Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.

βA4 secretion occurs upon processing of amyloid protein precursor (APP) by β‐secretase (N‐terminus of βA4) and γ‐secretase (C‐terminus). To determine the sequence of these activities and the processing intermediate of βA4, we expressed several truncated APP molecules in human HEK‐293 cells. Immunofluorescence and biotinylation studies indicated that full‐length APP or APP lacking the cytosolic domain both were located intracellularly, associated with the cell surface and secreted. APPs truncated after amino acid 40, 42, or 43 of βA4 were not inserted into cell membranes, were found intracellularly but not on the cell surface, and were efficiently secreted into the culture medium. The secretion of APP truncated at amino acid 40 of βA4 occurred without proteolytic processing. Neither βA4 nor P3 (the product of the α‐secretase) was secreted from any of the APP molecules truncated at the γ‐secretase sites. In sharp contrast to this, when the C‐terminal 100 amino acids of APP were expressed (APP truncated at the N‐terminus of βA4), a robust βA4 secretion was observed. Thus, the C‐terminal fragment of APP produced by β‐secretase activity is likely to be the processing intermediate of βA4. © 1996 Wiley‐Liss, Inc.

Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL201–66 isoform with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL201–55, CCL201–52, and a 12-aa C-terminal peptide CCL2059–70. Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1α and TNF-α in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular membranes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas.

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