Abstract 403: Expression of genes spanning a breast cancer susceptibility locus on 6q25.1 is modulated by epigenetic modification

Abstract

Abstract Single nucleotide polymorphisms within the region immediately upstream of the gene encoding ERα (ESR1) on 6q25.1, have been found to be associated with increased risk of breast cancer1,2. Three genes within this region, CCDC170, RMND1 and C6ORF211 are co-expressed with ESR1 in oestrogen receptor positive (ER+ve) breast cancers and CCDC170 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. We investigated methylation and chromatin structure in the 6q25.1 using methylation sensitive sequencing, chromatin immunoprecipitation (ChIP) and chromatin confirmation capture (3C). Gene expression of ESR1, the three genes and alternative ESR1 mRNA isoforms were analysed in vitro using a panel of breast cancer cell lines. Long-term oestrogen deprived (LTED) cell lines were also generated and used to model the effects of anti-oestrogen therapies used in the treatment of ER positive breast cancers. DNA methylation at the ESR1/CCDC170 locus, gene expression of the four transcripts and alternative ESR1 mRNA isoforms differed greatly between the breast cancer cell lines. ER negative breast cancer cell lines were similar to the ‘non-tumorigenic’ MCF10A cell line while ER positive cell lines were hypomethylated and showed higher expression relative to MCF10A. We also discovered two putative regulatory binding regions which were differentially methylated that correlated with gene expression in our cell lines; one negatively and one positively. Analysis of ER positive tumors found that oestrogen receptor positive tumors were hypomethylated relative to normal tissue. In tumors, two differentially methylated regions were shown to have a strong negative correlation with gene expression of ESR1 and the three genes. These gave correlation coefficients of -0.577, -0.593, -0.600 and -0.454 for ESR1, CCDC170, C6ORF211 and RMND1, respectively, using Pearson correlation analysis and correction for multiple testing. The differentially methylated regions in tumors 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 tumors could facilitate enhanced chromatin interactions and transcription leading to the co-expression of the transcriptional hub. These alterations seen in the 6q25.1 locus, both in vitro and in our dataset, distinguish ER positive tumors and may contribute to resistance to anti-oestrogen therapies.