EBV-EBNA1 constructs an immunosuppressive microenvironment for nasopharyngeal carcinoma by promoting the chemoattraction of Treg cells

Abstract

BackgroundNasopharyngeal carcinoma (NPC) is primarily caused by the Epstein-Barr virus (EBV) infection in NPC endemic areas. EBNA1 is an EBV-encoded nuclear antigen, which plays a critical role in the maintenance and replication of EBV genome. However, the mechanisms of EBNA1-promoted NPC immune escape are unknown. Regulatory T (Treg) cells are among the key regulators in restraining antitumor responses. However, the mechanisms of accumulation of Treg cells in NPC have not been defined. This study attempted to identify the detailed mechanisms of EBNA1 functions as a tumor accelerator to promote NPC immune escape by enhancing chemoattraction of Treg cells.MethodsmRNA profiles were determined by next-generation sequencing in NPC cells. In vitro and in vivo assays were performed to analyze the role of EBNA1 in regulation of recruitment of Treg cells. Colocation and coimmunoprecipitation analyzes were used to identify the SMAD3/c-JUN complex. Chromatin immunoprecipitation assay and dual luciferase reporter assays were designed to demonstrate c-JUN binding to miR-200a promoter and miR-200a targeting to CXCL12 3’Untranslated Regions. The hepatocellular carcinoma models were designed to demonstrate universality of the CXCL12-CXCR4-Treg axis in promoting immune evasion of various tumors.ResultA novel molecular mechanism was identified that involves EBV-EBNA1-stimulated chemotactic migration of Treg cells toward NPC microenvironment by upregulation of the transforming growth factor-β1 (TGFβ1)-SMAD3-PI3K-AKT-c-JUN-CXCL12-CXCR4 axis and downregulation of miR-200a. EBV-EBNA1 promotes the chemoattraction of Treg cells by governing the protein–protein interactions of the SMAD3/c-JUN complex in a TGFβ1-dependent manner in vitro and in vivo. TGFβ1 suppresses miR-200a by enhancing the SMAD3/c-JUN complex. miR-200a negatively regulates the CXCL12 chemokine by direct targeting of the CXCL12 3’UTR region. However, CXCL12 acts as the target gene of miR-200a and as an inhibitor of miR-200a transcription, and inhibition of miR-200a by CXCL12 is mediated by c-JUN, which directly binds to the miR-200a promoter and forms a c-JUN-miR-200a-CXCL12-c-JUN feedback loop. In addition, enhanced CXCL12 efficiently attracts CXCR4-positive Treg cells to remodel an immunosuppressive microenvironment.ConclusionsEBV-EBNA1 promotes chemotactic migration of Treg cells via the TGFβ1-SMAD3-PI3K-AKT-c-JUN-miR-200a-CXCL12-CXCR4 axis in the NPC microenvironment. These results suggest that EBV-EBNA1 may serve as a potential therapeutic target to reshape the NPC microenvironment.

The Stromal and Immune Landscape of Nasopharyngeal Carcinoma and Its Implications for Precision Medicine Targeting the Tumor Microenvironment

“…Type II latency is characterized by the expression of a set of latent genes in NPC, including Epstein-Barr nuclear antigen 1 (EBNA1), latent membrane protein 1 (LMP1), LMP2 and Epstein-Barr encoding region (EBER) RNAs (32,33). LMP1 and EBNA1 expressed by NPC cells are able to induce PD-L1 up-regulation via STAT3 and NF-kB signaling, Treg recruitment via CXCL12-CXCR4 chemotaxis, as well as expansion of myeloid-derived suppressor cells (MDSCs) (31,34,35). EBV can also infect B cells, however, the infiltrating B cells in the NPC microenvironment are uninfected, indicating that EBV infection on nasopharyngeal epithelial cells may occur prior to B-cell recruitment and accumulation (36).…”

Study On the Mechanism of LMP2A Maintaining Epstein-barr Virus Latency Infection Through Interaction With CXCR4

“…CXCR4 is highly expressed in various types of tumor cells and tissues, and can be involved in tumor growth, invasion, angiogenesis and metastasis, such as breast cancer, colorectal cancer, pancreatic cancer, etc. promote NPC immune escape [9]. In Xu's study, the results showed that in NPC, LMP1 affects cell motility and invasion by inducing CXCR4 [33].…”

Epstein–Barr Virus Epithelial Cancers—A Comprehensive Understanding to Drive Novel Therapies

“…Apart from LMP1, other EBV viral oncoproteins and miRNAs are also implicated in the shaping of TIME. In NPC cell lines, EBNA1 activates TGFb1-SMAD3 signaling and suppression of miR-200a, leading to increased CXCL12 expression and recruitment of CXCR4+ Tregs (121). EBNA1 also enhanced the production of CCL20, which is also important for Treg migration (122).Tumours with higher density of Treg infiltration correlate with EBNA1 expression and found to have a poorer survival.…”

“…CAFs have been associated with EMT transition and metastasis (1,11,146), and can be found around tumour cell nests, with varying amounts in different NPCs (138,139). Again, LMP1 has been shown in vitro to facilitate transition normal fibroblasts (NF) to CAFs (121,147). Alpha smooth muscle actin (aSMA), an immune marker for CAFs, was found to be correlated with CD34, an indicator of neoangiogenesis (121,147).…”

“…Again, LMP1 has been shown in vitro to facilitate transition normal fibroblasts (NF) to CAFs (121,147). Alpha smooth muscle actin (aSMA), an immune marker for CAFs, was found to be correlated with CD34, an indicator of neoangiogenesis (121,147). SDF-1/CXCL12, for which CXCR4 is the cognate receptor for (2), can be secreted by CAFs in various cancers to promote the growth of stromal fibroblasts.…”