Endocytosis of pro-cathepsin D into breast cancer cells is mostly independent of mannose-6-phosphate receptors.
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.
INSERM Unité Hormones et Cancer (U 148), Université de Montpellier 1, 34090 Montpellier, France.
V
V
Laurent-Matha
M R
MR
Farnoud
A
A
Lucas
C
C
Rougeot
M
M
Garcia
H
H
Rochefort
D000906:Antibodies / Q000378:metabolism; D000918:Antibody Specificity; D001693:Biological Transport, Active / Q000187:drug effects; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D002402:Cathepsin D / Q000032:analysis / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D004792:Enzyme Precursors / Q000032:analysis / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry / Q000201:enzymology; D008360:Mannosephosphates / Q000494:pharmacology; D011485:Protein Binding; D017527:Receptor, IGF Type 2 / Q000032:analysis / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured
D000906:Antibodies; D004792:Enzyme Precursors; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D013462:Sulfur Radioisotopes; C027693:mannose-6-phosphate; C058345:procathepsin D; D002402:Cathepsin D