LAUSR

Background: Forkhead box A1 (FOXA1) is a pioneer transcription factor (TF) for chromatin binding and function of other lineage-specific TFs essential for the normal development of endoderm-derived organs. Aberrant FOXA1 signaling, due to genetic amplification or mutations and/or overexpression, has been frequently detected in metastatic tumors of the breast, prostate, pancreas, bladder and thyroid, suggesting a general role and mechanism of FOXA1-driven tumorigenesis and disease progression. We recently reported that high levels of FOXA1 (H-FOXA1) promote endocrine-resistant (EndoR) and metastatic phenotypes in estrogen receptor (ER)+ breast cancer (BC) cells. Here we sought to uncover the role and the mechanisms by which H-FOXA1 promotes EndoR metastatic BC. Methods: Genomic sequencing data from an ER+/HER2- metastatic BC cohort (n=781, MSK-IMPACT; cBioportal) were used to compare mutations and copy number alterations of FOXA1 and ESR1. Genome-wide FOXA1-chromatin binding (cistrome) and distribution of the enhancer marks histone H3 lysine 27 acetylation (H3K27ac) and lysine 4 mono-methylation (H3K4me1) were analyzed by ChIP-seq in MCF7 cell model with inducible H-FOXA1. FOXA1 cistrome, H3K27ac distribution, and transcriptome of a FOXA1-overexpressing pancreatic ductal adenocarcinoma cell model (PDA-hT2) were obtained from NCBI GEO (GSE99311). The core regulatory circuitry (CRC) Mapper was used to identify auto-regulatory loop of TFs induced by H-FOXA1. Gene Ontology was used for gene set functional annotation. FOXA1-associated enhancers of ER+ metastatic vs. primary tumors were analyzed using the H3K27ac epigenome data (European Nucleotide Archive, PRJEB22757). Results: The FOXA1 and ESR1 genetic amplification and mutations displayed a largely mutually exclusive pattern in ER+/HER2- metastatic BC, suggesting a role of hyperactive FOXA1 signaling in promoting EndoR and metastatic BC distinct from that of the ESR1 mutations. FOXA1 overexpression in BC cells resulted in increased FOXA1 DNA binding and the establishment of more regions with gained H3K27ac and/or H3K4me1, suggesting a more accessible and active chromatin state. H-FOXA1-induced upregulated genes were enriched for the gained H3K27ac or H3K4me1, especially for the enhancers with both marks. An enhancer signature with gained and overlapped H3K27ac and H3K4me1 predicts genes enriched for proliferation, anti-apoptosis and developmental signaling. Upregulated genes induced by H-FOXA1 with gained enhancers were further enriched for pro-metastatic processes, sharing the same characteristics of cellular morphogenesis during embryonic development. Similar results were obtained using integrated data from the PDA-hT2 cell model, sharing enriched pro-metastatic genes predicted by the H-FOXA1-induced enhancer signature. A CRC auto-regulatory TF loop, comprising components of the AP-1 and SMAD families, was predicted to amplify the impact of this enhancer signature on activation of the pro-metastatic transcriptional programs. In line with our preclinical findings, epigenetic changes of active enhancers in ER+ metastatic vs. primary BC were associated with the H-FOXA1-induced enhancer signature. Conclusions: Our study suggests that in ER+ metastatic BC, genetic alterations of FOXA1 leading to hyperactive FOXA1 signaling involves epigenetic evolution to promote a pro-metastatic enhancer signature. This genome-wide H-FOXA1-induced enhancer signature supports the role of H-FOXA1 in unleashing oncogenic activities of lineage-specific TFs in many types of metastatic tumors. Developing therapeutics targeting FOXA1 itself or key components of the H-FOXA1-induced CRC is warranted to treat or prevent EndoR and metastatic BC effectively via targeting the entire aberrant transcriptional programs. Citation Format: Xiaoyong Fu, Resel Pereira, Carmine De Angelis, Sarmistha Nanda, Lanfang Qin, Jamunarani Veeraraghavan, Pier Selenica, Britta Weigelt, Jorge S Reis-Filho, Agostina Nardone, Rinath Jeselsohn, Myles Brown, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Identification of a high FOXA1-induced pro-metastatic enhancer signature in endocrine-resistant and metastatic breast cancer [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 PD7-01.