Short Hairpin RNA–Mediated Inhibition of Matrix Metalloproteinase-1 in MDA-231 Cells: Effects on Matrix Destruction and Tumor Growth

Abstract

Increased matrix metalloproteinase-1 (MMP-1) expression is associated with advanced stages of breast cancer and may be a predictive marker for the development of invasive disease. In this report, we used short hairpin RNA (shRNA) molecules to investigate whether MMP-1 production in MDA-231 breast cancer cells contributed to the degradation of a collagen matrix or tumor formation in nude mice. We created two groups of MDA-231 cell lines. MDA-231 cells containing a vector producing shRNA specific for MMP-1 had a >90% decrease in MMP-1 mRNA and protein compared with cells containing an empty vector, and blocking MMP-1 expression inhibited the in vitro collagenolytic activity of MDA-231 cells. When the cells were injected into the mammary fat pad, there was no difference in the frequency of tumor formation in mice. However, the average tumor size was larger in mice injected with cells containing the empty vector (1,216 F 334 mm 3 ) than in mice injected with cells expressing the MMP-1 shRNA (272 F 117 mm 3 ; P = 0.027). We conclude that MMP-1 expression is essential for the ability of MDA-231 cells to invade and destroy a collagen matrix and in vivo experiments suggest an important role for MMP-1 in breast tumor growth. (Cancer Res 2005; 65(23): 11101-8)

Regulation of matrix metalloproteinase gene expression by a retinoid X receptor–specific ligand

“…The collagen degradation assay was performed as previously described (24,25). Briefly, fibrillar collagen preparations were made from Vitrogen 100 bovine type I collagen (Cohesion Technologies, Palo Alto, CA) according to the manufacturer's instructions.…”

“…This did not result in the liberation of medium (data not shown). Other studies have shown that when the 1-ml collagen plug is fully degraded, ϳ2 ml of total medium can be recovered (25).…”

“…We then quantified the collagen breakdown by recovering the medium from each well and weighing it (24,25). Recovered media volumes greater than the 1 ml (i.e., 1 gm) that was originally added demonstrated collagen breakdown, with liberation of media previously embedded in the matrix.…”

“…In humans, the MMP family currently encompasses 23 unique members, which degrade all constituents of the extracellular matrix (ECM) (5). The MMPs can be subdivided into 6 main classes (1,4,5), consisting of the collagenases (MMPs 1, 8, and 13), the gelatinases (MMPs 2 and 9), the stromelysins (MMPs 3, 10, and 11), the matrilysins (MMPs 7 and 26), the membrane-type MMPs (MMPs 14,15,16,17,24,and 25), and a diverse subgroup.The collagenase subgroup is unique in its ability to cleave fibrillar collagens, and their enzymatic activity represents a rate-limiting step in the degradation of collagen-containing tissues. MMP-1 is expressed by a range of cell types, including chondrocytes and fibroblasts, and is most effective against type III collagen.…”

“…In humans, the MMP family currently encompasses 23 unique members, which degrade all constituents of the extracellular matrix (ECM) (5). The MMPs can be subdivided into 6 main classes (1,4,5), consisting of the collagenases (MMPs 1, 8, and 13), the gelatinases (MMPs 2 and 9), the stromelysins (MMPs 3, 10, and 11), the matrilysins (MMPs 7 and 26), the membrane-type MMPs (MMPs 14,15,16,17,24,and 25), and a diverse subgroup.…”