Oncogene (2007) 26, 7916–7920
& 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00
www.nature.com/onc
SHORT COMMUNICATION
The clock gene Per2 links the circadian system to the estrogen receptor
S Gery, RK Virk, K Chumakov, A Yu, HP Koeffler
Cedars-Sinai Medical Center, Division of Hematology/Oncology, UCLA School of Medicine, University of California,
Los Angeles, CA, USA
Circadian rhythms regulate diverse physiological processes including homeostatic functions of steroid hormones and their receptors. Estrogen receptor-a (ERa) is
essential for normal mammary gland physiology and is a
prognostic marker for the treatment of breast cancer. We
report that Per2, a core clock gene, links the circadian
cycle to the ERa signaling network. Binding of enhances
ERa degradation, while suppression of Per2 levels leads to
ERa stabilization. In turn, Per2 itself is estrogen inducible
in these cells, suggesting a feedback mechanism to attenuate
stimulation by estrogen. In addition, overexpression of Per2
in breast cancer cells leads to significant growth inhibition,
loss of clonogenic ability and apoptosis. Taken together,
these results further support a critical role for peripheral
circadian regulation in tissue homeostasis and suggest a
novel role for clock genes in estrogen receptor-positive
breast cancer.
Oncogene (2007) 26, 7916–7920; doi:10.1038/sj.onc.1210585;
published online 18 June 2007
Keywords: circadian rhythms; ERa; breast cancer;
Per2; transcriptional activation; estrogen
Most physiological processes in mammals are influenced
by circadian rhythms. These rhythms are driven by a
master clock within the hypothalamic suprachiasmatic
nuclei (SCN) that synchronizes numerous subsidiary
oscillators in peripheral tissues. The circadian clockwork
in both the SCN and the peripheral cells is composed of
transcription-translation feedback loops maintained by
a core set of clock genes (Shearman et al., 2000; Reppert
and Weaver, 2002; Schibler and Sassone-Corsi, 2002;
Ishida, 2007). Two transcription factors, Clock and
Bmal1, activate their targets, Period (Per1, 2 and 3) and
cryptochrome (Cry1 and Cry2); subsequently, the Per
and Cry proteins interfere with Bmal1:Clock activity
thereby forming the major negative circadian feedback
loop. The central clock, through neural, hormonal and
metabolic signals, synchronizes the peripheral oscillators, which in turn drive the expression of downstream
clock-controlled genes in a tissue-specific manner
(Panda et al., 2002; Storch et al., 2002; Miller et al.,
Correspondence: S Gery, Cedars-Sinai Medical Center, Davis Building
5066, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA.
E-mail: [email protected]
Received 26 March 2007; revised 8 May 2007; accepted 9 May 2007;
published online 18 June 2007
2007). Consequently in various tissues, circadian
rhythms impinge upon many physiological processes
and pathological conditions, including cancer (Fu et al.,
2002; Matsuo et al., 2003; Lowrey and Takahashi, 2004;
Ko and Takahashi, 2006). Recent studies suggested that
circadian disruption is associated with breast tumorigenesis (Hansen, 2001; Chen et al., 2005). The steroid
hormone estrogen is essential for normal mammary
gland physiology, and is also a potent mammary
mitogen (Sternlicht, 2006; Yager and Davidson, 2006).
Although the circadian clock has been linked to several
steroid hormone activities, the molecular mechanisms
underlying the function of core clock genes in mammary
tissue are largely unknown. We hypothesized that
circadian regulation may be implicated in hormone
homeostasis and hormone-related tumorigenesis in
breast epithelia cells.
Estrogen stimulation is thought to be a major factor
contributing to the development of breast cancer.
Deregulation of the core clock factor, Per2, has been
reported in several human malignancies including breast
cancer (Chen et al., 2005). We hypothesized that Per2
function in mammary epithelia cells could be linked to
the ERa signaling pathway. ERa regulates transcription
of target genes through an interaction with consensus
estrogen response elements (EREs). We performed
reporter assays with an ERE-luciferase reporter gene
to test the effect of Per2 on ERa transactivation
(Figure 1a). The ERa-positive breast cancer cell line,
MCF-7, was cotransfected with ERE-luciferase and
either Per2 or empty vector. While 17-b estradiol (E2)
induced high luciferase activity in the control cells, Per2
expression substantially reduced this activity. Per2 also
inhibited E2-activated ERE transcription in two additional ERa-positive cancer cell lines, T47D (breast) and
Ishikawa (endometrial). In contrast, silencing of Per2 by
small interfering RNA (siRNA) (as shown in Figure 1d)
in those cells enhanced the ERE reporter activity
(Figure 1a). We also examined whether Per2 can
endogenously suppress ERa-responsive genes (Figures
1b and c). MCF-7 cells transfected with either Per2 or a
control vector were treated with E2; and the mRNA
levels of the known ERa targets, pS2, cyclin D1 and
CCN1 were measured by real-time PCR (Figure 1b).
While expression of Per2 alone had little effect on
expression of those genes, it strongly inhibited E2-mediated
induction of pS2, and further reduced the levels of
cyclin D1 and CCN1 below basal level. Moreover,
Per2 siRNA enhanced E2-activation of the ERa target
Per2 links the circadian system to ER
S Gery et al
7917
siCon
siPer2
Cyclin D1
− + − +
Ishikawa
siCon
siPer2
CCN1
Cyclin D1
β-actin
100
E2
0
200
d
CCN1
100
T47D
Ishikawa
E2
0
−+ −+
− +− +
er2
MCF-7
+
−
+
−
+
−
+
Per2
on
− + − +
siP
− + − +
EV
Pe
r2
− + − +
−
siPer2
− + − +
T47D
0
200
siCon
− + − +
MCF-7
2
0
Per2
100
siC
Luciferase activity
(% of siCon+E2)
4
EV
siPer2
0
pS2
200
1
E2
E2
EV
Per2
c
siCon
2
b 300
Relative expression
Luciferase activity
(% of EV+E2)
a
β-actin
293T
MCF-7
Figure 1 Per2 suppresses ERa transcriptional activation. (a) MCF-7, T47D (breast) and Ishikawa (endometrial) cancer cell lines were
cotransfected with ERE-luciferase construct and either empty vector (EV) or Per2 (Per2, upper panel) or with either a control siRNA
(siCon) or Per2 siRNA (siPer2, bottom panel). Luciferase activity was measured either with or without treatment of cells with E2
(1 mM, 16 h). Results represent the percentages of luciferase activity with either E2-treated EV-transfected cells (upper panel) or E2treated siCon-transfected cells (bottom panel) set to 100%. Shown are the means7s.d. of triplicate samples. (b and c) MCF-7 cells
transfected with either empty vector or Per2 were selected with G418 for 5 days. Surviving cells were treated with E2 (1 mM, 16 h) and
harvested for RNA and whole-cell protein. In addition, MCF-7 cells were transfected with either siCon or siPer2. Two days later, cells
were treated with E2 (1 mM, 16 h) and harvested for RNA and whole-cell protein. (b) Real-time PCR analysis of the indicated genes. (c)
Western blot analysis of the indicated proteins. (d) 293T and MCF-7 cells were transfected with either siCon or siPer2. Nuclear lysates
were analysed for Per2 expression by immunoblotting. Cells culture, constructs, real-time PCR conditions and sources of antibodies
and regents have been described in the Supplementary Information.
genes (Figure 1b). Western blot analysis showed a
parallel effect of Per2 on the protein levels of cyclin D1
and CCN1 (Figure 1c). These results suggest that Per2
could be involved in breast cancer prevention by inhibiting E2-induced proliferation. Recently, Per2 was also
suggested to play a role in normal mammary cell differentiation (Metz et al., 2006).
In the absence of ligand, ERa is sequestered in the
cytoplasm in an inhibitory protein complex. Upon
binding to E2, ERa undergoes conformational changes
facilitating cofactor binding and nuclear localization
(McDonnell and Norris, 2002). Immunoprecipitation
experiments showed that Per2 associates with ERa in
293T and MCF-7 cells, and E2 stimulation enhances
the interaction (Figure 2a). Moreover, glutathione
S-transferase (GST) pull-down assays, with an in vitrotranslated Per2 and a GST-ERa fusion protein,
demonstrated direct binding of these proteins (Figure 2b).
As Per2 shuttles between the nucleus and the cytoplasm
(Yagita et al., 2002), it could change the subcellular
distribution of ERa. To test this possibility, 293T cells
cotransfected with ERa and V5-tagged mPer2, and
MCF-7 cells transfected with V5-tagged mPer2 were
treated with E2 and analysed by confocal microscopy.
Strong nuclear staining of ERa was detected in the 293T
cells (Figure 2c). Also, ERa localized mainly to the
nucleus in both nonexpressing and Per2-expressing
MCF-7 cells. These results indicate that Per2 does not
prevent nuclear localization of ERa. Colocalization of
Per2 and ERa was noted in both cell lines. Notably, Per2expressing MCF-7 cells exhibited changes in morphology
indicative of apoptosis, such as cell rounding and
detachment, while the 293T cells had normal morphology.
ERa is a short-lived protein and binding of E2 further
accelerates its degradation (Reid et al., 2002). While
Per2 expression had no effect on ERa mRNA levels
(Figure 2d), it markedly downregulated ERa protein
levels in MCF-7 cells (Figure 2e). PS-341, a specific
proteasome inhibitor, blocked Per2-stimulated downregulation of ERa (Figure 2f) showing that Per2mediated ERa degradation is through the proteasome
pathway. Moreover, suppression of endogenously
expressed Per2 by siRNA led to stabilization of ERa
(Figure 2g), suggesting that Per2 is necessary for
efficient proteasome-induced degradation of ERa. A
recent study showed that several nuclear receptors are
clock-controlled genes (Yang et al., 2006). Although
ERa does not exhibit a circadian expression pattern in
normal cells, it is downregulated in Clock mutant mice
(Miller et al., 2007), suggesting that core circadian
components have an important role in regulating ERa
expression. Protein degradation plays a role in the
circadian transcription-translation feedback loops, and
core clock factors modulate protein stability through the
proteasome pathway in other systems as well (Yagita
et al., 2002; Gallego and Virshup, 2007). Together, these
Oncogene
Per2 links the circadian system to ER
S Gery et al
7918
a
293T
+
−
E2
c
b
MCF-7
+
−
G
GS ST
TER
IgG
IgG
e
EV
Per2
200
ERα
100
β-actin
0
EV
f
−
E2
EV Per2
g
Per2
siCon
+
−
+
DNA
ERα relative
expression
ERα
IgG
ERα
ERα
IP
d
MCF-7
Per2
Per2
293T
siPer2
merge
ERα
β-actin
E2
PS-341
−
−
−
−
−
+
+
+
−
+
−
+
Figure 2 Per2 interacts with and induces degradation of ERa protein. (a) 293T and MCF-7 cells were transfected with Per2 and ERa
and treated with E2 (1 mM, 1 h). Protein lysates were immunoprecipitated (IP) either with ERa antibody or immunoglobulin G (IgG),
and analysed by western blot with Per2 antibody. (b) In vitro-translated Per2 was incubated with either GST-ERa fusion protein or
GST protein alone. GST-protein complexes were analysed by western blot with Per2 antibody. (c) 293T cells cotransfected with ERa
and V5-tagged Per2 and MCF-7 cells transfected with V5-tagged Per2 were treated with E2 (1 mM, 1 h) and triple-stained for ERa (ERa
antibody, red), Per2 (V5 antibody, green) and DNA (TO-PRO-3, blue). The merged images show colocalization of ERa and Per2
(yellow). (d and e) MCF-7 cells transfected with either empty vector (EV) or Per2 were selected with G418 for 5 days, treated with E2
(1 mM, 1 h) and harvested for RNA and proteins. (d) Real-time PCR analysis of ERa expression. (e) Western blot analysis of ERa
expression. (f) EV- and Per2-transfected MCF-7 cells were treated with the proteasome inhibitor PS-341 (100 nM, 16 h) either in the
presence or absence of E2 (1 mM, 1 h) and analysed for ERa expression by immunoblotting. (g) MCF-7 cells transfected with either
control siRNA (siCon) or Per2 siRNA (siPer2) were treated with E2 (1 mM, 1 h) and ERa expression was analysed by western blot.
studies suggest that control of protein turnover is an
important feature of circadian regulation.
Recent studies in rats showed Per2 is differentially
regulated by E2 in specific tissues (Nakamura et al.,
2005; Perrin et al., 2006). We found that Per2 mRNA
levels were induced by E2 in ERa-positive breast cancer
cell lines (Figure 3a), showing that the human Per2 gene
is E2-inducible in mammary epithelial cells. Analysing
the Per2 promoter, we identified a potential ERE
binding site at 365 bp. The ERE and the surrounding
nucleotides are conserved between mouse and human
Per2 promoters, suggesting functional significance
(Figure 3b). Electrophoretic mobility shift analyses
demonstrated protein-DNA binding between the ERE
element from the Per2 promoter and E2-treated MCF-7
nuclear extracts (Figure 3c). The protein binding was
specific as it could be competed with excess cold probe;
and the addition of ERa antibody resulted in a
supershift of the protein-DNA complex. No specific
complexes were detected using nuclear extracts from
control cells, nor in parallel experiments with a mutated
probe. Furthermore, chromatin immunoprecipitation
Oncogene
assays with MCF-7 cells detected the presence of ERa
on the upstream regulatory region of Per2 following E2
treatment (Figure 3d), demonstrating that endogenous
ERa binds to the Per2 promoter. Several additional
half-ERE sites were found within the Per2 promoter,
which may also contribute to the E2-induction of
Per2. Hormone-mediated gene expression is attenuated
rapidly; inducible expression of corepressors might
serve as a mechanism to control the magnitude of ERa
transcriptional activation. Indeed, other ERa corepressors including BRCA1 and SHARP are also E2
inducible (Romagnolo et al., 1998; Shi et al., 2001). In
this regard, regulation of circadian genes by E2 may
help maintain hormonal homeostasis.
Per2 mutant mice show increased susceptibility to
tumor development, suggesting that Per2 is a tumor
suppressor gene (Fu et al., 2002). Expression of Per2
dramatically reduced the growth of MCF-7 cells on
plastic and the ability of the cells to form colonies on
soft agar (Figures 4a and b). Per2-induced growth
inhibition was associated with a significant increase in
apoptotic death, measured by Annexin-V staining and
Per2 links the circadian system to ER
S Gery et al
c
MB-231
T47D
0
wtDNA
Anti-ERα
+
Competitor
+
E2
+ + +
Consensus ERE
Per2 promoter
Per2
EV
GGTCAnnnTGACC
GGTCAnnnTTTCC
Per2
d
EV
Per2
c
3%
PI
5
Human-365
GCGCGCGCGGTCACGTTTTCCACTATGTG
|||||||||||||||||||||||||||||
GCGCGCGCGGTCACGTTTTCCACTATGTG
Mouse-34
a
PARP
Per2
mDNA
+
+
+ + +
b 80
d
Input
ERα
IgG
E2
−
−
−
+
+
+
ERα
Figure 3 Per2 expression is estrogen inducible. (a) Breast cancer
cell lines were either untreated or treated with E2 (1 mM, 16 h) and
Per2 expression was analysed by real-time PCR. Data are presented
as fold increase in treated cells compared to untreated cells. (b)
Sequence alignment of the conserved region between the mouse
and human Per2 promoter. The potential ERE is underlined. The
nonconserved nucleotides between the Per2-ERE and consensuses
ERE are shown in bold. (c) Electrophoretic mobility shift analyses
(EMSA) was performed with nuclear extracts from MCF-7 cells
transfected with ERa and either untreated or treated with E2 (1 mM,
1 h). Extracts were incubated with oligonucleotides containing
either wild-type (wtDNA) or mutant (mDNA) ERE site from the
Per2 promoter. EMSA conditions have been described in the
Supplementary Information. Protein complexes and supershifted
protein complexes are indicated with arrow and an arrowhead,
respectively. (d) Chromatin immunoprecipitation analysis was
performed using MCF-7 cells cultured either without or with E2
(1 mM, 1 h) with ERa antibody. Samples were analysed by PCR
with Per2 promoter-specific primers. Input chromatin was included
as a positive control; immunoprecipitations with IgG antibody
were the negative control.
poly-(ADP-ribose)polymerase cleavage (Figures 4c and
d). A recent study also found that Per2 induced
apoptosis in murine lung and breast cancer cell lines
(Hua et al., 2006). These results show that Per2 has
antiproliferative effects in breast cancer cells and suggest
that dysregulation of this gene could play a role in
mammary tumorigenesis.
The circadian transcriptional network allows the
orchestration of physiology and behavior for optimal
adaptation of the organism to its environment. We
identified Per2 as a novel estrogen-inducible ERa
No. of colonies
10
MCF-7
Fold induction
b
15
EV
7919
a
63%
β-actin
Annexin
40
0
e
EV
Per2
ERα
Clock
Bmal1
E2
Per2 circadian
response
cycle
Figure 4 Per2 expression inhibits growth of MCF-7 cells. (a)
MCF-7 cells were transfected with either Per2 expression vector
(Per2) or empty vector (EV) and selected with G418. Shown are the
results of a representative experiment. (b and d) MCF-7 cells
transfected with either Per2 or empty vector were selected with
G418 for 5 days and used in subsequent assays. (b) Clonogenic
assays. (c) Apoptosis was analysed by Annexin-V/propidium iodide
staining. Late and early apoptotic populations are in the upper
right quadrant and lower right quadrant, respectively. (d) Western
blot analysis for Per2 and poly-(ADP-ribose)polymerase cleavage
expression. (e) A model showing that Per2 couples the circadian
cycle to the estrogen response; dysregulation in either pathway may
contribute to development of hormone-dependent breast cancer.
corepressor, suggesting a feedback mechanism coupling
the circadian clock to the estrogen pathway. Our results
further support a model wherein, while circadian
regulation helps maintain estrogen homeostasis in
normal mammary cells, disruption of this regulation
could play a role in the development of hormone-related
breast cancer (Figure 4e). Further, elucidating the
connections between clock genes and the ER network
could benefit the development of new therapeutic
strategies for breast cancers, as well as, provide insights
into chronotherapy, as a way to optimize current
therapies.
Acknowledgements
This work was supported by NIH grants, UCLA Cancer Gene
Medicine Training grant and also in part by the Parker Hughes
Trust, the Inger Foundation and the Mary Barry Foundation.
H Phillip Koeffler is a member of the UCLA Jonsson
Comprehensive Cancer Center and holds the endowed Mark
Goodson Chair of Oncology Research at Cedars-Sinai
Medical Center/UCLA School of Medicine.
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).
Oncogene