Patterns of protease production during prostate cancer progression: proteomic evidence for cascades in a transgenic model.

Extracellular proteases are recognized as critical factors in the progression of a number of carcinomas, including prostate cancer. Matrix metalloproteases (MMP) are important in processes of tumor growth, invasion and dissemination, but other classes of proteases, such as serine and cysteine proteases, also contribute. We utilized the TRAMP model for prostate cancer to elucidate proteases involved in prostate cancer progression. General proteomic analysis was performed on normal murine prostate, early TRAMP tumors and advanced TRAMP tumors, as well as normal and involved lymph nodes. Zymography and antigenic analyses revealed increased expression of mainly pro-MMP in early TRAMP tumors but substantial elaboration of activated MMP only in late TRAMP tumors. Progressive increase in cysteine, serine and certain membrane-bound proteases from normal to early to advanced prostate tumors, was also seen. Our results implicate pericellular proteases as initiators of major proteolytic cascades during tumor progression and suggest targets for maximal therapeutic effect.

T-antigen inhibits metalloproteinase expression and invasion in human placental cells transformed with temperature-sensitive simian virus 40.

Cellular transformation frequently induces invasive behavior in cells. The effects of simian virus (SV) 40 T-antigen on the relationship between metalloproteinase expression and cell invasion were tested in human placental trophoblast-like cells transformed with a temperature-sensitive form of the SV40 virus, tsA30. As a comparison, metalloproteinase expression was also tested in human fibroblasts transformed with wild-type SV40 T-antigen. When tsA30.1 cells were cultured at the nonpermissive temperature for T-antigen expression, 40 degrees C, they expressed abundant metalloproteinases, including the 72 kDa gelatinase A (MMP-2), the 92 kDa gelatinase B (MMP-9) and stromelysin-1 (MMP-3). In contrast, tsA 30.1 cells cultured at the permissive temperature of 33 degrees C produced T-antigen and showed markedly decreased amounts of these proteinases. A similar suppression was seen in the human fibroblasts transformed with wild-type T-antigen. The tsA30 cells cultured at either temperature expressed a similar amount of tissue inhibitor of metalloproteinases-1 and -2. Cell invasion assays were performed to determine whether the altered ratio of proteinases to inhibitors under these conditions affected the extracellular matrix-degrading and invasive characteristics of the cells. In their differentiated state at the nonpermissive temperature for T-antigen expression, tsA30.1 cells were highly invasive, whereas at the permissive temperature they were not invasive. Therefore, the expression of T-antigen suppressed metalloproteinase production and changed the cells from an invasive to a noninvasive phenotype. We conclude that in tsA30.1 cells, SV 40 T-antigen expression suppresses metalloproteinase production, thereby decreasing the rate of degradation of the extracellular matrix. Taken together, these data indicate that invasive behavior is related to proteinase gene expression rather than to transformation by T-antigen. Function-blocking antibody to beta 1 integrins did not affect adhesion of tsA30.1 cells but inhibited invasion at the nonpermissive temperature, even though they continued to secrete proteinases. This observation indicates that beta 1 integrin-mediated cell migration is required along with proteinases for cells to be invasive.

Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation.

Gelatinase B, a matrix metalloproteinase (MMP) of high specific activity, is highly expressed and activated by mouse blastocysts in culture, and inhibition of this enzyme activity inhibits lysis of extracellular matrix (Behrendtsen, O., Alexander, C. M. and Werb, Z. (1992) Development 114, 447-456). Because gelatinase B expression is linked to invasive potential, we studied the expression of gelatinase B mRNA and protein in vivo, in implanting trophoblast giant cells, and found that it was expressed and activated during colonization of the maternal decidua. mRNAs for several other MMPs (stromelysin-1, stromelysin-3 and gelatinase A) and MMP inhibitors (TIMP-1 and TIMP-2) were expressed in the undifferentiated stroma toward the outside of the decidua, and TIMP-3 mRNA was expressed in primary and some mature decidual cells during their differentiation. Both mRNA and TIMP-3 protein were present at high concentrations transiently, and declined from 6.5 days post coitum onward, as the cells underwent apoptosis during the main period of gelatinase B expression and ectoplacental growth and expansion. To assess the function of MMPs during implantation and decidual development, we either injected a peptide hydroxamate MMP inhibitor into normal mice or studied transgenic mice overexpressing TIMP-1. In both cases, decidual length and overall size were reduced, and the embryo was displaced mesometrially. Embryo orientation was less strictly regulated in inhibitor-treated deciduae than in control deciduae. Morphogenesis and development of oil-induced deciduomas were also slowed in the presence of the inhibitor. We conclude that administration of MMP inhibitors retards decidual remodeling and growth, and we suggest that the MMPs expressed in precursor stromal cells promote their differentiation and expansion.

Simian virus 40 transformation alters the actin cytoskeleton, expression of matrix metalloproteinases and inhibitors of metalloproteinases, and invasive behavior of normal and ataxia-telangiectasia human skin fibroblasts.

Alterations in the actin cytoskeleton of normal cells result in changes in cell shape and adhesiveness and induce expression of matrix-degrading matrix metalloproteinases. We examined the effect of simian virus 40 transformation of normal and ataxia-telangiectasia human skin fibroblasts, a process that produces actin reorganization, altered cell morphology, and altered cell behavior, on expression of genes of the matrix metalloproteinase and tissue inhibitor of metalloproteinases gene families. Simian virus 40 transformation induced collagenase-1 gene expression; in contrast, stromelysin-1, 72-kDa gelatinase (gelatinase A), tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 genes were repressed. Transformation also altered the response of the fibroblasts to 12-O-tetradecanoylphorbol-13-acetate. Collagenase mRNA was induced in 12-O-tetradecanoylphorbol-13-acetate treated transformed cells up to 50-fold more than in untreated transformed cells or in 12-O-tetradecanoylphorbol-13-acetate treated untransformed parent cells. In contrast, 12-O-tetradecanoylphorbol-13-acetate did not overcome the attenuated expression of stromelysin-1 in the simian virus 40 transformants. In addition, 92-kDa gelatinase (gelatinase B) was induced by 12-O-tetradecanoylphorbol-13-acetate only in the simian virus 40 transformants. The responses of gelatinase A and tissue inhibitor of metalloproteinases-1 to 12-O-tetradecanoylphorbol-13-acetate were unchanged. The pattern of altered proteinase expression after transformation was accompanied by a phenotypic alteration in cell invasion. The simian virus 40 transformants exhibited enhanced invasiveness through a basement-membrane-like matrix. These data demonstrate that enhanced invasiveness in simian virus 40 transformed cells is accompanied by changes in actin organization and expression of proteinases and inhibitors, as well as in the balance between proteinases and inhibitors in favor of proteinases.