Abstract A16: CHIP-seq of the estrogen receptor identifies new targetable estrogen regulated pathways in ovarian cancer
Authors: Cheng Georgina , Behbakht Kian , Spillman Monique A. 
Published: August 2014

Introduction: The estrogen receptor (ER) is found in about 60% of epithelial ovarian cancers; however, ovarian cancers are thought to be less estrogen responsive than breast cancers. Previous data has shown that long term estrogen responsive genes in ovarian cancer overlap with breast cancer genes less than 10% of the time. The question remains whether the estrogen receptor regulates the same signaling pathways in ovarian cancer as in breast cancer. Our hypothesis is that a unique set of estrogen responsive genes and proteins are activated by the ER in epithelial ovarian cancer as compared to breast cancer.

Methods: PEO4 cells were cultured in RPMI 1640-phenol red free media at 107 cells/plate and steroid depleted for four days. PEO4 cells were treated with and without 10-7 M 17 β-estradiol (Sigma) for 45min. Ethanol was used as a vehicle and control. Chromatin immunoprecipitation-sequencing (CHIP-seq) was used to determine the DNA-protein interactions of the estrogen receptor in the ovarian cancer cell line. DNA-protein interactions were crosslinked with formaldehyde, sheared and immunoprecipitated with the antibody ERalpha(HC-20):sc-543 (Santa Cruz). DNA was reversed crosslinked and purified. Biological triplicates were done. Library construction and Illumina HiSeq sequencing was done at the Genomics and Microarray Core at the University of Colorado. Reads were mapped using Novoalign. Identifying regions of enrichment, feature characterization, and motif analysis were performed using Homer (UCSD).

Results: 13,000 genes provided unique sequences by computer analysis in the cells treated with estrogen as compared to the ethanol control. 3,500 were uniquely identified as estrogen responsive genes. 2,800 genes were uniquely identified in control cells. Anticipated internal controls such as C-myc were appropriately identified by this analysis. A known estrogen responsive gene, the prolactin receptor, was identified as an estrogen responsive gene in our early timepoint. MicroRNAs 1260B and 3152, CUGBPa Elav-like family member 2, CSP-beta, STAU2 antisense RNA 1 were also seen in our analysis. Another more frequently identified sequence was CAPRIN1 (cell cycle associated protein 1), that is involved with cell proliferation. Additionally, DNA motif analysis of the ER crosslinked DNA revealed known estrogen responsive elements (EREs) and additionally found other DNA binding motifs such as those for p53, C-MYC, and PAX7. FOXA1, a major ER co-factor in breast cancer, was also enriched in our CHIP-seq analysis.

Conclusion: We have identified a unique set of directly ER modulated genes, microRNAs, and binding sites in an ovarian cancer cell line. These represent potential therapeutic targets for novel antiestrogens in ovarian cancer.

Citation Format: Georgina Cheng, Kian Behbakht, Monique A. Spillman. CHIP-seq of the estrogen receptor identifies new targetable estrogen regulated pathways in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A16.