A Mutated Cathepsin-D Devoid of Its Catalytic Activity Stimulates the Growth of Cancer Cells

A mutated cathepsin-D devoid of its catalytic activity stimulates the growth of cancer cells

Murielle Glondu1, Peter Coopman2, ValeÂ rie Laurent-Matha1, Marcel Garcia1, Henri Rochefort*,1 and Emmanuelle Liaudet-Coopman1

1INSERM U540 `Endocrinologie MoleÂculaire et Cellulaire des Cancers', UniversiteÂ de Montpellier 1, 60 rue de Navacelles, 34090 Montpellier, France; 2CNRS UMR 5539, UniversiteÂ de Montpellier 2, Place EugeÁne Bataillon, 34095 Montpellier, France

Cathepsin-D, a lysosomal aspartyl proteinase, is highly Introduction secreted by breast cancer cells and its over-expression by transfection stimulates cancer cell proliferation. The Initially, it was believed that the major role of mechanism by which this protease aects proliferation proteases in metastasis was to facilitate invasion of remains, however, unknown. In order to determine cancer cells by digesting the extra-cellular matrix and whether proteolytic activity is necessary, we abolished the basement membrane components (Liotta et al., its enzymatic activity using site-directed mutagenesis 1986). However, recent evidences suggest that proteases followed by stable transfection in 3Y1-Ad12 cancer cells. are also key regulators of tumor growth at both Substitution of the aspartic acid residue 231 by an primary and metastatic sites (reviewed by Chambers asparagine residue in its catalytic site abrogated the and Matrisian, 1997; Edwards and Murphy, 1998). cathepsin-D proteolytic activity but did not aect its Like proteases acting at a neutral pH, such as the expression level, processing or secretion. However, like plasminogen activator and the matrix metalloprotei- wild-type cathepsin-D, this mutated catalytically-inactive nases, the precursors of lysosomal proteases (e.g. pro- cathepsin-D retained its capacity to stimulate prolifera- cathepsin-B, D, and L) are also over-expressed and tion of cells embedded in Matrigel or collagen I hyper-secreted by carcinoma cells (Sloane and Honn, matrices, colony formation in soft agar and tumor 1984; Rochefort et al., 1987; Kane and Gottesman, growth in athymic nude mice. Addition on the mock- 1990). However, activation of these proteases requires transfected cells, of either conditioned media containing an acidic pH which is rather found intra-cellularly in the wild-type or the mutated pro-cathepsin-D, or of the endosomes, lysosomes or phagosomes. puri®ed mutated pro-cathepsin-D, partially mimicked the Cathepsin-D (cath-D) (EC 3.4.23.5) has been exten- mitogenic activity of the transfected cathepsin-D, sively studied in human breast cancer, and many indicating a role of the secreted pro-enzyme. Moreover, independent clinical studies have associated its over- addition of two anti-cathepsin-D antibodies on the expression with an increased risk of metastasis cathepsin-D transfected cells inhibited their proliferation, (Rochefort, 1992; Ferrandina et al., 1997; Foekens et suggesting an action of the secreted pro-cathepsin-D via al., 1999). Several reports have indicated that cath-D an autocrine loop. A synthetic peptide containing the 27- stimulates cell proliferation. The ®rst evidence came 44 residue moiety of the cathepsin-D pro-fragment was, from the puri®ed pro-cath-D from MCF-7 breast however, not mitogenic suggesting that a receptor for the cancer cells which doubled MCF-7 cell growth on pro-fragment was not involved. Furthermore, the cathe- plastic (Vignon et al., 1986). We further showed that psin-D mitogenicity was not blocked by inhibiting the 3Y1-Ad12 rat tumor cells transfected with the human interaction of pro-cathepsin-D with the mannose-6- cath-D cDNA grew more rapidly either at low or high phosphate receptors. Our results altogether demonstrate cell densities (Garcia et al., 1990). Moreover, Vetvicka that a mutated cathepsin-D devoid of catalytic activity is and colleagues reported that pro-cath-D is mitogenic in still mitogenic and suggest that it is acting extra- breast and prostate cancer cells (Fusek and Vetvicka, cellularly by triggering directly or indirectly a yet 1994; Vetvicka et al., 1999). unidenti®ed cell surface receptor. Oncogene (2001) 20, Dierent mechanisms have been proposed to be 6920 ± 6929. responsible for the cath-D mitogenicity. On one hand, they might involve its action as a ligand by interaction Keywords: cathepsin-D; mitogen; mutagenesis; cataly- of the mannose-6-phosphate (Man-6-P) moieties of tic site cath-D with the Man-6-P/ insulin-like growth factor-2 (IGF-2) receptor (Mathieu et al., 1990; De Leon et al., 1996) or by interaction of its pro-peptide with an unknown cell surface receptor (Fusek and Vetvicka, 1994; Vetvicka et al., 1999). Conversely, they might *Correspondence: H Rochefort; implicate its catalytic activity to activate growth factors E-mail: [email protected] Received 19 February 2001; revised 11 July 2001; accepted 16 July (Briozzo et al., 1991; Rifkin, 1997) or to prevent 2001 secretion of growth inhibitors (Liaudet et al., 1995). Cathepsin-D, a mitogenic ligand M Glondu et al 6921 One major question remains as to whether the catalytic activity of cath-D is necessary for its mitogenicity (reviewed by Rochefort and Liaudet- Coopman, 1999). To address this problem, we have generated an enzymatically-inactive cath-D by mutat- ing an aspartic acid residue of its catalytic site. Here, we show that, after transfection into the 3Y1-Ad12 rat tumor cells, catalytically-inactive cath-D facilitates cell growth as eciently as the wild-type cath-D both in vitro and in vivo. Our results indicate that cath-D mitogenic activity does not require its catalytic activity. Moreover, we show that exogenous addition of pro- cath-D on the mock-transfected cells partially mimicked the mitogenic activity of the transfected cath- D, whereas its pro-fragment alone was inecient. Furthermore, two antibodies speci®c to mature cath- D inhibit cell proliferation when added on cath-D transfected cells. Finally, we provide arguments indicating that neither the Man-6-P receptors nor a putative receptor speci®c for the cath-D pro-fragment were responsible for this mitogenic response.

Results

The cath-D catalytic site includes two aspartyl residues (33 and 231) respectively located on the 14 kDa and 34 kDa chains facing each other in their spatial con®guration (Metcalf and Fusek, 1993). By using oligonucleotide-directed mutagenesis, a single A to G nucleotide substitution was made in amino acid positions 33 and/or 231 of human cath-D, changing an aspartic acid to an asparagine as indicated in Figure 1a. To assess the importance of the cath-D catalytic activity, we stably transfected the wild-type cath-D, or the single Asn 33, Asn 231, or double Asn 33-231 Figure 1 Expression of the human cath-D mutants in 3Y1-Ad12 mutant cath-D constructs into 3Y1-Ad12 rat cancer transfected cells. (a) Schematic representation of the human pre- pro-cath-D sequence showing location of the introduced cells that do not secrete endogenous pro-cath-D Asp?Asn mutations in catalytic site. According to Faust et al. (Garcia et al., 1990). We ®rst quanti®ed the amount (1985), -64 corresponds to the ®rst amino acid of the translated of wild-type and mutated cath-D expressed by these pre-pro-cath-D and 1 to the ®rst amino acid of the mature cath- pooled transfected cells with an immunoradiometric D. Three Asp?Asn cath-D mutants were generated by replacing assay using monoclonal anti-human cath-D antibodies A by G in position 33 and/or 231. S=signal sequence. (b) Expression of the wild-type and of the three mutated human cath- (Figure 1b). The Asn 33 and Asn 33-231 mutants were D in 3Y1-Ad12-transfected cells. Cell extracts and conditioned expressed at a much lower level than the wild-type media under serum-free conditions for 24 h from the cath-D, Asn cath-D. The corresponding proteins might be either less 33, Asn 231 and Asn 33-231 transfectants were analysed for stable or not well recognised by the M1G8 or D7E3 human cath-D expression by an immunoradiometric assay. Cath- D was expressed as nanogram per microgram of DNA antibodies used in the immunoradiometric assay. (means+s.d., n=6). (c) Maturation and secretion of the wild- Western blot and immunoprecipitation experiments type and the Asn 231 mutated human cath-D in 3Y1-Ad12 cells. with the M1G8 antibody con®rmed the lower expres- Cells were labelled for 6 h with [S35]methionine. Cell lysates (C) sion of these two mutants which were, however, and media (S) samples were immunoprecipitated with the M1G8 normally processed and secreted (data not shown). In antibody, analysed on a 12% SDS ± PAGE and ¯uorographed as described in Materials and methods. Equal protein amounts were contrast, the Asn 231 mutant was expressed as loaded for the two transfected cell lines. Arrows indicate the eciently as the wild-type cath-D, both intra-cellularly migration of the three forms of cath-D. K=molecular mass in and as a secreted pro-enzyme. The cath-D concentra- kDa tion detected in cell extracts was in both cases 90+ 30 ng/mg DNA corresponding to 170+60 pmoles/mg cytosol protein, which is much higher than the median to 22 ng/mg DNA/24 h corresponding to a concentra- value used as a cut-o prognostic level in primary tion of 10 ± 15 nM pro-cath-D in the extra-cellular breast cancer (Rochefort, 1992; Ferrandina et al., 1997; medium. The Asn 231 mutation did not alter cath-D Foekens et al., 1999). The amount of the secreted pro- processing and pro-cath-D secretion. As shown in cath-D was also similar in both transfectants, e.g., 17 Figure 1c, the Asn 231 mutated pro-cath-D was

Oncogene Cathepsin-D, a mitogenic ligand M Glondu et al 6922 normally processed into the 48 kDa and 34 kDa forms, secreted (Figure 2b) Asn 231 mutated cath-D could and the 52 kDa pro-enzyme was secreted as eciently degrade [14C]methemoglobin at any pH (Figure 2c). as the wild-type pro-cath-D. The Asn 231 mutation, Thus, using this strategy, we generated an enzymati- however, completely inactivated the proteolytic activity cally-inactive, stable and normally processed and of cath-D (Figure 2). In contrast to the wild-type cath- secreted Asn 231 mutated cath-D. D, neither the intra-cellular (Figure 2a) nor the We next studied the eect of the Asn 231 mutation on the in vitro and in vivo cath-D mitogenic activities. Unlike the mock or non-transfected cells, both the wild-type and the Asn 231 mutated cath-D transfected cells grew eciently when embedded in Matrigel, a reconstituted basement membrane. This was shown by cell staining after 7 days of culture (Figure 3a, top panel) and by phase contrast microscopy after 4 days of culture (Figure 3a, bottom panel). In the latter case, higher magni®cation showed a stellate morphology of colonies with protrusions sprouting in the surrounding Matrigel. Comparable mitogenic activities of the wild- type and Asn 231 mutated cath-D were obtained when cells were embedded in a collagen I matrix (Figure 3b). As can be seen in Figure 4, time-course experiments in

Figure 2 Lack of proteolytic activity of the cellular and the secreted Asn 231 mutated human cath-D. (a) Cell extracts from the control (mock-transfected), cath-D and Asn 231 transfected cells were assayed for proteolytic activity at pH 3.5 with [14C]methemoglobin in the absence or presence of pepstatin. The intra-cellular human cath-D proteolytic activity was calculated by subtracting the TCA-soluble radioactivity obtained with pepstatin and with the control cell line corresponding to the endogenous rat cath-D proteolytic activity (approximately 1/20 of the total cath- D activity) and was expressed as c.p.m. per microgram of DNA. (b) Conditioned media from the control, cath-D, or Asn 231 transfected cells containing equivalent amount of proteins Figure 3 Mitogenic activities of the wild-type and the Asn 231 secreted by 24 h under serum-free conditions were assayed for mutated cath-D. Control (mock-transfected), cath-D, or Asn 231 proteolytic activity as in (a). The secreted cath-D proteolytic transfectants were embedded in a Matrigel matrix (a)orina activity was obtained by subtracting the TCA soluble radio- collagen I matrix (b). Upper rows: p-nitrotetrazolium violet cell activity obtained with pepstatin, no activity being measured in staining after 7 days' culture in a 24-well plate. Lower rows: phase control cells. (c) Eect of pH. Cell extracts from the control, cath- contrast optical photomicrographs after 4 days' culture. One D or Asn 231 transfected cells were incubated with [14C]methe- representative experiment out of four is shown for the Matrigel moglobin for 30 min at dierent pHs and the proteolytic activity matrix and out of two for the collagen I matrix. Bars (Ð 2 mm; was assayed as described in (a) ---- 50 mm)

Oncogene Cathepsin-D, a mitogenic ligand M Glondu et al 6923 Matrigel evidenced that the wild-type and Asn 231 the primary tumors. These data demonstrate that a mutated cath-D transfectants had the same exponential cath-D devoid of its catalytic activity remains mito- growth with a doubling time of 2 days. By contrast, genic both in vitro and in vivo. both the mock and the non-transfected cells did not grow in Matrigel (Figure 4). We veri®ed that a second pool of cath-D and Asn 231 transfectants presented the same growth rate (data not shown). Finally, both the Table 1 Soft agar colony formation of the wild-type and the wild-type and Asn 231 mutated cath-D transfected cells ASN 231 mutated cath-D transfectants formed colonies in soft agar and their number was Number of colonies signi®cantly increased as compared to the mock- Experiment no Control cath-D* Asn 231*8 transfected cells (Table 1) or non-transfected cells 120+2 330+36 298+20 (not shown). The mitogenic activity of the catalyti- 2 2.6+1.2 19+2.3 50+13 cally-inactive cath-D was also observed in vivo on 3 1.3+0.9 136+21 108+10 xenograft tumors in nude mice. In the cath-D and Asn Colonies in soft agar of the control (mock-transfected), cath-D and 231 groups, tumors became apparent 9 days after s.c. Asn 231 cells were stained with p-nitrotetrazolium violet after 14 injection of cells mixed with Matrigel and grew days' culture and photographed. The colony number was counted in exponentially with the same doubling time of 2 days triplicate on photographs. The data given from three independent (Figure 5a). After 16 days, the tumor sizes from the experiments are the means+s.d. of triplicate samples. *, P50.005 cath-D and Asn 231 groups reached approximately versus control; 8,NSversus cath-D (Kruskal Wallis test) 200 mm2, whereas the tumor sizes from the mock- transfected (control) cells reached only 30 mm2.At sacri®ce, the mean tumor weight was increased by 3.6- fold for both the cath-D and Asn 231 groups compared to the control group (Figure 5b). Local invasion of cutaneous tissue and muscle was identical for the cath- D and Asn 231 tumors and was more pronounced than for the control tumors. However, at the time of sacri®ce, no spontaneous metastases were detected macroscopically probably due to the rapid growth of

Figure 5 Tumor growth of the wild-type and the Asn 231 Figure 4 Matrigel outgrowth of the wild-type and the Asn 231 mutated cath-D transfected cells. Control (mock-transfected), mutated cath-D transfected cells. Control (mock-transfected), cath-D or Asn 231 transfected cells were subcutaneously injected cath-D or Asn231 transfectants and non-transfected 3Y1-Ad12 with Matrigel into nude mice (400 000 cells per site, two sites per cells were embedded in Matrigel and cells were recovered at the mouse and ®ve mice per group). (a) Tumor size was measured at indicated days by Matrisperse treatment. The DNA was the indicated time as described in Materials and methods and the quanti®ed as described in Materials and methods and the mean+s.e.mean is shown (n=10 per group). (b) Tumor weight Matrigel outgrowth was expressed as microgram of DNA determined at sacri®ce (day 16) (n=10 per group). Columns, (mean+s.d. of four independent experiments). Inset shows the means; bars, s.e.mean. **, P50.0025 versus control; *, P50.0005 logarithmic growth of cath-D and Asn 231 transfectants versus control (Student's t-test). Similar data were obtained in a (r=0.9764 for cath-D cells; r=0.9761 for Asn 231 cells) separate experiment using 40 000 cells per site

Oncogene Cathepsin-D, a mitogenic ligand M Glondu et al 6924 We then attempted to determine whether the tions in the conditioned media (Figure 6a). Moreover, secreted pro-enzyme or the intracellular enzyme were the puri®ed pro-cath-D-Asn 231 (elution fraction F2) responsible for this mitogenic activity. We ®rst tested added at a ®nal concentration of 2.5 nM on the mock- the eect of conditioned media from mock-transfected transfected cells stimulated their growth by 1.6-fold (control) cells, cath-D or Asn 231 transfectants on the while elution fraction F5, devoid of pro-cath-D-Asn growth of mock-transfected cells plated on a Matrigel 231, had no eect (Figure 7). Therefore, pro-cath-D matrix. Figure 6a shows that conditioned media secreted by the cath-D and Asn 231 transfectants containing the wild-type pro-cath-D or the pro-cath- partially mimicked the mitogenic activity of the D-Asn 231, added at a ®nal concentration of 10 nM transfected cath-D suggesting an autocrine mechanism. and 5 nM respectively, corresponding to optimal cath- To support this mechanism, we then tested the eects D mitogenic activity (Vignon et al., 1986), signi®cantly of anti-cath-D antibodies added to the medium, on the stimulated the growth of the mock-transfected cells by cath-D mitogenic activity. The D8F5 monoclonal 1.4-fold and 1.3-fold. We veri®ed by radioimmuno- antibody speci®c for total cath-D (52 kDa, 48 kDa metric assays that, after the 4 days incubation, the and 34 kDa forms) and described to prevent pro-cath- concentrations of pro-cath-D were unchanged. In this D binding and endocytosis (Freiss et al., 1988), bioassay, the growth of cath-D and Asn 231 inhibited the proliferation of cath-D transfectants by transfectants was increased respectively by 2.4-fold and twofold compared to the mock-transfected cells (Figure 6b), the corresponding secreted pro-enzyme concentrations being respectively 4.7 and 3.5 nM, which are in the range of the pro-enzyme concentra-

Figure 7 Eects of the puri®ed Asn 231 mutated pro-cath-D and of the cath-D pro-fragment on the mock-transfected cell Figure 6 Eects of the conditioned media from wild-type and growth. (a) Pro-cath-D-Asn 231 was puri®ed from the condi- Asn 231 mutated cath-D transfected cells on the mock-transfected tioned medium secreted by the Asn 231 transfectants under cell growth. (a) Conditioned media (CM) from control (mock- serum-free conditions for 24 h as described in Materials and transfected), cath-D and Asn 231 transfectants were prepared as methods. Inset shows the pro®le of the puri®ed pro-cath-D-Asn described in Materials and methods and the secreted wild-type 231 detected in the elution fraction F2 but not in the elution and Asn 231 mutated cath-D were quanti®ed by an immuno- fraction F5 by silver staining of a SDS ± PAGE. Arrow indicates radiometric assay. Control cells were plated on a Matrigel matrix the migration of the 52 kDa pro-cath-D-Asn 231. The puri®ed and control transfectants were treated with control, cath-D or pro-cath-D-Asn 231 was quanti®ed by an immunoradiometric Asn 231 CM (®nal concentration: pro-cath-D=10 nM, pro-cath- assay. The following products were added on the control (mock- D-Asn 231=5 nM). After 4 days, cells were recovered by transfected) cells plated on Matrigel: control=elution buer from Matrisperse treatment and the DNA was quanti®ed as described the puri®cation procedure; eluted fractions, F5=negative control in Materials and methods. Results are presented as mean+s.d. for F2 and F2=the puri®ed pro-cath-D-Asn 231 (2.5 nM); the (n=4 for control CM, n=3 for cath-D CM and n=2 for Asn 231 control peptide=negative control for cath-D pro-peptide CM). *, signi®cantly dierent from control at P50.025, **, (2.5 nM), the cath-D pro-peptide=amino acids 27-44 of cath-D signi®cantly dierent from control at P50.0025 (Student's t-test). pro-fragment (2.5 nM). After 4 days, cells were recovered by Similar results were obtained in another independent experiment. Matrisperse treatment and the DNA was quanti®ed as described (b) Cell growth of cath-D and Asn 231 transfectants plated on a in Materials and methods. Results are presented as mean+s.d. Matrigel matrix. DNA was quanti®ed as described in (a). Results (n=4 independent experiments for F2 and F5 obtained from two are presented as mean+s.d. (n=4). The ®nal concentrations of puri®cations, n=3 independent experiments for control and cath- the secreted pro-enzyme were: pro-cath-D=4.7 nM, pro-cath-D- D peptides. *, signi®cantly dierent from control at P50.0005 Asn 231=3.5 nM.(c-d) Representative phase contrast optical (Student's t-test). (b) Representative phase contrast optical photomicrographs. Control cells were treated as described in (a) photomicrographs. Control cells were treated as described in (a) and after 3 days, cells were photographed. Bars (- - - - 25 mm) and after 3 days, cells were photographed. Bars (- - - - 25 mm)

Oncogene Cathepsin-D, a mitogenic ligand M Glondu et al 6925 35.8%, almost to the level of the mock-transfected cells the entire cath-D pro-fragment (amino acids 1 ± 44) (Figure 8). By contrast, the M2E8 monoclonal anti- tested at a ®nal concentration of 20 nM, did not body speci®c for the 52 kDa pro-enzyme and described stimulate the proliferation of mock-transfected cells to be ineective to prevent pro-cath-D binding and seeded on plastic substratum in the conditions endocytosis (Freiss et al., 1988), was inecient (Figure described by Fusek and Vetvicka (1994) (data not 8). In addition, a rabbit polyclonal antibody speci®c shown). These data indicate that the receptor to this for total cath-D, which blocked pro-enzyme endocy- pro-fragment was unlikely to be responsible for the tosis (MR Farnoud, personal communication), inhib- mitogenic activity of cath-D observed in our condi- ited the proliferation of cath-D transfectants by 36.3%, tions. At present, the only identi®ed cell surface whereas a control rabbit serum had no eect (Figure receptors known to interact with the secreted pro- 8). Therefore, two antibodies that prevent pro-cath-D cath-D are the Man-6-P receptors. We tested the eect binding and endocytosis, neutralising the secreted pro- of an excess of free Man-6-P in conditions that totally enzyme and not the intracellular cath-D, also inhibited compete for cath-D binding and endocytosis via these the mitogenic activity of the transfected cath-D. receptors (Capony et al., 1987; Laurent-Matha et al., Together, these results show that the secreted pro- 1998). Neither Man-6-P nor free glucose-6-phosphate cath-D can mimic the mitogenic activity of the (Glu-6-P; negative control) prevented the Matrigel transfected cath-D and strongly suggest that pro-cath- outgrowth of cath-D or Asn 231 transfectants (Figure D is acting extra-cellularly via an autocrine loop 9a). Free Man-6-P did not mimic cath-D activity on possibly by triggering directly or indirectly a cell the mock-transfected cells since it had no eect on surface receptor. growth (Figure 9a). These results exclude that the We ®nally investigated which part of the pro-cath-D cath-D mitogenicity is transduced via the Man-6-P protein was implicated in this eect. It has been receptors in 3Y1-Ad12 cells. We ®nally studied proposed that the cath-D mitogenic activity is binding and endocytosis of the [35S]methionine-labeled mediated by the second half (amino acids 27 to 44) pro-cath-D secreted in the conditioned medium on of the cath-D pro-fragment (Fusek and Vetvicka, unlabeled recipient 3Y1-Ad12 cells in the absence or 1994; Vetvicka et al., 1999) that corresponds to amino the presence of an excess of Man-6-P (Figure 9b). acids 717 to 0 (numbering from Faust et al., 1985; SDS ± PAGE analysis of the 35S-labeled proteins see Figure 1a). However, a synthetic peptide contain- bound and taken up by cells revealed the 35S-labeled ing the 27 ± 44 residue moiety of the cath-D pro- 52 kDa pro-enzyme, and the formation of 35S-labeled fragment (cath-D pro-peptide), added at a ®nal 48 kDa and 34 kDa mature enzymes (Figure 9b, lane concentration of 2.5 nM, was ineective to stimulate 0), which are not observed in the conditioned medium the growth of the mock-transfected cells (Figure 7). by immunoprecipitation (Figure 9b, lane S). An excess Higher concentrations of the cath-D pro-peptide up to of Man-6-P, but not of Glu-6-P, only partially 50 nM were also ineective (not shown). In addition, inhibited pro-cath-D binding and endocytosis, indicating the presence of a Man-6-P independent receptor for endocytosis that might be related with the cath-D mitogenic activity (Figure 9).

Discussion

It is generally assumed that the mechanism of mitogenic activity of proteases is due to their proteolytic activity, facilitating either growth factor release (Rifkin, 1997), or growth inhibitor deterioration (Liaudet et al., 1995), or proteolytic activation of a mitogenic receptor as shown for the thrombin receptor (Carney and Cunningham, 1978). These eects are generally prevented by speci®c protease inhibitors (De Clerck and Imren, 1994). The active aspartyl protei- nases, which include the various pepsins, chymosins, cathepsin E and D, and renin consist of two lobes each Figure 8 Eect of anti-cath-D antibodies on the growth of cath- of which contains a key aspartate residue. Together D transfected cells. Control (mock-transfected) and cath-D cells they position the peptide substrate and activate its were plated on a Matrigel matrix. Cath-D cells were treated with either M2E8 or D8F5 monoclonal anti-cath-D antibodies (20 mg/ hydrolysis. Mutation of one of these active site ml), or with the control rabbit serum or with a rabbit anti-cath-D aspartate residues was sucient to eliminate enzymatic polyclonal antibody (dilution 1/200). After 4 days, cells were activity of pepsinogen (Lin et al., 1989), immunode®- recovered by Matrisperse treatment and the DNA was quanti®ed ciency virus (HIV) aspartyl proteinase (Kohl et al., as described in Materials and methods. Results are presented as in Figure 6b (mean+s.d. of three determinations). *, signi®cantly 1988; Seelmeier et al., 1988), and recombinant dierent from untreated cath-D transfectants at P50.001 baculovirus-expressed cath-D mutated in the aspartate (Student's t-test) 33 residue (Wittlin et al., 1999).

Oncogene Cathepsin-D, a mitogenic ligand M Glondu et al 6926

Figure 9 Eect of Man-6-P in excess on the growth of the wild-type and the Asn 231 mutated cath-D transfected cells (a) and on pro-cath-D endocytosis (b). (a) Matrigel outgrowth of the control (mock-transfected), cath-D, and Asn 231 cells in the presence of free Man-6-P. Cells were embedded in a Matrigel matrix in the absence or presence of 10 mM Man-6-P or 10 mM Glu-6-P (negative control) and after 7 days' culture in a 24-well plate, cells were stained with p-nitrotetrazolium violet. Similar results were obtained in another independent experiment and also with cells plated on the Matrigel matrix. (b) Eect of free Man-6-P on the internalization of 35S-labeled pro-cath-D via the Man-6-P receptors. Non-transfected 3Y1-Ad12 cells were incubated in the absence or presence of 10 mM Man-6-P or 10 mM Glu-6-P (negative control) for 24 h with 36106 c.p.m. TCA-precipitable proteins from 35S-labeled medium conditioned by cath-D transfectants. After washing, cell lysates were analysed by SDS ± PAGE after immunoprecipitation with the M1G8 antibody as described in Materials and methods. S=pro-cath-D immunoprecipitated from incubated labeled medium

First of all, we report that cath-D mutated in its tion obtained for breast cancer cell lines with puri®ed catalytic site at the position 231 lost its enzymatic pro-cath-D (Vignon et al., 1986). To our knowledge, activity (Figure 2) but retained its stability, processing this is the ®rst example of a protease remaining active and secretion (Figure 1). This mutant was used as a as a mitogen after inactivation of its catalytic site. tool to determine whether cath-D requires its enzy- These data are in accordance with our previous failure matic activity to be mitogenic. Here, we demonstrate to inhibit breast cancer cell proliferation by means of that cath-D remains mitogenic in the absence of the aspartyl protease inhibitor pepstatin (data not catalytic activity both in vitro and in vivo indicating shown). The extra-cellular action of cath-D as a that it does not operate as a protease to stimulate cell secreted protein, but not as a protease, is consistent proliferation in our experimental conditions (Figures with the fact that this protease requires a low-pH for 3 ± 5; Table 1). The growth of the dierent trans- its catalytic activity that is typically found in acidic fectants was monitored in absence of anchorage in cellular vesicles but rarely in the extra-cellular environ- 3-dimensional matrices (Matrigel, collagen I, soft agar). ment (Montcourrier et al., 1994, 1997). We show that cath-D induces Matrigel outgrowth of The Asn 231 mutant was as ecient as the wild-type cells that normally do not grow when embedded in cath-D in stimulating the growth of xenograft tumors such a matrix, suggesting that cath-D over-expression in athymic mice (Figure 5). Many independent clinical increases the transformed phenotype of these cells. studies reported that cath-D over-expression in human Indeed, cell growth in a 3-dimensional matrix might breast cancer is associated with an increased risk of optimise signal transduction between the extra-cellular developing clinical metastasis (Rochefort, 1992; Fer- proteins, cell membrane receptors and nuclear struc- randina et al., 1997; Foekens et al., 1999). It is tures mimicking more closely the in vivo situation therefore possible that this cath-D mitogenic activity (Bissell et al., 1999). In addition, culture in 3- also occurs in patients, thereby increasing the growth dimensional matrix is known to augment protein of micro-metastases that have spread before surgery. secretion as described for casein (Emerman et al., We do, however, not exclude that in vivo the wild-type 1977) and pro-cath-D in primary cultures of human cath-D acts both as a protease in acidic compartments breast cancer (Veith et al., 1983). and as an extra-cellular ligand at a physiological pH. Moreover, exogenous pro-cath-D can partially The relative contribution of these two mechanisms may mimic the mitogenicity of the transfected cath-D and depend upon the extra-cellular pH of the micro- antibodies interacting with mature cath-D inhibit cell environment where the pro-enzyme is secreted. proliferation, indicating a possible role of the secreted Finally, we show that the cath-D mitogenic activity pro-enzyme via an autocrine loop (Figures 6 ± 8). The is not directly transduced via the Man-6-P receptors level of growth stimulation observed with the puri®ed since free Man-6-P did not mimic the cath-D mitogenic pro-cath-D-Asn 231 was consistent with the stimula- activity on mock-transfected cells (Figure 9a). The

Oncogene Cathepsin-D, a mitogenic ligand M Glondu et al 6927 Man-6-P/IGF-2 receptor is a multifunctional receptor, cDNA isolated from a MCF-7 breast cancer library (Garcia binding IGF-2 and Man-6-P bearing molecules on et al., 1990) inserted into the XbaI-EcoRI site of the pSP64 distinct sites (reviewed by Vignon and Rochefort, vector (Promega). Oligonucleotides (Eurogentec) containing 1992). Our laboratory has previously demonstrated an AAC codon (asparagine) instead of a GAC codon that excess Man-6-P competes with Man-6-P bearing (aspartic acid) were used to mutate the Asp 33 and Asp 231 residues. Amino acid numbering is according to Faust et molecules but not with IGF-2 in Man-6-P/IGF-2 al. (1985). Mutations were con®rmed by DNA sequencing. receptor binding (Mathieu et al., 1990). Here, we can The full-length wild-type or mutated human pre-pro-cath-D exclude a cath-D action by displacement of IGF-2 cDNAs were then sub-cloned into the XbaI-EcoRI. site of from the Man-6-P/IGF-2 receptor to the mitogenic pcDNA3.1(-) vector (Invitrogen). insulin-like growth factor-1 (IGF-1) receptor since Man-6-P did not aect the growth of cath-D or Asn Cell lines, transfections and growth assays 231 transfectants on Matrigel (Figure 9a). This work, along with the evidence that pro-cath-D can be The 3Y1-Ad12 rat tumor cell line (Garcia et al., 1990) was endocytosed in breast cancer cells via a saturable cultured in RPMI 1640 medium with 5% fetal calf serum receptor dierent from the Man-6-P receptors (Laur- (FCS, GibcoBRL). Cells were transfected with the wild-type or mutated cath-D or the empty pcDNA3.1(-) vector alone ent-Matha et al., 1998), strongly suggests that cath-D is using Lipofectamine (GibcoBRL) in serum-free medium mitogenic independently of its binding and action (Optimem, GibcoBRL) and were then selected for resistance through the Man-6-P receptors. We ®nally show that to G418 (400 mg/ml, Sigma). All experiments using trans- pro-cath-D still binds to and is partially endocytosed in fected cells were performed from passages 4 ± 12 and the 3Y1-Ad12 cells even in the presence of an excess of free stability of human cath-D expression was controlled in this Man-6-P suggesting the existence of an alternative period. For Matrigel outgrowth assays, 100 000 sub-con¯uent receptor that might mediate the cath-D mitogenic cells were harvested and re-suspended in Matrigel (0.2 ml, activity (Figure 9b). Vetvicka and colleagues have 6.3 mg/ml) (Becton and Dickinson), an extra-cellular matrix described a mitogenic activity of the cath-D pro- extracted from Engelbreth-Holm-Swarm (EHS) mouse fragment moiety (amino acids 27 ± 44) on cancer cell tumor, at 48C and then quickly added to a pre-set Matrigel layer in 24-well plates as described (Thompson et al., 1992). lines maintained in serum-free conditions suggesting an The top Matrigel cell layers were gelled at 378C for 30 min, interaction of its pro-part with an unknown cell surface and then covered with culture medium with 5% FCS receptor (Fusek and Vetvicka, 1994; Vetvicka et al., (0.5 ml). For treatment of cells embedded in Matrigel, 1999). However, we have not been able to reproduce compounds (e.g. Man-6-P, Glu-6-P) were added to the this eect when the synthetic peptide 27 ± 44 was tested Matrigel layers and to the culture medium. For growth on on breast cancer cells seeded on plastic substratum in the Matrigel matrix, 100 000 sub-con¯uent cells were plated various conditions including that described by Fusek on a pre-set Matrigel layer (0.2 ml, 6.3 mg/ml) in 24-well and Vetvicka (1994) (unpublished experiments from plates and then covered with culture medium with 0.75% our laboratory) or on the 3Y1-Ad12 cells plated on FCS (0.5 ml). For treatment of cells plated on the Matrigel Matrigel (Figure 7) or on plastic (not shown). matrix, conditioned media (0.5 ml) or compounds (e.g. puri®ed pro-cath-D-Asn231, cath-D pro-peptide 27-44, con- Furthermore, the M2E8 antibody speci®c for the pro- trol peptide M20, anti-cath-D antibodies: M2E8, D8F5, enzyme did not inhibit cell proliferation in contrast rabbit polyclonal, or rabbit serum) were directly added after with D8F5 antibody interacting with the large chain attachment. To prepare the conditioned media, control, cath- (34 kDa) of cath-D (Figure 8), showing that inter- D or Asn 231 cells were plated at a low density and after ference with the pro-domain had no eect. Together, 24 h, cells were washed twice with culture medium without these observations indicate that a receptor to this pro- FCS and then cultured with 0.75% FCS. After 4 days in fragment was unlikely to be responsible for the 0.75% FCS, cells reached 70% con¯uence and the condi- mitogenic activity of cath-D detected in our conditions. tioned medium was removed and centrifuged at 800 g for In conclusion, our results demonstrate that a 10 min and ®nally stored at 7808C for subsequent mutated cath-D can stimulate cell growth without its experiments. The entire pro-fragment of 52 kDa pro-cath-D was provided by M. Fusek (Louisville, KY, USA) (Fusek and catalytic activity and suggest that pro-cath-D is acting Vetvicka, 1994). The cath-D pro-fragment peptide (amino as an extra-cellular ligand, but not as a protease, by acids 27-44) was synthesized by Eurogentec and its purity was triggering directly or indirectly a yet unidenti®ed cell veri®ed by HPLC. The control peptide M20 corresponds to surface receptor. This work might ultimately lead to amino acids 1793-1812 of the mouse BRCA1 (Santa Cruz the development of new therapeutic strategies designed Biotech.). Two mouse monoclonal antibodies raised against to block cath-D action in cancer cells by inhibiting its pro-cath-D were used: D8F5 (Garcia et al., 1985) and M2E8 interaction with a cell surface receptor coupled to a (Freiss et al., 1988), which interact speci®cally with total cath- mitogenic pathway rather than its proteolytic activity. D (52 kDa, 48 kDa and 34 kDa forms) and 52 kDa, respectively. The rabbit anti-cath-D antibody raised against total cath-D was purchased at DAKO (S.A.). Cells growing into or on Matrigel were recovered with 3 ml of Matrisperse (Becton and Dickinson), ®xed with methanol and their DNA Materials and methods content was determined by the diaminobenzoic acid ¯uores- cence assay (Vignon et al., 1986). Quanti®cation of cells in Generation of constructs Matrigel was checked to be linear from 100 000 to 5 000 000 Cath-D mutations were made by site-directed mutagenesis embedded cells and from 100 000 to 1 000 000 plated cells. (Stratagene) on the 2 kb full-length human pre-pro-cath-D For collagen I outgrowth, 100 000 sub-con¯uent cells were

Oncogene Cathepsin-D, a mitogenic ligand M Glondu et al 6928 embedded in a collagen I gel at 48C (0.2 ml, 0.5 mg/ml) adding cold extraction buer (10 mM Tris-HCl pH 7.4, (Becton and Dickinson) and then added on a pre-set rat tail 1.5 mM EDTA, 0.1% thioglycerol) followed by ®ve freeze- collagen I layer (0.2 ml, 0.5 mg/ml) in 24-well plates as thaw cycles. Total proteins from conditioned media (7 mg) or described (Veith et al., 1983) and supplemented with culture from cell extracts (50 mg) were incubated with 30 000 c.p.m. medium containing 5% FCS. Soft agar assays were [14C]methemoglobin (New England Nuclear, Boston, MA, performed using 20 000 cells per 35 mm Petri dish (Fang et USA), 2 mg of unlabeled methemoglobin (Sigma), in a citrate al., 1992). reaction buer (pH 2.5-4.5) (100 ml, ®nal volume) and in the absence or presence of 2 mM pepstatin (Sigma) (Capony et al., 1987). The proteolytic activity was calculated from the Protein labeling, immunoprecipitation, purification and TCA-soluble radioactivity per microgram of DNA extracted immunoassay from the corresponding cells. Cells were incubated in methionine-free DMEM supplemented with 200 mCi/ml [35S]methionine (41000 mCi/mmol, Tumor growth in animals Amersham) for 6 h. Labeled proteins were immunoprecipitated with the anti-cath-D antibody (M1G8, 80 mg/ml), Cells were trypsinized, washed, centrifuged, resuspended in analysed by 12% SDS ± PAGE and ¯uorography (Capony cold PBS (GibcoBRL) and mixed with an equal volume of et al., 1987). For pro-cath-D-Asn 231 puri®cation, Asn 231 cold Matrigel (10 mg/ml). A total volume of 0.3 ml contain- transfectants at 80% con¯uency were washed twice with ing (400 000) cells was subcutaneously injected into 6-week- culture medium without FCS and incubated for 24 h in old Balb/c athymic female nude mice (IFFA CREDO). absence of FCS. Conditioned medium was removed, Injected mice were examined every 2 days for tumor centrifuged at 800 g for 10 min and stored at 7808C. Pro- appearance and tumor sizes were estimated from the product cath-D-Asn 231 was puri®ed from the conditioned medium in of the two perpendicular diameters. the absence of detergent using a one-step procedure on a M1G8 Sepharose anity column (Capony et al., 1989) modi®ed as described (Laurent-Matha et al., 1998) and eluted with 20 mM lysine, pH 11. The eluate was then adjusted to pH 7.4 with 1 M Hepes buer and the purity of Acknowledgments the 52 kDa pro-cath-D recovered after elution was analysed We thank Dr F Vignon for helpful discussions and on a 12% SDS ± PAGE by silver staining (Pharmacia suggestions, Dr P Roger for pathology advice, V Gaubiac, Biotech). Intra-cellular and secreted human cath-D were M Gleizes and A Lucas for technical assistance, JY Cance quanti®ed using a cath-D immunoradiometric assay that does for photographs and drawing of ®gures, and N Kerdjadj not cross-react with rodent cath-D (Garcia et al., 1985). and E Moreno for secretarial assistance. This work was supported by the University of Montpellier I, the Ìnstitut National de la SanteÂ et de la Recherche MeÂ dicale,' the Proteolytic activity assays Àssociation pour la Recherche sur le Cancer,' the Ìnstitut Conditioned media were collected after 24 h incubation of de Recherches Servier' and the `Ligue Nationale contre le cells in serum-free medium and cell extracts were prepared by Cancer, ComiteÂ de l'HeÂ rault'.

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Oncogene