Biochemical and Biophysical Research Communications 474 (2016) 400e405

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

ERRF is essential for Estrogen-Estrogen Receptor alpha signaling

pathway in ER positive breast cancer cells
Ang Luo*, Xuan Zhang
Department of Genetics and Cell Biology, Nankai University College of Life Sciences, Tianjin, China

a r t i c l e i n f o a b s t r a c t

Article history: Estrogen-Estrogen Receptor alpha (ERa) belongs to one of the most important signaling pathways
Received 18 April 2016 controlling breast tissue development and progression of breast cancer. ERRF was recently identified as a
Accepted 25 April 2016 candidate breast cancer associated protein and showed positive association with ERa status in clinical
Available online 26 April 2016
samples and cell lines. To further explore the relationship between ERRF and ERa, we studied whether
ERRF plays any roles in estrogen-ERa pathway. Knockdown of ERRF in ER positive breast cancer cells T-
Keywords:
47D and BT-474 reduced the level of p-AKT, p-MAPK, and phosphorylation of ERa at Ser 118 and Ser 167,
ERRF
and the transcriptional activity of ERa was inhibited as well. Further mechanism study proved ERRF to be
Estrogen receptor alpha (ERa)
Breast cancer
an interacting partner of ERa. In total, these data revealed that ERRF is essential for the activity of E2-ERa
pathway.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction
As one of the major steroid hormones in mammalian, the roles
of estrogen in many tissues have been studied for many years, both
physiologically and pathologically [1]. Especially in the mammary
tissue, estrogen is central to the growth and development of the
mammary gland [2]. No wonder that the level of estrogen is a key
factor for the initiation and progression of breast cancer [3e6]. In
the mammary epithelial, estrogen controls many cellular activities
such as proliferation, differentiation and migration [7,8]. To exert its
functions in different tissues estrogen has to bind its receptors,
including alpha and beta (ERa and ERb), the former is the major one
involved in breast cancer and chosen as an important target for
endocrine therapy in clinic [3,9]. ERa mainly localizes in nuclear
region and minor in the cytoplasm and cell membrane, which de-
termines that ERa could function in different ways [10]. On one
hand, membrane-localized ERa usually forms different complexes
with proteins such as tyrosine kinase Src, PI3K, seven trans-
membrane G-protein-coupled receptor (GPCR-30) and epithelial
growth factor receptor (EGFR) [11]. Binding to estrogen quickly
leads to changes in the conformation of ERa in these protein
complexes, which further activate pathways such as MAPK and
AKT, resulting in the promotion of cellular proliferation. On the
* Corresponding author.
E-mail address: luoang973@mail.nankai.edu.cn (A. Luo).
other hand, binding with estrogen would trigger the phosphory-
lation of ERa at multiple sites, which could also be partly enhanced
by the activated MAPK and AKT. Phosphorylated ERa was able to
enter the nuclear region following dimerization, and by interacting
with other proteins, such as Fork head protein FOXA1, pre-B cell
leukemia homeobox 1 (PBX1), p300 and steroid receptor co-
activator family members (SRCs) to transcriptionally regulate the
expression of downstream genes by binding to the classic estrogen
response element (ERE) on their promoters [12e17]. The estrogen-
ERa complex could not only activate the expression of MYC,
cyclinD1 and GREB1 but also repress the expression of genes such as
p21, cyclinG2 and p27, the combined effect is the promotion of cell
proliferation and survival [18e20]. However because of the cross-
talk between estrogen-ERa and other pathways such as EGFRs and
IGF1-R, ERa can also be phosphorylated in the absence of estrogen,
which is considered to be related to endocrine therapy resistance
[21,22].
Estrogen receptor-related factor (ERRF) was previously identi-
fied in our lab as a candidate breast cancer related gene [23]. We
had shown that the expression of ERRF was significantly upregu-
lated in breast cancer samples and associated with clinical prog-
nosis. Of note, the expression of ERRF was positively associated
with ERa status both in breast cancer cell lines and clinical samples.
Considering the relevance between ERRF and ERa, and to further
validate its role as a therapeutic marker we deeply studied rela-
tionship between estrogen-ERa pathway and ERRF. In ER positive
breast cancer cells knockdown of ERRF decreased the

http://dx.doi.org/10.1016/j.bbrc.2016.04.132
0006-291X/© 2016 Elsevier Inc. All rights reserved.
 A. Luo, X. Zhang / Biochemical and Biophysical Research Communications 474 (2016) 400e405
phosphorylation of ERa at Ser118 and Ser167, and the level of ERa
was also reduced in a cell-type-specific manner. Besides, the tran-
scriptional activity of ERa was also inhibited following ERRF
reduction. Interestingly, ERRF was further proved to be an inter-
acting protein of ERa.
2. Material and methods
2.1. Cell culture and regents
Cell lines used were all from American Type Culture Collection
(ATCC, Manassas, Virginia). T-47D and BT-474 cells were cultured
RPMI160 (Gibco, Shanghai, China) supplemented with 10% fetal
bovine serum (FBS). MDA-MB-361 cells were cultured in L-15
(Gibco) supplemented with 10% FBS. 293 T cells and MCF7 cells
were cultured in DMEM (Gibco) supplemented with 10% FBS. For
17b-Estradiol (E2) treatment assays, cells were pre-cultured in
hormone starvation condition for 48 h: phenol-red-free RPMI1640
(Hyclone, Logan, Utah) supplemented with 5% charcoal-striped FBS
(Bioind, Kibbutz Beit Haemek, Israel) and 4 mM L-GlutaMAX
(Gibco) for T-47D and BT-474, phenol-red-free DMEM (Hyclone)
with 5% charcoal-striped FBS for MCF7, phenol-red-free L-15 with
10% charcoal-striped FBS for MDA-MB-361. 17b-Estradiol was from
Sigma-Aldrich (Shanghai, China) and dissolved in ethanol.
2.2. Plasmids, siRNAs and transfections
ORF sequence of ERRF was cloned from total cDNA of T-47D cells
by PCR and then sub-cloned into the relevant vectors. ERE-TK-Luc
reporter plasmid, pCDNA-3.0-FLAG-ERa, Control siRNA, siERRF-
1(siM1) and siERRF-2(siM2) were the same ones that have been
used in our lab previously [23,24]. siERRF-3 (sc-78852) was pur-
chased from SantaCruze Biotechnology (Shanghai, China). Plasmid
transfections in 293 T cells were done with EntransterTM-D
(Engreen Biosystem Co,Ltd, Beijing, China). siRNA transfections
were done with Lipofectamine RNAiMAX transfection reagent
(Invitrogen, Shanghai, China). Plasmid and siRNA co-transfections
were done with Lipofectamine 3000 Reagent (Invitrogen).
2.3. Western blot
Cell lysates were prepared in chilled cell lysis buffer (150 mM
NaCl, 50 mM Tris-HCl (pH7.4), 1% NP-40, 5% glycerin, 1 mM EDTA)
supplemented with cocktail protease inhibitor (Roche, Shanghai,
China) on ice for 30 min. After centrifugation the supernatant was
mixed with 4
loading buffer and boiled at 98  C for 8 min and
then used for loading. Proteins were separated in 8%e10% SDS-
PAGE and transferred to PVDF membrane (Millipore, Shanghai,
China) in wet conditions. The membrane was blocked in 5% non-fat
milk for two hours at room temperature, and then incubated with
primary antibody at 4  C overnight. After incubation in HRP-
conjugated secondary antibody for 2 h the membrane was
washed with TBST buffer for 3
5 min. The blot was developed
with WesternBright ECL HRP substrate (Advansta, Menlo Park,
California), and the image was captured by using ImageQuant LAS
4000mini (GE). Primary antibodies used in present study include:
b-Actin (A1978; Sigma), FLAG-tag antibody (F7425; Sigma), ERa
(04e820; Millipore), GFP (sc-8334; Santa Cruze Biotechnology).
Antibodies for AKT, p-AKT (Ser473), MAPK, p-MAPK, p-ERa (Ser118)
and p-ERa (Ser167) were all from Cell signaling Technology (Dan-
vers, Massachusetts).
2.4. Co-immunoprecipitation assay
Cells were seeded and transfected in 60 mm plates. For 293 T
401
cells, 24 h after transfection the cell lysates were prepared in cell
lysis buffer with protease inhibitor. The lysates were then incubated
overnight with ANTI-FLAG M2 affinity gel (A2220; Sigma) or
Agarose conjugated Anti-GFP D153-8 (MBL International, Woburn,
Massachusetts). The unspecific proteins were washed out with
chilled cell lysis buffer for five times. After wash the agarose was
resuspended in 1
SDS loading buffer, boiled for five minutes,
chilled on ice and centrifuged, the supernatant was used for SDS-
PAGE.
2.5. Luciferase reporter assay
Luciferase reporter assay were performed with Dual-Luciferase
Reporter Assay System (Promega, Madison, Wisconsin)) and in
TriStar LB 942 (Berthold, Bad Wildbad, Germany). Cells were
seeded in 24-well plates at densities of 2
104. After hormone
starvation, the cells were co-transfected with 200 ng plasmids and
50 nM siRNAs, and pRL-TK plasmid was co-transfected as the in-
ternal control. 48 h after tranfection, cells were treated with E2 or
ethanol for another 12 h and were then prepared for luciferase
activity determination.
2.6. RNA isolation and RT-PCR
Total RNA from cells was isolated by using TriPure Isolation
Reagent (Roche, Shanghai, China). 1 mg of RNA was reverse-
transcribed into 20 ml cDNA with PrimeScript™ RT Master Mix
(Takara, DaLian, China). The product of reverse transcription was
diluted by 1:5, and 2 ml diluent was used in each RT-PCR reaction.
Semiquantitative RT-PCR was done with 2
Es Taq MasterMiX
(CWbiotech, Beijing, China) and quantitative real-time PCR (qRT-
PCR) was done with SYBR® Premix Ex Taq II (2
) (Takara), and both
in a 20 ml reaction system. qRT-PCR was performed on Mastercycler
Realplex (Eppendorf, Shanghai, China). Primers for qRT-PCR used in
current study included: ERRF:50 -GAGCCAACCTCAAAGGC-30 (for-
ward) and 50 -CCGTGGGTGCAGTCAATA-30 (reverse); GAPDH: 50 -
GGTGGTCTCCTCTGACTTCAACA-30 (forward) and 50 -
GTTGCTGTAGCCAAATTCGTTGT-30 (reverse); PS2: 50 -AAA-
TAAGGGCTGCTGTTTCGACGAC-30 (forward) and 50 -
0
CAGAAGCGTGTCTGAGGTGTCCG-3 (reverse).
2.7. Statistical analysis
Statistical analysis was performed in Microsoft Excel. Student's t
test was used to compare the means of variables. P < 0.05 was
considered as statistical significant.
3. Results
3.1. ERRF knockdown inhibits the phosphorylation of AKT, MAPK
and ERa
Stimulation with E2 often causes a rapid response in the
extranuclear region of ER positive breast cancer cells, a process
which is associated with the activation of Src kinase, MAPK, AKT
and PI3K and is considered to play important roles in breast cancer
progression and metastasis [25]. To study whether ERRF plays any
roles in this process, we investigated the influences of ERRF
knockdown on the activation of AKT and MAPK. In addition to the
previously-used two ERRF siRNAs, here we added another ERRF
siRNA, namely siERRF-3. Because presently there's no suitable
antibody available to detect the endogenous ERRF protein, to
confirm the effect of ERRF siRNAs on ERRF protein we co-expressed
ERRF siRNAs and FLAG-ERRF plasmid in T-47D cells and BT-474 cells
and examined the efficiency of the siRNAs. It showed that the
402 A. Luo, X. Zhang / Biochemical and Biophysical Research Communications 474 (2016) 400e405

exogenous ERRF protein could be effectively reduced by ERRF siR-
NAs (Fig. 1A). In the condition of E2 treatment, ERRF knockdown led
to reduction of the levels of phosphorylated AKT (p-AKT) and
phosphorylated MAPK (p-MAPK) in T-47D cells, the efficiency of
ERRF siRNAs was verified by semiquantitative RT-PCR (Fig. 1B). As
phosphorylation of ERa was essential for its functions, we exam-
ined the influence of ERRF knockdown on two of the most well-
studied phosphorylation site of ERa, Ser118 and Ser167. It showed
the level of both p-ERa-S167 and p-ERa-S118 were reduced
following ERRF knockdown under E2 treatment, while the level of
ERa showed no significantly change (Fig. 1B). In addition, ERRF
knockdown could also reduce the level of p-AKT and p-MAPK in BT-
474 cells, but what's different from the case in T-47D cells was that
ERRF knockdown caused not only reduction of the level of phos-
phorylated ERa but also total ERa whereas the expression of ESR1
was not affected correspondingly ((Fig. 1C and data not shown). In
addition, we found in MCF7 cells that ERRF knockdown led to
similar changes on the level of ERa and its phosphorylation as that
in BT-474 cells but the level of p-AKT and p-MAPK was not affected
(Fig. S1).
3.2. ERRF knockdown inhibits the transcriptional activity of ERa
As E2-ERa pathway functions mainly through directly regu-
lating the transcription of its downstream genes, we tested the
effects of ERRF knockdown on the transcriptional activity of ERa. In
T-47D cells and BT-474 cells, both siERRF-1 and siERRF-2 was able
to reduce the ERE-TK-Luc reporter activity either in the presence or
the absence of E2 (Fig. 2A and data not shown). Then we examined
the mRNA level of PS2, a classic target of ERa, in T-47D, BT-474 and
MDA-MB-361 cells following ERRF knockdown by using qRT-PCR
(Figs. 2BeD). It showed that the expression of PS2 was reduced in
all the cell lines tested. In combination with the above data, it
suggests that ERRF plays important roles in maintaining activity of
E2- ERa pathway.
3.3. ERRF is an interacting protein of ERa
To explore the mechanism by which ERRF mediates the level of
ERa and its phosphorylation, we tested the interaction between
ERRF and ERa. ERRF and ERa were exogenously co-expressed in
293 T cells. Following co-immunoprecipitation, Western blot
showed that GFP-ERa was detected in the immunoprecipitates of
FLAG-ERRF but not in the control group (Fig. 3A). Similarly, FLAG-
tagged ERa was also proved to be able bind Myc-tagged ERRF,
indicating ERRF and ERa could interact with each other in 293 T
cells (Fig. 3B). Then, to define the regions of ERa that mediate its
interaction with ERRF, different regions of ERa were respectively
constructed into pEGFP-C1 vector and co-expressed with Myc-ERRF
in 293 T cells. Western blot following co-immunoprecipitation

Fig. 1. Knockdown of ERRF inhibits E2-ERa signaling. (A) T-47D cells and BT-474 cells were co-transfected with the indicated siRNAs and pcDNA3.0-FLAG-ERRF plasmid. 48 h after
transfection the cells were harvested and analyzed with the indicted antibodies by using Western blot (WB). (B) Hormone starved T-47D cells were transfected control siRNA (siCtrl)
or ERRF siRNAs for 48 h and then treated with ethanol or E2 (10 nM) for 1 h. The cells were harvested and analyzed with the indicated antibodies by using WB. The efficiency of ERRF
siRNAs was confirmed by using RT-PCR, the expression of GAPDH serves as internal control. (C) BT-474 cells were treated and analyzed as in (B).
 A. Luo, X. Zhang / Biochemical and Biophysical Research Communications 474 (2016) 400e405 403

Fig. 2. Knockdown of ERRF inhibits the transcriptional activity of ERa. (A) ERRF knockdown decreases the activity of ERE-reporter. Hormone starved T-47D cells were co-
transfected either with siCtrl or ERRF siRNAs together with ERE-Luc reporter plasmid for 48 h and then treated either with ethanol or E2. The luciferase activity was measured
12 h after E2 treatment. T-47D cells (B), BT-474 cells (C), MDA-MB-361 cells (D) were pre-cultured in hormone starvation condition for 48 h and then transfected with siCtrl or ERRF
siRNAs. 48 h after transfection the cells were treated with either ethanol or E2 for 12 h, and the expression of ERRF and PS2 were analyzed by using qRT-PCR. Values in A-D were
shown by mean ± SD, n ¼ 3. *, P < 0.05; **, P < 0.01.

assay showed that ERRF mainly interacted with the DNA binding
domain and ligand binding domain of ERa (Fig. 3C). What's more,
the interaction between ERRF and ERa was also tested in T-
47D cells. It showed that ERRF and ERa could interact with each
other regardless of the presence of E2 (Fig. 3D).
4. Discussion
It had been proved in the previous work that knockdown of ERRF
inhibited growth of ER-positive breast cancer cells and in the cur-
rent study we make it clear that the influence of ERRF on cell
growth is E2-independent (data not shown), but here we focused
on the condition with E2. ERRF knockdown impaired the activity of
E2-ERa pathway, which provided certain mechanism for how ERRF
knockdown inhibits cell proliferation in ER positive breast cancer
cells. Phosphorylation of ERa not only plays important functional
roles such as ligand binding, DNA binding and protein stability but
also has critical clinical significance [26,27]. Ser118 and Ser167 are
the two most well-studied phosphorylation sites of ERa and the
level of them are considered to be associated with breast cancer
biomarkers and clinical outcome. Here we demonstrated it in three
breast cancer cell lines that ERRF knockdown decreased the level of
p-ERa-Ser118 and p-ERa-Ser167, implying an universe role of ERRF
in maintaining the activity of E2-ERa pathway, which is also in
accordance with the positive association between ERRF and ERa in
breast cancer cell lines and samples [23]. What's more, we observed
ERRF knockdown resulted in the decrease of ERa protein in BT-474
and MCF7 cells but not in T-47D cells. The demonstration of the
interaction between ERRF and ERa in T-47D cells could provide
some hints for the mechanism, that the binding with ERRF may
help to maintain or promote the phosphorylation of ERa while
when ERRF is reduced the phosphorylation of ERa is also inhibited.
What's more interesting is that our unshown data proved that
knockdown of ERRF caused the increase of ERa ubiquitination in
BT474 cells, but we failed to detect the interaction between ERRF
and ERa. This also helps to explain why E2-ERa pathway is
compromised following ERRF knockdown. Besides, we can also see
from Fig. 1B that the reduction in phosphorylated ERa was more
obvious than the reduction of ERa itself. Therefore we tend to take
the opinion that ERRF affects E2-ERa pathway in two ways. To be
frank, the interaction between ERRF and ERa remains to be deeply
studied by using other assays such as in vitro GST-pulldown assay to
confirm whether their interaction is direct, and our work in this
aspect aims to provide some hints for explanation of the roles of
ERRF in E2-ERa pathway.
When studying the influence of ERRF knockdown on the tran-
scriptional activity of ERa we had also examined the expression of
other known downstream genes such as GREB1, CTSD, CyclinD1 and
cyclinG2 in T-47D and BT-474 cells (data not shown). We didn't
observed significant changes in the expression GREB1 and CTSD but
404 A. Luo, X. Zhang / Biochemical and Biophysical Research Communications 474 (2016) 400e405

Fig. 3. ERRF interacts with ERa. (A) 293 T cells were co-transfected with the pCDNA-3.0-FLAG-ERRF and pEGFP-C1-ERa for 24 h. The cell lysates were immunoprecipitated with
FLAG antibody and analyzed with the indicated antibodies by using WB. (B) 293 T cells were co-transfected with the pCDNA-3.0-FLAG- ERa and pCI-Myc-ERRF, treated and analyzed
with the indicated antibodies as in (A). (C) Left, domain structure of ERa and schematic representation of different ERa truncation constructs. Right, immunoprecipitation and WB
showing the domains of ERa that interact with ERRF in 293 T cells (D) Hormone starved T-47D cells were transfected with the indicated plasmids. 36 h after transfection the cells
were treated with ethanol or E2 for 2 h. The cell lysates were prepared and analyzed as in (A).

reduction in the expression of CyclinD1 in T-47D cells and upre-
gulation of cyclinG2 in BT-474 cells. This means the regulation of
ERRF on E2-ERa pathway is partly and cell type dependent.
Actually we have also tested the influence of ERRF over-
expression on cell proliferation and ERa phosphorylation in MCF7
cells (Fig. S2). Unexpectedly, overexpression of ERRF showed minor
or no effects on these two aspects. It may be because MCF7 is not a
suitable cell line to study ERRF overexpression or the role of ERRF is
confined to maintain some functions of E2-ERa pathway. Current
work has only preliminarily established an essential role of ERRF in
E2-ERa pathway, but the relevant details remain obscure. Hence
more work is yet to be done to deeply elucidate roles of ERRF in
breast cancer and cell biology.
Conflicts of interest
We declare that there are no conflicts of interest.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.bbrc.2016.04.132.
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