EBV-infected Epithelial Cells Research Articles

Helicobacter pylori and Epstein-Barr virus infection in cell polarity alterations.

The asymmetrical distribution of the cellular organelles inside the cell is maintained by a group of cell polarity proteins. The maintenance of polarity is one of the vital host defense mechanisms against pathogens, and the loss of it contributes to infection facilitation and cancer progression. Studies have suggested that infection of viruses and bacteria alters cell polarity. Helicobacter pylori and Epstein-Barr virus are group I carcinogens involved in the progression of multiple clinical conditions besides gastric cancer (GC) and Burkitt's lymphoma, respectively. Moreover, the coinfection of both these pathogens contributes to a highly aggressive form of GC. H. pylori and EBV target the host cell polarity complexes for their pathogenesis. H. pylori-associated proteins like CagA, VacA OipA, and urease were shown to imbalance the cellular homeostasis by altering the cell polarity. Similarly, EBV-associated genes LMP1, LMP2A, LMP2B, EBNA3C, and EBNA1 also contribute to altered cell asymmetry. This review summarized all the possible mechanisms involved in cell polarity deformation in H. pylori and EBV-infected epithelial cells. We have also discussed deregulated molecular pathways like NF-κB, TGF-β/SMAD, and β-catenin in H. pylori, EBV, and their coinfection that further modulate PAR, SCRIB, or CRB polarity complexes in epithelial cells.

The Comparison of Biomolecular Changes of Epstein-Barr Virus Infection in Nasopharyngeal Epithelial Cells Using Confocal Raman Microspectroscopy.

Due to its unique fingerprinting properties, Confocal Raman microscopy (CRM) can be used to examine the biomolecular changes of viruses invading and manipulating host cells. Recently, the biochemical changes due to the invasion and infection of B lymphocyte cells, nerve cells, and epithelial cells by Epstein-Barr virus (EBV) have been reported. However, biomolecular changes in nasopharyngeal epithelial cells that result from EBV infection are still poorly understood. In continuation of our prior investigation of EBV infection in nasopharyngeal epithelial cells, we tried to expound on biomolecular changes in EBV-infected nasopharyngeal epithelial cells using Raman microspectroscopy. EBV has two life cycles, latent infection and lytic replication. We have established latent and lytic infection models at the cellular level. In order to understand the characteristics of the two patterns of EBV infection, we used Raman spectroscopy to identify the changes in biomolecules of EBV latent cells (CNE2, CNE2-EBV) and lytic cells (NPEC1-BMI1-CN, NPEC1-BMI1-EBV). During latent infection, levels of glycogen, protein, and lipid molecules in the cell increased while levels of nucleic acid and collagen molecules decreased. Molecular levels of glycogen, proteins, and nucleic acids are reduced during lytic infection. We found that molecular levels of nucleic acid decreased during two different periods of infection, whereas levels of other biomolecules showed the opposite trend. Glycogen, proteins, lipids, nucleic acids, and other molecules are associated with alterations in cellular biochemical homeostasis. These changes correspond to unique Raman spectra in infected and uninfected cells associated with specific biomolecules that have been proven. These molecules are mainly responsible for cellular processes such as cell proliferation and apoptosis. The Raman signatures of these biomolecular changes depend on the different phases of viral infection. Therefore, by using CRM, it is possible to discern details in the progression of EBV infection in nasopharyngeal epithelial cells at the molecular level.

ΔNp63α promotes Epstein-Barr virus latency in undifferentiated epithelial cells.

Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and contributes to both B-cell and epithelial-cell malignancies. EBV-infected epithelial cell tumors, including nasopharyngeal carcinoma (NPC), are largely composed of latently infected cells, but the mechanism(s) maintaining viral latency are poorly understood. Expression of the EBV BZLF1 (Z) and BRLF1 (R) encoded immediate-early (IE) proteins induces lytic infection, and these IE proteins activate each other’s promoters. ΔNp63α (a p53 family member) is required for proliferation and survival of basal epithelial cells and is over-expressed in NPC tumors. Here we show that ΔNp63α promotes EBV latency by inhibiting activation of the BZLF1 IE promoter (Zp). Furthermore, we find that another p63 gene splice variant, TAp63α, which is expressed in some Burkitt and diffuse large B cell lymphomas, also represses EBV lytic reactivation. We demonstrate that ΔNp63α inhibits the Z promoter indirectly by preventing the ability of other transcription factors, including the viral IE R protein and the cellular KLF4 protein, to activate Zp. Mechanistically, we show that ΔNp63α promotes viral latency in undifferentiated epithelial cells both by enhancing expression of a known Zp repressor protein, c-myc, and by decreasing cellular p38 kinase activity. Furthermore, we find that the ability of cis-platinum chemotherapy to degrade ΔNp63α contributes to the lytic-inducing effect of this agent in EBV-infected epithelial cells. Together these findings demonstrate that the loss of ΔNp63α expression, in conjunction with enhanced expression of differentiation-dependent transcription factors such as BLIMP1 and KLF4, induces lytic EBV reactivation during normal epithelial cell differentiation. Conversely, expression of ΔNp63α in undifferentiated nasopharyngeal carcinoma cells and TAp63α in Burkitt lymphoma promotes EBV latency in these malignancies.

Virus–host interactions in carcinogenesis of Epstein-Barr virus-associated gastric carcinoma: Potential roles of lost ARID1A expression in its early stage

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a distinct molecular subtype of gastric cancer characterized by viral infection and cellular abnormalities, including loss of AT-rich interaction domain 1A (ARID1A) expression (lost ARID1A). To evaluate the significance of lost ARID1A in the development of EBVaGC, we performed in situ hybridization of EBV-encoded RNA (EBER) and immunohistochemistry of ARID1A in the non-neoplastic gastric mucosa and intramucosal cancer tissue of EBVaGC with in vitro infection analysis of ARID1A-knockdown and -knockout gastric cells. Screening of EBER by in situ hybridization revealed a frequency of approximately 0.2% EBER-positive epithelial cells in non-neoplastic gastric mucosa tissue samples. Six small foci of EBV-infected epithelial cells showed two types of histology: degenerated (n = 3) and metaplastic (n = 3) epithelial cells. ARID1A was lost in the former type. In intramucosal EBVaGC, there were ARID1A-lost (n = 5) and -preserved tumors (n = 7), suggesting that ARID1A-lost carcinomas are derived from ARID1A-lost precursor cells in the non-neoplastic mucosa. Lost ARID1A was also observed in non-neoplastic mucosa adjacent to an ARID1A-lost EBVaGC. In vitro experiments using siRNA knockdown and the CRISPR/Cas9-knockout system demonstrated that transient reduction or permanent loss of ARID1A expression markedly increased the efficiency of EBV infection to stomach epithelial cells. Taken together, lost ARID1A plays a role in initiating EBV-driven carcinogenesis in stomach epithelial cells, which develop to a distinct subtype of EBVaGC within the proper mucosal layer. Lost ARID1A is one of the constituents of virus-host interactions in the carcinogenesis of EBVaGC.

EBV downregulates the m6A “writer” WTAP in EBV-associated gastric carcinoma

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is characterized by the clonal growth of EBV-infected stomach epithelial cells. It has been reported that N6-methyladenosine (m6A) methylation can regulate the splicing, expression, decay and translation of mRNAs. Wilms' tumor 1-associating protein (WTAP) is an m6A “writer” with methyltransferase activity. An m6A RNA methylation quantification kit and immunofluorescence (IF) showed that the m6A total RNA methylation level of the Epstein-Barr virus-negative gastric carcinoma (EBVnGC) cell line (SGC7901) was higher than that in the EBVaGC cell line (GT38). To investigate the underlying mechanism of the downregulated expression of m6A RNA methylation, we analyzed the expression of WTAP. The results showed that the expression of WTAP protein in EBVaGC cell lines was significantly lower than that in EBVnGC cell lines according to western blotting and IF. Through plasmid overexpression and RNA interference technology, we further found that EBV-encoded small RNA1 (EBER1) could downregulate WTAP expression by activating the NF-κB signaling pathway. In addition, WTAP could increase proliferation and inhibit migration in gastric carcinoma cell lines. In summary, EBER1 of EBV potentially regulated WTAP by affecting the NF-κB signaling pathway and WTAP further affected cell proliferation and migration.

Molecular portrait of stomach cancer associated with the Epstein–Barr virus

Epstein–Barr virus (EBV) associated gastric carcinoma is a special form of gastric adenocarcinoma that arises against the background of clonal growth of EBV-infected epithelial cells of the gastric mucosa. This subtype of tumors has unique genetic and epigenetic features that determine its characteristic phenotype. Determination of the molecular features of EBV-associated gastric cancer made it possible to identify potential targets for drug therapy of this subtype of tumors. The review presents modern data on the epidemiology and pathogenesis of EBVassociated gastric cancer, describes its unique pathomorphological and molecular features. Particular attention is paid to the prognostic role of EBV infection and drug therapy potentially applicable to the treatment of EBV-positive gastric cancer.

Exosomes of Epstein-Barr Virus-Associated Gastric Carcinoma Suppress Dendritic Cell Maturation.

The Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is characterized by the infiltration of lymphocytes and a unique tumor microenvironment. Exosomes from cancer cells are essential for intercellular communication. The aims of this study were to investigate the secretion of EBVaGC exosomes and their physiological effect on dendritic cell maturation in vitro and to characterize dendritic cells (DCs) in EBVaGC in vivo. Western blotting analysis of CD63 and CD81 of exosomes from EBV-infected gastric cancer cell lines indicated an increase in exosome secretion. The fraction of monocyte-derived DCs positive for the maturation marker CD86 was significantly suppressed when incubated with exosomes from EBV-infected gastric cancer cell lines. Immunohistochemical analysis of GC tissues expressing DC markers (S100, Langerin, CD1a, CD83, CD86, and BDCA-2) indicated that the density of DCs was generally higher in EBVaGC than in EBV-negative GC, although the numbers of CD83- and CD86-positive DCs were decreased in the group with high numbers of CD1a-positive DCs. A low number of CD83-positive DCs was marginally correlated with worse prognosis of EBVaGC in patients. EBVaGC is a tumor with abundant DCs, including immature and mature DCs. Moreover, the maturation of DCs is suppressed by exosomes from EBV-infected epithelial cells.

Epstein-Barr Virus Infection of Pseudostratified Nasopharyngeal Epithelium Disrupts Epithelial Integrity.

Simple SummaryNasopharyngeal carcinoma is associated with Epstein-Barr virus (EBV) infection and originates junction of the oropharynx and nasal cavity, where stratified squamous epithelium and respiratory epithelium are the lining. To elucidate the mechanisms by which EBV transforms the nasopharyngeal epithelium, a pseudostratified multiple-layer model with cilia forming on the apical surface by air-liquid interface (ALI) culture of primary nasopharyngeal epithelial cells was established. We showed: (1) ALI cultures formed stratified epithelia and maintained the diversity of cells found in the airway epithelium, such as ciliated, muco-secretory, and basal cells. (2) Polarized stratified epithelium was more susceptible to EBV infection than monolayer cells. (3) EBV infection in ALI cultures was verified by showing EBV-encoded RNA expressions. (4) EBV infection disrupted the integrity of the epithelium. Thus, our model can be used not only to examine the pathogenesis of pre-neoplastic EBV-infected cells, but also to develop anti-EBV therapy or early stage NPC treatment.Epstein–Barr virus (EBV) is a human oncogenic virus that causes several types of tumor, such as Burkitt’s lymphoma and nasopharyngeal carcinoma (NPC). NPC tumor cells are clonal expansions of latently EBV-infected epithelial cells. However, the mechanisms by which EBV transforms the nasopharyngeal epithelium is hampered, because of the lack of good in vitro model to pursue oncogenic process. Our primary nasopharyngeal epithelial cell cultures developed pseudostratified epithelium at the air-liquid interface, which was susceptible to EBV infection. Using the highly sensitive RNA in situ hybridization technique, we detected viral infection in diverse cell types, including ciliated cells, goblet cells, and basal cells. EBV-encoded small RNA-positive cells were more frequently detected in the suprabasal layer than in the basal layer. We established the most physiologically relevant EBV infection model of nasopharyngeal epithelial cells. This model will advance our understanding of EBV pathogenesis in the development of NPC.

APX-115A, a pan-NADPH Oxidase Inhibitor, Induces Caspase-dependent Cell Death by Suppressing NOX4-ROS Signaling in EBV-infected Retinal Epithelial Cells

ABSTRACT Purpose Epstein-Barr virus is a γ-herpes virus that infects primary B cells and can transform infected cells into immortalized lymphoblastoid cell lines (LCL). The role of EBV in malignancies such as Burkitt’s lymphoma and nasopharyngeal carcinoma is well understood, however, its role in EBV-infected retinal cells remains poorly understood. Therefore, we investigated the effect of EBV on the growth of retinal cells. Methods Previously, we established and reported a cell line model to address the relationship between EBV infection and retinal cell proliferation that used adult retinal pigment epithelium (ARPE-19) and EBV infection. To determine the effect of EBV on ARPE-19 cells, cell death was measured by propidium iodine/annexin V staining and reactive oxygen species (ROS) were measured by FACS, and protein expression was evaluated using western blot analysis. Also, downregulation of LMP1 and NADPH oxidase 4 (NOX4) expression was accomplished using siRNA technology. Results We found that ROS were dramatically increased in EBV-infected ARPE19 cells (APRE19/EBV) relative to the parental cell line. Additionally, the expression level of NOX4, a main source of ROS, was upregulated by EBV infection. Interestingly, downregulation of LMP1, one of the EBV viral onco-proteins, completely decreased EBV-induced ROS accumulation and the upregulation of NOX4. Treatment with APX-115A, a pan-NOX inhibitor, induced apoptotic cell death of only the EBV-infected ARPE19 cells but not the parental cell line. Pretreatment with z-VAD, a pan-caspase inhibitor, inhibited NOX inhibitor-induced cell death in ARPE19/EBV cells. Furthermore, APX-115A-induced cell death mediated the activation of JNK and ERK. Finally, we confirmed the expression level of NOX4, and APX-115A induced cell death of EBV-infected human primary retina epithelial cells and the activation of JNK and ERK. Conclusion Taken together, these our results suggest that APX-115A could be a therapeutic agent for treating EBV-infected retinal cells or diseases by inhibiting LMP1-NOX4-ROS signaling.

Viral loads correlate with upregulation of PD-L1 and worse patient prognosis in Epstein-Barr Virus-associated gastric carcinoma.

Epstein–Barr virus (EBV)-associated gastric carcinoma (EBVaGC), one of four major gastric cancer types, consists of clonal growth of EBV-infected epithelial cells. However, the significance of viral loads in each tumor cell has not been evaluated. EBV-DNA is stably maintained in episomal form in the nucleus of each cancer cell. To estimate EBV copy number per genome (EBV-CN), qPCR of viral EBNA1 and host GAPDH, standardized by Namalwa DNA (one copy/genome), was applied to the formalin-fixed paraffin embedded (FFPE) surgically resected EBVaGC specimens (n = 43) and EBVaGC cell lines (SNU-719 and NCC-24). In surgical specimens, the cancer cell ratio (CCR) was determined with image analysis, and EBV-CN was obtained by adjusting qPCR value with CCR. Fluorescent in situ hybridization (FISH) was also applied to the FFPE sections using the whole EBV-genome as a probe. In surgical specimens, EBV-CN obtained by qPCR/CCR was between 1.2 and 185 copies with a median of 9.9. EBV-CN of SNU-719 and NCC-24 was 42.0 and 1.1, respectively. A linear correlation was observed with qPCR/CCR data up to 20 copies/genome (40 signals/nucleus), the limit of FISH analysis. In addition, substantial variation in the number of EBV foci was observed. Based on qPCR/CCR, high EBV-CN (>10 copies) correlated with PD-L1 expression in cancer cells (P = 0.015), but not with other pathological indicators. Furthermore, EBVaGC with high EBV-CN showed worse disease-specific survival (P = 0.041). Our findings suggest that cancer cell viral loads may contribute to expression of the immune checkpoint molecule and promotion of cancer progression in EBVaGC.

Inhibition of Epstein-Barr Virus Replication in Human Papillomavirus-Immortalized Keratinocytes

Epstein-Barr virus (EBV) is implicated in the pathogenesis of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OSCC). EBV-associated cancers harbor a latent EBV infection characterized by a lack of viral replication and the expression of viral oncogenes. Cellular changes promoted by HPV are comparable to those shown to facilitate EBV latency, though whether HPV-positive cells support a latent EBV infection has not been demonstrated. Using a model of direct EBV infection into HPV16-immortalized tonsillar cells grown in organotypic raft culture, we showed robust EBV replication in HPV-negative rafts but little to no replication in HPV-immortalized rafts. The reduced EBV replication was independent of immortalization, as human telomerase-immortalized normal oral keratinocytes supported robust EBV replication. Furthermore, we observed reduced EBV lytic gene expression and increased expression of EBER1, a noncoding RNA highly expressed in latently infected cells, in the presence of HPV. The use of human foreskin keratinocyte rafts expressing the HPV16 E6 and/or E7 oncogene(s) (HPV E6 and E7 rafts) showed that E7 was sufficient to reduce EBV replication. EBV replication is dependent upon epithelial differentiation and the differentiation-dependent expression of the transcription factors KLF4 and PRDM1. While KLF4 and PRDM1 levels were unaltered, the expression levels of KLF4 transcriptional targets, including late differentiation markers, were reduced in HPV E6 and E7 rafts compared to their levels in parental rafts. However, the HPV E7-mediated block in EBV replication correlated with delayed expression of early differentiation markers. Overall, this study reveals an HPV16-mediated block in EBV replication, through E7, that may facilitate EBV latency and long-term persistence in the tumor context.IMPORTANCE Using a model examining the establishment of EBV infection in HPV-immortalized tissues, we showed an HPV-induced interruption of the normal EBV life cycle reminiscent of a latent EBV infection. Our data support the notion that a persistent EBV epithelial infection depends upon preexisting cellular alterations and suggest the ability of HPV to promote such changes. More importantly, these findings introduce a model for how EBV coinfection may influence HPV-positive (HPV-pos) OSCC pathogenesis. Latently EBV-infected epithelial cells, as well as other EBV-associated head-and-neck carcinomas, exhibit oncogenic phenotypes commonly seen in HPV-pos OSCC. Therefore, an HPV-induced shift in the EBV life cycle toward latency would not only facilitate EBV persistence but also provide additional viral oncogene expression, which can contribute to the rapid progression of HPV-pos OSCC. These findings provide a step toward defining a role for EBV as a cofactor in HPV-positive oropharyngeal tumors.

NF-κB Signaling Regulates Epstein-Barr Virus BamHI-Q-Driven EBNA1 Expression.

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the few viral proteins expressed by EBV in nasopharyngeal carcinoma (NPC), most likely because of its essential role in maintaining the viral genome in EBV-infected cells. In NPC, EBNA1 expression is driven by the BamHI-Q promoter (Qp), which is regulated by both cellular and viral factors. We previously determined that the expression of another group of EBV transcripts, BamHI-A rightward transcripts (BARTs), is associated with constitutively activated nuclear factor-κB (NF-κB) signaling in NPC cells. Here, we show that, like the EBV BART promoter, the EBV Qp also responds to NF-κB signaling. NF-κB p65, but not p50, can activate Qp in vitro, and NF-κB signaling regulates Qp-EBNA1 expression in NPC cells, as well as in other EBV-infected epithelial cells. The introduction of mutations in the putative NF-κB site reduced Qp activation by the NF-κB p65 subunit. Binding of p65 to Qp was shown by chromatin immunoprecipitation (ChIP) analysis, while electrophoretic mobility shift assays (EMSAs) demonstrated that p50 can also bind to Qp. Inhibition of NF-κB signaling by the IκB kinase inhibitor PS-1145 resulted in the downregulation of Qp-EBNA1 expression in C666-1 NPC cells. Since EBNA1 has been reported to block p65 activation by inhibiting IKKα/β through an unknown mechanism, we suggest that, in NPC, NF-κB signaling and EBNA1 may form a regulatory loop which supports EBV latent gene expression, while also limiting NF-κB activity. These findings emphasize the role of NF-κB signaling in the regulation of EBV latency in EBV-associated tumors.

Epstein-Barr virus encoded latent membrane protein 1 suppresses necroptosis through targeting RIPK1/3 ubiquitination

Necroptosis is an alternative programmed cell death pathway that is unleashed in the absence of apoptosis and mediated by signaling complexes containing receptor-interating protein kinase 1 (RIPK1) and RIPK3. This form of cell death has recently been implicated in host defense system to eliminate pathogen-infected cells. However, only a few viral species such as herpes simplex virus (HSV) and cytomegalovirus (CMV) have evolved mechanisms inhibiting necroptosis to overcome host antiviral defense, which is important for successful pathogenesis. Here, we show that the γ-herpesvirus Epstein–Barr virus (EBV) blocks necroptosis in EBV-infected human nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells. Our findings indicate that EBV-encoded latent membrane protein 1 (LMP1), which lacks an RIP homotypic interaction motif (RHIM) domain, has mechanisms distinct from RHIM signaling competition to inhibit this necroptotic pathway. Intriguingly, LMP1 interacts directly with both RIPK1 and RIPK3 through its C-terminal activation region. More importantly, LMP1 can modulate the post-translational modification of the two receptor-interacting proteins. We then show that LMP1-mediated promotion of K63-polyubiquitinated RIPK1, suppression of RIPK1 protein expression and inhibition of K63-polyubiquitinated RIPK3 induced a switch in cell fate from necroptotic death to survival. These findings provide direct evidence for the suppression of necroptosis by EBV and define a mechanism of LMP1 to interrupt the initiation process of necroptosis before necrosome formation.

Gastritis-Infection-Cancer Sequence of Epstein-Barr Virus-Associated Gastric Cancer.

Epstein-Barr virus-associated gastric cancer (EBVaGC) is a representative EBV-infected epithelial neoplasm, which is now included as one of the four subtypes of The Cancer Genome Atlas molecular classification of gastric cancer. In this review, we portray a gastritis-infection-cancer sequence of EBVaGC. This virus-associated type of gastric cancer demonstrates clonal growth of EBV-infected epithelial cells within the mucosa of atrophic gastritis. Its core molecular abnormality is the EBV-specific hyper-epigenotype of CpG island promoter methylation, which induces silencing of tumor suppressor genes. This is due to the infection-induced disruption of the balance between DNA methylation and DNA demethylation activities. Abnormalities in the host cell genome, including phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit α (PIK3CA), AT-rich interaction domain 1A (ARID1A), and programmed death-ligand 1 (PD-L1), are associated with the development and progression of EBVaGC. Furthermore, posttranscriptional modulation affects the transformation processes of EBV-infected cells, such as epithelial mesenchymal transition and anti-apoptosis, via cellular and viral microRNAs (miRNAs). Once established, cancer cells of EBVaGC remodel their microenvironment, at least partly, via the delivery of exosomes containing cellular and viral miRNAs. After exosomes are incorporated, these molecules change the functions of stromal cells, tuning the microenvironment for EBVaGC. During this series of events, EBV hijacks and uses cellular machineries, such as DNA methylation and the miRNA delivery system. This portrait of gastritis-infection-cancer sequences highlights the survival strategies of EBV in the stomach epithelial cells and may be useful for the integration of therapeutic modalities against EBV-driven gastric cancer.

Regulation of tumour related genes by dynamic epigenetic alteration at enhancer regions in gastric epithelial cells infected by Epstein-Barr virus

Epstein-Barr virus (EBV) infection is associated with tumours such as Burkitt lymphoma, nasopharyngeal carcinoma, and gastric cancer. We previously showed that EBV(+) gastric cancer presents an extremely high-methylation epigenotype and this aberrant DNA methylation causes silencing of multiple tumour suppressor genes. However, the mechanisms that drive EBV infection-mediated tumorigenesis, including other epigenomic alteration, remain unclear. We analysed epigenetic alterations induced by EBV infection especially at enhancer regions, to elucidate their contribution to tumorigenesis. We performed ChIP sequencing on H3K4me3, H3K4me1, H3K27ac, H3K27me3, and H3K9me3 in gastric epithelial cells infected or not with EBV. We showed that repressive marks were redistributed after EBV infection, resulting in aberrant enhancer activation and repression. Enhancer dysfunction led to the activation of pathways related to cancer hallmarks (e.g., resisting cell death, disrupting cellular energetics, inducing invasion, evading growth suppressors, sustaining proliferative signalling, angiogenesis, and tumour-promoting inflammation) and inactivation of tumour suppressive pathways. Deregulation of cancer-related genes in EBV-infected gastric epithelial cells was also observed in clinical EBV(+) gastric cancer specimens. Our analysis showed that epigenetic alteration associated with EBV-infection may contribute to tumorigenesis through enhancer activation and repression.

Differentiation-Dependent LMP1 Expression Is Required for Efficient Lytic Epstein-Barr Virus Reactivation in Epithelial Cells.

Epstein-Barr virus (EBV)-associated diseases of epithelial cells, including tumors that have latent infection, such as nasopharyngeal carcinoma (NPC), and oral hairy leukoplakia (OHL) lesions that have lytic infection, frequently express the viral latent membrane protein 1 (LMP1). In lytically infected cells, LMP1 expression is activated by the BRLF1 (R) immediate early (IE) protein. However, the mechanisms by which LMP1 expression is normally regulated in epithelial cells remain poorly understood, and its potential roles in regulating lytic reactivation in epithelial cells are as yet unexplored. We previously showed that the differentiation-dependent cellular transcription factors KLF4 and BLIMP1 induce lytic EBV reactivation in epithelial cells by synergistically activating the two EBV immediate early promoters (Zp and Rp). Here we show that epithelial cell differentiation also induces LMP1 expression. We demonstrate that KLF4 and BLIMP1 cooperatively induce the expression of LMP1, even in the absence of the EBV IE proteins BZLF1 (Z) and R, via activation of the two LMP1 promoters. Furthermore, we found that differentiation of NOKs-Akata cells by either methylcellulose suspension or organotypic culture induces LMP1 expression prior to Z and R expression. We show that LMP1 enhances the lytic infection-inducing effects of epithelial cell differentiation, as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate treatment, in EBV-infected epithelial cells by increasing expression of the Z and R proteins. Our results suggest that differentiation of epithelial cells activates a feed-forward loop in which KLF4 and BLIMP1 first activate LMP1 expression and then cooperate with LMP1 to activate Z and R expression.IMPORTANCE The EBV protein LMP1 is expressed in EBV-associated epithelial cell diseases, regardless of whether these diseases are due to lytic infection (such as oral hairy leukoplakia) or latent infection (such as nasopharyngeal carcinoma). However, surprisingly little is known about how LMP1 expression is regulated in epithelial cells, and there are conflicting reports about whether it plays any role in regulating viral lytic reactivation. In this study, we show that epithelial cell differentiation induces LMP1 expression by increasing expression of two cellular transcription factors (KLF4 and BLIMP1) which cooperatively activate the two LMP1 promoters. We also demonstrate that LMP1 promotes efficient lytic reactivation in EBV-infected epithelial cells by enhancing expression of the Z and R proteins. Thus, in EBV-infected epithelial cells, LMP1 expression is promoted by differentiation and positively regulates lytic viral reactivation.

Vandetanib and ADAM inhibitors synergistically attenuate the pathological migration of EBV-infected retinal pigment epithelial cells by regulating the VEGF-mediated MAPK pathway.

The extracellular signals induced by vascular endothelial growth factor (VEGF) are implicated in choroidal neovascularization (CNV) and thus, are associated with vision-limiting complications in the human retina. Vandetanib is an oral anticancer drug that selectively inhibits the activities of VEGF receptor and epidermal growth factor receptor tyrosine kinase; however, the effects of vandetanib on VEGF in retinal pigment epithelial (RPE) cells have not yet been studied. In the present study, a combined treatment of vandetanib and a disintegrin and metalloproteinase (ADAM) protein inhibitors were used to assess the regulation of Epstein-Barr virus (EBV)-infected ARPE19 cells (ARPE19/EBV) migration as a model of CNV. Vandetanib suppressed the expression of the mesenchymal markers ADAM10 and ADAM17 in ARPE19/EBV cells, and also upregulated epithelial cell markers of the RPE cells, E-cadherin and N-cadherin. The migratory activity of ARPE19/EBV induced by VEGF was efficiently blocked by vandetanib. Furthermore, co-treatment with vandetanib and an ADAM10 inhibitor (GI254023X) or ADAM17 inhibitor (Marimastat) synergistically prevented migration and the expression of vimentin, Snail and α-smooth muscle actin by regulating extracellular signal-regulated kinase and p38 mitogen-activated protein kinase. These results suggest that a combination treatment of vandetanib and ADAM inhibitors may be developed as a novel therapeutic regimen to control retina neovascular disease.

Niclosamide inhibits lytic replication of Epstein-Barr virus by disrupting mTOR activation

Infection with the oncogenic γ-herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause several severe malignancies in humans. Inhibition of the lytic replication of EBV and KSHV eliminates the reservoir of persistent infection and transmission, consequently preventing the occurrence of diseases from the sources of infection. Antiviral drugs are limited in controlling these viral infectious diseases. Here, we demonstrate that niclosamide, an old anthelmintic drug, inhibits mTOR activation during EBV lytic replication. Consequently, niclosamide effectively suppresses EBV lytic gene expression, viral DNA lytic replication and virion production in EBV-infected lymphoma cells and epithelial cells. Niclosamide exhibits cytotoxicity toward lymphoma cells and induces irreversible cell cycle arrest in lytically EBV-infected cells. The ectopic overexpression of mTOR reverses the inhibition of niclosamide in EBV lytic replication. Similarly, niclosamide inhibits KSHV lytic replication. Thus, we conclude that niclosamide is a promising candidate for chemotherapy against the acute occurrence and transmission of infectious diseases of oncogenic γ-herpesviruses.

RETRACTED ARTICLE: Casticin inhibits epithelial-mesenchymal transition of EBV-infected human retina pigmental epithelial cells through the modulation of intracellular lipogenesis.

RETRACTED ARTICLE: Casticin inhibits epithelial-mesenchymal transition of EBV-infected human retina pigmental epithelial cells through the modulation of intracellular lipogenesis.

NF-κB Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma.

ABSTRACTEpstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinase inhibitor PS-1145 inhibited NF-κB activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-κB activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-κB signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-κB activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC. IMPORTANCE Nasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-κB signaling. EBV LMP1 is a potent activator of NF-κB signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-κB signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-κB signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-κB signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.