होम Life Sciences Genome-wide analysis of DNA methylation in UVB- and DMBA/TPA-induced mouse skin cancer models

Genome-wide analysis of DNA methylation in UVB- and DMBA/TPA-induced mouse skin cancer models

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10.1016/j.lfs.2014.07.031
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September, 2014
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Life Sciences 113 (2014) 45–54

Contents lists available at ScienceDirect

Life Sciences
journal homepage: www.elsevier.com/locate/lifescie

Genome-wide analysis of DNA methylation in UVB- and
DMBA/TPA-induced mouse skin cancer models
Anne Yuqing Yang a,b,c,1, Jong Hun Lee a,b,h,1, Limin Shu a,b, Chengyue Zhang a,b,c, Zheng-Yuan Su a,b,
Yaoping Lu a,d, Mou-Tuan Huang a,d, Christina Ramirez b,e, Douglas Pung b, Ying Huang a,b,c, Michael Verzi f,
Ronald P. Hart g, Ah-Ng Tony Kong a,b,d,⁎
a

Center for Cancer Prevention Research, Ernest Mario School of Pharmacy, Piscataway, NJ 08854, USA
Department of Pharmaceutics, Ernest Mario School of Pharmacy, Piscataway, NJ 08854, USA
c
Graduate Program in Pharmaceutical Sciences, Ernest Mario School of Pharmacy, Piscataway, NJ 08854, USA
d
Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Piscataway, NJ 08854, USA
e
Graduate Program in Cellular and Molecular Pharmacology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
f
Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
g
Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
h
Department of Food Science and Biotechnology, CHA university, Kyunggi, Korea
b

a r t i c l e

i n f o

Article history:
Received 6 May 2014
Accepted 21 July 2014
Available online 2 August 2014
Keywords:
DNA methylation
Epigenetics
MeDIP-Seq
UVB
DMBA/TPA

a b s t r a c t
Aims: Ultraviolet irradiation and carcinogens have been reported to induce epigenetic alterations, which potentially contribute to the development of skin cancer. We aimed to study the genome-wide DNA methylation profiles of skin cancers induced by ultraviolet B (UVB) irradiation and 7,12-dimethylbenz(a)anthracene (DMBA)/12O-tetradecanoylphorbol-1,3-acetate (TPA).
Main methods: Methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing was utilized to ascertain the;  DNA methylation profiles in the following common mouse skin cancer models: SKH-1 mice
treated with UVB irradiation and CD-1 mice treated with DMBA/TPA. Ingenuity® Pathway Analysis (IPA) software
was utilized to analyze the data and to identify gene interactions among the different pathways.
Key findings: 6003 genes in the UVB group and 5424 genes in the DMBA/TPA group exhibited a greater than 2-fold
change in CpG methylation as mapped by the IPA software. The top canonical pathways identified by IPA after the
two treatments were ranked were pathways related to cancer development, cAMP-mediated signaling, G proteincoupled receptor signaling and PTEN signaling associated with UVB treatment, whereas protein kinase A signaling
and xenobiotic metabolism signaling were associated with DMBA/TPA treatment. In addition, the mapped IL-6related inflammatory pathways displayed alterations in the methylation profiles of inflammation-related genes
linked to UVB treatment.
Significance: Genes with altered methylation were ranked in the UVB and DMBA/TPA models, and the molecular
interaction networks of those genes were identified by the IPA software. The genome-wide DNA methylation profiles of skin cancers induced by UV irradiation or by DMBA/TPA will be useful for future studies on epigenetic gene
regulation in skin carcinogenesis.
© 2014 Elsevier Inc. All rights reserved.

Introduction
Accumulating evidence suggests that epigenetic DNA alterations
play a crucial role in cancer initiation and development. Specifically,

Abbreviations: MeDIP, methylated DNA immunoprecipitation; UV, ultraviolet; UVA,
ultraviolet A; UVB, ultraviolet B; DMBA, 7,12-dimethylbenz(a)anthracene; TPA, 12-Otetradecanoylphorbol-1,3-acetate; IPA, Ingenuity® Pathway Analysis; IL, interleukin.
⁎ Corresponding author at: Rutgers, The State University of New Jersey, Ernest Mario
School of Pharmacy, Room 228, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA.
Tel.: +1 848 445 6369/8; fax: +1 732 445 3134.
E-mail address: kongt@pharmacy.rutgers.edu (A.-N.T. Kong).
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.lfs.2014.07.031
0024-3205/© 2014 Elsevier Inc. All rights reserved.

aberrant DNA methylation at the 5′-position of cytosine in CG dinucleotides in the cancer genome is postulated to be the most relevant epigenetic change in cancer (Esteller, 2008). DNA methylation affects gene
expression and therefore regulates a wide range of biological processes,
including proliferation, cell death, mutation and cancer initiation/promotion/progression (Momparler and Bovenzi, 2000; Zingg and Jones,
1997). Both global hypomethylation and regional hypermethylation
are characteristics of tumorigenesis (Baylin et al., 2000; Laird and
Jaenisch, 1996). DNA hypermethylation at promoter regions is a predominant epigenetic mechanism for reducing the expression of tumor
suppressor genes. Epigenetic silencing of tumor suppressor genes has
been reported in mouse models of malignant tumors (Chen et al.,
2003; Tommasi et al., 2005). Thus, the present study aimed to profile

46

A.Y. Yang et al. / Life Sciences 113 (2014) 45–54

the global DNA methylation changes that occur during carcinogenesis.
To address this aim, we conducted global profiling of DNA methylation
changes in two representative skin carcinogenesis models, ultraviolet B
(UVB)-exposed SKH-1 hairless mice and DMBA/TPA-induced carcinogenesis in CD-1 mice.
Ultraviolet irradiation has been reported to induce epigenetic alterations, which may contribute to the development of skin cancer
(Nandakumar et al., 2011). However, the precise mechanism by which
UV irradiation is related to carcinogenesis remains unclear. In addition,
a method for screening epigenetically modified genes in melanoma patients needs to be established (Koga et al., 2009; Kanavy and
Gerstenblith, 2011). Mutations in oncogenes and tumor suppressor
genes have been reported in melanoma. However, most of these mutations are not present in non-melanoma skin cancer (Hocker and Tsao,
2007), suggesting a specific role for UV irradiation in carcinogenesis.
In skin tumors, UV irradiation has been reported to induce epigenetic
modifications and may contribute to the development of skin cancer.
Epigenetic changes, such as DNA methylation and histone modification,
may play a crucial role in the initiation and development of certain types
of cancer (Ballestar and Esteller, 2008). Epigenetic alterations generally
represent the interface between the environment and the genome
(Jaenisch and Bird, 2003).
The multistage skin carcinogenesis model is established by applying
a sub-threshold dose of the carcinogen DMBA followed by repetitive
treatments with the tumor promoter TPA. With three well-defined
stages—initiation, promotion, and progression—this model is similar to
natural human tumor development. This model has been widely used
to investigate the anti-tumor efficacy of chemicals and the molecular
events that occur during each stage of tumor development. Several genetic alterations identified in human skin cancer patients have also been
described in the mouse multistage skin carcinogenesis model, such as
changes in cyclin D1 (Wilkey et al., 2009), TP53 (Saab et al., 2009),
CDKN21 (Nishijo et al., 2009) and PTEN (Zheng et al., 2008). The underlying similarity in the biology of cancer between mice and humans implies that genes related to mouse tumor development may also be
relevant to human tumor development.
In the present study, we used methylated DNA immunoprecipitation
(MeDIP) coupled with next-generation sequencing to profile the whole
genome DNA methylation patterns from the UVB and DMBA/TPA
models. The MeDIP-Seq results were evaluated by Ingenuity® Pathway
Analysis (IPA) to investigate genetic crosstalk and signal/function overlap. The present study included an initial assessment of genes with a
modified methylation profile and the identification of interacting molecular networks in skin carcinogenesis models.
Materials and methods

Newburyport, MA). The mice were assessed twice weekly for the appearance of papillomas and carcinomas. Skin papilloma and carcinoma
samples were collected, frozen in liquid nitrogen and stored at
−80 °C. The epidermises of age-matched untreated mice were isolated
from fresh skin as a control group.
Six-week-old female CD-1 mice were used for the DMBA/TPAinduced multistage carcinogenesis model. One day before treatment,
the backs of the mice were shaved. For tumor initiation, 200 nmol
DMBA in 200 μL of acetone was applied on the back skin of the mice.
Three days after DMBA treatment, 5 nmol TPA in 200 μL of acetone
was applied three times a week for 11 weeks to induce tumor promotion and progression.
Global analysis of methylated DNA
Genomic DNA (gDNA) was extracted from UV irradiation-induced
tumor samples from 3 female mice and from non-irradiated epidermis
samples from 3 female age-matched SKH-1 mice; from female CD-1
mice in the DMBA/TPA-induced carcinogenesis model for the MeDIPSeq assay. gDNA was extracted using a DNeasy Kit (Qiagen, Valencia,
CA) according to the manufacturer's protocol. The gDNA was electrophoresed on an agarose gel, and the OD ratios were determined to confirm
the purity and concentration of the gDNA prior to fragmentation by
Covaris (Covaris, Inc., Woburn, MA USA). Fragmented gDNA was evaluated for size distribution and concentration using an Agilent Bioanalyzer
2100 and a NanoDrop spectrophotometer.
MeDIP-Seq
MeDIP was performed to analyze genome-wide methylation. MeDIP
was performed using a MagMeDIP Kit from Diagenode according to the
manufacturer's instructions. Methylated DNA was recovered by immunoprecipitation with antibodies specific to methylated cytosine used to separate methylated DNA fragments from unmethylated fragments, and
Illumina libraries were created from the captured gDNA using NEBNext
reagents (catalog# E6040; New England Biolabs, Ipswich, MA, USA).
Enriched libraries were evaluated for size distribution and concentration
using an Agilent Bioanalyzer 2100. The samples were then sequenced on
an Illumina HiSeq2000 machine, which generated paired-end reads of 90
or 100 nucleotides (nt). The results were analyzed for data quality and
exome coverage using the platform provided by DNAnexus (DNAnexus,
Inc., Mountain View, CA, USA). Samples were sent to Otogenetics Corporation (Norcross, GA) for Illumina sequencing and alignment with the genome. The resulting BAM files were downloaded for analysis.
MeDIP alignments were compared with control samples using
Cuffdiff 2.0.2 with no length correction (Trapnell et al., 2012). Briefly,

Chemicals and antibodies

Mice and skin cancer induction
Two representative skin carcinogenesis models were utilized in the
present study. SKH-1 hairless female mice, 7–8 weeks old, were treated
with UVB (30 mJ/cm2) twice a week for 36 weeks. The ultraviolet (UV)
lamps (FS72T12-UVB-HO; National Biological, Twinsburg, OH) emitted
UVB (280–320 nm; 70–80% of the total energy) and ultraviolet A
(UVA) (320–375 nm; 20–75% of the total energy). The dose of UVB
was quantified using a UVB Spectra 305 dosimeter (The Daavlin Company, Bryan, OH). The radiation was further calibrated using a research radiometer/photometer (model IL-1700; International Light Inc.,

4500
4000

Number of probe sets
(Treatment/control)

The chemicals used in the current study were as follows: 7,12dimethylbenz(a)anthracene (DMBA; Sigma-Aldrich, MO, USA) and 12O-tetradecanoylphorbol-1,3-acetate (TPA; Alexis Co., CA, USA). The 5methylcytidine monoclonal antibody was purchased from Eurogentec.,
Belgium.

3500

4140 3781

UVB
DMBA-TPA

3000
2500
2000
1500

1863
1643

1000
500
0

Increased
Decreased
Log2 Fold‐Change

Fig. 1. Total number of significantly increased and decreased genes based on changes in
methylation (≥2-fold change in Log2) in the UVB and DMBA/TPA groups.

A.Y. Yang et al. / Life Sciences 113 (2014) 45–54

47

Table 1
Top 50 annotated genes with increased methylation ranked by log2 fold change. (A) UVB group. (B) DMBA/TPA group.
(A)
Rank

Symbol

Gene name

Log2 fold change
(UVB/control)

Location

Type(s)

1
2
3
4
5
6

RBFOX1
IMPG2
DGKK
MAD1L1
EVX2
PAN3

5.457
5.367
5.31
5.252
5.19
4.989

Cytoplasm
Extracellular space
Cytoplasm
Nucleus
Nucleus
Cytoplasm

Other
Other
Kinase
Other
Transcription regulator
Other

7
8
9
10
11
12
13
14
15
16

AAMP
ARHGAP18
ACAA2
OLFM1
TGS1
DYM
Hspg2
Kcnip2
TNS1
AGAP1

4.837
4.837
4.574
4.574
4.574
4.525
4.474
4.474
4.474
4.367

Plasma membrane
Cytoplasm
Cytoplasm
Cytoplasm
Nucleus
Cytoplasm
Extracellular space
Plasma membrane
Plasma membrane
Cytoplasm

Other
Other
Enzyme
Other
Enzyme
Other
Other
other
Other
Enzyme

17
18
19
20

CCDC180
EDN1
FOXE1
KCNN4

4.367
4.367
4.367
4.367

Other
Extracellular space
Nucleus
Plasma membrane

Other
Cytokine
Transcription regulator
Ion channel

21
22
23
24
25
26
27
28

LOXHD1
DCP2
DET1
DSC3
HOXD11
MAML2
MYO1E
PCBD1

4.367
4.252
4.252
4.252
4.252
4.252
4.252
4.252

Extracellular space
Nucleus
Nucleus
Plasma membrane
Nucleus
Nucleus
Cytoplasm
Nucleus

Other
Enzyme
Other
Other
Transcription regulator
Transcription regulator
Enzyme
Transcription regulator

29

WNT3

4.252

Extracellular space

Other

30
31
32
33

CENPF
Dos
FBXO11
GPR37

4.126
4.126
4.126
4.126

Nucleus
Other
Cytoplasm
Plasma membrane

Other
Other
Enzyme
G-protein coupled receptor

34
35
36
37
38
39
40
41
42
43
44

HPCA
LIMS2
LRRC8B
LTA4H
MEMO1
mir-221
mir-802
Olfr1323
PLN
PTPN23
SLC7A9

4.126
4.126
4.126
4.126
4.126
4.126
4.126
4.126
4.126
4.126
4.126

Cytoplasm
Cytoplasm
Other
Cytoplasm
Cytoplasm
Cytoplasm
Cytoplasm
Plasma membrane
Cytoplasm
Cytoplasm
Plasma membrane

Other
Other
Other
Enzyme
Other
MicroRNA
MicroRNA
G-protein coupled receptor
Transporter
Phosphatase
Transporter

45
46
47
48
49

VRK1
ZBTB34
ZNF622
SMYD2
DYSF

4.126
4.126
4.126
4.082
3.989

Nucleus
Nucleus
Nucleus
Cytoplasm
Plasma membrane

Kinase
Other
Other
Enzyme
Other

50

Ear2 (includes others)

RNA binding protein, fox-1 homolog (C. elegans) 1
Interphotoreceptor matrix proteoglycan 2
Diacylglycerol kinase, kappa
MAD1 mitotic arrest deficient-like 1 (yeast)
Even-skipped homeobox 2
PAN3 poly(A) specific ribonuclease subunit
homolog (S. cerevisiae)
Angio-associated, migratory cell protein
Rho GTPase activating protein 18
Acetyl-CoA acyltransferase 2
Olfactomedin 1
Trimethylguanosine synthase 1
Dymeclin
Heparan sulfate proteoglycan 2
Kv channel-interacting protein 2
Tensin 1
ArfGAP with GTPase domain, ankyrin repeat and PH
domain 1
Coiled–coil domain containing 180
Endothelin 1
Forkhead box E1 (thyroid transcription factor 2)
Potassium intermediate/small conductance
calcium-activated channel, subfamily N, member 4
Lipoxygenase homology domains 1
Decapping mRNA 2
De-etiolated homolog 1 (Arabidopsis)
Desmocollin 3
Homeobox D11
Mastermind-like 2 (Drosophila)
Myosin IE
Pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha
Wingless-type MMTV integration site family,
member 3
Centromere protein F, 350/400 kDa
Downstream of Stk11
F-box protein 11
G protein-coupled receptor 37 (endothelin receptor
type B-like)
Hippocalcin
LIM and senescent cell antigen-like domains 2
Leucine rich repeat containing 8 family, member B
Leukotriene A4 hydrolase
Mediator of cell motility 1
MicroRNA 221
MicroRNA 802
Olfactory receptor 1323
Phospholamban
Protein tyrosine phosphatase, non-receptor type 23
Solute carrier family 7 (amino acid transporter light
chain, bo, +system), member 9
Vaccinia related kinase 1
Zinc finger and BTB domain containing 34
Zinc finger protein 622
SET and MYND domain containing 2
Dysferlin, limb girdle muscular dystrophy 2B
(autosomal recessive)
Eosinophil-associated, ribonuclease A family,
member 2

3.989

Cytoplasm

Enzyme

(B)
Rank

Symbol

Gene name

Log2 fold change
(DMBA-TPA/control)

Location

Type(s)

1

FAM135A

5.974

Other

Enzyme

2
3

CADM2
VWC2L

5.231
4.877

Plasma membrane
Extracellular space

Other
Other

4
5
6

PTH2R
NPY
TNS1

Family with sequence similarity
135, member A
Cell adhesion molecule 2
von Willebrand factor C domain containing protein
2-like
Parathyroid hormone 2 receptor
Neuropeptide Y
Tensin 1

4.708
4.515
4.408

Plasma membrane
Extracellular space
Plasma membrane

G-protein coupled receptor
Other
Other
(continued
on on
next
page)
(continued
next
page)

48

A.Y. Yang et al. / Life Sciences 113 (2014) 45–54

Table 1 (continued)
(A)
(B)
Rank

Symbol

Gene name

Log2 fold change
(UVB/control)
(DMBA-TPA/control)

Location

Type(s)

7

PHYHIPL

4.408

Cytoplasm

Other

8
9
10
11
12
13
14
15
16

COX7C
CMYA5
HELB
WDR63
SIPA1L2
let-7
DSEL
ZNF521
TMTC2

4.408
4.408
4.351
4.292
4.292
4.292
4.292
4.167
4.167

Cytoplasm
Plasma membrane
Nucleus
Other
Other
Cytoplasm
Extracellular space
Nucleus
Cytoplasm

Enzyme
Other
Enzyme
Other
Other
MicroRNA
Enzyme
Other
Other

17
18
19
20
21

OTOL1
METTL21A
INTU
DHCR7
Cyp2a12/Cyp2a22

4.167
4.167
4.167
4.167
4.167

Other
Other
Cytoplasm
Cytoplasm
Cytoplasm

Other
Enzyme
Other
Enzyme
Enzyme

22
23

CTSC
CHORDC1

4.167
4.167

Cytoplasm
Other

Peptidase
Other

24
25

CBLN1
THAP1

4.167
4.029

Cytoplasm
Nucleus

Other
Other

26
27

RYBP
NDUFA12

4.029
4.029

Nucleus
Cytoplasm

Transcription regulator
Enzyme

28
29
30
31
32
33
34

Mug1/Mug2
MFSD10
KIF16B
GRID2
Gm4836 (includes others)
FOXN3
Cyp4f16/Gm9705

4.029
4.029
4.029
4.029
4.029
4.029
4.029

Extracellular space
Other
Cytoplasm
Plasma membrane
Cytoplasm
Nucleus
Cytoplasm

Transporter
Transporter
Enzyme
Ion channel
Other
Transcription regulator
Enzyme

35
36

CXorf22
Pag1

4.029
3.955

Other
Plasma membrane

Other
other

37
38
39
40

MSH6
ZFAT
Vmn1r188 (includes others)
TYW3

3.923
3.877
3.877
3.877

Nucleus
Nucleus
Plasma membrane
Other

Enzyme
Other
G-protein coupled receptor
Other

41
42
43
44
45

TPO
TLR4
THSD7B
SPINK5
SLC9A8

3.877
3.877
3.877
3.877
3.877

Plasma membrane
Plasma membrane
Other
Extracellular space
Cytoplasm

Enzyme
Transmembrane receptor
Other
Other
Transporter

46
47
48

SEC23A
RPS4Y1
PPP1R1C

3.877
3.877
3.877

Cytoplasm
Cytoplasm
Cytoplasm

Transporter
Other
Phosphatase

49
50

PARP8
NLRP4

Phytanoyl-CoA 2-hydroxylase interacting
protein-like
Cytochrome c oxidase subunit VIIc
Cardiomyopathy associated 5
Helicase (DNA) B
WD repeat domain 63
Signal-induced proliferation-associated 1 like 2
MicroRNA let-7a-1
Dermatan sulfate epimerase-like
Zinc finger protein 521
Transmembrane and tetratricopeptide repeat
containing 2
Otolin 1
Methyltransferase like 21A
Inturned planar cell polarity protein
7-dehydrocholesterol reductase
Cytochrome P450, family 2, subfamily a,
polypeptide 12
Cathepsin C
Cysteine and histidine-rich domain (CHORD)
containing 1
Cerebellin 1 precursor
THAP domain containing, apoptosis associated
protein 1
RING1 and YY1 binding protein
NADH dehydrogenase (ubiquinone) 1 alpha
subcomplex, 12
Murinoglobulin 1
Major facilitator superfamily domain containing 10
Kinesin family member 16B
Glutamate receptor, ionotropic, delta 2
Predicted gene 4836
Forkhead box N3
Cytochrome P450, family 4, subfamily f,
polypeptide 16
Chromosome X open reading frame 22
phosphoprotein associated with glycosphingolipid
microdomains 1
MutS homolog 6
Zinc finger and AT hook domain containing
Vomeronasal 1 receptor 217
tRNA-yW synthesizing protein 3 homolog
(S. cerevisiae)
Thyroid peroxidase
Toll-like receptor 4
Thrombospondin, type I, domain containing 7B
Serine peptidase inhibitor, Kazal type 5
Solute carrier family 9, subfamily A (NHE8, cation
proton antiporter 8), member 8
Sec23 homolog A (S. cerevisiae)
Ribosomal protein S4, Y-linked 1
Protein phosphatase 1, regulatory (inhibitor)
subunit 1C
Poly(ADP-ribose) polymerase family, member 8
NLR family, pyrin domain containing 4

3.877
3.877

Other
Cytoplasm

Other
Other

a list of overlapping regions of sequence alignment common to both
the immunoprecipitated and the control samples was created and
used to judge the quantitative enrichment in MeDIP samples over
control samples using Cuffdiff; statistically significant peaks at 5%
FDR and a minimum 4-fold difference using the Cummerbund package in R were selected (Trapnell et al., 2012). Peaks were matched
with adjacent annotated genes using ChIPpeakAnno (Zhu et al.,
2010).
Functional and pathway analysis by Ingenuity® Pathway Analysis (IPA)
We analyzed lists of genes with significant fold changes (based on the
P values; UV-induced tumor vs. control and DMBA/TPA treatment vs.

control) in the methylation pattern (increases and decreases)
ascertained in the MeDIP-Seq experiment using Ingenuity® Pathways
Analysis 4.0 (IPA 4.0, Ingenuity Systems, www.ingenuity.com). IPA utilized gene symbols that were identified as neighboring enriched methylation peaks by ChIPpeakAnno used for all of the analyses. IPA
mapped 6003 genes in the UVB group and 5424 genes in the
DMBA/TPA group with a ≥2-fold change compared with the control
correspondingly. Based on these fold change data, IPA identified biological functions and canonical pathways related to UVB-induced
cancer. The list of genes within canonical pathways was ranked
using the ratio of the number of genes mapped to each pathway
to the total number of genes in the corresponding pathway, which
is presented as observations/total in Table 3.

A.Y. Yang et al. / Life Sciences 113 (2014) 45–54

49

Table 2
Top 50 annotated genes with decreased methylation ranked by log2 fold change. (A) UVB group. (B) DMBA/TPA group.
(A)
Symbol

Gene name

Log2 fold change
(UVB/control)

Location

Type(s)

1
2
3
4
5
6
7

Nrxn3
SYN2
Mup1 (includes others)
KRT86
SULT1C3
ATP1A3
CHORDC1

−4.543
−4.378
−4.333
−4.24
−4.24
−4.191
−4.191

Plasma membrane
Plasma membrane
Extracellular space
Cytoplasm
Cytoplasm
Plasma membrane
Other

Other
Other
Other
Other
Enzyme
Transporter
Other

8
9

NRXN1
Htr6

−4.191
−4.141

Plasma membrane
Plasma membrane

Transporter
G-protein coupled receptor

10
11

PAM
SERPINB3

−4.141
−4.141

Plasma membrane
Cytoplasm

Enzyme
Other

12
13
14

TDRD3
Olfr1153
WFIKKN2

−4.141
−4.088
−4.088

Nucleus
Plasma membrane
Other

Transcription regulator
G-protein coupled receptor
Other

15
16
17
18
19
20
21

ABAT
ARHGAP6
AUH
CEP70
DGKH
GRIA1
SLC4A7

−4.034
−4.034
−4.034
−4.034
−4.034
−4.034
−4.034

Cytoplasm
Cytoplasm
Cytoplasm
Cytoplasm
Cytoplasm
Plasma membrane
Plasma membrane

Enzyme
other
Enzyme
Other
Kinase
Ion channel
Transporter

22
23

SLITRK1
Speer4a (includes others)

−4.034
−4.034

Other
Nucleus

Other
Other

24
25
26

CETN1
CYLC1
HS3ST5

−3.977
−3.977
−3.977

Nucleus
Cytoplasm
Cytoplasm

Enzyme
Other
Enzyme

27
28
29
30
31
32
33
34
35
36
37
38
39
40
41

Ott (includes others)
Scg5
CHM
Clvs2
MRPS30
Olfr1010
PPP1R12A
ZMYND11
ADCY10
Agtr1b
APAF1
AQP1
CDH10
CHRM3
GCNT2

−3.977
−3.977
−3.918
−3.918
−3.918
−3.918
−3.918
−3.918
−3.857
−3.857
−3.857
−3.857
−3.857
−3.857
−3.857

Other
Cytoplasm
Cytoplasm
Cytoplasm
Cytoplasm
Plasma membrane
Cytoplasm
Nucleus
Cytoplasm
Plasma membrane
Cytoplasm
Plasma membrane
Plasma membrane
Plasma membrane
Cytoplasm

Other
Other
Enzyme
Other
Enzyme
G-protein coupled receptor
Phosphatase
Other
Enzyme
G-protein coupled receptor
Other
Transporter
Other
G-protein coupled receptor
Enzyme

42

GNAI1

−3.857

Plasma membrane

Enzyme

43
44
45
46
47
48
49
50

SELENBP1
SP110
TMEM5
XIRP2
ZKSCAN2
1700009N14Rik
AGPAT9
ASCL1

Neurexin III
Synapsin II
Major urinary protein 1
Keratin 86
Sulfotransferase family, cytosolic, 1C, member 3
ATPase, Na+/K+transporting, alpha 3 polypeptide
Cysteine and histidine-rich domain (CHORD) containing 1
Neurexin 1
5-Hydroxytryptamine (serotonin) receptor 6, G
protein-coupled
Peptidylglycine alpha-amidating monooxygenase
Serpin peptidase inhibitor, clade B (ovalbumin),
member 3
Tudor domain containing 3
Olfactory receptor 1153
WAP, follistatin/kazal, immunoglobulin, kunitz and
netrin domain containing 2
4-Aminobutyrate aminotransferase
Rho GTPase activating protein 6
AU RNA binding protein/enoyl-CoA hydratase
Centrosomal protein 70 kDa
Diacylglycerol kinase, eta
Glutamate receptor, ionotropic, AMPA 1
Solute carrier family 4, sodium bicarbonate
cotransporter, member 7
SLIT and NTRK-like family, member 1
Spermatogenesis associated glutamate (E)-rich
protein 4a
Centrin, EF-hand protein, 1
Cylicin, basic protein of sperm head cytoskeleton 1
Heparan sulfate (glucosamine) 3-Osulfotransferase 5
Ovary testis transcribed
Secretogranin V
Choroideremia (Rab escort protein 1)
Clavesin 2
Mitochondrial ribosomal protein S30
Olfactory receptor 1010
Protein phosphatase 1, regulatory subunit 12A
Zinc finger, MYND-type containing 11
Adenylate cyclase 10 (soluble)
Angiotensin II receptor, type 1b
Apoptotic peptidase activating factor 1
Aquaporin 1
Cadherin 10, type 2 (T2-cadherin)
Cholinergic receptor, muscarinic 3
Glucosaminyl (N-acetyl) transferase 2, I-branching
enzyme (I blood group)
Guanine nucleotide binding protein (G protein),
alpha inhibiting activity polypeptide 1
Selenium binding protein 1
SP110 nuclear body protein
Transmembrane protein 5
Xin actin-binding repeat containing 2
Zinc finger with KRAB and SCAN domains 2
RIKEN cDNA 1700009N14 gene
1-Acylglycerol-3-phosphate O-acyltransferase 9
Achaete–scute complex homolog 1 (Drosophila)

−3.857
−3.857
−3.857
−3.857
−3.857
−3.793
−3.793
−3.793

Cytoplasm
Nucleus
Plasma membrane
Other
Nucleus
Other
Cytoplasm
Nucleus

Other
Other
Other
Other
Transcription regulator
Transporter
Enzyme
Transcription regulator

Rank

Symbol

Gene name

Log2 fold change
(DMBA-TPA/control)

Location

Type(s)

1
2
3
4
5
6
7
8

EBPL
PANX1
HES5
LHX4
GSG1L
PBX1
LRRC8B
ASAH2

Emopamil binding protein-like
Pannexin 1
Hairy and enhancer of split 5 (Drosophila)
LIM homeobox 4
GSG1-like
Pre-B-cell leukemia homeobox 1
Leucine rich repeat containing 8 family, member B

−5.292
−5.247
−4.632
−4.247
−4.247
−4.199
-4.199
−4.199

Cytoplasm
Plasma membrane
Nucleus
Nucleus
Plasma membrane
Nucleus
Other
Cytoplasm

Enzyme
Transporter
Other
Transcription regulator
Other
Transcription regulator
Other
Enzyme

(B)

(continued on next page)

50

A.Y. Yang et al. / Life Sciences 113 (2014) 45–54

Table 2 (continued)
(A)
(B)
Symbol

9
10
11
12
13
14
15
16
17
18
19
20
21
22

ALKBH3
ZNF518B
MAN1A1
TOX3
LSM11
TCEA3
OR7D2
KIAA0947
CBLB
NOS1AP
MACC1
ZNF277
Sp100
KCNA6

23
24
25
26
27
28
29
30
31
32
33

C19orf10
TMEM17
FAM92B
TBC1D5
Nrg1
GORASP1
AHRR
ADORA2A
RAB11A
ISY1-RAB43
GNE

34
35

FAM98A
ENPP4

36
37
38
39
40
41
42
43
44
45
46
47
48
49
50

CCDC43
ARHGEF10
TARSL2
SCARB1
RAB27A
L3MBTL3
Higd1a
GRIA1
GPC5
CLGN
CHKA
ASGR1
AMY2A
C1orf109
CADPS

Gene name
N-acylsphingosine amidohydrolase (non-lysosomal
ceramidase) 2
alkB, alkylation repair homolog 3 (E. coli)
Zinc finger protein 518B
Mannosidase, alpha, class 1A, member 1
TOX high mobility group box family member 3
LSM11, U7 small nuclear RNA associated
Transcription elongation factor A (SII), 3
Olfactory receptor, family 7, subfamily D, member 2
KIAA0947
Cbl proto-oncogene B, E3 ubiquitin protein ligase
Nitric oxide synthase 1 (neuronal) adaptor protein
Metastasis associated in colon cancer 1
Zinc finger protein 277
Nuclear antigen Sp100
Potassium voltage-gated channel, shaker-related
subfamily, member 6
Chromosome 19 open reading frame 10
Transmembrane protein 17
Family with sequence similarity 92, member B
TBC1 domain family, member 5
Neuregulin 1
Golgi reassembly stacking protein 1, 65 kDa
Aryl-hydrocarbon receptor repressor
Adenosine A2a receptor
RAB11A, member RAS oncogene family
ISY1-RAB43 readthrough
Glucosamine (UDP-N-acetyl)-2-epimerase/Nacetylmannosamine kinase
Family with sequence similarity 98, member A
Ectonucleotide pyrophosphatase/
phosphodiesterase 4 (putative)
Coiled–coil domain containing 43
Rho guanine nucleotide exchange factor (GEF) 10
Threonyl-tRNA synthetase-like 2
Scavenger receptor class B, member 1
RAB27A, member RAS oncogene family
l(3)mbt-like 3 (Drosophila)
HIG1 domain family, member 1A
Glutamate receptor, ionotropic, AMPA 1
Glypican 5
Calmegin
Choline kinase alpha
Asialoglycoprotein receptor 1
Amylase, alpha 2A (pancreatic)
Chromosome 1 open reading frame 109
Ca++-dependent secretion activator

Log2 fold change
(DMBA-TPA/control)
(UVB/control)

Location

Type(s)

−4.199
−4.100
−4.100
−3.993
−3.936
−3.877
−3.877
−3.877
−3.877
−3.816
−3.816
−3.752
−3.752
−3.752

Nucleus
Other
Cytoplasm
Other
Nucleus
Nucleus
Plasma membrane
Other
Nucleus
Cytoplasm
Nucleus
Nucleus
Nucleus
Plasma membrane

Enzyme
Other
Enzyme
Other
Other
Transcription regulator
G-protein coupled receptor
Other
Other
Other
Other
Transcription regulator
Transcription regulator
Ion channel

−3.752
−3.685
−3.650
−3.646
−3.614
−3.614
−3.614
−3.614
−3.540
−3.540
−3.540

Extracellular space
Extracellular space
other
Extracellular space
Extracellular space
Cytoplasm
Nucleus
Plasma membrane
Cytoplasm
Nucleus
Cytoplasm

Cytokine
Other
Other
Other
Growth factor
Other
Other
G-protein coupled receptor
Enzyme
Other
Kinase

−3.540
−3.540

Other
Other

Other
Enzyme

−3.540
−3.540
−3.462
−3.462
−3.462
−3.462
−3.462
−3.462
−3.462
−3.462
−3.462
−3.462
−3.462
−3.444
−3.430

Other
Cytoplasm
Other
Plasma membrane
Cytoplasm
Nucleus
Cytoplasm
Plasma membrane
Plasma membrane
Cytoplasm
Cytoplasm
Plasma membrane
Extracellular space
Other
Plasma membrane

Other
Enzyme
Enzyme
Transporter
Enzyme
Other
Other
Ion channel
Other
Peptidase
Kinase
Transmembrane receptor
Enzyme
Other
Other

Results
MeDIP-Seq results
Skin samples were collected from the UVB- and DMBA/TPA-induced
mouse skin cancer models, and gDNA was isolated from each sample. A
whole-genome DNA methylation analysis was performed on the DNA
samples using the described MeDIP-Seq method. The results were analyzed in a paired manner comparing the tumor to the normal skin tissue
samples for each model.
In the UVB group, 6003 genes were identified in the two samples with
a≥2-fold change in methylation. Compared with the control, 4140 genes
exhibited increased methylation, and methylation was decreased in 1863
genes (Fig. 1). However, in the DMBA/TPA treatment group, 5424 genes
were identified with a≥2-fold change in peak reads between the tumor
and the normal skin samples. Among these 5424 genes, the methylation
pattern was increased in 3781 genes and decreased in 1643 genes. To prioritize those changes, we ranked the top 50 increased (+, Table 1) and
decreased (−, Table 2) genes based on the log2 fold change from highest
to lowest, all with P values less than 0.05.

Table 3
Top 5 altered canonical pathways determined using Ingenuity Pathways Software in the
UVB and DMBA/TPA groups. The shared pathways are shown in bold.
Rank Name

Ratio

UVB/control
1
cAMP-mediated signaling
2
G-protein coupled receptor signaling
3
Molecular mechanisms of cancer
4
PTEN signaling
5
Role of osteoblasts, osteoclasts and
chondrocytes in rheumatoid arthritis

0.478 108/226
0.442 122/276
0.371 144/388
0.42
58/138
0.388
97/250

1.27E−09
5.24E−09
9.86E−07
5.21E−06
5.58E−06

0.352 144/409
0.325 126/338
0.351 101/288
0.342
67/196

5.5E−06
4.57E−05
7.59E−05
0.0011

0.394

0.0013

DMBA–TPA/control
1
Protein kinase A signaling
2
Molecular mechanisms of cancer
3
Xenobiotic metabolism signaling
4
Regulation of the epithelial–
mesenchymal transition pathway
5
Mouse embryonic stem cell
pluripotency

Observation/ p-Value
total

39/99

A.Y. Yang et al. / Life Sciences 113 (2014) 45–54

51

Fig. 2. (A) Genes mapped to the IL-6 pathway by IPA software. Red, increased methylation; green, decreased methylation; UVB irradiated vs. control. (B) List of genes mapped to the IL-6
pathway by IPA.

Pathway analysis by IPA

Discussion

To ascertain the significance of the methylation changes, 6003
genes in the UVB group and 5424 genes in the DMBA/TPA group
with a greater than 2-fold change in methylation were analyzed
using the Ingenuity® Pathway Analysis (IPA) software package.
The top 5 canonical signaling pathways were categorized (Table 3)
based on the ratio of the number of input genes to the total number
of genes in the corresponding pathway in the Ingenuity® Pathway
Analysis software. Fisher's exact test was used to calculate P values
to determine the significance of the associations of the input genes
with the canonical pathways.
IPA identified more than 50 signaling pathways containing genes
with significantly increased and decreased methylation. The
interleukin-6 (IL-6)-related signaling pathway was mapped by IPA
to the UVB group (Fig. 2); the methylation profile is presented in
red (increase) and green (decrease). Fig. 2B lists the genes involved
in the IL-6 signaling pathway that exhibited altered methylation (14
increased and 30 decreased) as mapped by IPA. It has been reported
that IL-6 protein expression is induced by UV irradiation (Vaid et al.,
2010). Correspondingly, the MeDIP-Seq data revealed that IL-6
increased methylation by 3.252-fold (log2). Based on the IPA functional pathways, skin cancer belongs to the category of mechanism
of cancer. The top 5 genes related to skin cancer based on IPA are
ranked by fold change in Table 4. This list includes genes with increased methylation and genes with decreased methylation that
are related to skin cancer pathways by IPA.

It was previously reported that hypermethylation of CpG islands in
tumor suppressor genes occurs in human squamous cell carcinoma
cell lines and in primary skin tumor tissues. Similar changes in DNA
methylation patterns were observed in the multistage mouse skin cancer model. In addition, loss of global genomic methylation has been
shown to be associated with increased aggressiveness of mouse skin
cancer cell lines. However, the precise mechanism by which UV irradiation promotes melanoma remains unclear. Furthermore, there is no
method for screening potential epigenetically modified genes involved
in promoting skin cancers (Kanavy and Gerstenblith, 2011; Jhappan
et al., 2003; Leiter and Garbe, 2008; Moan et al., 2008). Long-term exposure to UV irradiation is considered a major etiologic risk factor for the
development of melanoma and non-melanoma skin cancers. Epigenetic
alterations are generally considered to represent the interface between
the environment and the genome (Jaenisch and Bird, 2003). Ultraviolet
irradiation has been reported to induce epigenetic alterations, which
may contribute to the development of skin cancer. In the present
study, we performed global genome methylation screening using
MeDIP-Seq to identify genomic loci with aberrant methylation patterns
in cancer tissues.
One of the adverse effects of UV irradiation that has been observed in skin tumor development is a chronic and sustained inflammatory response. The relationship between inflammation and
epigenetic modifications in cancer is under active investigation
(Nile et al., 2008; Tekpli et al., 2013; Gasche et al., 2011). In this

52

A.Y. Yang et al. / Life Sciences 113 (2014) 45–54

B
Log2 Fold
Change
(UVB/Control)

Symbol

Gene Name

-3.421
-3.24
-3.141

IL1RAPL2
HSPB3
MAPK10

4

-3.141

PIK3C2G

5
6
7
8
9

-3.141
-2.918
-2.793
-2.793
-2.655

SOCS1
PIK3R1
IL1RL1
MAP3K7
CRP

10

-2.503

ABCB1

-2.503
-2.333
-2.141
-2.034

IL1R2
IL36B
MAP2K1
PIK3R4

interleukin 1 receptor accessory protein-like 2
heat shock 27kDa protein 3
mitogen-activated protein kinase 10
phosphatidylinositol-4-phosphate 3-kinase, catalytic
subunit type 2 gamma
suppressor of cytokine signaling 1
phosphoinositide-3-kinase, regulatory subunit 1 (alpha)
interleukin 1 receptor-like 1
mitogen-activated protein kinase kinase kinase 7
C-reactive protein, pentraxin-related
ATP-binding cassette, sub-family B (MDR/TAP),
member 1
interleukin 1 receptor, type II
interleukin 36, beta
mitogen-activated protein kinase kinase 1
phosphoinositide-3-kinase, regulatory subunit 4

Decreased
1
2
3

11
12
13
14
Increased
1

3.667

AKT3

2

3.667

PIK3CG

3
4
5
6

3.667
3.474
3.252
3.252

PTPN11
PIK3C3
COL1A1
IL6

7

3.252

RELA

8
9
10
11

3.169
2.989
2.989
2.989

IL1RL2
IL1RAP
MAP2K6
RRAS2

12

2.989

SRF

13

2.915

NFKB2

14
15
16
17

2.667
2.667
2.667
2.667

A2M
AKT1
CSNK2A1
HSPB1

18

2.667

IL6ST

19

2.667

MAP2K4

20

2.667

NFKBIA

21

2.474

PIK3C2A

22
23
24
25
26
27

2.252
2.252
2.252
2.252
2.252
2.252

FOS
IL1R1
LBP
MAPK1
MAPK8
MAPK13

28

2.252

NFKB1

29
30

2.252
2.252

SOS1
SOS2

v-akt murine thymoma viral oncogene homolog 3
phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic
subunit gamma
protein tyrosine phosphatase, non-receptor type 11
phosphatidylinositol 3-kinase, catalytic subunit type 3
collagen, type I, alpha 1
interleukin 6 (interferon, beta 2)
v-rel avian reticuloendotheliosis viral oncogene homolog
A
interleukin 1 receptor-like 2
interleukin 1 receptor accessory protein
mitogen-activated protein kinase kinase 6
related RAS viral (r-ras) oncogene homolog 2
serum response factor (c-fos serum response elementbinding transcription factor)
nuclear factor of kappa light polypeptide gene enhancer
in B-cells 2 (p49/p100)
alpha-2-macroglobulin
v-akt murine thymoma viral oncogene homolog 1
casein kinase 2, alpha 1 polypeptide
heat shock 27kDa protein 1
interleukin 6 signal transducer (gp130, oncostatin M
receptor)
mitogen-activated protein kinase kinase 4
nuclear factor of kappa light polypeptide gene enhancer
in B-cells inhibitor, alpha
phosphatidylinositol-4-phosphate 3-kinase, catalytic
subunit type 2 alpha
FBJ murine osteosarcoma viral oncogene homolog
interleukin 1 receptor, type I
lipopolysaccharide binding protein
mitogen-activated protein kinase 1
mitogen-activated protein kinase 8
mitogen-activated protein kinase 13
nuclear factor of kappa light polypeptide gene enhancer
in B-cells 1
son of sevenless homolog 1
son of sevenless homolog 2
Fig. 2 (continued).

study, a UV irradiation-induced abnormal inflammatory response
was suggested based on the IPA mapped IL-6 pathways, which included the methylation profiles of pro-inflammatory cytokines,

receptors and mitogen-activated protein kinases. Higher levels of
pro-inflammatory cytokines are associated with tumor development and progression (Mukhtar and Elmets, 1996; Tron et al.,

A.Y. Yang et al. / Life Sciences 113 (2014) 45–54
Table 4
Top 5 genes with altered methylation (increased or decreased) that were related to skin
cancer using the IPA Software Functional and Diseases analysis module in the UVB and
the DMBA/TPA groups. The shared genes are shown in bold.
Rank
UVB/control
Increased
1
2
3
4
5
Decreased
1
2
3
4
5
DMBA–TPA/control
Increased
1
2
3
4
5
Decreased
1
2
3
4
5

Mapped genes

Log2 fold change

RBFOX1
LOXHD1
EDN1
DYSF
NPSR1

5.457
4.367
4.367
3.989
3.837

SULT1C3
ABAT
GRIA1
PCSK1
SCN2A

−4.24
−4.034
−4.034
−3.726
−3.655

GRID2
NLRP4
EMR1
IL15
PCDH18

4.029
3.877
3.877
3.877
3.708

GRIA1
CADPS
BCL2L11
ACVR1C
GRM3

−3.462
−3.43
−3.292
−3.292
−3.292

1988). Interleukins (ILs) have different systemic functions and are
involved in inflammation. Nile et al. reported that the methylation
of CpGs in the promoter region of IL-6 affected the mRNA levels in
mononuclear cells (Nile et al., 2008). Tekpli et al. reported that the
methylation of CpGs near the IL-6 transcriptional start site is significantly higher in non-small cell lung cancer cells and is associated
with lower IL-6 mRNA expression (Tekpli et al., 2013). Our study
demonstrated that IL-6 gene methylation was significantly higher
in UV irradiation-induced skin tumors.
IL-6-Jak-Stat3 inflammatory signaling is also involved in cell survival
and provides a proliferative advantage in the two-stage chemical carcinogenesis model using DMBA as the tumor initiator and TPA as the
promoter. Phosphorylated-Stat3 overexpression in a papilloma cell
line leads to enhanced cell migration and invasion (Suiqing et al.,
2005). Transgenic mice with constitutive Stat3 expression have a
shorter latency period and increased tumor incidence compared with
non-transgenic littermates after DMBA/TPA treatment (Chan et al.,
2008). Moreover, mice with constitutively activated Stat3 bypassed
the premalignant stage and were initially diagnosed with carcinoma
in situ, which rapidly progressed to squamous cell carcinoma. In our
present study, we found 34 genes with altered DNA methylation in
total 124 genes involved in the IL-6 pathway in the UV group. Among
these genes with altered methylation, the SOCS1 (suppressor of
cytokine signaling 1) gene, which encodes a suppressor in the IL-6Jak-Stat3 loop, was hypermethylated in the tumor samples.
The top-ranked hypermethylated and hypomethylated genes could
enable the discovery of key genes in skin cancer development. For example, RBFOX1 is the top increased gene in terms of methylation status
change in UV-irradiated tumors compared with normal epidermis by
IPA (Fig. 1). RBFOX1 is an RNA-binding protein that is highly expressed
in the cytoplasm. RBFOX1 mutations were identified in colorectal cancer
cell lines (CRC), and RBFOX1 deletion was observed in a significant proportion of CRC cases (106/419) (Cancer Genome Atlas, N., 2012;
Sengupta et al., 2013). However, the role of RBFOX1 in skin cancer development is unclear. Surprisingly, tumor tissues from the DMBA/TPA
group exhibited a unique profile in terms of the top 50 genes with

53

increased or decreased methylation compared with the profile in the
UVB group. Cell adhesion molecule 2 (CADM2) was one of the top methylated genes in DMBA/TPA tumors. CADM2 belongs to a protein family
that participates in maintaining cell polarity and that has been considered to be a novel category of tumor suppressors (Chang et al., 2010).
Clinically, low CADM2 expression predicts a high risk of recurrence in
patients with hepatocellular carcinoma after hepatectomy (Yang et al.,
2014). It has also been reported that aberrant promoter hypermethylation and loss of CADM2 expression are associated with human renal cell
carcinoma progression (He et al., 2013). Our study is the first report to
suggest that CADM2 methylation could be involved in skin carcinogenesis. Moreover, we identified changes in the methylation patterns of
several genes encoding microRNAs, which are also involved in epigenetic regulation. This observation indicates that epigenetic changes may
occur at multiple levels with complex crosstalk in skin cancer development and progression.
The genes identified in this study demonstrated significant alterations in response to UV irradiation-induced inflammation and skin
cancer development. Although IPA revealed some overlapping signaling
changes in response to both UV irradiation and DMBA–TPA treatment and certain highly affected targets were common (including,
GRIA1 and TNS1), the top-ranked genes based on fold change differed markedly between the two treatments, indicating that distinct
epigenetic mechanisms trigger tumorigenesis after exposure to UV
or the DMBA carcinogen.
Conclusions
In this study, a comprehensive analysis of the DNA methylation patterns in the UVB or DMBA/TPA induced tumors compared with agematched normal skin was completed. Genes coding for inflammatory
cytokines were identified by IPA to exhibit altered methylation profiles
and may be associated with increased susceptibility to tumor development. Specifically, based on changes in methylation, molecular networks were identified that included genes encoding inflammatory
cytokines. Additional studies with a particular emphasis on epigenetic
alterations, such as DNA methylation, may lead to the development of
new strategies for the prevention of skin cancer and inflammationrelated skin disease.
Conflict of interest statement
The authors declare that there are no conflicts of interest.

Acknowledgments
The authors would like to thank the members of Tony Kong's laboratory for their helpful discussions. This work was supported in part by institutional funds.
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