Abstract P3-11-10: Estrogen receptor reprogramming by HOXB13 and GATA3 in ER-positive breast cancer cells

Abstract

Abstract Background: The Breast Cancer Index (BCI) is a gene expression-based signature comprising two functional biomarker panels. The Molecular Grade Index (MGI) is composed of five genes that measure tumor proliferation. BCI (H/I) is a ratio of the HOXB13 and IL17BR genes and measures estrogen signaling. Integration of MGI and BCI (H/I) provides a single prognostic score that quantifies the risk of both late (5-10 years) and overall (0-10 years) distant recurrence. BCI (H/I) has been validated to predict benefit from extended endocrine therapy across multiple adjuvant endocrine treatment backgrounds in several prospective-retrospective studies. Hormone receptor responses are dependent on genome-wide hormone receptor binding patterns. In hormone-dependent cancers, nuclear receptor binding patterns are frequently reprogrammed by other transcription factors. 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. Similarly, the transcription factor GATA3 was found to mediate enhancer accessibility at regulatory regions involved in estrogen receptor (ER)-mediated transcription. We have shown previously that HOXB13 overexpression reprograms and expands the ER binding pattern in breast cancer cells. In this study, we have further characterized the role of HOXB13 in reprograming the ER cistrome and evaluated potential interactions with GATA3. Methods: HOXB13 was expressed in MCF-7 and T47D cells by electroporating HOXB13 mRNA or eGFP mRNA as control. Cells were harvested after 18 hours and analyzed by western blot and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) using antibodies against ER, HOXB13, FOXA1, GATA3 and histone H3K27ac. Chromatin accessibility was assessed by ATAC-seq. After aligning reads with Bowtie2, peak calling, data integration and motif enrichment analysis was performed using HOMER v4.10. Results: ChIP-seq analysis of T47D cells expressing HOXB13 confirmed previous results in MCF-7 cells, where binding of HOXB13 to a significant number of genomic binding sites induced changes in binding of ER, FOXA1 and GATA3 compared to control cells. Integrative analysis of ATAC-seq and ChIP-seq data revealed that HOXB13-induced reprogramming results in open chromatin that frequently exhibits increased acetylation of histone H3K27, a hallmark of transcriptional activation. While both increased chromatin accessibility and H3K27 acetylation were associated with HOX and AP-1 motif enrichment of the underlying DNA sequences, newly open chromatin was specifically co-enriched for motifs for pioneering factors such as FOXA1 and GRHL1. Conclusion: This study lends further support to a model of HOXB13-mediated reprogramming of the ER cistrome in breast cancer. Motif analysis of HOXB13-induced chromatin opening suggests interactions with other pioneer transcription factors including FOXA1 and GRHL1, while HOXB13-induced transcriptional activation is associated with motifs for activating factors such as AP-1. These results will be expanded to inducible cell lines to further characterize the effects of HOXB13 expression on ER binding and function. Citation Format: Kai Treuner, Camila De Arruda Saldanha, Sven Heinz. Estrogen receptor reprogramming by HOXB13 and GATA3 in ER-positive breast cancer cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-10.