Figure 4. Olaparib activates the STING pathway more potently in HR-deficient TNBC cells compared to HR-proficient cells.

(A) The human TNBC isogenic cell line pair MDA-MB-436 control and BRCA1-reconstituted (+BRCA1) and a PARP inhibitor-resistant MDA-MB-436 clone were treated with DMSO (0 μM olaparib) or the indicated doses of olaparib for 72h and subjected to immunofluorescence with picogreen dye and anti-cGAS antibody. (Left panel) Quantification of micronuclei. Error bars represent SEM of 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Sidak’s post-hoc test. (Right panel) Representative images of picogreen (green) and cGAS (red) stained micronuclei are shown (60x magnification). Scale bar, 8 μm; (B) At 72h post-treatment with DMSO or olaparib, lysates from parental and PARP inhibitor-resistant MDA-MB-436 cells, as well as MDA-MB-436 control and BRCA1-reconstituted cells (+BRCA1) were subjected to ELISA for cGAMP production. cGAMP levels were expressed as fold-change versus DMSO. Error bars represent SEM of 2 independent experiments. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s post-hoc test; (C) The same cell lines were treated as in (B) and subjected to flow cytometric analysis of pTBK1^Ser172 expression. Cisplatin (Cis; 1 μM) was used as positive control. Error bars represent SEM of 2–4 independent experiments. Statistical analysis was performed using one-way ANOVA with Sidak’s post-hoc test (left panel) or two-way ANOVA with Tukey’s post-hoc (right panel); (D) Cell lines were treated with DMSO or the indicated concentrations of olaparib for 72h and lysates were subjected to immunoblotting for pTBK1^Ser172 and total TBK1 expression. Vertical black lines indicate points of blot cropping. (E) MDA-MB-436, HCC1937 and MDA-MB-231 human TNBC cell lines were treated with olaparib according to their 7-day IC₅₀ values and subjected to immunoblotting for pTBK1^Ser172 and total TBK1 expression. (F-G) Parental, PARP inhibitor-resistant, control and BRCA1-reconstituted MDA-MB-436 cells were subjected to flow cytometric analysis for pIRF3^Ser396 and pH2AX^Ser139 expression. Cisplatin (Cis; 1 μM) was used as a positive control. Error bars represent SEM of 2–4 independent experiments. Statistical analysis was performed using one-way ANOVA with Sidak’s post-hoc test or two-way ANOVA with Tukey’s post-hoc. (H) qPCR analysis of IFNβ, CCL5 and CXCL10 mRNA levels in parental, PARP inhibitor-resistant, control and BRCA1-reconstituted MDA-MB-436 cells, normalized to GAPDH internal control. Error bars represent SEM of 4 independent experiments. Statistical analyses were performed using Kruskal-Wallis test Dunn’s post-hoc test.