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.

REFERENCES

Brouillet
J. P.
,
Spyratos
F.
,
Hacene
K.
,
Fauque
J.
,
Freiss
G.
,
Dupont
F.
,
Maudelonde
T.
,
Rochefort
H.
(
1993
).
Immunoradiometric assay of pro-cathepsin D in breast cancer cytosol: Relative prognostic value versus total cathepsin D.
Eur. J. Cancer
29
,
1248
1251
Brouillet
J. P.
,
Dufour
F.
,
Lemamy
G.
,
Garcia
M.
,
Schlup
N.
,
Grenier
J.
,
Manni
J. C.
,
Rochefort
H.
(
1997
).
Increased cathepsin D level in serum of metastatic breast cancer patients detected with a specific pro-cathepsin-D immunoassay.
Cancer
79
,
2132
2136
Cailleau
R.
,
Young
R.
,
Olive
M.
,
Reeves
W. J.
(
1974
).
Breast tumor cell lines from pleural effusions.
J. Natl. Cancer Inst
53
,
661
674
Capony
F.
,
Morisset
M.
,
Barrett
A. J.
,
Capony
J.P.
,
Broquet
P.
,
Vignon
F.
,
Chambon
M.
,
Louisot
P.
,
Rochefort
H.
(
1987
).
Phosphorylation, glycosylation and proteolytic activity of the 52 kD estrogen-induced protein secreted by MCF7 cells.
J. Cell Biol
104
,
253
262
Capony
F.
,
Braulke
T.
,
Rougeot
C.
,
Roux
S.
,
Montcourrier
P.
,
Rochefort
H.
(
1994
).
Specific Mannose-6-Phosphate receptor-independant sorting of pro-cathepsin D in breast cancer cells.
Exp. Cell Res
215
,
154
163
Capony
F.
,
Rougeot
C.
,
Montcourier
P.
,
Cavailles
V.
,
Salazar
G.
,
Rochefort
H.
(
1989
).
Increased secretion, altered processing, and glycosylation of pro-cathepsin D in human mammary cancer cells.
Cancer Res
49
,
3904
3909
Chapman
R. E.
,
Munro
S.
(
1994
).
Retrieval of TGN proteins from the cell surface requires endosomal acidification.
EMBO J
13
,
2305
2312
Confort
C.
,
Rochefort
H.
,
Vignon
F.
(
1995
).
Insulin-like growth factors (IGFs) stimulate the release of 1-antichymotrypsin and soluble IGF-II/mannose-6-Phosphate receptor from MCF-7 breast cancer cells.
Endocrinology
136
,
3759
3766
Corvera
S.
,
Folander
K.
,
Clairmont
K. B.
,
Czech
M. P.
(
1988
).
A highly phosporylated subpopulation of insulin-like growth factor II/mannose 6-Phosphate receptors is concentrated in a clathrin-enriched plasma membrane fration.
Proc. Natl. Acad. Sci. USA
85
,
7567
7571
De Souza
A.
,
Hankins
G. R.
,
Washington
M. K.
,
Orton
T. C.
,
Jirtle
R. L.
(
1995
).
M6P/IGF2R gene is mutated in human hepatocellular carcinomas with loss of heterozygosity.
Nature Genetics
11
,
447
449
Diment
S.
,
Leech
M. S.
,
Stahl
P. D.
(
1988
).
Cathepsin D is membrane-associated in macrophages endosomes.
J. Biol. Chem
263
,
6901
6907
Freiss
G.
,
Vignon
F.
,
Rochefort
H.
(
1988
).
Characterization and properties of two monoclonal antibodies specific for the M r52,000 precursor of cathepsin D in human breast cancer cells.
Cancer Res
48
,
3709
3715
Fusek
M.
,
Vetvicka
V.
(
1994
).
Mitogenic function of human procathepsin D: the role of the propeptide.
Biochem J
303
,
775
780
Garcia
M.
,
Capony
F.
,
Derocq
D.
,
Simon
D.
,
Pau
B.
,
Rochefort
H.
(
1985
).
Characterization of monoclonal antibodies to the estrogen-regulated M r52,000 glycoprotein and their use in MCF-7 cells.
Cancer Res
45
,
709
716
Gottesman
M. M.
(
1990
).
Introduction: Do proteases play a role in cancer?.
Semin. Cancer Biol
1
,
97
98
Griffiths
J. R.
(
1991
).
Are cancer cells acidic?.
Br. J. Cancer
64
,
425
427
Harter
C.
,
Mellman
I.
(
1992
).
Transport of the lysosomal membrane glycoprotein Igp120 (Igp-A) to lysosomes does not require appearance on the plasma membrane.
J. Cell Biol
117
,
311
325
Isodoro
C.
,
Baccino
F. M.
,
Hasilik
A.
(
1997
).
Mis-sorting of procathepsin D in metastogenic tumor cells is not due to impaired synthesis of the phosphomannosyl signal.
Int. J. Cancer
70
,
561
566
Kiess
W.
,
Haskell
J. F.
,
Lee
L.
,
Greenstein
L. A.
,
Miller
B. E.
,
Aarons
A. L.
,
Rechler
M. M.
,
Nissley
S. P.
(
1987
).
An antibody that blocks insulin-like growth factor (IGF) binding to the type II IGF receptor is neither an agonist nor an inhibitor of IGF-stimulated biologic responses in L6 Myoblasts.
J. Biol Chem
262
,
12745
12751
Klumperman
J.
,
Hille
A.
,
Veenendaal
T.
,
Oorschot
V.
,
Stoorvogel
W.
,
Von Figura
K.
,
Geuze
H. J.
(
1993
).
Differences in the endosomal distribution of the two Mannose 6-Phosphate receptors.
J. Cell. Biol
121
,
997
1010
Liaudet
E.
,
Garcia
M.
,
Rochefort
H.
(
1994
).
Cathepsin D maturation and its stimulatory effect on metastasis are prevented by addition of KDEL retention signal.
Oncogene
9
,
1145
1154
Mathieu
M.
,
Vignon
F.
,
Capony
F.
,
Rochefort
H.
(
1991
).
Estradioldown-regulates the Mannose-6-Phosphate/insulin-like growth factor-II receptor gene and induces cathepsin-D in breast cancer cells: A receptor saturation mechanism to increase the secretion of lysosomal proenzymes.
Molecular Endocrinol
5
,
815
822
Maudelonde
T.
,
Rochefort
H.
(
1987
).
A 51 progestin-regulated protein secreted by human endometrial cells in primary culture.
J. Clin. Endocrinol. Metab
64
,
1294
1301
McIntyre
G. F.
,
Erickson
A. H.
(
1993
).
The lysosomal proenzyme receptor that binds procathepsin L to microsomal membranes at pH 5 is a 43-kDa integral membrane protein.
Proc. Nat. Acad. Sci. USA
90
,
10588
10592
Montcourrier
P.
,
Mangeat
P.H.
,
Valembois
C.
,
Salazar
G.
,
Sahuquet
J.
,
Duperray
C.
,
Rochefort
H.
(
1994
).
Characterization of very acidic phagosomes in breast cancer cells and their association with invasion.
J. Cell Sci
107
,
2381
2391
Pohlmann
R.
,
Wendland
M.
,
Boeker
C.
,
Von Figura
K.
(
1995
).
The two Mannose-6-Phosphate receptors transport distinct complements of lysosomal proteins.
J. Biol. Chem
270
,
27311
27318
Reilly
J. J.
Jr.
,
Chen
P.
,
Zenzius
L.
,
Sailor
,
Mason
R. W.
,
Chapman
H. A.
Jr.
(
1990
).
Uptake of extracellular enzyme by a novel pathway is a major determinant of cathepsin L levels in human macrophages.
J. Clin. Invest
86
,
176
183
Rijnboutt
S.
,
Kal
A.
,
Geuze
H.
,
Aerts
H.
,
Strous
G.
(
1991
).
Mannose-6-Phosphate-independent targeting of cathepsin D to lysosomes in hepG2 cells.
J. Biol. Chem
266
,
23586
23592
Rio
M. C.
,
Bellocq
J. P.
,
Gairard
B.
,
Rasmussen
U. B.
,
Krust
A.
,
Koehl
C.
,
Calderoli
H.
,
Schiff
V.
,
Renaud
R.
,
Chambon
P.
(
1987
).
Specific expression of the pS2 gene in subclasses of breast cancers in comparisonwith expression of the estrogen and progesterone receptors and the oncogene ERBB2.
Proc. Nat. Acad. Sci. USA
84
,
9243
9247
Rochefort
H.
,
Capony
F.
,
Garcia
M.
,
Cavailles
V.
,
Freiss
G.
,
Chambon
M.
,
Morisset
M.
,
Vignon
F.
(
1987
).
Estrogen-induced lysosomal proteases secreted by breast cancer cells. A role in carcinogenesis?.
J. Cell Biochem
35
,
17
29
Sloane
B. F.
(
1990
).
Cathepsin B and cystatins: evidence for a role in cancer progression.
Semin. Cancer Biol
1
,
137
152
Soule
H. D.
,
Vazquez
J.
,
Long
A.
,
Albert
S.
,
Bernnan
M. B.
(
1973
).
A human cell line from a pleural effusion derived from a breast carcinoma.
J. Nat. Cancer Inst
51
,
1409
1416
Unkeless
J. C.
,
Scigliono
E.
,
Freedman
F. H.
(
1988
).
Structure and function of human and murine receptors for IgG.
Ann. Rev. Immunol
6
,
251
281
Vetvicka
V.
,
Vetvickava
J.
,
Hilbery
I.
,
Voburka
Z.
,
Fusek
M.
(
1997
).
Analysis of the interaction of pro-cathepsin-D activation peptide with breast cancer cells.
Int. J. Cancer
73
,
403
409
Vignon
F.
,
Capony
F.
,
Chambon
M.
,
Freiss
G.
,
Garcia
M.
,
Rochefort
H.
(
1986
).
Autocrine growth stimulation of the MCF7 breast cancer cells by the estrogen-regulated 52K protein.
Endocrinology
118
,
1537
1545
Von Figura
K.
,
Hasilik
A.
(
1986
).
Lysosomal enzymes and their receptors.
Ann. Rev. Biochem
55
,
167
193
Westley
B.
,
Rochefort
H.
(
1980
).
A secreted glycoprotein induced by estrogen in human breast cancer cell lines.
Cell
20
,
352
362
Zhao
Y.
,
Escot
C.
,
Maudelonde
T.
,
Puech
C.
,
Rouanet
P.
,
Rochefort
H.
(
1993
).
Correlation between Mannose-6-Phosphate/IGFII receptor and cathepsin D RNA levels by in situ hybridization in benign and malignant mammary tumors.
Cancer Res
53
,
2901
2905
Zhu
Y.
,
Conner
G. E.
(
1994
).
Intermolecular association of lysosomal protein precursors during biosynthesis.
J. Biol. Chem
269
,
3846
3851
This content is only available via PDF.