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Background Dietary factors, such as consumption of preserved foods, fresh vegetables, and fruits, have been linked to the risk of nasopharyngeal carcinoma (NPC). However, little is known about associations between dietary patterns and the risk of NPC in NPC-endemic areas. Objectives We aimed to evaluate whether dietary patterns are associated with NPC risk. Methods We studied 2554 newly diagnosed NPC patients aged 20–74 y living in 3 endemic regions of southern China, and 2648 population-based controls frequency-matched to case patients by age, sex, and region, between 2010 and 2014. Dietary components were derived from food frequency data in adulthood and adolescence using principal component analysis. Four dietary components were identified and highly similar in adulthood and adolescence. We used multivariable unconditional logistic regression to calculate ORs with 95% CIs for the association between dietary patterns and NPC risk. Results Compared with the lowest quartile, individuals in the highest quartile of the “plant-based factor” in adulthood had a 52% (OR: 0.48; 95% CI: 0.38, 0.59) decreased risk of NPC, and those in the highest quartile of the “animal-based factor” had a >2-fold (OR: 2.26; 95% CI: 1.85, 2.77) increased risk, with a monotonic dose-response trend (P-trend

Background The potential role of occupational exposures in the development of nasopharyngeal carcinoma (NPC) remains unclear, particularly in high-incidence areas. Methods The authors conducted a population-based case-control study, consisting of 2514 incident NPC cases and 2586 randomly selected population controls, in southern China from 2010 to 2014. Occupational history and other covariates were self-reported using a questionnaire. Multivariate logistic regression was used to estimate odds ratios (ORs) with 95% confidence intervals (CIs) for the risk of NPC associated with occupational exposures. Restricted cubic splines were used to evaluate potentially nonlinear duration-response relations. Results Individuals who had exposure to occupational dusts (OR, 1.45; 95% CI, 1.26-1.68), chemical vapors (OR, 1.37; 95% CI, 1.17-1.61), exhausts/smokes (OR, 1.42; 95% CI, 1.25-1.60), or acids/alkalis (OR, 1.56; 95% CI, 1.30-1.89) in the workplace had an increased NPC risk compared with those who were unexposed. Risk estimates for all 4 categories of occupational exposures appeared to linearly increase with increasing duration. Within these categories, occupational exposure to 14 subtypes of agents conferred significantly higher risks of NPC, with ORs ranging from 1.30 to 2.29, including dust from metals, textiles, cement, or coal; vapor from formaldehyde, organic solvents, or dyes; exhaust or smoke from diesel, firewood, asphalt/tar, vehicles, or welding; and sulfuric acid, hydrochloric acid, nitric acid, and concentrated alkali/ammonia. Conclusions Occupational exposures to dusts, chemical vapors, exhausts/smokes, or acids/alkalis are associated with an excess risk of NPC. If the current results are causal, then the amelioration of workplace conditions might alleviate the burden of NPC in endemic areas. Lay summary The role of occupational exposures in the development of nasopharyngeal carcinoma (NPC) remains unclear, particularly in high-incidence areas. The authors conducted a population-based study with 2514 incident NPC cases and 2586 population controls in southern China and observed that occupational exposures were associated with an increased risk of NPC. Duration-response trends were observed with increasing duration of exposure. These findings provide new evidence supporting an etiologic role of occupational exposures for NPC in a high-incidence region.

Background: The potential effect of alcohol or tea intake on the risk of nasopharyngeal carcinoma (NPC) remains controversial. Methods: In a population-based case–control study in southern China, we assessed alcohol or tea intake from 2,441 histopathologically confirmed NPC cases and 2,546 controls. We calculated mean daily ethanol (g/day) and tea intake (mL/day). Fully adjusted ORs with 95% confidence intervals (CI) were estimated using logistic regression; potential dose–response trends were evaluated using restricted cubic spline analysis. Results: Compared with nondrinkers, no significantly increased NPC risk in men was observed among current alcohol drinkers overall (OR, 1.08; 95% CI, 0.93–1.25), nor among current heavy drinkers (OR for ≥90 g/day ethanol vs. none, 1.32; 95% CI, 0.95–1.84) or former alcohol drinkers. Current tea drinking was associated with a decreased NPC risk (OR, 0.73; 95% CI, 0.64–0.84). Compared with never drinkers, those with the low first three quintiles of mean daily current intake of tea were at significantly lower NPC risk (OR, 0.53, 0.68, and 0.65, respectively), but not significant for the next two quintiles. Current daily tea intake had a significant nonlinear dose–response relation with NPC risk. Conclusions: Our study suggests no significant association between alcohol and NPC risk. Tea drinking may moderately reduce NPC risk, but the lack of a monotonic dose–response association complicates causal inference. Impact: Tea drinking might be a healthy habit for preventing NPC. More studies on biological mechanisms that may link tea with NPC risk are needed.

Epstein–Barr virus (EBV)‐associated nasopharyngeal carcinoma (NPC) cells display conspicuous lipid accumulation. Here, we show that EBV‐encoded membrane protein 2A (LMP2A) induces lipid accumulation in NPC cells by inhibition of lipolytic gene, adipocyte triacylglycerol lipase (ATGL). Inhibition of ATGL results in enhanced lipid accumulation and migratory capacity in NPC cells. Loss of ATGL in NPC cells correlates with poor overall survival., Epstein–Barr virus (EBV)‐associated nasopharyngeal carcinoma (NPC) is one of the most common human cancers in South‐East Asia exhibiting typical features of lipid accumulation. EBV‐encoded latent membrane protein 2A (LMP2A) is expressed in most NPCs enhancing migration and invasion. We recently showed an increased accumulation of lipid droplets in NPC, compared with normal nasopharyngeal epithelium. It is important to uncover the mechanism behind this lipid metabolic shift to better understand the pathogenesis of NPC and provide potential therapeutic targets. We show that LMP2A increased lipid accumulation in NPC cells. LMP2A could block lipid degradation by downregulating the lipolytic gene adipose triglycerol lipase (ATGL). This is in contrast to lipid accumulation due to enhanced lipid biosynthesis seen in many cancers. Suppression of ATGL resulted in enhanced migration in vitro, and ATGL was found downregulated in NPC biopsies. The reduced expression level of ATGL correlated with poor overall survival in NPC patients. Our findings reveal a new role of LMP2A in lipid metabolism, correlating with NPC patient survival depending on ATGL downregulation.

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia, hence, identifying easily detectable biomarkers for NPC screening is essential for better diagnosis and prognosis. Using genome‐wide and targeted analyses based on next‐generation sequencing approaches, we previously showed that gene promoters are hypermethylated in NPC tissues. To confirm whether DNA methylation rates of genes could be used as biomarkers for NPC screening, 79 histologically diagnosed NPC patients and 29 noncancer patients were recruited. A convenient quantitative analysis of DNA methylation using real‐time PCR (qAMP) was carried out, involving pretreatment of tissue DNA, and circulating cell‐free DNA (ccfDNA) from nonhemolytic plasma, with methylation‐sensitive and/or methylation‐dependent restriction enzymes. The qAMP analyses revealed that methylation rates of RERG, ZNF671, ITGA4, and SHISA3 were significantly higher in NPC primary tumor tissues compared to noncancerous tissues, with sufficient diagnostic accuracy of the area under receiver operating characteristic curves (AUC). Interestingly, higher methylation rates of RERG in ccfDNA were statistically significant and yielded a very good AUC; however, those of ZNF671, ITGA4, and SHISA3 were not significant. Furthermore, the combination of methylation rates of RERG and ZNF671 in ccfDNA showed higher diagnostic accuracy than either of them individually. In conclusion, the methylation rates of specific genes in ccfDNA can serve as novel biomarkers for early detection and screening of NPC., The present study identified novel blood‐based noninvasive methylation biomarkers for nasopharyngeal carcinoma screening by a combination of RERG and ZNF671 methylation rates in circulating cell‐free DNA.

The dysregulation of epigenetic modification and energy metabolism cooperatively contribute to the tumorigenesis of nasopharyngeal carcinoma (NPC). However, the detailed mechanisms underlying their joint contribution to NPC development and progression remain unclear. Here, we investigate the role of Acy1 Coenzyme A Acyltransferases1 (ACAT1), a key enzyme in the metabolic pathway of ketone bodies, in the proliferation and metastasis of NPC and to elucidate the underlying molecular mechanisms. Ketogenesis, plays a critical role in tumorigenesis. Previously, we reported two enzymes involved in ketone body metabolism mediate epigenetic silencing and act as tumor suppressor genes in NPC. Here, we identify another key enzyme, Acetyl-CoA acetyltransferase 1 (ACAT1), and show that its transcriptional inactivation in NPC is due to promoter hypermethylation. Ectopic overexpression of ACAT1 significantly suppressed the proliferation and colony formation of NPC cells in vitro. The migratory and invasive capacity of NPC cells was inhibited by ACAT1. The tumorigenesis of NPC cells overexpressing ACAT1 was decreased in vivo. Elevated ACAT1 in NPC cells was accompanied by an elevated expression of CDH1 and a reduced expression of vimentin and SPARC, strongly indicating that ACAT1 is involved in regulating epithelial-mesenchymal transition (EMT). We also found that ACAT1 contributes to increased intracellular levels of β-hydroxybutyrate (β-HB). Exogenously supplied β-HB significantly inhibits the growth of NPC cells in a dose-dependent manner. In summary, ACAT1 may function as a tumor suppressor via modulation of ketogenesis and could thus serve as a potential therapeutic target in NPC. In summary, our data suggest that regulation of ketogenesis may serve as adjuvant therapy in NPC.

Background To assess the effects of Epstein–Barr virus (EBV) and human papillomavirus (HPV) infection on the tumor microenvironment, we examined the relationship between viral infection status, macrophage migration inhibitory factor (MIF), and tumor-associated macrophages in nasopharyngeal carcinoma (NPC). Methods A tissue microarray containing 150 cores from 90 patients with NPC and six with chronic inflammation was used. EBV and HPV status were detected using in situ hybridization with commercial EBER1 and HPV16/18 probes. Immunofluorescence double staining of MIF, pan-macrophage marker CD68, M1 macrophage marker CD11c, and M2 macrophage marker CD163 were analyzed using the same tissue microarray. The levels of these markers between NPC and inflammation cases and between tumor nests and stroma were compared. Correlations among these markers were analyzed. Results We found EBER1(+) cases in 90% of NPC patients, including 10% EBV/HPV co-infection. M1 macrophages mainly infiltrated the tumor nest, while M2 macrophages infiltrated the tumor stroma. We found a significant positive correlation between EBER1 levels and MIF levels in tumor nests and a significant positive correlation between HPV16/18 and CD11c(+) cell levels in NPC tissues. Conclusions It is suggested that MIF is associated with EBV, and M1 macrophage infiltration is affected by HPV status in NPC.

Background: Nasopharyngeal carcinoma (NPC) is a common tumor in head and neck and is prevailing in China. Although treatment methods continue to improve, the prognosis of advanced patients is still unsatisfactory. Kinesin family member 15 (KIF15) is a kind of protein, which regulates the process of cell mitosis and plays an important role in several types of human cancers. This study aims to investigate the role of KIF15 in NPC.Methods: First, the differential expression of KIF15 in NPC and para-carcinoma tissues was evaluated based on both data collected from Gene Expression Omnibus (GEO) database and immunohistochemical analysis on clinical specimens collected from in-house cohort. Next, cell lines C666-1 and CNE-2Z were selected for the construction of KIF15‑knockdown cell models. Then, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, wound healing, Transwell and clone formation assays were used to detect the proliferation, apoptosis, migration, invasion and colony formation of nasopharyngeal carcinoma cells in vitro. A mouse xenograft model was constructed for in vivo study. Furthermore, Human Apoptosis Antibody Array kit was used to screen possible targets of KIF15 in NPC. In the end, the potential molecular mechanisms underlying the effects of KIF15 was explored through western blot analysis.Results: The results showed that the expression of KIF15 in NPC tissues is higher than that in para-carcinoma tissues, and high levels of KIF15 expression are associated with low survival rates. In addition, knockdown of KIF15 inhibited cell proliferation, migration, invasion and colony formation ability, and promoted cell apoptosis. What’s more, in vivo xenograft experiments showed that down-regulation of KIF15 can inhibit NPC tumor growth. Moreover, the mechanism study demonstrated a variety of apoptosis-related proteins as well as PI3K/AKT and MAPK signaling pathways may be involved in KIF15-induced regulation of NPC.Conclusions: In short, we demonstrated that KIF15 is overexpressed and accelerated the progression of nasopharyngeal carcinoma. It can be used as a new prognostic indicator as well as a potential drug target for the treatment of NPC.

BACKGROUND Chemokines are important in inflammation, immunity, tumor progression, and metastasis. The purpose of this research was to find an integrated-RNA signature of chemokine family genes to predict the survival prognosis in head and neck squamous carcinoma (HNSC) patients. MATERIAL AND METHODS Relevant data of 504 HNSC patients were extracted from The Cancer Genome Atlas (TCGA) database. Through analyzing RNA sequencing data, the univariate Cox model was used to identify chemokine family genes associated with survival and then to develop a multiple-RNA signature in the training set. The prediction value of this multiple-RNA signature was further verified in the validation and entire sets. The receiver operating characteristic curves were used to assess the predictive value of this multiple-RNA signature. RESULTS Eleven chemokines were included in this prognostic signature. Based on this 11-chemokine signature, we further categorized patients as high or low risk. Compared with low-risk patients, high-risk patients had shorter overall survival (OS) time in the training set [hazard ratio (HR)=3.497, 95% confidence interval (CI)=2.142-5.711, p

Purpose: Caffeic acid phenethyl ester (CAPE) is the main polyphenol extracted from honeybee propolis, which inhibits the growth of several kinds of tumor. This study aimed to assess the inhibitory effect of CAPE in nasopharyngeal carcinoma (NPC), evaluate the synergistic action of CAPE in radiotherapy sensitivity of NPC cell lines and further elucidate the possible molecular mechanism involved. Materials and methods: CCK-8 assay was used to analyze cell proliferation ability. Colony formation assay was used to evaluate the clonogenic ability and radio-sensitiveness of NPC cells by CAPE treatment. Wound-healing and transwell assay were used to assess the motility of cells. The expression of key molecules of the epithelial-mesenchymal transition (EMT) was determined by western blot analysis and changes in radiation sensitivity were measured by colony-formation assay. cDNA microarray analysis was used to determine differentially expressed genes with and without CAPE treatment, with Gene Ontology enrichment of gene function and KEGG pathways determined. Cell cycle and apoptosis were detected by flow cytometry and western blot analysis. Results: CAPE suppressed the viability of NPC cell lines time- and dose-dependently. It induced apoptosis in NPC cells along with decreased expression of Bcl-XL and increased cleavage of PARP and expression of Bax. G1 phase arrest was induced by CAPE with ower expression of CDK4, CDK6, Rb and p-Rb. The migratory and invasive ability of NPC cells was decreased by the EMT pathway. The irradiation sensitivity of NPC cells was enhanced with CAPE treatment. CAPE specifically inhibited nuclear factor κB (NF-κB) signaling pathway by suppressing p65 subunit translocation from cytoplasm to nucleus. CAPE treatment was synergistic with chemotherapy and radiotherapy. Conclusion: CAPE may inhibit the proliferation and metastasis of NPC cells but enhance radiosensitivity in NPC therapy by inhibiting the NF-κB pathway. CAPE could be a potential therapeutic compound for NPC therapy.

The relationship between oral health and the risk of nasopharyngeal carcinoma (NPC) was previously established. However, the role of oral microbiome has not been evaluated in the disease in a large epidemiological study. This paper clearly establishes a difference in the oral microbiomes between NPC patients and healthy controls which cannot be explained by other confounding factors. It furthermore identifies a pair of closely related coexcluding organisms associated with the disease, highlighting the importance of modern methods for single-nucleotide resolution in 16S rRNA sequence characterization. To the best of our knowledge, this is one of the first examples of cancer-associated niche specialization of the oral microbiome., Oral health and changes in the oral microbiome have been associated with both local and systemic cancer. Poor oral hygiene is a known risk factor for nasopharyngeal carcinoma (NPC), a virally associated head and neck cancer endemic to southern China. We explored the relationship between NPC and the oral microbiome using 16S rRNA sequencing in a study of 499 NPC patients and 495 population-based age and sex frequency-matched controls from an area of endemicity of Southern China. We found a significant reduction in community richness in cases compared to that in controls. Differences in the overall microbial community structure between cases and controls could not be explained by other potential confounders; disease status explained 5 times more variation in the unweighted UniFrac distance than the next most explanatory variable. In feature-based analyses, we identified a pair of coexcluding Granulicatella adiacens amplicon sequence variants (ASVs) which were strongly associated with NPC status and differed by a single nucleotide. The G. adiacens variant an individual carried was also associated with the overall microbial community based on beta diversity. Co-occurrence analysis suggested the two G. adiacens ASVs sit at the center of two coexcluding clusters of closely related organisms. Our results suggest there are differences in the oral microbiomes between NPC patients and healthy controls, and these may be associated with both a loss of microbial diversity and niche specialization among closely related commensals. IMPORTANCE The relationship between oral health and the risk of nasopharyngeal carcinoma (NPC) was previously established. However, the role of oral microbiome has not been evaluated in the disease in a large epidemiological study. This paper clearly establishes a difference in the oral microbiomes between NPC patients and healthy controls which cannot be explained by other confounding factors. It furthermore identifies a pair of closely related coexcluding organisms associated with the disease, highlighting the importance of modern methods for single-nucleotide resolution in 16S rRNA sequence characterization. To the best of our knowledge, this is one of the first examples of cancer-associated niche specialization of the oral microbiome.

Kidney is an important organ for ketone body metabolism. However, the role of abnormal ketone metabolism and its possible function in tumorigenesis of clear cell renal cell carcinoma (ccRCC) have not yet been elucidated. Three differentially expressed key enzymes involved in ketone body metabolism, ACAT1, BDH2, and HMGCL, were screened out between ccRCC and normal kidney tissues using the GEO and TCGA databases.We confirmed that the transcription and protein expression of ACAT1, BDH2, and HMGCL were significantly lower in ccRCC by real-time RT-PCR and IHC assays. Those patients with lower expression of these three genes have a worse outcome. In addition, we demonstrated that ectopic expression of each of these genes inhibited the proliferation of ccRCC cells. The overexpressed ACAT1 and BDH2 genes remarkably impeded the migratory and invasive capacity of ccRCC cells. Furthermore, exogenous β-hydroxybutyrate suppressed the growth of ccRCC cells in vitro in a dose-dependent manner. Our findings suggest that ACAT1, BDH2, and HMGCL are potential tumor suppressor genes, and constitute effective prognostic biomarkers for ccRCC. Ketone body metabolism might thus be a promising target in a process for developing novel therapeutic approaches to treat ccRCC.

Objectives Given the role of exposures related to residence in the development of nasopharyngeal carcinoma (NPC) has not been well explored, present study aims to investigate the magnitude and pattern of associations for NPC with lifelong residential exposures. Materials and Methods We carried out a multi-center, population-based case-control study with 2533 incident NPC cases and 2597 randomly selected population controls in southern China between 2010 and 2014. We performed multivariate logistic regression to estimate odds ratios (ORs) with 95% confidence intervals (CIs) for the risk of NPC associated with residential exposures. Results Compared with those living in a building over lifetime, risk of NPC was higher for individuals living in a cottage (OR: 1.56; 95% CI: 1.34–1.81) or in a boat (3.87; 2.07–7.21). NPC risk was also increased in individuals using wood (1.34; 1.03–1.75), coal (1.70; 1.17–2.47), or kerosene (3.58; 1.75–7.36) vs. using gas/electricity as cooking fuel; using well water (1.57; 1.34–1.83), river water (1.80; 1.47–2.21), or spring/pond/stream water (2.03; 1.70–2.41) vs. tap water for source of drinking water; living in houses with smaller-sized vs. larger windows in the bedroom (3.08; 2.46–3.86), hall (1.89; 1.55–2.31) or kitchen (1.67; 1.34–2.08); and increasing exposure to cooking smoke [(1.53; 1.20–1.94) for high exposure)] or burned incense [(1.59; 1.31–1.95) for daily use)]. Weighted Cox regression analysis corroborated these results. Conclusion Poorer residential conditions and household air pollution are associated with an increased risk of NPC. Large-scale studies in other populations or longitudinal studies are warranted to further corroborate these findings.

The aim of this study was to investigate the inactivation of the MutS homolog human 3 (MSH3) gene by promoter methylation in nasopharyngeal carcinoma (NPC). Methylation-specific PCR, semi-quantitative reverse transcription PCR and immunohistochemical analysis were used to detect methylation and the mRNA and protein expression levels of MSH3 in 54 cases of NPC tissues and 16 cases of normal nasopharyngeal epithelial (NNE) tissues. The association between promoter methylation and mRNA expression, and the mRNA and protein expression of the gene and clinical factors was analyzed. The promoter methylation of MSH3 was detected in 50% (27/54) of the primary tumors, but not in the 16 NNE tissues. The mRNA and protein expression levels were significantly decreased in the 54 cases of human NPC as compared to the 16 NNE tissues (P0.05). On the whole, MSH3 was frequently inactivated by promoter methylation and its mRNA and protein expression correlated with the primary tumor stage in NPC.

Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) promotes the motility of nasopharyngeal carcinoma (NPC) cells. Previously, we have shown that the localization of integrin β4 (ITGβ4) is regulated by LMP2A, with ITGβ4 concentrated at the cellular protrusions in LMP2A-expressing NPC cells. In the present study, we aim to further investigate mechanisms involved in this process and its contribution to cell motility. We show that expression of LMP2A was correlated with increased epidermal growth factor receptor (EGFR) activation, elevated levels of intracellular Ca2+, calpain activation and accelerated cleavage of ITGβ4. Activation of EGFR and calpain activity was responsible for a redistribution of ITGβ4 from the basal layer of NPC cells to peripheral membrane structures, which correlated with an increased migratory capacity of NPC cells. Furthermore, we demonstrated that the calpain inhibitor calpastatin was downregulated in NPC primary tumors. In conclusion, our results point to LMP2A-mediated targeting of the EGFR/Ca2+/calpain/ITGβ4 signaling system as a mechanism underlying the increased motility of NPC cells. We suggest that calpain-facilitated cleavage of ITGβ4 contributes to the malignant phenotype of NPC cells., Summary: LMP2A expression in nasopharyngeal carcinoma cells increases EGFR activation and cytosolic Ca2+, subsequently stimulates calpain-dependent cleavage of ITGβ4 and enhances cell motility.

Background An association between a nonmedicinal herbal diet and nasopharyngeal carcinoma (NPC) has often been hypothesized but never thoroughly investigated. Methods This study enrolled a total of 2469 patients with incident NPC and 2559 population controls from parts of Guangdong and Guangxi Provinces in southern China between 2010 and 2014. Questionnaire information was collected on the intake of traditional herbal tea and herbal soup as well as the specific herbal plants used in soups and other potentially confounding lifestyle factors. Multivariate logistic regression models were used to estimate odds ratios (ORs) with 95% confidence intervals (CIs) for the NPC risk in association with herbal tea and soup intake. Results Ever consumption of herbal tea was not associated with NPC risk (OR, 1.03; 95% CI, 0.91-1.17). An inverse association was observed for NPC among ever drinkers of herbal soup (OR, 0.78; 95% CI, 0.67-0.90) but without any monotonic trend with an increasing frequency or duration of herbal soup consumption. Inverse associations with NPC risk were detected with 9 herbal plants used in herbal soup, including Ziziphus jujuba, Fructus lycii, Codonopsis pilosula, Astragalus membranaceus, Semen coicis, Smilax glabra, Phaseolus calcaratus, Morinda officinalis, and Atractylodes macrocephala (OR range, 0.31-0.79). Conclusions Consuming herbal soups including specific plants, but not herbal tea, was inversely associated with NPC. If replicated, these results might provide potential for NPC prevention in endemic areas.

Background 3-Hydroxybutyrate dehydrogenase type 2 (BDH2) is known to catalyse a rate-limiting step in the biogenesis of the mammalian siderophore and regulate intracellular iron metabolism. Here we aim to explore the expression and possible function of BDH2 in nasopharyngeal carcinoma (NPC). Methods The transcription and protein expression of BDH2 in NPC were determined by both real-time RT-PCR and immunohistochemistry staining assays. Cell proliferation, migration and invasion were evaluated by MTT assay, wound-healing assay and Transwell assay, respectively. The profile of genes regulated by restoring BDH2 expression in NPC cells was analysed by cDNA microarray. The level of iron in NPC cells was detected by iron colorimetric assay. Results The expression of BDH2 was significantly downregulated in NPC. Ectopic expression of BDH2 inhibited NPC cell proliferation and colony formation. Meanwhile, BDH2 suppressed the migration and invasion of NPC cells by reversing the epithelial–mesenchymal transition (EMT). In addition, a higher level of BDH2 decreased the growth and metastasis of NPC cells via reducing intracellular iron level. Conclusions Our findings suggest that BDH2 may be a candidate tumour-suppressor gene in NPC. Decreasing intracellular iron could be an effective therapeutic approach for NPC.

Rab11 family interacting protein 2 (Rab11-FIP2) is a conserved protein and effector molecule for the small GTPase Rab11. By interacting with Rab11 and MYO5B, Rab11-FIP2 regulates endosome trafficking of plasma membrane proteins, promoting cellular motility. The endosomal trafficking system in nasopharyngeal carcinoma (NPC) remains unclear. Here, an outlier analysis using the Oncomine database suggested that Rab11-FIP2 but not Rab11 and MYO5B was overexpressed in NPC. We confirmed that the transcription of Rab11-FIP2 was upregulated in NPC cell lines and primary tumor tissues as compared with a normal nasopharyngeal epithelial cell line and normal nasopharynx tissues. We further confirmed the elevated protein expression level of Rab11-FIP2 in NPC biopsies. Instead of regulating the epithelial-mesenchymal transition or Akt signaling pathway, knockdown of Rab11-FIP2 inhibited the migration and invasion ability of NPC cell lines by decreasing the expression of Rac and Cdc42. In summary, Rab11-FIP2 could be an oncogene in NPC, mainly contributing to metastatic capacity by activating Rho GTPase signaling.

Whether the association between body size or shape and nasopharyngeal carcinoma (NPC) risk exists or varies by age‐specific body size indicators is unclear. In a population‐based case–control study conducted in Southern China between 2010 and 2014, self‐reported height, weight, and body shape at age 20 and 10 years before interview were collected from 2448 histopathologically confirmed NPC cases and 2534 population‐based controls. Body mass index (BMI) was categorized according to the World Health Organization guidelines for Asian populations: underweight (

Glutaredoxin 3 (GLRX3) is antioxidant enzyme, maintaining a low level of ROS, thus contributing to the survival and metastasis of several types of cancer. However, the expression and functions of GLRX3 have not been addressed in nasopharyngeal carcinoma (NPC). In this study, we found that GLRX3 was overexpressed in NPC. Knockdown of GLRX3 in NPC cell lines inhibited proliferation in vitro, tumorignesis in vivo, and colony formation. In addition, GLRX3 knockdown decreased the migration and invasion capacity of NPC cells by reversing the epithelial-mesenchymal transition (EMT). Furthermore, stabilization of GLRX3 was positively related to with epidermal growth factor receptor (EGFR) expression and negatively with ROS generation. Phosphorylation of Akt, a key downstream effector, was induced by EGFR signaling but did not rely on increasing ROS level in NPC cells. GLRX3 might be an oncoprotein in NPC, playing important roles in increasing redox reaction and activating EGFR/ Akt signals, so it may be a therapeutic target for NPC.

// Shumin Wang 1, 2 , Yingxi Mo 1, 2 , Kaoru Midorikawa 1 , Zhe Zhang 2 , Guangwu Huang 2 , Ning Ma 3 , Weilin Zhao 1, 2 , Yusuke Hiraku 1 , Shinji Oikawa 1 , Mariko Murata 1 1 Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie, Japan 2 Department of Otolaryngology Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China 3 Faculty of Nursing Science, Suzuka University of Medical Science, Suzuka, Mie, Japan Correspondence to: Mariko Murata, e-mail: mmurata@doc.medic.mie-u.ac.jp Keywords: nasopharyngeal carcinoma, microRNA, Epstein-Barr virus, tumor suppressor, biomarker Received: July 03, 2015 Accepted: October 02, 2015 Published: October 14, 2015 ABSTRACT Nasopharyngeal carcinoma (NPC) is a malignancy with poor prognosis that is endemic to Southeast Asia. We profiled microRNAs (miRNAs) of NPCs using microarrays and confirmed the results by quantitative RT-PCR. The results revealed that seven miRNAs were significantly up-regulated, and six miRNAs were down-regulated, in NPC tissues relative to noncancerous nasopharyngeal epithelia (NNE). Expression of miR-497 was also significantly reduced in the plasma of NPC patients relative to the plasma of noncancerous control patients. The concordant down-regulation of miR-497 in tissues and plasma suggested that miR-497 could be used as a diagnostic biomarker for NPC. Functional analyses of the effect of miR-497 on cancer phenotypes revealed that transfection of miR-497 mimic into NPC cells suppressed cell growth and migration and induced apoptosis. Subcutaneous xenografts of transfected cells in nude mice demonstrated that miR-497 significantly inhibited tumor growth. Two potential targets of miR-497, ANLN (anillin, actin-binding protein) and HSPA4L (heat shock 70 kDa protein 4–like), both of which were overexpressed in NPC tissues, were negatively regulated by miR-497 mimic in NPC cell lines. Silencing of ANLN and HSPA4L suppressed cell proliferation and migration and induced apoptosis in NPC cells. Our findings indicate that miR-497 is a potent tumor suppressor that inhibits cancer phenotypes by targeting ANLN and HSPA4L in NPC.

// Xiaoying Zhou 1, 2, * , Jiazhang Wei 1, * , Fu Chen 3 , Xue Xiao 1 , Tingting Huang 1 , Qian He 1 , Shumin Wang 1 , Chunping Du 1 , Yingxi Mo 1, 4 , Longde Lin 5 , Ying Xie 1 , Lili Wei 1 , Ying Lan 1 , Mairiko Murata 4 , Guangwu Huang 1 , Ingemar Ernberg 2 , Liudmila Matskova 2 , Zhe Zhang 1 1 Department of Otolaryngology-Head & Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Guangxi, China 2 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden 3 Department of Radiation Oncology, Eye Ear Nose & Throat Hospital of Fudan University, Shanghai, China 4 Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie, Japan 5 School of Public Health, Guangxi Medical University, Guangxi, China * These authors have contributed equally to this work Correspondence to: Ingemar Ernberg e-mail: ingemar.ernberg@ki.se Zhe Zhang e-mail: zhangzhe@gxmu.edu.cn Keywords: UBE2L6, nasopharyngeal carcinoma, DNA methylation, tumor suppressor gene, ATGL Received: April 30, 2015 Accepted: August 20, 2015 Published: October 12, 2015 ABSTRACT We identified the UBE2L6 gene, encoding the ISG15-conjugating enzyme UbcH8, as one gene significantly downregulated by promoter hypermethylation in nasopharyngeal carcinoma (NPC). Reduced expression of the UbcH8 protein correlated with poor outcome in NPC patients. Restored expression of UBE2L6 suppressed proliferation and colony formation in NPC cells, while inducing apoptosis. Of particular interest, we found that aberrant lipid turnover was controlled by UbcH8 in NPC through ISG15-conjugation of valosin-containing protein (VCP). Tumor tissue and NPC cell lines showed conspicuously strong accumulation of lipid droplets (LDs) compared to control nasopharyngeal epithelium and non-cancerous cell lines. We demonstrated that UbcH8 counteracts degradation of adipocyte triglyceride lipase (ATGL), a key enzyme in lipid catabolism.

Collagen (COL) genes participate in tumor extracellular matrix (ECM)-receptor interactions and focal adhesion pathways, which play a crucial role in tumor invasion and metastasis. The prognostic value of COL genes has been shown for several malignancies. In the present study, we analyzed multiple microarray datasets using the Oncomine database to identify alterations of COL genes in gastric cancer (GC). Gene expression levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) in GC tissues and matched adjacent tissues. The prognostic value of differentially expressed COL genes in GC was evaluated by Kaplan-Meier survival analysis based on the complete mRNA transcriptomics data from The Cancer Genome Atlas (TCGA). We found that seven COL genes (COL1A2, COL4A1, COL4A2, COL6A1, COL6A2, COL6A3, and COL11A1) were elevated in GC. Among them, stepwise multivariate Cox regression was applied, and it was determined that COL4A1 and COL4A2 were signature and independent prognostic biomarkers in GC patients with obviously different overall survival (OS). High expression of COL4A1, COL4A2, COL6A1, COL6A2, and COL6A3 was correlated with poorer prognosis of GC patients treated by surgery only, while higher expression of COL4A1 and COL11A1 correlated with poorer survival of patients treated by 5-fluorouracil-based adjuvant therapy. Our results indicate that overexpression of COL genes might be utilized as novel prognostic markers for GC and assist with therapy selection.

Objects Nasopharyngeal carcinoma (NPC) incidence exhibits a remarkable sex disparity, with higher risk among males. Whether this pattern can be partly explained by female reproductive history is unclear. Methods A population-based case–control study of NPC was conducted in southern China between 2010 and 2014, including 674 histopathologically verified female NPC cases and 690 female controls randomly selected from population-based registries. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using logistic regression after adjusting for potential confounders. Results Women who had 3, 4, or ≥5 pregnancies compared with 2 pregnancies were at significantly increased risk for NPC (ORs 1.56, 1.45 and 1.88, respectively). History of deliveries was similarly associated with a greater risk of NPC. These positive associations were more prominent in women who were younger than 50 years, had less than 10 years of education, or were white-collar workers. Increasing time since menopause was associated with a diminished NPC risk (Ptrend = 0.010). Women more than 15 years after menopause had a 0.35-fold (95% CI: 0.16–0.75) NPC risk compared with those 0–3 years after menopause. Conclusion Contrary to our hypothesis, a history of pregnancy or delivery increased the risk of NPC and the risk decreased with increasing time since menopause. However, the non-linear relationship and no consistent risk patterns across strata indicate that the observed associations are unlikely to be causal, and may at least partially be ascribed to residual confounding by socioeconomic factors.

Background Chinese-style salted fish intake in early life is considered an established risk factor for nasopharyngeal carcinoma (NPC). However, results for adult intakes of salted fish and preserved foods are inconsistent. Objective The aim of this study was to ascertain the relations of Chinese-style hard and soft salted fish and preserved food intakes with NPC risk. Methods We conducted a population-based case-control study in southern China with 2554 NPC cases identified through a rapid case ascertainment system and 2648 healthy controls, frequency-matched on age, sex, and area. Subjects (aged 20-74 y) were interviewed via a food-frequency questionnaire, including information on portion size. Data were also collected on alcohol consumption and potential confounders. Food intake was grouped into 3-5 energy-adjusted intake levels during adulthood (10 y prior) and adolescence (16-18 y). For childhood (at age 10 y), intake frequency of selected food items was collected. Multivariate-adjusted ORs with 95% CIs were estimated via logistic regression. Results We found no association between NPC and intake of hard Chinese-style salted fish during adulthood, and an increased risk at the highest level of intake during adolescence (OR: 1.19; 95% CI: 1.03, 1.39). In contrast, we found a decreased risk for the middle intake level of soft salted fish during adulthood (OR: 0.68; 95% CI: 0.57, 0.81) and adolescence (OR: 0.71; 95% CI: 0.59, 0.85). Preserved foods showed contrasting risk profiles, e.g., the highest adult intake level of salted egg (OR: 1.51; 95% CI: 1.22, 1.87) and fermented black beans (OR: 0.67; 95% CI: 0.56, 0.80). Associations with NPC were weaker than previously reported, e.g., for weekly childhood intake of salted fish (OR: 1.56; 95% CI: 1.24, 1.97). Conclusions Hard and soft salted fish have different risk profiles. Salted fish and other preserved foods were at most weak risk factors for NPC in all periods and may play a smaller role in NPC occurrence than previously thought.

Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck malignancy with a high incidence in southern China. Previous studies have confirmed that taurine shows an anti-cancer effect on a variety of human tumors by inhibiting cell proliferation and inducing apoptosis. However, the underlying molecular mechanism of its anti-cancer effect on NPC is not well understood. To clarify these anti-cancer mechanisms, we performed cell viability and colony formation assays. Apoptotic cells were quantified by flow cytometry. The expression levels of apoptosis-related proteins were evaluated by Western blot. The results showed that taurine markedly inhibited cell proliferation in NPC cells, but only slightly in an immortalized normal nasopharyngeal cell line. Taurine suppressed colony formation and induced apoptosis of NPC cell lines in a dose-dependent manner. Furthermore, taurine increased the active form of caspase-9/3 in a dose-dependent manner. Taurine down-regulated the anti-apoptotic protein Bcl-xL and up-regulated the pro-apoptotic protein Bax and GRP78, a major endoplasmic reticulum (ER) chaperone. These results suggest the involvement of mitochondrial and ER stress signaling in apoptosis. In addition, taurine increased the levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10) and p53, and reduced phosphorylated Akt (protein kinase B). In conclusion, taurine may inhibit cell proliferation and induce apoptosis in NPC through PTEN activation with concomitant Akt inactivation.

Because persistent inflammation may render the nasopharyngeal mucosa susceptible to carcinogenesis, chronic ear-nose-throat (ENT) disease and its treatment might influence the risk of nasopharyngeal carcinoma (NPC). Existing evidence is, however, inconclusive and often based on methodologically suboptimal epidemiologic studies. In a population-based case-control study in southern China, we enrolled 2,532 persons with NPC and 2,597 controls, aged 20-74 years, from 2010 to 2014. Odds ratios were estimated for associations between NPC risk and history of ENT and related medications. Any history of chronic ENT disease was associated with a 34% increased risk of NPC. Similarly, use of nasal drops or aspirin was associated with approximately doubled risk of NPC. However, in secondary analyses restricted to chronic ENT diseases and related medication use at least 5 years prior to diagnosis/interview, most results were statistically nonsignificant, except a history of uncured ENT diseases, untreated nasal polyps, and earlier age at first diagnosis of ENT disease and first or most recent aspirin use. Overall, these findings suggest that ENT disease and related medication use are most likely early indications rather than causes of NPC, although the possibility of a modestly increased NPC risk associated with these diseases and related medications cannot be excluded.

Background Epigenetic changes, including DNA methylation, disrupt normal cell function, thus contributing to multiple steps of carcinogenesis. Nasopharyngeal carcinoma (NPC) is endemic in southern China and is highly associated with Epstein-Barr virus (EBV) infection. Significant changes of the host cell methylome are observed in EBV-associated NPC with cancer development. Epigenetic marks for NPC diagnosis are urgently needed. In order to explore DNA methylation marks, we investigated DNA methylation of candidate genes in EBV-associated nasopharyngeal carcinoma. Methods We first employed methyl-capture sequencing and cDNA microarrays to compare the genome-wide methylation profiles of seven NPC tissues and five non-cancer nasopharyngeal epithelium (NNE) tissues. We found 150 hypermethylated CpG islands spanning promoter regions and down-regulated genes. Furthermore, we quantified the methylation rates of seven candidate genes using bisulfite amplicon sequencing for nine NPC and nine NNE tissues. Results All seven candidate genes showed significantly higher methylation rates in NPC than in NNE tissues, and the ratios (NPC/NNE) were in descending order as follows: ITGA4 > RERG > ZNF671 > SHISA3 > ZNF549 > CR2 > RRAD. In particular, methylation levels of ITGA4, RERG, and ZNF671 could distinguish NPC patients from NNE subjects. Conclusions We identified the DNA methylation rates of previously unidentified NPC candidate genes. The combination of genome-wide and targeted methylation profiling by next-generation sequencers should provide useful information regarding cancer-specific aberrant methylation. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3482-3) contains supplementary material, which is available to authorized users.

Nasopharyngeal carcinoma (NPC) is endemic in southern China. In a genome-wide screen for genes inactivated by promoter hypermethylation, we identified Ras-related associated with diabetes (RRAD). Expression of RRAD was down-regulated in 83.3% (30/36) of the biopsies from NPC patients. RRAD was aberrantly methylated in 74.3% (26/35) of primary tumors, but not in normal nasopharyngeal epithelium. Ectopic RRAD expression in NPC cell lines inhibited the cell growth, colony formation, and cell migration. These results indicate that RRAD might act as a functional tumor suppressor and its epigenetic inactivation may play an important role in NPC development.

We investigated the transcription levels, promoter methylation status and role as a tumor suppressor gene (TSG) of the cadherin CDH4 in nasopharyngeal carcinoma (NPC). The expression of CDH4 was decreased in NPC cell lines, xenografts and primary tumor biopsies. Promoter hypermethylation of CDH4 was detected in all five NPC cell lines, both NPC xenograft lines and 94.3% of primary tumors but not in any of the 12 normal epithelial samples. Loss of CDH4 expression could be restored by the methyltransferase inhibitor 5-aza-2′-deoxycytidine in NPC cell lines. Ectopic expression of CDH4 in the NPC cell lines inhibits cell proliferation, colony formation, migration and elicit cell communication. CDH4 may be a novel putative TSG that can be frequently and tumor-specifically inactivated by its promoter methylation in NPC.

Secreted protein, acidic and rich in cysteine (SPARC) has been found to be involved in various stages of tumor progression such as migration, invasion, extracellular matrix deposition and angiogenesis. To obtain an insight into the role of SPARC in the progression of laryngeal and hypopharyngeal carcinoma, we investigated SPARC transcription levels and promoter methylation in carcinoma cell lines and primary tumors. Reverse transcription-PCR showed that SPARC was silenced in laryngeal and hypopharyngeal carcinoma cell lines, in which aberrant promoter methylation was detected. Hypermethylation of SPARC was detected in 56.1% (23/41) of laryngeal carcinoma and 70.0% (7/10) of hypopharyngeal carcinoma biopsies, but only in 11.1% (1/9) of normal epithelial specimens by a methylation-specific PCR assay. Bisulphite genomic sequencing indicated that CpG sites in the SPARC promoter were heavily methylated in cell lines and primary tumors. Moreover, pharmacological demethylation treatment rescued SPARC expression with 5-aza-2′-deoxycytidine (5-aza-dC) in the laryngeal carcinoma cell lines. SPARC promoter hypermethylation was significantly correlated with lymph node metastasis (p

CDH13 encodes a cell adhesion molecule, H-cadherin. In this study, we examined CDH13 methylation in nasopharyngeal carcinoma (NPC). Methylation specific PCR results showed that CDH13 was methylated in 20% (1/5) NPC cell lines, 100% (2/2) NPC xenografts and 89.7% (52/58) of the NPC primary tumors, while only methylated in 10% (1/10) normal nasopharyngeal epithelia (P0.05). CDH13 expression in NPC cell lines and NPC xenografts analyzed by RT-PCR showed that expressions of CDH13 were reversely correlated with their methylation status. In CDH13-silenced cell line, demethylating agent 5-aza-deoxycytidine could dramatically restore CDH13 expression. Taken together, CDH13 promoter is aberrantly methylated in NPC both in vitro and in vivo, and promoter methylation plays a pivotal role in the silencing of H-cadherin expression. Furthermore, the high sensitivity (81%) and specificity (0% false positives) of detecting CDH13 methylation from nasopharyngeal swabs suggest it could be utilized as a tool for early diagnosis.

RASSF2 can bind directly to K-Ras and function as a negative effector of Ras protein. RASSF2A is the only isoform of RASSF2 that contains CpG islands in its promoter and it has been reported to be inactivated by its promoter methylation in several human cancers. In the present study, we investigated the correlation of RASSF2A expression with its promoter methylation in nasopharyngeal carcinoma (NPC). Expression of RASSF2A was down-regulated in 80% (4/5) of NPC cell lines. Decreased RASSF2A expression was also observed in NPC primary tumors compared with normal nasopharyngeal epithelia. Promoter methylation of RASSF2A could be detected in all the RASSF2A-silenced cell lines (4/5) of the NPC cell lines and 50.9% (27/53) of primary tumors, but not in any of the normal epithelia. RASSF2A-methylated cases showed a significantly lower level of RASSF2A expression than unmethylated cases. Loss of RASSF2A expression can be greatly restored by the methyltransferase inhibitor 5-aza-dC in NPC cell lines. In addition, patients with methylated RASSF2A presented a higher frequency of lymph node metastasis (p < 0.05). Ectopic expression of RASSF2A in RASSF2A-silenced and -methylated NPC cell line CNE2 shows that RASSF2A could inhibit cell cycle progression, colony formation and cell migration, which provided further evidence that RASSF2A is a candidate tumor suppressor gene. In conclusion, RASSF2A, a candidate tumor suppressor gene (TSG), is frequently inactivated by its promoter methylation and this aberrant methylation correlates with lymph node metastasis in NPC.

This study aimed to investigate the inactivation of the parkin gene by promoter methylation and its relationship with genome instability in nasopharyngeal carcinoma. Parkin was considered as a tumor suppressor gene in various types of cancers. However, its role in nasopharyngeal carcinoma is unexplored. Genomic instabilities were detected in nasopharyngeal carcinoma tissues by the random amplified polymorphic DNA. The methylation-specific polymerase chain reaction, semi-quantitative reverse transcription polymerase chain reaction, and immunohistochemical analysis were used to detect methylation and mRNA and protein expression of parkin in 54 cases of nasopharyngeal carcinoma tissues and 16 cases of normal nasopharyngeal epithelia tissues, and in 5 nasopharyngeal carcinoma cell lines (CNE1, CNE2, TWO3, C666, and HONE1) and 1 normal nasopharyngeal epithelia cell line (NP69). mRNA expression of parkin in CNE1 and CNE2 was analyzed before and after methyltransferase inhibitor 5-aza-2-deoxycytidine treatment. The relationship between promoter methylation and mRNA expression, demethylation and mRNA expression, and mRNA and protein expression of the gene and clinical factors and genomic instabilities were analyzed. The mRNA and protein expression levels were significantly reduced in 54 cases of human nasopharyngeal carcinoma compared with 16 cases of normal nasopharyngeal epithelia. Parkin-methylated cases showed significantly lower mRNA and protein expression levels compared with unmethylated cases. After 5-aza-2-deoxycytidine treatment, parkin mRNA expression was restored in CNE1 and CNE2; 92.59% (50/54) of nasopharyngeal carcinoma demonstrated genomic instability. Parkin is frequently inactivated by promoter methylation, and its mRNA and protein expression correlate with lymph node metastasis and genomic instability. Parkin deficiency probably promotes tumorigenesis in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most prevalent malignant tumors with poor prognosis in Southern China and Southeast Asia. Angiogenesis-related molecules can be promising therapeutic targets in NPC. To investigate the relationships of cancer-associated fibroblasts (CAFs) and chemokine-related molecules with neoangiogenesis, we compared immunohistochemical analyses of alpha-smooth-muscle actin (α-SMA), stroma-derived factor-1 (SDF-1), and its receptor CXCR4 in primary NPC specimens and chronic nasopharyngitis tissues. In addition, we examined the expression of vascular endothelial growth factor (VEGF-A), and CD133- and VEGF- receptor-2 (VEGFR-2) double positive cells, as endothelial progenitor cells (EPCs). We also assessed CD34-positive microvessels. Significantly higher expression ofα-SMA was observed in fibroblasts in NPC stroma. The immunoreactive intensities of SDF-1 and CXCR4 were significantly higher in NPC cells. CXCR4-positive cells and CD133/VEGFR-2- double positive cells were observed in the stroma surrounding cancer nests, and VEGF was detected in both cancer and stromal cells. Microvessel density was significantly higher in the stroma of NPC tissues compared to chronic nasopharyngitis tissues. Our data suggest that CAFs and NPC tumor cells may enhance neoangiogenesis in a VEGF- and SDF-1-dependent manner by recruiting EPCs from the bone marrow into tumor stroma.

Cytochrome b5 reductase 2 (CYB5R2), a member of the flavoprotein pyridine nucleotide cytochrome reductase family, is associated with a number of physiological reactions. However, its role in cancer, especially nasopharyngeal carcinoma (NPC), has not been addressed. Here, we investigate the transcript levels and promoter methylation status of CYB5R2 in NPC derived cell lines and tumor biopsies and experimentally address its role as a tumor suppressor gene. We find that CYB5R2 transcript levels are decreased in NPC cell lines and tumor biopsies. Promoter hypermethylation of CYB5R2 was detected in all six tested NPC cell lines and in 84 % of primary NPC tumor biopsies but not in normal nasopharyngeal epithelium. Clinically, CYB5R2 methylation was associated with lymph node metastasis in NPC patients (P

Increasing evidence demonstrated that inactivation of tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event during carcinogenesis. Aiming at developing early diagnostic or prognostic tools for various tumors, we took an EBV-associated tumor, nasopharyngeal carcinoma (NPC), as a model and developed a powerful assay based on "multiplex methylation specific-PCR (MMSP)". The MMSP assay was designed to detect tumor-specific methylation status of several NPC-related genes and was capable of acquiring multiplex information simultaneously through a single PCR reaction with the tiny tumor DNA derived from the direct body fluid close to the primary tumor. In this study, we collected paired nasopharyngeal (NP) swabs and NPC biopsies from 49 NPC patients and twenty noncancerous controls. A panel of markers including two EBV, and two cellular TSG markers were applied in this NPC-specific-MMSP assay. We optimized the working condition of MMSP so that it provides information equal to that from the corresponding separate PCRs. The results showed that MMSP patterns of NPC swab were largely consistent with those of corresponding biopsies and significantly distinguished themselves from those of 20 noncancerous volunteers. Among the 69 samples (49 NPCs and 20 normal controls), the sensitivity of detecting NPC from NP swabs is 98%. The specificity is as high as 100%. In conclusion, being characterized by its noninvasiveness, high reproducibility and informativeness, MMSP assay is a reliable and potential diagnostic tool for NPC. It paves the way for the development of population screening and early diagnosis approaches for various tumor types.

Radiation-induced sarcoma in the head and neck (RISHN) is a rare condition whose clinical presentation and management remain difficult because of its low incidence. In this retrospective study, we analyzed the symptoms, diagnosis, and the treatment of 16,634 patients with head and neck disease, who received radiotherapy between 1960 and 2010 at the Affiliated Tumor Hospital and its predecessor, Guangxi Medical University, China. Among these patients, 16 with a first tumor of nasopharyngeal carcinoma (NPC) and 1 with squamous carcinoma of the tongue met the criteria of RISHN in the head and neck. Our epidemiological data showed that the incidence of RISHN rose from 0.06 to 0.17% from 1960 to 2010; the 3-year overall survival rate was 19.1%, and 3-year disease-free survival rate was 11.1%. The mean latency (SD) period was 93.2 (33) months. Based on the experiences at our institution, we suggest that RISHN is a rare complication after radiotherapy for head and neck tumors, especially NPC. Owing to its low incidence, it should not be a major factor affecting decisions about radiotherapy. Nevertheless, there may be a possibility of increasing incidence of RISHN after radiotherapy of NPC, as shown in our epidemiological results. Given the poor prognosis of RISHN, this possibility should be taken into serious consideration before determination of high-dose radiotherapy for patients with NPC and other head and neck tumors.

Background Epigenetic silencing of tumor suppressor genes plays an important role in nasopharyngeal carcinoma (NPC) tumorigenesis. In the present study, we explore a novel target gene of epigenetic silencing in NPC, Myocardin, which is inactivated by promoter hypermethylation. Methods Transcriptional expression levels of Myocardin were evaluated by reverse transcription–polymerase chain reaction (RT-PCR). Methylation status was addressed by methylation-specific PCR and bisulfite genomic sequencing. Results Myocardin mRNA expression was inactivated in 4 of 5 NPC cell lines. Myocardin was aberrantly methylated in 4 of 5 NPC cell lines (80%) and in 48 of 65 NPC primary tumors (73.8%, but not in any of the 12 normal nasopharyngeal tissues tested. Myocardin expression could be reactivated in NPC cells after treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC). Conclusions Epigenetic inactivation of Myocardin is a frequent and tumor-specific event in NPC. Our findings suggest that Myocardin is a candidate tumor suppressor gene in NPC. © 2010 Wiley Periodicals, Inc. Head Neck, 2011

Currently, there are no satisfactory biomarkers available to screen for nasopharyngeal carcinoma (NPC). Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), has been suggested to cause nitrative and oxidative stress, leading to the accumulation of 8-nitroguanine (8-NitroG) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) and the subsequent transversion mutation of DNA. The aim of this study was to evaluate iNOS expression and the status of nitrative and oxidative stress in NPC. Fifty-nine cases of NPC and 39 cases of chronic nasopharyngitis were investigated to examine the expression of iNOS and the formation of 8-NitroG and 8-OHdG, using double-immunofluorescent staining. The statistical differences in immunoreactivities were analyzed using the Mann–Whitney test. Thirty-six patients from the 57 cases of NPC and 36 healthy controls were investigated to examine the level of serum 8-OHdG, using enzyme-linked immunosorbent assay (ELISA). The statistical differences were analyzed using a t test. Strong DNA lesions were observed in the cancer cells of NPC patients. All cases of NPC were positive for 8-NitroG and 8-OHdG, and 54 (94.7%) were positive for iNOS. NPC samples exhibited significantly more intense staining for 8-NitroG, 8-OHdG and iNOS than those of chronic nasopharyngitis (P < 0.05, respectively). The mean value of serum 8-OHdG in the 36 NPC patients was 0.538 ± 0.336 ng/ml compared to 0.069 ± 0.059 ng/ml for the healthy controls. The difference in the serum levels of 8-OHdG between the NPC patients and controls was statistically significant (P < 0.05). Our present findings suggest that pathological stimulation of nasopharyngeal tissue, caused by bacterial, viral or parasitic inflammation, may lead to nitrative and oxidative DNA lesions, caused by NO. This may contribute to the cause and development of NPC. Thus, 8-NitroG and 8-OHdG could be potential biomarkers for evaluating the risk of NPC. Better understanding of the molecular mechanisms underlying nitrative and oxidative DNA damage may provide clues to molecular targets for new approaches of NPC prevention.

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. Recently, reactive nitrogen and oxygen species are considered to participate in inflammation-related carcinogenesis through DNA damage. In our study, we obtained biopsy and surgical specimens of nasopharyngeal tissues from NPC patients in southern China, and performed double immunofluorescent staining to examine the formation of 8-nitroguanine, a nitrative DNA lesion and 8-oxo-7,8-dihydro-2'-deoxyguanosine, an oxidative DNA lesion, in these specimens. Strong DNA lesions were observed in cancer cells and inflammatory cells in stroma of NPC patients. Intensive immunoreactivity of iNOS was detected in the cytoplasm of 8-nitroguanine-positive cancer cells. DNA lesions and iNOS expression were also observed in epithelial cells of EBV-positive patients with chronic nasopharyngitis, although their intensities were significantly weaker than those in NPC patients. In EBV-negative subjects, no or little DNA lesions and iNOS expression were observed. EGFR and phosphorylated STAT3 were strongly expressed in cancer cells of NPC patients, but NF-kappaB was not expressed, suggesting that STAT3-dependent mechanism is important for NPC carcinogenesis. IL-6 was expressed mainly in inflammatory cells of nasopharyngeal tissues of EBV-infected patients. EBV-encoded RNAs (EBERs) and latent membrane protein 1 (LMP1) were detected in cancer cells from all EBV-infected patients. In vitro cell system, nuclear accumulation of EGFR was observed in LMP1-expressing cells, and IL-6 induced phosphorylated STAT3 and iNOS. These data suggest that nuclear accumulation of EGFR and STAT3 activation by IL-6 play the key role in iNOS expression and resultant DNA damage, leading to EBV-mediated NPC.

Reactive nitrogen species are considered to participate in inflammation-related carcinogenesis through DNA damage. 8-Nitroguanine is a specific marker of inflammation-related carcinogenesis. Epstein-Barr virus infection-related nasopharyngeal carcinoma (NPC) is one of the most prevalent malignant tumors in southern China and Southeast Asia, and its prognosis has been poor for decades. Previously, we demonstrated that nitrative DNA damage, such as 8-nitroguanine formation, occurs in cancer cells of NPC patients (Ma et al. Int. J. Cancer 2008). In the present study, to investigate the involvement of stem cells in NPC, we performed immunohistochemical analyses to examine nitrative DNA lesions (8-nitroguanine) and several cancer stem / progenitor cell markers (CD44v6, CD24 and ALDH1A1) in nasopharyngeal tissues obtained from chronic nasopharyngitis and NPC patients. The staining intensity of 8-nitroguanine was significantly higher in cancer cells and inflammatory cells in stroma of NPC than in chronic nasopharyngitis tissues. Expression levels of CD44v6 and ALDH1A1 were significantly increased in cancer cells of primary NPC specimens in comparison to chronic nasopharyngitis tissues. 8-Nitroguanine was detected in CD44v6- or ALDH1A1-positive stem cells in NPC tissues. In the case of CD24 staining, there was no significant difference between NPC and chronic nasopharyngitis tissues. Intensive staining of CD44v6 and ALDH1A1 were also detected in NPC cell line HK1 compared to immortalized nasopharyngeal epithelial cells NP460 by a double immunofluorescence study and western blotting assay. In conclusion, CD44v6 and ALDH1A1 could be candidates of cancer stem cell markers for NPC. The present results indicate the possible mechanism by which inflammation causes NPC by inducing inflammatory processes and formation of 8-nitroguanine in CD44v6- and / or ALDH1A1-positive stem cells, and mutant stem cells participate in NPC development.

Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic head and neck cancer. However, mechanistic study of the invasion and metastasis of NPC has been hampered by the lack of proper in vivo models. We established an in vivo chick embryo chorioallantoic membrane (CAM) model to study NPC tumor biology. We found 100% micro-tumor formation 3 days after inoculation with NPC cell lines (4/4) or primary tumor biopsy tissue (35/35). The transplanted NPC micro-tumors grew on CAMs with extracellular matrix interaction and induced angiogenesis. In addition, the CAM model could be used to study the growth of transplanted NPC tumors and also several important steps of metastasis, including tumor invasion by detecting the extent of basement membrane penetration, tumor angiogenesis by analyzing the area of neo-vessels, and tumor metastasis by quantifying tumor cells in distant organs. We established and described a feasible, easy-to-manipulate and reliable CAM model for in vivo study of NPC tumor biology. This model closely simulates the clinical features of NPC growth, progression and metastasis and could help elucidate the biological mechanisms of the growth pattern and invasion of NPC cells and in quantitative assessment of angiogenesis and cell intravasation.

Interleukin (IL)-5 is thought to play an important role in asthmatic bronchial mucosal inflammation and is a potential therapeutic target. To investigate the effect of IL-5 on the infiltration of eosinophils in airway in vivo, we compared eosinophil counts and their activation status in airways without and after the topical instillation of recombinant human IL-5. Eight subjects with mild atopic asthma underwent initial bronchoscopy during which control bronchoalveolar lavage (BAL) fluid as well as bronchial mucosa were obtained, and at the same time, normal saline and IL-5 were administered to two sublobar segments separately. The second bronchoscopy were carried out and samples from challenged sites were taken 24 h later. It was found that the total eosinophils (BMK-13+ cells) and the activated eosinophils (EG2+ cells) in bronchial mucosa, the eosinophil numbers in BAL fluid, as well as eosinophil cationic protein (ECP) in BAL fluid from saline-challenged segments were not different from those in unchallenged segments. However, a significant eosinophilia was observed in bronchial mucosa and BAL fluid from IL-5-challenged sites. Eosinophil activation, as assessed by secretion of ECP, was also increased significantly in bronchial mucosa and BAL fluid. The results strongly suggested that IL-5 is capable of inducing eosinophil infiltration into the asthmatic airways, as well as the activation of infiltrating eosinophils.

Nasopharyngeal carcinoma (NPC) is a malignancy with ethnic and geographic distribution in southern China. The pathogenesis of NPC is a complicated process involving environment factors, genetic susceptibility, infection with the Epstein-Barr virus (EBV) and epigenetic changes. Unlike all other head neck squamous cell carcinomas, NPC is strongly associated with EBV infection, which can mediate nitrative and oxidative DNA damage and epigenetic changes. Reactive nitrogen and oxygen species are considered to participate in inflammation-related carcinogenesis through DNA damage. It is reported that nitric oxide (NO) can activate DNA methyltransferase (DNMT) and modify CpG island methylation. We have found promoter hypermethylation in several candidate genes for tumor suppressor genes in NPC. By examined the expression levels of EBV encoded small RNA (EBER) s, the formation of 8-nitroguanine and 8-hydroxy-2′-deoxyguanosine (8-OHdG), DMNT1 and 5-methyl-dC in NPC biopsies and non-cancerous controls, we studied the relationship between EBV infection, inflammation and epigenetic modification in NPC. Strong DNA lesions were observed in cancer cells and inflammatory cells in stroma of NPC tumor biopsies. Intensive immunoreactivity of iNOS was detected in the cytoplasm of 8-nitroguanine-positive cancer cells. Significantly higher levels of serum 8-OHdG, IL-6 and higher titers of EB-viral capsid antigen-IgA were observed in NPC patients than those of controls. Interestingly, the results of EBER in situ hybridization showed significant association with DNMT1 and 5-methyl-dC. These results indicate that EBV-mediated inflammation may play a role in cytosine methylation during NPC development.

Background Epigenetic silencing of tumor suppressor genes play important roles in NPC tumorgenesis. Tissue factor pathway inhibitor-2 (TFPI-2), is a protease inhibitor. Recently, TFPI-2 was suggested to be a tumor suppressor gene involved in tumorigenesis and metastasis in some cancers. In this study, we investigated whether TFPI-2 was inactivated epigenetically in nasopharyngeal carcinoma (NPC). Methods Transcriptional expression levels of TFPI-2 was evaluated by RT-PCR. Methylation status were investigated by methylation specific PCR and bisulfate genomic sequencing. The role of TFPI-2 as a tumor suppressor gene in NPC was addressed by re-introducing TFPI-2 expression into the NPC cell line CNE2. Results TFPI-2 mRNA transcription was inactivated in NPC cell lines. TFPI-2 was aberrantly methylated in 66.7% (4/6) NPC cell lines and 88.6% (62/70) of NPC primary tumors, but not in normal nasopharyngeal epithelia. TFPI-2 expression could be restored in NPC cells after demethylation treatment. Ectopic expression of TFPI-2 in NPC cells induced apoptosis and inhibited cell proliferation, colony formation and cell migration. Conclusions Epigenetic inactivation of TFPI-2 by promoter hypermethylation is a frequent and tumor specific event in NPC. TFPI-2 might be considering as a putative tumor suppressor gene in NPC.

Background Cytochrome b5 reductase 2 (CYB5R2) is a potential tumor suppressor that inhibits cell proliferation and motility in nasopharyngeal carcinoma (NPC). Inactivation of CYB5R2 is associated with lymph node metastasis in NPC. This study aimed to explore the mechanisms contributing to the anti-neoplastic effects of CYB5R2. Methods Polymerase chain reaction (PCR) assays were used to analyze the transcription of 84 genes known to be involved in representative cancer pathways in the NPC cell line HONE1. NPC cell lines CNE2 and HONE1 were transiently transfected with CYB5R2, and data was validated by real-time PCR. A chick chorioallantoic membrane (CAM) embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis. An immunohistochemical assay of the CAM model was used to analyze the protein expression of vascular endothelial growth factor (VEGF). Results In CYB5R2-transfected NPC cells, PCR assays revealed up-regulated mRNA levels of Fas cell surface death receptor (FAS), FBJ murine osteosarcoma viral oncogene homolog (FOS), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), integrin beta 3 (ITGB3), metastasis suppressor 1 (MTSS1), interferon beta 1 (IFNB1), and cyclin-dependent kinase inhibitor 2A (CDKN2A) and down-regulated levels of integrin beta 5 (ITGB5), insulin-like growth factor 1 (IGF1), TEK tyrosine kinase (TEK), transforming growth factor beta receptor 1 (TGFBR1), and VEGF. The angiogenesis in the CAM model implanted with CYB5R2-transfected NPC cells was inhibited. Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model. Conclusion CYB5R2 up-regulates the expression of genes that negatively modulate angiogenesis in NPC cells and down-regulates the expression of VEGF to reduce angiogenesis, thereby suppressing tumor formation.