ENDOCYTOSIS OF PRO-CATHEPSIN-D INTO BREAST-CANCER CELLS IS MOSTLY INDEPENDENT OF MANNOSE-6-PHOSPHATE RECEPTORS

Cathepsin D trafficking is altered in cancer cells, leading to increas ed secretion of the pro-enzyme, which can be reinternalized by the sam e cancer cells and by stromal cells. We studied pro-cathepsin D endocy tosis in two human breast cancer cell lines (MDA-MB231, MCF-7) and in human normal fibroblasts. Pro-enzyme uptake was studied indirectly thr ough immunofluorescence analysis of anti-pro-cathepsin D monoclonal an tibodies internalized in living cells. Both cancer cell lines internal ized the pro-cathepsin D-antibody complex into endosomal compartments in the presence of 10 mM mannose-6-phosphate, Non-malignant fibroblast s, which do not secrete pro-cathepsin D, only internalized anti-cathep sin D antibody when purified pro-cathepsin D was added and this endocy tosis was totally inhibited by mannose-6-phosphate, Cathepsin D endocy tosis in cancer cells was not mediated by lectins or another receptor binding the cathepsin profragment, It was not due to fluid endocytosis , since another protein pS2 secreted by MCF-7 was not endocytosed with its antibody in the same conditions. Double-immunofluorescence and co nfocal microscopy analyses revealed that antibodies specific to pro-ca thepsin D (M2E8) and to the mannose-6-phosphate/IGFII receptor were co -internalized independently in non-permeabilized MDA-MB231 cells and M CF-7 cells, but not in fibroblasts, Moreover, when metabolically label led pro-cathepsin D secreted by MCF-7 or MDA-MB231 cells was incubated with homologous or heterologous non-radioactive cells, the time-depen dent uptake and maturation of the pro-enzyme into fib ro blasts were t otally inhibited by mannose-6-phosphate, whereas they were not in the two breast cancer cell lines, The percentage of mannose-6-phosphate-in dependent binding of radioactively labelled pro-cathepsin D to MDA-MB2 31 cells at 16 degrees C was higher (7-8%) at low pro-cathepsin D conc entration than at high concentration (1.5%), indicating the presence o f saturable binding site(s) at the cell surface that are different fro m the mannose-6-phosphate receptors, We conclude that, in contrast to fibroblasts, breast cancer cells can endocytose the secreted pro-cathe psin D by a cell surface receptor that is different from the mannose-6 -phosphate receptors or other lectins, The nature of this alternative receptor and its significance in the action of secreted pro-cathepsin D remain to be elucidated.