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Epstein-Barr virus BRLF1 induces genomic instability and progressive malignancy in nasopharyngeal carcinoma cells.

ABSTRACT: Nasopharyngeal carcinoma (NPC) is a serious health problem in China and Southeast Asia. Relapse is the major cause of mortality, but mechanisms of relapse are mysterious. Epstein-Barr virus (EBV) reactivation and host genomic instability (GI) have correlated with NPC development. Previously, we reported that lytic early genes DNase and BALF3 induce genetic alterations and progressive malignancy in NPC cells, implying lytic proteins may be required for NPC relapse. In this study, we show that immediate early gene BRLF1 induces chromosome mis-segregation and genomic instability in the NPC cells. Similar phenomenon was also demonstrated in 293 and zebrafish embryonic cells. BRLF1 nuclear localization signal (NLS) mutant still induced genomic instability and inhibitor experiments revealed that BRLF1 interferes with chromosome segregation and induces genomic instability by activating Erk signaling. Furthermore, the chromosome aberrations and tumorigenic features of NPC cells were significantly increased with the rounds of BRLF1 expression, and these cells developed into larger tumor nodules in mice. Therefore, BRLF1 may be the important factor contributing to NPC relapse and targeting BRLF1 may benefit patients.

SUBMITTER: Huang SY

PROVIDER: S-EPMC5668011 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Epstein-Barr virus BRLF1 induces genomic instability and progressive malignancy in nasopharyngeal carcinoma cells.

Huang Sheng-Yen SY Wu Chung-Chun CC Cheng Yu-Jhen YJ Chou Sheng-Ping SP Jiang Yun-Jin YJ Chu Kuo-Chang KC Tsai Ching-Hwa CH Lin Su-Fang SF Chen Jen-Yang JY

Oncotarget 20170905 45

Nasopharyngeal carcinoma (NPC) is a serious health problem in China and Southeast Asia. Relapse is the major cause of mortality, but mechanisms of relapse are mysterious. Epstein-Barr virus (EBV) reactivation and host genomic instability (GI) have correlated with NPC development. Previously, we reported that lytic early genes DNase and BALF3 induce genetic alterations and progressive malignancy in NPC cells, implying lytic proteins may be required for NPC relapse. In this study, we show that imm ...[more]

PMID: 29108278

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Project description:BackgroundEpstein-Barr virus (EBV) infection is a crucial risk factor for nasopharyngeal carcinoma (NPC), but the mechanism for its elevated activation level in NPC endemic areas remains unclear. This study aims to identify the EBV natural variations contributed to the different reactivation potential between NPC endemic and non-endemic areas.Methods1030 subjects were recruited in China, including 303 healthy individuals from two NPC non-endemic areas, 483 healthy people from three endemic areas and 244 NPC patients. Among which, saliva DNA samples from 244 participants were sequenced for the EBV immediate early (IE) genes of BRLF1 and BZLF1, their promoters were included; the rest 786 subjects were used for the validation of significant variations among three different populations. Haplotype and population structure analysis were conducted. Dual-luciferase assay was used to detect the promoter activity.ResultsA total of 246 distinct variations were detected, 29 showed significant difference in the frequencies between healthy people from NPC endemic area and non-endemic area. Population structure analysis clustered EBV strains into 9 subgroups mostly in accordance with the geographical origin of samples. Interestingly, two EBV genotypes, Rp-V1 and Rp-V2, were identified according to the linkage relationship of the variations in BRLF1 promoter (Rp). Rp-V1 has higher frequency in NPC endemic areas than in non-endemic areas (52.38% vs 18.15%, P = 2.07 × 10-14), and was associated with higher oral EBV DNA levels (adjusted OR = 1.64, 95% CI = 1.21-2.24, P = .002), suggesting a more powerful activation ability of Rp-V1 than that of the prototype Rp-of the EBV strain; On the contrary, Rp-V2 has higher frequency in NPC non-endemic areas than in endemic areas (18.48% vs 0.38%, P = 1.17 × 10-7), might represent a reduced activation potential of EBV. Further dual-luciferase assay showed Rp-V1 has higher promoter activity while compared with Rp-V2 (P < .0001). Notably, Rp-V1 impaired the transcription repression effect of YY1 while Rp-V2 strengthened the transcription repression effect of EBF1 on Rp. In addition, significant differences of Rta 393-407 CTL epitope which may influence the recognition of Rta by CD8+ T cells were detected between healthy people from NPC endemic area and non-endemic area.ConclusionsThis study identified natural variations in cis-acting elements (YY1 and EBF1) of EBV Rp altering Rp transcription activities, which may contribute to the elevated EBV activation level in NPC endemic areas than non-endemic areas.

Project description:Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) infection in regions in which it is endemic, including Southern China and Southeast Asia. The high mortality rates of NPC patients with advanced and recurrent disease highlight the urgent need for effective treatments. While recent genomic studies have revealed few druggable targets, the unique interaction between the EBV infection and host cells in NPC strongly implies that targeting EBV may be an efficient approach to cure this virus-associated cancer. Key features of EBV-associated NPC are the persistence of an episomal EBV genome and the requirement for multiple viral latent gene products to enable malignant transformation. Many translational studies have been conducted to exploit these unique features to develop pharmaceutical agents and therapeutic strategies that target EBV latent proteins and induce lytic reactivation in NPC. In particular, inhibitors of the EBV latent protein EBNA1 have been intensively explored, because of this protein's essential roles in maintaining EBV latency and viral genome replication in NPC cells. In addition, recent advances in chemical bioengineering are driving the development of therapeutic agents targeting the critical functional regions of EBNA1. Promising therapeutic effects of the resulting EBNA1-specific inhibitors have been shown in EBV-positive NPC tumors. The efficacy of multiple classes of EBV lytic inducers for NPC cytolytic therapy has also been long investigated. However, the lytic-induction efficiency of these compounds varies among different EBV-positive NPC models in a cell-context-dependent manner. In each tumor, NPC cells can evolve and acquire somatic changes to maintain EBV latency during cancer progression. Unfortunately, the poor understanding of the cellular mechanisms regulating EBV latency-to-lytic switching in NPC cells limits the clinical application of EBV cytolytic treatment. In this review, we discuss the potential approaches for improvement of the above-mentioned EBV-targeting strategies.

Project description:UnlabelledUndifferentiated nasopharyngeal carcinoma (NPC) has a 100% association with Epstein-Barr virus (EBV). However, only three EBV genomes isolated from NPC patients have been sequenced to date, and the role of EBV genomic variations in the pathogenesis of NPC is unclear. We sought to obtain the sequences of EBV genomes in multiple NPC biopsy specimens in the same geographic location in order to reveal their sequence diversity. Three published EBV (B95-8, C666-1, and HKNPC1) genomes were first resequenced using the sequencing workflow of target enrichment of EBV DNA by hybridization, followed by next-generation sequencing, de novo assembly, and joining of contigs by Sanger sequencing. The sequences of eight NPC biopsy specimen-derived EBV (NPC-EBV) genomes, designated HKNPC2 to HKNPC9, were then determined. They harbored 1,736 variations in total, including 1,601 substitutions, 64 insertions, and 71 deletions, compared to the reference EBV. Furthermore, genes encoding latent, early lytic, and tegument proteins and glycoproteins were found to contain nonsynonymous mutations of potential biological significance. Phylogenetic analysis showed that the HKNPC6 and -7 genomes, which were isolated from tumor biopsy specimens of advanced metastatic NPC cases, were distinct from the other six NPC-EBV genomes, suggesting the presence of at least two parental lineages of EBV among the NPC-EBV genomes. In conclusion, much greater sequence diversity among EBV isolates derived from NPC biopsy specimens is demonstrated on a whole-genome level through a complete sequencing workflow. Large-scale sequencing and comparison of EBV genomes isolated from NPC and normal subjects should be performed to assess whether EBV genomic variations contribute to NPC pathogenesis.ImportanceThis study established a sequencing workflow from EBV DNA capture and sequencing to de novo assembly and contig joining. We reported eight newly sequenced EBV genomes isolated from primary NPC biopsy specimens and revealed the sequence diversity on a whole-genome level among these EBV isolates. At least two lineages of EBV strains are observed, and recombination among these lineages is inferred. Our study has demonstrated the value of, and provided a platform for, genome sequencing of EBV.

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Epstein-Barr virus BRLF1 induces genomic instability and progressive malignancy in nasopharyngeal carcinoma cells.

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Epstein-Barr virus BRLF1 induces genomic instability and progressive malignancy in nasopharyngeal carcinoma cells.

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