Activation of lytic cycle of Epstein-barr virus of histone deacetylaseinhibitors

published_or_final_versionPaediatrics and Adolescent MedicineMasterMaster of Philosoph

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

 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.published_or_final_versionPaediatrics and Adolescent MedicineDoctoralDoctor of Philosoph

Therapeutic Strategies against Epstein-Barr Virus-Associated Cancers Using Proteasome Inhibitors

Epstein-Barr virus (EBV) is closely associated with several lymphomas (endemic Burkitt lymphoma, Hodgkin lymphoma and nasal NK/T-cell lymphoma) and epithelial cancers (nasopharyngeal carcinoma and gastric carcinoma). To maintain its persistence in the host cells, the virus manipulates the ubiquitin-proteasome system to regulate viral lytic reactivation, modify cell cycle checkpoints, prevent apoptosis and evade immune surveillance. In this review, we aim to provide an overview of the mechanisms by which the virus manipulates the ubiquitin-proteasome system in EBV-associated lymphoid and epithelial malignancies, to evaluate the efficacy of proteasome inhibitors on the treatment of these cancers and discuss potential novel viral-targeted treatment strategies against the EBV-associated cancers

Autophagy-Dependent Reactivation of Epstein-Barr Virus Lytic Cycle and Combinatorial Effects of Autophagy-Dependent and Independent Lytic Inducers in Nasopharyngeal Carcinoma

Autophagy, a conserved cellular mechanism, is manipulated by a number of viruses for different purposes. We previously demonstrated that an iron-chelator-like small compound, C7, reactivates Epstein-Barr virus (EBV) lytic cycle by activating the ERK1/2-autophagy axis in epithelial cancers. Here, we aim to identify the specific stage of autophagy required for EBV lytic reactivation, determine the autophagy dependency of EBV lytic inducers including histone deacetylase inhibitor (HDACi) and C7/iron chelators, for EBV lytic reactivation and measure the combinatorial effects of these types of lytic inducers in nasopharyngeal carcinoma (NPC). Inhibition of autophagy initiation by 3-MA and autolysosome formation by chloroquine demonstrated that only autophagy initiation is required for EBV lytic reactivation. Gene knockdown of various autophagic proteins such as beclin-1, ATG5, ATG12, ATG7, LC3B, ATG10, ATG3 and Rab9, revealed the importance of ATG5 in EBV lytic reactivation. 3-MA could only abrogate lytic cycle induction by C7/iron chelators but not by HDACi, providing evidence for autophagy-dependent and independent mechanisms in EBV lytic reactivation. Finally, the combination of C7 and SAHA at their corresponding reactivation kinetics enhanced EBV lytic reactivation. These findings render new insights in the mechanisms of EBV lytic cycle reactivation and stimulate a rational design of combination drug therapy against EBV-associated cancers

Difference in structures and biological activities between hit compounds and classical lytic inducers.

<p>(a) AGS-BX1 cells were treated with the hit compounds for 24h. Hyperacetylation of histone proteins and PKCδ phosphorylation were detected by Western blotting. No significant increase in the level of acetylated histone 3 (Acetyl-H3) and phosphorylated PKCδ was observed. (b) AGS-BX1 cells were treated by compound E11 or C7 for 1, 2, 4, 8, 12 24h. Changes in the phosphorylation of JNK and p38 MAPK were examined in conjunction with changes in the expression level of EBV proteins. JNK phosphorylation increased and sustained during the treatment period while level of phosphorylated p38 MAPK fluctuated across the treatment period. (c) AGS-BX1 cells were pre-treated with specific inhibitors of PI3K (LY294002, 15 μM, LY), MEK (PD98059, 50μM, PD), JNK (SP600125, 50μM, SP), p38 MAPK (SB202190, 20μM, SB), PKCδ (Rottlerin, 10μM) and ATM (KU-55933, 10μM, Ku) kinases for 1h before the addition of E11 and C7. Cells were harvest after 24h for examination of lytic induction by Western blotting. Lytic induction by E11 was significantly inhibited by SP, the specific JNK inhibitor while both PD (MEK inhibitor) and SP affected the induction by C7. (d) Structure of common HDAC inhibitors, SAHA and romidepsin, and the phorbol ester TPA, used for lytic induction of EBV. The structure of the newly identified compounds differs greatly from the known lytic inducers.</p

EBV lytic induction of the 5 hit compounds in the EBV-positive GC cells.

<p>AGS-BX1 cells were treated with (a) various concentrations of the hit compounds or (b) the hit compounds at the concentrations which maximally induced lytic cycle (optimal concentrations) at various time points. Expression of various EBV lytic proteins were analysed with Western blotting with cellular β-actin as a loading control. (c) AGS-BX1 cells were treated with the hit compounds at their optimal concentrations for 72h. The expression of Zta was visualized by immunofluorescent staining (red; middle panel) and the nuclei were visualized by DAPI (blue; top panel). (d) AGS-BX1 cells were treated with the hit compounds at their optimal concentrations for 72h. The percentage of cells induced into lytic cycle, i.e. expressing the IE protein Zta, was quantified by flow cytometry. (e) Production of infectious virions in AGS-BX1 cells after treatment with the hit compounds. AGS-Bx1 cells, which are capable of producing GFP-tagged EBV virions, were first treated by the hit compounds for 5 days. The supernatants containing the virions produced were then collected and incubated with Daudi cells, which are susceptible to superinfection of EBV. Percentage of superinfected Daudi cells, which were GFP-positive, were shown on the graph. The grey area represented unstained Daudi cells incubated with culture medium only.</p

EBV lytic induction of the 5 hit compounds in several EBV-positive epithelial cell lines.

<p><b>(</b>a) HONE1-EBV cells, (b) C666-1 cells, (c) SNU-719 cells, and (d) YCCEL1 cells were treated with the hit compounds at various concentrations for the specified period. HONE1-EBV cells contain a recombinant Akata genome while C666-1 cells, SNU-719 cells and YCCEL1 cells all contain native EBV genomes. The expression of viral IE protein Zta after treatment was detected by western blotting. The compound coded C7 could induce EBV lytic cycle in all the cell lines tested.</p