Ceramide promotes lytic reactivation of Epstein-Barr virus in gastric carcinoma.

Epstein-Barr virus (EBV) has a lifelong latency period after initial infection. Rarely, however, when the EBV immediate early gene BZLF1 is expressed by a specific stimulus, the virus switches to the lytic cycle to produce progeny viruses. We found that EBV infection reduced levels of various ceramide species in gastric cancer cells. As ceramide is a bioactive lipid implicated in the infection of various viruses, we assessed the effect of ceramide on the EBV lytic cycle. Treatment with C6-ceramide (C6-Cer) induced an increase in the endogenous ceramide pool and increased production of the viral product as well as BZLF1 expression. Treatment with the ceramidase inhibitor ceranib-2 induced EBV lytic replication with an increase in the endogenous ceramide pool. The glucosylceramide synthase inhibitor Genz-123346 inhibited C6-Cer-induced lytic replication. C6-Cer induced extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB phosphorylation, c-JUN expression, and accumulation of the autophagosome marker LC3B. Treatment with MEK1/2 inhibitor U0126, siERK1&2, or siCREB suppressed C6-Cer-induced EBV lytic replication and autophagy initiation. In contrast, siJUN transfection had no impact on BZLF1 expression. The use of 3-methyladenine (3-MA), an inhibitor targeting class III phosphoinositide 3-kinases (PI3Ks) to inhibit autophagy initiation, resulted in reduced beclin-1 expression, along with suppressed C6-Cer-induced BZLF1 expression and LC3B accumulation. Chloroquine, an inhibitor of autophagosome-lysosome fusion, increased BZLF1 protein intensity and LC3B accumulation. However, siLC3B transfection had minimal effect on BZLF1 expression. The results suggest the significance of ceramide-related sphingolipid metabolism in controlling EBV latency, highlighting the potential use of drugs targeting sphingolipid metabolism for treating EBV-positive gastric cancer.IMPORTANCEEpstein-Barr virus remains dormant in the host cell but occasionally switches to the lytic cycle when stimulated. However, the exact molecular mechanism of this lytic induction is not well understood. In this study, we demonstrate that Epstein-Barr virus infection leads to a reduction in ceramide levels. Additionally, the restoration of ceramide levels triggers lytic replication of Epstein-Barr virus with increase in phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB. Our study suggests that the Epstein-Barr virus can inhibit lytic replication and remain latent through reduction of host cell ceramide levels. This study reports the regulation of lytic replication by ceramide in Epstein-Barr virus-positive gastric cancer.

The three-dimensional structure of the EBV genome plays a crucial role in regulating viral gene expression in EBVaGC.

Epstein-Barr virus (EBV) establishes lifelong asymptomatic infection by replication of its chromatinized episomes with the host genome. EBV exhibits different latency-associated transcriptional repertoires, each with distinct three-dimensional structures. CTCF, Cohesin and PARP1 are involved in maintaining viral latency and establishing episome architecture. Epstein-Barr virus-associated gastric cancer (EBVaGC) represents 1.3-30.9% of all gastric cancers globally. EBV-positive gastric cancers exhibit an intermediate viral transcription profile known as 'Latency II', expressing specific viral genes and noncoding RNAs. In this study, we investigated the impact of PARP1 inhibition on CTCF/Cohesin binding in Type II latency. We observed destabilization of the binding of both factors, leading to a disrupted three-dimensional architecture of the episomes and an altered viral gene expression. Despite sharing the same CTCF binding profile, Type I, II and III latencies exhibit different 3D structures that correlate with variations in viral gene expression. Additionally, our analysis of H3K27ac-enriched interactions revealed differences between Type II latency episomes and a link to cellular transformation through docking of the EBV genome at specific sites of the Human genome, thus promoting oncogene expression. Overall, this work provides insights into the role of PARP1 in maintaining active latency and novel mechanisms of EBV-induced cellular transformation.

Prognostic Effect of Tertiary Lymphoid Structures in Epstein-Barr Virus-Associated Gastric Carcinomas Measured by Digital Image Analysis.

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is characterized by prominent tumor-infiltrating lymphocytes (TILs) and has a favorable prognosis. Tertiary lymphoid structures (TLS), characterized by ectopic aggregated lymphocytes with high-endothelial venules (HEV), are associated with favorable outcomes in various solid tumors. We hypothesized that EBVaGC, characterized by intense TILs, may be closely associated with TLS or HEV. To test this hypothesis, we digitally analyzed the TLS, HEV, and TILs in 73 surgically resected advanced EBVaGCs. For HEV, dual MECA-79 and CD31 dual immunohistochemistry were performed, and the ectopic expression of MECA-79 in tumor cells was measured. In 73 patients with EBVaGC, a high-TLS ratio was found in 29 (39.7%) cases, high-tumor-associated HEV density in 44 (60.3%) cases, and high-CD8+ TIL density in 38 (52.1%) cases. Ectopic tumor expression of MECA-79 was observed in 36 patients (49.3%) cases. A low-TLS ratio and tumor-associated HEV density were significantly associated with lymph node metastasis (P = .005 and .042, respectively). Ectopic MECA-79 expression was significantly associated with lymph node metastasis (P = .003). Patients with a low-TLS ratio (P = .038), low-HEV density (P = .042), and ectopic tumor MECA-79 expression (P = .032) had significantly worse prognoses. In conclusion, TLS ratio and HEV density affect the survival of patients with EBVaGC and may be related to the immune response that interrupts lymph node metastasis.

Gastric cancer molecular classification based on immunohistochemistry and in-situ hybridisation and mortality.

BACKGROUND AND AIMS: Gastric cancers (GC) are divided into subtypes based on molecular profile: Epstein-Barr virus (EBV)-positive, microsatellite instability (MSI), chromosomal instability (CIN) and genomically stable (GS) tumours. The prognostic impact of this classification is unclear. The aim was to evaluate whether the molecular subtypes determined using in-situ hybridisation (ISH) and immunohistochemistry (IHC) are associated with clinicopathological parameters and prognosis. METHODS AND RESULTS: The study included 503 GC patients. Based on ISH (EBV) and IHC (MSI and TP53), tumours were divided into EBV-positive, MSI, CIN (EBVneg/MSS/TP53aberrant) and GS (EBVneg/MSS/TP53wild-type) subgroups. Survival analyses with intestinal- and diffuse-type tumours were examined separately. EBV-positive tumours associated with male sex. Both EBV-positive and MSI tumours associated with intestinal type. CIN tumours associated with intestinal-type and positive lymph node status. GS tumours associated with diffuse-type and negative lymph node status. In the total cohort, no significant differences in the 5-year survival were observed. In intestinal tumours, the 5-year survival was better in EBV-positive tumours compared with GS tumours [hazard ratio (HR) = 0.57, 95% confidence interval (CI) = 0.33-0.99]. In diffuse tumours, the 5-year survival was worse in CIN tumours compared with GS tumours (HR = 1.57, 95% CI = 1.14-2.18). In radically resected diffuse tumours, the 5-year survival was worse in MSI tumours compared with GS tumours (HR = 3.26, 95% CI = 1.20-8.82). CONCLUSIONS: The molecular classification is associated with histological type but not prognosis in GC. As the prognostic effects of molecular subtypes in intestinal- and diffuse-type cancers may differ, combining histological and molecular information is recommended for future studies.

Clinicopathological characteristics and prognosis of Epstein-Barr virus-associated gastric cancer.

OBJECTIVE: Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a distinct molecular subtype of gastric cancer (GC). At present, the clinical characteristics and prognostic implications of EBV infection and the potential clinical benefits of immune checkpoint blockade in GC remain to be clarified. Hence, this study was designed to analyze the clinical and pathological characteristics of GC patients with varying EBV infection states and compare their overall survival (OS). METHODS: A retrospective study was performed on 1031 consecutive GC patients who underwent gastrectomy at the Affiliated Hospital of Xuzhou Medical University from February 2018 to November 2022. EBV-encoded RNA (EBER) in situ hybridization (ISH) was used for EBV assessment, and immunohistochemical staining was used for evaluation of human epidermal growth factor receptor 2 (HER2), programmed death ligand 1 (PD-L1), and Ki67 expression. EBVaGC was defined as tumors with EBV positivity. In addition, EBV-negative GC (EBVnGC) patients were matched with EBVaGC patients based on seven clinicopathological parameters (age, gender, anatomic subsite, tumor size, Lauren classification, degree of differentiation, and tumor-node-metastasis [TNM] stage). The correlations of clinical features with HER2, PD-L1, and Ki67 expression were evaluated statistically. The survival of patients was assessed through medical records, telephone, or WeChat communication, and prognostic analysis was performed using the logrank test as well as univariable and multivariable regression analysis. RESULTS: Out of 1031 GC patients tested, 35 (3.4%) were diagnosed with EBVaGC. Notably, the EBVaGC group exhibited a distinct predominance of males and younger patients, significantly higher Ki67 and PD-L1 expression levels, and a lower prevalence of pericancerous nerve invasion than the EBVnGC group (P < 0.01). In the 35 EBVaGC cases, Ki67 expression was negatively correlated with age (P < 0.05), suggesting that a younger onset age was associated with higher Ki67 expression. In addition, PD-L1 expression was correlated with the degree of differentiation, T-stage, and clinical stage of the patient. Furthermore, PD-L1 expression was elevated in tumors with lower differentiation or at later stages (P < 0.05). Using univariate analysis, Ki67, PD-L1, and clinical stage were identified as significant factors influencing the overall survival (OS) of EBVaGC patients (P < 0.05). Moreover, multivariate survival analysis revealed that clinical stage and Ki67 expression were independent risk factors for the OS of the patients (P < 0.05), and the three-year OS rate of EBVaGC patients was 64.2%. CONCLUSION: EBV-ISH is a practical and valuable method to identify EBVaGC. Owing to its unique etiological, pathological, and clinical characteristics, patients with EBVaGC might benefit from immune checkpoint blockade therapy.

Ephrin receptor A2, the epithelial receptor for Epstein-Barr virus entry, is not available for efficient infection in human gastric organoids.

Epstein-Barr virus (EBV) is best known for infection of B cells, in which it usually establishes an asymptomatic lifelong infection, but is also associated with the development of multiple B cell lymphomas. EBV also infects epithelial cells and is associated with all cases of undifferentiated nasopharyngeal carcinoma (NPC). EBV is etiologically linked with at least 8% of gastric cancer (EBVaGC) that comprises a genetically and epigenetically distinct subset of GC. Although we have a very good understanding of B cell entry and lymphomagenesis, the sequence of events leading to EBVaGC remains poorly understood. Recently, ephrin receptor A2 (EPHA2) was proposed as the epithelial cell receptor on human cancer cell lines. Although we confirm some of these results, we demonstrate that EBV does not infect healthy adult stem cell-derived gastric organoids. In matched pairs of normal and cancer-derived organoids from the same patient, EBV only reproducibly infected the cancer organoids. While there was no clear pattern of differential expression between normal and cancer organoids for EPHA2 at the RNA and protein level, the subcellular location of the protein differed markedly. Confocal microscopy showed EPHA2 localization at the cell-cell junctions in primary cells, but not in cancer cell lines. Furthermore, histologic analysis of patient tissue revealed the absence of EBV in healthy epithelium and presence of EBV in epithelial cells from inflamed tissue. These data suggest that the EPHA2 receptor is not accessible to EBV on healthy gastric epithelial cells with intact cell-cell contacts, but either this or another, yet to be identified receptor may become accessible following cellular changes induced by inflammation or transformation, rendering changes in the cellular architecture an essential prerequisite to EBV infection.

Establishment and characterization of two Epstein-Barr virus-positive gastric cancer cell lines with epitheliotropic M81 strain undergoing distinct viral and altered cellular expression profiles.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a distinct subtype of gastric cancer (GC) distinguished by the presence of the EBV genome and limited viral gene expression within malignant epithelial cells. EBV infection is generally thought to be a relatively late event following atrophic gastritis in carcinogenesis, which implies the heterogeneity of EBVaGC. To facilitate the study of the role of EBV in EBVaGC, we established two EBV-positive GC cell lines (AGS-EBV and HGC27-EBV) with an epitheliotropic EBV strain M81 and characterized viral and cellular gene expression profiles in comparison to SNU719, a naturally derived EBV-positive GC cell line. Like SNU719, AGS-EBV and HGC27-EBV stably maintained their EBV genomes and expressed EBV-encoded small RNAs and nuclear antigen EBNA1. Comprehensive analysis of the expression of EBV-encoded miRNAs within the BamHI-A region rightward transcript region, and the transcripts of EBV latent and lytic genes in cell lines, as well as xenografts, reveals that AGS-EBV and HGC27-EBV cells undergo distinct viral expression profiles. A very small fraction of AGS-EBV and SNU719 cells can spontaneously produce infectious progeny virions, while HGC27-EBV does not. AGS-EBV (both M81 and Akata) cells largely mimic SNU719 cells in viral gene expression profiles, and altered cellular functions and pathways perturbed by EBV infection. Phylogenetic analysis of the EBV genome shows both M81 and Akata EBV strains are closely related to clinical EBVaGC isolates. Taken together, these two newly established EBV-positive GC cell lines can serve as models to further investigate the role of EBV in different contexts of gastric carcinogenesis and identify novel therapeutics against EBVaGC.

Epigenetic Regulation of MAP3K8 in EBV-Associated Gastric Carcinoma.

Super-enhancers (SEs) regulate gene expressions, which are critical for cell type-identity and tumorigenesis. Although genome wide H3K27ac profiling have revealed the presence of SE-associated genes in gastric cancer (GC), their roles remain unclear. In this study, ChIP-seq and HiChIP-seq experiments revealed mitogen-activated protein kinase 8 (MAP3K8) to be an SE-associated gene with chromosome interactions in Epstein-Barr virus-associated gastric carcinoma (EBVaGC) cells. CRISPRi mediated repression of the MAP3K8 SEs attenuated MAP3K8 expression and EBVaGC cell proliferation. The results were validated by treating EBVaGC cells with bromodomain and the extra-terminal motif (BET) inhibitor, OTX015. Further, functional analysis of MAP3K8 in EBVaGC revealed that silencing MAP3K8 could inhibit the cell proliferation, colony formation, and migration of EBVaGC cells. RNA-seq and pathway analysis indicated that knocking down MAP3K8 obstructed the notch signaling pathway and epithelial-mesenchymal transition (EMT) in EBVaGC cells. Further, analysis of the cancer genome atlas (TCGA) and GSE51575 databases exhibited augmented MAP3K8 expression in gastric cancer and it was found to be inversely correlated with the disease-free progression of GC. Moreover, Spearman's correlation revealed that MAP3K8 expression was positively correlated with the expressions of notch pathway and EMT related genes, such as, Notch1, Notch2, C-terminal binding protein 2 (CTBP2), alpha smooth muscle actin isotype 2 (ACTA2), transforming growth factor beta receptor 1 (TGFßR1), and snail family transcriptional repressors 1/2 (SNAI1/SNAI2) in GC. Taken together, we are the first to functionally interrogate the mechanism of SE-mediated regulation of MAP3K8 in EBVaGC cell lines.

Profiling of immune related genes silenced in EBV-positive gastric carcinoma identified novel restriction factors of human gammaherpesviruses.

EBV-associated gastric cancer (EBVaGC) is characterized by high frequency of DNA methylation. In this study, we investigated how epigenetic alteration of host genome contributes to pathogenesis of EBVaGC through the analysis of transcriptomic and epigenomic datasets from NIH TCGA (The Cancer Genome Atlas) consortium. We identified that immune related genes (IRGs) is a group of host genes preferentially silenced in EBV-positive gastric cancers through DNA hypermethylation. Further functional characterizations of selected IRGs reveal their novel antiviral activity against not only EBV but also KSHV. In particular, we showed that metallothionein-1 (MT1) and homeobox A (HOXA) gene clusters are down-regulated via EBV-driven DNA hypermethylation. Several MT1 isoforms suppress EBV lytic replication and release of progeny virions as well as KSHV lytic reactivation, suggesting functional redundancy of these genes. In addition, single HOXA10 isoform exerts antiviral activity against both EBV and KSHV. We also confirmed the antiviral effect of other dysregulated IRGs, such as IRAK2 and MAL, in scenario of EBV and KSHV lytic reactivation. Collectively, our results demonstrated that epigenetic silencing of IRGs is a viral strategy to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis of human gamma-herpesviruses (EBV and KSHV), considering that these IRGs possess antiviral activities against these oncoviruses.

Increasing frequency of gene copy number aberrations is associated with immunosuppression and predicts poor prognosis in gastric adenocarcinoma.

BACKGROUND: Patients with Epstein-Barr virus-positive gastric cancers or those with microsatellite instability appear to have a favourable prognosis. However, the prognostic value of the chromosomal status (chromosome-stable (CS) versus chromosomal instable (CIN)) remains unclear in gastric cancer. METHODS: Gene copy number aberrations (CNAs) were determined in 16 CIN-associated genes in a retrospective study including test and validation cohorts of patients with gastric cancer. Patients were stratified into CS (no CNA), CINlow (1-2 CNAs) or CINhigh (3 or more CNAs). The relationship between chromosomal status, clinicopathological variables, and overall survival (OS) was analysed. The relationship between chromosomal status, p53 expression, and tumour infiltrating immune cells was also assessed and validated externally. RESULTS: The test and validation cohorts included 206 and 748 patients, respectively. CINlow and CINhigh were seen in 35.0 and 15.0 per cent of patients, respectively, in the test cohort, and 48.5 and 20.7 per cent in the validation cohort. Patients with CINhigh gastric cancer had the poorest OS in the test and validation cohorts. In multivariable analysis, CINlow, CINhigh and pTNM stage III-IV (P < 0.001) were independently associated with poor OS. CIN was associated with high p53 expression and low immune cell infiltration. CONCLUSION: CIN may be a potential new prognostic biomarker independent of pTNM stage in gastric cancer. Patients with gastric cancer demonstrating CIN appear to be immunosuppressed, which might represent one of the underlying mechanisms explaining the poor survival and may help guide future therapeutic decisions.

Maintenance of Epstein-Barr virus latency through interaction of LMP2A with CXCR4.

Epstein-Barr virus (EBV) belongs to the subfamily Gammaherpesvirinae and was the first human tumor virus to be discovered. The global rate of EBV infection in adults exceeds 90%. EBV can participate in the regulation of multiple genes and signal pathways through its latency genes. Many studies have shown that CXCR4 is involved in the development of gastric cancer, but there have been few studies on the specific mechanisms involved in EBV-associated gastric cancer (EBVaGC). In this study, we explored the mechanism by which EBV-encoded products maintain latent EBV infection through interaction with CXCR4 and investigated the role of CXCR4 in EBV-positive cells. The results show that there is a positive feedback between the EBV-encoded products and CXCR4, and LMP2A can activate CXCR4 through the NF-κB pathway. In addition, CXCR4 can be fed back to LMP2A and EBNA1 through the ERK signaling pathway. At the same time, CXCR4 can promote the proliferation and migration of EBV-positive cells, reduce the expression of the immediate early protein BZLF1, the late protein EBV gp350, and the viral capsid antigen, and play an important role in maintaining the incubation period of EBV infection. These findings are applicable to the further targeted therapy of EBVaGC.

Epstein-Barr virus miR-BART4-3p regulates cell proliferation, apoptosis, and migration by targeting AXL in gastric carcinoma.

To investigate the role of miR-BART4-3p in EBV-associated gastric cancer (EBVaGC) and its regulation of cell proliferation, apoptosis, and migration by targeting AXL in GC. Quantitative real-time PCR and western blot were used to detect the expression of AXL. The methylation status of AXL gene promoter region was determined by bisulfite sequencing PCR. Luciferase reporter assay was used to detect whether miR-BART4-3p targets AXL. The key molecules of EMT and PI3K/AKT pathway were used to examine by western blot. CCK8, Transwell, and flow cytometry were used to detect the phenotypic gastric cancer cells after interference with AXL and miR-BART4-3p. EBV infection inhibited the expression of AXL in GC cells and the inhibition was not caused by the change of promoter methylation status. MiR-BART4-3p directly targeted AXL. Moreover, both inhibition of miR-BART4-3p and AXL inhibited cell proliferation and migration and promoted cell apoptosis. In addition, E-cadherin, Vimentin, ZEB1, and p-AKT were found to be the downstream molecules of the miR-BART4-3p/AXL pathway. The change of promoter methylation status was not the reason for the downregulation of AXL expression in EBV-positive cells. MiR-BART4-3p may inhibit the proliferation and migration and promote apoptosis of GC cells by directly targeting AXL.

ARID1A deficiency in EBV-positive gastric cancer is partially regulated by EBV-encoded miRNAs, but not by DNA promotor hypermethylation.

AT-rich interactive domain 1A (ARID1A), which is a tumor suppressor gene, is frequently mutated in Epstein-Barr virus-positive gastric cancer [EBV (+) GC]. While most ARID1A mutations in GC are truncating mutations, leading to loss of ARID1A protein expression, epigenetic modifications appear to contribute to ARID1A deficiency in EBV (+) GC harboring wild-type ARID1A. Based on the significant role of epigenetic modifications in EBV (+) GC that contributes to ARID1A deficiency, the methylation status of ARID1A was evaluated in EBV-infected cells and GC patients using a publicly available microarray and the Cancer Genome Atlas (TCGA) database. EBV-encoded miRNAs that potentially target ARID1A were identified as an additional epigenetic modulator by computational prediction. In vitro experiments were conducted to evaluate how EBV-encoded miRNAs affected ARID1A mRNA and protein levels. In clinical GC samples, the expression of predicted miRNAs and ARID1A and the mutation status of ARID1A was evaluated. As results, ARID1A was not hypermethylated in EBV (+) GC samples or EBV-infected GC cells. EBV infection did not alter ARID1A mRNA levels, suggesting that ARID1A protein deficiency was caused by post-transcriptional gene silencing in ARID1A-WT EBV (+) GC. Overexpression of miR-BART11-3p and miR-BART12, which were identified as miRNAs that potentially bind ARID1A, suppressed ARID1A protein expression in MKN7 and NCI-N87 cells. Highly expressed miR-BART11-3p and miR-BART12 were correlated with decreased ARID1A levels in GC tumors which did not harbor ARID1A mutations. The present findings revealed that ARID1A expression was epigenetically regulated by miR-BART11-3p and miR-BART12 in EBV (+) GC.

Critical review of Epstein-Barr virus microRNAs relation with EBV-associated gastric cancer.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is regarded as the most prevalent malignant tumor triggered by EBV infection. In recent years, increasing attention has been considered to recognize more about the disease process's exact mechanisms. There is accumulating evidence that showing epigenetic modifications play critical roles in the EBVaGC pathogenesis. MicroRNAs (miRNAs), as critical epigenetic modulators, are single-strand short noncoding RNA (length ~ <200 bp), which regulate gene expression through binding to the 3'-untranslated region (3'-UTR) of target RNA transcripts and either degrade or repress their activities. In the latest research on EBV, it was found that this virus could encode miRNAs. Mechanistically, EBV-encoded miRNAs are involved in carcinogenesis and the progression of EBV-associated malignancies. Moreover, these miRNAs implicated in immune evasion, identification of pattern recognition receptors, regulation of lymphocyte activation and lethality, modulation of infected host cell antigen, maintain of EBV infection status, promotion of cell proliferation, invasion and migration, and reduction of apoptosis. As good news, not only has recent data demonstrated the crucial function of EBV-encoded miRNAs in the pathogenesis of EBVaGC, but it has also been revealed that aberrant expression of exosomal miRNAs in EBVaGC has made them biomarkers for detection of EBVaGC. Regarding these substantial characterizes, the critical role of EBV-encoded miRNAs has been a hot topic in research. In this review, we will focus on the multiple mechanisms involved in EBVaGC caused by EBV-encoded miRNAs and briefly discuss their potential application in the clinic as a diagnostic biomarker.

Lymphocyte-activation gene 3 expression associates with poor prognosis and immunoevasive contexture in Epstein-Barr virus-positive and MLH1-defective gastric cancer patients.

Lymphocyte activation gene 3 (LAG-3) is a transmembrane immune checkpoint that facilitates immune escape via suppressing T-cell-mediated anti-tumor immunity. The role of LAG-3 in gastric cancer is little known. Consequently, we assessed the clinical significance of LAG-3 in gastric cancer. In our study, patients with gastric cancer from Zhongshan Hospital (n = 464) and data from the Asian Cancer Research Group (n = 300) were analyzed. LAG-3+ cell infiltration and other immune contexture in gastric cancer were detected by immunohistochemistry. Kaplan-Meier curves and log-rank test were used for survival analyses. Intratumoral LAG-3+ cells mainly accumulated in Epstein-Barr virus (EBV)-positive (EBV subtype) and MLH1-defective (dMLH1 subtype) gastric cancer. Furthermore, LAG-3+ cell infiltration was strongly associated with inferior clinical outcomes in patients with these two subtypes of gastric cancer. Moreover, we found intratumoral LAG-3+ cell high infiltration was associated with an immunoevasive contexture featured by decreased IFN-γ+ cells and perforin-1+ cells, but increased regulatory T cells and M2-like macrophages in EBV/dMLH1 subtype of gastric cancer. LAG-3 was a poor prognostic factor and might be a potential immunotherapeutic target in EBV-positive and MLH1-defective gastric cancer.

Activation of EHF via STAT3 phosphorylation by LMP2A in Epstein-Barr virus-positive gastric cancer.

Epstein-Barr virus (EBV) is associated with approximately 10% of gastric cancers (GCs). We previously showed that EBV infection of gastric epithelial cells induces aberrant DNA methylation in promoter regions, which causes silencing of critical tumor suppressor genes. Here, we analyzed gene expressions and active histone modifications (H3K4me3, H3K4me1, and H3K27ac) genome-widely in EBV-positive GC cell lines and in vitro EBV-infected GC cell lines to elucidate the transcription factors contributing to tumorigenesis through enhancer activation. Genes associated with "signaling of WNT in cancer" were significantly enriched in EBV-positive GC, showing increased active ß-catenin staining. Genes neighboring activated enhancers were significantly upregulated, and EHF motif was significantly enriched in these active enhancers. Higher expression of EHF in clinical EBV-positive GC compared with normal tissue and EBV-negative GC was confirmed by RNA-seq using The Cancer Genome Atlas cohort, and by immunostaining using our cohort. EHF knockdown markedly inhibited cell proliferation. Moreover, there was significant enrichment of critical cancer pathway-related genes (eg, FZD5) in the downstream of EHF. EBV protein LMP2A caused upregulation of EHF via phosphorylation of STAT3. STAT3 knockdown was shown to inhibit cellular growth of EBV-positive GC cells, and the inhibition was rescued by EHF overexpression. Our data highlighted the important role of EBV infection in gastric tumorigenesis via enhancer activation.

Gastric epithelial attachment of Helicobacter pylori induces EphA2 and NMHC-IIA receptors for Epstein-Barr virus.

Epstein-Barr virus (EBV)-associated gastric cancer belongs to 1 of the 4 subtypes of gastric cancer and accounts for 10% of total gastric cancers. However, most cases of gastric cancer have a history of Helicobacter pylori infection. Therefore, we investigated the possibility that H. pylori infection promotes the development of EBV-associated gastric cancer. H. pylori was exposed to principal EBV receptor, CD21, negative gastric epithelial cells, and then infected with EBV recombinant expressing enhanced green fluorescent protein. Changes in EBV infectivity due to prior H. pylori exposure were analyzed using flow cytometry. The treatment of gastric epithelial cells with H. pylori increased the efficiency of EBV infection. An increase was also observed when CagA-deficient, VacA-deficient, and FlaA-deficient H. pylori strains were used, but not when cag pathogenicity island-deficient H. pylori was used. The treatment of epithelial cells with H. pylori induced the expression of accessory EBV receptors, EphA2 and NMHC-IIA, and increased the efficiency of EBV infection depending on their expression levels. When gastric epithelial cells were treated with EPHA2 or NMHC-IIA siRNA, EBV infection via H. pylori attachment was decreased. The adhesion of H. pylori induced the expression of accessory EBV receptors in gastric epithelial cells and increased the efficiency of EBV infection.

EBV as a high infection risk factor promotes RASSF10 methylation and induces cell proliferation in EBV-associated gastric cancer.

Epstein-Barr virus (EBV) is the first identified human tumor-related DNA virus, and has a high infection among people worldwide. Recent studies have showed that nearly 10% of gastric cancers have shown EBV infection and this kind of gastric cancer has been identified as a new subtype: EBV associated Gastric cancer (EBVaGC). Furthermore, it has been reported that tumor related genes in the EBVaGC showed frequent methylation modifications compared to those in the EBV negative gastric cancer (EBVnGC). To fully understand the role of EBV in EBVaGC, we analyzed and found that 16.67% of gastric carcinoma samples showed positive EBER1 signals. Mechanically, EBV-encoded Latent membrane protein 1 (LMP1) inhibited the expression of RASSF10, and promoted tumorigenesis by recruiting DNMT1 and inducing the DNA methylation of RASSF10. Altogether, it allows us a better understanding of the possible mechanism of EBV-induced gene hypermethylation in gastric cancer genome. Targeting EBV-induced DNA methylation is a potential therapeutic modality of EBVaGC.

The impact of ICOS+ regulatory T cells and Helicobacter pylori infection on the prognosis of patients with gastric and colorectal cancer: potential prognostic benefit of pre-operative eradication therapy.

It remains unclear whether Helicobacter pylori (H. pylori), a major cause of gastric cancer (GC), is involved in other intestinal cancers. In our previous study, ICOS+ Foxp3+ CD4+ T cells (ICOS+ Tregs) in GC tumors were identified as effector Tregs and associated with H. pylori. In the present study, the impact of ICOS+ Tregs on not only GC, but also colorectal cancer (CRC) and their prognosis was investigated in association with H. pylori. Tissue-infiltrating lymphocytes (TILs) purified from fresh tumor and sera were obtained from GC and CRC patients prospectively. % ICOS+ Tregs were analyzed by flow cytometry and their production of anti-H. pylori antibody (Hp-Ab) in sera was detected by ELISA. % ICOS+ Tregs were higher in GC and CRC patients with Hp-Ab than in those without Hp-Ab, including eradicated patients. ICOS+ Tregs purified had higher potential to produce IL-10 than ICOS- Tregs. For prognostic analysis, immunohistochemical analysis and ELISA were performed using archival fixed specimens and frozen sera, respectively, obtained from GC and CRC patients. Overall survival was longer in patients with low % ICOS+ Tregs than in those with high % ICOS+ Tregs, and patients with Hp-Ab showed shorter recurrence-free survival than those without Hp-Ab. These results suggested that ICOS+ Tregs in GC and CRC patients were closely associated with H. pylori in gastric epithelium and their prognosis, and that pre-operative H. pylori eradication has potential as a novel immunotherapy for GC and CRC patients.

Alterations in cellular expression in EBV infected epithelial cell lines and tumors.

The Epstein Barr virus (EBV) is linked to the development of two major epithelial malignancies, gastric carcinoma and nasopharyngeal carcinoma. This study evaluates the effects of EBV on cellular expression in a gastric epithelial cell line infected with or without EBV and a nasopharyngeal carcinoma cell line containing EBV. The cells were grown in vitro and as tumors in vivo. The effects on cellular expression were determined using both 2D DIGE proteomics and high throughput RNA sequencing. The data identify multiple pathways that were uniquely activated in vitro. RNA sequences mapping to the mouse genome were identified in both the EBV positive and negative tumor samples in vivo, although, differences between the EBV positive and negative cells were not apparent. However, the tumors appeared to be grossly distinct. The majority of the identified canonical pathways based on two fold changes in expression had decreased activity within the tumors in vivo. Identification of the predicted upstream regulating factors revealed that in vitro the regulating factors were primarily protein transcriptional regulators. In contrast, in vivo the predicted regulators were frequently noncoding RNAs. Hierarchical clustering distinguished the cell lines and tumors, the EBV positive tumors from the EBV negative tumors, and the NPC tumors from the gastric tumors and cell lines. The delineating genes were changed greater than 4 fold and were frequently regulated by protein transcription factors. These data suggest that EBV distinctly affects cellular expression in gastric tumors and NPC and that growth in vivo requires activation of fewer cellular signaling pathways. It is likely that the broad changes in cellular expression that occur at low levels are controlled by regulatory viral and cellular RNAs while major changes are affected by induced protein regulators.