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An Epstein‐Barr virus (EBV)—encoded latent membrane protein 1 (LMP1) is a principal oncogene that plays a pivotal role in EBV‐associated malignant tumors including nasopharyngeal cancer (NPC). Recent genomic landscape studies revealed that NPC also contained many genomic mutations, suggesting the role of LMP1 as a driver gene for the induction of these genomic mutations. Nonetheless, its exact mechanism has not been investigated. In this study, we report that LMP1 alters the expression profile of APOBEC3s(A3s), host deaminases that introduce consecutive C‐to‐U mutations (hypermutation). In vitro, LMP1 induces APOBEC3B (A3B) and 3F(A3F), in a nasopharyngeal cell line, AdAH. Overexpression of LMP1, A3B, or A3F induces mtDNA hypermutation, which is also detectable from NPC specimens. Expression of LMP1 and A3B in NPC was correlated with neck metastasis. These results provide evidence as to which LMP1 induces A3s and mtDNA hypermutation, and how LMP1 facilitates metastasis is also discussed., EBV LMP1 induces host cytidine deaminases, APOBECs, in nasopharyngeal cells. TES2 domain is important for LMP1‐induced LMP1 hypermutates host mitochondrial genome.

Human papillomavirus (HPV) infection is now identified as a major etiologic factor for oropharyngeal cancer (OPC), and HPV positivity is well established better prognostic marker in OPC. Now, predictable markers for the prognosis of the patients who are stratified by HPV has been investigated in. Semaphorin 3A (SEMA3A) is a well-known axon guidance molecule in the nervous system. It is also known as a tumor suppressor in various cancers. In the present study, we examined the relationships between SEMA3A and clinicopathologic features, especially HPV status, and neoangiogenesis, and its prognostic significance for OPC patients. Thirty-two OPC patients and 17 normal patients were analyzed for SEMA3A expression by immunohistochemical analysis. We also analyzed 22 OPC specimens for CD34 expression as a marker of neoangiogenesis. SEMA3A was significantly downregulated in OPC compared with chronic tonsillitis tissues (p = 0.005). SEMA3A expression was negatively correlated with CD34 expression (r = &minus, 0.466, p = 0.033). Moreover, the higher SEMA3A expression cohort showed better survival than the lower SEMA3A expression cohort regardless of HPV status (p = 0.035). These results suggest that SEMA3A expression is a prognostic marker for survival regardless of HPV status and is associated with anti-angiogenesis in OPC.

Semaphorins were discovered as guidance signals that mediate neural development. Recent studies suggest that semaphorin 3A (Sema3A), a member of the semaphorin family, is involved in the development of several cancers. This study aimed to analyze the association of Sema3A with the clinical features of nasopharyngeal carcinoma (NPC), an Epstein−Barr virus-associated carcinoma, and the Epstein−Barr virus primary oncogene latent membrane protein 1 (LMP1). The expression of Sema3A and LMP1 was immunohistochemically examined in the 35 NPC specimens. The mean expression scores for Sema3A and LMP1 were 20.8% ± 14.5% and 13.9% ± 14.8%, respectively. The expression of Sema3A significantly correlated with that of LMP1 (r = 0.41, p = 0.014). In addition, the Sema3A high cohort showed significantly poorer prognosis than the Sema3A low cohort. Sema3A expression was higher in the LMP1-positive KH-1 and KR-4 cell lines compared to the LMP1-negative HeLa cells. Overexpression of LMP1 in the LMP1-negative AdAH cell line upregulated Sema3A expression, both at the transcriptional and translational level. Finally, Sema3A expression was associated with poor prognosis in patients with NPC. Our data suggest that LMP1 induces the expression of Sema3A, which may promote tumor progression in NPC.

Nasopharyngeal carcinoma (NPC) is very common in southern China and Southeast Asia. In regions where NPC is endemic, undifferentiated subtypes constitute most cases and are invariably associated with Epstein-Barr virus (EBV) infection, whereas the differentiated subtype is more common in other parts of the world. Undifferentiated NPC is a unique malignancy with regard to its epidemiology, etiology, and clinical presentation. Clinically, NPC is highly invasive and metastatic, but sensitive to both chemotherapy and radiotherapy (RT). Overall prognosis has dramatically improved over the past three decades because of advances in management, including the improvement of RT technology, the broader application of chemotherapy, and more accurate disease staging. Despite the excellent local control with modern RT, distant failure remains a challenging problem. Advances in molecular technology have helped to elucidate the molecular pathogenesis of NPC. This article reviews the contribution of EBV gene products to NPC pathogenesis and the current management of NPC.

Objective: Although Japan is a non-endemic area with nasopharyngeal carcinoma (NPC), the proportion of WHO type I NPC in Japan are different from that in non-endemic areas such as North America and Europe. Recently, it is said that not only Epstein-Barr virus (EBV) but also human papillomavirus (HPV) has an influence on NPC in non-endemic areas. The aim of this study is to clarify the influence of HPV on NPC in Japan. Methods: Paraffin-embedded tumor specimens were available for 59 patients with NPC diagnosed between 1996 and 2015. We detected the virus status by p16 immunohistochemistry, HPV PCR, and in situ hybridization for Epstein-Barr virus (EBV)-encoded RNA. Kaplan-Meier curves were used to compare the overall survival by viral status. Results: Among the 59 patients, 49 (83%) were EBV-positive/HPV-negative, 2 (3%) were EBV-positive/HPV-positive, and 8 (16%) were EBV-negative/HPV-negative. All HPV-positive NPCs were co-infected with EBV. There were no significant differences between the overall survival in the three groups (p=0.111). Conclusion: In Japan, HPV was detected in a few patients with NPC, and we suggest that HPV has no influence on NPC carcinogenesis in this population. © 2016 Elsevier Ireland Ltd., Embargo period 12 months

Conclusion: Reduced-RADPLAT for HPC achieved comparative survival and locoregional control rates with lower toxicities compared with concurrent chemoradiotherapies including original RADPLAT. S-1 adjuvant chemotherapy showed a survival benefit. Objectives: To evaluate the efficacy and toxicities of targeted intra-arterial (IA) infusion of cisplatin with concurrent radiotherapy with a reduced dose (reduced-RADPLAT) for resectable hypopharyngeal cancer (HPC). Methods: Between 1999–2012, 50 patients with stage II–IVA HPC primarily treated by reduced-RADPLAT were analyzed. They were treated by 2–5 courses of IA cisplatin infusion (100 mg per body) with simultaneous systemic infusion of sodium thiosulfate concurrent with conventional radiotherapy (66–70 Gy). After 2003, S-1, an oral fluoropyrimidine, adjuvant chemotherapy was administered to all eligible patients. Results: During a median follow-up of 48.6 months, the estimated 3- and 5-year overall survival (OS), progression-free survival (PFS), locoregional control, and laryngoesophageal dysfunction-free survival (LEDFS) rates were 76.0% and 62.0%, 58.0% and 50.0%, 66.0% and 62.0%, and 56.0% and 54.0%, respectively. Grade 3 toxicities were observed in 30.0%. No patient had grade 4 or higher toxicities. No patient required tube feeding or tracheotomy at 3 months after treatment. T4-lesions and S-1 administration were significant factors predicting poor and good OS, PFS, and LEDFS, respectively. © 2016 Informa UK Limited, trading as Taylor & Francis Group, Embargo Period 12 months

Conclusion: SPARC-expression is an indicator of the prognosis in terms of OS, independent of HPV-infection. HPV-negative patients with SPARC-Low show survival as favorable as HPV-positive patients, probably because of their higher salvage rate after relapse than SPARC-High patients. Objective: The objectives of the study were to clarify the correlation between the expression of secreted protein acidic and rich in cysteine (SPARC) and HPV-status, and to determine the prognostic value of SPARC-expression in oropharyngeal squamous cell carcinoma (OPSCC) patients. Methods: Fifty-three formalin-fixed and paraffin-embedded tissues were obtained from patients with OPSCC who underwent curative treatment. The SPARC protein was detected by immunohistochemistry. SPARC-expression level was divided into two categories, SPARC-High and SPARC-Low, according to the staining index. Results: Twenty-two out of the 53 OPSCC patients were HPV-positive. There was no significant correlation between the HPV-status and SPARC-expression level. Multivariate Cox proportional hazards regression analysis revealed that the HPV-status and SPARC-expression are independent prognostic indicators of favorable and unfavorable overall survival (OS) (p = 0.021 and p = 0.012), respectively. For disease-free survival, the HPV-status was the only predictive factor (p = 0.022). After stratification by the HPV-status, high SPARC-expression was a significant predictor of poor OS in HPV-negative OPSCC patients using Kaplan-Meier analysis and the log-rank test (p = 0.014). Ten out of 28 SPARC-Low patients relapsed, among which six patients (60%) were salvaged. However, 14 out of 25 SPARC-High patients relapsed, and only three patients (21.4%) were salvaged. © 2015 Taylor & Francis., Embargo Period 12 months

The free jejunum flap technique has been regarded as the optimal approach during circumferential pharyngolaryngectomy reconstruction. Although classical patency tests are available, an intraoperative guarantee of the patency of anastomoses and microcirculations is inevitable. Indocyanine green near-infrared angiography (ICGA) was intraoperatively performed in six patients after reconstruction using the free jejunum flap. An adequate arterial as well as venous phase was observed. In addition to classical patency tests and doppler, we have successfully monitored the flap after total pharyngolalyngectomy intraoperatively using the ICGA. Our preliminary results implicate that this novel technique offers secure intraoperative monitoring of a free jejunum graft. This technique will provide us with advantages over regular patency test in selected cases. © 2014 Springer-Verlag Berlin Heidelberg.

The contribution of the lymphatic system to tumor metastasis is being increasingly appreciated through studies of human cancers. As the biological behavior of nasopharyngeal carcinoma (NPC) depends on its nodal status, patients with advanced nodal status show a higher tendency toward a poor outcome. Here, we examined the role of lymphangiogenesis on lymphatic spread of NPC. We also evaluated the involvement of vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 (VEGFR3) signaling pathway on lymphangiogenesis in NPC. Furthermore, we tested whether Epstein-Barr virus (EBV)-latent membrane protein (LMP) 1 induces VEGF-C. Forty-one patients with NPC were evaluated for expressions of VEGF-C and its receptor, VEGFR3, and LMP1 proteins and lymphatic vessel counts (LVC) highlighted by anti-podoplanin employing immunohistochemistry. The VEGF-C induction by LMP1 was then tested with Western blotting and enzyme-linked immunosorbent assay in vitro. The LVC and VEGF-C expression were significantly higher in cases with advanced regional lymph node metastasis (N2,3) than those with no or limited lymph node involvement (N0,1) (p = 0.0380 and p = 0.0109, respectively). In VEGF-C/VEGFR3-positive cases, the LVC were significantly increased compared with VEGF-C/VEGFR3-negative cases (p = 0.0007). However, LMP1 expression did not show significant associations with LVC and VEGF-C-expression scores (p = 0.1210 and p = 0.1324, respectively). Induction of VEGF-C protein by LMP1 was not detected in vitro. These results suggest the involvement of the VEGF-C/VEGFR3 axis in the induction of lymphangiogenesis which results in lymphatic spread of NPC. However, EBV-LMP1 was not associated with the mechanism. © 2011 Elsevier Ltd. All rights reserved.

金沢大学医薬保健研究域医学系, Nasopharyngeal carcinoma (NPC) is a kind of rare head and neck cancer in Japan. However, NPC has some unique features. It is one of the most popular cancers in southern China, Southeast Asia, the Arctic, and the middle East/north Africa. This distinctive racial, ethnical, and geographic predisposition to NPC implies that both genetic susceptibility and environmental factors contribute to the development of this tumor. NPC is an Epstein-Barr virus - associated tumor. Consistent elevation of EBV antibody titers is a well-established risk factor of development of NPC. Not only pathophysiological relationship, but also molecular mechanism of EBV-mediated carcinogenesis has been enthusiastically investigated. LMP1, an EBV primary oncogene, upregulates each step of metastasis, and contribute to highly metastatic feature of NPC. A tumor suppressor gene p53 is mostly intact and overexpressed in NPC whereas expression of p16, a cyclin-dependent kinase inhibitory protein, is downregulated in 2/3 of NPC. Intention modulated radiotherapy (IMRT) is now getting prevalent for the treatment of NPC because of complicated structure and location of nasopharynx. A good therapeutic result can be achieved by distributing a high dose to the tumor while keeping down normal tissue complications by reducing radiation dose to normal tissues. Chemotherapy is important to control distant metastasis of chemoradiosensitive NPC, and thus, should play an important role. However, most effective combination of anti-tumor drugs, protocol of chemoradiotherapy has not well-established. Finally, molecular targeting therapy, including targeting EBV gene product, has been developing and on the way to the clinical use. © 2011 Elsevier Ireland Ltd. All rights reserved.

金沢大学医学部附属病院耳鼻咽喉科, In nasopharyngeal carcinoma (NPC), Epstein-Barr virus (EBV) infection is mainly latent, and the tumor cells contain episomal viral DNA. We have shown that the acyclic nucleoside phosphonate analog, cidofovir [(S)-1-(3-hydroxy-2- (phosphonylmethoxypropyl)cytosine] (HPMPC), inhibits growth of NPC xenografts in nude mice by causing apoptosis. The ribonucleotide reductase (RR) inhibitors, hydroxyurea and didox (3,4-dihydroxybenzohydroxamic acid), have been demonstrated to inhibit neoplastic growth and are used as antiviral and anticancer agents. Here we show that RR inhibitors enhance the antitumor effect of cidofovir in EBV-transformed epithelial cells. MTT assays indicate that hydroxyurea and didox enhance cidofovir-induced cell toxicity in NPC-KT cells, an EBV-positive epithelial cell line derived from NPC. The effect is due to enhancement of apoptosis through the caspase cascade as shown by pronounced cleavage of poly(ADP-ribose) polymerase. Finally, hydroxyurea strikingly enhanced the cidofovir-induced growth-inhibitory effect on NPC grown in athymic mice. The results suggest that RR inhibitors should enhance the antitumor effect of acyclic nucleoside phosphonate analogs on NPC. © 2005 Wiley-Liss, Inc.

It has emerged recently that exosomes are potential carriers of pro-tumorigenic factors that participate in oncogenesis. However, whether oncogenic transcription factors are transduced by exosomes is unknown. Hypoxia-inducible factor-1α (HIF1α) transcriptionally regulates numerous key aspects of tumor development and progression by promoting a more aggressive tumor phenotype, characterized by increased proliferation and invasiveness coupled with neoangiogenesis. It has been shown that the principal oncoprotein of Epstein–Barr virus (EBV), latent membrane protein 1 (LMP1), drives oncogenic processes and tumor progression of the highly invasive EBV malignancy, nasopharyngeal carcinoma (NPC). We now demonstrate that endogenous HIF1α is detectable in exosomes and that LMP1 significantly increases levels of HIF1α in exosomes. HIF1 recovered from exosomes retains DNA-binding activity and is transcriptionally active in recipient cells after exosome uptake. We also show that treatment of EBV-negative cells with LMP1-exosomes increases migration and invasiveness of NP cell lines in functional assays, which correlates with the phenotype associated with epithelial–mesenchymal transition (EMT). In addition, we provide evidence that HIF1α itself participates in exosome-mediated pro-metastatic effects in recipient cells, as exosome-mediated delivery of active and inactive forms of HIF1α results in reciprocal changes in the expression of E- and N-cadherins associated with EMT. Further, immunohistochemical analysis of NPC tumor tissues revealed direct correlation between protein levels of LMP1 and of the endosome/exosome marker tetraspanin, CD63, which suggests an increase in exosome formation in this EBV-positive malignancy. We hypothesize that exosome-mediated transfer of functional pro-metastatic factors by LMP1-positive NPC cells to surrounding tumor cells promotes cancer progression.

Objectives: Epstein-Barr virus (EBV)-related micoRNAs (miRNAs), BamHI-A rightward transcripts (BART)-miRNAs, are released in a stable form from viable cells, which are abundant in patients with EBV-positive nasopharyngeal carcinoma (NPC). We estimated copy numbers of circulating miR-BART2-5p, miR-BART17-5p, and miR-BART18-5p as well as BamHI-W DNA as biomarkers. Materials and Methods: Serums from 31 EBV-positive (confirmed by in situ hybridization for EBV-encoded small RNAs) NPC patients and 40 non-NPC controls were analyzed. Among the 31 NPC patients, serums at the initial diagnosis and three months after treatment were obtained from 20 patients, and serums only at three months after treatment were obtained from 11 patients. Results: The sensitivity/specificity of circulating BamHI-W DNA, miR-BART2-5p, miR-BART17-5p, and miR-BART18-5p for the diagnosis of NPC before treatment were 100/100, 85/85, 60/95, and 25/100%, respectively. For BamHI-W DNA, NPC patients with stage IV disease had significantly higher copy numbers than those with I-III. Copy numbers decreased significantly post-treatment. In contrast, copy numbers of the three BART-miRNAs showed no significant correlation with the clinical stage at diagnosis or any significant post-treatment change. After treatment, BamHI-W DNA and miR-BART17-5p were detected in 5 and 6 cases out of 11 patients with recurrent or residual tumors, respectively. However, BamHI-W DNA and miR-BART17-5p were absent in all 20 patients without relapse or residual tumors. Conclusion: The copy number of circulating BamHI-W DNA is a more useful biomarker for the initial diagnosis of NPC than the three BART-miRNAs examined. Post-treatment detection of miR-BART17-5p is a potential biomarker of a poor prognosis. © 2016 Hirai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

FGF-2, a potent angiogenic factor that is involved in tumor invasion, is known to be released extracellularly by a nonclassical secretory pathway. Recently it has become clear that Epstein-Barr virus, specifically its oncoprotein LMP1, can induce expression of angiogenic factors. Among these factors is FGF-2. LMP1 not only promotes expression of FGF-2, but also the release extracellularly of its 18-kDa isoform. We analyzed the mechanism of FGF-2 release induced by LMP1. Confocal immunofluorescence microscopy revealed colocalization of FGF-2 with LMP1 in small dots also stained positively for CD63 and cathepsin D, markers of late endosomes or multivesicular bodies. Biochemical analysis and immunoelectron microscopy of purified exosomal fractions from cotransfected cells demonstrated increased release of exosomes and the concentration of LMP1 and FGF-2 in these structures. Moreover, cotransfection appeared to induce partial redistribution of the Na(+)/K(+)-ATPase, which participates in FGF-2 release, from the plasma membrane to the intracellular LMP1/FGF-2 positive dots. Treatment with ouabain, which inhibits Na(+)/K(+)-ATPase activity, partially suppressed FGF-2 secretion via exosomes in a dose-dependent manner. The results suggest that exosomes may represent a previously unrecognized mechanism for FGF-2 release mediated by LMP1, and that this pathway involves the activity of Na(+)/K(+)-ATPase.