Epstein-Barr virus stably confers an invasive phenotype to epithelial cells through reprogramming of the WNT pathway.

Christine E Birdwell,Samantha Dykes,Kanchanjunga Prasai,Rona S Scott,Tawsha G.C Munroe,Yali Jia,Malgorzata Bienkowska-Haba

doi:10.18632/oncotarget.23824

Abstract

Epstein-Barr virus (EBV)-associated carcinomas, such as nasopharyngeal carcinoma (NPC), exhibit an undifferentiated and metastatic phenotype. To determine viral contributions involved in the invasive phenotype of EBV-associated carcinomas, EBV-infected human telomerase-immortalized normal oral keratinocytes (NOK) were investigated. EBV-infected NOK were previously shown to undergo epigenetic reprogramming involving CpG island hypermethylation and delayed responsiveness to differentiation. Here, we show that EBV-infected NOK acquired an invasive phenotype that was epigenetically retained after viral loss. The transcription factor lymphoid enhancer factor 1 (LEF1) and the secreted ligand WNT5A, expressed in NPC, were increased in EBV-infected NOK with sustained expression for more than 20 passages after viral loss. Increased LEF1 levels involved four LEF1 variants, and EBV-infected NOK showed a lack of responsiveness to β-catenin activation. Although forced expression of WNT5A and LEF1 enhanced the invasiveness of parental NOK, LEF1 knockdown reversed the invasive phenotype of EBV-infected NOK in the presence of WNT5A. Viral reprogramming of LEF1 and WNT5A was observed several passages after EBV infection, suggesting that LEF1 and WNT5A may provide a selective advantage to virally-infected cells. Our findings suggest that EBV epigenetically reprogrammed epithelial cells with features of basal, wound healing keratinocytes, with LEF1 contributing to the metastatic phenotype of EBV-associated carcinomas.

Highlights

Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma (SCC) prevalent in Southeastern Asia and Southern China [1].The World Health Organization has categorized nasopharyngeal carcinoma (NPC) into three types based on the tumor’s histological appearance
We focused on two members of the Wingless-Type MMTV Integration Site Family (WNT) signaling pathway, Lymphoid Enhancer Factor 1 (LEF1) and WNT5A, which were substantially increased after Epstein-Barr virus (EBV) infection
As EBV-infected cell lines were sorted on the basis of GFP, the absence of signal through the first 3 passages argues against clonal selection of a population of cells already expressing lymphoid enhancer factor 1 (LEF1) and WNT5A. These results suggest that EBV infection induced a slow cellular reprogramming that resulted in selection of stable expression of LEF1 and WNT5A that remained even after loss of the virus

Summary

Introduction

Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma (SCC) prevalent in Southeastern Asia and Southern China [1].The World Health Organization has categorized NPC into three types based on the tumor’s histological appearance. Epstein-Barr virus (EBV) is associated with a majority of undifferentiated. NPC cases and can be found in early pre-cancerous lesions, suggesting that the virus contributes to early events in NPC progression [2,3,4,5]. EBV has been associated with other undifferentiated carcinomas arising at other anatomical sites such as the stomach, tonsil, and thymus [8,9,10]. In EBV-associated carcinomas, a latent EBV infection is typically observed that includes expression of EBV nuclear antigen (EBNA) 1, latent membrane protein (LMP) 1, www.impactjournals.com/oncotarget

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