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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:586481 |
| Date | January 2013 |
| Creators | Bi, Jing |
| Contributors | Gilbert, Nicholas; Ramsahoye, Bernard |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/8076 |
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