Epigenetic alterations in nasopharyngeal carcinoma and Epstein-Barr virus
(EBV) associated gastric carcinoma: a lesson in contrasts
Hans Helmut Niller1, Ferenc Banati2, Janos Minarovits3
1Institute of Medical Microbiology and Hygiene, University of Regensburg, Franz-Josef-Strauss Allee 11, D-93053 Regensburg, Germany
2RT-Europe Nonprofit Research Ltd., Pozsonyi u. 88., H-9200 Mosonmagyaróvár, Hungary
3Department of Oral Biology and Experimental Dental Research, University of Szeged, Faculty of Dentistry, H-6720 Szeged, Tisza Lajos krt. 64, Hungary
Corresponding author: Hans Helmut Niller; Email: Hans-Helmut.Niller@klinik.uni-regensburg.de
Abstract: Epstein-Barr virus (EBV) is associated with diverse hematological and epithelial malignancies, such as Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and others. Upon infection of B‑lymphoid and epithelial cells, the virus adopts distinct gene expression patterns which depend on the cellular epigenetic machinery. Moreover, virus infection regularly induces modifications of the viral and host cell transcriptomes and epigenomes through the interaction of viral proteins with cellular epigenetic regulators. Viral latent and immediate early proteins may principally contribute to the reprogramming of the cellular epigenome. While EBV-immortalized B lymphoblastoid cell lines are characterized by a massive hypomethylation of the cellular genome and genome-wide reorganization and loss of heterochromatic histone marks, EBV associated malignancies are characterized by local hypermethylation of CpG islands (CGI) at specific gene sets characteristic for each tumor type. Groups of hypermethylated promoters may represent unique EBV associated epigenetic signatures in EBV-associated gastric carcinomas (EBVaGC) and nasopharyngeal carcinomas (NPC). Here, we review the similarities and differences between EBVaGC and NPC with an emphasis on the epigenetic perspective. Both tumors exhibit a CpG island methylator phenotype (CIMP) and a very high load of hypermethylated tumor suppressor genes, EBVaGC more so than the EBV-negative GC subtypes. However, according to present knowledge, there is only a very small set of hypermethylated gene loci which EBVaGC and NPC have in common. Constructio of whole genome comparative methylomes and genome-wide analysis of further epigenetic marks may illuminate the patho-epigenetics of these EBV-associated carcinomas.
Keywords: chromosomal band, CpG island, epigenetic field, hit and run oncogenesis, hypermethylation, latency, lytic cycle, methylator phenotype, methylome, nitrosamines, oncoprotein, pioneer transcription factor, tumor promoter, tumor suppressor
1.1 Epstein-Barr virus: the first human tumor virus
Epstein-Barr virus (EBV), a gammaherpesvirus infecting humans, has been discovered 50 years ago in cultured cells derived from Burkitt’s lymphoma (BL), an endemic childhood tumor mainly of equatorial Africa . In addition to BL, EBV plays a role in the initiation and progression of other lymphomas, too, and it is the causative agent of early onset posttransplant lymphoproliferative disorder (PTLD) and X-linked lymphoproliferative syndrome (reviewed in [2-4]). Due to its association with BL and its stunning ability to morphologically transform and immortalize B cells to lymphoblastoid cell lines (LCLs) , EBV was considered a purely lymphotropic virus. Finding viral DNA in cellular DNA from biopsies of anaplastic carcinomas of the nasopharynx (NPC) by DNA hybridization did not change that common view, because NPCs contain infiltrating lymphocytes which might have acted as carriers of persisting viral infection . Localizing the virus specifically to the malignant epithelial cells, but not the great many infiltrating lymphocytes, first established EBV infection of non-lymphatic cells and paved the way for the concept of EBV as an epithelial tumor virus [7, 8]. Consequently, the association of EBV with certain carcinomas of epidemiologic importance that develop in the aerodigestive tract was established: the anaplastic, undifferentiated subtype of nasopharyngeal carcinoma (NPC) is endemic in Southeast Asia, Tunisia and among Alaskan Inuit, whereas a subset of gastric carcinomas, EBV-associated gastric carcinoma (EBVaGC), is a sporadic neoplasm located predominantly to the upper stomach [9, 10], (reviewed in [3, 11]).
1.2 Molecular pathogenesis of EBV associated lymphomas
Compared with previous models for BL tumorigenesis which relied on EBV´s transforming functions expressed in latency class III (see below, paragraph 4) [12-14], another conceptual shift was brought about by our discovery of a binding site for the oncoprotein c‑Myc in the locus control region of EBV . A fundamental difference between the pathogenesis of LCL-like tumors on one side, and of primarily malignant EBV associated lymphomas on the other side became obvious: in our molecular model, LCL-like tumors, e.g. early onset PTLD, originate under conditions of severe immune suppression thereby depending on viral latency class III functions (see below), including the growth-transforming function of Epstein-Barr virus nuclear antigens (EBNAs) and latent membrane proteins (LMPs) that are expressed both in PTLDs invivo and in LCLs immortalized in vitro. Primarily malignant EBV associated B cell lymphomas, e.g. BL and Hodgkin lymphoma, originate under conditions of overstimulation of the lymphoid germinal center reaction, while they do not depend on viral latency class III functions which they mostly do not express [15-17]. The postulate of our model for a need to counter-balance the pro-apoptotic force of c‑Myc  through anti-apoptotic functions, either encoded by the viral genome or induced by virus infection, in order for an emerging BL to arise has recently been re-emphasized [19, 20]. Our differential pathogenesis model for EBV-associated lymphomas, although controversial at first [21, 22], (reviewed in ), has gained strong support by recent large scale epigenomic analyses: EBV-induced immortalization caused a massive demethylation of the B-cell genome affecting 2.18 GB of the genome and 1/3 of all genes . Contrary, EBV associated primarily malignant lymphomas are characterized by a local hypermethylation of selected genes in their cellular genomes [25-28].
1.3 Molecular pathogenesis ofEBV associated cancers
Not only primarily malignant EBV associated lymphomas, but also NPC and EBVaGC are monoclonal proliferations of neoplastic cells that carry latent EBV genomes [29-32] and display characteristic epigenetic alterations defined as CpG-island methylator phenotype (CIMP) that results in hypermethylation and silencing of a series of cellular promoters located to genomic regions enriched in CpG dinucleotides (reviewed in [3, 28, 33-37]). Gastric carcinoma (GC) is one of the most frequent cancers and even more so one of the leading causes of cancer-death worldwide. The majority of GC cases is associated with Helicobacter (H.) pylori infection, develop in the lower region of the stomach and belong to the intestinal subtype of common GC. Approximately 10% of GCs worldwide is associated with EBV infection which tends to locate to the upper, non-antral region of the stomach or to post-surgical gastric stump locations, preferentially afflicts men, and belongs to the diffuse subtype of common GC or to the lymphoepithelioma-like GC which histologically resembles NPC. Among gastric remnant carcinomas approximately 30% and among lymphoepithelioma-like GCs, approximately 80% are EBV-associated (reviewed in [34, 35]). Clinically, EBVaGC is currently not treated any differently from EBV-negative GC, however, it is less malignant and has a significantly better prognosis [38, 39]. Altogether, EBVaGC with an estimated more than 80.000 cases per year is the most frequent EBV-associated malignancy worldwide. Contrary to GC which occurs worldwide, NPC is an endemic tumor with a strong preference for South East Asia, especially Guangdong and Hong Kong, with an incidence rate of 20 to 30 cases per 100.000 persons per year. Again contrary to GC, virtually 100% of all nonkeratinizing and undifferentiated NPCs are EBV-associated. Here, we wish to comparatively overview the alternative scenarios of NPC and EBVaGC development with special regard to the genetic and epigenetic alterations that occur during tumorigenesis. We also wished to pinpoint the role or putative role of latent EBV proteins and RNAs in the carcinogenesis models suggested for NPC and EBVaGC.
2. Chromosomal aberrations in phenotypically normal epithelial cells of the nasopharynx: a genetic field for cancerization?
Lo et al. suggested that virological, genetic and environmental factors act in concert during the initiation and progression of NPC . According their collaborative model for NPC tumorigenesis , environmental carcinogens (e.g. nitrosamine from salted fish and preserved food) and local chronic inflammation might elicit DNA damage in histologically normal epithelia, resulting in frequent deletions at chromosome 3p and 9p as it was observed in the Southern Chinese population [40, 41]. The chromosomal deletions could be observed in the dysplastic lesions as well and according to a recent review of the literature the 3p-loss is characteristic for 20-75% of NPCs . Deletion of the short arm of chromosome 3 (3p) affects a series of tumor suppressor genes including RASSF1A (Ras association (RalGDS/AF-6) domain family member 1), a critical tumor suppressor gene located to 3p21.3 . Recently, aberrant methylation of RASSF1A was observed in EBV-negative dysplastic lesions of the nasopharynx that maintained 3p . This observation suggests that silencing of RASSF1A in nasopharyngeal epithelial cells may occur by a mechanism unrelated to the action of EBV LMP1 (latent membrane protein 1), a viral oncoprotein capable to suppress the activity of the RASSF1A. The RASSF1A protein plays an important role in the spatiotemporal regulation of mitosis  and its depletion caused premature activation of the anaphase-promoting complex as well as centrosome abnormalities and multipolar spindles . Thus, one may speculate that in cells with 3p- and 9p-loss or in other epithelial cells of the nasopharynx additional or alternative genetic changes may occur, due to the lack of RASSF1A protein, even before the acquisition of latent EBV genomes. In addition to LMP1, the EBV encoded nuclear antigen EBNA1 may also induce genomic instability in EBV infected host cells .
Based on comparative genomic hybridization (CGH) data, Huang et al. argued that NPC pathogenesis cannot be fully explained by a fixed linear progression model . Instead, based on chromosome abnormalities, NPC could be classified into two groups, with early chromosome imbalances ‒3p26-13 and +12p12 . The 3p-loss subclass could be further divided, there were 3p‒ NPCs with 1q+, 9p‒, 13q‒ or 14q‒, 16q‒, 9q‒, 1p‒ markers .
Loss of 9p affects several tumor suppressor genes including p16, a negative regulator of cyclin D signaling. Tsang et al. demonstrated that early premalignant lesions of nasopharyngeal epithelium overexpress cyclin D1 and this phenomenon may counteract EBV-induced cellular growth arrest and senescence, potentially contributing to a stable latent EBV infection of nasopharyngeal epithelial cells . Thus, 9p-loss, one of the chromosomal alterations characteristic for the genetic field of cancerization in the nasopharynx of an NPC-endemic population may permit, indirectly, stable EBV infection of normal or dysplastic epithelial cells. Alternatively, upregulation of cyclin D1 by other means (e.g. gene amplification) may also favour stable maintenance of latent EBV genomes . After the acquisition of latent EBV genomes, the EBV-encoded latent membrane protein 1 (LMP1) may also contribute to cyclin D1 upregulation by enhancing the activity of cyclin D1 promoter in NPC cells or their precursors . In samples of dysplastic nasopharyngeal mucosa (high grade dysplasia) and in tissue samples with carcinoma in situ clonal proliferations of EBV infected cells were detected by restriction-enzyme analysis of the fused terminal repeats of the viral genome [29, 31], (reviewed in ). By EBER hybridisation, EBV infection could be demonstrated in high grade dysplastic lesions and carcinoma in situ, but in contrast, EBV was absent from samples of normal nasopharyngeal tissue and low-grade dysplastic lesions [40, 41], (reviewed in ). The rare detection of preinvasive neoplasia (0.6%) and preinvasive neoplasia with nasopharyngeal carcinoma (3%) suggested that EBV-induced clonal proliferation can rapidly progress to NPC . It is worthy to note that EBV may enter epithelial cells including nasopharyngeal epithelial cells in complex with antiviral antibodies or via direct contact with EBV-infected B cells [51-53]. Such mechanisms may result in EBV infection of phenotypically normal, genetically or epigenetically unaltered host cells. Cocultivation-mediated EBV infection of a telomerase-immortalized nasopharyngeal epithelial cell line in vitro changed the growth properties and invasiveness of the cells during propagation and increased their resistance to starvation . The cells did not form tumors, however, after inoculation into nude mice. One may speculate that selection mechanisms absent in vitro may facilitate tumorigenesis and neoplastic progression in vivo. The alternative scenarios of NPC development are shown in Figure1.
3. Chromosomal changes in EBV associated gastric cancer
EBV-carrying gastric carcinomas frequently showed loss of chromosome 4p and 11p, unique chromosomal alterations that were absent from EBV-negative GCs, whereas loss of 18q was significantly more frequent in EBV-positive tumors than in their EBV-negative counterparts . Chromosome losses occuring in comparable frequencies in EBVaGC and EBV-negative GC included loss of 17p, 12q, 17q, 1p, 10p and 2q . Others observed gain of chromosome 11 and loss of 15q15 in EBVaGCs . Thus, the typical early chromosomal changes observed in NPC (loss of 3p and 9p) were absent from both EBVaGC and EBV-negative GC.
These data suggest that the genetic background of EBV target cells differs in the nasopharyngeal and gastric epithelium. Thus, 9p-loss and the consequent p16 gene deletion is not a characteristic feature of EBVaGC. Cyclin D1 overexpression may play a role, however, in the establishment of EBVaGC, because BARF1 (BamHI-A rigtward frame 1), a viral oncoprotein expressed in EBVaGC cells, but not in lymhoid cells carrying latent EBV genomes, can upregulate expression of cyclin D1 . Because BARF1 is expressed in nasopharyngeal carcinoma samples as well [57, 58] one may speculate that it may contribute to the upregulation of cyclin D1 expression already in the early, dysplastic and preinvasive lesions of the nasopharynx.
It is also worthy to note, that p16 inactivation by hypermethylation occurs in more than 90% of EBVaGC . Although there is a relationship between aberrant methylation of cellular genes and EBV infection (see below), one may speculate that methylation-mediated silencing of certain cellular genes may occur before the acquisition of latent EBV genomes by phenotypically normal or dysplastic gastric epithelial cells, similarly to the epigenetic field for cancerization  proposed for EBV-negative GCs that are associated with H. pylori infection.
Figure 1. Alternative scenarios for the development of nasopharyngeal carcinoma and EBV-associated gastric carcinoma.
In A)environmental carcinogens (NPC, EBVaGC) in themselves or in combination with mucosal injuries (EBVaGC) establish a genetic field of cancerization characterized by chromosomal alterations unique for the precursor cells of NPC or EBVaGC. Continued exposure to such factors results in premalignant cell populations susceptible for Epstein-Barr virus infection that reprograms the epigenotype of the target cells and alters their behaviour. Additional genetic and epigenetic events drive neoplastic progression (angiogenesis, metastasis). In B), the initial events are similar, but Epstein-Barr virus infection occurs only after the appearance of cells with a malignant phenotype and behaviour.
Similarly to NPC, certain environmental factors may facilitate the development of EBVaGC as well. In addition to high intake of nitrosamines, processed meat products, salt and salted foods, -exposure to metal dust, wood dust and metal filings tended also to be related to the development of EBVaGC [61-64]. These observations suggested that in addition to genotoxic agents, tumor promoters and potential inducers of the EBV lytic cycle, mechanical injuries of the gastric mucosa may also contribute to the development of EBVaGC. Such mechanical lesions include partial gastrectomy that is frequently associated with EBV-positive gastric remnant cancer (gastric stump cancer), as well as gastric ulcer, the latter representing a combination of chemical and mechanical injury [35, 65-67]. Kim et al suggested that mechanical or chemical damage of the stomach mucosa may facilitate EBV infection of gastric epithelial cells . In addition, increased cell proliferation and inflammation following injury may also favour tumorigenesis in the gastric epithelium, similarly to the changes elicited by H. pylori infection associated with the development of EBV-negative GC . The alternative scenarios of EBVaGC development are shown in Figure 1.
4. Epigenetic regulation of latent EBV genomes in EBV- associated carcinomas
EBV can establish latency in various cell types. The expression of the latent viral genome is highly restricted and cell type specific: different host cell types express various combinations of latent EBV proteins and non-translated RNAs corresponding to distinct EBV latency types. The latent proteins include Epstein-Barr virus nuclear antigens (EBNAs) and transmembrane proteins (latent membrane proteins, LMP1, LMP2A, LMP2B) whereas the non-translated RNAs comprise the constitutively expressed EBER1 and EBER2 and two clusters of EBV-encoded microRNAs. Viral gene expression patterns of NPC and EBVaGC correspond to latency classes I, II, or variants thereof. A variable expression of LMP1 is observed in NPC in addition to LMP2A which places the NPC latency pattern in between latency classes I and II [69, 70]. On the other hand, EBVaGC is characterized by a unique, modified latency type I expression pattern which includes the expression of BARF0 and BARF1and a variable expression of LMP2A [71-75], (reviewed in [23, 34]). It is worthy to note that BARF1, a protein expressed during productive EBV replication, is regularly expressed both in NPC and EBVaGC [75, 76]. BARF1 functions as an oncoprotein capable to induce malignant transformation of cells or act in synergy with other oncoproteins [77-79] . BARF1 expression may confer apoptosis resistance to tumor cells [80, 81], (reviewed in [57, 58]).
Out of six EBNAs, NPC and GC express only EBNA1, a DNA binding protein and pleiotropic regulator that has recognition sequences within oriP, the latent origin of EBV DNA replication and at the Q promoter (Qp, located to the BamHI Q fragment of the viral genome) where EBNA1 transcripts are initiated . In addition, EBNA1 also binds to a series of recognition sites present in the host cell DNA [20, 46, 83-88]. EBNA1 transcripts are initiated at Qp both in NPC and EBVaGC cells [30, 74, 89, 90]. Qp is invariably unmethylated independently of its activity, and it is associated with euchromatic histone marks in a nasopharyngeal cell lines actively using Qp [30, 89, 91, 92]. In B lymphoblastoid cell lines the unmethylated Qp is silenced by binding of a cellular repressor protein and it is devoid of activating histones [92-94]
Although both NPC and EBVaGC use Qp to generate EBNA1 transcripts, they differ regarding the activity of the latency promoters controlling LMP1, LMP2A and LMP2B transcription (reviewed in [11, 34]). The LMP1 promoter (LMP1p) and the co-regulated LMP2B promoter was active in the majority of NPC samples but silent in EBVaGC, whereas the distinct LMP2A promoter (LMP2Ap) is active in NPCs and in about half of the EBVaGC samples. CpG methylation plays a role in the silencing of LMP1p in both NPC and EBVaGC [30, 95]. Activation of LMP1p associated with an increased level of acetylated histones at the promoter and an increased overall level of phosphorylated histones in NPC cell lines [96, 97]. Silencing of LMP2Ap is also associated with CpG methylation in a nasopharyngeal carcinoma cell line . Because the linear double stranded EBV genomes are unmethylated at LMP1p and LMP2Ap , one may speculate that de novo methylation of LMP1p and LMP2Ap regulatory sequences in EBV-associated carcinomas occurs after EBV infection of the neoplastic cells or their precursors.
The EBV-encoded non-translated RNAs, EBER1 and EBER2 are the most abundant transcripts in a wide variety of host cells carrying latent EBV genomes. The transcription units of RNA polymerase III-transcribed EBER 1 and EBER2 were hypomethylated both in lymphoid cells and nude mouse passaged NPC lines . EBERs have an antiapoptotic function and induce an autocrine growth factor of NPC cells, insulin-like growth factor 1 (IGF-1) (reviewed in ). They also modulate innate immune responses because secreted EBERs may activate TLR3 (Toll-like receptor 3) that binds double-stranded RNA molecular . In additon, EBER2 was implicated in EBV
induced growth transformation of B cells in vitro.
The majority of EBV-encoded microRNAs that regulate the levels of both viral and cellular mRNA targets are processed from the introns of BamHI A righward transcripts (BARTs) expressed in a wide variety of EBV-infected cell types including NPC and EBVaGC(reviewed in ). They are dispensable for B cell immortalization, but may contribute to carcinogenesis by targeting the mRNAs of pro-apoptotic and tumor suppressor proteins. The promoters directing BART transcription are regulated by DNA methylation [104, 105].
5. The role of EBV encoded oncoproteins in reprogramming of the epigenome in NPC and EBVaGC
Transfection of the LMP1 gene into epithelial cells including an EBV-negative NPC cell line and the subsequent expression of the LMP1 oncoprotein activated cellular DNA methyltransferases and resulted in hypermethylation and silencing of the cellular E-cadherin promoter [106, 107]. In NPC cells the LMP1-mediated activation of the maintenance DNA methyltransferase DNMT1 involved c-Jun NH(2)-terminal kinase signaling and induced the formation of a repressor complex containing the maintenance DNA methyltransferase DNMT1, the de novo DNA methyltransferases DNMT3A and DNMT3B, as well as the methylcytosin-binding protein MeCP2, and the histone deacetylase HDAC1 on
the E-cadherin promoter . In NPC biopsy samples there was a moderate association between the LMP1 expression and DNMT1 expression as assessed by immunohistochemistry  and there was a significant correlation between the presence of EBV genomes and E-cadherin methylation in an independent study . In addition, expression of LMP1 in an EBV-negative nasopharyngeal carcinoma cell line upregulated the activity of DNA methyltransferases DNMT1, DNMT3A and DNMT3B and suppressed the growth-inhibitory effect of retinoic acid by inhibiting the avtivity of the retinoic acid receptor-β2 (RARB2) promoter via DNA methylation . There was a trend toward an increased frequency of hypermethylated cellular genes and LMP1 expression, as assessed by immunohistochemistry, in NPC samples derived from Tunisian patients . It is worthy to note that in Hodgkin lymphoma cell lines LMP1 upregulated Bmi-1, a Polycomb group (PcG) protein involved in gene silencing . It remains to be established whether LMP1 silences cellular genes using a similar mechanism in NPC cells, although there was a correlation between upregulation of EZH2, another PcG protein, and enhanced invasiveness of EBV-negative NPC cell lines . The LMP1 promoter is silent in GC, but LMP2A is expressed in about 50% of gastric carcinomas and may inactivate cellular genes via upregulation of DNMT, similarly to the LMP2A-induced, hypermethylation-mediated silencing of the PTEN promoter in GC cells . Thus, the induction of DNMTs through LMP1 via JNK signaling may be a general mechanism for the formation of repressive promoter complexes in NPCs [106, 107, 109]. On the other hand, the induction of DNMT1 through LMP2A via STAT3 phosphorylation, and the DNMT3A -induction by LMP2A may be a general way to generate promoter hypermethylation in EBVaGC [80, 113].
EBNA1, a DNA binding protein expressed in EBV infected cells independently of the host cell phenotype may also induce epigenetic alterations, similarly to „pioneer” transcription factors that activate and demethylate promoters located to heterochromatic regions and remain associated to mitotic chromatin marking genes for re-expression . In contrast to these expectations, binding of EBNA1 near to the start site of the divergently transcribed gastrokine 1 (GKN1) and gastrokine 2 (GKN2) tumor suppressor genes did not upregulate GKN1 and GKN2 transcription in the gastric carcinoma cell line AGS carrying an EBV bacmid . In contrast, following EBNA1 binding the methylation level of the putative regulatory sequence controlling the bidirectional promoter increased, as judged by methyl-DNA immunoprecipitation. In parallel, GKN1 and GKN2 transcription was repressed, compared to control AGS cells . EBNA1 was able, however, to access its recognition site even if it was organized into a nucleosomal structure , and up-regulated the activity of the survivin promoter by complexing with Sp1 or Sp1-like nuclear proteins in Burkitt lymphoma cells . Affecting cellular gene expression patterns by EBNA1 may certainly contribute to the enhanced tumor and metastasis formation of EBNA1-expressing NPC cells observed in nude mice . EBNA1 expression also increased the tumor forming capacity of gastric carcinoma cells in experimental animals . EBNA1 may affect the progression of NPC by binding to the promoters of c-Jun and ATF2 genes encoding key transcription factors that act as dimers, forming the heterodimeric AP-1 (activator protein 1) transcription factor . Activation of c-Jun and ATF2 transcription by EBNA1 resulted in upregulation of AP-1 targets including interleukin 8 (IL-8), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF1α), which play an important role in angiogenesis. Thus, EBNA1 may act as a transcriptional activator affecting a key step in neoplastic development.
6. Reactivation of latent Epstein-Barr virus genomes: a role in neoplastic progression?
In addition to permanently expressed viral latent proteins, a reactivation of the EBV genome or a limited expression of lytic cycle genes may also induce epigenetic changes, as indicated by the transient viral infection of a human lung carcinoma cell line . Because BZLF1 can bind to highly methylated viral sequences, Woellmer et al. identified it as a pioneer factor . Pioneer factor binding frequently results in local demethylation , a phenomenon observed at EBNA1, but not at BZLF1 binding sites. However, BZLF1 increased histone H3 acetylation at lytic EBV promoters , and induced the cellular early growth response 1 gene (EGR1), coding for a protein essential for mitogenic responses [124, 125]. In addition, BZLF binding to the highly methylated IL-13 promoter induced IL-13 expression, facilitating LCL growth in vitro, and enhanced BZLF1 expression was compatible with lymphoma development in a humanized mouse model . These data suggested a role for BZLF1 in lymphomagenesis.
Similarly to pioneer transcription factors and EBNA1, BZLF1 associated with mitotic chromosomes . In addition, two BZLF1 interacting cellular proteins, the acetyltransferase CBP (CREB-binding protein) and PAX5, a master regulator of B-cell differentiation were translocated and tethered by BZLF1 to the chromosomes, in parallel with an increase of acetylated histone H3 level . BZLF1 did not affect, however, the global CpG methylation pattern and expression of DNA methyltransferases in
NPC cells .
We suggest that early after the infection of host cells the transiently expressed BZLF1 may bind to to a set of inactive, highly methylated cellular promoters. BZLF1 binding may change the chromatin environment of the affected promoters, ensuring their permanent activation even after the cessation of BZLF1 expression. Such an epigenetic hit and run scenario, when the viral genomes are lost, or a hit and hide scenario, when the initially active viral genes are switched off [130, 131] may initiate EBV-mediated oncogenesis, a process promoted and maintained by the expression of viral oncoproteins and non-translated RNAs in the second phase of tumorigenesis. We speculate that initiation, i.e. epigenetic priming by BZLF1 may occur as a common early phase on the road to the development of EBV-associated lymphomas and carcinomas.
As we outlined above, environmental carcinogens (e.g. nitrosamine from salted fish and preserved food) and local chronic inflammation might elicit DNA damage and chromosomal aberrations in phenotypically normal epithelial cells of the nasopharynx , resulting in the formation of a genetic field of cancerization. In addition to Cantonese-style salted fish and preserved food, it was suggested that herbal medicines may also play a role in the initiation and progression of NPC (reviewed in ). The volatile N-nitrosamine compounds present in foodstuffs consumed in NPC endemic regions not only act as carcinogens and mutagens , but may also induce, especially when the exposition is repeated, the EBV lytic cycle or may have a synergistic effect on the activation of productive EBV replication by other chemicals including the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) and n-butyrate, a histone deacetylase inhibitor [134, 135]. One of the N-nitroso compounds, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) enhanced the transcriptional activity of the immediate early EBV promoters BRLF1p and BZLF1p in an EBV-positive nasopharyngeal carcinoma cell line . Activation and co-activation of productive EBV replication by MNNG apparently involved reactive oxygen species (ROS) and a p53-dependent mechanism . Repeated reactivation of productive EBV replication using MNNG, TPA and sodium butyrate caused genomic instability and increased the invasiveness and in vivo growth rate of NPC cells in SCID mice [135, 137]. BGLF5, the EBV-encoded DNase synthesized during the lytic cycle induced the formation of micronuclei and repressed DNA repair in epithelial cells including NPC cells . Thus, one of the lytic cycle EBV proteins could elicit genomic instability, a phenomenon that may facilitate the progression of NPC, provided that the cells survive productive EBV replication.
Recurrent EBV reactivation by N-nitrosamine compounds, phorbol esters or n-butyrate may result in excess production of virions that infect bystander cells initiating thereby new rounds of virus replication or malignant transformation. Such a process may facilitate infection of nasopharyngeal epithelial cells located to the genetic field of cancerization at the initial phase of carcinogenesis. In addition, after the stable acquisition of latent EBV genomes by NPC cells or their precursors, the production of cytokines, chemokines and secreted EBV proteins by neighbouring host cells supporting virus production may influence neoplastic progression in a paracrine manner. Reactivation of latent EBV episomes in a subpopulation of NPC cells or NPC precursors may have a similar, paracrine effect on the non-reactivated tumor or precursor cell population and may dysregulate host immune responses as well. ZEBRA, the immediate-early EBV protein not only activates a series of viral promoters but also induces – by binding to its recognition sequences located to cellular promoters – the transcription of cellular genes as well, resulting in increased local concentrations of interleukin 8 (IL-8), interleukin 10 (IL-10), transforming growth factor-βigh3 (TGF-βigh3) and TGF-β1 [139-141]. IL-8 (also called CXCL8) is a chemoattractant for leukocytes, acts as a growth factor for NPC cells and promotes angiogenesis and metastasis formation [141-143]. It is worthy to note that IL-8, an unfavourable prognostic factor, is upregulated by the EBV latency proteins EBNA1 and LMP1 as well [84, 142, 144, 145]. IL-10 as well as its viral homologue BCRF1 may counter immune defences . Cayrol and Flemington speculated that the EBV BCRF1 protein and the BZLF1-induced cellular TGF-β1 may enhance the production of IgA that facilitates the entry of EBV into epithelial cells .
7. Epigenetic changes in EBV associated gastric carcinoma
GC in general is characterized by a high level of CGI methylation frequently located to promoters of tumor suppressor genes which in the case of H. pylori-associated GC precedes cancerogenesis . This has led T. Ushijima to establish the concept of an “epigenetic field for cancerization” . In the case of EBVaGC, hypermethylation reaches even higher levels than in EBV-negative subtypes, it occurs genome-wide, but non-random, and frequently affects tumor suppressor genes, like CDKN2A (p16) [59, 148], TP73 (p73) , CDH1 (E-cadherin) [150, 151], and many other tumor suppressor genes and CGIs containing so called MINT (methylated in tumor) loci have been established as indicators for a CIMP [152-155]. However, hypermethylation is generally not detected in the surrounding mucosa of an EBVaGC [149, 155]. Thus, contrary to H. pylori-driven tumorigenesis, a preceding epigenetic field is mostly lacking in EBV-driven GC. Recently, MINT2, MINT31 and the tumor suppressor genes CDKN2A (p14ARF, p16INK4A), TP73, and RUNX3 , and in another study, the tumor suppressor genes TP73, BLU, FSD1, BCL7A, MARK1, SCRN1, and NKX3.1 have been found or confirmed as preferentially methylated in EBVaGC . Cellular pathways affected by hypermethylated CGIs include cell cycle regulation, DNA repair, cell adhesion, metastasis, angiogenesis, apoptosis and signal transduction (reviewed in [3, 23, 34]). A comprehensive review of hypermethylated CGIs in GC in general has recently been published .
Cancers of the gastrointestinal tract (GIT) including colorectal
carcinoma and gastric carcinoma (GC) are long known to exhibit an abnormal, mostly decreased expression of cell surface carbohydrate determinants which are normally expressed by GIT epithelia. Bisulfite sequencing of the methylation status of a comprehensive set of more than forty gene loci involved in carbohydrate synthesis in GC tissues from 78 patients revealed the frequent methylation of “glyco-promoters” in general. Hypermethylation of the CGIs at the B4GALNT2 and ST3GAL6 promoters was highly associated with EBVaGC and correlated with transcriptional silencing .
Regarding DNMT1 expression, contradictory results have been reported. While in one study suppression of DNMT RNAs was reported for EBVaGC , DNMT1 protein was found to be increased in EBVaGC in two other studies [159, 160]. DNMT3A could be induced in GC cell culture by LMP2A transfection .
Remarkably, hypermethylation of specific promoters was significantly associated with EBVaGC, but not EBV-negative GC, of genes such as TP73 and the homeobox gene HOXA10. The CGI of the promoter for CDKN2A (p16INK4A, p14ARF) was densely hypermethylated in EBVaGC, whereas hypermethylation patterns in EBV-negative GC were restricted to individual CpGs or variable . GSTP1 silencing by CGI methylation, although rare in GC, was almost exclusively found in EBVaGC . Recently, hypermethylation of RB, WNT5A and SSTR1 has also been preferentially found in EBVaGC.
By Illumina 27K BeadChip analysis of CpG island methylation, GC in general could be classified into three principal epigenotypes. Besides low and high-methylation EBV-negative subtypes, all EBVaGC have been shown to cluster to a third very high-methylation epigenotype. A surplus of 270 genes was found hypermethylated in EBVaGC which were not methylated in the other GC epigenotypes. Using highly stringent conditions, a surplus of 53 genes remained exclusive for the EBVaGC subtype. While PRC target genes were strongly enriched among the genes hypermethylated in the low and high-methylation epigenotypes, they were not enriched among the EBVaGC-specific hypermethylated genes . In GC cell culture, hypermethylation of numerous genes affecting several tumor suppressor pathways could be induced by EBV infection, which could not be attributed to a specific viral latency gene so far [169, 170]. Possibly, combinations of latency genes may be able to trigger CpG-methylation, or else, tumor suppressor genes may be hypermethylated by virus infection itself, implying a misdirected cellular defence mechanism against viral infection [161, 168, 170], (reviewed in ). For a complete comparative list of genes methylated in GC as reported by , see Table 1. Interestingly, TP73 and HOXA10 which previously have been described as highly associated with the EBVaGC subtype [149, 162], are not included in this list. Although TP73 and HOXA10 are de facto highly associated with EBVaGC, both genes did not fulfil the even more stringent requirements of the GC classification , (A. Kaneda, personal communication). Furthermore, the methylation of TFF1 was found to be induced by EBV infection of the already highly methylated GC cell line AGS , but was also not listed. The reason is that TFF1 belongs to the group of low-CpG promoter genes and is also methylated in normal gastric mucosa, thus it was excluded . Altogether, beyond the general tendency to CGI-methylation in GC, EBV may have found specific ways to leave additional epigenomic marks on the host cell as well. How the very high levels of CGI hypermethylation are attained, and how EBV-specific epigenetic marks are established, needs to be examined in greater depth.
8. Epigenetic alterations in nasopharyngeal carcinoma
Tumor suppressor or candidate tumor suppressor genes are regularly hypermethylated also in NPCs , especially in the two EBV associated subtypes which represent the great majority of NPCs [108, 173]. Comprehensive reviews of genes hypermethylated in NPC which are belonging to diverse gene ontology pathways have been provided by  and . Furthermore, tumor suppressor genes frequently hypermethylated in NPC are beeing reported at an almost monthly basis: LTF[174, 175], CMTM5, ADAMTS18, ADAMTS9, MYOCD, KIF1A, NOR1, CDH4, FBLN2, RRAD, PCDH8CYB5R2, CDK10, FEZF2, CACNA2D3, SOX11, HOXA2, ASS1, PTEN. For a comprehensive list of hypermethylated genes see Table 1 and references provided by  and .
In high risk populations, methylation sensitive PCR can be used as a means of early detection of NPC from the blood stream, in addition to elevated anti-EBV IgA antibody titers [194, 195]. Extended and increased levels of promoter hypermethylation also allow the prediction of radio- or chemotherapy resistance, e.g. against taxol  and 13-cis retinoic acid . The expression of the repressive PcG proteins and the increased presence of the repressive histone mark H3K27me3 were indicators for a poor prognosis both for NPC [197, 198], GC  and B cell lymphomas .
9. Comparison between EBVaGC and NPC
The specific role of EBV which is monoclonal in the tumor tissue and the sequential order of events in epithelial carcinogenesis remain to be clarified for both NPC and EBVaGC. Since virtually all tumor cells are EBV infected, EBV infection appears to be an important event, because terminal repeat (TR) analysis of the EBV genomes attested that both cancers are clonal proliferations harbouring a unique, tumor specific TR fragment [29-32, 201]. However, EBV has so far not been detected in normal nasopharyngeal or gastric epithelia, but only in dysplastic epithelia in both the nasopharyngeal and gastric locations [11, 170]. The key events of NPC cancerogenesis appear to be, in that temporal order, deletions at the 3p and 9p chromosomal loci, EBV infection, and hypermethylation at CGIs of multiple tumor suppressor gene loci, i.e. a CIMP is observed. It cannot be excluded, however, that repeated and at first possibly transient EBV infection may be the priming event of NPC cancerogenesis. The long-lasting primary EBV infection of some very young children in Southern China, as compared to Northern China, seems to play an important role in preparing the base for NPC tumorigenesis as well [202-204].
EBVaGC has a significantly better prognosis than EBV-negative GC [38, 39] and lower lymph node involvement [39, 205]. Furthermore, the different morphology of early EBVaGC [206, 207], and the strong tendency to a mutual exclusiveness of EBV association and MLH1 hypermethylation in GC makes EBVaGC a clinical and pathogenetic entity distinct from EBV-negative GC . Generally, EBER1/2 transcripts could not be detected in preneoplastic stages of GC suggesting that EBV could possibly infect only neoplastic gastric cells which would make EBV infection a late event in GC carcinogenesis [208, 209]. However, single cells at the surface of gastric pits of healthy gastric epithelium were sometimes EBV-infected [210, 211]. Although EBVaGC are higher methylated than the negative subtypes, hypermethylation is generally not detected in the surrounding mucosa of an EBVaGC [149, 155]. Thus, contrary to H. pylori-driven tumorigenesis, an epigenetic field is mostly lacking in EBV-driven GC, and EBV-driven epigenomic rearrangement must proceed rather quickly towards GC. It is also worthy to note that a case report by Au et al. documented a 50 day-transit from EBV gastritis to gastric carcinoma and demonstrated the presence of EBV in pre-malignant dysplastic cells, preceding CDH1 methylation in full-blown GC . One may speculate that the concerted action of lytic and latent viral proteins and RNAs may facilitate oncogenesis in vivo.
One important issue is the methylation of the DNA mismatch repair gene MLH1. Microsatellite instability (MSI) was regularly observed in GC , but not in EBVaGC [214, 215]. MSI in GC corresponded with MLH1 hypermethylation [216, 217]. Lack of MSI in EBVaGC corresponded with a complete lack of hypermethylation of the CGIs at the DNA repair genes MLH1 and MSH2 in EBVaGC [59, 153, 154, 218]. Although there have been examples to the contrary [150, 219], the principal mutual exclusiveness of EBVaGC and MLH1-methylation was recently confirmed . Contrary to EBVaGC, MLH1 is hypermethylated in about 40% of undifferentiated NPC which may lead to a MSI-associated mutator phenotype in those cases [220, 221].
We compared the genes identified – based on relatively less stringent criteria – as typically hypermethylated in EBVaGC with the list of genes hypermethylated in NPC and observed that a set of tumor suppressor genes including CDKN2A(p14ARF, p16INK4A)[59, 148, 163, 218, 220-223], CDKN2B (p15)[154, 220-222], TP73[149, 154, 157, 220], CDH1[150, 151, 154, 220, 221, 224, 225], DAPK1[154, 172, 221, 222], PTEN[113, 193], GSTP1[154, 164, 172], ZMYND10 (BLU)[157, 226-228] were hypermethylated both in EBVaGC and NPC, but not in EBV-negative GC.
In contrast, when we compared the highly stringent list of genes methylated in GC which was provided in  with the less stringent list of genes overexpressed or hypermethylated in NPC which was collected over time by many research groups, we found only a small intersection. The anti-apoptotic BCL2 oncogene promoter was generally hypermethylated in GC, while it was overexpressed in the majority of NPCs [229-231]. Thus, the anti-apoptotic requirement for GCs to emerge from the initial tumor stages must be provided by a gene different from BCL2. The metalloprotease gene ADAMTS18 was hypermethylated both in NPCs  and GC in general. ADAMTS18 contains a thrombospondin type 1 (THBS1) motif and its downregulation may contribute to the invasiveness of both tumor types. The THBS1 gene was frequently hypermethylated in NPC and in the high-methylation subgroup of GC, but was not found among the common low-methylation GC markers . Since THBS inhibits angiogenesis and cell migration, THBS1 suppression may contribute to tumor angiogenesis and metastasis. Finally, PTPRG was hypermethylated in NPC and solely in the EBVaGC subgroup with very high-methylation, but not in EBV-negative GCs . PTPRG codes for protein tyrosine phosphatase receptor-type γ. Since PTPRG is involved in cell cycle signaling, its suppression may contribute to the oncogenic transformation in both tumors.
Interestingly, a high proportion of hypermethylated genes listed in Table 1. is located close to the chromosome ends. The tendency towards the chromosome ends is slightly stronger for EBVaGC and NPC than for the EBV-negative GC subtypes. When summarizing hypermethylated genes on the outermost chromosomal bands only (i.e. 35/260 genes, ~13,5%), then seven genes are telomeric for EBV-negative low-methylation GC (AJAP1, 1p36.32; TCERG1L, 10q26.3; NTM, 11q25; GALR1, 18q23; ZNF154, 19p13.43; ZFP28, 19q13.43; NRSN2 (C20orf98), 20p13), eight genes for EBV-negative high-methylation GC, (PRR18 (MGC35308), 6q27; KCNK9, 8q24.3; IGF2-AS, 11p15.5; B3GAT1, 11q25; MMP17, 12q24.33; SCRT2, 20p13; SIRPA (PTPNS1), 20p13; ADAM33, 20p13), eleven genes for EBVaGC (HES6, 2q37.3; DOK7 (FLJ33718), 4p16.3; ZNF696, 8q24.3; ENTPD8, 9q34.3; HBA2, 16p13.3; HBA1, 16p13.3; METRN, 16p13.3; HIC1, 17p13.3; TIMP2, 17q25.3; HMHA1, 19p13.3; MAPK12, 22q13.33), and nine genes for NPC (TP73, 1p36.32; KIF1A, 2q37.3; SCGB3A1 (HIN1), 5q35.3; MGMT, 10q26.3; OPCML, 11q25; CHFR, 12q24.33; CDK10, 16q24.3; RASSF2A, 20p13; CDH4, 20q13.33). Since the oriP for EBV replication contains three functional telomer-like repeats [232, 233], EBV may have a transient affinity towards chromosome ends during its replication cycle.
Thus, although there are differences between both epithelial tumors, and the overlap of hypermethylated genes between both tumors appears to be surprisingly small, there is also common ground between NPC and EBVaGC. A similar highly stringent epigenomic analysis of EBV-associated NPC in comparison with EBV-negative NPC or in comparison with normal nasopharyngeal tissue would be very helpful. Furthermore, a whole genome methylome map of EBV associated NPC as it has been performed for LCLs  or BL  is highly desirable.
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Table 1. List of hypermethylated genes in three epigenotypes of gastric carcinoma and in nasopharyngeal carcinoma.
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