Breast Cancer Research and Treatment 16: 3-13, 1990. © 1990 KluwerAcademic Publishers. Printed in the Netherlands.

12th San Antonio Breast Cancer Symposium - Plenary lecture

Cathepsin D in breast cancer Henri Rochefort UnitO Hormones et Cancer (U148) INSERM, University of Montpellier 1, 60, rue de Navacelles, 34090 Montpellier France

Key words: estrogen, gene regulation, lysosomes, metastasis, prognostic marker, protease

Summary Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive and negative breast cancer cell lines, the mRNA coding for pro-cathepsin D is overexpressed and sorting and maturation of the pro-enzyme are altered, leading to accumulation of the active proteinase in large endosomes and to secretion of the precursor (52K protein). In MCF7 cells, the cathepsin D mRNA is induced directly and transcriptionally by estrogens and indirectly by growth factors. In vitro, pro-cathepsin D is an autocrine mitogen on breast cancer cells and can be auto-activated to degrade extracellular matrix and proteoglycans and to activate other proteinases in acidic microenvironments. In patients, there is a significant correlation between high cathepsin D concentrations in the cytosol of primary breast cancer and development of metastasis. This marker is independent of other prognostic factors and appears to be particularly useful in lymph node-negative tumors. These results suggest that overexpression and possible derouting of cathepsin D plays an important role in invasion and metastasis of breast cancer.

Introduction Human breast cancers are characterized by their high sensitivity to estrogens and antiestrogens (in 30 to 50% of patients) [1] and by the frequency of early metastasis. The understanding of molecular mechanisms controlling metastasis may lead to the development of new prognostic markers and new therapeutic approaches aimed at preventing or curing these metastases. We have used estrogen-responsive and hormone-resistant metastatic human breast cancer cell lines in culture, in order to define which secreted proteins may be important in controlling tumor growth and invasion. Since estrogens stimulate breast cancer growth in vivo, good candidates included secreted proteins induced by estrogens and inhibited by antiestrogens, having

the potential to act as autocrine and/or paracrine factors [2-4]. Since the early 1980s, several estrogen-induced proteins and peptides which are accumulated in or secreted by MCF7 cells have been described (Fig. 1). Among them, the '24K protein' has been identified as a 27K heat shock protein [5], and the pS2 protein as a member of a family of pancreatic spasmolytic peptides secreted by normal stomach and intestinal mucosa [6]. Our laboratory has been engaged in the study of a 52K secreted protein because this protein was initially found to be mitogenic following its purification [7], and also to be induced by the antiestrogen tamoxifen in several antiestrogen resistant cell lines, but not in the tamoxifen growth inhibited wild-type MCF7 cells [8].

Address for offprints: H. Rochefort, Unit6 Hormones et Cancer (U 148) INSERM, University of Montpellier 1, 60, rue de Navacelles, 34090 Montpellier, France


H Rochefort




o(t anti chym.




p $2 protein

Fig. 1. Estrogen-regulated proteins and pepfides in human breast cancer cells. Estrogens stimulate the synthesisof several proteins via their nuclear receptor (RE). Someproteins, whose functions are unknown, were first identified by labeling and SDS-PAGE analysisand designated by their molecularweight under denaturating conditions. In some cases, the corresponding mRNAs were also found to accumulate. Several of the secreted proteins and peptides recoveredin culture media conditioned by estradiol-treated-MCF7cellsare suspectedof being the agents that stimulate cell proliferation and tumor invasion. RP = progesterone receptor, a~ antichym= antichymotrypsin. Modified by permissionfrom Ref. 42. The 52K protein is a secreted pro-cathepsin D

The critical steps for identifying the 52K protein as a pro-cathepsin D (Fig. 2) were to prepare monoclonal antibodies [9], to purify this secreted protein [10] and related cellular proteins, and to study its co- and post-translational modifications (glycosylation and phosphorylation) [11]. As shown in Fig. 3, after cultured MCF7 cells were exposed to 32p, the 52K protein was intensely labeled. However, most of this label was removed by endoglycosidase-H treatment, which deletes two N-glycosylated chains of the protein. Mannose-6-phosphate signals were identified on these chains [11], indicating that the protein was a lysosomal hydrolase [12]. The purified secreted 52K protein and the corresponding cellular proteins (52K, 48K, 34K, 14K) were then identified as being different forms of cathepsin D [11, 13], based on their proteolytic activity at acidic pH, their inhibition by pepstatin, their immunoreactivities, and the sequence of the first 15 amino acids. Cathepsin D (E.C. is an aspartyl endo proteinase, like renin and pepsin [14]. It is an ubiq-

uitous and major lysosomal protease whose normal function is to degrade protein in lysosomes at an acidic pH. Its substrate specificity is broad, with a preference for high molecular weight proteins. Its optimal pH, although acidic, varies according to the nature of the substrate. Its activity is specifically inhibited by pepstatin, which binds to and blocks the active site. The amino acid structure of cathepsin D has been inferred by sequencing its cDNA from human liver and spleen [15]; its glycosylation is not precisely known, although it bears 2 N-linked oligosaccharide chains with mannose-6-phosphate signals at their extremities. These signals are responsible for binding to the mannose-6-phosphate receptors which carry lysosomal enzymes from the Golgi network to lysosomes via endosomes; there acidic pH enables the complex to dissociate, resulting in recycling the receptor to the Golgi and releasing cathepsin D into the lysosomes [12, 16]. Proteases have long been thought to play a role in the invasion and metastasis of cancer cells which over-produce and secrete a series of different proteases, involved in a cascade of proteolytic events [17-20]. One major difficulty is to define from these enzymes (tissue and urokinase type plasminogen activator, collagenases, other metallo proteinases, cathepsins, elastase, etc...) which proteases are causally involved in human carcinogenesis. It was surprising to find high secretion of procathepsin D in breast cancer cell lines, since neutral proteinase, rather than acidic lysosomal proteinase, has mainly been proposed as being a factor in carcinogenesis. Moreover, estrogen regulation of pro-cathepsin D secretion could have simply been an artefact of some permanent cell lines without any biological relevance to the in vivo situation in breast cancer patients. We therefore decided to use a multidisciplinary approach to study the biological and clinical significance of cathepsin D in breast cancer.

Structure of the cathepsin D of breast cancer cells compared to normal cathepsin D

In order to define the complete amino acid sequence of cathepsin D in MCF7 cells and compare

Cathepsin D


Man 6P Man

DI1E2 M~;A3 Man DTE3 ~.

M2E8 DgH8


-::2: ~




N 52K

> /,8K

pro- coth D

C 3~K + I L K


(tysosomes) ,> m a t u r e

( 2


Fig. 2. Structure and processing of the pro-cathepsin D (52K protein) of MCF7 cells. In the cell, pro-cathepsin is successively processed into an intermediate 48K active enzyme and a two chain (34K + 14K) mature enzyme, which normally function in the lysosomes. Two N-glycosylated oligosaccharide chains which bear an accessible mannose-6-P signal (Man-6-P) are represented. The sugar composition of each chain and the number of Man-6-Ps per chain are not known. The schematic position of the monoclonal antibodies used in ELISA or IRMA assays is represented. Total cathepsin D is currently assayed with D7E3 and M1G8 antibodies, and pro-cathepsin D with M2E8 and M1G8 antibodies. Modified by permission from Ref. 42.

it to that of normal human cells [15], we used monoclonal antibodies to the secreted 52K protein from MCF7 cells and a synthetic oligonucleotide obtained from partial sequencing of the protein, and screened a Xgt11 cDNA library of MCF7 cells, which was kindly given to us by Pierre Chambon (Strasbourg). Four clones were isolated and sequenced, covering the whole 52K mRNA coding sequence. Comparison of this sequence with that of normal human kidney pro-cathepsin D showed only 5 nucleotide changes, involving only one amino acid substitution (Ala to Val) in the pro-fragment [21]. It is not yet known whether this change is of general importance in cancer cells or if it is rather due to trivial polymorphism, since a different amino acid change has been found on cathepsin D cDNA cloned from the ZR-75-1 estrogen receptor positive cell line, (Fig. 3) [22]. The cathepsin D gene was found to be normally located at the extremity of the short arm of chromosome 11, close to the H-ras oncogene. Southern blot analysis did not show any obvious differences in the digestion pattern of cathepsin D of breast cancer vs. normal tissue (P. Augereau, unpublished results). However, since cathepsin D is a glycoprotein, differences on the glycosylated chains are possible as suggested by isoelectric focusing analysis of procathepsin D from normal and mammary cancer

cells, showing major microheterogeneities. 'Breast cancer' cathepsin D contains more acidic forms than 'normal' cathepsin D. The difference disappears following endoglycosidase H treatment, indicating that it is located on the N-glycosylated chains [11].

Altered processing of cathepsin D in breast cancer cells

Pro-cathepsin D processing in normal human mammary epithelial cells has been comparatively studied with that in breast cancer cells [23]. Normal mammary cells collected from patients undergoing reduction mammoplasty were purified after collagenase digestion and cultured on plastic at the same growth rate as MCF7 cells. According to pulsechase labeling experiments, and as in human fibroblasts, most of the precursor (52K) was found to be routed to lysosomes and processed rapidly into a mature form (34K + 14K) via production of an intermediate form (48K) (Fig. 2). Negligible amounts of the pro-form were accumulated in the cells or secreted. However, in several hormone dependent (MCF7, ZR-75-1) and independent (MDA-MB-231, BT29, etc.) breast cancer cell lines, processing was delayed, the proportion of cathepsin D secretion was markedly increased (up

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decreased activities of processing protease(s) involved in this maturation procedure.

Gene expression of cathepsin D and its regulation in breast cancer cells

66K52K 45K31K-



EH 32p





3HMan 35SMet

Fig. 3. Labelling of oligosaccharide and polypeptide components of the 52K protein. Estrogen-treated MCF7 cells were labeled with either 35S-methionine,32p H3PO4, or 3H-mannose. Media were immunoprecipitated with the M1G8 antibody to the 52K protein and analyzed by SDS-polyacrylamide gel electrophoresis. The immunoprecipitated secreted 52K protein was digested (+) or not ( - ) with endoglycosidase H (EH) and the TCA-precipitated proteins were electrophoresed. Reproduced by permission from Ref. 11.

to 50%), and 52K and 48K forms accumulated in the cells. The increased secretion of pro-cathepsin in cancer cells has also been observed with cathepsin L and B. A second major difference between normal and cancerous cells is that the cytosol concentration of total cathepsin D is generally markedly increased in cancer cells (see below). Derouting may be due to slight differences in the structure of pro-cathepsin D, to qualitative differences in the interaction with the mannose-6-P receptor, to the large increased production of procathepsin D that may overload the binding and transporting capacity of this receptor, and/or to the

Overproduction of cathepsin D in breast cancer cells was initially observed at the protein level using immunoperoxidase staining [24] and ELISA of breast cancer cytosols. Generally breast cancer cells produce from 2 to 30-fold more cathepsin D than normal mammary cells growing at the same rate [23]. Using a cDNA probe, similar overexpression was observed at the mRNA level [25, 26] (Fig. 4). In estrogen receptor positive breast cancer cell lines, there is a low constitutive accumulation of a 2.2 kb mRNA which is markedly increased by estrogen treatment. In estrogen receptor negative breast cancer cell lines, there is a high constitutive level of the mRNA, which may explain the absence of correlation between cathepsin D levels and estrogen receptor status in primary breast cancer tisshe.

Another difference between normal and cancer cells may involve the regulation of cathepsin D gene expression. The 52K pro-cathepsin D of breast cancer cells was initially described because of the dramatic effect of estrogen in stimulating its secretion [4]. To determine the level of this regulation, a cathepsin D cDNA probe was used to quantify cathepsin D mRNA by hybridization after Northern blotting. The accumulation of 2.2kb cathepsin D mRNA was increased approximately 10-fold by estradiol treatment of MCF7 cells, but not by tamoxifen [25]. The increase was direct, not inhibited by cycloheximide, and mostly due to the stimulation of mRNA synthesis, as demonstrated by run-on transcription assays. Estrogen receptor ligands were the only steroids to induce this mRNA, and their inductive activity parallelled their affinity for the estrogen receptor and their mitogenic activities. However, other mitogens, such as IGF-I, EGF, and basic FGF, are also able to increase steadystate concentrations of 2.2 kb cathepsin D mRNA in MCF7 cells [25]. The mechanism of induction by

Cathepsin D





11121 ,


i = = ,







52K-9 eDNA


b B r e a s t c a n c e r cell lines RE+










Normal, mammary cells



BT20 -





52K-9 36B4 -

Fig. 4. Increased expression of cathepsin D mRNA in breast cancer cells compared to normal mammary cells, a. The 52K-9 cDNA probe isolated from MCF7 cells is shown under cathepsin-D mRNA from normal kidney cells according to Faust et al. [15]. The open boxes represent the coding sequence and correspond to the signal sequence (1), the pro-sequence (2) and the sequence of the mature enzyme (3, 4). Coordinates of the coding sequence of normal cathepsin D (from 52 to 1287) and of the terminal nucleotides of the 52K-9 clone are indicated. The internal deletion (dotted line from 192 to 295) in this cDNA clone is a cloning artefact. Sequence analyses of 52K-9 and other clones indicate 99% homology with normal kidney cathepsin D [15]. b. Northern blot analysis of pro-cath-D mRNA from cancer and normal mammary cells. RNA was extracted from four breast cancer cell lines and from normal mammary epithelial cells obtained from 3 different patients. Breast cancer cells were cultured in medium without ( - ) or with (+) 10 nM estradiol (E2). Total RNA (40/zg) was analyzed by Northern blotting. Hybridization was done with 52K-9 cDNA to probe the 2.2 kb pro-cath-D mRNA. The fluorographs were exposed for 16 h (breast cancer) and 32 h (normal). The amount of RNA analyzed in each track was controlled using the constant cDNA probe 36B4. R E + : Two estrogen-receptor-positive cell lines. R E - : Two estrogen-receptor-negative cell lines. Partly reproduced by permission from Ref. 26.

growth factors differs from that by estradiol since it is inhibited by cycloheximide. This suggests that growth factors act indirectly by modulating the synthesis of a protein involved in the regulation of cathepsin D mRNA. Thus, in breast cancer cells, estrogens induce both growth factors, such as TGFa and IGF-I [3], and cathepsin D, which can also be induced by growth factors. The effect of antiestrogen tamoxifen on the cathepsin D gene varies according to the cell line studied [8]. In vitro, induction by tamoxifen was observed in three ER-positive antiestrogen-resistant variants (R27, RTx6, and LY2). However, tamoxifen was found to act in vivo as an estrogen-agonist

for cathepsin D gene expression within the first three weeks of treatment of post-menopausal patients [27]. This increase, probably due to the estrogenic (flare) activity of tamoxifen, indicates that cathepsin D is also regulated in vivo by estrogens in patients and suggests that this indication may play a role in mediating the effect of estrogens on growth and invasiveness of the tumor. It is interesting to note that in breast cancer cells and uterine cells, both of which contain functional estrogen and progesterone receptors, the nature of the steroid inducing cathepsin D is different. In normal human endometrium, in rat uterus, and in the Ishikawa human endometrial cancer cell line,


H Rochefort

estrogens are unable to stimulate cathepsin D expression [28, 29]. By contrast, progesterone is active both in the rat uterus and in human endometrium. These differences may indicate a difference in transcriptional factors or hormone-responsive elements controlling the pro-cathepsin D gene in mammary and uterine cells. Studies are in progress to define which hormone, estrogen or progesterone, could potentially stimulate cathepsin D gene expression in normal human mammary glands. A change of hormonal regulation of this gene from progesterone responsiveness to estrogen responsiveness could have major consequences on the extent of expression of the cathepsin D gene after menopause when no more progesterone is secreted, whereas some estrogens and adrenal androgens (5-androstenediol) acting on the estrogen receptor are still present.

Clinical prognostic value of cathepsin D in breast tumors

Due to the development of a panel of monoclonal antibodies, total cathepsin D and pro-cathepsin D (52K) are detectable in situ by immunoperoxidase staining of frozen tissue sections. They can also be assayed in the cytosol of tumor biopsies using a

solid-phase double-determinant immunoassay (ELISA or IRMA). It was therefore possible to verify whether cathepsin D concentration in primary breast cancer was correlated with the degree of aggressiveness of this cancer. It is known that at least 20% of node-negative breast cancer will recur and cause metastasis. Other prognostic markers are therefore needed in order to determine more accurately which patients will benefit from adjuvant therapy. Since no endogeneous cathepsin D inhibitors are known, contrary to the case for most neutral proteinases, the assay of cathepsin D may reveal its potential as an active protease.

Immunohistochemistry of frozen sections Using the D7E3 monoclonal antibody, cathepsin D was detected in frozen sections of several human tissues by the peroxidase-antiperoxidase technique of Sternberger [24]. Most of the staining was granular in the cytoplasm, due to lysosomes and possibly endosomes. Increased expression of cathepsin D was observed in some melanomas and breast cancers. Strong immunostaining was observed in 43% of 127 biopsies of benign mastopathies (mostly cysts over 3 mm in diameter and ductal hyperplasia). When histological types were pooled into pro-

Table 1. First clinical studies on the prognostic value of cytosolic cathepsin D in breast cancer.

1. 2. 3. 4. 5. 6. 7. 8.

Place and references

Number of patients


Montpellier Maudelonde et al. [33] Fibiger, Copenhagen Thorpe et al. [34] St-Cloud Spyratos et al. [35] San Antonio Tandon et al. [36] Montpellier Brouillet et al. [37] Dublin Duffy et al. [39] Nice Namer et al. [38] Marseille Romain et al. [40]


Independent from other prognostic markers


Shorter relapse-free survival in pre- and postmenopausal patients


Shorter metastasis-free survival in both node-negative and node-positive

200 140

Shorter relapse-free and overall survival in node-negative (34K mature form only) Independent of neu-erb-B-2 and int-2, correlated with c-myc


Correlated with urokinase plasminogen activator; shorter overall survival


Shorter overall survival in node-positive


Shorter survival in node-positive

Cathepsin D liferative (high-risk) and nonproliferative (lowrisk) lesions, according to Dupont and Page, we found a significant correlation between proliferation and total cathepsin D staining. Further studies with clinical follow up are required before being able to conclude that cathepsin D staining is useful in predicting high-risk mastopathies. Cathepsin D staining was also observed in breast cancer tissue, where it was generally less homogeneous than in proliferative benign mastopathies or in cells bordering the lumen of large cysts [30]. Cathepsin D staining was not correlated with steroid receptors. This absence of correlation was also demonstrated using fine-needle aspirates of breast carcinomas, by double immunohistochemical staining of the nuclear estrogen receptor and cytoplasmic cathepsin D in the same sample [31]. Cathepsin D was also found to be increased in human endometrium at the luteal phase, confirming its induction by progesterone, and in some invasive endometrial cancers. However, its concentration was much lower than in breast cancer [29].

Immunoassay of cathepsin D in the cytosol of primary breast cancer The first assay, an IRMA kit called ELSA-cath-D now commercialized by CIS International, is based on a solid phase sandwich assay using two monoclonal antibodies recognizing two different epitopes of the large chain (34K) of mature cathepsin D (Fig. 2). These antibodies also recognize the same epitopes in the intermediate chain (48K) and the precursor form (52K) of the enzyme, so that total cathepsin D concentration in cell extracts may be assayed [9, 32-35]. Approximately 90 to 98% of all forms of cathepsin D are quantitatively extracted by the homogenization procedure used in routine preparations of cytosol for estrogen and progesterone receptor assays (Tris EDTA buffer). Clinical studies performed in different cancer centers, and analyzed using the Cox multivariate test, have given two sets of information. The most important concerns the value of high cathepsin D concentration for predicting relapse and metastasis (Table 1). Most studies were performed using the


two-site immunoassay of total cathepsin D [32-35, 37-39]. The first study in Copenhagen [34], the second in St-Cloud [35], indicated that the predictive value of cathepsin D was more useful in node-negative than in node-positive patients. Independently, McGuire's group, using polyclonal antibodies to cathepsin D and quantifying the 34K mature form of cathepsin D by immunoblotting, came to similar conclusions [36]. Subsequently, using the commercially available ELSA-cath D kit, other groups also found a correlation between total cathepsin D level and overall survival, even though the significance was in some studies higher for node-positive patients [38, 40]. In all Cox multivariate studies, cathepsin D placed within the three most significant prognostic markers. The cut-off level of total cathepsin D concentration which best discriminated between breast cancer with good (low concentration) or bad (high concentration) prognosis varied depending on the study. It was 45 pmoles/mg protein in the study of the St-Cloud Cancer Center, and 35 pmoles/mg protein in the patients of the Nice Cancer Center [38]. The second set of information is that cathepsin D concentration and status is generally independent of classical prognostic markers such as axillary node invasion, tumor size, receptors, Scarff and Bloom histological grade, or age of patients [3335], as well as more recently used markers such as neu-erb-B-2 or int-2 oncogene amplification [37]. Cathepsin D slightly correlated with estrogen receptor status, only in premenopausal patients [34], which is consistent with its constitutive high production in estrogen receptor negative cell lines and with DNA ploidy [36]. The predictive value of cathepsin D therefore supplements that of other markers. These results also suggest that cathepsin D is associated with a stage of carcinogenesis which differs from that detected by other prognostic parameters of breast cancer. While the prognostic value of cathepsin D indicates that the overexpression of total cathepsin D (and the mature form) is correlated with the frequency of metastasis, we do not presently know whether the derouting or the altered processing of this protease has also some prognostic value. Such studies are in progress using a sandwich immunoas-


H Rochefort

say to exclusively measure pro-cathepsin D in primary breast cancer cytosol [41], and to compare its prognostic value to that of total cathepsin D. It is interesting that cathepsin D from breast cancer cells does not seem to increase the plasma level of cathepsin D in patients with metastasis and large tumors. This may be due to a normal secretion of cathepsin D into the blood through the liver. Hence, this marker is presently only valid as a tissue marker, but not as a circulating marker in breast cancer. Obviously, other independent studies are required in order to discriminate among all new prognostic markers those that will remain significant. Most of the clinical results currently available indicate that high cathepsin D concentration in primary breast cancer increases the risk of developing clinical metastasis [18, 21, 33] and is therefore associated with micrometastases, which are already present when primary tumors are removed. Cathepsin D therefore appears to be useful for determining which breast cancer patients will most likely need adjuvant systemic therapy after surgery in order to retard or prevent early recurrence.

Putative role of cathepsin D in mammary carcinogenesis

Clinical results on cathepsin D as a marker of metastasis indicate either that cathepsin D is merely associated with the metastatic process or, more excitingly, that it is actually responsible for at least part of this process [42]. At present, two biological activities of purified pro-cathepsin D have been described in vitro in human cell lines. First, both pro-cathepsin D and the mature enzyme have been shown to stimulate the growth of estrogen-deprived MCF7 cells [8]. This autocrine mitogenic activity does not reproduce the full effect of estrogens, suggesting that other autocrine growth factors are also required. The mechanism for this mitogenic activity of cathepsin D is currently unknown. Like other proteases, cathepsin D may act indirectly via its enzymatic activity by releasing growth factors from precursors or from extracellular matrix, and/or by activating growth fac-

tot receptors extracellularly or intracellularly, or by increasing the amount of amino acids available to make new proteins. The alternative is that cathepsin D is, like other growth factors, mitogenic by triggering a plasma membrane receptor. In fact, like other lysosomal enzymes, cathepsin D interacts with the mannose-6-phosphate IGF-II receptor [43]. Crosslinking and binding experiments have shown that pure pro-cathepsin D of breast cancer cells directly interacts with this receptor (M. Mathieu et al., submitted for publication). However, the coupling mechanism triggered by the activation of this receptor is unknown, and it has not yet been proven that cathepsin D acts as an IGF-II analog by stimulating this receptor. The second biological activity of cathepsin D, which may facilitate metastasis, is its proteolytic activity. Cathepsin D is secreted as an inactive proenzyme (52K) in breast cancer cells, and can be autoactivated at acidic pH by removing part of the N-terminal pro-fragment [11, 44]. Both the purified 52K pro-cathepsin D and conditioned media from estrogen-treated MCF7 cells digest in vitro extracellular matrix prepared from bovine corneal endothelial cells. Optimal activity occurs at acidic pH (4 to 5). The degradation of extracellular matrix by secreted proteases present in conditioned media of breast cancer cells is mostly due to cathepsin D, since it is completely inhibited by pepstatin but not by other inhibitors. Several epithelial cancer cell lines have been found to secrete a pepstatin-sensitive protease, an activity which correlates with cathepsin D antigen concentrations assayed by ELISA. It seems that autoactivation of the secreted pro-cathepsin D in vivo would require an acidic micro-environment, which is more frequently encountered within the cells (endosomes, lysosomes) than out of them. Large acidic vesicles containing both mature cathepsin D and endocytosed extracellular matrix have been found at much higher concentrations in breast cancer cells than in normal mammary cells, indicating that overproduction and derouting of cathepsin D may facilitate digestion of extracellular matrix following its internalisation by an endocytotic or phagocytotic process [45]. Whether cathepsin D and/or other proteinases are responsible for the extracellular digestion of

Cathepsin D extracellular matrix is not known. However, it is clear that in breast cancer ceils cathepsin D could potentially act in acidic intracellular compartments other than the lysosomes. Cathepsin D may also behave as a processing protease able to be autoactivated at high concentrations and low pH and to process and activate other proteases, such as the pro-cathepsin B secreted in ovarian cancers [46], thus initiating a proteolytic cascade. Direct proof that cathepsin D promotes some step in metastasis is still lacking. Using a mammalian expression vector carrying human pro-cathepsin D, attempts are in progress to transform transfected recipient cell lines, to develop transgenic mice overexpressing cathepsin D in mammary glands, and to study the role of cathepsin D on the occurrence of metastasis in nude mice. If the responsibility of cathepsin D is demonstrated in metastasis, the consequences could be considerable, particularly in directing new therapeutic approaches aimed at inhibiting the production, catalytic activity, or interactions of this protease.

Conclusions A series of experimental data favor the hypothesis that cathepsin D is an important protease in breast cancer for facilitating invasion and metastasis. It is induced by estrogens, and by antiestrogens in antiestrogen-resistant cells. It is also overproduced and secreted in cancer cells, compared to normal mammary cells growing at the same rate. It displays several biological activities which may facilitate development of metastasis. Also, high concentrations in breast cancer tissue are highly predictive of relapse and metastasis. Other proteases are also thought to play a role in cancer metastasis. However, very few retrospective clinical studies have shown a high correlation between concentrations of these proteases in the primary tumor and the occurrence of metastasis. To our knowledge, in breast cancer only the urokinase-type plasminogen activator appears to be correlated with metastasis [28]. In other cancers, other cathepsins (B and L) may be more important than


cathepsin D [20, 46, 47]. This would suggest that cancer cells have different strategies, depending on the state of differentiation of their tissues of origin, for reaching the same proliferation and dissemination goals. The nature of proteases involved in carcinogenesis may therefore vary according to the type of cancer, and possibly the species being considered. The example of cathepsin D in breast cancer underlines the importance of starting work on hormone-dependent human cancer cells in culture. This strategy enables the development of molecular probes (antibodies and cDNA), which could then be used directly on human samples. The clinical results obtained not only are useful in practice for improving the treatment of human cancer, but also are important for discriminating between laboratory artefacts and biologically significant facts which should be investigated further.

Acknowledgements I am grateful to members of the INSERM laboratory who have actively and cooperatively contributed to this venture, and to E. Barri6 for her skillful preparation of the manuscript. I thank SANOFI and CIS Laboratories and several clinical Centers in Copenhagen (Finsen Instituten, Dr S. Thorpe and C. Rose), MontpeUier (Prs H. Pujol, F. Laffargue), and St-Cloud (F. Spyratos, J. Rou~ssr), for clinical studies. This work was supported by the 'Institut National de la Sant6 et de la Recherche Mrdicale', the 'Association pour la Recherche sur le Cancer', the University of Montpellier 1, the 'Groupement des Entreprises Franqaises dans la Lutte contre le Cancer' and the 'Ligue Nationale Franqaise contre le Cancer'.

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H Rochefort

Hormonal control of breast cancer in cell culture. In: Iacobelli S, King RJB, Lindner HR, Lippman ME (eds) Hormones and Cancer. Raven Press, New York, 1980, pp 21-29 3. Lippman ME, Dickson RB, Bates S, Knabbe C, Huff K, Swain S, McManaway M, Bronzert D, Kasid A, Gelmann EP: Autocrine and paracrine growth regulation of human breast cancer. Breast Cancer Res Treat 7: 59-70, 1986 4. Westley B, Rochefort H: A secreted glycoprotein induced by estrogen in human breast cancer cell lines. Cell 20: 352-362, 1980 5. Fuqua SAW, Blum-Salingaros M, McGuire WL: Induction of the estrogen-regulated '24K' protein by heat shock. Cancer Res 49: 4126-4129, 1989 6. Rio MC, Bellocq JP, Daniel JY, Tomasetto C, Lathe R, Chenard MP, Batzenschlager A, Chambon P: Breast cancer-associated pS2 proteins: Synthesis and secretion by normal stomach mucosa. Science 241: 705-708, 1988 7. Vignon F, Capony F, Chambon M, Freiss G, Garcia M, Rochefort H: Autocrine growth stimulation on the MCF7 breast cancer cells by the estrogen-regulated 52K protein. Endocrinology 118: 1537-1545, 1986 8. Westley B, May FEB, Brown AMC, Krust A, Chambon P, Lippman ME, Rochefort H: Effects of antiestrogens on the estrogen regulated pS2 RNA, 52-kDa and 160-kDa protein in MCF7 ceils and two tamoxifen resistant sublines. J Biol Chem 259: 10030-10035, 1984 9. Garcia M, Capony F, Derocq D, Simon D, Pau B, Rochefort H: Monoclonal antibodies to the estrogen-regulated Mr 52,000 glycoprotein: Characterization and immunodetection in MCF7 cells. Cancer Res 45: 709-716, 1985 10. Capony F, Garcia M, Capdevielle J, Rougeot C, Ferrara P, Rochefort H: Purification and first characterization of the secreted and cellular 52-kDa proteins regulated by estrogens in human breast cancer cells. Eur J Biochem 161: 505-512, 1986 11. Capony F, Morisset M, Barrett AJ, Capony JP, Broquet P, Vignon F, Chambon M, Louisot P, Rochefort H: Phosphorylation, glycosylation and proteolytic activity of the 52K estrogen-induced protein secreted by MCF7 cells. J Cell Biol 104: 253-262, 1987 12. Von Figura K, Hasilik A: Lysosomal enzymes and their receptors. Ann Rev Biochem 55: 167-193, 1986 13. Morisset M, Capony F, Rochefort H: The 52-kDa estrogeninduced protein secreted by MCF7 cells is a lysosomalacidic protease. Biochem Biophys Res Commun 138: 102-109, 1986 14. Barrett AJ: Cathepsin D: Purification of isoenzymes from human and chicken liver. Biochem J 117: 601-607, 1970 15. Faust PL, Kornfeld S, Chirgwin JM: Cloning and sequence analysis of cDNA for human cathepsin D. Proc Natl Acad Sci USA 82: 4910--4914, 1985 16. Griffiths G, Hoflack B, Simons K, Mellman I, Kornfeld S: The mannose-6-phosphate receptor and the biogenesis of lysosomes. Cell 52: 329--341, 1988 17. Gordfarb RH: Proteolytic enzymes in tumor invasion and degradation of host extracellular matrices. In: Honn KV,

Powers WE, Sloane BF (eds) Mechanisms of Cancer Metastasis. Martinus Nijhoff Pub, Boston, 1986, pp 341-375 18. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz CM, Shafie S: Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 284: 67-68, 1980 19. Reich E: Activation of plasminogen: A widespread mechanism or generating localized extraeellular proteolysis. In: Ruddon RW (ed) Biological Markers of Neoplasia: Basic Applied Aspects. Elsevier, Amsterdam, 1978, pp 491 20. Gal S, Gottesman MM: The major excreted protein of transformed fibroblasts is an activable acid-protease. J Biol Chem 261: 1760-1765, 1986 21. Augereau P, Garcia M, Mattei MG, Cavaill6sV, Depadova F, Derocq D, Capony F, Ferrara P, Rochefort H: Cloning and sequencing of the 52K cathepsin D cDNA of MCF7 breast cancer cells and mapping on chromosome 11. Mol Endocrinol 2: 186-192, 1988 22. Westley BR, May FEB: Oestrogen regulates cathepsin D mRNA levels in oestrogen responsive human breast cancer cells. Nucl Acids Res 15: 3773-3786, 1987 23. Capony F, Rougeot C, Montcourrier P, Cavaill6s V, Salazar G, Rochefort H: Increased secretion, altered processing, and glycosylationof pro-cathepsin D in human mammary cancer cells. Cancer Res 49: 3904-3909, 1989 24. Garcia M, Salazar-Retana G, Pages A, Richer G, Domergue J, Pag6s AM, Cavali6 G, Martin JM, Lamarque JL, Pan B, Pujol H, Rochefort H: Distribution of the Mr 52,000 estrogen-regulated protein in benign breast diseases and other tissues of immunohistochemistry. Cancer Res 46: 3734-3738, 1986 25. Cavaill~s V, Augereau P, Garcia M, Rochefort H: Estrogens and growth factors induce the mRNA of the 52K-procathepsin-D secreted by breast cancer cells. Nucl Acids Res 16: 1903-1919, 1988 26. Rochefort H, Cavaill6sV, Augereau P, Capony F, Maudelonde T, Touitou I, Garcia M: Overexpression and hormonal regulation of pro-cathepsin D in mammary and endometrial cancer. J Steroid Biochem 34: 177-182, 1989 27. Maudelonde T, Domergue J, Henquel C, Freiss G, Brouillet JP, Franc~s D, Pujol H, Rochefort H: Tamoxifen treatment increases the concentration of 52K cathepsin D and its precursor in breast cancer tissue. Cancer 63: 1265-1270, 1989 28. Touitou I, Cavaill~s V, Garcia M, Defrenne A, Rochefort H: Differential regulation of 52K cathepsin D by estradiol and progesterone in mammary cancer and uterine cells. Mol Cell Endocrinol 66: 231-238, 1989 29. Maudelonde T, Martinez P, Brouillet JP, Laffargue F, Pag6s A, Rochefort H: Cathepsin D in human endometrium: Induction by progesterone and potential value as a tumor marker. J Clin Endocrin Metab 1989 70: 115--121,1990 30. Garcia M, Lacombe MJ, Duplay H, Cavaill~sV, Derocq D, Delarue JC, Krebs B, Contesso G, Sancho-Gamier H, Richer G, Domergue J, Namer M, Rochefort H: Immunohistochemical distribution of the 52-kDa protein in mare-

Cathepsin D









mary tumors: A marker associated with cell proliferation rather than with hormone responsiveness. J Steroid Biochem 27: 439-445, 1987 Cavaill6s V, Garcia M, Salazar G, Domergue J, Simony J, Pujol H, Rochefort H: Immunodetection of estrogen receptor and 52K protein in fine needle aspirates of breast cancer. J Natl Cancer Inst 79: 245-252, 1987 Rogier H, Freiss G, Besse MG, Cavali6-Barthez G, Garcia M, Pau B, Rochefort H, Paolucci F: Two-site immunoenzymometric assay of the 52-kDa-cathepsin D cytosols of breast cancer tissues. Clin Chem 35: 81-85, 1989 Maudelonde T, Khalaf S, Garcia M, Freiss G, Duport6 J, Benatia M, Rogier H, Paolucci F, Simony J, Pujol H, Pau B, Rochefort H: Immunoenzymatic assay of Mr 52,000 cathepsin D in 182 breast cancer cytosols. Low correlation with other prognostic parameters. Cancer Res 48: 462-466, 1988 Thorpe SM, Rochefort H, Garcia M, Freiss G, Christensen IJ, Khalaf S, Paolucci F, Pau B, Rasmussen BB, Rose C: Association between high concentrations of 52K cathepsin-D and poor prognosis in primary breast cancer. Cancer Res 49: 6008-6014, 1989 Spyratos F, Maudelonde T, Brouillet JP, Brunet M, Defrenne A, Andrieu C, Hacene K, Desplaces A, Rochefort H: Cathepsin D: An important marker predicting metastasis in primary breast cancer. Lancet 8672, 1115-1118, 1989 Tandon AK, Clark GM, Chamness GC, Chirgwin JM, McGuire WL: Cathepsin D and prognosis in breast cancer. N Engl J Med 322: 297-302, 1990 Brouillet JP, Theillet C, Maudelonde T, Defrenne A, Simony-Lafontaine J, Sertour J, Pujol H, Jeanteur P, Rochefort H: Cathepsin D assay in primary breast cancer and lymph nodes: relationship with c-myc, e-erb-B-2 and int-2 oncogene amplification and node invasiveness. Eur J Cancer Clin Oncol 1989 (in press) Namer M, Etienne MC, Fontana X, Ramaioli A, Lapalus F, Hrry M, Milano G: Prognostic value of total cathepsin D











in breast cancer (Abstract n° 10). Breast Cancer Res Treat 14: 135, 1989 Duffy MJ, Brouillet JP, Reilly D, Maudelonde T, Defrenne A, Rochefort H: Distribution and clinical significance of cathepsin D in breast cancer (Abstract). XIV International Congress of Clinical Chemistry (June 1990) Romain S, Muracciole X, Varette I, Bressac C, Brandone H, Martin PM: La cathepsine D: Un facteur pronostique indrpendant dans le cancer du sein. Bulletin du Cancer 1990 (in press) Freiss G, Vignon F, Pau B, Paolucci F, Rochefort H: A two-site immunoenzymometric assay of 52-kDa pro-cathepsin D, and its use in human breast diseases. Clin Chem 35: 234-237, 1989 Rochefort H, Capony F, Garcia M, Cavaill~s V, Freiss G, Chambon M, Morisset M, Vignon F: Estrogen-induced lysosomal proteases secreted by breast cancer cells. A role in carcinogenesis? J Cell Biochem 35: 17-29, 1987 Morgan DO, Edman JC, Standring DN, Fried VA, Smith MC, Roth RA, Rutter WJ: Insulin-like growth factor II receptor as a multifunctional binding protein. Nature 329: 301-307, 1987 Briozzo P, Morisset M, Capony F, Rougeot C, Rochefort H: In vitro degradation of extracellular matrix with Mr 52,000 cathepsin D secreted by breast cancer cells. Cancer Res 48: 3688-3692, 1988 Montcourrier P, Mangeat P, Salazar G, Morisset M, Sahuquet A, Rochefort H: Large acidic vesicles in cultured breast cancer cells contain cath-D and extracelhilar matrix. Submitted for publication Pagano M, Capony F, Rochefort H: La pro-cathepsine D peut activer in vitro la pro-cathepsine B srcrrtre par les cancers ovariens. C R Acad Sci Paris 309: 7-12, 1989 Sloane BF, Dunn JR, Honn KV: Lysosomal cathepsin B: Correlation with metastatic potential. Science 212: 11511153, 1981

Cathepsin D in breast cancer.

Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive and negative breast cancer cell lines, the mRNA coding...
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