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Correlation between the clinicopathological features and EBV status

BART15 and PHLPP1 are associated with NPC LN stage

Supplementary Data

Lymphatic metastasis is a common clinical symptom in nasopharyngeal carcinoma (NPC), the most common Epstein-Barr virus (EBV)-associated head and neck malignancy. However, the effect of EBV on NPC lymph node (LN) metastasis is still unclear. In this study, we demonstrated that EBV infection is strongly associated with advanced clinical N stage and lymphangiogenesis of NPC. We found that NPC cells infected with EBV promote LN metastasis by inducing cancer-associated lymphangiogenesis, whereas these changes were abolished upon clearance of EBV genomes. Mechanistically, EBV-induced VEGF-C contributed to lymphangiogenesis and LN metastasis, and PHLPP1, a target of miR-BART15, partially contributed to AKT/HIF1a hyperactivity and subsequent VEGF-C transcriptional activation. In addition, administration of anti-VEGF-C antibody or HIF1α inhibitors attenuated the lymphangiogenesis and LN metastasis induced by EBV. Finally, we verified the clinical significance of this prometastatic EBV/VEGF-C axis by determining the expression of PHLPP1, AKT, HIF1a, and VEGF-C in NPC specimens with and without EBV. These results uncover a reasonable mechanism for the EBV-modulated LN metastasis microenvironment in NPC, indicating that EBV is a potential therapeutic target for NPC with lymphatic metastasis.Implications:This research demonstrates that EBV induces lymphangiogenesis in NPC by regulating PHLPP1/p-AKT/HIF1a/VEGF-C, providing a new therapeutic target for NPC with lymphatic metastasis.

PDF file - 1299K,A, Western blot detection of CHK1 and CHK2 in CNE2 cells. B, Western blot detection of c-MYC, CHK1 and CHK2 in PKH26+ NPC cells after treated with c-Myc shRNA

PDF file - 3834K, A, Tumors from xenografts after treatment for 55 days. B, Representative IHC staining of tumor samples for Ki67 and Brdu after indicated treatments

PDF file - 174K,Include: Clonogenic survival assay;Patients and primary NPC samples;Ethics Statement;Quantitative real-time (qRT)-PCR;Alkaline comet assay ;Plasmids, oligonucleotides, and transfections;Luciferase assay;EdU labeling;c-MycBS prediction and ChIP assay;Immunohistochemistry

PDF file - 2945K, A, Flow cytometry of CD44 expression in PHK26 + cells. Data represent the mean�standard deviation (SD); n = 3. B, The delineation of CNE1 and CNE2 cells after labeling with PKH26 and treated with PI in Flow cytometry. C, RT-PCR validation of c-MYC, CHK1, CHK2, and ATM RNA levels in CNE2 cells. D, Western blot analysis of c-MYC, pCHK1, and pCHK2 markers in CNE2 cells. GAPDH served as a loading control

XLS file - 81K,Binding sites (c-MYCBSs) within the 2000bp promoter upstream of the CHK1 and CHK2 genes

PDF file - 68K, Supplemental Figure Legends

PDF file - 1504K,(A, B) The indicated amounts of PKH- and PKH+ cells from A549 cells were transplanted into 21-day old nude mice

Supplementary Tables 1-2 from MiR-26a Inhibits Cell Growth and Tumorigenesis of Nasopharyngeal Carcinoma through Repression of EZH2

Increased vascular permeability facilitates metastasis. Cancer-secreted exosomes are emerging mediators of cancer-host crosstalk. Epstein-Barr virus (EBV), identified as the first human tumor-associated virus, plays a crucial role in metastatic tumors, especially in nasopharyngeal carcinoma (NPC). To date, whether and how exosomes from EBV-infected NPC cells affect vascular permeability remains unclear. Here, we show that exosomes from EBV-positive NPC cells, but not exosomes from EBV-negative NPC cells, destroy endothelial cell tight junction (TJ) proteins, which are natural barriers against metastasis, and promote endothelial-to-mesenchymal transition (EndMT) in endothelial cells. Proteomic analysis revealed that the level of HMGA2 protein was higher in exosomes derived from EBV-positive NPC cells compared with that in exosomes derived from EBV-negative NPC cells. Depletion of HMGA2 in exosomes derived from EBV-positive NPC cells attenuates endothelial cell dysfunction and tumor cell metastasis. In contrast, exosomes from HMGA2 overexpressing EBV-negative NPC cells promoted these processes. Furthermore, we showed that HMGA2 upregulates the expression of Snail, which contributes to TJ proteins reduction and EndMT in endothelial cells. Moreover, the level of HMGA2 in circulating exosomes is significantly higher in NPC patients with metastasis than in those without metastasis and healthy negative controls, and the level of HMGA2 in tumor cells is associated with TJ and EndMT protein expression in endothelial cells. Collectively, our findings suggest exosomal HMGA2 from EBV-positive NPC cells promotes tumor metastasis by targeting multiple endothelial TJ and promoting EndMT, which highlights secreted HMGA2 as a potential therapeutic target and a predictive marker for NPC metastasis.

Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway.

Radiotherapy is the most successful nonsurgical treatment for nasopharyngeal carcinoma (NCP). Although NPCs initially respond well to a full course of radiation, recurrence and metastasis are frequent. In this study, we found that down-regulated c-MYB expression was associated with increased radiation resistance and DNA damage repair ability. Interestingly, c-MYB was over-expressed in cancer tissues but not in the adjacent tissues. Down-regulation of c-MYB expression inhibited cell proliferation, and led to cell cycle arrest at the M phase in NPC cells. Luciferase and chromatin immunoprecipitation assays demonstrated that c-MYB transactivated miR-143 through direct binding to its promoter. Based on these results, c-MYB might target miR-143 in order to regulate stem cell properties, cell growth, apoptosis, and DNA damage repair.

Cancer stem cells (CSCs) are considered to serve a key role in tumor progression, recurrence and metastasis. Tumorsphere culture is the most important method for enriching CSCs and is widely used in basic research and drug screening. However, the traditional suspension cell culture system has several disadvantages, including low efficiency, high cost and difficult procedure, making it difficult to produce tumorspheres on a large scale. In the present study, two biomaterials, methylcellulose (MC) and gellan gum (GG), were used to construct a novel culture system based on the traditional system. Subsequently, the characteristics of the novel three-dimensional (3D) culture system were evaluated, the design scheme was optimized, and the morphological and biological features of the tumorspheres cultured in this 3D system were compared with the traditional system. The results revealed that the tumorspheres cultured in the novel 3D system presented a higher seeding density and improved morphology, while maintaining stem-like properties. This evidence suggests that a simple, efficient and low-cost culture system that produces tumorspheres on a large scale was successfully constructed, which can be widely used in various aspects of stem cell research.

We report that the epidermal growth factor receptor (EGFR) pathway plays a critical role in regulating cancer stem-like cells (CSCs) in nasopharyngeal carcinoma (NPC), one of the most common malignant tumors in Southeast Asia. Effects of EGFR on maintaining CSCs are mainly mediated by AKT signaling, and β-catenin is responsible for governing CSC properties in response to EGFR/AKT activation. Significantly, CSCs are enriched by cisplatin and decreased by gefitinib in NPC xenograft models. Upon reimplantation in secondary mice, tumor cells derived from cisplatin-treated mice grew rapidly, whereas regrowth of tumor cells from gefitinib-treated mice was severely diminished. We further demonstrate that expression of EGFR correlates with expression of β-catenin and Nanog in primary tumor specimens from NPC patients. These findings provide mechanistic and preclinical evidence supporting the use of gefitinib alone or in combination with a chemotherapeutic agent in first-line therapy for patients with NPC. In addition, our results suggest that targeting β-catenin represents a rational clinical modality for patients whose tumors harbor activated EGFR or AKT.

To examine the expression patterns of short palate, lung and nasal epithelium clone 1 (SPLUNC1) gene in human tissues.In situ hybridization was used to detect the expression of SPLUNC1 gene in 37 different human tissues.We found that SPLUNC1 gene was not expressed in squamous epithelial cells of the palate, epidermis, esophagus, or the esophagus-cardia junction, metaplastic squamous cells in the nasopharynx, trachea, or uterus cervix, or tumor cells of esophageal squamous cell carcinoma or lung squamous cell carcinoma. SPLUNC1 gene was not expressed in the single layer columnar epithelia cells in the stomach, gallbladder, jejunum, colon, endometrium, or uterus cervix. SPLUNC1 expression was detected mainly in pseudostratified columnar epithelial cells in the nasopharynx, trachea and bronchi, and was gradually down-regulated from the upper to lower end of the respiratory tract, but was not detected in the lung tissues. SPLUNC1 expression was detected not only in the duct and serous gland cells in the parotid and submandibular glands, but also in cells of submucosal serous glands in the nasopharynx and lung, but not in the cells of the mucosal glands. The parietal cells of the gastric submucosa and epithelial cells of the lobula and ducts of the mammary glands expressed SPLUNC1. The adenocarcinoma cells in the lung, stomach, colon, mammary gland, uterus endometrium and cervix showed strong expressions of SPLUNC1 gene.SPLUNC1 expression is highly cell-specific in association with the cell functions.

Allelic loss of chromosome 3p, including the 3p21.3 region, is found in 95-100% of primary nasopharyngeal carcinoma (NPC) biopsies, suggesting that this region should harbor some tumor suppressor genes (TSGs) closely related to NPC development. Several TSGs located at 3p21.3, such as RASSF1A, LTF and BLU, have been demonstrated to be involved in NPC development. LARS2 (leucyl-tRNA synthetase 2, mitochondrial) is another gene located in the chromosome 3 common eliminated region-1 (C3CER1) at 3p21.3. In this study, we focussed on the epigenetic and genetic alterations of LARS2 in NPC. The mRNA expression of LARS2 was detected in 36 NPC and 8 chronic nasopharyngitis (NP) tissues by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. Subsequently, the mutation, allelic loss, and methylation status of LARS2 were analysed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), homozygous deletion (HD) analysis and methylation-specific polymerase chain reaction in primary NPC tissues. No expression or downregulation of LARS2 was observed in 78% of primary NPC tissues. No mutations, assessed by PCR-SSCP and DNA sequencing, were found in the promoter region and exon 1 of LARS2 in NPC tissues, whereas HD was detected in 28% of NPC specimens at the LARS2 locus. In addition, hypermethylation of LARS2 was found in 64% of NPC samples but only in 12.5% of NP biopsies. Our data indicate that inactivation of LARS2 by both genetic and epigenetic mechanisms may be a common and important event in the carcinogenesis of NPC.

RNA interference methodology suppresses specific gene expression, thus mimicking loss-of-function mutation and enabling in vitro and in vivo gene function analysis. Lipofectamine RNAiMAX, a new transfection reagent, has been confirmed high efficiency in delivering small interfering RNA (siRNA) into mesenchymal stem cells and neural stem cells. In this study, we used three transfection reagents (Lipofectamine RNAiMAX, Oligofectamine and Lipofectamine 2000) to deliver siRNA into human embryonic stem (hES) cells and compared the silencing efficiency of enhanced green fluorescent protein transgene and Oct4, a key regulator of pluripotency. As a result, siRNA can be delivered into hES cells more efficiently by Lipofectamine RNAiMAX compared with the other two transfection reagents and high efficient knockdown of target genes was obtained by Lipofectamine RNAiMAX even at a low concentration of siRNA. Quantitative real-time PCR showed approximately 90% knockdown of Oct4 transcript with cognate Oct4 siRNA transfection by Lipofectamine RNAiMAX compared to control siRNA. These results demonstrated that Lipofectamine RNAiMAX is an efficient and excellent transfection reagent for delivering siRNA into hES cells.

Matrix metalloproteinases (MMPs) play important roles in cancer initiation and development. Several polymorphisms in the promoters of a number of MMP genes, which can affect the respective MMP production in an allele-specific manner, have been well characterized. We examined whether these functional polymorphisms were related to the risk of nasopharyngeal carcinoma (NPC) in Chinese populations. Eight polymorphisms in the promoter of MMP1, MMP2, MMP3, MMP7, MMP9, MMP12, and MMP13 were genotyped in two independent case-control populations; one is from Guangxi province (593 patients with NPC and 480 controls), and the other is from Guangdong province (239 patients and 286 controls). We observed significantly increased susceptibility to NPC for the MMP2 -1306CC (rs243865:C>T) (odds ratio [OR] = 2.01, 95% confidence interval [CI] = 1.30-3.10) and -735CC (rs2285053:C>T) (OR = 1.56, 95% CI = 1.17-2.09) genotype carriers compared with noncarriers in the Guangxi population. This association was confirmed in the Guangdong population (for -1306CC: OR = 2.19, 95% CI = 1.21-3.96; for -735CC: OR = 1.60, 95% CI = 1.13-2.28). The C(-1306)-C(-735) haplotype was also significantly associated with increased susceptibility to NPC in both the Guangxi (OR = 1.64, 95% CI = 1.35-1.99) and Guangdong population (OR = 1.68, 95% CI = 1.29-2.19). Furthermore, stratified analysis indicated that the increased susceptibility to NPC related to the -1306CC and -735CC genotype and the C(-1306)-C(-735) haplotype was more pronounced in heavier smokers. Our findings suggest that the genetic polymorphisms or haplotype in the MMP2 promoter may play a role in mediating the susceptibility to NPC in Chinese populations.

Purpose: Mouse double minute 2 (MDM2) is a key negative regulator of the p53 activity. Recently, a polymorphism in the MDM2 intronic promoter, SNP309, was shown to influence MDM2 expression and p53 activity. We examined whether the SNP309 was related to the risk of developing nasopharyngeal carcinoma (NPC) among Chinese populations. Experimental Design: We genotyped the SNP309 in two independent case-control populations in southern China, one is from Guangxi province (including 593 NPC patients and 480 controls) and the other is from Guangdong province (including 239 patients and 286 controls), by PCR direct sequencing. Multivariate logistic regression analysis was used to calculate adjusted odds ratio (OR) and 95% confidence interval (95% CI). Results: We observed that compared with the TT genotype, the genotypes containing G allele (GT + GG genotype) were associated with significant increased susceptibility to NPC in both Guangxi (OR, 1.43; 95% CI, 1.04-1.91) and Guangdong population (OR, 1.53; 95% CI, 1.00-2.36). When these two sample sets were combined, the OR of the GT + GG genotype developing NPC was 1.45 (95% CI, 1.12-1.85) compared with the TT genotype. Furthermore, compared with the TT genotype, the GT + GG genotype was also significantly associated with the advanced lymph node metastasis (OR, 1.84; 95% CI, 1.09-3.05). Conclusions: Our findings suggest that the MDM2 SNP309 may be a risk factor for the occurrence and advanced neck lymph node metastasis of NPC in Chinese population.

Adult stem cells can be identified by label-retaining cell (LRC) approach based on their ability to retain nucleoside analog, such as bromodeoxyuridine (BrdU). We hypothesized that mouse nasopharynx contains a small population of epithelial stem/progenitor cells that may be detected by the LRC technique. To identify LRCs in mice nasopharyngeal epithelia, neonatal mice were intraperitoneally injected with BrdU twice daily for 3 consecutive days. After an 8-week chase, long-term BrdU-labeled LRCs (approximately 2% of cells) were detected in the adult mice nasopharyngeal epithelia by immunostaining with BrdU antibody and some of LRCs (approximately 12% of cells) were found to be recruited into the S phase of cell cycle with an additional radioactive thymidine-labeling technique, indicating that the stem cells also divide, most likely asymmetrically. To further investigate whether the LRCs existed in human nasopharyngeal carcinoma (NPC) tissues, three NPC cell lines (5-8F, 6-10B and TMNE) were labeled with BrdU in vitro and then individually engrafted into the back of nude mice, which developed tumors. Again, label-retaining stem cells were found in all the three kinds of NPC xenograft tumors (approximately 0.3% of cells), around 16% of which were also labeled with radioactive thymidine. Thus, this study has demonstrated for the first time the presence of epithelial LRCs in mouse nasopharynx and human NPC tissues and these stem-like LRCs are not completely quiescent, as they will be recruited into the cell cycle to participate physiological or pathological process at any moment. More importantly, our data showed that NPC also contained stem cells, which are most likely the cause for NPC spread, metastasis and recurrence.

The DLC-1 gene, located at the human chromosome region 8p22, behaves like a tumor suppressor gene and is frequently deleted in diverse tumors. The deletion of 8p22 is not an uncommon event in nasopharyngeal carcinoma (NPC), therefore we explored the expression levels of the DLC-1 gene in NPCs and NPC cell lines by reverse transcription-polymerase chain reaction. The results showed the mRNA level of DLC-1 was downregulated. To identify the mechanism of DLC-1 downregulation in NPC, we investigated the methylation status of the DLC-1 gene using methylation-specific PCR, and found that 79% (31 of 39) of the NPC tissues and two DLC-1 nonexpressing NPC cell lines, 6-10B and 5-8F, were methylated in the DLC-1 CpG island. Microsatellite PCR was also carried out, and loss of heterozygosity was found at four microsatellite sites (D8S552, D8S1754, D8S1790 and D8S549) covering the whole DLC-1 gene with ratios of 33% (4 of 12 informative cases), 18% (2 of 11), 5% (1 of 18), and 25% (3 of 12), respectively. Taken together, our results suggest that DLC-1 might be an NPC-related tumor suppressor gene affected by aberrant promoter methylation and gene deletion.

DNA polymerases with 3′-5′ proofreading function mediate high fidelity DNA replication but their application for mutation detection was almost completely neglected before 1998. The obstacle facing the use of exo + polymerases for mutation detection could be overcome by primer-3′-termini modification, which has been tested using allele-specific primers with 3′ labeling, 3′ exonuclease-resistance and 3′ dehydroxylation modifications. Accordingly, three new types of single nucleotide polymorphism (SNP) assays have been developed to carry out genome-wide genotyping making use of the fidelity advantage of exo + polymerases. Such SNP assays might also provide a novel approach for re-sequencing and de novo sequencing. These new mutation detection assays are widely adaptable to a variety of platforms, including real-time PCR, multi-well plate and microarray technologies. Application of exo + polymerases to genetic analysis could accelerate the pace of personalized medicine.

Nasopharyngeal carcinoma (NPC) is a malignancy with high incidence in Southern China and South-East Asia. Etiology studies indicate that chemical carcinogen promoters, such as 12-O- tetradecanoylphorbol-13-acetate (TPA), are important factors causing NPC development. However, the mechanism of the TPA effect on NPC remains unclear. In the present study, cells from a poorly differenti- ated squamous cell carcinoma NPC cell line, CNE2, were stimulated by TPA and proteomics technology was carried out to find protein discrepancies between control and TPA-treated cells. Results revealed that TPA treatment in CNE2 cells could upregulate the expression of "triosephosphate isomerase" and "14-3-3 protein sigma" and downregulate the expression of "reticulocalbin 1 precursor", "nucleophosmin", "mitochondrial matrix protein p1 precursor", and "stathmin". The changes in the expression of these genes suggested that TPA induced CNE2 cells to antiproliferation and to apoptosis, which was confirmed by subsequent apoptosis detection. Therefore, the effects of TPA on nasopharyngeal carcinoma cells were distinct from the effects on primary epithelial cells and we suggest reasons for these differences.

Objective: To compare gene expression profiles of nasopharyngeal carcinoma (NPC) tissue with that of control tissue by cDNA Array and to discuss possible reasons of TP53 accumulation in NPC tissue. Methods: (1) hybridization of Atlas Human Cancer cDNA Expression Array 7742-1; (2) analysis of Atlas Arrays using Atlasimage 1.01a; (3) verification of results of array by RT-PCR; (4) verification of protein expression alterations by immunohistochemistry. Results: (1) Of 588 tumor-related genes, 134 genes were upregulated, 88 downregulated; (2) Of 32 TP53-regulated genes, 13 genes were shown differential expression, 11 upregulated, 2 downregulated; (3) ATM and JNK2 were upregulated; (4) mRNA expression of ubiquitin-conjugating enzyme E2 (M74524) and ubiquitin-conjugating enzyme E2 (L22005) has no evident changes; Conclusion: (1) TP53 dysfunction exists in NPC tissues; (2) ATM and JNK might be the important causes of TP53 accumulation.

The invasive tumor front underlies the biological aggressiveness and epithelial-mesenchymal transition (EMT) in various human malignances. However, the molecular and biological characteristics of invasive tumor front in NPC have rarely been described. Additionally, the features of cancer stem cells (CSCs) in the invasive front of tumors and its correlation with EMT also remain elusive. Our study was to investigate the expression of CSCs marker aldehyde dehydrogenase 1 (ALDH1) in the invasive front of nasopharyngeal carcinoma (NPC) and its clinical significance. Immunohistochemistry was mainly used to detect ALDH1 expression in the invasive front of NPC. The relationship between ALDH1 expression and EMT-associated markers was also examined. ALDH1 expression in the invasive front correlated strongly with lymphatic invasion (p

To detect the prognostic significance of tumour budding and its expression of aldehyde dehydrogenase 1 (ALDH1) in nasopharyngeal carcinoma (NPC).Tumour budding was investigated in 105 patients with NPC by immunohistochemistry for pan-cytokeratin (AE1/AE3). The intensity of budding correlated strongly with T classification (P=0.008), lymphatic invasion (P0.001), vascular invasion (P=0.029), lymph node metastasis (P0.001), and clinical stage (P=0.010). Univariate analysis revealed that patients with high budding grade had poorer survival than those with low grade (P=0.002). Multivariate analysis showed that tumour budding was an independent predictor of survival (P=0.001). Furthermore, budding cells showed high-level expression of the cancer stem cell (CSC) marker ALDH1. Budding cells with high-level ALDH1 expression contributed to several aggressive behaviours and poor survival (P=0.000). We describe, for the first time, the presence of tumour budding and its correlation with aggressive tumour behaviour and poor patient survival in NPC. The degree of tumour budding could be a valuable predictive factor in NPC. In addition, we show, also for the first time, that budding cells in NPC might possess the invasive and metastatic properties of CSCs.

To investigate whether the neoplastic spindle cells in nasopharyngeal carcinoma (NPC) are associated with the process of epithelial-mesenchymal transition (EMT).We used immunohistochemistry to analyse the expression of cytokeratin, E-cadherin, β-catenin, vimentin, fibronectin, Snail1, Slug and aldehyde dehydrogenase 1 (ALDH1) in 115 cases of NPC in which there were neoplastic spindle cells; in 47 cases a neoplastic squamous cell component was also present. There was no significant difference in the expression of cytokeratin observed in the neoplastic spindle cells (P = 0.644), compared to the squamous component whereas E-cadherin expression was reduced. By contrast, the expression of β-catenin, vimentin, fibronectin, Snail1, Slug and ALDH1 was up-regulated in the spindle cells (all P = 0.000). Furthermore, E-cadherin expression was associated negatively with β-catenin (P0.001), vimentin (P0.001), fibronectin (P0.001), Slug (P0.001) and ALDH1 (P0.001) in neoplastic spindle cells, but did not correlate with Snail1 expression (P = 0.093).Our findings demonstrate for the first time that EMT might play an important role in the development of neoplastic spindle cells in NPC.

To investigate the aberrant expression of N-cadherin in nasopharyngeal carcinoma (NPC) and its prognostic significance.Immunohistochemical staining for N-cadherin protein was performed on tissue microarray (TMA) from 122 NPC patients. Cytoplasmic N-cadherin was observed in 42.6% and nuclear N-cadherin in 45.1% of NPC tissues. High expression of cytoplasmic and nuclear N-cadherin was associated with a majority of the clinicopathological variables, including lymph node metastasis, distant metastasis and clinical stage. Cytoplasmic N-cadherin was associated positively with nuclear N-cadherin expression (P = 0.000). In univariate analysis, cytoplasmic N-cadherin showed no significant impact on patient prognosis. In contrast, the overall survival was significantly shorter in patients with high nuclear N-cadherin than those with low levels of staining (P = 0.002). A high expression of nuclear N-cadherin predicted poorer survival in patients with late stage disease (P = 0.033), but not those with early tumour stage. In addition, multivariate analysis showed nuclear N-cadherin to bean independent prognostic marker for NPC patients (P = 0.024).Nuclear N-cadherin expression may represent a valuable prognostic marker in NPC patients, especially those with late stage disease.

Nasopharyngeal carcinoma (NPC) is a unique malignancy with high incidence in southern China and Southeast Asia. The etiology of NPC is associated with a complex interaction of genetic, viral, and environmental factors. Genetic susceptibility may be considered as the “first hit” for NPC development. Infection of Epstein–Barr virus (EBV) may be taken as the “second hit,” whereas environmental carcinogens may play a promoting role in NPC carcinogenesis. These factors working together drive the normal nasopharyngeal epithelial cells to preinvasive, then invasive tumor stages, as marked by various genetic and epigenetic abnormalities. Understanding of the unique molecular mechanisms of NPC has greatly increased in the last decade. This impressive achievement has led to the development of novel diagnostic and therapeutic strategies for NPC. However, the search for molecular-based treatments for NPC has only just started. Identification of molecular targets specific for NPC may provide better treatment strategies for NPC in the future.

To compare the gene expression profiles of nasopharyngeal carcinoma (NPC) cell line 5-8F-EGFP and the liver metastatic 5-8F-H3B-EGFP cells.The fluorescence-labeled cDNA were prepared separately from the total RNA extracted from the two cell lines and hybridized with Human_U133A2.0 Genechip (Affymetrix, USA) containing approximately 18 400 known gene. The gene expression profiles were analyzed with special software and cluster analysis.A total of 3767 genes were identified to have significant differential expressions between these two cell lines (P0.05), among which 281 genes showed twofold or higher differential expressions. Using MILANO software, we found 16 genes with probable close relation with liver metastasis of NPC.The 16 genes differentially expressed between the two cell lines can be of importance in the investigation of the molecular mechanism of NPC liver metastasis and identification of molecular markers for prognostic evaluation.

The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, while EGFR-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. To identify EGFR-regulated secreted proteins in NPC, we compared the secretome profiles of TGF-α-stimulated and unstimulated NPC cell line CNE-2. CNE-2 cells were cultured in the absence or presence of TGF-α for 24 h, and secreted proteins were obtained from conditioned serum-free media and enriched by ultrafiltration centrifugation. Using 2-DE and subsequent mass spectrometry, we identified 16 differential secreted proteins, among which the amyloid β-protein precursor (APP) was up-regulated and cystatin C was down-regulated after TGF-α stimulation. We further showed that the secretory changes of APP and cystatin C in CNE-2 after TGF-α stimulation could be abrogated by pretreatment of EGFR tyrosine kinase inhibitor PD153035 and PI3 kinase inhibitor Wortmannin, validating that APP and cystatin C are EGFR-regulated secreted proteins in NPC cells. Immunohistochemistry showed that the expression level of EGFR was positively correlated with the expression level of APP and negatively correlated with the expression level of cystatin C in NPC tissues, indicating that EGFR also regulates expression of APP and cystatin C in clinical NPC tissues. Furthermore, functional analysis showed that the growth and migration of CNE-2 cells decreased after neutralization of secretory APP in the medium using the anti-APP antibody. Our data provide substantial evidence that APP and cystatin C are target secreted proteins of EGFR in NPC, and upregulation of secretory APP by EGFR may be involved in the pathogenesis of NPC.

BACKGROUND AND OBJECTIVE Detection of label retaining cells (LRCs) has been a method to confirm existence of stem cells, and bromodeoxyuridine (BrdU) has commonly been used for labeling. In this study, to verify stem cells in nasopharyngeal carcinoma (NPC), LRCs were established and detected in NPC cell line 5-8F. METHODS The 5-8F cells were cultured with BrdU and inoculated subcutaneously into nude mice. By immunohistochemistry, immunocytochemistry, and immunofluorescence, BrdU was detected in 5-8F cells and xenograft tumors. RESULTS BrdU was strongly positive in cells on the 2nd and the 7th day after being added BrdU, while negative when cells were cultured without BrdU. However, only sporadic cells were positive on the 14th day after BrdU being washed out, and these cells were thought to be LRCs. The average percentage of LRCs was (0.67 +/- 0.32)%. After being cultivated with BrdU for 48 h, 5-8F cells were inoculated into nude mice subcutaneously. After chasing 8 weeks, only sporadic LRCs were detected in xenograft tumors, with a proportion of (0.55 +/- 0.36)%, and these LRCs were located at cancer margin. CONCLUSION The existence of LRCs in 5-8F cells indicates the existence of cancer stem cells in NPC.

Cisplatin (CDDP) is one of the most active drugs to treat nasopharyngeal carcinoma (NPC) patients. To further understand the mechanisms of CDDP-resistance in NPC, two CDDP-resistant sublines (CNE2-CDDP and CNE2-CDDP-5Fu) derived from parental NPC cell line CNE2 were established. It was found that at the IC50 level, the resistance of CNE2-CDDP and CNE2-CDDP-5Fu against CDDP was 2.63-fold and 5.35-fold stronger than that of parental CNE2, respectively. Of the four ABC transporters (ABCB1, ABCC1, ABCC2 and ABCG2) related to MDR, only ABCC2 was found to be elevated both in CDDP-resistant sublines, with ABCC2 located in nucleus of CNE2-CDDP-5Fu but not in CNE2-CDDP and parental CNE2. Further research showed that compared to untreated CNE2, the intracellular levels of CDDP were decreased by 2.03-fold in CNE2-CDDP and 2.78-fold in CNE2-CDDP-5Fu. After treatment with PSC833, a modulator of MDR associated transporters including ABCC2, the intracellular level of CDDP was increased in CDDP-resistant sublines, and the resistance to CDDP was partially reversed from 2.63-fold to 1.62-fold in CNE2-CDDP and from 5.35-fold to 4.62-fold in CNE2-CDDP-5Fu. These data indicate that ABCC2 may play an important role in NPC resistant to CDDP.

Point mutations in the p53 gene have been detected in a variety of human cancers; the mutations are clustered in four "hot-spots" located in the coding region of exons 5, 7, and 8, which coincide with the four most highly conserved regions of the gene. We report the finding of a heterozygous G----C mutation at codon 280 (exon 8), position 2, of the p53 gene in a nasopharyngeal carcinoma (NPC) cell line, originating from Guangdong, a province in the People's Republic of China that leads the world in NPC incidence. A survey of nasopharyngeal tissues and NPC biopsies revealed that 1 out of 12 NPC samples from Hunan, another province in the People's Republic of China with high NPC incidence, had the same heterozygous mutation at codon 280 of p53, and none of 10 biopsies from Taiwan showed a mutation within exons 5-8 of the p53 gene. No other alteration of gene structure, including gross rearrangement or loss of heterozygosity or abnormality of gene expression was detected in NPC cell lines or NPC biopsies. We conclude from this study that mutational or other alterations of the p53 gene are not common in nasopharyngeal carcinogenesis and that a codon-280 mutation of p53 may be involved in less than 10% of NPC cases. This result contrasts with the relatively high frequency of p53 mutations associated with several other human carcinomas and suggests the importance of other genes in NPC genesis.

To prepare an Epstein-Barr virus (EBV) microarray using known and predicted EBV-coded genes as the cDNA probes to detect the EBV gene expression in nasopharyngeal carcinoma (NPC) tissues.The EBV gene probes were amplified by PCR using a pair of primers designed in both sides of the multiple clone site (MCS) of the T/A vector. After purification of the PCR products, 85 EBV genes and 8000 human genes were printed onto the same slide as the detection chip consisting of both EBV and human genes. This genechip was used to detect the differential gene expression in NPC and non-cancerous nasopharynx (NP) tissues.Detection of the human gene expression profile using the prepared genechip resulted in the identification of numerous human genes in the tissue specimens. Some EBV genes were also detected in the tissues using the genechip, but the signals of the genes appeared rather weak without distinctly visible fluorescence, and were not comparable to the strong signal intensities of the human genes.The EBV microarray, though constructed successfully, can not meet the needs for clinical application due to the limited detection sensitivity and the relative small quantity of EBV gene expression in NPC samples. Further improvements of the research methods are warranted.

To establish a nasopharyngeal carcinoma (NPC) cell line with stable EIF4G1 gene silencing induced by small interfering RNA (siRNA).The EIF4G1 mRNA levels in 8 NPC cell lines including 5-8F, 6-10B, C666-1, CNE1, CNE2, HNE1, HONE1, and SUNE1 were detected by fluorescence quantitative RT-PCR (QRT-PCR). The recombinant lentivirus shRNA expression plasmid targeting EIF4G1 gene was packaged into mature lentivirus by 293FT cells and used to infect 5-8F cells. After blasticidin selection of NPC cells with constant expression of the EIF4G1-siRNA, the efficiency of EIF4G1 mRNA expression interference was determined using QRT-PCR.The 8 NPC cell lines showed differential expression of EIF4G1 mRNA, among which 5-8F cells had the highest EIF4G1 expression. The recombinant lentivirus plasmid pLenti6/BLOCK-iT-DEST/EIF4G1-shRNA was successfully constructed and verified by PCR and sequencing. The EIF4G1 mRNA level of 5-8F cells infected with shRNA-EIF4G1 lentivirus was significantly reduced as compared with the negative control and the blank control cells.The recombinant lentivirus vector pLenti6/BLOCK- iT-DEST/EIF4G1-shRNA we constructed results in marked downregulation of EIF4G1 mRNA expression and constant expression of EIF4G1-siRNA after infection of 5-8F cells.

Glutathione S-transferase M1 (GSTM1) deficiency may increase the risk of nasopharyngeal carcinoma (NPC). This study was to evaluate the correlation of the single nucleotide polymorphism (SNP) in the coding region of GSTM1 gene to NPC susceptibility in southern China population.In total 239 NPC patients and 286 age-matched healthy controls were entered into the study. Among them, 225 out of 239 NPC patients and 273 out of 286 controls were used for statistical analysis. SNP screening of all exons, relevant intron-exon boundaries, and the promoter region of GSTM1, in total 4739bp, was performed by PCR direct sequencing. The loci T1270533G and C1256088C were selected for the case-control study using the tetra-Primer ARMS-PCR, as well as the sequencing method.In total 29 SNPs of GSTM1 were identified by sequencing. Missense mutation occurred in the polymorphic loci of T1270533G and C1256088C. However, no evident relationships between the variants of T1270533G and clinical phenotypes of NPC were observed in the NPC group and healthy control group (OR = 0.170, 95%CI = 0.95-0.306 for homozygote TT). The deletion frequency of C1256088C was 45% (45/100) for NPC patients and 42% (42/100) for controls.The polymorphism of T1270533G does not affect the detoxification function of GSTM1. The T1270533G locus has no apparent association with genetic susceptibility to NPC in the southern China population. The loss rate of C1256088C is high in this study.