Estrogen receptor-α signaling in post-natal mammary development and breast cancers
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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Frequently Asked Questions (20)
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].