Increased Secretion, Altered Processing, and Glycosylation of Pro-Cathepsin D in Human Mammary...

transcript

(CANCER RESEARCH 49. 3904-3909. July 15. 1989]

Increased Secretion, Altered Processing, and Glycosylation of Pro-Cathepsin D inHuman Mammary Cancer Cells1

F. Capony, C. Rougeot, P. Montcourrier, V. Cavailles,2 G. Salazar, and H. Rochefort3

Institut National de la SantÃ©et de la Recherche MÃ©dicale,UnitÃ©Hormones et Cancer (U 148) (VAC 59 CNRS). 60 Rue de Navacelles, 34090 Montpellier, France

ABSTRACT

In human mammary cancer cells, pro-cathepsin D (pro-Cath-D) isinduced by estrogens and 50% of it is secreted. To determine whether itssecretion is characteristic of mammary cells or transformed cells, wecompared its production, processing, and glycosylation in primary cultures of normal mammary epithelial cells to those found in breast cancercell lines. The cytosolic concentration of total cathepsin D (precursor andmature enzyme) measured by enzyme-linked immunosorbent assay was8 times higher in cancer cells. Its inKNA level estimated by Northernblot analysis was 8 to SO times higher and its secretion was 30 timeshigher in cancer cells. Using pulse-chase labeling, the cellular processingof pro-Cath-D was altered in hormone-dependent and -independent breastcancer cells in comparison to normal cells. This alteration resulted in alower accumulation of mature enzyme, while the secretion and cyto-plasmic accumulation of pro-Cath-D were greater in breast cancer cellsthan in normal cells. M l,< 1 increased secretion of the proenzyme innormal cells but not in cancer cells. The secreted proenzyme was markedly heterogeneous and had a more acidic pi in MCF7 cells than innormal mammary cells. These acidic forms disappeared following endo-/?-/V-acetylglucosaminidase H treatment indicating that the structuraldifference between pro-Cath-D of normal and of cancer mammary cellswas located on high mannose or hybrid .V-linked oligosaccharides. Thisdifference may be responsible for the altered routing of the pro-Cath-Din breast cancer cells.

INTRODUCTION

Secreted proteases are thought to have important functionsin carcinogenesis, facilitating tumor invasion and growth (1,2),and some of them are reported to be secreted more abundantlyby cancer cells than by normal cells (3-5). In breast cancer celllines, a M, 52,000 protein, identified as a pro-cathepsin D (6),is induced by estrogens in estrogen receptor-positive cell lines(MCF7, ZR75-1) and is also produced constitutively at highlevels in hormone-independent cell lines (MDA-MB231,BT20). This protease can also be detected in primary breastcancer cells and assayed in cytosol using specific monoclonalantibodies (7). In vitro, the secreted M, 52,000 pro-Cath-D4

displays an autocrine mitogenic activity on MCF7 cells (8) andinteracts with the mannose 6-phosphate/IGFII receptor viamannose 6-phosphate signals (6, 9). At acidic pH in vitro, thisprotease can also degrade the extracellular matrix (10), suggesting that it may be involved in mammary carcinogenesis(11), as proposed for cathepsin L in other cancers (4).

Oncogene products are thought to be carcinogenic following

Received 2/1/89; revised 4/7/89; accepted 4/17/89.The costs of publication of this article were defrayed in pan by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by the Institut National de la SantÃ©et de la

Recherche MÃ©dicale,the Faculty of Medicine of Montpellier, the Associationpour la Recherche sur le Cancer, the Groupement des Entreprises FranÃ§aisesdans la Lutte contre le Cancer, and thÃ©Ligue Nationale contre le Cancer.

2Recipient of the MinistÃ¨re de la Recherche et de l'Enseignement SupÃ©rieur

Fellowship Grant.3To whom requests for reprints should be addressed.4The abbreviations used are: pro-Cath-D, cathepsin D precursor (M, 52,000);

Cath-D, cathepsin D (EC 3.4.23.5); DMEM, Dulbecco's modified Eagle's medium; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;Man-6-P, mannose 6-phosphate; cDNA, complementary DNA; IGF, insulin-likegrowth factor; Endo H, endo-ff-A'-acetylglucosaminidase H; Endo F, endo-ÃŸ-N-acetylglucosaminidase F.

alterations in the structure, regulation, or degree of expressionof normal protooncogenes (Ref. 12 and references therein). Wehave considered the possibility that pro-Cath-D also could favorsome steps of mammary carcinogenesis by acquiring differentproperties during or following transformation. The amino acidsequence of the pro-Cath-D of MCF7 cells, deduced fromsequencing its cloned cDNA (13), was found to be almostidentical (except for one Ala to Val change in the profragment)to that of the pro-Cath-D of normal human kidney (14). However, the production and secretion of this protease were abundant in the breast cancer cell lines studied, and in vivo thecellular Cath-D concentration as estimated by immunohisto-chemistry appeared to be elevated in 70% of primary breastcancers and in benign mastopathies compared to normal restingmammary glands (15). However, this difference in concentration might have been due to a higher proliferative rate of cancercells compared to normal mammary cells, which are mostlyquiescent in nonpregnant women (doubling time, 100 days).Moreover, the proportion of pro-Cath-D secreted varies considerably according to the cell type (16). Although it is low (5-10%) in fibroblasts (17) it can reach 50% in kidney cells (18),hepatocytes (19), and endothelial cells (20). There are no datain the literature on the processing and secretion of pro-Cath-Din normal mammary epithelial cells. It was therefore crucial tocompare the concentration and processing of pro-Cath-D innormal mammary cells and mammary cancer cells growing inculture at similar rates before concluding that differences inconcentration and/or secretion are linked to cell transformation.

We show in the present study that in cancer cells pro-Cath-D is produced in larger amounts and is processed differentlythan in normal mammary cells. We also report on the firstindication of structural differences related to posttranslationalmodifications producing more acidic ,/V-glycosylated chains.

MATERIALS AND METHODS

Cell Culture. MCF7 cells obtained from the Michigan Cancer Foundation (Detroit, MI; MCF7 R) and Marc E. Lippman (Bethesda, MD;MCF7 L) and MDA/MB231 cells (21) were cultured in DMEM withfetal calf serum (10%) as described previously (22). "Normal"5 mam

mary cells were prepared as described previously (23) with minormodifications from mammary tissues collected at surgery from patientsundergoing reduction mammoplasties. Histological diagnosis was performed in paraffin sections. Briefly, glandular epithelial structures(organoids) were isolated after digestion for 1-3 days with collagenaseIA (250 lU/ml; Sigma) and hyaluronidase IS (200 lU/ml; Sigma).Dissociated organoids, 40 to 300 urn, were then cultured in Hams' F-12/DMEM (1/1), supplemented with 10% fetal calf serum, penicillin-streptomycin (25 IU/ml), and insulin (0.5 ng/ml) in 25-cm2 cultureflasks or 2-cm2 24-well dishes (Primaria; Falcon). After 15-20 days of

culturing and before complete confluence, cells were analyzed for Cath-D content and secretion. Twelve of 33 samples were discarded for highcontamination with fibroblasts. In the 21 remaining cultures, the proportion of epithelial cells was 80 Â±17% (mean Â±SD) as checked bystaining with cytokeratin antibodies (Biosoft, Paris, France).

' "Normal" stands for nonmalignant since in a few patients benign breast

diseases such as fibrocysts or ductal or lobular hyperplasia were also present, asis usual in reduction mammoplasty tissue (44).

PRO-CATH-D PROCESSING IN HUMAN MAMMARY CELLS

Preparation of Conditioned Media and Cytosol and Immunoassay ofCath-D. Subconftuent cultures of normal mammary cells and cancercells in T25 flasks were washed three times for 30 min with Ham's F-

12 and DMEM, respectively, and conditioned in 2 ml of the samemedia for 6 or 18 h. The conditioned media were collected and centri-fuged at 1800 x g for 5 min. Cells were harvested with phosphate-buffered saline (Gibco-BRL) containing 1 mM EDTA and subsequentlycentrifuged at 1800 x g for 5 min.

Cytosols were prepared at 0-4Â°Cin Tris, EDTA, monothioglycerol

molybdate buffer from frozen tissues or from pelleted cells obtainedbefore or after culturing (24). Tissue pieces were homogenized usingan Ultraturrax device. Cell pellets and cultured cells were disrupted bysonication at 100 W 3 times for 10s followed by 60 s of cooling.Homogenates were then centrifuged at 15,000 x g for 30 min to obtainthe cytosols. The pellets were used for DNA assay. Quantitation oftotal Cath-D (including the precursor and the mature enzyme) in mediaand cytosols was performed by enzyme-linked immunosorbent assay(24, 25).

Radiolabeling of Cells and Immunoprecipitation of Cath-D. Subcon-fluent cells were washed twice for 30 min in methionine-free DMEM(MCF7, MDA/MB) or Ham's F-12 (normal mammary cells) and thenlabeled for 6 or 18 h with 200 ^Ci/ini of [35S]methionine in methionine-free medium. Pulse-chase experiments were performed as describedpreviously (26) except that the cells were preincubated for 30 min inmethionine-free medium prior to addition of [35S]methionine. Labeledmedia and Nonidet 1' 40 cell extracts were obtained as describedpreviously (6). The Cath-D was immunoprecipitated using protein A-Sepharose, as described (27) with 6 //g/ml purified D8F5 monoclonalantibody to Cath-D and 1% Nonidet P-40 (w/v).

RNA Preparation and Northern Blot Analyses. Total RNA was extracted by the method of Auffray and Rougeon (28), electrophoresedon a 1% agarose-formaldehyde denaturing gel, and then transferred tonitrocellulose. As a cDNA probe, we used the 52K-9 cDNA (13), whichcontains most of the coding sequence of pro-Cath-D; it was "P-labeled,using random primers (29), to a specific activity of 1 to 3 x 10' dpm/

Mg. Filters were hybridized and autoradiographed as described previously (13). The amount of RNA was determined by densitometricscanning of different exposures of the autoradiographs.

Treatment with Endoglycosidases. The secreted 35S-labeled pro-Cath-

D was purified by immunoprecipitation from MCF7 cells or normalmammary cells treated with NH,C1 and then treated for 24 h at 37Â°C

with the following enzymes: Endo H as described previously (6); EndoF (from Flavobacterium meningosepticum; Boehringer, Mannheim,France) at 3.6 units/ml in 100 mM sodium acetate (pH 7.0), 10 mMEDTA, 5 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride,and 2 /I.Mpepstatin A (Sigma). Control samples were incubated underthe same conditions.

Isoelectric Focusing. Analaytical isoelectric focusing in polyacryl-amide slab gels was performed on an FBE 3000 apparatus fromPharmacia (Uppsala, Sweden). The gel (1 mm thick) was a mixture of8 M urea, 2% (w/v) Nonidet P-40, 2% ampholines (range, pH 5-7) and5% acrylamide. The electrode solutions were l M H3PO4 (anode) andl N NaOH (cathode). Prefocusing was carried out at 8 W constantpower for 30 min and focusing at 16 W for 2.5 h. Protein isoelectricpoints (pi) were determined using the isoelectrofocusing pi calibrationkit of Pharmacia. Proteins in the gel were fixed and stained as recommended by Pharmacia (30). The gel was then processed for fluorographyas for SDS-PAGE.

Other Methods. SDS-PAGE (15% acrylamide) (31), sample preparation, and detection of the radioactivity on the gels were performed asdescribed previously (6, 31). Proteins were assayed by the method ofBradford (32) using -v-globulin as the standard. DNA content was

evaluated by diaminobenzoic acid fluorescence assay (33).

RESULTS

Immunoassay of Cath-D in Normal Mammary Cells Comparedto Breast Cancer Cells. In previous experiments using immunohistochemistry, 70% of the breast cancer tissues were positively stained for Cath-D (7) whereas normal resting mammaryglands collected from plastic surgery were negative (15). Since

this difference could be due to the much slower growth rate innormal mammary glands, we cultured these glands preparedfrom reduction mammoplasties under conditions allowing theirproliferation and assayed Cath-D in the corresponding cytosols.Table 1 indicates that there was no significant increase in Cath-D concentration following mammary gland isolation or primaryculturing. In primary culture, these cells grew with a doublingtime of 3.0 Â±1.4 days (mean of 3 experiments Â±SD) confirmingthe results of Yang et al. (34) and Stampfer et al. (35) andcontrasting with their doubling time in vivo (100 days). Cath-Dconcentration appeared to be similar in normal mammaryepithelial cells and connective tissue since there was no enrichment following isolation of epithelial organoid structures. Thecytosol concentration of Cath-D in normal mammary epithelialcells was in fact 8 to 16 times lower than in breast cancer cells(Table 2). A similar difference was found between the meanvalue of this protein in the cytosol of primary breast cancersdirectly collected from patients (24) and that of organoidsdirectly analyzed before culture.

Northern Blot Analysis of Pro-Cath-D mRNA. In an attemptto explain the higher cytosol concentration of Cath-D in cancercells, we estimated the level of its mRNA in both normal andcancer mammary cells. Fig. 1 shows that, in MCF7 cells, thesteady state level of the 2.2-kilobase Cath-D mRNA which isinduced 5-fold by estradiol treatment (Fig. 1, Lanes a-b) was 8to 50 times higher than in primary cultures of normal mammaryepithelial cells prepared from three different patients (Fig. 1,Lanes c-e). This indicated that the higher expression of cyto-solic Cath-D in human breast cancer cells is associated with anincrease of its mRNA concentration.

Lower Secretion of Pro-Cath-D in Normal Mammary CellsCompared to Breast Cancer Cells. The amount of pro-Cath-Dsecreted by normal mammary cells in primary culture was foundto be very low and in some cases undetectable. In 19 different

Table 1 Cath-D concentration in normal mammary cells

Mammary tissue was collected from reduction mammoplasties. Cytosols wereprepared directly from tissue (a), or from organoids (cells before culture) (b), orfrom epithelial cells cultured for 15-20 days (c). The cytosolic Cath-D concentration was determined by enzyme-linked immunosorbent assay, as described in"Materials and Methods."

fmol//ig DNApmol/mg PNo. of assaysTissue

(a)132

Â±736.1 Â±2.67Organoids

(b)140

Â±1049.4 Â±7.5

14Cells(c)1

30 Â±1744.2 Â±5.1

Table 2 Cath-D in cancer and normal mammary cells

Differences between values in normal and cancer cells are all highly significant:P< 10-'.

a. Cytosolic concentration. The concentration of total cytosolic Cath-D ofMCF7 R and normal cells was determined by ELISA" as described in "Materialsand Methods." Results are expressed as mean Â±SD.

b. Secretion into medium. Secretion of pro-Cath-D was estimated by ELISAof conditioned media or following 16 h of [3*S]methionine labeling and immunoprecipitation. The 100% value is the sum of labeled Cath-D recovered (intra-cellular plus extracellular).

P values were calculated with the Wilcoxon rank sum test.

a.CytosolMCF7Normal

cellsTimes increased

in cancerb.

SecretedMCF7

NonmalignantTimes increased

in cancerfmol/^g

DNA2230

Â±275 (5)130Â± 174(21)*

X16ELISA(fmol/Mg

DNA/6h)360

Â±220 (8)12.4 Â±16.6(19)

x29pmol/mg

Â±8.4 (6)4.2 Â±5.1 (21)*

xgLabeled

Cath-D(% oftotal)45

Â±10(3)1.4 Â±0.6 (5)

' ELISA, enzyme-linked immunosorbent assay.*Numbers in parentheses, results of Table 1.

abode ab

1 2 3NMC

Fig. 1. Northern blot analysis of pro-Cath-D mRNA from MCF7 cells andnormal mammary cells. RNA was extracted as described by Auffray and Rougeon(28) from MCF7 cells (Lanes a and ft) and from normal mammary cells obtainedfrom 3 different patients (NMC 1-3, Lanes c-e). The MCF7 cells were culturedin medium without (C) or with 10 nM estradici (f;) as described (22). Total RNA(40 ;<ni was analyzed by Northern blotting as described in "Materials andMethods." Hybridization was done with 52K-9 (13) cDNA to probe the 2.2-kilobase (kh) pro-Cath-D mRNAs. The fluorographs were exposed for 16 h (aand b) and 32 h (c-e). The amount of RNA analyzed in each track was verifiedto be equivalent using a constant cDNA probe (not shown).

patients, a mean of 12-14 fmol of pro-Cath-D per ^g of DNAper 6 h was found to be secreted by normal cells whereas breastcancer cells secreted 30 times more pro-Cath-D. A similar 30-fold difference was found after [15S]methionine labeling ofproteins followed by immunoprecipitation of pro-Cath-D (Table 2). The antibodies to Cath-D react with similar affinity withthe pro-Cath-D of MCF7 cells and that of normal cells6 indi

cating that the lower secretion by normal cells was real and notdue to an altered interaction of antibodies with different antigens. When analyzed by SDS-PAGE, the pattern of proteinslabeled by [15S]methionine and secreted by normal epithelial

cells in culture differed from that of breast cancer cells (Fig. 2,Lanes a and b) and from that of cellular proteins, indicatingprotein secretion rather than cell lysis (not shown). The pro-Cath-D was generally not visible in medium conditioned bynormal mammary cells in contrast with that produced by MCF7cells.

Altered Cellular Processing of Cath-D in Mammary CancerCells Compared to Normal Cells. The steady-state level of thethree forms of Cath-D can be visualized by silver staining ofSDS-PAGE following purification (Fig. 2, Lanes c and d). InMCF7 cells, all three forms were stained, with a high proportionof the nonprocessed precursor (M, 52,000) and of the intermediate (M, 48,000) form (Fig. 2, Lane c), whereas in normalmammary cells, only the mature M, 34,000 form was stained(Fig. 2, Lane d) which is consistent with a normal processingof Cath-D into lysosomes.

The altered processing of cellular pro-Cath-D was confirmedby pulse-chase experiments. About 45-59% of the precursorwas secreted by estradiol-treated MCF7 cells (Tables 2 and 3),whereas only 3-5% is secreted in the absence of estrogens (26).Two estrogen receptor-negative cell lines (MDA-MB231 andBT20) constitutively secreted high levels of precursor, indicating that this high secretion is not related to the estrogenresponsiveness of cancer cells. In normal mammary cells, thepercentage of the secreted precursor varied between 0 and 12%(Tables 2 and 3). It reached 20% in one benign ductal hyper-plasia (not shown). The increased secretion in cancer cells wasaccompanied by an altered iMiniceliular processing of pro-Cath-

34K- -â€¢' *

MCF7 NMC35S-Secreted

MCF7 NMC

CellularSilver-Stain

Fig. 2. SDS-PAGE of proteins from MCF7 and normal mammary cells. Lanesa and b, secreted labeled proteins. MCF7 cells grown in DMEM with 10% fetalcalf serum in 10-cm2 wells and normal mammary cells (NMC) grown in a T25flask were labeled with ["Sjmethionine for 6 h in serum-free medium as describedin "Materials and Methods." Secreted proteins amounting to 45.000 (MCF7)and 120,000 (NMC) trichloroacetic acid-precipitable cpm were analyzed corresponding, respectively, to 0.11 and 0.15 /ig of cellular DNA. Lanes c and d,steady-state levels of the cellular purified Cath-D. Subconfluent MCF7 cells andnormal mammary cells were cultured for 18 h in serum-free medium. The Cath-D of Nonidet P-40 cellular extracts was purified in parallel by sequential concan-avalin A-Sepharose and anti-Cath-D-Sepharose affinity chromatographies (45).Proteins were eluted from the immunoaffmity columns at pH 11, dialyzed, andconcentrated by lyophilization. The purified materials were analyzed by SDS-PAGE and revealed by silver staining. The normal mammary cell track (Lane d)shows more nonspecific staining than the MCF7 track (Lane c), since it wasloaded with 10 times as much material. The precursor (52K) and mature (4SK,34K) forms of Cath-D are arrowed. K, molecular weight in thousands.

Table 3 Processing of [3*S]pro-Cath-D in cancer and normal mammary celts

Quantification of the experiments shown in Fig. 3.

MCF7RL

MDA/MB231BT20NMC,*

NMC2Secreted

pro-Cath-DÂ°

at 10h(%)59

566640

122Cellular

pro-Cath-Dhalf-life*(h)63

4422M,

34,000r

at 10h(%)5-18

15100100

6 V. CavaillÃ¨s,unpublished results.

Â°The MS-labeled pro-Cath-D (M, 52,000) secreted following 10 h of chase was

immunoprecipitated with the D8F5 monoclonal antibody.* The cellular pro-Cath-D half-life was evaluated from Fig. 3.' M, 34,000 is the large chain of the mature Cath-D."â€¢' The 100% value is the number of densitometric units of cellular pro-Cath-

D at 0 h of chase.d NMC, and NMC2, normal mammary epithelial cells from two different

patients.

D. In MCF7, BT20, and MDA-MB231 cells, cellular pro-Cath-D decreased slowly with a half-life of 3-6 h, whereas the labeledM, 48,000 and M, 34,000 mature forms increased slowly withtime (Fig. 3, Lanes a-d). By contrast, the cellular processing ofpro-Cath-D was faster (half-life, 2 h) in normal mammary cells(Fig. 3, e-f). The proportion of the M, 34,000 mature form at10 h of chase was much higher in normal cells than in breastcancer cells. This was also observed by immunoprecipitation ofthe Cath-D following [15S]methionine labeling (Table 3; Fig. 3).

Ã„ ab e d

46 K-H52K

013 6 10 O 1 3 7

hours of chaseFig. 3. Pulse-chase experiments with normal mammary cells and cancer cells.

Normal mammary cells (NMC) and breast cancer cells (MCF7 L and R, BT20,and MDA/MB23I) were labeled for l h with |"S]methionine and chased for theindicated times with unlabeled methionine. The same amounts of Nonidet 1' 40cell extracts, based on trichloroacetic acid-precipitable counts, were immunopre-cipitated with the Cath-D monoclonal antibody. The immunoprecipitates wereanalyzed on a I S% acrylamide gel and the bands were quantified by scanning thefluorogram as described by Morisse! et al. (26). A. molecular weight in thousands.

Since the cellular processing of lysosomal enzymes and theirrouting to lysosomes is mostly mediated by their interactionwith Man-6-P receptors, a defect in the interaction of pro-Cath-D with this receptor was suspected. A pH increase in acidicorgandÃes following NH4C1 treatment of cells decreases theconcentration of available Man-6-P receptors leading to anincreased secretion of lysosomal enzymes (36,37). As expected,NHjCl markedly increased the secretion of pro-Cath-D bynormal mammary cells (Fig. 4A, Lanes a and />;Fig. 4Ã„,Lanesa and c). By contrast, NH4C1 had no effect on pro-Cath-Dsecretion in MCF7 and MDA-MB231 cells (Fig. 4A, Lanes cand ti: Fig. 4/f, Lanes b and d) which was already maximalbefore alkalinization. The NH4C1 resistance of MCF7 cellssuggests that the Cath-D interaction with Man-6-P receptors isalready modified in transformed cells. In MCF7 cells, theresistance to NH4C1 appeared to require a functional and activated estrogen receptor, since in estrogen-deprived cells, NH4C1was again effective in increasing the secretion of pro-Cath-D(Fig. 4, Lanes b and d).

Differences in the ./V-Glycosylation of Pro-Cath-D. Modifications in the intracellular routing to lysosomes could be due tostructural alterations of pro-Cath-D. Nucleotide sequencing ofthe complete cDNA cloned from MCF7 Cath-D (13) showed asingle amino acid (Ala to Val) modification compared to normalkidney Cath-D (14). This was not observed in another humanbreast cancer cell line, ZR75-1 (38) suggesting that it maycorrespond to polymorphism. Several sequences of clonedcDNA corresponded to a single sequence of the protein, butisoelectric focusing analysis of the secreted pro-Cath-D indicated a high heterogeneity of pis (Fig. 5) suggesting different

NH4CI: -

BÂ£IO

200-100-0a

2NMCâ€¢tÂ§1|Rvfe3â€¢KK1nnA

61|^Ã„^Â§r:1

i11Ã•sNHÂ¿CI

â€¢¿�â€¢-*-Â»-* -*-*10.5-0C

NMC117*-800400nd MCF78|sMDA

^Ã;(1

ig1Ns11Ã•"toJ]â€¢40200

^ NHÂ¿CI - *

Fig. 4. Effect of NH4CI on the secretion of the pro-Cath-D in normal mammary, MCF7. and MDA-MB 231 cells. A, normal mammary cells (NMC) andMCF7 cells grown in DMEM plus 10% fetal calf serum, were labeled for 18 hwith ["SJmethionine in the presence (+) or absence (â€”)of 10 mM NH4C1.Immunoprecipitates from the collected media were run on SDS-PAGE andrevealed by fluorography as in "Materials and Methods." B, same experiments as

above except for cultures 7 and 10 where cells were grown in DMEM plus 10%fetal calf serum treated with dextran-coated charcoal to remove steroid hormones.The cells were either unlabeled and Cath-D was assayed by enzyme-linkedimmunosorbent assay (Lanes a and b) or labeled as in A (Lanes c and d) andCath-D was immunoprecipitated as described by Morisse! et al. (26). Numbersabove columns, 4 cultures of NMC from different patients (Columns 1-4) and 6different passages of MCF7 cells (Columns 5-10). MDA-MB 231 (MDA) weretested in parallel. Note the different scale oÃordinales for MCF7 and MDA cellsand normal mammary cells.

posttranslational modifications. In the pro-Cath-D secreted byMCF7 cells, different isozymes were seen with pis of 5.2, 5.4,5.5, 5.6, 5.7, 6.0, and 6.2 (Fig. 5A, Lane c, and 5Ã„,Lane a). Innormal cells, the pro-Cath-D heterogeneity was consistentlydifferent with less of the acidic pi isoenzymes and more of thebasic species with pis of 6.2, 6.4 and 6.7 (Fig. 5/Ã•,Lanes b andd, and SB, Lane c). Since the amino acid modification wasneutral, these different electric charges were probably due tovariable posttranslational modifications of the protein.

Endo H treatment, which cleaved the high mannose andhybrid /V-glycosylated chains, markedly affected the isoelectricfocusing pattern of pro-Cath-D from MCF7 cells by displacingthe heterogeneous variants of pi 5.4 and 6.0 to more neutralcomponents of pi 6.2, 6.4, and 6.6 (Fig. 5Ã„,Lanes a and b).The effect of Endo H on pro-Cath-D of normal mammary cellswas weaker with a shift of bands towards higher pi (Fig. 5Ã„,Lanes c and d). The overall effect of Endo H treatment resultedin the disappearance of the differences observed between pro-Cath-D from normal and cancer cells. The effect of Endo F(not shown) was similar to that of Endo H, suggesting that thedifferences in electric charges are located on the high mannoseor hybrid /V-glycosylated chains. We conclude that the higheracidity of pro-Cath-D from MCF7 cells is mostly due to structural differences located on the high mannose and/or hybrid N-linked oligosaccharides.

DISCUSSION

Two types of proteins induced by estrogens and secreted bybreast cancer cells are thought to mediate the stimulatory effectof estrogens on the growth and invasiveness of breast cancercells. Growth factors such as transforming growth factor Â«andIGFI appear to be mostly associated with the cell growth ratesince their concentration is similar in normal proliferatingepithelial mammary cells and in breast cancer cells (39). Proteases such as plasminogen activator (40) and pro-Cath-D (11)

a c dP.i

â€¢¿�IM6-4-

NMC7NMC

O EH O EH

MCF7 NMCFig. 5. Isoelectric focusing of purified pro-Cath-D from cancer and normal

mammary cells. The proenzymes were purified by immunoprecipitation frommedia conditioned by |"S]methionine-labeled MCF7 and normal mammary cellsin the presence of 10 rriM NH4CI as described in "Materials and Methods." In A,

aliquots (=10.000 cpm) of each enzyme preparation were analyzed by isoelectricfocusing on a polyacrylamide slab gel. The gel was processed and fluorographedas indicated in "Materials and Methods." In B, aliquots containing 8,000-12.000cpm were treated with the enzyme Endo H (III] as described in "Materials andMethods." Control samples (0) were incubated under the same conditions in the

absence of enzyme. The treated pro-Cath-Ds were then analyzed as in A. MCF7samples are from different cultures and normal mammary cells (NMC) are fromdifferent patients, p. /., isoelectric points.

may be involved in both cell proliferation and tumor invasion.In the present study, we compared for the first time Cath-Dproduced by normal and cancerous mammary cells growing atsimilar rates. We show two differences, (a) The steady statecellular concentrations of Cath-D and its 2.2-kilobase mRNAare markedly increased in cancer cells, suggesting that theincrease is more associated with the transformation of mammary cells than with their proliferation. (Â¿>)There is an evenlarger increase in the secretion (30-fold) of pro-Cath-D bycancer cells. This increased secretion is associated with, andmay be the consequence of, altered processing of pro-Cath-D,which is totally processed into the mature form (M, 34,000) innormal mammary cells, whereas at least 45% of it is secretedas the proenzyme by breast cancer cells. The altered processingof Cath-D was observed in both the hormone-dependent and-independent breast cancer cell lines studied. This is also inagreement with the immunoassay of pro-Cath-D in the cytosolof mammary cells which represented an average of 10% of totalCath-D in 136 breast cancers (24), whereas it was undetectablein normal mammary epithelial cells.7 The molecular mechanism

for this altered processing may be related to the structure ofpro-Cath-D, since its secretion appears to be increased morethan those of other lysosomal enzymes.7 * The amino acidsequence of the pro-Cath-D of breast cancer cells (13) and theamino acid analysis of the purified secreted pro-Cath-D ofMCF7 cells9 are very similar to those of normal pro-Cath-D.By contrast, pro-Cath-D of MCF7 cells contains more acidicgroups located on W-glycosylated chains sensitive to Endo Hdigestion. Further work is required to identify the groups (sialicacids, phosphates, sulfates, etc.) responsible for the lower pi ofisoenzymes in cancer cells.

The derouting of a protease in mammary cells causing it tobe secreted at the periphery of the cells may have importantand general consequences in carcinogenesis. Cancer generallyresults from a loss of regulation and an overexpression ofproteins coded by oncogenes and involved in the control of cellproliferation and migration. The case of Cath-D in mammarycancer may illustrate another mechanism whereby a proteaseacting normally in lysosomes is excessively secreted from thecancer cells because of an altered processing of its precursor.After secretion, the pro-Cath-D acquires the capacity to actextracellularly as an autocrine mitogen (8) either indirectly viaits proteolytic activity or directly via the Man-6-P/IGFII receptor (41) and/or as a protease degrading basement membrane inan acidic environment (10) as described for osteoclast on bonematrix (42). This proposal is supported by clinical studiesshowing that high Cath-D concentrations are associated withpoor prognosis of breast cancer (24).lo- "

Increased production of proteases has previously been implicated in the transformation process (1, 43). Recently, the production and secretion of cathepsin L (4) and transin (5) werefound to be increased in NIH 3T3 fibroblasts after transformation by oncogenes or after growth factor treatment. It hasalso been shown that the precursor of cathepsin B is abnormallysecreted by cancer cells (43). Our results with Cath-D suggesta general characteristic of cancer cells that have acquired theability both to secrete more lysosomal proteases and to be moreinvasive. They also indicate that the kind of protease alteredvaries according to the type of tissue which is being transformed.

' F. Capony, unpublished results." T. Braulke and K. Von Figura, personal communication.* J. P. Capony and F. Capony, unpublished results.10S. Thorpe el al. High concentrations of 52K cathepsin D predict early

recurrence in primary breast cancer, submitted for publication." F. Spyratos et al. Cathepsin D concentration in primary breast cancer

predicts metastasis and survival, submitted for publication.

In mammary cancer, pro-Cath-D is the acidic protease mostabundantly secreted in conditioned media (10); whereas in othertransformed cells, the amount of cathepsin L or cathepsin B ismore important. Transfection with the full-length cDNA ofpro-Cath-D in normal and cancer cells will indicate whether ornot increased production and secretion of pro-Cath-D canfacilitate tumor invasion and may also reveal the mechanism ofthe altered processing of pro-Cath-D.

ACKNOWLEDGMENTS

We would like to thank Drs. J. P. Reynaud and A. de Ricard forkindly providing human mammary tissue, and Dr. Per Briand (FibigerInstitute, Copenhagen, Denmark) and Drs. F. Vignon and M. Garciafor critical reading of the manuscript. We are grateful to J. DuportÃ©,S. Khalaf, and D. Frances for the Cath-D enzyme-linked immunosor-bent assay and to M. EgÃ¨aand E. BarriÃ³for typing the manuscript.

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1989;49:3904-3909. Cancer Res F. Capony, C. Rougeot, P. Montcourrier, et al. Pro-Cathepsin D in Human Mammary Cancer CellsIncreased Secretion, Altered Processing, and Glycosylation of

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