Epigenetic silencing of the imprinted gene ZAC by DNA methylation is an early
event in the progression of human ovarian cancer
Tetsuya Kamikihara1,2, Takahiro Arima1, Kiyoko Kato1, Takao Matsuda1, Hidenori Kato1, Tsutomu Douchi2,
Yukihiro Nagata2, Mitsuyoshi Nakao3and Norio Wake1*
1Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation,
Kyusyu University, Oita, Japan
2Department of Obstetrics and Gynecology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan
3Department of Tumor Genetics and Biology, Kumamoto University School of Medicine, Kumamoto, Japan
ZAC is a paternally expressed, imprinted gene located on chromo-
some 6q24, within a region known to harbor a tumor suppressor
gene for several types of neoplasia, including human ovarian can-
cer (HOC). We have failed to identify genetic mutations in the
ZAC gene in tumor material. Many imprinted genes contain dif-
ferentially allele-specific-methylated regions (DMR) and harbor
promoter activity that is regulated by the DNA methylation. Aber-
rant DNA methylation is a common feature of neoplasia and
changes in DNA methylation at the ZAC locus have been reported
in some cases of HOC. We investigated the DNA methylation and
ZAC mRNA expression levels in a larger sample of primary HOC
material, obtained by laser capture microdissection. ZAC mRNA
expression was reduced in the majority of samples and this corre-
lated with hypermethylation of the ZAC-DMR. Treatment of
hypermethylated cells lines with a demethylating agent restored
ZAC expression. Our studies indicate that transcriptional silencing
of ZAC is likely to be caused by DNA methylation in HOC. Forced
expression of ZAC resulted in a reduction in proliferation and
marked induction of apoptotic cell death. The ZAC-mediated
apoptosis signal is p53-independent and eliminated by inhibitors
of caspase 3, 8 and 9. Reduced expression of ZAC would therefore
favor tumor progression. As there were no significant differences
in either DNA methylation or expression of ZAC mRNA between
localized and advanced tumors, our data indicates that loss of
ZAC is a relatively early event in HOC. (Supplementary material
for this article can be found on the International Journal of Cancer
' 2005 Wiley-Liss, Inc.
Key words: genomic imprinting; human ovarian cancer; ZAC;
DNA methylation; tumor suppressor gene
Genomic imprinting plays an important role in mammalian
development, growth and cell differentiation.1Mutations that
affect the epigenetic status of imprinted loci underlie a number of
diseases, including developmental abnormalities, congenital dis-
eases and malignant tumors.2Alterations in the expression of
imprinted genes is one of the most common changes seen in can-
cer.3,4Several imprinted genes including ARH1,5PEG36and
ZAC7,8function as tumor suppressor genes suggesting a direct link
between loss of imprinting, either by epigenetic changes or chro-
mosomal deletions, and failure of tumor suppressor mechanisms.
Global changes in DNA methylation occur during carcinogene-
sis. Although there is an overall decrease in DNA methylation,
some CpG island sequences become hypermethylated.9Hyperme-
thylation of CpG islands seems to be responsible for the transcrip-
tional silencing of critical genes, including caretaker genes and
suppressor genes. Transcriptional silencing of these genes may be
selected during the development and progression of a variety of
Methyl-CpG binding domain (MBD) proteins have been identi-
fied as candidate mediators for silencing methylated DNA.10
MeCP2 is postulated to form a5-mCpG-dependent transcriptional
repression complex with Sin3a and the histone deacetylase
(HDAC).11MBD2, which also binds methylated DNA, is part of
the NuRD (nucleosome remodeling and deacetylation) complex
containing HDAC, MBD3 and Mi-2.12The role of HDAC in
transcriptional silencing in cancer is unclear. For some genes,
treatment with trichostatin A (TSA), a HDAC inhibitor, is suffi-
cient to reverse repression associated with CpG island hyperme-
thylation, whereas for other genes, TSA treatment alone is unable
to restore gene expression.13Combined treatment with TSA and
an inhibitor of DNA methylation has been reported to trigger the
expression of silenced cancer genes carrying somatic CpG island
ZAC was identified originally, along with p53, in a functional
screen by their common ability to induce expression of the
PACAP (pituitary adenylate cyclase activating polypeptide)
Type I receptor gene.14ZAC and p53 are both pleiotropic regula-
tors that have a number of similar activities; both regulate cell
cycle, apoptosis and nuclear receptor functions and both interact
physically and functionally with CBP and p300 that serve as inte-
grators of multiple signaling pathway.15,16P53 has a pro-apoptotic
activity and recent experiments indicate that this activity mainly
involves the mitochondrial pathway that is dependent on the activ-
ity of ApafI and caspase 9.17ZAC functions to enhance the activ-
ity of p53 on ApafI and may itself be activated by p53.18,19
We identified ZAC in a screen for imprinted genes8and pro-
posed that deregulation of expression of ZAC may be a factor in
neonatal diabetes mellitus (TNDM). ZAC encodes an imprinted
zinc finger protein that localizes to the nuclear compartment and
functions as a transcription factor with anti-proliferative activity.14
ZAC is expressed only from the paternal allele and maps to human
chromosome 6q24. This region is involved frequently in allelic
losses in many tumors.20–22Loss of ZAC expression has been
reported in a number of tumor types.23–26Lot1 (Lost on transfor-
mation), the rat orthologue of ZAC, was cloned from rat ovarian
surface epithelial cells transformed spontaneously in vitro27sug-
gesting an association of ZAC with ovarian cancer. The frequent
LOH of 6q24 in ovarian cancer28and some preliminary data29
suggest that loss of ZAC expression may play a role in the initia-
tion and/or progression of human ovarian cancer.
In mice, imprinting of Zac1 may be regulated by a differentially
methylated CpG island (DMR) that partially overlaps the Zac1
and Hymai genes.29–31This region shows gamete-specific DNA
methylation that persists throughout pre- and post-implantation
development. Within this DMR, there is a region that exhibits a
high degree of homology between mouse and human that acts as a
strong transcriptional repressor when DNA methylated. We have
Grant sponsor: Ministry of Health and Welfare of Japan.
The first two authors contributed equally to this paper.
*Correspondence to: Department of Molecular Genetics, Division of
Molecular and Cell Therapeutics, Medical Institute of Bioregulation,
Kyusyu University, 4546, Tsurumihara, Beppu, Oita 874-0838, Japan.
Received 20 August 2004; Accepted after revision 29 November 2004
Published online 4 March 2005 in Wiley InterScience (www.interscience.
Int. J. Cancer: 115, 690–700 (2005)
' 2005 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
proposed that this DMR is an imprint control region (ICR) that
regulates expression of the imprinted genes within this domain,
including ZAC .32
Hypermethylation of the human ZAC locus has been reported in
some human ovarian cancers in a previous study.29Only 4 pri-
mary samples were examined and ZAC mRNA expression levels
not tested in these samples. ZAC mRNA expression levels was
found to be reduced in ovarian cancer cell lines but the authors did
not find a correlation between the increased methylation and
decreased ZAC mRNA expression levels in these samples. The
CpG rich region examined is not within the region we have pro-
posed as the DMR. We have now examined the DNA methylation
status of the DMR in a larger sample of ovarian cancer materials
and correlated its methylation status with ZAC mRNA expression
levels. We firstly examined human ovarian cancer (HOC) cell
lines. Our initial results suggested an association between
increased DNA methylation and loss of ZAC mRNA expression
levels in HOC. Because epigenetic changes can occur as a conse-
quence of in vitro culturing33we also carried out our analysis on
28 primary cancer tissues obtained by laser capture micro dissec-
tion (LMD) from surgically removed tissues. This method enables
small samples of tissue to be obtained from specific tissue sections
for RNA and DNA analysis. We report that the ZAC DMR, which
contains the ZAC promoter, is subject to epigenetic changes of
ovarian cancers and that the observed increase in DNA methyla-
tion correlates with loss of expression of ZAC mRNA in both
localized and advanced tumor groups. We suggest that the changes
in ZAC mRNA expression are relative early event in the progres-
sion of HOC. Furthermore, we have used 2 HOC cell lines to
experimentally address whether DNA methylation or histone ace-
tylation plays a role in repressing ZAC expression.
The function of ZAC in ovarian cells is unknown. We have
examined the effect of exogenous ZAC expression in HOC cell
lines that lack the endogenous ZAC. We examined tumorigenicity,
cell cycle regulation and apoptosis. Our studies suggest that
silencing of ZAC mRNA expression could contribute to ovarian
cancer development by allowing increased cellular proliferation
with a simultaneous escape from apoptosis.
Material and methods
Human ovarian cancer (HOC) cell lines (KK, TYK-nu, PA-1,
MH, KF, HTOA, SKOV-3, MCAS, HAC2, RMG and KM) were
used in our study. The source of these cells is as described.34They
were grown in either DMEM or RPMI1640 supplemented with
10% FBS before the isolation of DNA and RNA.
Northern blot analysis
We prepared total RNA from HOC cell lines using ISOGEN
(Nippon Gene, Tokyo, Japan). The ZAC cDNA (GenBank acces-
sion number AA463204) was used as a probe. Northern blot anal-
ysis was carried out as described.32The same membrane was
reprobed with a GAPDH probe (Clontech, Tokyo, Japan) as a con-
trol for loading of RNA.
Cancerous tissues and adjacent non-cancerous tissues were
excised from the patients during surgery, after informed consent had
been obtained. Frozen sections (8 ?m) of the samples were made
and mounted on glass slides covered for the micro dissection system
(Leica Microsystems, Tokyo, Japan) essentially as described previ-
ously.35Total RNA from these sections was extracted with RNeasy
mini kit (Qiagen, New York, NY) according to the manufacturer’s
protocol. The amplification of mRNA was made from total RNA
using Agilent Low RNA Input Fluorescent Linear Amplification kit
(Agilent Technologies, NY, USA). Amplified RNAs were treated
with DNAseI (Roche, Mannheim, Germany) and reverse transcribed
to single-stranded cDNAs using oligo (dT) primer and the RNA
PCR core kit (Roche, Piscataway, NJ). We prepared appropriate
dilutions of each cDNA for PCR amplification by monitoring
the GAPDH transcript. Primer sequences were as follows:
FIGURE 1 – Expression analysis
of human ZAC in normal human
ovary and human ovarian cancer
cell lines and tissue. (a) Expression
of ZAC in human ovary by in situ
expressed in the ovarian surface
epithelium (left). This is the devel-
opmental origin of most ovarian
cancers. It is also expressed in the
follicle epithelial cells (right). (b)
ZAC expression in 11 human ovar-
ian cancer cell lines by Northern
blot analysis. The level of expres-
sion of ZAC was reduced in 4 lines
(Lanes 4, 6, 8, 9) and completely
absent in 7 lines. Lanes: KK (1),
TYK-nu (2), PA1 (3), MH (4), KF
MCAS (8), HAC2 (9), RMG (10)
and KK (11). C1 and C2 are con-
GAPDH cDNA probe was used as
a control for RNA levels.
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