Misregulation of Stromelysin-1 Expression in Mouse Mammary Tumor Cells Accompanies Acquisition of Stromelysin-1-dependent Invasive Properties

Abstract

Stromelysin-1 is a member of the metalloproteinase family of extracellular matrix-degrading enzymes that regulates tissue remodeling. We previously established a transgenic mouse model in which rat stromelysin-1 targeted to the mammary gland augmented expression of endogenous stromelysin-1, disrupted functional differentiation, and induced mammary tumors. A cell line generated from an adenocarcinoma in one of these animals and a previously described mammary tumor cell line generated in culture readily invaded both a reconstituted basement membrane and type I collagen gels, whereas a nonmalignant, functionally normal epithelial cell line did not. The matrix metalloproteinases (MMPs) 1 are a family of extracellular matrix (ECM)-degrading enzymes that have been implicated in a variety of normal developmental and pathological processes, including tumorigenesis (1, 2). The MMP family comprises at least 15 members with different, albeit overlapping, substrate specificities. During activation of latent MMPs, their propeptides are cleaved and they are converted to a lower molecular weight form by other enzymes, including serine proteinases, and by autocatalytic cleavage.Among the MMPs, stromelysin-1 (SL1) possesses the broadest substrate specificity (1). Despite increasing knowledge about its enzymatic properties and the regulation of its expression, little is known about its function. We have generated transgenic animals that express an autoactivating mutant of rat SL1 targeted to the epithelial compartment of the mammary gland (3). Phenotypically, SL1 transgenic mice display increased branching morphogenesis and lactogenic differentiation at prepubertal stages and premature involution during late pregnancy (3, 4). Branching morphogenesis requires the invasion of epithelial cells into the adipose tissue, a process reminiscent of invasion of stromal compartments by tumor cells. Strikingly, a large number of SL1 transgenic animals also develop mammary tumors of various histotypes, including invasive adenocarcinomas (5). 2 Because tumor development is a late response of SL1 transgenic mice to overexpression of the transgene, it remains unclear whether SL1 plays a direct role in tumor growth and/or invasion or whether the observed tumors are a consequence of other molecular alterations in the microenvironment of the mammary gland before the onset of tumor growth. 3 Studies performed with synthetic inhibitors of MMP activity and tissue inhibitors of metalloproteinases (TIMPs) have shown that suppression of MMP activity also suppresses tumor growth and metastasis (6, 7). In many cases, the level of SL1 expression in tumors of the mammary gland and other tissues is positively correlated with the degree of malignancy (8, 9). However, the only direct evidence for the nature of the MMPs involved was provided by the demonstration that function-blocking antibodies against gelatinase A and antisense inhibition of matrilysin expression decreased the invasiveness of tumor cells in a reconstituted basement membrane assay (10, 11). The...

CCAAT/enhancer binding protein beta (C/EBPβ)-2 transforms normal mammary epithelial cells and induces epithelial to mesenchymal transition in culture

“…Stained cells were mounted in Cytoseal XYL (Stephens Scientific; Kalamazoo, MI, USA) and visualized by epifluorescence as described above. Vimentin expression was detected by indirect immunofluorescence as described previously (Lochter et al, 1997b), with the following exceptions. Confluent cells were grown on MatTek dishes, and the fluorescent-conjugated Alexa 546 goat anti-mouse secondary Ab was used at a final concentration of 2 mg/ml to indirectly detect vimentin expression.…”

370 The metastasis-promoting protein, S100A4, regulates mammary branching morphogenesis

“…Overexpression of MMP-3 in mammary gland has previously been shown to increase in vivo branching and cause increased incidence of preneoplastic lesions and invasive breast tumors compared to control mice (Sympson et al, 1995). It was later shown that MMP-3 also induced epithelial-mesenchymal transition (EMT) and malignant transformation in cultured cells (Lochter et al, 1997a;Lochter et al, 1997b;Lochter et al, 1999;Sternlicht et al, 1999). Altogether, it has been clearly demonstrated that deregulation of MMP-3 is a critical occurrence in breast tissue.…”

“…Culture medium conditioned by 3D Matrigel cultures for 2 days was processed for casein substrate gels as described previously (Lochter et al, 1997b;Talhouk et al, 1991) In brief, conditioned medium was mixed with zymogram sample buffer without reducing agents, incubated for 15 min at 37 °C, and separated on 8.8% sodium dodecyl sulfate (SDS)-polyacrylamide gels containing 1 mg/ml β-casein (Sigma St. Louis, MO, USA). After electrophoresis, gels were incubated for 30 min with 2.5% Triton X-100 and subsequently for 5 days at 37 °C in 100 mM Tris-HCl, pH 7.4, containing 15 mM CaCl 2 .…”

Inducible Expression of Chimeric EWS/ETS Proteins Confers Ewing's Family Tumor-Like Phenotypes to Human Mesenchymal Progenitor Cells

“…Moreover, EWS/ETS expression enhances the ability of UET-13 cells to invade Matrigel. This assay is thought to mimic the early steps of tumor invasion in vivo (34), and the ability to penetrate the Matrigel has been positively correlated with invasion potential in several studies. Therefore, we concluded that EWS/ETS expression could mediate a part of the feature of tumor transformation in human MPCs.…”