Affiliations of authors: Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD (AK, KUEO, NSF, LWF); Division of Geriatrics, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD (NSF)
Correspondence to: Abdullah Karadag, MD, PhD, MSc, Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, 9000 Rockville Pike, Bldg. 30, Rm. 228, Bethesda, MD 20892-4320 (e-mail: akaradag{at}dir.nidcr.nih.gov)
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ABSTRACT |
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INTRODUCTION |
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BSP is overexpressed by many malignant tissues, including breast (14), prostate (15), lung (16), and thyroid (17) cancers and melanoma (18). BSP expression has been associated with clinical severity and poor survival among patients with breast cancer (19) or with prostate cancer (15). Recently developed serum immunoassays for BSP and osteopontin show that serum from patients with breast, lung, colon, or prostate cancer had statistically significantly elevated levels of BSP and/or osteopontin (20). However, the role of BSP in these cancers is unclear.
Matrix metalloproteinases (MMPs), a class of zinc-dependent endopeptidases, are collectively capable of digesting all extracellular matrix components. In addition to their role in normal tissue development and remodeling, MMPs appear to play major roles in tumor cell invasion and metastasis (21). Although the mechanism by which tumors invade surrounding tissues is not completely understood, MMPs may play an important role by removing physical barriers to invasion. In particular, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) degrade extracellular matrix macromolecules in basement membranes and other interstitial connective tissues (22). Active MMP-2 can localize to the cell surface by binding directly to integrin v
3 (23), and proteolytically active MMP-9 can associate with CD44 (24), thereby focusing proteolytic activity on the cell membrane at the leading edge of the invasive cell.
The integrins are a family of transmembrane receptor proteins composed of heterodimeric complexes of and
chains (25). There are 18
and eight
chains, and these chains can dimerize to form at least 25 different complexes, each binding to a specific set of ligands. For example, integrin
v
3 binds to BSP, osteopontin, and DMP1. In addition to regulating cell adhesion to the extracellular matrix, integrins modulate many cellular processes including proliferation, apoptosis, migration, and invasiveness by activating various signaling pathways (26). Some integrins are overexpressed in malignant tumors. For example, integrin
v
3 is expressed at the invasive front of malignant melanoma cells and on angiogenic blood vessels (27). The level of integrin
v
3 expression in breast cancers is associated with the aggressiveness of the disease (28).
It is generally accepted that latent pro-MMPs are enzymatically activated by removal of their inhibitory propeptide. BSP, osteopontin, and DMP1 bind with nanomolar affinity to the latent and active forms of MMP-2, MMP-3, and MMP-9, respectively. When purified SIBLINGs are incubated with their pro-MMP partners, increased proteolytic activity is detected (29). Therefore, we hypothesize that one or more SIBLINGs increase the invasiveness of cancer cells by interacting with their specific MMP and integrin partners. To test this hypothesis, we used a modified Boyden chamber cell invasion assay, as described previously (30), to measure the invasiveness of various cancer cell lines through a layer of Matrigel (a mixture of basement membrane components), and we determined whether BSP increases the invasiveness of cancer cells by forming a trimolecular complex in which BSP acts as a bridge to link MMP-2 to integrin v
3.
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MATERIALS AND METHODS |
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Human breast cancer cell lines MDA-MB-231 (HTB-26), MDA-MB-435S (HTB-29), BT-474 (HTB-20), and MCF-7 (HTB-22); human prostate cancer cell lines PC-3 (CRL-1435), LNCaP (CRL-1740), and DU-145 (HTB-81); human thyroid cancer cell line SW-579 (HTB-107); human lung cancer cell line NCI-H520 (HTB-182); and human osteosarcoma cell lines SK-ES-1 (HTB-86), SaOS-2 (HTB-85), and MG-63 (CRL-1427) were obtained from the American Type Culture Collection (Manassas, VA). The mouse fibroblastic cell line NIH 3T3 was provided by Dr. Hynda Kleinman (National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD). Fetal bovine serum was purchased from Equitech-Bio (Kerrville, TX). RPMI-1640 medium, L-glutamine, 2-mercaptoethanol, sodium pyruvate, modified Eagle medium (MEM) nonessential amino acids, Hanks balanced salt solution (HBSS), phosphate-buffered saline (PBS), Versene (0.53 mM EDTA in PBS), and 10% zymogram gelatin gels were from Invitrogen (Carlsbad, CA). Matrigel was from Collaborative Research (Bedford, MA; provided by Dr. Hynda Kleinman). Transwell inserts and companion plates were purchased from BD Biosciences Discovery Labware (Bedford, MA). Calcein acetoxymethyl ester dye and the Alexa Fluor 488 protein labeling kit were purchased from Molecular Probes (Eugene, OR). Mouse anti-human vitronectin receptor monoclonal antibody immobilized on immunoaffinity gel matrix (GEM1976), vitronectin receptor complex in Triton X-100 (CC1018), and mouse anti-MMP-2 monoclonal antibody (MAB 13435) were obtained from CHEMICON International (Temecula, CA). Pro-MMP-2 and active MMP-2 were from Oncogene Research Products (Boston, MA). pBluescript II KS vector was purchased from Stratagene (La Jolla, CA). Digoxigenin labeling mixture was obtained from Roche Biochemicals (Indianapolis, IN). The in situ hybridization kit, which included 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT; product SH-3018-01), was from InnoGenex (San Ramon, CA). 1,10-Phenanthroline was from Sigma Chemical Co. (St. Louis, MO). MMP-2 inhibitor I, cis-9-octadecenoyl-N-hydroxylamideleoyl-N-hydroxylamide, and anti-integrin v
3 monoclonal antibody (LM609, MAB 1976Z) were obtained from Calbiochem (San Diego, CA). Rhodamine Redconjugated AffiniPure goat anti-rabbit immunoglobulin G (IgG; heavy- and light-chain) and Cy2-conjugated AffiniPure goat anti-mouse IgG (heavy- and light-chain) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Vectashield mounting medium for fluorescence microscopy with 4',6-diamidino-2-phenylindole (DAPI; product H-1200) was obtained from Vector Laboratories (Burlingame, CA). The ProbeQuant G-50 microcolumn was from Amersham Pharmacia Biotech (Piscataway, NJ). The Microcon YM-30 centrifugal filter device was from Millipore (Bedford, MA). In situ-ready human thyroid papillary adenocarcinoma serial paraffin sections (product 70452-3) were purchased from Novagen, (Madison, WI).
Cell Culture
The human cancer cell lines used, as described above, were first grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 5 mM 2-mercaptoethanol, 2 mM sodium pyruvate, and 0.1 mM MEM nonessential amino acids in a humidified atmosphere of 5% CO2/95% air at 37 °C. When the cells were approximately 80% confluent, they were used in the experiments described below or subcultured for up to 20 passages at a split ratio of 1 : 10.
SIBLING Production and Purification
Recombinant BSP, BSP-KAE (BSP in which the RGD sequence was replaced with the sequence KAE), osteopontin, and DMP1 with posttranslational modifications, including glycosylation, sulfation, and possibly phosphorylation, were made as described previously (9,11). Briefly, adenoviral constructs containing full-length human BSP (31), BSP-KAE (9), osteopontin (32), or bovine DMP1 (33) were subcloned into high-expression, replication-deficient adenovirus type 5 under the control of the elongation factor 1 (EF-1) promoter for BSP or the cytomegalovirus (CMV) promoter for BSP-KAE, osteopontin, and DMP1. The BSP-KAE constructs were made by in situ mutagenesis in pBluescript; the entire insert was checked for fidelity and then shuttled to the adenovirus plasmid (34). The adenoviruses were selected, purified, and expressed as previously described (9). Recombinant SIBLINGs were generated by infecting mid-passage subconfluent normal human bone marrow stromal fibroblasts (gift from Dr. P. Gehron Robey, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD). Harvested serum-free medium was subjected to anion-exchange chromatography, as described (9,11), to isolate SIBLINGs. The purity of each SIBLING was greater than 95%, as measured after sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis.
Modified Boyden Chamber Cell Invasion Assay
Invasiveness of each cancer cell line was measured by using a UV-opaque transwell polycarbonate membrane insert with a diameter of 6.4 mm and pore size of 8 µm in a modified Boyden chamber cell invasion assay. Transwell inserts were placed in a 24-well plate, precoated with Matrigel (510 µg in 50 µL per well), and dried overnight in a laminar airflow hood. Preconfluent cells were removed from culture dishes with 0.53 mM EDTA in PBS, washed twice in HBSS, and resuspended in serum-free RPMI-1640 culture medium at a final density of 4 x 105 cells per milliliter. Quadruplicate cultures of cells were briefly pretreated in a final volume of 250 µL of serum-free medium (containing 0.1% bovine serum albumin) with either buffer or SIBLINGs in 1.5-mL Eppendorf microcentrifuge tubes for 10 minutes and then placed in the upper compartment of a Boyden chamber. In some cases, cells were first treated for 20 minutes with inhibitors, blocking antibodies, or isotype control IgGs in the tube and then placed in the upper chamber. In the latter cases, buffer or recombinant SIBLING was then added directly to the chamber. To induce migration through the Matrigel layer, the lower chambers were filled with 750 µL of serum-free medium conditioned by mouse NIH 3T3 fibroblastic cells and containing 0.1% bovine serum albumin. Cells were then incubated in a humidified incubator at 37 °C for 624 hours, depending on the cell line. Cells that had not migrated through the barrier were removed from the top compartment, and inserts were moved to another 24-well plate in which each well contained 0.5 mL of the fluorescent dye calcein acetoxymethyl ester at 4 µg/mL. The plate was incubated at 37 °C for 45 minutes to allow the living cells to take up and activate the dye, and then the fluorescent intensity was read from the bottom of the insert with a fluorescence plate reader (Wallac 1420 VICTOR2 Multilabel Reader; PerkinElmer Life Sciences, Boston, MA). Fluorescence intensity was proportional to the number of cells migrating to the bottom of the UV-opaque membrane.
Immunoprecipitation Experiments
Commercial mouse anti-human vitronectin receptor (integrin v
3) monoclonal antibody immobilized on immunoaffinity gel matrix (i.e., beads) was washed three times in ice-cold Triton buffer (TB; 20 mM TrisHCl [pH 7.4], 150 mM NaCl, 0.2% Triton X-100, 2 mM MgCl2, and 0.1 mM CaCl2) and incubated in 1 mL of TB containing 1% bovine serum albumin at 4 °C for 30 minutes with gentle shaking. After washing three times with 1 mL of ice-cold TB, the beads were gently shaken with or without 10 µg of integrin
v
3 in 50 µL of TB at 4 °C for 10 minutes. The beads were then pelleted, the liquid was carefully removed, and the beads were washed in 1 mL of TB. The beads were then resuspended in 1 mL of buffer and separated into aliquots. An aliquot was gently shaken with buffer alone or buffer containing 500 nM BSP or 500 nM BSP-KAE (in a final volume of 50 µL) at 4 °C for 10 minutes. The beads were then pelleted, washed in 1 mL of TB, and incubated in 50 µL of TB containing 1 µg of pro-MMP-2 or 1 µg of active MMP-2 at 4 °C for 10 minutes. The beads were pelleted and washed with 1 mL of TB. The MMPs were eluted from the beads with 80 µL of 1x SDS sample buffer (2.5 mL of 0.5 M TrisHCl [pH 6.8], 2 mL of glycerol, 4 mL of 10% (wt/vol) SDS, and 0.5 mL of 0.1% bromophenol blue, adjusted to 20 mL with distilled water) and resolved by electrophoresis on a 10% gelatin zymogram gel.
SDSPolyacrylamide Gel Electrophoresis and Zymography
Samples in zymogram gel sample buffer were loaded on a 10% gelatin zymogram gel, subjected to electrophoresis, and processed as recommended by the manufacturer. Resulting Coomassie-stained gels were visualized with an EagleEye II imaging system (Stratagene, La Jolla, CA) by dynamic integrated exposure with an initial integration time of 3 seconds and an increment of 3 seconds (the camera sums frames of 1/30 second for a 3-second period, sends the image to the computer, collects light for 6 seconds, sends the image to the computer, and continues in this progression until integration is stopped).
Labeling of Purified Human Active MMP-2 and Pro-MMP-2 With Alexa 488 Dye
Latent (pro-MMP-2) or active MMP-2 was fluorescently labeled with the Alexa Fluor 488 protein labeling kit according to the manufacturer's protocol but was adjusted to the smaller amounts of protein being labeled. Briefly, shipping buffer from 50 µg of pro-MMP-2 or 50 µg of active MMP-2 was exchanged for the reaction buffer (PBS) on ProbeQuant G-50 microcolumns, and the resulting mixture was concentrated to approximately 50 µL with a prewashed Microcon YM-30 centrifugal filter device. Sodium bicarbonate (0.1 M, 5 µL) was added to raise the pH to 7.58.5 for efficient labeling. All steps were performed at 4 °C. The reactive dye was dissolved in 0.5 mL of PBS containing 0.1 M sodium bicarbonate, 50 µL of Alexa Fluor 488 dye was added to the MMP-2 solution, and the reaction mixture was stirred at 4 °C for 2 hours. The labeled MMP-2 protein was then separated from the unreacted dye on ProbeQuant G-50 microcolumns (in PBS) and stored as aliquots at 80 °C until use.
Flow Cytometry
Cells were detached from culture dishes with PBS containing 0.53 mM EDTA, washed twice in HBSS, and then incubated at 2 x 106 cells per milliliter with buffer alone or buffer containing 500 nM BSP or 500 nM BSP-KAE at room temperature for 10 minutes. For the studies involving the blocking anti-integrin v
3 antibody, cells were incubated with buffer alone or buffer containing anti-integrin
v
3 antibody (LM609, 4 µg/mL) or isotype control IgG1 (4 µg/mL) at room temperature for 10 minutes, and then the mixture was incubated with 500 nM BSP for 10 minutes. In the final step for all samples, cells were pelleted at 225g for 10 minutes at room temperature, washed once in HBSS, and then incubated at room temperature with Alexa Fluor 488labeled purified human pro-MMP-2 at 1 µg/mL or active MMP-2 at 1 µg/mL for 10 minutes. The cells were pelleted, washed once, resuspended in HBSS, and analyzed immediately with a FACSCalibur cell sorter equipped with a 488-nm argon laser and Cellquest software (BD Pharmingen, Bedford, MA).
Fluorescent Immunocytochemistry
To localize BSP, MMP-2, and integrin v
3 on individual cells, 1 mL containing 1 x 103 SW-579 cells was placed in each well of a two-well chamber slide and incubated at 37 °C for 24 hours. The cells were then washed with serum-free RPMI-1640 medium and incubated in this medium at 37 °C with no additions, with 100 nM BSP-KAE, or with 100 nM BSP for 24 hours. The cells were then washed and incubated at 37 °C with recombinant pro-MMP-2 (1 µg/mL per well) for 20 minutes. After three washes in PBS, the cells were fixed in absolute ethanol at 4 °C for 30 minutes, washed three times in PBS, and incubated in PBS with affinity-purified human anti-BSP polyclonal antibody (LF-84) and mouse anti-MMP-2 monoclonal antibody at the same time at 4 °C for 24 hours. The cells then were washed and incubated with Rhodamine Redcoupled AffiniPure goat anti-rabbit IgG and Cy2-coupled AffiniPure goat anti-mouse IgG secondary antibodies at room temperature for 30 minutes. The slides were detached from the chamber, washed three times with PBS, and immediately mounted in Vectashield mounting medium for fluorescence with DAPI for nuclear staining under a coverslip. The samples were analyzed with a fluorescence microscope that could simultaneously visualize both dye signals.
In Situ Hybridization
To generate strand-specific probes for in situ hybridization, a polymerase chain reactionamplified human MMP-2 cDNA fragment (317 bp) was subcloned into the BamHI site of pBluescript II KS vector. The oligonucleotides for amplification of the MMP-2specific probes were 5'-ATTAGGATCCGGTCACAGCTACTTCTTCAAG-3' (forward) and 5'-ATATGGATCCGCCTGGGAGGAGTACAG-3' (reverse). The BSP template was the full-length human BSP cDNA B6-5g (31). The human integrin v cDNA (1200 bp) insert originally cloned into the pUC12 vector was released with EcoRI and HindIII (35), and human integrin
3 cDNA (2275 bp) originally cloned into the pUC12 vector (36) was released with EcoRI. Both cDNA inserts were then subcloned in pBluescript II KS, a vector that contains the T3 and T7 RNA polymerase promoters for RNA probe synthesis. After the plasmids were linearized with the appropriate restriction enzymes, the probes were labeled with a digoxigenin-labeling mixture (1 mM ATP, 1 mM CTP, 1 mM GTP, 0.65 mM UTP, and 0.35 mM DIG-11-UTP [digoxigenin coupled to UTP at position 11], pH 7.5) to produce the specific digoxigenin-labeled single-stranded antisense and sense RNA fragments. In situ hybridization for thyroid papillary carcinoma serial sections was carried out with the InnoGenex in situ hybridization BCIP/NBT kit according to the manufacturer's instructions with minor modifications. Slides were deparaffinized in xylene and rehydrated through a graded ethanol series. After a 10-minute incubation in the kit's proteinase K solution, the slides were fixed in 1% formaldehyde for 10 minutes. Approximately 50 µL of hybridization buffer containing pre-titrated digoxigenin-labeled RNA probes (antisense or sense) were applied to each slide. The hybridization reaction included a 3-minute denaturation at 80 °C followed by overnight incubation at 37 °C. After hybridization, washes at room temperature consisted of rinsing with 2x PBS to remove the coverslip, followed by one 10-minute wash and two 5-minute washes in PBS. The sections were then treated with antibody-blocking solution (InnoGenex, product BS-1310-06) for 5 minutes at room temperature, and the blocking agent was gently removed. Biotinylated mouse anti-digoxigenin monoclonal antibody (InnoGenex, product AS-3000-06) was then applied to the sections for 5 minutes at 37 °C, washed for two 5-minute periods in PBS, and then incubated at 37 °C with alkaline phosphatase streptavidine conjugate (provided by the manufacturer) for 5 minutes. After washing twice with PBS, activation buffer was then applied to each section for 1 minute before incubating in BCIP/NBT substrate chromogen solution until satisfactory color reaction was observed (approximately 15 minutes). Sections were then counterstained with nuclear fast red, dehydrated through a graded series of alcohol and xylene, and mounted under a coverslip. Sections were photographed with an AxioCam MR-MRGrab camera imaging system (Carl Zeiss Vision, Munchen, Germany), which included an Axioplan2 microscope, an AxioCam MRm camera, and AxioVision 3.1 software.
Statistical Analysis
Data are the mean of quadruplicate determinations and its 95% confidence interval (CI). Each experiment was repeated at least two times. In each case, data from a single representative experiment are shown. Multiple comparisons were performed with a one-way analysis of variance followed by Dunnett's test for treatment versus control comparisons. Pairwise comparisons were carried out by performing a nonparametric MannWhitney U test. In each analysis, differences were considered statistically significant for P values less than .05. All statistical tests were two-sided.
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RESULTS |
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Recent reports (1420) that BSP is elevated in tumors and serum from patients with breast, prostate, lung, or thyroid cancers prompted us to investigate whether BSP has a role in the invasion of cancer cells. Invasiveness was measured with a modified Boyden chamber cell invasion assay. Treatment with BSP caused dose-dependent increases in the invasiveness of the breast cancer cell lines MDA-MB-231, MDA-MB-435S, and MCF-7; prostate cancer cell lines PC-3 and DU-145; lung cancer cell line NCI-H520; and thyroid cancer cell line SW-579. Results from a representative cell line for each cancer type are shown in Fig. 1. MDA-MB-231 cells showed a clear dose-response increase in their invasiveness through a Matrigel barrier, with a maximum increase of approximately 10-fold at 100 nM BSP (93.1 units [U; 1 U represents 1% of the maximum number of cells invaded], 95% CI = 86.6 to 99.6 U) compared with that of untreated cells (9.5 U, 95% CI = 6.8 to 12.2 U) (P<.001) (Fig. 1). MDA-MB-435S cells showed an approximately twofold increase at 100 nM BSP (84.7 U, 95% CI = 69.4 to 100.0 U) compared with that of untreated cells (43.7 U, 95% CI = 36.8 to 50.6 U) (P<.001). In addition, MCF-7 cells, which are usually not aggressive in Boyden chamber cell invasion assays, showed a statistically significant approximately ninefold increased invasiveness after treatment with 100 nM BSP (79.5 U, 95% CI = 59.0 to 100.0 U) compared with untreated cells (8.5 U, 95% CI = 3.0 to 14.0 U) (P<.001).
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The increase in the invasiveness of these cancer cell lines was specific to BSP, because the same dose range of osteopontin and DMP1, the two other members of the SIBLING family that can support cell attachment but cannot bind to MMP-2, did not increase the invasiveness of any of the cell lines tested (data not shown).
BSP-Enhanced Invasion, Integrin v
3, and MMP-2
The same invasiveness studies were performed with BSP-KAE, a recombinant BSP protein whose integrin-binding RGD sequence was replaced with the chemically similar but inactive tripeptide KAE. Treatment with BSP-KAE did not increase the invasiveness of any cell line that had previously responded to BSP compared with the invasiveness of untreated cells. Results from representative breast, prostate, thyroid, and lung cancer cell lines are shown in Fig. 2.
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In Vitro Interaction of BSP, MMP-2, and Integrin v
3
Because the BSP-enhanced invasion can be blocked by interfering with the activity of either integrin v
3 or MMP-2 and because BSP can form a complex with integrin
v
3 and a complex with MMP-2, we hypothesized that these three molecules form an RGD-dependent complex in which BSP acts as a bridge to link MMP-2 and integrin
v
3. We tested this hypothesis with immunoprecipitation experiments using purified components. Integrin
v
3 was incubated with immunoaffinity gel beads with covalently attached anti-integrin
v
3 monoclonal antibodies to allow integrin binding. The beads were washed to remove unattached integrins, and the washed beads were then incubated with buffer alone or buffer containing recombinant BSP or recombinant BSP-KAE. After washing to remove unbound proteins, beads were incubated with soluble active MMP-2 or inactive pro-MMP-2. The beads were washed again, and then the amount of bound MMP-2 activity was measured by use of gelatin zymogram electrophoresis. No MMP activity was detected when integrin
v
3-free beads were used, which showed that the immunoprecipitation assay had a very low background (data not shown). Beads with bound integrin
v
3 but no BSP bound a small but reproducible amount of pro-MMP-2 and an even smaller amount of active MMP-2 (Fig. 4, lane 2). This result confirms that of Brooks et al. (23) and suggests that some MMP-2 can bind directly to integrin
v
3. Addition of BSP-KAE, which lacks an active integrin-binding RGD sequence, did not increase the binding between integrin
v
3 and either MMP-2 or pro-MMP-2 (Fig. 4, lane 4). However, addition of BSP and then of MMP-2 (active MMP-2 or pro-MMP-2) to integrin
v
3-coated beads increased MMP activity associated with the beads, indicating that BSP stimulated the formation of a complex between integrin
v
3 and MMP-2, presumably by linking the two proteins (Fig. 4, lane 3).
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DISCUSSION |
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Because it is overexpressed in malignant tissues, BSP may play a role in the progression and invasion of a number of osteotropic cancers, including breast, prostate, lung, and thyroid cancers (1417,20). Addition of BSP stimulates the in vitro migration of breast cancer cells via a mechanism involving integrin v
3 (48,49). Strong and specific in vitro interactions (with nanomolar affinity) have been described (29) between three members of the SIBLING family (BSP, osteopontin, and DMP1) and specific MMPs (MMP-2, MMP-3, and MMP-9, respectively). Thus, the combination of BSP, MMP-2, and integrin
v
3 appears to play an important role in cancer cell invasion. This study demonstrated that BSP, but not osteopontin or DMP1, increased the Matrigel invasiveness of a large subset of breast, prostate, lung, and thyroid cancer cell lines. Addition of BSP-KAE, a recombinant form of BSP in which the RGD sequence was replaced with KAE, or addition of an antibody that blocks BSP binding to integrin
v
3 through RGD sequence blocked all BSP-enhanced invasive activity, suggesting that BSP acts through this integrin. The BSP-enhanced invasion by these cells was also inhibited by specific chemical inhibitors of MMP-2 and by an antibody for MMP-2. Formation of a complex containing BSP, integrin
v
3, and MMP-2 was demonstrated by immunoprecipitation experiments, immunofluorescence experiments, and flow cytometry. These results suggest that cells use BSP as a bridge to link MMP-2 to its cell surface receptor, integrin
v
3, which thereby increases their ability to invade basement membranes and other connective tissues. Fig. 8 shows BSP with an intact RGD bridging MMP-2 to its cell-surface receptor, integrin
v
3. When the integrin-binding RGD sequence is replaced with the chemically similar but inactive KAE sequence, MMP-2 may still bind to the BSP-KAE protein, but the complex between MMP-2 and BSP-KAE does not interact with cell-surface integrin.
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In conclusion, we have shown that recombinant BSP can enhance the invasiveness of some, but not all, breast, prostate, lung, and thyroid cancer cell lines in a modified Boyden chamber assay through formation of an RGD-dependent complex with MMP-2 and integrin v
3.
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NOTES |
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REFERENCES |
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1 Fisher LW, Torchia DA, Fohr B, Young MF, Fedarko NS. Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin. Biochem Biophys Res Commun 2001;280:4605.[CrossRef][ISI][Medline]
2 Fisher LW, Fedarko NS. Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins: special issue: 7th International Conference on the Chemistry and Biology of Mineralized Tissues. Connect Tissue Res 2003;44 Suppl 1:3340.[ISI][Medline]
3 Bianco P, Fisher LW, Young MF, Termine JD, Robey PG. Expression of bone sialoprotein (BSP) in developing human tissues. Calcif Tissue Int 1991;49:4216.[ISI][Medline]
4 MacDougall M. Refined mapping of the human dentin sialophosphoprotein (DSPP) gene within the critical dentinogenesis imperfecta type II and dentin dysplasia type II loci. Eur J Oral Sci 1998;106 Suppl 1:22733.[ISI][Medline]
5 Omigbodun A, Daiter E, Walinsky D, Fisher L, Young M, Hoyer J, et al. Regulated expression of osteopontin in human trophoblasts. Ann N Y Acad Sci 1995;760:3469.[ISI][Medline]
6 Hampel DJ, Sansome C, Romanov VI, Kowalski AJ, Denhardt DT, Goligorsky MS. Osteopontin traffic in hypoxic renal epithelial cells. Nephron Exp Nephrol 2003;94:e6676.[CrossRef][ISI][Medline]
7 Dhanireddy R, Senger D, Mukherjee BB, Mukherjee AB. Osteopontin in human milk from mothers of premature infants. Acta Paediatr 1993;82:8212.[ISI][Medline]
8 Gravallese EM. Osteopontin: a bridge between bone and the immune system. J Clin Invest 2003;112:1479.
9 Fedarko NS, Fohr B, Robey PG, Young MF, Fisher LW. Factor H binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack. J Biol Chem 2000;275:1666672.
10 Weber GF, Ashkar S, Glimcher MJ, Cantor H. Receptor-ligand interaction between CD44 and osteopontin (Eta-1). Science 1996;271:50912.[Abstract]
11 Jain A, Karadag A, Fohr B, Fisher LW, Fedarko NS. Three SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) enhance factor H's cofactor activity enabling MCP-like cellular evasion of complement-mediated attack. J Biol Chem 2002;277:137008.
12 Ue T, Yokozaki H, Kitadai Y, Yamamoto S, Yasui W, Ishikawa T, et al. Co-expression of osteopontin and CD44v9 in gastric cancer. Int J Cancer 1998;79:12732.[CrossRef][ISI][Medline]
13 Zohar R, Suzuki N, Suzuki K, Arora P, Glogauer M, McCulloch CA, et al. Intracellular osteopontin is an integral component of the CD44-ERM complex involved in cell migration. J Cell Physiol 2000;184:11830.[CrossRef][ISI][Medline]
14 Bellahcene A, Merville MP, Castronovo V. Expression of bone sialoprotein, a bone matrix protein, in human breast cancer. Cancer Res 1994;54:28236.[Abstract]
15 Waltregny D, Bellahcene A, Van Riet I, Fisher LW, Young M, Fernandez P, et al. Prognostic value of bone sialoprotein expression in clinically localized human prostate cancer. J Natl Cancer Inst 1998;90:10008.
16 Bellahcene A, Maloujahmoum N, Fisher LW, Pastorino H, Tagliabue E, Menard S, et al. Expression of bone sialoprotein in human lung cancer. Calcif Tissue Int 1997;61:1838.[CrossRef][ISI][Medline]
17 Bellahcene A, Albert V, Pollina L, Basolo F, Fisher LW, Castronovo V. Ectopic expression of bone sialoprotein in human thyroid cancer. Thyroid 1998;8:63741.[ISI][Medline]
18 Riminucci M, Corsi A, Peris K, Fisher LW, Chimenti S, Bianco P. Coexpression of bone sialoprotein (BSP) and the pivotal transcriptional regulator of osteogenesis, Cbfa1/Runx2, in malignant melanoma. Calcif Tissue Int 2003.
19 Bellahcene A, Menard S, Bufalino R, Moreau L, Castronovo V. Expression of bone sialoprotein in primary human breast cancer is associated with poor survival. Int J Cancer 1996;69:3503.[CrossRef][ISI][Medline]
20 Fedarko NS, Jain A, Karadag A, Van Eman MR, Fisher LW. Elevated serum bone sialoprotein and osteopontin in colon, breast, prostate, and lung cancer. Clin Cancer Res 2001;7:40606.
21 Matrisian LM. Cancer biology: extracellular proteinases in malignancy. Curr Biol 1999;9:R7768.[CrossRef][ISI][Medline]
22 Kleiner DE, Stetler-Stevenson WG. Matrix metalloproteinases and metastasis. Cancer Chemother Pharmacol 1999;43 Suppl:S4251.[CrossRef][ISI][Medline]
23 Brooks PC, Stromblad S, Sanders LC, von Schalscha TL, Aimes RT, Stetler-Stevenson WG, et al. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. Cell 1996;85:68393.[ISI][Medline]
24 Yu Q, Stamenkovic I. Localization of matrix metalloproteinase 9 to the cell surface provides a mechanism for CD44-mediated tumor invasion. Genes Dev 1999;13:3548.
25 Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992;69:1125.[ISI][Medline]
26 Aplin AE, Howe AK, Juliano RL. Cell adhesion molecules, signal transduction and cell growth. Curr Opin Cell Biol 1999;11:73744.[CrossRef][ISI][Medline]
27 Hood JD, Cheresh DA. Role of integrins in cell migration and invasion. Nature Rev Cancer 2002;2:91100.[CrossRef][ISI][Medline]
28 Gasparini G, Brooks PC, Biganzoli E, Vermeulen PB, Bonoldi E, Dirix LY, et al. Vascular integrin alpha(v)beta3: a new prognostic indicator in breast cancer. Clin Cancer Res 1998;4:262534.[Abstract]
29 Fedarko NS, Jain A, Karadag A, Fisher LW. Three small integrin binding ligand N-linked glycoproteins (SIBLINGs) bind and activate specific matrix metalloproteinases. FASEB J 2004;18:7346.
30 Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM, et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 1987;47:323945.[Abstract]
31 Fisher LW, McBride OW, Termine JD, Young MF. Human bone sialoprotein. Deduced protein sequence and chromosomal localization. J Biol Chem 1990;265:234751.
32 Kiefer MC, Bauer DM, Barr PJ. The cDNA and derived amino acid sequence for human osteopontin. Nucleic Acids Res 1989;17:3306.[ISI][Medline]
33 Hirst KL, Ibaraki-O'Connor K, Young MF, Dixon MJ. Cloning and expression analysis of the bovine dentin matrix acidic phosphoprotein gene. J Dent Res 1997;76:75460.[Abstract]
34 Becker TC, Noel RJ, Coats WS, Gomez-Foix AM, Alam T, Gerard RD, et al. Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol 1994;43:16189.[ISI][Medline]
35 Fitzgerald LA, Poncz M, Steiner B, Rall SC Jr, Bennett JS, Phillips DR. Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor alpha-subunits and platelet glycoprotein IIb. Biochemistry 1987;26:815865.[ISI][Medline]
36 Fitzgerald LA, Steiner B, Rall SC Jr, Lo SS, Phillips DR. Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. Identity with platelet glycoprotein IIIa and similarity to "integrin". J Biol Chem 1987;262:39369.
37 Oldberg A, Franzen A, Heinegard D, Pierschbacher M, Ruoslahti E. Identification of a bone sialoprotein receptor in osteosarcoma cells. J Biol Chem 1988;263:194336.
38 Collier IE, Wilhelm SM, Eisen AZ, Marmer BL, Grant GA, Seltzer JL, et al. H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen. J Biol Chem 1988;263:657987.
39 Pyke C, Ralfkiaer E, Huhtala P, Hurskainen T, Dano K, Tryggvason K. Localization of messenger RNA for Mr 72,000 and 92,000 type IV collagenases in human skin cancers by in situ hybridization. Cancer Res 1992;52:133641.[Abstract]
40 Monteagudo C, Merino MJ, San-Juan J, Liotta LA, Stetler-Stevenson WG. Immunohistochemical distribution of type IV collagenase in normal, benign, and malignant breast tissue. Am J Pathol 1990;136:58592.[Abstract]
41 Lochter A, Galosy S, Muschler J, Freedman N, Werb Z, Bissell MJ. Matrix metalloproteinase stromelysin-1 triggers a cascade of molecular alterations that leads to stable epithelial-to-mesenchymal conversion and a premalignant phenotype in mammary epithelial cells. J Cell Biol 1997;139:186172.
42 Belien AT, Paganetti PA, Schwab ME. Membrane-type 1 matrix metalloprotease (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter. J Cell Biol 1999;144:37384.
43 Ala-Aho R, Johansson N, Baker AH, Kahari VM. Expression of collagenase-3 (MMP-13) enhances invasion of human fibrosarcoma HT-1080 cells. Int J Cancer 2002;97:2839.[CrossRef][ISI][Medline]
44 Hotary K, Allen E, Punturieri A, Yana I, Weiss SJ. Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3. J Cell Biol 2000;149:130923.
45 Klein CE, Steinmayer T, Kaufmann D, Weber L, Brocker EB. Identification of a melanoma progression antigen as integrin VLA-2. J Invest Dermatol 1991;96:2814.[Abstract]
46 Deryugina EI, Bourdon MA, Luo GX, Reisfeld RA, Strongin A. Matrix metalloproteinase-2 activation modulates glioma cell migration. J Cell Sci 1997;110:247382.
47 Deryugina EI, Bourdon MA, Jungwirth K, Smith JW, Strongin AY. Functional activation of integrin alpha V beta 3 in tumor cells expressing membrane-type 1 matrix metalloproteinase. Int J Cancer 2000;86:1523.[ISI][Medline]
48 Sung V, Stubbs JT 3rd, Fisher L, Aaron AD, Thompson EW. Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the alpha(v)beta3 and alpha(v)beta5 integrins. J Cell Physiol 1998;176:48294.[CrossRef][ISI][Medline]
49 Byzova TV, Kim W, Midura RJ, Plow EF. Activation of integrin alpha(V)beta(3) regulates cell adhesion and migration to bone sialoprotein. Exp Cell Res 2000;254:299308.[CrossRef][ISI][Medline]
50 Xuan JW, Hota C, Shigeyama Y, D'Errico JA, Somerman MJ, Chambers AF. Site-directed mutagenesis of the arginine-glycine-aspartic acid sequence in osteopontin destroys cell adhesion and migration functions. J Cell Biochem 1995;57:68090.[ISI][Medline]
51 Chambers AF, Wilson SM, Kerkvliet N, O'Malley FP, Harris JF, Casson AG. Osteopontin expression in lung cancer. Lung Cancer 1996;15:31123.[CrossRef][ISI][Medline]
52 Tuck AB, O'Malley FP, Singhal H, Harris JF, Tonkin KS, Kerkvliet N, et al. Osteopontin expression in a group of lymph node negative breast cancer patients. Int J Cancer 1998;79:5028.[CrossRef][ISI][Medline]
53 Chaplet M, De Leval L, Waltregny D, Detry C, Fornaciari G, Bevilacqua G, et al. Dentin matrix protein 1 is expressed in human lung cancer. J Bone Miner Res 2003;18:150612.[ISI][Medline]
54 Albini A. Tumor and endothelial cell invasion of basement membranes. The matrigel chemoinvasion assay as a tool for dissecting molecular mechanisms. Pathol Oncol Res 1998;4:23041.[Medline]
Manuscript received September 3, 2003; revised April 14, 2004; accepted April 30, 2004.
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