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<p>Important candidate genes to human bone phenotypes are those involved in the regulation of hormonal action, such as the vitamin D receptor (VDR) and the estrogen receptor-α (ERα) genes and their cofactors. RIZ1 is a specific ERα cofactor proved to strongly enhance the function of the ERα. </p><p>The main focus of this thesis has been to study genetic variants in the VDR and RIZ genes and their associations to human bone phenotypes using candidate gene and functional approaches. Specifically, polymorphisms in the VDR 3’ untranslated region (UTR) and a deletion/insertion polymorphism of a proline in the RIZ gene were investigated.</p><p>The candidate gene approach was applied to large-scale population-based cohorts of pre-and post-menopausal women from Sweden and of elderly men from Sweden and Hong Kong. VDR 3’ UTR polymorphisms were associated with peak bone mass and body composition in young women. Further analysis of common VDR 3’ UTR haplotypes confirmed the association with BMD and risk of fractures in elderly men from Sweden and Hong Kong. The VDR polymorphisms were investigated for cis-acting effects, affecting allelic expression in the normal chromosomal context of human bone cells. The VDR allelic transcripts in the bone samples were unequally expressed, suggesting presence of regulatory variants in the 3’ UTR. </p><p>The polymorphism in the RIZ gene was strongly associated to BMD in pre- and postmenopausal women and in elderly men. The functional analyses included reporter constructs containing the RIZ polymorphic variants transfected in a cell line and its abilities in coactivating the ERα were examined. The variants were functionally different in coactivating the ERα-receptor complex. </p><p>To summarize, the results of this thesis show novel evidence for functional relevant polymorphisms in candidate genes to human bone phenotypes. These polymorphisms may contribute to the variation seen in BMD and risk of fractures in the population.</p>

Important candidate genes to human bone phenotypes are those involved in the regulation of hormonal action, such as the vitamin D receptor (VDR) and the estrogen receptor-α (ERα) genes and their cofactors. RIZ1 is a specific ERα cofactor proved to strongly enhance the function of the ERα. The main focus of this thesis has been to study genetic variants in the VDR and RIZ genes and their associations to human bone phenotypes using candidate gene and functional approaches. Specifically, polymorphisms in the VDR 3’ untranslated region (UTR) and a deletion/insertion polymorphism of a proline in the RIZ gene were investigated. The candidate gene approach was applied to large-scale population-based cohorts of pre-and post-menopausal women from Sweden and of elderly men from Sweden and Hong Kong. VDR 3’ UTR polymorphisms were associated with peak bone mass and body composition in young women. Further analysis of common VDR 3’ UTR haplotypes confirmed the association with BMD and risk of fractures in elderly men from Sweden and Hong Kong. The VDR polymorphisms were investigated for cis-acting effects, affecting allelic expression in the normal chromosomal context of human bone cells. The VDR allelic transcripts in the bone samples were unequally expressed, suggesting presence of regulatory variants in the 3’ UTR. The polymorphism in the RIZ gene was strongly associated to BMD in pre- and postmenopausal women and in elderly men. The functional analyses included reporter constructs containing the RIZ polymorphic variants transfected in a cell line and its abilities in coactivating the ERα were examined. The variants were functionally different in coactivating the ERα-receptor complex. To summarize, the results of this thesis show novel evidence for functional relevant polymorphisms in candidate genes to human bone phenotypes. These polymorphisms may contribute to the variation seen in BMD and risk of fractures in the population.

La protéine kinase D1, PKD1, est une nouvelle sérine/thréonine kinase activée par de nombreux mitogènes et dérégulée dans de nombreux types de cancers dont le cancer du sein, ce qui suggère un rôle de cette kinase dans la prolifération cellulaire et la tumorigenèse. Cependant, le rôle précis et les cibles de PKD1 ne sont pas encore bien connus. Au cours de ce travail, nous avons tout d’abord démontré que PKD1 est activée par les facteurs de croissance épidermique (EGF) et fibroblastique (FGF) et qu’elle régule la voie de signalisation de l’insulin-like Growth Factor-I (IGF-I). D’autre part, nos résultats démontrent que PKD1 favorise les propriétés pro-prolifératives et pro-tumorales des cellules MCF-7 dérivées d’un adénocarcinome mammaire humain estrogéno-dépendant. Ces mécanismes mettent en jeu des voies de signalisation dépendantes de protéines kinases (la voie MEK/ERK) et hormonales (la voie estrogène/REα). Ainsi, l’ensemble de ce travail fait apparaître PKD1 comme une nouvelle cible thérapeutique anti-tumorale potentielle. / Protein kinase D1, PKD1, is a novel serine/threonine kinase which can be activated by mitogens and whose expression is altered in many tumors such as breast cancer, suggesting a role for this kinase in cancer development. However, its precise role and targets are still unclear. Our study identified PKD1 as a new regulatory kinase implicated in the control of IGF-I signal transduction pathway. Furthermore, we showed that PKD1 enhances estrogen-dependent MCF-7 breast cancer cell proliferation and tumorigenesis through the regulation of MEK/ERK and estrogen/ERα pathways. Thus, this work may define PKD1 as a novel potential anti-tumor therapeutic target.

MyD88 est une protéine adaptatrice clé dans la signalisation des TLRs/IL-1R qui mène à l'activation de NF-KB et des MAPK, et à la production de cytokines inflammatoires. MyD88 participe à la tumorigénèse par le biais de son activité inflammatoire dans la signalisation des TLRs/IL-1R, et également via son interaction directe avec la kinase Erk dans la cellule cancéreuse. Dans cette thèse, nous identifions de nouveaux partenaires protéiques de MyD88 et nous examinons comment leurs interactions peuvent réguler sa fonction. Nous démontrons que MyD88 interagit avec Ubc9, ce qui conduit à sa sumoylation, et que cette modification posttraductionnelle régule négativement l'inflammation dépendante de MyD88. Nos résultats montrent également que MyD88 interagit avec le récepteur nucléaire, ER-α, et que cette interaction est nécessaire pour la réponse inflammatoire. Enfin, nous avons étudié l'importance de l'interaction MyD88/Erk dans le maintien de la transformation des tumeurs dépendant de l'oncogène Ras. Ces résultats pourraient éventuellement être exploités pour cibler MyD88 et ses interactions dans le traitement des maladies inflammatoires et le cancer / MyD88 is a protein that is at the interface between inflammation and cancer. It is the key adaptor protein used by TLRs/IL-1R to mediate their downstream signaling, resulting in NF-κB and MAPK activation, and inflammatory cytokine production. MyD88 also plays a role in tumorigenesis via two mechanisms, an inflammatory one dependent on its function in TLRs/IL- 1R signaling, and an intrinsic, cell-autonomous mechanism mediated by its interaction with the kinase Erk. Based on the different roles played by MyD88, this thesis work consisted in studying how MyD88 protein-protein interactions can regulate its function. We show that MyD88 interacts with Ubc9, resulting in its sumoylation and subsequent negative regulation of MyD88- mediated inflammation. We also demonstrate that MyD88 interacts with the nuclear receptor ER-α, an interaction necessary for the inflammatory response. Finally, we have studied the importance of the MyD88/Erk interaction in the maintenance of the transformed phenotype of Ras-dependent tumors. These findings could eventually be exploited to target MyD88 and its interactions in the treatment of inflammatory disorders and cancer

Στις μετεμμηνοπαυσιακές γυναίκες με καρκίνο μαστού, θετικό για οιστρογονικούς υποδοχείς, μετά από χειρουργική θεραπεία, η μακροχρόνια χορήγηση της ταμοξιφαίνης (SERM πρώτης γενεάς), έχει αποδειχθεί ευεργετική. Ο σκοπός της παρούσης μελέτης, ήταν να διαπιστώσει, εάν οι πολυμορφισμοί του γονιδίου των ER (PvuII & XbaI του ERα και οι RsaI & AluI του Erβ), οι οποίοι έχουν συσχετισθεί με καρκίνο μαστού, σχετίζονται με το στάδιο του καρκίνου του μαστού ή την ανταπόκριση του ενδομητρίου στην μακροχρόνια αγωγή με ταμοξιφαίνη, στις μετεμμηνοπαυσιακές γυναίκες με καρκίνο του μαστού. Η μελέτη περιέλαβε 87 μετεμμηνοπαυσιακές γυναίκες με καρκίνο μαστού θετικό για οιστρογονικούς υποδοχείς, στις οποίες χορηγήθηκε ταμοξιφαίνη. Η μέση ηλικία των ασθενών ήταν 58,7± 4,7 έτη, και η μέση διάρκεια της αγωγής με ταμοξιφαίνη ήταν 3,9 ± 1,1 έτη. Το γονιδιακό DNA απομονώθηκε από τα λευκά αιμοσφαίρια δειγμάτων περιφερικού αίματος με την κλασσική μέθοδο φαινόλης - χλωροφορμίου. Τα κλάσματα των γονιδίων των ERα και Erβ, τα οποία συμπεριελάμβαναν τις θέσεις των πολυμορφισμών, πολλαπλασιάσθηκαν με την αλυσιδωτή αντίδραση πολυμεράσης (PCR). Ο προσδιορισμός της παρουσίας των πολυμορφισμών στο DNA πραγματοποιήθηκε με την χρήση ενζύμων περιορισμού. Συμπερασματικά, στις Ελληνίδες μετεμμηνοπαυσιακές γυναίκες με καρκίνο του μαστού υπό αγωγή με ταμοξιφαίνη, οι πολυμορφισμοί των οιστρογονικών υποδοχέων, δεν συνδέθηκαν, ούτε με την παρουσία παθολογίας του ενδομητρίου, ούτε με το στάδιο του καρκίνου του μαστού. / In postmenopausal women with estrogen receptor (ER) positive breast cancer, after surgical treatment long term tamoxifen administration has been proved beneficial. The aim of the present study was to identify whether these ER gene polymorphisms are associated with breast cancer stage or endometrial responsiveness to long-term tamoxifen treatment in postmenopausal women with breast cancer. The study included 87 postmenopausal women with estrogen receptor positive breast cancer treated with tamoxifen. The mean age of patients was 58,7 ± 4,7 years and the mean duration of Tamoxifen treatment was 3.9 ± 1,1 years. Genomic DNA was extracted from peripheral blood leukocyte samples by the standard phenol/chloroform procedure. Fragments of the ERα and ERβ genes encompassing the polymorphic sites were amplified by the polymerase chain reaction (PCR). The determination of presence of polymorphisms in the DNA was realised with restriction endonucleases. Ιn conclusion, in Greek postmenopausal women with breast cancer under tamoxifen treatment, Estrogen Receptors polymorphisms were not linked to either the presence of endometrial pathology or the stage of breast cancer.