Aberrant CpG Island Methylation Of Major Tumor Suppressor PTEN And Its Underlying Mechanism In Nasopharyngeal Carcinoma

BackgroundNasopharyngeal carcinoma(NPC)is a malignancy particularly prevalent in the southern Chinese population of Guangdong,Inuits of Alaska and native Greenlanders.It is estimated that viral infections contribute to 15-20%of all human cancers.Epstein-Barr virus(EBV)infection has been long believed to be associated with many malignancies including Burkitt’s lymphoma,Hodgkin lymphoma,gastric cancer and NPC.Its infection belongs to the latency Ⅱ program in NPC,and is restricted to express several transcripts,such as EBV-determined nuclear antigen I(EBNA1),EBV encoded small RNA(EBER),latent membrane protein 2A(LMP2A),LMP1,and transcripts from the BamHI A region(BARFO).As an important hallmark for the latency program of EBV infection,LMP1 has the ability to transform rodent cells and render cell growth in soft agar.Recently,several studies have reported that LMP1 activates DNA methyltransferases(DNMTs)up-regulating the methylation of host genes,including TSG,through some signalings.As a tumor suppressor gene,one of the important functions of PTEN is regulation of the phosphatidylinositol 3-kinase/AKT pathway via lipid phosphatase activity.PTEN has also been found to be important in controlling a wide range of physiological and cellular activities,such as metabolism and chromosome stability.PTEN function in gene transcription modulation through interplay with histones and chromatin has also been demonstrated recently.The loss of function of PTEN leads to increases in cellular proliferation,survival and growth in many cancers.In NPC,silencing of tumor suppressor genes(TSGs)via deletion,insertion and mutation is an uncommon event in NPC carcinogenesis and aberrant methylation has been increasingly emerging as an alternative mechanism.Notably,CpG island hypermethylation has been recently identified as an alternative mechanism of PTEN inactivation in cancers including lung cancer,endometrial carcinoma,prostate cancer,brain tumors,EBV-associated gastric carcinoma,cervical neoplasm,malignant melanoma and hematologic malignancies.However,to date this mechanism of PTEN inactivation has not been reported in nasopharyngeal carcinoma.It augments DNMT1 expression via the c-Jun NH2-terminal kinase-activator protein-1 and the Rb-E2F signaling in NPC cells.It activates DNMT1,methylating and repressing Docking Protein 1(DOK1).In particular,the role of NF-κB in LMP1-dependent regulations becomes interesting.Enhanced NF-κB signaling is a critical facet of LMP1-associated signaling and many phenotypic changes associated with LMP1 expression are attributable to LMPl’s ability to activate NF-κB.These findings give a hypothesis that LMP1 may also promote the methylation of PTEN CpG island by means of some similar regulatory mechanism in NPC.Therefore,the aim of the present study is to clarify how EBV-encoded LMP1 can epigenetically alter the expression of host major TSG PTEN in NPC.We evaluated the PTEN expression in NPC specimens and control samples by quantitative qRT-PCR(qPCR)and examined the CpG island methylation status of PTEN using methylation-specific polymerase chain reaction(MSP)and sequencing.5-aza-dC treatment can lead to DNA demethylation via inhibition of DNA methyl transferase activity.We also treated NPC cell lines(HONE1,CNE1,6-10B)with 5-aza-dC followed by examining PTEN expression.Thus,we preliminarily demonstrate the role of PTEN methylation related with mechanism of PTEN inactivation in NPC.We provided cellular and clinical evidence revealing a close correlation among LMP1 expression,DNMt3b expression,and the methylation degree at PTEN CpG island,and further disclosed that LMP1-mediated NF-κB up-regulated DNMt3b transcription by binding to DNMt3b promoter region,leading to a higher methylation degree at PTEN CpG island,and ultimately silencing tumor suppressor PTEN in NPC.Materials and Methods1.Cell cultureThe human NPC cell lines 5-8F,6-10B,CNE1,CNE2,C666-1,HONE1,and SUNE1 obtained from Cancer Research Institute of Southern Medical University were cultured in RPMI-1640 with 10%calf serum.Two EBV-positive NPC cell lines(C666-1-EBV,HONE1-EBV)were kindly offered by Prof.George S.W.Tsao from the University of Hong Kong,All cells were cultured in a humidified atmosphere of 95%air and 5%CO2 at 37℃.2.Clinical specimensPrimary NPC biopsy specimens and normal biopsies of thenasopharynx were obtained from Nanfang Hospital(Southern Medical University,Guangzhou,China).Both tumor and normal tissues were histologically confirmed by H&E(hematoxylin and eosin)staining.All NPC specimens were classified as undifferentiated nonkeratinizing type.Informed consent was obtained from each patient,and the research protocols were approved by the Ethics Committee of Nanfang Hospital.3.Vector construction(1)LMP1:LMP1 vector were kindly offered by Prof.Zeng Mu-sheng from Sun Yatsen university cancer center,Guangzhou,China.(2)Wild-type(DNMT3B-pGL3-WT)or mutant(DNMT3B-pGL3-MUT)promoter of DNMT3b:A 960-bp fragment of DNMT3b promoter(-773,+187)was amplified by PCR and cloned downstream of the firefly luciferase gene in pGL3 vector.The vector was named DNMT3B-pGL3-WT.Site-directed mutagenesis of the NF-kB binding site in DNMT3b promoter was performed using GeneTailor Site-Directed Mutagenesis System and named DNMT3B-pGL3-MUT.(3)pcDNA3.1-p65 vector:Eukaryotic expression vectors of the wild type pcDNA3.1-p65 was constructed by Guangzhou Daengene.4.Real-time PCRTotal RNA and small RNA were extracted from cells and tissues.The RNA was reversely transcribed into cDNA.Quantitative real-time PCR(qPCR)was performed using SYBR Green PCR master mix.All samples were normalized to internal controls and fold changes were calculated through relative quantification(2-△△Ct).5.Western blotTotal protein was isolated and quantitated using BCA assay.The protein lysates were separated by 10%SDS-PAGE,and electrophoretically transferred to PVDF membrane.Then,the membrane was incubated with antibodies and detected by chemiluminescence.The intensity of protein fragments was quantified with the Quantity One software.6.Luciferase reporter assayC666 cells(1 × 104)were cultured in 24-well plates and co-transfected with 20 ng LMP1 vector or NC,5 ng of pRL-CMV Renilla luciferase reporter and 30 ng of luciferase reporter that contained the wild-type(DNMT3B-pGL3-WT)or mutant(DNMT3B-pGL3-MUT)promoter of DNMT3b.Transfections were performed in duplicate and repeated in three independent experiments.Forty-eight hours after transfection,the luciferase activities were analyzed with a Dual-Luciferase Reporter Assay System(Promega,Madison,WI,USA).7.MTT assayA total of 1×103 cells were seeded in a 96-well plate and then allowed to grow in normal medium for 4 days.For MTT assay,cells were incubated in 20μl of 5mg/ml solution of MTT at 37℃ for 4h and lysed in 150μl dimethyl sulfoxide(DMSO)at room temperature for 10min.The absorbance in each well was measured at 490 nm by a microplate reader.8.Methylation-specific PCRThe methylation status of the PTEN promoter region was determined by MSP using bisulfite-modified DNA.The targets of the promoter regions were one site.DNA was modified by the bisulfite reaction using an EpiTect Bisulfite kit(Qiagen).MSP experiments were performed at least in duplicate.9.Methylation-sensitive high resolution melting(MS-HRM)MS-HRM protocol was adapted from the method developed by Wojdacz et al.MS-HRM measures the fluorescence signal of an intercalating dye in double-stranded DNA.HRM analysis were carried out on LightScanner System(Idaho Technology Inc.).The reaction volume was 20 μl and contained 10μl mix(Takara Code:DRR420A),DNA binding dye LC Green Plus,forward/reverse primers and 5 ng bisulfite modified DNA.The melting curves were normalized and calculated using the software provided with the LightScanner System.10.Chromatin Immunoprecipitation(ChIP)AssaysChIP analysis was run on three 10 cm plates per treatment of C666 cells using the EZ-ChIPTM Chromatin Immunoprecipitation Kit(Millipore,Catalog#17-371)according to the manufacturer’s protocol.A ChIP grade NF-κB p65 antibody was used and purchased from Abeam,Inc.(Cambridge,MA,catalog no.ab2851).The IgG negative control antibody and the forward and reverse negative control GAPDH primers used were from the EZ-ChIPTM Chromatin Immunoprecipitation Kit.11.Statistical analysisStatistical analyses were performed with the SPSS 13.0 statistical software package(SPSS Inc.Chicago,IL,USA).The data are expressed as the mean ± s.e.m.from at least three independent experiments.Comparisons between two groups were performed using Student’s t-test,unless otherwise indicated.The association between LMP1,LMP2A and DNMT3b gene was analyzed using Spearman’s correlation coefficient.All statistical tests were two-sided,and p<0.05 was considered to be statistically significant.Results1.Aberrant CpG island methylation of PTEN is an early event in nasopharyngeal carcinoma and a potential diagnostic biomarkerWe first examined the expression level of PTEN in 45 NPC specimens and 22 non-tumor nasopharyngeal epithelial(NP)tissues.The average expression level of PTEN was significantly lower in NPC specimens compared with non-tumor NP tissues(p<0.0001).A panel of human NPC cell lines was also analyzed for the expression level of PTEN.Similarly,the expression level of PTEN was observed to be decreased in all 5 NPC cell lines compared with the non-tumor NP tissues.These data supported that PTEN was downregulated in NPC.In order to explore the potential role of CpG island methylation in the transcriptional silencing of the PTEN gene,we investigated the methylation status of PTEN in clinical specimens and NPC cell lines.As the 5’ region of PTEN contained many CpG islands spanning～3 kb,we focused only on its promoter region in the present study.Our MSP analysis showed that CpG islands in the PTEN promoter region were methylated in 80%(40/50)of NPC tissues,whereas the methylated PTEN appeared in only 5.3%(1/19)of the non-tumor NP tissues and in 80%(4/5)of NPC cell lines.The difference in the hypermethylation level between NPC tissues and non-tumor NP tissues was statistically significant(p<0.0001).To validate MSP results,we sequenced M-MSP and U-MSP products amplified from two NPC tissues using either M or U primers of PTEN.The sequencing results showed that all the cytosine residues in the M-MSP product were converted to thymines,except for those in CpG dinucleotides,indicating the presence of methylated cytosines in these CpG dinucleotides.To directly test the effect of promoter methylation on PTEN inactivation,we treated 3 NPC cell lines(HONE-1,CNE1 and 6-10B)with 5-aza-dC for 3 days,and then examined PTEN mRNA expression changes.We observed that PTEN mRNA expression was clearly upregulated in all 3 NPC cell lines after 5-aza-dC treatment.These results suggested that methylation of promoter region plays a regulatory role in silencing the expression of PTEN in NPC cells.2.PTEN CpG island hypermethylation in NPC cell lines and NPC specimens with EBV infectionWe examined the expression level of PTEN in EBV-positive and EBV-negative NPC cells.Both PTEN mRNA and protein were significantly down-expressed in EBV-positive NPC cells compared with EBV-negative NPC cells,implying the possible relevance of EBV infection to silenced PTEN expression.MSP assay was initially used to test the methylation status and showed that the CpG island of PTEN promoter was methylated in both EBV-positive and EBV-negative NPC cells.Subsequently,MS-HRM analysis,a high sensitive and reproducible approach for methylation quantitative detection,was conducted to assess the methylation degree at PTEN CpG island in EBV-positive and EBV-negative NPC cells.Notably,the shifted melting curves of MS-HRM products amplified from EBV-positive NPC cells were obviously higher than that of EBV-negative cells.To confirm the reliability of MS-HRM results,we sequenced the PCR products amplified from NPC cells using MS-HRM primer of PTEN.Consistently,the methylation frequency of CpG sites within this region was significantly higher in EBV-positive NPC cells than in EBV-negative NPC cells.Collectively,these results indicate that EBV infection is more closely associated with the degree of methylation at PTEN CpG island.As NPC is consistently associated with EBV infection,we replicated our cellular experiments in clinical NPC tissues and evaluated the relation of PTEN methylation with EBV infection.MSP results showed that the CpG islands in PTEN promoter was methylated in 80%(40/50)of NPC tissues.To evaluate the association of PTEN methylation with EBV infection,we compared the expression levels of EBNA1,LMP2A,and LMP1 between 40 methylated and 10 unmethylated NPC tissue samples.No significant difference of the expression levels of these viral proteins was observed between two NPC tissue gropus.Secondarily,we analyzed the DNA methylation degree of 40 NPC tissues that harbored the methylation of PTEN promoter using MS-HRM analysis.All methylated NPC samples were divided into the high and low methylation degree groups based on MS-HRM results.We compared the expression levels of EBNA1,LMP2A and LMP1 between high degree and low degree group.Interestingly,the average expression levels of LMP1 and LMP2A but not EBNA1 were significantly higher in high degree group than in low degree group.Taken together,these results suggest that LMP1 is clinically relevant to the methylation degree at PTEN CpG island.DNMTs always control the methylation of cellular promoters.To examine whether EBV-mediated DNA methylation is executed by DNMTs,we firstly examined DNMT gene expression in 50 NPC tissues.When comparing the expression levels of DNMT1,DNMT3a,and DNMt3b between 40 methylated and 10 unmethylated groups,we could not observed a significant association of DNMT expression with the DNA methylation at PTEN CpG island.Next,we continued to compare DNMT1,DNMT3a,and DNMt3b expression between high and low methylation degree groups.Notably,the average expression level of DNMt3b but not DNMT1 and DNMT3 a was significantly higher in high degree group,suggesting an important role of DNMt3b in facilitating a higher degree of DNA methylation at PTEN CpG island.3.Activation of DNA methyltransferase 3b by EBV Latent Membrane Protein 1 silences PTEN tumor suppressor gene through NF-κB signaling in nasopharyngeal carcinomaIf PTEN CpG island hypomethylation/hypermethylation status are indeed mediated by LMP1 and LMP2A,it is possible that this protein enhances DNA methyltransferase 3B expression.This would result in the elevation of DNA methyltransferase enzyme activity and hypermethylation of the PTEN CpG island.We correlated DNMT3B with LMP1 and LMP2A expression in the same NPC specimens.Statistical analysis revealed that the expression level of DNMT3b mRNA positive correlated with LMP2A(2-tailed Spearman’s correlation,r = 0.431;p<0.01)and LMP1(2-tailed Spearman’s correlation,r =0.365;p<0.05)mRNA expression in NPC tissues,respectively.LMP1-mediated DNMT3b upregulation were observed in the transient expression system in C666 and HONE1 cells.By quantitative RT-PCR,the mRNA expression level of the DNMT3b gene was shown to be increased 4.05-fold at 12 hours and 4.2-fold at 24 hours,and this level was sustained up to 48 hours after the transfection of LMP1 into C666 cells.The mRNA expression levels of DNMT3B in LMP1 gene-transfected HONE1 cells at the same level as that of C666 cells throughout the time course.LMP1-mediated PTEN CpG island methylation degree upregulation were observed in the transient expression system in C666 and HONE1 cells.The shifted melting curves of M-MSP products amplified from LMP1 gene-transfected NPC cell lines were higher than controls,it indicates that LMP1 play a important role in the hypermethylation of PTEN CpG island.Notably,confocal analysis showed that DNMT3B was highly expressed and accumulated in the nuclei of NPC cells upon exogenic expression of LMP1.To date,there are at least three well-defined signaling pathways,NF-κB,p38/MAPK,and JNK,which are elicited by LMP1 COOH-terminal domain.According to the Encyclopedia of DNA Elements at UCSC,computational prediction for transcription factor-DNA binding sites and the transcription factors spotted on Transcription Factors Arrays used,we have identified the NF-κB pathway,but not the JNK or p38/MAPK signaling pathway,which contributes to LMP1-mediated NPC cells DNMT3B gene activation.To further understand human DNMT3B transcriptional regulation via its cis-regulatory elements,approximately 1 kb of DNA sequence located 5’ of the start codon was scanned using prediction software and a putative NF-κB binding site(-159 to-150 bp)was found.To assess the significance of this binding site in the context of chromatin,C666 cell lines that maintained the DNMT3B promoter luciferase reporter containing either a wild-type or a mutant form of the NF-κB binding site were generated.The results showed that both basal and LMP1 gene-transfected reporter activities were higher in the wild type than in mutant cells,which,as we confirmed by PCR analysis,was not due to an unequal number of integrated reporter copies.To test the possibility that the NF-κB subunits directly regulate DNMT3B transcription by binding to the promoter region,ChIP assays were performed.The p65 subunit constitutively binds to the DNMT3B promoter region in C666 cells,indicated by the amplification of the promoter region that encompasses the κB site from chromatin samples immunoprecipitated with a p65 antibody but not IgG serum.After a 24-h LMP1 gene-transfected,the amount of p65 bound to the DNMT3B promoter region increases compared with basal levels,which is similar to p65 binding to the promoter of NFKBIA,the gene encoding IκBα(positive control).Furthermore,inhibition of LMP1-induced NF-κB activation by pretreatment with PDTC reduces the amount of p65 subunits bound to the DNMT3B promoter region to basal levels.Consistent with the increase in DNMT3B protein levels upon confocal analysis,western blot analysis showed that DNMT3B was highly expressed and accumulated in the nuclei of NPC cells upon exogenic expression of LMP1,however,LMP1-induced resulted in regulation in the levels of DNMT3B was inhibited when the NF-κB inhibitor PDTC was added.Taken together,these findings suggest that NF-κB regulation of DNMT3B occurs at the transcriptional level through direct binding to a functional NF-κB binding site on the DNMT3B promoter.Conclusions1.Aberrant CpG island methylation of PTEN is an early event in nasopharyngeal carcinoma and a potential diagnostic biomarker.2.PTEN CpG island hypermethylation in NPC cell lines and NPC specimens with EBV infection.3.Activation of DNA methyltransferase 3b by EBV Latent Membrane Protein 1 silences PTEN through NF-κB signaling in nasopharyngeal carcinoma.