Epstein-Barr Virus Nasopharyngeal Carcinoma Research Articles

Role of Peroxiredoxin 1 Induced by Epstein-Barr Virus Infection in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC), a common cancer in Southern China, is associated with Epstein-Barr Virus (EBV) infection. Although many therapies for NPC have been established, the definite role of EBV in NPC remains unclear. Therefore, this work focuses on LMP2A, a latent EBV gene, and investigates whether LMP2A is related to peroxiredoxin 1 (PRDX1) in EBV-positive NPC. The mRNA and protein expression levels of LMP2A, PRDX1, and beta-catenin were compared in patient samples. To identify molecular mechanisms, EBV-negative NP69 and EBV-positive C666-1 NPC cell lines were used. After making an agar cell block for cell slides, the intensity of LMP2A expression was observed visually. To measure the level of reactive oxygen species, both fluorescence microscope and flow cytometry were used. To investigate the intracellular signaling molecular mechanisms with and without the LMP2A gene, reverse transcription polymerase chain reaction and western blotting were used. Both patient samples and cells of nasopharyngeal carcinoma infected with EBV had increased expression of LMP2A compared with controls, and high ROS levels were identified. Cell viability assay showed that LMP2A promoted cell growth by regulating gene expression. Furthermore, LMP2A induced the expression of PRDX1 and beta-catenin. LMP2A also increased the expression of both cyclin B1 and cyclin D1. In NPC cells, PRDX1 and beta-catenin were regulated through LMP2A expression, which reduced cell growth through cell cycle-related gene expression. This study suggests that LMP2A could be a target molecule for inhibiting cancer progression in NPC cells infected with EBV.

The role of Epstein-Barr virus in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a metastasis-prone malignancy closely associated with the Epstein-Barr virus (EBV). Despite ubiquitous infection of EBV worldwide, NPC incidences displayed predominance in certain ethnic groups and endemic regions. The majority of NPC patients are diagnosed with advanced-stage disease, as a result of anatomical isolation and non-specific clinical manifestation. Over the decades, researchers have gained insights into the molecular mechanisms underlying NPC pathogenesis as a result of the interplay of EBV infection with several environmental and genetic factors. EBV-associated biomarkers were also used for mass population screening for the early detection of NPC. EBV and its encoded products also serve as potential targets for the development of therapeutic strategies and tumour-specific drug delivery. This review will discuss the pathogenic role of EBV in NPC and efforts in exploiting the potential of EBV-associated molecules as biomarkers and therapeutic targets. The current knowledge on the role of EBV and its associated products in NPC tumorigenesis, development and progression will offer a new outlook and potential intervention strategy against this EBV-associated malignancy.

A phase 1b/2 study of nanatinostat and valganciclovir in patients with advanced Epstein-Barr virus positive (EBV+) solid tumors and in combination with pembrolizumab in patients with recurrent/metastatic nasopharyngeal carcinoma (RM-NPC).

TPS6107 Background: Epstein-Barr virus (EBV) is linked to the development and pathogenesis of nasopharyngeal carcinoma (NPC). Most patients present with advanced stage disease at diagnosis (Li 2014); first-line chemoradiation is commonly followed by recurrence and poor prognosis emphasizing the need for new treatment options. Targeting EBV in NPC represents a novel therapeutic approach. EBV is predominantly latent in NPC; pre-clinical studies demonstrated that induction of the viral lytic phase by histone deacetylase inhibitors (HDACi) renders EBV+ tumor cells susceptible to the cytotoxic activity of ganciclovir (GCV) (Hui 2016). Nanatinostat (Nstat), a potent Class-I HDACi, induces the expression of the lytic BGLF4 protein kinase in EBV+ tumor cells, which activates the nucleoside analog GCV via phosphorylation. Phosphorylated GCV becomes incorporated into the cellular DNA causing chain termination and apoptosis. The all-oral combination of Nstat and valganciclovir (VGCV), a pro-drug of GCV, has demonstrated favorable safety and preliminary efficacy in a phase 1/2 study in patients with recurrent EBV+ lymphoma (NCT03397706). This phase 1b/2, open-label, multicenter study will evaluate the safety, tolerability, pharmacokinetics, and preliminary activity of Nstat + VGCV in patients with advanced EBV+ solid tumors. Additionally, the combination of pembrolizumab together with Nstat + VGCV will be evaluated in recurrent/metastatic NPC (RM-NPC) patients. NPC frequently exhibits high PD-L1 expression levels; however, PD-1 inhibitors resulted in limited response rates ranging from 20-30% in the RM-NPC setting. Methods: Phase 1b utilizes a 3+3 dose escalation design to determine the recommended Phase 2 dose (RP2D) of Nstat + VGCV in patients with EBV+ RM-NPC. In Phase 2, up to 60 patients with EBV+ RM-NPC will be randomized 1:1 to receive Nstat + VGCV at the RP2D with or without pembrolizumab to evaluate safety, tolerability, overall response rate, and potential pharmacodynamic markers of drug activity, including plasma EBV DNA levels. Additionally, patients with EBV+ solid tumors other than RM-NPC will receive Nstat + VGCV at the RP2D in a separate Phase 1b dose expansion cohort. Patients eligible for the phase 1b dose escalation and phase 2 will have EBV+ RM-NPC with 1-3 prior lines of platinum-based chemotherapy and no available curative therapies. Patients with advanced EBV+ non-NPC solid tumors (gastric cancer, lymphoepithelioma-like carcinoma, leiomyosarcoma) and no curative therapies are eligible for the phase 1b expansion cohort. All patients must have measurable disease per RECIST v1.1 and adequate bone marrow, liver, and renal function. Enrollment began in January 2022. Clinical trial information: NCT05166577.

F factor plasmid-mediated Epstein-Barr virus genome introduction establishes an EBV positive NPC cell model.

BackgroundMost Epstein-Barr virus (EBV)-positive cells lose the EBV episomes upon prolonged propagation. PurposeThe purposes of this study were to establish a simple cell model for nasopharyngeal carcinoma (NPC) research by introducing a plasmid with the EBV genome into NPC cells and then to investigate the resulting changes in malignant biological behaviour and NPC-associated signalling pathways. MethodsHONE1 NPC cells were transfected with F-factor plasmids including the EBV genome (HONE1-EBV cells). Then cell proliferation, migration, cell cycle distribution and apoptosis were evaluated in vitro by using CCK8, transwell and flow cytometry assays respectively. EBV-encoded proteins and cell signal tranducting proteins were detected by western blot assays. EBV-encoded RNAs were detected by in situ hybridization. EBV particles were assayed by transmission electron microscope (TEM). The morphology of cells were detected by immunofluorescence assays for alpha-tubulin. ResultsLatent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), Epstein-Barr nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) were successfully expressed in HONE1-EBV cells. No EBV particles were founded by TEM. Introduction of the EBV genome significantly promoted proliferation, cell cycle progression and migration and inhibited apoptosis in HONE1 cells. Immunofluorescence assays showed that the morphology of HONE1-EBV cells changed into spindle. Furthermore, EBV genome introduction significantly inhibited the JAK/STAT signalling pathway, while it activated the PI3K-AKT and NF-κB signalling pathways in HONE1 cells. ConclusionThese findings suggest that F-factor plasmid-mediated EBV genome introduction was successful in constructing an EBV positive cell model, which showed deteriorated biological behavior and activated NPC-associated signalling pathways. This model can serve as a good tool for studying EBV in NPC, but the subtle differences in cancer-associated pathways must be considered.

NF-κB Signaling Regulates Epstein-Barr Virus BamHI-Q-Driven EBNA1 Expression.

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the few viral proteins expressed by EBV in nasopharyngeal carcinoma (NPC), most likely because of its essential role in maintaining the viral genome in EBV-infected cells. In NPC, EBNA1 expression is driven by the BamHI-Q promoter (Qp), which is regulated by both cellular and viral factors. We previously determined that the expression of another group of EBV transcripts, BamHI-A rightward transcripts (BARTs), is associated with constitutively activated nuclear factor-κB (NF-κB) signaling in NPC cells. Here, we show that, like the EBV BART promoter, the EBV Qp also responds to NF-κB signaling. NF-κB p65, but not p50, can activate Qp in vitro, and NF-κB signaling regulates Qp-EBNA1 expression in NPC cells, as well as in other EBV-infected epithelial cells. The introduction of mutations in the putative NF-κB site reduced Qp activation by the NF-κB p65 subunit. Binding of p65 to Qp was shown by chromatin immunoprecipitation (ChIP) analysis, while electrophoretic mobility shift assays (EMSAs) demonstrated that p50 can also bind to Qp. Inhibition of NF-κB signaling by the IκB kinase inhibitor PS-1145 resulted in the downregulation of Qp-EBNA1 expression in C666-1 NPC cells. Since EBNA1 has been reported to block p65 activation by inhibiting IKKα/β through an unknown mechanism, we suggest that, in NPC, NF-κB signaling and EBNA1 may form a regulatory loop which supports EBV latent gene expression, while also limiting NF-κB activity. These findings emphasize the role of NF-κB signaling in the regulation of EBV latency in EBV-associated tumors.

Analysis of Epstein-Barr Virus Genomes and Expression Profiles in Gastric Adenocarcinoma.

Epstein-Barr virus (EBV) is a causative agent of a variety of lymphomas, nasopharyngeal carcinoma (NPC), and ∼9% of gastric carcinomas (GCs). An important question is whether particular EBV variants are more oncogenic than others, but conclusions are currently hampered by the lack of sequenced EBV genomes. Here, we contribute to this question by mining whole-genome sequences of 201 GCs to identify 13 EBV-positive GCs and by assembling 13 new EBV genome sequences, almost doubling the number of available GC-derived EBV genome sequences and providing the first non-Asian EBV genome sequences from GC. Whole-genome sequence comparisons of all EBV isolates sequenced to date (85 from tumors and 57 from healthy individuals) showed that most GC and NPC EBV isolates were closely related although American Caucasian GC samples were more distant, suggesting a geographical component. However, EBV GC isolates were found to contain some consistent changes in protein sequences regardless of geographical origin. In addition, transcriptome data available for eight of the EBV-positive GCs were analyzed to determine which EBV genes are expressed in GC. In addition to the expected latency proteins (EBNA1, LMP1, and LMP2A), specific subsets of lytic genes were consistently expressed that did not reflect a typical lytic or abortive lytic infection, suggesting a novel mechanism of EBV gene regulation in the context of GC. These results are consistent with a model in which a combination of specific latent and lytic EBV proteins promotes tumorigenesis.IMPORTANCE Epstein-Barr virus (EBV) is a widespread virus that causes cancer, including gastric carcinoma (GC), in a small subset of individuals. An important question is whether particular EBV variants are more cancer associated than others, but more EBV sequences are required to address this question. Here, we have generated 13 new EBV genome sequences from GC, almost doubling the number of EBV sequences from GC isolates and providing the first EBV sequences from non-Asian GC. We further identify sequence changes in some EBV proteins common to GC isolates. In addition, gene expression analysis of eight of the EBV-positive GCs showed consistent expression of both the expected latency proteins and a subset of lytic proteins that was not consistent with typical lytic or abortive lytic expression. These results suggest that novel mechanisms activate expression of some EBV lytic proteins and that their expression may contribute to oncogenesis.

Epstein-Barr Virus in Nasopharyngeal Carcinoma of Guatemalan and Brazilian Patients.

Nasopharyngeal carcinoma (NPC) is highly associated with Epstein-Barr virus (EBV), particularly the undifferentiated nonkeratinizing subtype. Prevalence of EBV in NPC in countries such as Guatemala and Brazil has not been studied. We analyzed 19 cases of NPC, 11 from Guatemala and 8 from Brazil, for the presence of EBV by in situ hybridization and immunohistochemistry. Additionally, 19 hyperplastic adenoids from children were analyzed for EBV by in situ hybridization, 12 from Guatemala and 7 from Brazil. All the NPC cases from Guatemala and 5 from Brazil were of the undifferentiated nonkeratinizing type. EBV-negative cases comprised 2 keratinizing NPC and 1 differentiated nonkeratinizing NPC. All undifferentiated nonkeratinizing NPC from both samples showed intense positivity for EBER, while LMP-1 only focally and scarcely expressed. EBER was positive in 75% and 43% of the adenoids from Guatemala and Brazil, respectively. All undifferentiated nonkeratinizing NPC irrespective of origin from Guatemala or Brazil were highly associated with EBV.

Human papillomavirus and Epstein-Barr virus in nasopharyngeal carcinoma in anon-endemic eastern european population.

Nasopharyngeal carcinoma (NPC) is arare malignancy in the Czech Republic and Slovakia, with the standardized incidence rate of < 1:100000 person-years. Viral status of NPC in these non-endemic Eastern European regions is currently unknown. In aretrospective study, we evaluated the presence of EBV and HPV in 62 NPC cases. EBV status was determined by the use of in situ hybridization (ISH) for EBV encoded small RNA 1 (EBER1). HPV status was examined with p16 immunohistochemistry, DNA ISH and DNA polymerase chain reaction. Sixty-one studied cases showed non-keratinizing morphology and one was keratinizing squamous cell carcinoma. Only one NPC with non-keratinizing morphology was scored as p16-positive (nuclear and cytoplasmic staining ≥ 70% of tumor cells). This case was positive for high-risk HPV by ISH and the DNA PCR confirmed the presence of HPV18 type. At the same time, this case was found negative for EBV. Remaining sixty-one cases that were scored as p16-negative were all found HPV-negative by ISH and the DNA PCR. EBV was detected in 85.5% (53/62) of cases and 9 cases were EBV-negative, including the case of keratinizing NPC. In contrast with previous reports on the prevalence of EBV-positivity in Caucasian patients with NPC, the majority of patients coming from this non-endemic region show EBV-positivity; therefore, they may be candidates for novel EBV-targeting therapies. Conversely, HPV-positive NPC is very rare and HPV does not seem to play asignificant role in the etiopathogenesis of NPC in these Eastern European populations.

Telomerase activity impacts on Epstein-Barr virus infection of AGS cells.

The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva.

Cancer-associated infectious agents and epigenetic regulation.

Infectious agents are one of the factors which contribute to cancer development. Few examples include human papilloma virus in cervical cancer, hepatitis virus in hepatocellular carcinoma, herpes virus in Kaposi's sarcoma, Epstein-Barr virus in nasopharyngeal carcinoma, human T-cell lymphotropic virus type-1 (HTLV-1) in T-cell leukemia and lymphoma, Helicobacter pylori in gastric cancer. These agents cause genomic instability in the host and most of them affect host immune system. Infectious agents may integrate in the host genome although their sit of integration is not fixed. Expression of some infectious agents involves epigenetic regulation by DNA methylation, histone modification, miRNA level alteration, and chromatin condensation. This chapter provides examples where epigenetic regulation has been reported in cancer-associated infectious agents. Epigenetic inhibitors and their potential in cancer control and treatment are also discussed.

Abstract 1512: Role of Epstein-Barr virus in nasopharyngeal carcinoma under metabolic stress.

Abstract Most nasopharyngeal carcinoma (NPC) biopsies contain a human pathogen named as Epstein-Barr virus (EBV). EBV infection is considered as an early event involved in the pathogenesis of NPC. However, the close association of EBV with NPC is a paradox. EBV infection is common that occurs in ≥95% of the world's population, yet the incidence rate of NPC is rare worldwide and is only seen in Cantonese speaking ethnic groups in Southern Asia. The mechanisms that regulate EBV infection of NPC/pre-malignant NP cells including the events involved in latency and lytic activation (either in vitro or in vivo) are not well-defined. Expressions of latent and lytic EBV genes have been shown to have variable effects on the biology and malignant properties of EBV-infected cells. Hypoxia and nutrient-starvation are common physiological stresses within tumours and may have impact on regulation of latent and lytic EBV infection in NPC cells. Here we report that the levels of hypoxia-inducible factor, HIF-1α protein, a prime hypoxic-stress responsive marker for mammalian cells, are higher in EBV-infected NPC/pre-malignant NP cell lines under hypoxia than their non-infected counterparts, which may imply an enhanced survival ability of EBV-infected NP cells. The alteration in gene expression profiles of host cells and expression of EBV genes observed in infected cells may be responsible for the stabilization of HIF-1α and vice versa. Moreover, EBV infection enhances the tumour growth rate and size of NPC cells xenografts in nude mice. The main stem of this study is to examine whether there is a differential response between EBV-infected and non-infected NP cells towards various metabolic stresses. Through studying this project, the mechanism and function of HIF-1α stability by EBV infection will be explored, which is expected to help characterizing the role of EBV in transformation of epithelial cells and how EBV infection may enhance NPC cells to survive inside a tumour core. Citation Format: VMY Lau, SW Tsao. Role of Epstein-Barr virus in nasopharyngeal carcinoma under metabolic stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1512. doi:10.1158/1538-7445.AM2013-1512 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Genomic Sequencing and Comparative Analysis of Epstein-Barr Virus Genome Isolated from Primary Nasopharyngeal Carcinoma Biopsy

Whether certain Epstein-Barr virus (EBV) strains are associated with pathogenesis of nasopharyngeal carcinoma (NPC) is still an unresolved question. In the present study, EBV genome contained in a primary NPC tumor biopsy was amplified by Polymerase Chain Reaction (PCR), and sequenced using next-generation (Illumina) and conventional dideoxy-DNA sequencing. The EBV genome, designated HKNPC1 (Genbank accession number JQ009376) is a type 1 EBV of approximately 171.5 kb. The virus appears to be a uniform strain in line with accepted monoclonal nature of EBV in NPC but is heterogeneous at 172 nucleotide positions. Phylogenetic analysis with the four published EBV strains, B95-8, AG876, GD1, and GD2, indicated HKNPC1 was more closely related to the Chinese NPC patient-derived strains, GD1 and GD2. HKNPC1 contains 1,589 single nucleotide variations (SNVs) and 132 insertions or deletions (indels) in comparison to the reference EBV sequence (accession number NC007605). When compared to AG876, a strain derived from Ghanaian Burkitt's lymphoma, we found 322 SNVs, of which 76 were non-synonymous SNVs and were shared amongst the Chinese GD1, GD2 and HKNPC1 isolates. We observed 88 non-synonymous SNVs shared only by HKNPC1 and GD2, the only other NPC tumor-derived strain reported thus far. Non-synonymous SNVs were mainly found in the latent, tegument and glycoprotein genes. The same point mutations were found in glycoprotein (BLLF1 and BALF4) genes of GD1, GD2 and HKNPC1 strains and might affect cell type specific binding. Variations in LMP1 and EBNA3B epitopes and mutations in Cp (11404 C>T) and Qp (50134 G>C) found in GD1, GD2 and HKNPC1 could potentially affect CD8+ T cell recognition and latent gene expression pattern in NPC, respectively. In conclusion, we showed that whole genome sequencing of EBV in NPC may facilitate discovery of previously unknown variations of pathogenic significance.

Detection of EBV in nasopharyngeal carcinoma by quantum dot fluorescent in situ hybridization

Aims Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia and is frequently associated with Epstein–Barr virus (EBV) infection. The primary aim of this study was to improve the method of EBV detection by exploring quantum dots in FISH detection, and compare QD-based FISH with conventional ISH. Materials and methods Biopsy specimens were retrospectively retrieved from 35 NPC patients as paraffin-embedded tissue blocks. QD-FISH was developed to detect the presence of EBV encoded small RNA (EBER) using biotin-labeled EBER oligonucleotide probe indirectly labeled with streptavidin-conjugated quantum dots. Conventional ISH was also performed using a commercial kit to assess concordance between the two methods. Results All the 35 NPC cases were nonkeratinizing carcinoma (7 differentiated and 28 undifferentiated subtypes). EBER-positive signals were detected in 91.43% (32/35) and 80% (28/35) cases by QD-FISH and ISH, respectively. There was no significant difference in the number of EBER-positive cases by the two methods. A moderate concordance was found between QD-FISH and ISH for EBER status (κ = 0.55). Four EBER-negative cases by ISH showed EBER-positive signals when detected by QD-FISH. Conclusions EBV is closely associated with NPC in Chinese patients. QD-FISH is a novel effective method for EBER detection, and has a moderate concordance with conventional ISH.

NOLC1, an Enhancer of Nasopharyngeal Carcinoma Progression, Is Essential for TP53 to Regulate MDM2 Expression

Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in South China, Singapore, and Taiwan. At present, its etiological factors are not well defined. To identify which genetic alterations might be involved in NPC pathogenesis, we identified genes that were differentially expressed in NPC cell lines and normal nasomucosal cells using subtractive hybridization and microarray analysis. Most NPC cell lines and biopsy specimens were found to have higher expression levels of the gene encoding nucleolar and coiled-body phosphoprotein 1 (NOLC1) as compared with normal cells. Severe combined immunodeficiency mice bearing NPC xenografts derived from NOLC1-short hairpin-RNA-transfected animals were found to have 82% lower levels of tumor growth than control mice as well as marked tumor cell apoptosis. Measuring the expression levels of genes related to cell growth, apoptosis, and angiogenesis, we found that the MDM2 gene was down-regulated in the transfectants. Both co-transfection and chromatin immunoprecipitation experiments showed that tumor protein 53-regulated expression of the MDM2 gene requires co-activation of NOLC1. These findings suggest that NOLC1 plays a role in the regulation of tumorigenesis of NPC and demonstrate that both NOLC1 and tumor protein 53 work together synergistically to activate the MDM2 promoter in NPC cells.

Regulation of IAPs gene family by interleukin‐1α and Epstein‐Barr virus in nasopharyngeal carcinoma

Inhibitors of apoptosis proteins (IAPs), which counteract apoptosis by potently inhibiting caspase activation, are promising targets of new anti-tumor therapy. However, their roles in the pathogenesis of nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-associated carcinoma, are not fully understood. Herein, we investigated the expression and regulation of IAPs in NPC. Using real-time quantitative polymerase chain reaction (PCR) analysis, we found that among the IAPs family only the transcription of survivin, HIAP-1, and HIAP-2 was consistently up-regulated in NPC and metastatic NPC tissues. Immunohistochemical staining showed that their proteins were more predominantly expressed in tumor cells nests. Noteworthy, these IAPs were upregulated by interleukin-1 alpha stimulation or EBV infection, and subsequently resulted in triggering rapid proliferation of NPC verified by strong Ki-67 staining. Survivin, HIAP-1, and HIAP-2 were distinctly upregulated in NPC, suggesting they may play significant roles in NPC tumorigenesis and serve as tumor markers with prognostic and therapeutic implications.

Hypermethylation of epithelial-cadherin gene promoter is associated with Epstein-Barr virus in nasopharyngeal carcinoma

Background: Epstein-Barr virus (EBV) is documented as the important etiologic agent of nasopharyngeal carcinoma (NPC) but the mechanism of development and pathogenesis induced by EBV is presently unclear. Hypermethylation of epithelial-cadherin (E-cadherin) promoter has been shown to be induced in NPC cell line by EBV LMP1 via DNA methyltransferase activation. EBV genomes and hypermethylation of E-cadherin promoter were investigated in NPC tissues to evaluate the role of EBV in the hypermethylation and pathogenesis of NPC. Methods: Methylation-specific polymerase chain reaction (MSP) was performed to detect E-cadherin promoter hypermethylation in paraffin embedded tissues from patients with NPC and normal nasopharyngeal tissues. EBV genomes were detected by PCR in the tissue samples. Results: Hypermethylation of E-cadherin promoter and EBV were predominantly detected in undifferentiated and non-keratinizing NPC compared to those in squamous cell NPC. Hypermethylation of E-cadherin was found in 28 of 38 (73.7%) patient samples. EBV was detected in 22 of the 28 (78.6%) NPC samples demonstrating E-cadherin hypermethylation. EBV genomes and hypermethylation were not detected in normal nasopharyngeal tissues. Significant association was found between E-cadherin hypermethylation and EBV genomes ( p < 0.001; Fisher's exact test). Hypermethylation of E-cadherin was more frequently detected in advanced stages compared to those in early stages of NPC ( p = 0.036; Fisher's exact test). Conclusions: The high incidence of EBV with the consistency of E-cadherin hypermethylation, particularly in undifferentiated and non-keratinizing NPC suggests the role of EBV in the hypermethylation. EBV exists at early stage of NPC that induces the hypermethylation and contributes to progression of the disease to the advanced stage of NPC.

Expression of VCA (viral capsid antigen) and EBNA1 (Epstein–Barr‐virus‐encoded nuclear antigen 1) genes of Epstein–Barr virus in Pichia pastoris and application of the products in a screening test for patients with nasopharyngeal carcinoma

EBV (Epstein-Barr virus) serological tests have been used for many years as accessory diagnostic predictors of NPC (nasopharyngeal carcinoma). To date, IF (indirect immunofluorescence) assays still serve as the 'gold standard' for EBV serodiagnosis. However, IF assays are time-consuming, unsuitable for automatic handling and difficult to standardize. This makes their application in mass screening of populations inconvenient. Some of the technical difficulties associated with IF have been overcome by the development of specific ELISAs, but, at present, high sensitivity and specificity cannot be achieved simultaneously by using recombinant protein-based ELISAs, as the diagnostic value of different fragments of EBV in NPC is different. In an attempt to determine a suitable recombinant EBV protein for diagnostic purposes, fragments of EBV VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes were expressed in the methylotrophic yeast Pichia pastoris, and a novel ELISA was established using P. pastoris-expressed VCA-BALF4 [aa (amino acids) 287-623; the BALF4 gene encodes the EBV glycoprotein gp125], EBNA1 (aa 390-641) and VCA-BFRF3 (the gene BFRF3 encodes a viral structural capsid protein or tegument protein VCA p18) proteins. Serum samples were collected from patients with NPC and healthy controls and were tested using this ELISA. The sensitivity of VCA-BFRF3, VCA-BALF4 and EBNA1 tests in the NPC sera were 65.0 (195/300), 76.3 (229/300) and 81.4% (244/300) respectively, whereas the specificity of normal individuals were 92 (460/500), 96 (480/500) and 95.8% (479/500). The optimum combination is VCA-BALF4 plus EBNA1, which identified 90.3% (271/300) of the NPC patients and had a specificity of 92.8% (464/500) for normal individuals. The results obtained from the evaluation of three antibodies to EBV as markers for detecting NPC suggests that a combination of EBNA1 (aa 390-641) and VCA-BALF4 (aa 287-623) assays would give better results in screening for NPC.

Oncogenic role of Epstein-Barr virus-encoded small RNAs (EBERs) in nasopharyngeal carcinoma

Objective Epstein-Barr virus (EBV) is a causative agent of nasopharyngeal carcinoma (NPC), and EBV gene expression is considered to be closely associated with the pathogenesis of NPC. Among EBV genes expressed in NPC, EBV-encoded non-polyadenylated RNAs, termed EBERs, are the most abundant transcripts of EBV in NPC. However, the role of EBERs still remains unclear. This study was designed to investigate the relevance of EBERs to the oncogenesis of NPC. Methods Two types of EBERs expression vectors (EBERs-high-expression vector and EBERs-low-expression vector) were constructed and transfected into EBV-negative cells, MDCK or EBV-negative clones of NPC-KT cells. Then, malignant transformation, represented by anchor independent growth, was evaluated between the EBERs-transfected cells and EBERs-negative cells using a soft agar colony formation assay. Apoptosis was induced by serum deprivation (0.1% concentration of fetal bovine serum) and interferon-α (IFN-α) (500 U/ml) treatment. Cell viability was evaluated with a trypan blue exclusion test. The activation of cellular transcriptional factor NF-κB was studied with the IL-8 promoter sequence using a luciferase reporter assay. Results EBERs-high-expression vector-transfected MDCK cells showed enhanced growth ability in soft agar compared with either EBERs-low-expression vector-transfected MDCK cells or EBERs-untransfected MDCK cells. However, they did not show the acquisition of any anti-apoptotic potential against either IFN-α or serum deprivation. Introduction of EBERs-low-expression vector into MDCK cells did not show anchor independent growth characteristics. Neither EBV-negative NPC-KT cells nor MDCK cells transfected with EBERs-high-expression vector showed any difference from EBERs-untransfected EBV-negative NPC-KT cells. Introduction of EBERs into MDCK cells did not transactivate the IL-8 promoter, indicating that neither NF-κB nor AP-1 was activated by EBERs. Conclusion EBERs are believed to induce the initial transformation of epithelial cells, thus contributing to the oncogenesis of NPC. Expression of abundant EBERs is considered to be critical for this transforming property of EBERs.

Functional assays of HLA A2-restricted epitope variant of latent membrane protein 1 (LMP-1) of Epstein-Barr virus in nasopharyngeal carcinoma of Southern China and Taiwan

Human leukocyte antigen (HLA) A2 was consistently associated with increased risk for nasopharyngeal carcinoma (NPC) in Chinese populations. Previously we have reported that an Epstein-Barr virus (EBV) strain carrying an HLA A2-restricted epitope variant of LMP-1 is prevalent in NPC in southern China and Taiwan (Lin et al., J. Gen. Virol. 85: 2023-2034, 2004). The variant has mutation selectively involved one of the two anchor residues in position 2 (125 L-->F) and an additional mutation in position 5 (129 M-->I). Functional assays of the epitope variant were carried out in the present work. The stabilization assay on T2 cells indicated that the variant peptide YFL (YFLEILWRL) prevalent in NPC binds to HLA A2 molecules less efficiently than the prototype peptide YLL (YLLEMLWRL). A dose-dependent binding of the HLA A2 molecules with added peptides was observed. In ex vivo cytotoxic T lymphocyte (CTL) assays with CD8-enriched effectors from A2-positive donors revealed that the YLL-specific CTL was able to lyse EBV-infected B cells expressing HLA A2, whereas the CTL recognition was abrogated with the peptide YFL. Cytokine (IFN-gamma) responses, measured both by intracytoplasmic staining and ELISPOT assays after peptide stimulation, also indicated that the variant epitope peptide failed to give an IFN-gamma response. The IFN-gamma response was almost entirely restricted to those tetramer-positive cells. These results show that EBV isolates from NPC of southern China and Taiwan is dominated by an HLA A2-restricted 'epitope-loss variants' of LMP-1, which would allow the virus to resist immune recognition and may in part contribute to the prevalence of NPC in these populations.

Amino acid changes in functional domains of latent membrane protein 1 of Epstein-Barr virus in nasopharyngeal carcinoma of southern China and Taiwan: prevalence of an HLA A2-restricted 'epitope-loss variant'.

Full-length sequences of the Epstein-Barr virus (EBV) gene for latent membrane protein (LMP)-1 from 22 nasopharyngeal carcinoma (NPC) biopsy specimens and 18 non-neoplastic counterparts (NPI) were determined. Relative to the B95-8 strain, the amino acid sequences of the toxic-signal and transformation domains were changed variably in NPC and NPI specimens; in contrast, no change was observed in the NF-kappaB (nuclear factor kappaB) activation domain. HLA typing revealed that 47 % of NPC and 31 % of NPI specimens were HLA A2-positive. A major A2-restricted epitope within LMP-1 (residues 125-133) was analysed. At residue 126, a change of L-->F was detected in 91 % (20/22) of NPC and 67 % (12/18) of NPI specimens. In addition, a deletion at residue 126 was detected in one NPC sample from Taiwan. At residue 129, a change of M-->I was observed in all samples, regardless of whether they were NPC or NPI. The changes in this peptide between NPC and NPI specimens, including mutation and deletion, are statistically significant (P<0.05). A recent report indicated that this variant sequence is recognized poorly by epitope-specific T cells. Genotyping results indicated that 96 % of NPC and 67 % of NPI samples carried a type A virus. By scanning the entire sequence of LMP-1, eight distinct patterns were identified. Detailed examination of these patterns revealed that type A strains are more prevalent in NPC than in NPI specimens and are marked by the loss of an XhoI site, the presence of a 30 bp deletion and the presence of a mutated, A2-restricted, T cell target epitope sequence. These results suggest that an EBV strain carrying an HLA A2-restricted 'epitope-loss variant' of LMP-1 is prevalent in NPC in southern China and Taiwan.