SPECIFIC MANNOSE-6-PHOSPHATE RECEPTOR-INDEPENDENT SORTING OF PRO-CATHEPSIN-D IN BREAST-CANCER CELLS

The secretion of pro-cathespin D (pro-cath-D) in some human metastatic breast cancer cells (MCF7, MDA/MB231), contrary to normal mammary cel ls, is not increased by ammonium chloride treatment, indicating a mann ose-6-phosphate-independent sorting to lysosomes. By studying a variet y of cell lines and lysosomal enzymes, we show that secretion of newly synthesized pro-cath-D was not mediated by the 46-kDa mannose-6-phosp hate receptor (MPR) and that its resistance to NH4Cl for secretion was specific to cath-D and not to other lysosomal enzymes. This resistanc e appeared to be correlated with the basal hypersecretion of pro-cath- D, but not with its overexpression. By contrast, pro-cath-D secretion was increased by (NHCl)-Cl-4 in fibroblasts and nontumoral epithelial mammary cells, suggesting a specificity for cancer cells. Immunofluore scence staining showed that pro-cath-D, but neither cathepsin B nor be ta-hexosaminidase, accumulated in intracytoplasmic vesicles of cells t reated with ammonium chloride. In pulse-chase experiments and by subce llular fractionation on Percoll gradient, cath-D was found to be sorte d into dense lysosomes whether cells were treated or not by NH4Cl. Tre atment of cells with NH4Cl, however, inhibited processing and maturati on of pro-cath-D, which was also observed in light vesicles in the abs ence of NH4Cl. Part of pro-cath-D, but not processed enzyme, was also found to be membrane associated in saponin-permeabilized cells. We con clude that in breast cancer cells, the MPR-independent pathway of pro- cath-D to lysosome is predominant compared to normal cells and other l ysosomal enzymes. This alternative pathway should therefore be conside red, in addition to MPR, to explain pro-cath-D sorting and activation in breast cancer cells. (C) 1994 Academic Press, Inc.