BRCA1 promoter methylation: the influence on gene expression and the effect of long term drug treatment
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Breast cancer is the most common type of cancer among woman all over the world, with over 1.67 million new cases in 2012. Heritable breast cancer is closely linked to mutations in the tumor suppressor gene BRCA1, with up to 80% lifetime risk for developing breast cancer among women harboring a mutation in this gene. However, most breast cancer cases are sporadic and somatic mutations of the BRCA1 gene are rare. Furthermore, some tumors show BRCAness, despite being BRCA1 wild-type. Thus, it is of great interest to assess alternative mechanisms for inactivation of the BRCA1 gene, and addressing the missing causality of many breast cancers. Furthermore, it is of great interest to assess the mechanisms of drug resistance, a major challenge in cancer treatment today, where BRCA1 may play an important role. The overall aim of this thesis is to increase the understanding of the biological role of BRCA1 promoter methylation in breast cancer. Three sub aims for the present project were outlined; 1) Quantify the BRCA1 α and β transcripts and the total BRCA1 protein levels and relate the expression data to the methylation pattern in the BRCA1 promoter region in a panel of breast cancer cell lines. 2) Investigate how the total expression levels, as well as the ratio between the α and β transcripts are affected by alterations in the α and β promoter region of BRCA1, including methylation of specific CpGs as well as the polymorphisms rs71361504 and rs799905. 3) Investigate the effect of long term treatment with the drugs olaparib and doxorubicin on the BRCA1 promoter methylation in SKBR3 breast cancer cells as a potential cause of drug resistance. The study showed a weak correlation between BRCA1 methylation pattern and BRCA1 mRNA expression. No correlation was observed between the methylation pattern and protein expressed or between mRNA levels and protein expression. Analysis of polymorphisms rs71361504 and rs799905 found in the BRCA1 promoter showed that the two variants seemed to counter-balance each other, giving equal luciferase expression levels when differing in two positions and lower expression levels when intermediate variants were studied. Finally, long term drug treatment of the cell line SKBR3 did not alter the methylation levels in the BRCA1 promoter, consequently demethylation seems not to be a mechanism for drug resistance in the experimental setup tested in this study.
PublisherThe University of Bergen
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