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

TLDR: 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.

Figures

Fig. 3 Hormonal context of the major stages of the post-natal mouse mammary development. From left to right: images of carmine-stained whole mounts from 6-week-old pubertal, 12-week-old adult virgin, 16-day-pregnant and 2-day lactating mice. The pubescent gland is characterized by the presence of terminal end buds (TEBs) at the tips of the growing ducts. The steroid hormones, estrogens and progesterone, are in red whereas the peptide hormones are in black. GH growth hormone. Bar: 0.25 mm

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 indicated using solid lines

Table 1 Major surface markers used to separate mouse mammary basal and luminal cells and enrich ERα-positive and ERα-negative luminal cell populations by flow cytometry after exclusion of endothelial and hemopoietic cells by CD31 and CD45 surface staining

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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 [1–6]. 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 [9–12]. In addition, ERα is routinely
used as a diagnosis marker supporting the molecular clas-
sification of breast cancers [13–15] 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 [16–18].
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) [38–41].
However, AF-1 can also be modified in response to E2 and
further stabilized following phosphorylation on serine 118
[42–44]. 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 [47–49].
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 [62–64].
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 [65–68]. 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 [73–75].
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 [81–83].
Studies using CHIP-Chip and CHIP-seq on MCF7 breast
cancer cells have revealed that ERα binds to 5000–10,000
locations [84–86]. 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
Gα
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)

Frequently asked questions

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. 

Q2. What have the authors stated for future works in "Estrogen receptor-α signaling in post-natal mammary development and breast cancers" ?

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. 

Q3. What are the main pathways of ER signaling in mammary cells?

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

Q4. What is the role of ER in mammary morphogenesis?

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

Q5. What is the role of estrogens in the development of the mammary gland?

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]. 

Q6. What was the effect of TET2 loss on mammary development?

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. 

Q7. What is the effect of IGF1R on tumor formation in vivo?

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

Q8. What is the effect of LOXL1 inhibition on tumor growth?

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

Q9. What is the effect of RSPO1 on mammary side branching?

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. 

Q10. What is the way to detect ER46?

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. 

Q11. What is the status of ER in luminal cells?

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

Q12. What are the main advantages of GEMMs?

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

Q13. What is the role of RIP140 in the development of the mammary gland?

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. 

Q14. What is the role of estrogen in the development of the pubertal duct?

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. 

Q15. What is the significance of ER loss in mammary cells?

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]. 

Q16. What is the role of ER in mammary gland development?

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

Q17. What is the effect of PI3KCA on luminal ER + mamm?

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

Q18. What is the role of FOXM1 in mammary luminal cell development?

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

Q19. What is the role of RSPO1 in the regulation of ER and Notch?

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

Q20. What is the role of ER in the development of the mammary gland?

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