Eleni Anastasiadou

Differential regulation of miR-21 and miR-146a by Epstein–Barr virus-encoded EBNA2

The discovery of microRNA (miR) represents a novel paradigm in RNA-based regulation of gene expression and their dysregulation has become a hallmark of many a tumor. In virally associated cancers, the host–pathogen interaction could involve alteration in miR expression. Epstein–Barr virus (EBV)-encoded EBNA2 is indispensable for the capacity of the virus to transform B cells in vitro. Here, we studied how it affects cellular miRs. Extensive miR profiling of the virus-infected and EBNA2-transfected B lymphoma cells revealed that oncomiR miR-21 is positively regulated by this viral protein. Conversely, Burkitt’s lymphoma (BL) cell lines infected with EBNA2 lacking P3HR1 strain did not show any increase in miR-21. EBNA2 increased phosphorylation of AKT and this was directly correlated with increased miR-21. In contrast, miR-146a was downregulated by EBNA2 in B lymphoma cells. Low miR-146a expression correlates with an elevated level of IRAK1 and type I interferon in EBNA2 transfectants. Taken together, the present data suggest that EBNA2 might contribute to EBV-induced B-cell transformation by altering miR expression and in particular by increasing oncomiR-like miR-21 and by affecting the antiviral responses of the innate immune system through downregulation of its key regulator miR-146a.

Epstein–Barr virus encoded LMP1 downregulates TCL1 oncogene through miR-29b

Epstein–Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is noted for its transforming potential. Yet, it also acts as a cytostatic and growth-relenting factor in Burkitts lymphoma (BL) cells. The underlying molecular mechanisms of the growth inhibitory property of LMP1 have remained largely unknown. In this study, we show that LMP1 negatively regulates a major oncogene, TCL1, in diffuse large B-cell lymphoma (DLBCL) and BL cells. MicroRNA (miR) profiling of LMP1 transfectants showed that among others, miR-29b, is upregulated. LMP1 diminished TCL1 by inducing miR-29b through C-terminus activation region 1 (CTAR1) and CTAR2. miR-29b locked nucleic acid (LNA) antisense oligonucleotide transfection into LMP1-expressing cells reduced miR-29b expression and consequently reconstituted TCL1, suggesting that LMP1 negatively regulates TCL1 through miR-29b upregulation. The miR-29b increase by LMP1 was due to an increase in the cluster pri-miR-29b1-a transcription, derived from human chromosome 7. Using pharmacological inhibitors, we found that p38 mitogen-activated protein kinase-activating function of LMP1 is important for this effect. The ability of LMP1 to negatively regulate TCL1 through miR-29b might underlie its B-cell lymphoma growth antagonistic property. As LMP1 is also important for B-cell transformation, we suggest that the functional dichotomy of this viral protein may depend on a combination of levels of its expression, lineage and differentiation of the target cells and regulation of miRs, which then directs the outcome of the cellular response.

EBV stimulates TLR- and autophagy-dependent pathways and impairs maturation in plasmacytoid dendritic cells: implications for viral immune escape.

Plasmacytoid DCs (pDCs) are crucial mediators in the establishment of immunity against most viruses, given their extraordinary capacity to produce a massive quantity of type I IFN. In this study we investigate the response of pDCs to infection with EBV, a γ‐herpes virus that persists with an asymptomatic infection in immunocompetent hosts, although in certain conditions it can promote development of cancers or autoimmune diseases. We show that high amounts of type I IFNs were released from isolated pDCs after exposure to EBV by a mechanism requiring TLRs and a functional autophagic machinery. We next demonstrate that EBV can infect pDCs via viral binding to MHC class II molecule HLA‐DR and that pDCs express EBV‐induced latency genes. Furthermore, we observe that EBV is able to induce activation but not maturation of pDCs, which correlates with an impaired TNF‐α release. Accordingly, EBV‐infected pDCs are unable to mount a full T‐cell response, suggesting that impaired pDC maturation, combined with a concomitant EBV‐mediated upregulation of the T‐cell inhibitory molecules B7‐H1 and ICOS‐L, could represent an immune‐evasion strategy promoted by the virus. These mechanisms might lead to persistence in immunocompetent hosts or to dysregulated immune responses linked to EBV‐associated diseases.

Cancer. Malicious exosomes.

Nanovesicles derived from cells of cancer patients carry microRNAs that initiate tumor growth in normal cells Nanovesicles known as exosomes are secreted from a variety of cell types and circulate in biological fluids such as urine and plasma. These exosomes “hijack” membrane components and cytoplasmic contents of these cells and play an important role in intercellular communication, often inducing physiological changes in recipient cells by transferring bioactive lipids, nucleic acids, and proteins (1). These tiny vesicles also have been implicated in a number of human diseases, including cancer, and are becoming an appreciated fundamental aspect of tumor progression and metastasis (2). Recently, Melo et al. (3) showed that exosomes from breast cancer cells transfer microRNAs (miRNAs) to normal cells and stimulate them to become cancerous. This potentially expands the mechanisms by which cancer spreads and may provide opportunities to develop exosome-based diagnostics and therapies.

EBNA2 interferes with the germinal center phenotype by downregulating BCL6 and TCL1 in non-hodgkin's lymphoma cells

ABSTRACT Epstein-Barr virus (EBV)-negative diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma-derived cell lines infected in vitro with a recombinant EBV expressed type II/III latency. High expression of EBNA2 inversely correlated with expression of germinal center (GC)-associated genes, BCL6 and TCL1. The decreased expression of BCL6 appeared to be dose dependent, with almost complete abrogation in highly EBNA2-expressing clones. The role of EBNA2 in negative regulation of these genes was confirmed by transfection and in a hormone-inducible EBNA2 cell system. LMP1 transfection reduced expression of TCL1, but not of BCL6, in DLBCLs. The GC-associated gene repression was at the transcriptional level and CBF1 independent. A decrease in HLA-DR, surface immunoglobulin M, and class II transactivator expression and an increase in CCL3, a BCL6 repression target, was observed in EBNA2-expressing clones. Since BCL6 is indispensable for GC formation and somatic hypermutations (SHM), we suggest that the previously reported lack of SHM seen in EBNA2-expressing GC cells from infectious mononucleosis tonsils could be due to negative regulation of BCL6 by EBNA2. These findings suggest that EBNA2 interferes with the GC phenotype.

Epstein-Barr virus infection leads to partial phenotypic reversion of terminally differentiated malignant B cells.

The B cell lymphomas associated with Epstein-Barr virus (EBV) are not limited to any specific stage of B cell differentiation but covers widely different B cell phenotypes. In vitro infection of the virus negative tumors with a recombinant EBV strain has provided important insights into virus-tumor interaction. Here, we investigated the interaction between EBV and terminally differentiated tumor derived B cells, namely multiple myeloma (MM). The in vitro EBV infected MM expressed restricted viral latency. Acquisition of the virus was accompanied by a partial reprogramming to a mature B cell phenotype. Thus, the plasma cell markers syndecan-1 (CD138), Blimp1 and MUM1 were downregulated, while expression of HLADR, CIITA and TCL1, which are normally not expressed in plasmacytoid cells, was upregulated. The silenced transcription factor gene encoding Pax5 and its target BLNK were activated. Significantly, the free lambda light chains secreted in the medium were reduced in EBV infected MM clones. Collectively, these results suggest that the restricted EBV latency can cause at least partial phenotypic reversion of terminally differentiated B tumor cells. We suggest that the restricted EBV latent gene expression may not only be the consequence but the cause of the mature B cell phenotype, actively participating in the virus persistence.

Epstein‐Barr virus infection induces miR‐21 in terminally differentiated malignant B cells

The association of Epstein‐Barr virus (EBV) with plasmacytoid malignancies is now well established but how the virus influences microRNA expression in such cells is not known. We have used multiple myeloma (MM) cell lines to address this issue and find that an oncomiR, miR‐21 is induced after in vitro EBV infection. The PU.1 binding site in miR‐21 promoter was essential for its activation by the virus. In accordance with its noted oncogenic functions, miR‐21 induction in EBV infected MM cells caused downregulation of p21 and an increase in cyclin D3 expression. EBV infected MM cells were highly tumorigenic in SCID mice. Given the importance of miR‐21 in plasmacytoid malignancies, our findings that EBV could further exacerbate the disease by inducing miR‐21 has interesting implications both in terms of diagnosis and future miR based therapeutical approaches for the virus associated plasmacytoid tumors.

MUC1 inhibition leads to decrease in PD-L1 levels via upregulation of miRNAs

The PD-L1/PD-1 pathway is a critical component of the immunosuppressive tumor microenvironment in acute myeloid leukemia (AML), but little is known about its regulation. We investigated the role of the MUC1 oncoprotein in modulating PD-L1 expression in AML. Silencing of MUC1 in AML cell lines suppressed PD-L1 expression without a decrease in PD-L1 mRNA levels, suggesting a post-transcriptional mechanism of regulation. We identified the microRNAs miR-200c and miR-34a as key regulators of PD-L1 expression in AML. Silencing of MUC1 in AML cells led to a marked increase in miR-200c and miR-34a levels, without changes in precursor microRNA, suggesting that MUC1 might regulate microRNA-processing. MUC1 signaling decreased the expression of the microRNA-processing protein DICER, via the suppression of c-Jun activity. NanoString (Seattle, WA, USA) array of MUC1-silenced AML cells demonstrated an increase in the majority of probed microRNAs. In an immunocompetent murine AML model, targeting of MUC1 led to a significant increase in leukemia-specific T cells. In concert, targeting MUC1 signaling in human AML cells resulted in enhanced sensitivity to T-cell-mediated lysis. These findings suggest MUC1 is a critical regulator of PD-L1 expression via its effects on microRNA levels and represents a potential therapeutic target to enhance anti-tumor immunity.

Immune and Epstein-Barr virus gene expression in cerebrospinal fluid and peripheral blood mononuclear cells from patients with relapsing-remitting multiple sclerosis

BackgroundGene expression analyses in paired cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) from patients with multiple sclerosis (MS) are restrained by the low RNA amounts from CSF cells and low expression levels of certain genes. Here, we applied a Taqman-based pre-amplification real-time reverse-transcription polymerase chain reaction (RT-PCR) (PreAmp RT-PCR) to cDNA from CSF cells and PBMC of MS patients and analyzed multiple genes related to immune system function and genes expressed by Epstein-Barr virus (EBV), a herpesvirus showing strong association with MS. Using this enhanced RT-PCR method, we aimed at the following: (1) identifying gene signatures potentially useful for patient stratification, (2) understanding whether EBV infection is perturbed in CSF and/or blood, and (3) finding a link between immune and EBV infection status.MethodsThirty-one therapy-free patients with relapsing-remitting MS were included in the study. Paired CSF cells and PBMC were collected and expression of 41 immune-related cellular genes and 7 EBV genes associated with latent or lytic viral infection were determined by PreAmp RT-PCR. Clinical, radiological, CSF, and gene expression data were analyzed using univariate and multivariate (cluster analysis, factor analysis) statistical approaches.ResultsSeveral immune-related genes were differentially expressed between CSF cells and PBMC from the whole MS cohort. By univariate analysis, no or only minor differences in gene expression were found associated with sex, clinical, or radiological condition. Cluster analysis on CSF gene expression data grouped patients into three clusters; clusters 1 and 2 differed by expression of genes that are related mainly to innate immunity, irrespective of sex and disease characteristics. By factor analysis, two factors grouping genes involved in antiviral immunity and immune regulation, respectively, accurately discriminated cluster 1 and cluster 2 patients. Despite the use of an enhanced RT-PCR method, EBV transcripts were detected in a minority of patients (5 of 31), with evidence of viral latency activation in CSF cells or PBMC and of lytic infection in one patient with active disease only.ConclusionsAnalysis of multiple cellular and EBV genes in paired CSF cell and PBMC samples using PreAmp RT-PCR may yield new information on the complex interplay between biological processes underlying MS and help in biomarker identification.

Epstein-Barr virus (EBV) positive classical Hodgkin lymphoma of Iraqi children: an immunophenotypic and molecular characterization of Hodgkin/Reed-Sternberg cells.

Classical Hodgkin lymphoma (cHL) in children is often associated with EBV infection, more commonly in developing countries.