Epstein-Barr virus-encoded EBNA1 inhibits the canonical NF-κB pathway in carcinoma cells by inhibiting IKK phosphorylation
© Valentine et al; licensee BioMed Central Ltd. 2010
Received: 9 October 2009
Accepted: 5 January 2010
Published: 5 January 2010
The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-κB.
In this report we demonstrate that EBNA1 inhibits the canonical NF-κB pathway in carcinoma lines by inhibiting the phosphorylation of IKKα/β. In agreement with this observation we find a reduction in the phosphorylation of IκBα and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-κB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-κB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-κB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied.
Inhibition of p65 NF-κB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-κB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-κB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.
Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is associated with both lymphoid and epithelial tumours , including undifferentiated NPC where there is a near 100% association with EBV infection. Whilst the pattern of EBV latent protein expression varies in different tumour types the EBV nuclear antigen, Epstein-Barr nuclear antigen-1 (EBNA1), is expressed in all EBV-associated malignancies due to its indispensable role in the maintenance and replication of the EBV genome via sequence-specific binding to the viral origin of replication, oriP . Furthermore, as a DNA binding protein EBNA1 interacts with viral gene promoters, thereby contributing to the transcriptional regulation of the EBNAs and of latent membrane protein 1 (LMP1) .
In addition to EBNA1's functions that depend on its binding to viral DNA, EBNA1 can also interact with host cell proteins, including the ubiquitin-specific protease USP7 which has been implicated in the destabilisation of p53 by binding with a higher affinity to the same region of USP7 as do p53 and MDM2. This suggests that EBNA1 can protect against either UV- or p53-induced apoptosis . Whilst 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 , other data are not supportive of such a role . Thus, studies using dominant-negative EBNA1 in an LCL with an integrated EBV genome revealed that EBNA1 had no effect on cell growth or cellular gene expression  whilst other work in which EBNA1 was expressed in Akata BL cells previously cleared of EBV infection demonstrated that EBNA1 expression alone is not sufficient to confer tumourigenic potential [8, 9]. However, in support of a role for EBNA1 in carcinogenesis we and others have demonstrated that EBNA1's transcription factor-like functions are not confined to the regulation of viral genes but also extend to the regulation of host cell gene expression. This has been demonstrated in the context of B-cells where EBNA1 has been shown to induce the expression of CD25, RAG1, RAG2 and CCL20 [10–12] whilst in epithelial cells we have established that expression of EBNA1 results in the differential regulation of cellular genes involved in translation, transcription and cell signalling [13, 14]. We have documented that EBNA1 enhances STAT1 expression which sensitises cells to interferon-induced STAT1 activation, modulates signalling in the TGFβ1 pathway, and increases AP-1 activity resulting in the enhancement of host cell mechanisms involved in angiogenesis and metastasis [13, 14]. The mechanism whereby EBNA1 enhances AP-1 activity was determined to be via EBNA1 binding to the promoters of the AP-1 subunits c-Jun and ATF2 . Furthermore, potential EBNA1 binding sites have been found in the promoters of numerous other cellular genes .
An in silico promoter analysis of gene expression microarray data from EBNA1-expressing carcinoma cells revealed that 15% (362 out of 2454) of the promoters of cellular genes differentially regulated by EBNA1 contained NF-κB DNA binding motifs [13, 14] (and unpublished data). It is well established that the EBV-encoded LMP1 activates the NF-κB cascade  and that the EBV-encoded latent membrane protein 2A (LMP2A) inhibits NF-κB activity in carcinoma cell models . However, the pattern of expression of these viral proteins varies in NPC biopsies whilst EBNA1 is always expressed due to its key role in EBV genome maintenance. Furthermore, dysregulation of the NF-κB pathway has been implicated and documented in the pathogenesis of a wide range of cancers . These observations coupled with reports that the functional homologues of EBNA1 (LANA and ORF73, encoded by KSHV and MuHV-4 respectively) inhibit NF-κB activity [18, 19] prompted us to investigate whether EBNA1 also influences NF-κB activity in carcinoma cells and if this may contribute to the development of EBV-associated epithelial cell tumours such as nasopharyngeal carcinoma (NPC).
Cell lines and tissue culture
Ad/AH (a human adenocarcinoma cell line derived from the nasopharynx), Hone1 (an EBV-negative NPC cell line), AGS (a human gastric-carcinoma derived cell line) and derivatives stably expressing EBNA1 at levels comparable to those found in EBV infection, Ad/AH cells stably infected with a recombinant EBV and C666-1 (an EBV-positive cell line derived from an undifferentiated EBV-positive NPC) were cultured as previously described [13, 14]. Neither Ad/AH cells stably infected with EBV or the C666-1 cell line express the EBV-encoded latent membrane protein 1 (LMP1). TNFα and IL-1β (Peprotech, London, UK) were reconstituted in serum-free growth medium to a concentration of 100 ng/ml, and stimulations carried out for 1 hour prior to harvesting.
Luciferase assays and transient transfections
Dual luciferase reporter assays were performed according to the manufacturer's instructions (Promega, Southampton, UK, cat. no. E1980) with cells cultured as previously described . Cells were transfected with the following plasmids using Lipofectamine (Invitrogen, Renfrew, UK) following the manufacturer's instructions: pSG5-EBNA1 , pGL3-basic (Promega), 3 enhancer-ConA (an NF-κB-dependent luciferase reporter construct in which transcription of the firefly luciferase gene is driven by three NF-κB binding sites) , dnEBNA1 (M15 EBNA1 dominant-negative mutant ), and a control Renilla luciferase plasmid (pRL-TK; Promega). All assays were carried out in biological and technical triplicate and are represented as the mean of three independent experiments.
Construction of wild-type and mutant EBNA1 lentivirus vectors
DNA encoding wild-type EBNA1 was excised from pSG5-EBNA1  using EcoRI and BsaHI and end-filled using Klenow DNA polymerase. Adenine nucleotides were added using Taq DNA polymerase and the resulting DNA was ligated into pCR8 (Invitrogen, cat. no. K2500-20), following the manufacturer's instructions. DNA encoding the dGA, dnEBNA1 (d395-450), d8-67, d41-376, d61-83 and d325-376 EBNA1 mutants  was amplified by PCR using the following primers specific to the flanking vector sequences; 5'GCCGGATCCCCCACTGCTTACTGGCTTAT-3' and 5'-GCCGTCGACGGCAAACAACAGATGGCTGGCAA-3'. These were inserted into pCR8 following the manufacturer's instructions. An empty vector control (Vector) was generated by self ligation of pCR8. The vectors derived above were recombined with pLenti6/R4R2/V5-DEST (Invitrogen, cat. no. K591-10 and K5910-00) and pENTR5 containing the human metallothionein II promoter, following the manufacturer's instructions. In the transient transfection experiments presented here zinc stimulation was not required as the metallothionein II promoter was found to exhibit inherent leakiness (data not shown).
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared according to the manufacturer's instructions (Pierce Biotechnology, Illionois, USA, cat. no. 78833) and EMSA analysis was carried out on 5 μg of nuclear protein according to the manufacturer's instructions (LI-COR Biosciences, Cambridge, UK, doc. 982-07487) using an NF-κB consensus probe (sense oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC-3') which was either 5' IRDye700 labelled or unlabelled for cold competition. EMSA gels were analysed and images were captured and quantified using the LI-COR Odyssey infrared laser imaging system. EMSAs were repeated for three independent biological replicates.
Nuclear protein extracts were isolated according to manufacturer's instructions (Active Motif, Rixensart, Belgium, cat. no. 40010). The NF-κB subunits p65 and p50 present in active dimers were measured using the ELISA based TransAM NF-κB family kit (Active Motif, cat. no. 43296) according to the manufacturer's instruction. Data are presented relative to the supplied internal Raji cell lysate control and are represented as the mean of three independent experiments.
RT-PCR, immunoblotting, immunofluorescence and immunohistochemistry
Oligonucleotide primers used in RT-PCR
RT-PCR primer oligonucleotides (5'-3')
Antibodies used in immunoblotting (IM), immunofluorescence (IF) and immunohistochemistry (IHC)
Santa Cruz (sc-7607)
Cell signalling (L570)
Santa Cruz (sc-8330)
Cell signalling (9242)
Cell signalling (3034)
Santa Cruz (sc-372)
Cell signalling (2859)
Cell signalling (2687)
Cell signalling (3031)
Where appropriate, statistical significance was calculated by performing a Sudent's t-test having first determined equal or unequal variance by using an F-test.
EBNA1 represses p65 NF-κB activity in carcinoma cells
EBNA1 inhibits the phosphorylation and nuclear translocation of p65 in carcinoma cells
EBNA1 inhibits the phosphorylation of IκBα and IKKα/β in carcinoma cells
The activation of the canonical NF-κB pathway is tightly regulated by signals such as pro-inflammatory cytokines, that stimulate the IκB kinase complex (IKK) to phosphorylate the inhibitory IκBs which marks them for ubiquitin-mediated degradation. This allows free NF-κB to translocate to the nucleus where it activates target genes. The IKK complex is composed of two catalytic subunits, IKKα and IKKβ, which contain N- terminal serine/threonine kinase domains and a regulatory subunit, IKKγ (NEMO), that does not exhibit kinase activity but is essential for IKK phosphorylation and activation of upstream kinases . Whilst both IKKα and IKKβ cooperate for IκB phosphorylation, IKKβ is indispensable for signalling via the canonical NF-κB pathway .
We then performed immunoblotting to determine whether EBNA1 achieved a reduction in IκBα and p65 phosphorylation by affecting components of the IKK complex. There was a marked reduction in the levels of serine phosphorylation within the activation loops of both IKKα/β, a prerequisite for activation, in Ad/AH and Hone1 cells stably expressing EBNA1, when compared with the neo control cell lines (Fig. 8B). In contrast, there was no appreciable difference in the total levels of IKKα, IKKβ or IKKγ (Fig. 8B) which demonstrated that the reduction in phospho-IKKα/β in EBNA1 expressing Ad/AH and Hone1 cells was not due to a reduction in the expression of these catalytic subunits.
Deletion of EBNA1 domains required for transactivation of EBV encoded genes abrogates the ability of EBNA1 to inhibit NF-κB activity
EBNA1 does not alter the expression of IL-1 or TNF receptors in carcinoma cells
EBNA1 does not inhibit NF-κB activity through down-regulation of pro-inflammatory cytokines or up-regulation of C/EBPβ or A20
We next sought to determine whether EBNA1 inhibited NF-κB activity by down-regulating the expression of IL-1 and TNFα. RT-PCR demonstrated that whilst TNFα and IL-1β expression could not be detected we surprisingly found IL-1α to be up-regulated by EBNA1 in both Ad/AH and Hone1 cells (Fig. 10B and microarray data not shown). This suggested that reduced stimulation of the NF-κB pathway is not responsible for the low levels of NF-κB activity observed in EBNA1 expressing epithelial cells. Intriguingly, however, elevated IL-1α is a marked feature of NPC suggesting that EBNA1 may contribute to this phenotype [29, 30].
The transcription factor C/EBPβ has been implicated in NF-κB inhibition by preventing p65 phosphorylation . RT-PCR revealed that EBNA1 expression in the nasopharyngeal cell lines was associated with C/EBPβ down-regulation and was therefore unlikely to be involved in the mechanism by which EBNA1 inhibited NF-κB activity in epithelial cells (Fig. 10B).
The microarray data used for the in silico promoter analysis that implicated EBNA1 as having a role in NF-κB modulation reported that expression of A20, which inhibits NF-κB machinery up-stream of the IKK complex, was up-regulated 2.5-fold. RT-PCR analysis, however, indicated that A20 was down-regulated in Ad/AH and Hone1 cells stably expressing EBNA1 and was therefore unlikely to be involved in the mechanism by which EBNA1 inhibited NF-κB activity in epithelial cells (Fig. 10B)
p65 is localised to the cytoplasm in NPC tumour cells
NF-κB signalling regulates a variety of major cellular processes, including cell growth, differentiation and apoptosis, and it is therefore not surprising that aberrant NF-κB signalling has been implicated and documented in the pathogenesis of a wide range of cancers. In addition, NF-κB signalling impacts upon both adaptive and innate immunity, the latter being crucial to the ability of host cells to mount effective defences against oncogenic viruses such as EBV . In this study we have demonstrated for the first time that NF-κB activity is repressed by both transient and stable expression of EBNA1 in a number of carcinoma cell lines; confirming that the phenomenon is not merely cell line specific or due to clonal variation. This study therefore reveals that, like its homologues from KSHV and MuHV-4 (LANA and ORF73, respectively) [18, 19], EBNA1 also has a role in inhibiting the NF-κB pathway and that this is therefore most likely a conserved function amongst these gammaherpesvirus nuclear proteins.
Whilst the mechanism for ORF73-mediated NF-κB inhibition has been determined to be via poly-ubiquitination and subsequent proteasomal-dependent nuclear degradation of p65, which is dependent upon a SOCs box motif present in ORF73, the mechanism for LANA-mediated NF-κB repression remains unknown, although it may be similar as LANA also contains a SOCs box [19, 35]. However, examination of EBNA1 reveals no obvious SOCs motif. In contrast, the findings presented here indicate that EBNA1 inhibits the canonical NF-κB pathway in carcinoma lines by inhibiting phosphorylation of the IKK complex upon which several pro-inflammatory signalling cascades converge, enabling EBNA1 to block NF-κB activation in response to a broad range of stimuli.
Whilst we have not fully elucidated the mechanism by which EBNA1 inhibits IKK phosphorylation our data do indicate that deletion of domains of EBNA1 reported to be essential to its ability to transactivate EBV encoded genes  abrogated the ability of EBNA1 to inhibit NF-κB activity, whereas deletion of the domain of EBNA1 known to bind with the cellular deubiquitinylating enzyme USP7  had no effect. These data therefore suggest that the ability of EBNA1 to inhibit IKK phosphorylation is most likely via EBNA1 regulating the expression of a cellular gene(s) involved in this process. Subsequent RT-PCR analysis determined that a reduction in IKKα/β phosphorylation was not likely to be as a result of inhibition of the expression of IL-1 or TNFα receptors or their ligands, or by up-regulation of the NF-κB inhibitors C/EBPβ or A20. Interestingly, RT-PCR analysis indicated that A20 was down regulated in Ad/AH and Hone1 cells stably expressing physiological levels of EBNA1, adding credence to our observations that EBNA1 inhibits NF-κB activity as A20 expression is positively regulated by NF-κB . In addition, preliminary observations suggest that EBNA1 does not bind with or relocalise IKKβ to the nucleus and so it is unlikely that this is the mechanism by which nuclear EBNA1 inhibits the phosphorylation and activity of the IKK complex (Figure S1, Additional file 1). The precise mechanism by which EBNA1 inhibits IKKα/β phosphorylation and NF-κB activity is therefore currently under investigation.
Chronic activation of NF-κB is associated with the development of a number of malignancies. Therefore on face value our observation that EBNA1 inhibits canonical NF-κB would appear counter intuitive with regard to the pathogenesis of NPC. However, within the context of epithelial cells it has been reported that NF-κB activation is growth inhibitory. For example, Seitz et al.  found that the expression of constitutively active p50 and p65 canonical NF-κB subunits in normal epithelial cells resulted in irreversible cell cycle arrest, whereas Gapuzan et al.  reported that p65 knockout fibroblasts have a transformed phenotype. In addition, expression of a dominant-negative IκBα "super repressor" in murine and human epidermis led to hyperplasia and the development of squamous cell carcinoma (SCC) . Furthermore, Dajee et al.  showed in SCC biopsies that p65 staining was predominantly cytosolic.
The ability of EBV-encoded LMP1 to activate both the canonical and non-canonical NF-κB pathway has been the subject of many, mostly in vitro, studies. However it is becoming increasingly evident that the ability of LMP1 to activate the NF-κB cascade in vivo where other EBV latent genes, including EBNA1, are expressed is not so well defined. Recent studies have gone some way to addressing this question by demonstrating in EBV-positive NPC biopsies and xenografts that p65 is located almost exclusively in the cytoplasm and that this is independent of LMP1 expression [32, 33]. In agreement with and expanding upon these data, we have demonstrated that whilst the level of p65 was elevated in 6 out of 11 NPC tumours examined, relative to matched normal control tissue from the same patient, in all cases p65 staining was cytoplasmic. Thornburg et al.  propose that inhibition of p65 in NPC may protect against growth arrest whilst p50/p50 and p50/BCL3 NF-κB could still maintain the tumourigenic effects of NF-κB. It is therefore interesting to speculate that EBNA1 may modulate the ability of LMP1 to activate specific aspects of NF-κB signaling and that this in turn may impact upon the pathogenesis of NPC and other EBV-related tumours. This clearly warrants further investigation.
Our findings suggest that EBNA1 may play a role in the inhibition of p65 NF-κB in NPC and that this could contribute to NPC pathogenesis by inducing tissue hyperplasia. Viruses have evolved an array of mechanisms to overcome the induction of NF-κB as a way of evading the innate immune response [34, 41–43]. We therefore propose that inhibition of canonical NF-κB by EBNA1 may not only contribute to the development of tissue hyperplasia but may also play a role in the pathogenesis of NPC via evasion of host immune responses during early EBV infection.
The EBNA1 M15 mutant was kindly provided by Jean-Claude Nicolas at the Service de Microbiologie, Hopital Rothschild, Paris. We thank Prof. Martin Rowe (School of Cancer Sciences, Cancer Research UK Cancer Centre, University of Birmingham, UK) for his advice and guidance. This work was supported by programme funding from Cancer Research UK and the European Commission's FP6 Life Sciences Health Programme (INCA project LSHC-CT-2005-018704).
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