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

Abstract

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.

Advances in targeted therapy mainly based on signal pathways for nasopharyngeal carcinoma

“…219 LINC00460 is highly associated with the EMT in NPC cells via binding miR-30a-3p to regulate the expression of Rasrelated protein 1A (Rap1A). 220 What's more, patients whose infiltrating lymphocytes are characterized by high expression of lncRNA AFAP1-AS1 incline to distant metastasis and poor prognosis, especially with positive PD1. 221 N,N′-dinitrosopiperazine (DNP) can promote proliferation, invasion, and metastasis of NPC by enhancing the expression of miR-149 which suppresses the level of PKP3.…”

Lytic Induction Therapy against Epstein–Barr Virus-Associated Malignancies: Past, Present, and Future

“…Table 1 summarizes the efficiencies of EBV lytic induction by the different compounds from multiple studies. In general, HDAC inhibitors such as NaB could reactivate 2–60% of EBV-positive B cells into lytic cycle, while SAHA could reactivate 30–65% of EBV-associated epithelial cells (AGS-BX1, HA, and HK1-EBV) into lytic cycle [ 45 , 46 , 47 ]. VPA could induce around 10% of AGS-EBV cells, while the percentage was low in LCL and C666-1 cells [ 48 ].…”

“…Novel compounds identified by our group such as C7, E11, C8, E7, and A10 could induce 30–60% of AGS-BX1 cells into lytic cycle [ 42 ]. Follow-up studies on C7, the best-performing compound identified, showed its ability to induce 6–12% of HA, C666-1, and NPC43 cells into lytic cycle [ 36 , 46 ]. Another new class of compounds, curcuminoids, were shown to induce 20–50% of AGS-BX1, C666-1, and HONE1-EBV cells into lytic cycle [ 41 ].…”

“…NaB was shown to induce lytic cycle in EBV-positive lymphoma cell lines including P3HR-1, B95.8, Raji, Daudi [ 51 ], and AK2003 [ 47 ], as well as in EBV-associated epithelial carcinoma cell line, AGS-BX1 [ 47 ], but does so very weakly in NPC cells [ 41 ]. SAHA could induce lytic cycle in AGS-BX1, HA [ 45 , 46 ], AK2003 [ 47 ], and C666-1 [ 42 ] cells, but not in NPC43 [ 46 ] and LCLs [ 47 ]. Similar results were found in chemotherapy agents such as 5-FU and cis-platinum, which could induce lytic cycle in Akata and AGS-EBV cells [ 27 ], but not in LCLs [ 17 ].…”

“…Indeed, a pilot study on the efficacy and safety of romidepsin in treating extranodal natural killer/T-cell lymphoma found a substantial increase in viremia in these patients [ 52 ]. The novel compound C7 [ 36 , 42 , 46 ] and anti-bacterial antibiotic, clofoctol [ 26 ], were found to induce the expression of immediately early and early lytic proteins, but not late lytic proteins. Moreover, EBV virions were not produced after lytic induction by these two compounds.…”