诊断

Review

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

 

 

Citation: Niller HH, Banati F, Minarovits J. Epigenetic alterations in nasopharyngeal carcinoma and Epstein-Barr virus(EBV) associated gastric carcinoma: a lesson in contrasts. J Nasopharyng Carcinoma, 2014, 1(9): e9. doi:10.15383/jnpc.9.

Competing interests:The authors have declared that no competing interests exist.

Conflict of interest: None.

Copyright:image001.gif2014 By the Editorial Department of Journal of Nasopharyngeal Carcinoma. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 

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. Introduction

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 [1]. 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) [5], 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 [6]. 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 [15]. 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[15]. 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 [18] 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 [23]), 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 [24]. 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 [11]. According their collaborative model for NPC tumorigenesis [11], 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 [42]. 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 [42]. Recently, aberrant methylation of RASSF1A was observed in EBV-negative dysplastic lesions of the nasopharynx that maintained 3p [36]. 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[43]. The RASSF1A protein plays an important role in the spatiotemporal regulation of mitosis [44] and its depletion caused premature activation of the anaphase-promoting complex as well as centrosome abnormalities and multipolar spindles [45]. 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 [46].

Based on comparative genomic hybridization (CGH) data, Huang et al. argued that NPC pathogenesis cannot be fully explained by a fixed linear progression model [47]. Instead, based on chromosome abnormalities, NPC could be classified into two groups, with early chromosome imbalances ‒3p26-13 and +12p12 [47]. The 3p-loss subclass could be further divided, there were 3p‒ NPCs with 1q+, 9p‒, 13q‒ or 14q‒, 16q‒, 9q‒, 1p‒ markers [48].

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 [49]. 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 [11]. 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 [50]. 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 [11]). 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 [11]). 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 [31]. 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 [53]. 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  [54]. Chromosome losses occuring in comparable frequencies in EBVaGC and EBV-negative GC included loss of 17p, 12q, 17q, 1p, 10p and 2q [54]. Others observed gain of chromosome 11 and loss of 15q15 in EBVaGCs [55]. 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 [56]. 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 [59]. 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 [60] 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 [67]. 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 [68]. 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 [82]. 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 [98]. Because the linear double stranded EBV genomes are unmethylated at LMP1p and LMP2Ap [99], 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 [100]. EBERs have an antiapoptotic function and induce an autocrine growth factor of NPC cells, insulin-like growth factor 1 (IGF-1) (reviewed in [101]). They also modulate innate immune responses because secreted EBERs may activate TLR3 (Toll-like receptor 3) that binds double-stranded RNA molecular [101].  In additon,  EBER2  was  implicated in EBV

induced growth transformation of B cells in vitro[102].

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 [103]). 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 [107]. In NPC biopsy samples there was a moderate association between the LMP1 expression and DNMT1 expression as assessed by immunohistochemistry [107] and there was a significant correlation between the presence of EBV genomes and E-cadherin methylation in an independent study [108]. 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 [109]. 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 [110]. It is worthy to note that in Hodgkin lymphoma cell lines LMP1 upregulated Bmi-1, a Polycomb group (PcG) protein involved in gene silencing [111]. 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 [112]. 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 [113]. 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 [114]. 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 [88]. 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 [88]. EBNA1 was able, however, to access its recognition site even if it was organized into a nucleosomal structure [115], and up-regulated the activity of the survivin promoter by complexing with Sp1 or Sp1-like nuclear proteins in Burkitt lymphoma cells [116]. 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 [117]. EBNA1 expression also increased the tumor forming capacity of gastric carcinoma cells in experimental animals [118]. 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 [119]. 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 [120]. Because BZLF1 can bind to highly methylated viral sequences, Woellmer et al. identified it as a pioneer factor [121]. Pioneer factor binding frequently results in local demethylation [122], a phenomenon observed at EBNA1, but not at BZLF1 binding sites. However, BZLF1 increased histone H3 acetylation at lytic EBV promoters [123], 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[126], and enhanced BZLF1 expression was compatible with lymphoma development in a humanized mouse model [127]. These data suggested a role for BZLF1 in lymphomagenesis.

Similarly to pioneer transcription factors and EBNA1, BZLF1 associated with mitotic chromosomes [128]. 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 [128]. BZLF1 did not affect, however, the global CpG methylation pattern and expression of DNA methyltransferases in

NPC cells [129].

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 [11], 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 [132]). The volatile N-nitrosamine compounds present in foodstuffs consumed in NPC endemic regions not only act as carcinogens and mutagens [133], 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 [134]. Activation and co-activation of productive EBV replication by MNNG apparently involved reactive oxygen species (ROS) and a p53-dependent mechanism [136]. 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 [138]. 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 [146]. 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 [139].

 

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 [147]. This has led T. Ushijima to establish the concept of an “epigenetic field for cancerization” [60]. 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) [149], 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 [156], 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 [157]. 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 [37].

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 [158].

Regarding DNMT1 expression, contradictory results have been reported. While in one study suppression of DNMT RNAs was reported for EBVaGC [152], 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 [161].

Remarkably, hypermethylation of specific promoters was significantly associated with EBVaGC, but not EBV-negative GC, of genes such as TP73[149] and the homeobox gene HOXA10[162]. 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 [163]. GSTP1 silencing by CGI methylation, although rare in GC, was almost exclusively found in EBVaGC [164]. Recently, hypermethylation of RB[165], WNT5A[166] and SSTR1[167] 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 [168]. 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 [171]). For a complete comparative list of genes methylated in GC as reported by [168], 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 [168], (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 [169], 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 [170]. 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 [172], 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 [33] and [36]. Furthermore, tumor suppressor genes frequently hypermethylated in NPC are beeing reported at an almost monthly basis: LTF[174, 175], CMTM5[176], ADAMTS18[177], ADAMTS9[178], MYOCD[179], KIF1A[180], NOR1[181], CDH4[182], FBLN2[183], RRAD[184], PCDH8[185]CYB5R2[186], CDK10[187], FEZF2[188], CACNA2D3[189], SOX11[190], HOXA2[191], ASS1[192], PTEN[193]. For a comprehensive list of hypermethylated genes see Table 1 and references provided by [33] and [36].

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 [196] and 13-cis retinoic acid [109]. 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 [199] and B cell lymphomas [200].

 

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 [34]. 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 [212]. 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 [213], 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 [168]. 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 [168] 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 [177] 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 [220]. 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 [225]. 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 [24] or BL [27] is highly desirable.

 

References

1. Epstein MA, Achong BG, Barr YM: Virus particles in cultured lymphoblasts from Burkitt´s Lymphoma. Lancet 1964, 1:702-703.

2. Niller HH, Wolf H, Minarovits J: Epstein-Barr Virus. In: Latency Strategies of Herpesviruses. Edited by Minarovits J, Gonczol E, Valyi-Nagy T. New York: Springer; 2007: 154- 91.

3. Niller HH, Wolf H, Minarovits J: Epigenetic dysregulation of the host cell genome in Epstein-Barr virus-   associated neoplasia. Semin Cancer Biol 2009, 19(3):158-164.

4. Saha A, Robertson ES: Epstein-Barr virus-associated B-cell lymphomas: pathogenesis and clinical outcomes. Clin Cancer Res 2011, 17(10):3056-3063.

5. Henle W, Diehl V, Kohn G, Zur Hausen H, Henle G: Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells. Science 1967, 157(3792):1064-1065.

6. zur Hausen H, Schulte-Holthausen H, Klein G, Henle W, Henle G, Clifford P, Santesson L: EBV DNA in biopsies of Burkitt tumours and anaplastic carcinomas of the nasopharynx. Nature 1970, 228(5276):1056-1058.

7. Wolf H, zur Hausen H, Becker V: EB viral genomes in epithelial nasopharyngeal carcinoma cells. Nat New Biol 1973, 244(138):245-247.

8. Desgranges C, Wolf H, De-The G, Shanmugaratnam K, Cammoun N, Ellouz R, Klein G, Lennert K, Munoz N, Zur Hausen H: Nasopharyngeal carcinoma. X. Presence of Epstein-Barr genomes in separated epithelial cells of tumours in patients from Singapore, Tunisia and Kenya. Int J Cancer 1975, 16(1):7-15.

9. Burke AP, Yen TS, Shekitka KM, Sobin LH: Lymphoepithelial carcinoma of the stomach with Epstein-Barr virus demonstrated by polymerase chain reaction. Mod Pathol 1990, 3(3):377-380.

10. Shibata D, Tokunaga M, Uemura Y, Sato E, Tanaka S, Weiss LM: Association of Epstein-Barr virus with undifferentiated gastric carcinomas with intense lymphoid infiltration. Lymphoepithelioma-like carcinoma. Am J Pathol 1991, 139(3):469-474.

11. Lo KW, Chung GT, To KF: Deciphering the molecular genetic basis of NPC through molecular, cytogenetic, and epigenetic approaches. Semin Cancer Biol 2012, 22(2):79-86.

12. Klein G: In defense of the “old” Burkitt Lymphoma scenario. In: Advances in Viral Oncology. Edited by Klein G. New York: Raven Press; 1987: 207-211.

13. Lenoir GM, Bornkamm GW: Burkitt’s Lymphoma, a human cancer model for the study of the multistep dvelopment of cancer: proposal for a new scenario. In: Advances in Viral Oncology. Edited by Klein G. New York: Raven Press; 1987: 173-206.

14. Rickinson AB, Kieff E: Epstein–Barr virus. In: Fields Virology. Volume 2, 4 edn. Edited by Knipe DM, Howley PM. Philadelphia: Lippincott, Williams & Wilkins; 2001: 2575–2627.

15. Niller HH, Salamon D, Ilg K, Koroknai A, Banati F, Bauml G, Rucker O, Schwarzmann F, Wolf H, Minarovits J: The in vivo binding site for oncoprotein c-Myc in the promoter for Epstein-Barr virus (EBV) encoding RNA (EBER) 1 suggests a specific role for EBV in lymphomagenesis. Med Sci Monit 2003, 9(1):HY1-HY9.

16. Niller HH, Salamon D, Ilg K, Koroknai A, Banati F, Schwarzmann F, Wolf H, Minarovits J: EBV-associated neoplasms: alternative pathogenetic pathways. Med Hypotheses 2004, 62(3):387-391.

17. Niller HH, Salamon D, Banati F, Schwarzmann F, Wolf H, Minarovits J: The LCR of EBV makes Burkitt's lymphoma endemic. Trends Microbiol 2004, 12(11):495-499.

18. Milner AE, Grand RJ, Waters CM, Gregory CD: Apoptosis in Burkitt lymphoma cells is driven by c-myc. Oncogene 1993, 8(12):3385-3391.

19. Mbulaiteye SM: Burkitt Lymphoma: beyond discoveries. Infect Agent Cancer 2013, 8(1):35.

20. Westhoff Smith D, Sugden B: Potential cellular functions of Epstein-Barr Nuclear Antigen 1 (EBNA1) of Epstein-Barr Virus. Viruses 2013, 5(1):226-240.

21. Rossi G, Bonetti F: EBV and Burkitt's lymphoma. N Engl J Med 2004, 350(25):2621.

22. Thorley-Lawson DA: EBV and Burkitt's lymphoma. N Engl J Med 2004, 350(25):2621.

23. Niller HH, Banati F, Ay E, Minarovits J: Epigenetic changes in virus-associated neoplasms. In: Patho-Epigenetics of Disease. Edited by Minarovits J, Niller HH. New York: Springer; 2012: 179-225.

24. Hansen KD, Sabunciyan S, Langmead B, Nagy N, Curley R, Klein G, Klein E, Salamon D, Feinberg AP: Large-scale hypomethylated blocks associated with Epstein-Barr virus-induced B-cell immortalization. Genome Res 2014, 24(2):177-184.

25. Martin-Subero JI, Ammerpohl O, Bibikova M, Wickham-Garcia E, Agirre X, Alvarez S, Bruggemann M, Bug S, Calasanz MJ, Deckert M et al: A comprehensive microarray-based DNA methylation study of 367 hematological neoplasms. PLoS One 2009, 4(9):e6986.

26. Martin-Subero JI, Kreuz M, Bibikova M, Bentink S, Ammerpohl O, Wickham-Garcia E, Rosolowski M, Richter J, Lopez-Serra L, Ballestar E et al: New insights into the biology and origin of mature aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling. Blood 2009, 113(11):2488-2497.

27. Kreck B, Richter J, Ammerpohl O, Barann M, Esser D, Petersen BS, Vater I, Murga Penas EM, Bormann Chung CA, Seisenberger S et al: Base-pair resolution DNA methylome of the EBV-positive Endemic Burkitt lymphoma cell line DAUDI determined by SOLiD bisulfite-sequencing. Leukemia 2013, 27(8):1751-1753.

28. Niller HH, Tarnai Z, Decsi G, Zsedenyi A, Szenthe K, Banati F, Minarovits J: The role of epigenetics in Epstein-Barr virus regulation and pathogenesis. Future Microbiol 2014, in press.

29. Raab-Traub N, Flynn K: The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell 1986, 47(6):883-889.

30. Imai S, Koizumi S, Sugiura M, Tokunaga M, Uemura Y, Yamamoto N, Tanaka S, Sato E, Osato T: Gastric carcinoma: monoclonal epithelial malignant cells expressing Epstein-Barr virus latent infection protein. Proc Natl Acad Sci U S A 1994, 91(19):9131-9135.

31. Pathmanathan R, Prasad U, Sadler R, Flynn K, Raab-Traub N: Clonal proliferations of cells infected with Epstein-Barr virus in preinvasive lesions related to nasopharyngeal carcinoma. N Engl J Med 1995, 333(11):693-698.

32. Gulley ML, Pulitzer DR, Eagan PA, Schneider BG: Epstein-Barr virus infection is an early event in gastric carcinogenesis and is independent of bcl-2 expression and p53 accumulation. Hum Pathol 1996, 27(1):20-27.

33. Li LL, Shu XS, Wang ZH, Cao Y, Tao Q: Epigenetic disruption of cell signaling in nasopharyngeal carcinoma. Chin J Cancer 2011, 30(4):231-239.

34.  Chen JN,  He D,  Tang F,  Shao CK: Epstein -Barr virus-

associated gastric carcinoma: a newly defined entity. J Clin Gastroenterol 2012, 46(4):262-271.

35. Iizasa H, Nanbo A, Nishikawa J, Jinushi M, Yoshiyama H: Epstein-Barr Virus (EBV)-associated gastric carcinoma. Viruses 2012, 4(12):3420-3439.

36. Lo KW, Chung GT, To KF: Acquired genetic and epigenetic alterations in nasopharyngeal carcinoma. In: Nasopharyngeal Carcinoma. Edited by Busson P. New York: Springer; 2013: 61-81.

37. Qu Y, Dang S, Hou P: Gene methylation in gastric cancer. Clin Chim Acta 2013, 424:53-65.

38. Matsunou H, Konishi F, Hori H, Ikeda T, Sasaki K, Hirose Y, Yamamichi N: Characteristics of Epstein-Barr virus-associated gastric carcinoma with lymphoid stroma in Japan. Cancer 1996, 77(10):1998-2004.

39. van Beek J, zur Hausen A, Klein Kranenbarg E, van de Velde CJ, Middeldorp JM, van den Brule AJ, Meijer CJ, Bloemena E: EBV-positive gastric adenocarcinomas: a distinct clinicopathologic entity with a low frequency of lymph node involvement. J Clin Oncol 2004, 22(4):664-670.

40. Chan AS, To KF, Lo KW, Mak KF, Pak W, Chiu B, Tse GM, Ding M, Li X, Lee JC et al: High frequency of chromosome 3p deletion in histologically normal nasopharyngeal epithelia from southern Chinese. Cancer Res 2000, 60(19):5365-5370.

41. Chan AS, To KF, Lo KW, Ding M, Li X, Johnson P, Huang DP: Frequent chromosome 9p losses in histologically normal nasopharyngeal epithelia from southern Chinese. Int J Cancer 2002, 102(3):300-303.

42. Chen J, Fu L, Zhang LY, Kwong DL, Yan L, Guan XY: Tumor suppressor genes on frequently deleted chromosome 3p in nasopharyngeal carcinoma. Chin J Cancer 2012, 31(5):215-222.

43. Man C, Rosa J, Lee LT, Lee VH, Chow BK, Lo KW, Doxsey S, Wu ZG, Kwong YL, Jin DY et al: Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures

and induces chromosomal aberrations in  human epithelial cells.

Oncogene 2007, 26(21):3069-3080.

44. Mathe E: RASSF1A, the new guardian of mitosis. Nat Genet 2004, 36(2):117-118.

45. Song MS, Song SJ, Ayad NG, Chang JS, Lee JH, Hong HK, Lee H, Choi N, Kim J, Kim H et al: The tumour suppressor RASSF1A regulates mitosis by inhibiting the APC-Cdc20 complex. Nat Cell Biol 2004, 6(2):129-137.

46. Gruhne B, Sompallae R, Marescotti D, Kamranvar SA, Gastaldello S, Masucci MG: The Epstein-Barr virus nuclear antigen-1 promotes genomic instability via induction of reactive oxygen species. Proc Natl Acad Sci U S A 2009, 106(7):2313-2318.

47. Huang Z, Desper R, Schaffer AA, Yin Z, Li X, Yao K: Construction of tree models for pathogenesis of nasopharyngeal carcinoma. Genes Chromosomes Cancer 2004, 40(4):307-315.

48. Wu LS: Construction of evolutionary tree models for nasopharyngeal carcinoma using comparative genomic hybridization data. Cancer Genet Cytogenet 2006, 168(2):105-108.

49. Tsang CM, Yip YL, Lo KW, Deng W, To KF, Hau PM, Lau VM, Takada K, Lui VW, Lung ML et al: Cyclin D1 overexpression supports stable EBV infection in nasopharyngeal epithelial cells. Proc Natl Acad Sci U S A 2012, 109(50):E3473-3482.

50. Xu Y, Shi Y, Yuan Q, Liu X, Yan B, Chen L, Tao Y, Cao Y: Epstein-Barr Virus encoded LMP1 regulates cyclin D1 promoter activity by nuclear EGFR and STAT3 in CNE1 cells. J Exp Clin Cancer Res 2013, 32(1):90.

51. Sixbey JW, Yao QY: Immunoglobulin A-induced shift of Epstein-Barr virus tissue tropism. Science 1992, 255(5051):1578-1580.

52. Imai S, Nishikawa J, Takada K: Cell-to-cell contact as an efficient mode of Epstein-Barr virus infection of diverse human epithelial cells. J Virol 1998, 72(5):4371-4378.

53. Tsang CM,  Zhang G,  Seto E,  Takada K, Deng W, Yip YL,

Man C, Hau PM, Chen H, Cao Y et al: Epstein-Barr virus infection in immortalized nasopharyngeal epithelial cells: regulation of infection and phenotypic characterization. Int J Cancer 2010, 127(7):1570-1583.

54. zur Hausen A, van Grieken NC, Meijer GA, Hermsen MA, Bloemena E, Meuwissen SG, Baak JP, Meijer CJ, Kuipers EJ, van den Brule AJ: Distinct chromosomal aberrations in Epstein-Barr virus-carrying gastric carcinomas tested by comparative genomic hybridization. Gastroenterology 2001, 121(3):612-618.

55. Chan WY, Liu Y, Li CY, Ng EK, Chow JH, Li KK, Chung SC: Recurrent genomic aberrations in gastric carcinomas associated with Helicobacter pylori and Epstein-Barr virus. Diagn Mol Pathol 2002, 11(3):127-134.

56. Wiech T, Nikolopoulos E, Lassman S, Heidt T, Schopflin A, Sarbia M, Werner M, Shimizu Y, Sakka E, Ooka T et al: Cyclin D1 expression is induced by viral BARF1 and is overexpressed in EBV-associated gastric cancer. Virchows Arch 2008, 452(6):621-627.

57. Takada K: Role of EBER and BARF1 in nasopharyngeal carcinoma (NPC) tumorigenesis. Semin Cancer Biol 2012, 22(2):162-165.

58. Hoebe EK, Le Large TY, Greijer AE, Middeldorp JM: BamHI-A rightward frame 1, an Epstein-Barr virus-encoded oncogene and immune modulator. Rev Med Virol 2013, 23(6):367-383.

59. Kang GH, Lee S, Kim WH, Lee HW, Kim JC, Rhyu MG, Ro JY: Epstein-Barr virus-positive gastric carcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype-positive gastric carcinoma. Am J Pathol 2002, 160(3):787-794.

60. Ushijima T: Epigenetic field for cancerization. J Biochem Mol Biol 2007, 40(2):142-150.

61. Chen CS, Pignatelli B, Malaveille C, Bouvier G, Shuker D, Hautefeuille A, Zhang RF, Bartsch H: Levels of direct-acting mutagens, total N-nitroso compounds in nitrosated fermented fish products, consumed in a high-risk area for gastric cancer in southern China. Mutat Res 1992, 265(2):211-221.

62. Koriyama C, Akiba S, Minakami Y, Eizuru Y: Environmental factors related to Epstein-Barr virus-associated gastric cancer in Japan. J Exp Clin Cancer Res 2005, 24(4):547-553.

63. Campos FI, Koriyama C, Akiba S, Carrasquilla G, Serra M, Carrascal E, Itoh T, Minakami Y, Eizuru Y: Environmental factors related to gastric cancer associated with Epstein-Barr virus in Colombia. Asian Pac J Cancer Prev 2006, 7(4):633-637.

64. Liu C, Russell RM: Nutrition and gastric cancer risk: an update. Nutr Rev 2008, 66(5):237-249.

65. Yamamoto N, Tokunaga M, Uemura Y, Tanaka S, Shirahama H, Nakamura T, Land CE, Sato E: Epstein-Barr virus and gastric remnant cancer. Cancer 1994, 74(3):805-809.

66. Nishikawa J, Yanai H, Hirano A, Okamoto T, Nakamura H, Matsusaki K, Kawano T, Miura O, Okita K: High prevalence of Epstein-Barr virus in gastric remnant carcinoma after Billroth-II reconstruction. Scand J Gastroenterol 2002, 37(7):825-829.

67. Kim RH, Chang MS, Kim HJ, Song KS, Kim YS, Choi BY, Kim WH: Medical history and lifestyle factors contributing to Epstein-Barr virus-associated gastric carcinoma and conventional gastric carcinoma in Korea. Anticancer Res 2010, 30(6):2469-2475.

68. Bechi P, Balzi M, Becciolini A, Maugeri A, Raggi CC, Amorosi A, Dei R: Helicobacter pylori and cell proliferation of the gastric mucosa: possible implications for gastric carcinogenesis. Am J Gastroenterol 1996, 91(2):271-276.

69. Brooks L, Yao QY, Rickinson AB, Young LS: Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. J Virol 1992, 66(5):2689-2697.

70. Busson P, McCoy R, Sadler R, Gilligan K, Tursz T, Raab-Traub N: Consistent transcription of the Epstein-Barr virus LMP2 gene in nasopharyngeal carcinoma. J Virol 1992, 66(5):3257-3262.

71. Hitt MM, Allday MJ, Hara T, Karran L, Jones MD, Busson P, Tursz T, Ernberg I, Griffin BE: EBV gene expression in an NPC-related tumour. EMBO J 1989, 8(9):2639-2651.

72. Gilligan KJ, Rajadurai P, Lin JC, Busson P, Abdel-Hamid M, Prasad U, Tursz T, Raab-Traub N: Expression of the Epstein-Barr virus BamHI A fragment in nasopharyngeal carcinoma: evidence for a viral protein expressed in vivo. J Virol 1991, 65(11):6252-6259.

73. Sadler RH, Raab-Traub N: Structural analyses of the Epstein-Barr virus BamHI A transcripts. J Virol 1995, 69(2):1132-1141.

74. Sugiura M, Imai S, Tokunaga M, Koizumi S, Uchizawa M, Okamoto K, Osato T: Transcriptional analysis of Epstein-Barr virus gene expression in EBV-positive gastric carcinoma: unique viral latency in the tumour cells. Br J Cancer 1996, 74(4):625-631.

75. zur Hausen A, Brink AA, Craanen ME, Middeldorp JM, Meijer CJ, van den Brule AJ: Unique transcription pattern of Epstein-Barr virus (EBV) in EBV-carrying gastric adenocarcinomas: expression of the transforming BARF1 gene. Cancer Res 2000, 60(10):2745-2748.

76. Decaussin G, Sbih-Lammali F, de Turenne-Tessier M, Bouguermouh A, Ooka T: Expression of BARF1 gene encoded by Epstein-Barr virus in nasopharyngeal carcinoma biopsies. Cancer Res 2000, 60(19):5584-5588.

77. Wei MX, Ooka T: A transforming function of the BARF1 gene encoded by Epstein-Barr virus. EMBO J 1989, 8(10):2897-2903.

78. Sheng W, Decaussin G, Sumner S, Ooka T: N-terminal domain of BARF1 gene encoded by Epstein-Barr virus is essential for malignant transformation of rodent fibroblasts and activation of BCL-2. Oncogene 2001, 20(10):1176-1185.

79. Jiang R, Cabras G, Sheng W, Zeng Y, Ooka T: Synergism of BARF1 with Ras induces malignant transformation in primary primate epithelial cells and human nasopharyngeal epithelial cells. Neoplasia 2009, 11(9):964-973.

80. Wang Q, Tsao SW, Ooka T, Nicholls JM, Cheung HW, Fu S, Wong YC, Wang X: Anti-apoptotic role of BARF1 in gastric cancer cells. Cancer Lett 2006, 238(1):90-103.

81. Seto E, Ooka T, Middeldorp J, Takada K: Reconstitution of nasopharyngeal carcinoma-type EBV infection induces tumorigenicity. Cancer Res 2008, 68(4):1030-1036.

82. Frappier L: Contributions of Epstein-Barr nuclear antigen 1 (EBNA1) to cell immortalization and survival. Viruses 2012, 4(9):1537-1547.

83. Wood VH, O'Neil JD, Wei W, Stewart SE, Dawson CW, Young LS: Epstein-Barr virus-encoded EBNA1 regulates cellular gene transcription and modulates the STAT1 and TGFbeta signaling pathways. Oncogene 2007, 26(28):4135-4147.

84. O'Neil JD, Owen TJ, Wood VH, Date KL, Valentine R, Chukwuma MB, Arrand JR, Dawson CW, Young LS: Epstein-Barr virus-encoded EBNA1 modulates the AP-1 transcription factor pathway in nasopharyngeal carcinoma cells and enhances angiogenesis in vitro. J Gen Virol 2008, 89(Pt 11):2833-2842.

85. Canaan A, Haviv I, Urban AE, Schulz VP, Hartman S, Zhang Z, Palejev D, Deisseroth AB, Lacy J, Snyder M et al: EBNA1 regulates cellular gene expression by binding cellular promoters. Proc Natl Acad Sci U S A 2009, 106(52):22421-22426.

86. d'Herouel AF, Birgersdotter A, Werner M: FR-like EBNA1 binding repeats in the human genome. Virology 2010, 405(2):524-529.

87. Lu F, Wikramasinghe P, Norseen J, Tsai K, Wang P, Showe L, Davuluri RV, Lieberman PM: Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1). Virol J 2010, 7:262.

88. Lu F, Tempera I, Lee HT, Dewispelaere K, Lieberman PM: EBNA1 binding and epigenetic regulation of gastrokine tumor suppressor genes in gastric carcinoma cells. Virol J 2014, 11:12.

89. Tao Q, Robertson KD, Manns A, Hildesheim A, Ambinder RF: The Epstein-Barr virus major latent promoter Qp is constitutively active, hypomethylated, and methylation sensitive. J

Virol 1998, 72(9):7075-7083.

90. Luo B, Wang Y, Wang XF, Liang H, Yan LP, Huang BH, Zhao P: Expression of Epstein-Barr virus genes in EBV-associated gastric carcinomas. World J Gastroenterol 2005, 11(5):629-633.

91. Bakos A, Banati F, Koroknai A, Takacs M, Salamon D, Minarovits-Kormuta S, Schwarzmann F, Wolf H, Niller HH, Minarovits J: High-resolution analysis of CpG methylation and in vivo protein-DNA interactions at the alternative Epstein-Barr virus latency promoters Qp and Cp in the nasopharyngeal carcinoma cell line C666-1. Virus Genes 2007, 35(2):195-202.

92. Fejer G, Koroknai A, Banati F, Gyory I, Salamon D, Wolf H, Niller HH, Minarovits J: Latency type-specific distribution of epigenetic marks at the alternative promoters Cp and Qp of Epstein-Barr virus. J Gen Virol 2008, 89(Pt 6):1364-1370.

93. Zhang L, Pagano JS: Interferon regulatory factor 2 represses the Epstein-Barr virus BamHI Q latency promoter in type III latency. Mol Cell Biol 1999, 19(4):3216-3223.

94. Salamon D, Takacs M, Ujvari D, Uhlig J, Wolf H, Minarovits J, Niller HH: Protein-DNA binding and CpG methylation at nucleotide resolution of latency-associated promoters Qp, Cp, and LMP1p of Epstein-Barr virus. J Virol 2001, 75(6):2584-2596.

95. Hu LF, Minarovits J, Cao SL, Contreras-Salazar B, Rymo L, Falk K, Klein G, Ernberg I: Variable expression of latent membrane protein in nasopharyngeal carcinoma can be related to methylation status of the Epstein-Barr virus BNLF-1 5'-flanking region. J Virol 1991, 65(3):1558-1567.

96. Nishikawa J, Kis LL, Liu A, Zhang X, Takahara M, Bandobashi K, Kiss C, Nagy N, Okita K, Klein G et al: Upregulation of LMP1 expression by histone deacetylase inhibitors in an EBV carrying NPC cell line. Virus Genes 2004, 28(1):121-128.

97. Li B, Huang G, Zhang X, Li R, Wang J, Dong Z, He Z: Increased phosphorylation of histone H3 at serine 10 is involved in Epstein-Barr virus latent membrane protein-1-induced carcinogenesis of nasopharyngeal carcinoma. BMC Cancer 2013,

13:124.

98. Gerle B, Koroknai A, Fejer G, Bakos A, Banati F, Szenthe K, Wolf H, Niller HH, Minarovits J, Salamon D: Acetylated histone H3 and H4 mark the upregulated LMP2A promoter of Epstein-Barr virus in lymphoid cells. J Virol 2007, 81(23):13242-13247.

99. Fernandez AF, Rosales C, Lopez-Nieva P, Grana O, Ballestar E, Ropero S, Espada J, Melo SA, Lujambio A, Fraga MF et al: The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer. Genome Res 2009, 19(3):438-451.

100. Minarovits J, Hu LF, Marcsek Z, Minarovits-Kormuta S, Klein G, Ernberg I: RNA polymerase III-transcribed EBER 1 and 2 transcription units are expressed and hypomethylated in the major Epstein-Barr virus-carrying cell types. J Gen Virol 1992, 73 ( Pt 7):1687-1692.

101. Iwakiri D, Takada K: Role of EBERs in the pathogenesis of EBV infection. Adv Cancer Res 2010, 107:119-136.

102. Wu Y, Maruo S, Yajima M, Kanda T, Takada K:

Epstein-Barr virus (EBV)-encoded RNA 2 (EBER2) but not EBER1 plays a critical role in EBV-induced B-cell growth transformation. J Virol 2007, 81(20):11236-11245.

103. Marquitz AR, Mathur A, Shair KH, Raab-Traub N: Infection of Epstein-Barr virus in a gastric carcinoma cell line induces anchorage independence and global changes in gene expression. Proc Natl Acad Sci U S A 2012, 109(24):9593-9598.

104. de Jesus O, Smith PR, Spender LC, Elgueta Karstegl C, Niller HH, Huang D, Farrell PJ: Updated Epstein-Barr virus (EBV) DNA sequence and analysis of a promoter for the BART (CST, BARF0) RNAs of EBV. J Gen Virol 2003, 84(Pt 6):1443-1450.

105. Kim DN, Song YJ, Lee SK: The role of promoter methylation in Epstein-Barr virus (EBV) microRNA expression in EBV-infected B cell lines. Exp Mol Med 2011, 43(7):401-410.

106. Tsai CN, Tsai CL, Tse KP, Chang HY, Chang YS: The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases. Proc Natl Acad Sci U S A 2002, 99(15):10084-10089.

107. Tsai CL, Li HP, Lu YJ, Hsueh C, Liang Y, Chen CL,

Tsao SW, Tse KP, Yu JS, Chang YS: Activation of DNA methyltransferase 1 by EBV LMP1 Involves c-Jun NH(2)-terminal kinase signaling. Cancer Res 2006, 66(24):11668-11676.

108. Niemhom S, Kitazawa S, Kitazawa R, Maeda S, Leopairat J: Hypermethylation of epithelial-cadherin gene promoter is associated with Epstein-Barr virus in nasopharyngeal carcinoma. Cancer Detect Prev 2008, 32(2):127-134.

109. Seo SY, Kim EO, Jang KL: Epstein-Barr virus latent membrane protein 1 suppresses the growth-inhibitory effect

of retinoic acid by inhibiting retinoic acid receptor-beta2 expression via DNA methylation. Cancer Lett 2008, 270(1):66-76.

110. Challouf S, Ziadi S, Zaghdoudi R, Ksiaa F, Ben Gacem R, Trimeche M: Patterns of aberrant DNA hypermethylation in nasopharyngeal carcinoma in Tunisian patients. Clin Chim Acta 2012, 413(7-8):795-802.

111. Dutton A, Woodman CB, Chukwuma MB, Last JI, Wei W, Vockerodt M, Baumforth KR, Flavell JR, Rowe M, Taylor AM et al: Bmi-1 is induced by the Epstein-Barr virus oncogene LMP1 and regulates the expression of viral target genes in Hodgkin lymphoma cells. 2007, 109(6):2597-2603.

112. Ma R, Wei Y, Huang X, Fu R, Luo X, Zhu X, Lei W, Fang J, Li H, Wen W: Inhibition of GSK 3beta activity is associated with excessive EZH2 expression and enhanced tumour invasion in nasopharyngeal carcinoma. PLoS One 2013, 8(7):e68614.

113. Hino R, Uozaki H, Murakami N, Ushiku T, Shinozaki A, Ishikawa S, Morikawa T, Nakaya T, Sakatani T, Takada K et al: Activation of DNA methyltransferase 1 by EBV latent membrane protein 2A leads to promoter hypermethylation of PTEN gene in gastric carcinoma. Cancer Res 2009, 69(7):2766-2774.

114. Niller HH, Minarovits J: Similarities between the Epstein-Barr Virus (EBV) Nuclear Protein EBNA1 and the Pioneer Transcription Factor FoxA: Is EBNA1 a "Bookmarking" Oncoprotein that Alters the Host Cell Epigenotype? Pathogens 2012, 1(1):37-51.

115. Avolio-Hunter TM, Lewis PN, Frappier L: Epstein-Barr nuclear antigen 1 binds and destabilizes nucleosomes at the viral origin of latent DNA replication. Nucleic Acids Res 2001, 29(17):3520-3528.

116. Lu J, Murakami M, Verma SC, Cai Q, Haldar S, Kaul R, Wasik MA, Middeldorp J, Robertson ES: Epstein-Barr Virus nuclear antigen 1 (EBNA1) confers resistance to apoptosis in EBV-positive B-lymphoma cells through up-regulation of survivin. Virology 2011, 410(1):64-75.

117. Sheu LF, Chen A, Meng CL, Ho KC, Lee WH, Leu FJ, Chao CF: Enhanced malignant progression of nasopharyngeal carcinoma cells mediated by the expression of Epstein-Barr nuclear antigen 1 in vivo. J Pathol 1996, 180(3):243-248.

118. Cheng TC, Hsieh SS, Hsu WL, Chen YF, Ho HH, Sheu LF: Expression of Epstein-Barr nuclear antigen 1 in gastric carcinoma cells is associated with enhanced tumorigenicity and reduced cisplatin sensitivity. Int J Oncol 2010, 36(1):151-160.

119. Yip YL, Pang PS, Deng W, Tsang CM, Zeng M, Hau PM, Man C, Jin Y, Yuen AP, Tsao SW: Efficient immortalization of primary nasopharyngeal epithelial cells for EBV infection study. PLoS One 2013, 8(10):e78395.

120. Queen KJ, Shi M, Zhang F, Cvek U, Scott RS: Epstein-Barr virus-induced epigenetic alterations following transient infection. Int J Cancer 2013, 132(9):2076-2086.

121. Woellmer A, Arteaga-Salas JM, Hammerschmidt W: BZLF1 governs CpG-methylated chromatin of Epstein-Barr Virus reversing epigenetic repression. PLoS Pathog 2012, 8(9):e1002902.

122. Zaret KS, Watts J, Xu J, Wandzioch E, Smale ST, Sekiya T: Pioneer factors, genetic competence, and inductive signaling: programming liver and pancreas progenitors from the endoderm. Cold Spring Harb Symp Quant Biol 2008, 73:119-126.

123. Ramasubramanyan  S,  Osborn K,  Flower K,  Sinclair AJ:

Dynamic chromatin environment of key lytic cycle regulatory regions of the Epstein-Barr virus genome. J Virol 2012, 86(3):1809-1819.

124. Chang Y, Lee HH, Chen YT, Lu J, Wu SY, Chen CW, Takada K, Tsai CH: Induction of the early growth response 1 gene by Epstein-Barr virus lytic transactivator Zta. J Virol 2006, 80(15):7748-7755.

125. Heather J, Flower K, Isaac S, Sinclair AJ: The Epstein-Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1. J Gen Virol 2009, 90(Pt 6):1450-1454.

126. Tsai SC, Lin SJ, Chen PW, Luo WY, Yeh TH, Wang HW, Chen CJ, Tsai CH: EBV Zta protein induces the expression of interleukin-13, promoting the proliferation of EBV-infected B cells and lymphoblastoid cell lines. Blood 2009, 114(1):109-118.

127. Ma SD, Yu X, Mertz JE, Gumperz JE, Reinheim E, Zhou Y, Tang W, Burlingham WJ, Gulley ML, Kenney SC: An Epstein-Barr Virus (EBV) mutant with enhanced BZLF1 expression causes lymphomas with abortive lytic EBV infection in a humanized mouse model. J Virol 2012, 86(15):7976-7987.

128. Adamson AL: Epstein-Barr virus BZLF1 protein binds to mitotic chromosomes. J Virol 2005, 79(12):7899-7904.

129. Chen YF, Tung CL, Chang Y, Hsiao WC, Su LJ, Sun HS: Analysis of global methylation using a Zta-expressing nasopharyngeal carcinoma cell line. Genomics 2011, 97(4):205-213.

130. Niller HH, Wolf H, Minarovits J: Viral hit and run-oncogenesis: Genetic and epigenetic scenarios. Cancer Lett 2011, 305(2):200-217.

131. Murata T, Tsurumi T: Switching of EBV cycles between latent and lytic states. Rev Med Virol 2013, 24(3):142-153.

132. Chang ET, Adami HO: The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev 2006, 15(10):1765-1777.

133. Radman M, Kinsella AR: Chromosomal events in carcinogenic initiation and promotion: implications for carcinogenicity testing and cancer prevention strategies. IARC Sci Publ 1980(27):75-90.

134. Huang SY, Fang CY, Tsai CH, Chang Y, Takada K, Hsu TY, Chen JY: N-methyl-N'-nitro-N-nitrosoguanidine induces and cooperates with 12-O-tetradecanoylphorbol-1,3-acetate/sodium butyrate to enhance Epstein-Barr virus reactivation and genome instability in nasopharyngeal carcinoma cells. Chem Biol Interact 2010, 188(3):623-634.

135. Fang CY, Huang SY, Wu CC, Hsu HY, Chou SP, Tsai CH, Chang Y, Takada K, Chen JY: The synergistic effect of chemical carcinogens enhances Epstein-Barr virus reactivation and tumor progression of nasopharyngeal carcinoma cells. PLoS One 2012, 7(9):e44810.

136. Huang SY, Fang CY, Wu CC, Tsai CH, Lin SF, Chen JY: Reactive oxygen species mediate Epstein-Barr virus reactivation by N-methyl-N'-nitro-N-nitrosoguanidine. PLoS One 2013, 8(12):e84919.

137. Fang CY, Lee CH, Wu CC, Chang YT, Yu SL, Chou SP, Huang PT, Chen CL, Hou JW, Chang Y et al: Recurrent chemical reactivations of EBV promotes genome instability and enhances tumor progression of nasopharyngeal carcinoma cells. Int J Cancer 2009, 124(9):2016-2025.

138. Wu CC, Liu MT, Chang YT, Fang CY, Chou SP, Liao HW, Kuo KL, Hsu SL, Chen YR, Wang PW et al: Epstein-Barr virus DNase (BGLF5) induces genomic instability in human epithelial cells. Nucleic Acids Res 2010, 38(6):1932-1949.

139. Cayrol C, Flemington EK: Identification of cellular target genes of the Epstein-Barr virus transactivator Zta: activation of transforming growth factor beta igh3 (TGF-beta igh3) and TGF-beta 1. J Virol 1995, 69(7):4206-4212.

140. Mahot S, Sergeant A, Drouet E, Gruffat H: A novel function for the Epstein-Barr virus transcription factor EB1/Zta: induction of transcription of the hIL-10 gene. J Gen Virol 2003, 84(Pt 4):965-974.

141. Hsu M, Wu SY, Chang SS, Su IJ, Tsai CH, Lai SJ, Shiau AL, Takada K, Chang Y: Epstein-Barr virus lytic transactivator Zta enhances chemotactic activity through induction of interleukin-8 in nasopharyngeal carcinoma cells. J Virol 2008, 82(7):3679-3688.

142. Li XJ, Peng LX, Shao JY, Lu WH, Zhang JX, Chen S, Chen ZY, Xiang YQ, Bao YN, Zheng FJ et al: As an independent unfavorable prognostic factor, IL-8 promotes metastasis of nasopharyngeal carcinoma through induction of epithelial-mesenchymal transition and activation of AKT signaling. Carcinogenesis 2012, 33(7):1302-1309.

143. Lo MC, Yip TC, Ngan KC, Cheng WW, Law CK, Chan PS, Chan KC, Wong CK, Wong RN, Lo KW et al: Role of MIF/CXCL8/CXCR2 signaling in the growth of nasopharyngeal carcinoma tumor spheres. Cancer Lett 2013, 335(1):81-92.

144. Yoshizaki T, Horikawa T, Qing-Chun R, Wakisaka N, Takeshita H, Sheen TS, Lee SY, Sato H, Furukawa M: Induction of interleukin-8 by Epstein-Barr virus latent membrane protein-1 and its correlation to angiogenesis in nasopharyngeal carcinoma. Clin Cancer Res 2001, 7(7):1946-1951.

145. Ren Q, Sato H, Murono S, Furukawa M, Yoshizaki T: Epstein-Barr virus (EBV) latent membrane protein 1 induces interleukin-8 through the nuclear factor-kappa B signaling pathway in EBV-infected nasopharyngeal carcinoma cell line. Laryngoscope 2004, 114(5):855-859.

146. Yao M, Ohshima K, Suzumiya J, Kume T, Shiroshita T, Kikuchi M: Interleukin-10 expression and cytotoxic-T-cell response in Epstein-Barr-virus-associated nasopharyngeal carcinoma. Int J Cancer 1997, 72(3):398-402.

147. Toyota M, Ahuja N, Suzuki H, Itoh F, Ohe-Toyota M, Imai K, Baylin SB, Issa JP: Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype. Cancer Res 1999, 59(21):5438-5442.

148. Osawa T, Chong JM, Sudo M, Sakuma K, Uozaki H, Shibahara J, Nagai H, Funata N, Fukayama M: Reduced expression and promoter methylation of p16 gene in Epstein-Barr virus-associated gastric carcinoma. Jpn J Cancer Res 2002, 93(11):1195-1200.

149. Ushiku T, Chong JM, Uozaki H, Hino R, Chang MS, Sudo M, Rani BR, Sakuma K, Nagai H, Fukayama M: p73 gene promoter methylation in Epstein-Barr virus-associated gastric carcinoma. Int J Cancer 2007, 120(1):60-66.

150. Wu MS, Shun CT, Wu CC, Hsu TY, Lin MT, Chang MC, Wang HP, Lin JT: Epstein-Barr virus-associated gastric carcinomas: relation to H. pylori infection and genetic alterations. Gastroenterology 2000, 118(6):1031-1038.

151. Sudo M, Chong JM, Sakuma K, Ushiku T, Uozaki H, Nagai H, Funata N, Matsumoto Y, Fukayama M: Promoter hypermethylation of E-cadherin and its abnormal expression in Epstein-Barr virus-associated gastric carcinoma. Int J Cancer 2004, 109(2):194-199.

152. Chong JM, Sakuma K, Sudo M, Ushiku T, Uozaki H, Shibahara J, Nagai H, Funata N, Taniguchi H, Aburatani H et al: Global and non-random CpG-island methylation in gastric carcinoma associated with Epstein-Barr virus. Cancer Sci 2003, 94(1):76-80.

153. Kusano M, Toyota M, Suzuki H, Akino K, Aoki F, Fujita M,

Hosokawa M, Shinomura Y, Imai K, Tokino T: Genetic, epigenetic, and clinicopathologic features of gastric carcinomas with the CpG island methylator phenotype and an association with Epstein-Barr virus. Cancer 2006, 106(7):1467-1479.

154. Chang MS, Uozaki H, Chong JM, Ushiku T, Sakuma K,

Ishikawa S, Hino R, Barua RR, Iwasaki Y, Arai K et al: CpG island methylation status in gastric carcinoma with and without infection of Epstein-Barr virus. Clin Cancer Res 2006, 12(10):2995-3002.

155. Enomoto S, Maekita T, Tsukamoto T, Nakajima T, Nakazawa K, Tatematsu M, Ichinose M, Ushijima T: Lack of association between CpG island methylator phenotype in human gastric cancers and methylation in their background non-cancerous gastric mucosae. Cancer Sci 2007, 98(12):1853-1861.

156. Saito M, Nishikawa J, Okada T, Morishige A, Sakai K, Nakamura M, Kiyotoki S, Hamabe K, Okamoto T, Oga A et al: Role of DNA methylation in the development of Epstein-Barr virus-associated gastric carcinoma. J Med Virol 2013, 85(1):121-127.

157. Okada T, Nakamura M, Nishikawa J, Sakai K, Zhang Y, Saito M, Morishige A, Oga A, Sasaki K, Suehiro Y et al: Identification of genes specifically methylated in Epstein-Barr virus-associated gastric carcinomas. Cancer Sci 2013, 104(10):1309-1314.

158. Kawamura YI, Toyota M, Kawashima R, Hagiwara T, Suzuki H, Imai K, Shinomura Y, Tokino T, Kannagi R, Dohi T: DNA hypermethylation contributes to incomplete synthesis of carbohydrate determinants in gastrointestinal cancer. Gastroenterology 2008, 135(1):142-151.

159. Etoh T, Kanai Y, Ushijima S, Nakagawa T, Nakanishi Y, Sasako M, Kitano S, Hirohashi S: Increased DNA methyltransferase 1 (DNMT1) protein expression correlates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multiple CpG islands in gastric cancers. Am J Pathol 2004, 164(2):689-699.

160. Han J, He D, Feng ZY, Ding YG, Shao CK: Clinicopathologic features and protein expression study of Epstein-Barr virus-associated gastric carcinoma in Guangzhou. Zhonghua Bing Li Xue Za Zhi 2010, 39(12):798-803.

161. Zhao J, Liang Q, Cheung KF, Kang W, Lung RW, Tong JH, To KF, Sung JJ, Yu J: Genome-wide identification of Epstein-Barr virus-driven promoter methylation profiles of human genes in gastric cancer cells. Cancer 2013, 119(2):304-312.

162. Kang GH, Lee S, Cho NY, Gandamihardja T, Long TI, Weisenberger DJ, Campan M, Laird PW: DNA methylation profiles of gastric  carcinoma characterized by quantitative  DNA

methylation analysis. Lab Invest 2008, 88(2):161-170.

163. Sakuma K, Chong JM, Sudo M, Ushiku T, Inoue Y, Shibahara J, Uozaki H, Nagai H, Fukayama M: High-density methylation of p14ARF and p16INK4A in Epstein-Barr virus-associated gastric carcinoma. Int J Cancer 2004, 112(2):273-278.

164. Kim J, Lee HS, Bae SI, Lee YM, Kim WH: Silencing and CpG island methylation of GSTP1 is rare in ordinary gastric carcinomas but common in Epstein-Barr virus-associated gastric carcinomas. Anticancer Res 2005, 25(6B):4013-4019.

165. Liu X, Tang X, Zhang S, Wang Y, Wang X, Zhao C, Luo B: Methylation and Expression of Retinoblastoma and Transforming Growth Factor-beta1 Genes in Epstein-Barr Virus-Associated and -Negative Gastric Carcinomas. Gastroenterol Res Pract 2012, 2012:906017.

166. Liu X, Wang Y, Wang X, Sun Z, Li L, Tao Q, Luo B: Epigenetic silencing of WNT5A in Epstein-Barr virus-associated gastric carcinoma. Arch Virol 2013, 158(1):123-132.

167. Zhao J, Liang Q, Cheung KF, Kang W, Dong Y, Lung RW, Tong JH, To KF, Sung JJ, Yu J: Somatostatin receptor 1, a novel EBV-associated CpG hypermethylated gene, contributes to the pathogenesis of EBV-associated gastric cancer. Br J Cancer 2013, 108(12):2557-2564.

168. Matsusaka K, Kaneda A, Nagae G, Ushiku T, Kikuchi Y, Hino R, Uozaki H, Seto Y, Takada K, Aburatani H et al: Classification of Epstein-Barr virus-positive gastric cancers by definition of DNA methylation epigenotypes. Cancer Res 2011, 71(23):7187-7197.

169. Ryan JL, Jones RJ, Kenney SC, Rivenbark AG, Tang W, Knight ER, Coleman WB, Gulley ML: Epstein-Barr virus-specific methylation of human genes in gastric cancer cells. Infect Agent Cancer 2010, 5:27.

170. Kaneda A, Matsusaka K, Aburatani H, Fukayama M: Epstein-Barr virus infection as an epigenetic driver of tumorigenesis. Cancer Res 2012, 72(14):3445-3450.

171. Matsusaka K,  Funata  S,  Fukayama  M,  Kaneda  A: DNA

methylation in gastric cancer, related to Helicobacter pylori and Epstein-Barr virus. World J Gastroenterol 2014, 20(14):3916-3926.

172. Kwong J, Lo KW, To KF, Teo PM, Johnson PJ, Huang DP: Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin Cancer Res 2002, 8(1):131-137.

173. Krishna SM, Kattoor J, Balaram P: Down regulation of adhesion protein E-cadherin in Epstein-Barr virus infected nasopharyngeal carcinomas. Cancer Biomark 2005, 1(6):271-277.

174. Yi HM, Li H, Peng D, Zhang HJ, Wang L, Zhao M, Yao KT, Ren CP: Genetic and epigenetic alterations of LTF at 3p21.3 in nasopharyngeal carcinoma. Oncol Res 2006, 16(6):261-272.

175. Zhang H, Feng X, Liu W, Jiang X, Shan W, Huang C, Yi H, Zhu B, Zhou W, Wang L et al: Underlying mechanisms for LTF inactivation and its functional analysis in nasopharyngeal carcinoma cell lines. J Cell Biochem 2011, 112(7):1832-1843.

176. Shao L, Cui Y, Li H, Liu Y, Zhao H, Wang Y, Zhang Y, Ng KM, Han W, Ma D et al: CMTM5 exhibits tumor suppressor activities and is frequently silenced by methylation in carcinoma cell lines. Clin Cancer Res 2007, 13(19):5756-5762.

177. Jin H, Wang X, Ying J, Wong AH, Li H, Lee KY, Srivastava G, Chan AT, Yeo W, Ma BB et al: Epigenetic identification of ADAMTS18 as a novel 16q23.1 tumor suppressor frequently silenced in esophageal, nasopharyngeal and multiple other carcinomas. Oncogene 2007, 26(53):7490-7498.

178. Lung HL, Lo PH, Xie D, Apte SS, Cheung AK, Cheng Y, Law EW, Chua D, Zeng YX, Tsao SW et al: Characterization of a novel epigenetically-silenced, growth-suppressive gene, ADAMTS9, and its association with lymph node metastases in nasopharyngeal carcinoma. Int J Cancer 2008, 123(2):401-408.

179. Chen F, Mo Y, Ding H, Xiao X, Wang SY, Huang G, Zhang Z, Wang SZ: Frequent epigenetic inactivation of Myocardin in human nasopharyngeal carcinoma. Head Neck 2011, 33(1):54-59.

180. Loyo M, Brait M, Kim MS, Ostrow KL, Jie CC, Chuang AY, Califano JA, Liegeois NJ, Begum S, Westra WH et al: A survey of methylated candidate tumor suppressor genes in nasopharyngeal carcinoma. Int J Cancer 2011, 128(6):1393-1403.

181. Li W, Li X, Wang W, Li X, Tan Y, Yi M, Yang J, McCarthy JB, Xiong W, Wu M et al: NOR1 is an HSF1- and NRF1-regulated putative tumor suppressor inactivated by promoter hypermethylation in nasopharyngeal carcinoma. Carcinogenesis 2011, 32(9):1305-1314.

182. Du C, Huang T, Sun D, Mo Y, Feng H, Zhou X, Xiao X, Yu N, Hou B, Huang G et al: CDH4 as a novel putative tumor suppressor gene epigenetically silenced by promoter hypermethylation in nasopharyngeal carcinoma. Cancer Lett 2011, 309(1):54-61.

183. Law EW, Cheung AK, Kashuba VI, Pavlova TV, Zabarovsky ER, Lung HL, Cheng Y, Chua D, Lai-Wan Kwong D, Tsao SW et al: Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma. Oncogene 2012, 31(6):728-738.

184. Mo Y, Midorikawa K, Zhang Z, Zhou X, Ma N, Huang G, Hiraku Y, Oikawa S, Murata M: Promoter hypermethylation of Ras-related GTPase gene RRAD inactivates a tumor suppressor function in nasopharyngeal carcinoma. Cancer Lett 2012, 323(2):147-154.

185. He D, Zeng Q, Ren G, Xiang T, Qian Y, Hu Q, Zhu J, Hong S, Hu G: Protocadherin 8 is a functional tumor suppressor frequently inactivated by promoter methylation in nasopharyngeal carcinoma. Eur J Cancer Prev 2012, 21(6):569-575.

186. Xiao X, Zhao W, Tian F, Zhou X, Zhang J, Huang T, Hou B,

Du C, Wang S, Mo Y et al: Cytochrome b5 reductase 2 is a novel candidate tumor suppressor gene frequently inactivated by promoter hypermethylation in human nasopharyngeal carcinoma. Tumour Biol 2013, 35(4):3755-3763.

187. You Y, Yang W, Wang Z, Zhu H, Li H, Lin C, Ran Y: Promoter hypermethylation contributes to the frequent suppression of the CDK10 gene in human nasopharyngeal carcinomas. Cell Oncol 2013, 36(4):323-331.

188. Shu XS, Li L, Ji M, Cheng Y, Ying J, Fan Y, Zhong L, Liu X, Tsao SW, Chan AT et al: FEZF2, a novel 3p14 tumor suppressor gene, represses oncogene EZH2 and MDM2 expression and is frequently methylated in nasopharyngeal carcinoma. Carcinogenesis 2013, 34(9):1984-1993.

189. Wong AM, Kong KL, Chen L, Liu M, Wong AM, Zhu C, Tsang JW, Guan XY: Characterization of CACNA2D3 as a putative tumor suppressor gene in the development and progression of nasopharyngeal carcinoma. Int J Cancer 2013, 133(10):2284-2295.

190. Zhang S, Li S, Gao JL: Promoter methylation status of the tumor suppressor gene SOX11 is associated with cell growth and invasion in nasopharyngeal carcinoma. Cancer Cell Int 2013, 13(1):109.

191. Li HP, Peng CC, Chung IC, Huang MY, Huang ST, Chen CC, Chang KP, Hsu CL, Chang YS: Aberrantly hypermethylated Homeobox A2 derepresses metalloproteinase-9 through TBP and promotes invasion in Nasopharyngeal carcinoma. Oncotarget 2013, 4(11):2154-2165.

192. Lan J, Tai HC, Lee SW, Chen TJ, Huang HY, Li CF: Deficiency in expression and epigenetic DNA Methylation of

ASS1 gene in nasopharyngeal carcinoma: negative prognostic impact and therapeutic relevance. Tumour Biol 2014, 35(1):161-169.

193. Li J, Gong P, Lyu X, Yao K, Li X, Peng H: Aberrant CpG island methylation of PTEN is an early event in nasopharyngeal carcinoma and a potential diagnostic biomarker. Oncol Rep 2014, 31(5):2206-2212.

194. Hutajulu SH, Indrasari SR, Indrawati LP, Harijadi A, Duin S, Haryana SM, Steenbergen RD, Greijer AE, Middeldorp JM: Epigenetic markers for early detection of nasopharyngeal carcinoma in a high risk population. Mol Cancer 2011, 10:48.

195. Zhang Z, Sun D, Hutajulu SH, Nawaz I, Nguyen Van D, Huang G, Haryana SM, Middeldorp JM, Ernberg I, Hu LF: Development of a non-invasive method, multiplex methylation specific PCR (MMSP), for early diagnosis of nasopharyngeal carcinoma. PLoS One 2012, 7(11):e45908.

196. Zhang X, Li W, Li H, Ma Y, He G, Tan G: Genomic methylation profiling combined with gene expression microarray reveals the aberrant methylation mechanism involved in nasopharyngeal carcinoma taxol resistance. Anticancer Drugs 2012, 23(8):856-864.

197. Cai MY, Tong ZT, Zhu W, Wen ZZ, Rao HL, Kong LL, Guan XY, Kung HF, Zeng YX, Xie D: H3K27me3 protein is a promising predictive biomarker of patients' survival and chemoradioresistance in human nasopharyngeal carcinoma. Mol Med 2011, 17(11-12):1137-1145.

198. Hwang CF, Huang HY, Chen CH, Chien CY, Hsu YC, Li CF, Fang FM: Enhancer of zeste homolog 2 overexpression in nasopharyngeal carcinoma: an independent poor prognosticator that enhances cell growth. Int J Radiat Oncol Biol Phys 2012, 82(2):597-604.

199. He LJ, Cai MY, Xu GL, Li JJ, Weng ZJ, Xu DZ, Luo GY, Zhu SL, Xie D: Prognostic significance of overexpression of EZH2 and H3k27me3 proteins in gastric cancer. Asian Pac J Cancer Prev 2012, 13(7):3173-3178.

200. van Kemenade FJ, Raaphorst FM, Blokzijl T, Fieret E, Hamer KM, Satijn DP, Otte AP, Meijer CJ: Coexpression of BMI-1 and EZH2 polycomb-group proteins is associated with cycling cells and degree of malignancy in B-cell non-Hodgkin lymphoma. Blood 2001, 97(12):3896-3901.

201. Iwasaki Y, Chong JM, Hayashi Y, Ikeno R, Arai K, Kitamura M, Koike M, Hirai K, Fukayama M: Establishment and characterization of a human Epstein-Barr virus-associated gastric carcinoma in SCID mice. J Virol 1998, 72(10):8321-8326.

202. Zeng Y: Seroepidemiological studies on nasopharyngeal carcinoma in China. Adv Cancer Res 1985, 44:121-138.

203. Zhu XX, Zeng Y, Wolf H: Detection of IgG and IgA antibodies to Epstein-Barr virus membrane antigen in sera from patients   with  nasopharyngeal  carcinoma   and  from  normal

individuals. Int J Cancer 1986, 37(5):689-691.

204. Zeng Y: EB virus and nasopharyngeal carcinoma. In: Etiology and pathogenesis of nasopharyngeal carcinoma. Edited by Zeng Y, Ou B. Beijing: The People’s Medical Publishing House; 1987: 18.

205. Tokunaga M, Land CE: Epstein-Barr virus involvement in gastric cancer: biomarker for lymph node metastasis. Cancer Epidemiol Biomarkers Prev 1998, 7(5):449-450.

206. Uemura Y, Tokunaga M, Arikawa J, Yamamoto N, Hamasaki Y, Tanaka S, Sato E, Land CE: A unique morphology of Epstein-Barr virus-related early gastric carcinoma. Cancer Epidemiol Biomarkers Prev 1994, 3(7):607-611.

207. Arikawa J, Tokunaga M, Satoh E, Tanaka S, Land CE: Morphological characteristics of Epstein-Barr virus-related early gastric carcinoma: a case-control study. Pathol Int 1997, 47(6):360-367.

208. zur Hausen A, van Rees BP, van Beek J, Craanen ME, Bloemena E, Offerhaus GJ, Meijer CJ, van den Brule AJ: Epstein-

Barr virus in gastric carcinomas and gastric stump carcinomas: a late event in gastric carcinogenesis. J Clin Pathol 2004, 57(5):487-491.

209. Truong CD, Feng W, Li W, Khoury T, Li Q, Alrawi S, Yu Y, Xie K, Yao J, Tan D: Characteristics of Epstein-Barr virus-associated gastric cancer: a study of 235 cases at a comprehensive cancer center in U.S.A. J Exp Clin Cancer Res 2009, 28:14.

210. Fukayama M, Hayashi Y, Iwasaki Y, Chong J, Ooba T, Takizawa T, Koike M, Mizutani S, Miyaki M, Hirai K: Epstein-Barr virus-associated gastric carcinoma and Epstein-Barr virus infection of the stomach. Lab Invest 1994, 71(1):73-81.

211. Fukayama M, Ushiku T: Epstein-Barr virus-associated gastric carcinoma. Pathol Res Pract 2011, 207(9):529-537.

212. Au WY, Pang A, Chan EC, Chu KM, Shek TW, Kwong YL: Epstein-Barr virus-related gastric adenocarcinoma: an early secondary cancer post hemopoietic stem cell transplantation. Gastroenterology 2005, 129(6):2058-2063.

213. Rhyu MG, Park WS, Meltzer SJ: Microsatellite instability occurs frequently in human gastric carcinoma. Oncogene 1994, 9(1):29-32.

214. Chong JM, Fukayama M, Hayashi Y, Takizawa T, Koike M, Konishi M, Kikuchi-Yanoshita R, Miyaki M: Microsatellite instability in the progression of gastric carcinoma. Cancer Res 1994, 54(17):4595-4597.

215. Chang MS, Lee HS, Kim HS, Kim SH, Choi SI, Lee BL, Kim CW, Kim YI, Yang M, Kim WH: Epstein-Barr virus and microsatellite instability in gastric carcinogenesis. J Pathol 2003, 199(4):447-452.

216. Leung SY, Yuen ST, Chung LP, Chu KM, Chan AS, Ho JC: hMLH1 promoter methylation and lack of hMLH1 expression in sporadic gastric carcinomas with high-frequency microsatellite instability. Cancer Res 1999, 59(1):159-164.

217. Fleisher AS, Esteller M, Wang S, Tamura G, Suzuki H, Yin J, Zou TT, Abraham JM, Kong D, Smolinski KN et al:

Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability. Cancer Res 1999, 59(5):1090-1095.

218. Vo QN, Geradts J, Gulley ML, Boudreau DA, Bravo JC, Schneider BG: Epstein-Barr virus in gastric adenocarcinomas: association with ethnicity and CDKN2A promoter methylation. J Clin Pathol 2002, 55(9):669-675.

219. Leung SY, Yuen ST, Chung LP, Chu KM, Wong MP,

Branicki FJ, Ho JC: Microsatellite instability, Epstein-Barr virus, mutation of type II transforming growth factor beta receptor and BAX in gastric carcinomas in Hong Kong Chinese. Br J Cancer 1999, 79(3-4):582-588.

220. Wong TS, Tang KC, Kwong DL, Sham JS, Wei WI, Kwong YL, Yuen AP: Differential gene methylation in undifferentiated nasopharyngeal carcinoma. Int J Oncol 2003, 22(4):869-874.

221. Wong TS, Kwong DL, Sham JS, Wei WI, Kwong YL, Yuen AP: Quantitative plasma hypermethylated DNA markers of undifferentiated  nasopharyngeal  carcinoma.  Clin  Cancer  Res

2004, 10(7):2401-2406.

222. Chang HW, Chan A, Kwong DL, Wei WI, Sham JS, Yuen AP: Evaluation of hypermethylated tumor suppressor genes as tumor markers in mouth and throat rinsing fluid, nasopharyngeal swab and peripheral blood of nasopharygeal carcinoma patient. Int J Cancer 2003, 105(6):851-855.

223. Lo KW, Cheung ST, Leung SF, van Hasselt A, Tsang YS, Mak KF, Chung YF, Woo JK, Lee JC, Huang DP: Hypermethylation of the p16 gene in nasopharyngeal carcinoma. Cancer Res 1996, 56(12):2721-2725.

224. Tsao SW, Liu Y, Wang X, Yuen PW, Leung SY, Yuen ST, Pan J, Nicholls JM, Cheung AL, Wong YC: The association of E-cadherin expression and the methylation status of the E-cadherin gene in nasopharyngeal carcinoma cells. Eur J Cancer 2003, 39(4):524-531.

225. Cheung AK, Lung HL, Hung SC, Law EW, Cheng Y, Yau WL, Bangarusamy DK, Miller LD, Liu ET, Shao JY et al: Functional analysis of a cell cycle-associated, tumor-suppressive gene, protein tyrosine phosphatase receptor type G, in nasopharyngeal carcinoma. Cancer Res 2008, 68(19):8137-8145.

226. Yau WL, Lung HL, Zabarovsky ER, Lerman MI, Sham JS, Chua DT, Tsao SW, Stanbridge EJ, Lung ML: Functional studies of the chromosome 3p21.3 candidate tumor suppressor gene BLU/ZMYND10 in nasopharyngeal carcinoma. Int J Cancer 2006, 119(12):2821-2826.

227. Liu XQ, Chen HK, Zhang XS, Pan ZG, Li A, Feng QS, Long QX, Wang XZ, Zeng YX: Alterations of BLU, a candidate tumor suppressor gene on chromosome 3p21.3, in human nasopharyngeal carcinoma. Int J Cancer 2003, 106(1):60-65.

228. Qiu GH, Tan LK, Loh KS, Lim CY, Srivastava G, Tsai ST, Tsao SW, Tao Q: The candidate tumor suppressor gene BLU, located at the commonly deleted region 3p21.3, is an E2F-regulated, stress-responsive gene and inactivated by both epigenetic and genetic mechanisms in nasopharyngeal carcinoma. Oncogene 2004, 23(27):4793-4806.

229. Lu JJ, Chen CL, Hsu TY, Chen JY, Su IJ, Yu WC, Yang CS: Expression of Epstein-Barr virus latent membrane protein 1 and B-cell leukemia-lymphoma 2 gene in nasopharyngeal carcinoma tissues. J Microbiol Immunol Infect 2002, 35(2):136-140.

230. Yu Y, Dong W, Li X, Yu E, Zhou X, Li S: Significance of c-Myc and Bcl-2 protein expression in nasopharyngeal carcinoma. Arch Otolaryngol Head Neck Surg 2003, 129(12):1322-1326.

231. Yip KW, Shi W, Pintilie M, Martin JD, Mocanu JD, Wong D, MacMillan C, Gullane P, O'Sullivan B, Bastianutto C et al: Prognostic significance of the Epstein-Barr virus, p53, Bcl-2, and survivin in nasopharyngeal cancer. Clin Cancer Res 2006, 12(19):5726-5732.

232. Niller HH, Glaser G, Knuchel R, Wolf H: Nucleoprotein complexes and DNA 5'-ends at oriP of Epstein-Barr virus. J Biol Chem 1995, 270(21):12864-12868.

233. Vogel M, Wittmann K, Endl E, Glaser G, Knuchel R, Wolf H, Niller HH: Plasmid maintenance assay based on green fluorescent protein and FACS of mammalian cells. Biotechniques 1998, 24(4):540-544.

234. Liu H, Zhang L, Niu Z, Zhou M, Peng C, Li X, Deng T, Shi L, Tan Y, Li G: Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells. BMC Cancer 2008, 8:253.

235. Hui AB, Lo KW, Kwong J, Lam EC, Chan SY, Chow LS, Chan AS, Teo PM, Huang DP: Epigenetic inactivation of TSLC1 gene in nasopharyngeal carcinoma. Molecular carcinogenesis 2003, 38(4):170-178.

236. Lung HL, Cheng Y, Kumaran MK, Liu ET, Murakami Y, Chan CY, Yau WL, Ko JM, Stanbridge EJ, Lung ML: Fine mapping of the 11q22-23 tumor suppressive region and involvement of TSLC1 in nasopharyngeal carcinoma. Int J Cancer 2004, 112(4):628-635.

237. Yu J, Ni M, Xu J, Zhang H, Gao B, Gu J, Chen J, Zhang L, Wu M, Zhen S et al: Methylation profiling of twenty promoter-CpG  islands of  genes  which may  contribute to hepatocellular

carcinogenesis. BMC Cancer 2002, 2:29.

238. Cheung HW, Ching YP, Nicholls JM, Ling MT, Wong YC, Hui N, Cheung A, Tsao SW, Wang Q, Yeun PW et al: Epigenetic inactivation of CHFR in nasopharyngeal carcinoma through promoter methylation. Molecular carcinogenesis 2005, 43(4):237-245.

239. Tong JH, Ng DC, Chau SL, So KK, Leung PP, Lee TL, Lung RW, Chan MW, Chan AW, Lo KW et al: Putative tumour-suppressor gene DAB2 is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma. BMC Cancer 2010, 10:253.

240. Seng TJ, Low JS, Li H, Cui Y, Goh HK, Wong ML, Srivastava G, Sidransky D, Califano J, Steenbergen RD et al: The major 8p22 tumor suppressor DLC1 is frequently silenced by methylation in both endemic and sporadic nasopharyngeal, esophageal, and cervical carcinomas, and inhibits tumor cell colony formation. Oncogene 2007, 26(6):934-944.

241. Kwong J, Chow LS, Wong AY, Hung WK, Chung GT, To KF, Chan FL, Daigo Y, Nakamura Y, Huang DP et al: Epigenetic inactivation of the deleted in lung and esophageal cancer 1 gene in nasopharyngeal carcinoma. Genes Chromosomes Cancer 2007, 46(2):171-180.

242. Kwong J, Lee JY, Wong KK, Zhou X, Wong DT, Lo KW, Welch WR, Berkowitz RS, Mok SC: Candidate tumor-suppressor gene DLEC1 is frequently downregulated by promoter hypermethylation and histone hypoacetylation in human epithelial ovarian cancer. Neoplasia 2006, 8(4):268-278.

243. Lo KW, Tsang YS, Kwong J, To KF, Teo PM, Huang DP: Promoter hypermethylation of the EDNRB gene in nasopharyngeal carcinoma. Int J Cancer 2002, 98(5):651-655.

244. Amanullah A, Azam N, Balliet A, Hollander C, Hoffman B, Fornace A, Liebermann D: Cell signalling: cell survival and a Gadd45-factor deficiency. Nature 2003, 424(6950):741-742.

245. Ying J, Srivastava G, Hsieh WS, Gao Z, Murray P, Liao SK, Ambinder R, Tao Q: The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors. Clin Cancer Res 2005, 11(18):6442-6449.

246. Lee KY, Geng H, Ng KM, Yu J, van Hasselt A, Cao Y, Zeng YX, Wong AH, Wang X, Ying J et al: Epigenetic disruption of interferon-gamma response through silencing the tumor suppressor interferon regulatory factor 8 in nasopharyngeal, esophageal and multiple other carcinomas. Oncogene 2008, 27(39):5267-5276.

247. Cheung AK, Lung HL, Ko JM, Cheng Y, Stanbridge EJ, Zabarovsky ER, Nicholls JM, Chua D, Tsao SW, Guan XY et al: Chromosome 14 transfer and functional studies identify a candidate tumor suppressor gene, mirror image polydactyly 1, in nasopharyngeal carcinoma. Proc Natl Acad Sci U S A 2009, 106(34):14478-14483.

248. Chan KC, Ko JM, Lung HL, Sedlacek R, Zhang ZF, Luo DZ, Feng ZB, Chen S, Chen H, Chan KW et al: Catalytic activity of Matrix metalloproteinase-19 is essential for tumor suppressor and anti-angiogenic activities in nasopharyngeal carcinoma. Int J Cancer 2011, 129(8):1826-1837.

249. Cui Y, Ying Y, van Hasselt A, Ng KM, Yu J, Zhang Q, Jin J, Liu D, Rhim JS, Rha SY et al: OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation. PLoS One 2008, 3(8):e2990. 250. Ying J, Li H, Seng TJ, Langford C, Srivastava G, Tsao SW,

Putti T, Murray P, Chan AT, Tao Q: Functional epigenetics identifies a protocadherin PCDH10 as a candidate tumor suppressor for nasopharyngeal, esophageal and multiple other carcinomas with frequent methylation. Oncogene 2006, 25(7):1070-1080.

251. Chang HW, Chan A, Kwong DL, Wei WI, Sham JS, Yuen AP: Detection of hypermethylated RIZ1 gene in primary tumor, mouth, and throat rinsing fluid, nasopharyngeal swab, and peripheral blood of nasopharyngeal carcinoma patient. Clin Cancer Res 2003, 9(3):1033-1038.

252. Kwong J, Lo KW, Chow LS, To KF, Choy KW, Chan FL, Mok SC, Huang DP: Epigenetic silencing of cellular retinol-binding proteins in nasopharyngeal carcinoma. Neoplasia 2005, 7(1):67-74.

253. Kwong J, Lo KW, Chow LS, Chan FL, To KF, Huang DP: Silencing of the retinoid response gene TIG1 by promoter hypermethylation in nasopharyngeal carcinoma. Int J Cancer 2005, 113(3):386-392.

254. Jin H, Wang X, Ying J, Wong AH, Cui Y, Srivastava G, Shen ZY, Li EM, Zhang Q, Jin J et al: Epigenetic silencing of a Ca(2+)-regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers. Proc Natl Acad Sci U S A 2007, 104(30):12353-12358.

255. Lo KW, Kwong J, Hui AB, Chan SY, To KF, Chan AS, Chow LS, Teo PM, Johnson PJ, Huang DP: High frequency of promoter hypermethylation of RASSF1A in nasopharyngeal carcinoma. Cancer Res 2001, 61(10):3877-3881.

256. Zhang Z, Sun D, Van do N, Tang A, Hu L, Huang G: Inactivation of RASSF2A by promoter methylation correlates with lymph node metastasis in nasopharyngeal carcinoma. Int J Cancer 2007, 120(1):32-38.

257. Wong TS, Kwong DL, Sham JS, Tsao SW, Wei WI, Kwong YL, Yuen AP: Promoter hypermethylation of high-in-normal 1 gene in primary nasopharyngeal carcinoma. Clin Cancer Res 2003, 9(8):3042-3046.

258. Wang S, Xiao X, Zhou X, Huang T, Du C, Yu N, Mo Y, Lin L, Zhang J, Ma N et al: TFPI-2 is a putative tumor suppressor gene frequently inactivated by promoter hypermethylation in

nasopharyngeal carcinoma. BMC Cancer 2010, 10:617.

259. Lung HL, Bangarusamy DK, Xie D, Cheung AK, Cheng Y, Kumaran MK, Miller L, Liu ET, Guan XY, Sham JS et al: THY1 is a candidate tumour suppressor gene with decreased expression in metastatic nasopharyngeal carcinoma. Oncogene 2005, 24(43):6525-6532.

260. Li L, Tao Q, Jin H, van Hasselt A, Poon FF, Wang X, Zeng MS, Jia WH, Zeng YX, Chan AT et al: The tumor suppressor UCHL1 forms a complex with p53/MDM2/ARF to promote p53 signaling and is frequently silenced in nasopharyngeal carcinoma. Clin Cancer Res 2010, 16(11):2949-2958.

261. Chan SL, Cui Y, van Hasselt A, Li H, Srivastava G, Jin H, Ng KM, Wang Y, Lee KY, Tsao GS et al: The tumor suppressor Wnt inhibitory factor 1 is frequently methylated in nasopharyngeal and esophageal carcinomas. Lab Invest 2007, 87(7):644-650.

262. Shi W, Bastianutto C, Li A, Perez-Ordonez B, Ng R, Chow KY, Zhang W, Jurisica I, Lo KW, Bayley A et al: Multiple dysregulated pathways in nasopharyngeal carcinoma revealed by gene expression profiling. Int J Cancer 2006, 119(10):2467-2475.

263. Hui AB, Or YY, Takano H, Tsang RK, To KF, Guan XY, Sham JS, Hung KW, Lam CN, van Hasselt CA et al: Array-based comparative genomic hybridization analysis identified cyclin D1 as a target oncogene at 11q13.3 in nasopharyngeal carcinoma. Cancer Res 2005, 65(18):8125-8133.

264. Or YY, Hui AB, Tam KY, Huang DP, Lo KW: Characterization of chromosome 3q and 12q amplicons in nasopharyngeal carcinoma cell lines. Int J Oncol 2005, 26(1):49-56.


 

Table 1. List of hypermethylated genes in three epigenotypes of gastric carcinoma and in nasopharyngeal carcinoma.

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Column A: CGIs hypermethylated in EBV-negative low-methylation GC, column B: CGIs hypermethylated in EBV-negative high-methylation GC, column C: CGIs hypermethylated in EBVaGC. Data in columns A, B and C are from [168]. Column D: CGIs hypermethylated in NPC, column E: names and chromosomal location for each gene locus, as provided by GeneCards (http://www.genecards.org/), column F: references for genes hypermethylated in NPC as listed in column D. Data in column D were adopted from two recent reviews [33, 36] and additionally from the following references: LTF[174, 175], CMTM5[176], ADAMTS18[177], ADAMTS9[178], MYOCD[179], NOR1[181], CDH4[182], FBLN2[183], RRAD[184], PCDH8[185], CYB5R2[186], CDK10[187], FEZF2[188], CACNA2D3[189], SOX11[190], KIF1A[180], HOXA2[191], ASS1[192], PTEN[193].

References in column F as provided by [33, 36]

BRD7                                       [234]

CADM1 (TSLC1)                     [235, 236]

CASP8                                     [220]

CDH1                                       [220, 221, 224, 225]

CDH13                                    [237]

CDKN2A, p16, ARF               [220-223]

CDKN2B, p15                          [220-222]

CHFR                                       [238]

DAB2 (DOC2)                         [239]

DAPK1                                   [172, 221, 222]

DLC1                                       [240]

DLEC1                                    [241, 242]

EDNRB                                    [243]

FHIT                                        [180]

GADD45G                              [244, 245]

GSTP1                                     [172]

IRF8                                        [246]

KIF1A                                     [180]

MGMT                                    [172, 220]

MIPOL1                                   [247]

MLH1                                      [220, 221]

MMP19                                   [248]

OPCML                                  [249]

PCDH10                                  [250]

PRDM2 (RIZ1)                      [251]

PTPRG                                   [180, 225]

RARB2                                  [172, 252]

RARRES1 (TIG1)                   [253]

RASAL1                                 [254]

RASSF1 (RASSF1A)              [172, 220, 222, 255]

RASSF2 (RASSF2A)               [256]

RBP1 (CRBP1)                         [252]

RBP7 (CRBP4)                         [252]

SCGB3A1 (HIN1)                     [257]

TFPI2                                        [258]

THBS1                                      [220]

THY1                                        [259]

TP73                                         [220]

UCHL1 (PGP9.5)                      [260]

WIF1                                         [261]

ZMYND10 (BLU)                    [226-228]

Overexpressed/amplified in NPC

BCL2                                        [262]

BMI1                                        [111]

CCND1                                   [263]

PIK3CA                                  [264]

 

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