LMP-1 Research Articles

EBV‑LMP1 is involved in vasculogenic mimicry formation via VEGFA/VEGFR1 signaling in nasopharyngeal carcinoma.

The Epstein-Barr virus latent membrane protein1 (EBV‑LMP1) is an oncoviral protein that plays an important role in oncogenic transformation in EBV‑associated nasopharyngeal carcinoma (NPC). Our previous studies demonstrated that LMP1 increased VEGFA expression and upregulated angiogenesis in NPC. Vasculogenic mimicry(VM) is a mechanism by which tumor cells can obtain nutrients to survive, and VM has been observed in numerous types of tumors. However, the occurrence and significance of VM in NPC and the relationship between LMP1 and VM have not yet been evaluated. In the present study, we observed that it was almost impossible for LMP1-negative NPC cells to form tubular structures, whereas LMP1-positive NPC cells were able to form tubular structures. Moreover, VEGFA was found to be involved in VM formation in LMP1-positive NPC cells. Knockdown of LMP1 or VEGFR1 distinctly disrupted tubular structures, whereas inhibition of VEGFR2 did not affect the process, indicating that VEGFR1 not VEGFR2 signaling was involved in LMP1-mediated VM formation. Furthermore, the data of immunohistochemistry (IHC) and CD34/PAS double staining in a tumor tissue array showed that LMP1 was positively correlated with VEGFR1 and VM. Meanwhile, after analyzing the clinicopathological features, we found that VM formation was associated with a poor prognosis in NPC patients. These results suggest that VM formation is increased by EBV‑LMP1 via VEGF/VEGFR1 signaling and provide additional information to clarify the role of EBV‑LMP1 in NPC pathophysiology.

Role of microRNAs and Exosomes in Helicobacter pylori and Epstein-Barr Virus Associated Gastric Cancers.

Emerging evidence suggests that chronic inflammation caused by pathogen infection is connected to the development of various types of cancer. It is estimated that up to 20% of all cancer deaths is linked to infections and inflammation. In gastric cancer, such triggers can be infection of the gastric epithelium by either Helicobacter pylori (H. pylori), a bacterium present in half of the world population; or by Epstein-Barr virus (EBV), a double-stranded DNA virus which has recently been associated with gastric cancer. Both agents can establish lifelong inflammation by evolving to escape immune surveillance and, under certain conditions, contribute to the development of gastric cancer. Non-coding RNAs, mainly microRNAs (miRNAs), influence the host innate and adaptive immune responses, though long non-coding RNAs and viral miRNAs also alter these processes. Reports suggest that chronic infection results in altered expression of host miRNAs. In turn, dysregulated miRNAs modulate the host inflammatory immune response, favoring bacterial survival and persistence within the gastric mucosa. Given the established roles of miRNAs in tumorigenesis and innate immunity, they may serve as an important link between H. pylori- and EBV-associated inflammation and carcinogenesis. Example of this is up-regulation of miR-155 in H. pylori and EBV infection. The tumor environment contains a variety of cells that need to communicate with each other. Extracellular vesicles, especially exosomes, allow these cells to deliver certain type of information to other cells promoting cancer growth and metastasis. Exosomes have been shown to deliver not only various types of genetic information, mainly miRNAs, but also cytotoxin-associated gene A (CagA), a major H. pylori virulence factor. In addition, a growing body of evidence demonstrates that exosomes contain genetic material of viruses and viral miRNAs and proteins such as EBV latent membrane protein 1 (LMP1) which are delivered into recipient cells. In this review, we focus on the dysregulated H. pylori- and EBV-associated miRNAs while trying to unveil possible causal mechanisms. Moreover, we discuss the role of exosomes as vehicles for miRNA delivery in H. pylori- and EBV-related carcinogenesis.

C-Myc Represses Transcription of Epstein-Barr Virus Latent Membrane Protein 1 Early after Primary B Cell Infection.

Recent evidence has shown that the Epstein-Barr virus (EBV) oncogene LMP1 is not expressed at high levels early after EBV infection of primary B cells, despite its being essential for the long-term outgrowth of immortalized lymphoblastoid cell lines (LCLs). In this study, we found that expression of LMP1 increased 50-fold between 7 days postinfection and the LCL state. Metabolic labeling of nascent transcribed mRNA indicated that this was primarily a transcription-mediated event. EBNA2, the key viral transcription factor regulating LMP1, and CTCF, an important chromatin insulator, were recruited to the LMP1 locus similarly early and late after infection. However, the activating histone H3K9Ac mark was enriched at the LMP1 promoter in LCLs relative to that in infected B cells early after infection. We found that high c-Myc activity in EBV-infected lymphoma cells as well as overexpression of c-Myc in an LCL model system repressed LMP1 transcription. Finally, we found that chemical inhibition of c-Myc both in LCLs and early after primary B cell infection increased LMP1 expression. These data support a model in which high levels of endogenous c-Myc activity induced early after primary B cell infection directly repress LMP1 transcription.IMPORTANCE EBV is a highly successful pathogen that latently infects more than 90% of adults worldwide and is also causally associated with a number of B cell malignancies. During the latent life cycle, EBV expresses a set of viral oncoproteins and noncoding RNAs with the potential to promote cancer. Critical among these is the viral latent membrane protein LMP1. Prior work suggests that LMP1 is essential for EBV to immortalize B cells, but our recent work indicates that LMP1 is not produced at high levels during the first few weeks after infection. Here we show that transcription of the LMP1 gene can be negatively regulated by a host transcription factor, c-Myc. Ultimately, understanding the regulation of EBV oncogenes will allow us to better treat cancers that rely on these viral products for survival.

CD40L-adjuvanted DNA vaccine carrying EBV-LMP2 antigen enhances anti-tumor effect in NPC transplantation tumor animal.

CD40L, a costimulatory molecule for dendritic cells (DCs) and B cells, can serve as an adjuvant for enhancing the specific immune response induced by DNA vaccine carrying tumor-associated antigens. In this study, we investigated the potential of CD40L as an adjuvant to enhance the anti-tumor effect mediated by a DNA vaccine based on the Epstein-Barr virus-latent membrane protein 2 (EBV-LMP2) antigen. The plasmids capable of expressing the fusion protein EBV-LMP2-CD40L were constructed. Expression vector pVAX1 and plasmid expressing the individual antigen EBV-LMP2 were used as control groups. These plasmids were used to immunize female BALB/c mice (4-6 weeks old) at days 0, 7 and 14. The results suggest that immunization with DNA vaccines carrying fusion gene EBV-LMP2-CD40L can induce specific immunity more effectively than the plasmid expression individual antigen EBV-LMP2. In order to evaluate the anti-tumor effect of this DNA vaccine, we constructed a tumor bearing mouse model. After immunization, the tumor bearing mouse model, DNA vaccination with EBV-LMP2-CD40L plasmid significantly inhibited tumor growth in the tumor bearing mouse model and enhanced the tumor inhibition rate. This study demonstrated that encoding the EBV-LMP2 tumor antigen within an EBV-LMP2-CD40L DNA vaccine generates an effective antitumor response against EBV tumor, which may be a promising method to improve the antitumor immunity of DNA vaccine.

LMP1-Positive Composite Tumour of Larynx: A Diagnostic Quandary

Malignant spindle cell tumours of the larynx are rare, of which the most common are Spindle Cell Squamous Carcinomas (SCSCs). Sarcomas are extremely infrequent in the larynx. Composite tumours, further, are quite unheard of, except for rare case reports. This case report describes a spindle cell malignancy of the larynx in an elderly male, which posed a diagnostic difficulty due to its unique morphological and immunohistochemical features. The tumour was superficial, polypoidal with the proliferation of malignant spindle cells and abundant mitoses under an ulcerated epithelium. Occasional squamous cell rests with keratin pearls were present. Spindle cells were strongly immunoreactive for vimentin, smooth muscle actin and squamoid cells were p63 positive. Desmin was negative. The dilemma in diagnosis was of a SCSC with smooth muscle differentiation against a composite tumour. The tumour also turned out to be positive for Epstein Barr Virus Latent Membrane Protein-1 (EBV-LMP1), establishing a noteworthy association.

LIMD1 is induced by and required for LMP1 signaling, and protects EBV-transformed cells from DNA damage-induced cell death.

LIMD1 (LIM domain-containing protein 1) is considered as a tumor suppressor, being deregulated in many cancers to include hematological malignancies; however, very little is known about the underlying mechanisms of its deregulation and its roles in carcinogenesis. Epstein-Barr Virus (EBV) is associated with a panel of malignancies of lymphocytic and epithelial origin. Using high throughput expression profiling, we have previously identified LIMD1 as a common marker associated with the oncogenic transcription factor IRF4 in EBV-related lymphomas and other hematological malignancies. In this study, we have identified potential conserved IRF4- and NFκB-binding motifs in the LIMD1 gene promoter, and both are demonstrated functional by promoter-reporter assays. We further show that LIMD1 is partially upregulated by EBV latent membrane protein 1 (LMP1) via IRF4 and NFκB in EBV latency. As to its role in the setting of EBV latent infection, we show that LIMD1 interacts with TRAF6, a crucial mediator of LMP1 signal transduction. Importantly, LIMD1 depletion impairs LMP1 signaling and functions, potentiates ionomycin-induced DNA damage and apoptosis, and inhibits p62-mediated selective autophagy. Taken together, these results show that LIMD1 is upregulated in EBV latency and plays an oncogenic role rather than that of a tumor suppressor. Our findings have identified LIMD1 as a novel player in EBV latency and oncogenesis, and open a novel research avenue, in which LIMD1 and p62 play crucial roles in linking DNA damage response (DDR), apoptosis, and autophagy and their potential interplay during viral oncogenesis.

N-acetylcysteine (NAC) ameliorates Epstein-Barr virus latent membrane protein 1 induced chronic inflammation.

Chronic inflammation results when the immune system responds to trauma, injury or infection and the response is not resolved. It can lead to tissue damage and dysfunction and in some cases predispose to cancer. Some viruses (including Epstein-Barr virus (EBV)) can induce inflammation, which may persist even after the infection has been controlled or cleared. The damage caused by inflammation, can itself act to perpetuate the inflammatory response. The latent membrane protein 1 (LMP1) of EBV is a pro-inflammatory factor and in the skin of transgenic mice causes a phenotype of hyperplasia with chronic inflammation of increasing severity, which can progress to pre-malignant and malignant lesions. LMP1 signalling leads to persistent deregulated expression of multiple proteins throughout the mouse life span, including TGFα S100A9 and chitinase-like proteins. Additionally, as the inflammation increases, numerous chemokines and cytokines are produced which promulgate the inflammation. Deposition of IgM, IgG, IgA and IgE and complement activation form part of this process and through genetic deletion of CD40, we show that this contributes to the more tissue-destructive aspects of the phenotype. Treatment of the mice with N-acetylcysteine (NAC), an antioxidant which feeds into the body’s natural redox regulatory system through glutathione synthesis, resulted in a significantly reduced leukocyte infiltrate in the inflamed tissue, amelioration of the pathological features and delay in the inflammatory signature measured by in vivo imaging. Reducing the degree of inflammation achieved through NAC treatment, had the knock on effect of reducing leukocyte recruitment to the inflamed site, thereby slowing the progression of the pathology. These data support the idea that NAC could be considered as a treatment to alleviate chronic inflammatory pathologies, including post-viral disease. Additionally, the model described can be used to effectively monitor and accurately measure therapies for chronic inflammation.

Characterization of a Suppressive Cis-acting Element in the Epstein-Barr Virus LMP1 Promoter.

Latent membrane protein 1 (LMP1) is a major oncogene encoded by Epstein–Barr virus (EBV) and is essential for immortalization of B cells by the virus. Previous studies suggested that several transcription factors, such as PU.1, RBP-Jκ, NFκB, EBF1, AP-2 and STAT, are involved in LMP1 induction; however, the means by which the oncogene is negatively regulated remains unclear. Here, we introduced short mutations into the proximal LMP1 promoter that includes recognition sites for the E-box and Ikaros transcription factors in the context of EBV-bacterial artificial chromosome. Upon infection, the mutant exhibited increased LMP1 expression and EBV-mediated immortalization of B cells. However, single mutations of either the E-box or Ikaros sites had limited effects on LMP1 expression and transformation. Our results suggest that this region contains a suppressive cis-regulatory element, but other transcriptional repressors (apart from the E-box and Ikaros transcription factors) may remain to be discovered.

Target-induced proximity ligation triggers recombinase polymerase amplification and transcription-mediated amplification to detect tumor-derived exosomes in nasopharyngeal carcinoma with high sensitivity

Tumor-derived exosomes (TEXs) are extracellular vesicles that are continuously released into the blood by tumor cells and carry specific surface markers of the original tumor cells. Substantial evidence has implicated TEXs as attractive diagnostic markers for cancer. However, the detection of TEXs in blood at an early tumor stage is challenging due to their very low concentration. Here, we established a method called PLA-RPA-TMA assay that allows TEXs to be detected with high sensitivity and specificity. Based on two proximity ligation assay (PLA) probes that recognize a biomarker on a TEX, we generated a unique surrogate DNA signal for the specific biomarker, which was synchronously amplified twice by recombinase polymerase amplification (RPA) coupled with transcription-mediated amplification (TMA), and then the products of the RPA-TMA reaction were quantitatively detected using a gold nanoparticle-based colorimetric assay. We established proof-of-concept evidence for this approach using TEXs from nasopharyngeal carcinoma (NPC) cells, with a detection limit of 102 particles/mL, and reported the measurement of plasma Epstein-Barr virus latent membrane protein 1 (LPM1)-positive (LMP1+, accuracy: 0.956) and epidermal growth factor receptor (EGFR)-positive (EGFR+, accuracy: 0.906) TEXs as potent early diagnostic biomarkers for NPC.

LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma.

Myeloid-derived suppressor cells (MDSCs) are expanded in tumor microenvironments, including that of Epstein–Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC). The link between MDSC expansion and EBV infection in NPC is unclear. Here, we show that EBV latent membrane protein 1 (LMP1) promotes MDSC expansion in the tumor microenvironment by promoting extra-mitochondrial glycolysis in malignant cells, which is a scenario for immune escape initially suggested by the frequent, concomitant detection of abundant LMP1, glucose transporter 1 (GLUT1) and CD33+ MDSCs in tumor sections. The full process has been reconstituted in vitro. LMP1 promotes the expression of multiple glycolytic genes, including GLUT1. This metabolic reprogramming results in increased expression of the Nod-like receptor family protein 3 (NLRP3) inflammasome, COX-2 and P-p65 and, consequently, increased production of IL-1β, IL-6 and GM-CSF. Finally, these changes in the environment of malignant cells result in enhanced NPC-derived MDSC induction. One key step is the physical interaction of LMP1 with GLUT1 to stabilize the GLUT1 protein by blocking its K48-ubiquitination and p62-dependent autolysosomal degradation. This work indicates that LMP1-mediated glycolysis regulates IL-1β, IL-6 and GM-CSF production through the NLRP3 inflammasome, COX-2 and P-p65 signaling pathways to enhance tumor-associated MDSC expansion, which leads to tumor immunosuppression in NPC.

Expression of interferon regulatory factor 7 correlates with the expression of Epstein-Barr Virus latent membrane protein 1 and cervical lymph node metastasis in nasopharyngeal cancer.

Interferon regulatory factor 7 (IRF7) has oncogenic properties in several malignancies such as Epstein-Barr virus (EBV)-associated lymphoma. However, there is no evidence whether IRF7 is associated with the oncogenesis of nasopharyngeal cancer (NPC), the pathogenesis of which is closely associated with EBV. Herein, we report that expression of IRF7 was increased in normal nasopharyngeal cells that expressed the EBV principal oncoprotein, latent membrane protein 1 (LMP1). In addition, IRF7 was mainly expressed in the nucleus in both normal nasopharyngeal cells and nasopharyngeal cancer cells that expresses LMP1. On immunohistochemical analysis, IRF7 was predominantly localized in the nucleus in biopsy samples of NPC tissues. In total, IRF7 expression was detected with 36 of 49 specimens of these tissues. Furthermore, the expression score of IRF7 correlated with the expression score of LMP1. Moreover, the expression score of IRF7 is associated with cervical lymph-node metastasis, which reflects the highly metastatic nature of this cancer. Taken together, our results suggest that expression of IRF7 is one of the metastatic effectors of LMP1 signalling in EBV-associated NPC.

Exposure to the Epstein-Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo.

Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20–50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

Abstract 5756: Effects of Epstein-Barr virus latent membrane protein 1 (LMP1) on cell invasiveness and expression of endogenous microRNAs in human cells in vitro

Abstract Infection by the Epstein-Barr virus (EBV) is associated with a variety of human cancers, notably Burkitt lymphoma and nasopharyngeal carcinoma (NPC). Most malignant cells within EBV-associated cancers are latently infected, and some viral products expressed during the latent phase of the viral cycle have well-described carcinogenicity, including the EBV latent membrane protein 1 (LMP1). Distinct viral genotypes described so far include the B95.8, the most studied viral strain originally found in an elderly patient with transfusion-induced infectious mononucleosis, and the M81 strain, isolated from a NPC case from a Chinese patient. Compared to B95.8, the EBV M81 strain infects epithelial cells more readily, and it is more likely to induce spontaneous viral lytic reactivation. However, whether these EBV strains have distinct biological features relevant for the progression of EBV-associated cancers remains to be elucidated. Thus, this study aimed to evaluate whether the expression of B95.1 and M81 variants of EBV LMP1 have different impact on cell migration and invasiveness of human cells lines in vitro, as well as on the expression of selected endogenous microRNAs (miRs) that were previously reported to have a role on cancer progression. Expression of EBV LMP1 was achieved in 293 and NP69 cells transfected with vectors encoding its B95.8 or M81 variants. In vitro migration and invasion rates were assessed by the scratch wound healing assay and the Boyden chamber assay, respectively. The expression of 91 selected miRs were evaluated by a customized qPCR-array approach. Cells expressing EBV LMP1 showed increased migration and invasion rates compared with mock-transfected cells. However, the differences observed between B95.8 and M81 variants did not achieve statistical significance. Conversely, 293 cells expressing the M81 variant of LMP1 showed hyperexpression of human miRs 497-5p, 17-3p, and 34a-5p, compared to B95.8. The in silico target prediction for these miRs showed enrichment of gene targets involved in the MAPK, NF-κB, and PI3K/AKT intracellular signaling pathways. This would reflect in negative regulation of cell proliferation and cell motility/adhesion, as well as higher levels of apoptosis. Based on these data, it is plausible to suggest that EBV LMP1 from M81 viral strain is somewhat less effective in terms of carcinogenicity, and this might be a reflect of the improved ability of EBV M81 to induce lytic viral infection compared to the B95.8 strain, as previously reported. In conclusion, the results indicate that cells expressing LMP1 from EBV isolates B95.8 and M81 showed differences in terms of miR expression signatures, which ultimately might impact the pathogenesis and progression of EBV-associated cancers. Citation Format: Bárbara Grasiele Müller-Coan, Ethel Cesarman, Deilson Elgui De Oliveira. Effects of Epstein-Barr virus latent membrane protein 1 (LMP1) on cell invasiveness and expression of endogenous microRNAs in human cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5756. doi:10.1158/1538-7445.AM2017-5756

Abstract 5759: Histopathologic patterns of nasopharyngeal malignancy in Lagos, Nigeria and its association with Epstein-Barr virus (EBV): A 10-year retrospective study

Abstract Background: Nasopharyngeal cancer (NPC) is a rare cancer worldwide. It is now found to be increasing in incidence in Nigeria, though the incidence is lower when compared to countries in the Mediterranean basin, North Africa and Southeast Asia. NPC is frequently associated with Epstein-Barr virus (EBV), but no previous study has been done in Lagos to document the association. Objective: To determine the prevalence, histologic types and age distribution of nasopharyngeal malignancy in Lagos University Teaching Hospital (LUTH) and its association with EBV over a period of 10 years, between January 2003 to December 2012. Materials and Methods: Slides and formalin-fixed paraffin-embedded (FFPE) tissue blocks, hospital request forms, referral cards, patients’ case notes and duplicate copies of histopathology reports of cases of histologically diagnosed nasopharyngeal malignancy in the Anatomic and Molecular Pathology department of LUTH within the study period were retrieved. The cases were reclassified into epithelial and non-epithelial malignancies. The epithelial malignancies were reclassified according to WHO classification. Immunohistochemistry (IHC) for EBV latent membrane protein-1 (LMP-1) was carried out on cases with viable FFPE to evaluate the prevalence of the virus in Nasopharngeal malignancy. Results: A total of 75 histologically confirmed cases of nasopharyngeal malignancy were seen during the study period, which constituted 1.1% of the total cancer recorded in this center. There were 47 males (63%) and 28 females (37%) with a male to female ratio of 1.7:1. The age ranged from 3 to 75 years, mean age was 44.1 years, the peak age group was 40 -49 years for males and 50 - 59 years for females. On histological classification, undifferentiated carcinoma (WHO type III) was the commonest (49.3%), differentiated non-keratinizing squamous cell carcinoma, SCC (WHO type II) and keratinizing SCC (WHO type I) accounted for 24% and 9.3% respectively. Thus, NPC accounted for 82.6% of the total cases of nasopharyngeal malignancy seen. Other histological types seen were non-Hodgkin lymphoma (NHL) 13.3%, well differentiated adenocarcinoma 2.7% and low grade mucoepidermoid carcinoma 1.3%. EBV LMP-1 IHC was positive in 30 (86%) out of the 35 cases studied. These consisted of 14 undifferentiated carcinoma, 5 NHL, 2 adenocarcinoma, 7differentiated non-keratinizing SCC (WHO type II) and the 2 cases of keratinizing SCC (WHO type 1). Conclusion: Nasopharyngeal malignancy is not uncommon in this environment. It occurred more frequently in males than females with the peak age of incidence at 5th decade of life, undifferentiated carcinoma (WHO type III) is the commonest histologic type and it is highly associated with EBV. Note: This abstract was not presented at the meeting. Citation Format: Oladipo Omoseebi, Olakanmi Akinde, Omobolade Obadofin, Nzechukwu Ikeri, Gabriel Ogun, Adekumbiola Banjo. Histopathologic patterns of nasopharyngeal malignancy in Lagos, Nigeria and its association with Epstein-Barr virus (EBV): A 10-year retrospective study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5759. doi:10.1158/1538-7445.AM2017-5759

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) increases IL-10 via Bruton’s tyrosine kinase and STAT3: Effect on T cell function

Abstract Data from our laboratory demonstrate that Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) promotes B cell lymphoma survival by enhancing IL-10 production, which has important implications in the treatment of EBV-associated B cell tumors. To determine the pathway used by LMP2A to increase IL-10, pharmacological inhibitors were added to LMP2A-expressing B cells and IL-10 was assessed. Our findings indicate for the first time, that LMP2A activates BTK to phosphorylate STAT3 in B cell tumors, which mediates the LMP2A-dependent increase in IL-10. Additionally, we assessed whether the LMP2A-mediated increase in IL-10 is immunosuppresssive to cytotoxic T cells that are critical for controlling the growth of EBV-associated tumors. Purified human CD8+ T cells activated with anti-CD3/CD28 mAbs were exposed to supernatants from either LMP2A-negative or –positive B cell lines for 48 hours. Our findings indicate that there is no difference in proliferation and IFN-γ secretion by activated CD8+ T cells exposed to supernatants from LMP2A-positive B cell lymphomas when compared to CD8+ T cells exposed to supernatants from LMP2A-negative B cell lymphomas. Although the LMP2A-mediated increase in IL-10 does not significantly influence the CD8+ T cell response, these findings highlight the possibility of using BTK and STAT3 inhibitors in clinical trials to treat EBV-associated lymphomas that express LMP2A. This work was supported by NIH grant 1R15CA149690-01.

Epstein-Barr virus infection and breast invasive ductal carcinoma in Egyptian women: A single center experience

BackgroundA controversy of the role of Epstein-Barr virus (EBV) infection in breast carcinomas has been reported in the literature. ObjectivesWe carried on this research to explore possible association between EBV infection and breast invasive ductal carcinoma (IDC) in Egyptian women attending our center. Study designThis study carried out at Sohag university hospital on 84 paraffin embedded samples of breast tissue, of them 42 breast IDC as the case group and 42 breast fibroadenomas as the control group. Nested PCRand immunohistochemistry (IHC) done separately for all samples to identify the Epstein-Barr nuclear antigen-1 (EBNA-1) gene and EBV latent membrane protein-1 (LMP-1) respectively, in breast cancer cells and controls. ResultsSpecimen considered positive when both (EBNA-1) gene and LMP-1 were detected using PCR and IHC separately for the same sample, this was achieved by 10/42 (23.81%) of breast IDC (case group) and 6/42 (14.29%) of breast fibro-adenomas (control group) (P-value=0.4). Nodal involvement was the only parameter that demonstrated a significant statistical relationship with EBV presence in cancerous tissue with p-value=0.003. ConclusionOur research could not find a significant statistical association between EBV infection and breast IDC in Egyptian women attending our center, but, there might be an association between the existence of EBV and tumor aggressiveness.

Epstein-Barr Virus Lytic Reactivation Activates B Cells Polyclonally and Induces Activation-Induced Cytidine Deaminase Expression: A Mechanism Underlying Autoimmunity and Its Contribution to Graves' Disease.

Graves' disease is an autoimmune disease that results in and is the most common cause of hyperthyroidism, and the reactivation of persisting Epstein–Barr virus (EBV) in B lymphocytes induces the differentiation of host B cells into plasma cells. We previously reported that some EBV-infected B cells had thyrotropin receptor antibodies (TRAbs) as surface immunoglobulins (Igs), and EBV reactivation induced these TRAb+EBV+ cells to produce TRAbs. EBV reactivation induces Ig production from host B cells. The purpose of the present study was to examine total Ig productions from B cell culture fluids and to detect activation-induced cytidine deaminase (AID), nuclear factor kappa B (NF-κB), and EBV latent membrane protein (LMP) 1 in culture B cells during EBV reactivation induction and then we discussed the mechanisms of EBV reactivation-induced Ig production in relation to autoimmunity. We showed that the EBV reactivation induces the production of every isotype of Ig and suggested that the Ig production was catalyzed by AID through LMP1 and NF-κB. The results that the amount of IgM was significantly larger compared with IgG suggested the polyclonal B cell activation due to LMP1. We proposed the pathway of EBV reactivation induced Ig production; B cells newly infected with EBV are activated by polyclonal B cell activation and produce Igs through plasma cell differentiation induced by EBV reactivation. LMP1-induced AID enabled B cells to undergo class-switch recombination to produce every isotype of Ig. According to this mechanism, EBV rescues autoreactive B cells to produce autoantibodies, which contribute to the development and exacerbation of autoimmune diseases.

Mouse model of Epstein–Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease

Epstein-Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

EBV LMP1 regulates cancer growth through IRF-7 in nasopharyngeal carcinoma.

The cellular Interferon (IFN) regulatory factor-7 (IRF-7) was originally identified in cells with Epstein-Barr virus (EBV) infection, which constituted of positive circuit with viral oncoprotein latent membrane protein 1 (LMP1), and both proteins are involved in neoplasia. EBV is known to be associated with the development of various cancers, including Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), Burkitt lymphoma, gastric lymphoma, and nasopharyngeal carcinoma (NPC). Although the exact role of IRF-7 in EBV-related oncogenesis is not clear, in this study, we demonstrated the role of IRF-7 in angiogenesis suppression via LMP1 down-regulation. Additionally, we employed knock down of IRF-7 and verified the inhibition of IRF-7 in NPC growth in vivo. These findings suggest that IRF-7 could be a potential therapeutic target in EBV-infected NPC tumors.

Positive regulation of TAZ expression by EBV-LMP1 contributes to cell proliferation and epithelial-mesenchymal transition in nasopharyngeal carcinoma.

The Epstein-Barr virus latent membrane protein 1 (LMP1) is an integral membrane protein. LMP1 has been reported to activate the NF-κB and mitogen-activated protein kinase pathways. However, these effects alone are unable to account for the profound oncogenic properties of LMP1. TAZ is one of the nuclear effectors of Hippo-related pathways and highly expressed in many human tumors. Here, we reported that TAZ was frequently expressed in LMP1-positive nasopharyngeal carcinoma. In NPC cell lines, we showed that LMP1 promoted TAZ expression. Gelsolin is an important inhibitor of TAZ activity. Our studies showed that LMP1 interacted with gelsolin, resulting in inhibition of Lats1/2 phosphorylation and improvement of TAZ stability. Furthermore, we revealed that TAZ is important for LMP1-mediated cell proliferation, cancer stem cell-like properties and epithelial-mesenchymal transition. These findings provide new insights into the carcinogenic roles of LMP1 and contribute to further understanding of its oncogenic mechanism.