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

// Christine E. Birdwell 1, 2, 4 , Kanchanjunga Prasai 1 , Samantha Dykes 1, 2, 3 , Yali Jia 1 , Tawsha G.C. Munroe 1 , Malgorzata Bienkowska-Haba 1, 2 and Rona S. Scott 1, 2 1 Department of Microbiology and Immunology, Louisiana State University Health Science Center-Shreveport, Shreveport, LA 71130, USA 2 Feist-Weiller Cancer Center, Shreveport, LA 71130, USA 3 Radiation Oncology Department, University of Florida, Gainesville, FL 32610, USA 4 Current address: Department of Microbial Pathogenesis and Immunology, Texas A & M University, College Station, TX 77843, USA Correspondence to: Rona S. Scott, email: rscott1@lsuhsc.edu Keywords: Epstein-Barr virus; epigenetics; WNT; LEF1; invasion Received: December 15, 2016      Accepted: December 18, 2017      Published: January 02, 2018 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.