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

Abstract

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 a ects proliferation remains, however, unknown. In order to determine whether proteolytic activity is necessary, we abolished its enzymatic activity using site-directed mutagenesis followed by stable transfection in 3Y1-Ad12 cancer cells. Substitution of the aspartic acid residue 231 by an asparagine residue in its catalytic site abrogated the cathepsin-D proteolytic activity but did not a ect its expression level, processing or secretion. However, like wild-type cathepsin-D, this mutated catalytically-inactive cathepsin-D retained its capacity 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 mocktransfected cells, of either conditioned media containing the wild-type or the mutated pro-cathepsin-D, or of the puri®ed mutated pro-cathepsin-D, partially mimicked the mitogenic activity of the transfected cathepsin-D, indicating a role of the secreted pro-enzyme. Moreover, addition of two anti-cathepsin-D antibodies on the cathepsin-D transfected cells inhibited their proliferation, suggesting an action of the secreted 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 the interaction of pro-cathepsin-D with the mannose-6-phosphate receptors. Our results altogether demonstrate that a mutated cathepsin-D devoid of catalytic activity is still mitogenic and suggest that it is acting extracellularly by triggering directly or indirectly a yet unidenti®ed cell surface receptor. Oncogene (2001) 20, 6920 ± 6929.

Down-regulation of cathepsin-D expression by antisense gene transfer inhibits tumor growth and experimental lung metastasis of human breast cancer cells

“…Previous studies in two cellular models, MCF-7 cells and rat embryo tumor cells, indicated that this mitogenic activity could be due to cath-D action as a protein ligand interacting with a mitogenic receptor (Fusek and Vetvicka, 1994;Glondu et al, 2001; reviewed by Rochefort and Liaudet-Coopman, 1999), or to the proteolytic activity of the enzyme. In rat tumor cells transfected with human cath-D cDNA, two distinct cath-D mitogenic activities were observed as a function of the cell density (Garcia et al, 1990).…”

“…The direct role of cath-D in cancer metastasis was first demonstrated in rat tumor cells in which transfectioninduced cath-D over-expression increased their metastatic potential in vivo (Garcia et al, 1990;Liaudet et al, 1994). In this rat tumor model, the cath-D mechanism responsible for metastasis stimulation seemed to have a positive effect on cell proliferation, favoring the growth of micro-metastases, rather than increasing the invasive potential (Garcia et al, 1990;Liaudet et al, 1994Liaudet et al, , 1995Glondu et al, 2001).…”

Cathepsin D is partly endocytosed by the LRP1 receptor and inhibits LRP1-regulated intramembrane proteolysis

“…We checked these results by investigating the effect of recombinant proteolytically inactive D231N pro-cath-D on the production of endogenous LRP1b-CTF in HMF .1(-) plasmids using Lipofectamine (Gibco-BRL, Life Technologies SAS, Villebon sur Yvette, France). pcDNA3.1(-)cath-D expression plasmid encoding human pre-pro-cath-D has previously been described (Vignon et al, 1986;Glondu et al, 2001Glondu et al, , 2002Berchem et al, 2002;Laurent-Matha et al, 2005;Hu et al, 2008). fibroblasts ( Figure 3C, panel a).…”

“…Cath-D affects both the cancer cells and the stromal cells of the tumor microenvironment. Human pre-pro-cath-D cDNA transfected in cancer cells promotes cancer cell proliferation, tumor angiogenesis, and tumor growth and metastasis (Glondu et al, 2001;Berchem et al, 2002). Human pre-pro-cath-D cDNA transfected in cath-DÀ/À MEF (mouse embryonic fibroblast) cells induces fibroblast outgrowth (LaurentMatha et al, 2005).…”

“…Human secreted pro-cath-D stimulates breast cancer cell proliferation (Vignon et al, 1986;Fusek and Vetvicka, 1994;Vetvicka et al, 1994;Ohri et al, 2008), fibroblast outgrowth (Laurent-Matha et al, 2005) and angiogenesis (Hu et al, 2008). We have shown that a mutated catalytically inactive version of cath-D (D231N) is still mitogenic for tumor cells and fibroblasts (Glondu et al, 2001;Berchem et al, 2002;Laurent-Matha et al, 2005). We recently discovered that pro-cath-D is the first ligand that binds to the extracellular domain of the b-chain of the LDL receptor-related protein-1 (LRP1) in fibroblasts (Beaujouin et al, 2010).…”