Abstract
BackgroundNormal 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.MethodsPrimary normal and DCIS-associated MECs, and normal (N-1089) and DCIS-modified myoepithelial (β6-1089) cell lines, were used to assess MMP-8 expression and function. β6-1089 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 α6β4 integrin to hemidesmosomes (HD), TGF-β 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.ResultsAssessment 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 β6-1089 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 β6-1089 led to greater localisation of α6β4 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-β signalling and gelatinolytic activity. MMP-8 knock-down enhanced TGF-β 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 β6-1089 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).ConclusionsThese data indicate MMP-8 is a vital component of the myoepithelial tumour-suppressor function. It restores MEC interaction with the matrix, opposes TGF-β 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.
Highlights
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
Many invasive breast cancers (IBC) develop through a pre-invasive stage known as ductal carcinoma in situ (DCIS) where the proliferative neoplastic cells are retained within the breast duct surrounded by an intact myoepithelial cell (MEC) layer lying in contact with basement membrane [1]
Transwells carried out using ER+ cell line MCF-7 showed no reduction in invasion – these cells exhibited a low degree of invasion and it can be speculated that the invasion could not be reduced any further. These results indicate that the invasionsuppressor activity exhibited by Matrix metalloproteinase-8 (MMP-8) wild-type MMP-8 (WT) MECs is dependent upon MMP-8 enzymatic activity, as MMP-8 Inactive mutant (EA) does not influence tumour cell invasion
Summary
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. Many invasive breast cancers (IBC) develop through a pre-invasive stage known as ductal carcinoma in situ (DCIS) where the proliferative neoplastic cells are retained within the breast duct surrounded by an intact myoepithelial cell (MEC) layer lying in contact with basement membrane [1]. MECs adhere tightly to the basement membrane using prominent hemidesmosome (HD) formation [12]. They show a high level of expression of anti-tumourigenic factors, such as proteinase inhibitors and anti-angiogenic mediators, and in vitro studies have demonstrated broad tumour-suppressor activities of normal primary MEC and MEC lines [13,14,15]. In DCIS, the MECs become altered, showing changes in gene expression, epigenetics and phenotype [13, 14, 16, 17], though the functional significance of these changes has not been established [9, 18,19,20]
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