A mutated cathepsin-D devoid of its catalytic activity stimulates the growth of cancer cells

Cathepsin-D, a lysosomal aspartyl proteinase, is highly secreted by breast cancer cells and its over-expression by transfection stimulates cancer cell proliferation. The mechanism by which this protease affects proliferation remains, however, unknown. In order to determine whether proteolytic activi ty is necessary, we abolished its enzymatic activity using site-directed mu tagenesis followed by stable transfection in 3Y1-Ad12 cancer cells. Substit ution of the aspartic acid residue 231 by an asparagine residue in its cata lytic site abrogated the cathepsin-D proteolytic activity but did not affec t its expression level, processing or secretion. However, like wild-type ca thepsin-D, this mutated catalytically-inactive cathepsin-D retained its cap acity to stimulate proliferation of cells embedded in Matrigel or collagen I matrices, colony formation in soft agar and tumor growth in athymic nude mice. Addition on the mock-transfected cells, of either conditioned media c ontaining the wild-ty pe or the mutated pro-cathepsin-D, or of the purified mutated pro-cathepsin-D, partially mimicked the mitogenic activity of the transfected cathepsin-D, indicating a role of the secreted pro-enzyme. More over, addition of two anti-cathepsin-D antibodies on the cathepsin-D transf ected cells inhibited their proliferation, suggesting an action of the secr eted pro-cathepsin-D via an autocrine loop. A synthetic peptide containing the 27-44 residue moiety of the cathepsin-D pro-fragment was, however, not mitogenic suggesting that a receptor for the pro-fragment was not involved. Furthermore, the cathepsin-D mitogenicity was not blocked by inhibiting th e interaction of pro-cathepsin-D with the mannose-6-phosphate receptors. Ou r results altogether demonstrate that a mutated cathepsin-D devoid of catal ytic activity is still mitogenic and suggest that it is acting extracellula rly by triggering directly or indirectly a yet unidentified cell surface re ceptor.