Introdução: Estimativas mostram que mais de dois terços das mulheres com câncer de mama possuem receptores hormonais positivos, e recebem terapia endócrina como tratamento, sendo tamoxifeno (TAM) o tratamento padrão (EBCTCG 2005; Davies et al., 2012). Porém, muitas pacientes se tornam resistentes com o passar do tempo. Estudos prévios mostraram que a expressão do receptor de estrogênio β (REβ) aumenta a resposta ao tratamento com TAM em células de câncer de mama, assim como a coexpressão de REα e REβ esta associada com maior ação proliferativa de TAM (Treeck et al., 2010; Sun et al., 2014). Também foi observada a existência de “cross-talk” entre os RE e a família do receptor do fator de crescimento epidérmico (HER) na resposta ao tratamento com TAM (Lindberg et al., 2011; Blows et al., 2010). Objetivo: Verificar a expressão do REβ, e suas interações com REα e receptores HER, durante o tratamento com TAM e em células resistentes ao TAM. Métodos: A expressão do REβ foi analisada em dois bancos de dados contendo informações de pacientes com câncer de mama. A expressão de RNAm dos RE, receptores HER e vias de sinalização PTEN, Akt e MAPK foram avaliadas após tratamento com TAM, em células resistentes ao TAM e em células silenciadas para os genes dos RE. Também foi avaliada a viabilidade celular após tratamento com TAM e nas células silenciadas para os genes dos RE. Resultados: Pacientes com câncer de mama apresentaram expressão reduzida do REβ, e os subtipos de câncer de mama REα positivos apresentaram baixa expressão do REβ quando comparados aos subtipos REα negativos. Células expressando níveis moderados de REβ apresentaram melhor resposta ao tratamento com TAM. Diminuição nos níveis dos RE é acompanhada por aumento nos níveis dos receptores ErbB2 e ErbB3, aumento de PTEN e diminuição de Akt e MAPK3 após tratamento com TAM. ERβ modula a ação antiproliferativa do TAM através da via de MAPK3. Células resistentes ao TAM apresentaram baixos níveis dos RE e altos níveis dos receptores EGFR, ErbB3 e ErbB4. Conclusão: Estes resultados demonstram que o REβ, e suas interações com REα e receptores HER, possuem papel importante na resposta ao tratamento com TAM. / Introduction: Approximately two-thirds of all breast cancer patients overexpress hormonal receptors, and are treated with endocrine therapy, being tamoxifen (TAM) the standard treatment. However many of initial responders to TAM as first-line experience relapse. Several mechanisms have been proposed to explain the occurrence of acquired TAM resistance. Previous studies showed that estrogen receptor β (ERβ) expression is associated with better response to tamoxifen treatment, as the co-expression of ERα and ERβ is associated with TAM antiproliferative effects. Moreover, there is growing interest about the cross-talk between ERs and ErbB family in response to endocrine therapy. Suggesting that TAM can acts through ERβ and/or ErbB family as compensatory pathways. Objective: To evaluate the expression of ERβ and the relation of ERβ with ERα and ErbB family in response to TAM treatment and in TAM resistant cells. Methods: ERβ expression was analyzed in two different databases of breast cancer patients. The mRNA levels of ER, HER receptors and PTEN, Akt and MAPK signal pathways were measured after TAM treatment, in TAM resistance cells and in cells silenced for ER genes. The cellular viability was also measured after TAM treatment, in TAM resistance cells and in cells silenced for ER genes. Results: Breast cancer patients presented reduced ERβ expression and the ERα-positive breast cancer subtypes presented lower ERβ levels when compared to ERα-negative breast cancer subtypes. Cells expressing moderates levels of ERβ presented better response to TAM treatment. Down-regulation of ERs induced by TAM treatment are accompanied with an increase in ErbB2 and ErbB3, reduced AKT and MAPK3 mRNA levels and increased PTEN levels. ERβ modulates TAM anti-proliferative effects through MAPK3 pathway. TAM– resistant cells expressed decreased ER mRNA levels and increased EGFR, ErbB3 and ErbB4 levels. Demonstrating that the cross-talk between ERs and HER family influence the response to TAM treatment. Conclusion: These results provide additional data indicating the importance of ERβ, and the relation with ERα and HER receptors, to predict TAM responsiveness.

Introdução: Estimativas mostram que mais de dois terços das mulheres com câncer de mama possuem receptores hormonais positivos, e recebem terapia endócrina como tratamento, sendo tamoxifeno (TAM) o tratamento padrão (EBCTCG 2005; Davies et al., 2012). Porém, muitas pacientes se tornam resistentes com o passar do tempo. Estudos prévios mostraram que a expressão do receptor de estrogênio β (REβ) aumenta a resposta ao tratamento com TAM em células de câncer de mama, assim como a coexpressão de REα e REβ esta associada com maior ação proliferativa de TAM (Treeck et al., 2010; Sun et al., 2014). Também foi observada a existência de “cross-talk” entre os RE e a família do receptor do fator de crescimento epidérmico (HER) na resposta ao tratamento com TAM (Lindberg et al., 2011; Blows et al., 2010). Objetivo: Verificar a expressão do REβ, e suas interações com REα e receptores HER, durante o tratamento com TAM e em células resistentes ao TAM. Métodos: A expressão do REβ foi analisada em dois bancos de dados contendo informações de pacientes com câncer de mama. A expressão de RNAm dos RE, receptores HER e vias de sinalização PTEN, Akt e MAPK foram avaliadas após tratamento com TAM, em células resistentes ao TAM e em células silenciadas para os genes dos RE. Também foi avaliada a viabilidade celular após tratamento com TAM e nas células silenciadas para os genes dos RE. Resultados: Pacientes com câncer de mama apresentaram expressão reduzida do REβ, e os subtipos de câncer de mama REα positivos apresentaram baixa expressão do REβ quando comparados aos subtipos REα negativos. Células expressando níveis moderados de REβ apresentaram melhor resposta ao tratamento com TAM. Diminuição nos níveis dos RE é acompanhada por aumento nos níveis dos receptores ErbB2 e ErbB3, aumento de PTEN e diminuição de Akt e MAPK3 após tratamento com TAM. ERβ modula a ação antiproliferativa do TAM através da via de MAPK3. Células resistentes ao TAM apresentaram baixos níveis dos RE e altos níveis dos receptores EGFR, ErbB3 e ErbB4. Conclusão: Estes resultados demonstram que o REβ, e suas interações com REα e receptores HER, possuem papel importante na resposta ao tratamento com TAM. / Introduction: Approximately two-thirds of all breast cancer patients overexpress hormonal receptors, and are treated with endocrine therapy, being tamoxifen (TAM) the standard treatment. However many of initial responders to TAM as first-line experience relapse. Several mechanisms have been proposed to explain the occurrence of acquired TAM resistance. Previous studies showed that estrogen receptor β (ERβ) expression is associated with better response to tamoxifen treatment, as the co-expression of ERα and ERβ is associated with TAM antiproliferative effects. Moreover, there is growing interest about the cross-talk between ERs and ErbB family in response to endocrine therapy. Suggesting that TAM can acts through ERβ and/or ErbB family as compensatory pathways. Objective: To evaluate the expression of ERβ and the relation of ERβ with ERα and ErbB family in response to TAM treatment and in TAM resistant cells. Methods: ERβ expression was analyzed in two different databases of breast cancer patients. The mRNA levels of ER, HER receptors and PTEN, Akt and MAPK signal pathways were measured after TAM treatment, in TAM resistance cells and in cells silenced for ER genes. The cellular viability was also measured after TAM treatment, in TAM resistance cells and in cells silenced for ER genes. Results: Breast cancer patients presented reduced ERβ expression and the ERα-positive breast cancer subtypes presented lower ERβ levels when compared to ERα-negative breast cancer subtypes. Cells expressing moderates levels of ERβ presented better response to TAM treatment. Down-regulation of ERs induced by TAM treatment are accompanied with an increase in ErbB2 and ErbB3, reduced AKT and MAPK3 mRNA levels and increased PTEN levels. ERβ modulates TAM anti-proliferative effects through MAPK3 pathway. TAM– resistant cells expressed decreased ER mRNA levels and increased EGFR, ErbB3 and ErbB4 levels. Demonstrating that the cross-talk between ERs and HER family influence the response to TAM treatment. Conclusion: These results provide additional data indicating the importance of ERβ, and the relation with ERα and HER receptors, to predict TAM responsiveness.

The signaling mechanisms involved in UVB-induced skin cancer are complex and although the scope of this work is inherently limited in focus, the findings may provide insight into how estrogen receptor signaling impacts cell growth, senescence, and apoptosis to protect keratinocytes. Additional signaling due to E2-activation of the estrogen receptor may provide back-up or redundant pathways in response to UVB.

The prevalence of obesity is dramatically increasing and thus constitutes a major risk factor for developing chronic diseases such as type 2 diabetes, dyslipidemia, cardiovascular diseases, and certain forms of cancer. High-caloric nutrition and a lack of physical activity are the main contributing factors for this global epidemic. Estrogen receptors (ERs) are recognized to be involved in many processes related to the control of energy homeostasis. In my studies, I investigated the impact of estrogens (17beta-estradiol (E2)) on energy homeostasis. Special emphasis was given to the effects of two synthetic ER subtype-selective agonists, 16alpha-LE2 (Alpha) and 8beta-VE2 (Beta), to determine to what extend the two distinct ER subtypes are involved in the underlying molecular mechanisms. Because of its estrogenic activity and also its widespread use as a nutritional supplement the influence of the isoflavone genistein (Gen) was examined. For this purpose two different female rat models were used: Wistar rats with nutrition-induced obesity and leptin resistant Zucker diabetic fatty (ZDF) rats. In both experiments, the animals were ovariectomized (OVX) and treated with vehicle (untreated controls) or the estrogenic compounds. The most important finding was that treatment of OVX animals with Beta enlarges soleus muscle fiber sizes in both animal models compared to untreated OVX animals. This anabolic effect may in turn improve the muscle/fat ratio of the body that enhances muscular uptake and utilization of fuels. By contrast, in the gastrocnemius muscle of OVX ZDF rats substitution with Alpha increased expression and distribution of the insulin-dependent glucose transporter 4 (GLUT4). Consequently, systemic insulin sensitivity in both animal models was improved by treatment with estrogenic compounds compared to untreated OVX animals. The strongest effect was observed in E2-treated rats that indicate an additive effect through activation of both pathways. In all OVX rats, treatment with either ER subtype-selective agonist showed an anti-lipogenic effect in adipose tissue, liver, and skeletal muscle of nutrition-induced obese Wistar rats in comparison to OVX animals without treatment. Decreased visceral fat mass, adipocyte sizes, serum leptin levels, triglyceride accumulation in liver and muscle as well as mRNA expression of genes that are involved in lipo-/adipogenesis reflected this. Therefore, the lower visceral fat mass as well as decreased accumulation of triglycerides in non-adipose tissues such as liver and skeletal muscle most likely contributes to the improved insulin sensitivity in such treated animals. Gen exerted effects similar to those of the ER beta-selective agonist (except on adipose tissue in Wistar rats). Especially, the similar ability to induce anabolic activity in the soleus muscle might be highly relevant. Gen-treated animals might have a more effective utilization of fuels compared to untreated OVX animals because they showed a lower TG content in muscle and liver as well as improved glucose metabolism. In conclusion, because of my studies and the fact that ER beta signaling is not involved in proliferation of uterus and mammary gland, an effective way to treat obesity and co-morbidities in postmenopausal women might be substances that only activate ER beta. A combination with physical activity may support the therapy of obesity and co-morbidities. The isoflavone Gen is able to activate both ER-subtypes. This compound is already placed on the market for treatment of postmenopausal complaints, although adverse effects of Gen cannot be excluded so far (e.g., increased risk of breast cancer). However, Gen might be a natural alternative – not only to the conventional hormone replacement therapy, but also as a strategy for treatment of obesity and co-morbidities – that deserves further research with respect to these new data. / Die dramatisch zunehmende Prävalenz der Adipositas und das damit verbundene Risiko für Folgeerkrankungen wie Diabetes mellitus, Hypertonie, Dyslipidämie und koronare Herzkrankheiten stellt eine große Herausforderung für das Gesundheitswesen dar. Als Hauptursache wird ein chronisches Missverhältnis der Energiehomöostase aufgrund permanenter Überernährung und Bewegungsmangel postuliert. Estrogene beeinflussen den Glukose- und Lipidstoffwechsel und sind somit in die Regulation des Energiehaushaltes involviert. Estrogene vermitteln ihre Effekte über zwei Estrogenrezeptor (ER)-Subtypen, den ER alpha und den ER beta. Ziel der vorliegenden Arbeit war es mittels tierexperimentellen Studien den Einfluss von Estrogenen, speziell 17beta-Estradiol, auf den Energiehaushalt zu untersuchen. Um einen tieferen Einblick in die zugrundeliegenden molekularen Mechanismen zu erhalten, wurden zwei Subtyp-selektive ER-Agonisten, 16alpha-LE2 (Alpha) and 8beta-VE2 (Beta), synthetischer Herkunft eingesetzt. Aufgrund der estrogenen Aktivität und der Verfügbarkeit als Nahrungsergänzungsmittel wurde des Weiteren der Einfluss des Isoflavons Genistein untersucht. Für die Studien wurden zwei Tiermodelle genutzt: zum einen weibliche Wistar-Ratten mit ernährungsinduzierter Adipositas und zum anderen weibliche leptinresistente „Zucker diabetic fatty“ (ZDF)-Ratten. Die Tiere wurden ovarektomiert (OVX) und entweder mit einem Vehikel (unbehandelte Kontrolltiere) oder mit der entsprechenden estrogenen Substanz behandelt. Die interessanteste Erkenntnis war, dass im Vergleich zu unbehandelten OVX-Tieren beider Tiermodelle die Behandlung mit Beta zur Vergrößerung der Faserquerschnitte im Soleusmuskel führte. Dieser anabole Effekt könnte die muskuläre Aufnahme und Verwertung von Brennstoffmolekülen verbessern und sich insgesamt positiv auf die Körperzusammensetzung auswirken. Den stärksten Effekt hinsichtlich einer erhöhten Expression und Translokation des insulinabhängigen Glukosetransporters 4 (GLUT4) in die Zellmembran des Gastrocnemiusmuskels zeigte sich dagegen durch die Behandlung von OVX ZDF-Ratten mit Alpha. Im Endergebnis zeigten die Tiere beider Modelle durch die Behandlung mit estrogenen Substanzen eine verbesserte systemische Insulinsensitivität im Vergleich zu unbehandelten Kontrolltieren. E2-behandelte Tiere tolerierten die Glukose am besten und lassen einen additiven Effekt aufgrund der Aktivierung beider Signalwege vermuten. Im Vergleich zu unbehandelten OVX Wistar-Ratten führte die Behandlung mit E2 oder mit jeweils einem der beiden ER-Subtyp-selektiven Agonisten zu einer geringeren viszeralen Fettmasse, kleineren Fettzellen, niedrigeren Leptinspiegeln im Serum und geringeren Triglyzeridwerten in Leber und Muskel. Auf der Ebene der Genexpression waren zudem geringere mRNA-Spiegel von lipo- und adipogenen Genen messbar. Somit scheinen beide ER-Subtypen in die antilipogene Wirkung von E2 involviert zu sein. Sowohl die reduzierte viszerale Fettmasse als auch die geringere Anreicherung von Triglyzeriden in Leber und Muskel tragen sehr wahrscheinlich ebenfalls zur verbesserten Insulinsensitivität bei. Die Behandlung von OVX Tieren mit Gen führte zu ähnlichen Ergebnissen wie die Behandlung mit Beta. Eine alleinige Ausnahme stellte das Fettgewebe dar, da hier eine Gen-Behandlung keine antilipogenen/-adipogenen Effekte zeigte. Speziell die Fähigkeit von Gen ebenfalls anabol zu wirken, könnte die molekulare Grundlage sein, weshalb Gen-behandelte Tiere im Vergleich zu unbehandelten Tiere eine verbesserte Toleranz gegenüber Glukose und eine geringere Anreicherung von Triglyzeriden in Muskel und Leber zeigten. Der ER beta ist nicht in die estrogenvermittelte Proliferation von Uterus und Brustdrüse involviert. Vor diesem Hintergrund lassen meine Ergebnisse vermuten, dass eine Behandlung mit ER beta-selektiven Substanzen eine effektive Möglichkeit darstellt, um Adipositas und deren Folgeerkrankungen in postmenopausalen Frauen zu behandeln, ohne deren Risiko für estrogenabhängige Krebsformen zu erhöhen. Eine Kombination mit regelmäßiger körperlicher Aktivität könnte die Erfolge bei der Behandlung von Adipositas und deren Folgeerkrankungen noch maximieren bzw. eine geringere Dosierung der verwendeten Substanz bei gleichbleibendem Behandlungserfolg ermöglichen. Das Isoflavon Gen mit seiner Fähigkeit beide ERs zu aktivieren ist eine bereits auf dem Markt befindliche Substanz und wird zur Behandlung von postmenopausalen Beschwerden eingesetzt, obwohl mögliche negative Effekte (z.B. ein erhöhtes Brustkrebsrisiko) noch nicht abschließend geklärt sind. Falls diese Risiken von Gen ausgeräumt werden können, könnte diese Substanz eventuell eine kostengünstige Alternative darstellen, um sowohl postmenopausale Beschwerden als auch Adipositas und deren Folgekrankheiten zu behandeln.

Thesis (PhD (Botany and Zoology))--Stellenbosch University, 2008. / Endocrine disruption is an alteration of the chemical messaging processes in the body. The value of studies‐ and monitoring of endocrine disruption using techniques included in the field of toxicogenomics is undoubtedly supported by scientific literature over the past four decades, as is demonstrated in Chapter 1 where I review relevant literature on the topic. Clearly, well sustained bio‐monitoring will include studies both in vitro and in vivo, and very well on transcriptional and translational levels. Animals are providing good models for in vivo studies to report or monitor endocrine disruption. It is imperative though to first understand such an animal’s biology, especially its endocrine system, and characterize what is considered “normal” for a species before engaging in endocrine disrupting exposures. A multitude of studies report endocrine disruption in relation to reproductive systems, with more recent work illustrating alteration of metabolism related to thyroidogenic disruption within the last decade. It is therefore essential to consider sex determination and ‐differentiation when studying sentinel species. Apart from the obvious academic interest in the matter of sex differentiation, altered patterns of sex differentiation in certain appropriate species provide for a very convincing endpoint in monitoring estrogenic endocrine disruption. As I approach to study a potential sentinel species for the southern African subcontinent, I set forward to study aspects of endocrine disruption influencing the reproductive system in a piece‐meal manner, starting with estrogenic endocrine disruption as this is the best studied facet of the endocrine disruption hypothesis to date. Yet, one learn from vast amounts of literature that in cases where sex is not exclusively determined by the genetic fraction of an individual, a number other characteristics may very well be used to determine estrogenic disruption in ecosystems. Quantitative production of the egg yolk precursor protein (vitellogenin) resides under these characteristics, and in the proposed sentinel, South African tilapiine, Oreochromis mossambicus phenotypic sex can be altered by environmental sex determination. The present study therefore targeted firstly the product most often used in tier I screening processes, vitellogenin (VTG). Specimens of O. mossambicus were cultured for this purpose from wild breeding stock, sampled at 5 day intervals and the transcription levels of vitellogenin gene (vtg) studied in those. Hereby, Chapter 2 describes the cloning of partial vtg gene and subsequent temporal expression of vtg quantitatively in O. mossambicus. To shed light on the state of gonadal differentiation sub‐samples were subjected to histology, illustrated in Chapter 3. In addition the quantitative vtg responses has been described in this study at a transcriptional level, both of adult males and juveniles subjected to low and very high levels of natural estrogens. In addition, a 3 kb 5’ flanking region of vtg was cloned and sequenced, and several putative binding sites identified for transcription factors of vtg, including several estrogen responsive elements (EREs). These indicate the expected regulational process of vtg by estrogens. Subsequently I measured the transcription levels of the only enzyme capable of aromatizing androgens into estrogens, Cytochrome P450 19 (cyp19) as has been characterized in Chapter 3. For stable binding of an estrogen to an ERE, binding of the ligand to its specific nuclear receptor (Estrogen receptor, ESR) is required. Since E2 is known to have different mechanisms of action in vertebrates, the expression levels of the ESRs were evaluated in our sample set after cloning 3 different homologues of ESR in O. mossambicus. The results on this matter is discussed in Chapter 4 and provides in addition to data on vtg and cyp19 a platform of “normal” transcription levels of these candidate genes involved in estrogenic endocrine disruption of O. mossambicus. Ultimately, characterization of those candidate genes involved extensively in phenotypic sex, contribute to our understanding of sex determination and differentiation in this species in a small way.

Oocyte maturation (OM) in teleosts is under precise hormonal control by estrogens and progestins. We show here that estrogens activate an epidermal growth factor receptor (EGFR) signaling pathway through the G protein-coupled estrogen receptor (GPER) to maintain meiotic arrest of full-grown zebrafish (Danio rerio) oocytes in an in vitro germinal vesicle breakdown (GVBD) bioassay. A GPER- specific agonist decreased OM and a GPER-specific antagonist increased spontaneous OM, whereas specific nuclear estrogen receptor (ERα and ERβ) agonists did not affect OM, which suggests the inhibitory action of estrogens on OM are solely mediated through GPER. Furthermore, a peptide-bound estrogen, which cannot enter the oocyte, decreased GVBD, showing that these estrogen actions are mediated through a membrane receptor. Treatment of oocytes with actinomycin D, a transcription inhibitor, did not block the inhibitory effects of estrogens on OM, indicating that estrogens act via a nongenomic mechanism to maintain oocyte meiotic arrest. EGFR mRNA was detected in denuded zebrafish oocytes by reverse transcription polymerase chain reaction (RT-PCR). Therefore, the potential role of transactivation of EGFR in estrogen inhibition of OM was investigated. The matrix metalloproteinase inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor (HB-EGF), increased spontaneous OM. Moreover, specific EGFR1 (ErbB1) inhibitors and inhibitors of extracellular-related kinase 1 and 2 (ERK1/2) increased spontaneous OM. Previously, estrogens have been shown to increase 3’-5’-cyclic adenosine mono phosphate (cAMP) levels through GPER in zebrafish oocytes during meiotic arrest. Taken together these present results suggest that estrogens also act through GPER to maintain meiotic arrest through a second signaling pathway involving transactivation of EGFR and activation of ERK 1 and 2. / text

The use of small molecules to probe the function of proteins is referred to as chemical genetics. The Proteolysis Targeting Chimera (PROTAC) is a chemical genetic tool that contains the ligand for a target protein of interest and the recognition motif for an E3 ubiquitin ligase attached by a linker. The PROTAC is capable of binding to and recruiting specific target proteins to the intracellular degradation system, the ubiquitin proteasome system (UPS). While the approach has had success it has not been optimized to be used on a broader scale. Optimization efforts focused on elucidating the ideal linker length between the ligand and the E3-ligase recognition motif, the preferred location for attachment of the linker to the two moieties, and the possibility for a dimeric PROTAC comprised of two ligands. An estrogen receptor (ER)-targeting PROTAC was chosen as a model for optimization attempts as the ER is known to have pathological significance in breast cancer. Optimization of the PROTAC technology will not only provide a novel tool to probe ER biology, but may also offer a novel approach to breast cancer therapies. The ER targeting PROTAC constitute the 17β-estradiol (E2), as the ligand for ER and a pentapeptide derived from HIF-1α as the E3-ligase recognition motif, joined by a linker. Following the successful synthesis and evaluation of a number of PROTACs, it was revealed that an optimum ER-targeting monomeric PROTAC (KC-3) has a spacer of 16 atoms between the E2 and HIF-1α pentapeptide. The spacer is attached at the C-7α position on E2 and at the N-terminus of the HIF-1α pentapeptide. It was also established that the PROTAC is capable of targeting the ER for degradation in a proteasome and E3- ligase dependent manner, which translated to a decrease in the proliferation of MCF-7 cells with an IC50 similar to that of tamoxifen. KC-3, in comparison with E2, displayed lower agonistic activity on an ER-regulated downstream target, the progesterone receptor (PR). A dimeric PROTAC more effectively binds and degrades the ER in a proteasome dependent manner, suggesting that the dimeric ligand approach may be applied to the design of other PROTACs.

This thesis investigates the biological activity of selected non-steroidal analogues of sex steroid hormones by examining two different effects of analogues on endogenous sex hormone activity. Non-steroidal analogues of sex hormones were synthesised to study their biological interactions with a sex steroid receptor and a sex steroid metabolising enzyme. Chapter One introduces the steroid hormones and their physiology, which leads to a review of the mechanisms by which steroids exert their effects. Their implication in disease is discussed, with particular emphasis on the sex steroids. As the biological activity of steroids is related to their chemical structure, the important features of steroid structure are identified, including the cyclopentanoperhydrophenanthrene nucleus, arrangement of ring substituents and ring junction conformation. The concept of non-steroidal analogues of steroids is introduced, and the harmful or beneficial effects analogues have on endogenous steroid activity are considered. Alteration of steroid activity and its consequences are focussed on two main areas; the potential adverse effects of environmental chemicals which mimic sex steroid activity, and the use of non-steroidal analogues in medicinal chemistry for treating sex steroid related disease. Chapter Two describes an investigation into the 17β-estradiol mimicking activity of non-steroidal analogues. Exogenous chemicals that mimic estradiol are of concern as they may alter endogenous estradiol activity and disrupt endocrine systems. Firstly, an introduction to the field of research concerned with environmental chemicals that mimic steroid hormones is given. The interaction of xenoestrogens with the estrogen receptor is described, as are the methods available for assessing the estrogen mimicking activity of xenoestrogens. The concern for insecticides mimicking estrogen activity is described by reviewing reported activities of insecticides, which leads into a discussion of work carried out as part of this thesis. Metabolites of the pyrethroid insecticides permethrin and cypermethrin, 2.14, 2.15, and 2.16 were synthesised while others were commercially obtained. The interaction of pyrethroid insecticide metabolites with the human estrogen receptor expressed in recombinant yeast (Saccharomyces cerevisiae) was studied, following the establishment and validation of the assay. Metabolites 2.11, 2.12, and 2.14 were found to weakly stimulate estrogen receptor-mediated estradiol responsive gene expression in the yeast assay (105 less active than 17β-estradiol). Since the activity of the metabolites using the yeast assay was greater than for the parent compounds, metabolic pathways need to be considered when assessing the impact of exposure to environmental estrogens. The low estrogenic activity suggests these compounds are not individually contributing significantly to the xenoestrogenic impact on humans, but will add to total xenoestrogen exposure. Chapter Three describes the inhibition of a sex steroid metabolising enzyme, steroid 5a-reductase, by novel non-steroidal compounds. Inhibitors of this enzyme are potentially useful therapeutic agents for regulating the activity of an androgen in prostate disorders. A review of the literature on non-steroidal inhibition of 5a-reductase identified three key structural features known to enhance inhibitor potency; ring substitution, position and nature of ring unsaturation and angular methyl group presence. These features were taken into account in the design of inhibitors synthesised in this thesis (3.55-3.57, 3.59, 3.61, 3.62, 3.110 and 3.111). Inhibitors consisting of non-steroidal 5- or 1-aryl pyridone scaffolds were synthesised to investigate SAR for 4'-substituents. The 5-aryl 1-methyl-2-pyridone/piperidone scaffold of compounds 3.55-3.57 and 3.59 was constructed by Suzuki cross coupling methodology, while the 1-aryl 2-methyl 2,3-dihydro-4-pyridone scaffold of 3.61 and 3.62 was constructed by aza Diels-Alder methodology. Long carbon chain olefin containing tethers 3.107 and 3.108 were synthesised for conjugation to inhibitor 3.57 by cross metathesis to give conjugates 3.110 and 3.111. Compounds 3.55-3.57, 3.59, 3.61, 3.62, 3.110 and 3.111 inhibited the type 1 5a-reductase isozyme expressed by HEK-I cells, with activities comparable to those of related literature compounds. The 1-aryl 2,3-dihydro-4-pyridone 3.62 inhibited both the type 1 and 2 isozymes (expressed by HEK-II cells) of 5a-reductase. The presence of bulky hydrophobic groups (benzoyl, long chain tethers) at the 4' position enhanced the potency of type 1 inhibition by 5-aryl pyridone type compounds in comparison to N,N-diisopropyl- and N-allylacetamide groups. This information provides further understanding of SAR within and across different classes of non-steroidal inhibitors of steroid 5a-reductase towards improved drug design.

<p>Lysine specific demethylase 1 (LSD1) is a FAD-dependent amine oxidase enzyme responsible for removing methyl groups from the side chain nitrogen of lysine within histones in order to regulate gene transcription. By its interaction with various transcriptional complexes, including those containing estrogen receptor &alpha; (ER&alpha;), LSD1 mediates expression of many genes important in cancer proliferation and progression. Herein, we report our efforts towards understanding the function of LSD1 in breast cancer. We have developed a straightforward method for the syntheses of 2-arylcyclopropylamines as irreversible mechanism-based inactivators of LSD1. We employed these small molecules as probes of LSD1 activity, and together with experiments involving the knockout of LSD1 by small interfering RNA (siRNA), we have shown that LSD1 activity is essential for both ER&alpha;-postive and ER&alpha;-negative breast cancer proliferation. LSD1 inhibitors induce a dramatic cell cycle arrest without causing apoptosis. </p><p>Furthermore, we observe that LSD1 and ER&alpha; work cooperatively to express certain estrogen-target genes through simultaneous recruitment to promoters; LSD1 inhibition diminishes ER&alpha; recruitment. Similarly, knockdown of CoREST, a binding partner of LSD1, results in comparable changes in gene expression. Although, we have not observed a direct interaction between LSD1 and ER&alpha;, we believe that CoREST may be facilitating this interaction. We have made efforts to inhibit the interaction between LSD1 and CoREST <italic>in vitro</italic> in hopes of targeting this interface in breast cancer cells in order to disrupt the necessary functional complex and prevent LSD1 activity.</p> / Dissertation

Le récepteur des oestrogènes (RE) peut moduler l’expression de gènes impliqués dans les processus de prolifération et d’apoptose cellulaires. Cette régulation est possible par le recrutement de complexes corégulateurs. Dans ces complexes, l’activité répressive s’explique essentiellement par la présence d’histones désacétylases (HDAC). Cette famille de protéines est composée de 18 membres classés en 4 groupes. Cette répartition est due aux similarités structurales et de fonctions de ces enzymes. Il y a la classe I (HDAC 1, -2, -3, -8), la classe II (HDAC 4, -5, -6, -7, -9, -10) et la classe IV (HDAC 11) qui ont une activité Zn2+ dépendante alors que la classe III (Sirt1-7) recense les HDAC avec une activité NAD+ dépendante. Des résultats récents du laboratoire ont montré, qu’au niveau ARNm, il y avait un important différentiel d’expression de HDAC9 entre les lignées cellulaires de cancer du sein RE positive et négative ou résistante au tamoxifène. Durant ma thèse, j’ai démontré que la régulation de HDAC9, au niveau de son expression, comme au niveau de ses fonctions, affecte la signalisation oestrogénique en modulant l’expression et l’activité transcriptionnelle de REα. De plus, de nombreuses études ont montré l’activité antiangiogénique d’inhibiteurs de HDAC (HDI) à large spectre comme la TSA (Tricostatin A). La conception et l’identification de HDI, potentiellement sélectifs, comme agents anti-tumoraux et/ou anti-métastatique représente une nouvelle approche de thérapie seule ou combinée avec les produits déjà utilisés dans le traitement du cancer. Ainsi, afin d’identifier et caractériser de nouveaux HDI, j’ai établi un outil bioluminescent pour la détection d’inhibiteurs sélectifs de HDAC. Plusieurs lignées cellulaires Gal4-VP16-HDAC ont été générées dans ce but / The estrogen receptor (ER) can modulate the gene expression with consequences in the cell proliferation, apoptosis. This modulation is possible by the recruitment of coactivator or corepressor complexes. The repression activity is in particular explained by the histones deacetylases (HDACs). This protein family is composed by eighteen members who have been classified in four groups. These HDACs are subdivided on structural and functional similarities. The class I isoforms (HDACs 1, 2, 3 and 8), class II (HDACs 4, 5, 6, 7, 9, 10) and class IV (HDAC11) are Zn-dependent enzymes, whereas class III HDACs (Sirtuins 1-7) are NAD+-dependent. Recent data from the laboratory have shown, at the mRNA level, there is an enormous expression differential of HDAC9 between breast cancer cell line ER positive and negative or OHT resistant cell line. During my thesis, I demonstrated that the regulations of the HDAC9 on the level of its expression as of its role in the various breast cancer cell lines were implicated in the estrogen signaling. This regulation takes place at the transcriptional level and in the ERet#945; activity.In addition, using broad spectrum HDAC inhibitors (HDIs) such as TSA (Tricostatin A), many studies have shown that these inhibitors had antiangiogenic activity. Thus, the design or the identification of selective and potent HDAC inhibitors as agents anti-tumoral and/or anti-metastatic can emerge in a novel opportunity used alone or in combination with the already existing agents for the treatment of cancers. In order to identify and characterize new HDIs, my thesis works consisted to establish bioluminescent cell lines for screening HDAC inhibitors. Different cell GAL4-VP16-HDACs chimeras' models were generated to determine the selectivity of HDIs for the different HDACs.