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We report on a stabilizer of the interaction between 14-3-3ζ and the Estrogen Receptor alpha (ERα). ERα is a driver in the majority of breast cancers and 14-3-3 proteins are negative regulators of this nuclear receptor, making the stabilization of this protein-protein interaction (PPI) an interesting strategy. The stabilizer (1) consists of three symmetric peptidic arms containing an arginine mimetic, previously described as the GCP motif. 1 stabilizes the 14-3-3ζ/ERα interaction synergistically with the natural product Fusicoccin-A and was thus hypothesized to bind to a different site. This is supported by computational analysis of 1 binding to the binary complex of 14-3-3 and an ERα-derived phosphopeptide. Furthermore, 1 shows selectivity towards 14-3-3ζ/ERα interaction over other 14-3-3 client-derived phosphomotifs. These data provide a solid support of a new binding mode for a supramolecular 14-3-3ζ/ERα PPI stabilizer.

U ovoj doktorskoj disertaciji razvijen je fluorescentni test u kvascu za identifikaciju potencijalnih prirodnih ili sintetičkih liganada ERα, ERβ ili AR i kvantifikaciju njihovog afiniteta vezivanja sa mogućnošću testiranja čitavih biblioteka modifikovanih steroida i ksenoestrogena. Takođe, opisana je primena optimizovanog biosenzora za procenu estrogenog potencijala sintetskih steroida i odabranih biljnih ekstrakata bogatih jedinjenjima fitoestrogenih osobina. U cilju potpunijeg sagledavanja mehanizma delovanja odabranih modifikovanih steroida ispitana je njihova antiproliferativna aktivnost prema ćelijskim linijama estrogen receptor pozitivnog kancera dojke (MCF-7) i kancera prostate (PC-3), dok su in silico metodom molekularnog dokinga predviđene energije i geometrije vezivanja ovih jedinjenja za ligand-vezujuće domene ERα i ERβ. Drugi deo ovog rada obuhvata razvoj testa za ispitivanje aktivnosti humanog enzima aromataze, heterologno eksprimiranog u ćelijama kvasca Saccharomyces cerevisiae i/ili bakterija Escherichia coli, u prisustvu ili odsustvu inhibitora. Interakcije modifikovanih steroida, odabranih na osnovu strukture, sa aromatazom ispitane su osetljivim spektroskopskim metodama, praćenjem promene spinskog stanja Fe iz hem grupe ili promene fluorescencije ostatka triptofana iz aktivnog centra usled konformacione promene proteina, izazvane interakcijom sa ligandom. Razvijeni in vitro testovi bez upotrebe radioaktivnih izotopa su, osim visoke efikasnosti i bezbednosti po korisnika i okolinu, pokazali specifičnost i ekonomičnost u preliminarnom skriningu liganada steroidnih receptora i inhibitora aromataze. Jedinjenja kod kojih je detektovana značajna biološka aktivnost mogu potencijalno poslužiti kao osnova za razvoj terapeutika u lečenju hormon-zavisnih bolesti i stanja, koja danas predstavljaju globalni zdravstveni problem., In this doctoral dissertation, a fluorescent assay in yeast was developed for identification of potential natural or synthetic ligands of ERα, ERβ or AR and quantification of their binding affinity, as well asevaluation of the estrogenic potential of synthetic steroids and selected plant extracts rich in phytoestrogen content. The assay could be used to screen libraries of modified steroids and xenoestrogens. In order to better understand the biomedical potential of selected modified steroids, results were compared to antiproliferative activity against estrogen receptor positive breast cancer (MCF-7) and prostate cancer (PC-3) cell lines. Binding energies and the geometry of binding of these compounds for ERα and ERβ ligand binding domains were predicted in silico by molecular docking methods. The second part of this study includes development of an assay for study of aromatase activity in the presence or absence of inhibitors by heterologous expression of human aromatase in Saccharomyces cerevisiae and/or Escherichia coli cells, as model-organisms. Furthermore, interactions between modified steroids, selected according to their structure, and aromatase were tested using sensitive spectroscopic methods based on ligand-induced changes in the spin state of Fe from the heme group or changes in the fluorescence of a tryptophan residue in the active site. The non-radioactive in vitro assays developed here, besides high efficiency, user and environmental safety, also have greater specificity and are more cost-effective for preliminary screening of steroid receptor ligands and aromatase inhibitors. Additionally, compounds identified to express significant biological activity can serve as a basis for the development of potential therapeutics in the treatment of hormone-dependent dise

Estrogen plays multiple roles in health and disease, exerting its effects through the classical estrogen receptors (ERα and ERβ) and the G protein‑coupled estrogen receptor (GPER). Current ER‑targeting ligands, including the therapeutic ERα antagonist tamoxifen, have been shown to cross‑activate GPER. This cross‑activation is hypothesized to contribute to clinically observed endocrine resistance in breast cancer, highlighting the potential benefit of truly ER‑selective antagonists. We report the identification of a novel class of ER‑selective ligands that lack cross‑reactivity towards GPER, identifying a truly ER‑selective agonist (AB‑1) and antagonist (AB‑82P). Importantly, AB‑82P degrades a clinically relevant ERα mutant and exhibits inhibitory effects in cellular models of endocrine resistance. This novel class of ER‑selective ligands can aid in improving our understanding of the individual estrogen receptors in estrogen biology and more importantly, provide a structural basis for the development of new, truly ER‑selective antagonists for the treatment of ERα‑positive breast cancers.

Estrogen receptor alpha (ERα) and retinoic acid receptors (RARs) play important and opposite roles in breast cancer growth. While exposure to ERα agonists such as 17β-estradiol (E2) is related to proliferation, RAR agonists such as all-trans retinoic acid (AtRA) induce anti-proliferative effects. Although crosstalk between these pathways has been proposed, the molecular mechanisms underlying this interplay are still not completely unraveled. The aim of this study was to evaluate the effects of AtRA on ERα-mediated signaling in the ERα positive cell lines MCF7/BUS and U2OS-ERα-Luc to investigate some of the possible underlying modes of action. To do so, this study assessed the effects of AtRA on different ERα-related events such as ERα-mediated cell proliferation and gene expression, ERα-coregulator binding and ERα subcellular localization. AtRA-mediated antagonism of E2-induced signaling was observed in the proliferation and gene expression studies. However, AtRA showed no remarkable effects on the E2-driven coregulator binding and subcellular distribution of ERα. Interestingly, in the absence of E2, ERα-mediated gene expression, ERα-coregulator binding and ERα subcellular mobilization were increased upon exposure to micromolar concentrations of AtRA found to inhibit cell proliferation after long-term exposure. Nevertheless, experiments using purified ERα showed that direct binding of AtRA to ERα does not occur. Altogether, our results using MCF7/BUS and U2OS-ERα-Luc cells suggest that AtRA, without being a direct ligand of ERα, can indirectly interfere on basal ERα-coregulator binding and basal ERα subcellular localization in addition to the previously described crosstalk mechanisms such as competition of ERs and RARs for DNA binding sites.

Depuis bon nombre d’années déjà, la question ne se pose plus ; l’utilisation des pesticides en agriculture est nécessaire puisque sans ces derniers les chances que les terres soient ravagées par les insectes, champignons ou petits rongeurs représenteraient un trop grand risque pour les agriculteurs. Cependant, cette utilisation massive de pesticides en agriculture apporte son lot de questionnements et d’inquiétudes face aux effets néfastes qu’ils pourraient avoir tant sur les humains que sur la faune et la flore. Bien que tous les pesticides utilisés au Canada soient homologués par Santé Canada, et sont donc jugés comme ne représentant pas de risques élevés ni pour l’environnement ni les humains, nul ne connait les effets d’un mélange de pesticides sur nos cellules, notre organisme. Ce projet de recherche vise donc à évaluer les effets d’un mélange de pesticides composé de cinq ingrédients actifs communément utilisés en agriculture sur les niveaux d’expression du gène CYP1A1, un gène cible du récepteur aux hydrocarbures d'aryle (AhR). Ce récepteur, bien qu’impliqué dans bon nombre d’autres réponses cellulaires, joue un rôle important dans la détoxification de l’organisme en activant la transcription de certains gènes dans la famille du cytochrome P450, dont le gène CYP1A1. Tel que mentionné précédemment, le but du présent projet de recherche est d’évaluer les effets d’un mélange composé d’au moins deux ingrédients actifs de pesticides sur l’activation de la voie de AhR. Les résultats du projet de recherche ont démontré que certaines combinaisons donnent lieu à une activation synergique de la voie AhR alors que d’autres donnent plutôt lieu à une activation additive. Dans le cas où la concentration des ingrédients actifs est élevée, on obtient plutôt un effet inhibiteur. N’est-ce pas paradoxal qu’à faibles doses, il y a un effet soit additif ou synergique alors qu’à de hautes concentrations, l’effet est plutôt inhibiteur ? Il ne faut alors pas croire que de fortes dos

Alternative splicing is a process that partly rejects the common definition of a gene – that one gene codes for one specific protein. By variable combination of coding regions (exons) and exclusion of non-coding regions (introns), formation of several different mRNA-transcripts, and consequently several different proteins, can derive from the same gene. Alternative splicing is an important condition for the development of complex life forms, but it is also a highly sensitive process and inaccurate splicing is the cause of approximately 15 % of mutations that cause genetic diseases. This article presents four genes, BRCA1, BRCA2, ERα and ERβ, and inaccurate splicing of these genes increases the risk of developing cancer, particularly breast cancer and ovarian cancer. Breast cancer is the second most common form of lethal cancer among women. After identifying the cancerogenic mutations, women of high-risk families can undergo genetic testing and preventive therapy can reduce the morbidity and mortality. The article also presents a short discussion around the ethical problems of genetic testing, and the social and psychological dilemmas women of high-risk families are facing when they are given the option to undergo genetic testing.

Stroke is a common and multifactorial disease influenced by genetic and environmental risk factors. It is a highly heterogeneous entity consisting of two main types, ischemic (80%) and hemorrhagic (20%) stroke. The most common form of hemorrhagic stroke is intracerebral hemorrhage (ICH). Ischemic stroke mainly results from thrombotic or embolic events, while ICH is caused by the rupture of an artery in the brain. The mean age of first-ever stroke is 75 years (73 vs. 78 years, for men and women, respectively) and the age-specific stroke incidence is higher for men as compared to women, suggesting that hormonal factors confer protection. A large body of experimental and observational studies shows that estrogens exert beneficial effects in the cardiovascular system. However, large, recent, clinical randomized trials have failed to demonstrate a lower risk of stroke with hormone replacement therapy (HRT) in elderly postmenopausal women. It is possible that HRT may only protect a subgroup of women. Here, genetic predisposition might be involved. Stroke incidence is 50% higher in northern compared to southern Sweden, suggesting a genetic predisposition in this population. This relatively homogeneous population displays founder effects, making it well suited for genetic studies. Since 1985, the MONICA and VIP projects have conducted large-scale cardiovascular health surveys in this population. Information about conventional stroke risk determinants and also DNA have been collected, and two prospective, nested case-referent cohorts (113 cases and 226 controls; 275 cases and 549 controls) have been sampled. To investigate whether genes of the estrogen signaling system may be important in stroke development, we performed genetic association studies, including specific functional single nucleotide polymorphisms in the genes for estrogen receptor alpha (ERα, ESR1), and its target genes osteoprotegerin (OPG, TNFRS11B) and interleukin-6 (IL-6, IL6). We found a significant associ

Stroke is a common and multifactorial disease influenced by genetic and environmental risk factors. It is a highly heterogeneous entity consisting of two main types, ischemic (80%) and hemorrhagic (20%) stroke. The most common form of hemorrhagic stroke is intracerebral hemorrhage (ICH). Ischemic stroke mainly results from thrombotic or embolic events, while ICH is caused by the rupture of an artery in the brain. The mean age of first-ever stroke is 75 years (73 vs. 78 years, for men and women, respectively) and the age-specific stroke incidence is higher for men as compared to women, suggesting that hormonal factors confer protection. A large body of experimental and observational studies shows that estrogens exert beneficial effects in the cardiovascular system. However, large, recent, clinical randomized trials have failed to demonstrate a lower risk of stroke with hormone replacement therapy (HRT) in elderly postmenopausal women. It is possible that HRT may only protect a subgroup of women. Here, genetic predisposition might be involved. Stroke incidence is 50% higher in northern compared to southern Sweden, suggesting a genetic predisposition in this population. This relatively homogeneous population displays founder effects, making it well suited for genetic studies. Since 1985, the MONICA and VIP projects have conducted large-scale cardiovascular health surveys in this population. Information about conventional stroke risk determinants and also DNA have been collected, and two prospective, nested case-referent cohorts (113 cases and 226 controls; 275 cases and 549 controls) have been sampled. To investigate whether genes of the estrogen signaling system may be important in stroke development, we performed genetic association studies, including specific functional single nucleotide polymorphisms in the genes for estrogen receptor alpha (ERα, ESR1), and its target genes osteoprotegerin (OPG, TNFRS11B) and interleukin-6 (IL-6, IL6). We found a significant associ

Stroke is a common and multifactorial disease influenced by genetic and environmental risk factors. It is a highly heterogeneous entity consisting of two main types, ischemic (80%) and hemorrhagic (20%) stroke. The most common form of hemorrhagic stroke is intracerebral hemorrhage (ICH). Ischemic stroke mainly results from thrombotic or embolic events, while ICH is caused by the rupture of an artery in the brain. The mean age of first-ever stroke is 75 years (73 vs. 78 years, for men and women, respectively) and the age-specific stroke incidence is higher for men as compared to women, suggesting that hormonal factors confer protection. A large body of experimental and observational studies shows that estrogens exert beneficial effects in the cardiovascular system. However, large, recent, clinical randomized trials have failed to demonstrate a lower risk of stroke with hormone replacement therapy (HRT) in elderly postmenopausal women. It is possible that HRT may only protect a subgroup of women. Here, genetic predisposition might be involved. Stroke incidence is 50% higher in northern compared to southern Sweden, suggesting a genetic predisposition in this population. This relatively homogeneous population displays founder effects, making it well suited for genetic studies. Since 1985, the MONICA and VIP projects have conducted large-scale cardiovascular health surveys in this population. Information about conventional stroke risk determinants and also DNA have been collected, and two prospective, nested case-referent cohorts (113 cases and 226 controls; 275 cases and 549 controls) have been sampled. To investigate whether genes of the estrogen signaling system may be important in stroke development, we performed genetic association studies, including specific functional single nucleotide polymorphisms in the genes for estrogen receptor alpha (ERα, ESR1), and its target genes osteoprotegerin (OPG, TNFRS11B) and interleukin-6 (IL-6, IL6). We found a significant associ

Non small cell lung carcinoma (NSCLC) occurs in two major histological types, squamous cell carcinoma (SCC) and adenocarcinoma (ADCA). There are well characterized differences in the carcinogenesis between these types. For example, epidermal growth factor receptor (EGFR) is frequently amplified and over expressed in SCC whilst mutations occur in ADCA. There are also gender associated differences in the relative incidence of SCC and ADCA. Estrogen receptor (ER) driven signalling have been proposed to play a role in this context. ER occurs in two different forms, α and β. The presence of ERα in NSCLC has been a matter of discussion. Immunohistochemical (IHC) analysis has reported divergent results, but the presence of ERα transcripts has been established to be widespread in NSCLC. ERα could be activated/phosphorylated in several ways, either ligand (estradiol) dependent or ligand independent. A potential crosstalk between EGFR and ERα has been proposed by a mechanism where EGFR signalling induces phosphorylation at specific sites within the ERα receptor. We investigated in the present study, in a material of 262 NSCLC, by IHC the presence of a phosphorylated form of ERα, pERαS118, and correlated these findings to EGFR over expression as well as an ERα co-activator, CREB, which has been associated to ligand independent phosphorylation of ERα at that specific site. We describe a widespread occurrence of pERαS118 in both SCC and ADCA. Furthermore, pERαS118is correlated to EGFR over expression in SCC. We propose a combined targeting of ERα and EGFR crosstalk as a possibility in the targeted therapy of SCC.

In non-small cell lung carcinoma (NSCLC) squamous cell carcinomas (SCC) occurs more frequently among men and adenocarcinomas (ADCA) in females. The presence of estrogen receptors (ERs) in NSCLC have been proposed as a factor contributing to the gender associated differences. HER-4, a member of the epidermal growth factor receptor family, has recently been identified as a co-activator to ER by action of the shredded intracellular domain, 4ICD. Our study was performed on tissue material from 262 NSCLC. In 137 cases, frozen material was available. HER-4/4ICD, ERα, PgR and aromatase was determined by immunohistochemistry. HER-4 gene copy number and HER-4 and ERα transcripts was also studied. Data was correlated to proliferation, histology, gender, smoking habits and clinical outcome. We report the presence of n4ICD, significantly more frequently occurring in ADCA than in SCC. Furthermore, n4ICD and ERα expression was associated with the concomitant expression of aromatase. n4ICD was negatively correlated to proliferation in ADCA. Interestingly, a strong correlation between never smoking and n4ICD positivity was found among females and in the ADCA group. In breast carcinomas, n4ICD is a biomarker for anti-ER treatment. A number of observations including our finding of n4ICD can constitute support for anti-ER treatment strategies in NSCLC.