Epstein-Barr viral product-containing exosomes promote fibrosis and nasopharyngeal carcinoma progression through activation of YAP1/FAPα signaling in fibroblasts

Abstract

Background The progression of nasopharyngeal carcinoma (NPC) is profoundly affected by Epstein-Barr virus (EBV) infection. However, the role of EBV in the intercommunication between NPC and surrounding stromal cells has yet to be explored. Methods NPC biopsies were obtained for immunohistochemical (IHC) analyses. Clinical correlations between the expression of active YAP1/FAPα and the fibrotic response and between YAP1/FAPα and the density of cytotoxic CD8a+ T lymphocytes were determined. Survival times based on IHC scores were compared between groups using Kaplan-Meier survival and log-rank tests. Independent prognostic factors for metastasis/recurrence-free survival and overall survival were identified using univariate and multivariate Cox regression models. Fibroblasts were isolated from human nasopharyngeal biopsies. Exosomes were purified from culture supernatants of EBV+-positive NPC cells. The effects of EBV product-containing exosomes on fibroblast activation, fibrotic response, tumor growth, immune response, and correlations between the expression of featured genes were investigated using gel contraction assays, ELISAs, EdU incorporation assays, real-time impedance assays, RNA sequencing, immunostaining, 3D cancer spheroid coculture systems, and an NPC xenograft model. Results NPC patients who developed metastasis had significantly higher levels of active YAP1 and FAPα in their tumor stroma, which was further correlated with tumor fibrosis and poorer metastasis-free survival. Exosomes released from EBV+-NPC cells contained abundant FAPα protein and EBV-encoded latent membrane protein 1. Viral product-containing exosomes markedly enhanced the fibrotic response and tumor growth in a mouse xenograft model. IHC analyses of human NPC and NPC xenografts revealed positive correlations between levels of active YAP1 and FAPα, YAP1 and the fibrotic response, and FAPα and the fibrotic response. Mechanistic studies showed that treatment of fibroblasts with viral product-containing exosomes promoted the characteristics of cancer-associated fibroblasts by stimulating YAP1 signaling and the production of the immunosuppressive cytokines IL8, CCL2, and IL6. Inhibition of YAP1 activation markedly reversed these exosome-mediated protumoral effects, resulting in reduced contractility, inactivation of YAP1 signaling, and decreased production of immunosuppressive cytokines in fibroblasts. Furthermore, fibroblasts stimulated with these viral product-containing exosomes promoted NPC resistance to T cell-mediated cytotoxicity within tumor spheroids. In NPC tissues, a significant negative correlation was found between YAP1/FAPα and the density of CD8a+ T lymphocytes with a granzyme B signature. Conclusion EBV orchestrates interactions with the host and surrounding stroma by stimulating the functions of YAP1 and FAPα in fibroblasts through exosome cargos to create a more immunosuppressive, proinvasive microenvironment.

Nasopharyngeal carcinoma ecology theory: cancer as multidimensional spatiotemporal “unity of ecology and evolution” pathological ecosystem

“…Consistent with the "seed and soil" theory of metastasis proposed by Paget in 1889 (78), the best predictors of the success of ecological dispersal are those variables that whether cancer cells have enough ecological adaptive and reproductive ability (79)(80)(81). In the primary sites, for example, NPC cells can secrete matrix metalloproteinases that physically alter their environments (82)(83)(84), contribute to angiogenesis via vascular endothelial growth factor (VEGF) production (85-87), manipulate the content and functions of extracellular vesicles (EVs) (88)(89)(90)(91)(92) or change tight vascular capillary endothelial walls by inducing endothelial-mesenchymal transition (EnMT) (93) of the organ ecosystem. This is well reflected in the detection of magnetic resonance imaging (MRI) with extensive local infiltration of adjacent soft tissues, erosion of skull base, etc.…”

Intercellular crosstalk between cancer cells and cancer-associated fibroblasts via extracellular vesicles

“… [ 173 ] TGF-β Mesothelioma Colorectal cancer Prostate cancer Bladder cancer Triger the myofibroblast differen-tiation [ 30 ] FAP and EBV-encoded latent membrane protein 1 (LMP1) Nasopharyngeal carcinoma Generate CAFs via enhancing YAP1 signalling and increasing FAP expression. [ 177 ] COL6A1 Osteosarcoma COL6A1-positive EVs generate CAFs expressing interleukins, α-SMA, and TGF-β [ 81 ] Gain-of-function p53 Ovarian cancer Instigate CAF phenotypes in fibroblasts through the Nrf2-dependent pathway [ 39 ] Annexin A1 Pancreatic cancer Induce myofibroblasts features in the fibroblasts and endothelial cells [ 82 ] Lin28B Pancreatic cancer Generate CAFs from pancreatic stellate cells through activating let-7/HMGA2/PDGFRβ axis. [ 83 ] Hyal1 Prostate cancer Stimulate fibroblast chemotaxis by the increased adhesion and activating FAK signalling.…”

Updates on Epstein–Barr Virus (EBV)-Associated Nasopharyngeal Carcinoma: Emphasis on the Latent Gene Products of EBV

“…The replication and the mitotic isolation of EBV episomes, as well as the safeguarding of EBV genomes in inert contaminated cells, all rely upon EBNA1. Additionally, it is demonstrated that EBNA1 upholds cell endurance after DNA harm by instigating genomic insecurity and initiating a few cell qualities through transcriptional enactment [ 74 , 75 ].…”

“…The EBV genome’s EBNA2-encoding (BamHI-WYH) region allows for strain diversity, allowing all viral isolates to be divided into type 1 (EBV-1, B95.8-like) and type 2 categories (EBV-2, Jijoye-like). Due to this chromosomal variance, the EBNA2 protein may exist in two antigenically different versions that only share 50% amino acid similarity [ 74 ].…”

“…EBV is also present in a subset of gastric adenocarcinomas and other carcinomas of the salivary glands. As a result, EBV epithelial infection can cause malignant transformation, requiring specific preventive and therapeutic treatments [ 74 , 76 ].…”