Overexpression of miR-142-5p and miR-155 in Gastric Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma Resistant to Helicobacter pylori Eradication

microRNAs (miRNAs) are small non-coding RNAs that can function as endogenous silencers of target genes and play critical roles in human malignancies. To investigate the molecular pathogenesis of gastric mucosa-associated lymphoid tissue (MALT) lymphoma, the miRNA expression profile was analyzed. miRNA microarray analysis with tissue specimens from gastric MALT lymphomas and surrounding non-tumor mucosae revealed that a hematopoietic-specific miRNA miR-142 and an oncogenic miRNA miR-155 were overexpressed in MALT lymphoma lesions. The expression levels of miR-142-5p and miR-155 were significantly increased in MALT lymphomas which do not respond to Helicobacter pylori (H. pylori) eradication. The expression levels of miR-142-5p and miR-155 were associated with the clinical courses of gastric MALT lymphoma cases. Overexpression of miR-142-5p and miR-155 was also observed in Helicobacter heilmannii-infected C57BL/6 mice, an animal model of gastric MALT lymphoma. In addition, miR-142-5p and miR-155 suppress the proapoptotic gene TP53INP1 as their target. The results of this study indicate that overexpression of miR-142-5p and miR-155 plays a critical role in the pathogenesis of gastric MALT lymphoma. These miRNAs might have potential application as therapeutic targets and novel biomarkers for gastric MALT lymphoma.     

Epstein-Barr virus small RNAs potentiate tumorigenicity of Burkitt lymphoma cells independently of an effect on apoptosis

The tumorigenic potential of the Burkitt lymphoma (BL) cell line Akata is dependent on the restricted latency program of Epstein-Barr virus (EBV) that is characteristically maintained in BL tumors. Within these cells, EBV-mediated inhibition of apoptosis correlates with an up-regulation of BCL-2 levels in concert with a down-regulation in c-MYC expression that occurs under growth-limiting conditions. Here we addressed whether EBV's effects on apoptosis and tumorigenicity are mediated by the EBV small RNAs EBER-1 and EBER-2. Stable expression of the EBERs in EBV-negative Akata BL cells, at levels comparable to those in EBV-positive cells, significantly enhanced the tumorigenic potential of EBV-negative BL cells in SCID mice, but did not fully restore tumorigenicity relative to EBV-positive Akata cells. Furthermore, wild-type or greater levels of EBER expression in EBV-negative Akata cells did not promote BL cell survival. These data therefore suggest that EBV can contribute to BL through at least two avenues: an EBER-dependent mechanism that enhances tumorigenic potential independent of a direct effect on apoptosis, and a second mechanism, mediated by an as-yet-unidentified EBV gene(s), that offsets the proapoptotic consequences of deregulated c-MYC in BL.     

Epstein-Barr virus-associated lymphoproliferative disorders

Epstein-Barr virus (EBV) is a ubiquitous member of the herpesvirus family that is associated with a variety of lymphomas and lymphoproliferative diseases. It encodes a multitude of genes that drive proliferation or confer resistance to cell death. Among these are two key viral proteins which mimic the effects of the activated cellular signaling proteins. EBV-associated lymphomas include Burkitt's lymphoma; natural killer (NK)/T-cell lymphoma, lymphoma and lymphoproliferative diseases in immunocompromized populations, and Hodgkin's lymphoma. The character of the viral association differs among these entities with some consistently associated with EBV in all populations and all parts of the world, and others associated with the virus only in particular circumstances. An example of the former is nasal NK/T-cell lymphoma, while an example of the latter is Burkitt's lymphoma. The pattern of viral gene expression also varies among tumor types with different viral genes playing key roles in different tumors and conferring sensitivity to immune surveillance. Thus some of the post-transplant lymphoproliferative diseases are exquisitely sensitive to CD8 T-cell immunosurveillance, while other tumors such as Burkitt's lymphoma may be nearly impervious to such surveillance. Knowledge of the EBV association is not only important for understanding the pathogenesis of these tumors, but is increasingly important for diagnosis, monitoring and treatment.     

Epstein-Barr Virus Contributes to the Malignant Phenotype and to Apoptosis Resistance in Burkitt’s Lymphoma Cell Line Akata

In the present study, we established an in vitro system representing the Burkitt’s lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.     

An Epstein-Barr Virus (EBV) mutant with enhanced BZLF1 expression causes lymphomas with abortive lytic EBV infection in a humanized mouse model

Immunosuppressed patients are at risk for developing Epstein-Barr Virus (EBV)-positive lymphomas that express the major EBV oncoprotein, LMP1. Although increasing evidence suggests that a small number of lytically infected cells may promote EBV-positive lymphomas, the impact of enhanced lytic gene expression on the ability of EBV to induce lymphomas is unclear. Here we have used immune-deficient mice, engrafted with human fetal hematopoietic stem cells and thymus and liver tissue, to compare lymphoma formation following infection with wild-type (WT) EBV versus infection with a "superlytic" (SL) mutant with enhanced BZLF1 (Z) expression. The same proportions (2/6) of the WT and SL virus-infected animals developed B-cell lymphomas by day 60 postinfection; the remainder of the animals had persistent tumor-free viral latency. In contrast, all WT and SL virus-infected animals treated with the OKT3 anti-CD3 antibody (which inhibits T-cell function) developed lymphomas by day 29. Lymphomas in OKT3-treated animals (in contrast to lymphomas in the untreated animals) contained many LMP1-expressing cells. The SL virus-infected lymphomas in both OKT3-treated and untreated animals contained many more Z-expressing cells (up to 30%) than the WT virus-infected lymphomas, but did not express late viral proteins and thus had an abortive lytic form of EBV infection. LMP1 and BMRF1 (an early lytic viral protein) were never coexpressed in the same cell, suggesting that LMP1 expression is incompatible with lytic viral reactivation. These results show that the SL mutant induces an "abortive" lytic infection in humanized mice that is compatible with continued cell growth and at least partially resistant to T-cell killing.     

Induction of lytic Epstein-Barr virus (EBV) infection by synergistic action of rituximab and dexamethasone renders EBV-positive lymphoma cells more susceptible to ganciclovir cytotoxicity in vitro and in vivo

The purposeful induction of the lytic form of Epstein-Barr virus (EBV) infection combined with ganciclovir (GCV) treatment has been advocated as a novel strategy for EBV-positive B-cell lymphoma. We demonstrated that rituximab had a synergistic effect with dexamethasone on induction of the lytic EBV infection in CD20-positive lymphoma cells. Addition of GCV to the dexamethasone/rituximab-treated cells was more effective than dexamethasone/rituximab alone in killing EBV-positive lymphoma cells in vitro and in lymphoma-bearing nude mice but not in EBV-negative cells. These data suggest that induction of the lytic EBV infection with dexamethasone/rituximab in combination with GCV could be a potential virally targeted therapy for EBV-associated B-cell lymphoma.     

Epstein-barr virus regulates c-MYC, apoptosis, and tumorigenicity in Burkitt lymphoma

Loss of the Epstein-Barr virus (EBV) genome from Akata Burkitt lymphoma (BL) cells is coincident with a loss of malignant phenotype, despite the fact that Akata and other EBV-positive BL cells express a restricted set of EBV gene products (type I latency) that are not known to overtly affect cell growth. Here we demonstrate that reestablishment of type I latency in EBV-negative Akata cells restores tumorigenicity and that tumorigenic potential correlates with an increased resistance to apoptosis under growth-limiting conditions. The antiapoptotic effect of EBV was associated with a higher level of Bcl-2 expression and an EBV-dependent decrease in steady-state levels of c-MYC protein. Although the EBV EBNA-1 protein is expressed in all EBV-associated tumors and is reported to have oncogenic potential, enforced expression of EBNA-1 alone in EBV-negative Akata cells failed to restore tumorigenicity or EBV-dependent down-regulation of c-MYC. These data provide direct evidence that EBV contributes to the tumorigenic potential of Burkitt lymphoma and suggest a novel model whereby a restricted latency program of EBV promotes B-cell survival, and thus virus persistence within an immune host, by selectively targeting the expression of c-MYC.     

The Epstein-Barr virus (EBV)-induced tumor suppressor microRNA MiR-34a is growth promoting in EBV-infected B cells

Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposi's sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts.     

Alterations of the p53 gene in Epstein-Barr virus-associated immunodeficiency-related lymphomas.

Mutations of the p53 tumor suppressor gene are among the most common genetic alterations found in many different human malignancies, including those of the colon, lung, and breast. Alterations in wild-type p53 lead to loss of the suppressor function and thus contribute to tumorigenesis. The potential role of p53 mutations in a sampling of B-cell lymphomas, the majority of which were associated with Epstein-Barr virus (EBV), was investigated. Twenty-six biopsy specimens from immunocompromised patients, including allograft recipients and patients with AIDS, Wiscott-Aldrich syndrome, and human T-cell leukemia virus type 1 infection, in comparison with three Burkitt lymphomas and four Burkitt lymphoma cell lines were analyzed. Mutation in p53 was detected in all four Burkitt lymphoma cell lines as well as the three Burkitt lymphoma biopsy specimens. In patients with AIDS, 5 of 10 lymphomas were EBV positive, and 1 had a mutation in p53. Mutation in p53 was not detected in 14 EBV-positive lymphomas which arose in transplant recipients. These data indicate that with the exception of Burkitt lymphomas, p53 mutations are not involved in the majority EBV-positive B-cell lymphomas which develop in immunocompromised patients.     

Differential tumorigenicity between Epstein-Barr virus genome-positive and genome-negative cell lines with t(11;14)(q13;q32) derived from mantle cell lymphoma.

Epstein-Barr virus (EBV) genome has been detected in several human lymphoproliferative diseases, but the oncogenic function of EBV is not fully understood. We previously established EBV-positive (SP-50B) and EBV-negative (SP-53) cell lines with the t(11;14)(q13;q32) chromosome abnormality from a single patient with mantle cell lymphoma. Monoclonal EBV DNA in a circular episomal form was demonstrated in the SP-50B cells by Southern blot hybridization with the EBV-terminal fragment probe. SP-50B cells were positive for not only EBV-encoded nuclear antigen-1 (EBNA1) but also latent membrane protein-1 and EBNA2. None of the EBV-encoded proteins was expressed in SP-53 cells. The isogenic EBV-infected and EBV-free cell lines of neoplastic clones made it possible to examine a tumorigenic role of EBV. Only EBV-positive SP-50B cells possessed malignant phenotypes, such as growth ability in low serum, colony formation in soft agarose, and tumorigenicity in nude mice. On the other hand, a lymphoblastoid B-cell line established by infecting the patient's normal B lymphocytes in vitro with exogenous EBV had no tumorigenicity. These results suggested that EBV infection, if it occurred in neoplastic lymphoma cells, could play a role in acquisition of malignant phenotypes.     

Hexose transport in Epstein-Barr virus (EBV) negative lymphoma lines and their EBV converted, virus genome carrying sublines

Increased hexose uptake is a marker for viral transformation, as has been shown in non-human fibroblasts transformed by oncogenic viruses. If this phenomenon is a general expression of viral induced transformation it should also apply on different oncogenic virus-cell systems. Recently two human EBV-negative lymphoma lines were converted to a stable EBV-positive state by infection with EBV. According to their biochemical and biological properties they enable us to study events associated with EBV-transformation. We analysed the uptake of (3H) glucosamine and (3H) 2-deoxy-D-glucose into BJAB and Ramos and their EBV-converted sublines and found a clear increase of the rate of uptake of both sugars in the EBV-positive sublines. Control experiments confirmed that the increased uptake was due to alterations on the level of the hexose membrane carriers and not due to increased metabolism. The observation of increased hexose uptake in the only presented available virus transformed human cell system is a strong argument for the general importance of this transformation-associated membrane change.     

Concomitant reduction of c-Myc expression and PI3K/AKT/mTOR signaling by quercetin induces a strong cytotoxic effect against Burkitt’s lymphoma

Burkitt’s lymphoma is an aggressive B cell lymphoma whose pathogenesis involves mainly c-Myc translocation and hyperexpression, in addition to antigen-independent BCR signaling and, in some cases, EBV infection. As result of BCR signaling activation, the PI3K/AKT/mTOR pathway results constitutively activated also in the absence of EBV, promoting cell survival and counterbalancing the pro-apoptotic function that c-Myc may also exert. In this study we found that quercetin, a bioflavonoid widely distributed in plant kingdom, reduced c-Myc expression and inhibited the PI3K/AKT/mTOR activity in BL, leading to an apoptotic cell death. We observed a higher cytotoxic effect against the EBV-negative BL cells in comparison with the positive ones, suggesting that this oncogenic gammaherpesvirus confers an additional resistance to the quercetin treatment. Besides cell survival, PI3K/AKT/mTOR pathway also regulates autophagy: we found that quercetin induced a complete autophagic flux in BL cells, that contributes to c-Myc reduction in some of these cells. Indeed, autophagy inhibition by chloroquine partially restored c-Myc expression in EBV-positive (Akata) and EBV-negative (2A8) cells that harbor c-Myc mutation. Interestingly, chloroquine did not affect the quercetin-mediated reduction of c-Myc expression in Ramos cells, that have no c-Myc mutation in the coding region, although autophagy was induced.These results suggest that mutant c-Myc could be partially degraded through autophagy in BL cells, as previously reported for other mutant oncogenic proteins.     

The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

MicroRNAs (miRNAs) are a family of small RNA molecules that negatively regulate the expression of protein-coding genes and play critical roles in orchestrating diverse cellular processes. This regulatory mechanism is also exploited by viruses to direct their life cycle and evade the host immune system. Epstein-Barr virus (EBV) is an oncogenic virus that is closely associated with multiple human diseases, including nasopharyngeal carcinoma (NPC), which is a highly metastatic type of tumor and is frequently reported in South Asia. Several viral proteins have been found to promote the migration and invasiveness of NPC cells. However, not all tumor tissues express these viral oncoproteins, suggesting that other mechanisms may contribute to the aggressive behavior of NPC tumor cells. A previous sequencing study by our group revealed that the EBV miRNA miR-BART9 was expressed at high levels in all EBV-positive NPC tissues. In the present study, we used gain- and loss-of-function approaches to investigate the effect of miR-BART9 in EBV-negative and EBV-positive NPC cells. We discovered that miR-BART9 promotes the migration and invasiveness of cultured NPC cells. The promigratory activity observed in vitro was manifested as an enhanced metastatic ability in vivo. Computational analysis revealed that miR-BART9 may target E-cadherin, a membrane protein that is pivotal in preserving cell-cell junctions and the epithelial phenotype. Through biochemical assays and functional rescue analysis, we confirmed that miR-BART9 specifically inhibits E-cadherin to induce a mesenchymal-like phenotype and promote the migration of NPC cells. These results indicated that miR-BART9 is a prometastatic viral miRNA and suggested that high levels of miR-BART9 in EBV-positive NPC cells may contribute to the aggressiveness of tumor cells.     

Constitutive autophagy contributes to resistance to TP53-mediated apoptosis in Epstein-Barr virus-positive latency III B-cell lymphoproliferations

The Epstein-Barr virus (EBV) is associated with various lymphoproliferative disorders and lymphomas. We have previously demonstrated that treating wild-type TP53-expressing B cell lines with the TP53 pathway activator nutlin-3 induced apoptosis in EBV-negative and EBV-positive latency I cells whereas EBV-positive latency III cells remained much more apoptosis-resistant. Here, we report a constitutively high level of autophagy in these resistant cells which express high levels of the proautophagic protein BECN1/Beclin 1 based, at least in part, on the activation of the NFKB signaling pathway by the viral protein LMP1. Following treatment with nutlin-3, several autophagy-stimulating genes were upregulated both in EBV-negative and EBV-positive latency III cells. However the process of autophagy was only triggered in the latter and was associated with an upregulation of SESN1/sestrin 1 and inhibition of MTOR more rapid than in EBV-negative cells. A treatment with chloroquine, an inhibitor of autophagy, potentiated the apoptotic effect of nutlin-3, particularly in those EBV-positive cells which were resistant to apoptosis induced by nutlin-3 alone, thereby showing that autophagy participates in this resistant phenotype. Finally, using immunohistochemical staining, clinical samples from various B cell lymphoproliferations with the EBV-positive latency II or III phenotype were found to harbor a constitutively active autophagy.