HDAC is indispensable for IFN-γ-induced B7-H1 expression in gastric cancer

Abstract

Background: B7 homolog 1 (B7-H1) overexpression on tumor cells is an important mechanism of immune evasion in gastric cancer (GC). Elucidation of the regulation of B7-H1 expression is urgently required to guide B7-H1-targeted cancer therapy. Interferon gamma (IFN-γ) is thought to be the main driving force behind B7-H1 expression, and epigenetic factors including histone acetylation are recently linked to the process. Here, we investigated the potential role of histone deacetylase (HDAC) in IFN-γ-induced B7-H1 expression in GC. The effect of Vorinostat (SAHA), a small molecular inhibitor of HDAC, on tumor growth and B7-H1 expression in a mouse GC model was also evaluated.

Results: RNA-seq data from The Cancer Genome Atlas revealed that expression of B7-H1, HDAC1-3, 6-8, and 10 and SIRT1, 3, 5, and 6 was higher, and expression of HDAC5 and SIRT4 was lower in GC compared to that in normal gastric tissues; that HDAC3 and HDAC1 expression level significantly correlated with B7-H1 in GC with a respective r value of 0.42 (p < 0.001) and 0.21 (p < 0.001). HDAC inhibitor (Trichostatin A, SAHA, and sodium butyrate) pretreatment suppressed IFN-γ-induced B7-H1 expression on HGC-27 cells. HDAC1 and HDAC3 gene knockdown had the same effect. SAHA pretreatment or HDAC knockdown resulted in impaired IFN-γ signaling, demonstrated by the reduction of JAK2, p-JAK1, p-JAK2, and p-STAT1 expression and inefficient STAT1 nuclear translocation. Furthermore, SAHA pretreatment compromised IFN-γ-induced upregulation of histone H3 lysine 9 acetylation level in B7-H1 gene promoter. In the grafted mouse GC model, SAHA treatment suppressed tumor growth, inhibited B7-H1 expression, and elevated the percentage of tumor-infiltrating CD8+ T cells.

Conclusion: HDAC is indispensable for IFN-γ-induced B7-H1 in GC. The study suggests the possibility of targeting B7-H1 using small molecular HDAC inhibitors for cancer treatment.

Keywords: B7-H1; Gastric cancer; HDAC; IFN-γ; Immune evasion.

Fig. 1

B7-H1 expression is correlated with HDAC in GC. a B7-H1 and HDAC1–3 expression was compared between normal gastric tissues (n = 34) and primary GC tissues (n = 415) from RNA-seq data of TCGA. The results were presented in box-and-whisker plots (median values (line), 25th–75th percentiles (box outline) and minimum and maximum values (whiskers)). b Spearman correlation analyses of B7-H1 and HDACs were performed using GC RNA-seq data of TCGA. c B7-H1 and HDAC1–3 protein expression was compared between 12 paired GC tissues and cancer-adjacent tissues collected from our institution. Shown were expression change between each pair of cancer and adjacent tissue. +, expression upregulation in cancer; −, expression downregulation in cancer. d B7-H1 and HDAC1–3 mRNA expression was compared between 12 paired GC tissues (cancer) and tumor-adjacent tissues (normal) collected from our institution. e H3K27Ac level in B7-H1 gene promoter region was assessed in seven cell lines using ChIP-seq data from ENCODE. *p < 0.05, **p < 0.001. NS, not significant

Fig. 2

HDAC inhibition suppressed IFN-γ-induced B7-H1 expression in GC. a HGC27 cells were treated with IFN-γ of 0 to 100 ng/ml for 24 h or of 50 ng/ml for 0 to 24 h before B7-H1 protein expression was determined. b As was shown in the treatment schedule, HGC27 cells were pretreated with either DMSO or different concentrations of HDACI (sodium butyrate, TSA, or SAHA) for 8 h, which was then removed, followed by treatment with 50 ng/ml IFN-γ for 24 h. c HGC27 cells were treated according to the above schedule; then, B7-H1 protein expression was determined. d HGC27 cells were treated according to the above schedule; then, B7-H1 mRNA expression was determined. e, f HGC27 cells were treated according to the above schedule; then, cell surface B7-H1 expression was determined by flow cytometry. c–e Experiments were repeated three times with similar results. *p < 0.05, **p < 0.001

Fig. 3

HDAC knockdown suppressed IFN-γ-induced B7-H1 expression in GC. a 36 h after transfection of negative control (NC) or HDAC siRNA, HDAC1, HDAC2, and HDAC3 protein expression in HGC27 cells was determined, respectively. b Following siRNA transfection, HGC27 cells were treated with 50 ng/ml IFN-γ for 24 h. Then, B7-H1 protein expression was determined. c Following siRNA transfection, HGC27 cells were treated with 50 ng/ml IFN-γ for 12 h. Then, B7-H1 mRNA expression were determined. a–c Experiments were repeated three times with similar results. **p < 0.001

Fig. 4

SAHA pretreatment or HDAC knockdown suppressed the expression of p-JAK1, p-JAK2, and p-STAT1. a HGC27 cells were pretreated with either DMSO or GLPG0634 (5 μM), fedratinib (5 μM), fludarabine (100 μM), and NSC 74589 (200 μM) for 8 h to inhibit JAK1, JAK2, STAT1, and STAT3, respectively, followed by treatment of 50 ng/ml IFN-γ for 24 h. Then, B7-H1 protein expression was determined. b HGC27 cells were treated according to the schedule shown in Fig. 2b; then, the expression of the phosphorylated and total protein of JAK1, JAK2, and STAT1 was determined. c Thirty-six hours after the transfection of negative control (NC) or HDAC siRNA, HGC27 cells were treated with 50 ng/ml IFN-γ for 3 h. Then, the expression of the phosphorylated and total protein of JAK1, JAK2, and STAT1 was determined. a–c Experiments were repeated three times with similar results

Fig. 5

HDAC inhibition or HDAC knockdown suppressed JAK2 expression. a HGC27 cells were treated with 0–2 μM SAHA for 24 h or with 0.5 μM SAHA for 0–24 h prior to JAK2 protein expression determination. b Thirty-six hours after transfection of NC or HDAC siRNA, JAK2 protein expression in HGC27 cells was determined. c Twenty-four hours after DMSO, SAHA (0.5 μM), or TSA (0.5 μM) treatment, JAK2 mRNA expression in HGC27 cells was determined. d Thirty-six hours after transfection of NC or JAK2 siRNA, HGC27 cells were treated with 50 ng/ml IFN-γ for 3 h. Then JAK2, p-JAK2, and p-STAT1 protein expression were determined. a–d Experiments were repeated three times with similar results. *p < 0.05, **p < 0.001

Fig. 6

SAHA pretreatment impaired STAT1 nuclear translocation. HGC27 cells were pretreated with either DMSO or 2 μM SAHA for 8 h before stimulation with 50 ng/ml IFN-γ for 1 h or 3 h. Immunofluorescence staining of STAT1 was performed. The experiment was repeated three times with similar results

Fig. 7

SAHA pretreatment impaired IFN-γ-induced histone acetylation of B7-H1 gene promoter. a HGC27 cells were pretreated with either DMSO or 2 μM SAHA for 8 h before stimulation with 50 ng/ml IFN-γ for 3 h. ChIP was performed using anti-H3K9Ac followed by quantification of immunoprecipitated B7-H1 gene promoter through PCR. H3K9Ac enrichment was determined by the percent input method. IFN-γ-induced upregulation of H3K9Ac was calculated as the ratio of H3K9Ac enrichment at post-stimulation to that at pre-stimulation. b The correlative relationship between enrichment of H3K9Ac at the promoters of B7-H1 gene and relative B7-H1 mRNA transcription in the same cell populations mentioned in A. X-axis stood for the mean value of enrichment of H3K9Ac at all the promoters of B7-H1 gene. Y-axis stood for B7-H1 mRNA expression relative to pre-stimulation level. a, b Experiments were repeated three times. **p < 0.001, *p < 0.05

Fig. 8

SAHA inhibited tumor growth and suppressed tumor B7-H1 expression in subcutaneously transplanted mouse GC model. a–c SAHA treatment group showed slower tumor growth and smaller tumor size compared to control group. d, e Tumor cell B7-H1 expression was lower in SAHA group than in the control group. f, g The percentage of tumor infiltrated CD8+ tumor cells was higher in the SAHA group than in the control group. Shown in d and f are representative results in one mouse. NS, not significant; **p < 0.001