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.

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.

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.

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.

Identification of a human estrogen receptor alpha-derived antiestrogenic peptide that adopts a polyproline II conformation.

Polyproline II (PPII) helix is an extended secondary structure present in a number of proteins. PPII-containing sequences mediate specific protein-protein interactions with partners containing appropriate cognate domains called PPII-recognizing domains (PRDs) and are involved in the activation of intracellular signaling pathways. Thus, the identification of PPII structures in proteins is of great interest, not only to explore molecular and physiological mechanisms, but also to elaborate new potential drugs. By revisiting X-ray crystal structures of liganded alpha-type human estrogen receptor (ERalpha), we have identified an 11-residue PPII-helical sequence (D(321)AEPPILYSEY(331)) in the ligand-binding domain of the receptor. The data recorded by far-ultraviolet circular dichroism (far-UV CD), vibrational Raman optical activity (ROA) and differential scanning calorimetry (DSC) show that the corresponding peptide (Ac-DAEPPILYSEY-NH(2)) is particularly well structured in PPII, with the same proportion of PPII as observed from X-ray structures (approximately 85%). In addition, studies carried out on ERalpha-negative Evsa-T breast cancer cells transiently co-transfected with a pcDNA3-ERalpha plasmid and a Vit-tk-Luc reporter gene revealed that the peptide antagonizes the estradiol-induced transcription providing perspectives for researching new molecules with antagonistic properties.

Calmodulin, a regulatory partner of the estrogen receptor alpha in breast cancer cells.

Although calmodulin (CaM) interaction with estrogen receptor alpha (ERalpha) has been known for more than two decades, it is only recently that the molecular mechanism of CaM-mediated regulation of ERalpha is beginning to emerge. Others and we have identified a putative calmodulin binding site (P(295)LMIKRSKKNSLALSTADQMVS(317)) in ERalpha, at the boundary between the hinge and the ligand binding domain. ERalpha mutations affecting its association with CaM have been reported to generate high basal, estrogen-independent transactivation activity, indicating that the P(295)-T(317) sequence has an inhibitory function. Moreover, we found that a synthetic peptide (ERalpha17p: P(295)-T(311)) containing residues crucial for CaM binding exerts estrogenic effects on breast carcinoma cells. Finally, computer-aided conformational studies revealed that the CaM binding site might associate with a region located downstream in ERalpha (the beta turn/H4 region), this association likely resulting in an auto-inhibitory folding of the receptor. Thus, we propose as a hypothesis that CaM acts as a positive regulator by relieving this ERalpha auto-inhibition.

Trophic effect in MCF-7 cells of ERalpha17p, a peptide corresponding to a platform regulatory motif of the estrogen receptor alpha--underlying mechanisms.

As yet, estrogen receptor alpha (ERalpha) inhibitors used in clinical practice target a unique site, i.e. the hormone-binding pocket. With the aim of discovering other potential therapeutic targets in the receptor, we studied its AF-2a domain, a site that proves to be critical for ligand-independent ERalpha activity. Previous studies from our laboratory highlighted an auto-inhibitory action associated with a site included in this domain, i.e. the P295-T311 sequence. Accordingly, a deletion of this sequence produces a constitutively activated receptor mutant. More interestingly, a synthetic peptide with the P295-T311 sequence (ERalpha17p) elicits in breast cancer cell lines estrogenic responses that may be ascribed to a competitive mechanism towards the P295-T311-associated auto-inhibition of ERalpha. In the present study, we show that ERalpha17p sustains MCF-7 cell growth in estrogen-depleted culture medium by inducing molecular events promoting G1/S phase transition. We demonstrate, moreover, that this proliferative activity is associated with receptor down regulation (acceleration of ERalpha degradation and repression of ESR1 gene transcription), similar to that induced by estrogen agonists. Complementary studies suggest that our observations may be, at least in part, relevant to a competitive inhibition affecting ERalpha-Hsp70 association. Hence, the design of drugs able to stabilize ERalpha-Hsp70 complexes - where the receptor is in an inactive conformation - may be of therapeutic value.

Fluorous tolerance of the estrogen receptor alpha as probed by 11-polyfluoroalkylestradiol derivatives.

The concern of this work was to try to delineate factors, inherent to fluorination, susceptible to influence estradiol binding to the estrogen receptor alpha (ERalpha). For this purpose, fluorinated chains were linked at 11beta position of the steroid (i.e., C(6)F(13), CH(2)CH(2)C(4)F(9), CH(2)CH(2)C(8)F(17)). Relative binding affinity (RBA) for ERalpha of these compounds and of other related fluorinated derivatives was compared to those of non-fluorinated analogs. Despite being relatively well accepted by the receptor, investigated compounds exhibited lower RBA values at 0 degrees C than their non-fluorinated counterparts. Nevertheless, heavily fluorinated chains were tolerated in so far as they are not too long (C-4) and insulated from the steroidal core by a two methylene spacer unit. Increase of the temperature of our binding assay (25 degrees C) failed to change the RBA values of two selected polyfluorohexyl derivatives while it drastically enhanced the value of the corresponding non-fluorinated analogs. Rigidity of the chain induced by fluorination as well as the oleophilic (fluorophobic) nature of the estradiol binding cavity of ERalpha is proposed to explain these properties.

Estrogens decrease gamma-ray-induced senescence and maintain cell cycle progression in breast cancer cells independently of p53.

PURPOSE: Sequential administration of radiotherapy and endocrine therapy is considered to be a standard adjuvant treatment of breast cancer. Recent clinical reports suggest that radiotherapy could be more efficient in association with endocrine therapy. The aim of this study was to evaluate the estrogen effects on irradiated breast cancer cells (IR-cells). METHODS AND MATERIALS: Using functional genomic analysis, we examined the effects of 17-beta-estradiol (E(2), a natural estrogen) on MCF-7 breast cancer cells. RESULTS: Our results showed that E(2) sustained the growth of IR-cells. Specifically, estrogens prevented cell cycle blockade induced by gamma-rays, and no modification of apoptotic rate was detected. In IR-cells we observed the induction of genes involved in premature senescence and cell cycle progression and investigated the effects of E(2) on the p53/p21(waf1/cip1)/Rb pathways. We found that E(2) did not affect p53 activation but it decreased cyclin E binding to p21(waf1/cip1) and sustained downstream Rb hyperphosphorylation by functional inactivation of p21(waf1/cip1). We suggest that Rb inactivation could decrease senescence and allow cell cycle progression in IR-cells. CONCLUSION: These results may help to elucidate the molecular mechanism underlying the maintenance of breast cancer cell growth by E(2) after irradiation-induced damage. They also offer clinicians a rational basis for the sequential administration of ionizing radiation and endocrine therapies.

Effects of (R,S)/(S,R)-4,5-bis(2-chloro-4-hydroxyphenyl)-2-imidazolines and (R,S)/(S,R)-2,3-bis(2-chloro-4-hydroxyphenyl)piperazines on estrogen receptor alpha level and transcriptional activity in MCF-7 cells.

4,5-Diaryl-2-imidazolines (Im(s)) and 2,3-diarylpiperazines (Pip(s)) belong to the type II class of estrogens. These compounds enhance ERalpha-mediated transcription of ERE-driven reporter genes in MCF-7 cells but do not compete with [(3)H]estradiol (E(2)) for receptor binding, because of distinct anchoring modes. The present study examined whether the estrogenic action of Im(s) and Pip(s) is associated with a down regulation of ERalpha, as reported for conventional agonists. Im and Pip derivatives displaying a large spectrum of activities in three distinct ERE-dependent transactivation systems were selected for that purpose. ERalpha immunostaining as well as Western blotting analysis revealed that both classes of compounds down regulated ERalpha with an efficiency closely related to their transactivation potency. MG-132 abrogated this down regulation, pointing to a proteasomal degradation process. Im(s) and Pip(s) with strong transactivation potency also altered [(3)H]E(2) binding parameters, leading to a progressive decrease of cellular estrogen binding capacity. This property occurred largely before ERalpha down regulation and persisted even in presence of MG-132, indicating that it did not result from ERalpha breakdown but rather from a conformational change of the receptor. The additional finding that the most active agonist tested in this study enhanced the capacity of a purified ERalpha recombinant to recruit LxxLL co-activators, while its inactive counterpart failed to do so confirmed this hypothesis. Altogether, our data indicate that the association of Im(s) and Pip(s) with ERalpha elicits similar responses to conventional agonists, even if they interact with distinct residues of the binding pocket.

Calmodulin-independent, agonistic properties of a peptide containing the calmodulin binding site of estrogen receptor alpha.

Calmodulin (CaM) contributes to estrogen receptor alpha (ER)-mediated transcription. In order to study the underlying mechanisms, we synthesized a peptide including the CaM binding site: ERalpha17p (P(295)-T(311)). This peptide inhibited ER-CaM association, unlike two analogs in which two amino acids required for CaM binding were substituted. Exposure of MCF-7 cells to ERalpha17p down regulated ER, stimulated ER-dependent transcription and enhanced the proliferation of ER-positive breast cancer cell lines. Interestingly, ERalpha17p analogs unable to bind to CaM induced similar responses, demonstrating that ERalpha17p-mediated effects are mainly relevant to mechanisms independent of ER-CaM dissociation. The P(295)-T(311) motif is indeed a platform for multiple post-translational modifications not necessarily CaM-dependent. The additional finding that deletion of the P(295)-T(311) sequence in ER produced a constitutive transcriptional activity revealed that this platform motif has autorepressive functions. With regard to cell function, association of CaM to ER would counteract this autorepression, leading thereby to enhanced ER-mediated transactivation.

Estrogen receptor alpha--identification by a modeling approach of a potential polyproline II recognizing domain within the AF-2 region of the receptor that would play a role of prime importance in its mechanism of action.

Estrogen receptors (ERs) behave not only as ligand-dependent transcriptional factors; they can also trigger non-genomic responses involving mitogen activated protein kinases (MAPKs), reported to be crucial in transduction cascades. MAPKs are partially activated by proteins with domains able to interact with polyproline II (PPII) regions. Recent studies have brought up the direct interaction of PPII-containing proteins with the alpha subtype human ER (ERalpha). Such observations suggest that ERalpha may contain a "PPII recognizing domain" (PRD). By sequence alignment, we identified such a potential PRD within the AF-2 region of ERalpha (residues 351-414). According to our modeling studies based on X-ray structural data, this PRD appears to be divided in two sub-regions known to interact with alpha-helix containing coactivators. Our data also reveal the potential existence of intramolecular interactions of this domain with a large PPII-rich region of the receptor (residues 301-330). Implication of these regulatory structural elements in both genomic and non-genomic responses seems likely.

Estrogen receptor alpha: impact of ligands on intracellular shuttling and turnover rate in breast cancer cells.

Estrogen receptors (alpha and beta) are members of the steroid/thyroid nuclear receptors superfamily of ligand-dependent transcription factors. Impact of the alpha isoform of estrogen receptor (ER) on breast cancer etiology and progression is now well established. Current therapeutic strategy to treat ER-positive breast cancer relies on the blockade of ER trancriptional activity by antiestrogens. Data accumulated during the last five years on the mechanism of action of ER enable one to foresee new strategies. These data indeed reveal that ER is not statically bound to DNA at promoter sites of genes regulating cell proliferation and/or differentiation, but rather behaves as a very mobile protein continuously shuttling between targets located within various cellular compartments (i.e. membrane, microsomes, nucleus...). This allows the receptor to generate both non-genomic and genomic responses. Ligands, growth factors and second messengers produced downstream of activated membrane receptors modulate ER-mediated responses by interfering with the traffic patterns of the receptor, as well as by locally blocking its transient anchorage. Changes in ER turnover rate associated with these regulatory processes seem also to strongly influence the ability of the receptor to mediate gene transactivation. The present paper surveys these biological data and analyzes how they may be integrated into new drug design programs aimed at expanding our therapeutic armamentarium against 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.

Opposite effects of estrogen receptors alpha and beta on MCF-7 sensitivity to the cytotoxic action of TNF and p53 activity.

We have investigated the effect of estrogen on p53 cellular location and its influence on tumor cell susceptibility to tumor necrosis factor (TNF)-mediated cytotoxic action. For this purpose, we have used the TNF-sensitive human breast adenocarcinoma MCF-7 and its derivative, the TNF-resistant 1001 clone. Our data indicate that although estrogen receptor (ER)alpha is present in both cell lines, estrogen treatment (1x10(-8) M) has an influence only on the MCF-7 cells and protects these cells from the TNF cytotoxicity. This protective effect is associated with translocation of p53 from the nucleus to the cytoplasm in p53 wild-type MCF-7 and not in p53-mutated 1001 cells. The translocation of p53 in MCF-7 cells results in a decrease in its transcriptional activity, as revealed by diminished p21(WAF1/CIP1) induction and an altered ratio of Bax and Bcl-2 proteins. The estrogen-induced effects are reversed by the selective estrogen inhibitor 182, 780 (1x10(-6) M). Interestingly, transient transfection of MCF-7 cells with ERbeta but not ERalpha cDNA encoding plasmid results in retention of p53 in the nucleus, a subsequent potentiation of its transcriptional activity, and in an increased MCF-7 sensitivity to TNF. The estrogen effects on p53 location and transcriptional activity may involve the mdm2 protein since both events were reversed following MCF-7 transfection with plasmid encoding the ARF cDNA. These studies suggest that estrogen-induced MCF-7 cell survival in the presence of TNF requires a transcriptionally active p53 and, more importantly, indicate that introduction of ERbeta can attenuate the estrogen effects on the p53 protein location, its transcriptional activity and also results in a potentiation of cell sensitivity to TNF-mediated cell death.

p53 and breast cancer, an update.

p53 plays a key role in mediating cell response to various stresses, mainly by inducing or repressing a number of genes involved in cell cycle arrest, senescence, apoptosis, DNA repair, and angiogenesis. According to this important function, p53 activity is controlled in a very complex manner, including several auto-regulatory loops, through the intervention of dozens of modulator proteins (the 'p53 interactome'). p53 mutations are observed in a significant minority of breast tumours. In the remaining cases, alterations of interactome components or target genes could contribute, to some extent, to reduce the ability of p53 to efficiently manage stress events. While the prognostic and predictive value of p53 is still debated, there is an increasing interest for p53-based therapies. The present paper aims to provide updated information on p53 regulation and function, with specific interest on its role in breast cancer.

Tangeretin inhibits extracellular-signal-regulated kinase (ERK) phosphorylation.

Tangeretin is a methoxyflavone from citrus fruits, which inhibits growth of human mammary cancer cells and cytolysis by natural killer cells. Attempting to unravel the flavonoid's action mechanism, we found that it inhibited extracellular-signal-regulated kinases 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent way. In human T47D mammary cancer cells this inhibition was optimally observed after priming with estradiol. The spectrum of the intracellular signalling kinase inhibition was narrow and comparison of structural congeners showed that inhibition of ERK phosphorylation was not unique for tangeretin. Our data add tangeretin to the list of small kinase inhibitors with a restricted intracellular inhibition profile.