Molecular characterisation and prophylactic treatment strategies among women with benign gynecological disorders

Background: Female reproduction is controlled by hormones in a timed and well-coordinated manner by the hypothalamuspituitary ovarian axis. In response to the ovarian hormones such as estrogen, and progesterone, endometrium, the inner lining of the uterus, undergoes repeated cycles of tissue growth, differentiation, tissue shedding and remodelling. However, dysregulation of those hormones, may generally cause uncontrolled cell proliferation, alteration of its invasive, migratory or angiogenic characteristics, leading to the displacement of endometrial tissues either outside of uterine cavity, for instance on ovary forming ovarian endometriosis; or inwardly into the myometrium causing adenomyosis. Several epidemiological studies as well as histological evidences have shown an increased risk of developing ovarian cancer among women with endometriosis known as endometriosis associated ovarian cancers (EAOC). Similarly, the risk of ovarian cancer is increased among carriers of BRCA1 or BRCA2 germline mutations due to error-prone DNA repair mechanisms. Aim: We aimed to investigate on early molecular alterations associated with cancer development among women with ovarian endometriosis. In addition, treatment or preventive strategies for reducing disease symptoms were explored among women with ovarian endometriosis, BRCA1 or BRCA2 mutations and adenomyosis, respectively. Methods and Results: Study I, we explored if there was a molecular link between endometriosis and ovarian cancer development, by analysing multipotent stem/stromal cells and tissues of endometrium and endometrioma among women with ovarian endometriosis. We investigated for intra-patient heterogeneity within stemand cancercellpathways using targeted PCR array as well as validated for their tumour initiating characteristics. We observed that a subgroup of women with endometriosis (4/30 endometrioma) exhibited dysregulation in estrogen receptor expression, upregulation of molecules related to epithelial-mesenchymal transition pathway such as KIT, HIF2a and E-cadherin as well as downregulation of tumour suppressor genes PTEN and ARID1A, thus supporting a link between above molecular changes and potential risk of EAOC. Study II, we investigated the molecular regulation of Syndecan-1 (SDC-1) and -4 (SDC-4) upon induced activation of TGF-b signalling in another cohort of ovarian endometrioma, to understand their interactions in the pathophysiology of endometriosis and potential EAOCs. Similar to Study I, we also identified molecular heterogeneity with aberrant activation of TGFb signalling as well as confirmed their anomalous behaviour using 3D spheroid and invasion assays in vitro. Interestingly, the above invasive phenotype could be altered by transient gene knockdown of either SDC-1 or SDC-4 during active TGF-b signalling. Moreover, we showed that the presence of high levels of TGF-b ligands control endometriotic cell proliferation and reduce its 3D-spheroid invasive potential in vitro. Thus, inhibition of SDCs among subjects with aberrant TGF-b signalling could be suggested as a potential treatment strategy to reduce inherent risk towards EAOC. In Study III, we examined the molecular action of selective progesterone receptor modulator, mifepristone among women with BRCA1 or BRCA2 mutations. Through in vitro studies, we confirmed the anti-proliferative action of mifepristone with inherent levels of progesterone among above cohort of BRCA1 or BRCA2 women, thus providing as an alternative preventive approach to avoid/delay the use of salpingo-oophorectomy in reducing ovarian cancer risk. In Study IV, we demonstrated the mechanism of action for bromocriptine in the first known human clinical trial for the management of adenomyosis. Bromocriptine provided a prolactin mediated potent growth inhibition, reduced heavy menstrual bleeding as well as exhibited reversal of fibrosis, thus could be further explored for reversing the pathogenesis of adenomyosis. Conclusion: To summarize, this thesis has demonstrated important molecular links underlying endometriosis and risk of EAOC. Also, this work has shown the possibilities for early detection and potential treatment strategies to reduce disease symptoms and/or inherent cancer risk. LIST OF SCIENTIFIC PAPERS I. Aberrant expression of genes associated with stemness and cancer in endometria and endometrioma in a subset of women with endometriosis Ponandai-Srinivasan S, Andersson KA, Nister M, Saare M, Hassan HA, Varghese SJ, Peters M, Salumets A, Gemzell-Danielsson K* and Lalitkumar PGL* Human Reproduction, Vol.33, No.10 pp1924-1938, 2018 II. Syndecan inhibition reverses the pre-malignant phenotype of endometrioma with molecular heterogeneity through TGF-b signalling Ponandai-Srinivasan S, Saare M, Bogavarappu NR, Ehrström S, Garcìa-Uribe PA, Rettkowski J, Iyengar A, Salumets A, Götte M, Lalitkumar PGL and Gemzell-Danielsson K. Manuscript III. Mifepristone mediates anti-proliferative effect on ovarian mesenchymal stem/stromal cells from female BRCA1−/2− carriers. Ponandai-Srinivasan S, Lalitkumar PGL, Garcia L, Varghese SJ, Carlson JW, GemzellDanielsson K*, Floter Radestad A*. Acta Obstet Gynecol Scand. 2019 Feb;98(2):250-261. IV. Bromocriptine induces endometrial growth inhibition in women with Adenomyosis. Andersson J*, Ponandai-Srinivasan S*, Pavone D, Lalitkumar PGL, Bogavarappu NR, GemzellDanielsson. Manuscript * Equal contribution CONTENTS