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About 70% of breast tumors express the estrogen receptor alpha (ERα). Antiestrogens (AEs) are used to treat all stages of ER+ breast cancer. There are two types of AEs: Selective Estrogen Receptor Modulators (SERMs) and Selective Estrogen Receptor Downregulators (SERDs). SERMs such as Tamoxifen (Tam) have tissue-specific partial agonist activity, while SERDs such as Fulvestrant or Faslodex (ICI182, 780) fully repress estrogen target genes regardless of the tissue and cell type. Previously, it has been reported that SERDs induce ERα ubiquitination and degradation. ERα is also SUMOylated in the presence of SERDs. Abrogating SUMOylation of ERα using a deSUMOylase (SENP1) resulted in a partial de-repression of estrogen target genes in the presence of SERDs. Mapping the domains using deletion mutagenesis in the presence of ICI 182,780 showed that C-terminal domain (CDEF regions) is required of the ICI induced modification but not the N-terminal domain (AB region). Thus, a detailed dissection of the structural determinants for the selective action of SERDs on ERα SUMOylation and ubiquitination remained unknown. Our work shows that pure antiestrogens like ICI182,780 induce SUMOylation and ubiquitination of ERα but not ERβ in live cells. Utilizing the fact that domains of ERα and ERβ display sequence homology, we designed chimeras to map the minimal domain required for ERα modification in the presence of antiestrogens. Interestingly, swapping domains between ERα and ERβ showed that the Ligand Binding Domain (LBD) of ERα is sufficient to confer the induction of ERα modification in the presence of AEs such as Raloxifene (Ral) and ICI182,780. Further dissecting this region, we also found that helices 3 to 6 (H3H6) located in the LBD region is sufficient to confer the induction of SUMOylation and ubiquitination in the ICI182,780. Importantly, the lysine residues in this region between ERα and ERβ are conserved, which suggests that conformational differences in the LBD determine the capacity of ICI182, 780 bound ERα to be modified by SUMO and ubiquitin. Replacement of Leucine at position 536 in helix 12 (H12) of ERα’s LBD by a Valine residue or mutating Aspartate at position 351 abolished the increase in SUMOylation and ubiquitination observed in the presence of Ral. This suggested that Ral, a SERM, required a different set of determinants than ICI182,780 present in the LBD of ERα. vi Our work has also showed that saturating concentrations (increasing the amount of drug added will not result in a higher response) of ICI 182,780 modified and fully repressed constitutively active mutations such as Y537C, N or S and D538G. Other mutation such V534E and L536R/Q mutants exhibited some residual activity and were not modified in the presence of saturating concentrations of ICI182,780. Interestingly, the loss of SUMOylation correlated with the partial resistance to AEs. Structure function analysis of residues at position 536 indicates amino acids with a bulky hydrophobic side chain residue at this position result in preservation of ERα modifications in the presence of ICI 182,780. However, Using BRET-FECT, we have demonstrated that ERαwt/L536R heterodimerize and have intermediate levels of SUMOylation compared to ERαwt in the presence of ICI 182,780. Our results shed light onto the molecular basis for the diverse pharmacological properties of antiestrogens and should help guide the design of novel SERDs for breast cancer treatment. / Environ 70% des cancers du sein expriment le récepteur des oestrogènes alpha (ERα). Les anti-oestrogènes (AEs) sont utilisés pour traiter tous les stades de cancer du sein ER+. Il y a deux types d’AEs : les Selective ER Modulators (SERMs) et les Selective ER Downregulators (SERDs). Les SERMs, comme le Tamoxifen (Tam), ont une activité agoniste partielle tissu-spécifique, alors que les SERDs, tel Fulvestrant ou Faslodex (ICI182,780), répriment entièrement les gènes cibles d’ER, quel que soit l’organe ou le type cellulaire. Il a précédemment été montré que les SERDs induisent l’ubiquitination et la dégradation d’ERα. ERα est aussi SUMOylé en présence des SERDs. Supprimer la SUMOylation d’ERα en utilisant une déSUMOylase (SENP1) résulte en une dérépression partielle des gènes cibles d’ER en présence de SERDs. La délétion successive des différents domaines d’ERα en présence d’ICI182,780 a révélé que la région C-terminale (domaines CDEF) est requise pour la modification induite par ICI, mais pas la région N-terminale (domaines AB). Ainsi, la dissection détaillée des déterminants structuraux responsables de l’activité sélective des SERDs pour la SUMOylation et l’ubiquitination d’ERα reste à entreprendre. Nos travaux montrent que les AEs purs comme ICI182,780 induisent la SUMOylation d’ERα, mais pas d’ERβ, dans des cellules en culture. Tirant profit de l’homologie de séquences des différents domaines d’ERα et ERβ, nous avons conçu des chimères pour cartographier la région minimale requise pour la modification d’ERα en présence d’AEs. De manière intéressante, l’interversion des domaines d’ERα et ERβ a montré que le domaine de liaison au ligand (LBD) d’ERα est suffisant pour permettre l’induction de la modification d’ERα en présence d’AEs tels le Raloxifene (Ral) et ICI182,780. En décortiquant davantage ce domaine, nous avons trouvé que les hélices 3 à 6 (H3H6) du LBD sont suffisantes pour induire la SUMOylation et l’ubiquitination d’ERα en présence d’ICI182,780. De manière importante, les résidus Lysine de cette région sont conservées entre ERα et ERβ, ce qui suggère que des différences conformationnelles entre les deux LBD déterminent la capacité d’ERα lié par ICI182,780 d’être modifié par SUMO et l’ubiquitine. La mutation de la Leucine à la position 536 dans l’hélice H12 du LBD d’ERα par une Valine, ou la mutation de l’Aspartate à la position 351 abolissent l’augmentation de la SUMOylation et l’ubiquitination observée en présence de iv Ral. Cela suggère que Ral, un SERM, requière différents déterminants structuraux du LBD d’ERα qu’ICI182,780. Nos travaux ont aussi montré que des concentrations saturantes (l’augmentation de la quantité de drogue ajoutée ne mènera pas à une réponse plus élevée) d’ICI182,780 modifient et répriment entièrement des mutants constitutivement actifs d’ERα comme Y537C, N ou S et D538G. D’autres mutants, tels V534E et L536R/Q, présentent une activité résiduelle et ne sont pas modifiés sous traitement avec des concentrations saturantes d’ICI182,780. De façon intéressante, la perte de SUMOylation corrèle avec la résistance partielle aux AEs. Une analyse structure – fonction des résidus à la position 536 indique que les acides aminés avec une chaine latérale hydrophobe volumineuse à cette position permettent de préserver les modifications d’ERα en présence d’ICI182,780. Cependant, en utilisant la technique BRET-FECT, nous avons démontré que les récepteurs ERα sauvage et L536R forment un hétérodimère qui présente des niveaux intermédiaires de SUMOylation en présence d’ICI182,780. Nos résultats révèlent les bases moléculaires des diverses propriétés pharmacologiques des AEs et devraient aider à guider la conception de nouveaux SERDs pour le traitement des cancers du sein.

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Invasiv äggstockscancer anses vara en av de mest dödligaste gynekologiska maligniteterna. Granulosacelltumör i äggstocken är en sällsynt subtyp av äggstockstumör, som utgör 1–5% av alla äggstockstumörer. De kan kännetecknas av den långsamma tillväxthastigheten och produktionen av höga östrogennivåer. Återfallsfrekvensen efter den första behandlingen är låg, men dödligheten på ett återfall är så hög som 80%. Det är därför av intresse att undersöka nya terapeutiska mål som kan användas för att behandla granulosacelltumör i framtiden. Granulosacelltumörer tros uppstå från de sena preovulatoriska granulosacellerna eftersom de delar liknande egenskaper; de uttrycker follikelstimulerande hormonreceptorer och producerar östrogen som respons på follikelstimulerandehormoner. Östrogen och dess motsvarande kärnreceptorer, östrogenreceptor α och β spelar en avgörande roll i utvecklingen av äggstocksfolliklarna under äggstockscykeln. Uttrycket av östrogenreceptorn α är ganska lågt i granulosacellerna, istället tros östrogenreceptorn β (ERβ/ESR2) spela den övervägande rollen att aktivera intracellulära signalvägar som främjar cellproliferation och överlevnad i äggstocken. Det finns också många varianter av östrogenreceptorn β, främst ERβ1, ERβ2 ibland kallad för ERβcx, ERβ3, ERβ4, och ERβ5. Deras roll i äggstocken är fortfarande okända och har varit svårt att studera eftersom de saknar ligand-bindningsdomänen. DNA-bindningsdomänen är fortfarande bevarad, så de kan fortfarande vara viktiga transkriptionsfaktorer i äggstocken. Nya ChIP-sekvenseringsresultat från en studie visade att ERβ och leverreceptorn homolog 1 (LRH-1/NR5A2) delar många av kromatinbindningsställena i en normal musäggstock. Detta resultat tyder på att dessa två transkriptionsfaktorer troligtvis interagerar med varandra fysiskt eller indirekt. ChIP-qPCR användes för att bekräfta ERβ-varianternas kromatinbindning i den mänskliga granulosatumörcellinjen COV434. Dubbel luciferasanalys användes för att undersöka om ERβ påverkar LRH-1s transaktivering aktivitet. Co-IP utfördes för att undersöka om ERβ och LRH-1 fysiskt interagerar med varandra. Vi kunde bekräfta att ERβ-varianterna ERβcx, ERβ5 och ERβ4 binder till LRP6 genen och att varianterna kan binda direkt till DNA. För att ytterligare bekräfta flera målgener för ERβ-varianterna måste DNA-proverna skickas för sekvensering. Resultaten från denna studie indikerar att ERβ undertrycker LRH-1s transaktiverings aktivitet med eller utan liganden östradiol, hur ERβ fungerar som repressor är fortfarande okänt. Vi kunde inte visa om ERβ fysiskt interagerar med LRH-1 i denna studie. / Invasive ovarian cancer is considered to be one of the most fatal gynecological malignancies. Granulosa cell tumor in the ovary is a rare subtype of ovarian tumor that makes up almost 1-5% of all ovarian tumors. It can be characterized by the slow growth rate and production of high estrogen levels. The recurrence rate after the first treatment is fairly low, but the mortality rate for the recurrence is as high as 80%. Therefore, it is interesting to investigate new therapeutic targets that can be used to treat granulosa cell tumors in the future. Granulosa cell tumors are thought to originate from the late preovulatory granulosa cells because they share similar features; they express follicle-stimulating hormone receptors and produce estrogen in response to follicle-stimulating hormone. Estrogen and its corresponding nuclear receptors, estrogen receptors α and β, play a crucial role in the development of the ovarian follicles during the ovarian cycle. The expression of the estrogen receptor α is relatively low in the nucleus of the granulosa cells; instead, estrogen receptor β (ERβ/ESR2) is thought to play the predominant role of activating intracellular signaling pathways that promote cell proliferation and survival in the ovary. There are also many splice variants of the estrogen receptor β, mainly ERβ1, ERβ2 or also sometimes called ERβcx, ERβ3, ERβ4, and ERβ5. Their role in the ovary is still unknown. Most of the splice variants lack the ligand-binding domain. However, the DNA-binding domain is still preserved, so they could be crucial transcriptional factors of different pathways in the ovary. Recent ChIP-sequencing results from a study showed that two nuclear receptors, ERβ and the liver receptor homolog 1 (LRH-1/NR5A2), share many chromatin binding sites in normal mouse ovary. This finding suggests that these two transcriptional factors may interact with each other physically or indirectly. ChIP-qPCR was used to confirm chromatin binding of ERβ’s splice variants in the human granulosa tumor cell line COV434. Dual luciferase assay was used to investigate if ERβ affects the transactivation activity of LRH-1. Co-IP was performed to investigate if ERβ and LRH-1 physically interact. We could confirm that LRP6 is a target gene of ERβ splice variants ERβcx, ERβ4 and ERβ5. We also demonstrated that these ERβ splice variants bind directly to DNA which has not been shown before. The DNA samples must be sent for further sequencing to confirm more target genes of the ERβ splice variants. The results from this study indicate that ERβ represses the transactivation activity of LRH-1, how ERβ acts as a repressor is still unknown. We could not show whether ERβ and LRH-1 physically interact in this study.

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Genome sequencing has resulted in the identification of >55,000 cytochrome P450 enzymes, many of which have an unknown function and regulation. In mammals, CYP3 genes appear in only one subfamily (CYP3A), which metabolize >50% of pharmaceuticals and some steroids in humans. Unlike mammals, fish contain genes in the CYP3A, CYP3B, CYP3C and CYP3D subfamilies. While it is commonly assumed that fish and mammalian CYP3A are functional similar, the function and regulation of fish CYP3 remains largely unknown. In this thesis, the receptors and compounds that regulate CYP3C genes in zebrafish were assessed. The induction of CYP3C genes in response to the aryl hydrocarbon (AHR) and estrogen receptor (ER) ligands, β-naphthoflavone and 17β-estradiol, was measured using quantitative PCR in intestine, liver and gonads. Zebrafish CYP3C genes were inducible by β-naphthoflavone and 17β-estradiol, implicating the aryl hydrocarbon and estrogen receptor in CYP3C gene regulation and suggesting that regulation of CYP3 genes in fish differs from that in mammals. To define the function of zebrafish CYP3A65 and CYP3C1, fluorogenic compounds which are specific markers of CYP1 and CYP3A activity in humans, were screened for metabolism by CYP3A65 and CYP3C1. Both CYP3A65 and CYP3C1 had the capacity to metabolize several of these compounds and the substrate profile overlapped with zebrafish CYP1A, suggesting that these compounds are not specific in fish. A high throughput approach was employed to screen ~4000 small biologically and pharmacologically active compounds for metabolism by CYP3A65 and CYP3C1, using NADPH consumption to assess catalytic activity. The substrate profiles of CYP3A65 and CYP3C1 largely overlapped and were different than mammalian CYP3A4. CYP3A65 and CYP3C1 appeared to have a bias for quinone-based compounds but further studies are required to confirm quinones as substrates and to assess a strong structure-activity relationship. Overall, this study provides insight on the regulation, function and evolution on CYP3 genes in fish. / Dissertation / Doctor of Philosophy (PhD) / Cytochrome P450 (CYP) enzymes break down compounds such as hormones and pharmaceuticals. While mammals have genes in the CYP3A subfamily, fish have unique subfamilies not found in mammals. The function and regulation of the CYP3 family in fish is unknown, but commonly assumed to be like human CYP3. The purpose of this thesis was to identify what receptors and compounds regulate CYP3C enzymes in zebrafish. We found that regulation of CYP3C enzymes in zebrafish is different than humans. Zebrafish CYP3C genes are regulated by the aryl hydrocarbon receptor and estrogen receptor, while human CYP3A is regulated by the pregnane-x-receptor. I used a high throughput approach to screen thousands of compounds to identify the function of CYP3A65 and CYP3C1 from zebrafish. CYP3A65 and CYP3C1 metabolize several plant-based and pharmaceutical compounds. CYP3A65 and CYP3C1 are more functionally similar to each other than to CYP3A in humans.