Disabled-2 (Dab2) is a mitogen-responsive adaptor protein playing a key role in multifaceted cellular functions, such as endocytosis, epithelial-mesenchymal transition (EMT), immune function, stem cell differentiation, oncogenesis, cell signaling, and inflammatory responses. The adenosine deaminase RNA-specific binding protein (ADAR1) is a multifunctional RNA-editing enzyme that can convert adenosine to inosine, which can modulate gene expression and cellular functions in multiple pathways, such as mRNA translation by changing codons and the subsequent protein sequence, pre-mRNA splicing by changing splice site sequences, RNA stability by altering sequence for nuclease recognition, and RNA structure-dependent functions by altering RNA-protein interactions. ADAR1 has displayed a largely pro-tumorigenesis role, especially its immunosuppressive function in cancer cells, which attributes ADAR1 as a potential novel immune checkpoint for cancer treatment. In our lab, we employed an F9 mouse teratocarcinoma stem cell differentiating model and confirmed that Dab2 is an indispensable element for retinoic acid (RA)-induced F9 cell differentiation. Interestingly, our new findings indicated that during the process of RA-induced F9 cell differentiation, both the protein levels of Dab2 and ADAR1 are significantly upregulated, and siRNA-mediated Dab2 silence results in the silence of ADAR1. In addition, results from EMT models and statistical analysis from the human TCGA database further indicated that there is a positive correlation between the expression of Dab2 and ADAR1. Our results imply that Dab2 and ADAR1 may cooperate with each other to modulate cellular functions, which will present a novel mechanism for the mechanistic study of Dab2 in tumorigenesis.

Angiogenesis is a crucial process under both physiological and pathological conditions. Vascular endothelial growth factor (VEGF) A and its cognate receptor, vascular endothelial growth factor receptor 2 (VEGFR2) are key regulators of angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by de novo synthesis, and by both exocytic and endocytic trafficking. VEGF-binding to VEGFR2 induces phosphorylation of key tyrosine residues located in the cytosolic domain of the receptor, followed by clathrin-mediated endocytosis and signal transduction leading to vascular morphogenesis. Disabled protein 2 (Dab2) is a cytosolic, clathrin-adaptor protein that is known to regulate endocytosis of certain cell surface receptors. Studies of Dab2 function have revealed its role in the development of embryonic vasculature. However, the mechanism of Dab2 function, particularly in conjunction with endosomal VEGFR2, remains poorly understood. Our results show that Dab2 interacts with VEGFR2 and that upon VEGF stimulation the two proteins co-localize within Rab5-positive early endosomes. Knockdown of Dab2 reduces levels of VEGF-induced phosphorylation of VEGFR2 at residue Y1175. This is significant because phosphorylation of VEGFR2-Y1175 is crucial for pro-angiogenic signal transduction. Moreover, knockdown of Dab2 causes an increased trafficking of VEGFR2 to late endosomes (LE). Finally, this altered VEGFR2 trafficking following Dab2 knockdown has major functional consequences for endothelial cells, as they are unable to undergo morphogenesis into tube-like structures in an in vitro assay of angiogenesis. Collectively, our data show that Dab2 plays a crucial role in VEGFR2 trafficking in the endocytic system and this impacts receptor signaling and endothelial cell morphogenesis during angiogenesis.

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Studies of prophylactic salpingectomy specimens from BRCA1/2 mutation carriers, at risk for tubal and ovarian high-grade serous carcinoma (SerCa), have consistently revealed occult carcinomas and putative histological cancer precursors in the distal fallopian tube epithelium (FTE), supporting the FTE as the source of SerCa. In this thesis I molecularly characterized and compared non-malignant FTE from mutation carriers (FTEb) and control patients (FTEn) to identify alterations that may predispose to malignant transformation. Gene expression profiling of laser capture microdissected FTEn, FTEb and SerCa indicated that SerCa have similar molecular profiles whether of presumed ovarian or tubal origin, supporting the notion they share a common cell of origin within the FTE. Furthermore, FTEb samples obtained during the post-ovulatory luteal phase showed gene expression profiles closely resembling SerCa samples, suggesting that the luteal phase milieu may contribute to serous carcinogenesis. An initial hypothesis was that FTEb may respond differently to luteal progesterone compared to FTEn, via differential expression of progesterone receptor (PR) isoforms. However, similar relative isoform expression in FTEn and FTEb samples suggested that a luteal phase-associated factor other than progesterone directs gene expression changes in FTEb. The possibility that FTEb respond differently to ovulation-associated inflammatory cytokines that are locally elevated during the luteal phase was next investigated. Importantly, FTEb specimens previously found to cluster with SerCa based on their global gene expression profiles showed evidence of increased nuclear factor-κB (NFκB)-dependent (pro-inflammatory) signalling and diminished glucocorticoid receptor (GR)-dependent (anti-inflammatory) signalling. Furthermore, I demonstrate that disabled homolog 2 (DAB2), an adaptor molecule decreased in SerCa and FTE luteal samples, enhances both GR-mediated transactivation and suppression of NFκB signalling, implicating DAB2 as a crucial determinant of inflammatory signalling and ovarian cancer risk. Altogether, this thesis identifies gene expression changes in FTE from BRCA mutation carriers during the post-ovulatory luteal phase that parallel those detected in SerCa. The data support a proposed novel testable model for predisposing events contributing to SerCa that centres on an altered ability to quickly resolve the pro-inflammatory environment created by the ovulatory event.

Studies of prophylactic salpingectomy specimens from BRCA1/2 mutation carriers, at risk for tubal and ovarian high-grade serous carcinoma (SerCa), have consistently revealed occult carcinomas and putative histological cancer precursors in the distal fallopian tube epithelium (FTE), supporting the FTE as the source of SerCa. In this thesis I molecularly characterized and compared non-malignant FTE from mutation carriers (FTEb) and control patients (FTEn) to identify alterations that may predispose to malignant transformation. Gene expression profiling of laser capture microdissected FTEn, FTEb and SerCa indicated that SerCa have similar molecular profiles whether of presumed ovarian or tubal origin, supporting the notion they share a common cell of origin within the FTE. Furthermore, FTEb samples obtained during the post-ovulatory luteal phase showed gene expression profiles closely resembling SerCa samples, suggesting that the luteal phase milieu may contribute to serous carcinogenesis. An initial hypothesis was that FTEb may respond differently to luteal progesterone compared to FTEn, via differential expression of progesterone receptor (PR) isoforms. However, similar relative isoform expression in FTEn and FTEb samples suggested that a luteal phase-associated factor other than progesterone directs gene expression changes in FTEb. The possibility that FTEb respond differently to ovulation-associated inflammatory cytokines that are locally elevated during the luteal phase was next investigated. Importantly, FTEb specimens previously found to cluster with SerCa based on their global gene expression profiles showed evidence of increased nuclear factor-κB (NFκB)-dependent (pro-inflammatory) signalling and diminished glucocorticoid receptor (GR)-dependent (anti-inflammatory) signalling. Furthermore, I demonstrate that disabled homolog 2 (DAB2), an adaptor molecule decreased in SerCa and FTE luteal samples, enhances both GR-mediated transactivation and suppression of NFκB signalling, implicating DAB2 as a crucial determinant of inflammatory signalling and ovarian cancer risk. Altogether, this thesis identifies gene expression changes in FTE from BRCA mutation carriers during the post-ovulatory luteal phase that parallel those detected in SerCa. The data support a proposed novel testable model for predisposing events contributing to SerCa that centres on an altered ability to quickly resolve the pro-inflammatory environment created by the ovulatory event.

L’hyperaldostéronisme primaire [HAP] résulte d’une hypersécrétion d’aldostérone d’origine surrénale. La compréhension de la pathogénie de cette maladie, dont la prévalence est estimée à 10% de la population hypertendue, est essentielle pour le développement de nouveaux outils diagnostiques et thérapeutiques. Dans ce contexte, ce travail de doctorat avait pour but d’identifier de nouvelles orientations thérapeutiques en testant un inhibiteur de l’aldostérone synthase et de rechercher de nouveaux marqueurs diagnostiques par l’étude du profil d’expression des microARN [miRs]. Dans une étude de phase II, 14 patients présentant un HAP ont reçu un inhibiteur de l’aldostérone synthase : le LCI699 pendant 4 semaines. Nous avons ainsi pu montrer que le LCI699 permet de diminuer les concentrations d’aldostérone de 70 à 80% et de normaliser la kaliémie chez tous les patients. En revanche, il n’a qu’un effet modéré sur la pression artérielle et sur l’élévation des concentrations de rénine, et n’est que partiellement sélectif pour l’aldostérone synthase. De plus son efficacité est moindre que celle de l’éplérénone, antagoniste minéralocorticoide administré aux mêmes patients au décours du LCI699. Nous avons ensuite étudié l’expression de 754 miRs dans des adénomes produisant de l’aldostérone [APA] et dans des surrénales contrôles. L’hypothèse était qu’une dérégulation de leur expression pouvait être impliquée dans la tumorigénèse et la surproduction d’aldostérone. L’objectif secondaire était d’identifier des miRs utilisables en tant que biomarqueurs. Cette analyse par carte microfluidique a révélé que 27 miRs sont significativement sous exprimés dans les APA et un seul miR est surexprimé. L’expression différentielle de deux de ces miRs : miR 137 et miR 375 a pu être confirmée dans une cohorte de validation de 36 APA: Des résultats préliminaires in vitro indiquent que le miR 375 pourrait induire une diminution de la synthèse d’aldostérone. Enfin, l’analyse de l’expression de ces miRs dans le plasma a permis de mettre en évidence une sous-expression du miR 375 chez les patients atteints d’HAP en comparaison à des sujets sains. En conclusion, le blocage de la biosynthèse de l’aldostérone représente une nouvelle option thérapeutiques, cependant il est nécessaire de développer une seconde génération de molécules : plus puissantes et plus sélectives. Les analyses effectuées sur les APA ouvrent de nouvelles perspectives pour l’identification de nouveaux biomarqueurs tels que les miRs circulants / Primary aldosteronism [PA] results from the hypersecretion of aldosterone by the adrenals. Understanding the pathogenesis of the disease is essential for identifying new diagnostic and therapeutic tools. In this context the purpose of my PHD was to investigate the effects of an aldosterone synthase inhibitor and second to investigate new diagnostic options by the extensive study of microRNA [miRNA]. In a phase II clinical study, 14 patients with PA were administered an aldosterone synthase inhibitor: LCI699. Four weeks of treatment lead to a 70 to 80% decrease in aldosterone concentration, associated with the cure of hypokalemia. However, there was only a mild effect on blood pressure and volemia (reflected by renin concentration). In addition, these results demonstrated an incomplete selectivity of LCI699 for aldosterone synthase in vivo, and showed that LCI699 is less potent than the blocker of the mineralocorticoid receptor: eplerenone . We also characterized the miRNA profile of Aldosterone producing adenomas [APA]. The hypothesis was that a dysregulation of the expression of miRNA could induce tumorigenesis and increase the production of aldosterone. The secondary aim of the study was to identify miRNA that could be measured in plasma as biomarkers. miRNA profiling of 754 miRNA using quantitative PCR Low Density array, revealed 28 miRNA whose expression was significantly different in APA. The differential expression of two miRNA: miRNA 137 and miRNA 375 was confirmed in a validation cohort of 36 APA. Preliminary in vitro studies showed that up-regulation of intracellular levels of miR 375 may reduce aldosterone secretion in H295R cells. Lastly, circulating plasma levels of miR 375 are differentially expressed between patients with PA and healthy volunteers. In conclusion, the blocking of the aldosterone pathway in hypertensive patients is a novel therapeutic option but second-generation drugs more potent and more selective of aldosterone synthase are required. Profiling miRNA in APA offers new prospect for the development of biomarkers, such as measuring circulating miRNA in plasma