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Invadopodia are actin-rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they were 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. This article is protected by copyright. All rights reserved.

Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer., The genomic characterisation of nasopharyngeal carcinoma (NPC) remains crucial. Here, the authors perform whole-genome sequencing for 70 NPCs with EBV gene expression, report the somatic alterations and EBV-mediated effects converging on NF-κB activation and immune escape and identify targetable homozygous MTAP deletions.

Nasopharyngeal cancer (NPC) is an Epstein-Barr virus (EBV)–positive epithelial malignancy with an extensive inflammatory infiltrate. Traditional RNA-sequencing techniques uncovered only microenvironment signatures, while the gene expression of the tumor epithelial compartment has remained a mystery. Here, we use Smart-3SEQ to prepare transcriptome-wide gene expression profiles from microdissected NPC tumors, dysplasia, and normal controls. We describe changes in biological pathways across the normal to tumor spectrum and show that fibroblast growth factor (FGF) ligands are overexpressed in NPC tumors, while negative regulators of FGF signaling, including SPRY1, SPRY2, and LGALS3, are down-regulated early in carcinogenesis. Within the NF-κB signaling pathway, the critical noncanonical transcription factors, RELB and NFKB2, are enriched in the majority of NPC tumors. We confirm the responsiveness of EBV-positive NPC cell lines to targeted inhibition of these pathways, reflecting the heterogeneity in NPC patient tumors. Our data comprehensively describe the gene expression landscape of NPC and unravel the mysteries of receptor tyrosine kinase and NF-κB pathways in NPC.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated cancer characterized by a high degree of recurrence, angiogenesis, and metastasis. The importance of alternative pro-angiogenesis pathways including viral factors has emerged after decades of directly targeting various signaling components. Using NPC as a model, we identified an essential oncogenic pathway underlying angiogenesis regulation that involves the inhibition of a tumor suppressor, Spry3, and its downstream targets by EBV-miR-BART10-5p (BART10-5p) and hsa-miR-18a (miR-18a). Overexpression of EBV-miR-BART10-5p and hsa-miR-18a strongly promotes angiogenesis in vitro and in vivo by regulating the expression of VEGF and HIF1-α in a Spry3-dependent manner. In vitro or in vivo treatment with iRGD-tagged exosomes containing antagomiR-BART10-5p and antagomiR-18a preferentially suppressed the angiogenesis and growth of NPC. Our findings first highlight the role of EBV-miR-BART10-5p and oncogenic hsa-miR-18a in NPC angiogenesis and also shed new insights into the clinical intervention and therapeutic strategies for nasopharyngeal carcinoma and other virus-associated tumors., Graphical Abstract, Wang et al. generated a novel oncogenic pathway underlying angiogenesis regulation that involves EBV-miR-BART10-5p and hsa-miR-18a inhibiting tumor suppressor Spry3 and its downstream targets. They established a synergistic role between virus and host microRNAs in the regulation of angiogenesis of virus-associated cancer.

Nasopharyngeal carcinoma (NPC) is an Epstein‐Barr virus (EBV)‐associated epithelial malignancy. The high expression of BART‐miRNAs (miR‐BARTs) during latent EBV infection in NPC strongly supports their pathological importance in cancer progression. Recently, we found that several BART‐miRNAs work co‐operatively to modulate the DNA damage response (DDR) by reducing Ataxia‐telangiectasia‐mutated (ATM) activity. In this study, we further investigated the role of miR‐BARTs on DDR. The immunohistochemical study showed that the DNA repair gene, BRCA1, is consistently down‐regulated in primary NPCs. Using computer prediction programs and a series of reporter assays, we subsequently identified the negative regulatory role of BART2‐3p, BART12, BART17‐5p and BART19‐3p in BRCA1 expression. The ectopic expression of these four miR‐BARTs suppressed endogenous BRCA1 expression in EBV‐negative epithelial cell lines, whereas BRCA1 expression was enhanced by repressing endogenous miR‐BARTs activities in C666‐1 cells. More importantly, suppressing BRCA1 expression in nasopharyngeal epithelial cell lines using miR‐BART17‐5p and miR‐BART19‐3p mimics reduced the DNA repair capability and increased the cell sensitivity to the DNA‐damaging chemotherapeutic drugs, cisplatin and doxorubicin. Our findings suggest that miR‐BARTs play a novel role in DDR and may facilitate the development of effective NPC therapies.

Nasopharyngeal carcinoma is an Epstein–Barr virus (EBV)-related malignancy. Recently, we found that the EBV-encoded miRNA BART2-5p was increased in the serum of patients with preclinical nasopharyngeal carcinoma and that the copy number positively correlated with disease progression. In this study, we established its role in nasopharyngeal carcinoma progression and explored underlying mechanisms and clinical significance. BART2-5p was an independent unfavorable prognostic factor for progression-free survival and its circulating abundance positively associated with distant metastasis. Ectopic expression of BART2-5p promoted migration and invasion of EBV-negative nasopharyngeal carcinoma cells, whereas genetic downregulation of BART2-5p in EBV-positive nasopharyngeal carcinoma cells decreased aggressiveness. Mechanistically, BART2-5p targeted RND3, a negative regulator of Rho signaling. Downregulation of RND3 phenocopied the effect of BART2-5p and reconstitution of RND3 rescued the phenotype. By suppressing RND3, BART2-5p activated Rho signaling to enhance cell motility. These findings suggest a novel role for EBV miRNA BART2-5p in promoting nasopharyngeal carcinoma metastasis and its potential value as a prognostic indicator or therapeutic target. Significance: This study shows that EBV-encoded BART2-5p miRNA suppresses expression of the RND3 Rho family GTPase, consequently promoting ROCK signaling, cell motility, and metastatic behavior of NPC cells.

Nasopharyngeal carcinoma (NPC) is a highly metastatic cancer that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we identify for the first time a role for monoamine oxidase A (MAOA) in NPC. MAOA is a mitochondrial enzyme that catalyzes oxidative deamination of neurotransmitters and dietary amines. Depending on the cancer type, MAOA can either have a tumour-promoting or tumour-suppressive role. We show that MAOA is down-regulated in primary NPC tissues and its down-regulation enhances the migration of NPC cells. In addition, we found that EBV infection can down-regulate MAOA expression in both pre-malignant and malignant nasopharyngeal epithelial (NPE) cells. We further demonstrate that MAOA is down-regulated as a result of IL-6/IL-6R/STAT3 signalling and epigenetic mechanisms, effects that might be attributed to EBV infection in NPE cells. Taken together, our data point to a central role for EBV in mediating the tumour suppressive effects of MAOA and that loss of MAOA could be an important step in the pathogenesis of NPC.

Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and contributes to both B-cell and epithelial-cell malignancies. EBV-infected epithelial cell tumors, including nasopharyngeal carcinoma (NPC), are largely composed of latently infected cells, but the mechanism(s) maintaining viral latency are poorly understood. Expression of the EBV BZLF1 (Z) and BRLF1 (R) encoded immediate-early (IE) proteins induces lytic infection, and these IE proteins activate each other’s promoters. ΔNp63α (a p53 family member) is required for proliferation and survival of basal epithelial cells and is over-expressed in NPC tumors. Here we show that ΔNp63α promotes EBV latency by inhibiting activation of the BZLF1 IE promoter (Zp). Furthermore, we find that another p63 gene splice variant, TAp63α, which is expressed in some Burkitt and diffuse large B cell lymphomas, also represses EBV lytic reactivation. We demonstrate that ΔNp63α inhibits the Z promoter indirectly by preventing the ability of other transcription factors, including the viral IE R protein and the cellular KLF4 protein, to activate Zp. Mechanistically, we show that ΔNp63α promotes viral latency in undifferentiated epithelial cells both by enhancing expression of a known Zp repressor protein, c-myc, and by decreasing cellular p38 kinase activity. Furthermore, we find that the ability of cis-platinum chemotherapy to degrade ΔNp63α contributes to the lytic-inducing effect of this agent in EBV-infected epithelial cells. Together these findings demonstrate that the loss of ΔNp63α expression, in conjunction with enhanced expression of differentiation-dependent transcription factors such as BLIMP1 and KLF4, induces lytic EBV reactivation during normal epithelial cell differentiation. Conversely, expression of ΔNp63α in undifferentiated nasopharyngeal carcinoma cells and TAp63α in Burkitt lymphoma promotes EBV latency in these malignancies., Author summary Epstein-Barr virus (EBV) is an important cause of both epithelial cell and B cell human cancers. EBV-infected tumors have predominantly latent viral infection, allowing them to escape the cell killing that occurs during lytic viral infection. EBV is highly lytic in normal differentiated oral epithelial cells. Thus, an important question is how the virus maintains the latent form of viral infection in EBV-associated epithelial cell tumors such as undifferentiated nasopharyngeal carcinoma (NPC). This study demonstrates that the cellular transcription factor ΔNp63ɑ, which is specifically expressed in undifferentiated basal epithelial cells and is over-expressed in NPC tumors, maintains EBV latency by inhibiting the activity of the viral immediate-early (IE) promoter (Zp) that drives expression of the BZLF1 IE protein. A related splice variant, TAp63α, found in some EBV+ lymphomas, has a similar inhibitory effect. Our findings reveal that ΔNp63ɑ and TAp63α contribute to EBV latency in both epithelial and B cell tumors. Furthermore, since differentiation results in loss of ΔNp63ɑ expression, our results help to explain why lytic EBV reactivation is promoted by epithelial cell differentiation.

Purpose We longitudinally evaluated the tumour growth and metabolic activity of two well-established nasopharyngeal carcinoma (NPC) animal models (C666-1, C17) and three novel models (Xeno76, Xeno23 and NPC43) using a microPET/MR system. With a better understanding of the interplay between tumour growth and metabolic characteristics of these NPC models, we aim to provide insights for the selection of appropriate NPC cell line/xenograft models to assist novel drug discovery and evaluation. Methods Mice were imaged by [18F]FDG microPET/MR twice a week for consecutive 3–7 weeks. [18F]FDG uptake was quantified by standardized uptake value (SUV) and presented as SUVmean tumour-to-liver ratio (SUVRmean). Longitudinal tumour growth patterns and metabolic patterns were recorded. SUVRmean and histological characteristics were compared across the five NPC models. Cisplatin was administrated to one selected optimal tumour model, C17 to evaluate our imaging platform. Results We found variable tumour growth and metabolic patterns across different NPC tumour types. C17 has an optimal growth rate and higher tumour metabolic activity compared with C666-1. C666-1 has a fast growth rate but is low in SUVRmean at endpoint due to necrosis as confirmed by H&E. NPC43 and Xeno76 have relatively slow growth rates and are low in SUVRmean, due to severe necrosis. Xeno23 has the slowest growth rate, and a relative high SUVRmean. Cisplatin showed the expected therapeutic effect in the C17 model in marked reduction of tumour size and metabolism. Conclusion Our study establishes an imaging platform that characterizes the growth and metabolic patterns of different NPC models, and the platform is well able to demonstrate drug treatment outcome supporting its use in novel drug discovery and evaluation for NPC.

The Epstein–Barr virus M81 strain, isolated from a nasopharyngeal carcinoma, induces potent spontaneous virus production in infected B cells. We found that the M81 non-coding Epstein–Barr-encoded RNA EBER2, which carries polymorphisms that are mainly restricted to viruses found in endemic nasopharyngeal carcinomas, markedly stimulated this process. M81 EBER2 increased CXCL8 expression, and this chemokine enhanced spontaneous lytic replication levels in M81-infected B cells. Both events resulted from the endocytosis of extracellular vesicles containing EBER2 that were generated by neighbouring M81-infected B cells, thereby generating a paracrine loop. These effects were strictly dependent on a functional Toll-like receptor 7 (TLR7), a sensor of single-stranded RNA located in the endosome of these cells. These unique properties of M81 EBER2 could be ascribed to its unusually high expression level and to the ability of its single-stranded region to activate TLR7; both of these properties were dependent on M81-specific polymorphisms. Thus, M81 induced chronic inflammation in its target cells and this resulted in increased virus production. These observations provide a mechanistic molecular link between M81 virus replication—a central viral function and a cancer risk factor—and the production of a chemokine involved in inflammation and carcinogenesis. The non-coding RNA EBER2 of the Epstein–Barr virus M81 strain potentiates virus lytic replication in B cells by generating a paracrine loop whereby the chemokine CXCL8 is released from infected cells via extracellular vesicles—which are taken up by neighbouring cells—thereby enhancing its own expression.

We and others have previously shown that the canonical nuclear factor kappa-B (NF-κB) pathway is essential to nasopharyngeal carcinoma (NPC) tumor development and angiogenesis, suggesting that the NF-κB pathway, including its upstream modulators and downstream effectors, are potential therapeutic targets for NPC. The inhibitor of upstream IκB kinase (IKK), PS1145, is a small molecule which can specifically inhibit the IκB phosphorylation and degradation and the subsequent nuclear translocation of NF-κB. The present study aims to determine the anti-tumor activity of PS1145 on NPC. Our results showed that PS1145 significantly inhibited the growth of tumorigenic NPC cell lines, but not in the normal nasopharyngeal epithelial cell line. Results in the in vivo study showed that low concentration of PS1145 (3 mg/kg) could significantly suppress the subcutaneous tumor formation in the nude mice bearing NPC xenografts. Apparent adverse effects were not observed in the animal study. Drug resistance against PS1145 seems to be associated with the increased levels of active NF-kB p65 and change of expression levels of kruppel-like factor 4. As can be seen, PS1145 appears to be a safe agent for animal experiments and its effects are tumor-specific, and the proteins associated with the drug resistance of PS1145 are implied.

Epstein-Barr virus (EBV) infection is strongly associated with nasopharyngeal carcinoma, a common cancer in Southeast Asia and certain regions of Africa. However, the dynamics of EBV episome maintenance in infected nasopharyngeal epithelial (NPE) cells remain largely undefined. Here, we report the establishment of a highly efficient cell-free EBV infection method for NPE cells. By using this method, we have defined some of the dynamic events involved in the early stage of EBV infection in NPE cells. We report, for the first time, a rapid loss of EBV copies from infected NPE cells during the first 12-72 h post-infection. The rate of EBV loss slowed at later stages of infection. Live cell imaging revealed that the freshly infected NPE cells were delayed in entry into mitosis compared with uninfected cells. Freshly infected NPE cells transcribed significantly higher levels of lytic EBV genes BZLF1 and BMRF1 yet significantly lower levels of EBER1/2 than stably infected NPE cells. Notably, there were very low or undetectable levels of protein expressions of EBNA1, LMP1, Zta and Rta in freshly infected NPE cells, whereas EBNA1 and LMP1 proteins were readily detected in stable EBV-infected NPE cells. The kinetics of EBV loss and the differential EBV gene expression profiles between freshly and stably infected NPE cells are in line with the suggestion of epigenetic changes in the EBV genome that affect viral gene expression and the adaptation of host cells to EBV infection to maintain persistent EBV infection in NPE cells.

EBV infection of preinvasive nasopharyngeal epithelium is believed to be an initiation step during pathogenesis of nasopharyngeal carcinoma (NPC), but the mechanisms remain poorly understood. Here we report a novel mechanism driving NPC metastasis through the EBV-encoded LMP1-mediated metabolic reprogramming, via activation of IGF1-mTORC2 signaling and nuclear acetylation of the Snail promoter by the PDHE1α, an enzyme involved in glucose metabolism. Mechanistically, EBV-LMP1 increases the cellular secretion of IGF1 which promotes phosphorylation of IGF1R to activate mTORC2/AKT signaling linking glucose metabolism to cell motility. LMP1 expression facilitates translocation of mitochondrial PDHE1α into the nucleus in a phosphorylation-dependent manner at Ser293 residue. Functionally, nuclear PDHE1α promotes H3K9 acetylation on the Snail promoter to enhance cell motility, thereby driving cancer metastasis. Importantly, the IGF1/mTORC2/PDHE1α/Snail axis correlates significantly with disease progression and poor prognosis in NPC patients. This study highlights the functional importance of IGF1-mTORC2-PDHE1α signaling mediated by EBV-LMP1 in NPC pathogenesis.

Deletion of Chromosome 3p is one of the most frequently detected genetic alterations in nasopharyngeal carcinoma (NPC). We reported the role of a novel 3p26.3 tumor suppressor gene (TSG) CHL1 in NPC. Down-regulation of CHL1 was detected in 4/6 of NPC cell lines and 71/95 (74.7%) in clinical tissues. Ectopic expressions of CHL1 in NPC cells significantly inhibit colony formation and cell motility in functional study. By up-regulating epithelial markers and down-regulating mesenchymal markers CHL1 could induce mesenchymal-epithelial transition (MET), a key step in preventing tumor invasion and metastasis. CHL1 could also cause the inactivation of RhoA/Rac1/Cdc42 signaling pathway and inhibit the formation of stress fiber, lamellipodia, and filopodia. CHL1 could co-localize with adhesion molecule Integrin-β1, the expression of CHL1 was positively correlated with Integrin-β1 and another known tumor suppressor gene (TSG) Merlin. Down-regulation of Integrin-β1 or Merlin was significantly correlated with the poor survival rate of NPC patients. Further mechanistic studies showed that CHL1 could directly interact with integrin-β1 and link to Merlin, leading to the inactivation of integrin β1-AKT pathway. In conclusion, CHL1 is a vital tumor suppressor in the carcinogenesis of NPC.

Background Recent genomic analyses revealed that druggable molecule targets were only detectable in approximately 6% of patients with nasopharyngeal carcinoma (NPC). However, a dependency on dysregulated CDK4/6–cyclinD1 pathway signaling is an essential event in the pathogenesis of NPC. In this study, we aimed to evaluate the therapeutic efficacy of a specific CDK4/6 inhibitor, palbociclib, and its compatibility with other chemotherapeutic drugs for the treatment of NPC by using newly established xenograft models and cell lines derived from primary, recurrent, and metastatic NPC. Methods We evaluated the efficacies of palbociclib monotherapy and concurrent treatment with palbociclib and cisplatin or suberanilohydroxamic acid (SAHA) in NPC cell lines and xenograft models. RNA sequencing was then used to profile the drug response–related pathways. Palbociclib-resistant NPC cell lines were established to determine the potential use of cisplatin as a second-line treatment after the development of palbociclib resistance. We further examined the efficacy of palbociclib treatment against cisplatin-resistant NPC cells. Results In NPC cells, palbociclib monotherapy was confirmed to induce cell cycle arrest in the G1 phase in vitro. Palbociclib monotherapy also had significant inhibitory effects in all six tested NPC tumor models in vivo, as indicated by substantial reductions in the total tumor volumes and in Ki-67 proliferation marker expression. In NPC cells, concurrent palbociclib treatment mitigated the cytotoxic effect of cisplatin in vitro. Notably, concurrent treatment with palbociclib and SAHA synergistically promoted NPC cell death both in vitro and in vivo. This combination also further inhibited tumor growth by inducing autophagy-associated cell death. NPC cell lines with induced palbociclib or cisplatin resistance remained sensitive to treatment with cisplatin or palbociclib, respectively. Conclusions Our study findings provide essential support for the use of palbociclib as an alternative therapy for NPC and increase awareness of the effective timing of palbociclib administration with other chemotherapeutic drugs. Our results provide a foundation for the design of first-in-human clinical trials of palbociclib regimens in patients with NPC.

One of the greatest unmet needs hindering the successful treatment of nasopharyngeal carcinomas (NPCs) is for representative physiological and cost-effective models. Although Epstein–Barr virus (EBV) infection is consistently present in NPCs, most studies have focused on EBV-negative NPCs. For the first time, we established and analyzed three-dimensional (3D) spheroid models of EBV-positive and EBV-negative NPC cells and compared these to classical two-dimensional (2D) cultures in various aspects of tumor phenotype and drug responses. Compared to 2D monolayers, the 3D spheroids showed significant increases in migration capacity, stemness characteristics, hypoxia and drug resistance. Co-culture with endothelial cells, which mimics essential interactions in the tumor microenvironment, effectively enhanced spheroid dissemination. Furthermore, RNA sequencing revealed significant changes at the transcriptional level in 3D spheroids compared to expression in 2D monolayers. In particular, we identified known (VEGF, AKT and mTOR) and novel (Wnt–β-catenin and Eph–ephrin) cell signaling pathways that are activated in NPC spheroids. Targeting these pathways in 3D spheroids using FDA-approved drugs was effective in monoculture and co-culture. These findings provide the first demonstration of the establishment of EBV-positive and EBV-negative NPC 3D spheroids with features that resemble advanced and metastatic NPCs. Furthermore, we show that NPC spheroids have potential use in identifying new drug targets.

Background. Nasopharyngeal carcinoma is a chemosensitive cancer characterized by prominent lymphocytic infiltration. However, recurrence remains a major challenge in treating this disease. Here, we examined the effects of idronoxil (IDX) on tumor-lymphocyte interactions. Methods The antitumor activity of cisplatin and/or IDX was tested in authentic EBV-positive NPC-derived cell lines (C666, C17 and NPC43). We analyzed 2D monolayer and 3D spheroids cocultures of NPC cell lines with immune cells to assess the infiltration, activation, phenotype and function of T cells toward human nasopharyngeal tumors in vitro. Flow cytometric and immunohistochemical analyses were carried out to evaluate infiltrating T cells in subcutaneous mice models. Results. Administration of low-dose of IDX (1-2 µM) not only sensitizes cancer cells to chemotherapy via apoptosis, but also promotes T cell proliferation and cytotoxicity. More importantly, combination treatment of IDX and cisplatin in cancer cells can enhance the expansion, trafficking and priming of CD4+, CD8+ and double-positive (DP) T cells. Mechanistically, we showed that the increase in migratory DP T cells was largely mediated by the CXCL10/type I interferon (IFN) axis, where IDX precipitates an elevation in IFNα levels and leads to CXCL10 induction. Additionally, IDX enhanced T-cell infiltration towards tumor spheroids with acquisition of increased tissue homing and functional abilities (attenuated CD62L and PD1 expression). Conclusions. Our findings underscore the potential of IDX to induce immunomodulatory responses, and in combination with conventional chemotherapies, enhance antitumor immunity.

The Wnt signaling pathway is one of the major signaling pathways used by cancer stem cells (CSC). Ecotropic Viral Integration Site 1 (EVI1) has recently been shown to regulate oncogenic development of tumor cells by interacting with multiple signaling pathways, including the Wnt signaling. In the present study, we found that the Wnt modulator ICG-001 could inhibit the expression of EVI1 in nasopharyngeal carcinoma (NPC) cells. Results from loss-of-function and gain-of-function studies revealed that EVI1 expression positively regulated both NPC cell migration and growth of CSC-enriched tumor spheres. Subsequent studies indicated ICG-001 inhibited EVI1 expression via upregulated expression of miR-96. Results from EVI1 3&prime, UTR luciferase reporter assay confirmed that EVI1 is a direct target of miR-96. Further mechanistic studies revealed that ICG-001, overexpression of miR-96, or knockdown of EVI1 expression could restore the expression of miR-449a. The suppressive effect of miR-449a on the cell migration and tumor sphere formation was confirmed in NPC cells. Taken together, the miR-96/EVI1/miR-449a axis is a novel pathway involved in ICG-001-mediated inhibition of NPC cell migration and growth of the tumor spheres.

Nasopharyngeal cancer (NPC), endemic in Southeast Asia, lacks effective diagnostic and therapeutic strategies. Even in high-income countries the 5-year survival rate for stage IV NPC is less than 40%. Here we report high somatostatin receptor 2 (SSTR2) expression in multiple clinical cohorts comprising 402 primary, locally recurrent and metastatic NPCs. We show that SSTR2 expression is induced by the Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) via the NF-κB pathway. Using cell-based and preclinical rodent models, we demonstrate the therapeutic potential of SSTR2 targeting using a cytotoxic drug conjugate, PEN-221, which is found to be superior to FDA-approved SSTR2-binding cytostatic agents. Furthermore, we reveal significant correlation of SSTR expression with increased rates of survival and report in vivo uptake of the SSTR2-binding 68Ga-DOTA-peptide radioconjugate in PET-CT scanning in a clinical trial of NPC patients (NCT03670342). These findings reveal a key role in EBV-associated NPC for SSTR2 in infection, imaging, targeted therapy and survival., Nasopharyngeal carcinoma (NPC) lacks effective diagnostic and therapeutic strategies, in particular at advanced stages. Here, the authors show that expression of the somatostatin receptor 2 is induced by Epstein-Barr virus in NPC and has a key role in the diagnosis, imaging, targeted therapies and prognosis of NPC.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein-Barr virus (EBV) infection. In NPC, miR-BARTs, the EBV-encoded miRNAs derived from BamH1-A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV-encoded miRNAs in a panel of NPC patient-derived xenografts and an EBV-positive NPC cell line by small RNA sequencing. Among the 40 miR-BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV-miRNAs, BART5-5p, BART7-3p, BART9-3p, and BART14-3p could negatively regulate the expression of a key DNA double-strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'-UTR. Notably, the expression of these four miR-BARTs represented more than 10% of all EBV-encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT-PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5-5p, BART7-3p, BART9-3p, and BART14-3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR-BARTs in EBV-positive NPC cells, we further demonstrated the novel function of miR-BARTs in inhibiting Zta-induced lytic reactivation. These findings imply that the four viral miRNAs work co-operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine-1-phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV-encoded latent genes (EBNA1, LMP1 and LMP2A) can up-regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is over-expressed in EBV-positive NPC patient-derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P-induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease.

In our previous whole-transcriptome sequencing analysis, downregulation of a long non-coding RNA, maternally expressed gene 3 (MEG3), was identified in NPC samples. This finding suggests the possible role of MEG3 as a tumor suppressor in this distinctive disease. In the present study, two MEG3 variants, AF119863 (MEG3-AF) and BX247998 (MEG3-BX), were found abundantly expressed in a normal nasopharyngeal epithelial cell line, NP69. Significant downregulation of MEG3-AF was further verified in a panel of NPC samples including xenografts and primary biopsies. MEG3 is an imprinted gene located within chromosome 14q32, a common deleted region in NPC. Both DNA copy number loss and aberrant promoter methylation contributed to MEG3 inactivation. Interestingly, MEG3 expression could successfully be rescued by the treatment of a demethylation agent. Besides, ectopic expression of MEG3 in NPC cell lines resulted in considerable repression of in vitro anchorage-independent growth and in vivo tumorigenicity, in addition to significant inhibition in cell proliferation, colony formation, and induction of cell cycle arrest. Finally, we revealed the association between MEG3 activity and the p53 signaling cascade. Our findings characterize MEG3 as a tumor suppressive long non-coding RNA in NPC and encourage the development of precise long non-coding RNA-targeted epigenetic therapy against this malignancy. © 2016 Wiley Periodicals, Inc.

Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients respond poorly to treatment. New treatments are therefore needed to improve the outcome of NPC. To better understand the molecular pathogenesis of NPC, here we used an NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways essential for NPC (i.e. dependence factors). This screen identified the Moz, Ybf2/Sas3, Sas2, Tip60 histone acetyl transferase complex, NF-κB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene as NPC dependence factors/pathways. Using gene knock out, complementary DNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.

The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC., The lack of appropriate models restricts pre-clinical research for nasopharyngeal carcinoma (NPC). Here the authors report the development and characterization of NPC patient-derived xenografts (PDXs), and EBV positive NPC cell line from patient tumor, and suggest their potential use in future NPC research.

NF-κB is a key regulator of inflammatory response and is frequently activated in human cancer including the undifferentiated nasopharyngeal carcinoma (NPC), which is common in Southern China including Hong Kong. Activation of NF-κB is common in NPC and may contribute to NPC development. The role of NF-κB activation in immortalization of nasopharyngeal epithelial (NPE) cells, which may represent an early event in NPC pathogenesis, is unknown. Examination of NF-κB activation in immortalization of NPE cells is of particular interest as the site of NPC is often heavily infiltrated with inflammatory cellular components. We found that constitutive activation of NF-κB signaling is a common phenotype in telomerase-immortalized NPE cell lines. Our results suggest that NF-κB activation promotes the growth of telomerase-immortalized NPE cells, and suppression of NF-κB activity inhibits their proliferation. Furthermore, we observed upregulation of c-Myc, IL-6 and Bmi-1 in our immortalized NPE cells. Inhibition of NF-κB downregulated expression of c-Myc, IL-6 and Bmi-1, suggesting that they are downstream events of NF-κB activation in immortalized NPE cells. We further delineated that EGFR/MEK/ERK/IKK/mTORC1 is the key upstream pathway of NF-κB activation in immortalized NPE cells. Elucidation of events underlying immortalization of NPE cells may provide insights into early events in pathogenesis of NPC. The identification of NF-κB activation and elucidation of its activation mechanism in immortalized NPE cells may reveal novel therapeutic targets for treatment and prevention of NPC.

Whether certain variants of Epstein-Barr virus (EBV) are linked to the pathogenesis of nasopharyngeal carcinoma (NPC), which shows a marked geographic restriction, remains an unresolved issue. We performed a case-control study comparing genomic sequences of EBV isolated from saliva samples of 142 population carriers with those from primary tumour biopsies derived from 62 patients with NPC of Hong Kong. Cluster analysis discovered five EBV subgroups 1A-C and 2A-B amongst the population carriers in contrast to the predominance of 1A and -B in the majority of NPC. Genome-wide association study (GWAS) identified a panel of NPC-associated single nucleotide polymorphisms (SNPs) and indels in the EBER locus. The most significant polymorphism, which can be found in 96.8% NPC cases and 40.1% population carriers of Hong Kong, is a four-base-deletion polymorphism downstream of EBER2 (EBER-del) from coordinates 7188-7191 (p = 1.91 × 10-7 ). In addition, the predicted secondary structure of EBER2 is altered with likely functional consequence in nearly all NPC cases. Using the SNPs and indels associated with NPC, genetic risk score is assigned for each EBV variant. EBV variants with high genetic risk score are found to be much more prevalent in Hong Kong Chinese than individuals of other geographic regions and in NPC than other EBV-associated cancers. We conclude that high risk EBV variants with polymorphisms in the EBER locus, designated as HKNPC-EBERvar, are strongly associated with NPC. Further investigation of the biological function and potential clinical application of these newly identified polymorphisms in NPC and other EBV-associated cancers is warranted.

Abnormal metabolism and uncontrolled angiogenesis are two important characteristics of malignant tumors. The occurrence of both events involves many key molecular changes including miRNA. However, EBV encoded miRNAs are rarely mentioned as capable of regulating tumor metabolism and tumor angiogenesis. Here, we reported that one of the key miRNAs encoded by EBV, EBV-miR-Bart1-5P, can significantly promote nasopharyngeal carcinoma (NPC) cell glycolysis and induces angiogenesis in vitro and in vivo. Mechanistically, EBV-miR-Bart1-5P directly targets the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1) and consequently regulates the AMPK/mTOR/HIF1 pathway which impelled NPC cell anomalous aerobic glycolysis and angiogenesis, ultimately leads to uncontrolled growth of NPC. Our findings provide new insights into metabolism and angiogenesis of NPC and new opportunities for the development of targeted NPC therapy in the future., Author summary The Epstein-Barr virus (EBV), the first reported human tumor virus found to encode miRNAs, which closely related to malignant progression of tumors. In our study, we have observed that EBV-miR-BART1-5P, an EBV-BARTs encoded miRNA, promotes glycolysis and induces angiogenesis in NPC. Interestingly, we showed that overexpression of EBV-miR -BART1-5P and restored PTEN at the same time, did not completely reverse the phenotypes of glycolysis, angiogenesis and proliferation, suggesting that EBV-miR-BART1-5P can mediate glycolysis and induction angiogenesis by a PTEN-independent manner. Further mechanism exploration demonstrated that EBV-miR-BART1-5P has important roles in cancer cell glucose metabolism and angiogenesis by inhibiting AMPKα1 and PTEN, which provides a molecular basis for the regulation of AMPK/mTOR/HIF1 and PTEN/FAK, Shc, AKT pathways, respectively.

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the few viral proteins expressed by EBV in nasopharyngeal carcinoma (NPC), most likely because of its essential role in maintaining the viral genome in EBV-infected cells. In NPC, EBNA1 expression is driven by the BamHI-Q promoter (Qp), which is regulated by both cellular and viral factors. We previously determined that the expression of another group of EBV transcripts, BamHI-A rightward transcripts (BARTs), is associated with constitutively activated nuclear factor-κB (NF-κB) signaling in NPC cells. Here, we show that, like the EBV BART promoter, the EBV Qp also responds to NF-κB signaling. NF-κB p65, but not p50, can activate Qp in vitro, and NF-κB signaling regulates Qp-EBNA1 expression in NPC cells, as well as in other EBV-infected epithelial cells. The introduction of mutations in the putative NF-κB site reduced Qp activation by the NF-κB p65 subunit. Binding of p65 to Qp was shown by chromatin immunoprecipitation (ChIP) analysis, while electrophoretic mobility shift assays (EMSAs) demonstrated that p50 can also bind to Qp. Inhibition of NF-κB signaling by the IκB kinase inhibitor PS-1145 resulted in the downregulation of Qp-EBNA1 expression in C666-1 NPC cells. Since EBNA1 has been reported to block p65 activation by inhibiting IKKα/β through an unknown mechanism, we suggest that, in NPC, NF-κB signaling and EBNA1 may form a regulatory loop which supports EBV latent gene expression, while also limiting NF-κB activity. These findings emphasize the role of NF-κB signaling in the regulation of EBV latency in EBV-associated tumors.

Undifferentiated nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. In addition, heavy infiltration of leukocytes is a common characteristic of EBV-associated NPC. It has long been suggested that substantial and interactive impacts between cancer and stromal cells create a tumor microenvironment (TME) to promote tumorigenesis. The coexistence of tumor-infiltrating lymphocytes with EBV-infected NPC cells represents a distinct TME which supports immune evasion and cancer development from the early phase of EBV infection. Intracellularly, EBV-encoded viral products alter host cell signaling to facilitate tumor development and progression. Intercellularly, EBV-infected cancer cells communicate with stromal cells through secretion of cytokines and chemokines, or via release of tumor exosomes, to repress immune surveillance and enhance metastasis. Although high expression of miR-BARTs has been detected in NPC patients, contributions of these more recently discovered viral products to the establishment of TME are still vaguely defined. Further investigations are needed to delineate the mechanistic linkage of the interplay between viral and host factors, especially in relation to TME, which can be harnessed in future therapeutic strategies.

To establish persistent infection in cells, viruses evolve strategies to alter host cellular pathways to regulate cell proliferation and energy metabolism which support viral infection. Epstein-Barr virus (EBV) undergoes both lytic and latent infection to achieve persistent and lifelong infection in human. EBV readily infects human B cells, driving their transformation to proliferative lymphoblastoid cell lines (LCL), and eventually establishes lifelong latent infection in memory B cells. In contrary, EBV undergoes lytic replication upon infection into normal epithelial cells which is essential for the replication of EBV genome and production of infectious viral particles for transmission through saliva. EBV shuttles between B cells and epithelial cells to complete its infection cycle. EBV infection is closely associated with nasopharyngeal carcinoma (NPC) and is present in practically 100% of undifferentiated NPC. In contrast to undergo lytic infection of normal pharyngeal epithelium, EBV establishes latent infection in NPC. The switch from lytic infection to latent infection may represent an early and essential step in the development of NPC. Recent studies in both B cells and NPC cells latently infected with EBV reveal alterations in cell metabolism to support persistent and latent EBV infection. Events underlying the switching of lytic to latent EBV infection in NPC cells are largely undefined. Molecular events and alterations of cell metabolism are likely to play crucial roles in switching EBV infection from lytic to latent in NPC cells. Latent EBV infection and expression of viral genes, including LMP1, LMP2, and possibly EBV-encoded micro RNAs, may play essential roles in alterations of cell metabolism to support NPC pathogenesis. Alteration of energy metabolism is an essential hallmark of cancer. The role of altered energy metabolism in host cells in modulating latent and lytic EBV infection in NPC cells is unclear. In this review, we will discuss the impact of genetic alterations in NPC to module cellular metabolism and its influence on latent infection and lytic reactivation of EBV infection in NPC cells. In particular, the role of EBV-encoded genes in driving glucose metabolism and their contribution to NPC pathogenesis will be discussed. This new perspective on the interplay between EBV infection and altered host metabolic pathways in NPC pathogenesis may offer novel and effective therapeutic strategies in the treatment of NPC and other EBV-associated malignancies.

Pan-histone deacetylase (HDAC) inhibitors, which inhibit 11 HDAC isoforms, are widely used to induce Epstein-Barr virus (EBV) lytic cycle in EBV-associated cancers in vitro and in clinical trials. Here, we hypothesized that inhibition of one or several specific HDAC isoforms by selective HDAC inhibitors could potently induce EBV lytic cycle in EBV-associated malignancies such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). We found that inhibition of class I HDACs, particularly HDAC-1, -2 and -3, was sufficient to induce EBV lytic cycle in NPC and GC cells in vitro and in vivo. Among a panel of selective HDAC inhibitors, the FDA-approved HDAC inhibitor romidepsin was found to be the most potent lytic inducer, which could activate EBV lytic cycle at ∼0.5 to 5 nM (versus ∼800 nM achievable concentration in patients' plasma) in more than 75% of cells. Upregulation of p21(WAF1) , which is negatively regulated by class I HDACs, was observed before the induction of EBV lytic cycle. The upregulation of p21(WAF1) and induction of lytic cycle were abrogated by a specific inhibitor of PKC-δ but not the inhibitors of PI3K, MEK, p38 MAPK, JNK or ATM pathways. Interestingly, inhibition of HDAC-1, -2 and -3 by romidepsin or shRNA knockdown could confer susceptibility of EBV-positive epithelial cells to the treatment with ganciclovir (GCV). In conclusion, we demonstrated that inhibition of class I HDACs by romidepsin could potently induce EBV lytic cycle and mediate enhanced cell death with GCV, suggesting potential application of romidepsin for the treatment of EBV-associated cancers.

Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1 and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future.

The TP53induced glycolysis and apoptosis regulator (TIGAR) is the protein product of the p53 target gene, C12orf5. TIGAR blocks glycolysis and promotes cellular metabolism via the pentose phosphate pathway; it promotes the production of cellular nicotinamide adenine dinucleotide phosphate (NADPH), which leads to enhanced scavenging of intracellular reactive oxygen species, and inhibition of oxidative stressinduced apoptosis in normal cells. Our previous study identified a novel nucleoside analog that inhibited cellular growth and induced apoptosis in nasopharyngeal carcinoma (NPC) cell lines via downregulation of TIGAR expression. Furthermore, the growth inhibitory effects of cMet tyrosine kinase inhibitors were ameliorated by the overexpression of TIGAR in the NPC cell lines. These results indicate a significant role for TIGAR expression in the survival of NPCs. The present study aimed to further define the function of TIGAR expression in NPC cells. In total, 36 formalinfixed, paraffinembedded NPC tissue samples were obtained for the immunohistochemical determination of TIGAR expression. The effects of TIGAR expression on cell proliferation, NADPH production and cellular invasiveness were also assessed in NPC cell lines. Overall, TIGAR was overexpressed in 27/36 (75%) of the NPC tissues compared with the adjacent noncancer epithelial cells. Similarly, TIGAR overexpression was also observed in a panel of six NPC cell lines compared with normal NP460 hTert and Het1A cell lines. TIGAR overexpression led to increased cellular growth, NADPH production and invasiveness of the NPC cell lines, whereas a knockdown of TIGAR expression resulted in significant inhibition of cellular growth and invasiveness. The expression of the two mesenchymal markers, fibronectin and vimentin, was increased by TIGAR overexpression, but reduced following TIGARknockdown. The present study revealed that TIGAR overexpression led to increased cellular growth, NADPH production and invasiveness, and the maintenance of a mesenchymal phenotype, in NPC tissues., published_or_final_version

The Epstein–Barr virus (EBV) is a ubiquitous virus that infects most of the human population. EBV infection is associated with multiple human cancers, including Burkitt’s lymphoma, Hodgkin’s lymphoma, a subset of gastric carcinomas, and almost all undifferentiated non-keratinizing nasopharyngeal carcinoma. Intensive research has shown that EBV triggers a DNA damage response (DDR) during primary infection and lytic reactivation. The EBV-encoded viral proteins have been implicated in deregulating the DDR signaling pathways. The consequences of DDR inactivation lead to genomic instability and promote cellular transformation. This review summarizes the current understanding of the relationship between EBV infection and the DDR transducers, including ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase), and discusses how EBV manipulates the DDR signaling pathways to complete the replication process of viral DNA during lytic reactivation.

Epstein-Barr virus (EBV) infects more than 90% of the adult human population. Undifferentiated nasopharyngeal carcinoma (NPC) is common in Southeast Asia, with a particularly high incidence among southern Chinese. The EBV genome can be detected in practically all cancer cells in undifferentiated NPC. The role of EBV in pathogenesis of undifferentiated NPC remains elusive. NPC cell lines are known to be difficult to establish in culture. The EBV+ve NPC cell lines, even if established in culture, rapidly lost their EBV episomes upon prolonged propagation. At present, the C666-1 NPC cell line, which is defective in lytic EBV reactivation, is the only EBV+ve NPC cell line available for NPC and EBV research. The need to establish new and representative NPC cell lines is eminent for NPC and EBV research. In this study, we report the use of the Rho-associated kinase inhibitor (Y-27632) has facilitated the establishment of a new EBV+ve NPC cell line from an earlier established NPC xenograft, C17. The C17 cell line was tumorigenic in immune-deficient mice (NOD/SCID). It retained the EBV episomes and could be induced to undergo productive lytic reactivation of EBV to generate infectious virus particles. The C17 cell line represents a new investigative tool for NPC and EBV studies. The ability of C17 to undergo lytic reactivation is unique and opens up the opportunity to examine regulation of latent and lytic infection of EBV and their contributions to NPC pathogenesis.

Epstein–Barr nuclear antigen 1 (EBNA1), a dimeric oncoprotein of the Epstein–Barr virus (EBV), is essential for both viral-genome maintenance and the survival of infected cells. Despite EBNA1’s potential as a therapeutic target, tools for the direct monitoring of EBNA1 in vitro and in vivo are lacking. Here, we show that a peptide-based inhibitor that luminesces when bound to EBNA1 inside the nucleus of EBV+ cells can regulate EBNA1 homodimer formation and selectively inhibit the growth of EBV+ tumours of nasopharyngeal carcinoma cells (C666-1 and NPC43) and Burkitt’s lymphoma Raji cells. We also show that the peptide-based probe leads to 93% growth inhibition of EBV+ tumours in mice. Our findings support the hypothesis that selective inhibition of EBNA1 dimerization can be used to afford better EBV-related cancer differentiation, and highlight the potential application of the probe as a new generation of biotracers for investigating the fundamental biological function of EBNA1 and for exploring its application as a therapeutic target. A peptide-based fluorescent inhibitor of the dimerization of an oncoprotein of the Epstein–Barr virus blocks the proliferation of tumours associated with the virus in mice.

To establish persistent infection in cells, viruses evolve strategies to alter host cellular pathways to regulate cell proliferation and energy metabolism which support viral infection. Epstein-Barr virus (EBV) undergoes both lytic and latent infection to achieve persistent and lifelong infection in human. EBV readily infects human B cells, driving their transformation to proliferative lymphoblastoid cell lines (LCL), and eventually establishes lifelong latent infection in memory B cells. In contrary, EBV undergoes lytic replication upon infection into normal epithelial cells which is essential for the replication of EBV genome and production of infectious viral particles for transmission through saliva. EBV shuttles between B cells and epithelial cells to complete its infection cycle. EBV infection is closely associated with nasopharyngeal carcinoma (NPC) and is present in practically 100% of undifferentiated NPC. In contrast to undergo lytic infection of normal pharyngeal epithelium, EBV establishes latent infection in NPC. The switch from lytic infection to latent infection may represent an early and essential step in the development of NPC. Recent studies in both B cells and NPC cells latently infected with EBV reveal alterations in cell metabolism to support persistent and latent EBV infection. Events underlying the switching of lytic to latent EBV infection in NPC cells are largely undefined. Molecular events and alterations of cell metabolism are likely to play crucial roles in switching EBV infection from lytic to latent in NPC cells. Latent EBV infection and expression of viral genes, including LMP1, LMP2, and possibly EBV-encoded micro RNAs, may play essential roles in alterations of cell metabolism to support NPC pathogenesis. Alteration of energy metabolism is an essential hallmark of cancer. The role of altered energy metabolism in host cells in modulating latent and lytic EBV infection in NPC cells is unclear. In this review, we will discuss the impact of genetic alterations in NPC to module cellular metabolism and its influence on latent infection and lytic reactivation of EBV infection in NPC cells. In particular, the role of EBV-encoded genes in driving glucose metabolism and their contribution to NPC pathogenesis will be discussed. This new perspective on the interplay between EBV infection and altered host metabolic pathways in NPC pathogenesis may offer novel and effective therapeutic strategies in the treatment of NPC and other EBV-associated malignancies.

Epstein–Barr virus (EBV) is associated with multiple types of human cancer, including lymphoid and epithelial cancers. The closest association with EBV infection is seen in undifferentiated nasopharyngeal carcinoma (NPC), which is endemic in the southern Chinese population. A strong association between NPC risk and the HLA locus at chromosome 6p has been identified, indicating a link between the presentation of EBV antigens to host immune cells and NPC risk. EBV infection in NPC is clonal in origin, strongly suggesting that NPC develops from the clonal expansion of a single EBV-infected cell. In epithelial cells, the default program of EBV infection is lytic replication. However, latent infection is the predominant mode of EBV infection in NPC. The establishment of latent EBV infection in pre-invasive nasopharyngeal epithelium is believed to be an early stage of NPC pathogenesis. Recent genomic study of NPC has identified multiple somatic mutations in the upstream negative regulators of NF-κB signalling. Dysregulated NF-κB signalling may contribute to the establishment of latent EBV infection in NPC. Stable EBV infection and the expression of latent EBV genes are postulated to drive the transformation of pre-invasive nasopharyngeal epithelial cells to cancer cells through multiple pathways. This article is part of the themed issue ‘Human oncogenic viruses’.

Nasopharyngeal carcinoma (NPC) is a common disease among southern Chinese. The major etiological factors proposed for NPC pathogenesis include genetic susceptibility, environment factors and EBV infection. In the high risk population, genetic susceptibility to NPC has been mapped to the HLA loci and adjacent genes in MHC region on chromosome 6p21. Consumption of preserved food including salted fish has been implicated in its etiology in earlier studies. Its contribution to pathogenesis of NPC remains to be determined. A decreasing trend of NPC incidence was observed in Hong Kong, Taiwan and Singapore in recent years which may be accounted by a change of dietary habits. A comprehensive epidemiological study will help to elucidate the relative importance of various risk factors in the pathogenesis of NPC. Despite the close association of EBV infection with NPC, the etiological role of EBV in NPC pathogenesis remains enigmatic. EBV infection in primary nasopharyngeal epithelial cells is uncommon and difficult to achieve. EBV does not transform primary nasopharyngeal epithelial cells into proliferative clones, which contrasts greatly with the well-documented ability of EBV to transform and immortalize primary B cells. Genetic alterations identified in premalignant nasopharyngeal epithelium may play crucial roles to support stable EBV infection. Subsequently, latent and lytic EBV gene products may drive clonal expansion and transformation of premalignant nasopharyngeal epithelial cells into cancer cells. Stromal inflammation in nasopharyngeal mucosa is believed to play an important role in modulating the growth and possibly drive the malignant transformation of EBV-infected nasopharyngeal epithelial cells. Furthermore, there are increasing evidences supporting a role of EBV infection to evade host immune surveillance. EBV-infected cells may have selective growth advantages in vivo by acquiring a stress-resistance phenotype. Understanding the etiological factors and pathogenesis of NPC will contribute effectively to the prevention and treatment of this disease.

Epstein–Barr virus (EBV) encodes at least 44 mature microRNAs (miRNAs), some of which are abundantly expressed in nasopharyngeal carcinoma cells. EBV-encoded miR-BART6 miRNA is known to undergo adenosine-to-inosine (A-to-I) RNA editing, which impacts on processing and function. Whether additional EBV miRNAs might be A-to-I edited remains to be determined. In this study, we have reported on A-to-I editing of EBV miR-BART3. The A-to-I editing enzyme was expressed abundantly in EBV-infected epithelial carcinoma cells. pri-miR-BART3 was found to be edited at four sites in these cells and in nasopharyngeal carcinoma samples. Whereas editing of the second site located within the seed region prevented the targeting of DICE1 mRNA, editing of the third site effectively crippled the biogenesis of mature miR-BART3. Thus, A-to-I editing perturbs biogenesis and targeting of miR-BART3 and may contribute to its differential expression and function in EBV-infected epithelial cells.

Nasopharyngeal carcinoma is a rare but highly invasive cancer. As options of agents for effective combination chemoradiotherapy for advanced nasopharyngeal carcinoma are limited, novel therapeutic approaches are desperately needed. The ubiquitin ligase CHFR is known to target PARP1 for degradation and is epigenetically inactivated in nasopharyngeal carcinoma. We present evidence that PARP1 protein is indeed overexpressed in nasopharyngeal carcinoma cells in comparison with immortalized normal nasopharyngeal epithelial cells. Tissue microarray analysis also indicated that PARP1 protein is significantly elevated in primary nasopharyngeal carcinoma tissues, with strong correlation with all stages of nasopharyngeal carcinoma development. We found that the PARP inhibitor AZD2281 (olaparib) increased DNA damage, cell-cycle arrest, and apoptosis in nasopharyngeal carcinoma cells challenged with ionizing radiation or temozolomide. Isobologram analysis confirmed that the cytotoxicity triggered by AZD2281 and DNA-damaging agents was synergistic. Finally, AZD2281 also enhanced the tumor-inhibitory effects of ionizing radiation in animal xenograft models. These observations implicate that PARP1 overexpression is an early event in nasopharyngeal carcinoma development and provide a molecular basis of using PARP inhibitors to potentiate treatment of nasopharyngeal carcinoma with radio- and chemotherapy. Mol Cancer Ther; 12(11); 2517–28. ©2013 AACR.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated tumor prevalent in southern China and southeast Asia, with the 3p14-p12 locus reported as a critical tumor suppressor gene (TSG) region during its pathogenesis. We identified a novel 3p14.2 TSG, FEZF2 (FEZ family zinc finger 2), for NPC. FEZF2 is readily expressed in normal tissues including upper respiratory epithelium, testis, brain and ovary tissues, as well as in immortalized nasopharyngeal epithelial cell line NP69, but it is completely silenced in NPC cell lines due to CpG methylation of its promoter, although no homozygous deletion of FEZF2 was detected. 5-Aza-2'-deoxycytidine treatment restored FEZF2 expression in NPC cell lines along with its promoter demethylation. FEZF2 was frequently downregulated in NPC tumors, with promoter methylation detected in 75.5% of tumors, but only in 7.1% of normal nasopharyngeal tissues. Restored FEZF2 expression suppressed NPC cell clonogenicity through inducing G2/M cell cycle arrest and apoptosis and also inhibited NPC cell migration and stemness. FEZF2 acted as a histone deacetylase-associated repressor downregulating multiple oncogenes including EZH2 and MDM2, through direct binding to their promoters. Concomitantly, overexpression of EZH2 was frequently detected in NPC tumors. Thus, we have identified FEZF2 as a novel 3p14.2 TSG frequently inactivated by promoter methylation in NPC, which functions as a repressor downregulating multiple oncogene expression.

Nasopharyngeal carcinoma (NPC), an endemic head and neck cancer found in Southeast Asia, is etiologically associated with the infection of an oncogenic virus, the Epstein-Barr virus (EBV). Here, we review literature findings on the potential/putative role of STAT3 signaling in its pathogenesis and discuss anti-STAT3 targeting as a therapeutic and possibly prevention strategy for NPC. Lastly, the potential involvement of STAT3 in other oncovirus-associated (e.g. the human papillomavirus) head and neck cancers have not been investigated and we hope that findings in EBV-associated NPC can prompt future investigations in these cancers, which are of increasing impact in the Western countries.

Latent infection with Epstein-Barr virus (EBV) is associated with several types of malignancies including nasopharyngeal carcinoma (NPC), which is particularly more prevalent in Southern China. EBV expresses at least 44 mature microRNAs (miRNAs) to modulate the activity of viral and cellular RNAs, but the targets of these EBV-encoded miRNAs in NPC are not well understood. In this report, we characterized DICE1 tumor suppressor to be a cellular target of EBV miR-BART3* miRNA. miR-BART3* was abundantly expressed in NPC cells. The target site of miR-BART3* located in the 3'-untranslated region of DICE1 transcript was identified and characterized. Enforced expression of miR-BART3* or its precursor pre-miR-BART3 led to down-regulation of endogenous DICE1 expression. Inhibition of endogenous miR-BART3* in NPC cells with anti-miR-BART3* oligonucleotide inhibitor resulted in increased expression of DICE1 protein. On the contrary, expression of miR-BART3* overcame the growth suppressive activity of DICE1 and stimulated cell proliferation. Consistent with its tumor suppressive function, DICE1 was underexpressed in EBV-expressing NPC tumor tissues. Taken together, our findings suggest that EBV encoded miR-BART3* miRNA targets DICE1 tumor suppressor to promote cellular growth and transformation in NPC.

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine-1-phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV-encoded latent genes (EBNA1, LMP1, and LMP2A) can up-regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is overexpressed in EBV-positive NPC patient-derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P-induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

In our previous whole-transcriptome sequencing analysis, downregulation of a long non-coding RNA, maternally expressed gene 3 (MEG3), was identified in NPC samples. This finding suggests the possible role of MEG3 as a tumor suppressor in this distinctive disease. In the present study, two MEG3 variants, AF119863 (MEG3-AF) and BX247998 (MEG3-BX), were found abundantly expressed in a normal nasopharyngeal epithelial cell line, NP69. Significant downregulation of MEG3-AF was further verified in a panel of NPC samples including xenografts and primary biopsies. MEG3 is an imprinted gene located within chromosome 14q32, a common deleted region in NPC. Both DNA copy number loss and aberrant promoter methylation contributed to MEG3 inactivation. Interestingly, MEG3 expression could successfully be rescued by the treatment of a demethylation agent. Besides, ectopic expression of MEG3 in NPC cell lines resulted in considerable repression of in vitro anchorage-independent growth and in vivo tumorigenicity, in addition to significant inhibition in cell proliferation, colony formation, and induction of cell cycle arrest. Finally, we revealed the association between MEG3 activity and the p53 signaling cascade. Our findings characterize MEG3 as a tumor suppressive long non-coding RNA in NPC and encourage the development of precise long non-coding RNA-targeted epigenetic therapy against this malignancy. © 2016 Wiley Periodicals, Inc.

Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer characterized by Epstein-Barr virus (EBV) infection and dense lymphocyte infiltration. The scarcity of NPC genomic data hinders the understanding of NPC biology, disease progression and rational therapy design. Here we performed whole-exome sequencing (WES) on 111 micro-dissected EBV-positive NPCs, with 15 cases subjected to further whole-genome sequencing (WGS), to determine its mutational landscape. We identified enrichment for genomic aberrations of multiple negative regulators of the NF-κB pathway, including CYLD, TRAF3, NFKBIA and NLRC5, in a total of 41% of cases. Functional analysis confirmed inactivating CYLD mutations as drivers for NPC cell growth. The EBV oncoprotein latent membrane protein 1 (LMP1) functions to constitutively activate NF-κB signalling, and we observed mutual exclusivity among tumours with somatic NF-κB pathway aberrations and LMP1-overexpression, suggesting that NF-κB activation is selected for by both somatic and viral events during NPC pathogenesis., Nasopharyngeal cancer is frequently characterized by Epstein-Barr virus infection. Here, using genomic analyses, the authors find that the tumours harbour mutations in genes involved in the NF-κB signalling pathway or overexpress a viral oncoprotein, latent membrane protein 1.