Epstein-Barr Virus In Gastric Carcinoma Research Articles

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.

Abstract 1252: Intracellular iron chelation by a novel compound, C7, reactivates Epstein-Barr virus (EBV) lytic cycle via the ERK-autophagy axis in EBV-positive epithelial cancers

Abstract Pharmaceutical reactivation of lytic cycle of Epstein-Barr virus (EBV) represents a potential therapeutic strategy against EBV-associated epithelial malignancies e.g. gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC). A novel lytic inducing compound, C7, which exhibits structural similarity to Dp44mT, a known chelator of intracellular iron, is found to reactivate EBV lytic cycle in GC and NPC. This study aims to investigate the role of intracellular iron chelation by C7 and other iron chelators in lytic reactivation of EBV in GC and NPC. Testing of 6 structural analogues of C7 revealed only those which had high affinity towards transition metals could induce EBV lytic cycle. Pre-complexing C7 and iron chelators to iron prior to treatment of the cells abolished EBV lytic reactivation. Though hypoxia signaling pathway was activated, it was not the only pathway associated with EBV reactivation. Specifically, C7 and iron chelators initiated autophagy by activating extracellular signal-regulated kinase (ERK1/2) to reactivate EBV lytic cycle since autophagy and EBV lytic reactivation were abolished in cells treated with ERK1/2 blockers whilst autophagy inhibition by 3-MA and ATG5 knockdown significantly abolished EBV lytic reactivation. In summary, we discover a novel mechanism of reactivation of EBV lytic cycle through intracellular iron chelation and induction of ERK-autophagy axis in EBV-positive epithelial malignancies, raising the question whether clinically available iron chelators can be combined with existing therapeutic modalities to treat these cancers. Note: This abstract was not presented at the meeting. Citation Format: Alan K S Chiang, Stephanie PT Yiu, Kwai F. Hui, Chung K. Choi, Richard YT Kao, Dan Yang. Intracellular iron chelation by a novel compound, C7, reactivates Epstein-Barr virus (EBV) lytic cycle via the ERK-autophagy axis in EBV-positive epithelial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1252.

Intracellular Iron Chelation by a Novel Compound, C7, Reactivates Epstein⁻Barr Virus (EBV) Lytic Cycle via the ERK-Autophagy Axis in EBV-Positive Epithelial Cancers.

Pharmaceutical reactivation of lytic cycle of Epstein–Barr virus (EBV) represents a potential therapeutic strategy against EBV-associated epithelial malignancies, e.g., gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC). A novel lytic-inducing compound, C7, which exhibits structural similarity to Di-2-Pyridyl Ketone 4, 4-Dimethyl-3-Thiosemicarbazone (Dp44mT), a known chelator of intracellular iron, is found to reactivate EBV lytic cycle in GC and NPC. This study aims to investigate the role of intracellular iron chelation by C7 and other iron chelators in lytic reactivation of EBV in GC and NPC. Testing of six structural analogs of C7 revealed only those which have high affinity towards transition metals could induce EBV lytic cycle. Precomplexing C7 and iron chelators to iron prior to treatment of the cells abolished EBV lytic reactivation. Though hypoxia signaling pathway was activated, it was not the only pathway associated with EBV reactivation. Specifically, C7 and iron chelators initiated autophagy by activating extracellular signal-regulated kinase (ERK1/2) to reactivate EBV lytic cycle since autophagy and EBV lytic reactivation were abolished in cells treated with ERK1/2 blockers whilst inhibition of autophagy by 3-Methyladenine (3-MA) and atg5 knockdown significantly abolished EBV lytic reactivation. In summary, we discovered a novel mechanism of reactivation of the EBV lytic cycle through intracellular iron chelation and induction of ERK-autophagy axis in EBV-positive epithelial malignancies, raising the question whether clinically available iron chelators can be incorporated into existing therapeutic regimens to treat these cancers.

Gastric Cancer and Epstein-Barr Virus Infection

Background: Epstein-Barr virus (EBV) has an important role in the oncogenesis of several malignant diseases. Reports even demonstrated the presence of Epstein-Barr virus in gastric carcinoma (GC). However, the pathogenic role of EBV in GC is uncertain. The present investigation was carried out to investigate a possible causal relationship between GC and EBV.
 Statistical Analysis: The method of the conditio sine qua non relationship was used to proof the hypothesis whether gastric cancer is a necessary condition (a conditio sine qua non) of the presence of EBV in human gastric tissues. In other words without GC no EBV in human stomach. The mathematical formula of the causal relationship k was used to proof the hypothesis, whether there is a cause effect relationship between gastric cancer and EBV. Significance was indicated by a p-value (two sided) of less than 0.05.
 Results: In toto 26 ISH based studies with a sample size of N = 11860 were re-analyzed. All the studies analyzed support the null-hypothesis without GC no EBV positivity in human stomach. In other words, gastric cancer itself is a conditio sine qua non of EBV positivity in stomach tissues while the cause effect relationship between gastric cancer and EBV was highly significant.
 Conclusions: Epstein-Barr virus is neither a cause, nor the cause of human gastric cancer.

Correlation of Fas/FasL expression to cell apoptosis in Epstein-Barr virus-associated gastric carcinoma (EBVaGC)

Epstein-Barr virus (EBV) has been detected in about 10% of gastric carcinomas. However, the pathogenetic role of EBV in gastric carcinoma is uncertain. This study was to explore the correlation of Fas/FasL expression to the apoptosis of tumor cells and tumor-infiltrating lymphocytes (TIL) in EBV-associated gastric carcinoma (EBVaGC). Fas/FasL expression in 49 specimens of EBVaGC, 20 specimens of EBV-negative gastric carcinoma (EBVnGC) and 12 specimens of normal gastric mucosa was detected by immunohistochemistry. The apoptotic index (AI) of cells was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). The positive rates of Fas were 91.7% in normal gastric mucosa and 76.8% in gastric carcinoma (P < 0.05); those of FasL were 16.7% in normal gastric mucosa and 58% in gastric carcinoma (P < 0.05). The positive rate of Fas was significantly lower in EBVaGC than in EBVnGC (71.4% vs. 90.0%, P < 0.05). The positive rate of FasL in EBVaGC was significantly higher than that in EBVnGC (63.2% vs. 45%, P < 0.05). The AI of EBVaGC cells was significantly lower than that of EBVnGC cells (P = 0.002). The number and AI of TIL in EBVaGC were higher than those in EBVnGC (P < 0.05). The AI of TIL was positively correlated with the level of FasL expression in tumor cells (r=0.237, P = 0.028). Up-regulation of FasL expression and decrease of TIL apoptosis in EBVaGC may facilitate the escape of tumor cells from the host immunosurveillance, and it might contribute to the development and progression of carcinoma.

CT appearance of Epstein-Barr virus-associated gastric carcinoma

Epstein-Barr virus (EBV) is associated with pathogenesis of several epithelial neoplasms and lymphoproliferative disorders. A certain percentage of gastric carcinomas are associated with EBV. EBV-associated gastric carcinoma is a distinct entity associated with good prognosis. Multiple carcinomas are highly prevalent in EBV-associated gastric carcinomas, and this warrants thorough endoscopic investigation before determining the extent of surgical resection. Therefore, it is important to consider the presence of Epstein-Barr virus in the preoperative CT evaluation of gastric carcinomas. The purpose of this article is to illustrate the CT features of Epstein-Barr virus-associated gastric carcinoma. The surgically resected gastric carcinomas at our institution from 1998 to 2004 were reviewed and were examined for the presence of EBV, and 13 EBV-associated gastric carcinoma lesions were found in 10 patients. The preoperative CT scans were reviewed in these patients. Radiologic features that may suggest the presence of Epstein-Barr virus in gastric carcinoma includes location in the upper part of the stomach, a large thickness-to-width ratio, and a bulky portion projecting from the gastric wall.

Expression of Epstein-Barr virus genes in EBV-associated gastric carcinomas

To understand the expression of latent and lytic genes of Epstein-Barr virus (EBV) in EBV-associated gastric carcinoma (EBVaGC) and to explore the relationship between EBV-encoded genes and development of EBVaGC at molecular level. One hundred and seventy-two gastric carcinoma tissues and 172 corresponding para-carcinoma tissues were tested for EBV genome by polymerase chain reaction (PCR)-Southern blotting. EBV-encoded small RNA (EBER) 1 of the PCR positive specimens was detected by in situ hybridization (ISH). Gastric carcinomas with positive EBER1 signals were classified as EBVaGCs. RT-PCR and Southern hybridization were applied to the detection of expression of nuclear antigen (EBNA) promoters (Qp, Wp and Cp), EBNA 1 and EBNA 2, latent membrane proteins (LMP) 1, 2A and 2B and lytic genes (immediate early genes BZLF1 and BRLF1, early genes BARF1 and BHRF1, late genes BcLF1 and BLLF1) in EBVaGCs. Eleven EBV positive samples existed in gastric carcinoma tissues (6.39%). No EBV positive sample was found in corresponding para-carcinoma tissues. The difference between EBV positivity in carcinoma tissues and corresponding para-carcinoma tissues was significant (chi(2) = 9.0909, P = 0.0026). Transcripts of Qp and EBNA1 were detected in all the 11 EBVaGCs, while both Wp and Cp were silent. EBNA2, LMP1 and LMP2B mRNA were absent in all the cases, while LMP2A mRNA was detected in 4 of the 11 cases. Of the 11 EBVaGCs, 7 exhibited BcLF1 transcripts and 2 exhibited BHRF1 transcripts. The transcripts of BZLF1 and BARF1 were detected in 5 cases, respectively. No BLLF1 and BRLF mRNA were detected. The latent pattern of EBV in gastric carcinoma corresponds to the latency I/II. Some lytic infection genes are expressed in EBVaGCs tissues. BARF1 and BHRF1 genes may play an important role in tumorigenesis of gastric carcinoma.

EBV-expressing AGS gastric carcinoma cell sublines present increased motility and invasiveness.

Tumor invasion marks a critical point in cancer progression; it is a harbinger of morbidity and mortality. Thus, the cellular events that enable the invasive phenotype are under intense investigation. Epstein-Barr virus (EBV) is associated with a number of cancers, including Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC) and is suspected to contribute to their tumorigenesis. On average, 8% of gastric carcinomas have been shown to carry this virus. To explore whether the presence of EBV in gastric carcinoma contributes to tumor progression in this predominantly invasive carcinoma, we examined a panel of 2 in vitro EBV-infected human gastric cancer cell line sublines and their mock-infected AGS parental control line. We found EBV infection caused a marked increase in transmigration of a Matrigel barrier (415% and 303%, p < 0.05, for the 2 infected lines). This correlated with increased motility of these sublines (233% and 140%, p < 0.05). As this pattern of increased motility leading to a more pronounced enhancement of invasion has been noted in other tumor cells, we explored the roles of autocrine signaling pathways previously implicated in carcinoma motility and invasion. Inhibitors to the epidermal growth factor receptor (EGFR) (PD153035), phospholipase C (PLC) (U73122), extracellular-signal regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) (PD089035) and PI-3 kinase (Wortmannin) were not informative. These data suggest that EBV increases migration of AGS cells by a mechanism independent of these autocrine growth factor-induced pathways. Instead, we found that the EBV-infected cells presented increased focal adhesion kinase (FAK) phosphorylation. These findings suggest a role for integrin-mediated signaling in promoting EBV-associated invasiveness.

Low rate of apoptosis and overexpression of bcl-2 in Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV) has been demonstrated in about 10% of gastric carcinomas. However, the pathogenetic role of EBV in gastric carcinoma is uncertain. We compared the rate of apoptotic cell death, cell proliferation and the expression of apoptosis-related proteins in gastric carcinomas with or without EBV. Epstein-Barr virus was detected in 40 gastric carcinomas by EBV-encoded small RNA-1 in-situ hybridization. Apoptotic cell death, MIB-1, p53, bcl-2 and bcl-x were examined by the terminal deoxynucleotidyl-mediated dUTP-nick end labelling method and immunohistochemistry. We also included 40 age-, sex- and disease stage-matched EBV-negative cases as a control. The number of apoptotic cells was significantly lower in EBV-positive (20 +/- 15. 1/1000 cells) and bcl-2-positive (17 +/- 12.9/1000 cells) tumours than in EBV-negative (43 +/- 37.1) and bcl-2-negative tumours (38 +/- 32.1, P < 0.001, P < 0.001, respectively). bcl-2 immunostaining was significantly higher in EBV-positive tumours (24 cases) than in EBV-negative tumours (12 cases, P < 0.05). There was no significant difference in bcl-x and p53 expression between EBV-positive and -negative tumours. The number of MIB-1-positive cells in EBV-positive tumours (237 +/- 161/1000) was significantly lower than in EBV-negative tumours (480 +/- 208/1000 cells, P < 0.001). A low rate of apoptosis and high bcl-2 expression were recognized in EBV-positive gastric carcinomas, suggesting that bcl-2 protein is the main inhibitor of apoptosis in EBV-positive carcinomas. In addition, the low apoptotic and proliferative activities may reflect a low biological activity in EBV-positive gastric carcinomas.

Epstein-Barr virus in gastric carcinoma in Suzhou, China and Osaka, Japan: association with clinico-pathologic factors and HLA-subtype.

Information on geographic differences of Epstein-Barr virus (EBV) positivity and association with HLA in gastric carcinoma are limited. Therefore, the association of gastric carcinoma with EBV was examined in 118 patients from Suzhou, China, where the incidence of nasopharyngeal carcinoma (NPC) is high, and in 216 patients from Osaka, Japan, where the incidence of NPC is low, NPC being one of the EBV-associated carcinomas. Expression of HLA-A2 was also examined in some of these cases. The EBV genome was evidenced by PCR and in the tumor cells by in situ hybridization in 7/90 and 9/151 of cases from Suzhou and Osaka, respectively. Immunohistochemistry revealed that cancer cells in all cases with EBV did not express latent membrane protein-I. Type A was found in all cases positive for EBV. Among several histologic and clinical factors, only age of patients showed a correlation with EBV positivity: patients over 60 showed a higher frequency than patients below 60 years of age (p < 0.05). Typing of the HLA-A locus was possible in 16 cases positive for the EBV genome: 3 of 4 cases from Suzhou and 4 of 7 from Osaka were positive for HLA-A2 products. Severe lymphoid cell infiltration was found in 2 of 7 cases and 1 of 4 cases with and without the HLA-A2 type, respectively. The reported frequency of EBV positivity in Chinese living in Taiwan and in Japanese living in Hawaii is higher than in Suzhou, China, and Osaka, Japan, respectively. Our findings suggest that EBV association with gastric carcinoma is influenced by environmental and cultural factors.

Epstein-Barr virus in gastric carcinoma in Suzhou, China and Osaka, Japan: Association with clinico-pathologic factors and HLA-subtype

Epstein-Barr virus in gastric carcinoma in Suzhou, China and Osaka, Japan: Association with clinico-pathologic factors and HLA-subtype

Epstein-Barr virus in gastric carcinoma and adjacent normal gastric and duodenal mucosa.

To look for Epstein-Barr virus (EBV) in a series of gastric cancers of common and rare histological types. Formalin sections from 19 cases of gastric carcinomas of different types were studied using in situ hybridisation with fluorescein conjugated EBER oligonucleotides and the avidin-biotin complex immunoperoxidase technique, using the monoclonal antibody Dako-EBV CS 1-4. The only positive tumour was a lymphoepithelioma-like gastric carcinoma. The remaining 18 cases, which included 11 consecutive cases of usual adenocarcinomas, three early gastric cancers, two adenosquamous carcinomas, and a case each of signet ring carcinoma and neuroepithelioma, were all negative. However, scattered EBV positive cells were seen in the normal gastric or duodenal mucosa bordering the tumours in seven out of 11 cases. Lymphoepithelioma-like gastric carcinoma seems to be the main, if not the only, EBV positive gastric cancer. The presence of EBV positive normal gastric and duodenal cells suggests that these cells may act as a reservoir for the virus in some people--a possibility that deserves wider investigation.

Epstein-Barr virus in gastric carcinoma with lymphoid stroma. Special reference to its detection by the polymerase chain reaction and in situ hybridization in 99 tumors, including a morphologic analysis

Epstein-Barr virus in gastric carcinoma with lymphoid stroma. Special reference to its detection by the polymerase chain reaction and in situ hybridization in 99 tumors, including a morphologic analysis