I have investigated epigenetic mechanisms of acquired endocrine-resistance in breast cancer using an in vitro model system based on estrogen-dependent MCF7 cells and their derivatives, LCC1 and LCC9. LCC1 cells, derived from MCF7 after passage in ovariectomised mice and routinely cultured in vitro in the absence of estrogen, exhibit estrogen-independent growth. They retain sensitivity to tamoxifen and fulvestrant. LCC9 cells, derived from LCC1 cells by growing them in increasing concentrations of fulvestrant, are completely estrogen-independent and are resistant to fulvestrant and cross-resistant to tamoxifen. When compared to MCF7 cells, LCC1 cells have marked up-regulation of the estrogen receptor α (ERα) protein that is not concomitant with increased estrogen receptor 1 (ESR1) transcription, suggesting a role for estrogen in controlling the proteasomal degradation of ERα. However, despite being grown in the same estrogen-deprived conditions, LCC9 cells do not have up-regulated ERα levels. As LCC1 cells retain sensitivity to tamoxifen and fulvestrant, these data suggest that LCC1 have developed estrogen-independence through ERα uncoupled from its ligand. However, LCC9 cells appear to have developed an alternative mechanism which is not dependent on ERα, presumably explaining their resistance to fulvestrant. I have studied global gene expression changes in the presence and absence of estrogen in these cell lines, using oligonucleotide microarrays, and correlated these data with global DNA methylation data derived from methylation arrays, which interrogate the methylation status of approximately 27,000 CpG dinucleotides in the genome. The analysis led to the discovery of more than 5,000 genes that were potentially either up-regulated or down-regulated by estrogen in MCF7 cells, either directly or indirectly. The transcriptional response to estrogen was generally muted in LCC1 and LCC9 compared with MCF7, but was not completely absent. I used various methods based on differential gene expression to parse the data, including gene ontology analysis, aiming to select genes for further mechanistic study. However, none of these methods led to the conclusive identification of a specific gene (or set of genes) that might have accounted for the physiological differences between the cell lines. In one strategy, I reasoned that, as the endocrine-resistant cells had lost their estrogen-dependence, genes involved might be regulated in an estrogen-dependent manner in MCF7 cells, without exhibiting misregulation in LCC9. This led to the identification of DUSP1 as a candidate gene, which was taken forward for mechanistic study because of its potential role in regulating ERα expression. However, when over-expressing DUSP1 in LCC9 cells, I could not demonstrate any effect on ERα levels. The final approach taken was to identify genes that might have been epigenetically deregulated, being both estrogen-regulated and deregulated in association with aberrant DNA methylation in the estrogen-independent cell lines. Surprisingly, given the phenotypic differences between the cell lines, only a very few genes were significantly methylated between cell lines. Of those that were differentially methylated between MCF7 cells and LCC1/9, only three exhibited the expected inverse correlation between methylation and expression. Of these, the gene CYBA was selected for further investigation. CYBA is a critical component of the NAPDH oxidase complex which is involved in generating oxygen free-radicals. My work suggests CYBA expression is estrogen-dependent, and that chronic estrogen deprivation leads to the epigenetic inactivation of CYBA in breast cancer cells. I speculate that the epigenetic suppression of CYBA may protect cells from the oxidant damage that results from estrogen deprivation and may be part of the mechanism that leads to acquired endocrine-resistance in previously sensitive cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:586470 |
| Date | January 2013 |
| Creators | Skerry, Benjamin James Oliver |
| Contributors | Ramsahoye, Bernard; Gilbert, Nick |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/8106 |
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