Review

Mechanisms that Increase Stability of Estrogen

Receptor Alpha in Breast Cancer
Angeles C. Tecalco-Cruz,1 Josu O. Ramrez-Jarqun2
Abstract
Estrogen receptor alpha (ER) is a transcriptional regulator that controls the expression of genes related to cellular
proliferation and differentiation in normal mammary tissue. However, the expression, abundance, and activity of this
receptor are increased in 70% of breast cancers. The ER upregulation is facilitated by several molecular mechanisms,
including protein stability, which represents an important strategy to maintain an active and functional repertoire of ER.
Several proteins interact and protect ER from degradation by the ubiquitin-proteasome system. Through diverse
mechanisms, these proteins prevent polyubiquitination and degradation of ER, leading to an increase in ER protein
levels; consequently, estrogen signaling and its physiologic effects are enhanced in breast cancer cells. Thus,
increased protein stability seems to be one of the main reasons that ER is upregulated in breast cancer. Here, we
highlight ndings on the proteins and mechanisms that participate directly or indirectly in ER stability and their
relevance to breast cancer.

Clinical Breast Cancer, Vol. -, No. -, --- 2016 Elsevier Inc. All rights reserved.
Keywords: ER, ER degradation, ER protein stability, ER upregulation, Estrogen signaling pathway

Introduction Furthermore, ER can bind genomic DNA regulatory elements

Estrogen receptor alpha (ER) is a protein of 595 amino acids,
classied as a nuclear receptor subfamily 3 group A member 1
(NR3A1).1,2 It is structurally organized into activation function
domains (AF-1 and AF-2), a DNA-binding domain (DBD), and a
ligand-binding domain (LBD) for the 17 beta estradiol hormone
(E2) (Figure 1, upper).1,3
The genomic or classical function of ER is triggered by the binding
of E2 to the LBD in the cytoplasm and/or the nucleus.4,5 As a result,
the conformation of ER is modied to form homodimers that directly
bind to estrogen responsive elements (ERE) within the intergenic
regions, enhancers, and promoters of E2-responsive genes.6,7
Importantly, transcription factors named Pioneer Factors open up
local chromatin to facilitate the binding of ER to ERE.8 Subse-
quently, ER recruits several coregulators through its AF-1 and AF-2
domains to modify chromatin structure9,10 and mediate the
formation of chromatin loops that affect genome organization to
control E2-dependent gene transcription (Figure 1A).11,12
1
Programa de Investigacin de Cncer de Mama, Instituto de Investigaciones Bio-
mdicas
2
Instituto de Fisiologa Celular, Universidad Nacional Autnoma de Mxico, Mxico
D.F., Mexico
Submitted: May 19, 2016; Revised: Jun 29, 2016; Accepted: Jul 20, 2016
Address for correspondence: Dr Angeles C. Tecalco-Cruz, PhD, Universidad Nacional
Autnoma de Mxico, Av Universidad s/n, 04510 Mexico DF, Mexico
E-mail contact: atecalco@iibiomedicas.unam.mx
indirectly through other transcription factors, such as AP-1, Sp1,
and NF-kB.1,5,13,14 In this way, ER acts as coregulator to modulate
gene expression induced by diverse signaling pathways and late-
regulated genes induced by E2 (Figure 1B).13,15-17 Additionally,
the crosstalk between ER and growth factor pathways can induce
ER phosphorylation and its subsequent transcriptional activity
(Figure 1B).13,14,18 Interestingly, ER can undergo a post-
translational modication named palmitoylation at C447 by
palmitoyl acyl transferases named DHHC7 and DHHC21.19 As a
result, ER interacts with caveolin 1 protein to be transported and
integrated to the plasma membrane.20 In this way, a small portion
of the total cellular ER content (approximately 5%) is membrane-
associated and can mediate rapid extranuclear signaling of E2.21
This non-genomic pathway of ER/E2 involves the activation of
kinases (ERK/MAPK, PI3K/AKT, PKC, and Src kinase) and
second messengers (Calcium, cAMP) (Figure 1C) that enable the
activation of other transcription factors associated to proliferation,
apoptosis, and differentiation.13,22 Moreover, genomic and non-
genomic pathways of ER can be strongly related; for example, ER
and its coactivators can be phosphorylated in response to
non-genomic pathways of E2 to regulate their transcription activity
(Figure 1A, B, C).13,22
Therefore, ER has a central role in the development and dif-
ferentiation of normal mammary glands. However, ER upregula-
tion is associated with the development of breast cancers.23 About
70% of breast cancer cases have high ER expression and activity

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 ER Stability in Breast Cancer
Figure 1 Estrogen Receptor (ER) Is an Important Regulator of Gene Transcription. Upper Panel, Schematic Representation of
Functional Domains of ER. The Amino-Terminal and Carboxy-Terminal Regions Contain the Transactivation Domains AF-1
and AF-2, Respectively, Which Recruit Both Coactivators and Corepressors. The DNA-Binding Domain (DBD) Is Required for
Specic Binding to Estrogen Response Elements (ERE) in Enhancers or Promoters. The Ligand-Binding Domain (LBD) Is
Recognized by the 17 Beta Estradiol Hormone (E2). The Hinge Domain Links LBD and DBD and Is Important for the
Conformational Changes in ER. Lower Panel, ER Is a Crucial Regulator in the Transcription of Genes: (A) ER Is a Transcription
Factor Activated by E2 Hormone. E2 Diffuses Into the Cell and Binds to ER in the Cytoplasm and/or Nucleus. Activated ER
Forms Homodimers, Which Recognize the ERE Sequence in Target Enhancers and Promoters and Recruits Coregulator (CoR)
Complexes to Induce Gene Transcription. Growth Factors can Also Induce ER activity as a Transcription Factor. (B) ER Is a
Coregulator for Some Transcription Factors (ie, AP-1, Sp1, and NF-kB) Through Indirect Binding to Genomic DNA Regulatory
Elements. (C) ER Is a Mediator in the activation of Other Transcription Factors. Cytoplasmic Membrane-Associated ER can
Induce Kinase-Dependent Signaling for the Activation of Some Transcription Factors

(ER-positive [ER] breast cancer), and hormonal therapy is
prescribed to inhibit its activation (selective estrogen receptor
modulators), synthesis (aromatase inhibitors), or stability (selective
estrogen receptor degrader). An example of this therapy is
tamoxifen (Tam), which is a selective estrogen receptor modulator
that competes with E2 by binding to ER.14,24,25 Nevertheless,
acquired resistance to Tam is a common problem in ER breast
cancer, and the mechanisms underlying this resistance are not
completely dened.24,26-29
The high expression of ER and the activation of its genomic and
nongenomic pathways have effects on breast cancer development
and Tam resistance. In this review, we focus on ER stability by
means of several proteins and mechanisms that inhibit the receptor
degradation via the ubiquitin-proteasome system (UPS).
Basis of the ER Degradation via UPS
The knowledge of the molecular basis of ER degradation by the
UPS is necessary to understand the importance of ER stability in
breast cancer. The UPS is an important mechanism for the down-
regulation of ER.14,30,31 This system includes several steps and
3 different enzymes known as E1 (ubiquitin activating enzyme), E2
(ubiquitin conjugating enzyme), and E3 (ubiquitin ligase).30,31 E3
ubiquitin ligases (ie, CHIP,32 E6AP,33 BRCA1,34 BARD1,35
SKP2,36 and MDM237) catalyze the covalent binding of the

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 Angeles C. Tecalco-Cruz, Josu O. Ramrez-Jarqun
protein ubiquitin to ER lysine residues to induce its degradation by
the 26S proteasome.
The UPS allows the rapid removal of ER to maintain a proper
cellular balance of ER protein levels in response to hormonal
stimulation. Thereby, E2 treatment induces ER degradation by the
UPS38-40 to control the intensity and duration of E2/ER
signaling.14,16,30
Conversely, the proteasome activity is also necessary to regulate
the transcriptional activity of ER. It has been proposed that
E2-liganded ER binds to ERE of its target genes and recruits both
coactivators as E3-ubiquitin ligases. Whereas the coactivators in-
crease the activity of ER, the E3-ubiquitin ligases are necessary for
its downregulation. These mechanisms allow a proper balance be-
tween the abundance and the ER functions.41,42 Furthermore, some
coactivators serve as E3-ubiquitin ligases for ER. For example, SKP2
is an E3 ligase for ER degradation with short E2 stimulation times.
However, SKP2 can also act as a coactivator of ER with longer
stimulation times that induce the transcription of late E2-target
genes linked to cell cycle progression.16 Thus, it is possible that
the UPS is linked to the transcriptional cycle of ER for a
coordinated and dynamic gene expression.
On the other hand, ER degradation by UPS has been associated
to its phosphorylation state. The interaction between ER and several
kinases, including CDK11p58,43 cyclin E-CDK2,16 Src,33 PKC,39
p38MAPK,36 and ERK7,44 is related to ER polyubiquitination.
Moreover, phosphorylation induced by E2 at ER residues S118,45
S294,36 S341,16 and Y53733 is required for ER degradation. For
instance, E2 induces the ER phosphorylation at residue Y537 by Src
kinase, then ER is polyubiquitinated by E6AP for its degradation.33
ER degradation is prevented by mutations at Y537 residue, and
these mutations have been associated with the development of the
hormonal resistance in breast cancer.17,46,47 Hence, ER phosphor-
ylation can lead to the recruitment of E3 ligases/coactivators to
modulate ER degradation and transcription of its target genes.
ER downregulation via the UPS is also observed in some hor-
monal therapies with selective estrogen receptor degraders such as
Fulvestrant.48,49 Similarly, novel drugs such as AZD9496,50 GDC-
0810,51 bazedoxifene,52 and RAD190153 have been synthesized as
ER degraders. Thus, degradation by the UPS has a primary role in
determining the abundance of ER and is regulated by various
stimuli.
Although ER degradation by UPS is central to maintaining the
cellular balance of ER levels and its transcription functions, the ER
lysine targets of ubiquitination are not completely known. A study
suggests that the residues K302 and K303 are indispensable for ER
polyubiquitination and degradation induced by E2 and Fulves-
trant.48 However, the same residues confer ER stability under
basal conditions of breast cancer cells.48 Altogether, these data
suggest: (1) a complex relationship between the degradation/sta-
bility and the transcriptional activity of ER and (2) that the
balance between these molecular processes may be lost in breast
cancer.
ER Protein Stability in Breast Cancer
Cells
Many proteins and mechanisms converge to maintain ER
stability by inhibiting its degradation via the UPS. Here, we describe
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each of these proteins and the molecular mechanisms for main-
taining ER stability in breast cancer cells.
Coactivators that Stabilize the Level Protein of ER
Mucin 1 (MUC1). MUC1 is a heterodimeric glycoprotein con-
sisting of the MUC1 N-terminal (MUC1-N) and MUC1 C-ter-
minal (MUC1-C) subunits.54,55 Interaction between the MUC1-C
subunit and DBD of ER has been reported at the promoters of E2
target genes. MUC1 acts as coactivator for ER by enhancing the
recruitment of proteins with histone acetyltransferase activity, such
as SRC1 and GRIP (p160 family proteins) to stimulate ER-
mediated transcription. Interestingly, ER levels decrease in
MUC1-knockdown MCF7 cells, but treatment with the protea-
some inhibitor MG132 recovers its levels. Hence, MUC1-C is an
oncoprotein that stabilizes ER to ERE of promoters and recruits
p160 family coactivators in breast cancer cells. ER stability
conferred by MUC1-C increases the expression of ER target genes
and, consequently, the growth and survival induced by E2 in breast
cancer cells.55 In the same way, MUC1-C overexpression induces
transformation, loss of cellular polarity, cellular proliferation and
migration anchorage-independent growth, and tumorigenicity in
transgenic mouse models.54,56-58
PIN1 (Protein Interacting With Never in Mitosis A). PIN1 is a
member of the peptidyl prolyl cis/trans isomerase (PPIase) family59
that is organized into an N-terminal WW domain and a C-terminal
parvulin-type catalytic PPIase domain, connected by a linker
domain.59 The WW domain interacts with pS/T-P motifs in target
proteins to convert one isomer to another by PPIase.60
PIN1 interacts with ER when this is phosphorylated at S118.10
This interaction enhances DNA binding and transcriptional activ-
ity of ER by inducing its cis/trans isomerization10,61 and conferring
protein stability.45 PIN1 associates with ER to prevent its interac-
tion with the E3 ligase, E6AP, and consequently, PIN1 inhibits the
polyubiquitination and degradation of ER.45 Downregulation of
PIN1 increases the E6AP-dependent ubiquitination status of ER
and its degradation. Furthermore, there is a correlation between
high levels of PIN1, increased ER levels, decreased E6AP levels, and
hormonal resistance in breast tumors.45 In this way, PIN1 is an ER
coactivator that is associated with ER stability in breast cancer cells.
The Kinases GSK3, LMTK3, and ABL Interact With and
Stabilize ER
Glycogen Synthase Kinase-3 (GSK3). GSK3 is a serine/threonine
kinase that exists in GSK3 alpha and GSK3 beta isoforms in
mammals.62 The GSK3 isoforms have homologous kinase domains,
but differ in their N- and C-terminal domains.62 Previous studies
have shown that GSK3 isoforms interact with and phosphorylate
ER.29,63,64 GSK3 association with ER mainly occurs in the cyto-
plasm in the absence of E2, and as result, GSK3 phosphorylates ER
at S102, S104, and S106 to stabilize it.29,64 However, GSK3 also
interacts with ER to phosphorylate S118 to enhance its transcrip-
tional activity in the nucleus of cells stimulated by E2.63,64 In
GSK3-knockdown breast cancer cells, ER levels are decreased
through degradation by UPS and are rescued in the presence of
MG132.63 GSK3 depletion has no effect on subcellular localization
of ER, but decreases phosphorylation, stability, and transcriptional
Clinical Breast Cancer Month 2016 -3
 ER Stability in Breast Cancer
activity of this receptor.63,64 Thus, ER stability is correlated with its
GSK3-dependent phosphorylation in breast cancer cells.
LMTK3 (Lemur Tyrosine Kinase 3). LMTK3 is another important
regulator of ER expression, as well as of ER stability.65 First,
LMTK3 enhances FOXO3-induced ER expression by decreasing
the activity of PKC and AKT. Second, LMTK3 has been shown to
phosphorylate ER in vitro, and its expression affects ER protein
levels. In LMTK3-knockdown MCF7 cells, the ER is highly
ubiquitinated and degraded, but proteasome inhibitors rescue ER
protein levels. In contrast, ER levels are higher in cells that
overexpress LMTK3. Therefore, LMTK3 interacts with and phos-
phorylates ER to protect it from UPS degradation and to stabilize it
in breast cancer cells.65
ABL Proto-Oncogene 1, Non-Receptor Tyrosine Kinase (ABL). One
more kinase that regulates the ER activity is ABL. It has been shown
that ER interacts with the ABL kinase, which stabilizes ER via
phosphorylation at Y52 and Y219.66 Consequently, the interaction
between ABL and ER leads to increased ER activity,67 although the
mechanism behind this stabilization remains undened, and more
studies on the importance of the phosphorylation of these residues
are required.66 Importantly, this functional relationship of ABL and
ER also stimulates resistance to Tam, and the overexpression of
these 2 proteins has been detected in breast tumor tissue samples.67
Non-Nuclear Mechanisms for Maintaining ER Stability
RNF31. RNF31 is a member of the RBR ligase family (RING-in
between rings (IBR)-RING) that contains 3 zinc ring-nger motifs,
a ubiquitin-associated domain and an RBR domain featured in
ubiquitin ligases.68 It has been suggested that RNF31 catalyzes the
monoubiquitination or linear polyubiquitination of its substrates to
stabilize them.69,70
Recently, RNF31 has been found to associate with ER in the
cytoplasmic compartments of MCF7 cells. This interaction specif-
ically controls ER protein stability, since RNF31 catalyzes ER
monoubiquitination and blocks its polyubiquitination, resulting in
high ER protein levels.70 RNF31 is not recruited to DNA and is not
a coactivator for ER. However, RNF31 is important for accumu-
lation of ER and increase of its transcriptional activity. Thus, when
RNF31 expression is reduced by siRNA, ER protein levels and the
expression of its target genes are decreased.70 The mechanistic de-
tails are unknown so far, but it is clear that competition between
monoubiquitination and polyubiquitination affects the stability and
degradation of ER. Thus, monoubiquitinated ER is more stable and
active, enhancing E2 signaling in breast cancer cells.
Retinoblastoma (RB). RB is a tumor suppressor that controls cell
cycle progression, survival, and differentiation.71 The N-terminal
domain (RB-N) and the C-terminal domain (RB-C) are important
for the formation of multiprotein complexes.72 Interestingly, the
N-terminal domain of the RB protein also interacts with ER in
breast cancer cells.72 The interaction between RB and ER allows the
assembly of an intermediate complex with proteins HSP90 and p23
in the cytoplasm. This complex (ER/HSP90/p23) is RB-dependent
and is very important because it protects ER from degradation by
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the UPS. Thus, ER is ubiquitinated and degraded in RB-
knockdown cells, but MG132 treatment restores its protein levels
in these cells. Therefore, RB stabilizes ER and protects it from
degradation by the UPS in a breast cancer context.72
ER Palmitoylation. Additionally, palmitoylation has been linked
to ER stability.73 For example, ER palmitoylation is required for the
association of ER with the cell plasma membrane.74 The membrane
associated ER induces rapid non-genomic signaling (PI3K/AKT and
ERK/MAPK pathways). Furthermore, these pathways converge
with the genomic pathway by means of the phosphorylation at S118
of the receptor and favoring its binding to DNA. This modication
protects ER from degradation, since it has been reported that ER
mutants that are unable to be palmitoylated are degraded via UPS,
decreasing ER activity in breast cancer cells.73
Recently, a new variant of ER has been reported; the ER36
which lacks both AF1 and AF2 domains of ER and is localized in
cellular membrane and cytoplasm. ER36 association with the
membrane seems to be palmitoylation-dependent. It has been
demonstrated that the chaperon gamma synuclein (SYN) protects
to ER from ubiquitination and degradation via UPS. This ER-SYN
interaction enhances the binding of E2 to LBD, which in turns
induces the non-genomic signaling. Furthermore, the glycoprotein
MGP96 interacts with ER36 to enhance the proliferation and
migration of breast cancer cells.75,76 Thus, processes of palmitoy-
lation are involved in ER and ER36 stability in breast cancer
cells.77
Other Proteins that Stabilize ER
The proteins MUC1, PIN1, GSK3, LMTK3, RNF31, RB, and
ABL are required to achieve ER stability in breast cancer (Table 1;
Figure 2). These proteins directly interact with ER to protect it from
degradation by UPS, through several mechanisms (Table 1). Spe-
cically, MUC1 and PIN1 induce the formation of stable tran-
scription complexes on the DNA45,55; RNF31 monoubiquitinates
ER; RB induces the assembly of ER with chaperone proteins72 and
GSK3 and LMTK3 phosphorylate ER.63,65
On the other hand, there are proteins that do not interact with
ER, but that can modulate its stability through indirect means
(Table 1). For instance, Src interacts and phosphorylates ER for the
recruitment of E3 ubiquitin ligase, E6AP, to induce receptor
degradation. This event is inhibited by PEBP4 (phosphatidyl
ethanolamine-binding protein 4) protein, which is overexpressed in
breast cancer cells.78,81 PEBP4 competes with ER for association
with the Src protein. In this way, PEBP4 interrupts the interaction
between ER and Src, and the ER degradation is decreased.78 Thus,
PEBP4 increases ER stability and its transcriptional activity in breast
cancer cells.
Another protein that appears to be involved in ER stability in
breast cancer cells is REGgamma (REG). It has been shown that ER
protein levels are decreased in REG-knockdown cells and that
treatment with MG132, recovers ER protein levels in these cells.
However, the mechanism behind this ER stabilization by REG is
unknown. Notably, a correlation between high levels of REG and
ER in breast tumors has been observed, as well as a direct correlation
between REG levels and poor clinical prognosis in patients with
breast cancer.79
 Angeles C. Tecalco-Cruz, Josu O. Ramrez-Jarqun
MUC1 overexpression may be useful as a biomarker of breast cancer
Table 1 Molecular Mechanisms Underlying ER Stability in
Breast Cancer Cells and as a predictor of poor response to Tam treatment.
Moreover, MUC1 has been proposed as a potential therapeutic
Type of target.82,84,85 It has been demonstrated that miR-125b,86
Interaction Protein/ Mechanisms for miR-145,87 and miR-122688 suppress MUC1 expression and the
on ER Modication ER Stability Reference
proliferation of breast cancer cells. In addition, the MUC1 deple-
55
Direct MUC1 Coactivator for ER. tion by using specic siRNAs recovers the Tam treatment sensi-
Assembly of
transcription tivity.82 MUC1 inhibitors, like Apigenin,85 GO-201,57 and
factors GO20382 have been designed and studied. Apigenin is a small
45
PIN1 Coactivator of molecule that blocks the dimerization and nuclear localization of
phosphorylated ER MUC1, resulting in apoptosis and reduced cell survival. Similarly,
at S118.
Formation of GO-201 decreases the tumorigenesis of breast cancer cells57,85
transcription factors through restoration of Rab31 negative regulation by Tam.82 In
GSK3 ER phosphorylation 63
the eld of breast cancer immunotherapy, MUC1 represents an
66
ABL ER phosphorilation important candidate target. Pre-clinical tests have demonstrated that
65
LMTK3 ER phosphorylation MUC1-based rBCG (Bacillus Calmette-Guerin) vaccines are able to
RB Assembly of ER with 72
generate anti-MUC immune responses.89 This vaccine causes a
chaperone proteins specic antitumor response to inhibit the growth of MUC1-positive
70
RNF31 ER monoubiquitination breast tumors in mice.89,90 These studies suggest that MUC1-based
78
Indirect PEBP4 Inhibition of ER-Src rBCG vaccines have great potential as a breast cancer treatment.
interaction required
for ER degradation On the other hand, PIN1 overexpression, a transcriptional
REGG Undetermined 79 coactivator of ER, has also been shown to stimulate the growth,
mechanisms. Correlation tumorigenicity, and epithelial-mesenchymal transition in breast
between REGG and ER cancer,91 as well as hyperplasia and the formation of malignant
protein levels
80 mammary tumors in mouse mammary glands.59 Furthermore,
Modications P300 ER acetylation
73 PIN1 polymorphisms have also been associated with the develop-
Palmitoylation Palmitoylation of ER
associated to membrane ment of breast cancer and with a poor clinical prognosis.60 In
cell signaling addition, PIN1 is targeted for breast cancer therapies with natural
47
Phosphorylation Phoshorylation of and synthetic inhibitors to control its activity.92-98 These strategies
sites involved in could affect PIN1 activity related to ER and consequently modulate
ER stability
46 its stability in breast cancer.
Mutations Point mutations of sites
associated to With respect to kinases, it has been reported that GSK3 cata-
phosphorylation and lytic activity affects the cellular growth and survival of cancer
polyubiquitination
cells.64,99-101 Further, sites of ER phosphorylated by GSK3 are
involved in ER
degradation directly associated with resistance to hormonal therapies.47
Recently, a novel GSK3 inhibitor, CG0009, decreases prolifera-
Abbreviations: ABL ABL proto-oncogene 1, nonreceptor tyrosine kinase; ER estrogen
receptor; GSK3 glycogen synthase kinase-3; LMTK3 lemur tyrosine kinase 3; MUC1
tion and survival of different breast cancer cell lines. This inhibitor
mucin 1; PEBP4 phosphatidyl ethanolamine-binding protein 4; PIN1 Protein interacting reduces cyclin D1 expression and activates p53, but it may also
with never in mitosis A; RB retinoblastoma; REGG REG gamma.
affect ER stability and offer a therapeutic approach to hormonal
resistance in breast cancer.102
One more posttranslational modication associated to ER It has been observed as well that LMTK3 overexpression stim-
stability is the acetylation. It has been suggested that P300 protein ulates cellular proliferation induced by E2 and xenotransplants of
interacts with and acetylates ER to enhance its stability in breast MCF7 cells in mice induce tumor formation while LMTK3 siRNA
cancer cells,80 but more studies are required to clarify this mecha- injection decreases tumor formation.65,103 In addition, LMTK3 is
nism and its effects. overexpressed in ER breast cancer tumors and correlates with
shorter survival times.65,104 Furthermore, intronic polymorphisms
ER Stability in the Diagnostics, in LMTK3 have been related to ER overexpression and suscepti-
Prognostics, and Therapy of Breast bility to ER breast cancer.65 Additionally, LMTK3 down-
Cancers regulation recovers responsiveness to Tam treatment. A genome-
Several reports have demonstrated that some of the proteins that wide analysis showed that the expression of several genes related
stabilize ER represent important biomarkers, predictor of the to Tam resistance was affected in LMTK3-knockdown cells but not
response to Tam treatment, and therapeutical targets. For example, in control cells. In a Tam-resistant xenograft breast-cancer mouse
the MUC1/ER complex regulates the expression of genes associated model, depletion of LMTK3 expression by siRNA restored the
with cellular growth and hormonal resistance.55,57,82 One of these antimitogenic effects of Tam and decreased tumor volume.105
genes is Rab31, which is frequently overexpressed in breast cancer Although LMTK3 is crucial for the ER stability, the mechanisms
cells that are insensitive to Tam.56 Furthermore, the overexpression that control its enzymatic activity are not known. However, it has
of MUC1 has been reported in 90% of breast cancer cases.83 Thus, been suggested that miR-34a is a modulator of the LMTK3

Clinical Breast Cancer Month 2016 -5

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 ER Stability in Breast Cancer
Figure 2 Mechanisms that Are Related to ER Stability. Many Proteins and Mechanisms Confer Stability to ER. A, the Proteins MUC1,
GSK3, LMTK3, RNF31, RB, and PIN1 Directly Interact With ER to Stabilize it. MUC1 and PIN1 Act as ER Coactivators; RB
Regulates the Assembly of an ER/HSP90/p23 Complex; RNF31 Monoubiquitinates ER; GSK3, LMTK3 and ABL Phosphorylate
ER; PEBP4 Inhibits the Interaction Between ER and Src. The Mutations of Residues Involved in ER Phosphorylation or
Ubiquitination and Posttranslational Modications Such as Palmitoylation (Palm) Are Indicated. Phosphorylation Are Marked
by Stars and Ubiquitination by Circles. Effect of Stabilization (S) or Degradation (D) Are Also Indicated. Sites of
Phosphorylation or Amino Acid Substitutions in ER that Have Been Identied in Breast-Cancer Biopsy Samples Are Indicated.
B, Together, These Mechanisms Prevent ER Degradation by the Ubiquitin-Proteasome System (UPS), Resulting in a Stable
and Active Repertoire of ER. As a Result, the Expression of E2-Dependent Genes Linked to Proliferation, Survival, and
Hormonal Therapy Resistance Are Increased in ER-Positive Breast Cancer

Abbreviations: ABL ABL proto-oncogene 1, nonreceptor tyrosine kinase; ER Estrogen receptor; GSK3 glycogen synthase kinase-3; LMTK3 lemur tyrosine kinase 3; MUC1 mucin 1;
PEBP4 phosphatidyl ethanolamine-binding protein 4; PIN1 Protein interacting with never in mitosis A; RB retinoblastoma.

expression and activity.103 Thus, LMTK3 may be a useful
biomarker and therapeutic target of breast cancer.
An interesting data is that RB expression and its functions are
altered in breast cancers.106-108 Hence, it has been proposed that the
loss of ER is related to the loss of RB expression in ER negative
(ER) breast cancers72 Consequently, the expression and activity of
RB may be important to understanding the development of
ER breast cancers. Furthermore, RB-knockdown in breast cancer
cell lines and xenograft assays leads to Tam resistance, and RB
dysfunction is related with poor responses to hormonal therapies in
patients.106,109 Thus, RB expression and function are linked to the
protein levels and activity of ER, as well the response to hormonal
therapy by breast cancer cells.
Furthermore, the enhancement of RNF31 expression positively
correlates with ER protein levels and the expression of its target
genes such as cyclin D1 and c-myc. Hence, RNF31 expression
produces an increase of the growth of breast cancer cells.70 Thereby,
this protein may be an important therapeutic target for the breast
cancer treatment.
On the other hand, palmitoylation has an important role in the
stability and localization in the cellular membrane of ER and ER36.
Both receptors can induce non-genomic signaling pathways that are
associated with endocrine resistance. Particularly, it has been sug-
gested that the binding of Tam and ER36 activates signaling
pathways that are involved with Tam resistance.76,77 In a manner
similar to ER, the proteins SYN and MGP96, that enhance ER36
stability, are important in the progression of breast cancer and
endocrine resistance. These non-genomic signaling pathways asso-
ciated to palmitoylation of ER and ER36 may be useful for breast
cancer treatments.

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 Angeles C. Tecalco-Cruz, Josu O. Ramrez-Jarqun
Thus, the expression and activity of the proteins linked to the
receptor stability are correlated with ER levels and to its activity in
breast tumors. For these reasons, the proteins and mechanisms that
modulate ER stability may be developed as possible therapeutic
targets and as biomarkers for breast cancer.
Mutations and Posttranslational
Modications Associated to the ER
Stability in Breast Tumors
It is important to mention the clinical relevance of the ER sta-
bility in the development of breast tumors. For instance, the Y537
residue is necessary for the phosphorylation and subsequent poly-
ubiquitination and degradation of ER.33 Surprisingly, the mutations
Y537N, Y537C, and Y537S have been identied in breast tumors
of patients with metastasis and resistance to Tam. Likewise, the
K303 is associated with ER polyubiquitination and degradation.48
Furthermore, the mutation K303R has been identied in breast
tumors of patients with poor survival outcome and prognosis.46
In addition, the residues S104, S106, S118, and S294 linked to
ER stability have been found phosphorylated in breast tumors
samples.17,47 Altogether, these data suggest that mutations and/or
phosphorylations of ER linked to its stability are frequently iden-
tied in breast tumors (Table 2; Figure 2A).
The evidence involving modications or mutations of ER ob-
tained from breast cancer primary tumor tissue samples of patients
indicate an imbalance between the degradation and stability pro-
cesses (Figure 2B). In this imbalance, ER stability is increased
because proteins and modications that inhibit its degradation are
enhanced, and the residues implicated in the E3 ligases recruitment
and polyubiquitination of the receptor are mutated and conse-
quently, ER degradation is decreased in tumors (Figure 2B). Thus,
several mechanisms could converge to increase ER stability and
decrease its degradation, and an interesting possibility implicates
that breast cancer cells become resistant to ER degradation due to
the upregulated systems that stabilize it.
Discussion
We have presented a current understanding of molecular mech-
anisms implicated in stabilizing ER in breast cancer. The proteins
MUC1, GSK3, LMTK3, RNF31, RB, and PIN1 regulate ER
stability in different subcellular compartments through various
mechanisms. MUC1 is a mucin glycoprotein and PIN1 is an
isomerase, both of which act as ER coactivators; GSK3 and LMTK3
phosphorylate ER; RNF31 is an atypical E3 ubiquitin ligase that
monoubiquitinates ER; and RB is a scaffold protein that regulates
the assembly of ER with chaperones. Each of these mechanisms
inhibit ER degradation, and, consequently, ER protein levels and
activity are enhanced. MUC1, LMTK3, and PIN1 stabilize ER in
the nucleus, whereas RNF31 and RB act on ER in the cytoplasm;
and GSK3 acts in both cellular compartments.
The expression of E2-dependent genes linked to proliferation,
survival, and hormonal therapy resistance are increased by ER sta-
bility in breast cancer cells in comparison with normal mammary
cells. Further, MUC1, LMTK3, RNF31, and PIN1 are relevant to
breast cancer, since they are increased in ER cells and
tumors.45,55,65,70 On the contrary, the tumor suppressor RB is
commonly dysfunctional in luminal B breast cancers or is lost in
ER breast cancers, and it is also associated with poor prognosis and
hormonal resistance.
These recent ndings clearly show the importance of ER stability
in breast cancer. Proteins associated with the receptor stability may
be useful as biomarkers and prognostic indicators of therapeutic
response and hormonal resistance, as well as new therapeutic targets
in breast cancer. For instance, MUC1 was the rst protein shown to
stabilize ER, in a report published in 2006.55 At the present, MUC1
is a biomarker, because more than 90% of ER breast cancers

Table 2 Amino Acids Residues Associated With ER Stability/Degradation

Aminoacid or Posttranslational Status in Breast

Domain Modicationa Protein Involved Tumor Effect on ER Reference(s)
66
Y52 P ABL N/A Stability
47,63
S104 P GSK3 P Stability
47,63
S106 P GSK3 P Stability
45,47,63
S118 P GSK3 P Stability/Degradation
66
Y219 P ABL N/A Stability
36
S294 P P38MAPK N/A Degradation
48
K302 Ub E3 ligases N/A Degradation
46,48
K303 Ub E3 ligases Mutated K303R Degradation
16
S341 P Cyclin E/CDK2 N/A Degradation
33,46
Y537 P Src Mutated Degradation
Y537C
Y537N
Y537S
65
N/A P LMTK3 N/A Stability
N/A P CDK11P58 N/A Degradation 43

44
LBD P ERK7 N/A Degradation

Abbreviations: ABL ABL proto-oncogene 1, nonreceptor tyrosine kinase; ER estrogen receptor; GSK3 glycogen synthase kinase-3; LBD ligand-binding domain; LMTK3 lemur tyrosine
kinase 3; N/A not available.
a
P denotes phosphorylation and Ub denotes ubiquitination at the indicated amino acid.

Clinical Breast Cancer Month 2016 -7

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 ER Stability in Breast Cancer
overexpress it.83 Furthermore, MUC1 is a therapeutic target for
inhibitors and vaccines in breast cancer treatment. Hence, more
studies on GSK3, LMTK3, RNF31, RB, and PIN1 and their roles
to stabilize ER promise results similar to MUC1.
In addition, there are other proteins and mechanisms associated
with the ER stability in breast cancer. Thus, proteins such as PEBP4
that compete with ER for components of the UPS, specic post-
translational modications (as palmitoylation, acetylation, and
phosphorylation) and mutations of sites linked to ER degradation
also regulate the ER stability in breast cancer cells.
The maintenance of high ER protein levels could augment the
expression of several genes linked to proliferation, induce the
transcription of new target genes, and/or enhance its nongenomic
functions110 These alterations on the ER/E2 signaling seems to be
involved with the development of tumor and hormonal
resistance.110,111
Thus, there is a system to control ER degradation, but the sta-
bility of the receptor is conferred by another system conformed by
several proteins and posttranslational modications, which could be
key in the progression of breast cancer.
Conclusion
Knowledge of the different mechanisms involved in the ER sta-
bility offers new opportunities to understand the development of
breast cancer. The proteins and mechanisms that stabilize ER could
be potential biomarkers and therapeutic targets for this pathology. It
remains to be elucidated whether ER stability is correlated with the
stage and grade of breast tumors. Therefore, the relationship be-
tween these dissimilar mechanisms for ER stability in breast cancer
demands attention and offers an open eld for research.
Acknowledgments
The authors acknowledge Programa de Investigacin de Cncer
Mama (PICM) and thank Dr. Armando I Gutirrez-Lerma for his
helpful review of the manuscript.
This work was supported by the DGAPAeUNAM (grant
numbers PAPIIT-IA200916).
Disclosure
The authors have stated that they have no conicts of interest.
References
1. Ng HW, Perkins R, Tong W, Hong H. Versatility or promiscuity: the estrogen
receptors, control of ligand selectivity and an update on subtype selective ligands.
Int J Environ Res Public Health 2014; 11:8709-42.
2. Germain P, Staels B, Dacquet C, Spedding M, Laudet V. Overview of nomen-
clature of nuclear receptors. Pharmacol Rev 2006; 58:685-704.
3. Kumar R, Zakharov MN, Khan SH, et al. The dynamic structure of the estrogen
receptor. J Amino Acids 2011; 2011:812540.
4. Manavathi B, Dey O, Gajulapalli VN, Bhatia RS, Bugide S, Kumar R. Derailed
estrogen signaling and breast cancer: an authentic couple. Endocr Rev 2013; 34:1-
32.
5. Vrtacnik P, Ostanek B, Mencej-Bedrac S, Marc J. The many faces of estrogen
signaling. Biochem Med (Zagreb) 2014; 24:329-42.
6. Hah N, Kraus WL. Hormone-regulated transcriptomes: lessons learned from
estrogen signaling pathways in breast cancer cells. Mol Cell Endocrinol 2014; 382:
652-64.
7. Hah N, Murakami S, Nagari A, Danko CG, Kraus WL. Enhancer tran-
scripts mark active estrogen receptor binding sites. Genome Res 2013; 23:
1210-23.
8. Manavathi B, Samanthapudi VS, Gajulapalli VN. Estrogen receptor coregulators
and pioneer factors: the orchestrators of mammary gland cell fate and develop-
ment. Front Cell Dev Biol 2014; 2:34.
8 - Clinical Breast Cancer Month 2016
Downloaded from ClinicalKey.com at Universitas Kristen Duta Wacana November 29, 2016.
For personal use only. No other uses without permission. Copyright 2016. Elsevier Inc. All rights reserved.
9. Hervouet E, Cartron PF, Jouvenot M, Delage-Mourroux R. Epigenetic regula-
tion of estrogen signaling in breast cancer. Epigenetics 2013; 8:237-45.
10. Rajbhandari P, Finn G, Solodin NM, et al. Regulation of estrogen receptor alpha
N-terminus conformation and function by peptidyl prolyl isomerase Pin1. Mol
Cell Biol 2012; 32:445-57.
11. He C, Wang X, Zhang MQ. Nucleosome eviction and multiple co-factor binding
predict estrogen-receptor-alpha-associated long-range interactions. Nucleic Acids
Res 2014; 42:6935-44.
12. Liu MH, Cheung E. Estrogen receptor-mediated long-range chromatin in-
teractions and transcription in breast cancer. Mol Cell Endocrinol 2014; 382:624-
32.
13. Acconcia F, Kumar R. Signaling regulation of genomic and nongenomic func-
tions of estrogen receptors. Cancer Lett 2006; 238:1-14.
14. Kerdivel G, Flouriot G, Pakdel F. Modulation of estrogen receptor alpha ac-
tivity and expression during breast cancer progression. Vitam Horm 2013; 93:
135-60.
15. Soltysik K, Czekaj P. Membrane estrogen receptors - is it an alternative way of
estrogen action? J Physiol Pharmacol 2013; 64:129-42.
16. Zhou W, Srinivasan S, Nawaz Z, Slingerland JM. ERalpha, SKP2 and E2F-1
form a feed forward loop driving late ERalpha targets and G1 cell cycle pro-
gression. Oncogene 2014; 33:2341-53.
17. Le Romancer M, Poulard C, Cohen P, Sentis S, Renoir JM, Corbo L. Cracking
the estrogen receptors posttranslational code in breast tumors. Endocr Rev 2011;
32:597-622.
18. Trevino LS, Weigel NL. Phosphorylation: a fundamental regulator of steroid
receptor action. Trends Endocrinol Metab 2013; 24:515-24.
19. Pedram A, Razandi M, Deschenes RJ, Levin ER. DHHC-7 and -21 are palmi-
toylacyltransferases for sex steroid receptors. Mol Biol Cell 2012; 23:188-99.
20. Acconcia F, Ascenzi P, Bocedi A, et al. Palmitoylation-dependent estrogen re-
ceptor alpha membrane localization: regulation by 17beta-estradiol. Mol Biol Cell
2005; 16:231-7.
21. Acconcia F, Marino M. The effects of 17beta-estradiol in cancer are mediated by
estrogen receptor signaling at the plasma membrane. Front Physiol 2011; 2:30.
22. Marino M, Galluzzo P, Ascenzi P. Estrogen signaling multiple pathways to
impact gene transcription. Curr Genomics 2006; 7:497-508.
23. Miyoshi Y, Murase K, Saito M, Imamura M, Oh K. Mechanisms of estrogen
receptor-alpha upregulation in breast cancers. Med Mol Morphol 2010; 43:193-6.
24. Cook KL, Shajahan AN, Clarke R. Autophagy and endocrine resistance in breast
cancer. Expert Rev Anticancer Ther 2011; 11:1283-94.
25. Magnani L, Brunelle M, Gevry N, Lupien M. Chromatin landscape and endo-
crine response in breast cancer. Epigenomics 2012; 4:675-83.
26. Johnson AB, OMalley BW. ERasing breast cancer resistance through the
kinome. Nat Med 2011; 17:660-1.
27. Muluhngwi P, Klinge CM. Roles for miRNAs in endocrine resistance in breast
cancer. Endocr Relat Cancer 2015; 22:R279-300.
28. Osborne CK, Schiff R. Mechanisms of endocrine resistance in breast cancer.
Annu Rev Med 2011; 62:233-47.
29. de Leeuw R, Neefjes J, Michalides R. A role for estrogen receptor phosphoryla-
tion in the resistance to tamoxifen. Int J Breast Cancer 2011; 2011:232435.
30. Zhou W, Slingerland JM. Links between oestrogen receptor activation and
proteolysis: relevance to hormone-regulated cancer therapy. Nat Rev Cancer 2014;
14:26-38.
31. Helzer KT, Hooper C, Miyamoto S, Alarid ET. Ubiquitylation of nuclear re-
ceptors: new linkages and therapeutic implications. J Mol Endocrinol 2015; 54:
R151-67.
32. Fan M, Park A, Nephew KP. CHIP (carboxyl terminus of Hsc70-interacting
protein) promotes basal and geldanamycin-induced degradation of estrogen re-
ceptor-alpha. Mol Endocrinol 2005; 19:2901-14.
33. Sun J, Zhou W, Kaliappan K, Nawaz Z, Slingerland JM. ERalpha phosphory-
lation at Y537 by Src triggers E6-AP-ERalpha binding, ERalpha ubiquitylation,
promoter occupancy, and target gene expression. Mol Endocrinol 2012; 26:1567-
77.
34. Eakin CM, Maccoss MJ, Finney GL, Klevit RE. Estrogen receptor alpha is a
putative substrate for the BRCA1 ubiquitin ligase. Proc Natl Acad Sci U S A 2007;
104:5794-9.
35. Hashizume R, Fukuda M, Maeda I, et al. The RING heterodimer BRCA1-
BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation.
J Biol Chem 2001; 276:14537-40.
36. Bhatt S, Xiao Z, Meng Z, Katzenellenbogen BS. Phosphorylation by p38
mitogen-activated protein kinase promotes estrogen receptor alpha turnover and
functional activity via the SCF(Skp2) proteasomal complex. Mol Cell Biol 2012;
32:1928-43.
37. Saji S, Okumura N, Eguchi H, et al. MDM2 enhances the function of estrogen
receptor alpha in human breast cancer cells. Biochem Biophys Res Commun 2001;
281:259-65.
38. Kocanova S, Mazaheri M, Caze-Subra S, Bystricky K. Ligands specify estrogen
receptor alpha nuclear localization and degradation. BMC Cell Biol 2010; 11:98.
39. Marsaud V, Gougelet A, Maillard S, Renoir JM. Various phosphorylation
pathways, depending on agonist and antagonist binding to endogenous estrogen
receptor alpha (ERalpha), differentially affect ERalpha extractability, proteasome-
mediated stability, and transcriptional activity in human breast cancer cells. Mol
Endocrinol 2003; 17:2013-27.
40. Valley CC, Solodin NM, Powers GL, Ellison SJ, Alarid ET. Temporal variation
in estrogen receptor-alpha protein turnover in the presence of estrogen. J Mol
Endocrinol 2008; 40:23-34.
 Angeles C. Tecalco-Cruz, Josu O. Ramrez-Jarqun
41. Lonard DM, Nawaz Z, Smith CL, OMalley BW. The 26S proteasome is
required for estrogen receptor-alpha and coactivator turnover and for efcient
estrogen receptor-alpha transactivation. Mol Cell 2000; 5:939-48.
42. Reid G, Hubner MR, Metivier R, et al. Cyclic, proteasome-mediated turnover of
unliganded and liganded ERalpha on responsive promoters is an integral feature
of estrogen signaling. Mol Cell 2003; 11:695-707.
43. Wang Y, Zong H, Chi Y, et al. Repression of estrogen receptor alpha by
CDK11p58 through promoting its ubiquitin-proteasome degradation. J Biochem
2009; 145:331-43.
44. Henrich LM, Smith JA, Kitt D, et al. Extracellular signal-regulated kinase 7, a
regulator of hormone-dependent estrogen receptor destruction. Mol Cell Biol
2003; 23:5979-88.
45. Rajbhandari P, Schalper KA, Solodin NM, et al. Pin1 modulates ERalpha levels
in breast cancer through inhibition of phosphorylation-dependent ubiquitination
and degradation. Oncogene 2014; 33:1438-47.
46. Thomas C, Gustafsson JA. Estrogen receptor mutations and functional conse-
quences for breast cancer. Trends Endocrinol Metab 2015; 26:467-76.
47. Murphy LC, Seekallu SV, Watson PH. Clinical signicance of estrogen receptor
phosphorylation. Endocr Relat Cancer 2011; 18:R1-14.
48. Berry NB, Fan M, Nephew KP. Estrogen receptor-alpha hinge-region lysines 302
and 303 regulate receptor degradation by the proteasome. Mol Endocrinol 2008;
22:1535-51.
49. Yeh WL, Shioda K, Coser KR, Rivizzigno D, McSweeney KR, Shioda T. Ful-
vestrant-induced cell death and proteasomal degradation of estrogen receptor
alpha protein in MCF-7 cells require the CSK c-Src tyrosine kinase. PLoS One
2013; 8:e60889.
50. De Savi C, Bradbury RH, Rabow AA, et al. Abstract 3650: Discovery of the
clinical candidate AZD9496: a potent and orally bioavailable selective estrogen
receptor downregulator and antagonist. Cancer Res 2015; 75:3650.
51. Lai A, Kahraman M, Govek S, et al. Identication of GDC-0810 (ARN-810), an
orally bioavailable selective estrogen receptor degrader (SERD) that demonstrates
robust activity in tamoxifen-resistant breast cancer xenografts. J Med Chem 2015;
58:4888-904.
52. Wardell SE, Nelson ER, Chao CA, McDonnell DP. Bazedoxifene exhibits
antiestrogenic activity in animal models of tamoxifen-resistant breast cancer:
implications for treatment of advanced disease. Clin Cancer Res 2013; 19:2420-
31.
53. Garner F, Shomali M, Paquin D, Lyttle CR, Hattersley G. RAD1901: a novel,
orally bioavailable selective estrogen receptor degrader that demonstrates antitumor
activity in breast cancer xenograft models. Anticancer Drugs 2015; 26:948-56.
54. Haddon L, Hugh J. MUC1-mediated motility in breast cancer: a review high-
lighting the role of the MUC1/ICAM-1/Src signaling triad. Clin Exp Metastasis
2015; 32:393-403.
55. Wei X, Xu H, Kufe D. MUC1 oncoprotein stabilizes and activates estrogen re-
ceptor alpha. Mol Cell 2006; 21:295-305.
56. Jin C, Rajabi H, Pitroda S, et al. Cooperative interaction between the MUC1-C
oncoprotein and the Rab31 GTPase in estrogen receptor-positive breast cancer
cells. PLoS One 2012; 7:e39432.
57. Raina D, Ahmad R, Joshi MD, et al. Direct targeting of the mucin 1 oncoprotein
blocks survival and tumorigenicity of human breast carcinoma cells. Cancer Res
2009; 69:5133-41.
58. Ghosh SK, Uchida M, Yoo B, et al. Targeted imaging of breast tumor progression
and therapeutic response in a human uMUC-1 expressing transgenic mouse
model. Int J Cancer 2013; 132:1860-7.
59. Lu Z, Hunter T. Prolyl isomerase Pin1 in cancer. Cell Res 2014; 24:1033-49.
60. Wang JZ, Liu BG, Zhang Y. Pin1-based diagnostic and therapeutic strategies for
breast cancer. Pharmacol Res 2015; 93:28-35.
61. Rajbhandari P, Ozers MS, Solodin NM, Warren CL, Alarid ET. Peptidylprolyl
Isomerase Pin1 Directly Enhances the DNA Binding Functions of Estrogen
Receptor alpha. J Biol Chem 2015; 290:13749-62.
62. Medina M, Wandosell F. Deconstructing GSK-3: the ne regulation of its ac-
tivity. Int J Alzheimers Dis 2011; 2011:479249.
63. Grisouard J, Medunjanin S, Hermani A, Shukla A, Mayer D. Glycogen synthase
kinase-3 protects estrogen receptor alpha from proteasomal degradation and is
required for full transcriptional activity of the receptor. Mol Endocrinol 2007; 21:
2427-39.
64. Medunjanin S, Hermani A, De Servi B, Grisouard J, Rincke G, Mayer D.
Glycogen synthase kinase-3 interacts with and phosphorylates estrogen receptor
alpha and is involved in the regulation of receptor activity. J Biol Chem 2005;
280:33006-14.
65. Giamas G, Filipovic A, Jacob J, et al. Kinome screening for regulators of the
estrogen receptor identies LMTK3 as a new therapeutic target in breast cancer.
Nat Med 2011; 17:715-9.
66. He X, Zheng Z, Song T, et al. c-Abl regulates estrogen receptor alpha tran-
scription activity through its stabilization by phosphorylation. Oncogene 2010; 29:
2238-51.
67. Dennis AP, Haq RU, Nawaz Z. Importance of the regulation of nuclear receptor
degradation. Front Biosci 2001; 6:D954-9.
68. Nakamura N. The role of the transmembrane RING nger proteins in cellular
and organelle function. Membranes (Basel) 2011; 1:354-93.
69. Grumati P, Dikic I. Germline polymorphisms in RNF31 regulate linear ubiq-
uitination and oncogenic signaling. Cancer Discov 2014; 4:394-6.
70. Zhu J, Zhao C, Kharman-Biz A, et al. The atypical ubiquitin ligase RNF31
stabilizes estrogen receptor alpha and modulates estrogen-stimulated breast cancer
cell proliferation. Oncogene 2014; 33:4340-51.
Downloaded from ClinicalKey.com at Universitas Kristen Duta Wacana November 29, 2016.
For personal use only. No other uses without permission. Copyright 2016. Elsevier Inc. All rights reserved.
71. Rizzolio F, Lucchetti C, Caligiuri I, et al. Retinoblastoma tumor-suppressor
protein phosphorylation and inactivation depend on direct interaction with
Pin1. Cell Death Differ 2012; 19:1152-61.
72. Caligiuri I, Toffoli G, Giordano A, Rizzolio F. pRb controls estrogen receptor
alpha protein stability and activity. Oncotarget 2013; 4:875-83.
73. La Rosa P, Pesiri V, Leclercq G, Marino M, Acconcia F. Palmitoylation regulates
17beta-estradiol-induced estrogen receptor-alpha degradation and transcriptional
activity. Mol Endocrinol 2012; 26:762-74.
74. Adlanmerini M, Solinhac R, Abot A, et al. Mutation of the palmitoylation site of
estrogen receptor alpha in vivo reveals tissue-specic roles for membrane versus
nuclear actions. Proc Natl Acad Sci U S A 2014; 111:E283-90.
75. Hou J, Deng M, Li X, et al. Chaperone gp96 mediates ER-alpha36 cell mem-
brane expression. Oncotarget 2015; 6:31857-67.
76. Panneerselvam M, Muthu K, Ramadas K. Structural insights into tumor-specic
chaperoning activity of gamma synuclein in protecting estrogen receptor alpha 36
and its role in tamoxifen resistance in breast cancer. Mol Biosyst 2015; 11:2998-
3010.
77. Lin SL, Yan LY, Zhang XT, et al. ER-alpha36, a variant of ER-alpha, promotes
tamoxifen agonist action in endometrial cancer cells via the MAPK/ERK and
PI3K/Akt pathways. PLoS One 2010; 5:e9013.
78. Liu H, Qiu J, Li N, Chen T, Cao X. Human phosphatidylethanolamine-binding
protein 4 promotes transactivation of estrogen receptor alpha (ERalpha) in hu-
man cancer cells by inhibiting proteasome-dependent ERalpha degradation via
association with Src. J Biol Chem 2010; 285:21934-42.
79. Chai F, Liang Y, Bi J, et al. REGgamma regulates ERalpha degradation via
ubiquitin-proteasome pathway in breast cancer. Biochem Biophys Res Commun
2015; 456:534-40.
80. Kim SH, Kang HJ, Na H, Lee MO. Trichostatin A enhances acetylation as well
as protein stability of ERalpha through induction of p300 protein. Breast Cancer
Res 2010; 12:R22.
81. Li H, Huang F, Fan L, et al. Phosphatidylethanolamine-binding protein 4 is
associated with breast cancer metastasis through Src-mediated Akt tyrosine
phosphorylation. Oncogene 2014; 33:4589-98.
82. Kharbanda A, Rajabi H, Jin C, Raina D, Kufe D. Oncogenic MUC1-C promotes
tamoxifen resistance in human breast cancer. Mol Cancer Res 2013; 11:714-23.
83. Lacunza E, Baudis M, Colussi AG, Segal-Eiras A, Croce MV, Abba MC. MUC1
oncogene amplication correlates with protein overexpression in invasive breast
carcinoma cells. Cancer Genet Cytogenet 2010; 201:102-10.
84. Alam M, Rajabi H, Ahmad R, Jin C, Kufe D. Targeting the MUC1-C onco-
protein inhibits self-renewal capacity of breast cancer cells. Oncotarget 2014; 5:
2622-34.
85. Zhou Y, Rajabi H, Kufe D. Mucin 1 C-terminal subunit oncoprotein is a target
for small-molecule inhibitors. Mol Pharmacol 2011; 79:886-93.
86. Rajabi H, Jin C, Ahmad R, McClary C, Joshi MD, Kufe D. Mucin 1 onco-
protein expression is suppressed by the miR-125b oncomir. Genes Cancer 2010;
1:62-8.
87. Sachdeva M, Mo YY. MicroRNA-145 suppresses cell invasion and metastasis by
directly targeting mucin 1. Cancer Res 2010; 70:378-87.
88. Jin C, Rajabi H, Kufe D. miR-1226 targets expression of the mucin 1 onco-
protein and induces cell death. Int J Oncol 2010; 37:61-9.
89. Yuan S, Shi C, Liu L, Han W. MUC1-based recombinant Bacillus Calmette-
Guerin vaccines as candidates for breast cancer immunotherapy. Expert Opin
Biol Ther 2010; 10:1037-48.
90. Yuan S, Shi C, Ling R, Wang T, Wang H, Han W. Immunization with two
recombinant Bacillus Calmette-Guerin vaccines that combine the expression of
multiple tandem repeats of mucin-1 and colony stimulating-factor suppress breast
tumor growth in mice. J Cancer Res Clin Oncol 2010; 136:1359-67.
91. Kotiyal S, Bhattacharya S. Breast cancer stem cells, EMT and therapeutic targets.
Biochem Biophys Res Commun 2014; 453:112-6.
92. Li X, Li L, Zhou Q, et al. Synthesis of the novel elemonic acid derivatives as Pin1
inhibitors. Bioorg Med Chem Lett 2014; 24:5612-5.
93. Potter A, Oldeld V, Nunns C, et al. Discovery of cell-active phenyl-imidazole
Pin1 inhibitors by structure-guided fragment evolution. Bioorg Med Chem Lett
2010; 20:6483-8.
94. Wei S, Kozono S, Kats L, et al. Active Pin1 is a key target of all-trans retinoic
acid in acute promyelocytic leukemia and breast cancer. Nat Med 2015; 21:457-
66.
95. Xu GG, Slebodnick C, Etzkorn FA. Cyclohexyl ketone inhibitors of Pin1 dock in
a trans-diaxial cyclohexane conformation. PLoS One 2012; 7:e44226.
96. Kim JA, Kim MR, Kim O, et al. Amurensin G inhibits angiogenesis and tumor
growth of tamoxifen-resistant breast cancer via Pin1 inhibition. Food Chem
Toxicol 2012; 50:3625-34.
97. Kim JH, Jung JH, Kim SH, Jeong SJ. Decursin exerts anti-cancer activity in
MDA-MB-231 breast cancer cells via inhibition of the Pin1 activity and
enhancement of the Pin1/p53 association. Phytother Res 2014; 28:238-44.
98. Moore JD, Potter A. Pin1 inhibitors: pitfalls, progress and cellular pharmacology.
Bioorg Med Chem Lett 2013; 23:4283-91.
99. Jacobs KM, Bhave SR, Ferraro DJ, Jaboin JJ, Hallahan DE, Thotala D. GSK-
3beta: a bifunctional role in cell death pathways. Int J Cell Biol 2012; 2012:
930710.
100. McCubrey JA, Davis NM, Abrams SL, et al. Diverse roles of GSK-3: tumor
promoter-tumor suppressor, target in cancer therapy. Adv Biol Regul 2014; 54:
176-96.
101. Mishra R. Glycogen synthase kinase 3 beta: can it be a target for oral cancer. Mol
Cancer 2010; 9:144.
Clinical Breast Cancer Month 2016 -9
 ER Stability in Breast Cancer
102. Kim HM, Kim CS, Lee JH, et al. CG0009, a novel glycogen synthase kinase 3
inhibitor, induces cell death through cyclin D1 depletion in breast cancer cells.
PLoS One 2013; 8:e60383.
103. Zhao G, Guo J, Li D, et al. MicroRNA-34a suppresses cell proliferation by
targeting LMTK3 in human breast cancer mcf-7 cell line. DNA Cell Biol 2013;
32:699-707.
104. Stebbing J, Filipovic A, Ellis IO, et al. LMTK3 expression in breast cancer: as-
sociation with tumor phenotype and clinical outcome. Breast Cancer Res Treat
2012; 132:537-44.
105. Stebbing J, Filipovic A, Lit LC, et al. LMTK3 is implicated in endocrine resis-
tance via multiple signaling pathways. Oncogene 2013; 32:3371-80.
106. Ertel A, Dean JL, Rui H, et al. RB-pathway disruption in breast cancer: differ-
ential association with disease subtypes, disease-specic prognosis and therapeutic
response. Cell Cycle 2010; 9:4153-63.
107. Trere D, Brighenti E, Donati G, et al. High prevalence of retinoblastoma
protein loss in triple-negative breast cancers and its association with a good
10 - Clinical Breast Cancer Month 2016
Downloaded from ClinicalKey.com at Universitas Kristen Duta Wacana November 29, 2016.
For personal use only. No other uses without permission. Copyright 2016. Elsevier Inc. All rights reserved.
prognosis in patients treated with adjuvant chemotherapy. Ann Oncol 2009; 20:
1818-23.
108. Witkiewicz AK, Knudsen ES. Retinoblastoma tumor suppressor pathway in
breast cancer: prognosis, precision medicine, and therapeutic interventions. Breast
Cancer Res 2014; 16:207.
109. Lehn S, Ferno M, Jirstrom K, Ryden L, Landberg G. A non-functional
retinoblastoma tumor suppressor (RB) pathway in premenopausal breast
cancer is associated with resistance to tamoxifen. Cell Cycle 2011; 10:956-
62.
110. Fowler AM, Solodin N, Preisler-Mashek MT, Zhang P, Lee AV, Alarid ET.
Increases in estrogen receptor-alpha concentration in breast cancer cells promote
serine 118/104/106-independent AF-1 transactivation and growth in the absence
of estrogen. FASEB J 2004; 18:81-93.
111. Fowler AM, Solodin NM, Valley CC, Alarid ET. Altered target gene regulation
controlled by estrogen receptor-alpha concentration. Mol Endocrinol 2006; 20:
291-301.