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Research Article Characterization of the Methylation Patterns in Human Papillomavirus Type 16 Viral DNA in Head and Neck Cancers Il-Seok Park 1,2 , Xiaofei Chang 1 , Myriam Loyo 1 , Gaosong Wu 1 , Alice Chuang 1 , Myoung Sook Kim 1 , Young Kwang Chae 3 , Sofia Lyford-Pike 1 , William H. Westra 4 , John R. Saunders 5 , David Sidransky 1,6 , and Sara Isabel Pai 1,6 Abstract Human papillomavirus (HPV) type 16 can integrate into the host genome, thereby rendering the viral coding genes susceptible to epigenetic modification. Using bisulfite genomic sequencing, we determined the methylation status of all 110 CpG sites within the viral epigenome in advanced stage III/IV HPV-16– associated head and neck cancers. We found that the viral genome was hypomethylated in the majority of head and neck cancers, in particular within the viral regulatory region, long control region (LCR), which controls transcription of the E6 and E7 oncogenes. The hypomethylation status of LCR correlated with detectable levels of E6 and E7 expression, which suggests that the tumors may still be dependent on these viral oncogenes to maintain the malignant phenotype. In addition to the methylation status of LCR, we report other potential factors which may influence intratumoral E6 and E7 expression including viral copy number and integration site. We were able to detect the viral epigenetic alterations in sampled body fluids, such as serum and saliva, which correlated with the changes observed in the primary tumors. Because viral epigenetic changes occur in the setting of viral integration into the human genome, the detection of methylated HPV genes in the serum and/or saliva may have diagnostic potential for early detection strategies of viral integration and assessment of risk for cancer development in high-risk individuals. Our findings also support continued targeting of the E6 and/or E7 antigens through various vaccine strategies against HPV-associated cancers. Cancer Prev Res; 4(2); 207–17. Ó2011 AACR. Introduction Epigenetics describes the regulation of gene expression through heritable changes in DNA methylation and chro- matin structure. DNA methylation can impact the tran- scription of genes by either physically impeding the binding of transcriptional proteins to the gene and/or by changing the chromatin structure to repress transcription. We now know that epigenetics plays an important role in tumorigenesis in mammals (1). DNA methylation occurs in cytosines (5-methylcytosine) that precede guanines in dinucleotide CpG sites. CpGs are asymmetrically distrib- uted into CpG-poor regions and dense regions called "CpG islands," which are located in the promoter regions. These CpG islands are usually unmethylated in normal cells, whereas the sporadic CpG sites in the rest of the genome are generally methylated (2). Methylation of CpG islands in promoter regions is often associated with gene silencing, and aberrant DNA methylation can occur in cancers, lead- ing to the silencing of tumor suppressor genes (1). Several oncogenic viruses can integrate within the host genome and become susceptible to modification by the host epigenetic machinery and, at times, can utilize the machinery to regulate its own viral gene expression. One such virus is the human papillomavirus (HPV). HPV type 16 (HPV-16) is the most common virus type associated with cervical and head and neck cancer and is present in greater than 90% of HPV-associated head and neck squa- mous cell carcinomas (HPV-HNSCC; ref. 3). After viral entry into a cell, episomal HPV-16 DNA can integrate into the host genome with resultant deletion of noncritical and regulatory viral genes. Late genes (L1 and L2) and some early genes (E1 and E2) are commonly deleted, and the viral oncogenes E6 and E7 are often the only open reading frames consistently expressed in cancer cell lines (4) and in primary HPV-associated cancers (5). Authors' Affiliations: 1 Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins Medical, Institutions, Baltimore, Maryland; 2 Department of Otolaryngology-Head and Neck Surgery, The Hallym University College of Medicine, Seoul, Korea; 3 Department of Medicine, Albert Einstein Medical Center, Philadelphia, Pennsylvania; 4 Department of Pathology, The Johns Hopkins Medical Institutions; 5 The Greater Baltimore Medical Center, Milton J. Dance, Jr., Head and Neck Center; and 6 Department of Oncology, The Sidney Kimmel Comprehensive Can- cer Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland Note: Supplementary data for this article are available at Cancer Preven- tion Research Online (http://cancerprevres.aacrjournals.org/). Corresponding Author: Sara I. Pai, Departments of Otolaryngology-Head and Neck Surgery and Oncology, The Johns Hopkins Medical Institutions, 601 N. Caroline Street, JHOC 6th floor, Baltimore, MD 21287. Phone: 410- 502-9825; Fax: 410-955-0035. E-mail: [email protected] doi: 10.1158/1940-6207.CAPR-10-0147 Ó2011 American Association for Cancer Research. Cancer Prevention Research www.aacrjournals.org 207 for Cancer Research. on June 27, 2021. © 2011 American Association cancerpreventionresearch.aacrjournals.org Downloaded from
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  • Research Article

    Characterization of the Methylation Patterns in HumanPapillomavirus Type 16 Viral DNA in Head and Neck Cancers

    Il-Seok Park1,2, Xiaofei Chang1, Myriam Loyo1, Gaosong Wu1, Alice Chuang1, Myoung Sook Kim1,Young Kwang Chae3, Sofia Lyford-Pike1, William H. Westra4, John R. Saunders5,David Sidransky1,6, and Sara Isabel Pai1,6

    AbstractHuman papillomavirus (HPV) type 16 can integrate into the host genome, thereby rendering the viral

    coding genes susceptible to epigenetic modification. Using bisulfite genomic sequencing, we determined

    the methylation status of all 110 CpG sites within the viral epigenome in advanced stage III/IV HPV-16–

    associated head and neck cancers. We found that the viral genome was hypomethylated in the majority of

    head and neck cancers, in particular within the viral regulatory region, long control region (LCR), which

    controls transcription of the E6 and E7 oncogenes. The hypomethylation status of LCR correlated with

    detectable levels of E6 and E7 expression, which suggests that the tumors may still be dependent on these

    viral oncogenes to maintain the malignant phenotype. In addition to the methylation status of LCR, we

    report other potential factors which may influence intratumoral E6 and E7 expression including viral copy

    number and integration site. We were able to detect the viral epigenetic alterations in sampled body fluids,

    such as serum and saliva, which correlated with the changes observed in the primary tumors. Because viral

    epigenetic changes occur in the setting of viral integration into the human genome, the detection of

    methylated HPV genes in the serum and/or saliva may have diagnostic potential for early detection

    strategies of viral integration and assessment of risk for cancer development in high-risk individuals. Our

    findings also support continued targeting of the E6 and/or E7 antigens through various vaccine strategies

    against HPV-associated cancers. Cancer Prev Res; 4(2); 207–17. �2011 AACR.

    Introduction

    Epigenetics describes the regulation of gene expressionthrough heritable changes in DNA methylation and chro-matin structure. DNA methylation can impact the tran-scription of genes by either physically impeding thebinding of transcriptional proteins to the gene and/or bychanging the chromatin structure to repress transcription.We now know that epigenetics plays an important role intumorigenesis in mammals (1). DNA methylation occurs

    in cytosines (5-methylcytosine) that precede guanines indinucleotide CpG sites. CpGs are asymmetrically distrib-uted into CpG-poor regions and dense regions called "CpGislands," which are located in the promoter regions. TheseCpG islands are usually unmethylated in normal cells,whereas the sporadic CpG sites in the rest of the genomeare generally methylated (2). Methylation of CpG islandsin promoter regions is often associated with gene silencing,and aberrant DNA methylation can occur in cancers, lead-ing to the silencing of tumor suppressor genes (1).

    Several oncogenic viruses can integrate within the hostgenome and become susceptible to modification by thehost epigenetic machinery and, at times, can utilize themachinery to regulate its own viral gene expression. Onesuch virus is the human papillomavirus (HPV). HPV type16 (HPV-16) is the most common virus type associatedwith cervical and head and neck cancer and is present ingreater than 90% of HPV-associated head and neck squa-mous cell carcinomas (HPV-HNSCC; ref. 3). After viralentry into a cell, episomal HPV-16 DNA can integrate intothe host genome with resultant deletion of noncritical andregulatory viral genes. Late genes (L1 and L2) and someearly genes (E1 and E2) are commonly deleted, and theviral oncogenes E6 and E7 are often the only open readingframes consistently expressed in cancer cell lines (4) and inprimary HPV-associated cancers (5).

    Authors' Affiliations: 1Department of Otolaryngology-Head and NeckSurgery, The Johns Hopkins Medical, Institutions, Baltimore, Maryland;2Department of Otolaryngology-Head and Neck Surgery, The HallymUniversity College of Medicine, Seoul, Korea; 3Department of Medicine,Albert Einstein Medical Center, Philadelphia, Pennsylvania; 4Departmentof Pathology, The Johns Hopkins Medical Institutions; 5The GreaterBaltimore Medical Center, Milton J. Dance, Jr., Head and Neck Center;and 6Department of Oncology, The Sidney Kimmel Comprehensive Can-cer Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland

    Note: Supplementary data for this article are available at Cancer Preven-tion Research Online (http://cancerprevres.aacrjournals.org/).

    Corresponding Author: Sara I. Pai, Departments of Otolaryngology-Headand Neck Surgery and Oncology, The Johns Hopkins Medical Institutions,601 N. Caroline Street, JHOC 6th floor, Baltimore, MD 21287. Phone: 410-502-9825; Fax: 410-955-0035. E-mail: [email protected]

    doi: 10.1158/1940-6207.CAPR-10-0147

    �2011 American Association for Cancer Research.

    CancerPreventionResearch

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  • To evaluate the role of epigenetic alterations in HPV-induced carcinogenesis, we mapped all potential DNAmethylation sites in the HPV-16 epigenome in patientswith head and neck cancer. Because the long control region(LCR) is a key regulatory site for viral gene expression, wefocused our analysis in this region and evaluated themethylation status of key inhibitory and activating tran-scriptional sites. Furthermore, we correlated the E6 and E7expression levels to the methylation status of LCR, and alsoevaluated other potential factors which may influence E6and E7 expression such as viral copy number and integra-tion status. We explored whether epigenetic alterationswithin the viral epigenome could be detected in bodyfluids, such as serum and saliva, which can have diagnosticsignificance for early detection strategies of virus integra-tion as well as assessment of risk for cancer development inhealthy individuals infected with the virus.

    Materials and Methods

    Cell lines, tissues, sera, and salivaCervical cancer cell lines, CaSki (�600 copies of inte-

    grated HPV-16 DNA per genome) and SiHa (2 copies ofintegrated HPV-16 DNA per genome) were purchased fromATCC (American Type Culture Collection) and cultured asper manufacturer’s instructions. Primary tumor tissue,serum, and saliva were collected from patients withHPV-positive, oropharyngeal squamous cell carcinoma(OPSCC) as determined by in situ hybridization with anHPV-16 specific probe. Primary tumor was obtained from22 patients with advanced stage III/IV HPV-associatedOPSCC. Tissue was microdissected to separate tumor fromstromal elements, yielding at least 80% cancer cells. Tissueblocks were stained with hematoxylin and eosin, andtumor areas were subsequently outlined, cut by a headand neck pathologist, and processed for DNA and RNAextraction. RNA was available and extracted from ninepatients. Matched saliva samples were available from 9patients prior to any therapy. Oral rinsing was performedby gargling twice in 20 mL of saline. Matched serumsamples were available from 10 patients prior to anytherapy. This study was approved by the InstitutionalReview Board of the Johns Hopkins University and writteninformed consent was obtained from all patients.

    DNA and RNA extractionTissue samples were centrifuged and digested in a solu-

    tion of SDS and proteinase K at 50�C overnight. GenomicDNA (gDNA) was isolated by phenol/chloroform extrac-tion, and precipitated in ethanol. The DNA pellet wasresuspended in TE buffer (EDTA, 2.5 mmol/L; Tris-HCl,10 mmol/L) and stored at �20�C. Total RNA was extractedfrom frozen tumor tissue using Trizol (Invitrogen). TheRNA integrity was assessed by agarose gel electrophoresis.

    Bisulfite treatmentBisulfite conversion of gDNA was performed using the

    EpiTect Bisulfite Kit (Qiagen) as per manufacturer’s

    recommendations. This bisulfite-modified DNA wassubsequently resuspended in 120 mL of TE buffer andstored at �80�C until use.

    Bisulfite sequencingThe modified DNA was amplified in the form of 18

    amplicons (Supplementary Table 1). Bisulfite-modifiedgDNA was amplified by PCR using 10� buffer [166mmol/L (NH4)2SO4, 670 mmol/L Tris Buffer (pH 8.8),67 mmol/L MgCl2, 0.7% 2-mercaptoethanol, 1% DMSO]and primer sets that were designed to recognize DNA basesafter bisulfite treatment. The conditions for PCR amplifica-tions were as follows: a 5-minute incubation at 95�C wasfollowed by 45 cycles of 1 minute at 95�C, 1 minute at54�C, and 2minutes at 72�C. A 7-minute elongation step at72�C completed the PCR amplification. For amplificationof some amplicons, touchdown PCR was performed asfollows: a 5-minute 95�C incubation step was followed by2 cycles of 1 minute at 95�C for denaturation, 1 minute at66�C for annealing, and 1 minute at 72�C for elongation.The annealing temperature was decreased by 2�C, and twoPCR cycles were run each time until the annealing tem-perature was 56�C. The PCR was run for 35 cycles, with anadditional 7 minutes at 72�C at the completion of thecycles for further elongation. PCR products were gelextracted (Qiagen) and sequenced with forward and reverseprimers using the ABI BigDye cycle sequencing kit (AppliedBiosystems).

    Methylation-specific PCRBisulfite-treated DNA was amplified with methylation-

    and unmethylation-specific (UnMSP) primer sets for eachindividual gene. Primer sequences are shown in Supple-mentary Table 2. PCR reactions were performed for 35cycles of 95�C for 30 seconds, 58�C for 30 seconds, and72�C for 1 minute.

    Quantitative PCRTo quantify the viral load of HPV-16, gDNA from

    patients was used for real-time PCR with primers andprobes sets specific for the E6 and E7 region of HPV-16DNA. PCR for b-actin was performed in parallel to normal-ize the input DNA. gDNA from the CaSki cell line was usedto develop standard curves for the HPV-16 viral load, as ithas been previously characterized to harbor 600 copies ofHPV-16 DNA per genome equivalent. gDNA from CaSkicells was serially diluted to the following concentrations:50, 5, 0.5, 0.05, and 0.005 ng. A standard curve wasdeveloped for b-actin which has 2 copies per genomeequivalent, using the same serial dilutions. All sampleswere run in triplicate. Taqman Fast Universal PCR MasterMix was used according to the manufacturer’s instructions(Applied Biosystems). Primer and probe sequences areshown in Supplementary Table 3.

    To evaluate the viral integration status, we used a pre-viously described real-time quantitative PCR assay (6). E2and E6 primers and probes were synthesized usingpublished sequences (6). The final primer and probe

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  • concentrations were 0.3 and 0.1 mmol/L, respectively, in atotal volume of 10 mL. A standard curve was obtained byamplification of a 10-fold dilution series of 0.3 to0.0003 ng of a HPV-16 plasmid clone (with a ratio ofE2:E6 ¼ 1:1). At least 3 water controls were included ineach run. All experiments were performed twice in dupli-cate with similar ratios. The integration status of HPV-16DNA was assessed by comparing the levels of detectedHPV-16 E2 and E6 genes and was expressed as an E2:E6ratio. Ratios of E2:E6 of less than 1 indicate the presence ofboth integrated and episomal forms. The ratio of E2:E6represents the amount of the episomal form in relation tothe integrated form.

    Quantitative RT-PCRTo examine the mRNA expression of E6 and E7, cDNA

    was synthesized from 1 mg of total RNA which was isolatedfrom patients’ samples, treated with DNase I, and subse-quently cleaned as recommended by the manufacturer(Qiagen). For cDNA preparation, 1mg of total RNA wastranscribed with random hexamers and oligodT using theSuperscript II reverse transcriptase (Invitrogen). The sameamount of RNA was processed without reverse transcrip-tase (RT) in the cDNA synthesis to assure that no gDNAwasamplified in the reaction. cDNA from the CaSki cell linewas used to develop standard curves for the E6 and E7transcripts. b-Actin was used as a loading control. Allsamples were run in triplicate. To examine the expressionlevels of E6 and E7, the mRNA levels of E6 and E7 wereseparately divided by the viral load within the tumor andmultiplied by 10,000.

    StatisticsPearson’s product–moment correlation coefficients and

    Spearman’s rank correlation coefficients were used to assessthe linearity and the rank association between methylationand expression levels. P value of 0.05 was used to assess thesignificance of the association.

    Results

    The HPV-16 epigenome is hypomethylated inadvanced stage HPV-related head and neck cancers

    The genome and epigenome map of HPV-16 DNA isshown in Figure 1A and B. We identified 110 CpG siteswithin the HPV-16 viral methylome. We evaluated themethylation status of all 110 sites in two establishedinvasive cervical cancer cell lines (CaSki and SiHa) andin 22 patients with advanced stage III/IV HPV-16–asso-ciated OPSCC by performing bisulfite-sequencinganalysis.

    We found that the CaSki cell line harbored dense CpGmethylation throughout the entire HPV-16 epigenome.Specifically, 94% of the genome was methylated and 5%of the genome was unmethylated in the E1 and E2 regions.In contrast, SiHa was methylated in 35% of the viralepigenome and 62% of the genome was unmethylated(Fig. 2).

    We evaluated the methylation status of the HPV-16 viralepigenome in 22 primary advanced stage head and neckcancers which consisted of one patient with stage III diseaseand 21 patients with stage IV disease. Interestingly, wefound that the methylation pattern in primary head andneck cancers was comparable with SiHA. Specifically, 12 ofthe 22 (54.5%) advanced stage OPSCC were unmethylatedin greater than 50% of the viral DNA CpG sites with areaslacking methylation clustered in the E2, LCR, and E6regions. Five of the 22 (22.7%) OPSCC were unmethylatedin less than 10% of the HPV-16 epigenome with areaslacking methylation clustered within LCR (Fig. 2).

    The LCR region is preferentially hypomethylated inadvanced stage head and neck cancers

    The LCR contains the origin of replication (ori), the E6enhancer as well as the E2-binding site (E2BS; refs. 6–8).Because this region is critical in transcriptional regulationof the viral genome, we were interested in further evaluat-ing the methylation status within this region. In the HPV

    Figure 1. A topography of theHPV-16 genome structure. A, theHPV-16 genome is a circular,double-stranded DNA, which is8 kb in length. It consists of anLCR, 6 early genes (E1, E2, E4–E7)encoding early proteins and 2 lategenes encoding L1 and L2. The E6and E7 oncoproteins are essentialfor HPV-mediated cellulartransformation. Enh, enhancer;Pro, promoter. B, a map of theCpG dinuleotides in the HPV-16genome. Each vertical barrepresents a CpG site. The thickbars indicate borders betweeneach gene region.

    E2 E5 L2 L1 LCR E1E7E6

    Replication & transcription

    Minor capsidprotein

    Major capsid protein

    Regulatory noncodong region

    Replication

    E4 P97 ori

    E6 Enh

    Early Pro

    Late Pro

    1 110

    A

    B

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  • cell lines, LCR was 100% unmethylated in SiHa and 100%methylated in CaSki. In advanced stage head and neckcancers, we found that 21 of 22 (95.5%) of the advancedstage tumors were unmethylated at or greater than 50% ofthe CpG sites within LCR. Interestingly, one tumor (sample3), which was methylated in greater than 90% of the wholeviral genome, was unmethylated in 40% of the CpG siteswithin LCR, suggesting preferential hypomethylationwithin this viral gene regulatory region (Fig. 3A).

    Both the E6 enhancer and the E2BS within LCR regulatetranscription of the viral oncoproteins, E6 and E7. There-fore, we evaluated whether there was preferential methyla-tion in either of these sites within LCR. The SiHa cell linewas 100% unmethylated in both the E6 enhancer and theE2BS and the CaSki cell line was 100% methylated in bothof these sites. We found that 19 of 22 (86%) of theadvanced stage III/IV OPSCC were unmethylated in greaterthan 50% of the CpG sites within the E6 enhancer region

    (Fig. 3B). Furthermore, 74% (14/19) of these tumors were100% unmethylated in this region. For the E2-bindingregion, 16 of 22 (73%) of the tumors were unmethylatedin greater than 50% of the CpG sites and 88% (14/16) ofthese tumors were 100% unmethylated within this region.Both the E6 enhancer and E2-binding region wereunmethylated in greater than 50% of the CpG sites in80% of the advanced stage cancers.

    Nine of 22 (41%) of the OPSCC demonstrated methyla-tion in greater than 50% of the CpG sites at either the E6enhancer region or the E2BS (Fig. 3B and C). Interestingly,the tumors which were heavily methylated in the E2BS(samples 3, 16, 5, 17, 19, and 4) were unmethylated in theE6 enhancer region and those tumors which were heavilymethylated in the E6 enhancer region (samples 2, 7, and 9)were unmethylated in the E2BS. Our results demonstratethat the E6 enhancer region and/or the E2BS are hypo-methylated in the majority of advanced stage OPSCC.

    CS

    E2 E5 L2 L1 LCR E6 E7 E1

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    Figure 2. DNAmethylation patternof the HPV-16 genome. Bisulfite-sequencing analysis of theHPV-16 genome was performedin 2 HPV-16 cell lines and 22HPV-16–associated stage III/IVOPSCC. Each bar represents aCpG site, and the thick black barsindicate the borders of each generegion. A blue line indicates anunmethylated site. A red lineindicates a methylated CpG site.A thin black indicates that site wasunable to be assessed. C, CaSkicell line; S, SiHa cell line.

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  • The methylation status within LCR correlates with E6and E7 expression levelsLCR contains the E6 enhancer and the E2BS both of

    which regulate viral E6 and E7 expression. Therefore, weevaluated whether the methylation status of LCR correlatedwith the expression levels of E6 and E7 in the cell lines aswell as 9 primary tumor samples for which RNA wasavailable. E6 and E7 expression levels were normalizedto b-actin. The CaSki cell line, which was 100%methylatedin LCR, expressed 16 copies of E6 per viral load (Fig. 4A andB). SiHa, which was 100% unmethylated in LCR, expressed361 copies of E6 per viral load. For the primary head andneck tumor samples, the majority of the cancers demon-strated a methylation pattern comparable with SiHa andwere unmethylated in greater than 50% of the CpG sites inLCR. E6 expression levels in the primary samples rangedfrom 7 to 2,220 copies of E6 per viral load. In 78% (7 of 9)

    of these tumors, E6 expression ranged between 51 to 282copies per viral load which is a higher level than CaSki butlower than SiHa (Fig. 4B). One patient (15) expressed2,220 copies of the E6 per viral load and one sample(14) expressed 7 copies of the E6 per viral load. Interest-ingly, both of these patient samples were unmethylated in93% to 100% of the CpG sites in LCR. Because the E2BSalso regulates E7 expression, we performed the same ana-lysis with E7 expression and found similar correlationsbetween methylation status and expression (Fig. 4A andSupplementary Fig. 1).

    Although there was a trend between methylation statusof LCR and E6 and E7 expression, there was no statisticalsignificance using Pearson correlation coefficients orSpearman’s rank correlation coefficients. The lack of sta-tistical significance may be attributed to the low number ofavailable samples (N ¼ 9) which could be evaluated.

    Figure 3. Frequency of CpGmethylation in the HPV-16 DNALCR, E6 enhancer, and E2BS inprimary head and neck cancersamples. Methylation frequenciesof CpG sites in the HPV-16 DNALCR (A), E6 enhancer (B), andE2BS (C) are depicted. Blue barrepresents the percentunmethylation (UnM) and the redbar represents the percentmethylation (M) of CpGs siteswithin each respective region.C, CaSki cell line; S, SiHa cell line.Numbers represent the patientsamples (Pts).

    (Pts) C S 3 14 24 16 2 6 11 7 8 1 5 17 20 18 21 22 10 23 19 4 9 15

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  • However, other factors may also be contributing to E6 andE7 expression and we explored these other potential con-founding factors.

    There is a variable viral load within head and neckcancers

    It has been previously published that the CaSki cell lineharbors between 500 and 600 copies of integrated HPV-16DNA in more than 11 chromosomal sites of the hyperdi-ploid genome. However, the CaSki cell line has only oneactive papillomaviral transcriptional center per cell thatmaps to a low tandem copy integration site at chromosome14. SiHa has 2 integrated copies of HPV-16 DNA at the13q21 locus of the homologous chromosomes and bothviral copies are transcriptionally active. On the basis of thisobservation, we evaluated the viral load in the primarycancers to determine if the viral copy number could influ-ence the expression levels of E6 and E7 in the primarytumors. The CaSki cell line served as our reference forcalculating the viral copy number in our tumors. In ourstudy, we found that CaSki contained 613 copies of HPV-16 DNA per genome and SiHa 6 copies of HPV-16 DNA pergenome. 78% (7/9) of the primary head and neck cancerscontained integrated viral copy numbers which rangedbetween 24 and 283 copies of HPV-16 DNA per genome(Supplementary Fig. 2). Outliers included patient sample15 which harbored 3 copies of HPV-16 DNA and patientsample 14 which harbored 1,866 integrated viral copies.

    A mixture of episomal and integrated forms of thevirus is present in OPSCC

    HPV DNA can exist in multiple forms within the gen-ome, including integrated and episomal forms. Therefore,we assessed the ratio of integrated to episomal forms of theviral DNA to determine if this factor could affect E6 and E7expression, independent of methylation status. Currentassays measuring HPV-16 integration are based on quanti-fication with real-time PCR of HPV-16 E6 relative to E2DNA because the E2 gene is often lost during the viralintegration process (6). Therefore, detection of a greaterquantity of HPV-16 E6 compared with E2 suggests thepresence of integrated HPV-16 DNA.We were able to assessthe viral integration status in 20 of 22 of the OPSCC casesbased on the availability of the tumor samples (Fig. 5). We

    found there were 8 of 20 (40%) tumors which contained anE2:E6 ratio greater than 1.0, suggesting the predominanceof the episomal forms of the virus, within the tumor(Fig. 5). There were 7 of 20 (35%) tumors with an E2:E6less than 0.5, suggesting predominance of the integratedforms of the virus. Despite the lack of episomal forms of thevirus within these tumors, therewas significant hypomethy-lation detected within LCR (Figs. 3 and 5), suggesting thatthere is preferential hypomethylation within this region inintegrated forms of the viral DNA.

    Methylation status within LCR is detected in serumand saliva

    We evaluated whether the HPV-16 DNA methylationstatus of LCR was detectable in the serum and saliva ofOPSCC patients. Therefore, we performed methylation-specific PCR (MSP) with primers designed in LCR ofHPV-16 DNA. Using these primers, we were able to detecta methylated allele in CaSki and an unmethylated allele inSiHa. An unmethylated DNA allele was observed in all ofthe serum and saliva samples from OPSCC patients testedexcept for case 7 in which greater than 50% of the CpG siteswere methylated in this region (Figs. 6 and 3). Within theprimary tumor 3, LCR was methylated in 60% of the CpGsites and both unmethylated and methylated alleles weredetected in the serum and saliva using this assay (Figs. 6and 3). Our results demonstrate that the LCR methylationstatus within the primary tumor is detectable in the salivaand serum of advanced stage HPV-16–associated OPSCCand can serve as a biomarker for viral DNA integration.

    Discussion

    The HPV-16 genome consists of an 8-kb circular, double-stranded DNA, which encodes 6 early expressed regulatorygenes (E1, E2, E4–E7) and 2 late expressed structural genes,L1 and L2. These two sets of viral coding regions areseparated by an LCR that contains the ori, P97, and non-coding cis-elements such as the E2BS, E6 enhancer, andpromoter regions (7–9). Binding of the E2 protein to theE2BS inhibits transcription factors from docking onto LCRand, thus, represses the transcription of the viral onco-genes, E6 and E7 (10, 11). Expression of the E6 and E7proteins are essential to HPV-mediated transformation due

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    Figure 4.Methylation frequency ofHPV-16 LCR and expression ofE6. A, the unmethylationfrequencies of CpG sites in theHPV-16 DNA LCR region isdepicted. B, real-time PCR wasperformed with cDNA from 9OPSCC patients. Relativeexpression of E6 per viral load wascalculated by comparing the ratiosof mRNA expression of E6 withviral load.

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  • to their binding and inhibition of the cellular gatekeepers,p53 and Rb proteins, which can result in dysregulation of avariety of cellular processes, including cell-cycle control(12). The integration of the viral DNA into the chromo-some of mammalian cells, as well as certain genetic orepigenetic alterations of the viral genome, can lead to loss

    of E2-mediated inhibition of E6 and E7 expression, whichis a key event in papillomaviral carcinogenesis.

    Integration of the viral DNA into the host genome,although requisite for carcinogenesis, makes it vulnerableto modification by the host’s methylation machinery. Thisphenomenon is highlighted when cultured primary human

    Figure 6. MSP analysis of theHPV-16 DNA. The methylationstatus of LCR in serum (A) andsaliva (B) was examined by MSPafter PCR with MSP and UnMSPprimers. Bisulfite-treated DNAfrom CaSki and SiHa were used ascontrols. b-Actin was used toconfirm the integrity of thebisulfite-treated DNA. Tumorsample 3 contained bothmethylated and unmethylatedforms of viral DNA which wasdetected in this assay in both theserum and saliva. Tumor sample 7contained greater than 70%methylation within LCR which wasdetected as a single methylatedband in the serum and saliva. C,CaSki cell line; S, SiHa cell line.Numbers represent the patientsamples.

    Saliva

    C S 1 2 3 4 5 6 7 8 9 10

    Serum

    β-Actin

    MSP

    UnMSP

    1 2 3 4 5 6 7 8 11

    A

    B

    β-Actin

    MSP

    UnMSP

    Figure 5. Assessment of viralintegration. The ratio of E2 to E6was measured for 20 HPV-16–associated OPSCC using real-time quantitative PCR. An E2:E6ratio of 0.5 indicates a mixture ofintegrated and episomal forms ofdetected HPV DNA. Valuesgreater than 1 indicatepredominance of the episomalform. Numbers represent thepatient samples.

    2.5

    2

    1.5

    E2

    : E6

    1

    0.5

    01 2 3 4 5 6 8 9 10 11

    Patients

    13 14 15 16 17 18 20 21 22 23

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  • foreskin keratinocytes are transfected with HPV-16 DNA(13, 14). In the preimmortal keratinocytes, the episomalHPV-16 DNA remains in an unmethylated state. However,the immortal descendant cells with integratedHPV-16 DNAacquire a densely methylated viral genome. Several hypoth-eses exist to explain this observation both in vitro and invivo. Some groups suggest that the host cell may actively besilencing the foreign genome as a cellular defense mechan-ism to inhibit the expression of nonself proteins which candisrupt normal cellular function. Other groups have sug-gested that the HPV-16 genome may not be a passivespectator in this process, but might actively participateby recruiting DNA methyltransferases (DNMT) and/orhistone deacetylases (HDAC) via the viral oncoprotein,E7, to strategically regulate viral gene expression duringthe viral life cycle to evade immune recognition by the host(15, 16).

    In human cancers, methylation of oncogenic viral DNAmay occur due to a combination of these factors. DNAmethylation impacts the transcription of genes by eitherphysically impeding the binding of transcriptional proteinsto the gene and/or altering the chromatin structure torepress transcription. We know that alterations in methyla-tion patterns play an important role in tumorigenesis inmammals (1). A hallmark of cancer is a paradoxical aberra-tion of normal DNA methylation patterns, with a globalloss of DNA methylation that coexists with regional hyper-methylation of certain genes. For example, the hyper-methylation of tumor suppressor genes has attractedsignificant attention, and DNA methylation inhibitorsare being tested as potential anticancer agents. However,emerging data also suggest that hypomethylation can play arole in activating pro-oncogenic genes which may berequired for metastasis and invasion. It has been proposedthat hypermethylation and hypomethylation in cancer areindependent processes, which target different cellular pro-grams at different stages of cancer development. Therefore,evaluating the methylation status at specific sites, asopposed to a more global genomic assessment, may yieldmore valuable and predictive information regarding cancerprogression.

    This concept is exemplified in studies performedwith theHPV-16 methylome in cervical lesions. Fernandez andcolleagues evaluated the DNA methylome of the HPV-16virus in a collection of human cervical samples at differentprogressive stages of disease (17). They found that the DNAmethylome evolved from an unmethylated to a highlymethylated state in association with disease progression,from asymptomatic healthy carriers, through chronicallyinfected tissues and premalignant lesions, to the develop-ment of invasive cervical cancer. Thus, the progression tocancer was associated with increasing numbers of methy-lated CpGs and increasing proportions of methylated HPV-16 genomes, which has been supported by other studies(18–20).

    However, other groups have evaluated the methylationstatus of specific regions within the HPV-16 methylome,which has resulted in conflicting data. Badal and colleagues

    evaluated 81 patients with HPV-16–associated cervicallesions and they found that the LCR and E6 genes ofHPV-16 DNA were hypermethylated in 52% of asympto-matic smears, 21.7% of precursor lesions, and 6.1% ofinvasive carcinomas (18). Bisulfite modification andsequencing analysis revealed that in most of the HPV-16genomes of the CaSki cell line and asymptomatic patients,all 11 CpG dinucleotides that overlap with the enhancerand the promoter regions in LCR weremethylated, whereasin the SiHa cells and cervical lesions, the same subset ofCpGs remained unmethylated (18). These results suggestthat neoplastic transformation may be suppressed by CpGmethylation, whereas hypomethylation seems to correlatewith neoplastic progression. In contrast, Kalantari andcolleagues evaluated 115 cervical samples to establishthe methylation patterns of 19 CpG dinucleotides withinLCR and the L1 gene by bisulfite modification and sequen-cing and reported that methylation of most sites washighest in carcinomas and methylation was lowest indysplasia (19).

    The discrepancy between these studies may be attributedto the differing methylation sites evaluated within LCR.LCR has binding sites for both inhibitory and activatingtranscription factors. For example, transcription starts atthe E6 promoter, P97, which is regulated by one bindingsite for Sp1 and two binding sites for the inhibitory HPV-encoded E2 protein (21, 22). The activity of P97 is stimu-lated by an enhancer with binding sites for several cellularfactors including AP1 (23), NF1 (24), and the progesteronereceptor (25). Other factors which have been reported to beable to bind the LCR region are HIV tat-1 (26), YY1 (27),Octa (28), and TEF-2 (28). In addition, two specificallypositioned nucleosomes can form over the enhancer andpromoter regions (29) to repress transcription when theyare modified by HDACs. Depending upon which of thesesites one analyzes, correlations between methylation statusand tumor progression might be directly or inverselyrelated.

    Because of the wide variance in the literature regardingHPV-associated cervical lesions, we were interested in char-acterizing the methylation status of the HPV-16 viral gen-ome in HPV-associated head and neck cancers (HPV-HNSCC). We performed bisulfite sequencing analysis ofall 110 CpG sites within the HPV-16 genome in 22 patientswith advanced stage III/IV HPV-16–associated HNSCC. Incontrast to the cervical lesions,we found that themajority ofthese advanced stage patients had significant hypomethyla-tion of the viral epigenome, especiallywithin LCRwhich is aregulatory region for viral oncogene expression.

    Because of the various regulatory sites within LCR, wefurther investigated this control region by focusing on themethylation status of the inhibitory E2BS as well as theactivating E6 enhancer region. We found that the E6enhancer region was hypomethylated in advanced stagehead and neck cancers, which facilitates binding of tran-scription factors to this region and, thus, allows for over-expression of the viral oncogenes. Interestingly, we foundthat the E2BS was also significantly hypomethylated.

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  • Previous studies reported that methylation of CpG dinu-cleotides within the binding site of HPV-16 E2 protein inLCR can directly inhibit the binding of E2 to the cognateDNA sequences in vitro; therefore, hypomethylation in thisregion would facilitate binding of E2 to the E2BS and,subsequently, inhibit E6 and E7 expression, which is con-trary to what one would expect in advanced stage cancers.However, an explanation for our findings might be foundwhen reviewing the events which occur upon viral integra-tion into the host genome. Specifically, the E2 gene is oftendisrupted or lost upon viral DNA integration. Without theinhibitor influence of the E2 protein, the selective pressureto methylate this site is lost, which is consistent with ourfindings of hypomethylation of this region in the primarytumors (30).We found a trend between themethylation status of LCR,

    E6 enhancer, and E2BS and levels of E6 and E7 expressionwithin the tumors. However, a statistical significance wasnot found which could be attributed to the low number oftumor samples available for the analysis. In addition, otherpotential factors may influence E6 and E7 expression levelsincluding the integrated viral copy number. Experimentaltransformation of keratinocytes with HPV-16 reveals aconsistent tendency toward reducing the number of tran-scriptionally active HPV genomes to 1 or 2 during thepassages and reactivation of the silent viral copies canoccur in the presence of the DNA methylation inhibitor5-azacytidine (14). The silencing of the redundant copiesmay be advantageous as well as critical in clonal selectionduring carcinogenesis. One could speculate that limitingthe number of actively transcribed viral oncogenes canprevent the accumulation of excess genomic instabilityof the host genome that could affect the growth andsurvival of the transformed cell. Our findings suggest thisexact phenomenonmay be occurring in vivo. We found thatthemajority of head and neck tumors harbored between 24and 283 integrated viral copies per genome and expressedbetween 51 and 282 copies of E6 per viral load and 5 and249 copies of E7 per viral load. However, we found onepatient who harbored 1,866 copies of HPV-16 DNA, butexpressed only 7 copies of E6 per viral load and 5 copies ofE7 per viral load. In contrast, a patient who harbored 3copies of HPV-16 DNA expressed 2,220 copies of E6 perviral load and 1,530 copies of E7 per viral load. We canhypothesize that cancers which contain a low number ofviral genomesmust keep them in an unmethylated form forcontinued oncogene transcription and maintenance of thecarcinogenic phenotype; whereas, those tumors with sig-nificant numbers of viral copies may limit the number oftranscribed viral oncogenes to maintain cell survival. Sup-port for this hypothesis is found in a study by Cohen andcolleagues, which evaluated the viral load in 35 HPV-16–associated HNSCC. They found that the patients with thehighest viral loads had an improved overall and disease-free survival (31). This finding is counterintuitive; however,based on our study, we propose that those tumors with thehighest viral loads could have significant silencing of theredundant copies through methylation and correspond-

    ingly low E6 and E7 expression levels, which could accountfor improved survival rates.

    An alternative factor which may influence E6 and E7expression, in addition to viral copy number and viralmethylation status, may be the site of integration of theviral genome, which may be permissive to either high orlow levels of viral gene transcription. Over 200 selectedHPV-16 and HPV-18 integration sites have been reported.These are widely distributed across the genome; however,there seems to be preferential integration near commonfragile sites (CFS), specific chromosomal loci that areparticularly prone to forming double-strand breaks(32–34). It has been reported that CaSki harbors between500 and 600 integrated copies of HPV-16 DNA. CaSki wasfound to be significantly methylated with only one activepapillomaviral transcriptional center per cell, which mapsto a low tandem copy integration site at chromosome 14.SiHa is significantly unmethylated and has 2 integratedcopies of HPV-16 DNA at the 13q21 locus of the homo-logous chromosomes and both viral copies are transcrip-tionally active. Therefore, for patients with low viral copynumbers, such as our patient with 3 viral copies, it mayhave integrated into a site which is permissive for the highlevels of E6 and E7 expression.

    A limitation to our study is that the current methodologydoes not allow the selective detection of the methylationpattern for the transcriptionally active copies of the viralgenome among the inactive ones. Rather, our assay reflectsthe cumulative status of all of the viral genomes, both activeas well as inactive, within the tumor. Despite this limita-tion, our data demonstrate that we are able to detect certainregions within the viral epigenome that are more likely tobe methylated or unmethylated and there is a trend towardhypomethylation of LCR with corresponding detectablelevels of E6 and E7 expression in advanced stage OPSCC.Another limitation to the study is the lack of evaluation ofearly stage disease which would allow us to determine howthe HPV-16 DNA methylation status may evolve withprogression of disease and/or stage. Because HPV-HNSCClocalizes to the tonsil and base of tongue which are areas ofthe head and neck that are more difficult to routinelyevaluate without a directed physical examination, HPV-HNSCC patients are typically diagnosed after lymphaticspread to the cervical nodes, resulting in a diagnosis at anadvanced stage of disease. Thus, we were not able toevaluate the HPV-16 methylation status of early stage Ior II HPV-HNSCC because patients rarely present withthese early stage lesions and tissue was not available foranalysis.

    The implications of our findings are several folds. Wereport that HPV-related head and neck cancers have regu-lated mechanisms of methylation of the viral epigenome.Our findings demonstrate that LCR is preferentially hypo-methylated in the majority of advanced stage HPV-16–associated head and neck cancers. Hypomethylationof LCR corresponds to detectable expression levels ofE6 and E7 within head and neck cancers, supportingthe feasibility of targeting these antigens for novel

    HPV-16 Viral Epigenome in Head and Neck Cancers

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  • immunotherapeutic strategies (35, 36). We demonstratethe feasibility of detecting methylated HPV genes in bodyfluids such as serum and saliva. Because viral epigeneticchanges occurs only in the setting of viral integration intothe human genome, the detection ofmethylatedHPV genesin the serum and/or salivamay help to identify the presenceof viral integration as compared with episomal forms andcan serve as a biomarker for HPV integration and maypotentially allow assessment of risk for cancer developmentin high-risk individuals. Further studies which distinguishpreferential sites of methylation within the HPV epigen-ome may be relevant and complement current assays forHPV integration.

    Disclosure of Potential Conflicts of Interest

    No potential conflicts of interest were disclosed.

    Grant Support

    NIH P50 CA19032 (W.H. Westra, D. Sidransky, S.I. Pai) and The Milton J.Dance, Jr., Head and Neck Center at the Greater Baltimore Medical Center (J.R.Saunders, S.I. Pai).

    The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

    Received June 30, 2010; revised December 9, 2010; accepted December16, 2010; published online February 3, 2011.

    References1. Jones PA, Baylin SB. The fundamental role of epigenetic events in

    cancer. Nat Rev Genet 2002;3:415–28.2. Jones PA, Takai D. The role of DNA methylation in mammalian

    epigenetics. Science 2001;293:1068–70.3. Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L,

    et al. Evidence for a causal association between human papilloma-virus and a subset of head and neck cancers. J Natl Cancer Inst 2000May;92:709–20.

    4. Nasseri M, Gage JR, Lorincz A, Wettstein FO. Human papillomavirustype 16 immortalized cervical keratinocytes contain transcriptsencoding E6, E7, and E2 initiated at the P97 promoter and expresshigh levels of E7. Virology 1991;184:131–40.

    5. Schwarz E, Freese UK, Gissmann L, Mayer W, Roggenbuck B,Stermlau A, et al. Structure and transcription of human papillomavirussequences in cervical carcinoma cells. Nature 1985;314:111–4.

    6. Peitsaro P, Johansson B, Syrjanen S. Integrated human papilloma-virus type 16 is frequently found in cervical cancer precursors asdemonstrated by a novel quantitative real-time PCR technique. J ClinMicrobiol 2002;40:886–91.

    7. Butel JS. Viral carcinogenesis: revelation of molecular mechan-isms and etiology of human disease. Carcinogenesis 2000;21:405–26.

    8. Zur Hausen H. Papillomaviruses and cancer: from basic studies toclinical application. Nat Rev Cancer 2002;2:342–50.

    9. Gatza ML, Chandhasin C, Ducu RI, Marriott SJ. Impact of transform-ing viruses on cellular mutagenesis, genome stability, and cellulartransformation. Environ Mol Mutagen 2005;45:304–25.

    10. Frattini MG, Hurst SD, Lim HB, Swaminathan S, Laimins LA. Abroga-tion of a mitotic checkpoint by E2 protines from oncogenic humanpapillomaviruses correlates with increased turnover of the p53 tumorsuppressor protein. EMBO J 1997;16:318–31.

    11. Howley PM, M€unger K. Human papillomaviruses and squamous cellcarcinomas. InParsonnet J, editor Microbes andMalignancy: Infectionas a Cause of Human Cancers. Oxford, UK: Oxford University Press.1999. p. 157–79.

    12. Wise-Draper TM,Wells SI. Papillomavirus E6 and E7 proteins and theircellular targets. Front Biosci 2008;13:1003–17.

    13. Steenbergen RD, Walboomers JM, Meijer CJ, van der Raaij-HelmerEM, Parker JN, Chow LT, et al. Transition of human papillomavirustype 16 and 18 transfected human foreskin keratinocytes towardsimmorality: activation of telomerase and allele losses at 3p, 10p,11q and/or 18q. Oncogene 1996;13: 1249–57.

    14. Van Tine BA, Kappes JC, Banerjee NS, Knops J, Lai L, SteenbergenRD, et al. Clonal selection for transcriptionally active viral oncogenesduring progression to cancer. J Virol 2004;78:11172–86.

    15. BurgersWA, Blanchon L, Pradhan S, de Launoit Y, Kouzarides T, FuksF. Viral oncoproteins target the DNA methyltransferases. Oncogene2007;26:1650–5.

    16. Zhang B, Laribee RN, Klemsz MJ, Roman A. Human papillomavirustype 16 E7 protein increases acetylation of histone H3 in humanforeskin keratinocytes. Virology 2004;329:189–98.

    17. Fernandez AF, Rosales C, Lopez-Nieva P, Grana O, Ballestar E,Ropero S, et al. Genome Res 2009;19:438–51.

    18. Badal V, Chuang LS, Tan EH, Badal S, Villa LL,Wheeler CM, et al. CpGmethylation of human papillomavirus type 16 DNA in cervical cancercell lines and in clinical specimens: genomic hypomethylation corre-lates with carcinogenic progression. J Virol 2003;77:6227–34.

    19. Kalantari M, Calleja-Macias IE, Tewari D, Hagmar B, Lie K, Barrera-Saldana HA, et al. Conserved methylation patterns of humanpapillomavirus type 16 DNA in asymptomatic infection and cervicalneoplasia. J Virol 2004;78:12762–72.

    20. Brandsma JL, Sun Y, Lizardi PM, Tuck DP, Zelterman D, Haines GK3rd, et al. Distinct human papillomavirus type 16 methylomes incervical cells at different stages of premalignancy. Virology 2009;389:100–7.

    21. Tan SH, Leong LE,Walker PA, Bernard HU. The human papillomavirustype 16 E2 transcription factor binds with low cooperativity to twoflanking sites and represses the E6 promoter through displacement ofSp1 and TFIID. J Virol 1994;68:6411–20.

    22. Garcia-Carrance A, Theirry F, Yaniv M. Interplay of viral and cellularproteins along the long control region of human papillomavirus type18. J Virol 1988;62:4321–30.

    23. Chan WK, Chong T, Bernard HU, Klock G. Transcription of thetransforming genes of the oncogenic human papillomavirus-16 isstimulated by tumor promoters through AP1 bindings sites. NucleicAcids Res 1990;18:763–9.

    24. Apt D, Chong T, Liu Y, Bernard HU. Nuclear factor I and epithelial cell-specific transcription of human papillomavirus type 16. J Virol1993;67: 4455–63.

    25. Chong T, Chan WK, Bernard HU. Transcriptional activation of humanpapillomavirus 16 by nuclear factor I, AP1, steroid receptors and apossibly novel transcription factor, PVF: amodel for the composition ofgenital papillomavirus enhancers. Nucleic Acids Res 1990;18:465–70.

    26. Vernon SD, Hart CE, Reeves WC, Icenogle JP. The HIV-1 tat proteinenhances E2-dependent human papillomavirus 16 transcription. VirusRes 1993;27:133–45.

    27. O’Connor MJ, Tan SH, Tan CH, Bernard HU. YY1 represses humanpapillomavirus type 16 transcription by quenching AP-1 activity. JVirol 1996;70:6529–39.

    28. Chong T, Apt D, Gloss B, Isa M, Bernard HU. The enhancer of humanpapillomavirus type 16: binding sites for the ubiquitous transcriptionfactors oct-1, NFA, TEF-2, NF1, and AP-1 participate in epithelial cell-specific transcription. J Virol 1991;65:5933–43.

    30. Stunkel W, Bernard HU. The chromatin structure of the long controlregion of human papillomavirus type 16 represses viral oncoproteinexpression. J Virol 1999;73:1918–30.

    29. Thain A, Jenkins O, Clarke AR, Gaston K. CpG methylation directlyinhibits binding of the human papillomavirus type 16 E2 protein tospecific DNA sequences. J Virol 1996;70:7233–5.

    31. Cohen MA, Basha SR, Reichenbach DK, Robertson E, Sewell DA.Increased viral load correlates with improved survival in HPV-16-asso-ciated tonsil carcinoma patients. Act Otolaryngol 2008;128: 583–9.

    Park et al.

    Cancer Prev Res; 4(2) February 2011 Cancer Prevention Research216

    for Cancer Research. on June 27, 2021. © 2011 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

    http://cancerpreventionresearch.aacrjournals.org/

  • 32. Dall KL, Scarpini CG, Roberts I, Winder DM, Stanley MA, MuralidharB, et al. Characterization of naturally occurring HPV16 integrationsites isolated from cervical keratinocytes under noncompetitiveconditions. Cancer Res 2008;68:8249–59.

    33. Durkin SG, Glover TW. Chromosome fragile sites. Annu Rev Genet2007;41:169–92.

    34. Thorland EC, Myers SL, Persing DH, Sarkar G, McGovern RM, Gost-out BS, et al. Human papillomavirus type 16 integrations in cervical

    tumors frequently occur in common fragile sites. Cancer Res2000;60:5916–21.

    35. Best SR, Peng S, Juang CM, Hung CF, Hannaman D, Saunders JR,et al. Administration of HPV DNA vaccine via electroporatino elicits thestrongest CD8þ T cell immune response compared to intramuscularinjection and intradermal gene gun delivery. Vaccine 2009;27:5450–9.

    36. Wu AA, Niparko KJ, Pai SI. Immunotherapy for head and neck cancer.J Biomed Sci 2008;15:275–89.

    HPV-16 Viral Epigenome in Head and Neck Cancers

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  • 2011;4:207-217. Cancer Prev Res Il-Seok Park, Xiaofei Chang, Myriam Loyo, et al. Papillomavirus Type 16 Viral DNA in Head and Neck CancersCharacterization of the Methylation Patterns in Human

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