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[Cancer Research 63, 256-262, January 2003]
© 2003 American Association for Cancer Research

Virology

Regulation of Matrix Metalloproteinase-1 by Epstein-Barr Virus Proteins1

Jean Lu, Huey-Huey Chua, Shao-Yin Chen, Jen-Yang Chen and Ching-Hwa Tsai2

Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix metalloproteinases (MMPs) play crucial roles in tumor progression. To investigate the roles of MMPs in the progression of nasopharyngeal carcinoma (NPC), the expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-12, MMP-13, MMP-14, and MMP-19 was explored by microarray assay. Among them, MMP-1 was significantly up-regulated in NPC biopsies. These results were confirmed further by real-time quantitative PCR in additional NPC biopsies and comparison with normal tissues and other head and neck cancers. Moreover, the use of RNA from different cellular constituents of NPC biopsies revealed that MMP-1 was detected predominantly in epithelial cells. Immunohistochemical staining of paraffin-fixed NPC sections confirmed that MMP-1 protein was expressed in the epithelial tumor cells. Because EBV is strongly associated with NPC formation, we sought a correlation between viral gene expression and MMP-1 up-regulation. The results showed clearly that the amounts of transcripts, proteins, and enzyme activities of MMP-1 were increased in cells expressing EBV proteins, LMP1 (latent membrane protein 1) and Zta (Z transactivator; also named as BZLF1 or ZEBRA) but not EBNA-1 (EBV nuclear antigen-1). Additionally, the mobility of LMP1 and Zta transfectants was increased in scrape-wound migration assays. The invasiveness and ability to survive in a three-dimensional collagen gel also were enhanced in LMP1- and Zta-expressing cells. Furthermore, anti-MMP-1 antibody and peptide inhibitors could block the invasiveness and survival properties of LMP1 and Zta transfectants, suggesting a real contribution of MMP-1 to cell mobility and survival. Taken together, our data show that the viral LMP1 and Zta proteins regulate the expression and activity of MMP-1, and thereby confer the invasive properties of the cells. This study presents the first evidence that viral proteins are capable of regulating MMP-1 and also provides clues for the role of EBV in NPC progression.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Head and neck cancers include laryngeal cancer, oropharynx cancer, hypopharynx, and NPC3 . Interestingly, NPC is distinguished from other human head and neck carcinomas by an unusually high metastatic rate, strong association with EBV and early occurrence in middle age (1, 2, 3) . However, the underlying mechanisms for these unique properties are unclear. Accumulated episerological data and pathological evidence have established a strong association between EBV and NPC development (1 , 4 , 5) . EBV is detected early in a monoclonal premaligant lesion of NPC but not in the surrounding polyclonal epithelial cells (6 , 7) . Additionally, >95% of NPC biopsies contain EBV transcripts or proteins (4) . Antibody titers against EBV proteins, as well as the copy numbers of viral genome in the sera, are not only elevated in NPC patients but also correlate with disease progression and remission (8, 9, 10, 11) . Furthermore, the presence of EBV transcripts or proteins such as EBNA-1, LMP1, LMP2, and Zta in tumor biopsies suggests the involvement of EBV in NPC formation (12, 13, 14, 15) .

The oncogenic potential of EBV has been proven both in vitro and in vivo (15) . EBV can immortalize primary primate B cells and epithelial cells in vitro (15, 16, 17) . In vivo, EBV induces B-cell lymphomas and promotes epithelial tumor cell growth in animal models (18 , 19) . Several EBV genes have been shown to be important for the oncogenic potential of the virus (15) . Expression of EBNA-1 protein can induce B-cell lymphomas in transgenic mice and enhances the metastasis of NPC cells in immunodeficient mice (20 , 21) . Transgenic mice expressing LMP1 may develop B-cell lymphomas or epidermal hyperplasia (22 , 23) . Zta, a crucial viral transactivator, can modulate viral and cellular gene expression through its AP-1 site binding activity or by dimerizing with other proteins such as p53, nuclear factor {kappa}B, and retinoid X receptor (24) . All of these pathways have the potential to regulate MMPs, which play crucial roles in tumor progression (25) .

The MMP family is involved in ECM degradation during physiological normal tissue remodeling and in pathological tumor progression (26) . ECM is the supporting tissue beneath epithelial cells and is composed mainly of collagens, proteoglycans, and laminins (27) . In addition to this supporting function, certain ECM components such as collagens can block inappropriate proliferation and prevent invasion of epithelial cells under normal circumstances (28 , 29) . However, during tissue remodeling or tumor formation, ECM is digested by corresponding MMPs, resulting in the release of nutrition, cytokines, and matrix-binding growth factors (27 , 30) . Under such circumstances, the physical barrier is removed and supporting molecules are released, thus the cells gain access to hyperplasia, angiogenesis, migration, and invasion (26 , 30) . Therefore, MMPs are important for many steps of tumor development (26 , 30) .

MMPs currently are classified into four groups, according to their substrate preference and subcellular localization: interstitial collagenases; gelatinases; stromelysins; and membrane-type MMPs (26 , 30 , 31) . To test the roles of MMPs in the development of NPC, microarray assay was used to screen the expression profiles of MMPs in NPC and control tissues. We found that MMP-1 is remarkably highly expressed in NPC biopsies and consecutive experiments revealed the effects of various EBV products on the expression of MMP-1. The effects and biological activities of EBV proteins and MMP-1 in NPC are discussed.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Biopsy Samples.
Twenty-nine cases of primary NPC and fifteen cases of other head and neck tumors, which had been validated pathologically, were obtained from the Department of Otolaryngology, National Taiwan University Hospital. As normal biopsy controls, thirteen samples of nasopharyngeal tissues, containing nonmalignant epithelium and lymphocytes, were obtained from patients with LH. Additional normal controls, seven samples of adenoid tissues, isolated from the same locations and with similar cellular constituents as LH, were obtained from pediatric patients.

RNA Extraction and cDNA Synthesis.
Total cellular RNA was extracted from biopsy tissues or cell lines using the TRIzol reagent (Life Technologies, Inc., Grand Island, NY) or REzol reagent (Protech, Taiwan, Republic of China), according to the manufacturer’s instructions. Twenty µl of cDNA was synthesized using 5 µg of total RNA as the template and random hexamers as primers, as described in the manufacturer’s protocol of reverse transcriptase (Life Technologies, Inc.). In Q-PCR analysis, the RNA was treated with DNase (Life Technologies, Inc.) before RT to avoid the contamination of DNA.

Microarray Assay.
A total of 1056 cDNA clones constructed in the pBluescript plasmid was amplified using the T3 (5'-AATTAACCCTCACTAAAGGG-3') and T7 (5'-TAATACGACTCACTATAGGG-3') primers. The PCR-amplified cDNA fragments were then blotted onto a membrane for a modified colorimetric catalyzed reporter deposition-microarray assay (32) . Briefly, total cellular RNA was extracted and reacted with oligo (dT)12–18, biotin-16-dUTP (Roche, Mannheim, Germany), and enzyme (Life Technologies, Inc.) for cDNA preparation. The membrane was prehybridized with hybridization solution (5x SSC, 0.1% N-lauroyl sarcosine, 0.02% SDS, 1% blocking reagent, and 50 µg/ml salmon sperm DNA) at 60°C for 1.5 h, and then incubated with 16 µl of hybridization solution containing cDNA probes, 5 µg of human Cot-1 DNA, and 5 µg oligo (dA)10 at 63°C overnight. After washing and blocking, the blot was reacted with streptavidin-peroxide (Roche) and amplification solution [0.1 M sodium tetraborate (pH 8.5), 0.0035% H2O2 and 20 µg/ml biotin-conjugated tyramine] and then streptavidin-ß-galactosidase (Life Technologies, Inc.). The blot was visualized after adding the 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside substrate and was scanned digitally. The results were analyzed by Image Quant 5.0 program (Molecular Dynamics, Sunnyvale, CA) in Dr. C-K. Chou’s laboratory at National Yang Ming University (Taiwan, Republic of China).

Q-PCR.
The Q-PCR reaction conditions were specified by the manufacturer (Perkin-Elmer, Foster, CA), except that the Taq polymerase was replaced by Dynazyme II (Finnzymes, Oy, Finland). Generally, the 25-µl reaction mixtures, containing 1 µl of cDNA, 0.3 mM deoxynucleotide triphosphate mix (dUTP, dATP, dTTP, dGTP), 0.2 µM primers (MMP-1: 5'-CTTGCACTGAGAAAGAAGACAAAGG-3' and 5'-ACACCCCAGAACAGCAGCA-3'; GAPDH: 5'-GAAGGTGAAGGTCGGAGTC-3' and 5'-GAAGATGGTGATGGGATTTC-3'), 0.125 µM probe (MMP-1: 5'-CAGTATGCACAGCTTTCCTCCACTGCTG-3'; GAPDH: 5'-CAAGCTTCCCGTTCTCAGCC-3'), 1x buffer [10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, and 0.1% Triton X-100]), 1 unit of uracil-DNA Glycosylase (MBI, Hanover, MD), and 1 unit of Dynazyme II were subjected to one unique cycle (50°C for 2 min, 10 min at 95°C) and then amplified for 40 cycles (95°C for 15 s, 60°C for 1 min). A reaction without reverse transcriptase served as a control to rule out the possibility of genomic DNA contamination. The relative amounts of MMP-1 transcripts were standardized against GAPDH and calculated as described previously (33) . Comparisons between each tested group were analyzed statistically using the F test and unpaired Student’s t test with the SAS program.

Isolation of NPC Cell Subpopulations.
Three major cell compartments of NPC, epithelial cells, CD4+ T cells, and CD8+ T cells were purified from seven fresh NPC tissue samples using specific antibody-coated paramagnetic beads (Dynal, Oslo, Norway; Ref. 34 ). The purity of each isolated subpopulation was determined by immunoflow cytometry and reached >95% that was shown previously (34) .

Immunohistochemical Assay.
Paraffin sections of NPC biopsies were deparaffinized sequentially in xylene and ethanol. The staining procedure using anti-MMP-1 antibody (0.1 µg/ml clone 41–1E5; Chemicon, Temecula, CA) or mouse immunoglobulin (Dako) was described previously (34) .

Cell Culture.
The immortalized primary human keratinocyte cell line, RHEK-1, was maintained in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (35) . The RHEK-1 transfectants used in this study, RHEK-EBNA-1, RHEK-LMP1, and RHEK-Z2 and RHEK-Z3, express EBNA-1, LMP1, and Zta, respectively (36 , 37) . For detection of MMP-1 transcripts, proteins, or activities, cells were starved in DMEM without fetal bovine serum for 24 h before 72 h of 50 nM collagen I (Life Technologies, Inc.) treatment.

MMP-1 Protein Assay.
To measure the total amount of MMP-1 protein, an ELISA assay was performed with 200 µl of tested culture supernatants and MMP-1 protein standards according to the manufacturer’s instructions (Amersham Pharmacia, Freiburg, Germany).

MMP-1 Activity Assay.
A specific MMP-1 proteolytic activity was assayed according to the manufacturer’s procedure using 200 µl of MMP-1 protein standards and tested culture supernatants (Amersham Pharmacia; Ref. 38 ).

Scrape-Wound Migration Assay.
Confluent cells for assay were scraped using a sterilized tip. The scraped area was observed and photographed using an inverted photomicroscope (Axiovert 10; Zeiss, Göttingen, Germany).

Three-Dimensional Collagen Gel Assay.
Cells (104 cells/10 µl) were resuspended in collagen gel mixture [70 µl of 3 mg/ml rat tail collagen I, 9 µl of 10x DMEM, 2 µl of 0.2 M HEPES (pH 7.3), 5 µl H2O, with the adjusted pH ~7.4]. Then the cells were seeded into a 96-well microtiter plate, and the gel was solidified for 30 min in a 37°C incubator with 5% CO2. Then, 100 µl of 1x DMEM containing 1% of FCS were added. For the inhibition assay, MMP-1-neutralizing antibody (Oncogene, 0.1 µg/ml) or MMP-blocking peptides (5 µM; Chemicon) were added to both the collagen gel and culture medium. After 7 or 14 days of incubation, the phenotype were observed and photographed using an inverted photomicroscope (Axiovert 10; Zeiss).

WST-1 Assay.
The experiment was carried out by adding 20 µl of WST-1 reagent (Roche) to the samples from collagen gel assay and incubating them in a 37°C incubator for 3 h. The absorbances of the samples were measured at absorbance 450 nm, with reference at 650 nm.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Investigation of Expression Profile of MMPs in NPC.
To explore the roles of MMPs in NPC, the expression profile of a panel of MMPs was screened in NPC biopsies by cDNA microarray because many MMPs are regulated at the transcriptional level (25) . Human biopsy samples, including LH tissues, adenoid tissues, other head and neck cancers, and NPC biopsies were used to generate probes to hybridize to the array membrane. Data from these microarrays indicated that of all of the MMPs, MMP-1 is the most significantly up-regulated one in NPC biopsies in comparison to LH tissues, adenoid tissues, and other head and neck cancers (Fig. 1)Citation . Moreover, the efficacy of this assay was shown by the detection of up-regulated IL-1ß and DDR2 expression in NPC, which are consistent with previous studies (data not shown; Refs. 34 , 37 ).



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  		Fig. 1. Examination of expression profiles of MMPs by microarray assay of NPC and control samples. Total cellular RNAs extracted from biopsy samples were labeled with biotin and subjected to microarray assay as described in "Materials and Methods." Hybridization signals are shown in the figure. Four kinds of biopsies, LH, adenoid, NPC, and other head and neck cancers were used in this assay. The source of cDNA on the microarray blots is indicated under each lane. The intensities of individual signals were quantified and labeled beneath each clone, and the intensity of each signal reflected the amount of each cDNA in the test samples. GAPDH was the positive control for standardizing the amounts of RNA detected. RCA (rubisco activase precursor), a plant gene, was served as a negative control for this hybridization assay.

 
To verify that MMP-1 is truly up-regulated in NPC, Q-PCR analysis was performed to measure the MMP-1 transcripts in 29 cases of NPC, 13 cases of LH, 7 cases of adenoid tissues, and 15 cases of other head and neck cancers. As shown in Fig. 2Citation , expression of MMP-1 was increased significantly in NPC ~124-, 27-, and 9-fold compared with LH, adenoid, and other head and neck cancers, respectively (t test; P < 0.001, P < 0.005, P < 0.05).



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  		Fig. 2. Estimate the amounts of MMP-1 transcripts in LH, adenoid, other head and neck cancers, and NPC biopsies. Q-PCR was performed to detect the expression of MMP-1 transcripts in 13 cases of LH, 7 cases of adenoid tissues, 15 cases of other head and neck cancers, and 29 cases of NPC. The RT served as a control to rule out the possibility of contamination with genomic DNA. GAPDH reaction was used as an internal control to standardize the total amount of input RNA. The Y axis shows the relative amounts of MMP-1 transcripts after correction by GAPDH and RT reaction. Each black dot represents the mean of triplicate tests in each case. Each test was carried out twice independently. The horizontal line among the dots indicates the mean of each group.

 
Detection of MMP-1 Transcripts and Proteins in NPC Biopsies.
Another pathological feature of NPC is the heavy infiltration of lymphocytes (5) . It would be of interest to ask whether the up-regulated MMP-1 originates from epithelial cells or lymphocytes. To address this issue, epithelial cell, CD4+ T cell, and CD8+ T cell populations were isolated using antibody-coated paramagnetic beads (34) . By Q-PCR analysis, MMP-1 transcripts were mostly found in the epithelial cells, with only one exception in T lymphocytes, among seven cases of NPC (Fig. 3)Citation . These data indicated that MMP-1 was expressed predominantly by the epithelial cells in NPC biopsies.



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  		Fig. 3. Detection of MMP-1 transcripts in the cellular subpopulations of NPC biopsies. Subpopulations of epithelial cells, CD4+ T cells, and CD8+ T cells were fractionated from NPC biopsies using antibody-coated, paramagnetic beads. Total RNA was extracted from fractionated cells and used for assay. The amounts of MMP-1 transcripts were measured by Q-PCR in epithelial cells (top panel), CD4+ T cells (middle panel), and CD8+ T cells (lower panel). The Y axis shows the relative amounts of MMP-1 transcripts after correction by GAPDH. The X axis indicates the assigned number of each biopsy, and the error bars indicate the SD of triplicate tests.

 
To confirm the Q-PCR results and to determine whether the MMP-1 transcripts were translated, the expression of MMP-1 proteins was examined by immunohistochemistry in 15 NPC biopsies. MMP-1 proteins were found predominantly in the epithelial tumor cells (Fig. 4BCitation and data not shown). Meanwhile, an irrelevant but same species immunoglobulin was used as an antibody negative control. No positive signals were detected (Fig. 4A)Citation . Furthermore, the specificity of the immunohistochemical reaction was demonstrated by a blocking assay, MMP-1 staining was abrogated when the MMP-1 antibody was preincubated with MMP-1 protein (Fig. 4C)Citation . Consistent with the results of Q-PCR, MMP-1 proteins were detected mostly in the epithelial cells. Therefore, we found that MMP-1 protein, as well as RNA, clearly were expressed in NPC biopsies.



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  		Fig. 4. Location of MMP-1 proteins in NPC by immunohistochemical staining. Paraffin-embedded NPC sections were incubated with the irrelevant immunoglobulin antibody control (A), anti-MMP-1 antibody (B), or MMP-1 antibody prereacted with MMP-1 protein (C). The samples were developed using a Histostain-Plus kit (Zymed) as described in "Materials and Methods," and counterstained with hematoxylin. The positive dark-brown signals were detected mostly in the cytoplasm of the tumor cells (T). The counterstaining was purple. Original magnification, x400.

 
Up-regulation of MMP-1 RNA, Proteins, and Activities by LMP1 and Zta Proteins.
It is well known that EBV is the most common factor in NPC formation. To elucidate whether viral proteins could regulate MMP-1, the effects of EBV EBNA-1, LMP1, and Zta proteins on MMP-1 expression were investigated. The expression of each EBV protein was confirmed by Western blot analysis (Refs. 36 , 37 and data not shown). MMP-1 transcripts were found by Q-PCR analysis to be increased in LMP1- and two Zta-expressing cells, Z2 and Z3, but neither in EBNA-1 transfectants nor in vector controls (Fig. 5A)Citation . The amounts of MMP-1 transcripts in LMP1, Z3, and Z2 cells increased ~47, 8, and 14 times, respectively, over the vector controls (Fig. 5A)Citation . Consistently, the results of conventional PCR using other MMP-1 primers confirmed the up-regulation of MMP-1 transcripts (data not shown). In addition, the total MMP-1 protein in the culture supernatants of EBNA-1-, LMP1-, and Zta-expressing cells was determined by ELISA. It is worth noting that the levels of MMP-1 protein were increased only in LMP1 and Zta transfectants (Fig. 5B)Citation . The MMP-1 enzyme activities also were augmented in LMP1 and Zta transfectants, according to a modified ELISA test, which detects MMP-1 enzyme activity specifically (Fig. 5CCitation ; Ref. 38 ). In summary, MMP-1 was up-regulated at the levels of RNA, protein, and enzyme activity in the presence of LMP1 or Zta protein, and the secreted MMP-1 proteins in the culture supernatant have enzyme activity. This suggests that LMP1 and Zta transfectants may secrete MMP-1 to digest the ECM and enhance mobility.



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  		Fig. 5. Up-regulation of MMP-1 in LMP1- and Zta-expressing cells. The amounts of transcripts, proteins, and enzyme activities of MMP-1 were evaluated in EBNA-1 (Lane EBNA1), LMP1 (Lane LMP1), or Zta (Lanes Z3, Z2) transfectants and vector controls (Lane V). Each transfectant and the corresponding vector control were tested at the same time. The error bars indicate the SD of triplicate tests. A, measurement of MMP-1 transcripts by Q-PCR. Total RNA was extracted from each cell clone. The Y axis shows the relative amounts of MMP-1 transcripts after correction by GAPDH and RT experiments. B, quantification of MMP-1 protein by ELISA. Sandwich ELISA was performed to determine the amount of secreted MMP-1 protein in culture supernatant. The MMP-1 protein concentrations of tested cells were calculated from the standard curve and are shown on the Y axis. C, measurement of MMP-1 enzyme activity. The enzyme activity of MMP-1 in the culture supernatants of tested cells was detected using a modified ELISA, as described in "Materials and Methods." This assay detects only the activity of MMP-1 enzyme. The MMP-1 activities were calculated from a standard curve, plotted using MMP-1 protein standards.

 
Demonstration of Mobility and Survival of LMP1- and Zta-expressing Cells.
Scrape-wound migration and three-dimensional collagen gel assays were carried out to address the biological function elicited by MMP-1 in LMP1- and Zta-expressing cells. The mobility of LMP-1 and Zta transfectants were increased in scrape-wound assay compared with the vector control cells (Fig. 6)Citation . Additionally, in the three-dimensional collagen gel assay, EBNA-1-expressing cells exhibited a spherical shape in the gel, similar to the vector control cells (Fig. 7, A, D, and E)Citation . On the other hand, both the LMP1- and Zta-expressing cells could extend into the collagen gel and exhibit a spindle-like shape in this three-dimensional environment (Fig. 7, B, C, and F)Citation . This observation suggested that LMP1- and Zta-expressing cells, but not the vector controls or EBNA-1 transfectants, could invade the type I collagen gel. Moreover, this invasiveness could be hampered in LMP1- and Zta-expressing cells by the presence of anti-MMP-1 antibody or MMP-1 peptide inhibitor but not by an irrelevant control antibody (Fig. 7, G, H, and ICitation ; data not shown). In addition, the survival rates of these cells were evaluated by WST-1 assay. The LMP1- and Zta- expressing cells, but not the vector controls, could survive in the collagen gel (Fig. 8)Citation . Interestingly, anti-MMP-1 antibody or MMP inhibitor could reverse the MMP-1-dependent survivability (Fig. 8Citation and data not shown). Thus, up-regulation of MMP-1 was required for the invasiveness and survival of the transfectants.



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  		Fig. 6. Detection of the mobility of LMP1- and Zta-expressing cells in scrape-wound migration assay. Confluent vector control cells (V), LMP1 transfectants (LMP1), and Zta transfectants (Z2, Z3) were scraped by tip. After 24 h of incubation, the cells were observed and photographed. Original magnification, x100.

 


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  		Fig. 7. Demonstration of the invasion ability of LMP1- and Zta-expressing cells in three-dimensional collagen gel. Various clones (1 x 104 cells) include Zta vector (A), Zta-transfectant Z3 (B), Zta-transfectant Z2 (C), EBNA-1 (D), LMP1 vector (E), and LMP1 (F) were embedded in a type-I collagen gel. After 7 days incubation, the invasion phenotypes were observed and are highlighted by arrows. To evaluate the role of MMP-1 activities in this invasion behavior, MMP-1 neutralizing antibody (H) or MMP blocking peptides (I) were incubated with LMP1 transfectants in the collagen gel assay. Irrelevant mouse immunoglobulin served as the negative antibody control (G). Original magnification, x200.

 


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  		Fig. 8. Survivability of LMP1-expressing cells in collagen gel. WST-1 assay was used to assess the survival of vector-transfected cells (V), LMP1 transfectants (LMP1), Zta transfectants (Z2, Z3), with or without the presence of immunoglobulin control (Ig), anti-MMP-1 antibody (anti-MMP), or MMP blocking peptides (MMP inhibitor), and WST-1 reagent was added to the sample of collagen assay as described previously. The absorbencies of the samples are shown on the Y axis. The error bars indicate the SD of triplicate tests.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The expression of MMPs, which can facilitate the processes of cell proliferation, angiogenesis, invasion, and metastasis, is now regarded as a crucial feature of tumor progression (26) . On the basis of the fact that the key regulation of MMPs is at the transcriptional level (25) , the expression profile of MMPs in NPC was examined by microarray assay. Among the eight MMPs examined, MMP-1 was most significantly up-regulated in NPC (Fig. 1)Citation . This up-regulation was confirmed by a Q-PCR assay, which compared the amounts of MMP-1 transcripts in NPC with those in LH, adenoid, and other head and neck biopsies (all P < 0.05; Fig. 2Citation ). This is the first evidence of MMP-1 expression in NPC.

By itself, MMP-1 is suggested to be involved in several stages of tumor progression. After degrading the growth factor binding proteins, MMP-1 can activate insulin-like growth factor, which has been shown to enhance cell propagation (39) . In vivo, expression of MMP-1 in a transgenic mouse leads to epidermal hyperplasia and increases susceptibility to tumor formation (40) . MMP-1 can promote the migration of endothelial cells during angiogenesis (41) . Furthermore, MMP-1 is required for keratinocyte migration on a type I collagen matrix and can promote MMP-2 activation at the posttranslational level (42 , 43) .

Because of the complex composition of the ECM and the substrate specificity of MMPs, cooperation between different types of MMP is critical in tumor progression (44 , 45) . After decomposition of the basement membrane by gelatinases MMP-2 or MMP-9 during the initial invasive step, the interstitial collagenases such as MMP-1 exert their protease activities by degrading the exposed interstitial connective tissues (31) . Then the tumor cells are able to grow by releasing matrix-bound growth factors and cytokines in the interstitial tissues and to metastasize by gaining access to the proximity of the vascular or lymphatic systems. MMP-1, the only interstitial collagenase detected in NPC in our study (Fig. 1)Citation , can degrade the major interstitial collagens, including collagen types I, II, and III (31) , thus facilitating tumor proliferation and migration. Because NPCs expressed more MMP-1 than other head and neck cancers, MMP-1 may contribute to the unique high metastatic rate of NPC.

From our findings and others discovered in the past, MMP-1, MMP-2, and MMP-9 were detected in NPC tissues (Fig. 1Citation and 2Citation ; Ref. 46 ). Functionally, MMP-1, MMP-2, and MMP-9 can degrade different types of collagens (26) . Also, they can enhance the protease activities of one another (43 , 47) . The collaborative network of MMP-1 with MMP-2 or MMP-9 might provide a profound contribution to NPC progression, which may distinguish NPC from other head and neck carcinomas.

In NPC, most of the MMP-1 and EBV transcripts and proteins were detected in the epithelial tumor cells (Figs. 3Citation and 4Citation ; Ref. 15 ). However, MMP-1 also was expressed in T-lymphocytes in one NPC case (Fig. 3)Citation . In studies of other human carcinomas, secretion of MMPs from normal stromal cells, but not from tumor cells, also could enhance tumor progression (26 , 30 , 31) . Thus, MMP-1 expressed in infiltrating T cells in NPC probably could promote migration or growth of the epithelial tumor cells.

Interestingly, MMP-1 transcripts were increased in LMP1- and Zta-expressing cells but not in EBNA-1 transfectants or vector controls (Fig. 5A)Citation . Furthermore, MMP-1 protein and MMP-1 enzyme activity were increased in the culture supernatants of LMP1- and Zta-expressing cells (Fig. 5, B and C)Citation . The expression of MMP-1 promotes the mobility and survival of these cells (Figs. 7Citation and 8Citation ). To our knowledge, this report first demonstrated that MMP-1 could be up-regulated by viral gene products. Additionally, as shown by the blockage of MMP-1 activity (Figs. 7Citation and 8Citation ; data not shown), it provides the first evidence of direct contribution of MMPs to cell survival and invasion in EBV proteins expressing cells.

Usually, only low levels of MMP-1 transcripts can be detected in resting cells, and the amounts are increased after stimulation by cytokines or growth factors such as IL-1{alpha}, IL-1ß, epidermal growth factor, and vascular endothelial growth factor, mediated by transcriptional activators such as AP-1, STAT, and Ets (25) . All of these activators might be regulated by LMP1 through direct or indirect pathways, and all of them were displayed in NPC biopsies (25 , 34 , 48, 49, 50, 51, 52, 53, 54, 55) . In addition to up-regulated MMP-1 in our study, LMP1 protein can up-regulate MMP-9 (46 , 56) . Therefore, the up-regulation of more than one MMP proteins by LMP1 may contribute to NPC progression.

In our previous study, Zta was shown to activate the MMP-1 upstream tyrosine kinase DDR2, also designated as TRK-related tyrosine kinase (37 , 57) . Both DDR2 and MMP-1 were found to be elevated in NPC (Fig. 2Citation ; Ref. 37 ). Therefore, Zta may transactivate MMP-1 indirectly through DDR2 or directly by binding to the AP-1 sites in MMP-1 promoter (24 , 58 , 59) . Interestingly, in NPC biopsies, the amounts of MMP-1 transcripts were correlated with Zta transcripts (correlation coefficient: 0.76; P <0.001 by the F test; data not shown). Therefore, Zta may up-regulate MMP-1 in NPC biopsies.

What is the advantage for EBV to up-regulate MMP-1 expression? It is hypothesized that activation of MMPs by EBV, which probably demolish the matrix barrier of tissues, may also benefit the spread of virus or virus-infected cells in the latent stage or lytic cycle. Although the major sites of EBV persistence are the nasopharyngeal region and lymphoid system, EBV has been detected in pathological tissues from various organs, including stomach, muscle, lung, breast, and the central nervous system (3 , 60 , 61) . Therefore, the induction of MMPs may facilitate the passage of EBV or EBV-infected cells through barriers such as the ECM or the blood-brain barrier. Supporting evidence comes from the fact that MMPs have been proposed to alter the blood-brain barrier and to facilitate the spread of HIV and human T-lymphotropic virus type I into the central nervous system (62 , 63) . Thus, the relationship in vivo between EBV spread and MMPs remains an interesting question awaiting additional research.

On the basis of these observations, developing specific MMPs or EB viral inhibitors may ameliorate the severity of EBV-associated diseases. For example, aspirin can block the invasiveness triggered by LMP1, which may act though inhibition of nuclear factor {kappa}B, AP-1 binding activity, and MMP-9 promoter activity (64) . In this study, we demonstrated that MMP-1 antibody or blocking peptide could prevent the invasion or survival of LMP1- and Zta-expressing cells in type I collagen (Figs. 6Citation 7Citation 8Citation and data not shown). Therefore, blockage of MMPs, in combination with other chemotherapeutic or antiviral agents, may inhibit or prevent the progression of NPC and other EBV-associated diseases.


   ACKNOWLEDGMENTS
 
We thank Dr. Konan Peck (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan) for technical assistance in microarray assay. We also thank Tzung-Shiahn Sheen (Department of Otolaryngology, National Taiwan University Hospital, Taipei, Taiwan) and Chi-Long Chen (Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan) for providing biopsies and analyzing the results of immunohistochemistry. We also thank Dr. Yu-Tzu Huang, Chiau-Jing Jung, and Shing-Fnn Huang for their proficient technical assistance and Tim J. Harrison of the Royal Free and University College Medical School of University College London (London, United Kingdom) for critically reviewing the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Science Council Grants NSC 89-2318-B-002-015-M51 and NSC 91-3112-B-002-016) and National Health Research Institutes Grants NHRI-EX90-9012BP and NHRI-EX91-9012BP. Back

2 To whom requests for reprints should be addressed, at Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Room 714, Number 1, Section 1, Jen-Ai Road, Taipei, Taiwan, Republic of China. Phone: 886-2-2312-3456, ext. 8298; Fax: 886-2-2391-5180; E-mail: chtsai{at}ha.mc.ntu.edu.tw Back

3 The abbreviations used are: NPC, nasopharyngeal carcinoma; EBNA-1, EBV nuclear antigen 1; MMP, matrix metalloproteinase; RT, reverse transcription; ECM, extracellular matrix; LH, lymphohyperplasia; Q-PCR, quantitative PCR; LMP, latent membrane protein; Zta, Z transactivator; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; DDR2, discoidin domain receptor 2; AP-1, activator protein 1; STAT, signal transducers and activators of transcription. Back

Received 7/30/02. Accepted 10/31/02.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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