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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.
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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
HPV-16 Viral Epigenome in Head and Neck Cancers
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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
3
Sta
ge
III/IV
Pat
ien
ts
17
7
5
9
15
16
24
2
1
20
419
8
21
18
22
10
23
11
6
14
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
M UnM
E2-binding region
(%)
E6 enhancer
0
50
100
(%)
LCR
0
50
100
(%)
(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
0
50
100
(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
A
B
C
HPV-16 Viral Epigenome in Head and Neck Cancers
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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
0
20
40
60
80
1002,3002,2002,1002,000
400300200100
0(Pts) C S 14 2 3 9 4 6 7 1 15
Un
met
hyl
atio
n (
%)
A B
E6
exp
/vir
al lo
ad
(Pts) C S 14 2 3 9 4 6 7 1 15
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
HPV-16 Viral Epigenome in Head and Neck Cancers
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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.
Park et al.
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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
www.aacrjournals.org Cancer Prev Res; 4(2) February 2011 217
for Cancer Research. on June 27, 2021. © 2011 American
Associationcancerpreventionresearch.aacrjournals.org Downloaded
from
http://cancerpreventionresearch.aacrjournals.org/
-
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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