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Matrix metalloproteinase expression in breast cancer
Journal of Surgical Research 110, 383–392 (2003) doi:10.1006/jsre.2003.6610
Matrix Metalloproteinase Expression in Breast Cancer John E. Bartsch, B.S., Edgar D. Staren, M.D., Ph.D., 1 and Hubert E. Appert, Ph.D. Department of Surgery, Medical College of Ohio, Toledo, Ohio 43614 Submitted for publication September 19, 2002
Background. Matrix metalloproteinases (MMPs) have been implicated as possible mediators of invasion and metastasis in some cancers. Our objective was to investigate which MMPs were constitutively expressed in breast tumor cells versus those that could be up-regulated by a number of agents known to affect MMP expression in other cell systems. Methods. We evaluated expression of MMPs 1–16 in breast tumor cell lines MDA-MB-231, T47D, and MCF-7 using semiquantitative RT-PCR and gelatin zymography. Exposure to 12-O-tetradecanoylphorbal-3-acetate (TPA), concanavalin-A (Con-A), the fibronectin-mimetic peptide GRGDSP (RGD), extracellular matrix (ECM) components, and anti-integrin antibodies was used to test for possible MMP up-regulation. Mitogen-activated protein kinase inhibitors (MAPK-I) were used to evaluate signal transduction pathways and regulation of MMP expression. Results. MMPs 1, 2, 7–11, 13, 14, and 16 were constitutively expressed in some tumor cell lines but not in normal breast epithelial cells. Administration of TPA, Con-A, and RGD increased the expression of MMPs 1, 2, 9, and 10. No MMP up-regulation was seen in MDAMB-231 or MCF-7 after exposure to ECM components or after exposure to anti-integrin antibodies. MAPK-I had no effect on constitutive MMP expression but reduced or abolished the TPA up-regulation of MMP-9 in MDA-MB-231 and MCF-7. Conclusions. Breast tumor cell lines constitutively express a number of MMPs. Because MMP expression can be up-regulated by Con-A, the fibronectin-mimetic peptide RGD, and TPA while being susceptible to inhibition by MAPK antagonists, MAPK signaling appears to play a role in this expression. © 2003 Elsevier Science (USA)
1 To whom correspondence should be addressed at Department of Surgery, Medical College of Ohio, 3065 Arlington Avenue, Toledo OH 43614 Fax: (419) 383-6636. E-mail: [email protected].
Key Words: breast cancer; fibronectin; integrin; matrix metalloproteinase; metastasis; mitogen-activated protein kinase; vitronectin. INTRODUCTION
Although substantial progress has been made in defining the gene expression changes that occur in breast cancer, much of even the most fundamental concepts, such as elucidation of the signal transduction pathways involved in tumor cell invasion and metastasis, remains largely unexplored. For breast cancer cells to manifest their malignant potential, they must develop the ability to break through and dissolve extracellular matrices (ECM), particularly the delimiting basement membrane (BM). The degradation of the pericellular BM and ECM is catalyzed by the concerted action of several classes of ECM-degrading enzymes. The cellular mechanisms controlling the release and activation of ECM-degrading enzymes are very complex and for the most part have not yet been identified [1]. One important class of ECM-degrading enzymes includes the matrix metalloproteinases (MMPs). Previous information suggests that both the constitutive and stimulated release of these enzymes are implicated in tumor cell metastasis [2– 4]. If it is possible to prevent their expression, release, and activation, it may be possible to significantly reduce the role of MMPs in tumor cell metastasis. MMPs are a family of zinc-dependent neutral endopeptidases which collectively are capable of degrading all components of the ECM. Currently the MMP family is known to contain at least 20 members, loosely classified into four groups: (1) interstitial collagenase (MMPs 1, 8, and 13); (2) type-IV collagenase (MMPs 2 and 9); (3) stromelysins (MMPs 3, 7, 10, 11, and 18); and (4) membrane-type MMPs (MMPs 14, 15, 16, 17, 24, and 25) [5, 6]. MMP-9 is an important collagenase contributing to the digestion of collagen type IV, the
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primary component of basement membranes. MMP-9 expression was previously shown to correlate with an invasive phenotype in rat embryo cells as well as a number of malignant tumors [3, 7]. In the normal mammary gland, constitutive expression of MMPs is low except during times of development, pregnancy, and involution [5, 8]. However, in pathologic states such as breast cancer, increased levels of MMPs have been reported in breast tumor cells as well as in the surrounding noncancerous breast tissue. Iwata et al. [3] showed MMPs 1, 2, and 9 to be highly expressed in human breast carcinoma cells. Kossakowska et al. [9] and Garbett et al. [4] showed MMPs 1, 2, 3, 9, and 11 to have a higher frequency of expression in breast cancer as opposed to normal breast tissue or benign breast disease. Furthermore, other investigators found that MMPs 1, 2, 9, 13, and 16 were expressed specifically in malignant or benign breast tissue [10 –13]. In this study, a number of different agents were used to up-regulate MMPs because previous investigators have shown that the expression of MMPs by tumor cells is influenced by multiple environmental factors, including growth factors and ECM components [8, 14, 15]. The effect of the tumor-promoting agents 12-Otetradecanoylphorbal-3-acetate (TPA) and concanavalin-A (Con-A) on MMP expression was evaluated based on our previous observation that they up-regulate MMP expression in pancreatic tumor cells [16]. Liu et al. [17] found that TPA and fibroblast growth factor (FGF) could up-regulate MMP-9 in MCF-7 cells. A fibronectin-mimetic peptide GRGDSP (RGD), ECM proteins, and anti-integrin antibodies were used so as to investigate integrin-induced expression and release of MMPs. Integrins are a family of cell surface receptors that play a key role in cell signal transduction from the ECM. Integrins do not directly activate signal transduction but do so as a result of their binding to linker proteins [18, 19]. Daemi et al. [20] found that the 4 subunit of integrins is responsible for stimulating the expression of MMP-2 in colon adenocarcinoma cells. Bafetti et al. [14] observed that the ECM protein vitronectin (VN) stimulates MMP-2 expression in melanoma cells and that this expression could be blocked by RGD. Finally, this study used the mitogen-activated protein kinase (MAPK) inhibitors genistein, PD 98059, and SB 203580 to elucidate which, if any, MAPK pathways are involved in MMP expression. Reddy et al. [15] found that epidermal growth factor (EGF)-induced MMP-9 secretion in the breast tumor cell line SKBR-3 could be greatly reduced by the use of MAPK inhibitors. MMP expression has been shown to be up-regulated by a number of tumor cell systems and has been shown to be related to tumor cell invasiveness. This study demonstrates constitutive expression of a number of MMPs in breast cancer cell lines, up-regulation of sev-
eral MMPs after exposure to TPA, Con-A, and RGD, and a role for MAPK in MMP expression. Nevertheless, the mechanisms that regulate the metastatic process are extremely complex and continue to prove difficult to analyze. This investigation may suggest new ways to prevent or reduce the role of MMPs in tumor cell metastasis. MATERIALS AND METHODS
Cells and Cell Culture Samples of breast tumor cell lines MDA-MB-231, T47D, and MCF-7 were obtained from ATCC (Manassas, VA) and also from Lombardi Cancer Center (Georgetown University, Washington, DC, courtesy of Shared Tissue Bank). The cells were maintained as described previously by Yang et al. [16]. Nontumor cells were used to isolate the mRNA used in this study.
Stimulatory/Inhibitory Agents Vitronectin (VN), fibronectin (FN), laminin, and collagen IV were purchased from Gibco BRL (Life Technologies, Rockville, MD). The monoclonal antibodies (MAb) were obtained from the following sources: anti-␣4 integrin (clone P4C2) and anti-␣5 integrin (clone B1D6) were obtained from Gibco BRL. Anti-␣v3 integrin (MAb 1976B) was purchased from Chemicon (Temucula, CA). Anti-1 integrin (MAb-13) was a gift from Dr. Kenneth Yamada (NCI, Bethesda, MD). Anti-4 integrin (MAb-9) was a gift from Dr. T.E. Carey (University of Michigan, Ann Arbor, MI). Concanavalin-A (Con-A) was purchased from EY Laboratories, Inc. (San Mateo, CA). 12-Otetradecanoylphorbal-3-acetate (TPA), genistein, and Coomassie blue were purchased from Sigma (St. Louis, MO). PD 98059 and SB 203580 were purchased from Cal Biochem (San Diego, CA). The peptide GRGDSP (RGD) was obtained from Bachem (Torrance, CA).
Isolation of Total Ribonucleic Acid (RNA) Total cellular RNA was isolated by a single step method using TRI reagent according to the manufacturer’s instructions. One milliliter TRIzol reagent was used per 10 cm 2 of culture flask. TRI reagent kits were purchased from Molecular Research Center, Inc. (Cincinnati, OH). Chloroform, isopropanol, and ethanol were purchased from Fisher Scientific; diethyl pyrocarbonate (DEPC) was purchased from Sigma. The mRNA from nontumor breast tissue was obtained from Clonetics (San Diego, CA).
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Using previously reported primers [16, 21], constitutive mRNA levels of MMPs were determined by RT-PCR using a SuperScript one-step RT-PCR system kit. Reactions were assembled by mixing 1.0 g of total RNA with every 25 l of 2⫻ Reaction Mix, 6 l of mixed primers (a final concentration of 0.2 M), 1 l of RT/Taq Mix, 6 l of 5 mM MgSO 4, and autoclaved distilled water to a final volume of 50 l. RT-PCR was performed in a TEMP 䡠 TRONIC DNA Thermal Cycler with 30 min incubation at 50°C, 2 min predenaturation at 94°C, and 30 or 40 cycles of amplification consisting of 94°C for 30 s (denaturation), 55°C for 30 s (annealing), and 72°C for 1 min (elongation). -actin was used as external control and normal adult female breast RNA (Stratogene, La Jolla, CA) was used as a control for MMP expression. The PCR product (10 l) for each sample was analyzed by electrophoresis in a 1% agarose gel containing ethidium bromide. Scion Image (Scion Corp., Frederick, MD) was used to obtain quantitative RT-PCR results of mRNA expression. SuperScript one-step RT-PCR system kits and primers were purchased
BARTSCH, STAREN AND APPERT: MMP EXPRESSION IN BREAST CANCER from Life Technologies Inc.; 100 bp DNA ladder was purchased from Gibco BRL; agarose was purchased from Sigma. The primer sequences were as follows: MMP-1 (786 bp): 5⬘-CGA CTC TAG AAA CAC AAG AGC AAG A-3⬘ (sense) 5⬘-AAG GTT AGC TTA CTG TCA CAC GCT T-3⬘ (antisense) MMP-2 (605 bp): 5⬘-GTG CTG AAG GAC ACA CTA AAG AAG A-3⬘ (sense) 5⬘-TTG CCA TCC TTC TCA AAG TTG TAG G-3⬘ (antisense) MMP-3 (729 bp): 5⬘-GAA CAA TGC ACA AAG GAT ACA ACA-3⬘ (sense) 5⬘-TTC TTC AAA AAC AGC ATC AAT CTT-3 (antisense) MMP-7 (373 bp): 5⬘-GGT CAC CTA CAG GAT CGT ATC ATA T-3⬘ (sense) 5⬘-CAT CAC TGC ATT AGG ATC AGA GGA A-3⬘ (antisense) MMP-8 (435 bp): 5⬘-GCT GCT TAT GAA GAT TTT GAC AGA G-3⬘ (sense) 5⬘-ACA GCC ACA TTT GAT TTT GCT TCA G-3⬘ (antisense) MMP-9 (243 bp): 5⬘-CAC TGT CCA CCC CTC AGA GC-3⬘ (sense) 5⬘-GCC ACT TGT CGG CGA TAA GG-3⬘ (antisense) MMP-10 (408 bp): 5⬘-CAC TCT ACA ACT CAT TCA CAG AGC T-3⬘ (sense) 5⬘-CTT GGA TAA CCT GCT TGT ACC TCA T-3⬘ (antisense) MMP-11 (326 bp): 5⬘-TAA AGG TAT GGA GCG ATG TGA C-3⬘ (sense) 5⬘-TGG GTA GCG AAA GGT GTA GAA G-3⬘ (antisense) MMP-12 (517 bp): 5⬘-TTC CCC TGA ACA GCT CTA CAA GCC TGG AAA-3⬘ (sense) 5⬘-GAT CCA GGT CCA AAA GCA TGG GCT AGG ATT-3⬘ (antisense) MMP-13 (330 bp): 5⬘-GTG GTG TGG GAA GTA TCA TCA-3⬘ (sense) 5⬘-GCA TCT GGA GTA ACC GTA TTG-3⬘ (antisense) MMP-14 (497 bp): 5⬘-CGC TAC GCC ATC CAG GGT CTC AAA-3⬘ (sense) 5⬘-CGG TCA TCA TCG GGC AGC ACA AAA-3⬘ (antisense) MMP-15 (454 bp): 5⬘-ACA ACC ACC ATC TGA CCT TTA GCA-3⬘ (sense) 5⬘-AGC TTG AAG TTG TCA ACG TCC TTC-3 (antisense) MMP-16 (652 bp): 5⬘-TTA CTT CTG GCG GGG CTT GCC TCC TAG TAT-3⬘ (sense) 5⬘-ACA GTA CAG TAT GTG GCG GGG TGT TCC TTT-3⬘ (antisense) TIMP-1 (667 bp): 5⬘-ATC CTG TTG TTG CTG TGG CTG ATA G-3⬘ (sense) 5⬘-TGC TGG GTG GTA ACT CTT TAT TTC A-3⬘ (antisense) TIMP-2 (405 bp): 5⬘-AAA CGA CAT TTA TGG CAA CCC TAT C-3⬘ (sense) 5⬘-ACA GGA GCC GTC ACT TCT CTT GAT G-3⬘ (antisense)
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Gelatin Zymography Determination of Secreted MMP2 and MMP-9 Gelatin zymography was performed using precast polyacrylamide gels containing 0.1% gelatin (Novex, San Diego, CA). Collected tissue culture medium was centrifuged at 1500 rpm for 3 min and 50 l of supernatant medium was mixed with 10 l of 2 ⫻ Tris-Glucine SDS sample buffer. Twenty-five microliters of mixed sample was loaded in 12-well gels and electrophoresis was performed at 100 V for 1.5 h. After electrophoresis, the gel was rinsed with renaturing 1 ⫻ buffer for 5 h at room temperature. The buffer was then switched to 1 ⫻ developing buffer and incubated overnight at 37°C. The gel was stained with Coomassie blue and then destained with distilled water. The unstained bands correspond to the areas of gelatin digestion. Bands were quantified using densitometry (Scion Image).
MMP Expression as Determined by RT-PCR Constitutive and up-regulated MMP expression at the mRNA level was studied by means of RT-PCR in breast tumor cell lines as compared with nontumor breast epithelial cells. MDA-MB-231, T47D, and MCF-7 were grown on T-75 tissue culture flasks in McCoy’s medium containing 10% fetal bovine serum (FBS) until they reached 90% confluency. McCoy’s medium was removed and cells were washed three times with serum-free medium. Cells were resuspended in 1 ml McCoy’s serum-free medium and incubated for 24 h with media alone or with either Con-A (50 g/ml), RGD (100 g/ml), or TPA (100 M). Subsequently total RNA was isolated using the primers and the RT-PCR procedure described previously.
EXPERIMENTAL DESIGN
Studies of the Constitutive vs Up-regulated MMP Expression Effects of ECM and anti-integrin antibodies on MMP-2 and MMP-9 enzyme release and activity determined by zymography. MCF-7 and MDA-MB-231 cell lines were plated on 12-well cell culture treated plates (Corning Inc., Life Sciences, Acton, MA) in 2 ml serumsupplemented McCoy’s medium and allowed to grow until they reached 90% confluency. McCoy’s medium was removed and cells were washed three times with serum-free medium. Cells were resuspended in 1 ml of McCoy’s serum-free medium, then incubated for 24 h in media to which the following were added: Con-A (50 g/ml), 200 g of collagen IV, 25 g of VN, 500 g of FN, 200 g of laminin, anti-integrin ␣4 (P4C2) (1:100 dilution), anti-integrin ␣5 (P1D6) (1:100), anti-integrin 1 (MAb-13) (1:200), anti-integrin 4 (MAb-9) (1:100), and anti-integrin ␣v3 (MAb1976B) (20 g/ml). Following a 24-h incubation period, the medium was removed and the cells were prepared for the zymographic determination of MMP-2 and MMP-9 as described previously. Effects of the MAPK inhibitors genistein, PD 98059, and SB 203580 on constitutive MMP expression as determined by RT-PCR. MDA-MB-231 was grown on T-75 tissue culture flasks in McCoy’s medium containing 10% FBS until it reached 90% confluency. The medium was then removed and cells were washed three times with serum-free medium. Cells were then
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incubated for 24 h with genistein (10 g/ml), PD 98059 (100 g/ml), or SB 203580 (20 M) in serum-free medium, and total RNA was isolated as described previously. RT-PCR was performed with primers for MMPs 1, 11, 13, 14, 15, and 16. Effects of the MAPK inhibitors genistein, PD 98059, and SB 203580 on the TPA-stimulated MMP-9 release and activity as determined by gelatin zymography. MCF-7 and MDA-MB-231 cell lines were plated on 12-well cell culture treated plates (Corning, Inc.) in 2 ml serum-supplemented McCoy’s medium and allowed to grow until they reached 90% confluency. McCoy’s medium was removed and cells were washed three times with serum-free medium. Cells were resuspended in 1 ml McCoy’s serum-free medium and incubated with TPA (100 M) either alone or with one of the following MAPK inhibitors: genistein (10 g/ml), PD 98059 (10 g/ml), or SB 203580 (20 M). RESULTS
Constitutive vs Stimulated MMP Expression The invasive capacities of MDA-MB-231, T47D, and MCF-7 tumor cells used in this investigation were previously shown to have high, medium, and low invasive capacities [16]. The mRNA isolated from the nontumor cells expressed only MMP-15. MMP unstimulated and stimulated expression in the MDA-MB-231 cell line. As shown in Table 1, MMPs 1, 2, 7–11, and 13–16, as well as the natural inhibitors of MMP, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2, were expressed in MDA-MB-231 in varying degrees. MMP-3 and MMP-12 could not be detected in MDA-MB-231 either prior to or after stimulation. There was a slight up-regulation of MMP-2 (60.5 kDa) with RGD (10 g/ml) (see also Fig. 1G, lane 3) and with TPA. There was also a slight increase in MMP-10 expression in MDA-MB-231 over that of baseline (lane 1) with Con-A (lane 2) (Fig. 1F). A moderate upregulation of MMP-9 in MDA-MB-231 was observed with exposure to TPA (Table 1). There were no other noticeable changes in any of the other MMPs after Con-A, RGD, or TPA exposure. Hence it can be seen that MMP-2 and MMP-9, in contrast to the other MMPs, can be susceptible to up-regulation. MMP unstimulated and stimulated expression in the T47D cell line. As shown in Table 2, prior to stimulation, MMPs 11, 13, 15, and 16 were expressed at high levels, while MMPs 1 and 7 were expressed at low levels. In contrast to the MDA-MB-231 cell line, MMPs 2, 3, 8, 9, 10, 12, and 14 were not expressed in the T47D cell line either before or after exposure to Con-A or RGD. There was an increase in MMP-1 in the T47D cell line after exposure to Con-A and RGD (Fig. 1D). This is in contrast to the initially higher expression of MMP-1
TABLE 1 Unstimulated and Stimulated MMP Expression in MDA-MB-231 as Determined by RT-PCR Primer
Control
Con-A
GRGDSP
TPA
Actin MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-10 MMP-11 MMP-12 MMP-13 MMP-14 MMP-15 MMP-16 TIMP-1 TIMP-2
⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹/0 — ⫹⫹ ⫹⫹ ⫹/0 ⫹⫹ ⫹⫹⫹⫹ — ⫹⫹/⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹/0 — ⫹⫹ ⫹⫹ ⫹/0 ⫹⫹⫹ ⫹⫹⫹⫹ — ⫹⫹/⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ — ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹⫹ — ⫹⫹/⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ — ⫹⫹ ⫹⫹ ⫹⫹ N.D. ⫹⫹⫹⫹ — ⫹⫹/⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
Note. Five grades of MMP expression were observed and categorized: ⫹⫹⫹⫹, strongest MMP expression; ⫹⫹⫹, strong MMP expression; ⫹⫹, moderate MMP expression; ⫹, weak expression; ⫹/0, questionable expression; —, no observed expression. MMPs 1, 2, 7–11, and 13–16, as well as TIMP-1 and TIMP-2, the natural inhibitors of MMP, were expressed in MDA-MB-231 to some extent both before and after stimulation. MMP-3 and MMP-12 were not expressed in MDA-MB-231 either prior to or after stimulation. After stimulation with Con-A, GRGDSP, and TPA, there was a slight up-regulation of MMP-2 with GRGDSP (see Fig. 1G) and TPA. There was also a slight change in MMP-10 expression in MDA-MB-231 with Con-A (see Fig. 1F). A moderate up-regulation of MMP-9 in MDA-MB-231 was observed with exposure to TPA. There were no other noticeable changes in any of the other MMPs after Con-A, GRGDSP, or TPA.
in MDA-MB-231 and lack of MMP-1 up-regulation in MDA-MB-231 cells. MMP expression in the MCF-7 cell line. As shown in Table 3, prior to stimulation, MMPs 14, 15, and 16 were expressed at high levels and MMPs 1 and 11 were expressed at low levels. Closely related to the findings observed with the T47D cell line, MMPs 2, 3, 7, 8, 10, and 12 were not expressed in the MCF-7 cell line either before or after stimulation with Con-A or RGD. Upregulation of MMP-11 was observed after stimulation with RGD and Con-A (Fig. 1B). Effects of ECM Components and Anti-integrin Antibodies on MMP-2 and MMP-9 Enzyme Release and Activity as Determined by Zymography Table 4A shows that the ECM components collagen IV, VN, FN, and laminin had no effect on the zymographic activity of MMP-2 and MMP-9 in MDA-MB231 and MCF-7 tumor cells. Also, as shown in Table 4B, no effect was observed on MMP zymographic activity after treatment with anti-integrins ␣4 (P4C2), ␣5
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FIG. 1. RT-PCR analysis of Con-A, and GRGDSP stimulated breast tumor lines in which some degree of regulation was observed. MDA-MB-231, T47D, and MCF-7 cells were cultured for 24 h in serum-free medium containing cells only (lane 1), Con-A (20 g/ml) (lane 2), and GRGDSP (100 g/ml) (lane 3). Total RNA was isolated followed by RT-PCR analysis of MMP expression. The expression of actin in MCF-7 (A), T47D (C), and MDA-MB-231 (E) served as external standards, and MMP-1 (D), MMP-2 (G), MMP-10 (F), and MMP-11 (B) are depicted. This figure shows that MMP-11, MMP-1, MMP-10, and MMP-2 can be up-regulated by agents that act on cell surface receptors of breast tumor cells.
(P1D6), 1 (MAb-13), 4 (MAb-9), and ␣v3 (MAb 1976B). Effects of the MAPK Inhibitors Genistein, PD 98059, and SB 203580 on Constitutive MMP Expression as Determined by RT-PCR As shown in Fig. 2, the MAPK inhibitors genistein, PD 98059, and SB 203580 had no effect on the constitutive expression of MMPs 1, 11, 13, 14, 15, and 16 in MDA-MB-231, as measured by RT-PCR. Effects of Genistein, PD 98059, and SB 203580 on TPA-Stimulated MMP-2 and MMP-9 Activity in MDA-MB-231 and MCF-7 Cells as Determined by Zymography Observations on MDA-MB-231 cells. Zymographic studies showed that TPA (100 M) treatment caused
the appearance of a 90 kDa band of activity which represented the active form of MMP-9 which was released from the MDA-MB-231 cells (Fig. 3A, lane 5), and this band could be reduced by about 50% with the addition of genistein (10 g/ml), PD 98059 (10 g/ml), or SB 203580 (20 M), as seen in lanes 6, 7, and 8, respectively in Fig. 3A. In Fig. 4B, dose-response effects could be demonstrated using TPA with 1, 5, and 10 g/ml of genistein (lanes 1, 2, and 3); TPA with PD 98059 with 1, 5, and 10 g/ml (lanes 4, 5, and 6), and with SB 203580, 2, 10, and 20 g/ml (lanes 7, 8, and 9). Observations on MCF-7 cells. As seen in Fig. 4A (lane 4), exposure of MCF-7 cells to TPA (100 M) resulted in a band of strong MMP-9 activity. The band resulting from exposure of TPA (100 M) was greatly reduced in the presence of 10 g/ml genistein, and to a lesser extent in the presence of PD 98059 (10 g) and
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TABLE 2 Unstimulated and Stimulated MMP Expression in T47D as Determined by RT-PCR Primer
Control
Con-A
GRGDSP
Actin MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-10 MMP-11 MMP-12 MMP-13 MMP-14 MMP-15 MMP-16 TIMP-1 TIMP-2
⫹⫹⫹⫹ ⫹ — — ⫹⫹ — — — ⫹⫹⫹ — ⫹⫹⫹ — ⫹⫹⫹⫹ ⫹⫹⫹⫹ — ⫹⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹ ⫹/0 — ⫹⫹ — — — ⫹⫹⫹ — ⫹⫹⫹ — ⫹⫹⫹⫹ ⫹⫹⫹⫹ — ⫹⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹ — — ⫹⫹ — — — ⫹⫹⫹ — ⫹⫹⫹ — ⫹⫹⫹⫹ ⫹⫹⫹⫹ — ⫹⫹⫹⫹⫹
Note. Five grades of MMP expression were observed and categorized: ⫹⫹⫹⫹, strongest MMP expression; ⫹⫹⫹, strong MMP expression; ⫹⫹, moderate MMP expression; ⫹, weak expression; ⫹/0, questionable expression; —, no observed expression. Prior to stimulation, MMPs 11, 13, 15, and 16 and TIMP-2 were expressed at high levels, while MMPs 1 and 7 were expressed at low levels. MMPs 2, 3, 8 –10, 12, and 14, as well as TIMP-1, were not expressed in the T47D cell line either before or after exposure to Con-A or GRGDSP. There was an increase in MMP-1 in the T47D cell line after exposure to Con-A and GRGDSP (also see Fig. 1D).
SB 203580 (20 M). Dose-response effects could be demonstrated with TPA (100 M) using genistein 1, 5, and 10 g, (Fig. 4B, lanes 1, 2, and 3), with PB 98059, 1, 5, and 10 g (Fig. 4B, lanes 4, 5, and 6), and with SB 203580, 2, 10, and 20 g/ml (Fig. 4B, lanes 7, 8, and 9). DISCUSSION
It has been shown that MMPs are expressed in increased levels in breast tumors as opposed to their expression in normal breast tissues [3–5, 8, 9]. Cassara et al. [22] demonstrated that 8701 BC tumor cells released MMP-containing vesicles into the tissue culture medium. Barsky et al. [23] demonstrated an increase in MMP-2 and MMP-9 immunoreactivity in invasive breast carcinoma, and Polette et al. [24] reported MMP-2 reactivity in two of six benign breast tumors and 13 of 17 malignant tumors. In situ hybridization studies indicate that MMP-2 is localized in stromal cells around the breast lesion, MMP-3 in the infiltrating T-lymphocytes, and MMP-9 in macrophages and neutrophils induced by the immune response to tumor cells [3, 25]. While some investigators suggest an almost exclusive stromal, rather than tumor cell, origin of MMPs, the current study, as well as studies by other investigators, demonstrates that a number of MMPs
are constitutively expressed in breast tumor cell lines in an environment completely isolated from stromal components. In this investigation, the tumor line MDA-MB-231 was shown by RT-PCR to constitutively express MMPs 1, 2, 7–11, and 13–16 (Table 1), while T47D expressed MMPs 1, 7, 11, 13, 15, and 16 (Table 2), and MCF-7 expressed MMPs 1, 11, 14, 15, and 16 (Table 3). Consistent with our findings, Balduyck et al. [26] and Lebeau et al. [27], reported increased MMP expression (e.g., MMPs 1, 3, and 13) in MDA-MB-231 cells. Balduyck et al. [26] did not, however, detect MMPs 2, 7, and 9 in MDA-MB-231, although we found those expressed at low levels. Our investigation demonstrated that MMPs 1, 2, 7–11, 13, 14, and 16 were, to some extent, overexpressed in one or more of the tumor cell lines studied. The differences in MMP expression are not surprising and could be a result of different cell culture conditions such as differences in growth factors in the composition of the serum added to the culture medium. Reddy et al. [15] showed that EGF can regulate the expression of MMP-9. This expression of multiple MMPs in tumor cells is in contrast to normal mammary tissue which expressed only MMP-15. In Table 1 it is shown by RT-PCR that incubation of the MDA-MB-231 cell line with RGD and TPA results in an up-regulation of the mRNAs for MMP-2 and MMP-9. We suspected that the increased constitutive expression of MMPs in tumor cells might be the result of the TABLE 3 Unstimulated and Stimulated MMP Expression in MCF-7 as Determined by RT-PCR Primer
Control
Con-A
GRGDSP
Actin MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-10 MMP-11 MMP-12 MMP-13 MMP-14 MMP-15 MMP-16
⫹⫹⫹⫹ ⫹⫹ — — — — — — ⫹⫹ — — ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹/⫹⫹ — — ⫹/0 ⫹/0 — — ⫹⫹ — ⫹/0 ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹/⫹⫹ — — ⫹/0 ⫹/0 — — ⫹⫹⫹ — ⫹/0 ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
Note. Five grades of MMP expression were observed and categorized: ⫹⫹⫹⫹, strongest MMP expression; ⫹⫹⫹, strong MMP expression; ⫹⫹, moderate MMP expression; ⫹, weak expression; ⫹/0, questionable expression; —, no observed expression. Prior to stimulation, MMPs 14, 15, and 16 were expressed at high levels and MMPs 1 and 11 were expressed at low levels. MMPs 2, 3, 7–10, and 12 were not expressed in the MCF-7 cell line either before or after stimulation with Con-A or GRGDSP. Slight up-regulation of MMP-11 was observed after stimulation with GRGDSP and Con-A (see also Fig. 1B).
BARTSCH, STAREN AND APPERT: MMP EXPRESSION IN BREAST CANCER
FIG. 2. Effects of the MAPK inhibitors genistein, PD 98059, and SB 203580 on constitutive MMP expression as determined by RTPCR. MDA-MB-231 cells were cultured for 24 h in serum-free medium containing cells only (lane 1), genistein (100 g/ml) (lane 2), PD 98059 (g/ml (lane 3), and SB 203580 (20 M) (lane 4). RNA was isolated followed by RT-PCR analysis of MMP-1 (A), MMP-11 (B), MMP-13 (C), MMP-14 (D), MMP-15 (E), and MMP-16 (F) as described in “Materials and Methods.” No noticeable change in constitutive MMP expression was observed after exposure to genistein, PD 98059, or SB 203580 in the concentrations used in this experiment; hence it appears that constitutive MMP expression is not under the control of MAPK signal transduction pathways.
cellular transformation of transcription factors related to mitogen-activated protein kinases. MAPKs consist of at least three distinct pathways in mammals, including the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and CSBP/p38/ RK/Mpk2 kinases [28]. These pathways appear to be activated in response to stimuli such as the binding of growth factors and cytokines, as well as exposure to stress such as ultraviolet radiation and osmotic shock. However, our results showed no change in MMPs 1, 11, or 13–16 that were constitutively expressed in the MDA-MB-231 cell line following the inhibition of MAPK pathways. Genistein (a nonspecific inhibitor of tyrosine kinases), SB 203580 (a specific p38 inhibitor), and PD 98059 (an inhibitor of MAP/ERK kinases in the ERK pathway) all failed to reduce the constitutive expression of MMPs, suggesting that constitutive MMP expression did not involve tyrosine kinase signaling pathways. In this investigation, the role exogenous agents play in MMP production by breast tumor cells was investigated using the tumor promoting agents 12-O-tetradecanoylphorbal-3-acetate (TPA) and concanavalin-A (Con-A). TPA activates protein kinase C (PKC) and indirectly activates numerous signal transduction
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pathways. Con-A causes the aggregation of cellular surface receptors and nonspecifically activates their signal transduction pathways. Our results show several MMPs to be regulated by either TPA or Con-A, as demonstrated in Tables 1–3 and in Fig. 1; specifically, MMPs 1, 2, 9, and 10 were up-regulated in one or more of the cell lines. These data support other investigators who have shown TPA and Con-A to stimulate the expression of MMPs 1, 9, and 14 in breast tumor cells [29 –31]. Although the current study observed no change in MMP-14 levels, Yu et al. [31] saw this increase only after quite sustained exposure to Con-A. TPA-induced MMP-9 expression was also investigated using specific MAPK inhibitors. This is a reasonable point to investigate because the promotor region of MMP-9 contains two TPA-responsive elements (TRE), one ⫺533 and the other ⫺79 upstream from the MMP-9 initiation site [32]. TREs serve as the binding site for the AP-1 dimers of the Jun and Fos families, both of which can be activated by the MAPK signal transduction pathways [32]. Reddy et al. [15] showed that the MAPK/ERK inhibitor PD 98059 could block the EGF-stimulated induction of MMP-9 in breast tumor cells, indicating that MAPK/ERK has a role in MMP-9 up-regulation. Simon et al. [33] showed that TPA can induce MMP-9 expression in squamous cell carcinoma cells and that the MAPK/p38 inhibitor SB 203580 can inhibit that expression. In this study, TPA was shown to stimulate increased MMP-9 in both the MDA-MB-231 and MCF-7 cell lines. TPA stimulation was inhibited by genistein, SB 203580, and PD 98059 in a dose-dependent manner (Figs. 3 and 4). This would suggest that both the MAPK/ERK and MAPK/p38 pathways may be involved in the TPA-induced upregulation of MMP-9 in breast carcinoma, although a similar effect was not demonstrated with respect to the constitutive expression of any MMPs. Previous investigators [14, 20, 34] demonstrated that interactions between integrin receptors and ECM proteins are capable of regulating MMPs. Therefore, we anticipated that ECM proteins might have regulatory effects on MMP expression in breast tumor cell lines. Such an effect of ECM proteins on MMP-2 and MMP-9 seems reasonable in light of the observations made by previous investigators [13, 15, 16, 35]. However, as indicated in Table 4, in the current investigation, the ECM proteins tested failed to show any distinct up-regulatory effect on the MMPs that were constitutively expressed prior to exposure to ECM proteins as detected by gelatin zymography. One possible explanation is that these MMPs were already being expressed at their near-maximal levels. The only regulation observed was a slight increase in MMP-11 expression detected by RT-PCR in the MCF-7 cell line after exposure to the FN-mimetic peptide fragment GRGDSP (RGD) (see Table 3). RGD consists of the binding site for integrins on ECM such as FN and VN
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FIG. 3. Zymographic gel patterns of MMPs 2 and 9 activity found in the tissue culture media obtained from MDA-MB-231. Upper 90 kDa band, MMP-9; lower 72 kDA band, MMP-7. lane 1A, ladder; lane 2A, MMP-9 standard; lane 3A, blank (medium containing 10% serum); lane 4A, MDA-MB-231 cells alone; lane 5A, TPA (100 M); lane 6A, TPA (100 M) and genistein (10 g/ml); lane 7A, TPA (100 M) and PD 98059 (10 g/ml); lane 8A, TPA (100 M) and SB 203580 (20 M). Figure 3B depicts the dose-response inhibition of MMP-9 activity in MDA-MB-231 cells. Lane 1B, TPA (100 M) and genistein (1 g/ml); lane 2B, TPA (100 M) and genistein (5 g/ml); lane 3B, TPA (100 M) and genistein (10 g/ml); lane 4B, TPA (100 M) and PD 98059 (1 g/ml); lane 5B, TPA (100 M) and PD 98059 (5 g/ml); lane 6B, TPA (100 M) and PD 98059 (10 g/ml); lane 7B, TPA (100 M) and SB 203580 (2 M); lane 8B, TPA (100 M) and SB 203580 (10 M); lane 9B, TPA (100 M) and SB 203580 (20 M). TPA caused an increased up-regulation of MMP-9 but had no effect on MMP-2 activity. In contrast to constitutive MMP expression, TPA stimulation can be inhibited by MAPK antagonists, but those antagonists had no effect on MMP-2. The fact that MMP-2 and MMP-9 could be detected in the medium indicates that those enzymes were released from the MDA-MB-231 cells.
FIG. 4. Zymographic gel patterns of MMPs 2 and 9 activity found in the tissue culture media obtained from MCF-7 showing doseresponse effects of inhibition of genistein and of the inhibition of SB 203580 on TPA-stimulated MMP-2 and MMP-9 up-regulation. 90 kDa band, MMP-9; 72 kDa band, MMP-2, not observed. Lane 1A, MMP-9 standard; lane 2A, blank; lane 3A, MCF-7 cells alone; lane 4A, TPA (100 M); lane 5A, TPA (100 M) and genistein (10 g/ml); lane 6A, TPA (100 M) and PD 98059 (10 g/ml); lane 7A, TPA (100 M) and SB 203580 (20 M). Figure 4B depicts the dose-response inhibition of MMP-9 activity in MCF-7 cells. Lane 1B, TPA (100 M) and genistein (1 g/ml); lane 2B, TPA (100 M) and genistein (5 g/ml); lane 3B, TPA (100 M) and genistein (10 g/ml); lane 4B, TPA (100 M) and PD 98059 (1 g/ml); lane 5B, TPA (100 M) and PD 98059 (5 g/ml); lane 6B, TPA (100 M) and PD 98059 (10 g/ml); lane 7B, TPA (100 M) and SB 203580 (2 M); lane 8B, TPA (100 M) and SB 203580 (10 M); lane 9B, TPA (100 M) and SB 203580 (20 M). TPA strongly up-regulated MMP-9 but failed to cause the appearance of MMP-2 in the culture medium (see lane 4). Genistein, PD 98059, and SB 203580 caused a dose-related inhibition of MMP-9. These dose-response effects provide further evidence that MAPK signaling can be involved in MMP up-regulation.
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TABLE 4 Effects of ECM Components and Anti-integrin Antibodies on MMP-2 and MMP-9 Enzyme Release and Activity as Determined by Gelatin Zymography A MMPs
Control
Collagen type IV
Vitronectin
Fibronectin
Laminin
MCF-7
MMP-2 MMP-9
— —
— —
— —
— —
— —
MDA-MB-231
MMP-2 MMP-9
— ⫹/0
— ⫹/0
— ⫹/0
— ⫹/0
— ⫹/0
B MMPs
Control
Anti-integrin ␣4
Anti-integrin ␣5
Anti-integrin 1
Anti-integrin 4
Anti-integrin ␣v3
MCF-7
MMP-2 MMP-9
— —
⫹/0 —
⫹/0 —
— —
⫹/0 —
— —
MDA-MB-231
MMP-2 MMP-9
— ⫹/0
⫹/0 ⫹/0
— ⫹/0
— ⫹/0
— ⫹/0
— ⫹/0
Note. Results indicated as follows: —, MMP not detected by zymography; ⫹/0, questionable amount of MMP detected by zymography with no up-regulation. MCF-7 and MDA-MB-231 cell lines were cultured for 24 h in serum-free medium containing ECM components (A) or anti-integrin antibodies (B), and conditioned medium was analyzed for MMP activity by gelatin zymography. Control represents conditioned medium incubated with cells alone. ECM components collagen IV, VN, FN, and laminin had no effect on the expression levels of MMPs 2 and 9 in MDA-MB-231 and MCF-7 tumor cells. No effect was observed after treatment with anti-integrin ␣4 (P4C2), anti-integrin ␣5 (P1D6), anti-integrin 1 (MAb-13), anti-integrin 4 (MAb-9), and ␣v3 (MAb 1976B). Bands were present in ␣4, ␣5, and 4; however, it is believed that they are from exogenous MMP-2 because of studies done analyzing MAb alone.
and has been shown to influence breast tumor cell attachment by means of ␣v3 and ␣v5 integrins [36]. Antiintegrin antibodies induced MMP up-regulation in other cell systems [37]. However, as illustrated in Fig. 4B, none of the anti-integrin antibodies tested in this study were shown to significantly induce MMP up-regulation as detected by gelatin zymography. It is possible that these results relate to a need for antibody-induced integrin receptor aggregation to occur before any receptordependent signal transduction reactions can be initiated. It was observed by previous investigators [14, 38] that receptor aggregation is an important step in the cell surface initiation of signal transduction. In summary, this investigation demonstrates that breast tumor cells can constitutively express a wide variety of MMPs at the mRNA levels whereas normal breast tissue does not express MMPs or expresses fewer of them at very low levels. The constitutive expression of MMPs in breast tumor cells can occur independent of the extracellular environment as evidenced by the fact that, in this investigation, MMP expression could be observed in tumor cells grown in vitro on plastic. RT-PCR studies showed minimal up-regulation of MMP-1, MMP-2, MMP10, and MMP-11 in one or more of the cell lines studied as a result of Con-A or of GRGDSP peptide stimulation. TPA, which bypassed cell surface receptor stimulation, could up-regulate the expression of MMP-2 and MMP-9. This up-regulation could be reduced by MAPK inhibitors
genistein, PD 98059, or SB 203580. This effect of TPA indicated that the tumor cells were not constitutively expressing maximal levels of MMPs, because MMP upregulation with TPA could occur. We conclude that signals originating from the cell surface can up-regulate MMP expression. The fact that TPA stimulation could up-regulate the expression of MMP-2 and MMP-9, which could not be significantly up-regulated by the ECM components used in this experiment, suggests that factors other than cell surface receptor stimulation by ECM components can regulate MMP expression. REFERENCES 1.
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Matrix Metalloproteinase Expression in Breast Cancer John E. Bartsch, B.S., Edgar D. Staren, M.D., Ph.D., 1 and Hubert E. Appert, Ph.D. Department of Surgery, Medical College of Ohio, Toledo, Ohio 43614 Submitted for publication September 19, 2002
Background. Matrix metalloproteinases (MMPs) have been implicated as possible mediators of invasion and metastasis in some cancers. Our objective was to investigate which MMPs were constitutively expressed in breast tumor cells versus those that could be up-regulated by a number of agents known to affect MMP expression in other cell systems. Methods. We evaluated expression of MMPs 1–16 in breast tumor cell lines MDA-MB-231, T47D, and MCF-7 using semiquantitative RT-PCR and gelatin zymography. Exposure to 12-O-tetradecanoylphorbal-3-acetate (TPA), concanavalin-A (Con-A), the fibronectin-mimetic peptide GRGDSP (RGD), extracellular matrix (ECM) components, and anti-integrin antibodies was used to test for possible MMP up-regulation. Mitogen-activated protein kinase inhibitors (MAPK-I) were used to evaluate signal transduction pathways and regulation of MMP expression. Results. MMPs 1, 2, 7–11, 13, 14, and 16 were constitutively expressed in some tumor cell lines but not in normal breast epithelial cells. Administration of TPA, Con-A, and RGD increased the expression of MMPs 1, 2, 9, and 10. No MMP up-regulation was seen in MDAMB-231 or MCF-7 after exposure to ECM components or after exposure to anti-integrin antibodies. MAPK-I had no effect on constitutive MMP expression but reduced or abolished the TPA up-regulation of MMP-9 in MDA-MB-231 and MCF-7. Conclusions. Breast tumor cell lines constitutively express a number of MMPs. Because MMP expression can be up-regulated by Con-A, the fibronectin-mimetic peptide RGD, and TPA while being susceptible to inhibition by MAPK antagonists, MAPK signaling appears to play a role in this expression. © 2003 Elsevier Science (USA)
1 To whom correspondence should be addressed at Department of Surgery, Medical College of Ohio, 3065 Arlington Avenue, Toledo OH 43614 Fax: (419) 383-6636. E-mail: [email protected].
Key Words: breast cancer; fibronectin; integrin; matrix metalloproteinase; metastasis; mitogen-activated protein kinase; vitronectin. INTRODUCTION
Although substantial progress has been made in defining the gene expression changes that occur in breast cancer, much of even the most fundamental concepts, such as elucidation of the signal transduction pathways involved in tumor cell invasion and metastasis, remains largely unexplored. For breast cancer cells to manifest their malignant potential, they must develop the ability to break through and dissolve extracellular matrices (ECM), particularly the delimiting basement membrane (BM). The degradation of the pericellular BM and ECM is catalyzed by the concerted action of several classes of ECM-degrading enzymes. The cellular mechanisms controlling the release and activation of ECM-degrading enzymes are very complex and for the most part have not yet been identified [1]. One important class of ECM-degrading enzymes includes the matrix metalloproteinases (MMPs). Previous information suggests that both the constitutive and stimulated release of these enzymes are implicated in tumor cell metastasis [2– 4]. If it is possible to prevent their expression, release, and activation, it may be possible to significantly reduce the role of MMPs in tumor cell metastasis. MMPs are a family of zinc-dependent neutral endopeptidases which collectively are capable of degrading all components of the ECM. Currently the MMP family is known to contain at least 20 members, loosely classified into four groups: (1) interstitial collagenase (MMPs 1, 8, and 13); (2) type-IV collagenase (MMPs 2 and 9); (3) stromelysins (MMPs 3, 7, 10, 11, and 18); and (4) membrane-type MMPs (MMPs 14, 15, 16, 17, 24, and 25) [5, 6]. MMP-9 is an important collagenase contributing to the digestion of collagen type IV, the
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primary component of basement membranes. MMP-9 expression was previously shown to correlate with an invasive phenotype in rat embryo cells as well as a number of malignant tumors [3, 7]. In the normal mammary gland, constitutive expression of MMPs is low except during times of development, pregnancy, and involution [5, 8]. However, in pathologic states such as breast cancer, increased levels of MMPs have been reported in breast tumor cells as well as in the surrounding noncancerous breast tissue. Iwata et al. [3] showed MMPs 1, 2, and 9 to be highly expressed in human breast carcinoma cells. Kossakowska et al. [9] and Garbett et al. [4] showed MMPs 1, 2, 3, 9, and 11 to have a higher frequency of expression in breast cancer as opposed to normal breast tissue or benign breast disease. Furthermore, other investigators found that MMPs 1, 2, 9, 13, and 16 were expressed specifically in malignant or benign breast tissue [10 –13]. In this study, a number of different agents were used to up-regulate MMPs because previous investigators have shown that the expression of MMPs by tumor cells is influenced by multiple environmental factors, including growth factors and ECM components [8, 14, 15]. The effect of the tumor-promoting agents 12-Otetradecanoylphorbal-3-acetate (TPA) and concanavalin-A (Con-A) on MMP expression was evaluated based on our previous observation that they up-regulate MMP expression in pancreatic tumor cells [16]. Liu et al. [17] found that TPA and fibroblast growth factor (FGF) could up-regulate MMP-9 in MCF-7 cells. A fibronectin-mimetic peptide GRGDSP (RGD), ECM proteins, and anti-integrin antibodies were used so as to investigate integrin-induced expression and release of MMPs. Integrins are a family of cell surface receptors that play a key role in cell signal transduction from the ECM. Integrins do not directly activate signal transduction but do so as a result of their binding to linker proteins [18, 19]. Daemi et al. [20] found that the 4 subunit of integrins is responsible for stimulating the expression of MMP-2 in colon adenocarcinoma cells. Bafetti et al. [14] observed that the ECM protein vitronectin (VN) stimulates MMP-2 expression in melanoma cells and that this expression could be blocked by RGD. Finally, this study used the mitogen-activated protein kinase (MAPK) inhibitors genistein, PD 98059, and SB 203580 to elucidate which, if any, MAPK pathways are involved in MMP expression. Reddy et al. [15] found that epidermal growth factor (EGF)-induced MMP-9 secretion in the breast tumor cell line SKBR-3 could be greatly reduced by the use of MAPK inhibitors. MMP expression has been shown to be up-regulated by a number of tumor cell systems and has been shown to be related to tumor cell invasiveness. This study demonstrates constitutive expression of a number of MMPs in breast cancer cell lines, up-regulation of sev-
eral MMPs after exposure to TPA, Con-A, and RGD, and a role for MAPK in MMP expression. Nevertheless, the mechanisms that regulate the metastatic process are extremely complex and continue to prove difficult to analyze. This investigation may suggest new ways to prevent or reduce the role of MMPs in tumor cell metastasis. MATERIALS AND METHODS
Cells and Cell Culture Samples of breast tumor cell lines MDA-MB-231, T47D, and MCF-7 were obtained from ATCC (Manassas, VA) and also from Lombardi Cancer Center (Georgetown University, Washington, DC, courtesy of Shared Tissue Bank). The cells were maintained as described previously by Yang et al. [16]. Nontumor cells were used to isolate the mRNA used in this study.
Stimulatory/Inhibitory Agents Vitronectin (VN), fibronectin (FN), laminin, and collagen IV were purchased from Gibco BRL (Life Technologies, Rockville, MD). The monoclonal antibodies (MAb) were obtained from the following sources: anti-␣4 integrin (clone P4C2) and anti-␣5 integrin (clone B1D6) were obtained from Gibco BRL. Anti-␣v3 integrin (MAb 1976B) was purchased from Chemicon (Temucula, CA). Anti-1 integrin (MAb-13) was a gift from Dr. Kenneth Yamada (NCI, Bethesda, MD). Anti-4 integrin (MAb-9) was a gift from Dr. T.E. Carey (University of Michigan, Ann Arbor, MI). Concanavalin-A (Con-A) was purchased from EY Laboratories, Inc. (San Mateo, CA). 12-Otetradecanoylphorbal-3-acetate (TPA), genistein, and Coomassie blue were purchased from Sigma (St. Louis, MO). PD 98059 and SB 203580 were purchased from Cal Biochem (San Diego, CA). The peptide GRGDSP (RGD) was obtained from Bachem (Torrance, CA).
Isolation of Total Ribonucleic Acid (RNA) Total cellular RNA was isolated by a single step method using TRI reagent according to the manufacturer’s instructions. One milliliter TRIzol reagent was used per 10 cm 2 of culture flask. TRI reagent kits were purchased from Molecular Research Center, Inc. (Cincinnati, OH). Chloroform, isopropanol, and ethanol were purchased from Fisher Scientific; diethyl pyrocarbonate (DEPC) was purchased from Sigma. The mRNA from nontumor breast tissue was obtained from Clonetics (San Diego, CA).
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Using previously reported primers [16, 21], constitutive mRNA levels of MMPs were determined by RT-PCR using a SuperScript one-step RT-PCR system kit. Reactions were assembled by mixing 1.0 g of total RNA with every 25 l of 2⫻ Reaction Mix, 6 l of mixed primers (a final concentration of 0.2 M), 1 l of RT/Taq Mix, 6 l of 5 mM MgSO 4, and autoclaved distilled water to a final volume of 50 l. RT-PCR was performed in a TEMP 䡠 TRONIC DNA Thermal Cycler with 30 min incubation at 50°C, 2 min predenaturation at 94°C, and 30 or 40 cycles of amplification consisting of 94°C for 30 s (denaturation), 55°C for 30 s (annealing), and 72°C for 1 min (elongation). -actin was used as external control and normal adult female breast RNA (Stratogene, La Jolla, CA) was used as a control for MMP expression. The PCR product (10 l) for each sample was analyzed by electrophoresis in a 1% agarose gel containing ethidium bromide. Scion Image (Scion Corp., Frederick, MD) was used to obtain quantitative RT-PCR results of mRNA expression. SuperScript one-step RT-PCR system kits and primers were purchased
BARTSCH, STAREN AND APPERT: MMP EXPRESSION IN BREAST CANCER from Life Technologies Inc.; 100 bp DNA ladder was purchased from Gibco BRL; agarose was purchased from Sigma. The primer sequences were as follows: MMP-1 (786 bp): 5⬘-CGA CTC TAG AAA CAC AAG AGC AAG A-3⬘ (sense) 5⬘-AAG GTT AGC TTA CTG TCA CAC GCT T-3⬘ (antisense) MMP-2 (605 bp): 5⬘-GTG CTG AAG GAC ACA CTA AAG AAG A-3⬘ (sense) 5⬘-TTG CCA TCC TTC TCA AAG TTG TAG G-3⬘ (antisense) MMP-3 (729 bp): 5⬘-GAA CAA TGC ACA AAG GAT ACA ACA-3⬘ (sense) 5⬘-TTC TTC AAA AAC AGC ATC AAT CTT-3 (antisense) MMP-7 (373 bp): 5⬘-GGT CAC CTA CAG GAT CGT ATC ATA T-3⬘ (sense) 5⬘-CAT CAC TGC ATT AGG ATC AGA GGA A-3⬘ (antisense) MMP-8 (435 bp): 5⬘-GCT GCT TAT GAA GAT TTT GAC AGA G-3⬘ (sense) 5⬘-ACA GCC ACA TTT GAT TTT GCT TCA G-3⬘ (antisense) MMP-9 (243 bp): 5⬘-CAC TGT CCA CCC CTC AGA GC-3⬘ (sense) 5⬘-GCC ACT TGT CGG CGA TAA GG-3⬘ (antisense) MMP-10 (408 bp): 5⬘-CAC TCT ACA ACT CAT TCA CAG AGC T-3⬘ (sense) 5⬘-CTT GGA TAA CCT GCT TGT ACC TCA T-3⬘ (antisense) MMP-11 (326 bp): 5⬘-TAA AGG TAT GGA GCG ATG TGA C-3⬘ (sense) 5⬘-TGG GTA GCG AAA GGT GTA GAA G-3⬘ (antisense) MMP-12 (517 bp): 5⬘-TTC CCC TGA ACA GCT CTA CAA GCC TGG AAA-3⬘ (sense) 5⬘-GAT CCA GGT CCA AAA GCA TGG GCT AGG ATT-3⬘ (antisense) MMP-13 (330 bp): 5⬘-GTG GTG TGG GAA GTA TCA TCA-3⬘ (sense) 5⬘-GCA TCT GGA GTA ACC GTA TTG-3⬘ (antisense) MMP-14 (497 bp): 5⬘-CGC TAC GCC ATC CAG GGT CTC AAA-3⬘ (sense) 5⬘-CGG TCA TCA TCG GGC AGC ACA AAA-3⬘ (antisense) MMP-15 (454 bp): 5⬘-ACA ACC ACC ATC TGA CCT TTA GCA-3⬘ (sense) 5⬘-AGC TTG AAG TTG TCA ACG TCC TTC-3 (antisense) MMP-16 (652 bp): 5⬘-TTA CTT CTG GCG GGG CTT GCC TCC TAG TAT-3⬘ (sense) 5⬘-ACA GTA CAG TAT GTG GCG GGG TGT TCC TTT-3⬘ (antisense) TIMP-1 (667 bp): 5⬘-ATC CTG TTG TTG CTG TGG CTG ATA G-3⬘ (sense) 5⬘-TGC TGG GTG GTA ACT CTT TAT TTC A-3⬘ (antisense) TIMP-2 (405 bp): 5⬘-AAA CGA CAT TTA TGG CAA CCC TAT C-3⬘ (sense) 5⬘-ACA GGA GCC GTC ACT TCT CTT GAT G-3⬘ (antisense)
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Gelatin Zymography Determination of Secreted MMP2 and MMP-9 Gelatin zymography was performed using precast polyacrylamide gels containing 0.1% gelatin (Novex, San Diego, CA). Collected tissue culture medium was centrifuged at 1500 rpm for 3 min and 50 l of supernatant medium was mixed with 10 l of 2 ⫻ Tris-Glucine SDS sample buffer. Twenty-five microliters of mixed sample was loaded in 12-well gels and electrophoresis was performed at 100 V for 1.5 h. After electrophoresis, the gel was rinsed with renaturing 1 ⫻ buffer for 5 h at room temperature. The buffer was then switched to 1 ⫻ developing buffer and incubated overnight at 37°C. The gel was stained with Coomassie blue and then destained with distilled water. The unstained bands correspond to the areas of gelatin digestion. Bands were quantified using densitometry (Scion Image).
MMP Expression as Determined by RT-PCR Constitutive and up-regulated MMP expression at the mRNA level was studied by means of RT-PCR in breast tumor cell lines as compared with nontumor breast epithelial cells. MDA-MB-231, T47D, and MCF-7 were grown on T-75 tissue culture flasks in McCoy’s medium containing 10% fetal bovine serum (FBS) until they reached 90% confluency. McCoy’s medium was removed and cells were washed three times with serum-free medium. Cells were resuspended in 1 ml McCoy’s serum-free medium and incubated for 24 h with media alone or with either Con-A (50 g/ml), RGD (100 g/ml), or TPA (100 M). Subsequently total RNA was isolated using the primers and the RT-PCR procedure described previously.
EXPERIMENTAL DESIGN
Studies of the Constitutive vs Up-regulated MMP Expression Effects of ECM and anti-integrin antibodies on MMP-2 and MMP-9 enzyme release and activity determined by zymography. MCF-7 and MDA-MB-231 cell lines were plated on 12-well cell culture treated plates (Corning Inc., Life Sciences, Acton, MA) in 2 ml serumsupplemented McCoy’s medium and allowed to grow until they reached 90% confluency. McCoy’s medium was removed and cells were washed three times with serum-free medium. Cells were resuspended in 1 ml of McCoy’s serum-free medium, then incubated for 24 h in media to which the following were added: Con-A (50 g/ml), 200 g of collagen IV, 25 g of VN, 500 g of FN, 200 g of laminin, anti-integrin ␣4 (P4C2) (1:100 dilution), anti-integrin ␣5 (P1D6) (1:100), anti-integrin 1 (MAb-13) (1:200), anti-integrin 4 (MAb-9) (1:100), and anti-integrin ␣v3 (MAb1976B) (20 g/ml). Following a 24-h incubation period, the medium was removed and the cells were prepared for the zymographic determination of MMP-2 and MMP-9 as described previously. Effects of the MAPK inhibitors genistein, PD 98059, and SB 203580 on constitutive MMP expression as determined by RT-PCR. MDA-MB-231 was grown on T-75 tissue culture flasks in McCoy’s medium containing 10% FBS until it reached 90% confluency. The medium was then removed and cells were washed three times with serum-free medium. Cells were then
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incubated for 24 h with genistein (10 g/ml), PD 98059 (100 g/ml), or SB 203580 (20 M) in serum-free medium, and total RNA was isolated as described previously. RT-PCR was performed with primers for MMPs 1, 11, 13, 14, 15, and 16. Effects of the MAPK inhibitors genistein, PD 98059, and SB 203580 on the TPA-stimulated MMP-9 release and activity as determined by gelatin zymography. MCF-7 and MDA-MB-231 cell lines were plated on 12-well cell culture treated plates (Corning, Inc.) in 2 ml serum-supplemented McCoy’s medium and allowed to grow until they reached 90% confluency. McCoy’s medium was removed and cells were washed three times with serum-free medium. Cells were resuspended in 1 ml McCoy’s serum-free medium and incubated with TPA (100 M) either alone or with one of the following MAPK inhibitors: genistein (10 g/ml), PD 98059 (10 g/ml), or SB 203580 (20 M). RESULTS
Constitutive vs Stimulated MMP Expression The invasive capacities of MDA-MB-231, T47D, and MCF-7 tumor cells used in this investigation were previously shown to have high, medium, and low invasive capacities [16]. The mRNA isolated from the nontumor cells expressed only MMP-15. MMP unstimulated and stimulated expression in the MDA-MB-231 cell line. As shown in Table 1, MMPs 1, 2, 7–11, and 13–16, as well as the natural inhibitors of MMP, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2, were expressed in MDA-MB-231 in varying degrees. MMP-3 and MMP-12 could not be detected in MDA-MB-231 either prior to or after stimulation. There was a slight up-regulation of MMP-2 (60.5 kDa) with RGD (10 g/ml) (see also Fig. 1G, lane 3) and with TPA. There was also a slight increase in MMP-10 expression in MDA-MB-231 over that of baseline (lane 1) with Con-A (lane 2) (Fig. 1F). A moderate upregulation of MMP-9 in MDA-MB-231 was observed with exposure to TPA (Table 1). There were no other noticeable changes in any of the other MMPs after Con-A, RGD, or TPA exposure. Hence it can be seen that MMP-2 and MMP-9, in contrast to the other MMPs, can be susceptible to up-regulation. MMP unstimulated and stimulated expression in the T47D cell line. As shown in Table 2, prior to stimulation, MMPs 11, 13, 15, and 16 were expressed at high levels, while MMPs 1 and 7 were expressed at low levels. In contrast to the MDA-MB-231 cell line, MMPs 2, 3, 8, 9, 10, 12, and 14 were not expressed in the T47D cell line either before or after exposure to Con-A or RGD. There was an increase in MMP-1 in the T47D cell line after exposure to Con-A and RGD (Fig. 1D). This is in contrast to the initially higher expression of MMP-1
TABLE 1 Unstimulated and Stimulated MMP Expression in MDA-MB-231 as Determined by RT-PCR Primer
Control
Con-A
GRGDSP
TPA
Actin MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-10 MMP-11 MMP-12 MMP-13 MMP-14 MMP-15 MMP-16 TIMP-1 TIMP-2
⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹/0 — ⫹⫹ ⫹⫹ ⫹/0 ⫹⫹ ⫹⫹⫹⫹ — ⫹⫹/⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹/0 — ⫹⫹ ⫹⫹ ⫹/0 ⫹⫹⫹ ⫹⫹⫹⫹ — ⫹⫹/⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ — ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹⫹ — ⫹⫹/⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ — ⫹⫹ ⫹⫹ ⫹⫹ N.D. ⫹⫹⫹⫹ — ⫹⫹/⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
Note. Five grades of MMP expression were observed and categorized: ⫹⫹⫹⫹, strongest MMP expression; ⫹⫹⫹, strong MMP expression; ⫹⫹, moderate MMP expression; ⫹, weak expression; ⫹/0, questionable expression; —, no observed expression. MMPs 1, 2, 7–11, and 13–16, as well as TIMP-1 and TIMP-2, the natural inhibitors of MMP, were expressed in MDA-MB-231 to some extent both before and after stimulation. MMP-3 and MMP-12 were not expressed in MDA-MB-231 either prior to or after stimulation. After stimulation with Con-A, GRGDSP, and TPA, there was a slight up-regulation of MMP-2 with GRGDSP (see Fig. 1G) and TPA. There was also a slight change in MMP-10 expression in MDA-MB-231 with Con-A (see Fig. 1F). A moderate up-regulation of MMP-9 in MDA-MB-231 was observed with exposure to TPA. There were no other noticeable changes in any of the other MMPs after Con-A, GRGDSP, or TPA.
in MDA-MB-231 and lack of MMP-1 up-regulation in MDA-MB-231 cells. MMP expression in the MCF-7 cell line. As shown in Table 3, prior to stimulation, MMPs 14, 15, and 16 were expressed at high levels and MMPs 1 and 11 were expressed at low levels. Closely related to the findings observed with the T47D cell line, MMPs 2, 3, 7, 8, 10, and 12 were not expressed in the MCF-7 cell line either before or after stimulation with Con-A or RGD. Upregulation of MMP-11 was observed after stimulation with RGD and Con-A (Fig. 1B). Effects of ECM Components and Anti-integrin Antibodies on MMP-2 and MMP-9 Enzyme Release and Activity as Determined by Zymography Table 4A shows that the ECM components collagen IV, VN, FN, and laminin had no effect on the zymographic activity of MMP-2 and MMP-9 in MDA-MB231 and MCF-7 tumor cells. Also, as shown in Table 4B, no effect was observed on MMP zymographic activity after treatment with anti-integrins ␣4 (P4C2), ␣5
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FIG. 1. RT-PCR analysis of Con-A, and GRGDSP stimulated breast tumor lines in which some degree of regulation was observed. MDA-MB-231, T47D, and MCF-7 cells were cultured for 24 h in serum-free medium containing cells only (lane 1), Con-A (20 g/ml) (lane 2), and GRGDSP (100 g/ml) (lane 3). Total RNA was isolated followed by RT-PCR analysis of MMP expression. The expression of actin in MCF-7 (A), T47D (C), and MDA-MB-231 (E) served as external standards, and MMP-1 (D), MMP-2 (G), MMP-10 (F), and MMP-11 (B) are depicted. This figure shows that MMP-11, MMP-1, MMP-10, and MMP-2 can be up-regulated by agents that act on cell surface receptors of breast tumor cells.
(P1D6), 1 (MAb-13), 4 (MAb-9), and ␣v3 (MAb 1976B). Effects of the MAPK Inhibitors Genistein, PD 98059, and SB 203580 on Constitutive MMP Expression as Determined by RT-PCR As shown in Fig. 2, the MAPK inhibitors genistein, PD 98059, and SB 203580 had no effect on the constitutive expression of MMPs 1, 11, 13, 14, 15, and 16 in MDA-MB-231, as measured by RT-PCR. Effects of Genistein, PD 98059, and SB 203580 on TPA-Stimulated MMP-2 and MMP-9 Activity in MDA-MB-231 and MCF-7 Cells as Determined by Zymography Observations on MDA-MB-231 cells. Zymographic studies showed that TPA (100 M) treatment caused
the appearance of a 90 kDa band of activity which represented the active form of MMP-9 which was released from the MDA-MB-231 cells (Fig. 3A, lane 5), and this band could be reduced by about 50% with the addition of genistein (10 g/ml), PD 98059 (10 g/ml), or SB 203580 (20 M), as seen in lanes 6, 7, and 8, respectively in Fig. 3A. In Fig. 4B, dose-response effects could be demonstrated using TPA with 1, 5, and 10 g/ml of genistein (lanes 1, 2, and 3); TPA with PD 98059 with 1, 5, and 10 g/ml (lanes 4, 5, and 6), and with SB 203580, 2, 10, and 20 g/ml (lanes 7, 8, and 9). Observations on MCF-7 cells. As seen in Fig. 4A (lane 4), exposure of MCF-7 cells to TPA (100 M) resulted in a band of strong MMP-9 activity. The band resulting from exposure of TPA (100 M) was greatly reduced in the presence of 10 g/ml genistein, and to a lesser extent in the presence of PD 98059 (10 g) and
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TABLE 2 Unstimulated and Stimulated MMP Expression in T47D as Determined by RT-PCR Primer
Control
Con-A
GRGDSP
Actin MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-10 MMP-11 MMP-12 MMP-13 MMP-14 MMP-15 MMP-16 TIMP-1 TIMP-2
⫹⫹⫹⫹ ⫹ — — ⫹⫹ — — — ⫹⫹⫹ — ⫹⫹⫹ — ⫹⫹⫹⫹ ⫹⫹⫹⫹ — ⫹⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹ ⫹/0 — ⫹⫹ — — — ⫹⫹⫹ — ⫹⫹⫹ — ⫹⫹⫹⫹ ⫹⫹⫹⫹ — ⫹⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹ — — ⫹⫹ — — — ⫹⫹⫹ — ⫹⫹⫹ — ⫹⫹⫹⫹ ⫹⫹⫹⫹ — ⫹⫹⫹⫹⫹
Note. Five grades of MMP expression were observed and categorized: ⫹⫹⫹⫹, strongest MMP expression; ⫹⫹⫹, strong MMP expression; ⫹⫹, moderate MMP expression; ⫹, weak expression; ⫹/0, questionable expression; —, no observed expression. Prior to stimulation, MMPs 11, 13, 15, and 16 and TIMP-2 were expressed at high levels, while MMPs 1 and 7 were expressed at low levels. MMPs 2, 3, 8 –10, 12, and 14, as well as TIMP-1, were not expressed in the T47D cell line either before or after exposure to Con-A or GRGDSP. There was an increase in MMP-1 in the T47D cell line after exposure to Con-A and GRGDSP (also see Fig. 1D).
SB 203580 (20 M). Dose-response effects could be demonstrated with TPA (100 M) using genistein 1, 5, and 10 g, (Fig. 4B, lanes 1, 2, and 3), with PB 98059, 1, 5, and 10 g (Fig. 4B, lanes 4, 5, and 6), and with SB 203580, 2, 10, and 20 g/ml (Fig. 4B, lanes 7, 8, and 9). DISCUSSION
It has been shown that MMPs are expressed in increased levels in breast tumors as opposed to their expression in normal breast tissues [3–5, 8, 9]. Cassara et al. [22] demonstrated that 8701 BC tumor cells released MMP-containing vesicles into the tissue culture medium. Barsky et al. [23] demonstrated an increase in MMP-2 and MMP-9 immunoreactivity in invasive breast carcinoma, and Polette et al. [24] reported MMP-2 reactivity in two of six benign breast tumors and 13 of 17 malignant tumors. In situ hybridization studies indicate that MMP-2 is localized in stromal cells around the breast lesion, MMP-3 in the infiltrating T-lymphocytes, and MMP-9 in macrophages and neutrophils induced by the immune response to tumor cells [3, 25]. While some investigators suggest an almost exclusive stromal, rather than tumor cell, origin of MMPs, the current study, as well as studies by other investigators, demonstrates that a number of MMPs
are constitutively expressed in breast tumor cell lines in an environment completely isolated from stromal components. In this investigation, the tumor line MDA-MB-231 was shown by RT-PCR to constitutively express MMPs 1, 2, 7–11, and 13–16 (Table 1), while T47D expressed MMPs 1, 7, 11, 13, 15, and 16 (Table 2), and MCF-7 expressed MMPs 1, 11, 14, 15, and 16 (Table 3). Consistent with our findings, Balduyck et al. [26] and Lebeau et al. [27], reported increased MMP expression (e.g., MMPs 1, 3, and 13) in MDA-MB-231 cells. Balduyck et al. [26] did not, however, detect MMPs 2, 7, and 9 in MDA-MB-231, although we found those expressed at low levels. Our investigation demonstrated that MMPs 1, 2, 7–11, 13, 14, and 16 were, to some extent, overexpressed in one or more of the tumor cell lines studied. The differences in MMP expression are not surprising and could be a result of different cell culture conditions such as differences in growth factors in the composition of the serum added to the culture medium. Reddy et al. [15] showed that EGF can regulate the expression of MMP-9. This expression of multiple MMPs in tumor cells is in contrast to normal mammary tissue which expressed only MMP-15. In Table 1 it is shown by RT-PCR that incubation of the MDA-MB-231 cell line with RGD and TPA results in an up-regulation of the mRNAs for MMP-2 and MMP-9. We suspected that the increased constitutive expression of MMPs in tumor cells might be the result of the TABLE 3 Unstimulated and Stimulated MMP Expression in MCF-7 as Determined by RT-PCR Primer
Control
Con-A
GRGDSP
Actin MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-10 MMP-11 MMP-12 MMP-13 MMP-14 MMP-15 MMP-16
⫹⫹⫹⫹ ⫹⫹ — — — — — — ⫹⫹ — — ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹/⫹⫹ — — ⫹/0 ⫹/0 — — ⫹⫹ — ⫹/0 ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹/⫹⫹ — — ⫹/0 ⫹/0 — — ⫹⫹⫹ — ⫹/0 ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
Note. Five grades of MMP expression were observed and categorized: ⫹⫹⫹⫹, strongest MMP expression; ⫹⫹⫹, strong MMP expression; ⫹⫹, moderate MMP expression; ⫹, weak expression; ⫹/0, questionable expression; —, no observed expression. Prior to stimulation, MMPs 14, 15, and 16 were expressed at high levels and MMPs 1 and 11 were expressed at low levels. MMPs 2, 3, 7–10, and 12 were not expressed in the MCF-7 cell line either before or after stimulation with Con-A or GRGDSP. Slight up-regulation of MMP-11 was observed after stimulation with GRGDSP and Con-A (see also Fig. 1B).
BARTSCH, STAREN AND APPERT: MMP EXPRESSION IN BREAST CANCER
FIG. 2. Effects of the MAPK inhibitors genistein, PD 98059, and SB 203580 on constitutive MMP expression as determined by RTPCR. MDA-MB-231 cells were cultured for 24 h in serum-free medium containing cells only (lane 1), genistein (100 g/ml) (lane 2), PD 98059 (g/ml (lane 3), and SB 203580 (20 M) (lane 4). RNA was isolated followed by RT-PCR analysis of MMP-1 (A), MMP-11 (B), MMP-13 (C), MMP-14 (D), MMP-15 (E), and MMP-16 (F) as described in “Materials and Methods.” No noticeable change in constitutive MMP expression was observed after exposure to genistein, PD 98059, or SB 203580 in the concentrations used in this experiment; hence it appears that constitutive MMP expression is not under the control of MAPK signal transduction pathways.
cellular transformation of transcription factors related to mitogen-activated protein kinases. MAPKs consist of at least three distinct pathways in mammals, including the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and CSBP/p38/ RK/Mpk2 kinases [28]. These pathways appear to be activated in response to stimuli such as the binding of growth factors and cytokines, as well as exposure to stress such as ultraviolet radiation and osmotic shock. However, our results showed no change in MMPs 1, 11, or 13–16 that were constitutively expressed in the MDA-MB-231 cell line following the inhibition of MAPK pathways. Genistein (a nonspecific inhibitor of tyrosine kinases), SB 203580 (a specific p38 inhibitor), and PD 98059 (an inhibitor of MAP/ERK kinases in the ERK pathway) all failed to reduce the constitutive expression of MMPs, suggesting that constitutive MMP expression did not involve tyrosine kinase signaling pathways. In this investigation, the role exogenous agents play in MMP production by breast tumor cells was investigated using the tumor promoting agents 12-O-tetradecanoylphorbal-3-acetate (TPA) and concanavalin-A (Con-A). TPA activates protein kinase C (PKC) and indirectly activates numerous signal transduction
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pathways. Con-A causes the aggregation of cellular surface receptors and nonspecifically activates their signal transduction pathways. Our results show several MMPs to be regulated by either TPA or Con-A, as demonstrated in Tables 1–3 and in Fig. 1; specifically, MMPs 1, 2, 9, and 10 were up-regulated in one or more of the cell lines. These data support other investigators who have shown TPA and Con-A to stimulate the expression of MMPs 1, 9, and 14 in breast tumor cells [29 –31]. Although the current study observed no change in MMP-14 levels, Yu et al. [31] saw this increase only after quite sustained exposure to Con-A. TPA-induced MMP-9 expression was also investigated using specific MAPK inhibitors. This is a reasonable point to investigate because the promotor region of MMP-9 contains two TPA-responsive elements (TRE), one ⫺533 and the other ⫺79 upstream from the MMP-9 initiation site [32]. TREs serve as the binding site for the AP-1 dimers of the Jun and Fos families, both of which can be activated by the MAPK signal transduction pathways [32]. Reddy et al. [15] showed that the MAPK/ERK inhibitor PD 98059 could block the EGF-stimulated induction of MMP-9 in breast tumor cells, indicating that MAPK/ERK has a role in MMP-9 up-regulation. Simon et al. [33] showed that TPA can induce MMP-9 expression in squamous cell carcinoma cells and that the MAPK/p38 inhibitor SB 203580 can inhibit that expression. In this study, TPA was shown to stimulate increased MMP-9 in both the MDA-MB-231 and MCF-7 cell lines. TPA stimulation was inhibited by genistein, SB 203580, and PD 98059 in a dose-dependent manner (Figs. 3 and 4). This would suggest that both the MAPK/ERK and MAPK/p38 pathways may be involved in the TPA-induced upregulation of MMP-9 in breast carcinoma, although a similar effect was not demonstrated with respect to the constitutive expression of any MMPs. Previous investigators [14, 20, 34] demonstrated that interactions between integrin receptors and ECM proteins are capable of regulating MMPs. Therefore, we anticipated that ECM proteins might have regulatory effects on MMP expression in breast tumor cell lines. Such an effect of ECM proteins on MMP-2 and MMP-9 seems reasonable in light of the observations made by previous investigators [13, 15, 16, 35]. However, as indicated in Table 4, in the current investigation, the ECM proteins tested failed to show any distinct up-regulatory effect on the MMPs that were constitutively expressed prior to exposure to ECM proteins as detected by gelatin zymography. One possible explanation is that these MMPs were already being expressed at their near-maximal levels. The only regulation observed was a slight increase in MMP-11 expression detected by RT-PCR in the MCF-7 cell line after exposure to the FN-mimetic peptide fragment GRGDSP (RGD) (see Table 3). RGD consists of the binding site for integrins on ECM such as FN and VN
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FIG. 3. Zymographic gel patterns of MMPs 2 and 9 activity found in the tissue culture media obtained from MDA-MB-231. Upper 90 kDa band, MMP-9; lower 72 kDA band, MMP-7. lane 1A, ladder; lane 2A, MMP-9 standard; lane 3A, blank (medium containing 10% serum); lane 4A, MDA-MB-231 cells alone; lane 5A, TPA (100 M); lane 6A, TPA (100 M) and genistein (10 g/ml); lane 7A, TPA (100 M) and PD 98059 (10 g/ml); lane 8A, TPA (100 M) and SB 203580 (20 M). Figure 3B depicts the dose-response inhibition of MMP-9 activity in MDA-MB-231 cells. Lane 1B, TPA (100 M) and genistein (1 g/ml); lane 2B, TPA (100 M) and genistein (5 g/ml); lane 3B, TPA (100 M) and genistein (10 g/ml); lane 4B, TPA (100 M) and PD 98059 (1 g/ml); lane 5B, TPA (100 M) and PD 98059 (5 g/ml); lane 6B, TPA (100 M) and PD 98059 (10 g/ml); lane 7B, TPA (100 M) and SB 203580 (2 M); lane 8B, TPA (100 M) and SB 203580 (10 M); lane 9B, TPA (100 M) and SB 203580 (20 M). TPA caused an increased up-regulation of MMP-9 but had no effect on MMP-2 activity. In contrast to constitutive MMP expression, TPA stimulation can be inhibited by MAPK antagonists, but those antagonists had no effect on MMP-2. The fact that MMP-2 and MMP-9 could be detected in the medium indicates that those enzymes were released from the MDA-MB-231 cells.
FIG. 4. Zymographic gel patterns of MMPs 2 and 9 activity found in the tissue culture media obtained from MCF-7 showing doseresponse effects of inhibition of genistein and of the inhibition of SB 203580 on TPA-stimulated MMP-2 and MMP-9 up-regulation. 90 kDa band, MMP-9; 72 kDa band, MMP-2, not observed. Lane 1A, MMP-9 standard; lane 2A, blank; lane 3A, MCF-7 cells alone; lane 4A, TPA (100 M); lane 5A, TPA (100 M) and genistein (10 g/ml); lane 6A, TPA (100 M) and PD 98059 (10 g/ml); lane 7A, TPA (100 M) and SB 203580 (20 M). Figure 4B depicts the dose-response inhibition of MMP-9 activity in MCF-7 cells. Lane 1B, TPA (100 M) and genistein (1 g/ml); lane 2B, TPA (100 M) and genistein (5 g/ml); lane 3B, TPA (100 M) and genistein (10 g/ml); lane 4B, TPA (100 M) and PD 98059 (1 g/ml); lane 5B, TPA (100 M) and PD 98059 (5 g/ml); lane 6B, TPA (100 M) and PD 98059 (10 g/ml); lane 7B, TPA (100 M) and SB 203580 (2 M); lane 8B, TPA (100 M) and SB 203580 (10 M); lane 9B, TPA (100 M) and SB 203580 (20 M). TPA strongly up-regulated MMP-9 but failed to cause the appearance of MMP-2 in the culture medium (see lane 4). Genistein, PD 98059, and SB 203580 caused a dose-related inhibition of MMP-9. These dose-response effects provide further evidence that MAPK signaling can be involved in MMP up-regulation.
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TABLE 4 Effects of ECM Components and Anti-integrin Antibodies on MMP-2 and MMP-9 Enzyme Release and Activity as Determined by Gelatin Zymography A MMPs
Control
Collagen type IV
Vitronectin
Fibronectin
Laminin
MCF-7
MMP-2 MMP-9
— —
— —
— —
— —
— —
MDA-MB-231
MMP-2 MMP-9
— ⫹/0
— ⫹/0
— ⫹/0
— ⫹/0
— ⫹/0
B MMPs
Control
Anti-integrin ␣4
Anti-integrin ␣5
Anti-integrin 1
Anti-integrin 4
Anti-integrin ␣v3
MCF-7
MMP-2 MMP-9
— —
⫹/0 —
⫹/0 —
— —
⫹/0 —
— —
MDA-MB-231
MMP-2 MMP-9
— ⫹/0
⫹/0 ⫹/0
— ⫹/0
— ⫹/0
— ⫹/0
— ⫹/0
Note. Results indicated as follows: —, MMP not detected by zymography; ⫹/0, questionable amount of MMP detected by zymography with no up-regulation. MCF-7 and MDA-MB-231 cell lines were cultured for 24 h in serum-free medium containing ECM components (A) or anti-integrin antibodies (B), and conditioned medium was analyzed for MMP activity by gelatin zymography. Control represents conditioned medium incubated with cells alone. ECM components collagen IV, VN, FN, and laminin had no effect on the expression levels of MMPs 2 and 9 in MDA-MB-231 and MCF-7 tumor cells. No effect was observed after treatment with anti-integrin ␣4 (P4C2), anti-integrin ␣5 (P1D6), anti-integrin 1 (MAb-13), anti-integrin 4 (MAb-9), and ␣v3 (MAb 1976B). Bands were present in ␣4, ␣5, and 4; however, it is believed that they are from exogenous MMP-2 because of studies done analyzing MAb alone.
and has been shown to influence breast tumor cell attachment by means of ␣v3 and ␣v5 integrins [36]. Antiintegrin antibodies induced MMP up-regulation in other cell systems [37]. However, as illustrated in Fig. 4B, none of the anti-integrin antibodies tested in this study were shown to significantly induce MMP up-regulation as detected by gelatin zymography. It is possible that these results relate to a need for antibody-induced integrin receptor aggregation to occur before any receptordependent signal transduction reactions can be initiated. It was observed by previous investigators [14, 38] that receptor aggregation is an important step in the cell surface initiation of signal transduction. In summary, this investigation demonstrates that breast tumor cells can constitutively express a wide variety of MMPs at the mRNA levels whereas normal breast tissue does not express MMPs or expresses fewer of them at very low levels. The constitutive expression of MMPs in breast tumor cells can occur independent of the extracellular environment as evidenced by the fact that, in this investigation, MMP expression could be observed in tumor cells grown in vitro on plastic. RT-PCR studies showed minimal up-regulation of MMP-1, MMP-2, MMP10, and MMP-11 in one or more of the cell lines studied as a result of Con-A or of GRGDSP peptide stimulation. TPA, which bypassed cell surface receptor stimulation, could up-regulate the expression of MMP-2 and MMP-9. This up-regulation could be reduced by MAPK inhibitors
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