Background: Forkhead box A1 (FOXA1) is an essential pioneer transcription factor (TF) evoking other key TFs-mediated lineage-specific transcriptional programs in several endoderm-derived organs. Aberrant FOXA1 augmentation, via genetic alterations, occurs in 10-15% of ER+ primary and metastatic breast cancer (BC). We have recently shown that high levels of FOXA1 (H-FOXA1) induces enhancer and transcriptional reprogramming to promote endocrine-resistant (EndoR) and pro-metastatic phenotypes. Using the core transcriptional regulatory circuitry (CRC) mapping method, we identified the AP-1 TF JUNB as a key CRC component in BC cells expressing H-FOXA1. In this study, we aimed to further characterize key AP-1 components that play a role in mediating H-FOXA1-induced transcriptional reprogramming in EndoR and metastatic BC. Methods: The ROSE and HOMER tools were used to identify super-enhancers (SEs) and the predicted SE-harboring TFs in MCF7-parental (P) cells with ectopic FOXA1 overexpression (OE), and in MCF7 tamoxifen-resistant (TamR) cells with endogenous FOXA1 amplification and OE. Cell growth, migration, and co-immunoprecipitation assays, were performed using ER+ BC P cells with ectopic FRA1 OE. A FOXA1 core gene signature (GS) was deduced using the BETA.plus algorithm to analyze our previously reported RNA-seq and ChIP-seq data derived from MCF7-P cells with ectopic FOXA1 OE. Additional RNA-seq analyses include MCF7-P cells with ectopic FRA1 OE, FOXA1 OE and simultaneous FRA1 siRNA knockdown (KD), and MCF7-TamR cells with FRA1 KD. We identified a FOXA1/FRA1-centered GS and its clinical relevance was examined using expression profiles of TCGA, METABRIC, and a metastatic biopsy study from cohort of patients with ER+ metastatic BC from Dana-Farber Cancer Institute.Results: We identified FRA1 as one of the top TFs selectively harboring SEs at their gene loci in MCF7-TamR vs. P cells. Both FRA1 and JUNB expression was elevated in TamR vs. P cells and altered concordantly with FOXA1 in P and TamR cells upon FOXA1 OE or KD, respectively. As we identified JUNB as a CRC component with binding sites enriched at the SEs in BC cells expressing H-FOXA1, we hypothesized that FRA1 and JUNB form a feed-forward transcriptional axis amplifying H-FOXA1-induced enhancer reprogramming. We found that JUNB co-immunoprecipitated with FRA1 in MCF7-TamR and MCF7-P cells with ectopic FRA1 OE, suggesting that FRA1 forms a heterodimer with JUNB to exert AP-1 activity. Ectopic FRA1 OE reduced P cell endocrine sensitivity, increased cell migration, and elicited a transcriptome enriched for the FOXA1-induced core GS. In P cells with ectopic FOXA1 OE, we identified a FRA1-dependent GS (n = 27) that is highly enriched for interferon signaling. This FOXA1/FRA1 GS was highly expressed in luminal B vs. A subtype of primary tumors, further elevated in ER+ metastases, where its expression was positively correlated with FRA1 mRNA levels. Notably, this FOXA1/FRA1 GS was not dependent on FRA1 in P cells without FOXA1 OE, suggesting its relevance in the context of H-FOXA1.Conclusions: Here we show that a FOXA1/FRA1-centered transcriptional axis induces an interferon signaling-enriched GS associated with poor outcome of ER+ BC and metastasis. A FRA1/JUNB AP-1 complex may form a feed-forward transcriptional axis to amplify H-FOXA1 signaling. The FOXA1/FRA1-centered GS could be used to stratify patients with ER+ BC who may need additional targeted therapies. Further studies are warranted to elucidate the interplay between FOXA1 and FRA1/JUNB in regulating interferon signaling, which may guide approaches to improve patient outcomes, possibly with immunotherapy using immune checkpoint inhibitors.

Citation Format: Xiaoyong Fu, Resel Pereira, Lanfang Qin, Carmine De Angelis, Sarmistha Nanda, Martin Shea, Agostina Nardone, Rinath Jeselsohn, Ofir Cohen, Nikhil Wagle, Mothaffar Rimawi, C Kent Osborne, Rachel Schiff. A FOXA1/FRA1-centered transcriptional axis regulates interferon signaling in high FOXA1-associated endocrine-resistant and metastatic breast cancer [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 PD8-03.