EBV-associated Gastric Carcinoma Research Articles

Latent membrane protein 2A inhibits expression level of Smad2 through regulating miR-155-5p in EBV-associated gastric cancer cell lines.

Epstein-Barr virus (EBV) infection is one of the causes of gastric cancer (GC). Besides, previous studies have demonstrated that EBV-encoded latent membrane protein 2A (LMP2A) influences the pathogenesis of EBV-associated gastric cancer (EBVaGC) through regulating several key pathways. In this study, the expression level of Smad2 was observed, which was reduced in EBVaGC cell lines, especially in the presence of LMP2A. Meanwhile, we found that LMP2A promoted the expression of miR-155-5p by activated nuclear factor-κB (NF-κB) signaling. After being treated with NF-κB inhibitor (BAY 11-7082), miR-155-5p sharply decreased. Western blot analysis proved that the overexpression of miR-155-5p could inhibit Smad2. Functional studies showed that the role of miR-155-5p might lead to good prognosis in EBV-positive GC through promoting cell apoptosis and cell cycle arrest, as well as inhibiting tumor proliferation. In addition, p-Smad2 protein was also reduced or induced by overexpression or knockdown, respectively, of miR-155-5p. Immunofluorescence analysis further indicated that LMP2A prevented p-Smad2 from transferring to the nucleus, which played a crucial role in transforming growth factor-β (TGF-β) signaling. In summary, our findings confirmed the relationship between LMP2A and Smad2 and provided a potential regulation of the TGF-β pathway in EBVaGC.

Clinicopathologic Characteristics of Epstein-Barr Virus-Associated Gastric Cancer Over the Past Decade in Japan.

Epstein–Barr virus (EBV) is a ubiquitous human herpes virus, but related with several types of malignancies. Among EBV-related malignancies, EBV-associated gastric carcinoma (EBVaGC) has the largest patient’s number. We screened for EBV infection in 1067 GC lesions of 1132 patients who underwent surgical resection from 2007 to 2017 in Japan and examined clinicopathological features of EBVaGC. EBV infection was detected by in situ hybridization with EBV-encoded small RNA 1(EBER-1 ISH). EBV was infected in 80 GC lesions (7.1%). Mean age was significantly lower in patients with EBVaGC than with EBV-negative GC. EBVaGC was more frequent in men than in women. EBVaGC was found twice as frequent in the upper or middle stomach as in the lower stomach. Early EBVaGC was more frequent, and submucosally invaded cases were dominant. The presence of lymphatic vessel invasion was less in EBVaGC, but frequency of lymph node metastasis was similar. Carcinoma with lymphoid stroma (CLS) was found in 3.8% (43/1132) of all lesions with 60.5% of EBV positivity. The synchronous or metachronous multiple GC was frequent in EBVaGC. We clarified clinicopathologic characteristics of EBVaGC over the past decade in Japan. EBV infection should be examined in gastric cancer cases showing these characteristics.

EBV down-regulates COX-2 expression via TRAF2 and ERK signal pathway in EBV-associated gastric cancer

Epstein-Barr virus-associated gastric cancer (EBVaGC) accounts for nearly 10% of gastric cancer. Cyclooxygenase-2 (COX-2) plays a crucial role in cancer progression. However, there is no experimental study on the regulation mechanism of EBV on COX-2 in EBVaGC. To understand more about the tumorigenic mechanism of EBVaGC, the study investigated the role of EBV encode latent membrane protein LMP1 and LMP2A in the regulation of COX-2. The expression of COX-2 was examined in EBVaGC and EBV negative gastric cancer (EBVnGC) cell lines. The plasmids were transfected in SGC7901 to overexpress LMP1/2A. Small interfering RNA (si-RNA) targeting LMP1/2A in GT38 and targeting TRAF2 in SGC7901 were used to detect the expression of COX-2. Furthermore, si-ERK1/2 and the MEK inhibitor PD0325901 were used to investigate whether p-ERK participate in the regulation of COX-2 in SGC7901. The overexpression of LMP1 or LMP2A in SGC7901 down-regulates both COX-2 and TRAF2 expression, and knockdown of LMP1 or LMP2A in GT38 resulted in a certain recovery of COX-2 and TRAF2 expression. Moreover, si-TRAF2 indicated that a sharp down-regulation of COX-2. And the decrease of p-ERK also mediates the inhibitory effect of LMP1 on COX-2. In summary, overexpression of LMP1 and LMP2A inhibits COX-2, which is mediated by a decrease of TRAF2, and p-ERK is involved in the inhibition of COX-2 by LMP1 in gastric cancer.

New Perspectives of Epstein–Barr Virus-Associated Gastric Cancer

Gastric cancer (CG) is one of the most common neoplasms in Peru. Recently the molecular subtypes have been typified in CG. A molecular subtype is associated with the Epstein Barr virus. This entity presents with a frequency of 10%, in a non-cardiac region, in male patients and with the intestinal histological subtype. The prognosis is better compared to the other types of CG. It presents its own molecular alterations that in the future could be susceptible to target therapies and immunotherapy.

Abstract 485: Exon 9 mutation of PIK3CA associated with poor survival in patients with Epstein-Barr virus-associated gastric cancer

Abstract PIK3CA would seem to play an important role in the prognosis of gastric cancer (GC), very few studies have reported on the clinical implications and prognostic effects of these mutations in patients with Epstein-Barr virus-associated gastric cancer (EBVaGC). This study attempted to identify the prognostic significance of PIK3CA mutations in a large number of EBVaGC patients and the relationship of these mutations to the clinicopathologic parameters associated with prognosis. After reviewing 1318 consecutive cases of surgically resected at our institution between January 2011 and November 2014, 120 patients were identified as EBV-positive using EBV-encoded RNA in situ hybridization. Among the 120 patients, 8 patients were excluded due to insufficient or unavailable material (6 patients) and technical issues (2 patients). The related medical files and pathologic reports were all reviewed to identify the clinical and demographic characteristics. PIK3CA mutations were identified in formalin-fixed and paraffin-embedded surgical specimens from 112 EBVaGC patients with available tumor tissue sample. A real-time polymerase chain reaction was used to evaluate hot-spot mutations of exons 1, 4, 7, 9, and 20 of PIK3CA. The frequency of PIK3CA mutations was 25.0% (n=28), and among the 28 patients harboring a PIK3CA mutation, most of the mutations were identified in exon 9 (n=21, 75%). The presence of PIK3CA mutation was also correlated with a higher T category (P<0.001) and N category (P<0.001), as well as the presence of perinueral invasion (P<0.001) and venous invasion (P<0.001). In a univariate analysis, PIK3CA mutation showed no association with overall survival (OS) (P=0.184) or disease-free survival (DFS) (P=0.150). The patients harboring exon 9 PIK3CA mutations did exhibit a significantly shorter OS(P=0.023) and DFS(P=0.013) than the patients lacking an exon 9 PIK3CA mutation, yet without statistical significance in the multivariate analysis. Notably, the exon 9 E542K mutation of PIK3CA was associated with the worst DFS (P=0.011). Plus, the PIK3CA-mutant tumors were not related to such immune-related factors as intratumoral (iTu) tumor-infiltrating lymphocytes (TILs), stromal (str) TILs, iTu-PD-L1, and str-PD-L1 (all P > 0.05). The PIK3CAmutations harbored in exon 9 were also strongly related to clinicopathologic parameters associated with an unfavorable prognosis. Although the exon 9 PIK3CA mutations were not identified as an independently robust prognostic factor, they could play an important role in carcinogenesis and tumor aggressiveness in EBVaGC. Consequently, these findings support the theory that exon 9 PIK3CA mutations can be a prognostic indicator for predicting patients’ outcomes and a rationale for therapeutic targeting in EBVaGC. Citation Format: Dongwon Baek, Jong Gwang Kim, Jin Ho Baek, Byung Woog Kang, Soo Jung Lee, Yee Soo Chae, Hee Jeong Cho. Exon 9 mutation of PIK3CA associated with poor survival in patients with Epstein-Barr virus-associated gastric cancer [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 485.

Abstract 3787: Reduced expression of INPP4B by promoter hypermethylation in Epstein-Barr virus-associated gastric carcinoma

Abstract Background: Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is attracting increasing attention due to distinct molecular and clinical characteristics. However, its carcinogenesis still remains unclear. Previous studies demonstrated that aberrant DNA methylation is one of the pathogenic mechanisms of EBVaGC. Inositol polyphosphate 4-phosphatase type II (INPP4B), as a component of PI3K/AKT pathway, is implicated in the regulation of crucial cellular processes and tumorigenesis. This study aims to reveal the correlation between INPP4B expression and EBV infection, as well as investigate the inactivation mechanism of INPP4B in EBVaGC. Methods: EBV status and INPP4B expression were examined in gastric carcinoma (GC) tissue microarray (301 cases) using EBV RNA probe and immunohistochemistry, respectively. The correlation of EBV status and INPP4B expression was analyzed in GC by Chi-Squared Test. The INPP4B promoter methylation was investigated in GC cell lines: SNU-719 (EBV positive), AGS (EBV negative) and SGC-7901 (EBV negative) with methylation-specific polymerase chain reaction (MSPCR). After treatment with 5-Aza-2′-deoxycytidine, the INPP4B promoter methylation and expression were detected using MSPCR, western blot, and qPCR. Results: INPP4B expression is decreased in 146 of 301 GC cases (48.5%). In 272 evaluable GC cases, EBV was present in 40 cases (14.71%). In addition, Lower expression of INPP4B is significantly correlated with EBV infection (p<0.05). There was a significantly decreased expression of INPP4B in EBV positive cell line (SNU-719) compared to EBV negative cell line (AGS and SGC-7901). Moreover, aberrant CpG island hypermethylation of INPP4B was found in SNU-719 rather than AGS and SGC-7901. After treatment with 5-Aza-2′-deoxycytidine, INPP4B protein and mRNA are dramatically up-regulated, while unmethylated DNA became detectable in SNU-719. Conclusions: Our results suggest that the reduction of INPP4B expression is commonly found in GC, particularly in EBVaGC. Promoter hypermethylation may be one of the mechanisms of INPP4B inactivation. Citation Format: Yiwen Ma, Jichao Tan, Weihong Dai, Yu Zhang, Yu Zhang, Rongjin Lin, Yan Xu. Reduced expression of INPP4B by promoter hypermethylation in Epstein-Barr virus-associated gastric carcinoma [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 3787.

Abstract PL04-01: Screening for nasopharyngeal carcinoma using plasma Epstein-Barr virus DNA: Technological and clinical insights

Abstract Epstein-Barr virus (EBV) DNA is released by a number of EBV-associated malignancies into the plasma. Examples of such malignancies include nasopharyngeal carcinoma (NPC), certain lymphomas and EBV-associated gastric carcinoma. Plasma EBV DNA in such malignancies exhibits a strong correlation with tumor stage, clinical progress and prognosis; and is thus an archetypal circulating tumor DNA species. Our group is interested in exploring the use of plasma EBV DNA to screen for NPC. We have recently completed a prospective population screening project involving over 20,000 middle aged men in Hong Kong. Using plasma EBV DNA as a screening tool, NPC could be found at a much earlier stage than was normally possible. Furthermore, the prognosis of the NPC patients identified through such screening was much more favorable than those diagnosed without such screening. The positive predictive value of this approach was 11%. False-positive results were observed a proportion of subjects without NPC. Factors that contributed to increased false-positive results included advancing age and surprisingly, the ambient temperature of the day when samples were collected. Hence, a higher percentage of false-positive results were observed when samples were collected in colder weather. We hypothesized that such results might be related to the fluctuations in the effectiveness of the immune system in certain individuals due to ambient temperature. Since the reporting of the above-mentioned results, we have been exploring approaches for further enhancing the positive predictive value. Through the use of targeted massively parallel paired-end sequencing, we found that the size profiles of plasma EBV DNA in subjects with NPC and those without were different. Indeed, by combining such size profile and the concentration of plasma EBV DNA as measured by sequencing, the positive predictive value could be further enhanced. Very recently, we investigated the epigenetic profile of plasma EBV DNA in subjects with NPC and those without cancer, in an attempt to further enhance the positive predictive value of the approach. We believe that the development of a robust approach for NPC screening using circulating EBV DNA would have major public health impact, allowing the earlier detection and treatment of NPC in high prevalence areas. We also believe that such developments would also guide the development of other circulating tumor DNA markers for early cancer detection. Citation Format: Yuk Ming Dennis Lo. Screening for nasopharyngeal carcinoma using plasma Epstein-Barr virus DNA: Technological and clinical insights [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 PL04-01.

Abstract 1497: Identification of discrepancy between CTLA4 expression and CTLA4 activation in gastric cancer

Abstract Objective: Recently, immune checkpoints blockers showed higher anti-tumor activity for advanced gastric cancer (GC). The purpose of the study is to find out biomarkers related to anti-CTLA4 therapy. Materials and Methods: Datasets of GEO, TCGA and GESA were extracted. Differential expression of CTLA4 between cancer tissues and normal mucosa, enrichment of WT (wild type) vs CTLA4_KO (knockout) upregulated gene set and clinical significance were analyzed. The expression of CTLA4,CD3 and granzyme A (GZMA) were validated on 30 cases of Chinese GC. MSI marker MLH1 and EBV marker EBER were examined on 30 cases of Chinese GC by immunohistochemistry and in situ hybridization. Results: CTLA4 was up-regulated in GC tissue relative to normal mucosa in datasets of GSE27342 (fold change=1.586, P<0.001) and GSE63089 (fold change=1.365, P<0.001). Increased CTLA4 expression was positively related to CTLA4 activation. EBV-associated GC (EBVaGC) and microsatellite instability GC (MSIGC) disclosed higher CTLA4 levels than other GCs. Genomic stability GC (GSGC) also showed higher enrichment score of CTLA4 activation. CTLA4 activation resulted in shorter overall survival in GC. The expression level of CTLA4 was well correlated to GZMA (R=0.701, P<0.001) and CD3 (R=0.750, P<0.001). Conclusions: GSGC was firstly identified as the potential GC subtypes responsive to anti-CTLA4 treatment. Note: This abstract was not presented at the meeting. Citation Format: Yingyan Yu. Identification of discrepancy between CTLA4 expression and CTLA4 activation in gastric cancer [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 1497.

Lentivirus-mediated RNA interference targeting EBNA1 gene inhibits the growth of GT-38 cells in vitro and in vivo.

Epstein-Barr virus nuclear antigen 1 (EBNA1) is associated with the pathogenesis of Epstein-Barr virus-associated gastric carcinoma (EBVaGC). However, the function of EBNA1 in the growth of EBVaGC cells remains unclear. In the present study, the effects of silencing EBNA1, by RNA interference (RNAi), on the growth of EBVaGC cells were investigated in vitro and in vivo. A lentivirus-mediated RNAi targeting EBNA1 was transfected into the EBVaGC cell line GT-38. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot analysis, MTT, colony formation and flow cytometry were performed to evaluate the biological behavior of GT-38 cells that were transfected with EBNA1 small interfering RNA (siRNA) in vitro. The effects of silencing EBNA1 on tumor growth were assessed in a tumor xenograft model using BALB/c nude mice. The results demonstrated that the proliferative and clonogenic abilities of GT-38 cells were significantly downregulated in response to EBNA1 siRNA (P<0.01). Furthermore, EBNA1 siRNA induced cell cycle arrest in the G0/G1 phase and promoted apoptosis of GT-38 cells (P<0.01). The tumorigenicity of GT-38 cells was significantly inhibited in the EBNA1 siRNA group. The results revealed that lentivirus-mediated RNAi of EBNA1 inhibited the growth of the EBVaGC cell line GT-38 in vitro and in vivo. Therefore, EBNA1 may be a potential target for gene therapy in EBVaGC.

Prospective observation: Clinical utility of plasma Epstein-Barr virus DNA load in EBV-associated gastric carcinoma patients.

Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs) may account for 8-9% of all gastric cancer (GC) patients. All previous reports on EBVaGC were retrospective. Prospective study is warranted to evaluate the exact role of EBV status in predicting the prognosis of GC. It is of special interest to figure out whether dynamic detection of plasma EBV-DNA load could be a feasible biomarker for the monitor of EBVaGC. From October 2014 to September 2017, we consecutively collected GC patients (n = 2,760) from Sun Yat-sen University Cancer Center for EBER examination. We detected EBV-DNA load in plasma and tissue samples of EBVaGC patients at baseline. Subsequently, plasma EBV-DNA load was dynamically monitored in EBVaGC patients. The overall prevalence of EBVaGC is 5.1% (140/2,760). The incidence rate of EBVaGC decreased with advanced AJCC 7th TNM stage (p < 0.001), with the corresponding percentages of 9.3, 9.9, 6.7 and 1.4% for Stage I, II, III and IV patients. EBVaGC patients were predominately young males with better histologic differentiation and earlier TNM stage than EBV-negative GC (EBVnGC) patients. EBVaGC patients were confirmed to had a favorable 3-year survival rate (EBVaGC vs. EBVnGC: 76.8% vs. 58.2%, p = 0.0001). Though only 52.1% (73/140) EBVaGC patients gained detectable EBV-DNA and 43.6% (61/140) reached a positive cutoff of 100 copies/ml, we found the plasma EBV-DNA load in EBVaGC decreased when patients got response, while it increased when disease progressed. Our results suggested that plasma EBV-DNA is a good marker in predicting recurrence and chemotherapy response for EBVaGC patients.

Clinicopathological and molecular characteristics of Epstein-Barr virus associated gastric cancer: a single center large sample case investigation

Epstein-Barr virus associated gastric cancer (EBVaGC) is different from the traditional gastric cancer (Epstein-Barr virus non-associated gastric cancer, EBVnGC), and has unique clinicopathological features. This study investigated the largest single center cancer series so as to establish the clinicopathological and molecular characteristics of EBVaGC in China. A retrospective analysis was conducted on EBVaGC and EBVnGC patients diagnosed at Peking University Cancer Hospital from 2003 to 2018 by comparing their clinicopathological features and prognosis. The gastric cancer (GC) dataset of public database was analyzed to obtain differentially expressed genes. The expression of important genes and their association with prognosis of GC were verified in GC tissues from our hospital. In this study, 3 241 GC patients were included, and a total of 163 EBVaGC (5.0%) patients were identified. Compared with EBVnGC, EBVaGC was higher in male and younger patients, and positively associated with remnant GC, poorly differentiated adenocarcinoma, and mixed type GC. EBVaGC was inversely related to lymph node metastasis. The 5-year survival rate of EBVnGC and EBVaGC was 59.6% and 63.2% respectively (P<0.05). In order to explore molecular features of EBVaGC, the Cancer Genome Atlas (TCGA) dataset was analyzed (n=240), and 7 404 significant differentially expressed genes were obtained, involving cell proliferation, apoptosis, invasion and metastasis. The down-regulated invasion/metastasis gene SALL4 and the up-regulated immune checkpoint gene PD-L1 were important molecular features of EBVaGC. Validation of these two genes in large GC series showed that the majority of the EBVaGC was SALL4 negative (1/92, 1.1%, lower than EBVnGC, 303/1 727, 17.5%), and that PD-L1 was mostly positive in EBVaGC (81/110, 73.6%, higher than EBVnGC, 649/2 350, 27.6%). GC patients with SALL4 negative and PD-L1 positive were often associated with better prognosis. EBVaGC is a unique subtype of GC with less metastasis and a good prognosis. It also has a distinct molecular background. The down-regulation of invasion/metastasis gene SALL4 and up-regulation of immune checkpoint gene PD-L1 are important molecular features.

LMP2A induces DNA methylation and expression repression of AQP3 in EBV-associated gastric carcinoma

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a unique type of gastric carcinomas that promoter hypermethylation of tumor-related genes is extremely frequent to be found. Aquaporin 3 (AQP3) is a small membrane transport protein that plays a crucial role in cancer progression and metastasis. However, there is no experimental study on the expression of AQP3 in EBVaGC and the regulation mechanism of EBV on AQP3. In this study, the loss of AQP3 was contributed by the hypermethylation status of AQP3 promoter in EBVaGC which was caused by elevated expression of DNMT3a. In addition, stable and transient transfection system in SGC7901 showed that viral latent membrane protein 2A (LMP2A) activated phosphorylated ERK and up-regulated DNMT3a. Taken together, LMP2A induced the phosphorylation of ERK, which activated DNMT3a transcription and caused AQP3 expression loss through CpG island methylation of AQP3 promoter in EBVaGC.

Epigenetic alternate promoter utilization and association with PD-L1 expression in Epstein–Barr virus positive gastric cancer.

e15509 Background: We recently elicited the role of epigenetic promoter alterations as a mechanism of immune-evasion and primary resistance to immune checkpoint inhibition in gastric cancer. High prevalence of epigenetic modifications are known to occur in Epstein-Barr virus associated gastric cancer (EBVaGC). EBVaGC has high response rates to anti-PD-1 immune checkpoint inhibitors and is associated with high levels of PD-L1 expression. However, not all EBVaGC express PD-L1 and mechanisms that mediate these phenomena are unknown. Methods: We performed NanoString profiling and PD-L1 immunohistochemistry (using Dako PD-L1 IHC 22C3) on tissue from gastric cancer patients undergoing primary tumor resections at Samsung Medical Centre, South Korea. NanoString panel was designed for 90 recurrent somatic alternate promoter-related genes, and immune-related genes including PD-L1. EBV status was determined using EBV-encoded RNA in situ hybridization and categorized as EBVaGC and EBV-negative. Samples in the top-quartile of alternate promoter utilization were classified as APhigh and the remaining APlow. Results: A total of 272 samples (EBVaGC n = 79; EBV-negative n = 193) were included in this study. EBVaGC had significantly higher PD-L1 expression (p &lt; 0.001) compared to EBV-negative samples. APhigh group (n = 67) consisted of 61 EBV-negative and 6 EBVaGC samples. EBVaGC APhigh tumors had significantly lower PD-L1 transcript expression compared to EBVaGC APlow tumors (p = 0.011, Wilcoxon-rank sum). Similar correlation was also found with PD-L1 IHC combined positive score (CPS)(median CPS score 1 vs 8, p = 0.047). There was a trend towards poorer survival for EBVaGC APhigh tumors (vs EBVaGC APlow; HR 0.23, 95% CI: 0.046 – 1.23, p = 0.087). EBV-negative APhigh tumors also had lower PD-L1 expression (vs EBV-negative APlow; p = 0.046, Wilcoxon-rank sum). Conclusions: Increased utilization of epigenetic alternate promoter isoforms correlates with lower transcriptomic and protein expression of PD-L1 in EBVaGC. Here we describe a potential mechanism of immune-evasion to explain low immune-infiltration and PD-L1 expression that occurs in a group of EBVaGC that is traditionally considered highly immunogenic.

Combination Therapy with a PI3K/mTOR Dual Inhibitor and Chloroquine Enhances Synergistic Apoptotic Cell Death in Epstein-Barr Virus-Infected Gastric Cancer Cells.

The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway is a promising target for gastric cancer (GC) treatment; however the efficacy of PI3K/mTOR dual inhibitors in GC has not yet been maximized. Additionally, the effect of autophagy regulation by PI3K/mTOR dual inhibitors has not been clearly elucidated in GC treatment. We aimed to show that our newly developed PI3K/mTOR dual inhibitor, CMG002, when combined with an autophagy inhibitor, chloroquine (CQ), potently induces effective cancer cell death in Epstein–Barr virus (EBV)-associated gastric cancer (EBVaGC) cells, where both the PI3K/AKT/mTOR and autophagy pathways play important roles in disease pathogenesis. EBV- and mock-infected AGS and NUGC3 GC cell lines were treated with CMG002 +/− CQ. PI3K/AKT/mTOR signaling pathway mediators, cellular apoptosis and autophagy markers were confirmed by Western blot assay. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. CMG002 effectively blocked the PI3K/AKT/mTOR pathway by markedly decreasing phosphorylation of AKT and its downstream mediator S6. CMG002 induced G0/G1 cell cycle arrest and enhanced apoptotic cell death in AGS and NUGC3 cells, particularly EBV-infected cells compared with mock-infected cells, as confirmed by flow cytometric analyses and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays. The combination of CMG002 plus CQ synergistically increased apoptotic cell death in EBV-infected GC cell lines when compared with CMG002 alone (P < 0.05). Our results suggest that the new PI3K/mTOR dual inhibitor, CMG002, when used in combination with the autophagy inhibitor, CQ, provides enhanced therapeutic efficacy against EBVaGC.

Current status and progress in the research for viral nfection-associated gastric cancer

Gastric cancer is one of the malignant tumors with high morbidity and high mortality in China. Research has shown that viral infection is closely related to the occurrence of gastric cancer. Epstein-Barr virus-associated gastric cancer characterized by EB virus infection has been classified as a subtype of gastric cancer, whose epidemiology, pathogenesis, clinical and histopathologic features have been studied in detail. At the same time, oncolytic viruses reveal the inhibitory effect of the virus on tumors, and their ability to target and kill tumor cells is used in the treatment of some advanced cancers. This article will review the research advances about relevance to gastric cancer of several viruses that have been reported and the latest progress in anticancer mechanisms and combined therapies for oncolytic viruses. Key words: Herpesvirus 4, human; Stomach neoplasms; Tumor virus infections; Correlation; Neoplastic processes; Oncolytic virus

Constitutive activation of the canonical NF-κB signaling pathway in EBV-associated gastric carcinoma

EBV-associated gastric carcinoma (EBVaGC) is a specific subgroup of gastric carcinoma, and the multifunctional transcriptional factor NF-κB may contribute to its tumorigenesis. In this study, we comprehensively characterized NF-κB signaling in EBVaGC using qRT-PCR, western blot, immunofluorescence assays, ELISA, and immunohistochemistry staining. NF-κB-signaling inhibitors may inhibit the growth of EBVaGC cells and induce significant apoptosis. IκBα is a key regulatory molecule, and repression of IκBα can contribute to aberrant NF-κB activation. Overexpression of LMP1 and LMP2A in the EBV-negative GC cell line SGC7901 could inhibit the expression of IκBα and induce NF-κB activation. These findings indicate that the canonical NF-κB signal is constitutively activated and plays an important role in EBVaGC tumorigenesis.

Exon 9 Mutation of PIK3CA Associated With Poor Survival in Patients With Epstein-Barr Virus-associated Gastric Cancer.

Epstein-Barr virus (EBV)-associated gastric cancer (GC) is known to harbor a significant enrichment of of phosphatidylinositol 4, 5-biphosphate 3- kinase catalytic subunit alpha isoform (PIK3CA). Therefore, this study investigated the clinical relevance and prognostic role of PIK3CA mutations in patients with EBV-GC. After reviewing 1,318 consecutive cases of surgically resected GC, 120 patients were identified as EBV-positive using EBV-encoded RNA in situ hybridization. PIK3CA mutations were identified in formalin-fixed and paraffin-embedded surgical specimens from 112 patients with EBV-GC with available tumor tissue samples. Real-time polymerase chain reaction was used to evaluate hot-spot mutations of exons 1, 4, 7, 9, and 20 of PIK3CA. Among the 112 patients, the frequency of PIK3CA mutations was 25.0% (n=28), and among the 28 patients harboring a PIK3CA mutation, most mutations were identified in exon 9 (n=21, 18.8%). The presence of PIK3CA mutation was also correlated with a higher T category (p<0.001) and N category (p<0.001), as well as the presence of perinueral invasion (p<0.001) and venous invasion (p<0.001). In a univariate analysis, PIK3CA mutation showed no association with overall survival (OS) (p=0.184) or disease-free survival (DFS) (p=0.150). Patients harboring exon 9 PIK3CA mutations exhibited a significantly shorter OS (p=0.023) and DFS (p=0.013) than the patients lacking an exon 9 PIK3CA mutation, yet without statistical significance in the multivariate analysis. Notably, exon 9 E542K mutation of PIK3CA was associated with the worst DFS (p=0.011). The current data show that PIK3CA mutations appear to play an important role in carcinogenesis and tumor aggressiveness in EBV-GC, and also support the concept that exon 9 mutation of PIK3CA is a prognostic indicator for predicting patient outcomes and a rationale for therapeutic targeting in EBV-GC.

Adenosine Induces EBV Lytic Reactivation through ADORA1 in EBV-Associated Gastric Carcinoma.

Cordyceps species are known to contain numerous bioactive compounds, including cordycepin. Extracts of Cordyceps militaris (CME) are used in diverse medicinal purposes because of their bioactive components. Cordycepin, one of the active components of CME, exhibits anti-proliferative, pro-apoptotic, and anti-inflammatory effects. Cordycepin structurally differs from adenosine in that its ribose lacks an oxygen atom at the 3′ position. We previously reported that cordycepin suppresses Epstein–Barr virus (EBV) gene expression and lytic replication in EBV-associated gastric carcinoma (EBVaGC). However, other studies reported that cordycepin induces EBV gene expression and lytic reactivation. Thus, it was reasonable to clarify the bioactive effects of CME bioactive compounds on the EBV life cycle. We first confirmed that CME preferentially induces EBV gene expression and lytic reactivation; second, we determined that adenosine in CME induces EBV gene expression and lytic reactivation; third, we discovered that the adenosine A1 receptor (ADORA1) is required for adenosine to initiate signaling for upregulating BZLF1, which encodes for a key EBV regulator (Zta) of the EBV lytic cycle; finally, we showed that BZLF1 upregulation by adenosine leads to delayed tumor development in the EBVaGC xenograft mouse model. Taken together, these results suggest that adenosine is an EBV lytic cycle inducer that inhibits EBVaGC development.

Morphological characteristics of vasculogenic mimicry and its correlation with EphA2 expression in gastric adenocarcinoma

Genetically deregulated tumor cells generate vascular channels by vasculogenic mimicry (VM) that is independent of endothelial blood vessels. The morphological characteristics of VM and the role of EphA2 in the formation of VM were evaluated in 144 clinical samples of gastric adenocarcinoma and AGS gastric cancer cell line. It has long been believed that VM consists of PAS-positive basement membrane and CD31/CD34-negative cells. Interestingly, we found that the luminal surface of gastric tumor cells that form VM channels showed PAS-positive reaction, and that the involvement of CD31/CD34-positive tumor cells in the formation of VM channels. Highly aggressive tumor cells that formed VM were found to express CD31 or CD34, implicating the angiogenic and vasculogenic potential of the genetically deregulated tumor cells. VM occurrence was positively correlated with high expression of EphA2 in our patient cohort, and the indispensable role of EphA2 in VM formation was identified by gene silencing in AGS cells. We also report that Epstein–Barr virus (EBV)-positive tumor cells were involved in the formation of VM channels in EBV-associated gastric cancer samples. Overall, our results suggest that EphA2 signaling promotes tumor metastasis by inducing VM formation during gastric tumorigenesis.

Epstein–Barr virus miR-BART3-3p promotes tumorigenesis by regulating the senescence pathway in gastric cancer

Epstein-Barr virus-associated gastric cancer (EBVaGC) accounts for about 10% of all gastric cancer cases and has unique pathological and molecular characteristics. EBV encodes a large number of microRNAs, which actively participate in the development of EBV-related tumors. Here, we report that EBV-miR-BART3-3p (BART3-3p) promotes gastric cancer cell growth in vitro and in vivo Moreover, BART3-3p inhibits the senescence of gastric cancer cells induced by an oncogene (RASG12V) or chemotherapy (irinotecan). LMP1 and EBNA3C encoded by EBV have also been reported to have antisenescence effects; however, in EBVaGC specimens, LMP1 expression is very low, and EBNA3C is not expressed. BART3-3p inhibits senescence of gastric cancer cells in a nude mouse model and inhibits the infiltration of natural killer cells and macrophages in tumor by altering the senescence-associated secretory phenotype (SASP). Mechanistically, BART3-3p directly targeted the tumor suppressor gene TP53 and caused down-regulation of p53's downstream target, p21. Analysis from clinical EBVaGC samples also showed a negative correlation between BART3-3p and TP53 expression. It is well known that mutant oncogene RASG12V or chemotherapeutic drugs can induce senescence, and here we show that both RASG12V and a chemotherapy drug also can induce BART3-3p expression in EBV-positive gastric cancer cells, forming a feedback loop that keeps the EBVaGC senescence at a low level. Our results suggest that, although TP53 is seldom mutated in EBVaGC, its expression is finely regulated such that EBV-encoded BART3-3p may play an important role by inhibiting the senescence of gastric cancer cells.