Fig 2: IFI16 mediates the type I IFN-induced upregulation of aromatase in SGBS cells. a SGBS cells were transfected with the indicated siRNAs. Twenty-four hours after transfection, the cells were treated with 10 ng/ml IFNß for 48 h. Expression level of aromatase was measured by qPCR. *P < 0.05 vs siControl with vehicle, #P < 0.05 as indicated (n = 3). The mRNA levels of ISGs are shown as control in Fig. S3. b SGBS cells were transfected with control siRNA or IFI16 siRNA. Twenty-four hours after transfection, the cells were treated with 10 ng/ml IFNß for 48 h. Expression levels of protein were analyzed by western blotting (top). Band intensities of aromatase and IFI16 in blots were quantified using ImageJ and normalized to that of Actin band (bottom). c–e SGBS cells were transfected with empty vector (0) or the indicated amount of Myc-IFI16 for 48 h. Expression level of aromatase was analyzed by western blotting (c, top). Band intensities of aromatase and IFI16 in blots were quantified using ImageJ and normalized to that of Actin band (c, bottom). The concentration of estradiol in the culture medium was measured by ELISA (d). Expression levels of the PI.4, PI.3, or PII-specific aromatase transcripts were measured by qPCR (e). *P < 0.05 vs empty vector (0) (n = 3)

Fig 3: E2-dependent growth of MCF7 is decreased in conditioned media obtained from the IFI16 KO SGBS preadipocytes. a Expression level of IFI16 in SGBS control or IFI16 KO cells was analyzed by western blotting (top). SGBS control or IFI16 KO cells were treated with 10 ng/ml IFNs for 48 h. Expression levels of mRNA and protein of aromatase were analyzed by qPCR (middle) and western blotting (bottom), respectively. *P < 0.05 vs vehicle, #P < 0.05 vs control treated with IFN (n = 4). b SGBS control or IFI16 KO cells were treated with 10 ng/ml IFNß for 48 h. The concentration of E2 in the culture supernatants was measured by ELISA. *P < 0.05 vs vehicle, #P < 0.05 vs control treated with IFNß (n = 3). c, d THP1 and MCF7 cells were cocultured as described in Fig. S2a. SGBS control or IFI16 KO cells were incubated with conditioned medium (CM) obtained from the THP1 and MCF7 coculture for 48 h. Expression levels of mRNA and protein of aromatase were analyzed by qPCR (top) and western blotting (bottom), respectively (c). The concentrations of E2 in the culture medium were measured by ELISA (d). *P < 0.05 vs DMEM/F12, #P < 0.05 vs control with coculture CM (n = 3). e THP1 cells pre-treated with 100 ng/ml PMA for 48 h, and SGBS cells were seeded in the bottom chamber of the transwell, as indicated in the scheme. MCF7 cells were seeded in the top chamber of the transwell insert. The number of viable MCF7 cells was counted using a hemocytometer. Statistical analysis was performed using two-way ANOVA followed by the Bonferroni posttest. ***P < 0.001 vs MCF7 cocultured with SGBS control with THP1 cells (filled square) (n = 4) (left). Total RNAs were obtained from the MCF7 cells at the day 5 and subjected to qPCR analysis. *P < 0.05 vs MCF7 monoculture, #P < 0.05 vs MCF7 cocultured with SGBS control with THP1 cells (filled square) (n = 3) (right)

Fig 4: Type I IFN leads to transcriptional activation of aromatase and E2 production in the SGBS cells. a MCF7 (4 × 105 cells) or SGBS cells (2 × 105 cells) were treated with vehicle (-), IFNa, or IFNß for 48 h. The doubling times of MCF7 and SGBS without IFN stimulation were about 25 h and 23 h, respectively. The mRNA level of aromatase was measured by qPCR. b SGBS cells (2 × 105 cells) were treated with vehicle (-) or IFNa for 48 h, as indicated. Expression level of aromatase was analyzed by western blotting (top). Band intensity of aromatase was quantified using ImageJ and normalized to that of Actin band (bottom). c MCF7 (4 × 105 cells) or SGBS (2 × 105 cells) cells were treated with vehicle (-), IFNa, or IFNß for 48 h, as indicated. The concentration of E2 in the culture supernatants was measured by ELISA. d Structure of aromatase gene promoters and primers used for measurement of promoter activity (top). SGBS cells were treated with vehicle (-), IFNa, or IFNß for 48 h. The levels of the PI.4, PI.3, or PII-specific aromatase transcripts were measured by qPCR (bottom). Data are presented as the mean ± SD (n = 3). *P < 0.05 vs vehicle

Fig 5: Clinical consequences of BC patients with different IFN expression levels. a The GSE21653 and the GSE6532 datasets were obtained and analyzed in the CTGS website (http://ctgs.biohackers.net/). Kaplan–Meier analysis was carried out in the group of patients stratified by ER status. Patients were categorized into a low (lower quartile) and a high (upper quartile) IFN expression groups. To analyze statistical differences, Log-rank (Mantel–Cox) tests were performed. b Gene expression analysis was performed using GSE22219 dataset obtained from the NCBI GEO website. Expression levels of IFNA1 and IFNB1 were analyzed in different grades of BC specimens stratified by ER status. Bars in red indicate the median expression level in each group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post hoc test. c Gene expression analysis was conducted using E-TABM-158 dataset obtained from ArrayExpress website. A case that lacked age information record was excluded from data analyses. Correlation between the expression of IFN and aromatase was analyzed in ER-positive BC patients by age group. Pearson’s correlation coefficient (r) was calculated