Search

Supplementary Tables 1-4. Supplementary Table 1. Odds ratios (ORs) and 95% confidence intervals (CIs) of nasopharyngeal carcinoma (NPC) associated with oral health among ever smokers in southern China (2010-2014). Supplementary Table 2. Stratified odds ratios (ORs) and 95% confidence intervals (CIs) of nasopharyngeal carcinoma (NPC) associated with number of teeth lost after age 20 years.* Supplementary Table 3. Stratified odds ratios (ORs) and 95% confidence intervals (CIs)of nasopharyngeal carcinoma (NPC) associated with frequency of tooth brushing.* Supplementary Table 4. Odds ratios (ORs) and 95% confidence intervals (CIs) of nasopharyngeal carcinoma (NPC) associated with oral health in southern China - restricted to cases interviewed within 30 days of diagnosis (2010-2014).

Background: The association between oral health and risk of nasopharyngeal carcinoma (NPC) is largely unknown. Further understanding could shed light on potential pathogenic mechanisms and preventive measures.Methods: We conducted a population-based case–control study in southern China between 2010 and 2014. We enrolled 2,528 incident NPC cases, aged 20–74 years, and 2,596 controls, randomly selected from the total population registers, with frequency matching to the 5-year age and sex distribution of the cases by geographic region. We interviewed subjects using a structured questionnaire inquiring about oral health indicators and potential confounding factors. We used unconditional logistic regression to estimate multivariate-adjusted ORs with 95% confidence intervals (CI).Results: A higher number of filled teeth was associated with an elevated risk of NPC. Individuals with 1 to 3 and more than 3 teeth filled versus none had adjusted ORs of 1.25 (95% CI, 1.06–1.49) and 1.55 (95% CI, 1.13–2.12), respectively (Ptrend = 0.002). Conversely, the adjusted OR for those who brushed teeth twice or more per day versus once or less per day was 0.62 (95% CI, 0.55–0.70). We detected a borderline significant positive association with earlier age at first adult tooth loss.Conclusion: Our study suggested a positive association between some indicators of poor oral health and risk of NPC. Further studies are needed to confirm whether the findings are causal and, if so, to further explain the underlying mechanisms.Impact: Improvement of oral hygiene might contribute to reducing NPC risk. Cancer Epidemiol Biomarkers Prev; 25(8); 1201–7. ©2016 AACR.

Growth differentiation factor-10 (GDF10) belongs to a member of the transforming growth factor-β (TGF-β) superfamily. Dysfunction of the TGF-β pathway can lead to carcinoma progression. Previous studies have shown that GDF10 acts as a tumor suppressor gene in some cancers. However, the molecular mechanisms of the association between GDF10 and cell functions in nasopharyngeal carcinoma (NPC) remain unclear. In this study, the expression and methylation levels of GDF10 were studied in human subjects and cell lines. Furthermore, overexpression of GDF10 was used to explore its biological function and potential mechanism in NPC cell lines. GDF10 was downregulated in NPC owing to its aberrant promoter methylation. After treatment with 5-aza-2′-deoxycytidine, the expression of GDF10 in NPC cells was reversed. We also confirmed that the overexpression of GDF10 significantly inhibited cell proliferation and tumor growth both in vitro and in vivo, respectively. Additionally, GDF10 overexpression in NPC cells attenuated migration and invasion and inhibited epithelial-to-mesenchymal transition with a decrease in nuclear Smad2 and NF-κB protein accumulation. GDF10 was silenced owing to its promoter hypermethylation, and it might originally act as a functional tumor suppressor via TGF-β/Smad and NF-κB signaling pathways in NPC.

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.

Background Epstein-Barr virus (EBV) reactivation from latent to lytic infection has been considered as a key step in nasopharyngeal carcinoma oncogenesis. However, epidemiological evidence regarding environmental risk factors for EBV reactivation on a population level remains largely lacking. Methods We enrolled 1916 randomly selected adults from the general population of Guangdong and Guangxi, China, from 2010 to 2014. Information on environmental factors was collected via a structured interview. Serum immunoglobulin A antibodies against EBV viral capsid antigen and nuclear antigen 1 were measured by enzyme-linked immunosorbent assay to evaluate EBV reactivation status. We used logistic regression to calculate odds ratios (ORs) with 95% confidence intervals (CIs) for the associations of EBV reactivation with various environmental factors. Results No associations were observed between EBV reactivation and extensive environmental factors, including alcohol or tea drinking, a history of chronic ear/nose/throat diseases, use of medications or herbs, consumption of salted fish or preserved foods, oral hygiene, sibship structure, and various residential and occupational exposures. Only cigarette smoking was associated with EBV reactivation (current smokers vs never smokers; OR = 1.37; 95% CI = 1.02–1.83), with positive exposure-response trends with increasing intensity, duration, and pack-years of smoking. Conclusions Consistent with previous studies, we found an association between cigarette smoking and EBV reactivation. Other examined exposures were not associated with EBV reactivation. These null results could suggest either more complex interactions between exposures and EBV reactivation or a predominant role of host and/or viral genetic variation.

Commensal microbes cross talk with their colonized mucosa. We show that microbes and their cell wall components induce an inflammatory response in cultured human mucosal cells derived from the nonmalignant nasopharyngeal epithelium (NNE) cells

Lipid is the building block and an important source of energy, contributing to the malignant behavior of tumor cells. Recent studies suggested that lipid droplets (LDs) accumulations were associated with nasopharyngeal carcinoma (NPC) progression. Solute carrier family 27 member 6 (SLC27A6) mediates the cellular uptake of long-chain fatty acid (LCFA), a necessary lipid component. However, the functions of SLC27A6 in NPC remain unknown. Here, we found a significant reduction of SLC27A6 mRNA in NPC tissues compared with normal nasopharyngeal epithelia (NNE). The promoter methylation ratio of SLC27A6 was greater in NPC than in non-cancerous tissues. The demethylation reagent 5-aza-2’-deoxycytidine (5-aza-dC) remarkably restored the mRNA expression of SLC27A6, suggesting that this gene was downregulated in NPC owing to DNA promoter hypermethylation. Furthermore, SLC27A6 overexpression level in NPC cell lines led to significant suppression of cell proliferation, clonogenicity in vitro, and tumorigenesis in vivo. Higher SLC27A6 expression, on the other hand, promoted NPC cell migration and invasion. In particular, re-expression of SLC27A6 faciliated epithelial-mesenchymal transition (EMT) signals in xenograft tumors. Furthermore, we observed that SLC27A6 enhanced the intracellular amount of triglyceride (TG) and total cholesterol (T-CHO) in NPC cells, contributing to lipid biosynthesis and increasing metastatic potential. Notably, the mRNA level of SLC27A6 was positively correlated with cancer stem cell (CSC) markers, CD24 and CD44. In summary, DNA promoter hypermethylation downregulated the expression of SLC27A6. Furthermore, re-expression of SLC27A6 inhibited the growth capacity of NPC cells but strengthened the CSC markers. Our findings revealed the dual role of SLC27A6 in NPC and shed novel light on the link between lipid metabolism and CSC maintenance.

The human microbiome has been reported to mediate the response to anticancer therapies. However, research about the influence of the oral microbiome on nasopharyngeal carcinoma (NPC) survival is lacking. We aimed to explore the effect of oral microbiota on NPC prognosis.Four hundred eighty-two population-based NPC cases in southern China between 2010 and 2013 were followed for survival, and their saliva samples were profiled using 16s rRNA sequencing. We analyzed associations of the oral microbiome diversity with mortality from all causes and NPC.Within- and between-community diversities of saliva were associated with mortality with an average of 5.29 years follow-up. Lower Faith's phylogenetic diversity was related to higher all-cause mortality [adjusted hazard ratio (aHR), 1.52 (95% confidence interval (CI), 1.06-2.17)] and NPC-specific mortality [aHR, 1.57 (95% CI, 1.07-2.29)], compared with medium diversity, but higher phylogenetic diversity was not protective. The third principal coordinate (PC3) identified from principal coordinates analysis (PCoA) on Bray-Curtis distance was marginally associated with reduced all-cause mortality [aHR, 0.85 (95% CI, 0.73-1.00)], as was the first principal coordinate (PC1) from PCoA on weighted UniFrac [aHR, 0.86 (95% CI, 0.74-1.00)], but neither was associated with NPC-specific mortality. PC3 from robust principal components analysis was associated with lower all-cause and NPC-specific mortalities, with HRs of 0.72 (95% CI, 0.61-0.85) and 0.71 (95% CI, 0.60-0.85), respectively.Oral microbiome may be an explanatory factor for NPC prognosis. Lower within-community diversity was associated with higher mortality, and certain measures of between-community diversity were related to mortality. Specifically, candidate bacteria were not related to mortality, suggesting that observed associations may be due to global patterns rather than particular pathogens.

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.

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.

Polygenic risk scores (PRS) have the potential to identify individuals at risk of diseases, optimizing treatment, and predicting survival outcomes. Here, we construct and validate a genome-wide association study (GWAS) derived PRS for nasopharyngeal carcinoma (NPC), using a multi-center study of six populations (6 059 NPC cases and 7 582 controls), and evaluate its utility in a nested case-control study. We show that the PRS enables effective identification of NPC high-risk individuals (AUC = 0.65) and improves the risk prediction with the PRS incremental deciles in each population (Ptrend ranging from 2.79 × 10−7 to 4.79 × 10−44). By incorporating the PRS into EBV-serology-based NPC screening, the test’s positive predictive value (PPV) is increased from an average of 4.84% to 8.38% and 11.91% in the top 10% and 5% PRS, respectively. In summary, the GWAS-derived PRS, together with the EBV test, significantly improves NPC risk stratification and informs personalized screening.

Background Acy1 Coenzyme A Acyltransferases1 (ACAT1) is a key enzyme in the metabolism of ketone bodies, but its expression and biological function in the pathogenesis of NPC remains underexplored. Methods The mRNA and protein expression levels of ACAT1 in NPC and normal control tissues were analyzed by qPCR and immunohistochemistry staining, respectively. GEO database was applied for meta-analysis of ACAT1 mRNA expression and DNA promoter methylation. The role of ACAT1 in NPC proliferation was examined by CCK8 and colony formation assays in vitro and tumorigenicity in vivo. The wound healing and transwell assays were used for analyzing the migratory and invasive ability. cDNA microarray analysis was performed to identify the genes involved in epithelial-mesenchymal transition and dysregulated by ACAT1. These changes were further confirmed by western blot. Results We found that ACAT1 is inactivated in NPC cell lines and primary tissues. DNA microarray data showed higher methylation in the CpG island region of ACAT1 in NPC than normal tissues. The demethylating reagent 5-aza-dC significantly restored the transcription of ACAT1 in NPC cell lines, suggesting that ACAT1 was inactivated by DNA promoter hypermethylation. Ectopic overexpression of ACAT1 remarkably suppressed the proliferation and colony formation of NPC cells in vitro. As well, the tumorigenesis of NPC cells overexpressing ACAT1 was decreased in vivo. In addition, the migratory and invasive capacities of NPC cells was inhibited by ACAT1 overexpression. Importantly, the higher level of ACAT1 was accompanied by an increased expression of CDH1, EPCAM, and a decreased expression of vimentin and SPARC. This strongly indicates that ACAT1 is able to affect the epithelial-mesenchymal transition in NPC, thereby controlling cellular motility. In addition, we found that ACAT1 expression increases the intracellular level of β-HB. Moreover, exogenous β-HB remarkably inhibits the growth of NPC cells in a dose-dependent manner. Conclusions We have discovered that the ketone body metabolism enzyme ACAT1 is epigenetically downregulated in NPC and acts as a potential tumor suppressor in NPC. Our findings highlight the possibility of using the modulation of ketone body metabolism as effective adjuvant therapy for NPC.

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.

BACKGROUND To the authors' knowledge, no studies to date have explored familial risks of nasopharyngeal carcinoma (NPC) in detail and quantified its lifetime risk in high-incidence populations. METHODS The authors conducted a population-based case-control study of 2499 NPC cases and 2576 controls randomly selected in southern China from 2010 through 2014. Unconditional logistic regression was used to estimate multivariable-adjusted odds ratios (ORs) with 95% confidence intervals (95% CIs) associated with a family history of NPC. In addition, the authors compiled a reconstructed cohort comprising 40,781 first-degree relatives of cases and controls to calculate the lifetime cumulative risk of NPC. RESULTS Individuals with a first-degree family history of NPC were found to be at a >4-fold risk of NPC (OR, 4.6; 95% CI, 3.5-6.1) compared with those without such a history, but had no excess risk of other malignancies. The excess risk was higher for a maternal than a paternal history and was slightly stronger for a sibling compared with a parental history, and for a sororal than a fraternal history. Among relatives of cases, the cumulative risk of NPC up to age 74 years was 3.7% (95% CI, 3.3%-4.2%), whereas that among relatives of controls was 0.9% (95% CI, 0.7%-1.2%). Cumulative risk was higher in siblings than in parents among relatives of cases, whereas no such difference was noted among relatives of controls. CONCLUSIONS Individuals with a family history of NPC have a substantially higher risk of NPC. These relative and cumulative risk estimates can guide the development of strategies for early detection and clinical consultation in populations with a high incidence of NPC. Cancer 2017;123:2716-25. © 2017 American Cancer Society.

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.

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.

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 (

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.

// 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.

// Weimin Ye 1 , Ellen T. Chang 2, 3 , Zhiwei Liu 1 , Qing Liu 4, 5 , Yonglin Cai 6, 7 , Zhe Zhang 8, 9 , Guomin Chen 10 , Qi-Hong Huang 11 , Shang-Hang Xie 4, 5 , Su-Mei Cao 4, 5 , Jian-Yong Shao 5 , Wei-Hua Jia 5 , Yuming Zheng 6, 7 , Jian Liao 12 , Yufeng Chen 9 , Longde Lin 9 , Liming Liang 13, 14 , Ingemar Ernberg 15 , Thomas L. Vaughan 16, 17 , Guangwu Huang 8, 9, * , Yi Zeng 10, * , Yi-Xin Zeng 5, 18, * and Hans-Olov Adami 1, 13, * 1 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden 2 Exponent, Inc., Health Sciences Practice, Menlo Park, CA, USA 3 Division of Epidemiology, Department of Health Research and Policy, Stanford University School of Medicine, Stanford, CA, USA 4 Department of Cancer Prevention Center, Sun Yat-sen University Cancer Center, Guangzhou, China 5 State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China 6 Department of Clinical Laboratory, Wuzhou Red Cross Hospital, Wuzhou, China 7 Wuzhou Health System Key Laboratory for Nasopharyngeal Carcinoma Etiology and Molecular Mechanism, Wuzhou, China 8 Department of Otolaryngology-Head & Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China 9 Key Laboratory of High-Incidence-Tumor Prevention & Treatment (Guangxi Medical University), Ministry of Education, Nanning, China 10 State Key Laboratory for Infectious Diseases Prevention and Control, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China 11 Sihui Cancer Institute, Sihui, China 12 Cangwu Institute for Nasopharyngeal Carcinoma Control and Prevention, Wuzhou, China 13 Department of Epidemiology, Harvard TH Chan School of Public Health, Boston, MA, USA 14 Department of Biostatistics, Harvard TH Chan School of Public Health, Boston, MA, USA 15 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden 16 Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 17 Department of Epidemiology, University of Washington, Seattle, WA, USA 18 Beijing Hospital, Beijing, China * These authors have contributed equally to this work Correspondence to: Weimin Ye, email: weimin.ye@ki.se Keywords: case-control study, nasopharyngeal carcinoma, china Received: April 26, 2017 Accepted: July 03, 2017 Published: July 29, 2017 ABSTRACT With its population of over 1.3 billion persons, China offers abundant opportunities to discover causes of disease. However, few rigorous population-based case-control studies have as yet been conducted in mainland China. We conducted a population-based case-control study of nasopharyngeal carcinoma in Guangdong Province and Guangxi Autonomous Region. We collected questionnaires and biospecimens from incident cases recruited between March 2010 and December 2013, and population-based controls between November 2010 and November 2014. Preparatory activities prior to subject enrollment required approximately 18 months. We enrolled a total of 2554 NPC cases and 2648 controls. Among all identified cases, 83.8% participated. For the participating cases, the median time between diagnosis and interview was 2 days. Among all contacted controls, 82.7% participated. From the enrolled cases, we collected 2518 blood specimens (provided by 98.6% of eligible cases), 2350 saliva specimens (92.0%), 2514 hair specimens (98.4%), and 2507 toenail/fingernail specimens (98.2%). From the enrolled controls, we collected 2416 blood specimens (91.2%), 2505 saliva specimens (94.6%), 2517 hair specimens (95.1%), and 2514 toenail/fingernail specimens (94.9%). We demonstrate that population-based epidemiologic research can successfully be conducted in southern China. The study protocols, databases, and biobank will serve as an extraordinarily valuable resource for testing future etiologic hypotheses.

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.

List of primary antibodies used in the study. (DOCX 16 kb)

Supplementary methods. (DOCX 14 kb)

To the authors' knowledge, no studies to date have explored familial risks of nasopharyngeal carcinoma (NPC) in detail and quantified its lifetime risk in high-incidence populations.The authors conducted a population-based case-control study of 2499 NPC cases and 2576 controls randomly selected in southern China from 2010 through 2014. Unconditional logistic regression was used to estimate multivariable-adjusted odds ratios (ORs) with 95% confidence intervals (95% CIs) associated with a family history of NPC. In addition, the authors compiled a reconstructed cohort comprising 40,781 first-degree relatives of cases and controls to calculate the lifetime cumulative risk of NPC.Individuals with a first-degree family history of NPC were found to be at a4-fold risk of NPC (OR, 4.6; 95% CI, 3.5-6.1) compared with those without such a history, but had no excess risk of other malignancies. The excess risk was higher for a maternal than a paternal history and was slightly stronger for a sibling compared with a parental history, and for a sororal than a fraternal history. Among relatives of cases, the cumulative risk of NPC up to age 74 years was 3.7% (95% CI, 3.3%-4.2%), whereas that among relatives of controls was 0.9% (95% CI, 0.7%-1.2%). Cumulative risk was higher in siblings than in parents among relatives of cases, whereas no such difference was noted among relatives of controls.Individuals with a family history of NPC have a substantially higher risk of NPC. These relative and cumulative risk estimates can guide the development of strategies for early detection and clinical consultation in populations with a high incidence of NPC. Cancer 2017;123:2716-25. © 2017 American Cancer Society.

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.

To discover the relationship of transcriptional levels and promoter methylation status of CHFR gene in human nasopharyngeal carcinoma,to discuss the significance and epigenetic mechanism of CHFR inactivation in NPC, and to evaluate the feasibility of detecting methylated CHFR in nasopharyngeal swab as a means for diagnosis of NPC.Transcriptional levels of CHFR was evaluated by RT-PCR. Methylation specific PCR was used to detect the methylation status of CHFR in NPC cells, normal nasopharyngeal epithelia, primary tumors and their paired nasopharyngeal swabs. Detailed methylation status was confirmed by bisulfite sequencing. NPC cells were treated by the methyltransferase inhibitor 5-aza-dC and the reactivation of CHFR was evaluated by RT-PCR.CHFR transcription was inactivated in NPC. The methylation frequency in NPC primary tumors and their paired swabs were 65.5% and 63.8%, respectively, with a 86.2% concordance. Bisulfite sequencing revealed a dense methylation in NPC cells and primary tumors, but all the normal nasopharyngeal epithelia were unmethylated. CHFR expression were restored after 5-aza-dC treatment.CHFR is epigenetically inactivated by promoter methylation in NPC. Detecting methylated CHFR can be served as a useful non-invasive means for diagnosis of NPC.

Gastric carcinoma is frequent, particularly in China, and therapy is often inefficient. Because cancer cells are partly or mainly dependent on glycolysis to generate adenosine triphosphate ATP (Warburg effect) and/or to produce precursors (of lipid, nucleotides, etc.) for building new cells, any inhibition of glycolysis may slow down the cell proliferation and/or may kill cells. The antitumor effect of citrate, an anti-glycolytic agent inhibiting phosphofructokinase (PFK) was tested on two human gastric carcinoma cell lines.Cell viability and morphology were assessed after 24-72 h exposure to citrate (5, 10, 220 mM). Apoptosis was assessed by annexin V-FITC/PI staining and Western immunobloting.A 3-day continuous exposure to citrate led to near destruction of the cell population in both cell lines, apoptotic cell death occurred through the mitochondrial pathway in a dose- and time-dependent manner, associated with the reduction of the anti-apoptotic Mcl-1 protein in both lines.Citrate demonstrates strong cytotoxic activity against two gastric cancer lines, leading to an early diminution of expression of Mcl-1 and to massive apoptotic cell death involving the mitochondrial pathway.

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.

Epiglottic laryngoplasty is technically feasible as a one-stage procedure with excellent functional results. Although the Kambic-Sedlacek-Tucker (K-S-T) technique of glottic reconstruction offers early extubation with an adequate airway, a subsequent wide neoglottis may increase the chance of aspiration and a poor voice. To better restore the laryngeal functions of closure and phonation, we made some modifications on the original K-S-T technique as follows: (1) One of the lateral margins of the epiglottis with the aryepiglottic fold is sutured to the arytenoid region of the cricoid rather than a thyroid cartilage remnant. A neo-arytenoid is formed. (2) The other lateral margin of the epiglottis with the aryepiglottic fold is sutured to the cut edge of the false and true cord instead of a thyroid ala remnant. Therefore both margins of the epiglottis with the aryepiglottic folds are lowered as much as possible to the level of the glottis. A new pseudocord is formed. (3) A cartilage cut is made at the anterior aspect of the epiglottis, leaving its laryngeal surface of mucoperichondrial intact. A new anterior commissure with a sharp angle is shaped by this maneuver. Nineteen hemilaryngectomies with modified epiglottic laryngoplasty have been performed by members of the Department of Otolaryngology of Guangxi Medical College since 1984. Results in this series are fairly good and indicate that the modified epiglottic laryngoplasty is effective in enhancing functional results in terms of respiration, deglutition, and phonation.

To investigate the effects of situation of misdiagnosis of nasopharyngeal carcinoma (NPC) on distant metastasis.The history of diagnosis and treatment of 85 newly diagnosed cases with nasopharyngeal carcinoma were studied by using itemized questionnaire purposely; 433 patients with different prognosis were analyzed retrospectively for the misdiagnoses and mistreatment, including surgical biopsy in the neck.(1) The rate of misdiagnosis of 85 patients was 72.64%, and the percentage decreased as the level of the hospitals increased; the majority of the patients (77.36%) were diagnoses within 1 month after the first symptom had appeared; the number of diseases misdiagnosed was 20, most common of which were lymphnoditis, tuberculosis of lymph node and secretory tympanitis; (2) Our data showed that among 433 patients analysed retrospectively, 60 cases had undergone surgical biopsy in the neck, 75% of whom had never received nasopharyngeal biopsy; 43 cases had underwent nasopharyngeal biopsy after the pathological diagnosis as metastatic carcinoma of neck biopsy (71.67%) and the rest (20.0%) received radiotherapy directly or after negative nasopharyngeal biopsy for merely 1 to 4 times; of those 43 cases who were diagnosed as NPC by nasopharyngeal biopsy, 79.17% got positive results at first sampling. (3) Rate of misdiagnosis and mistreatment including surgical biopsy in the neck of patients who had been tumor-free for 5 years or above was significantly lower than that of those who experienced distant metastasis after or before treatment (P0.05).Misdiagnosis and mistreatment including biopsy by surgery of neck is common even in high-grade hospitals; it is doctor that is responsible for this situation; the high occurrence rate of misdiagnosis and mistreatment, biopsy by neck surgery, especially the delayed treatment after the neck biopsy are the factors that contribute to distant metastasis of NPC.