Expression of the RERG gene is gender-dependent in hepatocellular carcinoma and regulated by histone deacetyltransferases.

Abstract

Ras-related, estrogen-regulated, and growth-inhibitory gene (RERG) is a novel gene that was first reported in breast cancer. However, the functions of RERG are largely unknown in other tumor types. In this study, RERG expression was analyzed in hepatocellular carcinomas of human patients using reverse transcriptase PCR analysis. In addition, the possible regulation of RERG expression by histone deacetyltransferases (HDACs) was studied in several cell lines. Interestingly, the expression of RERG gene was increased in hepatocellular carcinoma (HCC) of male patients (57.9%) but decreased in HCC of females (87.5%) comparison with paired peri-tumoral tissues. Moreover, RERG gene expression was increased in murine hepatoma Hepa1-6 cells, human breast tumor MDA-MB-231 cells, and mouse normal fibroblast NIH3T3 cells after treated by HDAC inhibitor, trichostatin A. Our results suggest that RERG may function in a gender-dependent manner in hepatic tumorigenesis and that the expression of this gene may be regulated by an HDAC-related signaling pathway.

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INTRODUCTION
Ras-related, estrogen-regulated, and growth-inhibitory
gene (RERG) is a novel gene that was first reported in breast
cancer (1). Like Ras, RERG protein exhibits intrinsic GDP/
GTP binding and GTP hydrolysis activity. However, unlike
Ras proteins, RERG lacks a known recognition signal for
COOH-terminal prenylation and is localized primarily in
the cytoplasm. RERG mRNA expression is induced rapidly
in MCF-7 breast carcinoma cells stimulated with beta-estra-
diol, whereas it is repressed by tamoxifen treatment. The
expression of RERG protein in these cells resulted in signif-
icant inhibition of both anchorage-dependent and anchorage-
independent growth in vitro and inhibited tumor formation
in nude mice. Moreover, RERG expression was decreased or
lost in a significant percentage of primary human breast tu-
mors with poor clinical prognosis. Importantly, high levels
of RERG expression correlate with the expression of a set of
genes that define an estrogen receptor-positive breast tumor
subtype and are associated with a slow rate of tumor cell pro-
liferation and a favorable prognosis for these cancer patients.
The study suggested that the loss of RERG expression con-
tributes to breast tumorigenesis. However, there have been
no further reports about this gene, and its functions in tumors
of other cell types are largely unknown.
Hepatocellular carcinoma (HCC) is the one of the world’s
most common malignancies (2). A general characteristic of
HCC across different geographical areas is the striking male
prevalence (3, 4). In addition, animal studies have indicated
that male rodents are more susceptible to hepatocarcinogen-
esis that either occurs spontaneously or is chemically induced,
as well as being more susceptible to chronic viral infection in
Ai-Guo Wang*, Wan Fang*,
Ying-Hao Han*, Sang-Mi Cho*,
Jong Young Choi
Wook Hwan Kim
Moon Gi Park¶, Dae-Yeul Yu*,
Nam-Soon Kim*, Dong-Seok Lee*
�, Kee Ho Lee
�, Jin Man Kim‖,
�,
,**
Laboratory of Human Genomics*, Korea Research
Institute of Bioscience and Biotechnology (KRIBB),
Daejeon; Department of Internal Medicine
St. Mary’s Hospital, The Catholic University of Korea,
Seoul; Laboratory of Molecular Oncology
Institute of Radiological and Medical Sciences, Seoul;
Depatment of Surgery
University, Suwon; Departments of Pathology‖,
College of Medicine, Chungnam National University,
Daejeon; Department of Surgery¶, SUN General
Hospital, Daejeon; Animal Resources Science**,
Kangwon National University, Chuncheon, Korea
�, Kangnam
�, Korea
�, School of Medicine Ajou
Address for correspondence
Dong-Seok Lee, Ph.D.
Animal Resources Science, Kangwon National
University, 192-1 Hyoja 2-dong, Chuncheon 200-701,
Korea
Tel : +82.33-250-8612, Fax : +82.33-251-7719
E-mail : dslee@kangwon.ac.kr
*This work was supported by the 21st century Frontier
Program in the Functional Human Genome Project of
Korea (Grant HGC0300324) and the Ministry of Health
and Welfare of Korea (Grant 02-PJ2-PG1-CH12-0002).
891
J Korean Med Sci 2006; 21: 891-6
ISSN 1011-8934
Copyright � The Korean Academy
of Medical Sciences
Expression of the RERG Gene is Gender-Dependent in Hepatocellular
Carcinoma and Regulated by Histone Deacetyltransferases
Ras-related, estrogen-regulated, and growth-inhibitory gene (RERG) is a novel gene
that was first reported in breast cancer. However, the functions of RERG are largely
unknown in other tumor types. In this study, RERG expression was analyzed in hep-
atocellular carcinomas of human patients using reverse transcriptase PCR analysis.
In addition, the possible regulation of RERG expression by histone deacetyltrans-
ferases (HDACs) was studied in several cell lines. Interestingly, the expression of
RERG gene was increased in hepatocellular carcinoma (HCC) of male patients
(57.9%) but decreased in HCC of females (87.5%) comparison with paired peri-tu-
moral tissues. Moreover, RERG gene expression was increased in murine hepa-
toma Hepa1-6 cells, human breast tumor MDA-MB-231 cells, and mouse normal
fibroblast NIH3T3 cells after treated by HDAC inhibitor, trichostatin A. Our results
suggest that RERG may function in a gender-dependent manner in hepatic tumori-
genesis and that the expression of this gene may be regulated by an HDAC-relat-
ed signaling pathway.
Key Words : Ras-Related Estrogen-Regulated and Growth-Inhibitory Gene (RERG); Carcinoma, Hepatocel-
lular; Sex; Histone Acetyltransferases
Received : 13 December 2005
Accepted : 27 February 2006

Page 2

experimental models (5, 6). Therefore, there appears to be a
gender-dependent regulation of common molecular mecha-
nisms that leads to a predominance of HCC in human and
rodent males. The mechanisms responsible for this gender
difference are not known. In addition, the genetic basis of
hepatocarcinogenesis is still poorly understood (7).
Histone deacetyltransferases (HDACs) participate in a vari-
etyof cellular processes, including transcription, DNA repli-
cation,and cell cycle progression. It has been suggested that
these enzymes are associated with proliferative diseases such
as cancer (8). Consequently, HDACs have recently attracted
interest as novel targets for anti-tumor therapies. HDAC in-
hibitors (HDACIs) have been shown to induce growth arrest,
differentiation, and apoptosis of cancer cells in vitro and in
vivo (9). In addition, few or no side effects were observed in
animal experiments and clinical trials of HDACIs within the
therapeutic range studied, which enhances the potential use
of these agents for therapeutic applications (10). However,
the changes in the genetic profile that are induced by HDACs
remain to be investigated.
In this study, the expression of the RERG gene in hepatic
tumorigenesis and the regulation of gene expression by HD-
ACs were studied. Gene expression was analyzed by RT-PCR
of human and mouse liver tumors as well as of several tumor
cell lines treated with the HDACI, trichostatin A (TSA).
MATERIALS AND METHODS
Patients
Primary HCC tumors and adjacent peri-tumoral tissues
were obtained from 27 patients (8 females and 19 males) at
the Catholic University of Korea (Department of Internal
Medicine, Seoul, Korea), Korea Institute of Radiological and
Medical Sciences (Laboratory of Molecular Oncology, Seoul,
Korea), School of Medicine Ajou University (Department of
Surgery, Suwon, Korea), College of Medicine, Chungnam
National University (Department of Pathology, Daejeon,
Korea), and College of Medicine, Yeungnam University (De-
partment of Biochemistry & Molecular Biology, Daegu, Ko-
rea). The tissue samples were snap-frozen and stored in liq-
uid nitrogen until RNA extraction. In all, 54 samples were
analyzed.
Mouse hepatic tumor samples
Transgenic animals expressing the H-ras12V oncogene,
which specifically induced the formation and growth of hep-
atic tumors, have been previously described (11). In the pre-
sent study, hepatic tumoral tissues and adjacentperi-tumoral
tissues from male (9 months of age) and female (14 months
of age) transgenic mice were analyzed for RERG gene expres-
sion. For control experiments, wild-type male and female
littermates were used. Tissue samples were snap-frozen and
stored in liquid nitrogen until RNA extraction.
Cell culture and treatment
The role of HDACs in regulating RERG gene expression
was investigated in murine hepatoma Hepa1-6 cells, human
breast tumor MDA-MB-231 cells, and mouse normal fibrob-
lastNIH3T3 cells. All cell lines were obtained from the Ame-
rican Type Cell Culture (ATCC, Rockville, MD, U.S.A.) and
were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) (Gibco, Carlsbad, CA, U.S.A.) containing 10% fetal
bovine serum (FBS). Almost all HDACs have approximately
the same sensitivity to different HDACIs, and TSA (Sigma,
St. Louis, MO, U.S.A.) is one of the most efficient HDACIs
identified thus far (10). For this reason, TSA (0, 0.5 M, or
1 M) (Sigma, St. Louis, MO, U.S.A.) was used in the present
study to inhibit HDAC activity. Before treatment, the cells
were re-plated and cultured for 24 hr. Subsequently, the medi-
umwas replaced with fresh medium containing the specified
concentration of TSA. After treatment for 24 hr, the medium
was washed out, and total RNA was immediately extracted.
Mice hepatectomy
Hepatectomy was performed according to previously des-
cribed methods (12), using the C57BL/6J inbred mouse as
the experimental animal model. After hepatectomy, regener-
atingliver tissues were sampled at 0-4, and 5 days. The hep-
atectomy experiments were performed independently for 3
times and 6 mice were hepatectomized for the 6 sample time
points at each time. The samples were snap-frozen and stored
in liquid nitrogen until RNA extraction.
RT-PCR analysis
Gene expression was analyzed by RT-PCR. Total RNA was
isolated from tissues or TSA-treated cells using the TRIzol
reagent (Invitrogen Life Technologies Inc., Carlsbad, CA,
U.S.A.). RT-PCR was carried out using a reverse transcrip-
tion system (Promega Corp., Madison, WI, U.S.A.) accord-
ing to the manufacturer’s instructions. The primers used to
detect RERG expression were: the sense primer, 5′ -ATGG-
CTAAAAGTGCGGAGG T-3′ , and the anti-sense primer,
5′ -CTAACTACTGATTTT GGTGA-3′ , for the human sam-
ples and the sense primer, 5′ -ATGGCTAAGAGCGCAGA-
GGTCAAG-3′ , and the anti-sense primer, 5′ -CTAACTA-
CTGATTTTGGTGAGCA TC-3′ , for the mouse samples.
The primers used for detecting variant ER- are shown in
Table 2. As a loading control, GAPDH expression was mea-
sured in a separate RT-PCR experiment. The production of
a single band of the expected size on RT-PCR was recognized
as amplification of the target genes.
892
A.-G. Wang, W. Fang, Y.-H. Han, et al.

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Gender-Dependent RERG Gene Expression in Hepatocellular Carcinoma
893
Statistical analysis
The differences between the experimental groups were test-
ed for statistical significance using the chi-squared test. p-
values <0.05 were considered to be significant.
RESULTS
Gender-dependent expression of the RERG gene in
human HCC
To determine whether the pattern of RERG expression is
the same in HCC as in the case of breast cancer (1), 27 paired
samples of tumoral and peritumoral tissue were collected from
human HCC patients, and RERG gene expression was exam-
ined by RT-PCR. In seven of eight (87.5%) female patients,
gene expression was decreased in hepatic tumor samples com-
pared with the expression in peri-tumoral tissues (Table 1,
Fig. 1). This result was consistent with the pattern of RERG
expression reported in breast cancer. However, the level of
RERG expression was increased compared with the level in
peri-tumoral tissues in 11 of 19 (57.9%) hepatic tumor tis-
sues from males. In 7 of 19 (36.8%) patients, there was no
difference in the expression level of RERG between tumoral
and peri-tumoral tissues (Table 1). This result was in contrast
to the expression pattern of RERG reported in breast cancer.
The RERG expression patterns in hepatic tumors of males
and females were significantly different (Table 1, Fig. 1). These
results indicate that RERG has different, gender-dependent
functions in hepatic tumorigenesis.
Expression of the estrogen receptor variant ER- in HCC
tissues of human patients
The estrogen receptor (ER) variant ER- has been suggested
to play important roles in breast cancer (13) and hepatocarci-
nogenesis (14). As RERG is an estrogen-regulated gene involv-
ed in breast cancer (1), the expression of the mutant ER- may
influence the regulation of RERG expression in HCC. This
hypothesis was tested by measuring ER- expression in tumors
of HCC patients. The results show that, while ER- was com-
monly expressed in the tumor and in peri-tumoral tissues of
HCC patients, the expression levels were usually higher in the
tumor tissues (Fig. 2). In addition, no significant difference
was found in ER- expression between females and males
(Table 3).
RERG expression is increased in hepatic tumors of male
but not female H-ras12V transgenic mice
To find out whether the pattern of RERG expression in
murine HCC resembles that in human HCC, the expression
of the gene was measured in hepatic tumors of H-ras12V
transgenic mice. The H-ras12V transgenic mice express the
H-ras12V oncogene and develop tumors in liver tissues, thereby
providing an animal model of HCC. Furthermore, hepatic
tumors in the transgenic mice occur predominately in males
(11). The results show that RERG expression in the peri-
tumoral tissues of transgenic females did not differ signifi-
cantly from that of control littermates, while the levels in
RERG
GAPDH
M NTNTNTNTNTNT
FemaleMale
Fig. 1. RERG expression in human hepatocellular carcinoma. RERG
expression was analyzed by RT-PCR. GAPDH was used as a qu-
antitative control.
M, size marker; N, adjacent peri-tumoral tissues; T, tumoral tissues.
439 bp
300 bp
Wild type ER
Variant type ER
NTNT
FemaleMale
Fig. 2. Expression of the estrogen receptor variant ER- in human
HCC. ER- expression was analyzed by RT-RCR.
T, tumoral tissues; N, adjacent peri-tumoral tissues.
N/T+N/TN+/T
Table 1. The expression level of RERG gene in human HCC
patients by RT-PCR
Female
Male
1/8 (12.5%)
11/19 (57.9%)*
0/8 (0%)
7/19 (36.8%)*
7/8 (87.5%)*
1/19 (5.3%)
N, peri-tumoral tissue; T, tumoral tissue; N/T+, the expression level
of RERG was higher in tumoral tissue thue than its peri-tumoral tis-
sue; N/T, the expression level of RERG was same in tumoral tissue
and peri-tumoral tissue; N+/T, the expression level of RERG was
higher in peri-tumoral tissue than its tumoral tissue. *Indicated signif-
icantdifference compared within the same column.
Forward primers Reverse primersWt sizeVt size
5′ -GGAGACATGAG
AGCTGCCAAC-3′
5′ -CCAGCAGCATGTC
GAAGATC-3′
439 bp300 bp
Table 2. The primers for variant type ER- determination
Wt, wild type; Vt, variant type; ER, estrogen receptor.
Females Males
Peri-tumoral tissues
HCC tissues
8/8 (100%)
8/8 (100%)
17/19 (89.4%)
18/19 (94.7%)
Table 3. Comparison of the expression of ER- variant type by
sex
hepatocellular carcinoma.

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894
A.-G. Wang, W. Fang, Y.-H. Han, et al.
tumor tissues were slightly lower (Fig. 3A). In males, two
of three transgenic mice showed increased expression of the
RERG gene in peri-tumoral liver and tumor tissues, while
the levels in the third mouse did not differ from those in nor-
mal littermates (Fig. 3B). These results indicate that RERG
expression in murine hepatic tumorigenesis is sex-dependent
in our H-ras12V transgenic mice.
RERG expression levels do not change during the normal
proliferation of hepatocytes
To examine the importance of RERG in normal hepatocyte
proliferation, the gene expression was measured in liver tis-
sues of hepatectomized mice. After hepatectomy, the remain-
inghepatocytes undergo rapid proliferation in order to restore
the liver to its original size. Thus, proliferating liver tissue is
an excellent system in which to study the proliferation process
and the expression of related genes. RERG expression was
measured in regenerating liver tissues on days 0-4 and 5 after
hepatectomy. However, no differences in expression level were
detected (Fig. 4) after hepatectomy. This result indicated that
RERG is not primarily involved in the proliferation of nor-
mal hepatocytes.
RERG gene expression is regulated by a HDAC-related
signaling pathway
HDACs are important in proliferation diseases, owing lar-
gely to the functions of these enzymes in regulating gene ex-
pression (8). It was therefore of interest to determine whether
the RERG gene is under the regulation of HDACs. Several
cell lines were treated with the HDAC inhibitor TSA, and
RERG expression was subsequently analyzed by RT-PCR.
The results show that RERG expression was regulated by
HDACs in NIH3T3, MDA-MB-231, and Hepa1-6 cells
(Fig. 5). We therefore propose that RERG plays an important
role in the inhibition of cell proliferation in several tumor cell
types, as has been reported in vitro and in vivo in breast can-
cer cells.
Previous reports have also shown that TSA treatment sig-
nificantly inhibits tumor cell proliferation (15, 16). We ob-
tained similar results in the tumor cell lines MDA-MB-231
and Hepa1-6, whereas no effect was observed in the normal
cell line NIH3T3 (data not shown). Thus, it is likely that the
inhibition of cell proliferation in response to TSA depends on
several factors.
DISCUSSION
RERG is novel gene that was first reported in breast can-
cer (1). Although RERG is thought to inhibit cell prolifera-
tion, its detailed biological functions are largely unknown.
In this study, the gender-dependent expression of RERG in
HCC and its regulation by HDACs were studied, yielding
novel insights into the function of this gene.
The expression pattern of the RERG gene in female HCC
patients is consistent with that previously described in breast
cancer (1). Like the breast, the liver is a hormone-sensitive
organ, and sex hormones are known to affect many functions
of the mammalian liver (17, 18). For example, both male and
female livers contain androgen receptors (19-21) as well as
high-affinity, low-capacity estrogen receptors (22, 23). Further-
RERG
GAPDH
M WtNT
B
A
Male Tg
RERG
GAPDH
M WtNT
Female Tg
Fig. 3. RERG expression in liver tissues of and H-ras12V transgenic
mice. RERG expression was analyzed by RT-PCR. Liver or tumor
tissues from 9-month-old male (A) and 14-month-old female (B)
H-ras12V transgenic mice were sampled.
RERG
GAPDH
M N-H012345
Fig. 4. RERG expression in liver tissues of hepatectomized mice.
RT-PCR analysis of RERG expression in liver tissues on days 0-4,
and 5 after hepatectomy. Three independent hepatectomy exper-
iments were performed. GAPDH was used as a quantitative con-
trol. M, size marker; Wt, normal control littermate; N, peri-tumoral
liver tissues; T, liver tumor tissues; Tg, H-ras12V transgenic mice;
N-H, non-hepatectomized mice.
RERG
GAPDH
RERG
GAPDH
RERG
GAPDH
NIH3T3
MDA-MB-231
Hepa1-6
MCo. 0.5 M 1 M
TSA treatment
Fig. 5. RERG expression in trichostatin A (TSA)-treated cells. Expres-
sion of the gene was analyzed by RT-PCR. NIH3T3, MDA-MD0-
231, and Hepa1-6 cells were treated with 0.5 M or 1 m of the
histone deacetyltransferases (HDAC) inhibitor TSA. GAPDH was
used as a quantitative control. M, size marker.

Page 5

more, variant type ER-
tissues. This variant has been suggested to play an important
role in breast cancer and hepatocarcinogenesis (12, 13). As
estrogen-dependent regulation of the RERG gene has been
suggested (1), it seems likely that the presence of the mutant
ER- in these two tumor types is related to the decrease in
RERG gene expression. Conversely, tumor cells must be able
to overcome an important barrier, given that RERG has been
suggested to play a role in inhibiting the proliferation of bre-
ast cancer cells. Taken together, the results suggest that the
RERG has the same growth-inhibiting function in female
hepatocarcinogenesis as in breast cancer.
By contrast, in male HCC patients, RERG expression in
tumor tissues was significantly increased, suggesting that the
gene does not inhibit but rather promotes hepatocarcinogen-
esis in males. Studies of the variant ER- showed that there
was no difference in the expression of the mutant receptor
between males and females. We therefore propose that RERG
has different, gender-dependent functions; however, the mech-
anismsexplaining this difference remain to be elucidated.
The remaining hepatocytes of hepatectomized mice prolif-
erate rapidly within 1 week in order to restore the liver its
original size. Thus, it is an excellent animal model to study
normal proliferation processes in vivo. Experiments measur-
ing RERG expression in hepatectomized mice were aimed
at determining the function of RERG in normal cellular pro-
liferation, based on the reported ability of this gene toinhib-
it tumor cell proliferation. It was surprising that RERG
expression remained constant during liver regeneration in he-
patectomized mice. This observation suggests that the RERG
may be not involved in regulating normal cell proliferation.
However, the protein level of RERG remained to be exam-
ined to find out the possible regulation in post-transcription
step.
In vitro experiments showed that RERG expression was
significantly increased in normal NIH3T3 cells treated with
TSA, an inhibitor of HDACs; however, the growth of these
cells was not inhibited. By contrast, in tumor cells treated
with TSA, growth was inhibited and RERG expression was
significantly increased. These results suggest that RERG
mainly functions in tumor cells, perhaps as a monitor that
responds to the abnormal status of the cells. This feature of
RERG may make it a target for tumor therapy in the future.
The results of the in vitro experiments also suggest that RERG
gene expression is regulated by a HDAC-related signaling
pathway that is distinct from the ER-regulated pathway. These
results provide new insight into the function of RERG. The
regulation of the gene by multiple pathways adds further sup-
portfor an important role of RERG in cells.
In conclusion, while the detailed mechanisms remain to be
elucidated, our study offers new insights into the function of
RERG. Our results are the first to show that RERG is regu-
lated by a HDAC-related signaling pathway. In addition, in
hepatic tumorigenesis, the function of RERG may be gender
was expressed in all female HCC
dependent. These unique features of RERG in tumorigenesis
may make it a new target for tumor therapy.
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