TRANSFECTED CATHEPSIN-D STIMULATES HIGH-DENSITY CANCER CELL-GROWTH BYINACTIVATING SECRETED GROWTH-INHIBITORS

Cathepsin D, a lysosomal protease, is overexpressed in primary breast cancer and associated with increased risk of metastasis. We have shown previously by transfection in rat tumor cells that overexpression of cathepsin D increased both experimental metastasis in nude mice and in vitro proliferation under low-serum conditions. In this study, we use d the transfected cell lines to investigate the mechanism by which cat hepsin D prevents density-dependent arrest of cell proliferation. This effect was not associated with a general alteration of cell-substratu m or cell-cell adhesiveness. As shown by coculture and conditioned med ia experiments, control cells reaching saturation density released inh ibitory activity that was able to prevent the growth of control or cat hepsin D transfectants and decreased the cloning efficiency of normal rat kidney fibroblasts in agar. By contrast, in media from two catheps in D-transfected cell lines, this inhibitory activity was markedly red uced. Cathepsin D overexpression did ndt affect cell sensitivity to th e inhibitor but modified the secretion of several proteins. The increa se in cell density appeared to be due to intracellular maturation of c athepsin D since it was reversed by amine treatment that neutralizes t he pH of acidic compartments within the cells. Moreover, the addition of secreted pro-cathepsin D was unable to increase the saturation dens ity of control clones. Finally, the inhibitory factor was partially ch aracterized as a heat-labile, secreted protein. We conclude that cathe psin D overexpression increases the growth of cancer cells to a higher density via an intracellular mechanism, leading to a decreased secret ion of growth inhibitor(s).