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Induction of matrix metalloproteinases by Epstein–Barr virus latent membrane protein 1 isolated from nasopharyngeal carcinoma
Biomedicine & Pharmacotherapy 61 (2007) 520e526 www.elsevier.com/locate/biopha
Induction of matrix metalloproteinases by EpsteineBarr virus latent membrane protein 1 isolated from nasopharyngeal carcinoma D.C.W. Lee a, D.T.T. Chua b,**, W.I. Wei c, J.S.T. Sham b, A.S.Y. Lau a,* a
Immunology Research Laboratory, Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, PR China b Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, PR China c Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, PR China Available online 14 September 2007
Abstract EpsteineBarr virus latent infection is associated with human malignancies including Burkitt’s lymphoma, gastric carcinoma and the highly invasive nasopharyngeal carcinoma (NPC). Increased expression of EBV latent membrane protein 1, LMP1, is correlated with tumor progression and metastasis in NPC. LMP1 induces cellular proteins including cytokines and matrix metalloproteinases (e.g., MMP1, MMP2 and MMP9). MMPs are endopeptidases involved in the degradation of extracellular matrix proteins; and their upregulation in cancer implicates their potential role in tumor metastasis. In light of the role of LMP1 in cytokine dysregulation and the fact that MMPs are regulated by cytokines, we examined whether LMP1 promotes NPC metastasis via the induction of MMPs. To delineate the oncogenic role of LMP1 in NPC, we first investigated the induction of MMP1, MMP2, MMP3 and MMP9 in LMP1-positive NPC tumor samples (n ¼ 15) by quantitative RT-PCR. We showed a significant induction of MMP1 and MMP3 transcripts in the EBV LMP1-positive NPC tissues, compared with biopsies obtained from the adjacent non-tumor tissues. To investigate the role of LMP1 in MMP expression in NPC, we cloned the LMP1 gene from NPC samples and transiently expressed it in MRC5 cells (human lung fibroblasts). Following transfection, a time-dependent elevation of endogenous MMP3 expression was found in the LMP1-transfectants by quantitative RT-PCR and Western analysis. Taken together, we observed that MMP3 is upregulated in LMP1-positive NPC tumors and LMP1-expression in fibroblasts is associated with MMP3 and cytokine expression. Our results suggest that LMP1 may contribute to invasiveness of NPC cells via the expression of MMP3 in fibroblasts. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Matrix metalloproteinase; Nasopharyngeal carcinoma; Latent membrane protein
1. Introduction EpsteineBarr virus (EBV) is a human herpesvirus that infects a large percentage of the world’s population and it appears to be the first human tumor virus [1,2]. EBV has been known to be highly associated with human malignancies, including Burkitt’s lymphoma, post-transplant lymphomas, AIDS-associated * Corresponding author. Tel.: þ86 852 2855 4269; fax: þ86 852 2855 1523. ** Corresponding author. E-mail addresses: [email protected] (D.T.T. Chua), asylau@hkucc. hku.hk (A.S.Y. Lau). 0753-3322/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2007.08.007
lymphoma, gastric carcinoma, and the highly invasive nasopharyngeal carcinoma (NPC), putatively related to and as a consequence of the latent infection state [1,3,4]. The elevated levels of EBV-specific IgG antiviral capsid antigen (VCA) and IgA anti-VCA antibodies in patient serum and the quantitative analysis of plasma EBV DNA provide useful information for diagnosis and monitoring the progression of NPC [5e7]. Specific EBV latent genes including LMP1, 2A, and 2B, as well as EBV nuclear antigen 1 (EBNA1) and EBV non-polyadenylated RNAs (EBER) are expressed in NPC [1,8]. LMP1 has been shown to have transforming properties in mouse fibroblasts, epithelial cells and primary human
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B-lymphocytes [9]. It also plays a significant role in the pathogenesis of NPC [10]. Increased expression of LMP1 and other EBV latent genes is correlated with tumor promotion and metastasis in the cancer [11]. Cancer metastasis is a complex process in which tumor cells spread from the primary lesion through circulation and lymphatic systems to distant organs, in addition to local invasion of the stroma. Tumor cell invasion involves multiple steps including stromal matrixetumor cell interaction, remodeling of extracellular matrix, intra- and extra-vascularization in the systemic circulation, and establishment of metastatic sites in distant organs [12,13]. Matrix metalloproteinases, with a total of 23 family members, are endopeptidases involved in the degradation of extracellular matrix proteins and their upregulation in cancer samples implicates their potential roles in tumor progression [14]. Selected MMPs including MMP1, MMP2, MMP3, MMP7, MMP9, MMP13 and MMP14 are highly expressed in malignant cancers, particularly in the areas of active invasion such as the interface of tumor and stroma [14,15]. For example, MMP3 or MMP7 cleaves E-cadherin, a prototype adhesion molecule in maintaining the intercellular matrix, at the cell surface to release the soluble E-cadherin, which results in the interference of the function of cell-bound E-cadherin resulting in the promotion of tumor cell invasion [16]. Previous studies have shown that MMPs activation is regulated by LMP1. LMP1-transfectants induce MMP1 expression leading to increases in cell motility in migration assays and invasiveness in collagen gels [17]. It has been shown that LMP1 induces MMP9 expression in epithelial cells [18,19]. Immunohistochemical studies showed that the expression of MMP9 is positively correlated with the expression of LMP1, as well as with the metastasis of NPC in patients [20]. To demonstrate the role of LMP1 in NPC metastasis, it was shown that the metastatic potential of NPC cells can be inhibited by LMP1targeted short hairpin RNA that is capable of inhibiting LMP1-expression [21]. LMP1 has been shown to play a regulatory role in the expression of proinflammatory cytokines including TNF and IL-6 as well as anti-inflammatory cytokines including IL-10 [22,23]. In light of the role of LMP1 in cytokine regulation and induction of MMPs are regulated by TNF and other proinflammatory cytokines [24e26], we investigated the role of LMP1 in the promotion of progression and metastasis of highly invasive NPC via the induction of MMPs. 2. Materials and methods 2.1. Patient tissue samples Fifteen tumor specimens were obtained from patients with newly diagnosed NPC who were treated in the Department of Clinical Oncology at Queen Mary Hospital. Clinical characteristics of the 15 patients were shown in Table 1. Non-tumor biopsy specimens adjacent to the location of the tumor were also obtained from the same patients with NPC. There were 10 male and five female patients, with a median age of 43
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Table 1 Clinical characteristics in 15 patients Characteristics Gender Male Female Age 50 >50 Median age (year) T stage T1 T2 T3 T4 N stage N0 N1 N2 N3 Stage grouping I II III IV Treatment received Radiotherapy Chemoradiotherapy
Number of patients 10 5 10 5 43 (range: 30e65) 3 7 4 1 3 2 5 5 1 3 6 5 5 10
years. The histology of the tumors was undifferentiated carcinoma in all cases. Ten patients had early T stage (T1e2) whereas five had advanced T stage (T3e4). Five patients had early N stage (N0e1), and 10 had advanced N stage (N2e3). Eleven patients had stage IIIeIV disease in accordance with the International Union Against Cancer (UICC) Staging System. Following the biopsy, all patients were treated by radical radiotherapy, and 10 also received concurrent chemotherapy with or without the addition of adjuvants. Twelve tumor samples from patients with non-NPC head-and-neck tumor were obtained from the Department of Surgery at Queen Mary Hospital. All patients gave informed consent in donating their biopsy materials for research work. 2.2. Detection of EBV LMP1 gene in NPC samples Genomic DNA was isolated from tumor samples of NPC patients using Qiagen DNA extraction kit (Qiagen). The LMP1 DNA was amplified in a final volume of 25 ml mixture containing 1X PCR buffer (Amersham Biosciences, UK), 0.2 mM dNTP, 5 pmol of each oligodeoxynucleotide primer and 1 unit of Taq DNA polymerase (Amersham Biosciences). Thirty-five and 25 cycles of PCR were performed at 94 C, 1 min; 55 C, 1 min and 72 C, 1 min for LMP1 and actin, respectively. The sequence of the primers were as follows: LMP1 forward, 50 -CTTTGGCTGCTTACTTGGTAAG-30 ; reverse, 50 -ATTTCCAGCAGAGTCGCTAGG-30 ; actin forward, 50 -TCACCCACACTGTGCCCATCT-30 ; and reverse, 50 -GAA CCGCTCATTGCCAATGG-30 . The PCR products were resolved by electrophoresis on an ethidium bromide stained agarose gels.
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2.3. Isolation of RNA and quantitative RT-PCR
2.6. Immunocytochemical staining
Total cellular RNA of the tumor tissue samples or transfected cells was extracted using TriZol (Invitrogen, USA) and reverse transcribed by using Taqman reverse transcription reagent kit (Applied Biosystems, USA) according to the manufacturer’s protocol. The level of MMP mRNA in sample was normalized with that of the 18S rRNA and analyzed by the comparative method as described previously [27,28]. The relative fold induction of MMP in tumor sample was compared with the corresponding adjacent non-tumor sample from the same patient. In LMP1-transfectants, the fold induction of MMP was compared to the cells transfected with the parental plasmid without the insertion of LMP1 DNA at the indicated time points.
Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.25% Triton X-100 for 5 min. The LMP1 protein was stained with anti-LMP1 mouse monoclonal antibody (DAKO, USA) and secondary anti-mouse-FITC antibody at 36 h post-transfection (Chemicon, USA). Nuclei were counterstained with 4,6-diamidine-2-phenylindole dihydrochloride (DAPI), and the immunocytochemical staining was examined under fluorescence microscopy. 2.7. Western analysis Total protein lysate (30 mg) of cytoplasmic protein was analyzed by Western blots using anti-MMP3 rabbit polyclonal
2.4. Isolation and cloning of EBV LMP1 gene in NPC samples The sequence of NPC EBV LMP1 gene was obtained from Genbank (accession no. X58140). The coding sequence of the LMP1 gene was amplified from an NPC tumor sample using the LMP1-specific primers: forward, 50 -CACACTACCCT GACCATGGAAC-30 ; and reverse, 50 -GGTTAGTCATAGTAG CTTAGC-30 . The PCR products were gel-purified, sequenced and cloned into the expression vector pcDNA3. 2.5. Cell culture and transfection The human lung fibroblasts, MRC5, were obtained from ATCC and the cells were cultured in MEM supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 mg/ml streptomycin. The LMP1-encoded plasmid or its parental counterpart without the LMP1 insert was transiently transfected into MRC5 by Lipofectamine 2000 according to the manufacturer’s protocol.
Fig. 1. Detection of EBV LMP1 gene in patients with nasopharyngeal carcinoma. (A) Tumor samples, no. 1e15, and (B) paired non-tumor tissue samples, no. 10 e150 , were obtained from 15 patients with newly diagnosed nasopharyngeal carcinoma. DNA was isolated and PCR amplification was performed with specific primers for LMP1 (35 cycles, upper panel) and actin (25 cycles, lower panel) as described in Section 2. Samples were resolved by electrophoresis in 1.5% agarose gels. M, 1 kb plus DNA ladder.
Fig. 2. Induction of matrix metalloproteinases in NPC tumor samples. (A) Total cellular RNA was extracted from tumor and adjacent paired non-tumor tissue samples as described in Section 2. Quantitative RT-PCR was performed for measuring the transcription levels of MMP1, MMP2, MMP3 and MMP9. The level of MMPs was normalized to that of the 18S rRNA. Fold induction of MMPs in tumor samples was plotted in the indicated column. Black dots represent the average of triplicate assays of the same tissue sample. The number in each column is the mean of fold induction of each group (* p < 0.001). (B) The level of MMP induction in patients with NPC was compared with that of the levels in 12 patients with other head-and-neck cancers. RT-PCR assay was performed three times independently in each of the RNA samples.
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antibodies (R&D Systems), anti-LMP1 mouse monoclonal antibody (DAKO) and anti-actin goat polyclonal antibodies (Santa Cruz, USA). The level of protein expression was revealed by using ECL solution (Amersham Biosciences, Buckinghamshire, UK). 3. Results 3.1. Detection of EBV LMP1 in nasopharyngeal carcinoma Previous studies have shown that oncogenic EBV LMP1 protein is associated with invasive cancers including nasopharyngeal carcinoma [19,20]. To investigate the tumorigenic role of LMP1 in NPC, we examined in the presence of LMP1 gene in tumor samples from NPC patients with newly diagnosed nasopharyngeal carcinoma by PCR. Histological examinations of the NPC tumor samples have shown undifferentiated carcinoma in all cases. Using EBV LMP1-specific primers, the LMP1 DNA was detected in 13 out of 15 NPC tumor tissues, but not in the adjacent non-tumor samples obtained in paired biopsies (Fig. 1A and B). Our results suggested that with its presence in most NPC samples, LMP1 may play a role in NPC pathogenesis. 3.2. Induction of matrix metalloproteinases in NPC tumor samples LMP1 has been shown to be associated with the enhancement of metastasis in NPC [29]. In addition, LMP1-induced
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expression of MMP1 and MMP9 has been shown to be associated with enhanced invasiveness of cancer cells resulting in tumor metastasis [17,20]. To investigate the induction of MMPs in the NPC tumor samples, we then measured the mRNA levels of MMP1, MMP2, MMP3 and MMP9 in the tumor and non-tumor paired biopsies by Taqman real-time RTPCR. The level of MMPs induction in tumor samples was compared to the corresponding non-tumor sample from the same patient. Our results showed that a significant upregulation of MMP1 and MMP3 was detected in the LMP1-positive biopsies, compared with the paired non-malignant nasopharyngeal tissues (Fig. 2A). In contrast, there was no differential induction of MMP2 and MMP9 between tumor and non-tumor samples (Fig. 2A). To further demonstrate the induction of MMP3 in NPC, we examined the levels of MMP3 mRNA in NPC and other headand-neck cancers. As shown in Fig. 2B, the induction of MMP3 was increased significantly in NPC, compared with other head-and-neck cancers ( p ¼ 0.0001). Consistent to previous studies, upregulation of MMP1 was also detected in the NPC samples ([17]; and our data not shown). 3.3. LMP1 regulates MMPs expression Following the expression profile of MMPs, we next delineated the role of LMP1 in the regulation of MMP3 expression in the tumor samples. We amplified the coding region of the LMP1 gene from a representative NPC tumor and cloned it into the pcDNA3 expression plasmid. The sequence of the
Fig. 3. Immunocytochemical staining of LMP1-expressing cells. The EBV LMP1 DNA was isolated from NPC tumor samples and cloned into the pcDNA3 expression vector. LMP1 protein was transiently expressed in MRC5 human lung fibroblasts using Lipofectamine 2000. (A) Expression of LMP1 was detected using anti-LMP1 monoclonal antibody at 36 h post-transfection. (B) Cells transfected with the parental plasmid only. Nuclei were counterstained with DAPI in cells transfected with the (C) LMP1-encoded or (D) parental plasmid. pcDNA3, parental expression plasmid. Magnification, 400.
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amplified LMP1 gene was compared to the sequence of NPC LMP1 DNA reported in GenBank [30]. Transient expression of LMP1 in MRC5 human lung fibroblasts was shown by immunocytochemical staining and Western blotting (Figs. 3 and 4B). We then analyzed the induction kinetics of endogenous MMPs in MRC5 cells transiently expressing LMP1. Our quantitative RT-PCR results showed that the levels of MMP3 mRNA increased significantly from 20 h to 24 h post-transfection, compared to the mock-transfected cells using the parental plasmid ( p < 0.05; Fig. 4A). In LMP1-expressing cells, the LMP1-induced MMP3 transcription was not blocked by the addition of cycloheximide, indicating that new protein synthesis was not required for the transcriptional activation of MMP3 (data not shown). Consistent to previous report, induction of MMP1 was also detected in our LMP1-expressing cells [17]. In contrast, induction of MMP2 and MMP9 was not found in the LMP1-expressing MRC5 cells (data not shown). Furthermore, expression of MMP3 protein in the LMP1expressing cells was shown by Western blotting at 48 h posttransfection (Fig. 4B) and the level of MMP3 protein increased from 24 h to 48 h post-transfection (Fig. 4C). As controls, LMP1 monoclonal antibody was used to determine the LMP1-expression levels by Western analysis. The input amount of protein samples was further controlled by anti-actin antibodies.
genetic studies have shown that a single adenine insertion/deletion polymorphism (5A/6A) in the MMP3 promoter region may contribute to an increased risk of metastasis among breast cancer patients [36,37]. LMP1 is a transmembrane protein with its C-terminal component containing two important signaling domains, CTAR1 and CTAR2. The interactions of CTAR domains with TRAFs or TRADD result in the activation of AP-1, NF-kB, p38 MAPK, JNK, PI3K and JAK/STAT signaling pathways [38e 41]. Two previous studies have shown that transcription of MMP3 is regulated by cellular factors including AP-1 and Ets [42,43]. Moreover, it has been shown that LMP1-induced MMP1 expression and MMP3 activation are via AP-1 and Ets. However, expression of the latent form of MMP3 was not
4. Discussion Matrix metalloproteinases have been shown to play critical roles in tumor metastasis including tumor-induced angiogenesis, tumor invasion, and establishment of metastatic foci at the secondary sites [14,15,31]. In this study, we found a differential induction of MMP1 and MMP3 transcripts in LMP1-positive NPC tumors comparing with the adjacent non-tumor tissues. With the confirmed induction of MMPs in NPC tumor samples and the reported oncogenic role of LMP1 in EBVassociated diseases, we next investigated the role of EBV LMP1 in the regulation of the MMPs induction in this highly invasive cancer. Previous studies have demonstrated that upregulation of MMP1 and MMP9 expression in LMP1-transfectants resulted in enhancement of the cell invasion in matrix collagen gels [17e20]. A subsequent study showed that aspirin treatment can suppress the MMP9-dependent invasive property of the LMP1-expressing cells [32]. Our results showed that a significant level of MMP3 induction was detected in NPC patients with LMP1-positive tumors, compared with other headand-neck cancer patients (Fig. 2A and B). Like other members of the MMP family, MMP3 can degrade numerous ECM substrates, release cellematrix interaction proteins, activate other MMPs, and inactivate several serine proteinase inhibitors [33]. MMP3 plays a critical role in organogenesis including mammary gland development [34]. However, constitutive or dysregulated expression of MMP3 may contribute to the initial stage of cancer development and tumor progression [16,35]. Furthermore, recent
Fig. 4. Kinetic studies of LMP1-induced MMP3 expression. (A) MRC5 human lung fibroblasts were transfected for transient expression of LMP1 and the level of MMP3 induction was measured by Taqman quantitative RT-PCR at the indicated time points. The values represent the mean SD of triplicates and are statistically analyzed by the two-tailed, paired t-test; * p < 0.001. (B) The expression of the exogenous LMP1 was examined by Western blotting with specific antibodies. MMP3 expression was detected in LMP1-expressing cells by Western blotting at 48 h post-transfection. The amounts of protein loaded on the gel were standardized by using anti-actin antibodies. (C) Kinetics of MMP3 protein expression in LMP1-transfectants using Western blotting. The levels of MMP3 protein increased over a time course of 48 h post-transfection. pcDNA3, parental expression plasmid.
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detected in LMP1-expressing cells [44]. Our results showed that LMP1 induces the expression of MMP3 in human lung fibroblasts (Fig. 4). The discrepancy of these two studies may be due to differences in cellular origins. To further elucidate the functions of MMP3 in tumor progression, we are investigating the roles of MMP3 in the invasive property of the LMP1-transfected cells. In addition, we will further investigate the upstream kinases that are responsible for mediating the LMP1-induced expression of MMPs and cytokines. Taken together, our results have clinical implications and may contribute to the design of novel therapeutics for the amelioration of tumor invasion to normal tissues in NPC and EBV-associated diseases. Acknowledgments This work was supported by grants to AS Lau from HKU CRCG Project Funding (Project no. 200607176145), and to AS Lau and DTT Chua from HKU Clinical Oncology Research Fund. References [1] Raab-Traub N. EpsteineBarr virus in the pathogenesis of NPC. Semin Cancer Biol 2002;12:431e41. [2] Pattle SB, Farrell PJ. The role of EpsteineBarr virus in cancer. Expert Opin Biol Ther 2006;6:1193e205. [3] Young LS, Murray PG. EpsteineBarr virus and oncogenesis: from latent genes to tumours. Oncogene 2003;22:5108e21. [4] Thompson MP, Kurzrock R. EpsteineBarr virus and cancer. Clin Cancer Res 2004;10:803e21. [5] Hadar T, Rahima M, Kahan E, Sidi J, Rakowsky E, Sarov B, et al. Significance of specific EpsteineBarr virus IgA and elevated IgG antibodies to viral capsid antigens in nasopharyngeal carcinoma patients. J Med Virol 1986;20:329e39. [6] Lo YM, Chan LY, Chan AT, Leung SF, Lo KW, Zhang J, et al. Quantitative and temporal correlation between circulating cell-free Epsteine Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 1999;59:5452e5. [7] Lo YM, Chan AT, Chan LY, Leung SF, Lam CW, Huang DP, et al. Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating EpsteineBarr virus DNA. Cancer Res 2000; 60:6878e81. [8] Wei WI, Sham JS. Nasopharyngeal carcinoma. Lancet 2005;365: 2041e54. [9] Rickinson A, Kieff E. EpsteineBarr virus. In: Fields B, Knipe D, Kieff E, editors. Fields virology. Philadelphia: Raven Press; 1996. p. 2397e446. [10] Tsao SW, Tramoutanis G, Dawson CW, Lo AK, Huang DP. The significance of LMP1 expression in nasopharyngeal carcinoma. Semin Cancer Biol 2002;12:473e87. [11] Sengupta S, den Boon JA, Chen IH, Newton MA, Dahl DB, Chen M, et al. Genome-wide expression profiling reveals EBV-associated inhibition of MHC class I expression in nasopharyngeal carcinoma. Cancer Res 2006;66:7999e8006. [12] Liotta LA, Kohn EC. The microenvironment of the tumourehost interface. Nature 2001;411:375e9. [13] Yang J, Mani SA, Weinberg RA. Exploring a new twist on tumor metastasis. Cancer Res 2006;66:4549e52. [14] Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002;2:161e74. [15] Deryugina EI, Quigley JP. Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev 2006;25:9e34.
525
[16] Noe V, Fingleton B, Jacobs K, Crawford HC, Vermeulen S, Steelant W, et al. Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1. J Cell Sci 2001;114:111e8. [17] Lu J, Chua HH, Chen SY, Chen JY, Tsai CH. Regulation of matrix metalloproteinase-1 by EpsteineBarr virus proteins. Cancer Res 2003;63: 256e62. [18] Yoshizaki T, Sato H, Murono S, Pagano JS, Furukawa M. Matrix metalloproteinase 9 is induced by the EpsteineBarr virus BZLF1 transactivator. Clin Exp Metastasis 1999;17:431e6. [19] Wakisaka N, Pagano JS. EpsteineBarr virus induces invasion and metastasis factors. Anticancer Res 2003;23:2133e8. [20] Horikawa T, Yoshizaki T, Sheen TS, Lee SY, Furukawa M. Association of latent membrane protein 1 and matrix metalloproteinase 9 with metastasis in nasopharyngeal carcinoma. Cancer 2000;89: 715e23. [21] Li XP, Li G, Peng Y, Kung HF, Lin MC. Suppression of EpsteineBarr virus-encoded latent membrane protein-1 by RNA interference inhibits the metastatic potential of nasopharyngeal carcinoma cells. Biochem Biophys Res Commun 2004;315:212e8. [22] Eliopoulos AG, Gallagher NJ, Blake SM, Dawson CW, Young LS. Activation of the p38 mitogen-activated protein kinase pathway by EpsteineBarr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production. J Biol Chem 1999; 274:16085e96. [23] Vockerodt M, Haier B, Buttgereit P, Tesch H, Kube D. The EpsteineBarr virus latent membrane protein 1 induces interleukin-10 in Burkitt’s lymphoma cells but not in Hodgkin’s cells involving the p38/SAPK2 pathway. Virology 2001;280:183e98. [24] Esteve PO, Chicoine E, Robledo O, Aoudjit F, Descoteaux A, Potworowski EF, et al. Protein kinase C-zeta regulates transcription of the matrix metalloproteinase-9 gene induced by IL-1 and TNF-alpha in glioma cells via NF-kappa B. J Biol Chem 2002;277:35150e5. [25] Nee LE, McMorrow T, Campbell E, Slattery C, Ryan MP. TNF-alpha and IL-1beta-mediated regulation of MMP-9 and TIMP-1 in renal proximal tubular cells. Kidney Int 2004;66:1376e86. [26] Hosseini M, Rose AY, Song K, Bohan C, Alexander JP, Kelley MJ, et al. IL-1 and TNF induction of matrix metalloproteinase-3 by c-Jun N-terminal kinase in trabecular meshwork. Invest Ophthalmol Vis Sci 2006; 47:1469e76. [27] Lee DC, Cheung CY, Law AH, Mok CK, Peiris M, Lau AS. p38 mitogenactivated protein kinase-dependent hyperinduction of tumor necrosis factor alpha expression in response to avian influenza virus H5N1. J Virol 2005;79:10147e54. [28] Cheung BK, Lee DC, Li JC, Lau YL, Lau AS. A role for double-stranded RNA-activated protein kinase PKR in Mycobacterium-induced cytokine expression. J Immunol 2005;175:7218e25. [29] Yoshizaki T. Promotion of metastasis in nasopharyngeal carcinoma by EpsteineBarr virus latent membrane protein-1. Histol Histopathol 2002;17:845e50. [30] Hu LF, Zabarovsky ER, Chen F, Cao SL, Ernberg I, Klein G, et al. Isolation and sequencing of the EpsteineBarr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma. J Gen Virol 1991;72(Pt 10):2399e409. [31] Stamenkovic I. Matrix metalloproteinases in tumor invasion and metastasis. Semin Cancer Biol 2000;10:415e33. [32] Murono S, Yoshizaki T, Sato H, Takeshita H, Furukawa M, Pagano JS. Aspirin inhibits tumor cell invasiveness induced by EpsteineBarr virus latent membrane protein 1 through suppression of matrix metalloproteinase-9 expression. Cancer Res 2000;60:2555e61. [33] Sternlicht MD, Bissell MJ, Werb Z. The matrix metalloproteinase stromelysin-1 acts as a natural mammary tumor promoter. Oncogene 2000;19:1102e13. [34] Sternlicht MD, Lochter A, Sympson CJ, Huey B, Rougier JP, Gray JW, et al. The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell 1999;98:137e46. [35] Lochter A, Sternlicht MD, Werb Z, Bissell MJ. The significance of matrix metalloproteinases during early stages of tumor progression. Ann N Y Acad Sci 1998;857:180e93.
526
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[36] Ghilardi G, Biondi ML, Caputo M, Leviti S, DeMonti M, Guagnellini E, et al. A single nucleotide polymorphism in the matrix metalloproteinase3 promoter enhances breast cancer susceptibility. Clin Cancer Res 2002;8:3820e3. [37] Krippl P, Langsenlehner U, Renner W, Yazdani-Biuki B, Koppel H, Leithner A, et al. The 5A/6A polymorphism of the matrix metalloproteinase 3 gene promoter and breast cancer. Clin Cancer Res 2004;10:3518e20. [38] Kieser A, Kaiser C, Hammerschmidt W. LMP1 signal transduction differs substantially from TNF receptor 1 signaling in the molecular functions of TRADD and TRAF2. EMBO J 1999;18:2511e21. [39] Gires O, Kohlhuber F, Kilger E, Baumann M, Kieser A, Kaiser C, et al. Latent membrane protein 1 of EpsteineBarr virus interacts with JAK3 and activates STAT proteins. EMBO J 1999;18:3064e73. [40] Eliopoulos AG, Young LS. LMP1 structure and signal transduction. Semin Cancer Biol 2001;11:435e44.
[41] Mainou BA, Everly Jr DN, Raab-Traub N. EpsteineBarr virus latent membrane protein 1 CTAR1 mediates rodent and human fibroblast transformation through activation of PI3K. Oncogene 2005;24:6917e24. [42] Jayaraman G, Srinivas R, Duggan C, Ferreira E, Swaminathan S, Somasundaram K, et al. p300/cAMP-responsive element-binding protein interactions with ets-1 and ets-2 in the transcriptional activation of the human stromelysin promoter. J Biol Chem 1999;274:17342e52. [43] Lewis M, Amento EP, Unemori EN. Transcriptional inhibition of stromelysin by interferon-gamma in normal human fibroblasts is mediated by the AP-1 domain. J Cell Biochem 1999;72:373e86. [44] Kondo S, Wakisaka N, Schell MJ, Horikawa T, Sheen TS, Sato H, et al. EpsteineBarr virus latent membrane protein 1 induces the matrix metalloproteinase-1 promoter via an Ets binding site formed by a single nucleotide polymorphism: enhanced susceptibility to nasopharyngeal carcinoma. Int J Cancer 2005;115:368e76.
Induction of matrix metalloproteinases by EpsteineBarr virus latent membrane protein 1 isolated from nasopharyngeal carcinoma D.C.W. Lee a, D.T.T. Chua b,**, W.I. Wei c, J.S.T. Sham b, A.S.Y. Lau a,* a
Immunology Research Laboratory, Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, PR China b Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, PR China c Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, PR China Available online 14 September 2007
Abstract EpsteineBarr virus latent infection is associated with human malignancies including Burkitt’s lymphoma, gastric carcinoma and the highly invasive nasopharyngeal carcinoma (NPC). Increased expression of EBV latent membrane protein 1, LMP1, is correlated with tumor progression and metastasis in NPC. LMP1 induces cellular proteins including cytokines and matrix metalloproteinases (e.g., MMP1, MMP2 and MMP9). MMPs are endopeptidases involved in the degradation of extracellular matrix proteins; and their upregulation in cancer implicates their potential role in tumor metastasis. In light of the role of LMP1 in cytokine dysregulation and the fact that MMPs are regulated by cytokines, we examined whether LMP1 promotes NPC metastasis via the induction of MMPs. To delineate the oncogenic role of LMP1 in NPC, we first investigated the induction of MMP1, MMP2, MMP3 and MMP9 in LMP1-positive NPC tumor samples (n ¼ 15) by quantitative RT-PCR. We showed a significant induction of MMP1 and MMP3 transcripts in the EBV LMP1-positive NPC tissues, compared with biopsies obtained from the adjacent non-tumor tissues. To investigate the role of LMP1 in MMP expression in NPC, we cloned the LMP1 gene from NPC samples and transiently expressed it in MRC5 cells (human lung fibroblasts). Following transfection, a time-dependent elevation of endogenous MMP3 expression was found in the LMP1-transfectants by quantitative RT-PCR and Western analysis. Taken together, we observed that MMP3 is upregulated in LMP1-positive NPC tumors and LMP1-expression in fibroblasts is associated with MMP3 and cytokine expression. Our results suggest that LMP1 may contribute to invasiveness of NPC cells via the expression of MMP3 in fibroblasts. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Matrix metalloproteinase; Nasopharyngeal carcinoma; Latent membrane protein
1. Introduction EpsteineBarr virus (EBV) is a human herpesvirus that infects a large percentage of the world’s population and it appears to be the first human tumor virus [1,2]. EBV has been known to be highly associated with human malignancies, including Burkitt’s lymphoma, post-transplant lymphomas, AIDS-associated * Corresponding author. Tel.: þ86 852 2855 4269; fax: þ86 852 2855 1523. ** Corresponding author. E-mail addresses: [email protected] (D.T.T. Chua), asylau@hkucc. hku.hk (A.S.Y. Lau). 0753-3322/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2007.08.007
lymphoma, gastric carcinoma, and the highly invasive nasopharyngeal carcinoma (NPC), putatively related to and as a consequence of the latent infection state [1,3,4]. The elevated levels of EBV-specific IgG antiviral capsid antigen (VCA) and IgA anti-VCA antibodies in patient serum and the quantitative analysis of plasma EBV DNA provide useful information for diagnosis and monitoring the progression of NPC [5e7]. Specific EBV latent genes including LMP1, 2A, and 2B, as well as EBV nuclear antigen 1 (EBNA1) and EBV non-polyadenylated RNAs (EBER) are expressed in NPC [1,8]. LMP1 has been shown to have transforming properties in mouse fibroblasts, epithelial cells and primary human
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B-lymphocytes [9]. It also plays a significant role in the pathogenesis of NPC [10]. Increased expression of LMP1 and other EBV latent genes is correlated with tumor promotion and metastasis in the cancer [11]. Cancer metastasis is a complex process in which tumor cells spread from the primary lesion through circulation and lymphatic systems to distant organs, in addition to local invasion of the stroma. Tumor cell invasion involves multiple steps including stromal matrixetumor cell interaction, remodeling of extracellular matrix, intra- and extra-vascularization in the systemic circulation, and establishment of metastatic sites in distant organs [12,13]. Matrix metalloproteinases, with a total of 23 family members, are endopeptidases involved in the degradation of extracellular matrix proteins and their upregulation in cancer samples implicates their potential roles in tumor progression [14]. Selected MMPs including MMP1, MMP2, MMP3, MMP7, MMP9, MMP13 and MMP14 are highly expressed in malignant cancers, particularly in the areas of active invasion such as the interface of tumor and stroma [14,15]. For example, MMP3 or MMP7 cleaves E-cadherin, a prototype adhesion molecule in maintaining the intercellular matrix, at the cell surface to release the soluble E-cadherin, which results in the interference of the function of cell-bound E-cadherin resulting in the promotion of tumor cell invasion [16]. Previous studies have shown that MMPs activation is regulated by LMP1. LMP1-transfectants induce MMP1 expression leading to increases in cell motility in migration assays and invasiveness in collagen gels [17]. It has been shown that LMP1 induces MMP9 expression in epithelial cells [18,19]. Immunohistochemical studies showed that the expression of MMP9 is positively correlated with the expression of LMP1, as well as with the metastasis of NPC in patients [20]. To demonstrate the role of LMP1 in NPC metastasis, it was shown that the metastatic potential of NPC cells can be inhibited by LMP1targeted short hairpin RNA that is capable of inhibiting LMP1-expression [21]. LMP1 has been shown to play a regulatory role in the expression of proinflammatory cytokines including TNF and IL-6 as well as anti-inflammatory cytokines including IL-10 [22,23]. In light of the role of LMP1 in cytokine regulation and induction of MMPs are regulated by TNF and other proinflammatory cytokines [24e26], we investigated the role of LMP1 in the promotion of progression and metastasis of highly invasive NPC via the induction of MMPs. 2. Materials and methods 2.1. Patient tissue samples Fifteen tumor specimens were obtained from patients with newly diagnosed NPC who were treated in the Department of Clinical Oncology at Queen Mary Hospital. Clinical characteristics of the 15 patients were shown in Table 1. Non-tumor biopsy specimens adjacent to the location of the tumor were also obtained from the same patients with NPC. There were 10 male and five female patients, with a median age of 43
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Table 1 Clinical characteristics in 15 patients Characteristics Gender Male Female Age 50 >50 Median age (year) T stage T1 T2 T3 T4 N stage N0 N1 N2 N3 Stage grouping I II III IV Treatment received Radiotherapy Chemoradiotherapy
Number of patients 10 5 10 5 43 (range: 30e65) 3 7 4 1 3 2 5 5 1 3 6 5 5 10
years. The histology of the tumors was undifferentiated carcinoma in all cases. Ten patients had early T stage (T1e2) whereas five had advanced T stage (T3e4). Five patients had early N stage (N0e1), and 10 had advanced N stage (N2e3). Eleven patients had stage IIIeIV disease in accordance with the International Union Against Cancer (UICC) Staging System. Following the biopsy, all patients were treated by radical radiotherapy, and 10 also received concurrent chemotherapy with or without the addition of adjuvants. Twelve tumor samples from patients with non-NPC head-and-neck tumor were obtained from the Department of Surgery at Queen Mary Hospital. All patients gave informed consent in donating their biopsy materials for research work. 2.2. Detection of EBV LMP1 gene in NPC samples Genomic DNA was isolated from tumor samples of NPC patients using Qiagen DNA extraction kit (Qiagen). The LMP1 DNA was amplified in a final volume of 25 ml mixture containing 1X PCR buffer (Amersham Biosciences, UK), 0.2 mM dNTP, 5 pmol of each oligodeoxynucleotide primer and 1 unit of Taq DNA polymerase (Amersham Biosciences). Thirty-five and 25 cycles of PCR were performed at 94 C, 1 min; 55 C, 1 min and 72 C, 1 min for LMP1 and actin, respectively. The sequence of the primers were as follows: LMP1 forward, 50 -CTTTGGCTGCTTACTTGGTAAG-30 ; reverse, 50 -ATTTCCAGCAGAGTCGCTAGG-30 ; actin forward, 50 -TCACCCACACTGTGCCCATCT-30 ; and reverse, 50 -GAA CCGCTCATTGCCAATGG-30 . The PCR products were resolved by electrophoresis on an ethidium bromide stained agarose gels.
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2.3. Isolation of RNA and quantitative RT-PCR
2.6. Immunocytochemical staining
Total cellular RNA of the tumor tissue samples or transfected cells was extracted using TriZol (Invitrogen, USA) and reverse transcribed by using Taqman reverse transcription reagent kit (Applied Biosystems, USA) according to the manufacturer’s protocol. The level of MMP mRNA in sample was normalized with that of the 18S rRNA and analyzed by the comparative method as described previously [27,28]. The relative fold induction of MMP in tumor sample was compared with the corresponding adjacent non-tumor sample from the same patient. In LMP1-transfectants, the fold induction of MMP was compared to the cells transfected with the parental plasmid without the insertion of LMP1 DNA at the indicated time points.
Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.25% Triton X-100 for 5 min. The LMP1 protein was stained with anti-LMP1 mouse monoclonal antibody (DAKO, USA) and secondary anti-mouse-FITC antibody at 36 h post-transfection (Chemicon, USA). Nuclei were counterstained with 4,6-diamidine-2-phenylindole dihydrochloride (DAPI), and the immunocytochemical staining was examined under fluorescence microscopy. 2.7. Western analysis Total protein lysate (30 mg) of cytoplasmic protein was analyzed by Western blots using anti-MMP3 rabbit polyclonal
2.4. Isolation and cloning of EBV LMP1 gene in NPC samples The sequence of NPC EBV LMP1 gene was obtained from Genbank (accession no. X58140). The coding sequence of the LMP1 gene was amplified from an NPC tumor sample using the LMP1-specific primers: forward, 50 -CACACTACCCT GACCATGGAAC-30 ; and reverse, 50 -GGTTAGTCATAGTAG CTTAGC-30 . The PCR products were gel-purified, sequenced and cloned into the expression vector pcDNA3. 2.5. Cell culture and transfection The human lung fibroblasts, MRC5, were obtained from ATCC and the cells were cultured in MEM supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 mg/ml streptomycin. The LMP1-encoded plasmid or its parental counterpart without the LMP1 insert was transiently transfected into MRC5 by Lipofectamine 2000 according to the manufacturer’s protocol.
Fig. 1. Detection of EBV LMP1 gene in patients with nasopharyngeal carcinoma. (A) Tumor samples, no. 1e15, and (B) paired non-tumor tissue samples, no. 10 e150 , were obtained from 15 patients with newly diagnosed nasopharyngeal carcinoma. DNA was isolated and PCR amplification was performed with specific primers for LMP1 (35 cycles, upper panel) and actin (25 cycles, lower panel) as described in Section 2. Samples were resolved by electrophoresis in 1.5% agarose gels. M, 1 kb plus DNA ladder.
Fig. 2. Induction of matrix metalloproteinases in NPC tumor samples. (A) Total cellular RNA was extracted from tumor and adjacent paired non-tumor tissue samples as described in Section 2. Quantitative RT-PCR was performed for measuring the transcription levels of MMP1, MMP2, MMP3 and MMP9. The level of MMPs was normalized to that of the 18S rRNA. Fold induction of MMPs in tumor samples was plotted in the indicated column. Black dots represent the average of triplicate assays of the same tissue sample. The number in each column is the mean of fold induction of each group (* p < 0.001). (B) The level of MMP induction in patients with NPC was compared with that of the levels in 12 patients with other head-and-neck cancers. RT-PCR assay was performed three times independently in each of the RNA samples.
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antibodies (R&D Systems), anti-LMP1 mouse monoclonal antibody (DAKO) and anti-actin goat polyclonal antibodies (Santa Cruz, USA). The level of protein expression was revealed by using ECL solution (Amersham Biosciences, Buckinghamshire, UK). 3. Results 3.1. Detection of EBV LMP1 in nasopharyngeal carcinoma Previous studies have shown that oncogenic EBV LMP1 protein is associated with invasive cancers including nasopharyngeal carcinoma [19,20]. To investigate the tumorigenic role of LMP1 in NPC, we examined in the presence of LMP1 gene in tumor samples from NPC patients with newly diagnosed nasopharyngeal carcinoma by PCR. Histological examinations of the NPC tumor samples have shown undifferentiated carcinoma in all cases. Using EBV LMP1-specific primers, the LMP1 DNA was detected in 13 out of 15 NPC tumor tissues, but not in the adjacent non-tumor samples obtained in paired biopsies (Fig. 1A and B). Our results suggested that with its presence in most NPC samples, LMP1 may play a role in NPC pathogenesis. 3.2. Induction of matrix metalloproteinases in NPC tumor samples LMP1 has been shown to be associated with the enhancement of metastasis in NPC [29]. In addition, LMP1-induced
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expression of MMP1 and MMP9 has been shown to be associated with enhanced invasiveness of cancer cells resulting in tumor metastasis [17,20]. To investigate the induction of MMPs in the NPC tumor samples, we then measured the mRNA levels of MMP1, MMP2, MMP3 and MMP9 in the tumor and non-tumor paired biopsies by Taqman real-time RTPCR. The level of MMPs induction in tumor samples was compared to the corresponding non-tumor sample from the same patient. Our results showed that a significant upregulation of MMP1 and MMP3 was detected in the LMP1-positive biopsies, compared with the paired non-malignant nasopharyngeal tissues (Fig. 2A). In contrast, there was no differential induction of MMP2 and MMP9 between tumor and non-tumor samples (Fig. 2A). To further demonstrate the induction of MMP3 in NPC, we examined the levels of MMP3 mRNA in NPC and other headand-neck cancers. As shown in Fig. 2B, the induction of MMP3 was increased significantly in NPC, compared with other head-and-neck cancers ( p ¼ 0.0001). Consistent to previous studies, upregulation of MMP1 was also detected in the NPC samples ([17]; and our data not shown). 3.3. LMP1 regulates MMPs expression Following the expression profile of MMPs, we next delineated the role of LMP1 in the regulation of MMP3 expression in the tumor samples. We amplified the coding region of the LMP1 gene from a representative NPC tumor and cloned it into the pcDNA3 expression plasmid. The sequence of the
Fig. 3. Immunocytochemical staining of LMP1-expressing cells. The EBV LMP1 DNA was isolated from NPC tumor samples and cloned into the pcDNA3 expression vector. LMP1 protein was transiently expressed in MRC5 human lung fibroblasts using Lipofectamine 2000. (A) Expression of LMP1 was detected using anti-LMP1 monoclonal antibody at 36 h post-transfection. (B) Cells transfected with the parental plasmid only. Nuclei were counterstained with DAPI in cells transfected with the (C) LMP1-encoded or (D) parental plasmid. pcDNA3, parental expression plasmid. Magnification, 400.
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amplified LMP1 gene was compared to the sequence of NPC LMP1 DNA reported in GenBank [30]. Transient expression of LMP1 in MRC5 human lung fibroblasts was shown by immunocytochemical staining and Western blotting (Figs. 3 and 4B). We then analyzed the induction kinetics of endogenous MMPs in MRC5 cells transiently expressing LMP1. Our quantitative RT-PCR results showed that the levels of MMP3 mRNA increased significantly from 20 h to 24 h post-transfection, compared to the mock-transfected cells using the parental plasmid ( p < 0.05; Fig. 4A). In LMP1-expressing cells, the LMP1-induced MMP3 transcription was not blocked by the addition of cycloheximide, indicating that new protein synthesis was not required for the transcriptional activation of MMP3 (data not shown). Consistent to previous report, induction of MMP1 was also detected in our LMP1-expressing cells [17]. In contrast, induction of MMP2 and MMP9 was not found in the LMP1-expressing MRC5 cells (data not shown). Furthermore, expression of MMP3 protein in the LMP1expressing cells was shown by Western blotting at 48 h posttransfection (Fig. 4B) and the level of MMP3 protein increased from 24 h to 48 h post-transfection (Fig. 4C). As controls, LMP1 monoclonal antibody was used to determine the LMP1-expression levels by Western analysis. The input amount of protein samples was further controlled by anti-actin antibodies.
genetic studies have shown that a single adenine insertion/deletion polymorphism (5A/6A) in the MMP3 promoter region may contribute to an increased risk of metastasis among breast cancer patients [36,37]. LMP1 is a transmembrane protein with its C-terminal component containing two important signaling domains, CTAR1 and CTAR2. The interactions of CTAR domains with TRAFs or TRADD result in the activation of AP-1, NF-kB, p38 MAPK, JNK, PI3K and JAK/STAT signaling pathways [38e 41]. Two previous studies have shown that transcription of MMP3 is regulated by cellular factors including AP-1 and Ets [42,43]. Moreover, it has been shown that LMP1-induced MMP1 expression and MMP3 activation are via AP-1 and Ets. However, expression of the latent form of MMP3 was not
4. Discussion Matrix metalloproteinases have been shown to play critical roles in tumor metastasis including tumor-induced angiogenesis, tumor invasion, and establishment of metastatic foci at the secondary sites [14,15,31]. In this study, we found a differential induction of MMP1 and MMP3 transcripts in LMP1-positive NPC tumors comparing with the adjacent non-tumor tissues. With the confirmed induction of MMPs in NPC tumor samples and the reported oncogenic role of LMP1 in EBVassociated diseases, we next investigated the role of EBV LMP1 in the regulation of the MMPs induction in this highly invasive cancer. Previous studies have demonstrated that upregulation of MMP1 and MMP9 expression in LMP1-transfectants resulted in enhancement of the cell invasion in matrix collagen gels [17e20]. A subsequent study showed that aspirin treatment can suppress the MMP9-dependent invasive property of the LMP1-expressing cells [32]. Our results showed that a significant level of MMP3 induction was detected in NPC patients with LMP1-positive tumors, compared with other headand-neck cancer patients (Fig. 2A and B). Like other members of the MMP family, MMP3 can degrade numerous ECM substrates, release cellematrix interaction proteins, activate other MMPs, and inactivate several serine proteinase inhibitors [33]. MMP3 plays a critical role in organogenesis including mammary gland development [34]. However, constitutive or dysregulated expression of MMP3 may contribute to the initial stage of cancer development and tumor progression [16,35]. Furthermore, recent
Fig. 4. Kinetic studies of LMP1-induced MMP3 expression. (A) MRC5 human lung fibroblasts were transfected for transient expression of LMP1 and the level of MMP3 induction was measured by Taqman quantitative RT-PCR at the indicated time points. The values represent the mean SD of triplicates and are statistically analyzed by the two-tailed, paired t-test; * p < 0.001. (B) The expression of the exogenous LMP1 was examined by Western blotting with specific antibodies. MMP3 expression was detected in LMP1-expressing cells by Western blotting at 48 h post-transfection. The amounts of protein loaded on the gel were standardized by using anti-actin antibodies. (C) Kinetics of MMP3 protein expression in LMP1-transfectants using Western blotting. The levels of MMP3 protein increased over a time course of 48 h post-transfection. pcDNA3, parental expression plasmid.
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detected in LMP1-expressing cells [44]. Our results showed that LMP1 induces the expression of MMP3 in human lung fibroblasts (Fig. 4). The discrepancy of these two studies may be due to differences in cellular origins. To further elucidate the functions of MMP3 in tumor progression, we are investigating the roles of MMP3 in the invasive property of the LMP1-transfected cells. In addition, we will further investigate the upstream kinases that are responsible for mediating the LMP1-induced expression of MMPs and cytokines. Taken together, our results have clinical implications and may contribute to the design of novel therapeutics for the amelioration of tumor invasion to normal tissues in NPC and EBV-associated diseases. Acknowledgments This work was supported by grants to AS Lau from HKU CRCG Project Funding (Project no. 200607176145), and to AS Lau and DTT Chua from HKU Clinical Oncology Research Fund. References [1] Raab-Traub N. EpsteineBarr virus in the pathogenesis of NPC. Semin Cancer Biol 2002;12:431e41. [2] Pattle SB, Farrell PJ. The role of EpsteineBarr virus in cancer. Expert Opin Biol Ther 2006;6:1193e205. [3] Young LS, Murray PG. EpsteineBarr virus and oncogenesis: from latent genes to tumours. Oncogene 2003;22:5108e21. [4] Thompson MP, Kurzrock R. EpsteineBarr virus and cancer. Clin Cancer Res 2004;10:803e21. [5] Hadar T, Rahima M, Kahan E, Sidi J, Rakowsky E, Sarov B, et al. Significance of specific EpsteineBarr virus IgA and elevated IgG antibodies to viral capsid antigens in nasopharyngeal carcinoma patients. J Med Virol 1986;20:329e39. [6] Lo YM, Chan LY, Chan AT, Leung SF, Lo KW, Zhang J, et al. Quantitative and temporal correlation between circulating cell-free Epsteine Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 1999;59:5452e5. [7] Lo YM, Chan AT, Chan LY, Leung SF, Lam CW, Huang DP, et al. Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating EpsteineBarr virus DNA. Cancer Res 2000; 60:6878e81. [8] Wei WI, Sham JS. Nasopharyngeal carcinoma. Lancet 2005;365: 2041e54. [9] Rickinson A, Kieff E. EpsteineBarr virus. In: Fields B, Knipe D, Kieff E, editors. Fields virology. Philadelphia: Raven Press; 1996. p. 2397e446. [10] Tsao SW, Tramoutanis G, Dawson CW, Lo AK, Huang DP. The significance of LMP1 expression in nasopharyngeal carcinoma. Semin Cancer Biol 2002;12:473e87. [11] Sengupta S, den Boon JA, Chen IH, Newton MA, Dahl DB, Chen M, et al. Genome-wide expression profiling reveals EBV-associated inhibition of MHC class I expression in nasopharyngeal carcinoma. Cancer Res 2006;66:7999e8006. [12] Liotta LA, Kohn EC. The microenvironment of the tumourehost interface. Nature 2001;411:375e9. [13] Yang J, Mani SA, Weinberg RA. Exploring a new twist on tumor metastasis. Cancer Res 2006;66:4549e52. [14] Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002;2:161e74. [15] Deryugina EI, Quigley JP. Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev 2006;25:9e34.
525
[16] Noe V, Fingleton B, Jacobs K, Crawford HC, Vermeulen S, Steelant W, et al. Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1. J Cell Sci 2001;114:111e8. [17] Lu J, Chua HH, Chen SY, Chen JY, Tsai CH. Regulation of matrix metalloproteinase-1 by EpsteineBarr virus proteins. Cancer Res 2003;63: 256e62. [18] Yoshizaki T, Sato H, Murono S, Pagano JS, Furukawa M. Matrix metalloproteinase 9 is induced by the EpsteineBarr virus BZLF1 transactivator. Clin Exp Metastasis 1999;17:431e6. [19] Wakisaka N, Pagano JS. EpsteineBarr virus induces invasion and metastasis factors. Anticancer Res 2003;23:2133e8. [20] Horikawa T, Yoshizaki T, Sheen TS, Lee SY, Furukawa M. Association of latent membrane protein 1 and matrix metalloproteinase 9 with metastasis in nasopharyngeal carcinoma. Cancer 2000;89: 715e23. [21] Li XP, Li G, Peng Y, Kung HF, Lin MC. Suppression of EpsteineBarr virus-encoded latent membrane protein-1 by RNA interference inhibits the metastatic potential of nasopharyngeal carcinoma cells. Biochem Biophys Res Commun 2004;315:212e8. [22] Eliopoulos AG, Gallagher NJ, Blake SM, Dawson CW, Young LS. Activation of the p38 mitogen-activated protein kinase pathway by EpsteineBarr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production. J Biol Chem 1999; 274:16085e96. [23] Vockerodt M, Haier B, Buttgereit P, Tesch H, Kube D. The EpsteineBarr virus latent membrane protein 1 induces interleukin-10 in Burkitt’s lymphoma cells but not in Hodgkin’s cells involving the p38/SAPK2 pathway. Virology 2001;280:183e98. [24] Esteve PO, Chicoine E, Robledo O, Aoudjit F, Descoteaux A, Potworowski EF, et al. Protein kinase C-zeta regulates transcription of the matrix metalloproteinase-9 gene induced by IL-1 and TNF-alpha in glioma cells via NF-kappa B. J Biol Chem 2002;277:35150e5. [25] Nee LE, McMorrow T, Campbell E, Slattery C, Ryan MP. TNF-alpha and IL-1beta-mediated regulation of MMP-9 and TIMP-1 in renal proximal tubular cells. Kidney Int 2004;66:1376e86. [26] Hosseini M, Rose AY, Song K, Bohan C, Alexander JP, Kelley MJ, et al. IL-1 and TNF induction of matrix metalloproteinase-3 by c-Jun N-terminal kinase in trabecular meshwork. Invest Ophthalmol Vis Sci 2006; 47:1469e76. [27] Lee DC, Cheung CY, Law AH, Mok CK, Peiris M, Lau AS. p38 mitogenactivated protein kinase-dependent hyperinduction of tumor necrosis factor alpha expression in response to avian influenza virus H5N1. J Virol 2005;79:10147e54. [28] Cheung BK, Lee DC, Li JC, Lau YL, Lau AS. A role for double-stranded RNA-activated protein kinase PKR in Mycobacterium-induced cytokine expression. J Immunol 2005;175:7218e25. [29] Yoshizaki T. Promotion of metastasis in nasopharyngeal carcinoma by EpsteineBarr virus latent membrane protein-1. Histol Histopathol 2002;17:845e50. [30] Hu LF, Zabarovsky ER, Chen F, Cao SL, Ernberg I, Klein G, et al. Isolation and sequencing of the EpsteineBarr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma. J Gen Virol 1991;72(Pt 10):2399e409. [31] Stamenkovic I. Matrix metalloproteinases in tumor invasion and metastasis. Semin Cancer Biol 2000;10:415e33. [32] Murono S, Yoshizaki T, Sato H, Takeshita H, Furukawa M, Pagano JS. Aspirin inhibits tumor cell invasiveness induced by EpsteineBarr virus latent membrane protein 1 through suppression of matrix metalloproteinase-9 expression. Cancer Res 2000;60:2555e61. [33] Sternlicht MD, Bissell MJ, Werb Z. The matrix metalloproteinase stromelysin-1 acts as a natural mammary tumor promoter. Oncogene 2000;19:1102e13. [34] Sternlicht MD, Lochter A, Sympson CJ, Huey B, Rougier JP, Gray JW, et al. The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell 1999;98:137e46. [35] Lochter A, Sternlicht MD, Werb Z, Bissell MJ. The significance of matrix metalloproteinases during early stages of tumor progression. Ann N Y Acad Sci 1998;857:180e93.
526
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[36] Ghilardi G, Biondi ML, Caputo M, Leviti S, DeMonti M, Guagnellini E, et al. A single nucleotide polymorphism in the matrix metalloproteinase3 promoter enhances breast cancer susceptibility. Clin Cancer Res 2002;8:3820e3. [37] Krippl P, Langsenlehner U, Renner W, Yazdani-Biuki B, Koppel H, Leithner A, et al. The 5A/6A polymorphism of the matrix metalloproteinase 3 gene promoter and breast cancer. Clin Cancer Res 2004;10:3518e20. [38] Kieser A, Kaiser C, Hammerschmidt W. LMP1 signal transduction differs substantially from TNF receptor 1 signaling in the molecular functions of TRADD and TRAF2. EMBO J 1999;18:2511e21. [39] Gires O, Kohlhuber F, Kilger E, Baumann M, Kieser A, Kaiser C, et al. Latent membrane protein 1 of EpsteineBarr virus interacts with JAK3 and activates STAT proteins. EMBO J 1999;18:3064e73. [40] Eliopoulos AG, Young LS. LMP1 structure and signal transduction. Semin Cancer Biol 2001;11:435e44.
[41] Mainou BA, Everly Jr DN, Raab-Traub N. EpsteineBarr virus latent membrane protein 1 CTAR1 mediates rodent and human fibroblast transformation through activation of PI3K. Oncogene 2005;24:6917e24. [42] Jayaraman G, Srinivas R, Duggan C, Ferreira E, Swaminathan S, Somasundaram K, et al. p300/cAMP-responsive element-binding protein interactions with ets-1 and ets-2 in the transcriptional activation of the human stromelysin promoter. J Biol Chem 1999;274:17342e52. [43] Lewis M, Amento EP, Unemori EN. Transcriptional inhibition of stromelysin by interferon-gamma in normal human fibroblasts is mediated by the AP-1 domain. J Cell Biochem 1999;72:373e86. [44] Kondo S, Wakisaka N, Schell MJ, Horikawa T, Sheen TS, Sato H, et al. EpsteineBarr virus latent membrane protein 1 induces the matrix metalloproteinase-1 promoter via an Ets binding site formed by a single nucleotide polymorphism: enhanced susceptibility to nasopharyngeal carcinoma. Int J Cancer 2005;115:368e76.