Epstein-barr virus BART lncRNAs induce IKZF3/Aiolos to maintain EBV latency and promote tumorigenicity in nasopharyngeal carcinoma

Abstract

Epstein-Barr virus (EBV) maintains latency in its associated tumors, including nasopharyngeal carcinoma (NPC), expressing very few viral proteins. However, EBV expresses abundant levels of noncoding RNAs (mainly EBERs and BART miRNAs and lncRNAs) in NPC cells. The functions of BART lncRNAs in EBV latency and pathogenesis of NPC remain elusive. We previously found that IKZF3/Aiolos is a downstream target of BART lncRNAs in the nasopharyngeal carcinoma cell line C666-1. Although IKZF3/Aiolos has been reported as an immune modulator in numerous studies on lymphoma, little is known about its functions in EBV-associated NPC. This study is designed to investigate: (1) how IKZF3/Aiolos is regulated in NPC cells; (2) the role of IKZF3/Aiolos in maintaining EBV latency in NPC cells; and (3) how IKZF3/Aiolos promotes oncogenesis of NPC. I found that transcription of IKZF3/Aiolos is highly upregulated in EBV-infected NPC cells and clinical tissue samples due to the action of EBV encoded BART lncRNAs. Meta-analysis of eight EBV-associated studies showed that IKZF3/Aiolos was consistently upregulated in EBV-infected NPC cells. Immunohistochemistry (IHC) analysis of NPC biopsy specimens further demonstrated that IKZF3/Aiolos was expressed at higher levels in late stage (stage Ⅲ & Ⅳ) NPC patients than in early stage (stage Ⅰ & Ⅱ) disease. Secondly, I also showed that EBV-infected NPC cell lines (NPC43-C7 EBV-M81 & NPC43-C7 EBV-M81-ΔmirBARTs) and immortalized nasopharyngeal epithelial (NPE) cells (NP460-hTERT EBV-M81 and NP361-hTERT EBV-M81) with high copy numbers of EBV episomes expressed higher levels of IKZF3/Aiolos than cells with fewer copies of EBV. Finally, I demonstrated that IKZF3/Aiolos was upregulated in BART-stimulated NPC cells but downregulated in BART-knockdown NPC cells. This study further indicated that IKZF3/Aiolos modulates transcription of EBV BZLF1 and the cellular gene, LRIG1, to maintain EBV latency and promote NPC tumorigenesis in vitro and in vivo. My results showed that the EBV lytic reactivator BZLF1 was upregulated in IKZF3/Aiolos knockout or Aiolos inhibitor-treated (lenalidomide) C666-1 and NPC43-C7 EBV-M81 cells. ChIP-qPCR, immunoprecipitation and immunofluorescence analyses further revealed that IKZF3/Aiolos induces H3K27 deacetylation to epigenetically silence expression of BZLF1 and inhibit activation of the EBV lytic cycle in NPC cells. Moreover, functional analyses and western blotting showed that IKZF3/Aiolos inhibited the tumor suppressor, LRIG1, to upregulate expression and phosphorylation of the cellular proto-oncogene ERBB2 for progression of NPC oncogenesis. Sphere- and colony-formation experiments demonstrated that IKZF3/Aiolos enhances growth of NPC cells in vitro and in vivo experiments further showed that IKZF3/Aiolos promotes tumorigenicity of C666-1 cells in mice. Mechanically, I found that EBV lytic function and the expression of EBV BZLF1 and cellular LRIG1 were negatively regulated by BART lncRNAs and Aiolos regulatory machinery in NPC cells, while ERBB2 was positively regulated. In summary, my study showed that EBV BART lncRNAs induce IKZF3/Aiolos to maintain EBV latency and promote NPC tumorigenesis in vitro and in vivo. IKZF3/Aiolos has been identified as a new biomarker for NPC and may lead to the development of novel diagnostic tests and treatments for NPC.