Abstract 4783: Amplification of distant estrogen response elements epigenetically deregulates target genes in ERα-mediated breast tumorigenesis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Since the first amplified gene, MYCN, was identified in neuroblastomas, considerable effort has been directed toward discovery of oncogenes in amplified genomic regions. However, there is no significant correlation between copy number gains and increased expression of corresponding genes within amplicons. We demonstrate that amplification of distant-acting regulatory elements, or enhancers, may be an alternative mechanism contributing to an oncogenic event. Enhancers comprise transcription factor binding sites known to remotely regulate transcription through chromatin looping or transvection. Using a “Seq-to-Seek” strategy integrating three next-generation sequencing approaches, we comprehensively mapped distant estrogen response elements (DEREs) that remotely control transcription of target genes through chromatin proximity. Surprisingly, a densely mapped DERE region located on chromosome 20q13 frequently amplifies in ERα-positive breast cancer with poor prognosis. Progressive accumulation of DERE copies was observed in normal breast progenitor and cancer cells chronically exposed to estrogen or estrogenic chemicals. Pharmacological studies with either an ERα antagonist or an ataxia telangiectasia mutated (ATM) kinase inhibitor further showed that ATM-dependent DNA repair pathway participates in ERα-driven increase of 20q13 DERE copies, suggesting involvement of breakage-fusion-bridge mechanism in DERE amplification. Furthermore, these aberrantly amplified DEREs altered chromatin interactions, leading to transcriptional repression of genes that are associated with tumor-suppressor and apoptosis pathways and potentially linked to cancer development and tamoxifen resistance. Correlation analyses of copy-number variation, occupancy of the repressive histone mark, H3K27me3, and methylation profiling of 20q13 DEREs in ERα-positive cancer cells support a model in which amplified DEREs preferentially induce repressive epigenetic modulation of target genes. In addition, interphase fluorescence in situ hybridization coupled with immunofluorescence analyses revealed that estrogen stimulation leads to DERE clustering in a transcriptional hub nearby in a heterochromatic region. These findings indicate that the 20q13 DERE region is a potential transcriptionally repressive domain whose aberrant amplification can result in suppressing expression of tumor suppressor genes. To determine the precise role of amplified DEREs in epigenetic transcription and their biological significance, our ongoing studies use CRISPR/Cas genomic editing system to delete candidate DEREs at 20q13. In summary, our findings suggest that amplification of DNA regulatory elements can profoundly alter target transcriptome during tumorigenesis and amplified DEREs can be used as potential prognostic markers for endocrine resistance. Citation Format: PEI-YIN HSU, HANG-KAI HSU, Victor X. Jin, Zelton D. Sharp, Tim H.-M. Huang. Amplification of distant estrogen response elements epigenetically deregulates target genes in ERα-mediated breast tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4783. doi:10.1158/1538-7445.AM2014-4783