The online version of this article (doi:10.1186/s13058-017-0822-9) contains supplementary material, which is available to authorized users.
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.
Primary 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.
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 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).
These 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.
Additional file 1: Table S1. Summary of breast tissue examined for MMP8. (DOC 27 kb)13058_2017_822_MOESM1_ESM.doc
Additional file 2: Table S2. Duct-by-duct analysis. (DOC 28 kb)13058_2017_822_MOESM2_ESM.doc
Additional file 3: Figure S1. Dual immunofluorescent staining of a normal breast duct showing MMP-8 (green Atlas, HPA02122,1:200) and p63 (red Abcam, Ab735, 1:50). The image shows predominant myoepithelial localisation of MMP-8. (TIF 7068 kb)13058_2017_822_MOESM3_ESM.tif
Additional file 4: Figure S2. Images of one representative organotypic gel fluorescently stained for myoepithelial marker p63 (green) (non-invading cell layer), nuclear marker DAPI (blue) and marker of invasive breast cancer cells Neso (red). From left to right images show gels comprising fibroblasts, MDA-MB-231 cells and MECs transfected with Empty Vector, MMP8 WT and MMP8 EA. The final panel shows a gel comprised of fibroblasts and MECs alone. Non-transfected MECs were used in the last panel. (TIF 986 kb)13058_2017_822_MOESM4_ESM.tif
Additional file 5: Figure S3. (i) Densitometry quantifying pSMAD2 versus tSMAD2 normalised to the loading control. MECs transfected with MMP-8 WT show a marked reduction of pSMAD2 compared to Empty Vector and MMP-8 EA at 5 minutes. (ii) Densitometry quantifying pSMAD2 versus tSMAD2 normalised to the loading control. MECs transfected with siRNA to MMP-8 demonstrated a markedly stronger pSMAD2 signal compared to control siRNA (siLUC). (TIF 336 kb)13058_2017_822_MOESM5_ESM.tif
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- Loss of MMP-8 in ductal carcinoma in situ (DCIS)-associated myoepithelial cells contributes to tumour promotion through altered adhesive and proteolytic function
Michael D. Allen
Dylan R. Edwards
J. Louise Jones
- BioMed Central
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