A Basic Review on Estrogen Receptor Signaling Pathways in Breast Cancer.

Breast cancer is the most common cancer and the deadliest among women worldwide. Estrogen signaling is closely associated with hormone-dependent breast cancer (estrogen and progesterone receptor positive), which accounts for two-thirds of tumors. Hormone therapy using antiestrogens is the gold standard, but resistance to these treatments invariably occurs through various biological mechanisms, such as changes in estrogen receptor activity, mutations in the ESR1 gene, aberrant activation of the PI3K pathway or cell cycle dysregulations. All these factors have led to the development of new therapies, such as selective estrogen receptor degraders (SERDs), or combination therapies with cyclin-dependent kinases (CDK) 4/6 or PI3K inhibitors. Therefore, understanding the estrogen pathway is essential for the treatment and new drug development of hormone-dependent cancers. This mini-review summarizes current literature on the signalization, mechanisms of action and clinical implications of estrogen receptors in breast cancer.

Codon adaptation by synonymous mutations impacts the functional properties of the estrogen receptor-alpha protein in breast cancer cells.

Oestrogen receptor-alpha (ERα) positivity is intimately associated with the development of hormone-dependent breast cancers. A major challenge in the treatment of these cancers is to understand and overcome the mechanisms of endocrine resistance. Recently, two distinct translation programmes using specific transfer RNA (tRNA) repertoires and codon usage frequencies were evidenced during cell proliferation and differentiation. Considering the phenotype switch of cancer cells to more proliferating and less-differentiated states, we can speculate that the changes in the tRNA pool and codon usage that likely occur make the ERα coding sequence no longer adapted, impacting translational rate, co-translational folding and the resulting functional properties of the protein. To verify this hypothesis, we generated an ERα synonymous coding sequence whose codon usage was optimized to the frequencies observed in genes expressed specifically in proliferating cells and then investigated the functional properties of the encoded receptor. We demonstrate that such a codon adaptation restores ERα activities to levels observed in differentiated cells, including: (a) an enhanced contribution exerted by transactivation function 1 (AF1) in ERα transcriptional activity; (b) enhanced interactions with nuclear receptor corepressor 1 and 2 [NCoR1 and NCoR2 (also known as SMRT) respectively], promoting repressive capability; and (c) reduced interactions with SRC proto-oncogene, non-receptor tyrosine kinase (Src) and phosphoinositide 3-kinase (PI3K) p85 kinases, inhibiting MAPK and AKT signalling pathway.

The different natural estrogens promote endothelial healing through distinct cell targets.

The main estrogen, 17ß-estradiol (E2), exerts several beneficial vascular actions through estrogen receptor α (ERα) in endothelial cells. However, the impact of other natural estrogens such as estriol (E3) and estetrol (E4) on arteries remains poorly described. In the present study, we report the effects of E3 and E4 on endothelial healing after carotid artery injuries in vivo. After endovascular injury, which preserves smooth muscle cells (SMCs), E2, E3, and E4 equally stimulated reendothelialization. By contrast, only E2 and E3 accelerated endothelial healing after perivascular injury that destroys both endothelial cells and SMCs, suggesting an important role of this latter cell type in E4's action, which was confirmed using Cre/lox mice inactivating ERα in SMCs. In addition, E4 mediated its effects independently of ERα membrane-initiated signaling, in contrast with E2. Consistently, RNA sequencing analysis revealed that transcriptomic and cellular signatures in response to E4 profoundly differed from those of E2. Thus, whereas acceleration of endothelial healing by estrogens had been viewed as entirely dependent on endothelial ERα, these results highlight the very specific pharmacological profile of the natural estrogen E4, revealing the importance of dialogue between SMCs and endothelial cells in its arterial protection.

Synergistic activation of genes promoting invasiveness by dual deprivation in oxygen and nutrients.

By depriving cancer cells of blood supplies of oxygen and nutrients, anti-angiogenic therapy is aimed at simultaneously asphyxiating and starving the cells. But in spite of its apparent logic, this strategy is generally counterproductive over the long term as the treatment seems to elicit malignancy. Since a defect of blood supply is expected to deprive tumours simultaneously of oxygen and nutrients naturally, we examine here these two deprivations, alone or in combination, on the phenotype and signalling pathways of moderately aggressive MCF7 cancer cells. Each deprivation induces some aspects of the aggressive and migratory phenotypes through activating several pathways, including HIF1-alpha as expected, but also SRF/MRTFA and TCF4/beta-catenin. Strikingly, the dual deprivation has strong cooperative effects on the upregulation of genes increasing the metastatic potential, such as four and a half LIM domains 2 (FHL2) and HIF1A-AS2 lncRNA, which have response elements for both pathways. Using anti-angiogenic agents as monotherapy is therefore questionable as it may give falsely promising short-term tumour regression, but could ultimately exacerbate aggressive phenotypes.

Hypoxia and ERα Transcriptional Crosstalk Is Associated with Endocrine Resistance in Breast Cancer.

Estrogen receptor-alpha (ERα) is the driving transcription factor in 70% of breast cancers and its activity is associated with hormone dependent tumor cell proliferation and survival. Given the recurrence of hormone resistant relapses, understanding the etiological factors fueling resistance is of major clinical interest. Hypoxia, a frequent feature of the solid tumor microenvironment, has been described to promote endocrine resistance by triggering ERα down-regulation in both in vitro and in vivo models. Yet, the consequences of hypoxia on ERα genomic activity remain largely elusive. In the present study, transcriptomic analysis shows that hypoxia regulates a fraction of ERα target genes, underlying an important regulatory overlap between hypoxic and estrogenic signaling. This gene expression reprogramming is associated with a massive reorganization of ERα cistrome, highlighted by a massive loss of ERα binding sites. Profiling of enhancer acetylation revealed a hormone independent enhancer activation at the vicinity of genes harboring hypoxia inducible factor (HIFα) binding sites, the major transcription factors governing hypoxic adaptation. This activation counterbalances the loss of ERα and sustains hormone-independent gene expression. We describe hypoxia in luminal ERα (+) breast cancer as a key factor interfering with endocrine therapies, associated with poor clinical prognosis in breast cancer patients.

Membrane estrogen receptor alpha (ERα) participates in flow-mediated dilation in a ligand-independent manner.

Estrogen receptor alpha (ERα) activation by estrogens prevents atheroma through its nuclear action, whereas plasma membrane-located ERα accelerates endothelial healing. The genetic deficiency of ERα was associated with a reduction in flow-mediated dilation (FMD) in one man. Here, we evaluated ex vivo the role of ERα on FMD of resistance arteries. FMD, but not agonist (acetylcholine, insulin)-mediated dilation, was reduced in male and female mice lacking ERα (Esr1-/- mice) compared to wild-type mice and was not dependent on the presence of estrogens. In C451A-ERα mice lacking membrane ERα, not in mice lacking AF2-dependent nuclear ERα actions, FMD was reduced, and restored by antioxidant treatments. Compared to wild-type mice, isolated perfused kidneys of C451A-ERα mice revealed a decreased flow-mediated nitrate production and an increased H2O2 production. Thus, endothelial membrane ERα promotes NO bioavailability through inhibition of oxidative stress and thereby participates in FMD in a ligand-independent manner.

Nuclear translocation of MRTFA in MCF7 breast cancer cells shifts ERα nuclear/genomic to extra-nuclear/non genomic actions.

The Myocardin-related transcription factor A [MRTFA, also known as Megakaryoblastic Leukemia 1 (MKL1))] is a major actor in the epithelial to mesenchymal transition (EMT). We have previously shown that activation and nuclear accumulation of MRTFA mediate endocrine resistance of estrogen receptor alpha (ERα) positive breast cancers by initiating a partial transition from luminal to basal-like phenotype and impairing ERα cistrome and transcriptome. In the present study, we deepen our understanding of the mechanism by monitoring functional changes in the receptor's activity. We demonstrate that MRTFA nuclear accumulation down-regulates the expression of the unliganded (Apo-)ERα and causes a redistribution of the protein localization from its normal nuclear place to the entire cell volume. This phenomenon is accompanied by a shift in Apo-ERα monomer/dimer ratio towards the monomeric state, leading to significant functional consequences on ERα activities. In particular, the association of Apo-ERα with chromatin is drastically decreased, and the remaining ERα binding sites are substantially less enriched in ERE motifs than in control conditions. Monitored by proximity Ligation Assay, ERα interactions with P160 family coactivators are partly impacted when MRTFA accumulates in the nucleus, and those with SMRT and NCOR1 corepressors are abolished. Finally, ERα interactions with kinases such as c-src and PI3K are increased, thereby enhancing MAP Kinase and AKT activities. In conclusion, the activation and nuclear accumulation of MRTFA in ERα positive breast cancer cells remodels both ERα location and functions by shifting its activity from nuclear genome regulation to extra-nuclear non-genomic signaling.

A Closer Look at Estrogen Receptor Mutations in Breast Cancer and Their Implications for Estrogen and Antiestrogen Responses.

Breast cancer (BC) is the most common cancer among women worldwide. More than 70% of BC cases express estrogen receptor alpha (ERα), a central transcription factor that stimulates the proliferation of breast cancer cells, usually in the presence of estrogen. While most cases of ER-positive BC initially respond to antiestrogen therapies, a high percentage of cases develop resistance to treatment over time. The recent discovery of mutated forms of ERα that result in constitutively active forms of the receptor in the metastatic-resistance stage of BC has provided a strong rationale for the development of new antiestrogens. These molecules targeting clinically relevant ERα mutants and a combination with other pharmacological inhibitors of specific pathways may constitute alternative treatments to improve clinical practice in the fight against metastatic-resistant ER-positive BC. In this review, we summarize the latest advances regarding the particular involvement of point mutations of ERα in endocrine resistance. We also discuss the involvement of synonymous ERα mutations with respect to co-translational folding of the receptor and ribosome biogenesis in breast carcinogenesis.

Apigenin, a Partial Antagonist of the Estrogen Receptor (ER), Inhibits ER-Positive Breast Cancer Cell Proliferation through Akt/FOXM1 Signaling.

Approximately 80% of breast cancer (BC) cases express the estrogen receptor (ER), and 30-40% of these cases acquire resistance to endocrine therapies over time. Hyperactivation of Akt is one of the mechanisms by which endocrine resistance is acquired. Apigenin (Api), a flavone found in several plant foods, has shown beneficial effects in cancer and chronic diseases. Here, we studied the therapeutic potential of Api in the treatment of ER-positive, endocrine therapy-resistant BC. To achieve this objective, we stably overexpressed the constitutively active form of the Akt protein in MCF-7 cells (named the MCF-7/Akt clone). The proliferation of MCF-7/Akt cells is partially independent of estradiol (E2) and exhibits an incomplete response to the anti-estrogen agent 4-hydroxytamoxifen, demonstrating the resistance of these cells to hormone therapy. Api exerts an antiproliferative effect on the MCF-7/Akt clone. Api inhibits the proliferative effect of E2 by inducing G2/M phase cell cycle arrest and apoptosis. Importantly, Api inhibits the Akt/FOXM1 signaling pathway by decreasing the expression of FOXM1, a key transcription factor involved in the cell cycle. Api also alters the expression of genes regulated by FOXM1, including cell cycle-related genes, particularly in the MCF-7/Akt clone. Together, our results strengthen the therapeutic potential of Api for the treatment of endocrine-resistant BC.

Tamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle Cells.

RATIONALE: Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an ER (estrogen receptor) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17ß-estradiol in other tissues such as arteries. However, the mechanisms of these tissue-specific actions remain unclear. OBJECTIVE: Here, we tested whether tamoxifen is able to accelerate endothelial healing and analyzed the underlying mechanisms. METHODS AND RESULTS: Using 3 complementary mouse models of carotid artery injury, we demonstrated that both tamoxifen and estradiol accelerated endothelial healing, but only tamoxifen required the presence of the underlying medial smooth muscle cells. Chronic treatment with 17ß-estradiol and tamoxifen elicited differential gene expression profiles in the carotid artery. The use of transgenic mouse models targeting either whole ERα in a cell-specific manner or ERα subfunctions (membrane/extranuclear versus genomic/transcriptional) demonstrated that 17ß-estradiol-induced acceleration of endothelial healing is mediated by membrane ERα in endothelial cells, while the effect of tamoxifen is mediated by the nuclear actions of ERα in smooth muscle cells. CONCLUSIONS: Whereas tamoxifen acts as an antiestrogen and ERα antagonist in breast cancer but also on the membrane ERα of endothelial cells, it accelerates endothelial healing through activation of nuclear ERα in smooth muscle cells, inviting to revisit the mechanisms of action of selective modulation of ERα.

Fine-tuning the metabolic rewiring and adaptation of translational machinery during an epithelial-mesenchymal transition in breast cancer cells.

BACKGROUND: During breast cancer progression, the epithelial to mesenchymal transition has been associated with metastasis and endocrine therapy resistance; however, the underlying mechanisms remain elusive. To gain insight into this process, we studied the transition undergone by MCF7-derived cells, which is driven by the constitutive nuclear expression of a MKL1 variant devoid of the actin-binding domain (MKL1 ΔN200). We characterized the adaptive changes that occur during the MKL1-induced cellular model and focused on regulation of translation machinery and metabolic adaptation. METHODS: We performed a genome-wide analysis at the transcriptional and translational level using ribosome profiling complemented with RNA-Seq and analyzed the expression of components of the translation machinery and enzymes involved in energy metabolism. NGS data were correlated with metabolomic measurements and quantification of specific mRNAs extracted from polysomes and western blots. RESULTS: Our results reveal the expression profiles of a luminal to basal-like state in accordance with an epithelial to mesenchymal transition. During the transition, the synthesis of ribosomal proteins and that of many translational factors was upregulated. This overexpression of the translational machinery appears to be regulated at the translational level. Our results indicate an increase of ribosome biogenesis and translation activity. We detected an extensive metabolic rewiring occurring in an already "Warburg-like" context, in which enzyme isoform switches and metabolic shunts indicate a crucial role of HIF-1α along with other master regulatory factors. Furthermore, we detected a decrease in the expression of enzymes involved in ribonucleotide synthesis from the pentose phosphate pathway. During this transition, cells increase in size, downregulate genes associated with proliferation, and strongly upregulate expression of cytoskeletal and extracellular matrix genes. CONCLUSIONS: Our study reveals multiple regulatory events associated with metabolic and translational machinery adaptation during an epithelial mesenchymal-like transition process. During this major cellular transition, cells achieve a new homeostatic state ensuring their survival. This work shows that ribosome profiling complemented with RNA-Seq is a powerful approach to unveil in-depth global adaptive cellular responses and the interconnection among regulatory circuits, which will be helpful for identification of new therapeutic targets.

Nuclear accumulation of MKL1 in luminal breast cancer cells impairs genomic activity of ERα and is associated with endocrine resistance.

Estrogen receptor (ERα) is central in driving the development of hormone-dependent breast cancers. A major challenge in treating these cancers is to understand and overcome endocrine resistance. The Megakaryoblastic Leukemia 1 (MKL1, MRTFA) protein is a master regulator of actin dynamic and cellular motile functions, whose nuclear translocation favors epithelial-mesenchymal transition. We previously demonstrated that nuclear accumulation of MKL1 in estrogen-responsive breast cancer cell lines promotes hormonal escape. In the present study, we confirm through tissue microarray analysis that nuclear immunostaining of MKL1 is associated with endocrine resistance in a cohort of breast cancers and we decipher the underlining mechanisms using cell line models. We show through gene expression microarray analysis that the nuclear accumulation of MKL1 induces dedifferentiation leading to a mixed luminal/basal phenotype and suppresses estrogen-mediated control of gene expression. Chromatin immunoprecipitation of DNA coupled to high-throughput sequencing (ChIP-Seq) shows a profound reprogramming in ERα cistrome associated with a massive loss of ERα binding sites (ERBSs) generally associated with lower ERα-binding levels. Novel ERBSs appear to be associated with EGF and RAS signaling pathways. Collectively, these results highlight a major role of MKL1 in the loss of ERα transcriptional activity observed in certain cases of endocrine resistances, thereby contributing to breast tumor cells malignancy.

The Basal Level of Gene Expression Associated with Chromatin Loosening Shapes Waddington Landscapes and Controls Cell Differentiation.

The baseline level of transcription, which is variable and difficult to quantify, seriously complicates the normalization of comparative transcriptomic data, but its biological importance remains unappreciated. We show that this currently neglected ingredient is essential for controlling gene network multistability and therefore cellular differentiation. Basal expression is correlated to the degree of chromatin loosening measured by DNA accessibility and systematically leads to cellular dedifferentiation as assessed by transcriptomic signatures, irrespective of the molecular and cellular tools used. Modeling gene network motifs formally involved in developmental bifurcations reveals that the epigenetic landscapes of Waddington are restructured by the level of nonspecific expression, such that the attractors of progenitor and differentiated cells can be mutually exclusive. This mechanism is universal and holds beyond the particular nature of the genes involved, provided the multistable circuits are correctly described with autonomous basal expression. These results explain the relationships long established between gene expression noise, chromatin decondensation and cellular dedifferentiation, and highlight how heterochromatin maintenance is essential for preventing pathological cellular reprogramming, age-related diseases, and cancer.

The tissue-specific effects of different 17ß-estradiol doses reveal the key sensitizing role of AF1 domain in ERα activity.

17ß-Estradiol (E2) action can be mediated by the full-length estrogen receptor alpha (ERα66), and also by the AF1 domain-deficient ERα (ERα46) isoform, but their respective sensitivity to E2 is essentially unknown. We first performed a dose response study using subcutaneous home-made pellets mimicking either metestrus, proestrus or a pharmacological doses of E2, which resulted in plasma concentrations around 3, 30 and 600 pM, respectively. Analysis of the uterus, vagina and bone after chronic exposure to E2 demonstrated dose-dependent effects, with a maximal response reached at the proestrus-dose in wild type mice expressing mainly ERα66. In contrast, in transgenic mice harbouring only an ERα deleted in AF1, these effects of E2 were either strongly shifted rightward (10-100-fold) and/or attenuated, depending on the tissue studied. Finally, experiments in different cell lines transfected with ERα66 or ERα46 also delineated varying profiles of ERα AF1 sensitivity to E2. Altogether, this work emphasizes the importance of dose in the tissue-specific actions of E2 and demonstrates the key sensitizing role of AF1 in ERα activity.

Membrane and Nuclear Estrogen Receptor Alpha Actions: From Tissue Specificity to Medical Implications.

Estrogen receptor alpha (ERα) has been recognized now for several decades as playing a key role in reproduction and exerting functions in numerous nonreproductive tissues. In this review, we attempt to summarize the in vitro studies that are the basis of our current understanding of the mechanisms of action of ERα as a nuclear receptor and the key roles played by its two activation functions (AFs) in its transcriptional activities. We then depict the consequences of the selective inactivation of these AFs in mouse models, focusing on the prominent roles played by ERα in the reproductive tract and in the vascular system. Evidence has accumulated over the two last decades that ERα is also associated with the plasma membrane and activates non-nuclear signaling from this site. These rapid/nongenomic/membrane-initiated steroid signals (MISS) have been characterized in a variety of cell lines, and in particular in endothelial cells. The development of selective pharmacological tools that specifically activate MISS and the generation of mice expressing an ERα protein impeded for membrane localization have begun to unravel the physiological role of MISS in vivo. Finally, we discuss novel perspectives for the design of tissue-selective ER modulators based on the integration of the physiological and pathophysiological roles of MISS actions of estrogens.

A model of dynamic stability of H3K9me3 heterochromatin to explain the resistance to reprogramming of differentiated cells.

Despite their dynamic nature, certain chromatin marks must be maintained over the long term. This is particulary true for histone 3 lysine 9 (H3K9) trimethylation, that is involved in the maintenance of healthy differentiated cellular states by preventing inappropriate gene expression, and has been recently identified as the most efficient barrier to cellular reprogramming in nuclear transfer experiments. We propose that the capacity of the enzymes SUV39H1/2 to rebind to a minor fraction of their products, either directly or via HP1α/ß, contributes to the solidity of this mark through (i) a positive feedback involved in its establishment by the mutual enforcement of H3K9me3 and SUV39H1/2 and then (ii) a negative feedback sufficient to strongly stabilize H3K9me3 heterochromatin in post-mitotic cells by generating local enzyme concentrations capable of counteracting transient bursts of demethylation. This model does not require direct molecular interactions with adjacent nucleosomes and is favoured by a series of additional mechanisms including (i) the protection of chromatin-bound SUV39H1/2 from the turnovers of soluble proteins, which can explain the uncoupling between the cellular contents in SUV39H1 mRNA and protein; (ii) the cooperative dependence on the local density of the H3K9me3 of HP1α/ß-dependent heterochomatin condensation and, dispensably (iii) restricted enzyme exchanges with chromocenters confining the reactive bursts of SUV39H1/2 in heterochromatin. This mechanism illustrates how seemingly static epigenetic states can be firmly maintained by dynamic and reversible modifications.

The AF-1-deficient estrogen receptor ERα46 isoform is frequently expressed in human breast tumors.

BACKGROUND: To date, all studies conducted on breast cancer diagnosis have focused on the expression of the full-length 66-kDa estrogen receptor alpha (ERα66). However, much less attention has been paid to a shorter 46-kDa isoform (ERα46), devoid of the N-terminal region containing the transactivation function AF-1. Here, we investigated the expression levels of ERα46 in breast tumors in relation to tumor grade and size, and examined the mechanism of its generation and its specificities of coregulatory binding and its functional activities. METHODS: Using approaches combining immunohistochemistry, Western blotting, and proteomics, antibodies allowing ERα46 detection were identified and the expression levels of ERα46 were quantified in 116 ERα-positive human breast tumors. ERα46 expression upon cellular stress was studied, and coregulator bindings, transcriptional, and proliferative response were determined to both ERα isoforms. RESULTS: ERα46 was expressed in over 70% of breast tumors at variable levels which sometimes were more abundant than ERα66, especially in differentiated, lower-grade, and smaller-sized tumors. We also found that ERα46 can be generated via internal ribosome entry site-mediated translation in the context of endoplasmic reticulum stress. The binding affinities of both unliganded and fully-activated receptors towards co-regulator peptides revealed that the respective potencies of ERα46 and ERα66 differ significantly, contributing to the differential transcriptional activity of target genes to 17ß estradiol (E2). Finally, increasing amounts of ERα46 decrease the proliferation rate of MCF7 tumor cells in response to E2. CONCLUSIONS: We found that, besides the full-length ERα66, the overlooked ERα46 isoform is also expressed in a majority of breast tumors. This finding highlights the importance of the choice of antibodies used for the diagnosis of breast cancer, which are able or not to detect the ERα46 isoform. In addition, since the function of both ERα isoforms differs, this work underlines the need to develop new technologies in order to discriminate ERα66 and ERα46 expression in breast cancer diagnosis which could have potential clinical relevance.

Changes in Gene Expression and Estrogen Receptor Cistrome in Mouse Liver Upon Acute E2 Treatment.

Transcriptional regulation by the estrogen receptor-α (ER) has been investigated mainly in breast cancer cell lines, but estrogens such as 17ß-estradiol (E2) exert numerous extrareproductive effects, particularly in the liver, where E2 exhibits both protective metabolic and deleterious thrombotic actions. To analyze the direct and early transcriptional effects of estrogens in the liver, we determined the E2-sensitive transcriptome and ER cistrome in mice after acute administration of E2 or placebo. These analyses revealed the early induction of genes involved in lipid metabolism, which fits with the crucial role of ER in the prevention of liver steatosis. Characterization of the chromatin state of ER binding sites (BSs) in mice expressing or not ER demonstrated that ER is not required per se for the establishment and/or maintenance of chromatin modifications at the majority of its BSs. This is presumably a consequence of a strong overlap between ER and hepatocyte nuclear factor 4α BSs. In contrast, 40% of the BSs of the pioneer factor forkhead box protein a (Foxa2) were dependent upon ER expression, and ER expression also affected the distribution of nucleosomes harboring dimethylated lysine 4 of Histone H3 around Foxa2 BSs. We finally show that, in addition to a network of liver-specific transcription factors including CCAAT/enhancer-binding protein and hepatocyte nuclear factor 4α, ER might be required for proper Foxa2 function in this tissue.

Envisioning metastasis as a transdifferentiation phenomenon clarifies discordant results on cancer.

Cancer is generally conceived as a dedifferentiation process in which quiescent post-mitotic differentiated cells acquire stem-like properties and the capacity to proliferate. This view holds for the initial stages of carcinogenesis but is more questionable for advanced stages when the cells can transdifferentiate into the contractile phenotype associated to migration and metastasis. Singularly from this perspective, the hallmark of the most aggressive cancers would correspond to a genuine differentiation status, even if it is different from the original one. This seeming paradox could help reconciling discrepancies in the literature about the pro- or anti-tumoral functions of candidate molecules involved in cancer and whose actual effects depend on the tumoral grade. These ambiguities which are likely to concern a myriad of molecules and pathways, are illustrated here with the selected examples of chromatin epigenetics and myocardin-related transcription factors, using the human MCF10A and MCF7 breast cancer cells. Self-renewing stem like cells are characterized by a loose chromatin with low levels of the H3K9 trimetylation, but high levels of this mark can also appear in cancer cells acquiring a contractile-type differentiation state associated to metastasis. Similarly, the myocardin-related transcription factor MRTF-A is involved in metastasis and epithelial-mesenchymal transition, whereas this factor is naturally enriched in the quiescent cells which are precisely the most resistant to cancer: cardiomyocytes. These seeming paradoxes reflect the bistable epigenetic landscape of cancer in which dedifferentiated self-renewing and differentiated migrating states are incompatible at the single cell level, though coexisting at the population level.

The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites.

Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene.