Loss of MMP-8 in ductal carcinoma in situ ( DCIS)-associated myoepithelial cells contributes to tumour promotion through altered adhesive and proteolytic function

Abstract

Background: Normal myoepithelial cells (MECs) play an important tumour-suppressor role in the breast but display an altered phenotype in ductal carcinoma in situ (DCIS), gaining tumour-promoter functions. Matrix metalloproteinase-8 (MMP-8) is expressed by normal MECs but is lost in DCIS. This study investigated the function of MMP-8 in MECs and the impact of its loss in DCIS. Methods: Primary normal and DCIS-associated MECs, and normal (N-1089) and DCIS-modified myoepithelial (a6-1089) cell lines, were used to assess MMP-8 expression and function. a6-1089 lacking MMP-8 were transfected with MMP-8 WT and catalytically inactive MMP-8 EA, and MMP-8 in N-1089 MEC was knocked down with siRNA. The effect on adhesion and migration to extracellular matrix (ECM), localisation of a6 beta 4 integrin to hemidesmosomes (HD), TGF-beta signalling and gelatinase activity was measured. The effect of altering MEC MMP-8 expression on tumour cell invasion was investigated in 2D and 3D organotypic models. Results: Assessment of primary cells and MEC lines confirmed expression of MMP-8 in normal MEC and its loss in DCIS-MEC. Over-expression of MMP-8 WT but not MMP-8 EA in a6-1089 cells increased adhesion to ECM proteins and reduced migration. Conversely, knock-down of MMP-8 in N-1089 reduced adhesion and increased migration. Expression of MMP-8 WT in a6-1089 led to greater localisation of a6 beta 4 to HD and reduced retraction fibre formation, this being reversed by MMP-8 knock-down in N-1089. Over-expression of MMP-8 WT reduced TGF-beta signalling and gelatinolytic activity. MMP-8 knock-down enhanced TGF-beta signalling and gelatinolytic activity, which was reversed by blocking MMP-9 by knock-down or an inhibitor. MMP-8 WT but not MMP-8 EA over-expression in a6-1089 reduced breast cancer cell invasion in 2D and 3D invasion assays, while MMP-8 knock-down in N-1089 enhanced cancer cell invasion. Staining of breast cancer cases for MMP-8 revealed a statistically significant loss of MMP-8 expression in DCIS with invasion versus pure DCIS (p = 0.001). Conclusions: These data indicate MMP-8 is a vital component of the myoepithelial tumour-suppressor function. It restores MEC interaction with the matrix, opposes TGF-beta signalling and MMP-9 proteolysis, which contributes to inhibition of tumour cell invasion. Assessment of MMP-8 expression may help to determine risk of DCIS progression.