Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer

Lung cancer is a worldwide health problem and a leading cause of cancer-related deaths. Silencing of potential tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event in the initiation and development of cancer. Thus, methylated cancer type-specific TSGs in DNA can serve as useful biomarkers for early cancer detection. We have now developed a “Multiplex Methylation Specific PCR” (MMSP) assay for analysis of the methylation status of multiple potential TSGs by a single PCR reaction. This method will be useful for early diagnosis and treatment outcome studies of non-small cell lung cancer (NSCLC). Genome-wide CpG methylation and expression microarrays were performed on lung cancer tissues and matched distant non-cancerous tissues from three NSCLC patients from China. Thirty-eight potential TSGs were selected and analyzed by methylation PCR on bisulfite treated DNA. On the basis of sensitivity and specificity, six marker genes, HOXA9, TBX5, PITX2, CALCA, RASSF1A, and DLEC1, were selected to establish the MMSP assay. This assay was then used to analyze lung cancer tissues and matched distant non-cancerous tissues from 70 patients with NSCLC, as well as 24 patients with benign pulmonary lesion as controls. The sensitivity of the assay was 99% (69/70). HOXA9 and TBX5 were the 2 most sensitive marker genes: 87% (61/70) and 84% (59/70), respectively. RASSF1A and DLEC1 showed the highest specificity at 99% (69/70). Using the criterion of identifying at least any two methylated marker genes, 61/70 cancer samples were positive, corresponding to a sensitivity of 87% and a specificity of 94%. Early stage I or II NSCLC could even be detected with a 100% specificity and 86% sensitivity. In conclusion, MMSP has the potential to be developed into a population-based screening tool and can be useful for early diagnosis of NSCLC. It might also be suitable for monitoring treatment outcome and recurrence.     

Cancer cell-secreted proteomes as a basis for searching potential tumor markers: nasopharyngeal carcinoma as a model

Nasopharyngeal carcinoma (NPC) is commonly diagnosed late due to its deep location and vague symptoms. To identify biomarkers for early NPC diagnosis, secreted proteomes of two NPC cell lines were analyzed. Proteins in the NPC cell-line cultured media were systematically identified by SDS-PAGE combined with MALDI-TOF MS. Twenty-three proteins were found in cultured media from both NPC cell lines. Among them, fibronectin, Mac-2 binding protein (Mac-2 BP), and plasminogen activator inhibitor 1 (PAI-1) were further confirmed by Western blot analysis. These three proteins were highly expressed in NPC biopsies, but weakly or not expressed in normal nasopharyngeal tissues. The serum levels of the three proteins were significantly higher in NPC patients (n = 46) than in normal controls (n = 47) (p < 0.01). NPC nude mice model (n = 9) also showed elevated levels of serum Mac-2 BP and PAI-1 compared with tumor-free mice (n = 9) (p < 0.01). Systematic analysis of cancer cell-secreted proteomes combined with animal tumor models can be a feasible, convenient strategy for searching multiple potential tumor markers. Furthermore, our work shows that fibronectin, Mac-2 BP, and PAI-1 may be potential markers for diagnosis of NPC.     

Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer

Lung cancer is a worldwide health problem and a leading cause of cancer-related deaths. Silencing of potential tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event in the initiation and development of cancer. Thus, methylated cancer type-specific TSGs in DNA can serve as useful biomarkers for early cancer detection. We have now developed a “Multiplex Methylation Specific PCR” (MMSP) assay for analysis of the methylation status of multiple potential TSGs by a single PCR reaction. This method will be useful for early diagnosis and treatment outcome studies of non-small cell lung cancer (NSCLC). Genome-wide CpG methylation and expression microarrays were performed on lung cancer tissues and matched distant non-cancerous tissues from three NSCLC patients from China. Thirty-eight potential TSGs were selected and analyzed by methylation PCR on bisulfite treated DNA. On the basis of sensitivity and specificity, six marker genes, HOXA9, TBX5, PITX2, CALCA, RASSF1A, and DLEC1, were selected to establish the MMSP assay. This assay was then used to analyze lung cancer tissues and matched distant non-cancerous tissues from 70 patients with NSCLC, as well as 24 patients with benign pulmonary lesion as controls. The sensitivity of the assay was 99% (69/70). HOXA9 and TBX5 were the 2 most sensitive marker genes: 87% (61/70) and 84% (59/70), respectively. RASSF1A and DLEC1 showed the highest specificity at 99% (69/70). Using the criterion of identifying at least any two methylated marker genes, 61/70 cancer samples were positive, corresponding to a sensitivity of 87% and a specificity of 94%. Early stage I or II NSCLC could even be detected with a 100% specificity and 86% sensitivity. In conclusion, MMSP has the potential to be developed into a population-based screening tool and can be useful for early diagnosis of NSCLC. It might also be suitable for monitoring treatment outcome and recurrence.     

一个定位在7q32染色体区域的鼻咽癌负相关EST

为了分离和克隆定位于７ｑ３２染色体区域的与鼻咽癌发病有关的抑瘤基因,检测了鼻咽癌活检组织及配对外周血标本中７ｑ３２微卫星ＤＮＡ多态性位点的基因型,发现在７ｑ３２区域存在３０％左右的杂合性丢失,从Ｉｎｔｅｒｎｅｔ中查询到定位于７ｑ３２区域的所有不同ＥＳＴ,对其中２０个ＥＳＴ进行分析,ＲＴ－ＰＣＲ和Ｎｏｒｔｈｅｒｎ杂交发现,ＡＡ０７０４３７,ＥＳＴ在鼻咽癌细胞株ＨＮＥ１中表达微弱,而在正常鼻咽上皮原代     

Reduced CTGF Expression Promotes Cell Growth,Migration, and Invasion in Nasopharyngeal Carcinoma

Background

The role of CTGF varies in different types of cancer. The purpose of this study is to investigate the involvement of CTGF in tumor progression and prognosis of human nasopharyngeal carcinoma (NPC).

Experimental design

CTGF expression levels were examined in NPC tissues and cells, nasopharynx (NP) tissues, and NP69 cells. The effects and molecular mechanisms of CTGF expression on cell proliferation, migration, invasion, and cell cycle were also explored.

Results

NPC cells exhibited decreased mRNA expression of CTGF compared to immortalized human nasopharyngeal epithelial cell line NP69. Similarly, CTGF was observed to be downregulated in NPC compared to normal tissues at mRNA and protein levels. Furthermore, reduced CTGF was negatively associated with the progression of NPC. Knocking down CTGF expression enhanced the colony formation, cell migration, invasion, and G1/S cell cycle transition. Mechanistic analysis revealed that CTGF suppression activated FAK/PI3K/AKT and its downstream signals regulating the cell cycle, epithelial-mesenchymal transition (EMT) and MMPs. Finally, DNA methylation microarray revealed a lack of hypermethylation at the CTGF promoter, suggesting other mechanisms are associated with suppression of CTGF in NPC.

Conclusion

Our study demonstrates that reduced expression of CTGF promoted cell proliferation, migration, invasion and cell cycle progression through FAK/PI3K/AKT, EMT and MMP pathways in NPC.     

Variable expression of latent membrane protein in nasopharyngeal carcinoma can be related to methylation status of the Epstein-Barr virus BNLF-1 5''-flanking region.

Seven virus-coded proteins, the nuclear proteins EBNA-1 to EBNA-6 and the latent membrane protein (LMP), are regularly expressed in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines. In nasopharyngeal carcinoma (NPC), only EBNA-1 is regularly expressed; LMP is detected in about 65% of the tumors. In Burkitt's lymphoma tumors only EBNA-1 is expressed. We have recently shown that the methylation patterns of the EBV genome varied between these cell types. In virally transformed lymphoblastoid cell lines of normal origin, the EBV DNA is completely unmethylated. In contrast, in the Burkitt's lymphoma-derived cell line Rael and in a nude mouse-passaged NPC tumor, C15, there was an extensive methylation of CpG pairs. The methylation extended into the coding regions of the two expressed genes, EBNA-1 (in both tumor types) and LMP (in C15). Two presumptive control regions were exempted from this overall methylation: the oriP that contains both an origin of DNA replication and an EBNA-1-dependent enhancer and the 5'-flanking region of the BNLF-1 open reading frame that codes for LMP. The latter was only exempted in the LMP expressing NPC. We have now investigated the relation between expression of LMP and methylation of DNA in the 5'-flanking 1 kb region of BNLF-1, coding for LMP. LMP was methylated in 3 of 12 NPC biopsies that did not express LMP but was partially or totally unmethylated in the remaining 9 that expressed the protein. The three BNLF-1 exons were highly methylated in all the tumors. The oriP region was unmethylated in all the tumors, as in the previously studied Rael cell line and nude mouse-passaged NPC. Also, the BamHI W enhancer region involved in the expression of EBNA nuclear proteins was methylated. None of the biopsies expressed EBNA-2. Our data show that the EBV genomes are highly methylated in NPC tumors. The strong reverse correlation between the methylation of the putative control region of the LMP gene and the expression of LMP suggests that methylation has a role in the regulation of this gene.     

DNAJC10基因在鼻咽癌中的表达及表达下调机制的研究

运用RT-PCR检测候选抑瘤基因DNAJC10在鼻咽癌组织中的表达,运用甲基化特异性PCR(MSPCR)技术、LOH和测序等技术分别检测DNAJC10基因在鼻咽癌组织中的甲基化状况、LOH和启动子突变情况.结果表明,DNAJC10基因在肿瘤组织中较对照慢性鼻咽炎组织表达明显下调(P<0.05).DNAJC10在鼻咽癌中不表现高甲基化,其LOH的缺失率为6.25%,突变率为66.7%.因此,DNAJC10基因的表达下调主要是其遗传改变(LOH和突变)所致.     

鼻咽癌下调新基因NOR1相互作用蛋白的筛选和鉴定

NOR1基因是新的鼻咽癌相关基因,该基因在鼻咽癌细胞系HNE1和鼻咽癌组织中表达下调．在鼻咽癌细胞HNE1中恢复NOR1基因表达抑制了鼻咽癌细胞的生长和增殖能力．为了探讨NOR1基因的生物学功能,以NOR1基因为诱饵运用酵母双杂交技术在人胎脑文库中筛选其交互作用蛋白,挑选阳性克隆,进行DNA序列分析和同源检索,阳性克隆编码7个不同的蛋白质,其中一个阳性克隆编码线粒体ATP合成酶亚基OSCP蛋白．瞬时转染pCMV-myc-NOR1质粒进入鼻咽癌5-8F细胞,通过密度梯度离心法分离线粒体蛋白,Western blot检测表明myc-NOR1蛋白分布于线粒体与胞浆．免疫荧光检测表明在鼻咽正常上皮细胞NP69中内源性NOR1蛋白与线粒体存在明显共定位．随后采用特异性酵母双杂交、免疫荧光共定位、免疫共沉淀技术证实了NOR1与OSCP在线粒体内存在交互作用．提示,NOR1是一个新的线粒体蛋白,可能通过结合OSCP蛋白调控细胞能量代谢,为深入探讨其功能提供了重要线索．     

Expression of human leukocyte antigen G is associated with prognosis in nasopharyngeal carcinoma

Human leukocyte antigen G (HLA-G) has multiple immune regulatory functions including the induction of immune tolerance in malignancies. The roles of HLA-G have not been investigated in nasopharyngeal carcinoma (NPC). This study is aimed to evaluate the role of HLA-G as prognostic factor for NPC patients as well as its role in the immune regulation. Western assays showed high HLA-G expression in NPC cell lines, but low in the immortalized nasopharyngeal epithelial cell line NP69. HLA-G protein was further detected in 79.2% of 552 NPC specimens with immunohistochemistry (IHC), but not in normal nasopharyngeal epithelium tissue. Moreover, high expression of HLA-G predicted poor survival of NPC patients and positively correlated with tumor N classification and recurrence or metastasis. Multivariate analysis indicated that HLA-G was an independent and unfavorable prognostic factor. Furthermore, the presence of CD68+ macrophages and IL-10 were also examined, which are two prognostic markers of NPC and important factors for regulating immune surveillance. The correlations of HLA-G with these two immune factors were revealed in NPC tissues. Taken together, our results suggest that HLA-G is an independent biomarker for NPC prognosis, and HLA-G might contribute to NPC progression, which might jointly regulate immune surveillance in NPC together with macrophages and IL-10.     

Inactivation of 14-3-3 σ by promoter methylation correlates with metastasis in nasopharyngeal carcinoma

14-3-3 σ, the downstream target of p53, is a negative regulator of cell cycle G2-M phase checkpoint in response to DNA damage. Our previous comparative proteomics study showed that 14-3-3 σ was downregulated or lost in nasopharyngeal carcinoma (NPC) tissue compared with non-cancerous nasopharyngeal epithelial tissue (NNET). In this study, we further investigated for the epigenetic mechanism of 14-3-3 σ inactivation. Methylation-specific PCR showed 14-3-3 σ promoter methylation in 100% of analyzed NPC cell lines (4/4) but not in immortalized human nasopharyngeal epithelial cell line NP69. Treatment of the four NPC cell lines with the methyltransferase inhibitor 5-aza-2′-dC resulted in the demethylation and upregulation of 14-3-3 σ. In tissues, 14-3-3 σ promoter methylation occurred at a higher frequency in NPC, 63/75 (84%), compared to adjacent NNET, 7/25 (28%), and fully methylated 14-3-3 σ promoter was detected in NPC but not in any of adjacent NNET. RT-PCR, Western blotting, and immunohistochemistry showed that 14-3-3 σ expression was downregulated or lost in NPC with methylation, and there was a negative correlation between the expression levels and methylation statuses of 14-3-3 σ gene. In addition, the patients with methylated 14-3-3 σ presented a higher frequency of lymph node and distant metastasis, and an advanced clinical stage, and overexpression of 14-3-3 σ in NPC cell line 5-8F with high metastatic potential was able to inhibit its in vitro invasive ability. Our data are the first to show that 14-3-3 σ is frequently inactivated by promoter methylation in NPC and this aberrant methylation correlates with lymph node and distant metastasis. J. Cell. Biochem. 106: 858–866, 2009. © 2009 Wiley-Liss, Inc.     

Self-collected mid-turbinate swabs for the detection of respiratory viruses in adults with acute respiratory illnesses

Background

The gold standard for respiratory virus testing is a nasopharyngeal (NP) swab, which is collected by a healthcare worker. Midturbinate (MT) swabs are an alternative due to their ease of collection and possible self-collection by patients. The objective of this study was to compare the respiratory virus isolation of flocked MT swabs compared to flocked NP swabs.

Methods

Beginning in October 2008, healthy adults aged 18 to 69 years were recruited into a cohort and followed up for symptoms of influenza. They were asked to have NP and MT swabs taken as soon as possible after the onset of a fever or two or more respiratory symptoms with an acute onset. The swabs were tested for viral respiratory infections using Seeplex® RV12 multiplex PCR detection kit. Seventy six pairs of simultaneous NP and MT swabs were collected from 38 symptomatic subjects. Twenty nine (38%) of these pairs were positive by either NP or MT swabs or both. Sixty nine (91%) of the pair results were concordant. Two samples (3%) for hCV OC43/HKU1 and 1 sample (1%) for rhinovirus A/B were positive by NP but negative by MT. One sample each for hCV 229E/NL63, hCV OC43/HKU1, respiratory syncytial virus A, and influenza B were positive by MT but negative by NP.

Conclusions

Flocked MT swabs are sensitive for the diagnosis of multiple respiratory viruses. Given the ease of MT collection and similar results between the two swabs, it is likely that MT swabs should be the preferred method of respiratory cell collection for outpatient studies. In light of this data, larger studies should be performed to ensure that this still holds true and data should also be collected on the patient preference of collection methods.     

OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation

Background

Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. Loss of heterozygosity at 11q25 is common in multiple tumors including nasopharyngeal carcinoma (NPC). OPCML, located at 11q25, is one of the downregulated genes we identified through digital expression subtraction.

Methodology/Principal Findings

Semi-quantitative RT-PCR showed frequent OPCML silencing in NPC and other common tumors, with no homozygous deletion detected by multiplex differential DNA-PCR. Instead, promoter methylation of OPCML was frequently detected in multiple carcinoma cell lines (nasopharyngeal, esophageal, lung, gastric, colon, liver, breast, cervix, prostate), lymphoma cell lines (non-Hodgkin and Hodgkin lymphoma, nasal NK/T-cell lymphoma) and primary tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues. Pharmacological and genetic demethylation restored OPCML expression, indicating a direct epigenetic silencing. We further found that OPCML is stress-responsive, but this response is epigenetically impaired when its promoter becomes methylated. Ecotopic expression of OPCML led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells with endogenous silencing.

Conclusions/Significance

Thus, through functional epigenetics, we identified OPCML as a broad tumor suppressor, which is frequently inactivated by methylation in multiple malignancies.     

Repression of DERL3 via DNA methylation by Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV)-associated invasive malignancy. Increasing evidence indicates that epigenetic abnormalities, including DNA methylation, play important roles in the development of NPC. In particular, the EBV principal oncogene, latent membrane protein 1 (LMP1), is considered a key factor in inducing aberrant DNA methylation of several tumour suppressor genes in NPC, although the mechanism remains unclear. Herein, we comprehensively analysed the methylome data of Infinium BeadArray from 51 NPC and 52 normal nasopharyngeal tissues to identify LMP1-inducible methylation genes. Using hierarchical clustering analysis, we classified NPC into the high-methylation, low-methylation, and normal-like subgroups. We defined high-methylation genes as those that were methylated in the high-methylation subgroup only and common methylation genes as those that were methylated in both high- and low-methylation subgroups. Subsequently, we identified 715 LMP1-inducible methylation genes by observing the methylome data of the nasopharyngeal epithelial cell line with or without LMP1 expression. Because high-methylation genes were enriched with LMP1-inducible methylation genes, we extracted 95 high-methylation genes that overlapped with the LMP1-inducible methylation genes. Among them, we identified DERL3 as the most significantly methylated gene affected by LMP1 expression. DERL3 knockdown in cell lines resulted in significantly increased cell proliferation, migration, and invasion. Lower DERL3 expression was more frequently detected in the advanced T-stage NPC than in early T-stage NPC. These results indicate that DERL3 repression by DNA methylation contributes to NPC tumour progression.     

Epstein-Barr viral antigens used in the diagnosis of nasopharyngeal carcinoma

The major antigen complexes of Epstein-Barr virus (EBV) include the latent infectious proteins, early antigens, membrane antigens and viral capsid antigens. The various polypeptides within each antigen complex have been identified and isolated through gene-cloning technology. These polypeptides are exploited to be used as serological markers for the diagnosis of nasopharyngeal carcinoma (NPC) through enzyme-linked immunosorbent assay. This paper reviews the recent studies on the profile of antibodies in patients with NPC towards these EBV polypeptides of each antigen complex. The sensitivity and specificity of each polypeptide when used as serological markers to NPC patients' sera are summarized.     

Demethylation of E-cadherin gene in nasopharyngeal carcinoma could serve as a potential therapeutic strategy

E-cadherin has been proven to be widely down-regulated and tightly associated with tumour invasion and metastasis in multiple human cancer types. Recent research demonstrated that aberrant methylation around gene promoter region attributes to E-cadherin silencing. However, the detailed information about this epigenetic inactivation in nasopharyngeal carcinoma (NPC) is rare. The aim of this study was to probe more into the basic mechanism of E-cadherin methylation in NPC and elucidate the application of demethylating agents to restore E-cadherin expression. To address this question, we initially studied E-cadherin methylation status in NPC primary tumours and cell lines by methylation-specific PCR, and compared it with E-cadherin expression. Methylated E-cadherin was detected in 13 of 20 (65%) NPC clinical specimens and 2 of 2 (100%) NPC cell lines (HNE-1 and CNE-2), which was inversely correlated with E-cadherin expression. The detailed methylation profile at individual CpGs within CpG island of E-cadherin promoter region was confirmed by bisulphite sequencing. E-cadherin gene could be demethylated and reactivated in HNE-1 and CNE-2 cells upon treatment with 5-aza-dC, a DNA demethylating agent. Our findings indicate that frequent aberrant methylation of E-cadherin may play an important role in downregulation of E-cadherin, and demethylation therapy could serve as a promising strategy for NPC patients. Furthermore, a high frequency of E-cadherin methylation (9/20, 45%) in peripheral blood of NPC patients suggests its potential clinical application as an early diagnostic or predictive marker.     

The tumor supressor function of STGC3 and its reduced expression in nasopharyngeal carcinoma

STGC3 is a novel candidate tumor suppressor gene that was found to be associated with nasopharyngeal carcinoma (NPC) via the cDNA cloning and RACE processes. The biological function of the STGC3 protein and its expression level in nasopharyngeal carcinoma remain unknown. This study aimed to evaluate the STGC3 protein expression level in NPC and to investigate the inhibitory function of STGC3 as a candidate tumor suppressor gene. We assessed the expression of the STGC3 protein in NPC biopsies and normal control specimens via Western blot and immunohistochemical analysis. The expression of STGC3 as induced by doxycycline (Dox) via a tetracycline (Tet)-regulated system in human nasopharyngeal carcinoma cell line CNE2 was also established, and the effect of STGC3 restoration on the biological behavior of CNE2 was observed. A reduced level of STGC3 expression (0.978 ± 0.213 versus 0.324 ± 0.185, P < 0.05) was detected in NPC versus normal nasopharyngeal tissue by Western blot assay. Immunohistochemical assays for STGC3 detected positive staining in the nuclei and cytoplasm of epithelial cells, and the positive expression rate in NPC, 8 of 21 (38%), was lower than that in normal nasopharynx samples, 16 of 22 (72%). After STGC3 expression was restored, the growth capacity and clone formation potential of CNE2 cells in soft agar were significantly suppressed, and the cell percentage in G₀/G₁ phase increased, while the percentage of cells entering the S and G₂ phases decreased. This indicates that an abnormality in STGC3 expression is associated with nasopharyngeal carcinogenesis and that it may play an important role in controlling cell growth and regulating the cell cycle.     

Blood diagnostic biomarkers for major depressive disorder using multiplex DNA methylation profiles: discovery and validation

Aberrant DNA methylation in the blood of patients with major depressive disorder (MDD) has been reported in several previous studies. However, no comprehensive studies using medication-free subjects with MDD have been conducted. Furthermore, the majority of these previous studies has been limited to the analysis of the CpG sites in CpG islands (CGIs) in the gene promoter regions. The main aim of the present study is to identify DNA methylation markers that distinguish patients with MDD from non-psychiatric controls. Genome-wide DNA methylation profiling of peripheral leukocytes was conducted in two set of samples, a discovery set (20 medication-free patients with MDD and 19 controls) and a replication set (12 medication-free patients with MDD and 12 controls), using Infinium HumanMethylation450 BeadChips. Significant diagnostic differences in DNA methylation were observed at 363 CpG sites in the discovery set. All of these loci demonstrated lower DNA methylation in patients with MDD than in the controls, and most of them (85.7%) were located in the CGIs in the gene promoter regions. We were able to distinguish patients with MDD from the control subjects with high accuracy in the discriminant analysis using the top DNA methylation markers. We also validated these selected DNA methylation markers in the replication set. Our results indicate that multiplex DNA methylation markers may be useful for distinguishing patients with MDD from non-psychiatric controls.     

Promoter hypermethylation of tumor suppressor genes in serum as potential biomarker for the diagnosis of nasopharyngeal carcinoma

Purpose: Promoter hypermethylation of tumor suppressor genes may serve as a promising biomarker for the diagnosis of cancer. Cell-free circulating DNA (cf-DNA) shares hypermethylation status with primary tumors. This study investigated promoter hypermethylation of five tumor suppressor genes as markers in the detection of nasopharyngeal carcinoma (NPC) in serum samples. Methods: cf-DNA was extracted from serum collected from 40 NPC patients and 41 age- and sex-matched healthy subjects. The promoter hypermethylation status of the five genes (RASSF1, CDKN2A, DLEC1, DAPK1 and UCHL1) was assessed by methylation-specific PCR after sodium bisulfite conversion. Differences in the methylation status of these five genes between NPC patients and healthy subjects were compared. Results: The concentration of cf-DNA in the serum of NPC patients was significantly higher than that in normal controls. The five tumor suppressor genes – RASSF1, CDKN2A, DLEC1, DAPK1 and UCHL1 – were found to be methylated in 17.5%, 22.5%, 25.0%, 51.4% and 64.9% of patients, respectively. The combination of four-gene marker – CDKN2A, DLEC1, DAPK1 and UCHL1 – had the highest sensitivity and specificity in predicting NPC. Conclusion: Screening DNA hypermethylation of tumor suppressor genes in serum was a promising approach for the diagnosis of NPC.     

Epstein-Barr Virus-Encoded LMP2A Induces an Epithelial–Mesenchymal Transition and Increases the Number of Side Population Stem-like Cancer Cells in Nasopharyngeal Carcinoma

It has been recently reported that a side population of cells in nasopharyngeal carcinoma (NPC) displayed characteristics of stem-like cancer cells. However, the molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. Epstein-Barr virus was the first identified human tumor virus to be associated with various malignancies, most notably NPC. LMP2A, the Epstein-Barr virus encoded latent protein, has been reported to play roles in oncogenic processes. We report by immunostaining in our current study that LMP2A is overexpressed in 57.6% of the nasopharyngeal carcinoma tumors sampled and is mainly localized at the tumor invasive front. We found also in NPC cells that the exogenous expression of LMP2A greatly increases their invasive/migratory ability, induces epithelial–mesenchymal transition (EMT)-like cellular marker alterations, and stimulates stem cell side populations and the expression of stem cell markers. In addition, LMP2A enhances the transforming ability of cancer cells in both colony formation and soft agar assays, as well as the self-renewal ability of stem-like cancer cells in a spherical culture assay. Additionally, LMP2A increases the number of cancer initiating cells in a xenograft tumor formation assay. More importantly, the endogenous expression of LMP2A positively correlates with the expression of ABCG2 in NPC samples. Finally, we demonstrate that Akt inhibitor (V) greatly decreases the size of the stem cell side populations in LMP2A-expressing cells. Taken together, our data indicate that LMP2A induces EMT and stem-like cell self-renewal in NPC, suggesting a novel mechanism by which Epstein-Barr virus induces the initiation, metastasis and recurrence of NPC.