RESUMEN
PURPOSE: Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) constitutes the largest subpopulation in EBV-associated tumors worldwide. To date, 44 mature EBV-encoded microRNAs (EBV miRNAs) have been identified, but their roles in EBVaGC development are still poorly understood. In this study, we aimed to investigate the roles and targets of ebv-miR-BART10-3p (BART10-3p) and ebv-miR-BART22 (BART22) in EBVaGC. METHODS: EBV miRNA expression in EBVaGCs was evaluated by deep sequencing and qRT-PCR, and relationships between BART10-3p or BART22 expression and clinicolpathological characteristics and survival rates of patients with EBVaGC were analyzed. The roles of BART10-3p and BART22 and their underlying mechanisms were further investigated through exogenous overexpression or silencing in EBVaGC cells, and validated in clinical EBVaGC tissue samples. RESULTS: BART10-3p and BART22 were found to be highly expressed in the EBVaGC cell lines SNU719 and YCCEL1. Higher expression of BART10-3p or BART22 in primary EBVaGC samples was significantly associated with lymph node metastasis and a worse 5-year overall survival. BART10-3p and BART22 promoted cell migration and invasion by targeting adenomatous polyposis coli (APC) and Dickkopf 1 (DKK1), thereby activating the Wnt signaling pathway and, consequently, upregulating downstream Twist and downregulating downstream E-cadherin. In 874 primary gastric carcinoma samples, APC and DKK1 were found to be lower expressed in EBVaGC than in EBV-negative samples, and their expression levels were inversely correlated with those of BART10-3p and BART22 in 71 EBVaGC samples. CONCLUSIONS: From our data we conclude that BART10-3p and BART22 play vital roles in promoting EBVaGC metastasis by targeting APC and DKK1 and, subsequently, activating the Wnt signaling pathway, thereby providing novel prognostic biomarkers and potential therapeutic targets for EBVaGC.
Asunto(s)
Herpesvirus Humano 4/genética , MicroARNs/metabolismo , Neoplasias Gástricas/patología , Neoplasias Gástricas/virología , Vía de Señalización Wnt , Secuencia de Bases , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática/patología , Masculino , MicroARNs/genética , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/genética , Análisis de SupervivenciaRESUMEN
This study evaluated the associations of HER2 protein, HER2 gene amplification, and positivity for p-AKT, p-ERK, and p-PLCγ proteins with clinicopathological status and overall survival (OS) of patients who had Epstein-Barr virus-associated gastric cancer (EBVaGC; nâ¯=â¯58) or EBV-negative GC (EBVnGC; nâ¯=â¯329). Tissue samples were subjected to immunohistochemistry and fluorescence in situ hybridization (FISH). Results showed that EBVaGC less expressed HER2 and amplified HER2 gene. p-AKT (pâ¯=⯠0.035) and p-ERK (pâ¯=⯠0.001) were inhibited in EBVaGC than in EBVnGC, while p-PLCγ (pâ¯=⯠0.034) was upregulated. Among EBVaGC patients, p-ERK positivity was associated with Lauren classification (pâ¯=â¯0.023), and p-PLCγ positivity was inversely associated with TNM stage (pâ¯=â¯0.041) and lymph node metastasis (pâ¯=â¯0.041). In contrast, among EBVnGC patients, HER2 expression was associated with distant metastasis (pâ¯=â¯0.043) and p-AKT positivity was associated with intestinal subtype (pâ¯<â¯0.004), lymph node metastasis (pâ¯=â¯0.031), distant metastasis (pâ¯<â¯0.001), and elder age (>60y, pâ¯<â¯0.004). Overall analysis showed that EBVaGC patients presented better OS than EBVnGC patients (pâ¯=â¯0.044). Among EBVaGC patients, p-AKT positivity (pâ¯=â¯0.008) was associated with worse OS; as well as, HER2 high expression (pâ¯<â¯0.001), p-AKT positivity (pâ¯=â¯0.010), and p-PLCγ (pâ¯<⯠0.001) were associated with worse OS in EBVnGC patients. Multivariate analysis showed that distant metastasis (95% CI: 1.559 to 4.028, pâ¯<⯠0.001), HER2 high expression (95% CI: 1.058 to 2.454, pâ¯=⯠0.026), and p-PLCγ positivity (95% CI: 1.056 to 2.435, pâ¯=â¯0.027) were independent prognostic predictors of OS in EBVnGC patients. Our results indicated that p-AKT positive patients presented worse OS than p-AKT negative ones in EBVaGC, as well as, HER2, p-AKT, and p-PLCγ are prognostic biomarkers for OS in EBVnGC patients.
Asunto(s)
Adenocarcinoma/metabolismo , Adenocarcinoma/virología , Receptor ErbB-2/biosíntesis , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/virología , Adenocarcinoma/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Infecciones por Virus de Epstein-Barr/complicaciones , Femenino , Herpesvirus Humano 4 , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Fenotipo , Fosfolipasa C gamma/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias Gástricas/mortalidadRESUMEN
Spasmolytic polypeptide-expressing metaplasia (SPEM) and intestinal metaplasia (IM) have been recognized as neoplastic precursors in gastric carcinogenesis. We explored the relationship between SPEM and IM in Epstein-Barr virus-associated (EBVaGC) and Epstein-Barr virus-negative (EBVnGC) gastric cancer. Sixty-four EBVaGC and one hundred and fifty-four EBVnGC patients were included. EBV positivity was identified using Epstein-Barr virus-encoded RNA-1 in situ hybridization. SPEM was subclassified into absent, early, and advanced SPEM. Acute and chronic inflammation was graded as absent, mild, moderate, and marked. Univariate and multivariate logistic regression analyses were conducted to analyze the correlation between SPEM, IM, and inflammation. Our study revealed that SPEM was detected in 87.5% EBVaGC and 85.1% EBVnGC patients. Distribution of patients according to the SPEM classification was significantly different between EBVaGC and EBVnGC groups (P=.038). IM was observed less frequently in EBVaGC when compared with EBVnGC patients (P<.001). No difference was observed between EBVaGC and EBVnGC in the levels of acute and chronic inflammation. A positive correlation between IM and SPEM status was observed in both EBVaGC and EBVnGC patients. Furthermore, advanced SPEM was an independent influential factor to IM in EBVnGC (P=.013). In conclusion, SPEM was associated with both EBVaGC and EBVnGC more frequently than IM. Moreover, advanced SPEM had a stronger association with IM than early SPEM in EBVnGC. These results suggest that identification of SPEM should be used as a high-risk indicator for detecting early gastric carcinoma, and should be brought to the attention of pathologists and clinicians.
Asunto(s)
Adenocarcinoma/química , Biomarcadores de Tumor/análisis , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/aislamiento & purificación , Péptidos/análisis , Lesiones Precancerosas/química , Neoplasias Gástricas/química , Estómago/química , Adenocarcinoma/patología , Adenocarcinoma/cirugía , Adenocarcinoma/virología , Biopsia , Distribución de Chi-Cuadrado , China , Femenino , Gastritis/metabolismo , Gastritis/patología , Gastritis/virología , Herpesvirus Humano 4/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular , Estimación de Kaplan-Meier , Modelos Logísticos , Masculino , Metaplasia , Persona de Mediana Edad , Análisis Multivariante , Oportunidad Relativa , Lesiones Precancerosas/patología , Lesiones Precancerosas/cirugía , Lesiones Precancerosas/virología , Modelos de Riesgos Proporcionales , ARN Viral/genética , Estómago/patología , Estómago/virología , Neoplasias Gástricas/patología , Neoplasias Gástricas/cirugía , Neoplasias Gástricas/virologíaRESUMEN
As a special subtype of gastric carcinoma, Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) has distinct clinicopathological features. The Cancer Genome Atlas Research Network revealed that EBVaGC also has distinct molecular features: PIK3CA mutations, DNA hypermethylation, and JAK2, PD-L1, and PD-L2 amplification. Here, we evaluated PIK3CA, JAK2, PD-L1, and PD-L2 expression in 59 EBVaGC and 796 EBV-negative gastric carcinoma (EBVnGC) cases using immunohistochemistry and found that PIK3CA, JAK2, PD-L1, and PD-L2 were highly expressed in 75.9% and 48.8% (P<.001), 81.8% and 71.1% (P=.091), 92.5% and 84.8% (P=.132), and 98.1% and 89.7% (P=.049) of the EBVaGC and EBVnGC cases, respectively. However, the expression of PIK3CA, JAK2, PD-L1, or PD-L2 was not significantly associated with clinicopathological features or patient outcomes in EBVaGC. In contrast, in EBVnGC, high PIK3CA expression was significantly associated with indolent clinicopathological features and independently predicted better 5-year overall survival (57.8% versus 33.4%, P<.001). Our study indicated that the protein expression of the 4 characteristic molecules of EBVaGC was basically consistent with their genetic alterations, making them potential characteristic protein biomarkers and therapeutic targets of EBVaGC. The favorable impact of PIK3CA overexpression on survival found in this study gives us new insight into the clinical significance of PIK3CA in EBVnGC.
Asunto(s)
Adenocarcinoma/enzimología , Antígeno B7-H1/análisis , Biomarcadores de Tumor/análisis , Infecciones por Virus de Epstein-Barr/virología , Janus Quinasa 2/análisis , Fosfatidilinositol 3-Quinasas/análisis , Proteína 2 Ligando de Muerte Celular Programada 1/análisis , Neoplasias Gástricas/enzimología , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adenocarcinoma/virología , Distribución de Chi-Cuadrado , China , Fosfatidilinositol 3-Quinasa Clase I , ADN Viral/genética , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/diagnóstico , Infecciones por Virus de Epstein-Barr/mortalidad , Femenino , Herpesvirus Humano 4/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis Multivariante , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , ARN Viral/genética , Factores de Riesgo , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Neoplasias Gástricas/virologíaRESUMEN
Approximately 10% of gastric carcinomas are associated with Epstein-Barr virus (EBV) and are defined as EBV-associated gastric carcinomas (EBVaGCs). EBVaGCs are known to be accompanied by massive CD8(+) cytotoxic T cell (CTL) infiltration; however, adoptive cellular immunotherapy based on EBV-specific CD8(+) CTLs has been explored with limited success. Because regulatory T cells (Tregs) are regarded as a critical hurdle in anti-tumour immunity, we assessed the distribution of Tregs in 45 cases of EBVaGC and 45 cases of EBV-negative gastric carcinoma (EBVnGC) with matched clinicopathological parameters by immunohistochemistry. We showed that Tregs were significantly increased in EBVaGC compared to EBVnGC (15.92 ± 11.45/HPF vs. 8.45 ± 6.16/HPF, p = 0.001). In addition, we explored the accumulation mechanisms of Tregs in EBVaGC by using EBV (+) gastric carcinoma cell lines SNU719 and GT39 as ex vivo models. When peripheral blood mononuclear cells (PBMCs) were co-cultured with EBV (+) gastric carcinoma cell lines, the Treg frequency increased, and they underwent phenotypic and functional changes. The enhanced recruitment by CCL22 produced by EBVaGC cells, the decreased emigration due to CCR7 downregulation on the Treg surface, the higher proliferation rate, and the lower apoptosis rate of Tregs at tumour sites may promote the accumulation of Tregs in EBVaGC.
Asunto(s)
Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Neoplasias Gástricas/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Apoptosis/inmunología , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Quimiocina CCL22/inmunología , Quimiocina CCL22/metabolismo , Técnicas de Cocultivo , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Femenino , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunohistoquímica , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Receptores CCR7/inmunología , Receptores CCR7/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/virología , Linfocitos T Reguladores/metabolismoRESUMEN
Alterations in global DNA methylation and specific regulatory gene methylation are frequently found in cancer, but the significance of these epigenetic changes in EBV-associated gastric carcinoma (EBVaGC) remains unclear. We evaluated global DNA methylation status in 49 EBVaGC and 45 EBV-negative gastric carcinoma (EBVnGC) tissue samples and cell lines by 5-methylcytosine immunohistochemical staining and methylation quantification. We determined promoter methylation status and protein expression for the p16, FHIT, CRBP1, WWOX, and DLC-1 genes in tissues and studied the correlation between CpG island methylator phenotype (CIMP) class and clinicopathological characteristics. Changes in gene methylation and mRNA expression in EBVaGC cell line SNU-719 and in EBVnGC cell lines SGC-7901, BGC-823, and AGS were assessed after treatment with 5-aza-2'-deoxycytidine (5-aza-dC), trichostatin A (TSA), or a combination of both, by methylation-specific PCR and quantitative real-time RT-PCR. Global genomic DNA hypomethylation was more pronounced in EBVnGC than in EBVaGC. Promoter methylation of all five genes was more frequent in EBVaGC than in EBVnGC (p < 0.05). p16 and FHIT methylation was reversely correlated with protein expression in EBVaGC. Most (41/49) EBVaGC exhibited CIMP-high (CIMP-H), and the prognosis of CIMP-H patients was significantly worse than that of CIMP-low (p = 0.027) and CIMP-none (p = 0.003) patients. Treatment with 5-aza-dC and/or TSA induced upregulation of RNA expression of all five genes in SNU-719; meanwhile, individual gene expression increased in EBVnGC cell lines. In summary, EBV-induced hypermethylation of p16, FHIT, CRBP1, WWOX, and DLC-1 may contribute to EBVaGC development. Demethylation therapy may represent a novel therapeutic strategy for EBVaGC.
Asunto(s)
Biomarcadores de Tumor/genética , Metilación de ADN , Infecciones por Virus de Epstein-Barr/genética , Regulación Neoplásica de la Expresión Génica , Regiones Promotoras Genéticas/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Ácido Anhídrido Hidrolasas/genética , Apoptosis , Proliferación Celular , Islas de CpG/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina , ADN de Neoplasias/genética , Epigénesis Genética/genética , Infecciones por Virus de Epstein-Barr/mortalidad , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Femenino , Estudios de Seguimiento , Proteínas Activadoras de GTPasa/genética , Herpesvirus Humano 4/patogenicidad , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Oxidorreductasas/genética , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Ácido Retinoico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/virología , Tasa de Supervivencia , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Oxidorreductasa que Contiene Dominios WWRESUMEN
To explore HER2 expression in Epstein-Barr virus-associated gastric carcinoma (EBVaGC) and the possible mechanisms causing down-regulation of HER2 expression in EBVaGC, we first evaluated HER2 and LMP2A expression on a clinicopathological-features matched cohort including 78 EBVaGC and 216 EBV-negative gastric carcinoma (EBVnGC) cases by immunohistochemistry. Cases with high HER2 expression in EBVaGC were significantly less than in EBVnGC (5.1% versus 23.7%; p<0.001), and none of the 34 LMP2A+ EBVaGC showed high HER2 expression. Further, overexpressing LMP2A in EBV-negative SGC7901 cells significantly decreased HER2, TWIST and YB-1 mRNA by 36.1%±8.1%, 87.6%±14.0% and 83.8%±5.7%, and protein by 44%, 57% and 49%, respectively. Additionally, the nucleus/cytoplasm ratios of TWIST and YB-1 were also decreased by 85% and 80%, respectively. Silencing LMP2A by siRNA in EBV-positive SNU719 cells for 48 h significantly increased HER2, TWIST and YB-1 mRNA to 276.7%±14.6%, 1284.8%±38.2% and 332.0%±15.5% and protein to 212%, 457% and 232%, respectively. The nucleus/cytoplasm ratios of TWIST and YB-1 were up-regulated by 4.00- and 3.57-fold, respectively, following LMP2A down-regulation. Moreover, LMP2A+/HER2low EBVaGC cases presented the best overall survival compared with LMP2A-/HER2low and LMP2A-/HER2high cases (p=0.003, log-rank test). These results suggest that LMP2A may suppress the HER2 expression through the TWIST/YB-1 axis in EBVaGC.