Abstract P4-04-03: Hyperactive FOXA1 activates super-enhancer-engaged HIF2α/EPAS1 to promote endocrine-resistant metastatic ER-positive breast cancer
Authors: Fu X , Pereira R , De Angelis C , Veeraraghavan J , Shea MJ , Nanda S , Feng Q , Jeselsohn R , O'Malley BW , Brown M , Osborne CK , Schiff R 
Published: February 2018

Background: We have recently reported that acquired endocrine resistance (Endo-R) in multiple ER+ breast cancer (BC) Endo-R cell models is driven by high levels of FOXA1 (High-FOXA1), via gene amplification and/or overexpression (OE), leading to coordinated reprogramming of the FOXA1 genomic binding (cistrome) and transcriptome. Forced FOXA1 OE in parental (P) cells induced similar transcriptional reprogramming leading to Endo-R and metastasis. Recent clinical data showing enrichment of FOXA1 amplification in ER+ metastases further support the clinical importance of our findings. However, the molecular components and the mechanism of High-FOXA1-induced transcriptional reprogramming in Endo-R and metastasis are unknown.

Methods: High-FOXA1-containing MCF7 tamoxifen-resistant (TamR) and P/FOXA1-OE cells were used in this study. An integrative multi-OMICS approach was employed to analyze transcriptome (RNA-seq), FOXA1 cistrome, and histone H3K27 acetylation (ac) (ChIP-seq). Intersection of High-FOXA1-induced transcriptome and distinct FOXA1 cistrome-predicted genes defined a High-FOXA1 core gene signature (CGS). Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) were used for functional annotation. Cell growth and migration/invasion were measured by a bright-field automated cell counter and Transwell insert system. Altered gene expression was measured by RT-qPCR. High-FOXA1 signaling inhibition included gene knockdown (siRNA) or pharmacologic blockade (the EPAS1 inhibitor PT2385). The predictive role of EPAS1 and the associated gene signature were analyzed using publicly available BC datasets.

Results: FOXA1 OE reprogrammed the FOXA1 cistrome in P cells to resemble that of the TamR cells. The FOXA1 cistrome was significantly correlated with the deposition of H3K27ac in TamR vs. P cells (P&lt;2.2e-16). Similarly, the differentially expressed genes in TamR vs. P cells were enriched for FOXA1 binding at enhancers demarcated by H3K27ac (P=8e-125). The FOXA1-CGS was linked to multiple metastasis-related GO terms including “hypoxia response”, enriched for the cancer secretome gene set (P=4.1e-16), and highly represented in the Endo-R transcriptome across our multiple cell models (MCF7, 600MPE, and CAMA1) (P&lt;0.01). Integrative analysis of H3K27ac-defined super-enhancers (SEs) and altered cistrome/transcriptome upon High-FOXA1 nominated EPAS1, a hypoxia-inducible transcription factor (TF), as a top candidate of SE-activated TFs amplifying High-FOXA1 signaling. EPAS1 blockade markedly repressed the secretome genes (e.g., IL8 and S100P) and cell migration and invasion in TamR cells. Primary ER+ tumors (TCGA) with high EPAS1 are enriched for a cancer secretome gene set (P=3e-4). High EPAS1 predicts poor distant metastasis-free survival in ER+ BC treated with endocrine therapy (P=.034).

Conclusions: High-FOXA1 induces transcriptional reprogramming by coordinating histone enhancer marks to activate EPAS1 via an SE mechanism, which in turn mediates transcriptional reprogramming, partly via inducing a pro-metastatic secretome, to promote Endo-R and metastasis. Targeting the High-FOXA1/EPAS1 axis to block transcriptional reprogramming may offer a new therapeutic strategy to prevent and treat Endo-R metastatic ER+ BC.

Citation Format: Fu X, Pereira R, De Angelis C, Veeraraghavan J, Shea MJ, Nanda S, Feng Q, Jeselsohn R, O'Malley BW, Brown M, Osborne CK, Schiff R. Hyperactive FOXA1 activates super-enhancer-engaged HIF2α/EPAS1 to promote endocrine-resistant metastatic ER-positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-03.