Epstein-Barr Virus Positive Nasopharyngeal Carcinoma Research Articles

Phosphoribosyl pyrophosphate amidotransferase: Novel biomarker and therapeutic target for nasopharyngeal carcinoma.

Cancer cells show a dynamic metabolic landscape, requiring a sufficient supply of nucleotides to proliferate. They are highly dependent on de novo purine biosynthetic pathways for their nucleotide requirements. Phosphoribosyl pyrophosphate amidotransferase (PPAT), catalyzing the first step of de novo purine biosynthesis, is highly expressed in various cancers. We observed an increased expression of PPAT in nasopharyngeal carcinoma (NPC). Moreover, our ribonucleic acid sequencing analysis showed high PPAT expression in Epstein-Barr virus-positive NPC, which was supported by invitro analysis. Through a gene knockdown study, we showed that the suppression of PPAT expression reduced the proliferation and invasion of NPC cells. We also demonstrated the regulation of PPAT by glutamine, a cosubstrate for PPAT. A glutamine antagonist, 6-diazo-5-oxo-L-norleucine, blocked glutamine-mediated induction of PPAT and reduced NPC cell proliferation. Immunohistochemical analysis of PPAT in NPC tissues revealed increased expression of PPAT with disease progression, which was significantly associated with poor prognosis. In summary, this study highlighted the biological function of PPAT in NPC, establishing its potential as a novel prognostic biomarker for aggressive NPC and a promising therapeutic target.

Abstract LB412: BRAFp.G469E hotspot mutation promotes nasopharyngeal carcinoma tumorigenesis with potential druggability

Abstract Nasopharyngeal Carcinoma (NPC) is a distinct type of Epstein-Barr Virus (EBV)-associated head & neck cancer. It is most prevalent in Southeast Asia with a high incidence rate of ~15/100,000. While mutations of the MAPK pathways are found in nearly one-fifth of head & neck squamous cell carcinoma (HNSCC) in Western countries (TCGA-HNSCC Firehose cohort, N=512), and some of which are druggable in HNSCC setting (e.g. MAPK1 and HRAS mutations), we sought to determine the cumulative mutational rate of this potentially druggable pathway in 164 micro-dissected NPC cases using recently published next-generation sequencing data (98 whole-exome and 63 whole-genome-sequenced cases). An overall rate of 9.32% cases of NPC harbored MAPK pathway mutations (16 mutations in 15 tumors). Four genes are recurrently mutated: 4 NRAS (all Q61R/L hotspot mutations), 3 HRAS (G12S, Q61R, S106L), 2 BRAF (R726H, Y647*), and 2 SHC4 (Q519R, A552T) mutations. Among which, both patients bearing BRAF mutations died of NPC, suggestive of the biological importance of BRAF mutations in NPC progression. Prompted by an independent clinical report on an advanced NPC patient with somatic co-mutations of BRAFp.G469E and KRASp.G12D being sensitive to the B-raf/C-raf inhibitor sorafenib, we first examined the biological functions and potential druggability of this particular BRAFp.G469E mutation in NPC, as it represents a hotspot mutation site across 20 cancer types (BRAFp.G469E/A/R/V/* found in 137/70655 pan-cancer samples, cBioPortal). Based on the NPC clinical report, we hypothesized that this BRAFp.G469E hotspot mutation could drive NPC tumorigenesis and might be potentially sensitive to the B-raf inhibitor sorafenib. By ectopic expression of BRAFp.G469E mutation, BRAF-wildtype (wt) and EGFP-control gene into a recently developed Epstein-Barr virus-positive nasopharyngeal carcinoma [EBV(+)NPC] cell line, NPC43, we determined the biological functions of this mutation in NPC. We showed that NPC43-BRAFp.G469E-expressing cells significantly drove a 4-fold increase in 3D tumoroid growth vs. BRAF-wt and EGFP-control cells on day 7 (flat-bottom low attachment plates). Further, wound healing assay demonstrated a significantly higher migration rate of NPC43-BRAFp.G469E cells achieving ~80% wound healing (at 96h) vs. 30% and 10% healing in BRAF-wt and EGFP-control cells. Lastly, our preliminary drug study showed that NPC43-BRAFp.G469E cells displayed higher sensitivity to low dose sorafenib (33.3nM and 100nM) than BRAF-wt and EGFP-control cells, achieving ~20% and 30% growth inhibition at 72h, vs. no growth inhibition in control cells. Thus, BRAFp.G469E hotspot mutation can drive NPC tumorigenesis, and may confer sensitivity to raf inhibitors such as sorafenib in NPC. Potential druggability of other BRAF mutations in NPC should be investigated. Acknowledgements: The study was supported by Research Impact Fund (R4015-19F), Research Grants Council, University Grants Committee, Hong Kong SAR during 6/29/20-9/7/21. Citation Format: Chun Ho Law, Zoey Wing Yee Liu, Franky Leung Chan, Tin Yu Samuel Law, Vivian W.Y. Lui. BRAFp.G469E hotspot mutation promotes nasopharyngeal carcinoma tumorigenesis with potential druggability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB412.

Identification of DNA aptamers that specifically targets EBV+ nasopharyngeal carcinoma via binding with EphA2/CD98hc complex

Nasopharyngeal carcinoma (NPC) is one of the Epstein-Barr virus (EBV)-associated malignancies and has a distinct geographical distribution. The high mortality rates of NPC patients with advanced and recurrent disease highlight the urgent need for biomarkers for early diagnosis and effective treatments. In this study, we developed DNA aptamers that specifically bind to EBV positive NPC cells by the Cell-SELEX procedure. We further identified the EphA2 (ephrin type-A receptor 2)/CD98hc (CD98 heavy chain) complex as the potential target of the aptamer EA-3 by combining aptamer-based separation and mass spectrometry analysis. Our results revealed for the first time that EphA2 colocalized with CD98hc at the plasma membrane and EphA2 coimmunoprecipitated with CD98hc, which may serve as a starting point for exploring the potential functions of the complex of EphA2 and CD98hc in NPCs. Here, we demonstrated that aptamers can be useful for the identification of protein complexes on the surface of cancer cells.

Effect of exosomes derived from human Epstein-Barr virus-positive nasopharyngeal carcinoma cells on lymphangiogenesis and lymph node metastasis

To investigate the effect of exosomes derived from Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) cells on lymphangiogenesis and lymph node metastasis of NPC. Exosomes from NP69 cells and EBV-positive HK1 (HK1-EBV) cells were obtained by ultracentrifugation and identified by Western blotting and nanoparticle tracking analysis. Dio dye phagocytosis test was performed to observe exosome uptake by lymphatic endothelial cells. Lymphatic endothelial cells were treated with exosomes from nasopharyngeal epithelium (NP69), HK1-EBV, and C666-1 cells or exosome-free supernatant of HK1-EBV and C666-1 cells, and tube formation and migration of the cells were observed. In a nude mouse model of popliteal lymph node metastasis of NPC, the effects of normal saline, NP69 cell-derived exosomes, HK1-EBV cell-derived exosomes, exosome-free supernatant of HK1-EBV cells, and HK1-EBV exosome-free supernatant protein on lymphangiogenesis and lymph node metastasis of the tumor were observed. The exosomes obtained by ultracentrifugation contained abundant exosome-specific proteins and showed a normal size range. The exosomes from NPC cells and NP69 cells could be taken up by lymphatic endothelial cells. Compared with the blank control and exosomes form NP69 cells, exosomes derived from HK1-EBV and C666-1 cells significantly promoted tube formation and migration of lymphatic endothelial cells (P < 0.05), and the exosomes and exosome-free supernatant of HK1-EBV and C666-1 cell produced similar effects (P > 0.05). In the tumor-bearing nude mice, exosomes derived from HK1-EBV cells significantly promoted metastasis of NPC cells and local lymphangiogenesis compared with the blank control, NP69 cell-derived exosomes and exosome-free supernatant of HK1-EBV cells (P < 0.05). Exosomes from EBV-positive NPC cells can significantly promote lymphangiogenesis and lymph node metastasis of NPC.

Prognostic Value of Programmed Cell Death-Ligand 1 Expression in Tumor-Infiltrating Lymphocytes and Viral Load in Peripheral Blood Mononuclear Cells for Epstein-Barr Virus-Positive Nasopharyngeal Carcinoma.

Epstein-Barr virus (EBV) infection has a role in the development and progression of nasopharyngeal carcinoma (NPC); however, it is unclear whether EBV load correlates with tumor prognosis or the need for immunotherapy. This study evaluated whether the EBV DNA concentration in peripheral blood mononuclear cells (PBMC) or programmed cell death-ligand1 (PD-L1) expression in tumor-infiltrating lymphocytes (TIL) could predict the clinical outcomes of patients with NPC. Clinicopathological parameters of 198 patients with NPC were analyzed retrospectively from June 2012 to May 2018. Patients' EBV loads were determined by droplet digital PCR. TIL PD-L1 was analyzed by immunohistochemistry. A log value of 1.98 log IU/mL for PBMC EBV DNA and a percentage of PD-L1 expression of 15% in TILs marked distinguishing cutoffs in NPC prognosis. The 5-year progression-free survival (PFS) rates in patients with high vs low log (PBMC EBV DNA) were 68.2% and 93.1%, respectively (P = 0.002). The 5-year PFS rates in patients with high vs low TIL PD-L1 expression were 66.3% and 33.7%, respectively (P = 0.03). The 5-year PFS rates of the high-risk group (high log [PBMC EBV DNA] and low TIL PD-L1), low-risk group (low log [PBMC EBV DNA] and high TIL PD-L1), and those in between (intermediate group) were 0%, 91.9%, and 71.4%, respectively (P < 0.001). Concentrations of PBMC EBV DNA and TIL PD-L1 expression can be used as prognostic markers in NPC. The combination of both an increased EBV DNA concentration and suppressed TIL PD-L1 expression is associated with metastasis or relapse.

Evaluation of the effect of 5-aminolevulinic acid hexyl ester (H-ALA) PDT on EBV LMP1 protein expression in human nasopharyngeal cells.

Nasopharyngeal carcinoma (NPC) is of high prevalence in Hong Kong and southern China. The pathogenesis of NPC is closely associated with Epstein-Barr virus (EBV) infection via regulation of viral oncoprotein latent membrane protein 1 (LMP1). The conventional treatment for NPC is chemo-radiotherapy, but the prognosis remains poor for advanced stage, recurrent and metastatic NPC. Photodynamic therapy (PDT) is a therapeutic approach to combat tumors. PDT effectiveness depends on the interaction of photosensitizers, light and molecular oxygen. 5- aminolevulinic acid hexyl derivative (H-ALA) is one of the photosensitizers derived from 5-ALA. H-ALA with improved lipophilic properties by adding a long lipophilic chain (hexyl group) to 5-ALA, resulted in better penetration into cell cytoplasm. In this study, the effect of H-ALA-PDT on NPC cells (EBV positive C666-1 and EBV negative CNE2) was investigated. The H-ALA mediated cellular uptake and cytotoxicity was revealed via flow cytometry analysis and MTT assay respectively. H-ALA PDT mediated protein modulation was analysed by western blot analysis. Our finding reported that the cellular uptake of H-ALA in C666-1 and CNE2 cells was in a time dependent manner. H-ALA PDT was effective to C666-1 and CNE2 cells. EBV LMP1 proteins was expressed in C666-1 cells only and its expression was responsive to H-ALA PDT in a dose dependent manner. This work revealed the potential of H-ALA PDT as a treatment regiment for EBV positive NPC cells. Understanding the mechanism of H-ALA mediated PDT could develop improved strategies for the treatment of NPC.

F factor plasmid-mediated Epstein-Barr virus genome introduction establishes an EBV positive NPC cell model.

BackgroundMost Epstein-Barr virus (EBV)-positive cells lose the EBV episomes upon prolonged propagation. PurposeThe purposes of this study were to establish a simple cell model for nasopharyngeal carcinoma (NPC) research by introducing a plasmid with the EBV genome into NPC cells and then to investigate the resulting changes in malignant biological behaviour and NPC-associated signalling pathways. MethodsHONE1 NPC cells were transfected with F-factor plasmids including the EBV genome (HONE1-EBV cells). Then cell proliferation, migration, cell cycle distribution and apoptosis were evaluated in vitro by using CCK8, transwell and flow cytometry assays respectively. EBV-encoded proteins and cell signal tranducting proteins were detected by western blot assays. EBV-encoded RNAs were detected by in situ hybridization. EBV particles were assayed by transmission electron microscope (TEM). The morphology of cells were detected by immunofluorescence assays for alpha-tubulin. ResultsLatent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), Epstein-Barr nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) were successfully expressed in HONE1-EBV cells. No EBV particles were founded by TEM. Introduction of the EBV genome significantly promoted proliferation, cell cycle progression and migration and inhibited apoptosis in HONE1 cells. Immunofluorescence assays showed that the morphology of HONE1-EBV cells changed into spindle. Furthermore, EBV genome introduction significantly inhibited the JAK/STAT signalling pathway, while it activated the PI3K-AKT and NF-κB signalling pathways in HONE1 cells. ConclusionThese findings suggest that F-factor plasmid-mediated EBV genome introduction was successful in constructing an EBV positive cell model, which showed deteriorated biological behavior and activated NPC-associated signalling pathways. This model can serve as a good tool for studying EBV in NPC, but the subtle differences in cancer-associated pathways must be considered.

Abstract 56: Establishment and characterization of new tumor xenografts and cancer cell lines from EBV positive nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is rare worldwide but common in southern China, including Hong Kong. The endemic NPC among southern Chinese is typically non-keratinizing carcinoma, which is almost 100% associated with Epstein-Barr virus (EBV) infection. Patient-derived xenografts (PDXs), given their close resemblance with patient tumors, serve as important models in preclinical evaluation for novel therapeutic drugs. For unclear reasons, it has been difficult to establish NPC PDXs in vivo and cell lines in vitro. Currently, there are few NPC PDXs available for research, including X666, X2117, C15, C17 and C18. However, all of them have been passaged in nude mice for over 25 years and may deviate from their original NPC tumors in patients. In vitro, C666-1 is the only EBV-positive (EBV+ve) NPC cell line which has been used extensively in investigations. C666-1 was established from the NPC xenograft (X666), which had been propagated for a long period of time. Most if not all the other previously reported NPC cell lines have lost their EBV episomes upon in vitro propagation. Furthermore, many of these reported NPC cell lines have been shown with genetic contamination of HeLa cells. Hence, their applications in NPC studies are limited. The lack of representative nasopharyngeal carcinoma (NPC) models has also seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. In this study, we report the successful establishment and comprehensive characterization of four new NPC PDXs (all EBV+ve) and three NPC cell lines (one EBV+ve; two EBV-ve). These newly established EBV+ve NPC PDXs and cell lines significantly recapitulate the mutation profiles of their original NPC tumors, and harbor common genetic alterations reported in NPC, which supports their potential applications in the investigations of NPC pathogenesis. We postulate that lytic EBV reactivation may be an intrinsic barrier to the successful establishment of EBV+ve NPC PDXs and cell lines. The ROCK inhibitor (Y-27632) was shown to be able to suppress epithelial differentiation and lytic reactivation of EBV in infected NPC cells. Establishment of a new EBV-positive NPC cell line, NPC43, was achieved directly from patient NPC tissues by including Y-27632 in culture medium. NPC43 cells exhibited tumorigenicity in immunodeficient mice, and could be induced to undergo EBV lytic reactivation with production of infectious visions. The establishment and characterization of new NPC PDXs and cell lines will provide valuable experimental tools for NPC and EBV research. Funding support: Research Grant Council, Hong Kong: AoE NPC grant (AoE/M-06/08), Theme-based Research Scheme grant (T12-401/13-R) and Collaborative Research Fund (C7027-16G), General Research Fund (106140069, 106160185, 17111516, 17110315) and HMRF grant (04151726, 13142201). Citation Format: Weitao Lin, Yim Ling Yip, Lin Jia, Wen Deng, Kwok Wai Lo, Chin Man Tsang, Maria Li Lung, George Sai Tsao. Establishment and characterization of new tumor xenografts and cancer cell lines from EBV positive nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 56.

Low PD-L1 Expression Strongly Correlates with Local Recurrence in Epstein-Barr Virus-Positive Nasopharyngeal Carcinoma after Radiation-Based Therapy.

The prognostic value of programmed death-ligand 1 (PD-L1) expression in nasopharyngeal carcinoma (NPC) is controversial, with previous studies showing conflicting results. Most NPCs in endemic areas are Epstein-Barr virus (EBV)-positive. Our aim was to evaluate the clinical significance of PD-L1 expression in EBV-positive NPC. We retrospectively analyzed PD-L1 expression on tumor cells (TCs) and immune cells (ICs) by immunohistochemistry in 208 EBV-positive NPC patients who underwent radiotherapy (203 with concurrent chemotherapy). The percentages of TCs and ICs expressing PD-L1 were evaluated respectively. There was a strong correlation between local recurrence and low PD-L1 expression on ICs (p = 0.0012), TCs (p = 0.013) or both (p = 0.000044), whereas all clinical parameters had no influence on local recurrence. Using multivariate analysis, low PD-L1 expression on ICs was an independent adverse prognostic factor (p = 0.0080; HR = 1.88; 95% CI = 1.18–3.00) for disease-free survival. High PD-L1 expression on both ICs and TCs was an independent favorable prognostic factor (p = 0.022; HR = 0.46; 95% CI = 0.24–0.89) for overall survival. We show for the first time that low PD-L1 expression on ICs and TCs strongly correlates with local recurrence in EBV-positive NPC patients after radiation-based therapy. A simple immunohistochemical study for PD-L1 can identify patients prone to local recurrence, and such patients might benefit from more aggressive treatment in future clinical trials.

Downregulation of EB virus miR-BART4 inhibits proliferation and aggressiveness while promoting radiosensitivity of nasopharyngeal carcinoma.

This study aims to explore the role of Epstein-Barr virus (EBV) miR-BART4 in occurrence and progression of nasopharyngeal carcinoma (NPC) and its effect on radiosensitivity. The expressions of EBV and miR-BART4 in 108 cases of NPC tissues and 97 cases of chronic nasopharyngeal inflammation tissues were determined by real time quantitative polymerase chain reaction (PCR), and the relationship between the expression of miR-BART4 and the clinicopathological features of NPC was analyzed. Cell lines, HONEl, CNEl, CNE2, C666-1, 6-10B, and NP-69 were used to compare the expression of miR-BART4, in which the CNE2 cells were selected for further experiments. CNE2 cells were grouped into blank group, negative control (NC) group, miR-BART4 inhibitors group and miR-BART4 mimics group. Cells in above groups were under radiation of 6 Gy X ray for 12 h before grouped into control group, 6 Gy group, NC + 6 Gy group, miR-BART4 inhibitors + 6 Gy group and miR-BART4 mimics + 6 Gy group. Cell proliferation, clone formation ability, cell apoptosis, invasion and migration ability were measured by MTT assay, clone formation assay, flow cytometry (FCM), Transwell assay and scratch test, respectively. Western blot analysis was used to detect the expression of apoptosis-related proteins (cleaved caspase-3, Bax and Bcl-2) and epithelial-mesenchymal transition (EMT) marker protein E-cadherin and Vimentin. mRNA and protein expression of PTEN were detected by qRT-PCR and western blot. Bioinformatics software and luciferase activity experiments were used to verify the targeting relationship between miR-BART4 and PTEN. Positive rate of EBV in NPC tissues (93.5%) was remarkably higher than that in chronic nasopharyngeal inflammation tissues (21.6%). miR-BART4 was highly expressed and mRNA and protein expression of PTEN was lowly expressed in EBV positive NPC tissues compared with EBV negative NPC tissues and chronic nasopharyngeal inflammation tissues. The expression of miR-BART4 was related to the clinical stage, lymph node metastasis and differentiation degree of NPC. Expression of miR-BART4 in CNE2, CNEl, HONEl, C666-1, 6-10B, 5-8F cells was higher than that in NP-69 cells. In CNE2 and C666-1 cell experiments, compared with blank group and NC group, miR-BART4 inhibitors group had decreased miR-BART4 expression, increased mRNA and protein expression of PTEN, cell survival rate, invasion and migration ability and increased cell apoptosis rate, which is totally contrary to the observation in miR-BART4 mimics group. The radiosensitive NPC tissues had higher miR-BART4 expression than that in radio-resistance NPC tissues. In comparison to 6 Gy group and NC + 6 Gy group, cell survival rate and clone number was inhibited, but the cell apoptosis rate was increased in miR-BART4 inhibitors +6 G group, in contrary to the observation in miR-BART4 inhibitors + 6 Gy group. Bioinformatics software and luciferase activity experiments confirmed that miR-BART4 could inhibit the expression of PTEN. EBV may promote development and progression of NPC by up-regulating miR-BART4 expressions, consequently inhibiting its radiosensitivity, whose effect may be related to the targeting inhibition of PTEN expression.

Genomic Landscapes of EBV-Associated Nasopharyngeal Carcinoma vs. HPV-Associated Head and Neck Cancer.

Epstein-Barr virus-positive nasopharyngeal carcinoma (EBV(+) NPC), and human papillomavirus-positive head and neck squamous cell carcinoma (HPV(+) HNSCC) are two distinct types of aggressive head and neck cancers with early age onsets. Their recently identified genomic landscapes by whole-exome sequencing (WES) clearly reveal critical roles of: (1) inflammation via NF-kB activation, (2) survival via PI3K aberrations, and perhaps (3) immune evasion via MHC loss in these cancers as summarized in this review. Immediate outcomes of these WES studies include the identification of potential prognostic biomarkers, and druggable events for these cancers. The impact of these genomic findings on the development of precision medicine and immunotherapies will be discussed. For both of these cancers, the main lethality comes from metastases and disease recurrences which may represent therapy resistance. Thus, potential curing of these cancers still relies on future identification of key genomic drivers and likely druggable events in recurrent and metastatic forms of these intrinsically aggressive cancers of the head and neck.

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.

Prognostic role of tumor infiltrating lymphocytes in EBV positive and EBV negative nasopharyngeal carcinoma

ObjectivesTumor infiltrating lymphocytes (TILs) correlate with both better and worse prognosis in solid tumors. As therapeutic modalities for nasopharyngeal carcinoma (NPC) are limited, immunotherapy could be a potential alternative. Up till now there is limited prognostic data on the role of TILs in NPC, so we assessed the prognostic role of TILs in Epstein-Barr-virus (EBV) positive and negative NPC. MethodsTissue of 92 NPCs was assessed for CD3, CD4, CD8, PD1 and PDL1 expression in the tumor’s micro-environment. Correlations between clinicopathological characteristics was assessed using the Pearson X2 test, Fisher’s exact test and ANOVA. Survival was analyzed with the Kaplan-Meier method and Cox regression. Differences in CD3, CD4, CD8, PD1, PDL1 counts/(co)expression between EBV positive and negative NPCs were evaluated using the Mann-Whitney U test. Two-tailed P values below 0.05 were considered statistically significant. ResultsEBV positive NPC contains significantly more CD3, CD4 and CD8 TILs than EBV negative NPC. In the whole NPC group, increased CD8 count is associated with better overall survival (OS) (HR 0.219 (95%CI 0.075–0.640)), but also in cases with PDL1 co-expression (HR 0.073 (95%CI 0.010–0.556)). In EBV positive NPC co-expression of CD8 and PDL1 showed better disease free survival (HR 0.407 (95%CI 0.195–0.850)) and OS (HR 0.170 (95%CI 0.037–0.787)). ConclusionsAlthough TILs are significantly different between EBV positive and negative NPCs, it is especially composition of the infiltrate which determines prognosis. Effects of PD1 and CD8 need more study, because these findings show much potential in using immunotherapeutic modalities in NPC treatment.

EBV based cancer prevention and therapy in nasopharyngeal carcinoma

Epstein–Barr virus is an important cancer causing virus. Nasopharyngeal carcinoma is an infection-related cancer strongly driven by Epstein–Barr virus. In this cancer model, we identified the major host targets of latent membrane protein 1 which is a driving oncogene encoded by Epstein–Barr virus in latency infection. latent membrane protein 1 activates several oncogenic signaling axes causing multiple malignant phenotypes and therapeutic resistance. Also, Epstein–Barr virus up-regulates DNA methyltransferase 1 and mediates onco-epigenetic effects in the carcinogenesis. The collaborating pathways activated by latent membrane protein 1 constructs an oncogenic signaling network, which makes latent membrane protein 1 an important potential target for effective treatment or preventive intervention. In Epstein–Barr virus lytic phase, the plasma level of Epstein–Barr virus DNA is considered as a distinguishing marker for nasopharyngeal carcinoma in subjects from healthy high-risk populations and is also a novel prognostic marker in Epstein–Barr virus-positive nasopharyngeal carcinoma. Now the early detection and screening of the lytic proteins and Epstein–Barr virus DNA have been applied to clinical and high-risk population. The knowledge generated regarding Epstein–Barr virus can be used in Epstein–Barr virus based precision cancer prevention and therapy in the near future.

Integrated miRNA-mRNA analysis of Epstein-Barr virus-positive nasopharyngeal carcinoma.

This study aims to identify the crucial miRNAs in Epstein-Barr virus-positive nasopharyngeal carcinoma (NPC) and their target genes. Gene expression profile data (GSE12452) that included 31 NPC and 10 normal nasopharyngeal tissue specimens were downloaded. Differentially expressed genes (DEGs) were identified using significance analysis of microarrays. The underlying function of DEGs was predicted via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The miRNA sequencing dataset GSE14738 was also downloaded, and expression levels of miRNA were calculated by the number of reads mapped to each miRNA. The selected miRNAs were integrated into the miRecords database to obtain their target genes. Target genes associated with DEGs were used to construct the interaction network via Cytoscape. A total of 1437 DEGs between NPC and control were identified, most of which were enriched in cell cycle and extracellular matrix-receptor interaction signaling pathways. Furthermore, 112 miRNAs were considered upregulated in NPC samples. A total of 2228 relationships between 39 miRNAs and 1247 target genes were obtained, of which 182 relationships between 32 miRNAs and 97 target genes were chosen to construct an interaction network. The interactions between DEGs and the let-7 or miR-29 families appeared strongest in this network, where CDC25A, COL3A1, and COL1A1 were regulated by several let-7 family members, while COL4A1 and COL5A2 were regulated by several miR-29 family members. The let-7 and miR-29 families may be related to the development of NPC by regulating the genes involved in cell cycle and ECM-receptor interaction.

EBV-driven LMP1 and IFN-γ up-regulate PD-L1 in nasopharyngeal carcinoma: Implications for oncotargeted therapy.

PD-L1 expression is a feature of Epstein-Barr virus (EBV) associated malignancies such as nasopharyngeal carcinoma (NPC). Here, we found that EBV-induced latent membrane protein 1 (LMP1) and IFN-γ pathways cooperate to regulate programmed cell death protein 1 ligand (PD-L1). Expression of PD-L1 was higher in EBV positive NPC cell lines compared with EBV negative cell lines. PD-L1 expression could be increased by exogenous and endogenous induction of LMP1 induced PD-L1. In agreement, expression of PD-L1 was suppressed by knocking down LMP1 in EBV positive cell lines. We further demonstrated that LMP1 up-regulated PD-L1 through STAT3, AP-1, and NF-κB pathways. Besides, IFN-γ was independent of but synergetic with LMP1 in up-regulating PD-L1 in NPC. Furthermore, we showed that PD-L1 was associated with worse disease-free survival in NPC patients. These results imply that blocking both the LMP1 oncogenic pathway and PD-1/PD-L1 checkpoints may be a promising therapeutic approach for EBV positive NPC patients.

FosPeg® PDT alters the EBV miRNAs and LMP1 protein expression in EBV positive nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) is one of the top ten cancers highly prevalent in Hong Kong and South China. Epstein-Barr virus (EBV) infection contributes to the tumorigenesis of NPC through the expression of different viral proteins. Among these, Latent Membrane Protein 1(LMP1) is the major oncoprotein expressed by EBV. Foscan® (Biolitec AG), m-tetrahydroxyphenylchlorin (mTHPC)-based photosensitizing drug, has been used in the photodynamic therapy (PDT) for head and neck cancers. FosPeg® (Biolitec AG) is a new formulation of mTHPC contained in PEGylated liposomes with optimized distribution properties. In this in vitro study, the potential of FosPeg®-PDT on human EBV positive NPC cell (c666-1) and EBV negative cells (HK1 and CNE2) were investigated. Effects of FosPeg®-PDT on the expression of EBV BART miRNAs (EBV miRNA BART 1-5p, BART 16, and BART 17-5p), LMP1 mRNA and proteins on c666-1 cells were also elucidated.The killing efficacy of FosPeg®-PDT on NPC cells were determined by MTT assay after LED activation. Effects of FosPeg®-PDT on the expression of LMP1 mRNA and protein were examined by real time PCR and western blot analysis.FosPeg®-PDT demonstrated its antitumor effect on c666-1 cells in a drug and light dose dependent manner. LD30, LD50 and LD70 were achieved by applying LED activation (3J/cm2) at 4h post incubated cells with 0.05μg/ml, 0.07μg/ml and 0.3μg/ml FosPeg®, respectively. Up-regulation of both LMP1 mRNA and protein were observed after FosPeg®-PDT in a dose dependent manner.FosPeg®-PDT exerted antitumor effect on c666-1 cells through up-regulation of LMP1 protein. Understanding the mechanism of FosPeg®-PDT may help to develop better strategies for the treatment of NPC.

Minicircle-oriP-IFNγ: A Novel Targeted Gene Therapeutic System for EBV Positive Human Nasopharyngeal Carcinoma

BackgroundNonviral vectors are attractively used for gene therapy owing to their distinctive advantages. Our previous study has demonstrated that transfer of human IFNγ gene into nasopharyngeal carcinoma (NPC) by using a novel nonviral vector, minicircle (mc), under the control of cytomegalovirus (CMV) promoter was effective to inhibit tumor growth. However, therapies based on CMV promoter cannot express the targeted genes in cancer tissues. Previous studies indicated that the development of human NPC was closely associated with Epstein-Barr virus (EBV) and demonstrated the transcriptional enhancer function of oriP when bound by EBV protein. Therefore, the present study is to explore the targeted gene expression and the anti-tumor effect of a novel tumor-specific gene therapeutic system (mc-oriP-IFNγ) in which the transgene expression was under the transcriptional regulation of oriP promoter.Methodology/Principal FindingsDual-luciferase reporter assay and ELISA were used to assess the expression of luciferase and IFNγ. WST assay was used to assess the cell proliferation. RT-PCR was used to detect the mRNA level of EBNA1. RNAi was used to knockdown the expression of EBNA1. NPC xenograft models in nude mice were used to investigate the targeted antitumor efficacy of mc-oriP-IFNγ. Immunohistochemistry was used to detect the expression and the activity of the IFNγ in tumor sections. Our results demonstrated that mc-oriP vectors mediated comparable gene expression and anti-proliferative effect in the EBV-positive NPC cell line C666-1 compared to mc-CMV vectors. Furthermore, mc-oriP vectors exhibited much lower killing effects on EBV-negative cell lines compared to mc-CMV vectors. The targeted expression of mc-oriP vectors was inhibited by EBNA1-siRNA in C666-1. This selective expression was corroborated in EBV-positive and -negative tumor models.Conclusions/SignificanceThis study demonstrates the feasibility of mc-oriP-IFNγ as a safe and highly effective targeted gene therapeutic system for the treatment of EBV positive NPC.

Inhibition of nasopharyngeal carcinoma growth by RTA‐expressing baculovirus vectors containing oriP

Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Activation of latent EBV into lytic replication by introducing viral lytic gene BRLF1 (RTA) has been shown to be a potential approach to suppress the growth of EBV-associated NPC tumor. The baculovirus vectors with RTA expression cassette (BV-R), RTA and the EBV latent replication origin (OriP, BV-RO), or RTA, OriP and EBNA-1 gene (BV-ROE-CMV), were constructed and examined for their ability to mediate RTA expression, initiate lytic replication and induce cell death in EBV-positive cell lines Hone1-EBV, HK1-EBV and C666-1 in vitro. Their effect to inhibit the growth of EBV-positive NPC tumors was also evaluated in nude mice. The baculovirus vectors BV-RO and BV-ROE-CMV mediated efficient expression of RTA in EBV-positive NPC cells. Lytic EBV replication and cell death were observed in these infected cells. Both vectors also significantly inhibited the growth of EBV-positive NPC tumor in nude mice. EBV early lytic gene expression and tumor cell death were observed in these treated tumors. The presence of OriP improved the performance of the RTA-expressing baculovirus vectors to induce EBV lytic replication and cell death in vitro, and suppress the growth of EBV positive NPC tumors in vivo. By confining RTA and EBNA-1 expression to EBV-positive cells, BV-RO is expected to be a better candidate in application than BV-ROE-CMV in the long term.

Association of immunoescape mechanisms with Epstein‐Barr virus infection in nasopharyngeal carcinoma

We have investigated the association of immunoescape mechanisms in nasopharyngeal carcinoma (NPC) lesions with Epstein-Barr virus (EBV) infection and clinical course of the disease. Tumor biopsy specimens obtained from 36 Japanese NPC patients were examined for antigen processing machinery component and HLA class I antigen expression, CD8(+) T cell infiltration, and Fas, Fas ligand (FasL) and IL-10 expression using immunohistochemical staining. The results were correlated with the histopathological characteristics of the lesions, the clinical course of the disease and EBV infection. LMP2, TAP1, tapasin and HLA class I antigens were downregulated in more than 65% of the lesions tested, while FasL, Fas and IL-10 were expressed in at least 60% of the lesions. Statistical analysis showed that (i) HLA class I antigen expression was significantly correlated with LMP2 and tapasin expression (r = 0.39 and 0.45, respectively); (ii) CD8(+) T cell infiltration into tumor lesions was significantly correlated with HLA class I antigen, LMP2 and Fas expression (r = 0.34, 0.49 and 0.44, respectively); (iii) LMP2 and FasL expression was significantly correlated with IL-10 expression (r = 0.49 and 0.52, respectively); (iv) IL-10 expression was significantly associated with EBERs and EBV oncoprotein LMP1 expression (p = 0.00078 and 0.015, respectively) and (v) FasL overexpression was significantly associated with reduced patients' survival (p = 0.033). Multivariate analysis identified FasL overexpression as an independent unfavorable prognostic marker. These results suggest that NPC cells may utilize multiple immunoescape mechanisms, including dysfunction of HLA class I antigens and Fas/FasL apoptosis pathways. Furthermore, FasL expression appears to be associated with IL-10 upregulation in EBV positive NPC cells.