Regulatory Mechanisms Modulating the Co-Expression of ESR1 and Three Upstream Genes in Breast Cancer
|dc.contributor.author||White, Jackie Anna|
|dc.identifier.citation||White, J. A. (2013). Regulatory Mechanisms Modulating the Co-Expression of ESR1 and Three Upstream Genes in Breast Cancer (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/4369||en|
|dc.description.abstract||Breast cancer is the most prevalent form of cancer diagnosed in women with oestrogen receptor α (ERα) positive breast cancers accounting for approximately 80% of all malignancies. Several genome wide association studies have associated breast cancer risk with a number of SNPs located in the 6q25.1 locus just upstream of the ESR1 gene which encodes ERα. More recently, ESR1 was found to be co-expressed with three uncharacterised genes, C6ORF97, C6ORF211 and RMND1 (collectively C6ORFs) situated immediately upstream from ESR1, in ER positive cell lines and tumours. These genes remained highly correlated after treatment with an ERα antagonist, so are unlikely to be regulated via ERα. RMND1 is also transcribed on the opposite strand, excluding the possibility of a single transcript. Alteration of DNA methylation is a common observation in breast cancer. Therefore, this project aimed to determine whether changes in DNA methylation occur at the 6q25.1 locus and whether this influences gene expression. This study hypothesised that changes in DNA methylation would correlate with gene expression of ESR1 and the three C6ORFs. Gene expression of ESR1, the three C6ORFs and alternative ESR1 mRNA isoforms were analysed in vitro using breast cancer cell lines. Long-term oestrogen deprived (LTED) cell lines were also generated and, together with fulvestrant resistant cell lines, were used to model the effects of anti-oestrogen therapies used in the treatment of ER positive breast cancers. These analyses revealed that DNA methylation in the ESR1/C6ORF97 locus, gene expression of the four transcripts and alternative ESR1 mRNA isoforms differed greatly between breast cancer cell lines. ER negative breast cancer cell lines were similar to the ‘non-tumourigenic’ MCF10A cell line while ER positive cell lines were hypomethylated and showed higher expression relative to MCF10A. Additionally, changes were seen in the ER positive cell lines during adaptation to oestrogen deprivation or fulvestrant treatment with the T47D LTED and fulvestrant resistant cell lines displaying an ER negative phenotype. Two putative regulatory binding regions were also discovered which were differentially methylated and correlated with gene expression in the cell lines; one negatively and one positively. To further examine this correlation in a clinically relevant setting, a public breast cancer dataset was analysed. ER positive tumours were found to be hypomethylated relative to normal tissue. In tumours, two differentially methylated regions were shown to have a strong negative correlation with gene expression of ESR1 and the three C6ORFs. These gave correlation coefficients of -0.577, -0.593, -0.600 and -0.454 for ESR1, C6ORF97, C6ORF211 and RMND1, respectively, using Pearson correlation analysis and correction for multiple testing. Further analysis of a normal breast tissue dataset revealed a very uniform DNA methylation pattern and ESR1 expression did not correlate with expression of the three C6ORFs. The differentially methylated regions in tumours coincided with binding sites for ERα and cohesin and long range chromatin interaction sites involving RNA polymerase II. Therefore, reduced DNA methylation seen in the ER positive tumours could facilitate enhanced chromatin interactions and transcription leading to the co-expression of ESR1 and the three C6ORFs. These alterations seen in the 6q25.1 locus, both in vitro and in the dataset, distinguish ER positive tumours and may contribute to resistance to anti-oestrogen therapies.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.title||Regulatory Mechanisms Modulating the Co-Expression of ESR1 and Three Upstream Genes in Breast Cancer|
|thesis.degree.name||Master of Science|
|thesis.degree.grantor||University of Otago|
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