EBV-infected Nasopharyngeal Carcinoma Cells Research Articles

Abstract 465: Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors

Abstract Most humans infected with Epstein-Barr virus (EBV) remain asymptomatic throughout the whole life. However, EBV is linked to several human cancers, including Burkitt's lymphoma, Hodgkin's disease, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma (NPC), and gastric carcinoma. Among these EBV-associated tumors, EBV genome is present in 100% of NPC tumor cells. In NPC cells, EBV establishes a latent infection, expressing limited number of viral proteins but elevated levels of non-coding RNAs transcribed from the BamHI-A region of the EBV genome, also known as BamHI-A rightward transcripts (BARTs). The family of EBV BART RNAs includes BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). Although various functions of BART miRNAs have been identified, the role of EBV lnc-BARTs remains largely unknown. In this study, we provide evidence demonstrating that lnc-BARTs functions as a regulatory long non-coding RNA, referred to as lnc-BARTs. These lnc-BARTs play keys role in modulating the core networks that maintains EBV latency and promotes NPC oncogenesis through epigenetic mechanisms. Our study found that the apoptosis rate of NPC cells is significantly increased following the knockdown of EBV lnc-BARTs, indicating their anti-apoptotic role in EBV-associated tumors. Mass spectrometry analysis identified several host factors, including transcriptional regulators BRD4 and SC35’ interact with EBV lnc-BARTs in NPC cells. RNA-FISH assays showed lnc-BARTs co-localized with BRD4 and SC35 complexes in nuclear s peckles and such interaction were disrupted by JQ1, a BRD4 competitive inhibitor. Binding of lnc-BARTs to BRD4 RNA was confirmed using immunoprecipitation and pulldown assays. ATAC sequencing and transcriptome analyses showed that BART knockdown in EBV-harboring NPC cell line C666-1 led to reduced chromatin accessibility, downregulation of oncogenes, and decreased EBV episome replication and maintenance. Quantitative qPCR and DNA FISH analyses of cell samples treated with shRNA targeting lnc-BARTs or BRD4 reduced EBV copy numbers and EBNA1 RNA expression levels in EBV-infected NPC cells. Finally, ChIP sequencing analysis revealed that lnc-BARTs is critical for BRD4 binding to the oriP region of the EBV genome. These findings support a mechanism by which EBV lnc-BARTs epigenetically regulate host gene expression through functional interactions with elongation regulatory machinery and contribute to the maintenance of EBV latent genome by interaction with BRD4 complex. Citation Format: Jiayan Liu, Bobo Wing-Yee Mok, Honglin Chen. Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 465.

The immune escape mechanism of nasopharyngeal carcinoma.

Tumor cells are known for being able to evade immune system surveillance, a hallmark of malignancy. Complicated immune escape mechanisms in the tumor microenvironment (TME) provide favorable conditions for tumor invasion, metastasis, treatment resistance, and recurrence. Epstein-Barr virus (EBV) infection is closely related to the pathogenesis of nasopharyngeal carcinoma (NPC), and the co-existence of EBV-infected NPC cells and tumor-infiltrating lymphocytes represents a distinctive, highly heterogeneous, and suppressive TME that supports immune escape and promotes tumorigenesis. Understanding the complex interaction between EBV and NPC host cells and focusing on the immune escape mechanism of TME may help to identify specific immunotherapy targets and to develop effective immunotherapy drugs.

Abstract 3748: Epstein-Barr virus BART lncRNAs induce IKZF3/Aiolos to maintain EBV latency and promote tumorigenicity in nasopharyngeal carcinoma

Abstract Epstein-Barr virus (EBV) maintains latency in its associated tumors, including nasopharyngeal carcinoma (NPC), expressing very few viral proteins but abundant levels of noncoding RNAs (mainly EBERs and BARTs) in NPC cells. We found that IKZF3/Aiolos is a downstream target of BART lncRNAs in NPC cells. The functions of BART lncRNAs and IKZF3 in EBV latency and pathogenesis of NPC remain elusive. In this study, meta-analysis of eight EBV-associated studies showed that IKZF3/Aiolos was consistently upregulated in EBV-infected NPC cells. Our study further showed that transcription of IKZF3/Aiolos is highly upregulated in EBV-infected NPC cells and clinical tissue samples due to the action of EBV encoded BART lncRNAs, with IKZF3/Aiolos was expressed at higher levels in late stage (stage III & IV) NPC patients than in early stage (stage I & II) disease. Secondly, we found that IKZF3/Aiolos was upregulated in BART-activated NPC cells but downregulated in BART-knockdown NPC cells. In addition, this study further demonstrated that IKZF3/Aiolos modulates transcription of EBV BZLF1 and the cellular gene, LRIG1, to maintain EBV latency and promote NPC tumorigenesis in vitro and in vivo. Our results showed that the EBV lytic reactivator BZLF1 was upregulated in IKZF3/Aiolos knockout or Aiolos inhibitor-treated NPC cells. ChIP-qPCR, immunoprecipitation and immunofluorescence analyses further revealed that IKZF3/Aiolos induces H3K27 deacetylation to silence expression of BZLF1 and maintain EBV latency in NPC cells. Moreover, functional analyses and western blotting showed that IKZF3/Aiolos inhibited the tumor suppressor, LRIG1, to upregulate expression and phosphorylation of the cellular proto-oncogene Erb-B2 for NPC pathogenesis. Sphere- and colony-formation assays demonstrated that IKZF3/Aiolos enhances growth of NPC cells in vitro, and in vivo experiments further showed that IKZF3/Aiolos promotes tumorigenicity of NPC cells in NOD mice. Mechanically, we found that the expression of EBV lytic reactivator BZLF1 and cellular LRIG1 were negatively regulated by BART lncRNA/IKZF3/Aiolos regulatory machinery in NPC cells, while Erb-B2 was positively regulated, which indicated that IKZF3/Aiolos is a new biomarker for NPC and may lead to the development of novel diagnostic tests and treatments for NPC. Citation Format: Songtao He, Jiayan Liu, Bobo Wing-Yee Mok, Sai Wah Tsao, Honglin Chen. Epstein-Barr virus BART lncRNAs induce IKZF3/Aiolos to maintain EBV latency and promote tumorigenicity in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3748.

Impact of Epstein Barr Virus Infection on Treatment Opportunities in Patients with Nasopharyngeal Cancer

Chemical, physical, and infectious agents may induce carcinogenesis, and in the latter case, viruses are involved in most cases. The occurrence of virus-induced carcinogenesis is a complex process caused by an interaction across multiple genes, mainly depending by the type of the virus. Molecular mechanisms at the basis of viral carcinogenesis, mainly suggest the involvement of a dysregulation of the cell cycle. Among the virus-inducing carcinogenesis, Epstein Barr Virus (EBV) plays a major role in the development of both hematological and oncological malignancies and importantly, several lines of evidence demonstrated that nasopharyngeal carcinoma (NPC) is consistently associated with EBV infection. Cancerogenesis in NPC may be induced by the activation of different EBV "oncoproteins" which are produced during the so called "latency phase" of EBV in the host cells. Moreover, EBV presence in NPC does affect the tumor microenvironment (TME) leading to a strongly immunosuppressed status. Translational implications of the above-mentioned statements are that EBV-infected NPC cells can express proteins potentially recognized by immune cells in order to elicit a host immune response (tumor associated antigens). Three immunotherapeutic approaches have been implemented for the treatment of NPC including active, adoptive immunotherapy, and modulation of immune regulatory molecules by use of the so-called checkpoint inhibitors. In this review, we will highlight the role of EBV infection in NPC development and analyze its possible implications on therapy strategies.

Exosomal HMGA2 protein from EBV-positive NPC cells destroys vascular endothelial barriers and induces endothelial-to-mesenchymal transition to promote metastasis

Increased vascular permeability facilitates metastasis. Cancer-secreted exosomes are emerging mediators of cancer-host crosstalk. Epstein-Barr virus (EBV), identified as the first human tumor-associated virus, plays a crucial role in metastatic tumors, especially in nasopharyngeal carcinoma (NPC). To date, whether and how exosomes from EBV-infected NPC cells affect vascular permeability remains unclear. Here, we show that exosomes from EBV-positive NPC cells, but not exosomes from EBV-negative NPC cells, destroy endothelial cell tight junction (TJ) proteins, which are natural barriers against metastasis, and promote endothelial-to-mesenchymal transition (EndMT) in endothelial cells. Proteomic analysis revealed that the level of HMGA2 protein was higher in exosomes derived from EBV-positive NPC cells compared with that in exosomes derived from EBV-negative NPC cells. Depletion of HMGA2 in exosomes derived from EBV-positive NPC cells attenuates endothelial cell dysfunction and tumor cell metastasis. In contrast, exosomes from HMGA2 overexpressing EBV-negative NPC cells promoted these processes. Furthermore, we showed that HMGA2 upregulates the expression of Snail, which contributes to TJ proteins reduction and EndMT in endothelial cells. Moreover, the level of HMGA2 in circulating exosomes is significantly higher in NPC patients with metastasis than in those without metastasis and healthy negative controls, and the level of HMGA2 in tumor cells is associated with TJ and EndMT protein expression in endothelial cells. Collectively, our findings suggest exosomal HMGA2 from EBV-positive NPC cells promotes tumor metastasis by targeting multiple endothelial TJ and promoting EndMT, which highlights secreted HMGA2 as a potential therapeutic target and a predictive marker for NPC metastasis.

EBV infection-induced GPX4 promotes chemoresistance and tumor progression in nasopharyngeal carcinoma

Epstein–Barr virus (EBV) was the first oncogenic virus identified in humans. It is primarily associated with multiple lymphoid and epithelial cancers, including nasopharyngeal carcinoma (NPC). However, its association with ferroptosis and its role in cancer therapy resistance have not been fully elucidated. Here, we show that EBV infection reduces the sensitivity of NPC cells to ferroptosis by activating the p62-Keap1-NRF2 signaling pathway in conjunction with upregulation of SLC7A11 and GPX4 expression. Knockdown of endogenous GPX4 or blockade of GPX4 using a specific inhibitor enhanced the chemosensitivity of EBV-infected NPC cells. Functional studies revealed that GPX4 knockdown suppresses the proliferation and colony formation of NPC cells. Mechanistically, GPX4 interacts with the TAK1-TAB1/TAB3 complex, regulates TAK1 kinase activity, and further activates downstream MAPK-JNK and NFκB pathways. High GPX4 expression is correlated with poor clinical outcomes in patients with NPC and other cancer types. Taken together, our findings suggest that EBV infection has important effects on redox homeostasis, revealing a previously unappreciated role for GPX4 in tumor progression. This novel mechanism provides a potential new target for the treatment of EBV-related tumors.

Interaction of phospholipid scramblase 1 with the Epstein-Barr virus protein BZLF1 represses BZLF1-mediated lytic gene transcription

Human phospholipid scramblase 1 (PLSCR1) is strongly expressed in response to interferon (IFN) treatment and viral infection, and PLSCR1 has been suggested to play an important role in IFN-dependent antiviral responses. In this study, we showed that the basal expression of PLSCR1 was significantly elevated in Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC). PLSCR1 was observed to directly interact with the EBV immediate-early transactivator BZLF1 in vitro and in vivo, and this interaction repressed the BZLF1-mediated transactivation of an EBV lytic BMRF1 promoter construct. In addition, PLSCR1 expression decreased the BZLF1-mediated up-regulation of lytic BMRF1 mRNA and protein expression in WT and PLSCR1-knockout EBV-infected NPC cells. Furthermore, we showed that PLSCR1 represses the interaction between BZLF1 and CREB-binding protein (CBP), which enhances the BZLF1-mediated transactivation of EBV lytic promoters. These results reveal for the first time that PLSCR1 specifically interacts with BZLF1 and negatively regulates its transcriptional regulatory activity by preventing the formation of the BZLF1-CBP complex. This interaction may contribute to the establishment of latent EBV infection in EBV-infected NPC cells.

Epstein–Barr virus noncoding RNAs from the extracellular vesicles of nasopharyngeal carcinoma (NPC) cells promote angiogenesis via TLR3/RIG-I-mediated VCAM-1 expression

Viral noncoding RNAs (Epstein–Barr virus-encoded RNAs, EBERs) are believed to play a critical role in the progression of lymphoma and nasopharyngeal carcinoma (NPC). However, the accurate mechanisms accounting for their oncogenic function have not been elucidated, especially in terms of interaction between tumor cells and mesenchymal cells. Here, we report that, in addition to NPC cells, EBERs are also found in endothelial cells in Epstein–Barr virus (EBV)-infected NPC parenchymal tissues, which implicates NPC-derived extracellular vesicles (EVs) in transmitting EBERs to endothelial cells. In support of this hypothesis, we first ascertained if EBERs could be transferred to endothelial cells via EVs isolated from NPC culture supernatant. Then, we clarified that EVs-derived EBERs could promote angiogenesis through stimulation of VCAM-1 expression. Finally, we explored the involvement of EBER recognition by TLR3 and RIG-I in NPC angiogenesis. Our observations collectively illustrate the significance and mechanism of EVs-derived EBERs in angiogenesis and underlie the interaction mechanisms between EBV-infected NPC cells and the tumor microenvironment.

Abstract 1108: M2-polarized macrophages increase invasiveness of EBV-associated nasopharyngeal carcinoma by inducing invadopodia formation

Abstract Cancer metastasis is a major cause of mortality, accounting for approximately 90% of cancer related deaths. Emerging evidences suggest that tumour microenvironment plays an indispensable role in cancer metastasis. Recruitment of innate and adaptive immune cells to the tumour results in chronic inflammation which favours tumour growth and progression. Particularly, tumour associated macrophages (TAMs) are found to promote the initiation, growth, and metastasis in several types of cancers. Nasopharyngeal carcinoma (NPC) is one of the highly invasive and metastatic cancers and is closely associated with EBV infection. We postulate that TAMs may increase the invasiveness of NPC by enhancing the formation of invadopodia, which are membranous actin-rich protrusions with digestive ability of extra-cellular matrix. In this study, monocyte THP-1 cells were polarized to M2-macrophages, and co-cultured with nasopharyngeal cells in a contained chamber-insert. The stimulated nasopharyngeal cells then formed massive numbers of invadopodia and digested larger area of gelatin compared with the unstimulated counterparts. A cytokine antibody array was used to identify the upregulated cytokines in the co-culture medium of the M2-macrohphages and the NPC cells. Tumour necrosis factor alpha (TNFα) was revealed to be the responsible cytokine to induce the formation of invadopodia and digestion of gelatin. Besides, western blot analysis identified that several actin-associated proteins such as Src, Erk, and cortactin were involved in the TNFα-mediated invadopodia formation. We also found that the expression of latent membrane protein 1 (LMP1), an EBV protein that has similar structure to TNF receptor was elevated in NPC cells after co-culturing with the M2-polarized macrophages. Through transient transfection and stable expression of LMP1 in the NPC cells, LMP1 could also promote the formation of invadopodia through cdc42 activation. Taken together, these data indicate macrophages can promote the invasiveness of EBV-infected NPC cells by enhancing their ability in forming invadopodia through an activated TNFα signaling axis and LMP1 upregulation. Citation Format: Wilson Wing Chung Tang, Chan Ping You, Gareth E. Jones, George Sai-Wah Tsao, Anna Chi Man Tsang. M2-polarized macrophages increase invasiveness of EBV-associated nasopharyngeal carcinoma by inducing invadopodia formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1108.

Baicalein inhibits growth of Epstein-Barr virus-positive nasopharyngeal carcinoma by repressing the activity of EBNA1 Q-promoter

Epstein-Barr virus (EBV) can establish a life-long latent infection in the host and is associated with various human malignancies, including nasopharyngeal carcinoma (NPC), the most common cancer originated from nasopharynx. EBV nuclear antigen 1 (EBNA1) is the only viral protein absolutely demanded for segregation, replication, transcription and maintenance of EBV viral genome in host cells. Baicalein, a bioactive flavonoid compound purified from the root of Scutellariae baicaleinsis, displays anti-inflammatory, immunosuppressive, and anti-tumor properties. In this study, the therapeutic effects and functional mechanism of baicalein on EBV-positive human NPC were determined. Cell Counting Kit-8 assays and cell formation colony were performed to investigate that baicalein can suppress proliferation of EBV-infected human NPC cells. Flow cytometric and hoechst 33258 staining results indicated that baicalein induced cell cycle arrest and apoptosis. Western blotting results demonstrated that baicalein down-regulates EBNA1 expression but not reduces the stability and half-life of EBNA1 in EBV-infected NPC cells. Additionally, the mRNA level of EBNA1 was examined by real time-PCR, the activity of EBNA1 Q promoter (Qp) was determined by dual luciferase reporter assay. Considering that transcription factor specificity protein 1 (Sp1) can maintain EBNA1 Qp active. Further analyses also elucidated that baicalein inhibits the expression of Sp1 while knock-down Sp1 by specific shRNAs decreases the expression and transcription levels of EBNA1. Therefore, the results suggested that baicalein may decrease EBNA1 expression level in EBV-positive NPC cells via inhibiting the activity of EBNA1 Q-promoter while over-expression of EBNA1 attenuate the inhibitory effect of baicalein. Finally, it was found that baicalein may strongly reduce growth of tumor in the mouse xenograft model of EBV-positive NPC. These results indicated that baicalein inhibits growth of EBV-positive NPC by repressing the activity of EBNA1 Q-promoter. Baicalein may be used as a therapeutic agent to treat EBV-positive NPC.

Interplay of Viral Infection, Host Cell Factors and Tumor Microenvironment in the Pathogenesis of Nasopharyngeal Carcinoma.

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.

Suppression of Epstein-Barr virus DNA load in latently infected nasopharyngeal carcinoma cells by CRISPR/Cas9

Epstein-Barr virus (EBV) infects more than 90% of the world’s adult population. Once established, latent infection of nasopharyngeal epithelial cells with EBV is difficult to eradicate and might lead to the development of nasopharyngeal carcinoma (NPC) in a small subset of individuals. In this study we explored the anti-EBV potential of CRISPR/Cas9 targeting of EBV genome in infected NPC cells. We designed gRNAs to target different regions of the EBV genome and transfected them into C666-1 cells. The levels of EBV DNA in transfected cells were decreased by about 50%. The suppressive effect on EBV DNA load lasted for weeks but could not be further enhanced by re-transfection of gRNA. Suppression of EBV by CRISPR/Cas9 did not affect survival of C666-1 cells but sensitized them to chemotherapeutic killing by cisplatin and 5-fluorouracil. Our work provides the proof-of-principle for suppressing EBV DNA load with CRISPR/Cas9 and a potential new strategy to sensitize EBV-infected NPC cells to chemotherapy.

NF-κB Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma.

ABSTRACTEpstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinase inhibitor PS-1145 inhibited NF-κB activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-κB activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-κB signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-κB activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC. IMPORTANCE Nasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-κB signaling. EBV LMP1 is a potent activator of NF-κB signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-κB signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-κB signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-κB signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

The role of Epstein-Barr virus infection in the pathogenesis of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. EBV episomes are detected in almost all NPC cells. The role of EBV in NPC pathogenesis has long been postulated but remains enigmatic. In contrast to infection of B lymphocytes, EBV infection does not directly transform nasopharyngeal epithelial cells into proliferative clones with malignant potential. EBV infection of normal pharyngeal epithelial cells is predominantly lytic in nature. Genetic alterations in premalignant nasopharyngeal epithelium, in combination with inflammatory stimulation in the nasopharyngeal mucosa, presumably play essential roles in the establishment of a latent EBV infection in infected nasopharyngeal epithelial cells during the early development of NPC. Establishment of latent EBV infection in premalignant nasopharyngeal epithelial cells and expression of latent viral genes, including the BART transcripts and BART-encoded microRNAs, are crucial features of NPC. Expression of EBV genes may drive further malignant transformation of premalignant nasopharyngeal epithelial cells into cancer cells. The difficulties involved in the establishment of NPC cell lines and the progressive loss of EBV epsiomes in NPC cells propagated in culture strongly implicate the contribution of host stromal components to the growth of NPC cells in vivo and maintenance of EBV in infected NPC cells. Defining the growth advantages of EBV-infected NPC cells in vivo will lead to a better understanding of the contribution of EBV infection in NPC pathogenesis, and may lead to the identification of novel therapeutic targets for NPC treatment.

Modulation of B-cell exosome proteins by gamma herpesvirus infection.

The human gamma herpesviruses, Kaposi sarcoma-associated virus (KSHV) and EBV, are associated with multiple cancers. Recent evidence suggests that EBV and possibly other viruses can manipulate the tumor microenvironment through the secretion of specific viral and cellular components into exosomes, small endocytically derived vesicles that are released from cells. Exosomes produced by EBV-infected nasopharyngeal carcinoma cells contain high levels of the viral oncogene latent membrane protein 1 and viral microRNAs that activate critical signaling pathways in recipient cells. In this study, to determine the effects of EBV and KSHV on exosome content, quantitative proteomics techniques were performed on exosomes purified from 11 B-cell lines that are uninfected, infected with EBV or with KSHV, or infected with both viruses. Using mass spectrometry, 871 proteins were identified, of which ∼360 were unique to the viral exosomes. Analysis by 2D difference gel electrophoresis and spectral counting identified multiple significant changes compared with the uninfected control cells and between viral groups. These data predict that both EBV and KSHV exosomes likely modulate cell death and survival, ribosome function, protein synthesis, and mammalian target of rapamycin signaling. Distinct viral-specific effects on exosomes suggest that KSHV exosomes would affect cellular metabolism, whereas EBV exosomes would activate cellular signaling mediated through integrins, actin, IFN, and NFκB. The changes in exosome content identified in this study suggest ways that these oncogenic viruses modulate the tumor microenvironment and may provide diagnostic markers specific for EBV and KSHV associated malignancies.

Deregulation of lipid metabolism pathway genes in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a unique tumour of epithelial origin with a distinct geographical distribution, closely associated with the Epstein-Barr virus (EBV). EBV-encoded RNAs (EBERs) are small non-polyadenylated RNAs that are abundantly expressed in latent EBV-infected NPC cells. To study the role of EBERs in NPC, we established stable expression of EBERs in HK1, an EBV-negative NPC cell line. Cells expressing EBERs consistently exhibited an increased growth rate. However, EBERs did not confer resistance towards cisplatin-induced apoptosis or promote migration or invasion ability in the cells tested. Using microarray gene expression profiling, we identified potential candidate genes that were deregulated in NPC cells expressing EBERs. Gene Ontology analysis of the data set revealed that EBERs upregulate the cellular lipid metabolic process. Upregulation of low-density lipoprotein receptor (LDLR) and fatty acid synthase (FASN) was observed in EBER-expressing cells. NPC cells exhibited LDL-dependent cell proliferation. In addition, a polyphenolic flavonoid compound, quercetin, known to inhibit FASN, was found to inhibit proliferation of NPC cells.

EBV up‐regulates cytochrome c through VDAC1 regulations and decreases the release of cytoplasmic Ca2+ in the NPC cell line

EBV (Epstein-Barr virus) is considered to be a major factor that causes NPC (nasopharyngeal carcinoma), which is one of the sneakiest cancers frequently occurring in Southeast Asia and Southern China. Apoptosis and pro-apoptotic signals have been studied for decades; however, few have extended the prevailing view of EBV to its impact on NPC in perspective of apoptosis. One of the important proteins named VDAC1 (voltage-dependent anion protein 1) on the mitochondrial outer membrane controls the pro-apoptotic signals in mammalian cells. The impact of EBV infection on VDAC1 and related apoptotic signals remains unclear. In order to study the VDAC1's role in EBV-infected NPC cells, we employ siRNA (small interfering RNA) inhibition to analyse the release of Ca2+ and Cyto c (cytochrome c) signals in the cytoplasm, as they are important pro-apoptotic signals. The results show a decrease of Ca2+ release and up-regulation of Cyto c with EBV infection. After siRNA transfection, the dysregulation of Cyto c is neutralized, which is evidence that the level of Cyto c release in virus-infected NPC cells is the as same as that of non-infected NPC cells. This result indicates that EBV infection changes the cytoplasmic level of Cyto c through regulating VDAC1. In summary, this study reports that EBV changes the release of Ca2+ and Cyto c in the cytoplasm of NPC cells, and that Cyto c changes are mediated by VDAC1 regulation.

Cancer du nasopharynx dans l’Ouest algérien : résultats à long terme et facteurs pronostiques dans une cohorte de 200 cas

Nasopharyngeal carcinoma (NPC) arises from a unique anatomical site with heavy infiltration of leucocytes, predominantly T-lymphocytes, together with other stromal cells. Undifferentiated NPC is the prevalent histological subtype (>98%) in endemic areas in Southern China including Hong Kong. Epstein–Barr virus (EBV) infection is closely associated with undifferentiated NPC. EBV infection in NPC is generally latent in nature. Expression of viral gene products may facilitate the malignant transformation of premalignant nasopharyngeal epithelium to NPC cells. Among the latent EBV gene products, the high expression of EBV-encoded BART-microRNAs in NPC is strongly implicated in NPC pathogenesis. A unique tumor microenvironment (TME) infiltrated heavily with T-lymphocytes and other stromal cells is believed to be involved to support growth of NPC in patients and latent EBV infection in NPC. Various cytokines secreted by EBV-infected NPC cells and the infiltrated stromal cells in the TME are involved in the recruitment of the rich cellular components to the tumor stroma. The dynamic and reciprocal interaction of cancer cells with stromal cells in the TME has been postulated to support EBV infection and modulate growth and invasive properties of NPC cells. Delineating the complex cancer cell–stromal cell interaction in NPC may reveal novel therapeutic targets effective for NPC treatment.

Multiplexed microRNA detection by capillary electrophoresis with laser-induced fluorescence

In this study, we developed a novel assay that simultaneously detects multiple miRNAs (microRNAs) within a single capillary by combining a tandem adenosine-tailed DNA bridge-assisted splinted ligation with denaturing capillary gel electrophoresis with laser-induced fluorescence. This proposed method not only represents a significant improvement in resolution but also allows for the detection of multiple miRNAs within a single capillary based on the length differences of specified target bridge DNA. The assay's linear range covers three orders of magnitude (1.0 nM to 1.0 pM) with a limit of detection (S/N = 3) as low as 190 fM (2.5 zmol). Five miRNAs of Epstein-Barr virus (EBV) were also detected in EBV-infected nasopharyngeal carcinoma cells, while they did not appear in non-virus infected cells. Moreover, the electropherogram indicated that the screening of isomiRs (isomer of miRNA) of BART2 by CE-LIF is feasible by our proposed method. The developed electrophoresis-based method for miRNA detection is fast, amplification-free, multiplexed and cost-effective, making it potentially applicable to large-scale screening of isomiRs.

Extra-cellular release and blood diffusion of BART viral micro-RNAs produced by EBV-infected nasopharyngeal carcinoma cells

BackgroundNasopharyngeal carcinoma (NPC) is a human epithelial malignancy consistently associated with the Epstein-Barr virus. The viral genome is contained in the nuclei of all malignant cells with abundant transcription of a family of viral microRNAs called BART miRNAs. MicroRNAs are well known intra-cellular regulatory elements of gene expression. In addition, they are often exported in the extra-cellular space and sometimes transferred in recipient cells distinct from the producer cells. Extra-cellular transport of the microRNAs is facilitated by various processes including association with protective proteins and packaging in secreted nanovesicles called exosomes. Presence of microRNAS produced by malignant cells has been reported in the blood and saliva of tumor-bearing patients, especially patients diagnosed with glioblastoma or ovarian carcinoma. In this context, it was decided to investigate extra-cellular release of BART miRNAs by NPC cells and their possible detection in the blood of NPC patients. To address this question, we investigated by quantitative RT-PCR the status of 5 microRNAs from the BART family in exosomes released by NPC cells in vitro as well as in plasma samples from NPC xenografted nude mice and NPC patients.ResultsWe report that the BART miRNAs are released in the extra-cellular space by NPC cells being associated, at least to a large extent, with secreted exosomes. They are detected with a good selectivity in plasma samples from NPC xenografted nude mice as well as NPC patients.ConclusionsViral BART miRNAs are secreted by NPC cells in vitro and in vivo. They have enough stability to diffuse from the tumor site to the peripheral blood. This study provides a basis to explore their potential as a source of novel tumor biomarkers and their possible role in communications between malignant and non-malignant cells.