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Ovarian cancer is the most fatal of all gynecological malignancies. Epithelial ovarian cancer (EOC) accounts for about 90% of malignant ovarian tumours and is thought to originate mostly from ovarian surface epithelium (OSE) cells. Epidemiological data suggest that hormone replacement treatment (HRT) users have a higher risk of ovarian cancer, which is related to the use of oestrogen-only HRT. In addition, EOC is oestrogen responsive. This thesis reveals the capacity for production and metabolism of oestrogen in normal OSE and malignant primary EOC cells, and describes the action of oestrogen in the development of EOC at three levels. First, the expression of the genes encoding oestrogen production and metabolism and oestrogen receptor (ER) was investigated in OSE and EOC cells at RNA and protein levels. Immunohistochemistry revealed that steroid sulphatase (STS), oestrogen sulfotransferase (EST), 17βhydroxysteroid dehydrogenase (17βHSD) 2 and 17βHSD5 proteins were present in pre-menopausal, post-menopausal and inclusion cystic OSE as well as EOC cells. Taqman qRT-PCR revealed STS, EST, 17βHSD1, 17βHSD2, 17βHSD5, ERα, ERβ and oestrone sulphate (E1S) transporters such as organic anion transporting polypeptide (OATP)-B, OATP-D and OATP-E mRNAs were expressed in pre-menopausal OSE and EOC at different levels. When basal mRNA levels were compared among untreated samples of pre-menopausal OSE and EOC, EST mRNA expression was significantly higher in the OSE compared to EOC cells (P<0.05) while OATP-B mRNA level was the opposite (P<0.05). Radiometric enzyme activity assays demonstrated different metabolism patterns of E1S and oestrone (E1) between normal and malignant cells, indicating overall activities of STS and 17βHSD1 or 17βHSD5 to be higher than the overall activities of EST and 17βHSD2 in cancer cells while enzyme activities in OSE cells were opposite to this. Second, the impact of inflammation on oestrogen production, metabolism and action was compared in OSE and EOC cells by testing the response of target genes to a panel of pro-inflammatory cytokines. The data revealed that in OSE cells, EST (P<0.01) and 17βHSD2 (P<0.001) mRNAs were decreased while ERα mRNA (P<0.001) was increased by IL-1α. In addition, EST mRNA was inhibited by IL-4 (P<0.05). In SKOV-3 (EOC cell line) cells, IL-1α stimulated STS mRNA (P<0.001)and enzyme activity (P<0.05). Moreover, IL-4 inhibited (P<0.05) while IL-8 and IL- 10 enhanced (P<0.01) ERα mRNA levels. Finally, the effect of oestrogenic components of HRT medication (equilin and equilin-sulphate) on the expression of cancer-associated genes was compared to that of 17β-oestradiol (E2) in PEO-1 (an oestrogen-responsive EOC cell line) cells. Expression of the oestrogen-responsive genes FN1 and IGFBP3 mRNA expression was similarly inhibited by E2 and equilin (P<0.05) as well as E1S and sodium equilin-sulphate (P<0.05). In conclusion, this thesis presents evidence that intracrine oestrogen formation and metabolism differs between OSE and EOC cells, such that E2 formation is inhibited in normal OSE but is promoted in EOC. Inflammatory cytokines also influence the local production of E2 by regulating genes encoding oestrogen production and metabolism and receptors. Finally, local HRT metabolites can regulate cancer-associated gene expression in EOC. Together, these data suggest a role for local oestrogen production and action in inflammation-associated development of EOC. Conversely, differential regulation of the same parameters in OSE cells from premenopausal women minimizes oestrogen formation and ‘protects’ against the promotion of EOC.

Breast cancer is a common disease in women and has major impacts on health and quality of life. About 70% of breast cancers over express ERα, and are classified as ER positive breast cancer. Oestrogen receptor alpha (ERα) belongs to the nuclear receptor superfamily and is responsible for many effects of oestrogen on normal and cancerous breast tissue. Endocrine therapies that block the function of ERα or the synthesis of oestrogen have been a mainstay of ERα positive breast cancer treatment. However, their efficacy is limited by intrinsic and acquired drug resistance overtime, and endocrine resistance remains one of the biggest challenges in breast cancer treatment. In order to investigate the underlying mechanisms of acquired drug resistance, and to develop new strategies for breast cancer therapy, I generated a novel long-term oestrogen deprived cell line (DH) in serum-free condition. As DH cells are cultured in a defined media with known concentrations of growth factors, it provides an ideal system to identify and dissect changes in signalling pathways in response to hormones and inhibitors in vitro. At the same time, DH cells are representative of ER positive breast cancers treated with drugs that reduce the level of oestrogen. It enables the identification of survival pathways that could be activated during oestrogen deprivation. By using this cell model, I find that oestrogen stimulation enables cells to up-regulate the EGFR level and simultaneously reduces ERα expression at both mRNA and protein levels. Once up-regulated, EGFR expression is maintained despite oestrogen withdrawal indicating a stable transcriptional re-programming at the EGFR promoter. By using the whole genome expression microarrays, I identified a list of genes that also show stable changes in gene expression in response to oestrogen, suggesting that the oestrogen promotes transcriptional re-programming at multiple pathways in cells. In terms of signalling pathways, oestrogen activates the growth promoting MAPK pathway in an EGFR dependent manner and a 5-day oestrogen pulse substantially increases the resistance of cells to tamoxifen, while cells remain sensitive to the EGFR inhibitor, demonstrating a functional switch between ERα and EGFR survival pathways. Furthermore, microarray analysis of ERα and EGFR downstream target genes shows that there is a general activation of MAPK gene signature after 5 days of oestrogen stimulation in DH cells. In this thesis, I also investigate the molecular mechanism of oestrogen induced EGFR up-regulation in ER positive breast cancer cells. c-Myb is an oestrogen responsive transcription factor whose expression is regulated by ERα in breast cancer cells. I demonstrate that oestrogen treatment leads to ERα dependent c-Myb up-regulation in DH cells. I also find that c-Myb transiently locates upstream of the EGFR promoter to enhance its expression. As the up-regulation of EGFR in ER positive breast cancer could lead to survival pathway switching and endocrine therapy resistance, c-Myb could be a good drug target to prevent the likelihood these switches and subsequent relapse on endocrine therapies. The expression of EGFR remains high after the removal of oestrogen suggesting there may be epigenetic changes, which maintain the transcriptional re-programming stimulated by c-Myb. Bisulphite sequencing however demonstrates EGFR promoter DNA methylation pattern is not affected by oestrogen. Meanwhile, ChIP microarrays with four different histone modifications show no significant changes around the promoter area of EGFR in response to oestrogen. These observations suggest that alternative epigenetic modifications or epigenetic alternations at other genes may subsequently lead to the stable expression of EGFR in response to oestrogen.

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