Abstract PS17-32: Characterization of HOXB13-induced estrogen receptor reprogramming in breast cancer cells

Abstract

Abstract Background: The Breast Cancer Index (BCI) is a gene expression-based signature that consists of two functional biomarker panels, HOXB13/IL17BR (H/I) and Molecular Grade Index (MGI), that interrogate important proliferation and estrogen signaling pathways in breast cancer. The BCI prognostic score reports individualized risk of overall and late distant recurrence and is based on the algorithmic combination of the H/I ratio and MGI, while the predictive component, BCI (H/I), reports a categorical prediction of high versus low likelihood of benefit from extended endocrine therapy.The homeobox transcription factor HOXB13 has previously been shown to reprogram genome-wide binding of the androgen receptor (AR) during prostate cancer tumorigenesis, where it colocalizes with FOXA1 at reprogrammed AR binding sites. The aim of the current study was to characterize the potential role of HOXB13 in estrogen receptor (ER) reprogramming by analyzing changes in the global ER binding pattern induced by transient overexpression of HOXB13 in breast cancer cells. In addition, gene ontology (GO) analysis was performed to delineate the functional role of HOXB13 in modulating estrogen signaling and response to endocrine therapy.Methods: HOXB13 was overexpressed in MCF-7 cells by electroporating HOXB13 mRNA, or eGFP mRNA as control. Cells were harvested at different time points and analyzed by western blot and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) using antibodies against ER, HOXB13, FOXA1 and H3K27ac. After aligning reads with Bowtie2, peak calling and data integration were performed using HOMER v4.10. Gene ontology (GO) analysis was performed using the Genomic Regions Enrichment of Annotations Tool (GREAT v4.0.4) (http://great.stanford.edu/) to identify annotations enriched among genes near ER genomic binding sites.Results: ChIP-seq analysis revealed substantial binding of HOXB13 to a large number of genomic binding sites compared to the eGFP control. HOXB13 overexpression in ER+ breast cancer cells significantly increased ER binding with a majority of ER binding sites being co-bound by FOXA1 and HOXB13. GO analysis of both proximal and distal genomic regions showed significant enrichment of genes associated with mammary gland development, such as “mammary gland epithelium development”, “mammary gland development”, and “mammary gland epithelial cell differentiation”.Conclusion: Findings from this analysis show that transient HOXB13 overexpression reprograms and expands the ER binding pattern in breast cancer cells. Genomic regions newly bound by ER are enriched for genes involved in mammary gland and epithelial differentiation suggesting that HOXB13 activates ER transcriptional programs that link oncogenic and developmental pathways. These results will be compared to ongoing experiments using an inducible HOXB13 expression system to assess the effects of HOXB13 expression on ER binding and function in response to estradiol and tamoxifen. Citation Format: Kai Treuner, Dominic Schenone, Christopher Benner, Catherine A Schnabel, Sven Heinz. Characterization of HOXB13-induced estrogen receptor reprogramming in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-32.