The polymorphism rs975484 in the protein arginine methyltransferase 1 gene modulates expression of immune checkpoint genes in hepatocellular carcinoma

Abstract

Protein arginine methyltransferase 1 (PRMT1) is a key regulator of hepatic immune responses. Recently, we reported that PRMT1 regulates the tumor immune response in hepatocellular carcinoma (HCC). Here we found that PRMT1 expression in human HCC correlates with that of programmed cell death 1 ligand 1 (PD-L1), PD-L2, and other checkpoint genes. PRMT1 deletion in mice reduced PD-L1 and PD-L2 expression in tumors and reduced the efficiency of PD-1 antibody treatment in a diethylnitrosamine-induced HCC mouse model, suggesting that PRMT1 regulates the hepatic immune checkpoint. Mice had reduced PD-L1 and PD-L2 expression when PRMT1 was specifically deleted in tumor cells or macrophages, but PRMT1 deletion in dendritic cells did not alter PD-L1 and PD-L2 expression. rs975484 is a common polymorphism in the human PRMT1 gene promoter, and we found that it alters PRMT1 expression in blood monocytes and tumor-associated macrophages in human HCC. PRMT1 expression was higher in individuals with a GG genotype than in individuals with a CC genotype, and heterozygous carriers had intermediate expression. Luciferase reporter assays indicated that this differential expression is due to an extra C/EBPβ-binding site in the PRMT1 promoter of individuals carrying the minor G allele. The rs975484 genotype also correlated with PRMT1 target expression in HCC. Individuals with the GG genotype had significantly higher levels of the PRMT1 targets PD-L1, PD-L2, and VISTA than those with the CC genotype. We conclude that PRMT1 critically controls immune checkpoints in mice and humans and that the PRMT1 polymorphism rs975484 affects checkpoint gene expression in HCC.

Keywords: CCAAT-enhancer-binding protein (C/EBP); PD-L2; SNP; checkpoint control; inflammation; macrophage; programmed cell death 1 ligand 1 (PD-L1); protein arginine methyltransferase 1 (PRMT1); rs975484; tumor immunology.

Figure 1.

PRMT1 gene polymorphisms regulate its expression in human blood monocytes. A, map of the genomic region containing the PRMT1 gene. Shown are transcription factor ChIP sequencing clusters (161 factors) from ENCODE, with transcription (Txn) factor binding sites shown in green. Also shown are the location and linkage disequilibrium for the indicated SNPs. B, PRMT1 mRNA expression in blood monocytes from individuals with the indicated genotypes. n = 67. *, p < 0.05; **, p < 0.01. C, PRMT1 mRNA expression in blood monocyte–derived macrophages from individuals with the indicated genotypes. n = 26. *, p < 0.05.

Figure 2.

PRMT1 gene polymorphism rs975484 regulate its expression through C/EBPβ binding. A, PRMT1 promoter–driven luciferase construct with C or G in the position of rs975484. B, relative luciferase mRNA expression in THP-1 cells transfected with C- or G-containing constructs or a control plasmid. n = 4 independent experiments; expression was normalized to C construct expression. *, p < 0.05, paired t test on the raw expression values. C, PRMT1 promoter sequence aligned with the CEBPB binding motif MA0466.2 (JASPAR database) and CEBPB binding sites in IL-6 and CCR5 gene promoters. D, relative luciferase mRNA expression in THP-1 cells transfected with C- or G-containing constructs or a control plasmid in the presence of a shRNA control plasmid or CEBPB-specific shRNAs. n = 3 independent experiments; expression was normalized to C (blue columns) or G (black columns) construct expression. *, p < 0.05, paired t test on the raw expression values. E, ChIP using anti-C/EBPβ antibody or IgG as a negative control in THP-1 cells expressing control vector or the C or G PRMT1 promoter construct. Data are presented as mean enrichment (over IgG) ± S.D. n = 3.

Figure 3.

PRMT1 gene polymorphisms regulate its expression in tumor-associated macrophages. A, PRMT1 mRNA expression in whole-tissue mRNA of HCC specimens from individuals with the indicated genotypes. n = 36. B, representative images of immunohistochemistry staining of HCC sections from individuals with the GG and CC genotypes using anti-MRC1 or anti-CD68 to detect TAMs and anti-PRMT1 antibodies. Arrows indicate TAMs. C, number of PRMT1-positive TAMs counted by a blinded person unaware of the genotype of the samples in HCC sections from individuals with the GG (n = 3), GC (n = 6), or CC (n = 3) genotypes. MRC1-positive or CD68-positive TAMs double-positive for PRMT1 were counted as PRMT1+ TAMs. *, p < 0.05; **, p < 0.01. D, Left, number of PRMT1-positive TAMs (MRC1-positive cells) in HCC specimens of individuals with the indicated genotypes. n = 12. *, p < 0.05; **, p < 0.01. Right, multivariate analysis of PRMT1-positive TAMs correlation with indicated SNPs.

Figure 4.

PRMT1 regulates immune checkpoint gene expression in mice and humans. A, correlation between PRMT1 and CD274 (PD-L1) gene expression in HCC samples or liver tumor samples from the GEPIA database (A, The Cancer Genome Atlas (TCGA) samples, n = 369 HCC) or ProteinAtlas database (B, n = 365 liver tumors). p < 0.00001. B, PRMT1 floxed mice were injected with DEN (75 mg/kg) four times weekly starting at 4 weeks of age. At 6 months of age, mice were given AAV.CMV.CRE or AAV.control vectors at 10¹¹ gc/mouse i.p. At 8 months of age, livers were analyzed. Liver sections containing tumor nodules were stained using anti-PRMT1 antibodies (top panel) or anti-PD-L1, anti-PD-L2 antibodies (bottom panel). Individual tumors were excised and analyzed for whole-tumor mRNA expression of genes coding for PD-L1 (Cd274), PD-L2 (Pdcd1lg2), and B7-H3 (Cd276). n = 3 mice. *, p < 0.05. C, PRMT1 mRNA expression in livers of mice correlates with tumor number. PRMT1 floxed mice were injected with DEN (75 mg/kg) four times weekly starting at 4 weeks of age. At 6 months of age, mice were given AAV.CMV.CRE or AAV.control vectors at 10¹¹ gc/mouse i.p. Pairs of littermates, WT or KO, received four injections biweekly, starting at 6 months, of age of PD-1–specific antibody (12.5 mg/kg) or vehicle control. Left panel, surface tumor number was analyzed at 8 months of age. *, p < 0.05. Right panel, representative H&E staining of liver sections of WT and knockout mice). No tumor nodules were found in WT mice treated with PD-1 antibody. D, PRMT1 floxed mice were injected with DEN (10 mg/kg) at 2 weeks of age. At 6 months of age, mice were given AAV.TBG.CRE or AAV.control vectors at 10¹¹ gc/mouse i.p. At 8 months of age, livers were analyzed. Individual tumors were excised and analyzed for whole-tumor mRNA expression. n = 3 mice. *, p < 0.05. E, relative mRNA in tumors of PRMT1 flox/flox LysM Cre mice and PRMT1 flox/flox WT littermates. Mice were injected with DEN (10 mg/kg) at 2 weeks of age. At 9 months of age, livers were analyzed. n = 3 mice. *, p < 0.05. F, PRMT1 flox/flox cd11c-Cre mice and PRMT1 flox/flox WT littermates were injected with DEN (10 mg/kg) at 2 weeks of age. At 9 months of age, livers were analyzed. Individual tumors were excised and analyzed for whole-tumor mRNA expression. n = 5–7 mice. *, p < 0.05.

Figure 5.

PRMT1 regulates PD-L1 and PD-L2 expression in tumor cells and macrophages. A, liver sections from WT or hepatocyte-specific PRMT1 knockout mice as in Fig. 4D were stained using anti-PD-L1 and anti-PD-L2 antibodies. Relative staining intensity is presented on the right. n = 3 mice. *, p < 0.05. B, liver sections from WT or myeloid-specific PRMT1 knockout littermates treated with DEN and analyzed at 6 months of age were stained using anti-PD-L1 and anti-PD-L2 antibodies. Relative staining intensity is presented on the right. n = 3 mice. *, p < 0.05. C, liver sections from WT or DC-specific PRMT1 knockout littermates treated with DEN and analyzed at 9 months of age were stained using anti-PD-L1 and anti-PD-L2 antibodies.

Figure 6.

The rs975484 genotype correlates with expression of the PRMT1 targets PD-L1, PD-L2, and VISTA in human HCC. A, mRNA expression in whole-tissue mRNA of HCC specimens from individuals with the indicated genotypes. n = 36. *, p < 0.05. B and C, liver sections containing HCC were stained for PD-L1, PD-L2, VISTA, B7-H3, and TIM3. B, relative staining intensity in individuals with the indicated genotypes. n = 3 (GG), 5 (GC), and 5 (CC). *, p < 0.05. C, representative images of staining in individuals with the GG and CC genotypes.