BRRIAR lncRNA alters breast cancer risk by modulating interferon signaling in cis and in trans through BHLHE40 and RIG-I

Interferons (IFNs) are key regulators of cell proliferation and anti-tumor immunity. We identified a breast cancer-associated long noncoding RNA (lncRNA), BRRIAR, that modulates IFN signaling in estrogen receptor-positive (ER+) breast cancer. BRRIAR is transcribed from an 11 kb enhancer cluster at 3p26, and its reduced expression is linked to breast cancer GWAS risk variants. Primarily expressed in ER+ breast tumors, BRRIAR exhibits dual functionality, acting both in cis and in trans. Nuclear BRRIAR regulates BHLHE40 expression through its enhancer, while cytoplasmic BRRIAR binds to the pattern recognition receptor RIG-I, modulating its activation. BRRIAR RNA overexpression activates RIG-I signaling, inducing IFN responses that selectively trigger apoptosis in ER+ breast tumor cells in vitro and in vivo, while promoting immune activation in human peripheral blood mononuclear cells. These findings emphasize the complex regulatory mechanisms at GWAS risk regions, reveal the critical role of lncRNAs as modulators of tumor immunity and identify BRRIAR as a promising RNA-based therapeutic for ER+ breast cancer. Overall design: H3K27ac ChIPseq data in T47D cells after CRISPRi-BRRIAR or using gRNAs targeted to the 3' end of the enhancer cluster. T47D cells were cross-linked with 1% formaldehyde (Merck) at 37oC for 10 min, rinsed with phosphate buffered saline (PBS; TMO) containing 5% bovine serum albumin (BSA; Scientifix) and harvested in PBS containing protease inhibitor cocktail (PIC; Roche). Harvested cells were centrifuged for 2 min at 3,000 rpm. Cell pellets were resuspended in lysis buffer (1% sodium dodecyl sulfate (SDS; Merck), 10 mM EDTA (TMO), 50 mM Tris-HCl, pH 8.1, PIC) and sonicated three times for 15 s at 70% duty cycle (Branson SLPt) then centifuged at 13,000 rpm for 15 min. Supernatants were resuspended in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1). Antibodies detecting H3K27ac (2 µg; Abcam) or an IgG control (2 µg; Abcam) were prebound for 6 h to protein G Dynabeads (TMO), then incubated with the chromatin for 16 h. The complexes were then washed six times in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate (Merck), 0.1% SDS, 1 mM DTT, PIC). To reverse cross-links, the complexes were incubated at 65oC for 16 h in elution buffer (1% SDS, 0.1 M NaHCO3), then DNA fragments purified using the QIAquick spin kit (Qiagen). Libraries were prepared using the NEBNext Ultra II DNA library prep kit (NEB), according to manufacturer's instructions.