ABSTRACT
Viral noncoding RNAs (Epstein-Barr virus-encoded RNAs, EBERs) are believed to play a critical role in the progression of lymphoma and nasopharyngeal carcinoma (NPC). However, the accurate mechanisms accounting for their oncogenic function have not been elucidated, especially in terms of interaction between tumor cells and mesenchymal cells. Here, we report that, in addition to NPC cells, EBERs are also found in endothelial cells in Epstein-Barr virus (EBV)-infected NPC parenchymal tissues, which implicates NPC-derived extracellular vesicles (EVs) in transmitting EBERs to endothelial cells. In support of this hypothesis, we first ascertained if EBERs could be transferred to endothelial cells via EVs isolated from NPC culture supernatant. Then, we clarified that EVs-derived EBERs could promote angiogenesis through stimulation of VCAM-1 expression. Finally, we explored the involvement of EBER recognition by TLR3 and RIG-I in NPC angiogenesis. Our observations collectively illustrate the significance and mechanism of EVs-derived EBERs in angiogenesis and underlie the interaction mechanisms between EBV-infected NPC cells and the tumor microenvironment.
Subject(s)
DEAD Box Protein 58/genetics , Extracellular Vesicles/genetics , Herpesvirus 4, Human/genetics , RNA, Untranslated/genetics , Toll-Like Receptor 3/genetics , Vascular Cell Adhesion Molecule-1/genetics , Capillaries/metabolism , Capillaries/virology , Cell Line, Tumor , Cells, Cultured , Gene Expression Profiling/methods , Herpesvirus 4, Human/physiology , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/virology , Humans , Nasopharyngeal Carcinoma/genetics , Nasopharyngeal Carcinoma/pathology , Nasopharyngeal Carcinoma/virology , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/virology , RNA, Viral/genetics , Receptors, ImmunologicABSTRACT
Pathogen-induced inflammation has been one of the intensive research areas in carcinogenesis. EBV encoded RNAs (EBERs) have been suggested to play roles in anti-apoptosis and growth-promotion in lymphoid and immune disorders. However, pathological roles of EBERs in solid tumors of epithelia origin remain to be elucidated. Given their characteristic dsRNA structures, recent studies provided evidences for the activation of some pattern recognition receptors (PRR) by EBERs, which is fundamental in the process of pathogenesis. Here, we show that EBERs induce inflammatory response in nasopharyngeal carcinoma (NPC) cells through Toll-like receptor 3 (TLR3), mainly featured by high level of TNFα production. Interestingly, EBERs and EBV latent membrane protein 1 (LMP1) form a positive regulatory loop with NF-κB as a key node that amplifies the inflammatory signals in EBV infected epithelial cells. We demonstrate in vivo that EBERs can interact with TLR3 and induce tumor cells to produce cytokines in B16 synergetic tumor and human NPC xenograft models, in which macrophages are recruited and activated, leading to a favorable microenvironment for solid tumor growth. Lastly, we verify a positive association between EBER and TNFα levels in NPC clinical samples and the combination of EBER and TNFα expressions provides a predictor of poor survival of NPC patients. In conclusion, EBERs play a pivotal role in inflammation-to-oncogenesis transition in NPC development.
Subject(s)
Gene Expression Regulation, Neoplastic , Herpesvirus 4, Human/genetics , Nasopharyngeal Neoplasms/metabolism , RNA, Viral/metabolism , Toll-Like Receptor 3/metabolism , Animals , Apoptosis , Carcinoma , Cell Adhesion , Cell Line, Tumor , Cell Transformation, Neoplastic , Epstein-Barr Virus Infections/metabolism , Female , Gene Expression Regulation, Viral , Humans , Inflammation , Macrophages/metabolism , Melanoma, Experimental , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Nasopharyngeal Carcinoma , Neoplasm Transplantation , RNA, Double-Stranded/metabolism , Signal Transduction , Treatment Outcome , U937 Cells , Viral Matrix Proteins/metabolismABSTRACT
EBERs (EBER1 and EBER2) are suggested to be involved in cellular transformation and tumor growth. Cytoplasmic pattern recognition receptor-RIG-I, which is characterized by the recognition of viral dsRNAs, could efficiently trigger the downstream pathways of innate immunity. Although some previous reports have shown that EBERs and RIG-I associate with hematological malignancies, the role of EBERs-RIG-I signaling in solid tumors remains to be clarified. Here we demonstrate that EBER mediation of the inflammatory response via RIG-I contributes to NPC development in vitro and in vivo. We first verified that the expression level of RIG-I was associated with EBER transcription in a dose-dependent manner in NPC cells and specimens from NPC patients. Furthermore, pro-inflammatory cytokine transcription and release were sharply reduced after RIG-I knockdown compared with the control shRNA group in the presence of EBERs, accompanied by an attenuation of the NF-κB and MAPK signaling pathways. Consequently, the tumor burden was greatly alleviated in the RIG-I knockdown group in a xenograft model. In addition, macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein (MCP-1), which promote the maturation and attraction of tumor-associated macrophages, were stimulated upon the introduction of EBERs, and this upregulation conceivably led to the tumor-promoting subset transition of the macrophages. Taken together, our results reveal that EBERs could promote NPC progression through RIG-I-mediated cancer-related inflammation.
