The pioneer transcription factor FOXA1 is a critical determinant for estrogen receptor (ER) function in hormone-dependent breast cancers. Upon estrogen stimulation, liganded ER binds to poised enhancer regions across the genome that are demarcated by FOXA1 and histone modifications such as H3K4me1 and H3K27ac. In a recent publication, we show that proinflammatory signaling, caused by the cytokine TNFa, drives FOXA1 to latent enhancer binding sites to promote chromatin accessibility for subsequent ER binding upon estrogen ligation. These latent enhancers, activated by the combined treatment of estrogen and TNFa, induced the expression of a unique transcriptome with clinical significance. The effects of TNFa treatment on FOXA1 chromatin redistribution and subsequent gene expression occur within 40 minutes, which points to a rapid signaling cascade that culminates in either changes in FOXA1's posttranslational modifications (PTMs) or its binding partners. To understand how proinflammatory TNFa signaling can redirect FOXA1 to new sites across the genome, we started by characterizing the posttranslational modifications (PTMs) of FOXA1. We immunoprecipitated FOXA1 from MCF-7 breast cancer cells that were treated by E2, TNFa or E2+ TNFa, and then examined their posttranslational status using semi-quantitative and quantitative mass spectrometry approaches. Several phosphorylation sites and acetylation sites have been identified near the DNA binding domain of FOXA1, and acetylation of lysine 295 (K295) was found specifically enriched in TNFa treatment. To test if acetylation of FOXA1 at K295 changes its binding preference and genomic distribution, we used the programmable properties of CRISPR/Cas9 to create specific knockin mutations to mimic or prevent acetylation of K295 in MCF-7 cells. More specifically, we mutated K295 to glutamine (K295Q) to mimic acetylation and essentially “lock” FOXA1 into a permanently acetylated state and, for comparison, we created another cell line where K295 was mutated to arginine (K295R) to prevent acetylation of FOXA1. Our preliminary data shows changes in the genomic redistribution of FOXA1 in the knock-in cell lines resulting in altered gene expression programs. These data suggest that inflammation-based acetylation of FOXA1 can affect estrogen signaling pathways in breast cancer cells by altering the enhancer landscape of FOXA1 and consequently the estrogen receptor.

Supported by a grant from the NIH/NCI (R00 CA204628) to H.L.F

Citation Format: Shen Li, Raul Mendez-Giraldez, Joseph P. Garay, Kamila Wisniewska, Colby A. Tubbs, Charles M. Perou, Hector L. Franco. Cytokine-induced post-translational modifications of FOXA1 affect enhancer selection and estrogen signaling in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4496.