Association of epigenetic inactivation of the WRN gene with anticancer drug sensitivity in cervical cancer cells.
ABSTRACT The Werner (WRN) gene codes for a DNA helicase that contributes to genomic stability and has been identified as the gene responsible for progeria. Recent studies have shown reduced WRN expression due to aberrant DNA hypermethylation in cancer cells. Furthermore, WRN expression is thought to affect sensitivity to DNA topoisomerase I inhibitors in cancer therapy. In this study, we examined the relationship between aberrant DNA hypermethylation of WRN and the sensitivity of cervical cancer cells to anticancer drugs. DNA was extracted from samples from 22 patients with primary cervical cancer and 6 human cervical cancer-derived cell lines. Aberrant DNA hypermethylation was analyzed by methylation-specific PCR. WRN expression in cultured cells before and after addition of 5-aza-2-deoxycytidine, a demethylating agent, was examined using RT-PCR. The sensitivity of cells to anticancer drugs was determined using a collagen gel droplet embedded culture drug sensitivity test (CD-DST). siRNA against WRN was transfected into a cervical cancer-derived cell line with high WRN expression. Changes in drug sensitivity after silencing WRN were determined by CD-DST. Aberrant DNA hypermethylation and decreased expression of WRN were detected in 7/21 cases of primary cervical cancer and in two cervical cancer-derived cell lines. These two cell lines showed high sensitivity to CPT-11, a topoisomerase I inhibitor, but became resistant to CPT-11 after treatment with 5-aza-2-deoxycytidine. Transfection of siRNA against WRN increased the sensitivity of the cells to CPT-11. Aberrant DNA hypermethylation of WRN also increased the sensitivity of cervical cancer cells to CPT-11. Therefore, epigenetic inactivation of this gene may be a biomarker for selection of drugs for the treatment of cervical cancer. This is the first report to show a relationship between the methylation of the WRN gene and sensitivity to CPT-11 in gynecological cancers.
- SourceAvailable from: Miho Iida[Show abstract] [Hide abstract]
ABSTRACT: Cervical cancer ranks high among the causes of female cancer mortalities and is an important disease in developing and developed countries. Current diagnosis of cervical cancer depends on colposcopy, pathological diagnosis and preoperative diagnosis using methods, including magnetic resonance imaging and computed tomography. Advanced cervical cancer has a poor prognosis. The tumor marker squamous cell carcinoma is conventionally used for screening, but recent studies have revealed the mechanisms of carcinogenesis and the factors associated with a poor prognosis in cervical cancer. These include epigenetic biomarkers, with the methylation level of the checkpoint with forkhead and ring finger gene being potentially useful for predicting the malignancy of cervical cancer and sensitivity to treatment with paclitaxel. The extent of methylation of the Werner DNA helicase gene is also useful for determining sensitivity to an anticancer agent, CPT-11. In addition to epigenetic changes, the expression levels of hypoxia-inducible factor 1α subunit, epidermal growth factor receptor and cyclooxygenase-2 have been reported as possible biomarkers in cervical cancer. Novel prognostic factors, including angiogenic factors, fragile histidine triad, thymidylate synthase, glucose-related protein 58 and mucin antigens, have also been described, and hemoglobin and platelets may also be significant prognostic biomarkers. Utilization of these biomarkers may facilitate personalized treatment and improved outcomes in cervical cancer.Molecular and Clinical Oncology 09/2014; 2(5):647-655.
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ABSTRACT: The KLF4 gene has been shown to be inactivated in cervical carcinogenesis as a tumor suppressor. However, the mechanism of KLF4 silencing in cervical carcinomas has not yet been identified. DNA methylation plays a key role in stable suppression of gene expression. The methylation status of the KLF4 promoter CpG islands was analyzed by bisulfite sequencing (BSQ) in tissues of normal cervix and cervical cancer. KLF4 gene expression was detected by RT-PCR, immunohistochemistry and western blot. KLF4 promoter methylation in cervical cancer cell line was determined by BSQ and methylation-specific polymerase chain reaction (MS-PCR). Cell proliferation ability was detected by cell growth curve and MTT assay. The methylated allele was found in 41.90% of 24 cervical cancer tissues but only in 11.11% of 11 normal cervix tissues (P<0.005). KLF4 mRNA levels were significantly reduced in cervical cancer tissues compared with normal cervix tissues (P<0.01) and KLF4 mRNA expression showed a significant negative correlation with the promoter hypermethylation (r = -0.486, P = 0.003). Cervical cancer cell lines also showed a significant negative correlation between KLF4 expression and hypermethylation. After treatment with the demethylating agent 5-Azacytidine (5-Aza), the expression of KLF4 in the cervical cancer cell lines at both mRNA and protein levels was drastically increased, the cell proliferation ability was inhibited and the chemosensitivity for cisplatin was significantly increased. KLF4 gene is inactivated by methylation-induced silencing mechanisms in a large subset of cervical carcinomas and KLF4 promoter hypermethylation inactivates the gene's function as a tumor suppressor in cervical carcinogenesis.PLoS ONE 02/2014; 9(2):e88827. · 3.53 Impact Factor
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ABSTRACT: Cervical cancer is the third most common cancer worldwide, and the development of new diagnosis, prognostic, and treatment strategies is a major interest for public health. Cisplatin, in combination with external beam irradiation for locally advanced disease, or as monotherapy for recurrent/metastatic disease, has been the cornerstone of treatment for more than two decades. Other investigated cytotoxic therapies include paclitaxel, ifosfamide and topotecan, as single agents or in combination, revealing unsatisfactory results. In recent years, much effort has been made towards evaluating new drugs and developing innovative therapies to treat cervical cancer. Among the most investigated molecular targets are epidermal growth factor receptor and vascular endothelial growth factor (VEGF) signaling pathways, both playing a critical role in cervical cancer development. Studies with bevacizumab or VEGF receptor tyrosine kinase have given encouraging results in terms of clinical efficacy, without adding significant toxicity. A great number of other molecular agents targeting critical pathways in cervical malignant transformation are being evaluated in preclinical and clinical trials, reporting preliminary promising data. In the current review, we discuss novel therapeutic strategies which are being investigated for the treatment of advanced cervical cancer.Journal of Cancer. 01/2014; 5(2):86-97.
ONCOLOGY REPORTS 28: 1146-1152, 2012
Abstract. The Werner (WRN) gene codes for a DNA helicase
that contributes to genomic stability and has been identified as
the gene responsible for progeria. Recent studies have shown
reduced WRN expression due to aberrant DNA hypermethyl-
ation in cancer cells. Furthermore, WRN expression is thought
to affect sensitivity to DNA topoisomerase I inhibitors in cancer
therapy. In this study, we examined the relationship between
aberrant DNA hypermethylation of WRN and the sensitivity of
cervical cancer cells to anticancer drugs. DNA was extracted
from samples from 22 patients with primary cervical cancer
and 6 human cervical cancer-derived cell lines. Aberrant DNA
hypermethylation was analyzed by methylation-specific PCR.
WRN expression in cultured cells before and after addition of
5-aza-2-deoxycytidine, a demethylating agent, was examined
using RT-PCR. The sensitivity of cells to anticancer drugs was
determined using a collagen gel droplet embedded culture drug
sensitivity test (CD-DST). siRNA against WRN was transfected
into a cervical cancer-derived cell line with high WRN expres-
sion. Changes in drug sensitivity after silencing WRN were
determined by CD-DST. Aberrant DNA hypermethylation and
decreased expression of WRN were detected in 7/21 cases of
primary cervical cancer and in two cervical cancer-derived cell
lines. These two cell lines showed high sensitivity to CPT-11, a
topoisomerase I inhibitor, but became resistant to CPT-11 after
treatment with 5-aza-2-deoxycytidine. Transfection of siRNA
against WRN increased the sensitivity of the cells to CPT-11.
Aberrant DNA hypermethylation of WRN also increased the
sensitivity of cervical cancer cells to CPT-11. Therefore, epigen-
etic inactivation of this gene may be a biomarker for selection of
drugs for the treatment of cervical cancer. This is the first report
to show a relationship between the methylation of the WRN gene
and sensitivity to CPT-11 in gynecological cancers.
The incidence of cervical cancer has decreased in advanced
countries due to more frequent health checkups and the devel-
opment of vaccines. In the United States in 2010, there were
12,200 cases of cervical cancer and 4,210 deaths related to this
disease, reflecting a decreasing tendency (1). However, cases
of cervical cancer worldwide have increased from 378,000 in
1980 to 454,000 in 2010 (2). Death due to cervical cancer has
decreased, but there were still about 200,000 deaths worldwide
in 2010, including 46,000 females aged 15-49 years in devel-
oping countries (2).
Cervical cancer occurs due to infection with the human papil-
lomavirus (HPV) (3). More than 100 genotypes of HPV have
been detected and about 40 have been found to infect the genital
tract. HPV is classified into high-risk types causing cervical
cancer, including HPV16 and HPV18, and low-risk types causing
conditions other than cancer, such as a polyp. HPV16, the most
frequent genotype, is detected in approximately half of patients
with cervical cancer (4). After infecting cells, HPV produces
oncoproteins E6 and E7, which inhibit controlling the cell cycle
and apoptosis, and therefore have important roles in onco-
genesis. E6 is associated with p53 and induces p53 degradation
by E3 ubiquitin ligase via the function of E6-associated protein
(E6AP). E7 inactivates the retinoblastoma tumor suppressor
gene product, pRb and its family members (5).
Epigenetic DNA methylation of tumor suppressor genes in
the promoter region is generally important in carcinogenesis
(6-8). In cervical cancer, carcinogenesis is related to aberrant
methylation of CpG islands of p16, FHIT, retinoic acid receptor β,
E-cadherin, death-associated protein kinase, HIC-1, APC, and
Ras association domain family 1A genes (9-12). Methylation of
CpG islands in the WRN promoter region is related to carcino-
genesis in various cancers (13). The chromosomal WRN locus
on the short arm of chromosome 8, is composed of 35 exons,
and has a length >250 kb (14). WRN encodes the WRN protein,
which is a member of the RecQ helicase family and is also an
exonuclease. Loss of WRN causes abnormalities in DNA repair,
replication, and telomere maintenance.
Werner syndrome (WS) is an autosomal recessive genetic
disease that is caused by mutation of the WRN gene. WS symp-
toms include aging at an early stage and various secondary
symptoms associated with aging, including bilateral cataract,
Association of epigenetic inactivation of the WRN gene
with anticancer drug sensitivity in cervical cancer cells
KENTA MASUDA, KOUJI BANNO, MEGUMI YANOKURA, KOSUKE TSUJI, YUSUKE KOBAYASHI,
IORI KISU, ARISA UEKI, WATARU YAMAGAMI, HIROYUKI NOMURA,
EIICHIRO TOMINAGA, NOBUYUKI SUSUMU and DAISUKE AOKI
Department of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo, Japan
Received April 24, 2012; Accepted June 8, 2012
Correspondence to: Dr Kouji Banno, Department of Obstetrics and
Gynecology, School of Medicine, Keio University, Shinanomachi 35,
Shinjuku-ku, Tokyo 160-8582, Japan
Key words: WRN, cervical cancer, DNA hypermethylation, CPT-11,
MASUDA et al: WRN AND CERVICAL CANCER
skin change, short stature and graying hair; in addition, diabetes
mellitus, osteoporosis, atherosclerosis and cancer also develop
frequently (15). Malignant complications include sarcomas of
mesenchymal origins, including soft-tissue sarcoma and osteo-
sarcoma, suggesting that the mechanism of carcinogenesis in
WS differs from that in carcinogenesis in other cancers (16). The
mean age at death of WS patients is 46-54 years and one of major
causes is the high prevalence of malignancy (17,18).
Several studies have shown a relationship between WRN
expression and malignancy and have indicated that epigenetic
inactivation of WRN is of importance in carcinogenesis. In
many tumors, loss of heterozygosity is detected in chromo-
some 8p, in which WRN is located, but WRN somatic mutation
has not been found, suggesting that epigenetic control has a
significant effect (19,20). Epigenetic DNA methylation in the
promoter region of WRN and a methylation-induced decrease
in WRN expression have been found in colorectal, lung, gastric,
prostate and breast cancer. The methylation-induced decrease
in WRN expression increases chromosomal instability (13).
Reduced WRN expression is also related to sensitivity to
camptothecin (CPT-11), a topoisomerase I (Top-I) inhibitor
(13,21). CPT-11 is an alkaloid found in plants such as Campto-
theca acuminata. A single-strand break (SSB) occurs after Top I
binds to DNA and generates a Top I-DNA cleavable complex.
CPT-11 stabilizes this complex and inhibits reconnection of
the SSB, resulting in inhibition of DNA synthesis (22). CPT-11
also inhibits replication fork progression, resulting in DNA
double-strand breaks (DSBs) and apoptosis (23). Inactivation
of WRN in cancer cells increases the effect of CPT-11 (13,21),
and overall survival of patients with colorectal cancer treated
with irinotecan, a camptothecin analogue, is dependent on the
methylation status of CpG islands in the WRN promoter (13).
Cisplatin is a key drug in chemotherapy for cervical cancer
(24). CPT-11 is a similarly important drug and has a high response
rate of 24% (25). Evaluation of WRN expression as a marker
of sensitivity to CPT-11 may be clinically useful in treatment
of cervical cancer. Thus, in this study, the associations among
cervical cancer, WRN expression, and cancer cell sensitivity to
CPT-11 were investigated.
Materials and methods
Subjects and cytologic specimens. Samples were obtained from
21 cervical cancer smears collected using a ThinPrep system
(Cytyc, Boxborough, MA, USA) and kept in preservation
fluid (PreservCyt Solution, Cytyc) (26). Informed consent was
obtained before collection. Pathological diagnosis was performed
by cervical histology, and the cytological and histological results
were consistent for all smears. Of the 21 cervical cancer smears,
10 were squamous carcinoma and 11 were adenocarcinoma. The
histological type and stage were determined according to the
General Rules for Clinical Cervical Cancer in Japan published
by the Japan Society of Obstetrics and Gynecology.
Cultured cell lines. The human cervical squamous cell
carcinoma-derived cell lines, SKG-I, SKG-II, SKG-IIIa and
SKG-IIIb, and the human cervical adenocarcinoma-derived cell
lines, HeLa and TCO-I, were used in the study. HeLa cells were
incubated in DMEM (Sigma, St. Louis, MO, USA) with 10%
fetal bovine serum (FBS) (Sanko Junyaku Co., Tokyo, Japan)
and TCO-I cells were incubated in MEM medium (Sigma) with
10% FBS. All other cell lines were incubated in F12 medium
(Sigma) with 10% FBS. Cells were incubated in 10-cm dishes at
37˚C in a 5% CO2 atmosphere.
DNA extraction and methylation-specific PCR (MSP) assay of
the WRN gene. DNA was extracted from 21 cervical smears and
6 cervical carcinoma-derived cell lines using a Get Pure DNA
kit (Dojin Glocal, Kumamoto, Japan). DNA (1 µg) extracted
from cervical smears was diluted with 50 µl of distilled water
and incubated in 5.5 µl of 3 N NaOH at 37˚C for 15 min. To
this solution, 30 µl of 10 mM hydroquinone (Sigma) and 520 µl
of 3 M sodium bisulfite (prepared at pH 5.5 with 10 N NaOH,
Sigma) were added with mixing. Mineral oil was laid over the
solution to prevent evaporation, and the solution was incubated
overnight at 50˚C. The lower layer of the reaction solution was
mixed with 1 ml of Clean-up Resin (Promega, Madison, WI,
USA) and then injected into a column. After rinsing with 2 ml of
80% isopropanol, the mixture was centrifuged at 15,000 rpm for
3 min to remove isopropanol. Hot (70˚C) distilled water (50 µl)
was added and the mixture was centrifuged at 15,000 rpm for
2 min to elute DNA. The DNA was then incubated with 5.5 µl
of 2 N NaOH at 37˚C for 20 min. Next, 66 µl of 5 N ammo-
nium acetate and 243 µl of 95% ethanol were added and the
mixture was incubated at -80˚C for one hour and centrifuged
at 15,000 rpm for 30 min to precipitate DNA. Supernatant
exceeding 50 µl was removed, 1 ml of 60% ethanol was added,
and the mixture was centrifuged at 15,000 rpm for 30 min and
rinsed. The precipitated DNA was dried in air and dissolved
in 20 µl of distilled water. DNA solution (2 µl) was used as the
MSP template. In the PCR assay, AmpliTaq Gold and 10X PCR
buffer/MgCl2 with dNTP (Applied Biosystems, Foster City, CA,
USA) were used and the results were analyzed with a GeneAmp
PCR System 9700 (Applied Biosystems).
The primer sequences were 5'-CGGGTAGGGGTATCG
TTCGC-3' (sense) and 5'-AACGAAATCCACCGCCCGCC-3'
(antisense), 159 bp. Primer sequences for the unmethylated
reaction were 5'-GTAGTTGGGTAGGGGTATTGTTTGT-3'
(sense) and 5'-AAACAACCTCCACCACCCACCCC-3' (anti-
sense), 165 bp. PCR was performed for 35 cycles (95, 65 and
72˚C for 30 sec, respectively). DNA extracted from the cultured
cell lines was prepared similarly for use in MSP analysis of the
RNA extraction and RT-PCR assay of WRN expression. Total
RNA from 6 cervical cancer-derived cell lines was extracted
using an RNeasy mini-kit (Qiagen, Valencia, CA, USA). cDNA
was synthesized from 1 µg of total RNA using SuperScriptII
Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). WRN
expression was analyzed in an RT-PCR assay using 1 µl of first-
strand cDNA as template. AmpliTaq Gold and 10X PCR buffer/
MgCl2 with dNTP were used in the PCR assay, with analysis using
a GeneAmp PCR System 9700 (Applied Biosystems). The primer
sequences were 5'-GCATGTGTTCGGAAGAGTGTTT-3'
(sense) and 5'-TGACATGGAAGAAACGTGGAA-3' (anti-
sense), 258 bp. PCR was performed for 30 cycles (94, 57 and
72˚C for 30 sec, respectively).
Demethylation treatment. Cervical carcinoma-derived SKGII
and TCO-I cells with aberrant methylation of WRN were plated
ONCOLOGY REPORTS 28: 1146-1152, 2012
on 10-cm dishes at 106 cell/dish and incubated for 72 h. 5-Aza-2-
deoxycytidine (5-aza) (Sigma), a demethylating agent, was then
added at a final concentration of 1 µM in culture medium. After
48 h of incubation, 5-aza was added again and DNA and RNA
were extracted 48 h after the second addition of 5-aza.
In vitro test of sensitivity to anticancer agents. The sensitivity of
cervical carcinoma-derived cell lines to anti-cancer agents was
determined using the collagen gel droplet-embedded culture
drug sensitivity test (CD-DST) (27). Cells were pretreated with
cell dispersion enzyme EZ (Nitta Gelatin Inc., Tokyo, Japan) for
2 h, followed by centrifugation to collect the cells. In a flask
containing collagen gel, the cells were pre-incubated for 24 h
and surviving cells that adhered to collagen gel were collected.
Cellmatrix Type CD solution was added to the collected cells,
and the suspension of cells and collagen gel was dropped onto
a 6-well plate to prepare 3 drops of 30 µl each. The suspension
was left to stand in an incubator at 37˚C in a 5% CO2 atmosphere
for 1 h for gelling and then overlaid with 4 ml/well of medium.
Cisplatin (CDDP), doxorubicin (ADM), or CPT-11 was then
added to the suspension at final concentrations of 0.2, 0.02 and
0.03 µg/ml, respectively. After 24 h, the anticancer agents were
removed by rinsing and the cells were incubated without serum
at 37˚C in 5% CO2 for 7 days. The cells were dyed with neutral
red, fixed with formalin, and dried. Images were collected by
scanning using an image analyzer and the ratio of the volume
of living cancer cells in the treated group (T) to that in the
control group (C) (T/C ratio) was determined. In general, cells
are considered to be highly sensitive to the agent when the T/C
ratio is ≤50% (28).
Transfection of small interfering RNA (siRNA). SKG-IIIb cells
were plated on 60-mm dishes at 4x105 cell/dish and transfected
48 h later with siRNA using siFector (B-Bridge International
Inc., Cupertino, CA, USA). In this procedure, 4.5 µl of siRNA
stock solution (100 µM) and 295.5 µl of serum-free MEM were
mixed in a test tube. In another tube, 13.5 µl of siFector and 286.5
µl of serum-free MEM were mixed. The solutions from the two
tubes were mixed and incubated at room temperature for 30 min.
Each dish containing SKG-IIIb cells was rinsed twice with 2 ml
of serum-free MEM and 2.4 ml of serum-free MEM was then
added. The incubated siRNA mixture was added to the dish at 0.6
ml/dish and incubated at 37˚C in 5% CO2 for 6 h. After incuba-
tion, 3 ml of MEM containing 20% serum was added to the dish.
A negative control siRNA was used as designed by B-Bridge
International Inc. The siRNA sequence corresponding to the
WRN gene was 5'-GUUCUUGUCACGUCCUCUGdTdT-3'. The
expression levels of mRNA and protein were determined 48 h
after siRNA addition. Anticancer agents were added 48 h after
siRNA addition and the sensitivity of the cells to each agent was
analyzed using the CD-DST.
Immunoblotting. siRNA-transfected SKG-IIIb cells were
rinsed with PBS, trypsinized, and centrifuged at 15,000 rpm
for 5 min at 4˚C. Protein was extracted using a Mammalian
Cell Extraction kit (BioVision Research Products, Mountain
View, CA, USA). The sample (200 µg of protein) was mixed
with sample buffer (Bio-Rad Laboratories, Hercules, CA, USA)
containing the equivalent volume of 5% β-mercaptoethanol
(Bio-Rad Laboratories) and the mixture was boiled for 5 min.
After boiling, the mixture was electrophoresed on a 10%
polyacrylamide gel and the proteins were transferred to nitro-
cellulose membranes (Bio-Rad Laboratories). The membranes
were soaked in PBS containing 1% BSA and 0.1% Tween-20 and
incubated at room temperature for 1 h for blocking. They were
then reacted with anti-β-actin antibody (AC-74, Sigma-Aldrich,
St. Louis, MO, USA; 5000-fold diluted) and anti-WRN antibody
(4H12, Abcam, Cambridge, UK; 500-fold diluted) at 4˚C over-
night, followed by rinsing three times with PBS containing 0.1%
Tween (PBS-T) for 10 min each. The anti-β-actin and anti-WRN
antibodies were reacted with anti-mouse IgG antibody (PK-6102,
Vector Laboratories, Burlingame, CA, USA) and anti-goat IgG
antibody (BA-5000, Vector Laboratories; 250-fold diluted),
respectively, at room temperature for 1 h. The membranes were
rinsed with PBS-T three times and reacted with ABC complex
(PK-6102, Vector Laboratories; pre-reacted at 4˚C for 30 min) at
room temperature for 1 h, then rinsed with PBS-T twice and PBS
once, and visualized with DAB (Sigma).
Figure 1. Analysis of aberrant DNA hypermethylation of the WRN gene in
cervical cancer cytologic specimens. (A) Cervical cancer specimens and
methylation status of WRN. CC, cervical cancer; SCC, squamous cell carci-
noma; MAD, mucinous adenocarcinoma; M, methylated; U, unmethylated.
(B) MSP analysis of WRN in cervical cancer specimens. P, positive control;
N, negative control.
MASUDA et al: WRN AND CERVICAL CANCER
Cell cycle analysis using flow cytometry. The cell cycle was
evaluated 96 h after siRNA addition. Cells were trypsinized and
rinsed twice with PBS. Supernatant was separated from the cell
pellets by centrifugation at 1,000 rpm for 5 min, and 450 µl of
PBS was added to the pellets and the mixture was pipetted well.
As the mixture was vortexed, 1 ml of cool 100% ethanol was
added. The mixture was then incubated at room temperature for
30 min for cell fixation. The cells were rinsed twice with PBS
and 500 µl of RNase was added to the pellets after supernatant
removal. The cells were then incubated at room temperature
for 20 min. Subsequently, 500 µl of propidium iodide solution
was added, the mixture was poured into a cell strainer, and the
cell cycle was determined by flow cytometry using an Epics XL
MCL (Beckman Coulter, Inc., Fullerton, CA, USA).
Aberrant methylation of the WRN gene in cervical cancer was
examined using specimens collected for cytology (Fig. 1).
Aberrant methylation was detected in 7 (33.3%) of 21 patients,
including in 2 (20%) of 10 cases of squamous cell carcinoma
and 5 (45.5%) of 11 cases of adenocarcinoma. Among 6 cervical
cancer-derived cell lines, aberrant methylation was detected in
2 cell lines, SKG-II and TCO-I (Fig. 2A), and mRNA and protein
levels for WRN were lower in these cells (Fig. 2B and C).
Changes in WRN mRNA levels in cervical cancer-derived
cell lines were analyzed before and after treatment with 5-aza,
a demethylating agent (Fig. 3A). After administration of 5-aza,
WRN mRNA increased in SKG-II and TCO-I cells, in which
aberrant methylation of WRN was found. Sensitivity to anti-
cancer drugs before and after treatment with 5-aza was analyzed
by CD-DST, based on the T/C ratio (Fig. 3B). Sensitivity to
CDDP and ADM did not change in 4 cell lines after adminis-
tration of 5-aza. For CPT-11, the T/C ratio increased to >50% in
SKG-II and TCO-I cells after administration of 5-aza, showing
decreased sensitivity to CPT-11.
Introduction of siRNA for WRN in SKG-IIIb cells decreased
the levels of WRN mRNA and protein (Fig. 4A and B). The
sensitivity of the cells to CPT-11 was increased by siRNA
treatment based on the marked decrease in the T/C ratio in the
CD-DST (Fig. 4C). Flow cytometry indicated that the percentage
of S-phase cells increased from 28.6 to 34.3% after siRNA for
WRN was introduced into SKG-IIIb cells (Fig. 4D).
Figure 2. (A) MSP analysis of the WRN gene in cervical cancer-derived cell lines. Aberrant DNA hypermethylation of WRN was observed in SKG-II and TCO-1
cells. (B) Analysis of WRN expression in cervical cancer-derived cell lines using RT-PCR. WRN expression was decreased in SKG-II and TCO-1 cells, which
had aberrant DNA hypermethylation of the WRN gene. (C) Western blot analysis of WRN in cervical cancer-derived cell lines. The WRN protein level was
decreased in SKG-II and TCO-1 cells, which had aberrant DNA hypermethylation of the WRN gene.
Figure 3. (A) Demethylation analysis of the WRN gene in cervical cancer-
derived cell lines using RT-PCR. Treatment with a demethylating agent (5-aza)
reactivated WRN expression in SKG-II and TCO-1 cells, which had aberrant
DNA hypermethylation of the WRN gene. (B) Changes in sensitivity (T/C ratio)
of cervical cancer-derived cell lines to anticancer agents after treatment with
5-aza. M, methylated; U, unmethylated.
ONCOLOGY REPORTS 28: 1146-1152, 2012
This study provided the first evidence of a relationship of
WRN promoter methylation with cervical cancer, with aberrant
methylation of WRN detected in 33.3% of specimens of cervical
cancer and in two cervical cancer-derived cell lines. Decreased
WRN mRNA and protein levels were also found in both cell lines.
These results suggested that aberrant methylation of WRN plays
an important role in cervical cancer. The sensitivity to CPT-11
of cervical cancer cells with aberrant methylation of WRN was
decreased by treatment with a demethylating agent. This treat-
ment also increased the level of WRN mRNA, consistent with
the general effect of demethylating agents on expression of many
Selective downregulation of WRN expression using siRNA
increased the sensitivity of cervical cancer cells to CPT-11, but
not to other anticancer agents. Several previous studies have
shown that WRN inactivation increases the anticancer effect of
CPT-11 (13,21). CPT-11 acts on the covalent complex of topoi-
somerase I (Top-I) and DNA, and inhibits DNA replication
and causes strand breaks (29-31). CPT-11 acts in S-phase and
causes activation of S-phase checkpoint function via the ATR
(ataxia and Rad-related protein)-CHK1 (checkpoint kinase-1)
pathway (32-34). WRN is involved in the ATR-CHK1 pathway
by recognizing the Top-I-DNA complex and detecting replica-
tion-derived DNA structures or unresolved positive supercoils
(35,36). Thus, if ATR, CHK1 and WRN have reduced activity,
cells are hypersensitive to CPT-11, and administration of CPT-11
to inactivated WS cells increases S-phase DSBs and unresolved
recombination structures. A similar effect does not occur with
etoposide, a topoisomerase II inhibitor (37). The S-phase check-
point is activated upon DNA damage and is regulated by ataxia
telangiectasia mutated (ATM) and ATM and Rad3-related
protein (ATR) kinases. WRN is related to both kinases (38,39).
Cells with inactivated WRN proceed to S-phase earlier than
wild-type cells; however, the slow progress in late S-phase for
cells with inactivated WRN causes the final S-phase lengths to
be equal (40). In this study, siRNA for WRN produced a small,
but insignificant, increase in the number of S-phase cells.
In chemotherapy for cervical cancer, the response rate to
cisplatin is 20-30% (24) and that to monotherapy with CPT-11
has been found to be 24% (25). A combination of cisplatin
and CPT-11 has a good response rate of 59% and is effective
therapy (41). Taxanes also play an important role in chemo-
therapy for cervical cancer, with response rates of 17% for
taxane monotherapy (42) and 46% for cisplatin and paclitaxel
(TP) combination therapy for recurrent or advanced cervical
cancer, making this regimen the current standard of care
(43). We previously showed a relationship between aberrant
methylation of CHFR and sensitivity to taxanes, and suggested
that aberrant methylation of CHFR could serve as a molecular
marker for the sensitivity of cervical cancer to anticancer drugs
(44). Thus, examination of aberrant methylation of CHFR and
WRN in specimens collected for cytology may be useful for
prediction of treatment outcome before administration of anti-
The standard treatment for cervical cancer is concurrent
chemoradiotherapy from stages IB bulky to IIB (45). Neoadjuvant
chemotherapy (NAC) is a promising approach for reduction
of tumor size to increase the number of patients indicated for
Figure 4. (A) RT-PCR after WRN knockdown by siRNA in SKG-IIIb cells. (B) Western blot analysis after WRN knockdown by siRNA in SKG-IIIb cells. (C) siRNA-
induced changes in sensitivity (T/C ratio) of SKG-IIIb cells to anticancer agents. After suppression of WRN expression, only sensitivity to CPT-11 was increased.
(D) Cell cycle analysis of SKG-IIIb cells using flow cytometry. After suppression of WRN expression, the percentage of cells in the S-phase increased.
MASUDA et al: WRN AND CERVICAL CANCER
surgery and reduce distant metastasis through an effect on
micrometastasis (46). It has been suggested that NAC could be
used in stages IB2 to IIIB (47), but the efficacy of NAC remains
inconclusive (48). A disadvantage of NAC is that the tumor may
grow prior to the main therapy in cases that are non-responsive
to NAC. However, this concern may be avoided by choice of the
most effective chemotherapy based on evaluation of methylation
of CHFR and WRN in specimens collected before initiation of
NAC. This approach may represent a new therapeutic strategy
for cervical cancer.
In conclusion, our data suggest that aberrant methylation of
WRN plays an important role in carcinogenesis and sensitivity
to CPT-11 of cervical cancer. This is the first report to show a
relationship between the methylation of the WRN gene and sensi-
tivity to CPT-11 in gynecologic cancer.
The authors gratefully acknowledge grant support from the
Japan Society for the Promotion of Science (JSPS) through a
Grant-in-Aid for Scientific Research (KAKENHI), a Grant-
in-Aid for Scientific Research (B) (22390313), a Grant-in-Aid
for Scientific Research (C) (22591866), and a Grant-in-Aid
for Young Scientists (B) (21791573); the Ichiro Kanehara
Foundation; Kobayashi Foundation for Cancer Research; and
the Keio University Medical Science Fund through a Research
Grant for Life Sciences and Medicine.
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