvβ6-integrin, which is associated with loss of suppressor activity. Preliminary observations indicated that there is also loss of MMP-8 expression in DCIS-MEC. The aim of this study is to investigate the impact of loss of MEC derived MMP-8 on MEC function and how this might modulate tumour progression. To generate a model of DCIS MEC, an αvβ6 over-expressing cell line (Myo-β6) was generated from normal MECs (Myo-Puro). These cells were found to lose MMP-8 expression. To dissect gain-of-function effect, MMP-8 was re-introduced into Myo-β6 (MMP-8 WT). A proteolytic inactive form of MMP-8 was used to dissect the dependence of function on proteolysis. In-vitro analysis demonstrated that MMP-8 WT but not inactive MMP-8 significantly up-regulates MEC adhesion (p=0.0001) and spread (p=0.0003) on ECM, but reduces migration towards ECM proteins including Collagen-I (p=0.006) and fibronectin (p=0.01). Furthermore MMP-8 WT results in reduced numbers of filopodia/retraction fibres (p=0.01) and reduced protrusion length (p=0.0001) on MEC cell surface. MMP-8 promotes the localisation of α6β4-integrin to hemidesmosomal adhesive structures (p=0.003), and inhibits MEC gelatinase (p=0.002) and TGF-β activity. Conversely, knock-down of endogenous MMP-8 in Myo-Puro MECs promotes migration and filopodia/retraction fibre formation (p=0.05), increases gelatinase activity (p=0.007) and TGF-β signalling. To analyse the paracrine effect of MEC-derived MMP-8 on breast cancer cell invasion, MDA MB 231 or SUM159 cells were co-cultured with modified Myo-β6 cells in Boyden chamber invasion assays. A significant reduction in breast cancer cell invasion was observed only in the presence of MMP-8 WT (p=0.004) but not with inactive MMP-8. In contrast, MMP-8 knock-down in Myo-Puro MECs significantly enhanced breast cancer cell invasion (p=0.001). In order to recapitulate the DCIS stromal micro-ecology, 3D-organotypic cultures were constructed. In these systems there was a significant reduction in invasion only in the presence of MMP-8 WT MECs (p<0.001). Conversely, in the absence of Myo-Puro-derived MMP-8 breast cancer cell invasion was significantly up-regulated (p=0.007). These results suggest that MMP-8 does contribute to MEC tumour suppressor function via mechanisms dependent upon its proteolytic activity. These data support the hypothesis that loss of MMP-8 may contribute to the progression of DCIS to invasive disease.
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