Modulatory effects of steroid hormones on actin cytoskeleton

Steroid hormones act as chemical messengers, control diverse physiologic and cellular processes and affect almost all aspects of eukaryotic physiology, from sexual differentiation, growth, and reproduction to immunity, metabolism, and behaviour. Sex steroids imbalance have been widely described to be associated with several human diseases, including hormone-dependent tumours, cardiovascular disease, endometriosis, metabolic syndrome and polycystic ovarian syndrome among others. Their actions are mediated by steroid hormone receptors through two major mechanism called genomic and non-genomic pathways. The aim of this thesis was to investigate the effects of oestrogens, androgens and progesterone on the modulation of cellular processes that involve cell migration, actin cytoskeleton rearrangement and cell proliferation in 2D and 3D cell cultures. In the first part of chapter II we focused on the study of estetrol as a selective oestrogen receptor modulator (SERM) and it comparation with 4-OH-tamoxifen, a well-known SERM on breast cancer T47D monolayer and mammosphere culture. We observed some differences between both models and a similar SERM activity of estetrol compared to 4-OH-tamoxifen in presence of physiological concentrations of oestradiol on 2D/3D growth, migration, KI-67 expression and MMP9 activity. On the second part, we investigated whether testosterone, dihydrotestosterone and dehydroepiandrosterone, may regulate T47D cell motility and invasion through the modulation of Moesin phosphorylation and actin remodelling. We found that androgens promote cell migration and invasion through Moesin activation and actin cytoskeleton rearrangement. Also, we show that testosterone and dehydroepiandrosterone exert their actions via androgen and oestrogen receptor, whereas dihydrotestosterone only through the first one. Next, in chapter III we evaluated if estetrol may exert a modulatory action on the expression of PAI-1, u-PA and t-PA, proteins that are involved in the fibrinolytic system and its relationship with Human umbilical vein endothelial cell migration. We saw that estetrol increased the expression of t-PA, u-PA and PAI-1, but to a lesser extent than equimolar concentration of oestradiol. Moreover, the expression of PAI-1, u-PA and t-PA is necessary for endothelial cell migration stimulated with both oestrogens. Finally, in chapter IV we studied on primary cell culture whether Ulipristal acetate could affect the morphology of human endometrial stromal cells by interfering progesterone or oestradiol stimulus; as well as its effects on actin cytoskeleton rearrangement and FAK/Paxillin/Moesin activation. We found that ulipristal acetate interferes with actin cytoskeletal rearrangement induced by physiological mounts of oestradiol and progesterone by decreasing FAK and Moesin phosphorylation, interfering with Vinculin and Paxillin focal adhesion dots formation on the proximity of cell membrane and affecting endometrial stromal cells migration. In summary, the work presented in this thesis describe for the first time the effects of estetrol on mammosphere model and its SERM activity compared to 4-OH-tamoxifen. In addition, we are the first in evaluate the combination of Tmx and E4 as a treatment, under the hypothesis of combine 4-OH-tamoxifen efficacy with the SERM properties of E4 that could reduce sides effects observed by Tmx therapy. On the other hand, we first describe the modulation of actin rearrangement by Ulipristal acetate on endometrial stromal cells isolated from healthy women. The results presented in this thesis contribute to the understanding of steroid hormones signalling on breast cancer, endothelial and endometrial stromal cells and highlight the importance of studying the mechanism of action of selective oestrogen/progesterone receptor modulator to improve and/or develop new therapeutic strategies for sex steroid dependent human diseases.