Induction of lytic reactivation by hypoxia in an authenticated nasopharyngeal carcinoma cell line, NPC43, harbouring endogenous Epstein-Barr Virus

Abstract

Recent establishment of an endogenously EBV-infected nasopharyngeal carcinoma (NPC) cell line, NPC43, provides relevant cell model to explore the pathogenic role of EBV in NPC research. Previous scarcities of authenticated NPC cell systems and patient-derived xenografts were due to loss of EBV episomes upon in vitro propagation and viral infection-mediated growth inhibition. Tumour hypoxia, which arises when cell proliferation outbalances vascular supply, is a clinically significant phenomenon due to its association with poor prognosis in various cancer types, including NPC. Furthermore, hypoxia is a well-established lytic stimulus in EBV-infected B cells and unauthenticated NPC cells, the latter of which have been reported to be contaminated by genetic components of HeLa cells. HIF-1α-stabilising hypoxia mimetic agents, including iron-chelating agents and proteasome inhibitors, have demonstrated lytic reactivation potency in preclinical studies. The lack of authentic EBV-infected NPC cell line model responsive to hypoxia has impeded investigations into the relevance of these studies to NPC. This study aims to: 1) Demonstrate that hypoxia induces lytic replication in an authenticated EBV-positive NPC cell system, which closely recapitulates patient tumour cell phenotype 2) Dissect cellular mediators of lytic induction by hypoxia in EBV-infected NPC cells 3) Explore whether hypoxia enhances susceptibility to other classes of lytic inducers being studied Hypoxia was found to induce immediate-early Zta and early EA-D expressions in NPC43 cells through immunoblot analysis. RT-qPCR analysis showed transcriptional upregulation of late viral gene, BLLF1, by hypoxia, suggesting productive rather than abortive lytic reactivation in our NPC cell system. This was supported by the increase in EBV genome copy number per cell with time of hypoxia exposure. Failure to reproduce these results in other two endogenously infected NPC cell lines, C666-1 and C17, may be explained by differential epigenetic modification of viral genome. Examining the role of HIF-1α in regulating expression of lytic EBV protein is a major focus of this study. HIF-1α-expressing plasmid transfection only induced Zta and EA-D synthesis weakly, while siRNA knockdown of HIF-1α led to significant attenuation of lytic protein expression, indicating that HIF-1α is necessary but not sufficient to induce expression of lytic EBV proteins in NPC cells. Co-expressions of HIF-1α and viral lytic proteins were consistently observed in NPC PDXs X2117, Xeno23 and Xeno32 via immunohistochemical analysis. Accumulation of cellular differentiation regulator BLIMP-1 alongside lytic proteins in hypoxia-treated NPC43 cells strongly supports its role in hypoxia-driven EBV reactivation. siRNA knockdown of BLIMP-1 achieved comparable fold reduction in lytic protein synthesis as HIF-1α. Finally, NPC43 cells under chemical-induced hypoxia displayed hypersensitivity to lytic reactivating Stat3 inhibitors and ER stress inducers. In conclusion, this study illustrates the capacity of NPC43 cells to undergo productive lytic reactivation in response to a physiologically relevant lytic stimulus. Loss of this capacity at late passage may be accounted for by epigenetic modification of viral genome and poorly defined host factors. This study has established the use of an authenticated EBV-infected NPC model to study hypoxia-induced lytic reactivation, providing a platform to test hypoxia mimetic agents, alone or synergistically, in lytic induction therapy.