IFNγ-induced PD-L1 expression in ovarian cancer cells is regulated by JAK1, STAT1 and IRF1 signaling

Abstract

Expression of the immune checkpoint programmed death ligand-1 (PD-L1) is increased in ovarian cancer (OC) and correlates with poor prognosis. Interferon-γ (IFNγ) induces PD-L1 expression in OC cells, resulting in their increased proliferation and tumor growth, but the mechanisms that regulate the PD-L1 expression in OC remain unclear. Here, we show that the IFNγ-induced PD-L1 expression in OC cells is associated with increased levels of STAT1, Tyr-701 pSTAT1 and Ser-727 pSTAT1. Suppression of JAK1 and STAT1 significantly decreases the IFNγ-induced PD-L1 expression in OC cells, and STAT1 overexpression increases the IFNγ-induced PD-L1 expression. In addition, IFNγ induces expression of the transcription factor interferon regulatory factor 1 (IRF1) and IRF1 suppression attenuates the IFNγ-induced gene and protein levels of PD-L1. Chromatin immunoprecipitation results show that IFNγ induces PD-L1 promoter acetylation and recruitment of STAT1, Ser-727 pSTAT1 and IRF1 in OC cells. Together, these findings demonstrate that the IFNγ-induced PD-L1 expression in OC cells is regulated by JAK1, STAT1, and IRF1 signaling, and suggest that targeting the JAK1/ STAT1/IRF1 pathway may provide a leverage to regulate the PD-L1 levels in ovarian cancer.

Keywords: Interferon-γ; Ovarian cancer; PD-L1; STAT1; Transcriptional regulation.

Fig. 1.

IFNγ induces STAT1 signaling in OC cells. Real time qRT-PCR of PD-L1 and STAT1 mRNA in SKOV3 (A) and OVCAR3 (B) cells incubated with 50 ng/mL IFNγ. Western blotting of PD-L1, STAT1, Tyr701-pSTAT1, Ser727-pSTAT1 and control actin analyzed in whole cell extracts (WCE) of SKOV3 (C) and OVCAR3 (D) cells incubated with 50 ng/mL IFNγ. The values represent the mean +/−SE (n = 3); an asterisk denotes a statistically significant (* p < 0.05) change compared to control untreated (UT) cells.

Fig. 2.

IFNγ induced PD-L1 expression in OC cells is dependent on JAK1. Gene expression of JAK1 (A) and PD-L1 (B) measured by RT-PCR in SKOV3 cells transfected with control and JAK1 siRNA and incubated with 50 ng/mL IFNγ. (C) Western blotting of JAK1, PD-L1, Tyr701- pSTAT1, Ser727-pSTAT1, STAT1, and control actin analyzed in WCE of SKOV3 cells transfected with control and JAK1 siRNA and incubated with 50 ng/mL IFNγ. The values represent the mean +/−SE (n = 3); an asterisk denotes a statistically significant (* p < 0.05) change compared to cells transfected with control siRNA.

Fig. 3.

IFNγ induced PD-L1 expression in OC cells is dependent on STAT1. Gene expression of STAT1 (A) and PD-L1 (B) measured by RT-PCR in SKOV3 cells transfected with control and STAT1 siRNA and incubated with 50 ng/mL IFNγ. (C) Western blotting of STAT1, PD-L1 and actin analyzed in WCE of SKOV3 cells transfected with control and STAT1 siRNA and incubated with 50 ng/mL IFNγ. (D) Densitometric evaluation of PD-L1 protein levels shown in panel C; PD-L1 densities were normalized to actin and expressed as % compared with control UT cells. (E) Western blotting and (F) densitometric evaluation of PD-L1 levels in SKOV3 cells transfected with control and STAT1 activation plasmids and incubated 24 h with 50 ng/mL IFNγ. The values represent the mean +/−SE (n = 3); an asterisk denotes a statistically significant (* p < 0.05) change compared to cells transfected with control siRNA or plasmid.

Fig. 4.

IFNγ induced PD-L1 expression in OC cells is dependent on IRF1. Gene expression of IRF1 (A) and PD-L1 (B) measured by RT-PCR in SKOV3 cells transfected with control and IRF1 siRNA and incubated with 50 ng/mL IFNγ. (C) Western blotting of IRF1, PD-L1 and actin analyzed in WCE of SKOV3 cells transfected with control and IRF1 siRNA and incubated with 50 ng/mL IFNγ. (D) Densitometric evaluation of PD-L1 protein levels shown in panel C; PD-L1 densities were normalized to actin and expressed as % compared with control UT cells. The values represent the mean +/−SE (n = 3); an asterisk denotes a statistically significant (* p < 0.05) change compared to cells transfected with control siRNA.

Fig. 5.

IFNγ induces PD-L1 promoter acetylation and occupancy by STAT1, Ser727-pSTAT1 and IRF1 in OC cells. (A) Schematic illustration of STAT1 and IRF1 binding sites in human PD-L1 promoter. Occupancy of STAT1 (B) and IRF1 (C) binding sites in human PD-L1 promoter by STAT1, Ser727-pSTAT1, IRF1, histone H3 and ac-histone H3 analyzed by chromatin immunopredpitation (ChIP) in SKOV3 cells incubated with 50 ng/mL IFNγ. The data are presented as fold difference in occupancy of the particular protein at the particular locus compared with the negative control human IGX1A (SA Biosciences) locus, which does not contain any transcription factors binding sites. The values represent the mean ± SE (n = 3); an asterisk denotes a statistically significant (* p < 0.05) change compared with UT cells.