Abstract P4-04-03: High-throughput epigenetic screening in hormone receptor positive (HR+) breast cancer cells

Abstract

Abstract Background & Objective: Substantial progress has been made in treating HR+ metastatic breast cancer patients with CDK4/6, mTOR and PIK3CA inhibitors (all FDA approved), but there are no current available therapies to directly restore sensitivity to endocrine therapy once patients’ cells develop epigenetic suppression of ER pathways. CCCTC-binding factor (CTCF), which regulates higher-order chromatin organization and estrogen reception (ER) transcription, is altered by hemizygous deletion or mutation in more than 50% of HR+ luminal type breast tumors. The goal of this project is to characterize an epigenetic signature in CTCF-altered breast cancers to identify novel epigenetic targets for therapeutics development. Targeting CTCF influenced pathways with or without its chaperones may restore sensitivity to endocrine therapy in patients with HR+ breast cancer. Methods: We identified patient-derived cell lines from the Cancer Cell Line Encyclopedia (CCLE) with known CTCF gene copy number. Selected cell lines were tested for proliferation with a custom siRNA library targeting 1,300 genes involved in epigenetic regulation while in the presence of different concentrations of estradiol, tamoxifen and fulvestrant. We confirmed presence of CTCF protein by western blot and mRNA expression by real-time PCR. Proliferation of the lines in vitro was quantified by the MTT assay. Further functional annotation and pathway enrichment analysis of the identified genes were performed by using the g:ProfileR package. Results: We have screened more than 60 breast cancer cell lines and focused on six HR+ luminal (3 each “a” and “b” type) lines with different levels of CTCF copy number; a normal breast epithelial line was used as control. We observed that cell growth responded to anti-estrogen therapy differently based on the copy number of CTCF. Cells with high copy number of CTCF are resistant to endocrine therapy, the sensitivity was restored by silencing CTCF using siRNA. We then selected two cell lines with low and high copy number of CTCF for further high-throughput screening with the epigenetic siRNA library. A total of 67 and 35 candidate sensitizer target genes, and 56 and 44 candidate enhancer target genes were prioritized in cells with low and high copy number of CTCF, respectively. Functional annotation and pathway enrichment analysis of the identified genes showed that cells with low copy number of CTCF had apoptosis related hits, such as BCL family and caspase genes; however, cells with high copy number of CTCF were found to relate to other epigenetic regulators such as DNMTs and CHD. This suggests that CTCF might form complexes with other epigenetic proteins to form abnormal chromatin configuration with abnormal regulation of ER pathway as consequence. Conclusions: To our knowledge, this is the first epigenetic library screen of HR+ luminal breast cancer cells with responses categorized by differential CTCF copy number status. We have confirmed CTCF as an important protein in predicting response to endocrine therapy and identified possible interactions between CTCF, DNMT and CHD. We conclude that development of therapeutics against targets identified in our screen to reverse endocrine resistance may be achievable for treatment of luminal breast cancers that undertake epigenetic deregulation. Keywords: Breast cancer; Hormone receptor positive; epigenetic screening Citation Format: Fengting Yan, Olga Nikolova, VK Gadi. High-throughput epigenetic screening in hormone receptor positive (HR+) breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-04-03.