Specific expression of matrix metalloproteinases 1, 3, 9 ... - Springer Link

Download PDF
2 downloads 0 Views 116KB Size Report
Comment
1Laboratoire de Biochimie, Hôpital Claude Huriez, 59037 Lille Cedex, ... Concord Street, Boston, MA 02118, USA; 3Unité INSERM 377, place de Verdun,.
Clinical & Experimental Metastasis 18: 171–178, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

171

Specific expression of matrix metalloproteinases 1, 3, 9 and 13 associated with invasiveness of breast cancer cells in vitro Malika Balduyck1 , Farid Zerimech1 , Val´erie Gouyer1 , Raphael Lemaire2 , Brigitte Hemon3 , Georges Grard1 , Carole Thiebaut1 , V´eronique Lemaire1 , Evelyne Dacquembronne1 , Th´er`ese Duhem1 , Anne Lebrun1 , Marie-Jos´e Dejonghe3 & Guillemette Huet1,3 1 Laboratoire

de Biochimie, Hôpital Claude Huriez, 59037 Lille Cedex, France; 2 Boston University School of Medicine, The Arthritis Center – Bldg. K-5, 80E. Concord Street, Boston, MA 02118, USA; 3 Unit´e INSERM 377, place de Verdun, 59045 Lille Cedex, France Received 26 June 2000; accepted in revised form 5 September 2000

Key words: metalloproteinases, breast cancer cells, invasiveness, tissue inhibitors of metalloproteinases, synthetic inhibitors of metalloproteinases

Abstract Several matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) were studied in highly invasive (MDAMB-231) and slightly invasive (MCF-7, T47D, BT-20) breast cancer cell lines. Investigations were carried out at the protein level and/or at the mRNA level, either in cells cultured as monolayers on plastic, or in cells seeded on a thin layer of Matrigel basement membrane matrix. Analysis of MMP expression by RT-PCR showed expression of MMP-1, MMP-3, and MMP-13 in highly invasive MDA-MB-231 cells, but not in slightly invasive cell lines. The extracellular secretion of MMP-1 and MMP-3 by MDA-MB 231 cells could be also shown by ELISA. TIMP-1 and TIMP-2 mRNAs were found in all cell lines, however, the extracellular secretion of both TIMPs was much higher in MDA-MB-231 cells than in the other cell lines. When the cells were cultured on Matrigel matrix, MMP-9 expression was induced in MDA-MB-231 cells only, as assessed by RT-PCR and zymography experiments. The invasive potential of MDA-MB-231 cells evaluated in vitro through Matrigel was significantly inhibited by the MMP inhibitor BB-2516, by 25% and 50% at the concentrations of 2 × 10−6 M and 10−5 M, respectively. In conclusion, our data show that highly invasive MDA-MB-231 cells but not slightly invasive T47D, MCF-7 and BT-20 cells express MMP-1, MMP-3, MMP-9 and MMP-13. MMP-9 which is specifically up-regulated by cell contact to Matrigel, may play a key role in the invasiveness of MDA-MB-231 cells through basement membranes.

Introduction Mortality from breast cancer is due to dissemination of tumor cells and formation of metastasis. The invasive potential of malignant cells is linked to their capacity to degrade basement membranes and extracellular matrix. In that field, the matrix metalloproteinases (MMPs) display more or less specific proteolytic activity against components of these matrices [1]. MMPs are a family of zinc-binding endopeptidases classified into four general classes: interstitial collagenases, gelatinases (type IV collagenases), stromelysins and membrane-type MMPs (MTMMPs). Interstitial collagenases (MMP-1/collagenase-1, MMP-8, and MMP-13/collagenase-3) degrade the fibrillar collagens type I, II, III, VII and X. Gelatinases (MMP-2/72 kDa type IV collagenase/gelatinase A, and MMP-9/92 kDa type IV collagenase/gelatinase B) cleave the collagens type IV, V, VII, and X, fibronectin and elastin. The stromelysins MMP-3/stromelysin-1, MMP-10/stromelysin-2 and MMPCorrespondence to: Dr Guillemette Huet, Laboratoire de Biochimie, Hôpital Claude Huriez, rue Michel Polonovski, 59037 Lille Cedex, France. Tel: +33-20446154; Fax: +33-20445693; E-mail: [email protected]

7/matrilysin display a proteolytic activity upon collagen types III, IV, V, IX, proteoglycans and other matrix glycoproteins, whereas the MMP-11/stromelysin 3 has the serine proteinase inhibitors α1-proteinase inhibitor (α1-PI) and α2antiplasmin as unique known substrates [2, 3]. However, the latter MMP was recently reported to increase the tumorigenicity of breast cancer cells via a proteolytic activity [4]. The membrane-type MT1-MMP has been described as an activator of pro-MMP2 [5–7]. In breast cancer, overexpression of several MMPs has been described and is generally associated with breast tumor progression: MMP-2, MMP-9, MMP-11, MMP-13 and MT1-MMP [8–14]. The expression level of MT1-MMP was found correlated with the activation of proMMP-2 and with the presence of lymph node and distant metastases [13]. MMP-11 mRNA has been particularly described as an independent prognostic factor for relapse-free survival [10, 11]. Despite their function of inhibition of enzymatic activities of MMPs, the increase in the expression of TIMP-1 and TIMP-2 in breast cancers is associated to poor prognosis and development of metastasis [15–17]. Investigations by in situ hybridization on human tumors have shown that MMPs can

172 be produced by cancer cells or stromal cells. In breast cancer, several MMPs (MMP-1, -2, -3, -11 and -14) are expressed by stromal fibroblasts, according to different expression patterns in the tumor stroma [18–22]. MMP-9 is expressed by tumor infiltrating macrophages and vascular pericytes [19, 21, 23]. MMP-7 and MMP-13 are specifically produced by normal and/or neoplastic epithelial cells of breast tumors [19, 21]. TIMP-1 and TIMP-2 were found expressed in tumor and/or stromal cells [14, 21]. The expression of MMPs has been studied in many cell lines, particularly in the study of Giambernardi et al. [24] including 84 cell lines of different origin. No secreted MMPs was detected in MCF-7 and BT-20 cell lines, while T-47D cells expressed MMP-7 and MMP-10, and MDA-MB-231 cells MMP-1, -2, -7, -9, and -10. However, MDA-MB231 cells were also previously reported to express MMP-1, MMP-3 and MMP-9 but not MMP-2 [25], while in the study of Gilles et al. [26], MMP-1 but not MMP-3 could be detected. Expression of the membrane-type MT1-MMP was also found in BT-20, T47D and MDA-MB-231 cells [24, 27]. Some of these MMPs have been identified in the extracellular medium of breast cancer cells in culture, and in particular MMP-1, MMP-2 and MMP-9 for MDA-MB-231 cells [25, 28–32], MMP-2 and MMP-9 in MCF-7 cells [30]. The secretion of TIMP-1 and TIMP-2 by MDA-MB-231 cells was also reported [32]. However, to our knowledge, a comparative study of MMPs and TIMPs at a quantitative protein level in invasive or non-invasive breast cancer cells has not been previously reported. The contribution of the MMPs in cellular invasion and metastatic potential can be evaluated in in vitro assays through reconstituted basement membrane, using specific synthetic inhibitors of MMPs [31, 33]. Our aim was first to compare the expression and secretion of MMPs and TIMPs in cell lines of different invasive and estrogen receptor-expressing phenotypes. We used the highly invasive estrogen receptor (ER)-negative MDA-MB231 cell line, and the slightly invasive ER-negative (BT-20) or ER-positive (MCF-7, T47D) cell lines. Investigations were successively carried out in cells cultured as monolayers on plastic, and in cells cultured on a thin layer of Matrigel in order to study the regulation by this extracellular matrix. We have then examined the modulation by the synthetic metalloproteinase inhibitor BB-2516 (British Biotech).

Materials and methods Cell monolayer culture Four cell lines MDA-MB-231, T47D, MCF-7, BT-20 were comparatively analyzed. Each cell line was cultured in triplicate. Cells were seeded in 75 cm2 flasks in minimum essential medium (MEM) with Earle’s salts and without phenol red (Gibco), containing 10% fetal calf serum, at 37 ◦ C under 5% CO2 /95% air atmosphere. Cells were cultured up to 80% confluence. A triplicate culture was used for the study at the mRNA level by RT-PCR. For the investigation of secreted MMPs and TIMPs by ELISA, another triplicate was

M. Balduyck et al. seeded, and cultured in serum – free medium for 24 hours before collection of the cell conditioned culture media. The effect of the synthetic metalloproteinase inhibitor BB-2516 upon viability and proliferation of the breast cancer cells was studied at different concentrations of the drug with each cell line (0.5, 2, 5 and 10 µM). The drug was added in the culture medium all over the cell culture from day 2 after seeding. After BB-2516 treatment, the cells were trypsinized. Aliquots of the cell suspensions were used for evaluation of cell viability and for cell counting. Cell viability, evaluated after BB-2516 treatment, was always > 94%, by Trypan Blue exclusion, and was not affected by BB-2516 treatment. The amounts of cells obtained after culture in the presence of BB-2516 were always similar to the amounts of control cells obtained after culture in the absence of the drug, whatever the concentration used between 0.5 to 10 µM. Cell culture on a thin layer of Matrigel basement membrane Matrigel invasion chamber Cells in MEM containing 0.1% BSA were seeded on Biocoat Matrigel six chamber plates (7 × 105 cells per chamber) (Becton Dickinson). Conditioned cell culture medium of cultured 3T3 fibroblasts filled the lower compartment as chemoattractant. The invasion assay through Matrigel was carried out at 37 ◦ C under 5% CO2 / 95% air atmosphere up to 24 hours. For the analysis of modulation by BB-2516, the Matrigel coated inserts were first incubated with the drug dissolved in the culture medium for 2 hours at room temperature, and then seeded with the cells suspended in the same drug-enriched medium. Each invasion assay was performed in duplicate. After that, the invading cells were countered in each chamber by two different operators using 10 areas. The results were then statistically analyzed. We also checked that the percentage of viable cells after culture on Matrigel was not altered by BB-2516 treatment. In another experiment, cells were seeded in the same conditions on Matrigel coated inserts and incubated up to 24 or 48 hours. The upper supernatant was collected, centrifuged and analyzed by zymography. Both adhesive cells, and cells embedded in the Matrigel, were then isolated by mean of Matrisperse (Becton Dickinson), and processed for RNA isolation as described later. Cell culture on Matrigel coated dishes Cells (1.4 × 106) were also seeded on a thin layer of Matrigel (100 micrograms/cm2) coated on 35 mm dishes (Becton Dikinson) and cultured for 24 hours. Culture media and cells were processed as previously described. RT-PCR RNA expression of MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-11, MMP-13) and TIMPs (TIMP1, TIMP-2, TIMP-3, TIMP-4) was investigated by RT-PCR. Investigations were carried out from cell monolayers and from cells removed from the Matrigel matrix by mean of Matrisperse. Duplicate RT-PCR analyses were carried out on RNA isolated from the triplicate cell monolayer cultures

MMP-1,-3,-9 and -13 secreted by invasive breast cancer cells

173

(six experiments). Duplicate RT-PCR analyses were carried out for cells isolated from Matrigel both in the invasion chamber assay and in the coated dishes. Total cellular RNA was isolated after cell lysis in guanidium isothiocyanate and ultracentrifugation through a cesium chloride gradient. The amount of isolated RNA was quantified by measuring its absorbance at 260 nm. The same RNA samples were used for MMP or TIMP analysis. Reverse transcription of RNA was performed using the SuperscriptTM RNA H− reverse transcriptase kit (Gibco), and PCR was carried out with specific primers (Eurogentec), on a GeneAmp 2400 system (Perkin Elmer). Specific primers were as follows: MMP1, 50 -ATTGGAGCAGCAAGAGGCTGGGA-30 and 50 TTCCAGGTATTTCTGGACTAAGT-30 [34]; MMP-2, 50 GGGGCCTCTCCTGACATT-30 and 50 -TCACAGTCCGCC AAATGAA-30 [35]; MMP-3, 50 -GCATAGAGACAACATAG AGCT-30 and 50 -TTCTAGATATTTCTGAACAAGG-30 [34]; MMP-7, 50 -AGATGTGGAGTGCCAGATGT-30 and 50 TAGACTGCTACCATCCGTCC-30 [36]; MMP-9, 50 -GGCC CTTCTACGGCCACT-30 and 50 -CAGAGAATCGCCAGTA CTT-30 [35]; MMP-11, 50 -CGCAACCGACAGAAGAGGTT CG-30 and 50 -GCAGGAAGTAGGCATAGCCATCAGC-30; MMP-13, 50 -TGCTGCATTCTCCTTCAGGA-30 and 50 ATGCATCCAGGGGTCCTGGC-30 [37]; TIMP-1, 50 CTTCCACAGGTCCCACAACC-30 and 50 -CAGCCCTGG CTCCCGAGGC-30 ; TIMP-2, 50 -GGTCTCGCTGGACGTT GGAG-30 and 50 -GGAGCCGTCACTTCTCTTG-30 [35]; TIMP-3, 50 -CGCTGGTCTACACCATCAAGC-30 and 50 CAGGAGGATAGTTCCCAATAAACC-30 ; TIMP-4, 50 CAGTGAGAAGGTAGTTCCGGCC-30 and 50 -TCTTATTA GCTGGCAGCAAGAGG-30 . RT-PCR analyses monitoring Figure 1. RT-PCR analysis of MMP-1, MMP-3, and MMP-13, in MCF-7, expression of MMPs and TIMPs was performed using 30 T47D, BT-20 and MDA-MB-231 breast cancer cells cultured as cell monolayers. amplification cycles. Glyceraldehyde phosphate deshydrogenase (GAPDH) was amplified as internal control (G3PDH Control Amplimer Set, Clontech). The PCR products were Zymography then analyzed by electrophoresis in 2% agarose gels, with Secreted MMPs were analyzed by zymography. Cell culture detection by ethidium bromide. medium (20 µl) collected from cell monolayers in 75 cm2 flasks or from cells seeded on Matrigel was migrated on a ELISA 10%/SDS-PAGE containing 0.1% gelatin as substrate. The Cells were homogeneized in EORTC buffer. Protein con- gel was incubated for 48 hours at 37 ◦ C in a 50 mM Tris HCl tents in the cell lysates were determined by the BCA protein buffer containing 5 mM CaCl2 , 1 µM ZnCl2 , 1% Triton, assay from Pierce. The matrix metalloproteinases MMP- 0.02% NaN3 , and 1 mM p-aminophenylmercuric acetate, 1, and MMP-3 and the inhibitors TIMP-1 and TIMP-2 and then stained by Coomassie blue. were measured in the cell culture media by commercially available ELISA kits (from Amersham-Pharmacia-Biotech, Statistical analysis Buckinghamshire, UK). MMP-1 and MMP-3 assays recognize all MMP1 and MMP-3 forms, respectively, free MMPs, All values are given as mean ± standard derivation MMPs complexed with inhibitors, pro-MMPs. TIMP-1 as- (m ± SD). The non-parametric test (U Mann–Whitney test) say recognizes all TIMP-1 forms, respectively, free TIMP-1, was used to compare treated and control cells. Differences TIMP-1 complexed with the active form of MMPs, TIMP-1 were considered statistically significant when P was < 0.05. complexed with ProMMP-9. TIMP-2 assay recognizes free TIMP-2, TIMP-2 complexed with the active form of MMPs, but not TIMP-2 complexed with pro-MMP-2. All the values result from duplicate ELISA made from three different cell cultures (6 determinations). The results were expressed in ng per mg of cell proteins.

174

M. Balduyck et al. MMP-1 and MMP-3, that were found expressed in MDA-MB-231 cells, were evaluated in cell-conditioned media by immunoassay. The results are given as the mean value of duplicates on three different cultures (Table 1). The secretion of MMP-1 (204 ng/mg) and MMP-3 (265 ng/mg) was shown in the extracellular medium of MDA-MB-231 cells but not of the other cell lines. Although TIMP-1 and TIMP-2 were found expressed in all cell lines by RT-PCR, the corresponding proteins were found at a much higher level in MDA-MB-231 cell culture media (Table 1). Protease analyses after culture of breast cancer cells on a thin layer of Matrigel basement membrane matrix

Figure 2. RT-PCR analysis of the expression of TIMPs in MCF-7, T47D, BT-20 and MDA-MB-231 breast cancer cells cultured as cell monolayers.

Results Comparative analysis of MMPs and TIMPs in invasive and non invasive breast cancer cell monolayers We first studied the expression of MMPs and TIMPs by RTPCR in the four breast cancer cell lines, MCF-7, T47D, BT-20 and MDA-MB-231. MMP amplification products were only observed in MDA-MB-231 cell line, and Figure 1 shows the amplification products obtained among the panel of MMPs studied by RT-PCR analysis. Gelatinase mRNA, MMP-2 and MMP-9, and stromelysin mRNA, MMP-7 and MMP-11, were not detected in either cell type studied. Positive control amplifications for MMP-2, -7, -9, -11 were obtained using RNAs from colon cancer and lung cancer tissues. The collagenases MMP-1 and MMP-13, and the stromelysin MMP-3 were expressed in MDA-MB-231 cells (Figure 1). Figure 2 shows RT-PCR analysis of TIMPs in the four breast cancer cell lines. All cell lines expressed TIMP-1, TIMP-2 and TIMP-3, while TIMP-4 mRNA was only detected in MDA-MB-231 cells (Figure 2). Besides, an additional band to the expected amplification product of TIMP-2 was observed in MCF-7, T47D and BT-20 cell lines but not in MDA-MB-231 cells.

In vitro invasion assays in Matrigel invasion chambers were carried out for the four cell lines. Only MDA-MB-231 cells displayed a high invasive capacity on Matrigel, in accordance with previously published results [38]. With regards to the fact that Matrigel is mainly constituted by the collagen type IV, the expression of the gelatinases MMP-2 and MMP-9 was investigated in the cells cultured on the Matrigel matrix. RT-PCR with the specific primers of MMP-9 revealed the presence of an amplification product at the expected size (134 bp) in MDA-MB-231 cells only (Figure 3). In contrast, a band at a higher molecular size was seen for the three other slightly invasive cell lines. No MMP-2 amplification product could be detected in MDA-MB-231 cells, as well as in the other cell lines. In an attempt to gain more knowledge upon the specificity of expression of MMP-9 by MDA-MB-231 cells versus the three other cell lines, the upper cell culture media of the four cell lines were collected after the invasion assay for a zymography analysis on gelatin substrate SDS-PAGE. Interestingly zymography analysis showed the presence of a gelatinolytic activity at about 92 kDa in the Matrigel supernatant of MDA-MB-231 cells only (Figure 3). In order to confirm this observation, the four cell lines were then cultured on a thin layer of Matrigel coated on plastic dishes. Amplification with the specific primers for MMP-9 confirmed the above mentioned results. Likewise, the 92 kDa gelatinolytic activity was only visualized in the culture supernatant of MDA-MB-231 cells (data not shown). Effects of BB-2516 in the invasion assay The effects of the drug upon the invasive potential of MDAMB-231 cells was studied in the Matrigel invasion chambers over a period of 24 hours. BB-2516 was found to inhibit significantly the invasive capacity of MDA-MB-231 cells. The inhibition was of 25% at the concentration of 2 × 10−6 M, and of 50% at the concentration of 10−5 M (Figure 4).

Discussion In an attempt to highlight MMPs involved in breast cancer metastasis, we have compared the production and secretion of MMPs and TIMPs in the highly invasive ER-negative

MMP-1,-3,-9 and -13 secreted by invasive breast cancer cells

175

Figure 3. Analysis of MMP-9 expression after culture of T47D, MCF-7, BT-20 and MDA-MB-231 breast cancer cells on Matrigel. (A) RT-PCR analysis of MMP-9 expression in cells detached from Matrigel after 24-hour invasion assay of cells setted on Biocoat Matrigel. (B) Zymography of the upper cell culture media collected after 24 hour invasion assay of cells setted on Biocoat Matrigel.

Figure 4. Inhibition assays of MDA-MB-231 cell invasiveness in Matrigel invasion chambers with BB-2516 at the concentrations of 2 × 10−6 M and 10−5 M.

176

M. Balduyck et al. Table 1. MMP-1, MMP-3, TIMP-1 and TIMP-2 levels evaluated by ELISA in cell culture media of breast cancer cells cultured as cell monolayers.

MMP-1 MMP-3 TIMP-1 TIMP-2

BT-20

MCF-7

MDA-MB-231

T47D

9.1 ± 3.0 4.2 ± 0.2 318.2 ± 21.5 445.7 ± 203.5

4.7 ± 1.8 3.2 ± 0.2 51.4 ± 21.5 372.4 ± 43.0

203.8 ± 250.0 265.0 ± 250.0 2743.0 ± 594.0 9765.5 ± 4191.2

3.0 ± 1.5 6.7 ± 0.3 101.8 ± 0.2 297.3 ± 79.0

Results are expressed in ng per mg of cell proteins (mean value ± SD).

MDA-MB-231 cell line in comparison to the slightly invasive ER-negative (BT-20) or ER-positive (MCF-7, T47D) cell lines. Investigations were carried out in cells cultured as monolayers on plastic or on Matrigel in order to study the regulation by this extracellular matrix. Then, we have studied the modulation of cell invasiveness in the highly invasive MDA-MB-231 cell line by the metalloproteinase synthetic inhibitor BB-2516. The comparison between the highly invasive cell line (MDA-MB-231) and the three slightly invasive cell lines (MCF-7, T47D and BT-20) showed strong differences in the expression and release of MMPs. Analysis of MMP expression by RT-PCR in cells cultured on plastic showed expression of MMP-1, MMP-3, and MMP-13 in MDAMB-231 cells, but not in the other cell lines tested. Some discrepancies were observed in the expression pattern of MMPs in MDA-MB-231 cells in comparison to the RTPCR study of Giambernadi et al. [24], and they likely result from differences in culture conditions. On the other hand, by Northern blot, expression of MMP-1 by MDAMB-231 cells was also previously shown, while expression of MMP-3 was not detected [26]. The secretion of MMP1 and MMP-3 could be also evidenced in the extracellular medium of MDA-MB-231 cells but not of the other cell lines, in accordance to the expression pattern observed by RT-PCR. Although MDA-MB-231 cells were previously reported to produce MMP-9 [25], MMP-9 mRNA could not be detected in MDA-MB-231 cells cultured on plastic in our conditions, but its expression was specifically induced by culture of MDA-MB-231 cells on Matrigel. Furthermore, a gelatinase activity was specifically visualized in the upper cell culture medium of MDA-MB-231 cells seeded on Matrigel invasion chamber, as well as in the cell culture medium of these cells cultured on a thin layer of Matrigel. Therefore, the contact between the breast cancer cells studied and Matrigel results in the stimulation of the expression and secretion of MMP-9 in the invasive MDA-MB-231 cells only. In contrast, the gelatinase MMP-2, did not appear to be significantly induced in these conditions. MMP-9 thus appeared as a protease specifically expressed and externalized through the contact of MDA-MB-231 cells with extracellular matrix components. Expression of MMP genes is primarily regulated at the transcriptional level by growth factors, cytokines and contact with ECM [39]. Concerning the regulation of MMPs by cell contact to ECM, data have been particularly reported for the MMP-1 gene expression in fibroblast-like synoviocytes and keratinocytes [40-42]. Collagen type 1 was

also reported to induce the expression of membrane type 1 MT1-MMP and the activation of MMP-2 in fibroblasts and invasive human breast carcinoma cell lines, and these mechanisms might be involved in tumor invasion and metastasis [43]. Matrigel represents an artificially reconstituted basement membrane which contains proteins expressing the RGD sequence and particularly laminin. Our data suggest a specific activation of the expression of MMP-9 in MDA-MB-231 cells via integrins. Monoclonal anti-integrin antibodies anti-α3 and anti-β1 induce MMP-9 expression in human keratinocytes [44]. A monoclonal anti-β4 integrin antibody was recently reported to increase the expression of MMP-2 in human colon adenocarcinoma cells, and the invasiveness of these cells [45]. Matrigel invasion chambers provide a tool to evaluate cell invasiveness in vitro. Only MDA-MB-231 cells displayed a significant invasive potential in this invasion assay. In our assay in serum-free conditions u-PA is not likely playing a major role in Matrigel invasion as the activity of u-PA in MDA-MB-231 cell invasiveness has been previously shown to be strictly dependent upon the presence of plasminogen [33]. Previous in vitro invasion assays on radiolabelled subendothelial basement membrane [33] or on a thin layer of Matrigel [31] using different types of protease inhibitors have shown that synthetic MMP inhibitors generated the most important inhibition. Our current results highlight the potential role of MMP-1, MMP-3, MMP-9 and MMP-13, as they are the MMPs significantly expressed by MDA-MB-231 cells in comparison to the other non-invasive cells. A functional proteolytic activity against Matrigel is clearly shown in the invasion assay by MDA-MB-231 cells, despite the fact that these cells secrete high levels of TIMP1 and TIMP-2 and also specifically expressed TIMP-4 in comparison to the non-invasive breast cancer cells. The inhibition potential exerted by the synthetic metalloproteinase inhibitor BB-2516 upon cell invasion suggests that the secreted TIMPs cannot circumvented the enzymatic activity of secreted MMPs, at least in focalized areas at the periphery of the cells. Such hypothesis was also suggested as a possible in vivo mechanism in tumors [32]. Furthermore, it must be also emphasized that, in addition to their inhibition potential towards MMPs, TIMPs are known to play other functions, such as in the regulation of the activation of proMMP-2 by the membrane-type 1 MMP (MT1-MMP) [7, 46, 47], or as potential growth factor activity as TIMP-1 displays a high homology with erythroid-potentiating activity [48].

MMP-1,-3,-9 and -13 secreted by invasive breast cancer cells The enzymatic specificity of MMP-9 towards type IV collagen suggests that this MMP could be potentially responsible for the proteolysis of the Matrigel matrix, which is comprised, in part, by type IV collagen. Furthermore proMMP-9 has been shown to be activated by active MMP13 [49] or MMP-3 [50, 51] in vitro, and these two MMPs are specifically expressed by MDA-MB 231 cells in comparison to the other cell lines here studied. The activation of proMMP-9 in a cell culture system was obtained by the addition of both plasminogen and proMMP-3 [32]. The activation of proMMP-3 occurred via plasmin generated by uPA produced by MDA-MB-231 cells [32]. However, the authors reported that the activation of the secreted proMMP-9 was obtained with MDA-MB-231 cells overexpressing MMP9 by transfection, but not with standard MDA-MB-231 cells, because the high extracellular level of TIMP-1 dampened the activation of proMMP-9 by binding to proMMP-9 [32]. Indeed, the gelatinase activity here visualized by the zymography experiment with the cell culture medium of MDA-MB-231 cells seeded on Matrigel exhibited an apparent molecular weight corresponding to that of the latent proMMP-9 form. A similar observation was made for MMP1, as no collagenolytic activity on native collagen (from calf skin) could be evidenced in the cell culture medium of MDA-MB-231 cells as of other cell lines (data not shown). In the culture conditions of MDA-MB-231 cells upon Matrigel, the expression and secretion of proMMP-9 is induced, and it may be suggested that a part of proMMP-9 could be locally activated by mean of the other secreted proMMPs, the latter being activated via the plasmin or the membrane type MMP (MT-MMP) cascades [5, 51, 52]. Indeed Matrigel matrix is known to contain a low level of plasminogen, which could lead to the production of plasmin through u-PA which is secreted at a high level by MDA-MB-231 cells in comparison to the other cell lines (data not shown). MDAMB-231 cells also express MT1-MMP [24, 27], and the induction of MT1-MMP expression was previously shown to be correlated with MMP-2 activation [27, 43]. In conclusion, our data show that the highly invasive MDA-MB-231 cells differentiate from the slightly invasive T47D, MCF-7 and BT-20 cells by the expression of MMP1, MMP-3, MMP-9, and MMP-13. Our results also suggest that the proteolytic activity of MMP-9 plays an important role in the invasiveness of MDA-MB-231 cells through Matrigel. Multiple mitogen-activated protein kinase (MAPK) signaling pathways regulate MMP gene expression, allowing specific responses to different signals [39]. The activity of the p38 MAPK has been recently shown to mediate the expression of MMP-9 in squamous cell carcinoma cells, and also the invasive potential of these cells through Matrigel [53]. Perhaps this MAPK is stimulated in invasive breast cancer cells upon contact with laminin via integrin receptors. Acknowledgements This work was supported by a grant from the Delegation à la Recherche du CHRU de Lille (no. 9714). The authors are very grateful to British Biotech Pharmaceuticals, Oxford,

177 UK, for the gift of the MMP inhibitor BB-2516, and to Dr R. Lafyatis (Boston University School of Medicine) for critical reading of the manuscript. We are also very grateful to Dr J.Ph Peyrat and Mrs M Louchez (Laboratoire d’Oncologie Moléculaire, Centre Oscar Lambret, Lille) for the culture of the cell lines.

References 1. 2. 3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Liotta LA, Stetler-Stevenson WG. Metalloproteinases and cancer invasion. Semin Cancer Biol 1990; 1: 99–106. Woessner JFJ. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991; 5: 2145–54. Pei D, Majmudar G, Weiss SJ. Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3. J Biol Chem 1994; 269: 25849–55. Noel A, Boulay A, Kebers F et al. Demonstration in vivo that stromelysin-3 functions through its proteolytic activity. Oncogene 2000; 19: 1605–12. Sato H, Takino T, Okada Y et al. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 1994; 370: 61–5. Sato H, Takino T, Kinoshita T et al. Cell surface binding and activation of gelatinase A induced by expression of membrane-type-1-matrix metalloproteinase (MT1-MMP). FEBS Lett 1996; 385: 238–40. Strongin AY, Collier I, Bannikov G et al. Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J Biol Chem 1995; 270: 5331–8. Monteagudo C, Merino MJ, San-Juan J et al. Immunohistochemical distribution of type IV collagenase in normal, benign, and malignant breast tissue. Am J Pathol 1990; 136: 585–92. Onisto M, Riccio MP, Scannapieco P et al. Gelatinase A/TIMP-2 imbalance in lymph-node-positive breast carcinomas, as measured by RT-PCR. Int J Cancer 1995; 63: 621–6. Engel G, Heselmeyer K, Aver G et al. Correlation between stromelysin-3 mRNA level and outcome of human breast cancer. Int J Cancer 1994; 58: 830–5. Ahmad A, Hanby A, Dublin E et al. Stromelysin 3: An independent prognostic factor for relapse-free survival in node-positive breast cancer and demonstration of novel breast carcinoma cell expression. Am J Pathol 1998; 152: 721–8. Freije JM, Diez-Itza I, Balbin M et al. Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. J Biol Chem 1994; 269: 16766–73. Ueno H, Nakamura H, Inoue M et al. Expression and tissue localization of membrane-types 1, 2, and 3 matrix metalloproteinases in human invasive breast carcinomas. Cancer Res 1997; 57: 2055–60. Jones JL, Glynn P,Walker RA. Expression of MMP-2 and MMP9, their inhibitors, and the activator MT1-MMP in primary breast carcinomas. J Pathol 1999; 189: 161–8. Ree AH, Florenes VA, Berg JP et al. High levels of messenger RNAs for tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in primary breast carcinomas are associated with development of distant metastases. Clin Cancer Res 1997; 3: 1623–8. McCarthy K, Maguire T, McGreal G et al. High levels of tissue inhibitor of metalloproteinase-1 predict poor outcome in patients with breast cancer. Int J Cancer 1999; 84: 44–8. Remacle A, McCarthy K, Noel A et al. High levels of TIMP-2 correlate with adverse prognosis in breast cancer. Int J Cancer 2000; 89: 118–21. Basset P, Bellocq JP, Wolf C et al. A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 1990; 348: 699–704. Wolf C, Rouyer N, Lutz Y et al. Stromelysin 3 belongs to a subgroup of proteinases expressed in breast carcinoma fibroblastic cells and possibly implicated in tumor progression. Proc Natl Acad Sci USA 1993; 90: 1843–7. Okada A, Bellocq J, Rouyer N et al. Membrane-type matrix metalloproteinase (MT-MMP) gene is expressed in stromal cells of human

178

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

M. Balduyck et al. colon, breast, and head and neck carcinomas. Proc Natl Acad Sci USA 1995; 92: 2730–4. Heppner KJ, Matrisian LM, Jensen RA et al. Expression of most matrix metalloproteinase family members in breast cancer represents a tumor-induced host response. Am J Pathol 1996; 149: 273–82. Nakopoulou L, Giannopoulou I, Gakiopoulou H et al. Matrix metalloproteinase-1 and -3 in breast cancer: Correlation with progesterone receptors and other clinicopathologic features. Hum Pathol 1999; 30: 436–42. Nielsen B, Sehested M, Kjeldsen L et al. Expression of matrix metalloprotease-9 in vascular pericytes in human breast cancer. Lab Invest 1997; 77: 345–55. Giambernardi TA, Grant GM, Taylor GP et al. Overview of matrix metalloproteinase expression in cultured human cells. Matrix Biol 1998; 16: 483–96. Mackay AR, Ballin M, Pelina MD et al. Effect of phorbol ester and cytokines on matrix metalloproteinase and tissue inhibitor of metalloproteinase expression in tumor and normal cell lines. Invasion Metastasis 1992; 12: 168–84. Gilles C, Polette M, Birembaut P et al. Expression of c-ets-1 mRNA is associated with an invasive, EMT-derived phenotype in breast carcinoma cell lines. Clin Exp Metastasis 1997; 15: 519–26. Pulyaeva H, Bueno J, Polette M et al. MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells. Clin Exp Metastasis 1997; 15: 111–20. Nutt JE, Lunec J. Induction of metalloproteinase (MMP1) expression by epidermal growth factor (EGF) receptor stimulation and serum deprivation in human breast tumour cells. Eur J Cancer 1996; 32A: 2127–35. Kim J, Yu W, Kovalski K et al. Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell 1998; 94: 353–62. Shao ZM, Wu J, Shen ZZ et al. Genistein exerts multiple suppressive effects on human breast carcinoma cells. Cancer Res 1998; 58: 4851– 7. Kolkhorst V, Sturzebecher J, Wiederanders B. Inhibition of tumour cell invasion by protease inhibitors: Correlation with the protease profile. J Cancer Res Clin Oncol 1998; 124: 598–606. Hahn-Dantona E, Ramos-DeSimone N, Sipley J et al. Activation of proMMP-9 by a plasmin/MMP-3 cascade in a tumor cell model. Regulation by tissue inhibitors of metalloproteinases. Ann NY Acad Sci 1999; 878: 372–87. Stonelake PS, Jones CE, Neoptolemos JP et al. Proteinase inhibitors reduce basement membrane degradation by human breast cancer cell lines. Br J Cancer 1997; 75: 951–9. Yang M, Kurkinen M. Different mechanisms of regulation of the human stromelysin and collagenase genes. Analysis by a reversetranscription-coupled-PCR assay. Eur J Biochem 1994; 222: 651–8. Nuovo GJ, MacConnell PB, Simsir A et al. Correlation of the in situ detection of polymerase chain reaction-amplified metalloproteinase complementary DNAs and their inhibitors with prognosis in cervical carcinoma. Cancer Res 1995; 55: 267–75. Yoshimoto M, Itoh F, Yamamoto H et al. Expression of MMP7(PUMP-1) mRNA in human colorectal cancers. Int J Cancer 1993; 54: 614–8. Reboul P, Pelletier JP, Tardif G et al. The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes

38.

39. 40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

but not by synoviocytes. A role in osteoarthritis. J Clin Invest 1996; 97: 2011–9. Thompson EW, Paik S, Brunner N et al. Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines. J Cell Physiol 1992; 150: 534–44. Westermarck J, Kahari VM. Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J 1999; 13: 781–92. Tremble P, Damsky CH, Werb Z. Components of the nuclear signaling cascade that regulate collagenase gene expression in response to integrin-derived signals. J Cell Biol 1995; 129: 1707–20. Sudbeck BD, Pilcher BK, Welgus HG et al. Induction and repression of collagenase-1 by keratinocytes is controlled by distinct components of different extracellular matrix compartments. J Biol Chem 1997; 272: 22103–10. Sarkissian M, Lafyatis R. Integrin engagement regulates proliferation and collagenase expression of rheumatoid synovial fibroblasts. J Immunol 1999; 162: 1772–9. Gilles C, Polette M, Seiki M et al. Implication of collagen type Iinduced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma. Lab Invest 1997; 76: 651–60. Larjava H, Lyons JG, Salo T et al. Anti-integrin antibodies induce type IV collagenase expression in keratinocytes. J Cell Physiol 1993; 157: 190–200. Daemi N, Thomasset N, Lissitzky JC et al. Anti-beta4 integrin antibodies enhance migratory and invasive abilities of human colon adenocarcinoma cells and their MMP-2 expression. Int J Cancer 2000; 85: 850–6. Will H, Atkinson SJ, Butler GS et al. The soluble catalytic domain of membrane type 1 matrix metalloproteinase cleaves the propeptide of progelatinase A and initiates autoproteolytic activation. Regulation by TIMP-2 and TIMP-3. J Biol Chem 1996; 271: 17119–23. Pei D, Weiss SJ. Transmembrane-deletion mutants of the membranetype matrix metalloproteinase-1 process progelatinase A and express intrinsic matrix-degrading activity. J Biol Chem 1996; 271: 9135–40. Docherty AJ, Lyons A, Smith BJ et al. Sequence of human tissue inhibitor of metalloproteinases and its identity to erythroid-potentiating activity. Nature 1985; 318: 66–69. Knauper V, Smith B, Lopez-Otin C et al. Activation of progelatinase B (proMMP-9) by active collagenase-3 (MMP-13). Eur J Biochem 1997; 248: 369–73. Nagase H, Enghild JJ, Suzuki K et al. Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl)mercuric acetate. Biochemistry 1990; 29: 5783–89. Ogata Y, Itoh Y, Nagase H. Steps involved in activation of the pro-matrix metalloproteinase 9 (progelatinase B)-tissue inhibitor of metalloproteinases-1 complex by 4-aminophenylmercuric acetate and proteinases. J Biol Chem 1995; 270: 18506–11. Knauper V, Will H, Lopez-Otin C et al. Cellular mechanisms for human procollagenase-3 (MMP-13) activation – evidence that MT1MMP (MMP-14) and gelatinase A (MMP-2) are able to generate active enzyme. J Biol Chem 1996; 271: 17124–31. Simon C, Goepfert H, Boyd D. Inhibition of the p38 mitogenactivated protein kinase by SB 203580 blocks PMA-induced Mr 92,000 type IV collagenase secretion and in vitro invasion. Cancer Res 1998; 58: 1135–9.