Induction of epstein-barr virus (EBV) lytic cycle and its cellular consequences in EBV-positive epithelial malignancies

Abstract



In Epstein-Barr virus (EBV)-associated malignancies, the virus is harbored in every tumor cell and persists in a tightly latent form (latency I, II or III) expressing a limited number of viral latent proteins. Induction of EBV lytic cycle, which triggers expression of a much larger number of viral proteins, may lead to therapeutic effects against EBV-associated cancers. We previously found that suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, induced EBV lytic cycle and mediated enhanced cell death in EBV-positive gastric carcinoma cells (latency II). In this thesis, we sought to investigate SAHA’s induction of EBV lytic cycle and its cellular consequences in EBV-associated epithelial malignancies, with particular focus on nasopharyngeal carcinoma (NPC) due to its strong association with EBV and high prevalence in southern Chinese populations.

SAHA effected strong induction of EBV lytic cycle in EBV-positive epithelial malignancies, including gastric carcinoma and NPC, as evidenced by strong expression of EBV lytic proteins, replication of viral DNA and production of infectious viral particles. Immunofluorescent staining revealed that up to 70% EBV-positive epithelial cancers expressed EBV lytic proteins following treatment with micromolar concentrations of SAHA. However, SAHA could not induce EBV lytic cycle in NK lymphoma cells (both NPC and NK lymphoma express EBV latency II pattern), indicating preferential viral lytic induction in epithelial rather than lymphoid malignancies. EBV lytic cycle induction in NPC by SAHA required activation of protein kinase C-delta (PKC-) and acetylation of non-histone protein but required neither phosphatidylinositol 3’-kinase (PI3K), MAPK/ERK kinase (MEK), c-Jun aminoterminal kinase (JNK) nor p38 stress mitogen-activated protein kinase (MAPK) signaling pathway.

Conflicting observations regarding the effect of EBV lytic cycle induction on apoptosis were reported. Thus, we investigated the relationship between EBV lytic cycle induction and apoptosis in NPC following treatment with SAHA. EBV-positive NPC showed a higher percentage of apoptosis and proteolytic cleavage of PARP, caspases-3, -7 and -9 over EBV-negative NPC and greater than 85% of NPC cells co-expressed EBV immediate-early (Zta), early (BMRF1) or late (gp350/220) lytic proteins and cleaved caspase-3. Tracking of expression of these lytic proteins over time demonstrated that NPC proceeded to apoptosis following EBV lytic cycle induction, contrary to the previously reported anti-apoptotic effect of EBV lytic proteins in Burkitt lymphoma. Analyses of cleaved caspase-3 expression upon RNAi knockdown and exogenous expression of Zta further supported that EBV lytic cycle directly led to apoptosis of EBV-positive NPC cells. Interestingly, inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA’s induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. Finally, in vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were observed in NPC tumors established in nude mice.

In conclusion, activation of EBV lytic cycle from latent cycle in EBV-positive epithelial malignancies including NPC by SAHA effected apoptosis and tumor growth suppression of the cancer cells and provided experimental evidence for virus-targeted therapy against EBV-positive cancers.