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

Abstract

Overexpression of cathepsin-D in primary breast cancer has been associated with rapid development of clinical metastasis. To investigate the role of this protease in breast cancer growth and progression to metastasis, we stably transfected a highly metastatic human breast cancer cell line, MDA-MB-231, with a plasmid containing either the full-length cDNA for cathepsin-D or a 535 bp antisense cathepsin-D cDNA fragment. Clones expressing antisense cathepsin-D cDNA that exhibited a 70 -80% reduction in cathepsin-D protein, both intra-and extracellularly compared to controls, were selected for further experiments. These antisense-transfected cells displayed a reduced outgrowth rate when embedded in a Matrigel matrix, formed smaller colonies in soft agar and presented a significantly decreased tumor growth and experimental lung metastasis in nude mice compared with controls. However, manipulating the cathepsin-D level in the antisense cells has no effect on their in vitro invasiveness. These studies demonstrate that cathepsin-D enhances anchorage-independent cell proliferation and subsequently facilitates tumorigenesis and metastasis of breast cancer cells. Our overall results provide the first evidence on the essential role of cathepsin-D in breast cancer, and support the development of a new cathepsin-D-targeted therapy.

Stress-induced apoptosis is impaired in cells with a lysosomal targeting defect but is not affected in cells synthesizing a catalytically inactive cathepsin D

“…Or, conversely, has it got some mitogenic functions as noticed in cancer cells. 50,52 Clearly, further investigations will be required to answer this question. Seemingly conflicting reports on the role of cathepsin D in apoptosis refer to different systems.…”

“…51 Conversely, downregulation of cathepsin D expression by antisense gene transfer was described to inhibit tumor growth and experimental lung metastasis of human breast cancer cells. 52 In general accordance with these studies, the present work demonstrates that stress-induced cell death is not affected in cells isolated from cathepsin D-deficient sheep or in human cells treated with pepstatin A, a well-known inhibitor of cathepsin D. Specifically, we show that cathepsin D is implicated neither in cell death induced by death receptors (TNF or CD95) nor after addition of other classes of apoptotic inducers (doxorubicin, vinblastine, ara-C, gamma radiation, staurosporine, sphingosine, ceramide or naphthazarin). These findings are in good agreement with recent observations that lysosomal cathepsin D is not positively connected with cell death, 22,28,30 but conflict with other reports mentioning that inhibition of cathepsin D suppresses cell death [17][18][19]24 and most particularly with data obtained on fibroblasts from cathepsin D-deficient mice.…”

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).…”

The GH–IGF-I axis and breast cancer

“…It is also unknown whether GH may exert IGF-1 independent effects in this tissue, although GH has been shown to stimulate transcription of c-myc, a proto-oncogene. IGF-1 also regulates the expression of several genes associated with breast tumourigenisis; c-myc, VEGF, progesterone receptor (PgR) and cathepsin D 77 .…”