Proteinases and extracellular matrix degradation in breast cancer.

Abstract

A variety of proteases have been shown to promote the progression of cancer by virtue of their ability to degrade extracellular proteinaceous barriers, such as basement membrane and interstitial stroma. At the outset of this study available evidence strongly implicated cathepsin D in breast cancer metastasis. It was envisaged that an antibody inhibitory to the activity of this enzyme might retard invasion, and restrain a tumour from spreading. To this end anti-peptide antibodies were generated against a peptide sequence derived from the substrate capturing "flap" of the enzyme. Inhibition of enzyme activity by these antibodies could not be demonstrated, probably due to the lack of a suitably sensitive enzyme assay. However, the rationale of this study and the expertise gained from it could be applied, in the future, to enzymes that have since been found to be more relevant to tumour invasion. A feature of many transformed cells is an anomalous lysosomal enzyme trafficking system, and concomitant hyper-secretion of some enzymes. The distribution of low pH compartments and lysosomal enzyme-containing compartments was investigated in human breast epithelial cells, and their c-Ha-ras- transformed counterparts. Immunofluorescence and immunoelectron microscopy showed that these compartments have a more peripheral cellular distribution with respect to normal cells, and cathepsins B and D were cell surface-associated. Studies were undertaken to reveal the extracellular matrix degrading ability of c-Ha- ras-transformed cells. Transformed cells exhibited increased degradation of fluorescein-labelled extracellular matrix in serum free medium, and increased motility, and degradation and disruption of extracellular matrix in serum-containing medium. In vitro invasion through artificial basement membrane by transformed cells was investigated using scanning electron microscopy, and was further used to preliminarily identify the proteases involved in invasion by specific inhibition. By this means, greatest inhibition of in vitro invasion was obtained using a specific metalloproteinase inhibitor. Overexpression by transformed cells of a metalloproteinase was detected by gelatin zymography. Together these results suggest that the increased invasive capacity of ras-transformed breast epithelial cells may be largely due to increased metalloproteinase activity.