The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
PES1 promotes breast cancer by differentially
regulating ERα and ERβ
Long Cheng,1,2 Jieping Li,1,3 Yongjian Han,1 Jing Lin,4 Chang Niu,1 Zhichao Zhou,1 Bin Yuan,1
Ke Huang,5 Jiezhi Li,1 Kai Jiang,1 Hao Zhang,1 Lihua Ding,1 Xiaojie Xu,1 and Qinong Ye1,2
1Department of Medical Molecular Biology, Beijing Institute of Biotechnology, Beijing, People’s Republic of China. 2Institute of Cancer Stem Cell, Cancer Center,
Dalian Medical University, Liaoning, People’s Republic of China. 3Department of Clinical Laboratory, Fuzhou General Hospital of Fujian Corps,
CAPF, Fuzhou, People’s Republic of China. 4Department of Clinical Laboratory, First Affiliated Hospital, and 5Department of Obstetrics and Gynecology,
Chinese PLA General Hospital, Beijing, People’s Republic of China.
The initiation of breast cancer is associated with increased expression of tumor-promoting estrogen receptor α
(ERα) protein and decreased expression of tumor-suppressive ERβ protein. However, the mechanism underly-
ing this process is unknown. Here we show that PES1 (also known as Pescadillo), an estrogen-inducible protein
that is overexpressed in breast cancer, can regulate the balance between ERα and ERβ. We found that PES1
modulated many estrogen-responsive genes by enhancing the transcriptional activity of ERα while inhibiting
transcriptional activity of ERβ. Consistent with this regulation of ERα and ERβ transcriptional activity, PES1
increased the stability of the ERα protein and decreased that of ERβ through the ubiquitin-proteasome path-
way, mediated by the carboxyl terminus of Hsc70-interacting protein (CHIP). Moreover, PES1 transformed
normal human mammary epithelial cells and was required for estrogen-induced breast tumor growth in nude
mice. Further analysis of clinical samples showed that expression of PES1 correlated positively with ERα
expression and negatively with ERβ expression and predicted good clinical outcome in breast cancer. Our data
demonstrate that PES1 contributes to breast tumor growth through regulating the balance between ERα and
ERβ and may be a better target for the development of drugs that selectively regulate ERα and ERβ activities.
The association between estrogen and breast cancer was recognized
over 100 years ago. Estrogen exerts its function through its 2 nucle-
ar receptors, estrogen receptor α (ERα) and ERβ (1, 2). ER belongs
to a superfamily of ligand-activated transcription factors that share
structural similarity characterized by several functional domains.
N-terminal estrogen-independent and C-terminal estrogen-depen-
dent activation function domains (AF1 and AF2, respectively) con-
tribute to the transcriptional activity of the 2 receptors. The DNA-
binding domain of the ERs is centrally located. The ligand-binding
domain, overlapping AF2, shows 58% homology between ERα and
ERβ. The DNA-binding domain is identical between the 2 recep-
tors, except for 3 amino acids. However, the AF1 domain of ERβ
has only 28% homology with that of ERα. The binding of estrogen
to ER leads to ER dimerization and its recruitment to the estrogen-
responsive elements (EREs) on the promoters of ER target genes,
thereby either enhancing or repressing gene activation.
The development of breast cancer is associated with dysregu-
lation of ER expression (3–8). Compared with that in normal
breast tissues, the proportion of cells expressing ERα is increased,
whereas ERβ expression is reduced, in hormone-dependent breast
tumors. The ratio of ERα/ERβ expression is higher in breast
tumors than in normal tissues, and ERα and ERβ are antagonis-
tic to each other. ERα mediates the tumor-promoting effects of
estrogens, whereas ERβ inhibits breast cancer cell growth. ERβ
reduces cell proliferation induced by ERα activation. Although
ERα and ERβ have been shown to have a yin-yang relationship in
breast tumorigenesis, the molecular mechanism underlying this
process remains unclear.
In this study, we show that PES1 (also known as Pescadillo)
plays an essential role in estrogen-induced breast tumor growth
through regulation of the yin-yang balance between ERα and ERβ
and is the first such gene to be identified to our knowledge. PES1,
a breast cancer–associated gene 1 (BRCA1) C-terminal (BRCT)
domain-containing protein, is estrogen inducible, and its expres-
sion gradually increases during breast cancer development and
progression (9–11). Theoretically, in the treatment of patients
with ERα-positive breast cancer, in which ERβ is antagonistic
to ERα, a drug that decreases transcriptional activity of ERα but
increases that of ERβ should be better than the currently used
endocrine drugs tamoxifen or fulvestrant, which decrease both
ERα and ERβ transactivation (12, 13). We show that, through
the ubiquitin-proteasome pathway, PES1 enhances ERα levels
but reduces ERβ protein levels, correlating with their respective
physiological activities in breast cancer. Thus, PES1 may repre-
sent a very promising target for the development of better drugs
for breast cancer endocrine therapy.
PES1 differentially regulates transcriptional activity of ERα and ERβ as
well as their target genes. To define the exact role of PES1 in breast
tumor growth, we investigated whether PES1 regulates estrogen
signaling. PES1 overexpression in ERα- and ERβ-positive MCF7
cells (Figure 1A), ERα-positive and ERβ-negative ZR75-1 and
T47D cells (Supplemental Figure 1, A and B; supplemental mate-
rial available online with this article; doi:10.1172/JCI62676DS1),
and ERα- and ERβ-negative SKBR3 (Figure 1B) breast cancer cells
increased transcription of a luciferase reporter construct con-
taining the ERE in response to the ERα-specific agonist propyl-
pyrazole triol (PPT) but decreased ERE reporter transcription in
response to the ERβ-specific agonist diarylpropionitrile (DPN).
This effect was PES1 specific because expression of the known
Authorship note: Long Cheng and Jieping Li contributed equally to this work.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2012;122(8):2857–2870. doi:10.1172/JCI62676.
Related Commentary, page 2771
2858 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
PES1 differentially regulates transcriptional activity of ERα and ERβ and expression of
their target genes. (A and B) Luciferase reporter assays of ERα and ERβ transcriptional
activity in (A) MCF7 or (B) SKBR3 cells transiently transfected with ERE-LUC and PES1,
SRC1, GRIP1, XRCC1, or BARD1 with or without ERα or ERβ and 24-hour treatment
with 10 nM E2, 1 nM PPT, or 1 nM DPN. Results shown are mean ± SD of 3 independent
experiments. ‡P < 0.01, *P < 0.01, #P < 0.01, †P < 0.01 versus empty vector in the (A)
absence or (B) presence of ERα or ERβ with vehicle (–), E2, PPT, and DPN, respectively.
(C) Luciferase reporter assays in MCF7 cells stably transfected with PES1 siRNA or PES1
siRNA plus siRNA-resistant PES1 (PES1-R) and treated as above. Immunoblot analysis of
PES1 expression is shown. Results shown are mean ± SD of 3 independent experiments.
*P < 0.01, #P < 0.01, †P < 0.01 versus control siRNA with E2, PPT, and DPN, respectively.
(D) Real-time RT-PCR analysis of 47 genes identified by cDNA microarray in our study and
4 genes identified in other studies (CCND1, CTSD, E2F1, and C-FOS) in PES1 knockdown
MCF7 cells treated or not treated with E2 (+E2 or –E2, respectively) for 24 hours. Data
shown are mean ± SD of triplicate measurements that have been repeated 3 times with
similar results. *P < 0.05, #P < 0.01 versus control siRNA without E2. ‡P < 0.05, †P < 0.01
versus control siRNA with E2. (E) Immunoblot analysis of estrogen-responsive gene expres-
sion in PES1 knockdown MCF7 cells.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
ER cofactors, steroid receptor coactivator-1 (SRC1) or glutamate
receptor-interacting protein 1 (GRIP1), did not oppositely regu-
late ERα and ERβ transactivation, and other BRCT domain–con-
taining proteins, X-ray repair complementing defective repair in
Chinese hamster cells 1 (XRCC1) and BRCA1-associated RING
domain protein 1 (BARD1), had no effect. As expected, SRC1 and
GRIP1 increased the transcriptional activity of both ERα and ERβ
(Figure 1A). In contrast, siRNA knockdown of endogenous PES1
reduced transcriptional activity of ERα and enhanced that of ERβ
in MCF7 cells (Figure 1C). These effects could be rescued by PES1
reexpression in PES1 knockdown MCF7 cells.
Next, we performed cDNA microarray analysis to monitor gene
expression profiles in PES1 knockdown MCF7 cells. In the pres-
ence of 17β-estradiol (E2), PES1 regulated the expression of 256
genes, including over 127 previously reported E2-regulated genes
(refs. 14–22, Supplemental Figure 2, and Supplemental Tables
1–3). Real-time RT-PCR confirmed the PES1-mediated expression
of 47 genes identified in our study and 4 well-known E2-regulated
genes (cyclin D1 [CCND1], cathepsin D [CTSD], E2F transcription
factor 1 [E2F1], and C-FOS) identified in other studies (refs. 14–22
and Figure 1D). The expression of many estrogen-responsive genes
known to have important functions in DNA replication (23, 24)
and cell cycle regulation (24) was found to be downregulated by
PES1 knockdown, including replication factor C (RFC), minichro-
mosome maintenance genes, proliferating cell nuclear antigen
(PCNA), E2F1, E2F2, MYB, MYC, cyclin-dependent kinase 1 (CDC2),
CCND1, and survivin (BIRC5). Interestingly, some of these genes,
such as MYC, CCND1, PCNA, E2F1, and BIRC5, were reported to
be activated by ERα but to be repressed by ERβ (14–22). Consis-
tent with the results of PES1 knockdown, PES1 overexpression
increased the transcription of E2F1, CCND1, cyclin E2 (CCNE2),
and CTSD in the presence and/or absence of E2, with higher
magnitude in the presence of E2 (Supplemental Figure 3A). The
expression of 10 representative estrogen-responsive genes was fur-
ther confirmed by immunoblotting (Figure 1E).
PES1 differentially regulates the dimerization and promoter occupancy
of ERα and ERβ. Dimerization is a regulatory mechanism of con-
trolling transcription factor activity. Upon dimerization, tran-
scription factors bind to promoter sequences of target genes. ERα
and ERβ homodimerization is thought to be critical for ERα and
ERβ transcriptional activity, whereas ERα-ERβ heterodimeriza-
tion facilitates inhibition of ERα transactivation by ERβ (25). In
agreement with the findings that ERα and ERβ transcriptional
activity was oppositely regulated by PES1, overexpression of PES1
increased ERα homodimerization (Figure 2A) and decreased ERβ
homodimerization and ERα-ERβ heterodimerization (Figure 2B).
Like ERα and ERβ, PES1 was recruited to the estrogen-responsive
CTSD, CCND1, E2F1, and CCNE2 promoters but not to an unrelat-
ed β-actin promoter (Figure 2C). In addition, unlike ERα and ERβ
(26, 27), PES1 was not recruited to the distal enhancers of CTSD
and CCND1 (Figure 2C). Importantly, consistent with the results
of ERα and ERβ transactivation, which was oppositely regulated
by PES1, PES1 knockdown decreased ERα promoter occupancy
but increased that of ERβ (Figure 2D).
PES1 has been shown to directly interact with the cadmium
response element (CdRE) of the heme oxygenase-1 (HO1) promot-
er (28). We searched potential CdREs of the CTSD, CCND1, E2F1,
and CCNE2 genes and found that CCND1 and E2F1 had putative
CdREs. The results of EMSA demonstrated that PES1 bound
indeed to the CdRE of the HO1 promoter (Figure 2E). However,
PES1 did not bind to the putative CdREs of CCND1 and E2F1,
suggesting that PES1 may regulate estrogen-responsive gene tran-
scription through EREs.
PES1 oppositely modulates ERα and ERβ protein stability. To inves-
tigate how PES1 increases transactivation of ERα but decreases
that of ERβ, we examined the effects of PES1 on ERα and ERβ
expression. In the absence or presence of E2, PES1 knockdown
reduced ERα protein expression but enhanced ERβ protein lev-
els in MCF7 cells (Figure 3A). Reexpression of PES1 in the knock-
down cells rescued this effect. PES1 knockdown did not alter ERα
and ERβ mRNA levels in MCF7 cells (Supplemental Figure 3B).
Similar trends were obtained in ZR75-1 cells and normal human
mammary epithelial cells (HMECs) (Supplemental Figure 3, C–F),
suggesting that PES1 regulates ERα and ERβ expression at the
Recent studies show that at least 3 ERβ isoforms, including the
wild-type ERβ (ERβ1), ERβ2, and ERβ5, are expressed in breast
cancer (29, 30). ERβ is the only fully functional isoform. ERβ2
and ERβ5 can not bind estrogen. All 3 ERβ isoforms can inhibit
ERα transcriptional activity (31). Western blot analysis with anti-
ERβ or anti-MYC showed that ERβ1/ERβ was indeed expressed
in MCF7 cells, because the location of the endogenous band was
similar to that of MYC-tagged ERβ1 but not MYC-tagged ERβ2
and ERβ5 (Supplemental Figure 4A). The anti-ERβ used recog-
nized all 3 ERβ isoforms (Supplemental Figure 4A, right panel).
In addition, PES1 downregulated ERβ, ERβ2, and ERβ5 (Supple-
mental Figure 4B).
Since PES1 modulates ERα and ERβ expression at the posttran-
scriptional level, we first determined the half-life of ERα protein in
PES1 knockdown MCF7 cells. In the absence or presence of E2, the
half-life of ERα protein in PES1 knockdown cells was reduced from
more than 12 hours to approximately 4 hours and from 3 to 2 hours,
respectively (Figure 3B). Because ERβ is usually expressed at low lev-
els in breast cancer cell lines, we determined the half-life of ERβ by
ERβ overexpression in HEK293T cells. In the absence and presence of
E2, PES1 overexpression decreased the half-life of ERβ from approxi-
mately 12 to 6 hours and from 9 to 3 hours, respectively (Figure 3C).
Next, we determined effects of PES1 on the protein levels of ERα
and ERβ domains. In transfected HEK293T cells, PES1 increased
the protein levels of the ERα AF2 domain but reduced the lev-
els of the ERβ AF2 domain (Supplemental Figure 5, A and B).
Domain-swapping experiments in which the ERα AF2 domain was
exchanged with the ERβ AF2 domain abolished the ability of PES1
to regulate ERα and ERβ protein levels (Supplemental Figure 5C),
indicating important roles of the AF2 domains of ERα and ERβ in
the regulation of ERα and ERβ protein levels by PES1.
To define the region of PES1 that modulates ERα and ERβ pro-
tein levels, we transfected HEK293T cells with a series of PES1
deletion mutants. The 221–322 region of PES1 (PES1Δ221–322),
but not other regions tested, enhanced ERα protein expression,
whereas the 311–588 region of PES1 (PES1Δ311–415) decreased ERβ
protein levels (Supplemental Figure 5, D and E). As expected,
PES1Δ221–322 and PES1Δ311–415 did not change the expression of ERα
and ERβ, respectively, but PES1Δ221–322 reduced ERβ expression,
and PES1Δ311–415 increased ERα expression (Figure 3, D and E). In
MCF7 cells, PES1Δ221–322 decreased ERβ transcriptional activity,
and PES1Δ311–415 increased ERα transcriptional activity, suggesting
that the alteration of ERα and ERβ protein levels by PES1Δ221–322
and PES1Δ311–415 correlates with their effects on ERα and ERβ
transactivation (Figure 3F).
2860 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
The E3 ubiquitin ligase CHIP is important for PES1 modulation of ERα
and ERβ protein stability. The finding that PES1 regulates ERα and
ERβ protein stability suggests that the ubiquitin-proteasome
pathway may be involved in this process. Indeed, addition of the
proteasome inhibitor MG132 or lactacystin blocked PES1 knock-
down–mediated ERα degradation and PES1 overexpression–medi-
ated ERβ degradation (Figure 4, A and B, and data not shown).
Overexpression of PES1 or PES1Δ311–415, which increases ERα
protein levels, reduced ERα ubiquitination, whereas expression
of PES1Δ211–322, which does not increase ERα protein levels, did
not (Supplemental Figure 6A). Likewise, overexpression of PES1
or PES1Δ211–322, which decreases ERβ protein levels, increased ERβ
PES1 differentially modulates the dimerization and promoter occupancy of ERα and ERβ. (A and B) Coimmunoprecipitation analysis of ERα and
ERβ homodimerization and ERα-ERβ heterodimerization. HEK293T cells transiently transfected with the indicated HA-, FLAG-, and MYC-tagged
constructs were immunoprecipitated with (A) anti-FLAG or (B) anti-MYC antibodies, followed by immunoblotting as indicated. (C) ChIP analysis
of the occupancy of PES1, ERα, or ERβ on the indicated estrogen-responsive proximal promoters (PPs) or distal enhancers (DEs) in MCF7 cells
treated with 10 nM E2 for 1 hour. The β-actin promoter was included as a negative control. IgG, normal serum. Data shown are mean ± SD of trip-
licate measurements that have been repeated 3 times with similar results. *P < 0.05, †P < 0.01 versus respective IgG without E2. #P < 0.01 versus
respective IgG with E2. (D) ChIP analysis of the occupancy of ERα or ERβ on CTSD and CCND1 promoters in MCF7 cells stably transfected
with control siRNA or PES1 siRNA and treated as in C. Data shown are mean ± SD of triplicate measurements that have been repeated 3 times
with similar results. *P < 0.01 versus respective control siRNA without E2. #P < 0.01 versus respective control siRNA with E2. (E) EMSA using
the in vitro–translated PES1 and the biotin-labeled CdRE or mutated CdRE (mCdRE) probe. Cold probe was used for competition experiments.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
Modulation of ERα and ERβ stability by PES1 correlates with their respective transcriptional activity. (A) Immunoblot analysis of MCF7 cells stably
transfected with PES1 siRNA or PES1 siRNA plus siRNA-resistant PES1 and treated with E2 for 24 hours. (B) Immunoblot analysis of ERα in MCF7
cells stably transfected with control siRNA or PES1 siRNA at the indicated times after exposure to the protein synthesis inhibitor cycloheximide (20
mg/ml) in the absence or presence of 10 nM E2. Graphs show quantification of immunoblot data. (C) Immunoblot analysis of ERβ in HEK293T cells
transiently transfected with FLAG-tagged ERβ (FLAG-ERβ) and MYC-tagged PES1 (MYC-PES1) and treated as in B. (B and C) Data shown are mean
± SD of 3 independent experiments. (D and E) Immunoblot showing (D) ERα and (E) ERβ protein levels in HEK293T cells transiently transfected with
ERα or ERβ and FLAG-PES1, FLAG-PES1Δ221–322, or FLAG-PES1Δ311–415. (F) Luciferase reporter assays of ERα and ERβ transcriptional activity in
MCF-7 cells transiently transfected with ERE-LUC and FLAG-tagged PES1, PES1Δ221–322, or PES1Δ311–415 and treated with 10 nM E2, 1 nM PPT, or 1
nM DPN for 24 hours. Results shown are mean ± SD of 3 independent experiments. *P < 0.01, #P < 0.01, †P < 0.01 versus empty vector with E2, PPT,
and DPN, respectively. Immunoblot analysis of FLAG-tagged PES1, PES1Δ221–322, or PES1Δ311–415 in the presence of 10 nM E2 is shown.
2862 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
ubiquitination, whereas PES1Δ311–415, which does not decrease ERβ
protein levels, did not (Supplemental Figure 6B). Importantly,
PES1 knockdown increased the ubiquitination of endogenous
ERα but decreased the ubiquitination of endogenous ERβ in
MCF7 cells (Figure 4, C and D).
ERα and ERβ are substrates of carboxyl terminus of Hsc70-inter-
acting protein (CHIP) (32–34), an E3 ubiquitin ligase. Intriguingly,
PES1 or PES1Δ311–415, but not PES1Δ211–322, reduced CHIP-mediated
ERα ubiquitination (Figure 4E). Likewise, PES1 or PES1Δ211–322, but
not PES1Δ311–415, increased CHIP-mediated ERβ ubiquitination (Fig-
ure 4F). CHIP knockdown greatly inhibited the ability of PES1 to
regulate ERα and ERβ ubiquitination (Supplemental Figure 6, C and
D). Furthermore, consistent with the ubiquitination results, CHIP
knockdown almost abolished the effects of PES1 on ERα and ERβ
degradation (Figure 4, G and H). These data suggest that CHIP plays
a key role in the modulation of ERα and ERβ protein levels by PES1.
PES1 oppositely regulates ERα and ERβ stability through the CHIP-mediated ubiquitin-proteasome pathway. (A) Immunoblot analysis of MCF7
cells stably transfected with PES1 siRNA or control siRNA and treated with 10 nM E2 or 10 nM E2 plus the proteasome inhibitor MG132 (10 μM).
(B) Immunoblot analysis of HEK293T cells transiently transfected with FLAG-PES1 and MYC-ERβ and treated as in A. (C and D) Ubiquitination of
endogenous ERα and ERβ. Cell lysates from MCF-7 cells stably transfected with PES1 siRNA were immunoprecipitated with antibodies specific
for (C) ERα or (D) ERβ, followed by immunoblotting with the indicated antibodies. Ub, ubiquitin. (E and F) Effects of PES1 on CHIP-mediated
ERα and ERβ ubiquitination. Coimmunoprecipitation was performed in HEK293T cells transiently transfected with the indicated constructs. (G
and H) Effects of PES1 on CHIP-mediated degradation of ERα and ERβ. Western blot analysis of MCF-7 cells transfected with (G) CHIP siRNA
and PES1 siRNA or with (H) CHIP siRNA and FLAG-PES1 and treated with 10 nM E2 for 24 hours.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
PES1 forms a complex with CHIP and ERβ but not with CHIP and
ERα. Based on our findings that PES1 regulates CHIP-dependent
ERα and ERβ degradation, we tested whether PES1 physically
interacts with ER and CHIP. Indeed, GST pull-down and coim-
munoprecipitation experiments showed that exogenous PES1 pro-
tein associated with exogenous ERα and ERβ proteins as well as
exogenous CHIP protein, both in vitro and in vivo (Supplemental
Figure 7, A and B, and Supplemental Figure 8, A and B). Impor-
tantly, in the absence or presence of E2, endogenous ERα, ERβ,
or CHIP from MCF7 cell lysates specifically coimmunoprecipi-
tated with endogenous PES1 (Figure 5, A–C). Moreover, confocal
immunofluorescence analysis of MCF7 cells revealed that PES1
also colocalized with ERα and ERβ in the absence or presence of
E2 (Supplemental Figure 7, C and D). These data strongly indicate
that PES1 interacts with ERα, ERβ, and CHIP.
To map interaction regions of ERα and ERβ in PES1, we per-
formed coimmunoprecipitation experiments using transfected
HEK293T cells. PES11–110, PES1111–220, PES1221–322, and PES1311–588
interacted with ERα and ERβ, whereas PES1415–588 did not (Sup-
plemental Figure 7, E and F). On the other hand, coimmunopre-
cipitation assays showed that PES1 interacted with the AF1 and
AF2 domains, but not the DNA-binding domain, of ERα but only
with the AF2 domain of ERβ (Supplemental Figure 7, G and H).
To define the region of CHIP that interacts with ERα, ERβ, and
PES1, we used CHIP deletion mutants in coimmunoprecipita-
tion assays. ERβ and PES1 interacted with both CHIP1–126, con-
taining the tetratricopeptide repeat domain, and CHIP127–304,
containing the U-box domain, but ERα interacted only with
CHIP1–126 (Supplemental Figure 8, C–E). Importantly, PES1
and CHIP formed a complex with ERβ but not with ERα, pos-
sibly because ERβ has more interaction regions in CHIP than
ERα (Figure 5D). Based on these observations, we tested
whether PES1 affects the interaction of CHIP with ERα and
ERβ. Consistent with the effects of PES1 on CHIP-mediat-
ed ERα and ERβ degradation, PES1 or PES1Δ311–415 reduced
the interaction between CHIP and ERα, whereas PES1Δ211–322
did not (Figure 5E and Supplemental Figure 9A). Likewise, PES1
or PES1Δ211–322 increased the interaction between CHIP and ERβ,
PES1 and CHIP formed a complex with ERβ but not with ERα. (A–C) Coimmunoprecipitation analysis of the endogenous interaction of PES1
with (A) ERα, (B) ERβ, or (C) CHIP. Cell lysates from MCF-7 cells were immunoprecipitated with antibodies specific for (A) ERα, (B) ERβ, or (C)
CHIP, followed by immunoblotting with the indicated antibodies. (D) PES1, ERβ, and CHIP formed a complex. HEK293T cells transfected with the
indicated plasmids were immunoprecipitated with anti-FLAG (1st IP). The immune complexes were eluted with FLAG peptide and reimmunopre-
cipitated (Re-IP) with anti-HA or normal IgG, followed by immunoblotting with the indicated antibodies. (E and F) Effects of PES1 on the interaction
of CHIP with (E) ERα and (F) ERβ. Coimmunoprecipitations were carried out in HEK293T cells transiently transfected with the indicated constructs.
2864 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
whereas PES1Δ311–415 did not (Figure 5F and Supplemental Fig-
ure 9B). These effects are specific for CHIP, because PES1 did not
change the interaction of ERα and ERβ with E6-associated pro-
tein, another E3 ubiquitin ligase for ERα and ERβ (refs. 35, 36, and
Supplemental Figure 9, C and D).
PES1 is required for estrogen-mediated breast tumor growth. Next,
we determined the effect of PES1 on breast cancer cell growth.
In assays of anchorage-dependent growth, MCF7 cells trans-
fected with PES1 grew faster than those transfected with
PES1Δ211–322, PES1Δ311–415, or empty vector, and MCF7 cells
transfected with PES1Δ211–322 or PES1Δ311–415 grew faster than
those transfected with empty vector (Figure 6A). In contrast,
PES1 knockdown almost completely abolished E2-mediated
growth stimulation of MCF7 cells (Figure 6B), and this phe-
notype was rescued by PES1 reexpression. Similar results were
observed in ZR75-1 and T47D cells (Supplemental Figure 10, A
and B). PES1 knockdown also greatly inhibited anchorage-inde-
pendent growth of MCF7, ZR75-1, and T47D cells (Figure 6C
and Supplemental Figure 10, C and D), and again, the observed
effects were rescued by PES1 reexpression in MCF7 cells (Figure
6C). Furthermore, all mice inoculated with MCF7 or ZR75-1
cells expressing control siRNA developed tumors in the pres-
ence of E2 but not in the absence of E2 (Figure 6D, Supple-
mental Figure 10E, and data not shown), suggesting that both
MCF7 and ZR75-1 cell lines are estrogen dependent. In con-
trast, in mice inoculated with MCF7 or ZR75-1 cells expressing
PES1 siRNA, only 1 or 3, respectively, out of 8 mice developed
tumors in the presence of E2, and these showed late latency and
a much smaller tumor size (Figure 6D and Supplemental Figure
10E). The tumors in mice inoculated with MCF7 cells express-
ing PES1 siRNA had reduced protein levels of ERα, progester-
one receptor (PR), MYC, CCND1, and survivin, and increased
levels of ERβ (Figure 6D). With the exception of those concern-
ing ERβ, similar effects were observed in ERβ-negative ZR75-1
cells (Supplemental Figure 10E).
Using growth in soft agar as an index of transformation, we
examined the effect of PES1 on transformation of HMECs.
HMECs with elevated PES1 expression levels, equivalent to those
of MCF7 cells, could grow in soft agar, whereas HMECs expressing
green fluorescent protein or empty vector could not (Figure 6E).
PES1 overexpression increased expression of ERα but decreased
that of ERβ.
Correlation of PES1 with ERα and ERβ in patients with breast cancer.
The implication of ERα in breast cancer has been widely investi-
gated with high-quality ERα antibodies. Thus, we examined the
specificity of anti-ERβ and anti-PES1 antibodies. We confirmed
the specificity of the antibodies by immunoblotting of lysates
from MCF7 cells transfected with ERβ siRNA or PES1 siRNA
(Supplemental Figure 11A), immunofluorescence analysis of ERβ
or PES1 knockdown MCF7 cells (Supplemental Figure 11B), and
immunohistochemical staining of breast cancer samples incubat-
ed with anti-ERβ or anti-PES1 antibodies preincubated with their
respective antigens (Supplemental Figure 11C). Intriguingly, con-
sistent with our findings that PES1 upregulated ERα and down-
regulated ERβs (ERβ, ERβ2, and ERβ5), immunohistochemical
staining of 116 breast cancer tissues and 92 normal tissues adja-
cent to breast cancer, using antibodies with confirmed specific-
ity, showed that PES1 expression correlated positively with ERα
expression (P < 10–4) but negatively with expression of ERβs
(P < 10–4) (Figure 7, A and B). These data strongly suggest impor-
tant pathological roles of PES1 in breast cancer.
PES1 predicts clinical outcome of tamoxifen therapy. To determine
the clinical relevance of PES1 modulation of ER, we analyzed
the survival follow-up information of 65 subjects. PES1 overex-
pression was significantly associated with better disease-free sur-
vival (P = 0.001) and overall survival (P = 0.002) in patients with
breast cancer who received tamoxifen treatment (Figure 7, C and
D). To verify the effects of PES1 on tamoxifen sensitivity, we per-
formed animal experiments. Overexpression of PES1 in ZR75-1
cells increased ERα expression and caused increased sensitivity to
tamoxifen (Supplemental Figure 10F).
An increasing number of studies show that estrogen signaling
depends principally on the balance between ERα and ERβ expres-
sion (3, 4). The disturbance of such balance and, especially, a pre-
dominance of proliferative ERα protein over antiproliferative ERβ
protein may cause cancer in estrogen-responsive organs. Thus, elu-
cidating the regulation of the balance between ERα and ERβ expres-
sion may not only provide novel mechanistic insights into estrogen-
induced tumorigenesis but also improve current endocrine therapy
for estrogen-related cancers. Our work reveals for what we believe
to be the first time that PES1 plays an essential role in estrogen-
induced breast tumor growth through regulation of the yin-yang
balance between ERα and ERβ (Figure 7E). First, we demonstrated
that PES1 enhances transcriptional activity of ERα and reduces
that of ERβ and modulates many estrogen-responsive genes. Sec-
ond, consistent with this regulation of ERα and ERβ transcrip-
tional activity, PES1 increased the stability of the ERα protein and
decreased that of ERβ through CHIP-mediated ubiquitin-protea-
some pathway. Third, PES1 can transform normal HMECs through
increased ERα protein and decreased ERβ protein and is required
for estrogen-induced breast tumor growth in nude mice. Fourth,
expression of PES1 correlates positively with ERα expression and
negatively with ERβ expression in patients with breast cancer. Since
PES1 transforms normal HMECs and is required for estrogen-
induced breast carcinogenesis. (A) Anchorage-dependent growth
assays in MCF-7 cells transiently transfected with FLAG-tagged
PES1, PES1Δ221–322, or PES1Δ311–415. The transfection efficiency is
approximately 30%. Cell viability was assessed at the indicated
times. *P < 0.01 versus empty vector on day 4. #P < 0.05, †P < 0.01
versus PES1 on day 4. Immunoblot analysis with anti-FLAG is
shown. (B) Anchorage-dependent growth assays in MCF-7 cells
stably transfected with PES1 siRNA or PES1 siRNA plus siRNA-
resistant PES1. Cells were treated with 10 nM E2 and analyzed as
in A. *P < 0.01 versus control siRNA without E2 on day 4. #P < 0.01
versus control siRNA with E2 on day 4. Immunoblot analysis with
anti-PES1 is shown. (C) Anchorage-independent growth assays in
MCF-7 cells stably transfected as in B. Scale bar: 50 μm. (A–C)
Data are shown as mean ± SD of 3 independent experiments.
*P < 0.01 versus control siRNA without E2. #P < 0.01 versus control
siRNA with E2. (D) Volume of xenograft tumors derived from MCF-7
cells expressing control siRNA or PES1 siRNA. Data are shown
as mean ± SD (n = 8 for control siRNA; n = 1 for PES1 siRNA due
to absence of visible tumors in the other 7 mice). *P < 0.01 ver-
sus control siRNA. Representative tumor tissues were subjected
to immunoblot analysis with indicated antibodies. (E) Anchorage-
independent growth assays in HMECs infected with recombinant
lentivirus carrying GFP or PES1. Scale bar: 50 μm. Immunoblotting
with the indicated antibodies is shown.
2866 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
Estrogen has been shown to stimulate the expression of PES1
protein (9–11). Our cDNA microarray analysis indicated that PES1
regulates the expression of 256 genes in MCF7 breast cancer cells,
including over 127 previously reported estrogen-responsive genes
(14–22). Moreover, PES1 knockdown almost abolishes estrogen-
mediated anchorage-dependent and -independent growth of
breast cancer cells, and most of mice inoculated with PES1 knock-
down breast cancer cells do not develop tumors, even in the pres-
ence of estrogen. These results suggest that PES1 is a key mediator
of estrogen signaling and that a positive feedback loop of estro-
gen/PES1 promotes malignant growth of breast cancer cells.
PES1 has been demonstrated to play important roles in embry-
onic development (43), ribosome biogenesis (44–46), cell cycle reg-
ulation (47), and chromosome stability (45), although molecular
mechanisms underlying these processes remain largely unknown.
We show that PES1 modulates many estrogen-responsive genes
that are known to have important functions in DNA replication,
cell cycle regulation, and DNA repair. For example, PES1 can reg-
ulate the expression of a large number of cell cycle–related genes,
including cyclin A2 (CCNA2), cyclin B2 (CCNB2), CCND1, CCNE2,
cyclin-dependent kinase 1 (CDK1), CDK2, E2F1, E2F2, cell-division
cycle gene 20 (CDC20), PCNA, MYC, MYB, and minichromosome
maintenance genes (MCM2–MCM8 and MCM10). In normal
human cells, cellular division is an ordered, tightly regulated
process, involving multiple cell cycle checkpoints that ensure
genomic integrity. Altered regulation of the cell cycle is a hall-
mark of human cancers (48). Cyclins and their associated CDKs
are the central machinery that governs cell cycle progression.
Overexpression of cyclin D1, the major regulatory subunit for
CDK4, is common in human cancers of epithelial cell origin (49).
Approximately 50% of human breast cancers express abnormally
high levels of cyclin D1, which is maintained throughout subse-
quent stages of breast cancer progression, from in situ carcinoma
to invasive carcinoma. Both cyclin D1 and CDK2 are required for
mammary tumorigenesis induced by the ErbB-2 oncogene (50).
Like cyclin D1, MYC and MYB are also overexpressed in breast
tumors (51, 52). Overexpression of MYC contributes to breast
cancer development and progression and is associated with poor
clinical outcome. MYC regulates cell cycle at the G1/S transition
through activation of downstream targets such as cyclin E/CDK2.
In addition, MYC promotes cell cycle progression through activa-
tion of cyclin D1, CDK4, E2F1, and E2F2. The expression of MYB
protein was shown to be important for estrogen-stimulated pro-
liferation of breast cancer cells (52). MYB controls G2/M cell cycle
transition by direct regulation of cyclin B1 expression. Like cyclin
D1, MYC, and MYB, E2F1 is also an estrogen-inducible protein
(53). E2F1 is necessary for estrogen regulation of breast cancer
cell proliferation. Interestingly, a large number of genes involved
in the control of the cell cycle contain regulatory binding sites for
E2F1 (54) (e.g., CCNA2, cyclin D3, CCNE2, CDK1, MYC, and PCNA
as well as E2F1 itself). DNA replication takes place in the S phase
of the cell cycle. The highly orchestrated process of DNA replica-
tion ensures the accurate inheritance of genetic information from
one cell generation to the next. The MCM proteins are essential
for the process of DNA replication (55, 56). Loss of MCM func-
tion results in DNA damage and genome instability. The fact that
PES1 can regulate many key molecules of cell cycle, DNA replica-
tion, and DNA repair suggests the importance of PES1 in breast
tumorigenesis and as a therapeutic target. It will be interesting to
investigate how PES1 regulates these molecules.
dysregulation of the balance between ERα and ERβ has also been
reported to be associated with other cancers, such as colon cancer
and thyroid cancer (37, 38), our data suggest that PES1 may not
only act as a determinant of breast tumorigenesis but also play an
important role in the development of other cancers. PES1 may be a
useful target for hormone-related cancer therapy.
Estrogen stimulates breast cancer cell growth through ERα, and
use of antiestrogens blocks this stimulating response (3, 39, 40).
As approximately 70% to 80% of all breast cancers are ERα positive
at the time of diagnosis, ERα expression has considerable implica-
tions for cancer biology and therapy. Tamoxifen is an antiestrogen
drug that was developed over 30 years ago and has been widely used
in the treatment of all stages of ERα-positive breast cancer (39, 40).
Tamoxifen binds and blocks ERα on the surface of cells, prevent-
ing estrogens from binding and activating the cell. Due to the dis-
covery of a second form of the ER, ERβ, in 1996, the role of tamoxi-
fen in breast cancer endocrine therapy appears to be complicated.
Tamoxifen also binds ERβ and inhibits ERβ transcriptional activ-
ity. However, many lines of evidence demonstrate that ERβ has an
antiproliferative function when reintroduced into ERα-positive
breast cancer cells (3, 5–7). In many ways, ERβ seems to oppose the
action of ERα. The same problem remains for another endocrine
drug, fulvestrant (41, 42). It is indicated for the treatment of ERα-
positive metastatic breast cancer in postmenopausal women after
treatment with other antiestrogens. Fulvestrant binds to ER and
prevents structural changes that are necessary for ER to initiate
gene transcription. On the other hand, fulvestrant degrades ER,
which also reduces gene transcription. Like tamoxifen, fulvestrant
also inhibits both ERα and ERβ transcriptional activity. Ideally,
in the treatment of patients with ERα-positive breast cancer, in
which ERβ is antagonistic to ERα, a drug that reduces transcrip-
tional activity of ERα but enhances that of ERβ should be better
than the currently used endocrine drugs tamoxifen or fulvestrant.
We demonstrate that PES1 increases ERα but decreases ERβ pro-
tein levels, correlating with their respective physiological activities
in breast cancer. Thus, PES1 represents a very promising target
for the development of better drugs for endocrine cancer therapy.
Association of PES1 with ERα and ERβ in breast cancer. (A and B)
Expression of ERα, ERβ, and PES1 in (A) human breast cancer tis-
sues and (B) normal tissues adjacent to breast cancer. Representative
immunohistochemical staining of PES1, ERα, and ERβ is shown at top.
Original magnification, ×20. Scale bar: 100 μm. A summary of (A) 116
breast cancer tissues or (B) 92 normal breast tissues is shown below,
with tissues categorized by low and high expression of PES1 and ERα or
ERβ. Case 1 and case 2 refer to 2 representative samples. The P value
was generated using the χ2 test. (C and D) Kaplan-Meier estimate of (C)
disease-free survival and (D) overall survival in 65 patients with breast
cancer who received tamoxifen treatment. Marks on graph lines represent
censored samples. High PES1 and low PES1 refer to samples with high
and low levels of PES1 expression, respectively. (E) Proposed model for
PES1 modulation of the balance between ERα and ERβ. The estrogen-
inducible protein PES1 blocks interaction of ERα with CHIP through its
interaction with CHIP and instead forms a complex with ERβ and CHIP,
leading to reduced degradation of ERα and increased degradation of
ERβ by CHIP. In turn, PES1 enhances transcriptional activity of ERα
but reduces that of ERβ through increased ERα homodimerization and
decreased ERβ homodimerization and ERα-ERβ heterodimerization.
Disruption of the balance between ERα and ERβ by PES1 contributes to
2868 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
immunoprecipitated with anti-FLAG (Sigma-Aldrich), anti-ERα (Millipore),
or anti-ERβ (Novus Biologicals) antibodies as described previously (60). The
immunocomplexes were subjected to immunoblot analysis with antibodies
specific for MYC or ubiquitin (both from Santa Cruz Biotechnology Inc.).
GST pull-down assay. GST fusion proteins were expressed in pGEX-KG and
purified according to the manufacturer’s instructions (Amersham Pharma-
cia Biotech). HEK293T extracts expressing ERα, ERβ, or CHIP were mixed
with 10 μg of GST derivatives bound to glutathione-Sepharose beads, and
the adsorbed proteins were analyzed as previously described (61).
Coimmunoprecipitation. Cell extracts were prepared, immunoprecipitated,
and analyzed as previously described (61). Immunoprecipitation was per-
formed with anti-FLAG M2 Affinity Gel (Sigma-Aldrich), anti-MYC Affini-
ty Gel (Sigma-Aldrich), anti-ERα (Millipore), anti-ERβ (Novus Biologicals),
or anti-CHIP (Santa Cruz Biotechnology Inc.).
Immunofluorescence. Immunofluorescence was performed as previously
described (57). Briefly, cells grown on glass coverslips were fixed, permeabi-
lized, and blocked in normal goat serum. The coverslips were then incubated
with rabbit anti-PES1 (Bethyl Laboratories) and mouse anti-ERα (Santa Cruz
Biotechnology Inc.) or mouse anti-PES1 (Santa Cruz Biotechnology Inc.) and
rabbit anti-ERβ (Millipore), followed by incubation with corresponding sec-
ondary antibodies. Nuclei were counterstained with DAPI. Confocal images
were collected using a Radiance2100 confocal microscope (Bio-Rad).
EMSA. The probes for EMSA were labeled with the Biotin 3′-End DNA
Labeling Kit (Pierce) as instructed by the manufacturer. The sequences
of the labeled oligonucleotides for the CdRE of the HO-1 gene (28), the
putative CdRE of the CCND1 gene, and the putative CdRE of the E2F1
gene were 5′-AATTCGGCGGATTTTGCTAGATTTTGCG-3′ (CdRE-HO-1),
and 5′-TTTGAACCTGATGCTAGATCTTTTTATTTT-3′ (CdRE-E2F1),
respectively (the core sequence is underlined). The mutated sequences
were 5′-AATTCGGCGGATTTTGCTGAATTTTGCG-3′ (mCdRE-HO-1),
and 5′-zTTTGAACCTGATGCTGAATCTTTTTATTTT-3′ (mCdRE-E2F1).
EMSA was performed using in vitro–translated protein or the same
amount of unprogrammed lysate (Promega) with LightShift Chemilu-
minescent EMSA Kits (Pierce). For competition experiments, a 100-fold
molar excess of unlabeled CdRE was mixed with the biotin-labeled probe.
The resulting protein-DNA complexes were analyzed by electrophoresis on
a polyacrylamide gel, followed by chemiluminescent detection.
Anchorage-dependent and -independent growth assays. Anchorage-dependent
cell proliferation was analyzed by a crystal violet assay as described pre-
viously (59). For anchorage-independent growth (57), 1 × 104 cells were
plated on 6-cm plates containing a bottom layer of 0.6% low-melting-tem-
perature agar in DMEM and a top layer of 0.3% agar in DMEM. Colonies
were scored after 3 weeks of growth.
Animal experiments. Two days after implantation of estrogen pellets (E2,
0.36 mg/pellet, 60-day release) (Innovative Research of America), 1 × 107
tumor cells were injected into the abdominal mammary fat pad of 6-week-
old female nude mice. When tumors reached the volume of approxi-
mately 100 mm3, we randomly allocated the mice to groups in which they
received placebo or tamoxifen pellets (Innovative Research of America).
Tumor growth was monitored by caliper measurements. Excised tumors
were weighed, and portions were frozen in liquid nitrogen or fixed in 4%
paraformaldehyde for further study.
Immunohistochemistry. Immunohistochemical staining was performed as
described previously (59) using rabbit anti-ERα (Millipore), rabbit anti-ERβ
(Millipore), and rabbit anti-PES1 (Bethyl Laboratories) as primary antibodies.
Statistics. Differences among variables were assessed by χ2 analysis or
2-tailed Student’s t test. Estimation of disease-free and overall survival was
performed using the Kaplan-Meier method, and differences between sur-
Plasmids and siRNAs. The estrogen-responsive reporter construct ERE-Luc
and eukaryotic expression vectors for FLAG-tagged ERα and ERβ have
been described previously (57–59). Other mammalian expression vectors
encoding FLAG-, MYC-, or HA-fusion proteins tagged at the amino termi-
nus were constructed by inserting PCR-amplified fragments into pcDNA3
(Invitrogen) or pIRESpuro2 (Clontech). Plasmids encoding GST fusion
proteins were generated by cloning PCR-amplified sequences into pGEX-
KG (Amersham Pharmacia Biotech). The cDNA target sequences of siRNAs
for PES1 and CHIP were ACACAAGAAGAAGGTTAAC and GCACGA-
CAAGTACATGGCGGA, respectively, and were inserted into pSilencer2.1-
U6neo (Ambion) and pSIH-H1-puro (System Biosciences). Expression
vectors for siRNA-resistant PES1 containing a silent mutation in the 3′
nucleotide of a codon in the middle of the siRNA-binding site were gener-
ated by recombinant PCR. Recombinant lentivirus vectors for PES1 or GFP
were made by inserting PCR-amplified fragments into pCDH-EF1-MCS-
T2A-puro (System Biosciences).
Cell culture, transfection, and luciferase reporter assay. HEK293T embryonic
kidney cells and MCF7, ZR75-1, T47D, and SKBR3 breast cancer cells were
routinely cultured in DMEM (Invitrogen) containing 10% FBS (Hyclone).
Normal HMECs (Invitrogen) were cultured in HMEC medium (Invitrogen).
For hormone treatment experiments, cells were cultured in medium
containing phenol red–free DMEM supplemented with 10% charcoal/
dextran-treated FBS (Hyclone). Lipofectamine 2000 reagent was used for
transfections following the manufacturer’s protocol (Invitrogen). Lentivi-
ruses were produced by cotransfection of HEK293T cells with recombinant
lentivirus vectors and pPACK Packaging Plasmid Mix (System Biosciences)
using Megatran reagent (Origene). Lentiviruses were collected 48 hours
after transfection and added to the medium of target cells with 8 μg/ml
polybrene (Sigma-Aldrich). Stable cell lines were selected in 500 μg/ml
G418 or 1 μg/ml puromycin for approximately 2 months. Pooled clones
or individual clones were screened by standard immunoblot protocols
and produced similar results. PES1 knockdown stable cell lines grew very
slowly and could only be passaged several times. Luciferase reporter assays
were performed as described previously (59).
cDNA microarray analysis. cDNA generated from RNA was labeled with
Cy3 (PES1 siRNA) and Cy5 (control siRNA), mixed, and hybridized to
human oligo chip 35 k v2.0 containing 35,000 human gene elements (Cap-
italBio). The chip was scanned by LuxScan 10K/A (CapitalBio), and data
were analyzed using MAS 3.0 Software (CapitalBio). All results were given
as the gene expression ratio (ratio of the intensity of Cy3 to that of Cy5).
Genes with more than or equal to 2-fold intensity change were considered
of interest and subjected to further investigation by gene ontology analy-
sis and pathway analysis based on the Kyoto Encyclopedia of Genes and
Real-time RT-PCR. Total RNA was isolated using TRIzol Reagent
(Invitrogen) and reverse transcribed using SuperScript II Reverse Tran-
scriptase (Invitrogen). Real-time PCR was performed with the primers
listed in Supplemental Table 4A as described previously (57).
ChIP assay. ChIP assays were performed as described previously (57) using
anti-ERα (Millipore), anti-ERβ (Novus Biologicals), and anti-PES1 (Bethyl
Laboratories). The primers used for ChIP are listed in Supplemental Table 4B.
Cycloheximide chase assay. Transfected cells were cultured in phenol red–
free DMEM medium supplemented with 10% charcoal-stripped FBS for
3 days. Cells were treated with 20 μg/ml cycloheximide for different time
periods in the presence or absence of 10 nM E2. Cell lysates were analyzed
by immunoblotting with antibodies specific for ERα (Sigma-Aldrich),
FLAG (Sigma-Aldrich), and GAPDH (Santa Cruz Biotechnology Inc.).
Ubiquitination assay. Transfected cells were treated with the proteasome
inhibitor MG132 (10 μM) for 4 hours. The cells were lysed in RIPA buffer and
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
Program (2011CB504202 and 2012CB945100), National Natu-
ral Science Foundation (81072173, 30625035, and 30530320),
and National Key Technologies R&D Program for New Drugs
Received for publication December 30, 2011, and accepted in
revised form May 31, 2012.
Address correspondence to: Qinong Ye, Department of Medical
Molecular Biology, Beijing Institute of Biotechnology, 27 Tai-
Ping Lu Rd., Beijing 100850, China. Phone: 8610.6818.0809; Fax:
8610.6824.8045; E-mail: email@example.com.
vival curves were determined with the log-rank test. Statistical calculations
were performed using SPSS 13.0. P values of less than 0.05 were considered
Study approval. Animal studies were approved by the Institutional Animal
Care Committee of Beijing Institute of Biotechnology. Breast cancer sam-
ples were obtained from Chinese PLA General Hospital, with the informed
consent of patients and with institutional approval for experiments from
Chinese PLA General Hospital and Beijing Institute of Biotechnology.
We thank Rong Li and Haian Fu for helpful discussions. This
work was supported by Major State Basic Research Development
1. Deroo BJ, Korach KS. Estrogen receptors and
human disease. J Clin Invest. 2006;116(3):561–570.
2. Yager JD, Davidson NE. Estrogen carcinogenesis in
breast cancer. N Engl J Med. 2006;354(3):270–282.
3. Thomas C, Gustafsson JA. The different roles of
ER subtypes in cancer biology and therapy. Nat Rev
4. Matthews J, Gustafsson JA. Estrogen signaling: a
subtle balance between ERα and ERβ. Mol Interv.
5. Helguero LA, Faulds MH, Gustafsson JA, Haldosen
LA. Estrogen receptors alfa (ERα) and beta (ERβ)
differentially regulate proliferation and apoptosis
of the normal murine mammary epithelial cell line
HC11. Oncogene. 2005;24(44):6605–6616.
6. Paruthiyil S, Parmar H, Kerekatte V, Cunha GR,
Firestone GL, Leiteman DC. Estrogen receptor β
inhibits human breast cancer cell proliferation and
tumor formation by causing a G2 cell cycle arrest.
Cancer Res. 2004;64(1):423–428.
7. Omoto Y, Eguchi H, Yamamoto-Yamaguchi Y,
Hayashi S. Estrogen receptor (ER) β1 and ERβcx/β2
inhibit ERα function differently in breast cancer cell
line MCF7. Oncogene. 2003;22(32):5011–5020.
8. Roger P, Sahla ME, Makela S, Gustafsson JA, Baldet
P, Rochefor H. Decreased expression of estrogen
receptor β protein in proliferative preinvasive mam-
mary tumors. Cancer Res. 2001;61(6):2537–2541.
9. Charpentier AH, et al. Effects of estrogen on global
gene expression: identification of novel targets of
estrogen action. Cancer Res. 2000;60(21):5977–5983.
10. Li J, et al. Down-regulation of pescadillo inhibits
proliferation and tumorigenicity of breast cancer
cells. Cancer Sci. 2009;100(12):2255–2260.
11. Zhang H, et al. The antibody preparation and
expression of human Pescadillo. Sci China C Life Sci.
12. Nilsson S, Koehler KF, Gustafsson JA. Development
of subtype-selective oestrogen receptor-based thera-
peutics. Nat Rev Drug Discov. 2011;10(10):778–792.
13. Shanle EK, Xu W. Selectively targeting estrogen
receptors for cancer treatment. Adv Drug Deliv Rev.
14. Welboren WJ, Sweep FC, Span PN, Stunnenberg
HG. Genomic actions of estrogen receptor α: what
are the targets and how are they regulated? Endocr
Relat Cancer. 2009;16(4):1073–1089.
15. Williams C, Edvardsson K, Lewandowski SA, Strom
A, Gustafsson JA. A genome-wide study of the
repressive effects of estrogen receptor β on estrogen
receptor α signaling in breast cancer cells. Oncogene.
16. Bourdeau V, Deschenes J, Laperriere D, Aid M,
White JH, Mader S. Mechanisms of primary and
secondary estrogen target gene regulation in breast
cancer cells. Nucleic Acids Res. 2008;36(1):76–93.
17. Chang EC, Frasor J, Komm B, Katzenellenbogen
BS. Impact of estrogen receptor β on gene networks
regulated by estrogen receptor α in breast cancer
cells. Endocrinology. 2006;147(10):4831–4842.
18. Carroll JS, et al. Genome-wide analysis of estro-
gen receptor binding sites. Nat Genet. 2006;
19. Carroll JS, et al. Chromosome-wide mapping of
estrogen receptor binding reveals long-range regu-
lation requiring the forkhead protein FoxA1. Cell.
20. Lindberg MK, et al. Estrogen receptor (ER)-β reduc-
es ERα-regulated gene transcription, supporting a
“ying yang” relationship between ERα and ERβ in
mice. Mol Endocrinol. 2003;17(2):203–208.
21. Frasor J, Danes JM, Komm B, Chang KC, Lyttle
CR, Katzenellenbogen BS. Profiling of estrogen
up- and down-regulated gene expression in human
breast cancer cells: insights into gene networks
and pathways underlying estrogenic control of
proliferation and cell phenotype. Endocrinology.
22. Coser KR, Chesnes J, Hur J, Ray S, Isselbacher KJ,
Shioda T. Global analysis of ligand sensitivity
of estrogen inducible and suppressible genes in
MCF7/BUS breast cancer cells by DNA microarray.
Proc Natl Acad Sci U S A. 2003;100(24):13994–13999.
23. Santen R, et al. Estrogen mediation of breast tumor
formation involves estrogen receptor-dependent,
as well as independent, genotoxic effects. Ann N Y
Acad Sci. 2009;1155:132–140.
24. Foster JS, Henley DC, Ahamed S, Wimalasena J.
Estrogens and cell-cycle regulation in breast cancer.
Trends Endocrinol Metab. 2001;12(7):320–327.
25. Fox EM, Davis RJ, Shupnik MA. ERβ in breast can-
cer—Onlooker, passive player, or active protector?
26. Bretschneider N, Kangaspeska S, Seifert M, Reid
G, Gannon F, Denger S. E2-mediated cathepsin
D (CTSD) activation involves looping of distal
enhancer elements. Mol Oncol. 2008;2(2):182–190.
27. Eeckhoute J, Carroll JS, Geistlinger TR, Torres-
Arzayus MI, Brown M. A cell-type-specific tran-
scriptional network required for estrogen regu-
lation of cyclin D1 and cell cycle progression in
breast cancer. Genes Dev. 2006;20(18):2513–2526.
28. Sikorski EM, Uo T, Morrison RS, Agarwal A. Pesca-
dillo interacts with the cadmium response element of
the human heme oxygenase-1 promoter in renal epi-
thelial cells. J Biol Chem. 2006;281(34):24423–24430.
29. Shaaban AM, et al. Nuclear and cytoplasmic expres-
sion of ERβ1, ERβ2, and ERβ5 identifies distinct
prognostic outcome for breast cancer patients. Clin
Cancer Res. 2008;14(16):5228–5235.
30. Honma N, et al. Clinical importance of estrogen
receptor-β evaluation in breast cancer patients
treated with adjuvant tamoxifen therapy. J Clin
31. Peng B, Lu B, Leygue E, Murphy LC. Putative func-
tional characteristics of human estrogen receptor-β
isoforms. J Mol Endocrinol. 2003;30(1):13–29.
32. Tateishi Y, et al. Ligand-dependent switching of
ubiquitin–proteasome pathways for estrogen
receptor. EMBO J. 2004;23(24):4813–4823.
33. Fan M, Park A, Nephew KP. CHIP (Carboxyl
terminus of hsc70-interacting protein) pro-
motes basal and geldanamycin-induced degra-
dation of estrogen receptor-α. Mol Endocrinol.
34. Tateishi Y, et al. Turning off estrogen receptor
β-mediated transcription requires estrogen-
dependent receptor proteolysis. Mol Cell Biol. 2006;
35. Li L, Li Z, Howley PM, Sacks DB. E6AP and
calmodulin reciprocally regulate estrogen receptor
stability. J Biol Chem. 2006;281(4):1978–1985.
36. Picard N, et al. Phosphorylation of activation func-
tion-1 regulates proteasome-dependent nuclear
mobility and E6-associated protein ubiquitin
ligase recruitment to the estrogen receptor beta.
Mol Endocrinol. 2008;22(2):317–330.
37. Chen GG, Vlantis AC, Zeng Q, van Hasselt CA.
Regulation of cell growth by estrogen signaling
and potential targets in thyroid cancer. Curr Cancer
Drug Targets. 2008;8(5):367–377.
38. Bardin A, Boulle N, Lazennec G, Vignon F, Pujol
P. Loss of ERβ expression as a common step in
estrogen-dependent tumor progression. Endocr
Relat Cancer. 2004;11(3):537–551.
39. Riggs BL, Hartmann LC. Selective estrogen-
receptor modulators—mechanisms of action
and application to clinical practice. N Engl J Med.
40. Jordan VC. Selective estrogen receptor modulation:
concept and consequences in cancer. Cancer Cell.
41. Robertson JF. Fulvestrant (Faslodex)—how to make
a good drug better. Oncologist. 2007;12(7):774–784.
42. Croxtall JD, McKeage K. Fulvestrant: a review of its
use in the management of hormone receptor-pos-
itive metastatic breast cancer in postmenopausal
women. Drugs. 2011;71(3):363–380.
43. Allende ML, Amsterdam A, Becker T, Kawakami
K, Gaiano N, Hopkins N. Insertional mutagenesis
in zebrafish identifies two novel genes, pescadillo
and dead eye, essential for embryonic development.
Genes Dev. 1996;10(24):3141–3155.
44. Lapik YR, Fernandes CJ, Lau LF, Pestov DG. Physi-
cal and functional interaction between Pes1 and
Bop1 in mammalian ribosome biogenesis. Mol Cell.
45. Killian A, et al. Inactivation of the RRB1-Pes-
cadillo pathway involved in ribosome biogen-
esis induces chromosomal instability. Oncogene.
46. Grimm T, et al. Dominant-negative Pes1 mutants
inhibit ribosomal RNA processing and cell prolif-
eration via incorporation into the PeBoW-complex.
Nucleic Acids Res. 2006;34(10):3030–3043.
47. Kinoshita Y, et al. Pescadillo, a novel cell cycle regu-
latory protein abnormally expressed in malignant
cells. J Biol Chem. 2001;276(9):6656–6665.
48. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144(5):646–674.
49. Musgrove EA, Caldon CE, Barraclough J, Stone A,
Sutherland RL. Cyclin D as a therapeutic target in
cancer. Nat Rev Cancer. 2011;11(8):558–572.
2870 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
50. Ray D, Terao Y, Christov K, Kaldis P, Kiyokawa H.
Cdk2-null mice are resistant to ErbB-2–induced
mammary tumorigenesis. Neoplasia. 2011;
51. Meyer N, Penn LZ. Reflecting on 25 years with
MYC. Nat Rev Cancer. 2008;8(12):976–990.
52. Ramsay RG. Gonda TJ. MYB function in normal
and cancer cells. Nat Rev Cancer. 2008;8(7):523–534.
53. Stender JD, Frasor J, Komm B, Chang K, Kraus WL,
Katzenellenbogen BS. Estrogen-regulated gene net-
works in human breast cancer cells: involvement of
E2F1 in the regulation of cell proliferation. Mol
54. Chen HZ, Tsai SY, Leone G. Emerging roles of E2Fs
in cancer: an exit from cell cycle control. Nat Rev
55. Maiorano D, Lutzmann M, Mechali M. MCM
proteins and DNA replication. Curr Opin Cell Biol.
56. Bailis JM, Forsburg SL. MCM proteins: DNA dam-
age, mutagenesis and repair. Curr Opin Genet Dev.
57. Ding L, et al. Human four-and-a-half LIM fam-
ily members suppress tumor cell growth through
a TGF-β-like signaling pathway. J Clin Invest.
58. He X, et al. c-Abl regulates estrogen receptor α tran-
scription activity through its stabilization by phos-
phorylation. Oncogene. 2010;29(15):2238–2251.
59. Zhang H, et al. Stimulatory cross-talk between
NFAT3 and estrogen receptor in breast cancer cells.
J Biol Chem. 2005;280(52):43188–43197.
60. Han J, et al. Hepatitis B virus X protein and the
estrogen receptor variant lacking exon 5 inhibit
estrogen receptor signaling in hepatoma cells.
Nucleic Acids Res. 2006;34(10):3095–3106.
61. Ding L, et al. Ligand-independent activation of
estrogen receptor α by XBP-1. Nucleic Acids Res.