Sequential Interferon β-Cisplatin Treatment Enhances the Surface Exposure of Calreticulin in Cancer Cells via an Interferon Regulatory Factor 1-Dependent Manner

Abstract

Immunogenic cell death (ICD) refers to a unique form of cell death that activates an adaptive immune response against dead-cell-associated antigens. Accumulating evidence indicates that the efficacy of conventional anticancer agents relies on not only their direct cytostatic/cytotoxic effects but also the activation of antitumor ICD. Common anticancer ICD inducers include certain chemotherapeutic agents (such as anthracyclines, oxaliplatin, and bortezomib), radiotherapy, photodynamic therapy (PDT), and oncolytic virotherapies. However, most chemotherapeutic reagents are inefficient or fail to trigger ICD. Therefore, better understanding on the molecular determinants of chemotherapy-induced ICD will help in the development of more efficient combinational anticancer strategies through converting non- or relatively weak ICD inducers into bona fide ICD inducers. In this study, we found that sequential, but not concurrent, treatment of cancer cells with interferon β (IFNβ), a type I IFN, and cisplatin (an inefficient ICD inducer) can enhance the expression of ICD biomarkers in cancer cells, including surface translocation of an endoplasmic reticulum (ER) chaperone, calreticulin (CRT), and phosphorylation of the eukaryotic translation initiation factor alpha (eIF2α). These results suggest that exogenous IFNβ may activate molecular determinants that convert cisplatin into an ICD inducer. Further bioinformatics and in vitro experimental analyses found that interferon regulatory factor 1 (IRF1) acted as an essential mediator of surface CRT exposure by sequential IFNβ-cisplatin combination. Our findings not only help to design more effective combinational anticancer therapy using IFNβ and cisplatin, but also provide a novel insight into the role of IRF1 in connecting the type I IFN responses and ICD.

Keywords: bioinformatics; chemotherapy; endoplasmic reticulum stress; immunogenic cell death; interferon.

Figure 1

Effects of interferon β (IFNβ) and cisplatin treatment protocols on cell viability and surface calreticulin expression in HeLa cells. (A) HeLa cells were cotreated with 100 ng/mL IFNβ and 2 μg/mL cisplatin for 24 h, or sequentially treated with 100 ng/mL IFNβ for 24 h and 2 μg/mL cisplatin for another 24 h. Surface calreticulin (CRT) (ecto-CRT) staining was performed and analyzed by flow cytometry. (B) The mean fluorescence of ecto-CRT in (A) was quantified and plotted. p < 0.05 (* or ^#), p < 0.01 (** or ^##) and p < 0.001 (***) indicate significant differences compared to control samples or the indicated group. (C) HeLa cells were sequentially treated with 100 ng/mL IFNβ for 24 h and 2 μg/mL cisplatin for another 4 h. Protein expression was analyzed by Western blotting. (D–F) For the concurrent treatment (cotreatment) protocol (D), HeLa cells were treated with 100 ng/mL IFNβ and the indicated doses of cisplatin for 72 h. For IFNβ-cisplatin sequential treatment protocol (E), HeLa cells were treated with 100 ng/mL IFNβ for 24, 48, or 72 h, and then cells were replated in 96-well plates and treated with the indicated doses of cisplatin for 72 h. Cell viability was examined by MTT assay. For the cisplatin-IFNβ sequential treatment protocol (F), HeLa cells were treated with 0.25 μg/mL cisplatin for 24 h, and then cells were replated in 96-well plates and treated with the indicated doses of IFNβ for 72 h.

Figure 2

Effects of interferon β (IFNβ) and cisplatin treatment protocols on cell viability and surface calreticulin expression in cancer cells. (A) SiHa, SKOV3, TC-1, and MOSEC cells were cotreated with 100 ng/mL IFNβ and the indicated doses of cisplatin for 72 h. Cell viability was examined by MTT assay. (B) SiHa, SKOV3, TC-1, and MOSEC cells were treated with 100 ng/mL IFNβ for 48 h, and then cells were replated in 96-well plates and treated with the indicated doses of cisplatin for 72 h. Cell viability was examined by MTT assay. (C) SiHa, SKOV3, TC-1, and MOSEC cells were sequentially treated with 100 ng/mL IFNβ for 24 h and 2 μg/mL cisplatin for another 24 h. Surface CRT (ecto-CRT) staining was performed and analyzed by flow cytometry. The mean fluorescence of ecto-CRT was quantified and plotted. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (*** or ^###) indicate significant differences compared to control samples or the indicated group.

Figure 3

Gene set enrichment analysis (GSEA) for cisplatin- and oxaliplatin-treated A2870 cells. (A) The microarray data set for oxaliplatin- and cisplatin-treated A2780 human ovarian cancer cells (GSE8057) was obtained from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. GSEA was performed for the enrichment of 50 cancer hallmarks. The enriched hallmarks with p < 0.01 and false discovery rate (FDR) with p < 0.25 are shown in the Venn diagram. (B) The enrichment plots for the apoptosis hallmark in oxaliplatin- and cisplatin-treated A2780 cells. The leading edge genes are highlighted in the red squares. The overlapping, oxaliplatin-specific, and cisplatin-specific genes were analyzed using the STRING database for network construction.

Figure 4

Gene set enrichment analysis (GSEA) for doxorubicin- and cisplatin-treated HeLa cells. (A) The enrichment plots for the apoptosis hallmark in doxorubicin- and cisplatin-treated HeLa cells. The leading edge genes are highlighted in the red squares. (B) The leading edge genes in doxorubicin-treated HeLa cells were analyzed using the STRING database for network construction.

Figure 5

Effects of JUN knockdown on sequential interferon β (IFNβ)-cisplatin treatment-induced cell viability inhibition and immunogenic cell death (ICD) biomarker expression in HeLa cells. (A) HeLa cells were transfected with JUN siRNA (si-JUN) or the non-targeting control siRNA (si-NC) for 24 h, and then sequentially treated with 100 ng/mL IFNβ for 24 h and 2 μg/mL cisplatin for another 4 h. Protein expressions were analyzed by Western blotting. (B) JUN siRNA-transfected HeLa cells were treated with 100 ng/mL IFNβ for 48 h, and then cells were replated in 96-well plates and treated with the indicated doses of cisplatin for 72 h. Cell viability was examined by MTT assay. (C) JUN siRNA-transfected HeLa cells were sequentially treated with 100 ng/mL IFNβ for 24 h and 2 μg/mL cisplatin for another 24 h. Surface CRT (ecto-CRT) staining was performed and analyzed by flow cytometry. The mean fluorescence intensity in each treatment (the red line) was compared with that in untreated si-NC-transfected HeLa cells (the black line). (D) The mean fluorescence of ecto-CRT in (C) was quantified and plotted. p < 0.001 (***) indicates significant differences compared to control samples.

Figure 6

Effects of interferon regulatory factor 1 (IRF1) knockout on sequential interferon β (IFNβ)-cisplatin treatment-induced cell viability inhibition and immunogenic cell death (ICD) biomarker expression in HeLa cells. (A) The IRF1 expression in IRF1-knockout (IRF1-KO#1 and IRF1-KO#2) and parental wildtype (WT) HeLa cells was analyzed by Western blotting. (B) IRF1-knockout (IRF1-KO#1 and IRF1-KO#2) and parental (WT) HeLa cells were treated with 100 ng/mL IFNβ for 48 h, and then cells were replated in 96-well plates and treated with the indicated doses of cisplatin for 72 h. Cell viability was examined by MTT assay. (C) IRF1-knockout (IRF1-KO#1) and parental (WT) HeLa cells were sequentially treated with 100 ng/mL IFNβ for 24 h and 2 μg/mL cisplatin for another 24 h. Surface CRT (ecto-CRT) staining was performed and analyzed by flow cytometry. The mean fluorescence intensity in each treatment (the red line) was compared with that in untreated parental HeLa cells (the black line). (D) The mean fluorescence of ecto-CRT in (C) was quantified and plotted. p < 0.05 (*), p < 0.01 (** or ^##) and p < 0.001 (***) indicate significant differences compared to control samples or the indicated group. (E) IRF1-knockout (IRF1-KO#1) and parental (WT) HeLa cells were sequentially treated with 100 ng/mL IFNβ for 24 h and 2 μg/mL cisplatin for another 4 h. Protein expressions were analyzed by Western blotting.