A 3-gene signature comprising CDH4, STAT4 and EBV-encoded LMP1 for early diagnosis and predicting disease progression of nasopharyngeal carcinoma.

PURPOSE: Nasopharyngeal carcinoma is highly metastatic but difficult to detect in its early stages. It is critical to develop a simple and highly efficient molecular diagnostic method for early detection of NPC in clinical biopsies. METHODS: The transcriptomic data of primary NPC cell strains were used as a discovery tool. Linear regression approach was used to define signatures distinctive between early and late stage of NPC. Expressions of candidates were validated with an independent set of biopsies (n = 39). Leave-one-out cross-validation technique was employed to estimate the prediction accuracy on stage classification. The clinical relevance of marker genes was verified using NPC bulk RNA sequencing data and IHC analysis. RESULTS: Three genes comprising CDH4, STAT4, and CYLD were found to have a significant differentiating power to separate NPC from normal nasopharyngeal samples and predicting disease malignancy. IHC analyses showed stronger CDH4, STAT4, and CYLD immunoreactivity in adjacent basal epithelium compared with that in tumor cells (p < 0.001). EBV-encoded LMP1 was exclusively expressed in NPC tumors. Using an independent set of biopsies, we showed that a model combining CDH4, STAT4, and LMP1 had a 92.86% of diagnostic accuracy, whereas a combination of STAT4 and LMP1 had a 70.59% accuracy for predicting advanced disease. Mechanistic studies suggested that promoter methylation, loss of DNA allele, and LMP1 contributed to the suppressive expression of CDH4, CYLD, and STAT4, respectively. CONCLUSION: A model combining CDH4 and STAT4 and LMP1 was proposed to be a feasible model for diagnosing NPC and predicting late stage of NPC.

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

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.

Epstein-Barr Virus Induces Adipocyte Dedifferentiation to Modulate the Tumor Microenvironment.

The most frequent location of metastatic EBV+ nasopharyngeal carcinoma (NPC) is the bone marrow, an adipocyte-dominant region. Several EBV-associated lymphoepithelioma-like carcinoma (LELC) types also grow in the anatomical vicinity of fat tissues. Here we show that in an adipose tissue-rich tumor setting, EBV targets adipocytes and remodels the tumor microenvironment. Positive immunoreactivity for EBV-encoded early antigen D was detected in adipose tissue near tumor beds of bone marrow metastatic NPC. EBV was capable of infecting primary human adipocytes in vitro, triggering expression of multiple EBV-encoded mRNA and proteins. In infected adipocytes, lipolysis was stimulated through enhanced expression of lipases and the AMPK metabolic pathway. The EBV-mediated imbalance in energy homeostasis was further confirmed by increased release of free fatty acids, glycerol, and expression of proinflammatory adipokines. Clinically, enhanced serum levels of free fatty acids in patients with NPC correlated with poorer recurrence-free survival. EBV-induced delipidation stimulated dedifferentiation of adipocytes into fibroblast-like cells expressing higher levels of S100A4, a marker protein of cancer-associated fibroblasts (CAF). IHC analyses of bone marrow metastatic NPC and salivary LELC revealed similar structural changes of dedifferentiated adipocytes located at the boundaries of EBV+ tumors. S100A4 expression in adipose tissues near tumor beds correlated with fibrotic response, implying that CAFs in the tumor microenvironment are partially derived from EBV-induced dedifferentiated adipocytes. Our data suggest that adipose tissue serves as an EBV reservoir, where EBV orchestrates the interactions between adipose tissues and tumor cells by rearranging metabolic pathways to benefit virus persistence and to promote a protumorigenic microenvironment. SIGNIFICANCE: This study suggests that Epstein-Barr virus hijacks adipocyte lipid metabolism to create a tumor-promoting microenvironment from which reactivation and relapse of infection could potentially occur.