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Cancer Research UK Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK
Correspondence
John D. O'Neil
ONeilJD{at}bham.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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in response to EBNA1 expression, which enhances microtubule formation in an in vitro angiogenesis assay. Furthermore, we confirm elevation of VEGF and the phosphorylated isoforms of c-Jun and ATF2 in NPC biopsies. These findings implicate EBNA1 in the angiogenic process and suggest that this viral protein might directly contribute to the development and aggressively metastatic nature of NPC.
Present address: Laboratory of Cancer Biology, University of Oxford, Room 1501, Women's Centre, John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, UK. 
Three supplementary figures and a supplementary table are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The pattern of EBV latent protein expression differs in these tumours, with the EBV nuclear antigen EBNA1 alone being expressed in Burkitt's lymphoma (BL) while EBNA1 and two membrane proteins (LMP1 and LMP2A/B) are expressed in Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC) (Kieff & Rickinson, 2001; Young & Rickinson, 2004; Raab-Traub, 2002). The consistent expression of EBNA1 in all EBV-related malignancies is a result of the indispensable role that EBNA1 plays in maintenance and replication of the EBV genome via sequence-specific binding to the viral origin of replication, oriP (Raab-Traub, 2002). In addition to the role that EBNA1 plays in viral genome maintenance, it also interacts with viral gene promoters, thereby contributing to the transcriptional regulation of the EBNAs and of LMP1. Recent studies have also demonstrated that EBNA1 can interact with the ubiquitin-specific protease USP7, which has been implicated in the stabilization of p53. Holowaty & Frappier (2004) showed that EBNA1 can bind with a higher affinity to the same region of USP7 as p53 and MDM2 and suggest that, as a consequence, EBNA1 can protect against either UV- or p53-induced apoptosis. A more direct involvement of EBNA1 in carcinogenesis has been suggested by the ability of B-cell-directed EBNA1 expression to produce B-cell lymphomas in transgenic mice (Wilson et al., 1996). However, dominant-negative EBNA1 (dnEBNA1) studies in a lymphoblastoid cell line with an integrated EBV genome revealed no effect of EBNA1 on cell growth or cellular gene expression (Kang et al., 2001). Furthermore, studies in which EBNA1 has been expressed in Akata BL cells previously cleared of EBV infection demonstrated that EBNA1 expression alone is not sufficient to confer tumorigenic potential (Komano et al., 1998; Ruf et al., 2000).
Whilst EBNA1's contribution to the development of EBV-related tumours remains controversial, the ability of EBNA1 to modulate viral gene expression suggested that it was likely that EBNA1 could also influence cellular gene expression. This has been demonstrated in the context of B cells, where EBNA1 has been shown to induce CD25 expression in an EBV-negative HL cell line and to upregulate RAG1 and RAG2 expression in a BL cell line (Srinivas & Sixbey, 1995; Kube et al., 1999). We have recently established that EBNA1 expression in a carcinoma cell line resulted in the upregulation of 113 cellular genes and the downregulation of 49 genes (Wood et al., 2007). Furthermore, validation of a number of these changes revealed that EBNA1 influences the expression of a range of cellular genes, including those involved in translation, transcription and cell signalling. We found that EBNA1 expression enhanced STAT1 expression, which sensitized cells to interferon-induced STAT1 activation and repressed the TGFβ1 signalling pathway. As these data revealed that EBNA1 can influence cellular gene transcription, resulting in effects that may contribute to the development of EBV-associated tumours such as NPC, we sought to determine whether EBNA1 could also modulate cellular gene expression by influencing other key transcriptional pathways, where aberrations have been implicated in oncogenesis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). HMEC-1 cells (Cancer Research UK cell line repository) were cultured in Biocoat Endothelial Cell Growth Environment medium (BD Biosciences) amended with 8 % FCS and 1 % penicillin–streptomycin. All cells were cultured at 37 °C with 5 % CO2 and 20 % O2, unless stated otherwise. Hypoxic growth conditions were achieved by incubating cells at 37 °C with 5 % CO2 and 1 % O2 for 24 h.
Luciferase assays and transient transfection.
Dual luciferase reporter assays were performed according to manufacturer's instructions (Promega) with cells cultured in RPMI 1640 supplemented with 0.5 % FCS, 2 mM L-glutamine, and 1 % penicillin–streptomycin solution (Sigma-Aldrich) throughout. Cells were transfected with the following plasmids using Lipofectamine (Invitrogen) following the manufacturer's instructions: pSG5-EBNA1 (Sample et al., 1992), pSG5-LMP1 (Eliopoulos et al., 1997), pGL3-basic (Promega), AP-1-luciferase reporter (BD Biosciences), interleukin (IL)-8-luc and IL-8-mut reporters (Eliopoulos et al., 1999), c-Jun promoter luciferase reporter (Wei et al., 1998), AP-1 wild-type and mutant decoy oligonucleotides (Ahn et al., 2002), dnEBNA1 (Marechal et al., 1999), dominant-negative c-Jun (dnc-Jun) (Ham et al., 1995) and a control Renilla luciferase plasmid (pRL-TK; Promega). 12-O-tetradecanoyl-phorbal-acetate (TPA) stimulation was achieved by the addition of 60 ng TPA ml–1 for 16 h prior to harvesting. Endothelial growth factor (EGF) stimulation was achieved by the addition of 100 ng EGF ml–1 for 3 h. All assays were carried out in biological and technical triplicate and are represented as the mean of three independent experiments.
Quantitative RT-PCR (qRT-PCR) and immunoblotting analysis.
RNA was extracted using EZ-RNA total RNA isolation kit (Geneflow) and amplification by qRT-PCR was performed using an ABI 7500 real-time PCR machine following standard procedures. qRT-PCR Taqman primer and probe sets for c-Jun, ATF2 and IL-8 were purchased from Applied Biosystems (Hs99999141_s1, Hs00153179_m1 and Hs00174103_m1, respectively). Taqman hypoxia inducible factor (HIF)-1 primers and probe set were as follows: 5'-ATGAACATAAAGTCTGCAACATGGA-3' (forward), 5'-CTGAGGTTGGTTACTGTTGGTATCATATA-3' (reverse) and 5'-TTGCACTGCACAGGCCACATTCAC-3' (probe). Standard immunoblotting procedures (Young & Rickinson, 2004) were used to detect HIF-1 (1 µg mouse MAB1536 ml–1; R&D Systems).
TransAM analysis.
Nuclear protein extracts were isolated following the manufacturer's instruction (Active Motif). The AP-1 subunits present in active AP-1 dimers were measured using the ELISA based TransAM AP-1 family and ATF2 kit (Active Motif) according to the manufacturer's instruction.
Cytokine analysis.
ELISA analysis for secreted IL-8 and vascular endothelial growth factor (VEGF) was performed following the manufacturers' instructions (Sanquin Reagents and R&D Systems, respectively).
Electrophoretic mobility shift assay (EMSA).
Nuclear extracts were prepared and EMSA analysis was carried out on 5 µg nuclear protein according to the manufacturer's instructions (LI-COR Biosciences) using a wild-type AP-1 probe (sense oligonucleotide 5'-IRDye700-CGCTTGATGACTCAGCCGGAA-3') and a mutant AP-1 probe (sense oligonucleotide 5'-IRDye700-CGCTTGATGACTTGGCCGGAA-3'); nucleotides in bold type indicate the AP-1 binding motif. EMSA gels were analysed and images were captured using the LI-COR Odyssey infrared laser imaging system. EMSAs were repeated for three independent biological replicates.
Chromatin immunoprecipitation (ChIP) assays.
ChIP assays were performed following the protocol provided by Upstate Biotechnology (catalogue no. 17-371), using an EBNA1 antibody (chEBNA1), the conditions described by Chau & Lieberman (2004) and a rabbit isotype control antibody (Santa Cruz). Real-time quantitative PCR was performed on ChIP DNA using the Taqman primers and probe sets detailed in Supplementary Table S1 (available in JGV Online) using an ABI 7500 real-time PCR machine. Statistical significance was determined using Student's t-test.
In vitro microtubule formation assay.
Conditioned growth medium was harvested from Ad/AH cells cultured in RPMI 1640 supplemented with 0.5 % FCS, 2 mM L-glutamine and 1 % penicillin–streptomycin solution (Sigma-Aldrich) for 48 h. The microtubule formation assay was performed using HMEC-1 cells resuspended in control (RPMI 1640 supplemented with 5 % FCS, 2 mM L-glutamine and 1 % penicillin–streptomycin solution) or conditioned growth media layered onto Matrigel according to the manufacturer's instructions (BioCoat Endothelial Cell Tube Formation Angiogenesis System; BD Biosciences). Images were captured after 6–10 h of incubation at 37 °C with 5 % CO2 and 20 % O2. The number and length of tubes were determined using AxioVision image analysis software (Zeiss Imaging Solutions). At least 100 tubes were measured over at least three fields of view. Statistical significance was determined using Student's t-test.
Immunohistochemistry.
Sections of paraffin-embedded Chinese NPC biopsies were processed using the agitated low temperature epitope retrieval (ALTER) method (Hussain et al., 2007), using the following primary antibodies: p-c-Jun (mouse monoclonal SC-822, 1 : 100; Santa Cruz), p-ATF2 (mouse monoclonal sc-8398, 1 : 100; Santa Cruz) and VEGF (mouse monoclonal VG-1, 1 : 100; Cancer Research UK). Sections were counterstained with haematoxylin.
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RESULTS |
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; unpublished data) were screened for host cell transcription factor DNA-binding motifs using the promoter analysis and interaction network toolset (PAINT) (Vadigepalli et al., 2003). This in silico approach revealed that 20 % (495 of 2453) of the promoters of cellular genes differentially regulated by EBNA1 contained AP-1-binding sites, including the promoters of IL-8 and VEGF (data not shown). As it had been reported that functional homologues of EBNA1 from Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) and human papillomavirus (HPV) (E2) (An et al., 2002; Behren et al., 2005) modulate AP-1 activity, we set out to determine whether the ability of EBNA1 to influence cellular gene expression was mediated by the AP-1 transcription factor. Initially, we performed luciferase reporter assays in a range of epithelial cells using a synthetic AP-1 reporter. Transient expression of EBNA1 in HEK293, Ad/AH, HONE1 and AGS cells resulted in an increase in AP-1 activity that was comparable to that achieved by TPA stimulation or transfection with LMP1 (Fig. 1a). To assess whether enhanced AP-1 activity required a functional EBNA1, dnEBNA1 was titrated against wild-type EBNA1 expressed at a concentration comparable to that seen during EBV infection in Ad/AH cells, and AP-1 luciferase activity was measured. Increasing doses of dnEBNA1 resulted in almost complete abrogation of the ability of wild-type EBNA1 to enhance AP-1 activity (Fig. 1b). Expression of dnEBNA1 alone did not result in enhanced AP-1 activity (Supplementary Fig. S1).
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). Furthermore, AP-1 activity was assayed in two additional Ad/AH stable EBNA1 clones, yielding results comparable to those presented here (Supplementary Fig. S2, available in JGV Online). In addition, EMSA analysis demonstrated enhanced binding of an AP-1-specific DNA probe with nuclear protein from stable EBNA1-expressing Ad/AH, HONE1 and AGS cells. The degree of enhanced DNA binding in the EBNA1 expressing cells was comparable to that seen upon TPA stimulation of the Neo control cells. In contrast, a mutant AP-1 probe resulted in almost undetectable levels of DNA binding, which demonstrated the high degree of specificity within the assay (Fig. 1d).
EBNA1 enhances IL-8 promoter activity and IL-8 expression in an AP-1-dependent manner in Ad/AH cells
The ability of EBNA1 to enhance AP-1 activity prompted us to examine whether this resulted in changes in cellular gene expression. As microarray analysis had identified upregulation of IL-8, a well-documented target of AP-1, in EBNA1-expressing Ad/AH cells, we performed IL-8 luciferase assays and qRT-PCR to determine whether EBNA1 expression influenced IL-8 expression in an AP-1-dependent manner. Transfection of the stable EBNA1-expressing Ad/AH cells and their Neo counterpart with a wild-type IL-8 luciferase reporter construct revealed a 2.5-fold increase in reporter activity in the EBNA1 cells (Fig. 2a). Furthermore, the ability of EBNA1 to elevate IL-8 reporter activity was attenuated by deletion of the AP-1 site (Fig. 2a). The ability of EBNA1 to elevate IL-8 expression was confirmed using qRT-PCR in Ad/AH cells transiently transfected with increasing doses of EBNA1 (to a maximum of approximately threefold) as well as in Ad/AH cells stably expressing EBNA1 (Fig. 2b). In addition, abrogation of AP-1 activity by transfecting Ad/AH cells with an EBNA1 plasmid and increasing doses of dnc-Jun plasmid demonstrated that the ability of EBNA1 to induce IL-8 transcription was significantly reduced by high doses of dnc-Jun (Fig. 2c).
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), we sought to determine whether EBNA1 could contribute to the secretion of angiogenic cytokines, many of which are regulated by AP-1. ELISA demonstrated that IL-8 secretion was elevated by 1.6-fold and VEGF was elevated by 1.4-fold in the Ad/AH cells stably expressing EBNA1, with a statistical significance of P<0.05 (Fig. 2d). Furthermore, there was a significant (P<0.05) increase in the secretion of IL-8 and VEGF (2.8- and 1.9-fold, respectively) in Ad/AH cells stably infected with recombinant EBV.
EBNA1 enhances the expression of the subunit of HIF-1
The major stimulus that initiates angiogenesis and neovascularization of solid tumours is hypoxia, and a key transcription factor that orchestrates angiogenesis in response to hypoxia is HIF-1 (Choi et al., 2003). We had demonstrated that EBNA1 enhanced the secretion of IL-8 and VEGF, both of which are transcriptional targets of HIF-1, and our microarray data indicated that the HIF-1 subunit HIF-1, a transcriptional target of AP-1, was upregulated in Ad/AH EBNA1 cells. Therefore, we examined whether EBNA1 expression impacted upon the expression of HIF-1. qRT-PCR demonstrated that expression of HIF-1 was enhanced in both the EBNA1-expressing cells and Ad/AH cells stably infected with EBV in response to hypoxic growth conditions (2.25- and 2.36-fold, respectively) (Fig. 3a). Immunoblot analysis validated these findings at the protein level and also indicated that the phenomenon was abrogated by transfection with dnc-Jun (Fig. 3b).
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). In addition we found that total ATF2 protein was elevated by approximately fourfold in the EBNA1-expressing Ad/AH cells (Supplementary Fig. S3, available in JGV Online). In order to determine whether EBNA1 expression influenced the dimer composition of transcriptionally competent AP-1, we used TransAM analysis, which revealed that stable EBNA1 expression in Ad/AH cells resulted in an increase in the amount of phosphorylated c-Jun, JunB, JunD, FosB and ATF2 present in AP-1 dimers bound to the AP-1 probe (a 1.6-, 1.5-, 1.2-, 1.4- and 1.58-fold increase relative to the Neo control cells, respectively) (Fig. 4b). The enrichment of active AP-1 subunits afforded by EBNA1 expression was not observed using a mutated AP-1 probe (data not shown). Furthermore, ChIP analysis using an EBNA1-specific antibody revealed a 4.8- and 7.6-fold and statistically significant enrichment of c-Jun and ATF2 promoter DNA, respectively, relative to the isotype antibody control, indicating that EBNA1 was present at the c-Jun and ATF2 promoters in cells stably expressing EBNA1 (Fig. 4c). No significant enrichment was observed using ChIP qPCR oligonucleotides specific for the GAPDH promoter (negative control) or for the AP-1 subunits JunB, JunD, c-Fos, FosB, Fra1 or Fra2. In addition, transfection of Ad/AH cells stably expressing EBNA1 with a wild-type c-Jun promoter luciferase reporter indicated enhanced reporter activity comparable to that seen upon stimulation of the Neo control cells with EGF (positive control) (Fig. 4d).
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, we wanted to assess whether EBNA1 could contribute to neovascularization in this carcinoma cell model. We therefore assayed the ability of endothelial cells (HMEC-1) to form microtubules in Matrigel when cultured in control medium (5 % FCS as an inducer of microtubule formation) or reduced serum growth medium (0.5 % FCS) harvested from cells after 48 h of incubation (conditioned growth medium). We observed that conditioned growth medium from EBNA1-expressing cells (Fig. 5a, bottom left) stimulated the formation of a more comprehensive network of microtubules when compared with the conditioned medium harvested from Neo control cells (Fig. 5a, top left). Furthermore, the degree of microtubule formation approached that which was seen using conditioned growth medium from Ad/AH cells stably infected with EBV (Fig. 5a, bottom right) or the 5 % FCS control (Fig. 5a, top right).
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). The mean tube length was significantly greater in the control 5 % FCS medium compared with the conditioned medium from the Neo control cells. Conditioned media from both EBV-infected and EBNA1-transfected Ad/AH cells resulted in a significant increase in mean tube length compared with the Neo control, reaching levels similar to that of the FCS control.
VEGF, p-c-Jun and p-ATF2 are elevated in vivo in NPC biopsies
The observation that EBNA1 enhanced the expression of c-Jun and ATF2 in Ad/AH cells and that AP-1 dimers bound to DNA were enriched for the phosphorylated and therefore transcriptionally active isoforms of these subunits led us to examine the expression of p-c-Jun and p-ATF2 in NPC biopsies. In addition, we chose to validate VEGF expression in NPC biopsies, as we had demonstrated that EBNA1 enhanced VEGF secretion in Ad/AH cells. Strong staining in tumour cells was observed in 12 of 19 cases (63 %) for p-c-Jun, 7 of 20 cases (35 %) for p-ATF2 and 12 of 24 cases (50 %) for VEGF, compared with weak/absent staining in the surrounding infiltrate and stroma. Representative examples of the NPC biopsies studied are given in Fig. 6.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Not surprisingly, aberrant AP-1 activity has been implicated in a wide range of cancers where it impacts upon these tightly regulated processes and can therefore contribute to cellular transformation (Hess et al., 2004). In addition, AP-1 complexes are commonly activated during a range of viral infections (Ludwig et al., 2003; Panteva et al., 2003; Tyler et al., 2001), including EBV infection, where LMP1 has been demonstrated to activate c-Jun/AP-1 and ATF2 (Kieser et al., 1997; Eliopoulos et al., 1999). Here, for the first time, we have demonstrated enhanced AP-1 luciferase activity in several epithelial cell lines in response to EBNA1 expression. Furthermore, the enhancement of AP-1 activity in Ad/AH cells stably expressing EBNA1 was not confined to a single EBNA1 clone, confirming that the phenomenon was not merely due to clonal variation. The increase in AP-1 activity afforded by EBNA1 was considered to be substantial as it was comparable to that seen upon TPA stimulation (a potent activator of the AP-1 pathway) and separately by transfection with a plasmid expressing LMP1 (an EBV-encoded protein known to activate the AP-1 pathway). The observation that EBNA1 expression not only enhanced AP-1 luciferase activity but also led to enhanced AP-1 EMSA probe binding in multiple cell lines demonstrated that the ability of EBNA1 to enhance AP-1 activity was not restricted to an individual epithelial cell line or EBNA1-expressing isolate and suggested that the phenomenon could be applicable to epithelial cells in general. The possibility that AP-1 activity was elevated due to EBNA1 causing a general cellular stress response was addressed by transfecting EBNA1-expressing Ad/AH cells with a dnEBNA1 plasmid. This demonstrated that the ability of EBNA1 to enhance AP-1 activity was dependent on domains required for the trans-activation of viral genes and maintenance of the viral episome.
Whilst the classical view is that elevated AP-1 activity is oncogenic, the roles that individual AP-1 subunits play in tumorigenesis are complex, as different dimer combinations influence key cell fate decisions and can be considered as anti-oncogenic or oncogenic in a stimulus- and cell context-specific manner (Eferl & Wagner, 2003). Nevertheless, studies in mice and humans have allowed generalizations to be made regarding the transcriptional potency and relative contributions of dimer combinations to tumorigenesis (Eferl & Wagner, 2003; Hess et al., 2004). We found that stable EBNA1 expression in the Ad/AH cell line resulted in enhanced transcription of c-Jun and ATF2, that active AP-1 dimers bound to AP-1 probes were enriched for the phosphorylated and therefore active forms of c-Jun and ATF2, demonstrated by ChIP assays showing that EBNA1 was present at the promoters of c-Jun and ATF2, and that EBNA1 enhanced the c-Jun promoter luciferase activity. In addition, we observed elevated levels of phosphorylated AP-1 subunits JunB, JunD and FosB in the EBNA1 stable cell line. However, as ChIP analysis revealed that JunB, JunD and FosB promoter DNA was not statistically significantly enriched in EBNA1-expressing cells (though there was a non-significant fourfold enrichment of FosB promoter DNA) it was considered that the regulation of these subunits was likely to be more complex. Therefore, the influence of EBNA1 on the expression or activation of these subunits via mechanisms other than binding at their promoters, for example as a result of the EBNA1-mediated enhancement of general AP-1 activity impacting upon their expression, warrants further investigation. In agreement with our TransAM data, EBNA1 was found not to be present at the promoters of c-Fos, Fra1 or Fra2, which correlated with a lack of enrichment for the transcriptionally active isoforms of these subunits. In addition to the in vitro study, we report strong staining for p-c-Jun in NPC tumour cells from biopsy samples, which is in agreement with recently published observations (Tsai et al., 2006) and, for the first time, we demonstrate strong staining for p-ATF2 in NPC tumour cells. The inability to detect p-ATF2 in all NPC cases may reflect methodological problems (e.g. antigen retrieval, antibody detection sensitivity) or variability between cases that may be relevant to tumour stage and grade.
Though we have not elucidated whether EBNA1 binds directly with promoter DNA or indirectly by associating with other proteins, we nevertheless propose that this promoter binding is the mechanism by which EBNA1 enhances the transcription of these two AP-1 subunits, resulting in elevated AP-1 activity in the NPC cell model studied. However, whilst we observed a correlation between EBNA1 binding at the ATF2 promoter and elevated expression of ATF2 mRNA and protein, it is difficult to draw comparisons between the degree of promoter DNA enrichment and the level of ATF2 expression, as promoter occupancy does not necessarily directly correlate with the magnitude of the resulting changes in gene expression. Similarly, it is difficult to draw comparisons between the strength of EBNA1 binding at the promoter of c-Jun and the resulting enhancement of mRNA expression or reporter activity. It is tempting to draw parallels between the ability of both LMP1 (Eliopoulos et al., 1999) and EBNA1 to enhance the expression and activation of the key AP-1 subunits c-Jun and ATF2, particularly as these components exhibit strong DNA-binding affinity and potent oncogenic potential. Given that JunB, JunD and FosB have all generally been characterized as being antagonists of the oncogenic potential of c-Jun, an understanding of the precise effects of EBNA1 on the balance of AP-1 activity is essential (Eferl & Wagner, 2003).
In order to alleviate the hypoxic conditions commonly found in solid tumours, such as NPC, the oxygen deficit stimulates neovascularization by inducing the release of angiogenic cytokines – a process that is orchestrated by regulating the expression and activity of the -subunit of the HIF-1 transcription factor (Minet et al., 1999; Salceda & Caro, 1997). We demonstrate here that EBNA1 specifically enhances both IL-8 promoter activity and expression in Ad/AH cells. Elevated IL-8 expression has been correlated with enhanced angiogenesis and metastasis and thus a generally more aggressive tumour phenotype in a wide range of solid tumours, including NPC (Yoshizaki et al., 2001; Gokhale et al., 2005). In addition, and in agreement with published data, we have demonstrated strong positive staining for VEGF in a high proportion (50 %) of NPC cases studied (Krishna et al., 2006). Elevated VEGF expression and secretion have been reported to be associated with enhanced angiogenesis and poor prognosis both in NPC cell models and in NPC biopsies (Yoshizaki et al., 2001; Krishna et al., 2006; Qian et al., 2000). Moreover, the regulation of VEGF expression is controlled, at least in part, by AP-1 (Josko & Mazurek, 2004).
The promoter of HIF-1 contains AP-1/ATF2-binding sites, and hypoxia-induced HIF-1 activity has been shown to enhance the expression of IL-8 and VEGF in carcinoma cells (Minet et al., 1999; Choi et al., 2003; Wakisaka et al., 2004). We found that the expression of HIF-1 RNA and protein is elevated by both EBNA1 expression and EBV infection in Ad/AH cells, an effect that is enhanced by hypoxia. This is in agreement with published data reporting that the HIF-1 protein is elevated in NPC (Chan et al., 2007; Wakisaka & Pagano, 2003) and that HIF-1 transcription is elevated in approximately 50 % of gastric cancers, which correlated with VEGF-induced angiogenesis (Ma et al., 2007). In addition, abrogation of the ability of EBNA1 to enhance HIF-1 protein expression using dnc-Jun indicates that HIF-1 expression is regulated by AP-1 in the NPC cell model used in this study. Interestingly, it has recently been demonstrated that KSHV LANA, a functional homologue of EBNA1, interacts directly with the HIF-1 protein, resulting in its nuclear localization in U2OS cells (Cai et al., 2007). Furthermore, LANA leads to accumulation of HIF-1 in B lymphoma tumour cells latently infected with KSHV, by targeting VHL and p53 (both HIF-1 suppressors) for degradation (Cai et al., 2006). Our findings that EBNA1 not only enhances the expression of IL-8, VEGF and HIF-1 but also stimulates enhanced microtubule formation in an in vitro angiogenesis assay adds credence to our hypothesis that EBNA1 plays a role in angiogenesis, and that this role is multi-faceted and could be of greater significance in the natural tumour setting where hypoxic conditions are prevalent.
In light of these observations, we are currently elucidating the exact composition(s) of AP-1 dimers in the Ad/AH cell line and other NPC cell lines. Furthermore, the ability of multiple EBV latent genes to impact upon AP-1 regulation and transcriptional activity suggests that targeting the AP-1 pathway could be of therapeutic value in the treatment of NPC.
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ACKNOWLEDGEMENTS |
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Received 21 April 2008;
accepted 14 July 2008.
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