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

SynopsisIn addition to secreted growth factors, acting as autocrine or paracrine mitogens, breast cancer cells secrete other proteins whose function and significance in mammary carcinogenesis may be important. Among them, proteases are particularly interesting since it has been suggested that they play a role in metastatic process, and since at least two of them, the tissue type plasminogen activator and pro-cathepsin D, the precursor of a lysosomal protease, are induced by oestrogens and secreted in excess in some mammary cancer cells.In oestrogen-receptor-positive human breast cancer cell lines (MCF7, ZR75–1), oestrogens stimulate cell proliferation and specifically increase the secretion into the culture medium of a 52,000-dalton (52-kDa) glycoprotein identified as the secreted precursor of a cathepsin D bearing mannose-6-phosphate signals, which is routed to lysosomesviamannose-6-phosphate-IGF-II receptors. We have determined the structure of this procathepsin D by sequencing its complete cDNA sequence, its chromosomal localisation and its transcriptional regulation by oestrogens and other mitogens. In breast cancer cells, pro-cathepsin D production and secretion is much higher and its processing is altered compared to normal mammary epithelial cells in culture.In vitro, pro-cathepsin D acts as an autocrine mitogen on breast cancer cells and can be activated at acidic pH to degrade extracellular matrix, suggesting a role in mediating the effect of oestrogens on tumour growth and invasion. Retrospective clinical studies indicate a significant correlation between high 52-kDa cathepsin D concentrations in the cytosol of primary breast cancer and poor prognosis (Danish Breast Cancer Group, S. Thorpe, Copenhagen). We propose that among the proteases secreted by cancer cells, 52-kDa cathepsin D is important both as a tissue marker in breast cancer and as a potential factor involved in carcinogenesis.

Download Full-text

Endocytosis of pro-cathepsin D into breast cancer cells is mostly independent of mannose-6-phosphate receptors

Journal of Cell Science ◽

10.1242/jcs.111.17.2539 ◽

1998 ◽

Vol 111 (17) ◽

pp. 2539-2549 ◽

Cited By ~ 1

Author(s):

V. Laurent-Matha ◽

M.R. Farnoud ◽

A. Lucas ◽

C. Rougeot ◽

M. Garcia ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Cathepsin D ◽

Breast Cancer Cells ◽

Cancer Cell Lines ◽

Breast Cancer Cell Lines ◽

Mannose 6 Phosphate ◽

Mcf 7

Cathepsin D trafficking is altered in cancer cells, leading to increased secretion of the pro-enzyme, which can be reinternalized by the same cancer cells and by stromal cells. We studied pro-cathepsin D endocytosis in two human breast cancer cell lines (MDA-MB231, MCF-7) and in human normal fibroblasts. Pro-enzyme uptake was studied indirectly through immunofluorescence analysis of anti-pro-cathepsin D monoclonal antibodies internalized in living cells. Both cancer cell lines internalized the pro-cathepsin D-antibody complex into endosomal compartments in the presence of 10 mM mannose-6-phosphate. Non-malignant fibroblasts, which do not secrete pro-cathepsin D, only internalized anti-cathepsin D antibody when purified pro-cathepsin D was added and this endocytosis was totally inhibited by mannose-6-phosphate. Cathepsin D endocytosis 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 confocal microscopy analyses revealed that antibodies specific to pro-cathepsin D (M2E8) and to the mannose-6-phosphate/IGFII receptor were co-internalized independently in non-permeabilized MDA-MB231 cells and MCF-7 cells, but not in fibroblasts. Moreover, when metabolically labelled pro-cathepsin D secreted by MCF-7 or MDA-MB231 cells was incubated with homologous or heterologous non-radioactive cells, the time-dependent uptake and maturation of the pro-enzyme into fibroblasts were totally inhibited by mannose-6-phosphate, whereas they were not in the two breast cancer cell lines. The percentage of mannose-6-phosphate-independent binding of radioactively labelled pro-cathepsin D to MDA-MB231 cells at 16 degrees C was higher (7–8%) at low pro-cathepsin D concentration than at high concentration (1.5%), indicating the presence of saturable binding site(s) at the cell surface that are different from the mannose-6-phosphate receptors. We conclude that, in contrast to fibroblasts, breast cancer cells can endocytose the secreted pro-cathepsin 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.

Download Full-text

Specific Mannose-6-Phosphate Receptor-Independent Sorting of Pro-Cathepsin D in Breast Cancer Cells

Experimental Cell Research ◽

10.1006/excr.1994.1327 ◽

1994 ◽

Vol 215 (1) ◽

pp. 154-163 ◽

Cited By ~ 43

Author(s):

Françoise Capony ◽

Thomas Braulke ◽

Christian Rougeot ◽

Sylvie Roux ◽

Philippe Montcourrie ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Cathepsin D ◽

Breast Cancer Cells ◽

Mannose 6 Phosphate

Download Full-text

Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells.

The Journal of Cell Biology ◽

10.1083/jcb.104.2.253 ◽

1987 ◽

Vol 104 (2) ◽

pp. 253-262 ◽

Cited By ~ 102

Author(s):

F Capony ◽

M Morisset ◽

A J Barrett ◽

J P Capony ◽

P Broquet ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Hormonal Regulation ◽

Cathepsin D ◽

Normal Tissue ◽

Breast Cancer Cells ◽

Immunoperoxidase Staining ◽

Mcf7 Cells ◽

Lysosomal Hydrolases ◽

Mannose 6 Phosphate

We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.

Download Full-text

Large Pore Mesoporous Silica and Organosilica Nanoparticles for Pepstatin A Delivery in Breast Cancer Cells

Molecules ◽

10.3390/molecules24020332 ◽

2019 ◽

Vol 24 (2) ◽

pp. 332 ◽

Cited By ~ 9

Author(s):

Saher Rahmani ◽

Jelena Budimir ◽

Mylene Sejalon ◽

Morgane Daurat ◽

Dina Aggad ◽

...

Keyword(s):

Breast Cancer ◽

Mesoporous Silica ◽

Silica Nanoparticles ◽

Cancer Cells ◽

Cathepsin D ◽

Breast Cancer Cells ◽

Potent Inhibitor ◽

Mesoporous Silica Nanoparticles ◽

Sol Gel ◽

Pepstatin A

(1) Background: Nanomedicine has recently emerged as a new area of research, particularly to fight cancer. In this field, we were interested in the vectorization of pepstatin A, a peptide which does not cross cell membranes, but which is a potent inhibitor of cathepsin D, an aspartic protease particularly overexpressed in breast cancer. (2) Methods: We studied two kinds of nanoparticles. For pepstatin A delivery, mesoporous silica nanoparticles with large pores (LPMSNs) and hollow organosilica nanoparticles (HOSNPs) obtained through the sol–gel procedure were used. The nanoparticles were loaded with pepstatin A, and then the nanoparticles were incubated with cancer cells. (3) Results: LPMSNs were monodisperse with 100 nm diameter. HOSNPs were more polydisperse with diameters below 100 nm. Good loading capacities were obtained for both types of nanoparticles. The nanoparticles were endocytosed in cancer cells, and HOSNPs led to the best results for cancer cell killing. (4) Conclusions: Mesoporous silica-based nanoparticles with large pores or cavities are promising for nanomedicine applications with peptides.

Download Full-text