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J Cancer Res Clin Oncol
DOI 10.1007/s00432-014-1620-8
ORIGINAL ARTICLE - CANCER RESEARCH
GPER functions as a tumor suppressor in triple‑negative breast
cancer cells
Christine Weißenborn · Tanja Ignatov · Hans‑Joachim Ochel · Serban Dan Costa ·
Ana Claudia Zenclussen · Zoya Ignatova · Atanas Ignatov
Received: 5 February 2014 / Accepted: 10 February 2014
© Springer-Verlag Berlin Heidelberg 2014
factors, such as radiation, and GPER amount inversely cor-
related with the p53 expression level.
Conclusions Overall, our results establish the protective
role in breast cancer tumorigenesis, and the cell surface
expression of GPER makes it an excellent potential thera-
peutic target for triple-negative breast cancer.
Keywords GPER · GPR30 · Breast cancer ·
Tumor suppression · TNBC
Introduction
Several distinct subtypes of invasive breast cancers, the
most common cancer types in women, have been identified
using microarray-based approach (Perou et al. 2000). One
of them, the triple-negative breast cancer (TNBC), is nega-
tive for estrogen, progesterone, and HER2 receptors, which
limits the options for optimal adjuvant therapy. The iden-
tification of potential therapeutic targets for this subtype
remains a clinical challenge (Perou et al. 2000; Schneider
et al. 2008).
More than 50 % of breast cancer patients express high
levels of the orphan G-protein-coupled receptor, GPR30,
a membrane-bound estrogen receptor (GPER) (Arias-
Pulido et al. 2010; Filardo et al. 2006; Ignatov et al. 2011;
Kuo et al. 2007; Liu et al. 2009; Tu et al. 2009), whose
expression is favorable for patients’ survival (Arias-Pulido
et al. 2010; Ignatov et al. 2011). Similar results were also
obtained for ovarian cancer patients (Ignatov et al. 2013a).
The function of GPER, however, in the disease pathology
is controversial and still a subject of intense debate. The
identification and first functional studies on GPER pro-
posed rapid nongenomic effects of estrogen (Prossnitz et al.
2008). Studies with high-affinity non-steroidal receptor
Abstract
Background The orphan, membrane-bound estrogen
receptor (GPER) is expressed at high levels in a large frac-
tion of breast cancer patients and its expression is favorable
for patients’ survival.
Methods We investigated the role of GPER as a poten-
tial tumor suppressor in triple-negative breast cancer cells
MDA-MB-231 and MDA-MB-468 using cell cycle analy-
sis and apoptosis assay. The constitutive activity of GPER
was investigated.
Results GPER-specific activation with G-1 agonist inhib-
ited breast cancer cell growth in concentration-dependent
manner via induction of the cell cycle arrest in G2/M phase,
enhanced phosphorylation of histone H3 and caspase-3-me-
diated apoptosis. Analysis of the methylation status of the
GPER promoter in the triple-negative breast cancer cells
and in tissues derived from breast cancer patients revealed
that GPER amount is regulated by epigenetic mechanisms
and GPER expression is inactivated by promoter methyla-
tion. Furthermore, GPER expression was induced by stress
C. Weißenborn · T. Ignatov · S. D. Costa · A. Ignatov (*)
Department of Obstetrics and Gynecology, University
of Magdeburg, Gerhart-Hauptmann Str 35, Magdeburg, Germany
e-mail: atanas.ignatov@gmail.com
C. Weißenborn · A. C. Zenclussen
Department of Experimental Obstetrics and Gynaecology,
University of Magdeburg, Magdeburg, Germany
H.-J. Ochel
Department of Radiotherapy, University of Magdeburg,
Magdeburg, Germany
Z. Ignatova
Department of Biochemistry and Biology, University of Potsdam,
Potsdam, Germany
J Cancer Res Clin Oncol
1 3
agonist G-1 and antagonist G-15 (Bologa et al. 2006; Den-
nis et al. 2009) suggest that GPER mediates the prolifera-
tive effects of estrogen in many estrogen-related cancers
(Albanito et al. 2007; Filardo et al. 2000; Ignatov et al.
2010b, c; Vivacqua et al. 2006). Conversely, studies in vari-
ous cell cultures show that GPER can act as an inhibitor
on cell growth and proliferation (Ariazi et al. 2010; Chan
et al. 2010; Gao et al. 2011; Holm et al. 2011; Wang et al.
2012). Thereby, GPER blocks the cell cycle progression
in G1 or G2/M phase of estrogen receptor-positive breast
cancer cells without any apoptotic effect (Ahola et al.
2002; Ariazi et al. 2010). In vivo, the GPER expression is
down-regulated during breast cancer progression, suggest-
ing the potential role of GPER in tumor suppression (Igna-
tov et al. 2013b). The influence of various environmental
and milieu-specific factors, including the presence or the
absence of estrogen (Fan et al. 2009; Ignatov et al. 2010c;
Leblanc et al. 2007), may explain in part those polarized
observations. An alternative plausible explanation might
be the fact that GPER-induced stimulation of cell prolif-
eration is mainly observed after receptor stimulation with
non-specific GPER agonists such as estrogen and tamox-
ifen (Albanito et al. 2008; Bologa et al. 2006; Filardo et al.
2000, 2002; Girgert et al. 2012; Ignatov et al. 2010c; Mag-
giolini et al. 2004). Importantly, the TNBC is negative for
estrogen receptor implying that underlying mechanisms
that control the GPER expression may significantly differ
than in other type of breast cancer.
Here, we demonstrate that GPER mediates the inhibi-
tory effect of G-1 on triple-negative breast cancer cells via
induction of the cell cycle arrest and cell apoptosis. GPER
expression is regulated by epigenetic mechanisms both in
vitro and in vivo. Moreover, GPER expression is stimulated
via radiation which acts as DNA-damaging agent. Our
results propose that GPER may act as a potential therapeu-
tic target in TNBC.
Materials and methods
Cell culture and treatment
MDA-MB-231, MDA-MB-468, MCF-7, and HEK-293
cells were obtained from Cell Lines Services (Germany)
and routinely cultured in DMEM/F12 (PAN Biotech)
supplemented with 10 % FBS, 100 U/ml penicillin, and
100 μg/μl streptomycin at 37 °C in a humidified 5 % CO2
atmosphere. If necessary, the cells were synchronized by
estrogen withdrawal for 24 h using phenol red-free medium
supplemented with 10 % charcoal-stripped steroid-depleted
FBS. Thereafter, the cells were treated as indicated in the
figure legends, and the cell count was measured with a
Coulter counter as described (Filardo et al. 2006).
MTT viability assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide)-viability assay was performed as already
described (Ignatov et al. 2003). Briefly, 2,000 cells per well
were seeded and cultured in a 96-well plate in the growth
medium. After 24 h, the cells were stimulated for 3 days
with G-1 or DMSO as a control. Thereafter, MTT was
added for 3 h in the dark. The supernatants were removed,
cells were lysed in 150 μl lysis buffer (isopropanol con-
taining 4 mM HCl and 0.1 % NP-40), and the absorbance
at 570 nm was recorded.
Cell cycle analysis and apoptosis assay
Cells were treated with 1 μM G-1 or medium for 1–3 days.
Cell cycle distribution was analyzed by propidium iodide
(PI) staining using flow cytometry. Apoptosis was deter-
mined with FITC Annexin V Apoptosis Detection Kit (BD,
Heidelberg) following the manufacturer’s protocol.
Real-time RT-PCR
RNA isolation and quantitative RT-PCR were performed
as previously described (Schumacher et al. 2009). We used
the following primers for GPER identification: forward,
5′-AGTCGGATGTGAGGTTCAG-3′ and reverse, 5′-TCT-
GTGTGAGGAGTACAAG-3′. GPER mRNA levels were
normalized to the β-actin transcript levels. All reactions
were carried out on an iCycler (BioRad, Germany).
Western blot analysis
The following primary antibodies were used: sc-48524-R
for GPER (1:500 dilution; Santa Cruz), ab13847 for cas-
pase-3 (1:1,000 dilution; Abcam), OP43 for p53 (1:2,000
dilution; Calbiochem), #4138 for cyclin B1 (dilution
1:1,000; Cell signalling), #9112 for Cdc2 (dilution 1:2,000;
Cell signalling); #9701 phosphoH3 (Ser10) (dilution
1:2,000; Cell signalling), and A5441 for β-actin (dilution
1:10,000; Sigma-Aldrich). Peroxidase-conjugated anti-
rabbit or anti-mouse antibodies (Thermo Scientific), diluted
1:2,000 or 1:5,000, respectively, were used as secondary
antibodies.
siRNA treatment
GPER knockout experiments were performed as already
described (Ignatov et al. 2010c) after transfection of the
cells with GPER-specific siRNA (sc-60743, Santa Cruz),
p53-specific siRNA (sc-29435, Santa Cruz), or scram-
bled control siRNA (sc-37007, Santa Cruz). The trans-
fection was performed with Lipofectamine 2000 (Life
J Cancer Res Clin Oncol
1 3
Technologies) according to the manufacturer’s protocol.
Cells were analyzed 48 h after transfection.
Methylation analysis of GPER promoter region in breast
cancer cells and tissue
The methylation status of the GPER promoter region was
determined by methylation-specific PCR (MSP) as previ-
ously described (Ignatov et al. 2008, 2010a). The following
primers, designed with the MethPrimer software (Li and
Dahiya 2002), were used:
me thylated forward 5′-GGTTAGTAGGGGGCGTAT
TC-3′;
me thylated reverse 5′-TAATTACGAATTTCACAATCT
CGTA-3′;
un methylated forward 5′-TAAATGGTTAGTAGGGGGT
GTATTT-3′;
un methylated reverse 5′-CATAATTACAAATTTCACAA
TCTCATA-3′.
Breast cancer cells, breast cancer tissues, and normal
breast specimens were analyzed. DNA was isolated with
NucleoSpin® DNA extraction kit (Macherey & Nagel).
The initial bisulfite reaction that converts unmethylated
cytosines to uracils was performed with the CpGenome
DNA modification Kit (S7820, Chemicon). The MSP con-
sists of two individual PCRs with distinct primers specific
for methylated and unmethylated DNA sequences. We used
100 nmol/l specific forward and reverse primers, 2 μl of
2 mmol/l dNTPs, 5 μl PCR buffer, 0.75 U of GoTaq poly-
merase (Promega), and 100 ng of template DNA in a final
volume of 25 μl. The PCR conditions were as follows:
95 °C for 10 min; followed by 40 cycles of denaturation at
94 °C for 1 min, annealing at 55 °C for 1 min, and amplifi-
cation at 72 °C for 1 min. The PCR products were analyzed
on 3 % agarose gel stained with ethidium bromide.
Methylated and unmethylated DNA sequences were
always included in the analysis. Gels were interpreted only
in case of working positive control. The methylation of
GPER promoter was made using yes/no decision.
Radiation treatment of breast cancer cells
MDA-MB-231, MDA-MB-468, and MCF-7 cells were
grown on culture plates to 70–80 % confluence and irra-
diated with 0, 2, 5, and 10 Gy at room temperature. The
experiments were performed after 48-h incubation.
Bioinformatic tools and statistical analysis
The following software tools were used to predict methyla-
tion sites in the 5′-regions upstream of the transcription start:
http://www.ebi.ac.uk/Tools/emboss/cpgplot/index.html;
http://bio.dfci.harvard.edu/Methylator/.
The curve fit and nonlinear regression analysis of the
dose–response curves were performed with the GraphPad
Prism software. Data are present as mean ± SD. In all sta-
tistical analyses, two-sided tests were applied. Statistical
significance was determined by Student’s t-test, and p val-
ues <0.05 were considered as statistically significant.
Results
GPER stimulation with G-1 inhibited the growth
of triple-negative breast cancer cells via cell cycle arrest
in the M-phase and caspase-dependent cell apoptosis
Triple-negative MDA-MB-231 and MDA-MB-468 breast
cancer cell lines were stimulated with increasing concen-
trations of GPER-specific agonist G-1 for 3 days. G-1
caused a concentration-dependent inhibition of the cell
growth with an IC50 value of 0.1 μM for MDA-MB-231
and 0.3 μM for MDA-MB-468 cells (Fig. 1a). Notably,
MDA-MB-231 cells expressed lower levels of GPER
mRNA than the MDA-MB-468 cells (Fig. 1b). To con-
firm the specificity of GPER agonist G-1, we next down-
regulated the GPER expression with siRNA. Intriguingly,
the down-regulation of GPER in the absence of G-1 was
associated with increased cell growth in MDA-MB-231
and MDA-MB-468 cells. These data demonstrated the
constitutive activity of GPER. In both MDA-MB-231 and
MDA-MB-468 cells, the specific knockdown of the GPER
expression abrogated the inhibitory effect of G-1 (Fig. 1c).
The effect was specific to the triple-negative MDA-
MB-231 and MDA-MB-468 breast cancer cells; no effect
was observed in the control HEK-293 cells lacking GPER
(Fig. 1c).
To assess the effect of G-1-stimulated GPER on the cell
cycle, we next analyzed the distribution of the cells in dif-
ferent phases of the cell cycle using propidium iodide (PI)
staining. The stimulation of MDA-MB-231 and MDA-
MB-468 cells with 1 μM G-1 led to a significant accumula-
tion of cells in G2/M phase compared to the control cells
(Fig. 2a and b). The cell cycle arrest in the G2/M phase
was already detectable after 24 h of G-1 stimulation and
slightly decreased after 48 h (Fig. 2a and b). Notably, a
subpopulation of cells accumulated in the apoptotic sub-G1
phase upon G-1 treatment; the effect was stronger in MDA-
MB-468 than in MDA-MB-231 cells (Fig. 2a and b).
To disentangle the nature of GPER-induced cell cycle
arrest in more details, we next investigated the expression
of G2/M-phase-specific regulatory proteins cyclin B1 and
Cdc2 and the phosphorylation of histone H3. G-1-induced
cell cycle arrest enhanced the phosphorylation of histone
J Cancer Res Clin Oncol
1 3
H3, which is indicative of cells in mitotic phase. The effect
was detectable even 6 h after G-1 stimulation of the cells
and reached the highest value between 12 and 24 h (Fig. 2c
and d). However, the levels of cyclin B1 and Cdc2 protein
remained unchanged (Fig. 2c and d). GPER knock down
by siRNA abrogated the G-1-induced cell cycle arrest and
apoptosis (data not shown), suggesting again that G-1
effects are mediated by GPER.
The observed accumulation of a sizeable fraction of
cells in the apoptotic sub-G1 phase (Fig. 2a and b) led
us determining the effect of GPER on cell apoptosis.
The number of apoptotic cells significantly increased in
both MDA-MB-231 and MDA-MB-468 cells upon GPER
stimulation with G-1 compared to the background of
apoptotic cells among the non-stimulated cells (Fig. 3a
and b): The number of apoptotic cells increased to
13.95 % in MDA-MB-231 cells (Fig. 3a) and to 24.34 %
in MDA-MB-468 cells (Fig. 3b). In addition, pro-cas-
pase-3 and caspase-3 increased in both MDA-MB-231
and MDA-MB-468 cells (Fig. 3c and d). Taken together,
these results suggest that G-1-induced GPER activation
inhibits the proliferation of triple-negative breast cancer
cells via cell cycle arrest in the M-phase and caspase-
dependent cell apoptosis. These analyses were performed
in four additional cell lines: MCF-7, SK-BR-3, BT-2, and
MDA-MB-453. The G-1 inhibitory effect with cell cycle
arrest and stimulation of cell apoptosis was observed in
all cell lines investigated.
Cell growth (% of control)
0
50
100
150
-4-6-8-10
MDA-MB-231
MDA-MB-468
A
0
1
2
Relative mRNA expression
levels of GPER
MDA-MB-231 MDA-MB-468
B
C
Cell number (x105l)
0
100
300
400
+1µM G-1
HEK-293MDA-MB-231
p= 0.0013 p= 0.0087
MDA-MB-468
+1µM G-1+1µM G-1
G-1 concetration, log [M]
MDA-MB-468
MDA-MB-231
GPER
β-actin
GPER
β-actin
200
p= 0.0413
p= 0.0140
Fig. 1 G-1 inhibits the growth of the triple-negative breast cancer
cells. a MTT viability assay of MDA-MB-231 and MDA-MB-468
cells treated with different concentrations of G-1 for 3 days. The
number of proliferating cells is normalized to the untreated (control)
cells. b qRT/PCR of the mRNA expression level of GPER presented
as means of three independent replicates ±SD. GPER signal was nor-
malized to β-actin transcript levels. c Silencing of the GPER expres-
sion abrogate the inhibitory effect of G-1. MDA-MB-231 and MDA-
MB-486 cells were transiently transfected with GPER-specific siRNA
and followed by treatment with 1 μM G-1 for 72 h. Thereafter, cell
number was counted by cell counter. The expression level was nor-
malized to cells transfected with scrambled siRNA (control) and is
presented as means of three experiments ±SD. HEK-293 cells lack-
ing GPER were treated in the same way and served as control. Inset
cells transfected with scrambled or GPER-specific siRNA. β-Actin
was used as a loading control. Each experiment was repeated at least
three times
J Cancer Res Clin Oncol
1 3
Promoter methylation controls GPER expression
The observed inhibition of cell proliferation of the triple-
negative breast cancer cells raised the intriguing question as
to whether GPER can act as a potential tumor suppressor in
breast cancer. The clear dose-dependent inhibitory effect of
the GPER on the cell growth suggested to us that tuning the
promoter by methylation may determine the GPER expres-
sion levels in the cell. We used 5-Aza-2′-deoxycytidine
(5-Aza) to inhibit the DNA-methyltransferase, which is
involved in the methylation of the promoter. Intriguingly,
the treatment with 5-Aza enhanced the GPER mRNA level
in both MDA-MB-231 and MDA-MB-468 cells in a time-
dependent manner (Fig. 4a and b).
To address whether methylation-dependent increase in the
GPER expression has the same effect on cell proliferation as
the G-1-induced GPER expression, we pre-treated the two
cell lines with 5 μM 5-Aza for 24 h and then incubated them
with 1 μM G-1 for 72 h. Pre-treatment with 5-Aza showed
synergistic inhibitory effect to G-1 abrogation of the cell
growth: The growth of MDA-MB-231 and MDA-MB-468
cells was reduced down to ~5 % by combined treatment with
5-Aza and G-1 compared to the treatment with a single drug.
These data clearly suggest that the inhibitory effect of the
GPER is proportional to its expression level and significantly
decreases upon promoter methylation.
Next, we analyzed the 5′-region upstream of the transla-
tion start site of GPER using MethPrimer software (Li and
B
A
0
20
40
60
80
Sub G1
G-1 stimulation (h)
024 48 72 0244872024 48 72 0244872
G0/G1S G2/M
p= 0.0032
p= 0.0002
p= 0.0084
p= 0.0404
p= 0.0032
p= 0.0239
p= 0.0129
p= 0.0287
p= 0.0424
p= 0.0369
p= 0.0071
0
20
40
60
80
p= 0.0258
p= 0.0171
C
C
cyclin B1
cdc2
G-1CG-1C G-1
0h 24h 48h72h
β-actin
CG-1 CG-1 CG-1
0h 24h48h 72h
C
phospho-H3
β -actin
G-1C G-1C G-1
0h 1h 6h 24h
CG-1 CG-1 CG-1
0h 1h 6h 24h
Percentage of cells
Percentage of cells
Sub G1
G-1 stimulation (h)
024 48 72 0244872024 48 72 0244872
G0/G1S G2/M
D
cyclin B1
cdc2
β -actin
phospho-H3
β -actin
p= 0.0009
11.06 0.94 0.98 2.73 1.42 6.72 10.90 1.85 0.72 4.16 0.41 2.10
11.11 1.44 0.97 1.17 1.04 1.13
11.16 1.52 0.78 1.13 0.59 0.52
11.11 1.26 0.97 1.17 1.04 1.08
11.09 1.14 0.96 0.94 0.75 0.79
Fig. 2 G-1-stimulated GPER inhibits the cell cycle in triple-negative
breast cancer cells. Flow cytometry analysis of the distribution of
MDA-MB-231 (a) and MDA-MB-468 (b) cells in different phases
of the cell cycle after treatment with 1 μM G-1 or control for dif-
ferent times. The results are presented as means of three independ-
ent experiments ±SD. Western blot analysis of expression of cyclin
B1 and Cdc2 and phosphorylation of histone 3 in MDA-MB-231 (c)
and MDA-MB-468 (d) cell stimulated with 1 μM G-1 or control for
different times. β-Actin was used as a loading control. Each Western
blot is a representative example of three independent experiments.
The numbers above the blots represent the relative expression units
compared to the control. The control was set as 1
J Cancer Res Clin Oncol
1 3
Dahiya 2002) to find any potential CpG island(s). The hyper-
methylation of CpG islands in promoter region of tumor sup-
pressor genes is associated with their inactivation (Esteller
et al. 2001). In the region between −4,365 and −4,797 nt
upstream of the transcription start site, we identified a single
433-bp-long CpG-reach island (Fig. 5a). Methylation-specific
PCR approach confirmed experimentally the methylation
within the GPER promoter region in both breast cancer cell
lines and primary breast cancer tissue (Fig. 5b). We analyzed
the breast tissue of 30 breast cancer patients and compared it
to a cohort of healthy individuals of the same size. In 17 out
of 30 patients (56.7 %), the GPER promoter was methylated,
while the normal breast tissues contained only non-methyl-
ated GPER promoters. Furthermore, we also tested whether
5-Aza alters the expression of GPER because of changing the
methylation status of the GPER promoter. Notably, pre-treat-
ment with 5-Aza led to a significant decrease in GPER meth-
ylation in MDA-MB-231 (p = 0.010) and MDA-MB-468
cells (p = 0.009) (Fig. 5c), thus corroborating the enhanced
GPER expression we observed in both triple-negative breast
cancer cells (Fig. 4b and c).
Stress-induced expression of GPER in breast cancer cells
Important function of the tumor suppressor genes is to
induce cell apoptosis and/or to arrest cell cycle progression
in response to DNA-damaging agents, such as radia-
tion (Sun and Yang 2010). We therefore hypothesized that
GPER as a potential tumor suppressor may also play a role
in this processes. We next exposed the MDA-MB-231 and
MDA-MB-468 cells to various doses of γ-radiation for
48 h and measured the levels of GPER mRNA. In both
cell lines, we observed a clear dose-dependent increase
in the GPER mRNA levels (Fig. 6a); the effect was much
stronger in MDA-MB-231 cells. In addition, the expres-
sion of the tumor suppressor p53 protein was also elevated
upon radiation in both cell lines (Fig. 6b). Note that MDA-
MB-231 and MDA-MB-468 cells express a non-functional
mutant of p53 (Kastan et al. 1991; Lim et al. 2009). Thus,
we included in the analysis MCF-7 breast cancer cells that
express wild-type p53 protein (Lim et al. 2009). Intrigu-
ingly, in MCF-7 cells, we observed the opposite effect:
Upon radiation the, GPER expression decreased progres-
sively with the increasing dosage of radiation (Fig. 6a).
Thus, we hypothesized that p53 may modulate the expres-
sion pattern of GPER in an opposite direction. To test this
hypothesis, p53 gene was silenced with a specific siRNA in
MCF-7 cells, and the expression of GPER upon exposure
of the cells to γ-radiation was monitored. p53 knockdown
led to a significant increase in GPER mRNA expression
after radiation (Fig. 6a), implying the direct role of wild-
type p53 in GPER expression. In addition, incubation with
072
Cell apoptosis (%)
p = 0.0007
p = 0.0064
p = 0.0264
0
15
30
45
A
C
B
Pro-caspase-3
Caspase-3
β-actin
24 48
07
2
Cell apoptosis (%)
p = 0.0026
p = 0.0015 p = 0.0005
0
15
30
45
24 48
Time (h)Time (h)
CG-1 CG-1
0h 24h 48h
MDA-MB-231 MDA-MB-468
D
Pro-caspase-3
Caspase-3
β-actin
CG-1 CG-1
0h 24h48h
Control
G-1
Control
G-1
10.98 1.20.93 2.3111.71.33.6
Fig. 3 G-1 induces caspase-dependent apoptosis in triple-negative
breast cancer cells. The fraction of apoptotic cells increases in MDA-
MB-231 (a) and MDA-MB-468 (b) cells upon treatment with 1 μM
G-1 for different times. The apoptotic cells were stained with annexin
V/propidium iodide, counted by flow cytometry, and normalized to
the number of untreated cells. The results are presented as means of
three independent replicates ±SD. The level of pro-caspase-3 and
caspase-3 was enhanced in MDA-MB-231 (c) and MDA-MB-468 (d)
cells upon treatment with 1 μM G-1 for different times as indicated
by Western blot. Time zero represents the basal expression of cas-
pase-3 before the addition of medium (c) and G-1 to the cells. β-actin
was used as loading control. Each Western blot is a representative
example of three independent experiments. The numbers above the
blots represent the relative expression units compared to the control.
The control was set as 1
J Cancer Res Clin Oncol
1 3
G-1 raised the p53 expression level in all three cell lines
(Fig. 6c), corroborating the functional association between
p53 and GPER.
Discussion
Here, we present an analysis on the effect of GPER on tri-
ple-negative breast cancer cells. Our observations clearly
suggest that (1) GPER-specific agonist inhibited breast can-
cer cell proliferation in concentration-dependent manner,
(2) GPER activation leads to a cell cycle arrest and apop-
tosis in cell-dependent fashion, (3) GPER expression is
down-regulated in breast cancer tumorigenesis, (4) aberrant
GPER expression is caused by a reversible promoter meth-
ylation both in vivo and in vitro, and (5) GPER expression
is induced by stress factors such as radiation.
GPER, a member of the GPCR family, is implicated in
breast, endometrial, and ovarian cancers (Filardo et al.
2006; Fujiwara et al. 2012; Smith et al. 2007, 2009). A large
number of GPCR receptors, which are involved in various
types of human cancers, are associated with an increased
cell proliferation and tumor progression (Dorsam and
Gutkind 2007). Our data clearly suggest that GPER acti-
vation in triple-negative breast cancer cells resembles the
growth-specific alterations described for GPER in other
cancer types (Chan et al. 2010; Chimento et al. 2013; Gao
et al. 2011; Holm et al. 2011; Ignatov et al. 2013a; Wang
et al. 2012). The cell growth suppression through enhanced
GPER expression occurs via specific cell cycle arrest in the
M-phase and caspase-3-mediated apoptosis. The effect is
potentiated by the G-1 agonist, which is highly specific for
the triple-negative breast cancer cells and is not a result of
general cytotoxic effect as no growth defect was observed in
GPER-1-negative HEK293 cells. Although Wang et al. have
very recently demonstrated that G-1-induced ovarian cancer
cell inhibition is GPER-independent (Wang et al. 2013), our
observation suggested a GPER-dependent activity of G-1,
and these results have been confirmed by various resent
observations (Ariazi et al. 2010; Chan et al. 2010; Chimento
et al. 2013; Holm et al. 2011; Ignatov et al. 2013a). Unlike
the estrogen receptor-positive MCF-7 breast cancer cells,
for which G-1-induced growth inhibition through G1-phase
block has been described (Fujiwara et al. 2012; Gao et al.
2011), we did not observe any indications of G1-phase alter-
ations in the triple-negative breast cancer cells. Our results
0
2
4
6
Relative mRNA expression
levels of GPR30
8
B
p= 0.0322
p= 0.0163
p= 0.0116
A
0
2
4
6
Relative mRNA expression
levels of GPR30
0244872
Time (h)
p= 0.0297 p= 0.0313
0
50
100
150
Cell proliferation
(% of control)
p= 0.0338
p= 0.0489
5-Aza -+ -+
G-1 --+ +
D
p= 0.0016
p= 0.0157
C
Control
5-Aza
Control
5-Aza
0244872
Time (h)
0
50
100
150
Cell proliferation
(% of control)
5-Aza -+ -+
G-1 --+ +
Fig. 4 Inhibition of promoter methylation enhances GPER level.
qRT-PCR quantification of the mRNA expression of GPER in MDA-
MB-231 (a) and MDA-MB-468 (b) cells upon treatment with 5 μM
5-Aza for different times. Control cells were treated the same way
in a medium without 5-Aza. MTT-viability assay of MDA-MB-231
(c) and MDA-MB-468 (d) cells treated with 5 μM 5-Aza for 48 h,
followed by stimulation with 1 μM G-1 for 72 h. Non-treated cells
served as a control, and their viability was arbitrarily set as 100 %.
The results are presented as means of three independent replicates
±SD
J Cancer Res Clin Oncol
1 3
clearly show that the growth inhibition in MDA-MB-231
and MDA-MB-468 occurs rather through alterations of
the G2/M phase through an increase in the phosphoryla-
tion of histone H3, an important step occurring during G2
to M transition (Hans and Dimitrov 2001). The expression
of the other G2/M-phase-specific regulatory proteins cyclin
B1 and Cdc2 remains unchanged, which is in contrast to the
findings in estrogen receptor-positive breast cancer cells and
prostate cancer cells (Ariazi et al. 2010; Chan et al. 2010).
The increased mitotic duration, which perturbs mitotic pro-
gression, triggers cell apoptosis via caspase-3 cleavage—an
effect that has been already described for both microtubule
inhibitors with anticancer activity, taxol, and nocodazole
(Choi et al. 2011; Toh et al. 2010). We also observed a
G-1-induced apoptosis in MCF-7, an effect that has not been
described yet for these cells, implying that apoptosis might
be a general mechanism to cease cell growth and prolifera-
tion in cancer cells upon activation of the GPER expression.
GPER is ubiquitously encoded in normal breast tissues,
but its expression is silenced through a hypermethylation of
the promoter. Our systematic analysis on the methylation
status of the promoter in cell culture and primary breast
tissue derived from breast cancer patients revealed that
the GPER expression is tightly regulated by epigenetic
mechanisms through methylation and demethylation of
the promoter. Although we have performed the analysis
with a sizeable cohort of patients, to further confirm the
link between promoter methylation and GPER expression
and extract the clinical significance, much larger cohorts of
patients need to be analyzed.
Further important finding of our study is the radiation-
induced alterations in the GPER expression in different
breast cancer cells; the radiation-induced expression is a
characteristic feature of various tumor suppressor genes
(Fei and El Deiry 2003). Strikingly, GPER expression
showed opposing expression in different cells: upregu-
lated GPER expression in MDA-MB-231 and MDA-
MB-468 cells and down-regulated in MCF-7 cells. This
pattern is linked most likely to the form of the p53 protein
expressed in each cell. While MDA-MB-231 and MDA-
MB-468 cells express a non-functional p53 gene, MCF-7
cells have normal wild-type p53 (Kastan et al. 1991; Tam
B
UM-Primer
A
ATG
-4797
5‘
CpG island (433bp)
3‘
1 3
-4365
2
5‘
M (for) -4659
UM (for) -4664
M (rev) -4543
UM (rev) -4541
C1UM M234 56
M-Primer
M-Primer
UM-Primer
CUMM
MDA-MB-231
MDA-MB-468
C
MDA-MB-468
M-Primer
UM-Primer
C5-Aza
M-Primer
MDA-MB-231
C5-Aza
0
1
2
p=0.010
0
1
2p=0.009
Relative
expression
Relative
expression
200
100
200
100
200
100
200
100
Fig. 5 GPER promoter is methylated in triple-negative breast can-
cer cells and in primary breast cancer tissue. a Schematic of the
GREP gene. The putative 433-bp-long CpG island between positions
−4,365 and −4,797 is indicated. Gray boxes delineate exon 1, 2, and
3; arrows denote the positions of the MSP specific primers; verti-
cal lines mark the individual CpG sites. The analysis was performed
with a DNA sequence corresponding to the region between 1126443
to 1133451 nt of the chromosome 7 (NCBI Reference sequence
NC_000007.13). b Representative examples of the methylation
analysis of GPER promoter in breast cancer cells and tissue. c Meth-
ylation status of GPER promoter in MDA-MB-231 (left) and MDA-
MB-468 (right) cells upon pre-treatment with 5 μM 5-Aza for 48 h.
The numbers on the left denote the DNA ladder in bp. M-primer,
primer specific for the methylated CpG sites; UM-primer, primer
specific for unmethylated CpG sites. C, negative control; M, standard
methylated control; UM, standard unmethylated control; lanes 1–6 in
panel C, six randomly chosen exemplary cases of primary breast can-
cer tissues. 1, 3, and 6, examples of methylated GPER promoter; 2, 4,
and 5, examples of unmethylated GPER poromoter
J Cancer Res Clin Oncol
1 3
et al. 1994). siRNA-induced knockdown of the wild-type
p53 in MCF-7 cells reverses the GPER expression phe-
notype to those of the MDA-MB-231 and MDA-MB-468
cells. Thus, intact wild-type p53 exerts a protective role: It
counteracts the GPER-induced expression upon radiation.
Moreover, G-1-induced activation of the breast cancer cell
lines enhances the expression of p53. Therefore, we sug-
gest a feedback mechanism between the expression of p53
and GPER: GPER activation increases the p53 expression,
which in turn down-regulates the expression of GPER. In
case of a non-functional p53, GPER expression remains
high and an alternative emergency mechanism is activated,
which induces cell apoptosis. The cell surface expression
of GPER makes it an excellent target for TNBC.
Acknowledgments This work was supported by Deutsche
Krebshilfe.
Conflict of interest This work was supported by a grant from
Deutsche Krebshilfe to A.I.
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