EBV-encoded Latent Membrane Protein Research Articles

3D-Q-FISH/Telomere/TRF2 Nanotechnology Identifies a Progressively Disturbed Telomere/Shelterin/Lamin AC Complex as the Common Pathogenic, Molecular/Spatial Denominator of Classical Hodgkin Lymphoma.

The bi- or multinucleated Reed-Sternberg cell (RS) is the diagnostic cornerstone of Epstein-Barr Virus (EBV)-positive and EBV-negative classical Hodgkin lymphoma (cHL). cHL is a germinal center (GC)-derived B-cell disease. Hodgkin cells (H) are the mononuclear precursors of RS. An experimental model has to fulfill three conditions to qualify as common pathogenic denominator: (i) to be of GC-derived B-cell origin, (ii) to be EBV-negative to avoid EBV latency III expression and (iii) to support permanent EBV-encoded oncogenic latent membrane protein (LMP1) expression upon induction. These conditions are unified in the EBV-, diffuse large B-Cell lymphoma (DLBCL) cell line BJAB-tTA-LMP1. 3D reconstructive nanotechnology revealed spatial, quantitative and qualitative disturbance of telomere/shelterin interactions in mononuclear H-like cells, with further progression during transition to RS-like cells, including progressive complexity of the karyotype with every mitotic cycle, due to BBF (breakage/bridge/fusion) events. The findings of this model were confirmed in diagnostic patient samples and correlate with clinical outcomes. Moreover, in vitro, significant disturbance of the lamin AC/telomere interaction progressively occurred. In summary, our research over the past three decades identified cHL as the first lymphoid malignancy driven by a disturbed telomere/shelterin/lamin AC interaction, generating the diagnostic RS. Our findings may act as trailblazer for tailored therapies in refractory cHL.

Modeling NK-cell lymphoma in mice reveals its cell-of-origin and microenvironmental changes and identifies therapeutic targets

Extranodal NK/T-cell lymphoma (ENKTCL) is an Epstein-Barr virus (EBV)-related neoplasm preferentially involving the upper aerodigestive tract. Here we show that NK-cell-specific Trp53 disruption in mice leads to the development of NK-cell lymphomas after long latency, which involve not only the hematopoietic system but also the salivary glands. Before tumor onset, Trp53 knockout causes extensive gene expression changes, resulting in immature NK-cell expansion, exclusively in the salivary glands. Both human and murine NK-cell lymphomas express tissue-resident markers, suggesting tissue-resident NK cells as their cell-of-origin. Murine NK-cell lymphomas show recurrent Myc amplifications and upregulation of MYC target gene signatures. EBV-encoded latent membrane protein 1 expression accelerates NK-cell lymphomagenesis and causes diverse microenvironmental changes, particularly myeloid propagation, through interferon-γ signaling. In turn, myeloid cells support tumor cells via CXCL16-CXCR6 signaling and its inhibition is effective against NK-cell tumors in vivo. Remarkably, KLRG1-expressing cells expand in the tumor and are capable of repopulating tumors in secondary recipients. Furthermore, targeting KLRG1 alone or combined with MYC inhibition using an eIF4 inhibitor is effective against NK-cell tumors. Therefore, our observations provide insights into the pathogenesis and highlight potential therapeutic targets, including CXCL16, KLRG1, and MYC, in ENKTCL, which can help improve its diagnostic and therapeutic strategies.

Study on the regulatory mechanism of latent membrane protein 2A on GCNT3 expression in nasopharyngeal carcinoma.

O-Glycan synthesis enzyme glucosaminyl (N-acetyl) transferase 3 (GCNT3) is closely related to the occurrence and development of various cancers.However, the regulatory mechanism and function of GCNT3 in nasopharyngeal carcinoma (NPC) are still poorly understood. This study aims to explore the regulatory mechanism of EBV-encoded latent membrane protein 2A (LMP2A) on GCNT3 and the biological role of GCNT3 in NPC.The results show that LMP2A can activate GCNT3 through the mTORC1 pathway, and there is a positive feedback between the mTORC1 and GCNT3. GCNT3 regulates EMT progression by forming a complex with ZEB1 to promote cell migration. GCNT3 can also promote cell proliferation. These findings indicate that targeting the LMP2A-mTORC1-GCNT3 axis may represent a novel therapeutic target in NPC.

Epstein-Barr virus-encoded Latent Membrane protein-1(LMP-1) as a Prognostic marker in OSCC and OPMDs

Epstein-Barr virus-encoded Latent Membrane protein-1(LMP-1) as a Prognostic marker in OSCC and OPMDs

CD30 protects EBV-positive diffuse large B-cell lymphoma cells against mitochondrial dysfunction through BNIP3-mediated mitophagy

Epstein-Barr virus (EBV) positive diffuse large B-cell lymphoma (EBV+ DLBCL) predicts poor prognosis and CD30 expression aggravates the worse consequences. Here, we reported that CD30 positivity was an independent prognostic indicator in EBV+ DLBCL patients in a retrospective cohort study. We harnessed CRISPR/Cas9 editing to engineer the first loss-of-function models of CD30 deficiency to identify that CD30 was critical for EBV+ DLBCL growth and survival. We established a pathway that EBV infection mediated CD30 expression through EBV-encoded latent membrane protein 1 (LMP1), which involved NF-κB signaling. CRISPR CD30 knockout significantly repressed BCL2 interacting protein 3 (BNIP3) expression and co-IP assay indicated a binding between CD30 and BNIP3. Moreover, silencing of CD30 induced mitochondrial dysfunction and suppressed mitophagy, resulting in the accumulation of damaged mitochondria by depressing BNIP3 expression. Additionally, CRISPR BNIP3 knockout caused proliferation defects and increased sensitivity to apoptosis. All the findings reveal a strong relationship between mitophagy and adverse prognosis of EBV+ DLBCL and discover a new regulatory mechanism of BNIP3-mediated mitophagy, which may help develop effective treatment regimens with anti-CD30 antibody brentuximab vedotin to improve the prognosis of CD30+ EBV+ DLBCL patients.

Establishment of Epstein-Barr Virus (EBV) Latent Gene-Expressing T-Cell Lines with an Expression Vector Harboring EBV Nuclear Antigen 1.

Chronic active Epstein-Barr virus (EBV) infection (CAEBV) is characterized by chronic or recurrent infectious mononucleosis-like symptoms and is associated with EBV-associated T/natural killer (NK)-cell lymphoproliferative disorders, which frequently lead to the development of life-threatening complications, such as virus-associated hemophagocytic syndrome and EBV-positive apparent leukemia/lymphoma mainly in T- and NK-cell lineages. In order to clarify the EBV genes responsible for the diseases, we introduced the plasmid coding sequences of EBV-encoded small RNAs (EBERs) and/or latent membrane protein (LMP) 1 into human T-lymphocyte virus-I-negative human T-cell lines using a gene expression vector harboring EBV nuclear antigen 1, established the G418-resistant transformants of five T-cell lines, and quantitatively examined the expression of EBERs and LMP1 using real-time reverse transcriptase-polymerase chain reaction. The expression levels of EBERs in T-cell transformants with EBER DNA paralleled those in EBV-positive human T- and NK-cell lines, SNTK cells. The expression of LMP1 mRNA varied in SNTK cells and in human T-cell transformants, and the expression of LMP1 mRNA in T-cell lines expressing both EBERs and LMP1 was much lower than that in the same cell line expressing LMP1 mRNA alone. The currently employed gene expression system and currently obtained transformants may be useful for the analyses of the pathophysiology of CAEBV and EBV-positive T/NK-cell lymphoproliferative disorders.

Anoikis resistance and immune escape mediated by Epstein-Barr virus-encoded latent membrane protein 1-induced stabilization of PGC-1α promotes invasion and metastasis of nasopharyngeal carcinoma

BackgroundEpstein-Barr virus (EBV) is the first discovered human tumor virus that is associated with a variety of malignancies of both lymphoid and epithelial origin including nasopharyngeal carcinoma (NPC). The EBV-encoded latent membrane protein 1 (LMP1) has been well-defined as a potent oncogenic protein, which is intimately correlated with NPC pathogenesis. Anoikis is considered to be a physiological barrier to metastasis, and avoiding anoikis is a major hallmark of metastasis. However, the role of LMP1 in anoikis-resistance and metastasis of NPC has not been fully identified.MethodsTrypan blue staining, colony formation assay, flow cytometry, and TUNEL staining, as well as the detection of apoptosis and anoikis resistance‐related markers was applied to evaluate the anoikis-resistant capability of NPC cells cultured in ultra-low adhesion condition. Co-immunoprecipitation (Co-IP) experiment was performed to determine the interaction among LMP1, PRMT1 and PGC-1α. Ex vivo ubiquitination assay was used to detect the ubiquitination level of PGC-1α. Anoikis- resistant LMP1-positive NPC cell lines were established and applied for the xenograft and metastatic animal experiments.ResultsOur current findings reveal the role of LMP1-stabilized peroxisome proliferator activated receptor coactivator-1a (PGC-1α) in anoikis resistance and immune escape to support the invasion and metastasis of NPC. Mechanistically, LMP1 enhances PGC-1α protein stability by promoting the interaction between arginine methyltransferase 1 (PRMT1) and PGC-1α to elevate the methylation modification of PGC-1α, thus endowing NPC cells with anoikis-resistance. Meanwhile, PGC-1α mediates the immune escape induced by LMP1 by coactivating with STAT3 to transcriptionally up-regulate PD-L1 expression.ConclusionOur work provides insights into how virus-encoded proteins recruit and interact with host regulatory elements to facilitate the malignant progression of NPC. Therefore, targeting PGC-1α or PRMT1-PGC-1α interaction might be exploited for therapeutic gain for EBV-associated malignancies.

STIM1-regulated exosomal EBV-LMP1 empowers endothelial cells with anaggressive phenotype by activating the Akt/ERK pathway in nasopharyngeal carcinoma.

Stromal interaction molecule 1 (STIM1)-mediated Ca2+ signaling regulates tumor angiogenesis in nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-related human malignancy. However, the mechanism by which STIM1 modulates endothelial functional phenotypes contributing to tumor angiogenesis remains elusive. NPC cell-derived exosomes were isolated via differential centrifugation and observed using transmission electron microscopy. Exosome particle sizes were assessed by nanoparticle tracking analysis (NTA). Uptake of exosomes by recipient ECs was detected by fluorescent labeling of the exosomes with PKH26. Tumor angiogenesis-associated profiles were characterized by determining cell proliferation, migration, tubulogenesis and permeability in human umbilical vein endothelial cells (HUVECs). Activation of the Akt/ERK pathway was assessed by detecting the phosphorylation levels using Western blotting. A chick embryo chorioallantoic membrane (CAM) xenograft model was employed to study tumor-associated neovascularization in vivo. We found that NPC cell-derived exosomes harboring EBV-encoded latent membrane protein 1 (LMP1) promoted proliferation, migration, tubulogenesis and permeability by activating the Akt/ERK pathway in ECs. STIM1 silencing reduced LMP1 enrichment in NPC cell-derived exosomes, thereby reversing its pro-oncogenic effects in an Akt/ERK pathway-dependent manner. Furthermore, STIM1 knockdown in NPC cells blunted tumor-induced vascular network formation and inhibited intra-tumor neovascularization in the chorioallantoic membrane (CAM) xenograft model. STIM1 regulates tumor angiogenesis by controlling exosomal EBV-LMP1 delivery to ECs in the NPC tumor microenvironment. Blocking exosome-mediated cell-to-cell horizontal transfer of EBV-associated oncogenic signaling molecules may be an effective therapeutic strategy for NPC.

Expression of soluble CD27 in extranodal natural killer/T-cell lymphoma, nasal type: potential as a biomarker for diagnosis and CD27/CD70-targeted therapy.

The engagement of CD27 on lymphocytes with its ligand, CD70, on tumors is believed to mediate tumor immune evasion and the elevation of serum soluble CD27 (sCD27) levels in patients with CD70-positive malignancies. We previously showed that CD70 is expressed in extranodal natural killer/T-cell lymphoma, nasal type (ENKL), an Epstein-Barr virus (EBV)-related malignancy. However, little is known about serum sCD27 expression and its association with the clinical characteristics of, and the CD27/CD70 interaction in, ENKL. In the present study, we show that serum sCD27 is significantly elevated in the sera of patients with ENKL. The levels of serum sCD27 provided excellent diagnostic accuracy for discriminating patients with ENKL from healthy subjects, correlated positively with the levels of other diagnostic markers (lactate dehydrogenase, soluble interleukin-2 receptor, and EBV-DNA), and decreased significantly following treatment. Elevated serum sCD27 levels also correlated significantly with advanced clinical stage and tended to correspond with shorter survival, in patients with ENKL. Immunohistochemistry indicated that CD27-positive tumor-infiltrating immune cells exist adjacent to CD70-positive lymphoma cells. In addition, serum sCD27 levels in patients with CD70-positive ENKL were significantly higher than those in patients with CD70-negative ENKL, suggesting that the intra-tumoral CD27/CD70 interaction boosts the release of sCD27 in serum. Furthermore, the EBV-encoded oncoprotein latent membrane protein 1 upregulated CD70 expression in ENKL cells. Our results suggest that sCD27 may serve as a novel diagnostic biomarker and also may serve as a tool for evaluating the applicability of CD27/CD70-targeted therapies by predicting intra-tumoral CD70 expression and CD27/CD70 interaction in ENKL.

Latent membrane protein 1 and macrophage-derived TNFα synergistically activate and mobilize invadopodia to drive invasion of nasopharyngeal carcinoma.

Invadopodia are actin-rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they have been visualized as localized and dot-like structures in different types of cancer cells on top of a 2D matrix. In this investigation of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), a highly invasive cancer frequently accompanied by neck lymph node and distal organ metastases, we revealed a new form of invadopodium with mobilizing features. Integration of live-cell imaging and molecular assays revealed the interaction of macrophage-released TNFα and EBV-encoded latent membrane protein 1 (LMP1) in co-activating the EGFR/Src/ERK/cortactin and Cdc42/N-WASP signaling axes for mobilizing the invadopodia with lateral movements. This phenomenon endows the invadopodia with massive degradative power, visualized as a shift of focal dot-like digestion patterns on a 2D gelatin to a dendrite-like digestion pattern. Notably, single stimulation of either LMP1 or TNFα could only enhance the number of ordinary dot-like invadopodia, suggesting that the EBV infection sensitizes the NPC cells to form mobilizing invadopodia when encountering a TNFα-rich tumor microenvironment. This study unveils the interplay of EBV and stromal components in driving the invasive potential of NPC via unleashing the propulsion of invadopodia in overcoming matrix hurdles. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cyclophilin A binds to AKT1 and facilitates the tumorigenicity of Epstein-Barr virus by mediating the activation of AKT/mTOR/NF-κB positive feedback loop.

The AKT/mTOR and NF-κB signalings are crucial pathways activated in cancers including nasopharyngeal carcinoma (NPC), which is prevalent in southern China and closely related to Epstein-Barr virus (EBV) infection. How these master pathways are persistently activated in EBV-associated NPC remains to be investigated. Here we demonstrated that EBV-encoded latent membrane protein 1 (LMP1) promoted cyclophilin A (CYPA) expression through the activation of NF-κB. The depletion of CYPA suppressed cell proliferation and facilitated apoptosis. CYPA was able to bind to AKT1, thus activating AKT/mTOR/NF-κB signaling cascade. Moreover, the use of mTOR inhibitor, rapamycin, subverted the activation of the positive feedback loop, NF-κB/CYPA/AKT/mTOR. It is reasonable that LMP1 expression derived from initial viral infection is enough to assure the constant potentiation of AKT/mTOR and NF-κB signalings. This may partly explain the fact that EBV serves as a tumor-promoting factor with minimal expression of the viral oncoprotein LMP1 in malignancies. Our findings provide new insight into the understanding of causative role of EBV in tumorigenicity during latent infection.

Are Viral Infections Key Inducers of Autoimmune Diseases? Focus on Epstein-Barr Virus.

It is generally accepted that certain viral infections can trigger the development of autoimmune diseases. However, the exact mechanisms by which these viruses induce autoimmunity are still not understood. In this review, we first describe hypothetical mechanisms by which viruses induce some representative autoimmune diseases. Then, we focus on Epstein–Barr virus (EBV) and discuss its role in the pathogenesis of rheumatoid arthritis (RA). The discussion is mainly based on our own previous findings that (A) EBV DNA and its products EBV-encoded small RNA (EBER) and latent membrane protein 1 (LMP1) are present in the synovial lesions of RA, (B) mRNA expression of the signaling lymphocytic activation molecule-associated protein (SAP)/SH2D1A gene that plays a critical role in cellular immune responses to EBV is reduced in the peripheral T cells of patients with RA, and (C) EBV infection of mice reconstituted with human immune system components (humanized mice) induced erosive arthritis that is pathologically similar to RA. Additionally, environmental factors may contribute to EBV reactivation as follows: Porphyromonas gingivalis peptidylarginine deiminase (PAD), an enzyme required for citrullination, engenders antigens leading to the production of citrullinated peptides both in the gingiva and synovium. Anti-citrullinated peptides autoantibody is an important marker for diagnosis and disease activity of RA. These findings, as well as various results obtained by other researchers, strongly suggest that EBV is directly involved in the pathogenesis of RA, a typical autoimmune disease.

Epstein-Barr viral product-containing exosomes promote fibrosis and nasopharyngeal carcinoma progression through activation of YAP1/FAPα signaling in fibroblasts

BackgroundThe progression of nasopharyngeal carcinoma (NPC) is profoundly affected by Epstein-Barr virus (EBV) infection. However, the role of EBV in the intercommunication between NPC and surrounding stromal cells has yet to be explored.MethodsNPC biopsies were obtained for immunohistochemical (IHC) analyses. Clinical correlations between the expression of active YAP1/FAPα and the fibrotic response and between YAP1/FAPα and the density of cytotoxic CD8a+ T lymphocytes were determined. Survival times based on IHC scores were compared between groups using Kaplan-Meier survival and log-rank tests. Independent prognostic factors for metastasis/recurrence-free survival and overall survival were identified using univariate and multivariate Cox regression models. Fibroblasts were isolated from human nasopharyngeal biopsies. Exosomes were purified from culture supernatants of EBV+-positive NPC cells. The effects of EBV product-containing exosomes on fibroblast activation, fibrotic response, tumor growth, immune response, and correlations between the expression of featured genes were investigated using gel contraction assays, ELISAs, EdU incorporation assays, real-time impedance assays, RNA sequencing, immunostaining, 3D cancer spheroid coculture systems, and an NPC xenograft model.ResultsNPC patients who developed metastasis had significantly higher levels of active YAP1 and FAPα in their tumor stroma, which was further correlated with tumor fibrosis and poorer metastasis-free survival. Exosomes released from EBV+-NPC cells contained abundant FAPα protein and EBV-encoded latent membrane protein 1. Viral product-containing exosomes markedly enhanced the fibrotic response and tumor growth in a mouse xenograft model. IHC analyses of human NPC and NPC xenografts revealed positive correlations between levels of active YAP1 and FAPα, YAP1 and the fibrotic response, and FAPα and the fibrotic response. Mechanistic studies showed that treatment of fibroblasts with viral product-containing exosomes promoted the characteristics of cancer-associated fibroblasts by stimulating YAP1 signaling and the production of the immunosuppressive cytokines IL8, CCL2, and IL6. Inhibition of YAP1 activation markedly reversed these exosome-mediated protumoral effects, resulting in reduced contractility, inactivation of YAP1 signaling, and decreased production of immunosuppressive cytokines in fibroblasts. Furthermore, fibroblasts stimulated with these viral product-containing exosomes promoted NPC resistance to T cell-mediated cytotoxicity within tumor spheroids. In NPC tissues, a significant negative correlation was found between YAP1/FAPα and the density of CD8a+ T lymphocytes with a granzyme B signature.ConclusionEBV orchestrates interactions with the host and surrounding stroma by stimulating the functions of YAP1 and FAPα in fibroblasts through exosome cargos to create a more immunosuppressive, proinvasive microenvironment.

Noninvasive Diagnosis of Nasopharyngeal Carcinoma Based on Phenotypic Profiling of Viral and Tumor Markers on Plasma Extracellular Vesicles.

Nasopharyngeal carcinoma (NPC) is a malignant tumor commonly associated with Epstein-Barr virus (EBV) infection, and its early diagnosis as well as its differentiation from nasopharyngitis (NPG) remains challenging due to the insufficient sensitivity of routine screening methods in clinical practice. To date, circulating extracellular vesicles (EVs, 40-1000 nm) have shown appealing potential in liquid biopsy for cancer diagnosis and prognosis. Herein, nanoflow cytometry (nFCM) capable of single EV analysis was applied to examine the expression of surface proteins with very low copy numbers on individual EVs as small as 40 nm. The particle concentrations of five EV subsets exposing EBV-encoded latent membrane proteins (LMP1 and LMP2A) and tumor markers (PD-L1, EGFR, and EpCAM) in plasma were determined rapidly via single-particle enumeration. We identified a five-marker panel named EVSUM5 (an unweighted sum of the concentration of the five individual EV subsets) that significantly surpassed the traditional VCA-IgA assay in discriminating NPC patients from both healthy donors and NPG patients with accuracies of 96.3 and 83.1%, respectively. Moreover, EVSUM2 (an unweighted sum of virus-specific LMP1- and LMP2A-positive EVs) could achieve the diagnosis of NPG with an accuracy of 82.6%. Collectively, the work presented a rapid, reliable, and noninvasive method as well as two diagnostic markers to help more accurately differentiate NPC from NPG patients and healthy donors in clinical practice.

Abstract 3192: EBV-products containing exosomes remodel NPC tumor microenvironment by activating fibroblasts

Abstract Epstein-Barr virus (EBV) infection is intimately associated with type III nasopharyngeal carcinoma (NPC). Most NPCs respond well to chemoradiation. Nevertheless, approximately 20% of patients experience recurrence and distant metastasis. Tumor desmoplastic responses and high densities of cancer-associated fibroblasts (CAFs) within tumors have been correlated with poorer prognosis. In this study, we demonstrate that EBV activates fibroblasts via exosomes to remodel a pro-tumoral microenvironment. Exosomes derived from NPC cells contained a high level of fibroblast activation protein alpha (FAPα) and EBV-encoded latent membrane protein 1 (LMP1). Treating primary fibroblasts with these exosomes induced functional changes resembling CAFs. Transcriptomic analysis on exosome-treated fibroblasts revealed genes involved in fibrosis-associated pathways were enriched. Noticeably, treating with EBV products-containing exosomes in fibroblasts enhanced the production of several immune-suppressive cytokines, including IL6, IL8, and MCP-1. Moreover, stimulation with these exosomes enhanced activation of YAP1 signaling, a key pathway linking to fibrosis. The in vivo mouse xenografted NPC studies showed that EBV products-containing exosomes markedly enhanced the severity of tissue fibrosis and tumor growth. IHC analysis on mouse NPC xenografts and human NPC biopsies showed a positive correlation between levels of nuclear YAP1 and FAPα, which were further correlated with an enhanced fibrotic response within tumors. Pharmaceutical intervention with YAP1 inhibitors blocked the exosome-mediated effects on fibroblasts. Collectively, our data suggest that EBV products-containing exosome is a causal factor in CAF formation, which constitutes a pro-tumoral microenvironment to benefit the survival of both tumor and virus itself. Citation Format: Po-Ju Lee, Yun-Hua Sui, Chen-Han Huang, Tzu-Tung Liu, Ngan-Ming Tsang, Shu-Chen Liu. EBV-products containing exosomes remodel NPC tumor microenvironment by activating fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3192.

Epstein-Barr virus infection patterns in nodular lymphocyte-predominant Hodgkin lymphoma.

To investigate Epstein-Barr virus (EBV) latency types in 19 cases of EBV-positive nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), as such information is currently incomplete. Immunohistochemistry (IHC) for CD20, CD79a, PAX5, OCT2, CD30, CD15, CD3 and programmed cell death protein 1 was performed. For EBV detection, in-situ hybridisation (ISH) for EBV-encoded RNA (EBER) was employed combined with IHC for EBV-encoded latent membrane protein (LMP)-1, EBV-encoded nuclear antigen (EBNA)-2, and EBV-encoded BZLF1. In 95% of the cases, neoplastic cells with features of Hodgkin and Reed-Sternberg (HRS) cells were present, mostly showing expression of CD30. In all cases, the B-cell phenotype was largely intact, and delineation from classic Hodgkin lymphoma (CHL) was further supported by myocyte enhancer factor 2B (MEF2B) detection. All tumour cells were EBER-positive except in two cases. EBV latency type II was most frequent (89%) and type I was rare. Cases with latency type I were CD30-negative. Five cases contained some BZLF1-positive and/or EBNA-2-positive bystander lymphocytes. As HRS morphology of neoplastic cells and CD30 expression are frequent features of EBV-positive NLPHL, preservation of the B-cell transcription programme, MEF2B expression combined with NLPHL-typical architecture and background composition facilitate distinction from CHL. EBER ISH is the method of choice to identify these cases. The majority present with EBV latency type II, and only rare cases present with latency type I, which can be associated with missing CD30 expression. The presence of occasional bystander lymphocytes expressing BZLF1 and/or EBNA-2 and the partial EBV infection of neoplastic cells in some cases could indicate that EBV is either not primarily involved or is only a transient driver in the pathogenesis of EBV-positive NLPHL.

Epstein-Barr Virus-Encoded Latent Membrane Protein 2A Downregulates GCNT3 via the TGF-β1/Smad-mTORC1 Signaling Axis.

Increasing evidence shows that Epstein-Barr virus (EBV) infection is closely related to various lymphoid and epithelioid malignancies. However, the underlying mechanisms are unclear. GCNT3 (core 2β-1,6-acetylglucosaminyltransferase) is a new type of core mucin synthase, and its expression in EBV-associated gastric cancer (EBVaGC) is lower than that in EBV-negative gastric cancer (EBVnGC). EBV-encoded latent membrane protein 2A (LMP2A) is a transmembrane protein with tumorigenic transformation properties. Here, we demonstrated that LMP2A inhibited the transcription of GCNT3 by inhibiting Smad2/3 and Smad4. LMP2A restrained the activation of the mTORC1 pathway by inactivating the TGF-β1/Smad pathway and then downregulated GCNT3 expression. The mTORC1-GCNT3 pathway promoted cell proliferation and migration and inhibited G0/G1 cell arrest. Related proteins involved in epithelial-mesenchymal transition (EMT) were downstream molecules of the TGF-β1/Smad-mTORC1-GCNT3 pathway. GCNT3 inhibited autophagy by inducing mTORC1 phosphorylation. These findings indicate that targeting the TGF-β1/Smad-mTORC1-GCNT3 axis may represent a novel therapeutic target in GC.ImportanceEpstein-Barr virus (EBV) is an opportunistic pathogen, and the latent membrane protein 2A (LMP2A) encoded by EBV plays a key role in ensuring the incubation period of EBV. Glycosylation modification is an important marker of cancer cells, and recent studies have reported that it is related to EBV. Our conclusions provide deeper theoretical support for the role of LMP2A and TGF/Smad-mTORC1-GCNT3 in EBVaGC and help to understand glycosylation abnormalities in cancer. Our results may provide novel therapeutic targets for the treatment of gastric cancer against the TGF/Smad-mTORC1-GCNT3 signaling cascade.

LMP2A suppresses the role of AHR pathway through ERK signal pathway in EBV-associated gastric cancer

ObjectiveTo investigate the function of the aryl hydrocarbon receptor (AHR) pathway in Epstein-Barr Virus (EBV)-associated gastric cancer (EBVaGC) and to explore the relationship between EBV and AHR expression. MethodsThe expression of AHR in EBVaGC and EBV negative GC (EBVnGC) tissues was detected by immunohistochemistry (IHC). Real-time qPCR (RT-qPCR) and Western blot analysis were used to examine the expression of AHR, cytochrome P450 1A1 (CYP1A1), and cytochrome P450 1B1 (CYP1B1) in gastric cancer cells. The cell proliferation and migration assay were tested by CCK8 and transwell analysis. EBV-encoded latent membrane protein 2A (LMP2A) was over-expressed in SGC7901 cells and silenced in AGS-EBV cells to further identify its role in EBV positive GC cells. ResultsIt was found that EBV infection inhibited the expression of AHR in gastric cancer tissues and cell lines. We also found that the activation of AHR pathway can promote cell proliferation and migration. However, the function was restricted in EBVaGC cell lines compared with EBVnGC. LMP2A can suppress AHR expression and pathway activation by activating phosphorylation of extracellular signal-regulated kinase (ERK) in EBV positive GC cell lines. ConclusionEBV-encoded LMP2A regulated the function of the AHR pathway by activating the ERK signal pathway in EBV positive GC cell lines.

The Role of EBV-Encoded LMP1 in the NPC Tumor Microenvironment: From Function to Therapy.

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. It is also characterized by heavy infiltration with non-malignant leucocytes. The EBV-encoded latent membrane protein 1 (LMP1) is believed to play an important role in NPC pathogenesis by virtue of its ability to activate multiple cell signaling pathways which collectively promote cell proliferation and survival, angiogenesis, invasiveness, and aerobic glycolysis. LMP1 also affects cell-cell interactions, antigen presentation, and cytokine and chemokine production. Here, we discuss how LMP1 modulates local immune responses that contribute to the establishment of the NPC tumor microenvironment. We also discuss strategies for targeting the LMP1 protein as a novel therapy for EBV-driven malignancies.

EBV as a high infection risk factor promotes RASSF10 methylation and induces cell proliferation in EBV-associated gastric cancer

Epstein-Barr virus (EBV) is the first identified human tumor-related DNA virus, and has a high infection among people worldwide. Recent studies have showed that nearly 10% of gastric cancers have shown EBV infection and this kind of gastric cancer has been identified as a new subtype: EBV associated Gastric cancer (EBVaGC). Furthermore, it has been reported that tumor related genes in the EBVaGC showed frequent methylation modifications compared to those in the EBV negative gastric cancer (EBVnGC). To fully understand the role of EBV in EBVaGC, we analyzed and found that 16.67% of gastric carcinoma samples showed positive EBER1 signals. Mechanically, EBV-encoded Latent membrane protein 1 (LMP1) inhibited the expression of RASSF10, and promoted tumorigenesis by recruiting DNMT1 and inducing the DNA methylation of RASSF10. Altogether, it allows us a better understanding of the possible mechanism of EBV-induced gene hypermethylation in gastric cancer genome. Targeting EBV-induced DNA methylation is a potential therapeutic modality of EBVaGC.