The Epstein-Barr Virus (EBV) Oncogene Latent Membrane Protein 1 (LMP1) Regulates B Cell MicroRNAs that Target PI3K/Akt/mTOR Survival Signaling

Abstract

EBV+ Post-transplant Lymphoproliferative Disorder (PTLD) continues to be a serious, often fatal complication of bone marrow and solid organ transplantation; indeed, post-transplant cancers are projected to surpass cardiovascular complications as a leading cause of death in transplant recipients in the next twenty years. EBV+ PTLD are usually associated with expression of the EBV oncogene, latent membrane protein 1 (LMP1). However, the mechanisms by which LMP1 contributes to the development and progression of EBV-associated PTLD remain to be determined, and constitute potential therapeutic targets for the treatment of PTLD. We previously demonstrated that both EBV infection and LMP1 signaling can modulate expression of host cell microRNAs (miRs), small non-coding RNAs that regulate gene expression. Altered expression of miRs is associated with a variety of human malignancies and, in the case of EBV infection and related malignancies, may promote viral persistence and/or host cell survival. Additionally, we have shown that LMP1 activates the PI3K/Akt/mTOR pathway to promote survival of EBV+ PTLD-derived cell lines both in vitro and in a xenogeneic model of PTLD. The purpose of this study was to determine how LMP signaling regulates cellular miR expression and whether these LMP1-regulated miRs promote a dependence on PI3K/Akt/mTOR signaling in EBV+ PTLD. Using an inducible, chimeric LMP1 signaling system expressed in EBV negative B lymphoma cells and small molecule inhibitors of PI3K/Akt (LY294002) and mTOR (rapamycin), we analyzed the expression of 667 unique human miRs by Taqman qPCR array. Array data was normalized to internal control, then to control cells in which LMP1 signaling was not activated. Activation of LMP1 results in the modulation of 62 miRs (23 up, 39 down) to a minimum 2-fold change. Of these 62 miRs, 34 showed a 2-fold or greater change in expression in the presence of either signaling inhibitor, eighteen miRs were specific to one of the two pathways, while 10 miRs were not affected by inhibition of either pathway. Using the miR target predictor TargetScan, we found that LMP1 signaling regulates several miRs predicted to target the 3‘-UTR of members of the PI3K/Akt/mTOR pathway. For example, miR-155 is predicted to target 5 positive regulators of this pathway (PI3K p85a, PDK1, Rheb, Raptor, and Rictor). A number of LMP1-regulated miRs are also predicted to target negative regulators of this pathway, including miR-367 (PTEN, PHLPP2, TSC1), miR-613 (PHLPP2, P70S6K), and miR-451 (TSC1). This suggests that LMP1-induced miR can further augment PI3K/Akt/mTOR activation to effect B lymphoma cell survival and growth. In conclusion, LMP1 and PI3K/Akt/mTOR constitute an important signaling node in the induction of host cell miR in EBV+ B cell lymphomas. Moreover, alteration of virally-induced miR constitutes a novel mechanism whereby mTOR inhibitors such as rapamycin can affect growth and survival of EBV+ B cell lymphomas.