Figure 1. BRCA1 loss leads to transcriptional reprogramming of ovarian cancer cells and results in overexpression of the DS/IFN pathway.

(A) (Upper) UWB1.289 cell lines: UWB1.289 BRCA1^mut with concomitant loss of wild-type (WT) allele through LOH and WT BRCA1-reconstituted isogenic cell line. (Lower) Western blot (WB) analysis of BRCA1 in nuclear (N) extracts of BRCA1^mut and BRCA1^WT cells.

(B) Heatmap of hallmark signatures with significantly different enrichment score between BRCA1^mut and BRCA1^WT isogenic cell lines (adjusted p value of <0.05 after linear regression) at the proteomics level.

(C) Volcano plot of differentially expressed proteins in the BRCA1^mut and BRCA1^WT cell lines by MS. Immune-related proteins are highlighted in blue; selected genes of interest are highlighted in red. The position on the right side of the plot indicates higher expression in the BRCA1^mut cell line. Black curves represent significance cutoff (t test permutation-based false discovery rate [FDR] < 0.01, S0 = 0.3).

(D) WB analysis for STING and IFI16 in cytoplasmic (CYT) and nuclear (NUC) extracts of BRCA1^mut and BRCA1^WT cells (n = 3). The signal obtained for each protein was normalized to that of housekeeping β-actin and lamin B in CYT and NUC, respectively. Data are presented as mean ± SEM. p values were calculated by an unpaired t test.

(E) Pre-ranked gene set enrichment analysis using the difference in the number of enhancers between BRCA1^mut and BRCA1^WT cell lines as a ranking factor. Pathways enriched in BRCA1^WT are in red. All of the others were found in the BRCA1^mut. Right panels display the three most enriched pathways in BRCA1^mut.

(F) Association of differentially expressed genes and the presence of enhancers in the BRCA1^mut versus the BRCA1^WT cells by RNA-seq, Hi-C, and H3K27ac ChIP-seq. Genes in red are those implicated in the DS/IFN pathway.

(G) WB analysis for pSTAT1 and total STAT1 in the cytoplasmic (C) and nuclear (N) fractions of BRCA1^mut and BRCA1^WT cells treated with liposomes or poly(I:C)- or poly(dA:dT)-loaded liposomes. β-Actin and lamin B were used as protein loading controls in C and N, respectively.

(H) RT-PCR analysis of CCL5, CXCL9, IFNA1, TNFA, CXCL10, and IFNB1 in BRCA1^mut and BRCA1^WT cells treated with liposomes or poly(I:C)- or poly(dA:dT)-loaded liposomes (n = 3). Data are presented as mean ± SEM. p values were calculated by an unpaired t test.

(I)WB analysis of BRCA1, STING, IFI16, NF-κB, pIRF3, pSTAT1, and total STAT1 in the C and N fractions of OVCAR5 BRCA1^kd and BRCA1^WT cells treated with liposomes or poly(I:C)- or poly(dA:dT)-loaded liposomes.

(J)Immunofluorescence (IF) analysis of pSTAT1 (green), γH2AX (red), and DAPI (blue) in BRCA1^kd and BRCA1^WT cells 48 h after irradiation (10 Gy). Scale bars, 25 mm (10 μm in insets).

See also Figure S1 and Tables S1 and S2.