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Oncology Research, Centocor, Inc., Malvern, PA
Requests for reprints: Li Yan, Oncology Research, Centocor, Inc., 145 King of Prussia Road, Mail Stop R-4-2, Radnor, PA 19087. Phone: (610) 240-8108; Fax: (610) 889-4418. E-mail: LYan2{at}cntus.jnj.com
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Abstract |
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Key Words: cancer • cleavage • fibroblast cells • extracellular matrix • metalloproteinase inducer • matrix metalloproteinase • positive feedback • proteinase • remodeling • stroma
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Interestingly, recent studies of clinical tumor specimens provided evidence that most MMPs are generated by peritumoral fibroblasts in the stromal compartment, instead of by tumor cells themselves. Peritumor stromal cells are indeed responsible for the production of tumor-associated interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), stromelysin-3 (MMP-11), and gelatinase A (MMP-2), in breast, colon, lung, skin, and head and neck cancers (13–16). The induction of MMP production is, at least in part, mediated by tumor-stromal cell interaction via a tumor cell surface glycoprotein, extracellular matrix metalloproteinase inducer (EMMPRIN; 17, 18).
EMMPRIN was originally purified from the plasma membrane of cancer cells as a glycoprotein of Mr 58,000, and was designated tumor collagenase stimulating factor (TCSF) because of its ability to stimulate fibroblast synthesis of collagenase-1 (MMP-1; 19, 20). Subsequent studies further demonstrated that EMMPRIN also induced fibroblast synthesis of MMP-2, MMP-3, as well as the membrane-type 1 MMP (MT1-MMP) and MT2-MMP that function as endogenous activators of MMP-2 (17, 21, 22). Therefore, EMMPRIN functions as an upstream modulator of MMP production in tumor local environment. EMMPRIN-positive tumor cells stimulate neighboring fibroblast cells to express MMPs and therefore facilitate tumor invasion and metastasis.
This rationale for the function of EMMPRIN has been supported by results from several clinical studies that demonstrated high levels of EMMPRIN expression in tumor compartments as compared to peritumoral stromal tissues. These tumors include the lung (16), breast (16), bladder (23, 24), and glioma (25). Examination of EMMPRIN expression in these clinical samples by a variety of means, including Northern blot, in situ hybridization, and immunostaining, revealed that in most cases, EMMPRIN is expressed by tumor cells, but not by the neighboring stromal cells. In contrast, MMPs are expressed by peritumoral stromal cells.
The biological significance of increased expression of EMMPRIN in tumor cells was investigated by in vitro studies using recombinant EMMPRIN or native EMMPRIN purified from tumor cells. EMMPRIN has been shown to stimulate the expression of various MMPs produced by fibroblasts (17, 18). The induction occurred at the transcription level and is at least in part mediated by a mitogen-activated protein kinase (MAPK) p38 kinase signaling pathway (18). The role of EMMPRIN in tumor growth and metastasis was directly illustrated using EMMPRIN-overexpressing human breast cancer cells. MDA MB 463 cells are normally slow-growing cells when they are implanted into nude mice. However, when these cells were transfected with EMMPRIN, they adopted a more aggressive growth pattern, with both accelerated growth rate and metastatic phenotypes (26).
To further study the interplay between tumor cell-associated EMMPRIN and fibroblast cells, we used recombinant engineering to generate MDA MB 231 human breast cancer cells expressing different levels of EMMPRIN. We discovered a novel positive feedback regulatory mechanism controlling the interaction between tumor and fibroblast cells. Our results demonstrate that EMMPRIN positively regulates its own gene expression, in addition to stimulating MMP expression. Furthermore, we also detected generation of soluble EMMPRIN in the coculture of tumor and fibroblast cells that occurs via an MMP-dependent cleavage. On the basis of these new findings, we propose a new model in which the interplay between tumor and stromal compartments is regulated by both tumor cell surface EMMPRIN, as well as by soluble EMMPRIN, generated from MMP-dependent cleavage.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Generation of MDA MB 231 Tumor Cells Expressing Different Levels of EMMPRIN
To further explore the potential regulatory mechanism of EMMPRIN gene expression in fibroblast cells, especially in the context of tumor-fibroblast cell-cell interaction, we used recombinant DNA engineering technology to generate cancer cells that express different levels of EMMPRIN. As described in "Materials and Methods," MDA MB 231 human breast cancer cells were transfected with expression vectors harboring the full-length coding region of human EMMPRIN in either sense or antisense orientations. Stably transfected cells were cloned and EMMPRIN expression level in these cells was determined by Western blot analysis. As shown in Fig. 3
, EMMPRIN protein expression in one of sense transfected cell clones S1-3 was approximately 2-fold higher as compared to that of wild-type control cells. In contrast, EMMPRIN protein expression was substantially inhibited in two antisense transfected cell clones, AS1-5 and AS2-5, to only 47% and 62% of that in wild-type cells. Changes in EMMPRIN protein expression in these engineered cells were also independently confirmed by ELISA analysis (Fig. 4
). In addition, changes in EMMPRIN protein expression also occurred on cell surface in sense and antisense transfected cells, respectively, as determined by fluorescence-activated cell (FAC) analysis (data not shown).
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Stimulation of Both Cell-Associated and Soluble EMMPRIN Expression in Coculture of Tumor Cells and Fibroblast Cells
In monoculture of tumor cells or fibroblast cells, only low levels of EMMPRIN protein were detected in cell pellets or conditioned medium by ELISA analysis. Transfection with sense or antisense expression constructs resulted in substantial changes in cell-associated EMMPRIN expression, with an increase from 3.32 ng/ml in wild-type to 7.2 ng/ml in sense transfected S1-3 cells, and a decrease to 0.85 and 1.43 ng/ml in antisense transfected AS1-5 and AS2-5 cells, respectively (Fig. 4A). Only low levels of soluble EMMPRIN were detected in conditioned medium of these cells in monoculture. Transfection of tumor cells with sense and antisense expression constructs resulted in only minor changes in soluble EMMPRIN protein levels (Fig. 4B).
The expression of cell-associated EMMPRIN was readily induced when tumor cells were cultured together with fibroblast cells. In coculture, EMMPRIN was detected at 10 ng/ml as compared to 0.8 ng/ml in fibroblast cells and 3.32 ng/ml in wild-type tumor cells in monoculture (Fig. 4A). This increase in EMMPRIN expression in coculture was mediated by tumor cell-associated EMMPRIN. When EMMPRIN expression in tumor cells was inhibited by antisense transfection in AS1-5 and AS2-5 cells, the increase in EMMPRIN expression stimulated by coculture was concomitantly reduced to minimal levels (Fig. 4A).
It is known that coculture of EMMPRIN-positive tumor cells with fibroblast cells stimulates MMP expression (22, 27). These MMPs can in turn cleave cell surface proteins to generate soluble proteins. We therefore postulated that there may be an increase in soluble EMMPRIN protein level in conditioned medium of these cells in coculture. In monoculture, the levels of soluble EMMPRIN were extremely low in conditioned medium of fibroblast cells (0.31 ng/ml) and wild-type tumor cells (0.53 ng/ml; Fig. 4B). However, coculture of tumor cells with fibroblast cells induced the release of substantial amounts of soluble EMMPRIN, 7.48 ng/ml by wild-type and fibroblast coculture and 10.39 ng/ml by S1-3 and fibroblast coculture (Fig. 4B). Similar to cell-associated EMMPRIN, the increase in soluble EMMPRIN expression was also correlated with the degree of EMMPRIN expression in tumor cells. Inhibition of EMMPRIN expression by antisense transfection virtually abolished the generation of soluble EMMPRIN in coculture (Fig. 4B).
Taken together, these results demonstrate a positive feedback regulatory mechanism of EMMPRIN gene expression during tumor-fibroblast cell-cell interaction. In addition to stimulating EMMPRIN expression and MMP expression, tumor cell-associated EMMPRIN also mediates the release of soluble EMMPRIN during tumor-fibroblast cell interactions.
MMP-Dependent Generation of Soluble EMMPRIN in Coculture of EMMPRIN-Positive Tumor Cells and Fibroblast Cells
Western blot analyses using antibodies against different regions of EMMPRIN were performed to further characterize soluble EMMPRIN generated in the coculture of tumor cells and fibroblast cells. Antibodies against the extracellular portion of EMMPRIN molecule detected a single band of approximately Mr 52,000, consistent with the size of intact protein, in total cell lysates. Meanwhile, this same antibody detected a slightly smaller molecule in the conditioned medium from coculture, indicating a loss of a portion of EMMPRIN protein (Fig. 5A
). Interestingly, antibodies specific to the carboxyl terminus of EMMPRIN only detected EMMPRIN in total cell lysates, but failed to detect any EMMPRIN protein in conditioned medium (Fig. 5A). Taken together, these data suggest that soluble EMMPRIN secreted into coculture medium of tumor and fibroblast cells is likely a cleaved product of the full-length transpanning form of membrane EMMPRIN.
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These results demonstrate that the generation of soluble EMMPRIN stimulated by tumor fibroblast cell-cell interactions is the result of proteolytic cleavage of membrane-associated EMMPRIN by increased MMP activities in coculture.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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EMMPRIN is normally expressed as a glycoprotein on tumor cell surface that migrates at an apparent molecular size of Mr 58,000 (19). The domain structure of EMMPRIN consists of an extracellular portion which contains two immunoglobulin-like domains, a transmembrane domain, and a short cytoplasmic domain (19). It has been suggested that tumor cell-associated EMMPRIN exerts its MMP stimulatory effects on fibroblast cells via close cell-to-cell contact. However, the production of soluble EMMPRIN by tumor cells has recently been indicated in several studies (29, 30). In these studies, tumor cells affect fibroblast cells in the absence of physical contact in an EMMPRIN-dependent fashion, resulting in increased MMP-2 expression and activation (29), or enhanced cell migration or invasion (30). In addition, soluble full-length EMMPRIN has been purified from concentrated culturing medium of breast cancer cells, presumably secreted in shed vesicles (31). However, secreted soluble EMMPRIN in cancer cell monoculture is estimated to account for only 2–3% of EMMPRIN produced by these cells (31). In the present study, we have demonstrated the release of a substantial amount of soluble EMMPRIN into cell culture medium of cocultured tumor and fibroblast cells. Because fibroblast cells respond to EMMPRIN stimulation in producing increased levels of EMMPRIN, it is likely that the soluble EMMPRIN detected in coculture is derived from both tumor and fibroblast cells. In addition, the soluble EMMPRIN generated in coculture lacks the intracellular carboxyl terminus as determined using an antibody against the intracellular region of the molecule. In contrast, this carboxyl terminal region was preserved in cell-associated EMMPRIN. More interestingly, generation of soluble EMMPRIN in coculture could be efficiently blocked via inhibiting MMP activity. Therefore, this soluble EMMPRIN likely represents the proteolytic product of membrane-associated EMMPRIN.
The generation of soluble EMMPRIN at the interface of tumor-host interaction may have profound biological significance. The soluble EMMPRIN could diffuse away from local tumor invasion site and stimulate stromal cells at distant sites. We and others have shown that soluble EMMPRIN consisting of only the extracellular portion of the molecular is functional in stimulating MMP expression in fibroblast and endothelial cells (17, 32). Increased MMP activity in the stromal compartment in turn promotes tumor invasion and angiogenesis.
The receptor on fibroblast cells that is responsible for EMMPRIN-mediated stimulation of MMP production has remained elusive (17, 21). It has been suggested that EMMPRIN may serve as its own counter-receptor in cancer cells, thus stimulating MMP via a homophilic interaction (33). One could speculate that EMMPRIN on fibroblast cells could also serve as counter-receptors for tumor cell-associated EMMPRIN in stimulating MMP expression. However, this hypothesis is not supported by the fact that only extremely low levels of EMMPRIN expression could be detected in normal fibroblast cells. Our finding of a novel positive feedback regulatory mechanism of EMMPRIN expression provides an alternative explanation for the potential role of EMMPRIN as its own counter-receptor in fibroblast cells. When fibroblast cells were exposed to EMMPRIN stimulus, EMMPRIN expression is up-regulated in these cells. Newly synthesized EMMPRIN could then be presented on the cell surface and serves as the counter-receptor for EMMPRIN-dependent signaling between tumor and fibroblast cells. This novel regulatory mechanism is supported by recent findings of EMMPRIN mRNA expression in peritumoral fibroblasts in ovarian carcinoma (34). However, this homophilic interaction likely only accounts for EMMPRIN-mediated tumor-host interaction in some tumors because EMMPRIN expression has not been detected in the stroma of many other tumors including melanoma (35), or lung and breast cancer (16). In these samples, despite of a lack of EMMPRIN expression in stromal tissue, MMP expression is stimulated in stromal tissues adjacent to EMMPRIN-positive tumor cells, suggesting that there might be an unknown receptor(s) on stromal cell surface mediating these interactions.
In conclusion, we have identified a novel positive feedback regulatory mechanism of EMMPRIN expression in fibroblast cells and discovered a soluble form of EMMPRIN. These findings suggest an amplification of EMMPRIN-mediated, MMP-dependent signaling events at the tumor and host interface, as well as at distant sites (Fig. 6 ).
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Materials and Methods |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Cell Treatment and MMP-1 Activity Assay
Recombinant EMMPRIN corresponding to the extracellular domain of human EMMPRIN protein was produced in NSO cells (R&D Systems, Minneapolis, MN). MMP-1 activity in serum-free medium conditioned by fibroblast cells treated with different amounts of recombinant EMMPRIN protein was quantitatively determined using an MMP-1 Activity Assay Kit according to product manual (R&D Systems). Briefly, MMP-1 contained in 150 µl of standards or samples was captured by anti-MMP-1 antibodies immobilized on the bottom of assay wells. Captured MMP-1 was subsequently activated by 4-aminophenylmercuric acetate (APMA). MMP substrate added into each well was cleaved by activated MMP-1 and the resulting fluorescence was determined using SpectraFluor Plus Plate Reader (TECAN, Research Triangle Park, NC) with the following parameters: excitation wavelength at 320 nm and emission wavelength at 405 nm.
Generation of Sense and Antisense Expression Constructs and Establishment of Stable Transfectants
To study the function of EMMPRIN in mediating tumor and stromal fibroblast cell interaction, the cDNA sequence corresponding to human EMMPRIN open reading frame (ORF) was PCR amplified and subcloned into pcDNA3.1TOPO vectors (Invitrogen, Carlsbad, CA). The EMMPRIN open reading frame was cloned in both sense and antisense orientations for cytomegalovirus promoter-driven expression in mammalian cells. Transfection of MDA MB 231 human breast carcinoma cells was performed using Effectene transfection reagents according to manufacturer's recommendations (Qiagen, Valencia, CA). Stably transfected cells were selected using Genicitin at 1000 µg/ml (Life Technologies). Individual transfectant colonies were established by cloning antibiotic-resistant cell colonies. Effects of sense and antisense transfection on EMMPRIN protein expression were determined using Western blot analysis and ELISA.
RNA Sample Preparation and TaqMan Quantitative Reverse Transcription-PCR
Total RNA was extracted from cells using RNeasy Kit (Qiagen) following manufacturer's instructions. Five micrograms of total RNA were used in the reverse transcription (RT) reactions which were carried out in a 50-µl total volume using Superscript First-Strand Synthesis System for RT-PCR kit following manufacturer's instructions (Invitrogen). Briefly, RNA samples were mixed with 0.5 µg/µl Oligo(dT), 0.4 mM dNTP mix in a total volume of 25 µl and were incubated at 65°C for 5 min, followed by quick cooling on ice for 2 min. The tubes were spun briefly and added with 5 µl 10x RT buffer, 10 µl 25 mM MgCl2, 2 µl 0.1 M DTT, 1 µl RNaseOUT, 1 µl (50 units) of Superscript II RT, and 6 µl nuclease-free water. The tubes were gently mixed and incubated at 42°C for 50 min. The reaction was terminated at 70°C for 15 min followed by quick cooling on ice for 2 min. The tubes were spun briefly and 1 µl of RNase H was added and incubated for 20 min at 37°C before proceeding to amplification of the target gene.
Real-time PCR analyses were performed in 50 µl volume containing 2x TaqMan Universal PCR Master mix (Perkin-Elmer, Norwalk, CT), appropriate amounts of cDNA or genomic DNA, and 200 nM of TaqMan primers and 400 nM of TaqMan probes. Primer pairs and probes used in the study were designed using Primer Express software (Perkin-Elmer), with sequences listed in Table 1. Probes were labeled at the 5'-end with fluorescent reporter dye Fam and at the 3'-end with fluorescent quencher dye Tamra by Synthegen (Houston, TX) to allow direct detection of PCR products. Real-time PCR amplification and detection were performed in 96-well optical plates using ABI 7900HT sequence detector (PE Biosystems, Norwalk, CT). PCR conditions included thermal cycles of 30 s of 95°C for denaturing, 30 s of 60°C for annealing, and 60 s of 68°C for elongation. Relative gene copy numbers were calculated using a standard curve generated using PCR standards, serially diluted human genomic DNA (Clontech, Palo Alto, CA).
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EMMPRIN ELISA
EMMPRIN protein concentration in conditioned medium was determined using EMMPRIN ELISA. Briefly, 96-well Nunc Immuno Plates (Nunc, Denmark) were coated overnight at 4°C with 50 µl of anti-EMMPRIN antibody (R&D Systems) at 5 µg/ml in buffer A (pH 7.4, 20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 10 µM MnCl2). After one wash in buffer A, nonspecific binding sites were blocked with 150 µl of blocking buffer [3% bovine serum albumin (BSA) in buffer A] for 1 h at 37°C. Wells were then rinsed two times with binding buffer (buffer A with 0.3% BSA and 0.2% Tween 20). Either recombinant human EMMPRIN or conditioned medium was added into each well and was incubated at 37°C for 2 h. After three washes with binding buffer at RT, plates were blocked with 150 µl of blocking buffer (3% BSA in buffer A) at 37°C for 1 h. Fifty microliters of 1:5000 diluted biotin-conjugated anti-EMMPRIN antibodies (RDI-147, Research Diagnostics) were added into each well and incubated at 37°C for 2 h. After three washes with binding buffer, 50 µl of horseradish peroxidase-conjugated streptavidin 1:10,000 diluted in binding buffer were added into each well, and were incubated at 37°C for 1 h. After three washes with binding buffer at RT, 50 µl of developing buffer (1 mg/ml O-phenylenediamine, 0.1 M citric acid, 0.2 M sodium phosphate, 0.01% H2O2) were added to each well and incubated at RT until color develops. Colorimetric reactions were stopped by adding 25 µl of 4 N H2SO4. ELISA data acquisition was performed using VersaMax Tunable MicroPlate Reader (Molecular Devices, Sunnyvale, CA) at 490 nm. Data were analyzed using Softmax Pro 3.1 software (Molecular Devices).
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Acknowledgements |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Received August 7, 2003; revised December 12, 2003; accepted January 5, 2004.
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, NHDF; 
, WT; 
, WT + NHDF; 
, WT + NHDF + 0.1 µM 1,10 PA; 
, WT + NHDF + 0.5 µM 1,10 PA; 
, WT + NHDF + 1.0 µM 1,10 PA; *, P < .05 compared to WT + NHDF.