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Estrogen receptor-α signaling in post-natal mammary development and breast cancers

TL;DR: In this paper, the authors summarize the current understanding of the complex ERα signaling pathways that involve either classical nuclear or membrane non-genomic actions and regulate in concert with other hormones the different stages of mammary development.
Abstract: 17β-estradiol controls post-natal mammary gland development and exerts its effects through Estrogen Receptor ERα, a member of the nuclear receptor family. ERα is also critical for breast cancer progression and remains a central therapeutic target for hormone-dependent breast cancers. In this review, we summarize the current understanding of the complex ERα signaling pathways that involve either classical nuclear “genomic” or membrane “non-genomic” actions and regulate in concert with other hormones the different stages of mammary development. We describe the cellular and molecular features of the luminal cell lineage expressing ERα and provide an overview of the transgenic mouse models impacting ERα signaling, highlighting the pivotal role of ERα in mammary gland morphogenesis and function and its implication in the tumorigenic processes. Finally, we describe the main features of the ERα-positive luminal breast cancers and their modeling in mice.

Summary (4 min read)

Introduction

  • The mammary gland is an exocrine gland of ectodermal origin whose primary function is to produce milk for the nourishment of offspring.
  • Prolactin, GH and oxytocin are peptide hormones of pituitary origin, whereas estrogens and progesterone are steroid hormones primarily produced by ovaries during reproductive life.
  • Pioneering works showing that ovariectomized and ERα-deficient mice were unable to develop mammary gland at puberty have indicated that signaling through estrogens is crucial for the post-natal mammary development [9–12].
  • Here, the authors review the current understanding of the mechanisms of ERα actions, derived from different studies on mammary development, stem cell function and tumorigenesis.

ERα and its modes of action

  • In humans and rodents, two distinct estrogen receptors, ERα and ERβ, have been identified.
  • ESR1 gene spans over 300 kb and consists of nine coding exons and seven introns (Fig. 1).
  • AF-1 can also be modified in response to E2 and further stabilized following phosphorylation on serine 118 [42–44].
  • The D domain is a hinge region that provides flexibility between the DBD and the LBD (E/ F) domains.
  • Moreover, ERα also interacts with some corepressors, such as the repressor of estrogen receptor activity (REA) repressor which binds on the LBD domain in a liganddependent manner [54] or RIP140 (receptor interacting protein) through a direct competition with SCR-1 [55].

Natural isoforms of ERα

  • In addition to the “classic” full-length isoform of ERα (ERα66 kDa) which contains the two AF-1 and AF-2 activation functions, there is a shorter 46 kDa isoform lacking the first 173 amino acids and, therefore, the AF-1 function (Fig. 1).
  • The females are sterile, with uterine atrophy while they conserved several vasculoprotective actions of E2 [62–64].
  • Western blot with antibodies directed against the C-terminal domain is the unique procedure to detect the ERα46 isoform since ERα46 and ERα66 share identical aminoacid sequences that cannot be distinguish by immunohistochemistry.
  • It was found expressed in various cell types such as vascular endothelial cells and macrophages [65–68].
  • ERα46 is also expressed in breast cancer cells including tamoxifenresistant cells [69] and in more than 70% of human breast tumors with highly variable expression levels, sometimes even more abundant than the ERα66 protein [70].

The nuclear actions of ERα

  • As a member of the nuclear receptor family, ERα mainly functions as a ligand-activated transcription factor through different mechanisms (Fig. 2).
  • Most of these ERBs are distally located from targets genes and function as distal-cis-regulatory elements, generating a complex numbers of loops and anchors to bring the receptor binding sites closer to the transcription initiation site [85, 88].
  • These modifications were shown to be particularly essential for the genomic effects of ERα, in particular for the recruitment of transcriptional co-activators [93–96].
  • Thus, phosphorylation integrates these signaling pathways, such as epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) into a complex cross-talk network with estrogen signaling. [92, 97].
  • The crucial role of these pioneer factors for the ERα response was demonstrated when FOXA1 and AP2gamma binding to several sites is decreased upon ERα silencing [103] (see also Chapter 4.2 for their roles in the mammary gland development).

The membrane “non‑genomic” actions of ERα

  • A small fraction of the ERα is found at the plasma membrane where it activates the so-called “rapid”, “nongenomic”, or MISS for “Membrane-Initiated Steroid Signaling”, which induces multiple signaling pathways [49, 104] and creates cross-talk between membrane and nuclear signaling [21, 22] (Fig. 2).
  • Membrane ERα has near identical affinity for E2 than nuclear ERα and originates from the same transcript, but its abundance is very low (around 3% as compared to nuclear ERα) [108].
  • Membrane ERα effects were studied using transgenic mouse models mutated either for the palmitoylation site (ERα-C451A, murine counterpart of human C447) [114, 115], or the methylation site (R264A, murine counterpart of human R260) [116].
  • Rapid signaling was also blocked by overexpression of a peptide that prevents ERs from interacting with the scaffold protein striatin (the disrupting mouse peptide) [117].
  • In view of these studies, it is, therefore, difficult to functionally dissociate these two actions of estrogenic signaling.

Mammary development and cell lineages

  • Overview of the post‑natal mammary development and its hormonal context Comprehensive reviews on mammary development have been recently published [4–6].
  • At sexual maturity, TEBs regress and ductal elongation ceases.
  • Around parturition, progesterone levels abruptly drop down resulting in induction of labor.
  • Ovariectomy of prepubertal females impedes mammary development, whereas administration of exogeneous estrogens restores its growth, resulting in morphological changes similar to those observed at puberty [9, 134].
  • A Schematic representation of mammary duct and alveolus and main specific markers of the basal myoepithelial, ERα-positive and –negative luminal cell lineages.

Mammary basal and luminal lineages

  • It is now established that stem cells drive the post-natal mammary development.
  • Pioneering orthotopic transplantation studies have shown that basal cells isolated from the adult mammary epithelium were able to regenerate bilayered ducts and alveoli, even at single cell level, whereas luminal cells had no significant regenerative potential [139–141].
  • Characteristics of the ERα luminal cell lineage Distribution of ERα + luminal cells within the developing mammary epithelium Immunohistochemical (IHC) studies have shown that ERα is expressed in the nuclei of both mammary epithelial and stromal cells [9].
  • Absent at birth, ERα was detected in about half of luminal cells at post-natal day 7, a proportion maintained during the pubertal growth [138, 145].
  • This percentage decreases to about 5% at the end of pregnancy, the remaining positive cells being primarily located in ducts.

Mouse models mutated for ERα

  • The transgenic mouse models used to dissect the role of ERα signaling in mammary development and function are presented in Table 2.
  • Of note, the ERα-KO mouse completely lacks ERα transcript expression, whereas the ERαNeoKO was found later to retain a substantial ERα function, by producing a spliced mRNA that gives rise to a receptor lacking part of the ligand-independent AF-1 domain, a form reminiscent of that from the ERα-AF1° deficient mice [62, 200].
  • Collectively, the data obtained from mouse models revealed the complex status of ERα expression in the mammary epithelium and the multiple implications of ERα signaling in the control of mammary development.
  • Collectively, these studies revealed a complex interplay between ERα, GATA3 and FOXA1 [181].
  • As SRC-2, SRC-3 is not essential for E2-stimulated ductal growth in virgin mice, 1 3 but is required for progesterone-stimulated cellular proliferation and glandular differentiation during pregnancy [230].

ERα‑positive luminal breast cancers

  • Considerable interest has focused on luminal cells in the context of mammary gland development and tumorigenesis, as most breast cancers are thought to originate from deregulated luminal cells, either negative or positive for ERα [4, 241].
  • The most frequent special histological subtype is the invasive lobular carcinoma (ILC) that clusters with luminal A and B subtypes and is characterized by a loss of E-cadherin expression [15].
  • Most ERα-positive breast cancers depend on estrogen for their growth and ERα expression is predictive for responsiveness to endocrine therapies targeting the E2/ERα 1 3 pathway.
  • Hence, ERα-positive tumors are highly heterogeneous with a broad range of ERα expression spanning from 1% to nearly 100%.
  • In addition, an important proportion of the patients do not respond to endocrine therapies and up to 50% acquire resistance under treatment [245].

Exposure to estrogens and breast cancers

  • The impact of estrogens on breast cancer was first demonstrated more than a century ago by the British surgeon George Beatson who observed regression of a breast tumor following ovariectomy [246].
  • Nowadays, early and prolonged exposure to endogenous or exogenous estrogens during a woman’s life is recognized as being a factor of major risk in developing a breast cancer, in particular an ERα-positive subtype [247, 248].
  • Early menarche, late menopause, nulliparity or late first pregnancy are viewed as risk factors while breast feeding is considered as a protective factor [247, 249, 250].
  • This absolute increase in risk remains low but rises with longer durations of use [248, 251].
  • More recent analyses have shown that the levels of risks varied between types of hormonal replacement therapies, with higher risks when progestins were used in the combination with estrogens (as compared to the natural progesterone), and again, for longer duration of use [254].

Mutations of ESR1 in human breast tumors

  • The most frequent mutated genes in ERα-positive breast cancers are PIK3CA, GATA3, MAP3K1, KMT2C and TP53.
  • Mutation of CDH1 (encoding E-cadherin) or loss of alleles are common in the lobular subtype (reviewed in [15, 255]).
  • In contrast, ESR1 mutations are rare (less < 1%) in primary ERα-positive breast cancers [256] but between 20 and 40% of ESR1 mutations are observed in metastatic breast cancer and influence response to hormone therapy (reviewed in [256–260]).
  • This mutated tyrosine Y537 has been particularly involved in the growth of mammary cancer cells and xenografts following phosphorylation by Src tyrosine kinases (p56lck and p60c−src) [263–267].
  • These ESR1 fusion genes not only led to endocrine resistance but also induced epithelial–mesenchymal transition (EMT) leading to metastasis.

Models of ERα‑positive breast cancers

  • Establishing in vivo models mimicking the complex biology of ERα-positive breast cancers remains an active field of research (reviewed in [279].
  • Nonetheless, the broadly used MMTVPyMT mouse model that expresses polyoma middle T (PyMT) oncogenic protein in the mammary epithelium recapitulates some aspects of ERα-positive breast cancers.
  • The use of specific promoters for addressing pertinent oncogenic mutations in the ERα + luminal cell lineage should lead to the design of novel GEMMs, providing further insights into initiation and progression of the ERα + luminal breast cancers.
  • Finally, many PDX models have been successfully established for pre-clinical breast cancer research, however, the take rates of ERα-positive tumor samples transplanted in the mammary fat pad of immunocompromised mice were noticeably low [289, 290].
  • The same strategy was further used to design a model of ERα-positive ILC and test novel therapeutic approaches [293].

Conclusion

  • Since the cloning of ESR1 in 1986, the field has made considerable advances in deciphering the molecular mechanisms of ERα signaling through genomic and non-genomic actions and in addition, piecing together the role of ERα in luminal cells and in mammary gland development and 1 3 function.
  • The target cells of the non-genomic membrane actions of ERα signaling within the mammary epithelium remain to be precisely identified.
  • Funding Some of the work summarized here performed at I2MCINSERM U1297 was supported by Institut National de la Santé et de la Recherche Médicale, Université et CHU de Toulouse, Faculté de Médecine Toulouse-Rangueil, Fondation pour la Recherche Médicale, Association pour la Recherche Contre le Cancer (PJA 20141201844 and PJA 20161204764 to F.L.) and La ligue Contre le Cancer- AriègeHaute-Garonne-Tarn.
  • MR was supported by a grant from the Agence Nationale de la Recherche- (BENEFIT to F.L.).

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Estrogen receptor-α signaling in post-natal mammary
development and breast cancers
Mariam Rusidzé, Marine Adlanmérini, Elodie Chantalat, I. Raymond-Letron,
Surya Cayre, Jean-François Arnal, Marie-Ange Deugnier, Françoise Lenfant
To cite this version:
Mariam Rusidzé, Marine Adlanmérini, Elodie Chantalat, I. Raymond-Letron, Surya Cayre, et al..
Estrogen receptor-α signaling in post-natal mammary development and breast cancers. Cellular and
Molecular Life Sciences, Springer Verlag, 2021, �10.1007/s00018-021-03860-4�. �hal-03268888�

Vol.:(0123456789)
1 3
Cellular and Molecular Life Sciences
https://doi.org/10.1007/s00018-021-03860-4
REVIEW
Estrogen receptor‑α signaling inpost‑natal mammary development
andbreast cancers
MariamRusidzé
1
· MarineAdlanmérini
1
· ElodieChantalat
1
· I.Raymond‑Letron
2
· SuryaCayre
3
·
Jean‑FrançoisArnal
1
· Marie‑AngeDeugnier
3
· FrançoiseLenfant
1
Received: 25 January 2021 / Revised: 12 May 2021 / Accepted: 19 May 2021
© The Author(s) 2021
Abstract
17β-estradiol controls post-natal mammary gland development and exerts its effects through Estrogen Receptor ERα, a
member of the nuclear receptor family. ERα is also critical for breast cancer progression and remains a central therapeutic
target for hormone-dependent breast cancers. In this review, we summarize the current understanding of the complex ERα
signaling pathways that involve either classical nuclear “genomic” or membrane “non-genomic” actions and regulate in con-
cert with other hormones the different stages of mammary development. We describe the cellular and molecular features of
the luminal cell lineage expressing ERα and provide an overview of the transgenic mouse models impacting ERα signaling,
highlighting the pivotal role of ERα in mammary gland morphogenesis and function and its implication in the tumorigenic
processes. Finally, we describe the main features of the ERα-positive luminal breast cancers and their modeling in mice.
Keywords Mammary gland· 17β-estradiol· ERα-positive luminal cells· Lineage specification· Stem cells
Introduction
The mammary gland is an exocrine gland of ectodermal
origin whose primary function is to produce milk for the
nourishment of offspring. In humans as in most mammals,
mammary morphogenesis is initiated during the embryonic
period but the most important part of mammary develop-
ment and remodeling occurs after birth, throughout puberty,
pregnancy, lactation and involution [16]. Despite some dif-
ferences, the human and mouse mammary epithelium shares
strong similarities in developmental processes, cellular
organization and signaling molecules [4, 7]. Mouse mod-
els are, therefore, widely used to decipher the molecular
mechanisms controlling the development and homeostasis
of the mammary gland, and analyze their deregulation upon
tumorigenic processes.
The post-natal development of the mammary gland and its
function are controlled by a hormonal network that mainly
comprises estrogens, progesterone, prolactin, growth hor-
mone (GH) and oxytocin [3, 8]. Prolactin, GH and oxytocin
are peptide hormones of pituitary origin, whereas estrogens
and progesterone are steroid hormones primarily produced
by ovaries during reproductive life. Pioneering works show-
ing that ovariectomized and ERα-deficient mice were unable
to develop mammary gland at puberty have indicated that
signaling through estrogens is crucial for the post-natal
mammary development [912]. In addition, ERα is routinely
used as a diagnosis marker supporting the molecular clas-
sification of breast cancers [1315] and remains an essential
therapeutic target for hormone-dependent breast cancers, in
particular through administration of tamoxifen (TAM) and/
or aromatase inhibitors (AI), that both are very efficient in
reducing the risk of cancer recurrence [1618].
As member of the nuclear receptor family, ERα has a
well-established transcription factor activity and controls
the expression a large spectrum of target genes [19, 20].
Cellular andMolecular Life Sciences
Marine Adlanmérini and Elodie Chantalat have contributed
equally.
* Françoise Lenfant
francoise.lenfant@inserm.fr
1
INSERM U1297, Institut Des Maladies Métaboliques et
Cardiovasculaires, Université de Toulouse - UPS, CHU,
Toulouse, France
2
LabHPEC et Institut RESTORE, Université de Toulouse,
CNRS U-5070, EFS, ENVT, Inserm U1301, Toulouse,
France
3
Department ofCell Biology andCancer, Institut
Curie, PSL Research University, Sorbonne University,
CNRSUMR144Paris, France

M.Rusidzé et al.
1 3
However, estrogens and ERα can also act at the cell mem-
brane level to induce non-genomic events [21, 22]. The
recent development of new transgenic mouse models and
omics-based analyses has allowed to better characterize the
ERα-positive luminal cell lineage and to further dissect the
complex signaling events triggered by estrogens in the mam-
mary epithelium. Here, we review the current understanding
of the mechanisms of ERα actions, derived from different
studies on mammary development, stem cell function and
tumorigenesis.
ERα andits modes ofaction
In humans and rodents, two distinct estrogen receptors, ERα
and ERβ, have been identified. They show large sequence
homology and similar binding affinity for 17β-estradiol (E2),
the predominant form of circulating estrogens [19, 23]. Esr1
(ESR1 in human) encoding ERα was first identified in 1986
[24, 25] and located on a different chromosome than Esr2
coding for ERβ, identified later in 1996 [26]. ERα is believed
to be the ancestral steroid receptor originating 400–500 mil-
lion years ago [27] and its complex modes of action and gene
organization remain abundantly studied [28]. Invivo, per-
turbation of ERα signaling has a major impact on mammary
development [11, 12], whereas ERβ loss does not result in
a deleterious mammary phenotype and impaired function
[29, 30].
ESR1 gene spans over 300kb and consists of nine cod-
ing exons and seven introns (Fig.1). The first eight exons
encode the major full-length 66kDa isoform of ERα [31].
The promoter region (over 150kb) contains several promoter
sequences named A to T that drives its specific expression in
target tissues [32, 33]. ESR1 gene expression is tightly regu-
lated by multiple regulatory elements, including transcrip-
tion factors, chromatin environment, autocrine, paracrine
and endocrine secreted factors, and multiple environment
factors (cell–cell and cell–matrix interactions, mechanical
forces) [34]. In addition, the 3’UTR region of ERα contains
several regulatory elements specific for miRNAs, such as
miR18a, miR22, miR206 and miR221/22, that control ERα
stability or translocation [35].
ERα is composed of six structural domains namely A to
F, including two binding domains, one to DNA (DBD, C
domain) and the other to ligand (LBD, E domain) [19, 21].
It also includes a ligand-independent (AF1) and a ligand-
dependent (AF-2) subdomain, mapping to the A/B and E
domains, respectively [36, 37] (Fig.1). The AF-1 transac-
tivation domain is mainly ligand independent, its stimula-
tion relying on the phosphorylation of serine 104/106, 118
or 167 by kinases activated downstream of growth factors
such as EGF (Epidermal Growth Factor), IGF-1 (Insulin-like
Growth Factor-1), or TGFα (Tumor Growth Factor) [3841].
However, AF-1 can also be modified in response to E2 and
further stabilized following phosphorylation on serine 118
[4244]. The A domain interacts with the C-terminal domain
to allow repression in absence of ligand [45]. The D domain
is a hinge region that provides flexibility between the DBD
and the LBD (E/ F) domains. The mutation of this D region
affects the synergy between the AF-1 and AF-2 functions of
ERα [46]. AF1 and AF2 display distinct activation functions
that are specifically involved in the recruitment of cofac-
tors. These coregulators are not only proteins that link the
receptor and the transcription machinery but rather have
enzymatic activities that induce chromatin modification and
remodeling, and control initiation of transcription [4749].
Among the coregulators that bind to the AF-2 domain
exposed following E2 binding, there are members of the
p160 family that includes three analogous factors SRC-1,
SRC-2 and SRC-3 (Steroid Receptor Coactivator, part of
histone deacetylase) [50, 51]. Other well-known cofactors
comprise CBP/p300 and MED1. Interestingly, p160 proteins
also interact with the NH2-terminal domain of ERα, in par-
ticular the AF1 domain, and p300 allows a functional syn-
ergy between AF1 and AF2 [40, 52]. This was confirmed by
the recent quaternary structure of an active ERα-coregulator
Fig. 1 Structure of the ESR1
gene and the different isoforms
of ERα. On the top, the coding
exons are annotated following
the nomenclature published in
[32]. Alternative splicing that
generates the shorter ERα46
and ERα36 isoforms are indi-
cated using solid lines
h-ERα
66kDa
178
36kDa
178
DBD
599
A/B Domain
E
C
D
F
AF1
AF2
LBD
DBD
599
AF2
LBD
DBD
457
LBD
27aa
46kDa
Ex1
5’UTR
ATG1
ATG2
Ex2
Ex3
Ex4
Ex5
Ex6
Ex7
Ex8
IRES
TGA
TGA
Ex9
B
A

Estrogen receptor-α signaling inpost-natal mammary development andbreast cancers
1 3
complex on DNA identified using cryoelectron microscopy
[53]. Moreover, ERα also interacts with some corepressors,
such as the repressor of estrogen receptor activity (REA)
repressor which binds on the LBD domain in a ligand-
dependent manner [54] or RIP140 (receptor interacting
protein) through a direct competition with SCR-1 [55].
Natural isoforms ofERα
In addition to the “classic” full-length isoform of ERα (ERα-
66kDa) which contains the two AF-1 and AF-2 activation
functions, there is a shorter 46kDa isoform lacking the first
173 amino acids and, therefore, the AF-1 function (Fig.1).
Although the prominent, if any, mechanisms accounting for
the expression of the ERα46 isoform still remain to be clari-
fied, three possible processes of generation were reported: (i)
an alternative splicing that generated a mRNA deficient in
the nucleotide sequence corresponding to exon 1 encoding
the A/B domain generation [56]; (ii) proteolysis [57, 58];
and (iii) initiation of translation at a downstream ATG which
encodes methionine 174 in the human ERα66 by an IRES
(Internal Ribosome Entry Site) located within the full-length
mRNA [59]. A recent study showed that the expression of
ERα46 is due to the action of the oncoprotein HMGA1a
(High Mobility Group A protein1a) that regulates the alter-
native splicing of ESR1 in MCF7 breast cancer cells [60].
Overexpression of ERα46 in proliferating MCF7 cells pro-
vokes a cell cycle arrest in G0/G1 phases and inhibits the
ERα66-mediated estrogenic induction of all AF-1-sensitive
reporters: c-fos and cyclin D1 as well as estrogen-respon-
sive element-driven reporters [56, 61]. The role of the AF-
1-deficient ERα46 isoform has also been questioned invivo
using a “knock in” strategy. These mice (named ERαAF-1
0
)
only express a short 49kDa isoform that lacks 441 nucleo-
tides from exon 1 and is functionally similar to ERα46 [62].
The females are sterile, with uterine atrophy while they
conserved several vasculoprotective actions of E2 [6264].
Studies on mammary gland development are reported later
in chapter4.1.
Western blot with antibodies directed against the C-ter-
minal domain is the unique procedure to detect the ERα46
isoform since ERα46 and ERα66 share identical aminoacid
sequences that cannot be distinguish by immunohistochemis-
try. Although the ERα46 isoform has not been studied exten-
sively, it was found expressed in various cell types such as
vascular endothelial cells and macrophages [6568]. ERα46
is also expressed in breast cancer cells including tamoxifen-
resistant cells [69] and in more than 70% of human breast
tumors with highly variable expression levels, sometimes
even more abundant than the ERα66 protein [70]. Impor-
tantly, higher amounts of ERα46 proteins were associated
with highly differentiated tumors of lower grade and smaller
size [70].
In 2005, another shorter 36kDa isoform of ERα was
identified from a human endometrium cDNA library [71].
This ERα36 isoform is transcribed from an alternative pro-
moter located in the first intron of the ESR1 gene and is
encoded by exons 1, 2–6, and 9 (Fig.1). ERα-36 thus lacks
the transactivation functions AF-1 and AF-2 but retains the
DNA-binding domain of ERα66 and its partial dimeriza-
tion and ligand-binding domains. It also contains a unique
27 amino acids at the C-terminus that replaced the last 138
aminoacids encoded by exons 7 et 8 and can be detected by
specific antibodies. ERα36 contains three potential myris-
toylation sites which are conserved in the full-length ERα66.
These are residues 25–30 (GVWSCE), 76–81 (GMMKGG)
and 171–176 (ELLTNL) [71]. Myristoylation being a post-
translational modification allowing anchoring to the plasma
membrane, ERα-36 was suggested to be mainly localized
at the plasma membrane where it could relay rapid estro-
gen signaling and inhibit the transcriptional activity of ERα
66kDa, probably by competition at DNA-binding sites [71,
72]. The ERα36 receptor is not expressed in mice. How-
ever, it was found largely expressed in both ERα-positive
and ERα-negative breast cancers, at a proportion that varies
between 40 and 50% according to cohort studies [7375].
ERα36 is mainly described in the literature to be involved
in the acquired resistance to anti-estrogen drugs, such as
tamoxifen and in the progression of mammary tumors in
response to chemotherapy [76].
Complexity ofactions ofERα signaling
ERα activation is a complex process involving many signal-
ing pathways that trigger either classical nuclear “genomic”
or membrane “non-genomic” actions (Fig.2).
The nuclear actions ofERα
As a member of the nuclear receptor family, ERα mainly
functions as a ligand-activated transcription factor through
different mechanisms (Fig.2). Estrogen binding to the LBD
induces dissociation from the Hsp90/Hsp70-multi-protein
chaperone machinery, receptor dimerization and nuclear
entry. Crystal structure revealed that the LBD has 12 alpha
helices and E2-binding repositionnes helix 12, such that
activation function AF-2 is exposed, allowing interactions
with coregulators [77]. ERα is then stabilized in its active
state and binds directly to specific DNA sites to estrogen-
response elements (ERE = 5’GGTCAnnnTGACC3’ palin-
dromic sequences) [78].
About 25% of estrogen-regulated genes lack complete
ERE sequences in their promoter regions [79]. Moreo-
ver, ERα can bind to DNA by indirect tethering to other

M.Rusidzé et al.
1 3
transcription factors such as the Stimulating protein 1
(SP1) on sites rich in GC, the jun/c-fos proteins which
form a dimeric complex binding to “Activator Protein 1”
(AP-1) sites [80] and Nuclear factor–κβ (NF-κβ). Genome-
wide analysis of ERα DNA-binding sites has identified
not only rigorously dissociate the genomic and, but also
PITX1 whose binding motif was found present in 28% of
genome-wide ERα-binding sites [8183].
Studies using CHIP-Chip and CHIP-seq on MCF7 breast
cancer cells have revealed that ERα binds to 5000–10,000
locations [8486]. However, only < 5% of these ERα bind-
ing sites (ERBs) are located in the proximal region of ERα
TKR
Kinases
PR
AREG
AP1
E2
Hsp90/70
TKR
NON-GENOMIC/
MISS
IGF-1
EGF
TGFα
ERK1-2
Direct tethering
Indirect tethering
GENOMIC
IGF-1
EGF
TGFα
ERα
P
ERα
Caveolin
Strian
ERα
ERα
P
P
EDC
PaPEs
PI3K
i
ERE
ERBs
ERα
Ligand-
independent
ER - Estrogen receptor
P
TKR
TKR-Tyrosine Kinase Receptor
Phosphorylaon
Palmitoylaon
Methylaon
ERα
Src
IκB
AKT
P
P
DBD
AF1
AF1
LBD
LBD
Dimer ER
ERα
ERα
P
P
ERα
ERα
P
P
ERBs
Ligand-dependent
Pioneer factors
ERα
ERα
P
P
P
NF-κB
STAT2
P
G/C
Fos/Jun
SP1
Fig. 2 Estrogen receptor ERα signaling. Classic ERα signaling
leads to genomicactions through ligand-receptor binding, leading to
dimerization of ERα that binds directly to specific DNA sites (called
estrogen response elements, ERE) that activate transcription. ERα can
also bind by indirect tethering to other transcription factors, such as
AP1 or SP1 (blue line). The ERα can also be activated in a ligand-
independent manner through downstream events of receptor tyrosine
kinases (RTKs) activated by growth factors in the mammary gland,
such as IGF-1, EGF (blue dotted line, in particular through phospho-
rylation of serine residues in the AF-1 domain). Induction of tran-
scriptional response depends on the chromatin remodeling, induced
by pioneer factors such as FoxA1 and GATA-3 in the mammary
gland, and is modulated by the specific recruitment of coregulators.
Non-genomic, membrane-initiated steroid signaling (MISS) actions
involve a small pool of ERα located on the extracellular compart-
ment or close to the membrane, at least in part through direct inter-
action with caveolin-1 in response to post-translational modifications
such as palmitoylation. Transient methylation of arginine 260 has
also been observed to induce ERα interaction with the p85 subunit
of PI3K and Src, Upon E2 binding, these non-genomic activations
activate the subsequent interaction of ERα with protein kinases (Src
and PI3K), G-coupled protein I, leading to activation of signaling
cascades (Akt, ERK1/2) and further shuttle of these phosphorylated
transcription factors in the nucleus. These non-genomic signaling
pathways are rapidly activated and further induce genomic activations
(orange dotted line)

Citations
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Journal ArticleDOI
TL;DR: The mammary gland is a compound, branched tubuloalveolar structure and a major characteristic of mammals as discussed by the authors , and mammary development begins during embryogenesis as a rudimentary structure that grows into an elementary branced ductal tree and is embedded in one end of a larger mammary fat pad at birth.
Abstract: The mammary gland is a compound, branched tubuloalveolar structure and a major characteristic of mammals. The mammary gland has evolved from epidermal apocrine glands, the skin glands as an accessory reproductive organ to support postnatal survival of offspring by producing milk as a source of nutrition. The mammary gland development begins during embryogenesis as a rudimentary structure that grows into an elementary branched ductal tree and is embedded in one end of a larger mammary fat pad at birth. At the onset of ovarian function at puberty, the rudimentary ductal system undergoes dramatic morphogenetic change with ductal elongation and branching. During pregnancy, the alveolar differentiation and tertiary branching are completed, and during lactation, the mature milk-producing glands eventually develop. The early stages of mammary development are hormonal independent, whereas during puberty and pregnancy, mammary gland development is hormonal dependent. We highlight the current understanding of molecular regulators involved during different stages of mammary gland development.

14 citations

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TL;DR: In this paper , the authors focus on the involvement of NRs in oral tumor pathogenesis and highlight the importance of targeting NRs using various agonists and antagonists that could serve as a potential strategy for the prevention and treatment of oral malignancies.

9 citations

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TL;DR: The molecular structure features and biological functions of DDX5/DDX17 and their effects on tumorigenesis and tumor progression, as well as their potential clinical application for tumor treatment are discussed.
Abstract: DEAD-box (DDX)5 and DDX17, which belong to the DEAD-box RNA helicase family, are nuclear and cytoplasmic shuttle proteins. These proteins are expressed in most tissues and cells and participate in the regulation of normal physiological functions; their abnormal expression is closely related to tumorigenesis and tumor progression. DDX5/DDX17 participate in almost all processes of RNA metabolism, such as the alternative splicing of mRNA, biogenesis of microRNAs (miRNAs) and ribosomes, degradation of mRNA, interaction with long noncoding RNAs (lncRNAs) and coregulation of transcriptional activity. Moreover, different posttranslational modifications, such as phosphorylation, acetylation, ubiquitination, and sumoylation, endow DDX5/DDX17 with different functions in tumorigenesis and tumor progression. Indeed, DDX5 and DDX17 also interact with multiple key tumor-promoting molecules and participate in tumorigenesis and tumor progression signaling pathways. When DDX5/DDX17 expression or their posttranslational modification is dysregulated, the normal cellular signaling network collapses, leading to many pathological states, including tumorigenesis and tumor development. This review mainly discusses the molecular structure features and biological functions of DDX5/DDX17 and their effects on tumorigenesis and tumor progression, as well as their potential clinical application for tumor treatment.

7 citations

Journal ArticleDOI
01 Mar 2022-Animals
TL;DR: In this article , microRNA expression profiles in female ICR mice's mammary glands at the virgin stage (V), day 16 of pregnancy (P16d), day 12 of lactation (L12d) and day 1 of forced weaning (FW1d) were analyzed.
Abstract: Simple Summary Milk synthesis is vital for maintaining the normal growth of newborn animals. Abnormal mammary gland development leads to a decrease in female productivity and the overall productivity of animal husbandry. This study characterized the dynamic miRNA expression profile during the process of mammary gland development, and identified a novel miRNA regulating expression of β-casein—an important milk protein. The results are valuable for studying mammary gland development, increasing milk production, improving the survival rate of pups, and promoting the development of animal husbandry. Abstract Mammary gland morphology varies considerably between pregnancy and lactation status, e.g., virgin to pregnant and lactation to weaning. Throughout these critical developmental phases, the mammary glands undergo remodeling to accommodate changes in milk production capacity, which is positively correlated with milk protein expression. The purpose of this study was to investigate the microRNA (miRNA) expression profiles in female ICR mice’s mammary glands at the virgin stage (V), day 16 of pregnancy (P16d), day 12 of lactation (L12d), day 1 of forced weaning (FW1d), and day 3 of forced weaning (FW3d), and to identify the miRNAs regulating milk protein gene expression. During the five stages of testing, 852 known miRNAs and 179 novel miRNAs were identified in the mammary glands. Based on their expression patterns, the identified miRNAs were grouped into 12 clusters. The expression pattern of cluster 1 miRNAs was opposite to that of milk protein genes in mammary glands in all five different stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the predicted target genes of cluster 1 miRNAs were related to murine mammary gland development and lactation. Furthermore, fluorescence in situ hybridization (FISH) analysis revealed that the novel-mmu-miR424-5p, which belongs to the cluster 1 miRNAs, was expressed in murine mammary epithelial cells. The dual-luciferase reporter assay revealed that an important milk protein gene—β-casein (CSN2)—was regarded as one of the likely targets for the novel-mmu-miR424-5p. This study analyzed the expression patterns of miRNAs in murine mammary glands throughout five critical developmental stages, and discovered a novel miRNA involved in regulating the expression of CSN2. These findings contribute to an enhanced understanding of the developmental biology of mammary glands, providing guidelines for increasing lactation efficiency and milk quality.

3 citations

Journal ArticleDOI
TL;DR: In this article , the authors summarize the current understanding of extra-nuclear, membrane-initiated functions of ERs with a specific focus on ERα and discuss the perspectives and future challenges opened by the integration of ERα signaling in physiology and pathology of estrogens.

3 citations

References
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Journal ArticleDOI
TL;DR: Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.
Abstract: The purpose of this study was to classify breast carcinomas based on variations in gene expression patterns derived from cDNA microarrays and to correlate tumor characteristics to clinical outcome. A total of 85 cDNA microarray experiments representing 78 cancers, three fibroadenomas, and four normal breast tissues were analyzed by hierarchical clustering. As reported previously, the cancers could be classified into a basal epithelial-like group, an ERBB2-overexpressing group and a normal breast-like group based on variations in gene expression. A novel finding was that the previously characterized luminal epithelial/estrogen receptor-positive group could be divided into at least two subgroups, each with a distinctive expression profile. These subtypes proved to be reasonably robust by clustering using two different gene sets: first, a set of 456 cDNA clones previously selected to reflect intrinsic properties of the tumors and, second, a gene set that highly correlated with patient outcome. Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.

10,791 citations

Journal ArticleDOI
15 Dec 1995-Cell
TL;DR: This research presents a new probabilistic procedure called ‘spot-spot analysis’ to characterize the response of the immune system to the presence of E.coli.

6,818 citations

Journal ArticleDOI
TL;DR: It is concluded that clone 29 cDNA encodes a novel rat ER, which is suggested be named rat ERbeta to distinguish it from the previously cloned ER (ERalpha) from rat uterus.
Abstract: We have cloned a novel member of the nuclear receptor superfamily. The cDNA of clone 29 was isolated from a rat prostate cDNA library and it encodes a protein of 485 amino acid residues with a calculated molecular weight of 54.2 kDa. Clone 29 protein is unique in that it is highly homologous to the rat estrogen receptor (ER) protein, particularly in the DNA-binding domain (95%) and in the C-terminal ligand-binding domain (55%). Expression of clone 29 in rat tissues was investigated by in situ hybridization and prominent expression was found in prostate and ovary. In the prostate clone 29 is expressed in the epithelial cells of the secretory alveoli, whereas in the ovary the granuloma cells in primary, secondary, and mature follicles showed expression of clone 29. Saturation ligand-binding analysis of in vitro synthesized clone 29 protein revealed a single binding component for 17beta-estradiol (E2) with high affinity (Kd= 0.6 nM). In ligand-competition experiments the binding affinity decreased in the order E2 > diethylstilbestrol > estriol > estrone > 5alpha-androstane-3beta,17beta-diol >> testosterone = progesterone = corticosterone = 5alpha-androstane-3alpha,17beta-diol. In cotransfection experiments of Chinese hamster ovary cells with a clone 29 expression vector and an estrogen-regulated reporter gene, maximal stimulation (about 3-fold) of reporter gene activity was found during incubation with 10 nM of E2. Neither progesterone, testosterone, dexamethasone, thyroid hormone, all-trans-retinoic acid, nor 5alpha-androstane-3alpha,I7beta-diol could stimulate reporter gene activity, whereas estrone and 5alpha-androstane-3beta,17beta-diol did. We conclude that clone 29 cDNA encodes a novel rat ER, which we suggest be named rat ERbeta to distinguish it from the previously cloned ER (ERalpha) from rat uterus.

4,782 citations

Journal ArticleDOI
14 Apr 2004-JAMA
TL;DR: The use of conjugated equine estrogen (CEE) increases the risk of stroke, decreases therisk of hip fracture, and does not affect CHD incidence in postmenopausal women with prior hysterectomy over an average of 6.8 years, indicating no overall benefit.
Abstract: Author(s): Anderson, Garnet L; Limacher, Marian; Assaf, Annlouise R; Bassford, Tamsen; Beresford, Shirley AA; Black, Henry; Bonds, Denise; Brunner, Robert; Brzyski, Robert; Caan, Bette; Chlebowski, Rowan; Curb, David; Gass, Margery; Hays, Jennifer; Heiss, Gerardo; Hendrix, Susan; Howard, Barbara V; Hsia, Judith; Hubbell, Allan; Jackson, Rebecca; Johnson, Karen C; Judd, Howard; Kotchen, Jane Morley; Kuller, Lewis; LaCroix, Andrea Z; Lane, Dorothy; Langer, Robert D; Lasser, Norman; Lewis, Cora E; Manson, JoAnn; Margolis, Karen; Ockene, Judith; O'Sullivan, Mary Jo; Phillips, Lawrence; Prentice, Ross L; Ritenbaugh, Cheryl; Robbins, John; Rossouw, Jacques E; Sarto, Gloria; Stefanick, Marcia L; Van Horn, Linda; Wactawski-Wende, Jean; Wallace, Robert; Wassertheil-Smoller, Sylvia; Women's Health Initiative Steering Committee | Abstract: Despite decades of use and considerable research, the role of estrogen alone in preventing chronic diseases in postmenopausal women remains uncertain.To assess the effects on major disease incidence rates of the most commonly used postmenopausal hormone therapy in the United States.A randomized, double-blind, placebo-controlled disease prevention trial (the estrogen-alone component of the Women's Health Initiative [WHI]) conducted in 40 US clinical centers beginning in 1993. Enrolled were 10 739 postmenopausal women, aged 50-79 years, with prior hysterectomy, including 23% of minority race/ethnicity.Women were randomly assigned to receive either 0.625 mg/d of conjugated equine estrogen (CEE) or placebo.The primary outcome was coronary heart disease (CHD) incidence (nonfatal myocardial infarction or CHD death). Invasive breast cancer incidence was the primary safety outcome. A global index of risks and benefits, including these primary outcomes plus stroke, pulmonary embolism (PE), colorectal cancer, hip fracture, and deaths from other causes, was used for summarizing overall effects.In February 2004, after reviewing data through November 30, 2003, the National Institutes of Health (NIH) decided to end the intervention phase of the trial early. Estimated hazard ratios (HRs) (95% confidence intervals [CIs]) for CEE vs placebo for the major clinical outcomes available through February 29, 2004 (average follow-up 6.8 years), were: CHD, 0.91 (0.75-1.12) with 376 cases; breast cancer, 0.77 (0.59-1.01) with 218 cases; stroke, 1.39 (1.10-1.77) with 276 cases; PE, 1.34 (0.87-2.06) with 85 cases; colorectal cancer, 1.08 (0.75-1.55) with 119 cases; and hip fracture, 0.61 (0.41-0.91) with 102 cases. Corresponding results for composite outcomes were: total cardiovascular disease, 1.12 (1.01-1.24); total cancer, 0.93 (0.81-1.07); total fractures, 0.70 (0.63-0.79); total mortality, 1.04 (0.88-1.22), and the global index, 1.01 (0.91-1.12). For the outcomes significantly affected by CEE, there was an absolute excess risk of 12 additional strokes per 10 000 person-years and an absolute risk reduction of 6 fewer hip fractures per 10 000 person-years. The estimated excess risk for all monitored events in the global index was a nonsignificant 2 events per 10 000 person-years.The use of CEE increases the risk of stroke, decreases the risk of hip fracture, and does not affect CHD incidence in postmenopausal women with prior hysterectomy over an average of 6.8 years. A possible reduction in breast cancer risk requires further investigation. The burden of incident disease events was equivalent in the CEE and placebo groups, indicating no overall benefit. Thus, CEE should not be recommended for chronic disease prevention in postmenopausal women.

4,298 citations

Journal ArticleDOI
TL;DR: An international Expert Panel that conducted a systematic review and evaluation of the literature and developed recommendations for optimal IHC ER/PgR testing performance recommended that ER and PgR status be determined on all invasive breast cancers and breast cancer recurrences.
Abstract: Purpose To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers. Methods The American Society of Clinical Oncology and the College of American Pathologists convened an international Expert Panel that conducted a systematic review and evaluation of the literature in partnership with Cancer Care Ontario and developed recommendations for optimal IHC ER/PgR testing performance. Results Up to 20% of current IHC determinations of ER and PgR testing worldwide may be inaccurate (false negative or false positive). Most of the issues with testing have occurred because of variation in preanalytic variables, thresholds for positivity, and interpretation criteria. Recommendations The Panel recommends that ER and PgR status be determined on all invasive breast cancers and breast cancer recurrences. A testing algorithm that relies on accurate, reproducible assay performance is proposed. Elements to reliably reduce assay variation are specified. It is recommended that ER and PgR assays be considered positive if there are at least 1% positive tumor nuclei in the sample on testing in the presence of expected reactivity of internal (normal epithelial elements) and external controls. The absence of benefit from endocrine therapy for women with ER-negative invasive breast cancers has been confirmed in large overviews of randomized clinical trials.

3,902 citations

Frequently Asked Questions (20)
Q1. What have the authors contributed in "Estrogen receptor-α signaling in post-natal mammary development and breast cancers" ?

In this review, the authors summarize the current understanding of the complex ERα signaling pathways that involve either classical nuclear “ genomic ” or membrane “ non-genomic ” actions and regulate in concert with other hormones the different stages of mammary development. The authors describe the cellular and molecular features of the luminal cell lineage expressing ERα and provide an overview of the transgenic mouse models impacting ERα signaling, highlighting the pivotal role of ERα in mammary gland morphogenesis and function and its implication in the tumorigenic processes. Finally, the authors describe the main features of the ERα-positive luminal breast cancers and their modeling in mice. 

An important direction for future research is to further define the niche of ERα + luminal cells and identify niche signals regulating the development and homeostasis of this lineage. 

classical and nonclassical progesterone signaling pathways through nuclear and membrane receptors have been identified in mammary epithelial and cancer cells [153]. 

In addition, non-genomic effects of ERα signaling that modulate intercellular communications participate in the regulation of mammary morphogenesis. 

In particular, estrogens induce the expression of progesterone receptor (PR) and prolactin receptor (PRLR) transcripts, highlighting the pivotal role of ERα signaling in the hormonal response of the developing mammary epithelium [136–138]. 

TET2 loss led to a decreased expression of ERα, FOXA1 and GATA3 expression both at protein and mRNA levels that profoundly perturbed the luminal lineage commitment and the balance between the basal and the luminal lineages and thereby altered mammary development. 

overexpression of IGF1R in epithelial cells in mice leads to abnormal development of the ducts (hyperplasia) and tumor formation in vivo [239]. 

LOXL1 inhibition through a pan LOX inhibitor was found to reduce tumor growth and metastasis by human lobular cell lines injected intraductally. 

Using a luminal cell-specific Rspo1-deficient transgenic mouse model, the authors found that loss of RSPO1 resulted in reduced mammary side branching in adult virgin females, with a decreased ERα expression and signaling activity in luminal cells. 

Western blot with antibodies directed against the C-terminal domain is the unique procedure to detect the ERα46 isoform since ERα46 and ERα66 share identical aminoacid sequences that cannot be distinguish by immunohistochemistry. 

Whether ERαhigh and ERαlow cells represent mature and progenitor cells or reflect a continuous gradient in ERα expression levels remains to be determined. 

GEMMs have contributed significantly to the field of breast cancer research and translational oncology, however, most of them develop ERα-negative mammary tumors [280]. 

RIP140 acts as a coregulator of ERα and is recruited to a number of its target gene promoters/ enhancers, such as Areg, Pgr, Ccnd1 and Stat5a. 

Estrogens acts in concert with other growth factorsNumerous data have demonstrated that estrogens act in concert with growth factors and the cooperation between estrogens and growth hormone (GH) in governing pubertal development has been particularly studied. 

the maintenance of early alveolar progenitors, potentially analogous to the so-called parity-identified mammary epithelial cells that express WAP and survive involution might be affected by ERα loss either directly or indirectly [202]. 

The data demonstrated that mutation of the palmitoylation site of ERα was necessary in promoting intercellular communications essential for mammary gland development. 

PR + mammary tumors while its expression in the whole luminal population gave rise to luminal ERα + mammary tumors and basal-like ERα- PRtumors. 

The chromatin complex formed by ESR1, GATA3, and FOXA1 thus coordinately orchestrates mammary luminal lineage commitment and estrogen response. 

ERα and Notch1 expression in post-natal luminal cells is mutually exclusive [144], suggesting a negative cross-talk between Notch and ERα signaling. 

This model confirmed that estrogen-induced activation of ERα is crucial for the development of female reproductive tract and mammary gland [211].