EBV gene variation and epigenetic alterations in asian nasopharyngeal carcinoma and potential clinical applications

University dissertation from Stockholm : Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology

Abstract: The incidence of nasopharyngeal carcinoma (NPC), an epithelial tumor, varies remarkably in different geographic regions and human populations. The Epstein-Barr virus (EBV), other environmental factors and genetic susceptibility are all considered to be involved in the etiology of this form of cancer and the EBV has been extensively studied in this context. The present work focuses on three aspects of this field: genetic variations in EBV isolated from Asian/Vietnamese patients with NPC and their functional consequences; antisense therapy directed against both the EBNA1 (EB Nuclear Antigen 1) and the LMP1 (Latent Membrane Protein 1) genes of EBV; and epigenetic changes in cellular and EBV genes. Genetic variations in tumor viruses might contribute to geographic differences in the prevalence of virus-associated cancers. Inside cells, EBV expresses the EBNA1, LMP1 and LMP2 proteins, which play important roles in connection with infection. In order to investigate the possible existence of genetic variants of this virus in patients with NPC in Asia, where the frequency of EBV-associated NPC is high, we analyzed the nucleotide sequences encoding the entire LMP1 (including the promoter region), the functional domains (N and C-termini) of EBNA1, the N-terminus of LMP2A and binding sites for EBNA1, including the FR (family of repeats) and Q locus, in virus isolated from NPC biopsies. In comparison to the B95-8 strain of EBV, base substitutions were detected in all of these sequences, with the exception of the Q locus. The LMP1 sequence in specimens from three patients was almost identical to the common China 1 variant, whereas in the other two cases analyzed the sequence encoding the C-terminus of this protein was similar to the corresponding sequence in the Thai SEA-2 variant. In these latter two cases, one carried EBV type 1 and the other type 2. The majority of samples contained the V-val-v1 (Vvv1) subtype of EBNA1, while only one contained the Vvv2 subtype. Moreover, substitutions, deletions and variable numbers of repeats were frequently detected in the FR region of oriP. In contrast, sequence analysis of LMP2A revealed only minor polymorphisms. As assessed employing a luciferase reporter system, EBNA1 and FR containing 20 repeats each 30-bp in length derived from an Asian isolate supported a higher rate of transcription than that observed in the prototype B95-8 strain. In contrast, repression of the negative regulatory element of Qp by the Asian Vvv1 variant of EBNA1 was attenuated in comparison to the corresponding repression in B95-8. These differences indicate that viral genetic variation may influence the risk for EBV-associated disease, in particular NPC. Antisense oligonucleotides can effectively inhibit gene expression at the mRNA level and some are presently being tested in clinical trials. Here, antisense oligonucleotides directed against EBNA1 and LMP1 were transfected into cell lines employing the lipofectin procedure. Expression of EBNA1 and LMP1 was thereby down-regulated by 90% and 85% respectively, as assessed on the basis of FR-luciferase activity driven by EBNA1 and NF-kB-luciferase activity driven by LMP1. The inhibition was maximal 24 hours after transfection and persisted for more than 60 hours. Combined transfection with both EBNA1 and LMP1 antisense oligonucleotides synergistically inhibited the expression of these genes. The growth of C15 NPC xenografts transplanted to mice was suppressed by subcutaneous injection of these antisense molecules, opening the possibility that this approach might provide a valuable complement to established strategies for treatment and prevention of NPC. An increasing body of evidence indicates that epigenetic changes such as DNA methylation and histone modifications play important roles during the early stages of carcinogenesis. Here, we found that the promoter region of the human CDH13 gene, which encodes the cell adhesion molecule H-cadherin, was methylated in 89.7% of our samples of primary NPC, whereas such methylation was only detected in 10% of samples of normal nasopharyngeal epithelia (p<0.05). Our detection of CDH13 methylation in 81% of nasopharyngeal swabs from patients with NPC and none of the control swabs suggests that this might be a valuable marker for early diagnosis.

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