Abstract P4-06-11: Expression of genes spanning a breast cancer susceptibility locus on 6q25.1 is modulated by epigenetic modification

Abstract

Abstract Background: Genome wide association studies have identified single nucleotide polymorphisms around C6ORF97, an open reading frame (ORF) immediately upstream of the gene encoding ERα (ESR1) on 6q25.1, to be associated with increased risk of breast cancer1,2. C6ORF97 and two neighbouring genes, RMND1 and C6ORF211, are also co-expressed with ESR1 in oestrogen receptor positive (ER+ve) breast cancers and C6ORF97 expression is inversely associated with proliferation and recurrence free survival in a tamoxifen-treated patient cohort3. Epigenetic modification represents one potential mechanism through which expression of co-localised genes could be modulated. Aim: To investigate the role of epigenetic modification in the co-expression of ESR1, RMND1, C6ORF211 and C6ORF97. Methods: Methylation status and expression of genes in the ESR1/C6ORFs locus was assessed in postmenopausal breast cancers and ER+ve cell lines. Two ER+ve cell lines, MCF7 and T47D, were cultured in oestrogen-depleted media and samples were taken fortnightly for 6 months. DNA and RNA from ER+ve tumours was also analysed in addition to control ER-ve cell lines. Methylation analysis of CpG Islands within the ESR1/C6ORFs locus was carried out using methylation sensitive sequencing analysis. Expression levels of ESR1, RMND1, C6ORF211 and C6ORF97 were determined using quantitative real-time PCR. Results: Variation in methylation patterns was observed at a number of key CpG islands in the ESR1/C6ORFs region. ER+ve cell lines and highly ER+ve tumours were hypomethylated. Hypomethylation was associated with increased expression of ESR1, RMND1, C6ORF211 and C6ORF97. Temporal changes in methylation and expression of these genes were also observed over the period of oestrogen deprivation in both MCF7 and T47D. Conclusions: Methylation at the ESR1/C6ORFs locus is associated with expression levels of ESR1, RMND1, C6ORF211 and C6ORF97. This suggests that methylation is involved in modulating the expression of these four genes and that it could partially explain the previously observed co-expression of the genes. Our finding that hypomethylation in this region occurs in tumours with high ERα expression suggests that it could be a mechanism through which tumours increase ERα levels to maximise growth stimulation from oestradiol. Our observation that RMND1, C6ORF211 and C6ORF97 also increase in expression suggests that the proteins encoded by these genes may contribute to the phenotype exhibited by ER+ve breast tumours.