Epstein-Barr virus (EBV) infection contributes to human carcinogenesis, particularly in the development of lymphoma, nasopharyngeal cancer (NPC) and gastric cancer (1). EBV is a γ-herpes virus and infects B cells of the immune system and epithelial cells. EBV latently persists in the host B cells for life (2) and thus, EBV is estimated to infect >90% of the population worldwide (2). In a small number of infected cell populations, EBV infection can transform B cells to lymphoma cells (3) or epithelial cells to nasopharyngeal cancer cells (4). Furthermore, ~10% of gastric cancer worldwide was associated with EBV infection and this subtype of gastric cancer is termed EBV-associated gastric cancer (EBVaGC) (5,6). However, the molecular mechanism by which EBV infection causes these malignancies remains to be defined.

Molecularly, the BamHI-A rightward frame 1 (BARF1) is an EBV gene that is expressed early in the EBV lytic cycle and shares 38% primary amino acid sequence homology with the bcl-2 proto-oncogene product (7). The constitutive expression of BARF1 protein was able to immortalize lymphoblast cells and prolong cell survival (8). The carcinogenic activity of the oncogene c-myc is obligatory for the development of Burkitt lymphoma and previous studies suggested that BARF1 expression is required to inhibit c-myc-induced apoptosis and exhibits a synergic role in mediating the effect of Bcl-2 and c-myc during B cell transformation (9–13). BARF1 protein is also involved in the regulation of microvessel density (MVD) and micro-lymphatic vessel density (MLVD) (14,15). Furthermore, MVD and MLVD are strongly associated with cancer metastasis and with the survival of cancer patients (14). In addition, during EBV latency, there are >8 EBV encoded proteins and several non-coding RNAs expressed in cells (e.g., two EBV encoded small RNAs, termed EBER1 and EBER2), nuclear antigens and membrane proteins (16). Latent membrane protein 1 (LMP1) and LMP2A are two EBV latent membrane proteins, which function as constitutively active receptors independent of ligand binding and manipulate the signaling pathways for B cell activation and differentiation in order to sustain the long-life of EBV-positive cells (17). The carboxyl terminus of LMP1 contains consensus tumor necrosis factor receptor-associated factor (TRAF)-binding domains, which can constitutively activate signal transducers and activators of transcription (STAT), janus kinase (JNK), and nuclear factor (NF)-κB pathways for cell survival and growth (18). Although, the oncogenic role of LMP1 is well established, its roles in microvessel and micro-lymphatic vessel formation are less clear. Thus, the present study assessed the EBV infection in NPC and gastric cancer tissue samples, and then analyzed the levels of MVD and MLVD to identify their association with the clinicopathological features of the patients. This study may provide a novel insight into the oncogenic role of EBV in NPC and gastric cancer.

For in situ hybridization, an ISH-5022 EBER kit was used (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc., Beijing, China). A labeled oligonucleotide probe complementary to EBER1 was used to detect the EBER1 as positive REBV infection. Briefly, tissue sections were deparaffinized and rehydrated, and then hybridized to a labeled probe according to the manufacturer's instructions. Tissue sections without probe hybridization were used as negative controls. For immunohistochemistry, tissue sections were deparaffinized by 2 times incubation in xylenol for 10 min at room temperature and dehydrated in a series of ethanol (100, 75 and 50%). Sections were then incubated in 3% H₂O₂ for 10 min at room temperature and subsequently subjected twice to antigen repair in 0.01 M acid buffer (pH 6.0) using a microwave at 92–98°C for 5 min, with a 10 min break between incubations. Sections were washed in phosphate-buffered saline (PBS), incubated with normal goat serum for 15 min at room temperature, and further incubated with primary antibodies at 4°C overnight. The primary antibodies used were rabbit monoclonal anti-LMP1 (cat. no. ZM-0386), rabbit monoclonal anti-BHRF1 (cat. no. ZA-0627), rabbit monoclonal anti-VEGF-C (cat. no. ZA-0266), mouse monoclonal anti-LYVE-1 (cat. no. ZA-0483) and mouse monoclonal anti-CD34 (cat. no. ZM-0046) (dilution used for all was 1:200; Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.). The sections were washed 3 times with PBS and incubated with biotinylated anti-mouse (cat. no. BA1001) or anti-rabbit (cat. no. BA1003) secondary antibodies (dilution used for all was 1:200; Boster Biological Technology, Ltd., Beijing, China) for 30 min at room temperature, and subsequently visualized by incubation of tissue sections with a 3,3′-diaminobenzidine solution (Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.). Sections were counterstained with H&E, mounted with a coverslip and visualized using the Olympus CX23 light microscope (Olympus Corporation, Tokyo, Japan).

All tissue samples were reviewed and scored blindly by two pathologists, and the pathology of each tissue section was confirmed. To assess the in situ hybridization data, negative controls were indicated as the sections without any staining, and positive controls were indicated as the stained sections with appropriate nuclear localization. To score immunohistochemical data, the two pathologists reviewed at least ten x400 fields and counted staining intensity and percentage of positive cells vs. total cells. The staining intensity was judged as no staining, −; light brown, +; brown, ++; and strong brown, +++. The percentage of positive cells <10% was considered as negative and the detailed information is as follows: <10%, −; 11–25%, +; 26–50%, ++; >50%, +++. These two scores were added together to form a total score, high (score ≤2+) vs. low expression of a protein.

EBVaGC is a recently identified cancer type that may be caused by EBV infection (19). The present study further demonstrated that EBV infection contributes to the development of a small percentage (5%) of gastric cancers, although previous studies have shown that 10% of worldwide gastric cancers were EBVaGC (6,20). By contrast, the frequency of EBV infection in NPC was notably higher, effecting 85% of the patients observed. Although the frequency of EBV infection in EBVaGC and NPC was different, the molecular mechanism of EBV infection in different types of cancer may be the same. Thus, in this study expression of the EBV-associated proteins LMP1 and BARF1 was further assessed in NPC and EBVaGC tissue specimens for association with clinicopathological data and with MVD and MLVD. It was demonstrated that BARF1 expression was associated with lymph node metastasis and MLVD in patients with EBVaGC. In NPC, LMP1 expression was associated with TNM stage (P=0.011) and lymph node metastasis (P=0.041). Only 13.3% cases were BARF1 positive and MLVD was significantly higher in LMP1-positive cases than in LMP1-negative cases. The data from the current study indicate that although EBV infection is involved in the development of gastric cancer and NPC, expression of EBV-associated proteins LMP1 and BARF1 may have differential functions during the tumorigenesis of these two types of cancer.

Generally, primary EBV infection occurs via the oral route and establishes a lifelong virus carrier state, termed latent infection (21). In latent infection, infected cells only express a limited set of viral genes, but can provide a survival advantage to the infected cell (22). The latent infection can be further divided into different subgroups due to specific viral proteins (23). EBVaGC is considered as a latency I EBV infection, while NPC can be both latency I and II EBV infections (24,25). Latency I infection is characterized by expression of EBV nuclear antigen 1 (EBNA1), EBER1 and 2, and BamHI-A rightward transcripts (BART) (4,26). In addition to latency I transcripts, latency II infection can also express latent membrane protein 1 (LMP1) (4,26). Our current data are consistent with previous findings (4,26). Only one patient with weak LMP1 expression was identified in the 30 EBVaGC cases. By contrast, 38 NPC tissues in these 61 NPCs exhibited LMP1 expression. This confirmed that the latency of EBV infection between EBVaGC and NPC was different. Therefore, due to the different expression of viral proteins, oncogenic mechanisms of EBV in EBVaGC and NPC may differ.

Furthermore, tumor metastasis is the leading cause of cancer-related mortality (27,28). A greater understanding of the molecular mechanism underlying tumor metastasis may aid the development of effective cancer therapies. The initial site of cancer metastasis is usually the regional lymph nodes (29,30). Clinical and experimental data suggested that lymphoangiogenesis can greatly facilitate the migration of tumor cells into the lymph nodes (31–33). The present study demonstrated that MLVD was associated with NPC lymph node metastasis and that EBV infection was associated with MLVD. The association between EBV infection and MLVD was consistent in EBVaGC and NPC. These data suggest that EBV infection may have a common effect on the regulation of lymphoangiogenesis, although the exact molecular mechanisms remain unknown. Further investigation is required to clarify how EBV infection contributes to an increase in MLVD.

EBV-positive epithelial malignancies show selective and abundant expression of a viral gene that encodes BARF1 protein (34). BARF1 expression was usually low in lymphomas, but more frequent in EBV-associated carcinomas. BARF1 may function as an oncogene in NPC, parallel to the more widely investigated viral protein LMP1 (35). In EBV-positive gastric cancer, BARF1 was expressed in the absence of LMP1, possibly functioning as the predominant EBV oncogene in this disease (36). BARF1 expression was able to immortalize and transform epithelial cells of different origins by acting as a mitogenic growth factor, inducing cyclin-D expression and upregulating anti-apoptotic Bcl-2, and in turn stimulating host cell growth and survival (37). In the current study, 13% of NPCs were positive for BARF1, whereas BARF1 was expressed in 56.7% of EBVaGCs. This finding suggests that BARF1 and LMP1 may have redundant functions in promoting tumorigenesis in gastric cancer and in NPC.

The current study does have certain limitations. For example, it is only a proof-of-principle descriptive study and additional mechanistic data are required to support the current findings. It remains to be determined how these two viral proteins function differentially in these two types of human cancer or whether EBV is involved in the development of EBV-positive gastric cancer since EBV is estimated to infect >90% of the worldwide population and EBV infection may be just bystander in these gastric types of cancer.