Epstein-Barr Virus-encoded Latent Membrane Protein Research Articles

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.

EPSTEIN-BARR VIRUS–POSITIVE MUCOCUTANEOUS ULCER: A CASE REPORT

Epstein-Barr virus–positive mucocutaneous ulcer (EBVMCU) is a newly recognized clinicopathologic entity included in the group of mature B-cell neoplasms in the 2017 revision of World Health Organization Classification for Lymphoproliferative Disorders. A nonsmoking 92-year-old man presented with an ulcerated lesion on the maxillary alveolar ridge. An incisional biopsy was performed and reported an undifferentiated malignancy composed of cell clusters with scarce cytoplasm and very pleomorphic nuclei. The immunohistochemical profile expressed CD20, CD30, CD15, CD45 and Epstein-Barr virus-encoded latent membrane protein (EBV-LMP) antigens, consistent with EBVMCU. Within 2 weeks, the lesion became painful and the palate was infiltrated. Tomography showed loss of bone tissue and permeative aspect in the right maxilla. After 8 weeks of rituximab, the patient was asymptomatic and presented regression of the lesion, maintaining only the bone exposed area. This case highlights that proper clinical history and ancillary studies can avoid misinterpretation and overtreatment of EBVMCU oral lesions. Epstein-Barr virus–positive mucocutaneous ulcer (EBVMCU) is a newly recognized clinicopathologic entity included in the group of mature B-cell neoplasms in the 2017 revision of World Health Organization Classification for Lymphoproliferative Disorders. A nonsmoking 92-year-old man presented with an ulcerated lesion on the maxillary alveolar ridge. An incisional biopsy was performed and reported an undifferentiated malignancy composed of cell clusters with scarce cytoplasm and very pleomorphic nuclei. The immunohistochemical profile expressed CD20, CD30, CD15, CD45 and Epstein-Barr virus-encoded latent membrane protein (EBV-LMP) antigens, consistent with EBVMCU. Within 2 weeks, the lesion became painful and the palate was infiltrated. Tomography showed loss of bone tissue and permeative aspect in the right maxilla. After 8 weeks of rituximab, the patient was asymptomatic and presented regression of the lesion, maintaining only the bone exposed area. This case highlights that proper clinical history and ancillary studies can avoid misinterpretation and overtreatment of EBVMCU oral lesions.

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 and B-Cell Growth Transformation Induce Lipogenesis through Fatty Acid Synthase.

Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro Several B-cell malignancies are associated with latent LMP1-positive EBV infection, including Hodgkin's and diffuse large B-cell lymphomas. We have previously reported that promotion of B cell proliferation by LMP1 coincided with an induction of aerobic glycolysis. To further examine LMP1-induced metabolic reprogramming in B cells, we ectopically expressed LMP1 in an EBV-negative Burkitt's lymphoma (BL) cell line preceding a targeted metabolic analysis. This analysis revealed that the most significant LMP1-induced metabolic changes were to fatty acids. Significant changes to fatty acid levels were also found in primary B cells following EBV-mediated B-cell growth transformation. Ectopic expression of LMP1- and EBV-mediated B-cell growth transformation induced fatty acid synthase (FASN) and increased lipid droplet formation. FASN is a crucial lipogenic enzyme responsible for de novo biogenesis of fatty acids in transformed cells. Furthermore, inhibition of lipogenesis caused preferential killing of LMP1-expressing B cells and significantly hindered EBV immortalization of primary B cells. Finally, our investigation also found that USP2a, a ubiquitin-specific protease, is significantly increased in LMP1-positive BL cells and mediates FASN stability. Our findings demonstrate that ectopic expression of LMP1- and EBV-mediated B-cell growth transformation leads to induction of FASN, fatty acids, and lipid droplet formation, possibly pointing to a reliance on lipogenesis. Therefore, the use of lipogenesis inhibitors could be used in the treatment of LMP1+ EBV-associated malignancies by targeting an LMP1-specific dependency on lipogenesis.IMPORTANCE Despite many attempts to develop novel therapies, EBV-specific therapies currently remain largely investigational, and EBV-associated malignancies are often associated with a worse prognosis. Therefore, there is a clear demand for EBV-specific therapies for both prevention and treatment of virus-associated malignancies. Noncancerous cells preferentially obtain fatty acids from dietary sources, whereas cancer cells will often produce fatty acids themselves by de novo lipogenesis, often becoming dependent on the pathway for cell survival and proliferation. LMP1- and EBV-mediated B-cell growth transformation leads to induction of FASN, a key enzyme responsible for the catalysis of endogenous fatty acids. Preferential killing of LMP1-expressing B cells following inhibition of FASN suggests that targeting LMP-induced lipogenesis is an effective strategy in treating LMP1-positive EBV-associated malignancies. Importantly, targeting unique metabolic perturbations induced by EBV could be a way to explicitly target EBV-positive malignancies and distinguish their treatment from EBV-negative counterparts.

In this Issue

Cancer ScienceVolume 111, Issue 3 p. 764-765 IN THIS ISSUEOpen Access In this Issue Volume 111, Issue 3, March 2020 First published: 07 March 2020 https://doi.org/10.1111/cas.14047AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Discovery of chemical probes that suppress Wnt/β-catenin signaling through high-throughput screening Abnormalities in the Wnt/β-catenin signaling pathway have been noted in a variety of cancers, including colorectal carcinoma (CRC), endometrial carcinoma, esophageal carcinoma, and diffuse large B-cell lymphoma. The frequency of these abnormalities varies, but in cancers like CRC it has been reported as high as 90%. The Wnt/β-catenin signaling pathway and Wnt target genes are known to be proto-oncogenes, cell cycle regulators, and stem cell markers, but there is growing evidence that the pathway also plays a significant role in the immune system. Further improvements in cancer immunotherapy may depend on increased targeting of the Wnt/β-catenin signaling pathway. In this study, Yamaguchi et al comprehensively review the current state of Wnt inhibitors. They cover the six classes, including tankyrase inhibitors, porcupine inhibitors, CK1α activators, inhibitors of β-catenin-TCF interaction, inhibitors of transcriptional coactivators of β-catenin, and inducers of β-catenin degradation. They also discuss the different models of reporter assays that can be used in high-throughput screening (HTS). https://onlinelibrary.wiley.com/doi/10.1111/cas.14297 YES1 activation induces acquired resistance to neratinib in HER2-amplified breast and lung cancers Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that acts with other members of the ErbB family of proteins to induce signaling pathways like PI3K-AKT and MAPK to promote cell proliferation and survival. Several cancers, breast cancer in particular, can be driven by the overexpression of HER2. HER2-targeted drugs, like trastuzumab, have improved outcomes in breast cancer patients, but many of these patients recur because resistance develops. There are already new drugs like neratinib, an irreversible tyrosine kinase inhibitor, that can treat these resistant tumors, but eventually resistance will also develop to neratinib. In this study, Takeda et al have developed neratinib-resistant cells in order to study how this resistance develops. They found that YES1 was upregulated in the neratinib-resistant cell lines and appeared to be responsible for the resistance. Further, they showed that dasatinib, a SRC inhibitor that targets YES1, overcame the resistance. This provides valuable insight into the development of resistance and provides a solution when the resistance develops. https://onlinelibrary.wiley.com/doi/10.1111/cas.14289 Epstein-Barr virus-encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan-2 and synaptotagmin-like-4 in nasopharyngeal carcinoma cells Nasopharyngeal carcinoma (NPC) is particularly prevalent in Southeast Asia and in southern China. Progression of NPC is strongly correlated with the presence of Epstein-Barr virus (EBV). EBV’s oncogenic properties have been attributed to the protein, EBV-encoded latent membrane protein 1 (LMP1). Transmission of extracellular vesicles laden with EBV-encoded LMP1 activates many signaling pathways that promote tumor invasion and progression in the tumor microenvironment (TME). In this study, Liao et al examined the molecular mechanism by with LMP1 promotes EV secretion. They confirmed that LMP1 does in fact promote EV secretion and determined that this was due to the upregulation of syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4). SDC2 promoted formation of EVs, while SYTL4 promoted secretion of EVs. Furthermore, EVs induced by LMP1 led to tumor growth in vivo. This study provides valuable mechanistic information into the progression and metastasis of NPC. https://onlinelibrary.wiley.com/doi/10.1111/cas.14305 From our sister journal—A Foxp3 variant, neutral at steady state, enhances antitumor immunity Immune surveillance of tumors and efficacy of tumor immunotherapies are restricted by natural mechanisms of immune tolerance, and highly variable between individuals. The role of host-, rather than tumor-genetics underlying this variance has been overlooked. In this issue, Almeida-Santos et al. tested three tumor models with different immunogenicity and invasiveness in mice carrying the commonly used Foxp3fGFP allele, which is neutral at steady state in reference strains. The inspection of Foxp3fGFP allele revealed the cryptic immunogenicity of a primary tumor otherwise fully tolerated, delayed metastasis dissemination of an invasive tumor, and transformed anti-CTLA4 therapy of a resistant melanoma from inefficient to efficacious. These findings reinforce the notion that experimental variations can be related to reporter alleles, and highlight that identification of weak hypomorphs in genes related to immune regulation may guide cancer prognostics and therapeutic strategies. Reprinted with Permission: Eur J Immunol 2020, https://doi.org/10.1002/eji.201948251 References Almeida-Santos J, Bergman ML, Amendoeira Cabral I, Correia V, Caramalho Í, Demengeot J. Eur. J. Immunol. 2020. https://doi.org/10.1002/eji.201948251 Volume111, Issue3March 2020Pages 764-765 ReferencesRelatedInformation

Epstein-Barr virus-encoded latent membrane protein 1 induces epithelial to mesenchymal transition by inducing V-set Ig domain containing 4 (VSIG4) expression via NF-kB in renal tubular epithelial HK-2 cells

The epithelial to mesenchymal transition (EMT), a hallmark of chronic kidney disease, is a key event in the conversion from tubular epithelial cells to myofibroblasts in renal fibrosis. Epstein-Barr virus (EBV) is a γ-herpes oncovirus associated with chronic kidney disease. However, the relationship between EBV and the EMT process in renal tubular epithelial cells is not well understood. Among EBV-latent genes, EBV-encoded latent membrane protein 1 (LMP1) induces EMT by regulating a variety of molecules in EBV-induced oncogenic transformation. In this study, we investigated EBV-encoded LMP1 and EMT process markers in human proximal tubule epithelial cell line HK-2. LMP1 overexpression induces cell morphological changes via the epithelial to mesenchymal process in HK-2 cells, and these changes accelerate cell proliferation, cell motility, and invasion. Furthermore, VSIG4 upregulation by EBV-LMP1 induced LMP1-mediated EMT, cell motility, and invasion. VSIG4 upregulation by LMP1 was regulated at the transcriptional level via the NF-kB signaling axis. These results suggest that EBV-encoded LMP1 regulates EMT through the NF-kB-VSIG4 axis in HK-2 cells, and VSIG4 is a potential target in EBV-induced chronic kidney diseases.

The anecdote of viral etiopathogenia in ameloblastoma and odontogenic keratocyst: Why don’t we let it go?

BackgroundAmeloblastoma (AM) and odontogenic Keratocyst (OKC) are destructive odontogenic lesions of the gnathion. Although their exact pathogeneses are not yet totally understood, the viral etiopathogenesis in AM and KCOT has been proposed. True to syndromic keratocystic odontogenic tumor (sKCOT) and non-syndromic OKC is the high recurrence rate. ObjectivesGiven that shared pathways trailed by AM and by sKCOT/OKC have been suggested, this study, however, contrasts the expression of AM and OKC for viral antibodies. MethodA total of archival 80 paraffin blocks of cases of parakeratinized odontogenic keratocyst (non-syndromic KCOTs) and of ameloblastomas (n=40 for each) were included in this study to be sectioned and stained for two immunohistochemical markers: anti-human papillomavirus and Epstein-Barr virus-encoded latent membrane protein. ResultsAll the submitted cases of AM and parakeratinized OKC were negative for both markers: anti-HPV and anti-LMP-1. ConclusionsAlthough results could have been biased, given the same ethnic group and territory examined in this study, all cases were negative for both markers. Therefore, the viral contribution to the etiopathogenesis in AM and OKC could not be established in this study.

Novel roles and therapeutic targets of Epstein-Barr virus-encoded latent membrane protein 1-induced oncogenesis in nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) was first discovered 50 years ago as an oncogenic gamma-1 herpesvirus and infects more than 90% of the worldwide adult population. Nasopharyngeal carcinoma (NPC) poses a serious health problem in southern China and is one of the most common cancers among the Chinese. There is now strong evidence supporting a role for EBV in the pathogenesis of NPC. Latent membrane protein 1 (LMP1), a primary oncoprotein encoded by EBV, alters several functional and oncogenic properties, including transformation, cell death and survival in epithelial cells in NPC. LMP1 may increase protein modification, such as phosphorylation, and initiate aberrant signalling via derailed activation of host adaptor molecules and transcription factors. Here, we summarise the novel features of different domains of LMP1 and several new LMP1-mediated signalling pathways in NPC. When then focus on the potential roles of LMP1 in cancer stem cells, metabolism reprogramming, epigenetic modifications and therapy strategies in NPC.

Epstein-Barr virus-encoded latent membrane protein 2A promotes the epithelial-mesenchymal transition in nasopharyngeal carcinoma via metastatic tumor antigen 1 and mechanistic target of rapamycin signaling induction.

Epstein-Barr virus-encoded latent membrane protein 2A (LMP2A) promotes the epithelial-mesenchymal transition (EMT) of nasopharyngeal carcinoma (NPC), thereby increasing tumor invasion. Recently, the dysregulation of metastatic tumor antigen 1 (MTA1) was found to enhance tumor metastasis in a variety of cancers. A molecular connection between these two proteins has been proposed but not firmly established. In this study, we reported the overexpression of MTA1 in 29/60 (48.3%) NPC patients, and the overexpression of MTA1 significantly correlated with tumor metastasis. The overexpression of MTA1 promoted EMT via the Wnt1 pathway and β-catenin activation. We demonstrated that LMP2A reinforces the expression of MTA1 via the mechanistic target of rapamycin (mTOR) pathway to promote EMT in NPC. Furthermore, by knocking down 4EBP1 in combination with the new mTOR inhibitor INK-128 treatment, we discovered that LMP2A expression activates the 4EBP1-eIF4E axis and increases the expression of MTA1 at the translational level partially independent of c-myc. These findings provided novel insights into the correlation between the LMP2A and MTA1 proteins and reveal a novel function of the 4EBP1-eIF4E axis in EMT of nasopharyngeal carcinoma. Importance: Prevention of the recurrence and metastasis of NPC is critical to achieving a successful NPC treatment. As we all know, EMT has a vital role in metastasis of malignancies. LMP2A, an oncoprotein of Epstein-Barr virus, a well-known NPC activator, induces EMT and has been proved to exert a promoting effect in tumor metastasis. Our study demonstrated that LMP2A could induce EMT by activating MTA1 at the translational level via activating mTOR signaling and the 4EBP1-eIF4E axis. Taken together, our findings bridge the gap between the NPC-specific cell surface molecule and the final phenotype of the NPC cells. Additionally, our findings indicate that LMP2A and mTOR will serve as targets for NPC therapy in the future.

Expression of Epstein-Barr Virus-encoded Latent Membrane Protein (LMP-1), p16 and p53 Proteins in Nonendemic Nasopharyngeal Carcinoma (NPC): A Clinicopathological Study

Although the latent membrane protein type 1 (LMP1) is frequently expressed in Epstein-Barr virus (EBV) malignancies, its contribution to the pathogenesis of nasopharyngeal carcinoma (NPC) is not fully defined. LMP1 functions as a viral mimic of the TNFR family member engaging a number of signaling pathways that induce morphological and phenotypic alterations. This study aimed to investigate the LMP1 expression and EBV infection in relation to clinical outcome and survival in a series of Mexican NPC patients. We also studied expression of p16 and p53 proteins. We analyzed in 25 tumor specimens the expression of LMP1, p16 and p53 by immunohistochemistry (IHC) and EBV presence by IHC/in situ hybridization. Differences in clinical outcome and survival in relation to protein expression were correlated through χ(2) statistics and Kaplan-Meier survival curves. Our results showed a rate of 92% (23/25) of EBV infection. The expressions of LMP-1, p16 and p53 proteins were 40.0, 44.0 and 40.0%, respectively. LMP-1 immunoexpression was more common in older patients (>50 vs. <50 years old, p = 0.02) and with parapharyngeal space invasion (p = 0.02). The presence of metastatic disease at diagnosis (p = 0.03), distant recurrence disease (p = 0.006) and shorter distance recurrence-free survival (p = 0.05) was associated with lack of p16. In our series, EBV infection rates are particularly high for nonendemic NPC, although without a statistically significant difference in overall survival, LMP1 and p16 expression was correlated with poorer clinical prognosis. Probably, LMP1 and p16 detection identify a worse clinical prognosis in NPC patient subgroup.

Epstein–Barr virus‐encoded LMP1 triggers regulation of the ERK‐mediated Op18/stathmin signaling pathway in association with cell cycle

The MAPKs are activated by a variety of cellular stimuli to participate in a series of signaling cascades and mediate diverse intracellular responses. One potential target of the MAPKs is Op18/stathmin, a molecule that acts as an integrator of diverse cell signaling pathways and regulates the dynamics of microtubules, which are involved in modulating a variety of cellular processes, including cell cycle progression and cell growth. Our study focused on the regulation of the MAPK-mediated Op18/stathmin signaling pathway, which is triggered by the Epstein-Barr virus-encoded latent membrane protein 1 ( LMP1) oncogene in nasopharyngeal carcinoma cells. The results showed that the activity of MAPK, which was induced by LMP1, varied with cell cycle progression; LMP1 upregulated phosphorylation of ERK during the G(1)/S phase, but negatively regulated phosphorylation of ERK during the G(2)/M phase. We found that the regulation of Op18/stathmin signaling by LMP1 was mainly mediated through ERK. The inhibition of LMP1 expression attenuated the interaction of ERK with Op18/stathmin and promoted microtubule depolymerization. These findings indicate the existence of a new cell cycle-associated signaling pathway in which LMP1 regulates ERK-mediated Op18/stathmin signaling.

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 (LMP1) and LMP2A Function Cooperatively To Promote Carcinoma Development in a Mouse Carcinogenesis Model

The Epstein-Barr virus (EBV) proteins latent membrane proteins 1 and 2 (LMP1 and LMP2) are frequently expressed in EBV-associated lymphoid and epithelial cancers and have complex effects on cell signaling and growth. The effects of these proteins on epithelial cell growth were assessed in vivo using transgenic mice driven by the keratin 14 promoter (K14). The development of papillomas and carcinomas was determined in the tumor initiator and promoter model using dimethyl benzanthracene (DMBA), followed by repeated treatments of 12-O-tetradecanoyl phorbol 13-acetate (TPA). In these assays, LMP1 functioned as a weak tumor promoter and increased papilloma formation. In contrast, mice expressing LMP2A did not induce or promote papilloma formation. Transgenic LMP1 mice had slightly increased development of squamous cell carcinoma; however, the development of carcinoma was significantly increased in the doubly transgenic mice expressing both LMP1 and LMP2A. DMBA treatment induces an activating mutation in the Harvey-ras (H-ras(61)) oncogene, and this mutation was identified in most papillomas and carcinomas although several papillomas and carcinomas in K14-LMP1 and K14-LMP1/LMP2A mice lacked the mutation. Analysis of signaling pathways that are known to be activated by LMP1 and/or LMP2 indicated that all genotypes had high levels of activated extracellular signal-regulated kinase (ERK) and Stat3 in carcinomas with significantly higher activation in the doubly transgenic carcinomas. These findings suggest that, in combination, LMP1 and LMP2 contribute to carcinoma progression and that this may reflect the combined effects of the proteins on activation of multiple signaling pathways. This study is the first to characterize the effects of LMP2 on tumor initiation and promotion and to identify an effect of the combined expression of LMP1 and LMP2 on the increase of carcinoma development.

Latency Type-Dependent Modulation of Epstein-Barr Virus-Encoded Latent Membrane Protein 1 Expression by Type I Interferons in B Cells

We report that type I interferons (IFNs) upregulate latent membrane protein 1 (LMP-1) expression by direct activation of the ED-L1 promoter in several Epstein-Barr virus (EBV)-carrying Burkitt's lymphoma lines. In EBV-infected primary B cells, IFN-α transiently upregulates LMP-1 mRNA, but not protein levels, followed by downregulation of both, suggesting a novel antiproliferative mechanism of type I IFNs. Furthermore, our results may explain the expression of LMP-1 in memory B cells of systemic lupus erythematosus patients.

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 (LMP1) and LMP2A Function Cooperatively To Promote Carcinoma Development in a Mouse Carcinogenesis Model

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 (LMP1) and LMP2A Function Cooperatively To Promote Carcinoma Development in a Mouse Carcinogenesis Model

MiRNA and nasopharyngeal carcinoma

MicroRNAs (miRNAs) constitute a family of small non-coding RNA molecules 22–25 nucleotides in length. miRNAs control the expression of target genes at the post-transcriptional level by inhibiting translation or by degrading target mRNA through binding to complementary sequences in the 3′-untranslated regions. Recent studies have demonstrated that miRNAs are intimately involved in processes leading to nasopharyngeal carcinoma, such as the Epstein-Barr virus-encoded latent membrane protein 1 activated signal transduction pathways, gene-regulatory networks, mitosis, tumor angiogenesis, invasion and migration. Exploring the relationship between miRNAs and the development of nasopharyngeal carcinoma, will further the understanding of this cancer and provide new avenues for diagnosis and treatment.

EBV-encoded LMP1 increases nuclear β-catenin accumulation and its transcriptional activity in nasopharyngeal carcinoma

This paper aimed to study whether Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) regulates β-catenin signaling pathway in nasopharyngeal carcinoma (NPC). Western blotting, immunofluorescence, luciferase reporter assay, co-immunoprecipitation assay, and immunohistochemistry staining were used. LMP1 increased β-catenin transcriptional activity in NPC cell lines. The upregulation of β-catenin transcriptional activity induced by LMP1 was much higher in poorly differentiated NPC cell line CNE2 than that in well-differentiated NPC cell line CNE1. Immunofluorescence staining and Western blotting also showed that LMP1 increased nuclear β-catenin accumulation in NPC cell lines. Moreover, LMP1 expression was closely related to abnormal β-catenin expression in NPC tissues by immunohistochemistry. LMP1 may be involved in nasopharyngeal carcinogenesis via β-catenin signaling pathway.

This article has been retracted: Epstein-Barr virus-encoded latent membrane protein 1 activates β-catenin signaling in B lymphocytes

This article has been retracted at the request of Editor-in-Chief & Author. The following article from Cancer Science: Mariko Tomita, Zahidunnabi Dewan, Naoki Yamamoto, Akira Kikuchi and Naoki Mori. Epstein–Barr virus-encoded latent membrane protein 1 activates β-catenin signaling in B lymphocytes, Cancer Science 2009; 100: 807–812, (doi: 10.1111/j.1349-7006.2009.01121.x), published online on 20 March 2009 on Wiley Interscience (http://www.interscience.wiley.com), now Wiley Online Library (http://onlinelibrary.wiley.com/), has been retracted by agreement between the authors, the journal Editor-in-Chief, Yusuke Nakamura, and Blackwell Publishing Asia Pty Ltd. All authors wish to retract this paper due to repeated use of images in the same gel in two figures within the article. Yusuke NakamuraEditor-in-ChiefCancer Science

The MYND domain-containing protein BRAM1 inhibits lymphotoxin beta receptor-mediated signaling through affecting receptor oligomerization

MYND (myeloid-Nervy-DEAF-1) domains exist in a large number of proteins that are functionally important in development or associated with cancers. We have previously demonstrated that a MYND domain-containing protein, the bone morphogenesis protein receptor-associated molecule 1 (BRAM1), is able to interact with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1), which acts as a constitutively activated tumor necrosis factor receptor (TNFR). Herein we further demonstrated that BRAM1 additionally associates with the TNFR-superfamily member, the lymphotoxin beta receptor (LTβR), and hence inhibits LTβR-mediated function. Using the yeast two-hybrid assay, we demonstrated that BRAM1 interacts with LTβR mainly through the self-association domain of LTβR (aa 336–398). The co-immunoprecipitation experiment further revealed that BRAM1 as well as MYND domain-containing proteins, MTG8 and DEAF-1, interacts with LTβR via their MYND domains. The BRAM1-LTβR interaction impedes the self-association of LTβR and the recruitment of TNFR-associated factors 2 and 3 (TRAF2 and TRAF3), leading to abolishment of LTβR-induced NF-κB signaling, JNK activation, and caspase-dependent cell death. In sum, our data demonstrate that the MYND-containing protein BRAM1 abrogates LTβR function through a protein–protein interaction. These findings may provide a direction for the treatment of dysregulation of LTβR-mediated signaling.

Influence of suppression of Epstein-Barr Virus-encoded latent membrane protein 1 by rAAV vector mediated RNA interference on metastatic ability of nasopharyngeal cancer cells in vivo

To study the effect of Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP-1) on the tumor growth and metastasis of nasopharyngeal carcinoma (NPC) cells in vivo and its possible mechanism. To construct two recombinant adeno-associated virus (rAAV): rAAV-shRNA-LMP-1 and rAAV-EGFP (enhanced green fluorescent protein). Multiplicity of infection (MOI) was confirmed by using different titre of rAAV-EGFP to transfect a NPC cell line, C666-1. Then C666-1 cells were transfected by rAAV-shRNA-LMP-1 at MOI titre and the inhibiting efficiency of target gene's expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). C666-1 cells treated by RNAi on LMP-1 in vitro was directly inoculated into the liver via laparotomy under direct vision to establish the animal model of NPC xenograft in liver and lung metastasis from the liver. The biological effect and its mechanism after "gene silencing" of LMP-1 on NPC cells tumorigenesis and metastasis were observed by the primary intrahepatic tumor formation and lung metastasis and the expression of matrix metalloproteinases-9 (MMP-9) revealed by immunohistochemistry. The transfection efficiency was higher than 95% with 5 x 10(4) virus genome (v.g)/cell with rAAV-EGFP. The expression of target gene was inhibited more than 90%, assessed by RT-PCR after transfection with rAAV-shRNA-LMP-1 at a dose of 5 x 10(4) v.g/cell. The primary tumor volume implanted in the liver of rAAV-shRNA-LMP-1 treatment was (0.2527 +/- 0.1152) cm3, with no significant difference in comparrison with rAAV-EGFP control group [(0.2533 +/- 0.0754) cm3, P>0.05]. But the rate of intrahepatic tumor formation was 50.0% and the rate of lung metastasis was 33.3% of the rAAV-shRNA-LMP-1 group, significantly lower than those in the rAAV-EGFP group (P<0.05). The survival time (15.50 +/- 2.47) d of rAAV-shRNA-LMP-1 group was significantly longer than that in the rAAV-EGFP group [(11.50 +/- 1.22) d, P<0.05]. Immunohistochemistry indicated suppression of LMP-1 by rAAV-shRNA-LMP-1 can down-regulate the expression of MMP-9 significantly. The expression of LMP-1 can be suppressed effectively by rAAV mediated RNA interference. The suppression of LMP-1 expression has no effect on cell growth but can inhibit the metastasis in vivo, probably through down-regulating the expression of MMP-9.

Granulomatous Inflammatory Pseudotumor of the Spleen

A 74-year-old woman with a clinical history of invasive ductal carcinoma of the breast was found to have a splenic mass during a routine radiographic survey. Splenectomy revealed a 3-cm well-demarcated lesion, which on histopathologic examination consisted of heterogeneous inflammatory cells. A striking feature of the lesion was the presence of innumerable well-formed non-necrotizing granulomas. Immunohistochemical studies confirmed the lesion to be composed mainly of mixed T and B lymphocytes, histiocytes, and plasma cells. No spindle cell component was evident on light microscopic examination or by immunohistochemical staining for smooth muscle actin, anaplastic lymphoma kinase, or follicular dendritic cell markers CD21 and CD35. Interestingly, Epstein-Barr virus-encoded RNA and latent membrane protein were detected by in situ hybridization and immunohistochemistry in numerous lymphohistiocytic cells within the lesion, but not in surrounding uninvolved splenic tissue. To our knowledge, this case represents a rare example of splenic inflammatory pseudotumor with exuberant granulomatous reaction in association with Epstein-Barr viral infection.

THIS ARTICLE HAS BEEN RETRACTED Epstein–Barr virus‐encoded latent membrane protein 1 activates β‐catenin signaling in B lymphocytes

The Epstein-Barr virus-encoded latent membrane protein 1 is considered the Epstein-Barr virus oncogene based on its importance in Epstein-Barr virus-induced B-lymphocyte transformation. Beta-catenin is a potential oncogene, and its accumulation has been implicated in a variety of human cancers. Here, we found that beta-catenin protein was highly expressed in Epstein-Barr virus-immortalized B-cell lines compared with peripheral blood mononuclear cells from healthy donors. Beta-catenin expression in Epstein-Barr virus-immortalized B-cell line decreased following treatment with LY294002, an inhibitor of phosphatidylinositol 3-kinase. Treatment with LY294002 or knockdown of beta-catenin by small interfering RNA reduced the growth of Epstein-Barr virus-immortalized B-cell line. Transient transfection of latent membrane protein 1 expression plasmid increased beta-catenin protein expression and beta-catenin-dependent transcription. Latent membrane protein 1 deletions mutants lacking the carboxyl-terminal activating region 1 domain failed to enhance beta-catenin protein expression and beta-catenin-dependent transcriptional activity. They also failed to increase phosphorylated AKT expression. Dominant-negative AKT suppressed latent membrane protein 1-induced beta-catenin-dependent transcriptional activity. These results suggest that latent membrane protein 1 activates beta-catenin through the phosphatidylinositol 3-kinase/AKT signaling pathway. Activation of the beta-catenin pathway by Epstein-Barr virus may contribute to the lymphoproliferation characteristic of Epstein-Barr virus-infected B-cells.