Mapping the FOXA1 Interactome in ER+ Breast Cancer Cells using Proximity Labeling Reveals Novel Interactions with the Orphan Nuclear Receptor NR2C2 [RNA-Seq]

FOXA1 is a pioneer transcription factor essential for chromatin accessibility and transcriptional regulation in hormone-driven cancers. In breast cancer, FOXA1 plays a central role in facilitating nuclear receptor binding, reprogramming enhancer landscapes, and promoting transcriptional changes associated with therapy resistance. While FOXA1's function has been primarily studied in the context of estrogen receptor-a (ER), its broader protein interaction network remains incompletely defined. Here, we systematically map FOXA1-interacting proteins in ER-positive breast cancer cells using proximity-dependent biotin labeling (miniTurbo) combined with quantitative LC-MS/MS proteomics. We engineered MCF-7 cell lines stably expressing miniTurbo-tagged FOXA1 at either the N-terminus or C-terminus to ensure comprehensive coverage of interaction interfaces. This approach recovered known FOXA1 partners, including AR, MLL3, YAP1, and GATA3, and identified 157 previously unreported FOXA1 interactors. Notably, 42 of these novel partners, including NR2C2, were significantly associated with poor relapse-free survival in ER+ breast cancer patients. To demonstrate the utility of this resource, we characterized the FOXA1-NR2C2 interaction in depth. Integrating ChIP-seq and RNA-seq, we show that FOXA1 and NR2C2 co-occupy a subset of genomic regions and drive co-regulated transcriptional programs involved in tumor progression. Our study reveals an expanded FOXA1 interactome and new insights into its functional network in breast cancer, providing candidate proteins for further exploration as biomarkers or therapeutic targets. Overall design: To comprehensively interrogate the transcriptomic proflie of NR2C2 in ER+ breast cancer cells, we compared MCF-7 and T47D cells treated with 30pmol siRNA against NR2C2 or Scramble (control) for 72 hours. Biological tripilicates of each sample, for a total of 12 samples, were submitted to the Puerto rico OMIC Center (PROMIC). PROMIC performed sequencing using 1ug of RNA per sample, performed the library prep using Illumina Stranded mRNA prep kit and sequenced using Illumina NextSeq 550.