Lymph Node Metastasis In Nasopharyngeal Carcinoma Research Articles

MiR-2110 induced by chemically synthesized cinobufagin functions as a tumor-metastatic suppressor via targeting FGFR1 to reduce PTEN ubiquitination degradation in nasopharyngeal carcinoma.

Tumor cell metastasis is the key cause of death in patients with nasopharyngeal carcinoma (NPC). MiR-2110 was cloned and identified in Epstein-Barr virus (EBV)-positive NPC, but its role is unclear in NPC. In this study, we investigated the effect of miR-2110 on NPC metastasis and its related molecular basis. In addition, we also explored whether miR-2110 can be regulated by cinobufotalin (CB) and participate in the inhibition of CB on NPC metastasis. Bioinformatics, RT-PCR, and in situ hybridization were used to observe the expression of miR-2110 in NPC tissues and cells. Scratch, Boyden, and tail vein metastasis model of nude mouse were used to detect the effect of miR-2110 on NPC metastasis. Western blot, Co-IP, luciferase activity, colocalization of micro confocal and ubiquitination assays were used to identify the molecular mechanism of miR-2110 affecting NPC metastasis. Finally, miR-2110 induced by CB participates in CB-stimulated inhibition of NPC metastasis was explored. The data showed that increased miR-2110 significantly suppresses NPC cell migration, invasion, and metastasis. Suppressing miR-2110 markedly restored NPC cell migration and invasion. Mechanistically, miR-2110 directly targeted FGFR1 and reduced its protein expression. Decreased FGFR1 attenuated its recruitment of NEDD4, which downregulated NEDD4-induced phosphatase and tensin homolog (PTEN) ubiquitination and degradation and further increased PTEN protein stability, thereby inactivating PI3K/AKT-stimulated epithelial-mesenchymal transition signaling and ultimately suppressing NPC metastasis. Interestingly, CB, a potential new inhibitory drug for NPC metastasis, significantly induced miR-2110 expression by suppressing PI3K/AKT/c-Jun-mediated transcription inhibition. Suppression of miR-2110 significantly restored cell migration and invasion in CB-treated NPC cells. Finally, a clinical sample assay indicated that reduced miR-2110 was negatively correlated with NPC lymph node metastasis and positively related to NPC patient survival prognosis. In summary, miR-2110 is a metastatic suppressor involving in CB-induced suppression of NPC metastasis.

Diabetes can increase the prevalence of EBV infection and worsen the prognosis of nasopharyngeal carcinoma

Epstein‒Barr virus (EBV) infection is a primary oncogenic factor of nasopharyngeal carcinoma (NPC) that elicits epithelial-mesenchymal transition (EMT). Although diabetic patients are more susceptible to various infectious diseases, the pathological association with virus-related NPC has not yet been clarified. Herein, we evaluated the influence of diabetes on the clinicopathological changes of 70 patients with NPC. Disease-specific survival (DSS) modified by viral infection was also analysed. The proportion of NPC patients with diabetes was 32.9% (23/70 cases), and 91.3% (21/23 cases) were infected with EBV detected by EBER-I in situ hybridisation. NPC with diabetes showed an effect on EMT evaluated by immunostaining for E-cadherin and vimentin, which was correlated with HbA1c levels. Receiver operating characteristic (ROC) curve analysis determined a HbA1c level of 6.5% as the cut-off value for primary disease death at 2 years [area under the curve (AUC) 0.76; sensitivity 0.64; and specificity 0.81]. High HbA1c levels (≥6.5%) significantly increased the number of lymph node metastases in NPC compared to low HbA1c levels (<6.5%, p<0.01). Diabetic NPC patients had a significantly poorer prognosis than all non-diabetic patients (DSS, 72 months vs not reached, p<0.05). Diabetic EBV-positive NPC patients had a significantly poorer prognosis than non-diabetic EBV-positive patients (DSS, 35 months vs not reached, p<0.01). Multivariate analysis using the Cox proportional hazards model also suggested that HbA1c ≥6.5% was a significant factor in poor prognosis, with a hazard ratio of 6.84 (p<0.05). Collectively, our results revealed for the first time a high prevalence of EBV infection, poor prognosis and the importance of proper glycaemic control in diabetic NPC patients.

Re-evaluation of the prognostic significance of retropharyngeal node metastasis in nasopharyngeal carcinoma patients treated with intensity-modulated radiotherapy.

To investigate the prognostic value of retropharyngeal lymphadenopathy in nasopharyngeal carcinoma (NPC) after intensity-modulated radiotherapy. Retrospective studies were performed in a total of 1197 patients. We evaluated the incidence of the retropharyngeal node (RPN) metastasis and the characteristics of the metastatic RPN including laterality, size, necrosis, and extranodal neoplastic spread. RPN metastasis occured in 86.3% of patients. The RPN and level II metastasis shared similar survival outcomes. RPN metastasis was an independent prognostic factor for distant failure (hazard ratio =1.615; 95%confidence interval, 1.063-2.452; P=0.025), in which the laterality of RPN metastasis significantly influences both the distant failure (P=0.006) and disease progression (P=0.001). In N1 disease, the occurrence of unilateral and bilateral RPN metastasis resulted in significantly different outcomes of the disease-specific survival (P=0.045) and progression-free survival (P=0.049). The co-occurrence of bilateral RPN and cervical lymph nodes (CLN) metastasis was an independent adverse prognostic factor (P<0.01) for distant failure and disease progression but not for locoregional recurrence. Both the RPN and level II are the first stations of NPC lymph node metastasis. For N1-stage NPC patients, RPN metastasis, especially co-occurrence of bilateral RPN and CLN metastasis, have an adverse influence on survival outcomes.

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

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

Lymph-node Epstein-Barr virus concentration in diagnosing cervical lymph-node metastasis in nasopharyngeal carcinoma.

Cervical lymph-node (CLN) metastasis commonly occurs in patients with nasopharyngeal carcinoma (NPC) metastasis. The presence of Epstein-Barr virus (EBV) genomes in neck lymph nodes may diagnose CLN. This research was designed to appraise the diagnostic value of EBV concentration for cervical lymph nodes in NPC. Two hundred and fifty-three NPC patients with 276 CLNs were enrolled. MRI was performed to detect CLN metastasis, and plasma EBV concentration was measured by quantitative PCR before treatment. Ultrasonography (US) and US-FNA were subsequently performed in the suspicious lymph nodes. Fifteen patients (22 lymph nodes) underwent fine-needle aspiration cytology (FNAC), and the remaining 242 patients (254 lymph nodes) underwent core needle biopsy (CNB) for CLNs at the clinician's demand. The aspiration needle was rinsed with 1ml of normal saline for EBV detection. The method of lymph-node EBV measurement was consistent with that for plasma. The MRI results and EBV concentrations in plasma and lymph nodes were recorded and analyzed. Plasma EBV concentrations ≥ 4000 copies/ml were regarded as positive. CLN-EBV concentrations ≥ 787.5 copies/ml were regarded as positive according to receiver-operating characteristic curve analysis. The AUC of the EBV (0.925) concentration in CLN metastasis was significantly larger than the AUC of MRI (0.714) (P < 0.001). The sensitivity and specificity were 94.09% and 48.72% for MRI in lymph-node metastasis and 95.36% (P > 0.05) and 84.62% (P < 0.01) for EBV DNA in CLN metastasis, respectively. The sensitivity and specificity of EBV in plasma were 77.2% and 71.8%, respectively. The diagnostic specificity and AUC of EBV in CLNs were higher than those of MRI and plasma EBV (P < 0.005). Ultrasound-guided CLN FNA to obtain EBV concentrations may provide a new method to diagnose CLN metastasis with high sensitivity and specificity.

LMP1 Up-regulates Calreticulin to Induce Epithelial-mesenchymal Transition via TGF-β/Smad3/NRP1 Pathway in Nasopharyngeal Carcinoma Cells.

Background: Latent membrane protein 1 (LMP1) is known as an oncogenic protein encoded by the EBV genome. The purpose of this study was to investigate the mechanism of LMP1-induced cell epithelial-mesenchymal transition (EMT).Methods: The NP69 cell line of nasopharyngeal epithelial cells with high expression of LMP1 was established to observe the effect of high expression of LMP1 on cell growth, proliferation, cycle, apoptosis, migration and invasion. We used proteomics to screen and identify differentially expressed proteins related to LMP1-mediated epithelial cell transformation. Then, we analyzed the expression and significance of differentially expressed calreticulin (CRT) in nasopharyngeal carcinoma (NPC), and observed the effect of CRT expression on EMT in CNE2 cells of NPC. Finally, the expression of neuropilin-1 (NRP1), which is a protein downstream of the EMT-related signaling pathway TGF-β (transforming growth factor β), was detected.Results: LMP1 promoted NP69 cells proliferation, inhibited apoptosis and induced EMT. We identified 22 differentially expressed proteins associated with LMP1-induced EMT. Among them, CRT expression level was significantly increased in NPC compared with adjacent tissues, and was interrelated with TNM staging and lymph node metastasis of NPC. After knockdown of CRT expression, the phenomenon of cell EMT was reduced and the ability of cell migration and invasion was weakened. CRT regulated NRP1 expression by affecting SMAD3 phosphorylation.Conclusion: LMP1 induced cell EMT via TGF-β/Smad3/NRP1 pathway, which promoted migration and invasion of NPC cells.

Genetic polymorphisms in interleukin 13 gene with the susceptibility to nasopharyngeal carcinoma in a Chinese population

Although inflammation is emerging as a candidate risk factor in tumorigenesis of nasopharyngeal carcinoma (NPC). In particular, Interleukin (IL) 13 involved inflammatory diseases and cancers. Single nucleotide polymorphisms in IL-13 have been associated with multiple cancers. The study analyzed genetic polymorphisms in IL-13 aiming to investigate its’ potential susceptibility with the NPC. The genotyping of polymorphisms (rs20541, rs1295687 and rs2069744) was examined by Snapshot SNP and DNA sequencing. All SNPs were within Hardy-Weinberg equilibrium and each appeared in three genotypes in NPC and controls. Adjusted logistic regression showed that the TT genotype of rs20541 increased the risk of lymph node metastasis (TT vs. CC: OR = 2.87, 95%CI, 1.33–6.18, P = 0.007). CT/CC genotypes were associated with the decreased the risk of lymph node metastasis in NPC (CT/CC vs. TT: OR = 0.32, 95%CI, 0.16–0.65, P = 0.002). The concentration of IL-13 was significantly elevated in NPC patients compared with controls (P = 0.012). Moreover, significant differences were detected in the T-C-T haplotype distribution between NPC patients and controls (OR = 2.47, 95%CI, 1.06–5.78, P = 0.031). Our results, the first report, provide evidence that rs20541 polymorphisms may affect the lymph node metastasis of NPC patients in Chinese population.

Long non-coding RNA ZFAS1 promotes nasopharyngeal carcinoma through activation of Wnt/β-catenin pathway.

To investigate the expression characteristics of long non-coding RNA (lncRNA) ZFAS1 in nasopharyngeal carcinoma (NPC) and to further investigate the role and mechanism of ZFAS1 in the occurrence and development of NPC. 76 surgical excision samples from newly diagnosed NPC patients and 76 cases of paracancerous tissues were selected. The expression of ZFAS1 was detected by qRT-PCR (quantitative real-time polymerase chain reaction), and the relationship between the expression of ZFAS1 and the clinical prognosis of patients with NPC was analyzed. After the knockdown, the expression of ZFAS1 in nasopharyngeal carcinoma cell line CNE-1 by small interfering RNA, the effect of ZFAS1 on the biological function of NPC cells was analyzed by cell counting kit-8 (CCK8), colony formation assay, flow cytometry and apoptosis assay. The effect of ZFAS1 on the Wnt/β-catenin pathway was analyzed by Western blot. After analyzing the clinical data of enrolled patients, we found that ZFAS1 was overexpressed in NPC patients, which was closely correlated with tumor size and lymph node metastasis of NPC. Moreover, ZFAS1 was negatively correlated to the survival of NPC patients. After interfering with the expression of ZFAS1, the proliferation of NPC cells was significantly decreased, the cell cycle was inhibited and the apoptosis rate was increased. Western Blot showed that ZFAS1 promoted the occurrence of NPC by activating the Wnt/β-catenin pathway. ZFAS1 was overexpressed in NPC and was significantly related to tumor size, lymph node metastasis and poor prognosis of NPC. ZFAS1 could promote NPC by activating Wnt/β-catenin pathway.

A novel N staging system for NPC based on IMRT and RTOG guidelines for lymph node levels: Results of a prospective multicentric clinical study.

The present study aimed to investigate the cervical lymph node metastasis of nasopharyngeal carcinoma (NPC) and to establish a novel N staging standard for NPC, based on intensity modulated radiation therapy (IMRT) via a prospective multicenter clinical trial. Between January 2006 and December 2009, a total of 492 patients with NPC without distant metastasis were included in the present study. All patients were treated with IMRT. According to Radiation Therapy Oncology Group division standards, the present study proposed a novel N staging system following the review of magnetic resonance images in comparison with the 7th edition of Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC) staging system. Retropharyngeal lymph nodes, cervical lymph node level and cervical lymph node laterality were independent prognostic factors used in multivariate analyses. According to the results of the risk variety, the present study suggested that the novel N staging system included: N0 (no lymph node metastasis), N1 [retropharyngeal or/and unilateral upper cervical (I, II, III, Va, VIIb, VIII, IX and X regions) lymph node metastasis], N2 [bilateral upper cervical (I, II, III, Va, VIIb, VIII, IX and X regions) lymph node metastasis] and N3 (lymph node metastasis in IVa and Vb regions and their lower regions). The novel N staging system proposed in the present study performs better in risk difference and distribution balance. Furthermore, the differences of 5-year curves of distant metastasis-free survival and overall survival had greater statistically significant differences compared with the 7th edition of the UICC/AJCC staging system. The present study suggested a novel N staging system for cervical lymph node metastasis of NPC, which may predict the prognosis of patients with NPC in a more objective and accurate way.

Overexpression of osteopontin promotes cell proliferation and migration in human nasopharyngeal carcinoma and is associated with poor prognosis.

Nasopharyngeal carcinoma (NPC), a malignant tumor at the top and side of the nasopharyngeal cavity, highly occurs in the southern region of China. Cancer cell metastasis is one of the leading causes of death in NPC patients. Osteopontin (OPN), is a phosphorylated extracellular matrix protein with a variety of functions, was found to be overexpressed in many cancers. However, the expression and role of OPN in patients with NPC in Guangxi, China are unclear. Here, we observed that NPC patients had upregulated OPN at mRNA protein and levels. Immunochemistry (IHC) analysis of OPN expression in 68 NPC clinical specimens indicated that high expression of OPN had positive correlation with NPC lymph node metastasis (P=0.012), distant metastasis (P=0.001) and TNM staging (P=0.018). Moreover, compared with relatively low OPN, NPC patients with higher expression of OPN showed a poorer overall survival rate (P=0.001, log rank test). Multivariate analysis showed that OPN expression in NPC was an independent prognostic marker. The proliferation, apoptosis and migration ability of CEN-2Z cancer cells in NPC were determined by MTT, flow cytometry and wound-healing assays, respectively. Upregulation of OPN in CEN-2Z cancer cells promoted cancer cell proliferation and migration, and suppressed apoptosis. In sum, our result suggests OPN could be used as a valuable oncoprotein and show that overexpression of OPN in NPC may serve as a potential prognostic marker.

Cyclooxygenase-2 expression is positively associated with lymph node metastasis in nasopharyngeal carcinoma.

BackgroundAccumulating evidence has demonstrated that cyclooxygenase-2 (COX-2) is involved in head and neck cancers, especially in nasopharyngeal carcinoma (NPC). However, the association between COX-2 expression and lymph node metastasis in NPC remains uncertain. This systematic review and meta-analysis meta-analysis investigated the relationship between COX-2 expression and lymph node metastasis and other signs of disease progression in NPC.MethodsPreviously published studies assessing COX-2 expression and lymph node metastasis in NPC were identified in four English databases and three Chinese ones (Pubmed, Embase, Cochrane Database of Systematic Reviews, Web of Science, China National Knowledge Infrastructure, Wanfang, Vip Journal Integration Platform) up to November 2016. Quality of all eligible studies was assessed using the Newcastle-Ottawa Quality Assessment Scale (NOS). Pooled odds ratios (OR) and their 95% confidence intervals (95%CI) were calculated with fixed-effects or random-effects model to evaluate the effects of COX-2 expression on lymph node metastasis.ResultsA total of 27 studies with 1797 NPC patients met the inclusion criteria. The expression of COX-2 was significantly higher in patients with nasopharyngeal carcinoma than those without the carcinoma, with a combined OR of 21.17 (95%CI = 15.02–29.85, I2 = 35.1%, Pheterogeneity = 0.070). A statistically significant association between COX-2 expression and lymph node metastasis in NPC patients, with an OR of 4.44 (95%CI = 3.46–5.70, I2 = 38.3%, Pheterogeneity = 0.024), and with other indicators of disease progression. Subgroup analyses based on COX-2 assay and staging criteria of TNM showed no significant heterogeneity.ConclusionsThe results suggest that expression of COX-2 is associated with lymph node metastasis and disease progression in NPC, indicating a potential role in evaluation of prognosis and in treatment decisions. COX-2 inhibitors have potential in the treatment of NPC that should be further investigated.

Upregulation of flotillin-1 promotes invasion and metastasis by activating TGF-β signaling in nasopharyngeal carcinoma.

Metastasis is the main cause of cancer-related deaths. Nasopharyngeal carcinoma (NPC) is characterized by severe local invasion and high incidence of regional lymph node metastasis, which represents poor prognosis. However, the underlying mechanism that induces lymph node metastasis of NPC remains largely unknown. Herein, we report that flotillin-1 (FLOT1), a component of lipid raft, which was reported to be involved in tumor progression, was robustly upregulated in the NPC samples with lymph node metastasis. High FLOT1 expression was significantly associated with N classification as well as poorer overall and disease-free survivals in 169 archived clinical NPC samples. Overexpression of FLOT1 enhanced the migratory and invasive abilities of NPC cells in vitro, and more importantly, promoted invasion into the surrounding tissues and metastasis to lymph nodes in vivo. Whereas silencing of endogenous FLOT1 in NPC cells decreased the local invasion and metastasis to lymph nodes. Furthermore, FLOT1 induced the expression and secretion of TGF-β1, facilitated the activation of TGF-β/Smad3 signaling to effectuate epithelial-mesenchymal transition. Our findings present new evidence that FLOT1 plays an important role in promoting aggressive behavior of NPC and provide new insights into the regulatory mechanism of TGF-β signaling.

Argonaute 2 and nasopharyngeal carcinoma: a genetic association study and functional analysis.

BackgroundArgonaute 2 (AGO2), a central component of RNA-induced silencing complex, plays critical roles in cancer. We examined whether the single nucleotide polymorphisms (SNPs) of AGO2 were related to the risk of nasopharyngeal carcinoma (NPC).MethodsTwenty-five tag SNPs within AGO2 were genotyped in Guangxi population consisting of 855 NPC patients and 1036 controls. The SNPs significantly associated with NPC were further replicated in Guangdong population consisting of 996 NPC patients and 972 controls. Functional experiments were conducted to examine the biologic roles of AGO2 in NPC.ResultsA significantly increased risk of advanced lymph node metastasis of NPC was identified for the AGO2 rs3928672 GA + AA genotype compared with GG genotype in both the Guangxi and Guangdong populations (combined odd ratio = 2.08, 95 % confidence interval = 1.44-3.01, P = 8.60 × 10−5). Moreover, the AGO2 protein expression levels of rs3928672 GA + AA genotype carriers were higher than the GG genotype carriers in the NPC tissues (P = 0.041), and AGO2 was significantly over-expressed in NPC tissues compared with non-cancerous nasopharyngeal tissues (P = 0.011). In addition, AGO2 knockdown reduced cell proliferation, induced apoptosis, and inhibited migration of NPC cells. Furthermore, gene expression microarray showed that genes altered following AGO2 knockdown were clustered in tumorigenesis and metastasis relevant pathways.ConclusionsOur findings suggest that the genetic polymorphism in AGO2 may be a risk factor for the advanced lymph node metastasis of NPC in Chinese populations, and AGO2 acts as an oncogene in the development of NPC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1895-4) contains supplementary material, which is available to authorized users.

Overexpression of acylglycerol kinase is associated with poorer prognosis and lymph node metastasis in nasopharyngeal carcinoma.

Acylglycerol kinase (AGK) has been reported to promote a malignant phenotype and enhance the development of cancer stem cells. However, the clinical value of AGK in cancer remains unclear. This study aimed to investigate the expression and clinicopathological significance of AGK in nasopharyngeal carcinoma (NPC). AGK was significantly upregulated in NPC cell lines and clinical specimens as indicated by real-time PCR and Western blotting. Among the AGK-positive cases, 52/114 (45.6 %) of the archived human NPC specimens expressed high levels of AGK. High expression of AGK was associated with significantly shorter overall and disease-free survival (P < 0.001 and P = 0.002; log-rank test) and was an independent prognostic factor for overall survival (P = 0.041; multivariate Cox analysis). High AGK expression was associated with lymph node metastasis (P < 0.001; chi-squared test) and was an independent predicted factor for lymph node metastasis in NPC (P = 0.032; multivariate logistic analysis). AGK is overexpressed and associated with disease progression and lymph node metastasis in NPC. AGK has potential as a novel prognostic factor for overall survival in NPC.

Cytochrome b5 reductase 2 suppresses tumor formation in nasopharyngeal carcinoma by attenuating angiogenesis.

BackgroundCytochrome 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.MethodsPolymerase 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).ResultsIn 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.ConclusionCYB5R2 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.

Increased expression of flotillin-2 protein as a novel biomarker for lymph node metastasis in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a head and neck malignant tumor rare throughout most of the world but common in Southeast Asia, especially in Southern China. Flotillin-2 (Flot-2) is not only an important component of cellular membrane, but also involves in various cellular processes such as membrane trafficking, T cell and B cell activation, regulation of several signaling pathways associated with cell growth and malignant transformation, keeping structure and junction of epidermal cells and formation of filopodia. Although such molecular effects of Flot-2 have been reported, whether the expression of Flot-2 protein is associated with clinicopathologic implication for NPC has not been reported. The purpose of this research is to investigate the expression of Flot-2 protein in NPC and control nasopharyngeal epithelial tissues by immunohistochemistry and elucidate the association between the expression of Flot-2 protein and clinicopathological characteristics of NPC. The results showed that the positive percentage of Flot-2 expression in the NPC, nasopharyngeal epithelia with atypical hyperplasia and in the control nasopharyngeal mucosa epithelia was 88.8% (119/134), 76.9% (10/13) and 5.7% (5/88), respectively. There was significantly higher expression of Flot-2 protein in NPC and nasopharyngeal epithelia with atypical hyperplasia compared to the control nasopharyngeal mucosa epithelia (P<0.001, respectively). The positive percentage of Flot-2 protein expression in NPC patients with lymph node metastasis was significantly higher than those without lymph node metastasis. Increasing of Flot-2 expression was obviously correlated with clinical stages of NPC patients. The expression of Flot-2 was proved to be the independent predicted factor for lymph node metastasis by multivariate analysis. The sensitivity of Flot-2 for predicting lymph node metastasis of NPC patients was 93%. Taken together, our results suggest that the increased expression of Flot-2 protein is a novel higher sensitivity biomarker that can predict lymph node metastases in NPC.

Blocking PI3K/Akt signaling attenuates metastasis of nasopharyngeal carcinoma cells through induction of mesenchymal-epithelial reverting transition.

In the present study, we evaluated the role of phosphatidylinositol-3 OH kinase/protein kinase B (PI3K/Akt) signaling on changes to epithelial-to-mesenchymal reverting transition (EMrT) in nasopharyngeal carcinoma (NPC). Protein expression levels of p-Akt (Ser473), and the epithelial‑to-mesenchymal transition (EMT) markers E-cadherin, vimentin, α smooth muscle actin (α-SMA), were examined in clinical samples from 130 cases of undifferentiated non-keratinizing NPC, and 20 cases of benign nasopharyngitis. The relationship between protein expression levels and the statue of NPC lymph node metastasis was analyzed. The poorly‑differentiated NPC cell line CNE2Z was treated with various concentrations of the PI3K inhibitor, LY294002, and western blotting and quantitative polymerase chain reaction assays were used to analyze the activation of PI3K/Akt signaling and expression of E-cadherin, vimentin and α-SMA. The ability of cellular migration and invasion was assessed using Transwell assays. The in vivo effects of LY294002 on metastasis and expression of EMT markers in CNE2Z cells was evaluated using tumor xenograft experiments. The expression levels of p-Akt (Ser473) in NPC samples were higher than those in nasopharyngitis. There were reduced levels of membrane E-cadherin protein expression, and increased cytosol vimentin and α-SMA expression levels in NPC samples compared with those in nasopharyngitis samples. High expression levels of p-Akt (Ser473), vimentin, and α-SMA, and low expression levels of E-cadherin were positively associated with lymph node metastasis of NPC cells. Treating CNE2Z cells with LY294002 inhibited p-Akt (Ser473), vimentin and α-SMA expression but upregulated E-cadherin expression, leading to significantly attenuated cell invasion and migration. Administration of mice with LY294002 resulted in upregulation of membrane E-cadherin, and downregulation of vimentin and α-SMA in CNE2Z xenografts, with reduced pulmonary metastasis. Our findings suggest that inhibiting the PI3K/Akt pathway using LY294002 attenuated NPC metastasis via induction of EMrT.

3,3'-Diindolylmethane inhibits the invasion and metastasis of nasopharyngeal carcinoma cells in vitro and in vivo by regulation of epithelial mesenchymal transition.

Nasopharyngeal carcinoma (NPC) is characterized by silent progression and atypical early symptoms. Early metastasis to the neck lymph nodes is common. However, conventional chemoradiotherapy is limited and unable to effectively control cervical lymph node metastasis of NPC. In addition, toxicities caused by chemoradiotherapy often induce damage to normal tissues and organs. Thus, the aim of this study was to investigate the ability of 3,3′-diindolylmethane (DIM) to inhibit the invasion and metastasis of NPC cells in vitro and in vivo. The migration and invasive abilities of the 5–8F human NPC cell line were detected using a Transwell assay. Lymph node metastasis in nude mice was observed following the implantation of xenograft tumors for 8 weeks. In addition, western blot analysis was used to detect the expression levels of epithelial mesenchymal transition (EMT)-associated key proteins in NPC cells treated with DIM in vitro and in vivo. The results demonstrated that DIM effectively inhibited the migration and invasion of NPC cells in vitro and the effect was concentration-dependent. In addition, DIM significantly delayed and reduced the occurrence of lymph node metastasis in the animal model. The expression levels of a number of key proteins associated with EMT were affected by DIM treatment. In the animal model, there were no signs of toxicity in the vital organs, including the heart, liver and kidney, of animals fed a diet containing DIM. Therefore, the results of the present study indicate that DIM affects the expression levels of a number of EMT-associated key proteins and induces the inhibition of invasion and metastasis of NPC cells in vitro and in vivo.

Expression and significance of hOT7T175 in nasopharyngeal carcinoma

Objective To investigate the expression and clinical significance of hOT7T175 in nasopharyngeal carcinoma (NPC).Methods hOT7T175 expression levels were detected in nasopharyngeal carcinoma tissues and nontumorous nasopharyngeal tissues by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).Categorical date were analyzed by x2 test,multi-factor analysis using logistic regression analysis.Results The positive rate of hOT7T175 protein in NPC was 40.9％ (45/110) and 70.5％ (31/44) in nontumorous nasopharyngeal tissue,and the difference was statistically significant (x2 =10.976,P =0.001).The expression of hOT7T175 protein closely correlated with lymph node metastasis in NPC,and the smallest concomitant probability was 0.003 and the largest Wald statistic was 8.813.It was illustrated that lymph node metastasis was an important factor of affecting hOT7T175 protein expression.The positive rate of hOT7T175 mRNA in NPC was 47.1％ (16/34) and 88.2％ (15/17) in nontumorous nasopharyngeal tissue,and the difference was statistically significant (x2 =6.426,P =0.011).The expression of hOT7T175 mRNA correlated with the age of NPC patient and lymph node metastasis.The concomitant probability of lymph node metastasis (0.001) was the smallest and the largest Wald statistic was 11.647.It was illustrated that lymph node metastasis was an important factor of affecting hOT7T175 mRNA expression.Conclusion hOT7T175 highly expresses in nontumorous nasopharyngeal tissue and lowly expresses in NPC.The low expression of hOT7T175 is correlated with lymph node metastasis of NPC,which indicates that hOT7T175 may play an important role in the metastasis of NPC. Key words: Nasopharyngeal neoplasms; Genes; hOT7T175

A Functional Polymorphism in MIR196A2 Is Associated with Risk and Progression of Nasopharyngeal Carcinoma in the Chinese Population

Genetic variations in microRNAs may alter their processing, expression, and binding to target mRNAs, consequently affecting many cancer-related biological processes. Recently, a polymorphism rs11614913 in MIR196A2 was shown to affect the processing of the precursor microRNA into its mature forms and the repertoire of target mRNAs with which it interacts. We examined whether this polymorphism was relevant to the risk of occurrence or progression of nasopharyngeal carcinoma (NPC) in the Chinese population. We genotyped the MIR196A2 rs11614913 in a case-control study of 1084 patients with NPC and 1036 cancer-free controls using the TaqMan assay. The genetic associations with the risk of occurrence and progression of NPC were analyzed by logistic regression. We observed a significantly increased occurrence of NPC associated with the rs11614913 T allele (odds ratio [OR]=1.15, 95% confidence interval [CI]=1.02-1.32, p=0.026) compared with the C allele. The T allele was also significantly associated with the advanced local tumor invasion (T₃+T₄ vs. T₁+T₂; OR=1.27, 95% CI=1.04-1.54, p=0.015) and advanced lymph node metastasis (N₂+N₃ vs. N₀+N₁; OR=1.23, 95% CI=1.02-1.49, p=0.031) of NPC compared with the C allele. Furthermore, stratified analysis indicated that the increased susceptibility to advanced lymph node metastasis of NPC related to the T allele was more pronounced in patients with a positive family history (N₂+N₃ vs. N₀+N₁; p=0.016, test for homogeneity). Our study suggests that the functional polymorphism rs11614913 in the MIR196A2 gene may contribute to the risk of occurrence and progression of NPC in the Chinese population.