Natural Anti-Estrogen Receptor Alpha Antibodies Able to Induce Estrogenic Responses in Breast Cancer Cells: Hypotheses Concerning Their Mechanisms of Action and Emergence.

The detection of human anti-estrogen receptor α antibodies (ERαABs) inducing estrogenic responses in MCF-7 mammary tumor cells suggests their implication in breast cancer emergence and/or evolution. A recent report revealing a correlation between the titer of such antibodies in sera from patients suffering from this disease and the percentage of proliferative cells in samples taken from their tumors supports this concept. Complementary evidence of the ability of ERαABs to interact with an epitope localized within the estradiol-binding core of ERα also argues in its favor. This epitope is indeed inserted in a regulatory platform implicated in ERα-initiated signal transduction pathways and transcriptions. According to some experimental observations, two auto-immune reactions may already be advocated to explain the emergence of ERαABs: one involving probably the idiotypic network to produce antibodies acting as estrogenic secretions and the other based on antibodies able to abrogate the action of a natural ERα inhibitor or to prevent the competitive inhibitory potency of released receptor degradation products able to entrap circulating estrogens and co-activators. All of this information, the aspect of which is mainly fundamental, may open new ways in the current tendency to combine immunological and endocrine approaches for the management of breast cancer.

ERE-dependent transcription and cell proliferation: Independency of these two processes mediated by the introduction of a sulfone function into the weak estrogen estrothiazine.

The synthetic coumestrol derivative 6,12-dihydro-3-methoxy-1-benzopyrano[3,4-b][1,4]benzothiazin-6-one (estrothiazine, ESTZ) has been identified as a weak estrogen receptor α (ERα) ligand unable to compete with tritiated estradiol. The biological activity of this compound, supported by a methoxy group in position 3, seems mainly to result from its capacity to activate ERα dimerization without any participation of coactivators. In support of this view and referring to conventional estrogens, an ESTZ metabolism study conducted with hepatic human microsomes failed to provide any argument in favour of an estrogenic activity dependent on a metabolic conversion of the compound into hydroxylated metabolites with strong receptor activation ability. Interestingly, we failed to detect any oxidation of the sulfur atom of the compound. In the light of pharmacological literature data concerning sulfonylation, we assessed ERα-mediated activities generated by two sulfonylated ESTZ derivatives in which the methoxy group that plays a key role in its mechanism of action was maintained or removed. Sulfonylated ESTZ, even in its demethoxylated form, induced ERE-mediated transcriptions in MCF-7 breast cancer cells, without affecting the ERα turnover rate. In contrast to ESTZ, this compound failed to enhance the proliferation of ERα-positive breast cancer cells, suggesting that its sulfone function confers upon the receptor a capacity to elicit some of the known characteristics associated with estrogenic responses. Moreover, we demonstrated that this sulfone may contribute to ERα dimerization without any requirement of the methoxy group. Nevertheless, it seems to cooperate with this group, as reflected by a weak ability of the sulfonylated form of ESTZ to compete with tritiated estradiol for ERα-binding. Assessment of the docking of this compound within the ligand-binding domain of the receptor by molecular dynamics provided an explanation for this observation since the sulfone is engulfed in a small hydrophobic pocket involving the residues Leu-346, Leu-349, Ala-350 and Leu-384, also known to recruit coactivators. This work not only reports the sulfone functional group as a pharmacophore for estrogenic activity, but also opens new perspectives for the development of estrogenic molecules with therapeutic purpose and devoid of proliferative side effects.

ERα dimerization: a key factor for the weak estrogenic activity of an ERα modulator unable to compete with estradiol in binding assays.

Estrothiazine (ESTZ) is a weak estrogen sharing structural similarities with coumestrol. ESTZ failed to compete with [3H]17ß-estradiol ([3H]17ß-E2) for binding to the estrogen receptor α (ERα), questioning its ability to interact with the receptor. However, detection by atomic force spectroscopy (AFS) of an ESTZ-induced ERα dimerization has eliminated any remaining doubts. The effect of the compound on the proliferation of ERα-positive and negative breast cancer cells confirmed the requirement of the receptor. The efficiency of ESTZ in MCF-7 cells was weak without any potency to modify the proliferation profile of estradiol and coumestrol. Growth enhancement was associated with a proteasomal degradation of ERα without substantial recruitment of LxxLL coactivators. This may be related to an unusual delay between the acquisition by the receptor of an ERE-binding capacity and the subsequent estrogen-dependent transcription. A complementary ability to enhance TPA-induced AP-1 transcription was observed, even at concentrations insufficient to activate the ERα, suggesting a partly independent mechanism. ESTZ also rapidly and transiently activated ERK1/2 likely through membrane estrogenic pathways provoking a reorganization of the actin network. Finally, the systematic absence of biological responses with an ESTZ derivative unable to induce ERα dimerization stresses the importance of this step in the action of the compound, as reported for conventional estrogens. In view of the existence of many other ERα modulators (endocrine disruptors such as, for example, pesticides, environmental contaminants or phytoestrogens) with extremely weak or similar apparent lack of binding ability, our work may appear as pilot investigation for assessing their mechanism of action.

The sequence Pro295-Thr311 of the hinge region of oestrogen receptor α is involved in ERK1/2 activation via GPR30 in leiomyoma cells.

The ERα (oestrogen receptor α)-derived peptide ERα17p activates rapid signalling events in breast carcinoma cells under steroid-deprived conditions. In the present study, we investigated its effects in ELT3 leiomyoma cells under similar conditions. We show that it activates ERK1/2 (extracellular-signal-regulated kinase 1/2), the Gαi protein, the trans-activation of EGFR (epidermal growth factor receptor) and, finally, cell proliferation. It is partially internalized in cells and induces membrane translocation of ß-arrestins. The activation of ERK1/2 is abolished by the GPR30 (G-protein-coupled receptor 30) antagonist G15 and GPR30 siRNA. When ERα is down-regulated by prolonged treatment with E2 (oestradiol) or specific ERα siRNA, the peptide response is blunted. Thus the simultaneous presence of GPR30 and ERα is required for the action of ERα17p. In addition, its PLM sequence, which interferes with the formation of the ERα-calmodulin complex, appears to be requisite for the phosphorylation of ERK1/2 and cell proliferation. Hence ERα17p is, to our knowledge, the first known peptide targeting ERα-GPR30 membrane cross-talk and the subsequent receptor-mediated biological effects.

Interactions of isoflavones and other plant derived estrogens with estrogen receptors for prevention and treatment of breast cancer-considerations concerning related efficacy and safety.

Phytoestrogens are natural endocrine disruptors that interfere with estrogenic pathways. They insert directly within the hormone-binding domain of ERα and ß, with a preference for the ß isoform of which the concentration predominates in the normal mammary epithelium. Since ERß antagonizes the growth promoting effect of ERα, which is mainly expressed in estrogen-sensitive tumor cells, a potential protective action against breast cancer incidence has been ascribed to phytoestrogens. The fact that Asian women living in far-east countries who consume isoflavone-rich food are less subjected to breast cancer emergence than their congeners in the USA as well as Caucasian women has been advocated to justify such a concept. Overview of data concerning the mechanism of action phytoestrogens reveals that such a view is an oversimplification: Such compounds interfere with a huge panel of regulatory proteins, giving rise to both promoting and antagonizing carcinogenic effects. Moreover, various physiological and pathological factors able to amplify these effects are not often sufficiently taken into account, which increases the difficulty to interpret data. Nevertheless, this overview of data established that chemical structures and concentrations modulate such effects: at the micromolar level, isoflavones activate ERα-mediated transcription and breast cancer cell proliferation while flavones fail to induce any significant promoting effects. At higher doses, both classes of compounds may display an antitumor activity. Reasons for such distinct behaviors as well as their potential impact in therapeutic applications are analyzed here. Ability of isoflavones and flavones to antagonize the association of calmodulin to ERα, which is required for its enhanced transcriptional activity is evoked to justify the antitumor activity ascribed to some flavones. Finally, a suspicion that peculiar classes of phytoestrogens may adopt a SERM-like conformation is addressed in a context of selection and synthesis of compounds with non-equivocal therapeutic value. This article is part of a Special Issue entitled "Phytoestrogens".

Whole transcriptome analysis of the ERα synthetic fragment P295-T311 (ERα17p) identifies specific ERα-isoform (ERα, ERα36)-dependent and -independent actions in breast cancer cells.

ERα17p is a peptide corresponding to the sequence P295LMIKRSKKNSLALSLT311 of the estrogen receptor alpha (ERα) and initially found to interfere with ERα-related calmodulin binding. ERα17p was subsequently found to elicit estrogenic responses in E2-deprived ERα-positive breast cancer cells, increasing proliferation and ERE-dependent gene transcription. Surprisingly, in E2-supplemented media, ERα17p-induced apoptosis and modified the actin network, influencing cell motility. Here, we report that ERα17p internalizes in breast cancer cells (T47D, MDA-MB-231, SKBR3) and induces a massive early (3 h) transcriptional activity. Remarkably, about 75% of significantly modified transcripts were also modified by E2, confirming the pro-estrogenic profile of ERα17p. The different ER spectra of the used cell lines allowed us to identify a specific ERα17p signature related to ERα as well as its variant ERα36. With respect to ERα, the peptide activates nuclear (cell cycle, cell proliferation, nucleic acid and protein synthesis) and extranuclear signaling pathways. In contrast, through ERα36, it mainly triggers inhibitory actions on inflammation. This is the first work reporting a detailed ERα36-specific transcriptional signature. In addition, we report that ERα17p-induced transcripts related to apoptosis and actin modifying effects of the peptide are independent from its estrogen receptor(s)-related actions. We discuss our findings in view of the potential use of ERα17p as a selective peptidomimetic estrogen receptor modulator (PERM).

Electronic effects of 11ß substituted 17ß-estradiol derivatives and instrumental effects on the relative gas phase acidity.

Numerous studies have highlighted the role of the proton donor characteristics of the phenol group of 17ß-estradiol (E(2)) in its association with the estrogen receptor alpha (ERα). Since the substitutions at position C((11)) have been reported to modulate this association, we hypothesized that such substitutions may modify the phenol acidity. Hence, phenol gas-phase acidity of nine C((11))-substituted E(2)-derivatives were evaluated using the extended Cooks' kinetic method, which is a method widely used to determine thermochemical properties by mass spectrometry. To enhance accuracy in data collection we recorded data from several instruments, including quadrupole ion trap, triple quadrupole, and hybrid QqTOF. Indeed, we report for the first time the use of the QqTOF instrument to provide a novel means to improve data accuracy by giving access to an intermediate effective temperature range. All experimental gas-phase acidity values were supported by theoretical calculations. Our results confirmed the ability of distant substituents at C((11)) to modulate the phenol acidity through electrostatic interactions, electron withdrawing inductive effects, and mesomeric effects. However, no relationship was found between the phenol gas-phase acidity of investigated steroids and their binding affinity for ERα assessed in solution. Thus, our results highlight that the intrinsic properties of the hormone do not influence sufficiently the stabilization of the hormone/ERα complex. It is more likely that such stabilization would be more related to factors depending on the environment within the binding pocket such as hydrophobic, steric as well as direct intermolecular electrostatic effects between ERα residues and the substituted steroidal estrogens.

ERα17p, a peptide reproducing the hinge region of the estrogen receptor α associates to biological membranes: A biophysical approach.

Recently, we identified a peptide (ERα17p, P(295)LMIKRSKKNSLALSLT(311)) that corresponds to the 295-311 sequence of the estrogen receptor α (ERα, hinge region) and which exerts a panel of pharmacological effects in breast cancer cells. Remarkably, these effects can result from the interaction of ERα17p with the plasma membrane. Herein, we show that ERα17p adopts a ß-sheet secondary structure when in contact with anionic phospholipids and that it is engulfed within the lipid bilayer. While ERα17p increases the fluidity of membrane mimics, it weakly internalizes in living cells. In light of the above, one may evoke one important role of the 295-311 region of the ERα: the corresponding peptide could be secreted/delivered to the extracellular medium to interact with neighboring cells, both intracellularly and at the membrane level. Finally, the 295-311 region of ERα being in proximity to the cystein-447, the palmitoylation site of the ERα raises the question of its involvement in the interaction/stabilization of the protein with the membrane.

Palmitoylation regulates 17ß-estradiol-induced estrogen receptor-α degradation and transcriptional activity.

The estrogen receptor-α (ERα) is a transcription factor that regulates gene expression through the binding to its cognate hormone 17ß-estradiol (E2). ERα transcriptional activity is regulated by E2-evoked 26S proteasome-mediated ERα degradation and ERα serine (S) residue 118 phosphorylation. Furthermore, ERα mediates fast cell responses to E2 through the activation of signaling cascades such as the MAPK/ERK and phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog 1 pathways. These E2 rapid effects require a population of the ERα located at the cell plasma membrane through palmitoylation, a dynamic enzymatic modification mediated by palmitoyl-acyl-transferases. However, whether membrane-initiated and transcriptional ERα activities integrate in a unique picture or represent parallel pathways still remains to be firmly clarified. Hence, we evaluated here the impact of ERα palmitoylation on E2-induced ERα degradation and S118 phosphorylation. The lack of palmitoylation renders ERα more susceptible to E2-dependent degradation, blocks ERα S118 phosphorylation and prevents E2-induced ERα estrogen-responsive element-containing promoter occupancy. Consequently, ERα transcriptional activity is prevented and the receptor addressed to the nuclear matrix subnuclear compartment. These data uncover a circuitry in which receptor palmitoylation links E2-dependent ERα degradation, S118 phosphorylation, and transcriptional activity in a unique molecular mechanism. We propose that rapid E2-dependent signaling could be considered as a prerequisite for ERα transcriptional activity and suggest an integrated model of ERα intracellular signaling where E2-dependent early extranuclear effects control late receptor-dependent nuclear actions.

Calcium-induced activation of estrogen receptor alpha--New insight.

Calcium being an important modulator in multiple regulatory processes, we overviewed reported investigations concerning its potential influence on ERα transcriptional activity in breast cancer cells. Three main activating mechanisms depending on either intra- or extracellular calcium are described. At physiological intracellular concentration (µM), Ca(++) activates calmodulin and promotes its association with ERα; the resulting complex stably interacts with EREs at promoter sites, giving rise to enhanced transcription of estrogen target genes. Hypercalcemic concentrations (mM) produce a similar response through a direct association of the ion with the ligand binding domain of the receptor, this binding of calcium conferring an active conformation to ER. In contrast to these intracellular processes, very high extracellular concentrations of Ca(++) (>10mM) detected in bones at time of tumor metastasis operate via a signal transduction pathway initiated at the cell membrane through a specific activation of the calcium-sensing receptor.

Biophysical studies of the interaction between calmodulin and the R²87-T³¹¹ region of human estrogen receptor α reveals an atypical binding process.

The transcriptional activity of human estrogen receptor ERα is modulated by a number of coregulatory proteins among which calmodulin (CaM). Segment 295-311 in the hinge region of ERα has previously been proposed to be the CaM binding site. In this work, we investigate the molecular mechanism of the interaction of CaM with peptides derived from the hinge region of ERα, using a biophysical approach combining isothermal titration calorimetry, fluorescence, CD and NMR. The ERα17p peptide, corresponding to the previously identified 295-311 region of ERα, recruits mainly the C-terminal domain of Ca(4)CaM, as shown by NMR spectroscopy. In contrast, a longer peptide, ERα25p, extended on the N-terminal side (residues 287-311) interacts with both N- and C-terminal domains of Ca(4)CaM. These results lead to a new delineation of the CaM binding site, encompassing residues 287-294. In particular, fluorescence spectroscopy reveals that the conserved W(292) residue is engaged within hydrophobic pockets on Ca(4)CaM. ITC results show that ERα25p binds Ca(4)CaM with an atypical 2:1 stoichiometry and a dissociation constant in the micromolar range. Based on the NMR titration of Ca(4)CaM by ERα25p showing a biphasic behavior for several residues, we suggest that concerted conformational changes of CaM domains may be required to accommodate the binding of a second peptide. CD spectra indicate that ERα25p partially folds into an α-helix upon binding to Ca(4)CaM. Hence, ERα25p is a new CaM-binding ligand that could be appropriate for the synthesis of derivatives able to control ER-dependent transcription, particularly in the context of hormone-dependent breast tumors.

ERα36, a new variant of the ERα is expressed in triple negative breast carcinomas and has a specific transcriptomic signature in breast cancer cell lines.

Triple negative breast cancer is deprived of estrogen receptor alpha (ERα), progesterone receptor (PR) and HER-2 protein. It constitutes the most heterogeneous and aggressive group of breast carcinomas, for which identification of novel characteristics and characterization of putative targets becomes very demanding. In the present work we have assayed the expression of ERα36, a recently identified ERα variant of 36kDa, in a series of triple negative breast cancers, in relation to the clinical behavior and other clinico-pathological features of the tumors. While widely expressed within the cytoplasm in almost all tumors, we found that exclusively the membrane/submembrane expression of the receptor exhibits a correlation with patient's survival. Moreover, membrane ERα36 correlates in an inverse manner with the expression of miRNA210, a pro-angiogenic miR, with high prognostic relevance in triple negative carcinomas. A thorough transcriptomic, pharmacological-based approach in breast cancer cell lines, revealed an early (direct) transcriptional signature of the receptor activation, related to immune system processes and T-cell differentiation, RNA biosynthesis, regulation of metabolism, VEGF signaling and regulation of the cell cycle, with a down-regulation of CREB, NFκB and STATs transcription factors. Finally, ERα36 expression is not limited within breast cancer epithelial linen, but is equally identified in tumor vasculature, peritumoral fat tissue, lymphocytic infiltrate and stromal fibroblasts. In light of the above, ERα36 could represent a major counterpart in triple negative breast cancer.

Recent insights into the effect of natural and environmental estrogens on mammary development and carcinogenesis.

The present work reviews recent findings related to the action of steroidal (physiological) estrogens on normal mammary gland development and carcinogenesis, as well as effects of related environmental mediators (phyto- and xeno-estrogens), the role of which remains controversial. Orchestration by estrogen receptors (i.e. ERα and ERß) and coregulators of growth, apoptosis and differentiation of epithelial cells, directed our analysis. The bidirectional coordination between epithelium and stroma in parallel with maintenance of stemness are also investigated. The relevance of nuclear and extranuclear localization of ERs and other eventual estrogen binding sites, mediating differential actions in regard to these various topics, is critically addressed to delineate the importance of direct and indirect activation procedures and delicate feedback loops (ligand-induced or/and cross-talk activation, respectively). The inclusion of the outlined regulatory concepts in drug design programs for the prevention and treatment of breast cancer may have potent effects.

ERα17p, an ERα P295 -T311 fragment, modifies the migration of breast cancer cells, through actin cytoskeleton rearrangements.

Recently, our knowledge on estrogen receptor alpha (ERα) functions and fate has progressed: ERα enters in repeated transcription-modulating cycles (nucleus/cytoplasm/membrane trafficking processes and proteasomal degradation) that are governed by specific protein-protein interactions. Receptor fragments, especially those resulting from the proteolysis of its ligand binding domain, as well as corresponding synthetic peptides, have been studied with respect to their estrogenic/antiestrogenic potency. A peptide, corresponding to the human ERα P(295) -T(311) sequence (ERα17p) has been shown to alter breast cancer cell fate, triggering proliferation, or apoptosis. The aim of this work was to explore the effect of ERα17p on breast cancer cell migration and actin cytoskeleton dynamics and further analyze the mechanism of its membrane action. We show that ERα17p increases (MCF-7 and SK-BR-3 cells) or decreases (T47D and MDA-MB-231 cells) migration of breast cancer cells, in an ERα-independent manner, by mechanism(s) depending on Rho/ROCK and PI3K/Akt signaling pathways. Moreover, the peptide enhances the association of both estrogens and androgens to membranes and modifies cell migration, induced by E(2) -BSA. Additionally, initial evidence of a possible agonistic action of the peptide on GPR30 is also provided. ERα17p can be considered as a cell migration-modulator and could therefore constitute a therapeutic challenge, even in anti-estrogen-resistant tumors.

A critical view of the effects of phytoestrogens on hot flashes and breast cancer risk.

The increased risk of breast cancer recently observed with some specific estro-progestin associations has raised concerns about the harmful effects of menopausal hormone replacement therapy (HRT). It has been proposed that phytoestrogens (PEs), which have a similar chemical structure to estrogens, could be used as HRT. The main selling points of these preparations concern the management of hot flashes and their potential beneficial effects on breast tissue. In this review, we will address the effects of PE on hot flashes and breast cancer risk as well as the questions raised on a chemical point of view. We conclude that the efficacy of a PE rich diet or nutritional supplements is not clearly established. The use of PE as an alternative for HRT cannot be advocated for now, due to insufficient and conflicting data on efficacy and safety. Moreover, due to the hormone dependence of breast cancer, PE use must be contraindicated in breast cancer survivors.

Peptides targeting estrogen receptor alpha-potential applications for breast cancer treatment.

Activation of the estrogen receptor alpha (ERα) is of prime importance for the development of hormone-dependent breast cancers. Hence, drugs able to impede the emergence of an active folding of ERα have been used for a long time as a first line therapeutic strategy. Aromatase inhibitors that block estradiol synthesis and / or antiestrogens that compete with hormone binding to the receptor are routinely prescribed. Unfortunately, emergence of tumor resistance almost invariably results from currently used antihormonal approaches. One may anticipate that a "multi-target" strategy affecting key regulatory domains distinct from ligand binding pocket of ERα may help to circumvent this problem. To reach this goal, the synthesis of peptides that may specifically inhibit intra- or inter-molecular interactions has been proposed. This paper describes functional motifs potentially suitable for the design of such antagonists. Activity of available peptidic and non-peptidic mimics of these motifs is also reviewed.

The estrogen receptor alpha-derived peptide ERα17p (P(295)-T(311)) exerts pro-apoptotic actions in breast cancer cells in vitro and in vivo, independently from their ERα status.

In recent years, our knowledge on estrogen receptors (ER) has been modified profoundly with the identification and the deciphering of the role of its protein effectors, as well as with the deeper insight of its molecular structure/function dynamics, characteristics associated with its nucleo-cytoplasmic-membrane shuttling properties. Also, significant progress has been made concerning its turn-over and associated final proteasomal degradation processes. These advances could lead in the near future to the design and the synthesis of novel receptor-interacting drugs. Recently, a number of receptor-related peptides acting as specific ER ligands have been identified and extensively studied with respect to their estrogenic/antiestrogenic activities. Among them, ERα17p, a synthetic analog of the P(295)-T(311) sequence of ERα, has been shown to exert pseudo-estrogenic effects by interacting in the close vicinity of its hinge region (BF3 domain). Remarkably, this sequence appears as the epicenter of a number of post-transcriptional modifications as well as of the recruitment of co-regulators, suggesting that it would play a key role in ERα functions. Here, we provide evidence that ERα17p induces apoptosis in ERα-positive (MCF-7, T47D) and -negative (MDA-MB-231, SK-BR-3) breast cancer cells by an ERα-independent membrane mechanism, triggering major pro-apoptotic signaling cascades. Finally, ERα17p induces the regression of breast ERα-negative cancer tumor xenografts, without apparent toxicity, suggesting that it could represent a new attractive tool for the development of future promising therapeutic approaches, and providing a novel insight to ER regulation of cell fate.

Lack of sufficient information on the specificity and selectivity of commercial phytoestrogens preparations for therapeutic purposes.

Phytoestrogens (PEs) are polyphenols of plant origin among which flavones, flavanones, isoflavones, coumarins, chalcones, lignans and stilbenes are the best representatives. By interacting with specific residues of the estradiol-binding pocket of estrogen receptors (ERs), they induce estrogenic responses, supporting the concept that they could be of benefits against the menopausal disorders due to endogenous estradiol depletion. According to literature data, PEs target a panel of proteins, suggesting that their effects are not limited to ER-dependent transcription pathways. In this regard, commercial preparations usually contain a mixture of compounds of which nature and concentration are not specified. Such mixtures being freely accessible and escaping thereby medical survey, they could exert unwanted effects, depending on their qualitative and quantitative composition as well as the physiopathological status of the women. This work outlines the necessity to inform consumers of the exact nature of these PEs preparations. Moreover, women who want to take PEs should inform their practitioner to be under strict medical survey. In the case of hormone-dependent cancer antecedents or predispositions, use of PEs is extremely inadvisable.