Tumor-intrinsic PRMT5 upregulates FGL1 via methylating TCF12 to inhibit CD8+ T-cell-mediated antitumor immunity in liver cancer

Abstract

Protein arginine methyltransferase 5 (PRMT5) acts as an oncogene in liver cancer, yet its roles and in-depth molecular mechanisms within the liver cancer immune microenvironment remain mostly undefined. Here, we demonstrated that disruption of tumor-intrinsic PRMT5 enhances CD8⁺ T-cell-mediated antitumor immunity both in vivo and in vitro. Further experiments verified that this effect is achieved through downregulation of the inhibitory immune checkpoint molecule, fibrinogen-like protein 1 (FGL1). Mechanistically, PRMT5 catalyzed symmetric dimethylation of transcription factor 12 (TCF12) at arginine 554 (R554), prompting the binding of TCF12 to FGL1 promoter region, which transcriptionally activated FGL1 in tumor cells. Methylation deficiency at TCF12-R554 residue downregulated FGL1 expression, which promoted CD8⁺ T-cell-mediated antitumor immunity. Notably, combining the PRMT5 methyltransferase inhibitor GSK591 with PD-L1 blockade efficiently inhibited liver cancer growth and improved overall survival in mice. Collectively, our findings reveal the immunosuppressive role and mechanism of PRMT5 in liver cancer and highlight that targeting PRMT5 could boost checkpoint immunotherapy efficacy.

Keywords: Antitumor immunity; FGL1; Liver cancer; PRMT5; TCF12.

Graphical abstract

Figure 1

PRMT5 disruption promotes CD8⁺ T-cell-mediated antitumor immunity suppressing liver cancer growth. (A) GO analysis of differentially expressed genes between control and PRMT5-KD HepG2 cells, as determined by RNA-seq (n = 3). (B) Tumor appearance in nude and C57BL/6 mice post-inoculation with Hepa1-6 (Ctrl) or Hepa1-6 (PRMT5 KD) cells (n = 5). (C) Excised tumor weights (left) and tumor volume changes (right) for each group, with volume measurements every 3 days (n = 5). (D) Representative immunofluorescence images and quantitative analysis of MDSC (CD11b⁺ Ly6c⁺), cDC (CD11c⁺ MHCII⁺), M1-Mϕ (F4/80⁺ CD86⁺), and NK cell (NK1.1⁺ CD3^–) in Ctrl and PRMT5-KD tumors of C57BL/6 mice. Scale bar, 50 μm or 20 μm. (E) Frequencies of MDSCs (CD45⁺ CD11b⁺ Gr1⁺), cDCs (CD45⁺ CD3^– NK1.1^–CD11c⁺ MHCII⁺), M1-Mϕs (CD45⁺ CD11b⁺ F4/80⁺ CD86⁺), and NK cells (CD45⁺ CD3^– NK1.1⁺) within CD45⁺ cells in Ctrl and PRMT5-KD tumors from C57BL/6 mice, as quantified by flow cytometry. (F) Representative immunohistochemistry (IHC) images and quantification of PRMT5, CD3⁺, CD4⁺, CD8⁺, and GzmB⁺ T cells in tumor tissues of C57BL/6 mice (n = 5). Scale bar, 50 μm or 20 μm. (G) Representative IHC images and correlation analysis of PRMT5 levels and CD3⁺ T cells in clinical liver cancer samples (n = 55). Scale bar, 50 μm. (H) Tumor weights of C57BL/6 mice across eight groups: IgG, PRMT5 KD + IgG, anti-CD4, PRMT5 KD + anti-CD4, anti-CD8, PRMT5 KD + anti-CD8, anti-CD4 + anti-CD8, and PRMT5 KD + anti-CD4 + anti-CD8 (n = 6). (I) Survival analysis across 8 groups (n = 8). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns: no significance. GO: Gene Ontology, KD: knockdown, MDSC: myeloid-derived suppressor cell, cDC: conventional dendritic cell, M1-Mϕ: M1 macrophage, NK: natural killer, GzmB: granzyme B, TGI: tumor growth inhibition value.

Figure 2

PRMT5 inhibition enhances CD8⁺ T-cell antitumor immunity via downregulating FGL1 expression in vivo and in vitro. (A) OT-1 CD8⁺ T cells were co-cultured with Hepa1-6-OVA cells treated with Ctrl or PRMT5 KD for 48 h. The proportion of GzmB⁺ cells within CD8⁺ T cells was quantified by flow cytometry. (B) T-cell-mediated tumor cell killing assays were performed by co-culturing OT-1 CD8⁺ T cells with the indicated Hepa1-6-OVA cells for 24 h, followed by quantification of surviving cell intensities. (C) CD8⁺ T cells were co-cultured with Hepa1-6 cells treated with Ctrl or PRMT5 KD for 48 h. The proportion of GzmB⁺ cells within CD8⁺ T cells was quantified by flow cytometry. (D) T-cell-mediated tumor cell killing assays were performed by co-culturing CD8⁺ T cells with the indicated Hepa1-6 cells for 72 h, followed by quantification of surviving cell intensities. (E) Heatmap displaying top 10 DEGs among Ctrl and PRMT5 KD treated HepG2 cells. (F) The mRNA and protein levels of PRMT5 and FGL1 in HepG2 cells treated with siCtrl and siPRMT5 (50 or 100 nmol/L). (G) The mRNA levels of PRMT5 and FGL1 in HepG2 cells treated with DMSO, GSK591 (5 μmol/L), or GSK591 (10 μmol/L), alongside the protein levels of FGL1, PRMT5, H4R3me2s, and Rme2sy in each treatment group. (H) Correlation analysis of mRNA (n = 53) and protein levels (n = 55) between PRMT5 and FGL1 in human liver cancer tissues, using Pearson's correlation coefficient. (I–K) CD8⁺ T cells were co-cultured with HepG2 or Huh7 cells treated with Ctrl, FGL1 KD, PRMT5 KD, or PRMT5 KD + FGL1 OE. (I, J) Flow cytometry analysis of the proportions of Ki67⁺ CD8⁺ T cells (I) and GzmB⁺ CD8⁺ T cells (J). (K) IL-2 expression in the co-culture supernatant was measured after 48 and 72 h using ELISA. (L) T-cell-mediated tumor cell killing assays conducted by co-culturing CD8⁺ T cells with either HepG2 or Huh7 cells for 72 h. Representative images of surviving tumor cells visualized with crystal violet staining (upper), and quantification of surviving cell intensities (lower). (M) Tumor appearance and weights in BALB/c mice following inoculation with Ctrl, FGL1-KD, PRMT5-KD, and PRMT5-KD + FGL1-OE H22 cells (n = 6). (N) Intratumoral CD8⁺ T-cell density across various groups, as detected by IHC staining (n = 6). (O) Intratumoral proportions of GzmB⁺ CD8⁺ T cells across various groups, as detected by flow cytometry. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns: no significance. KD: knockdown, OE: overexpression, GzmB: granzyme B.

Figure 3

TCF12 transcriptionally upregulates FGL1 expression in liver cancer. (A, B) Protein and mRNA levels of FGL1 in HepG2 and Huh7 cells following TCF12 knockdown (A) and overexpression (B). (C) Luciferase activities of FGL1 promoter reporter vectors in Cas9 control and TCF12-KO HepG2 cells. (D) Luciferase activities of FGL1 promoter reporter vectors in HepG2 and Huh7 cells transfected with either Flag-vector or Flag-TCF12 plasmids. (E) ChIP analysis of TCF12 binding to FGL1 promoter in HepG2 cells. Different regions of the FGL1 promoter containing different putative TCF12 binding sites are shown in Fig. S3J. (F) Correlation analysis of TCF12 and FGL1 mRNA levels in human liver cancer tissues using Pearson's correlation coefficient (n = 53). (G) Representative IHC images and correlation analysis of TCF12 and FGL1 protein levels in human liver cancer tissues, using Pearson's correlation coefficient (n = 55). Scale bar, 50 μm. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns: no significance. KO: knockout, WT: wide type, OE: overexpression.

Figure 4

PRMT5 catalyzes symmetric dimethylation of TCF12 at the R554 residue. (A) Diagram illustrating the binding of PRMT5 with TCF12 (upper). Residues around the protein–protein interaction interface form numerous hydrophobic interactions (lower). (B) Confocal microscopy showed the co-localization of PRMT5 and TCF12 (indicated by black arrows). Scale bar, 2 μm. (C) GST pulldown assay analyzing the direct binding between PRMT5 and TCF12 proteins. (D) Co-immunoprecipitation analysis was conducted to detect the interaction between endogenous TCF12 with PRMT5, and the Rme2sy levels of TCF12 in HepG2 and Huh7 cells. (E) Immunoprecipitation analysis of TCF12 methylation in HepG2 and Huh7 cells treated with siCtrl, siPRMT5 or siPRMT9. (F) Immunoprecipitation analysis of TCF12 methylation in HepG2 and Huh7 cells treated with DMSO, GSK591 or HLCL61. (G) Immunoprecipitation analysis was performed to detect the methylation of ectopic TCF12 in 293T cells transfected with Flag-TCF12, either alone or in conjunction with HA-PRMT5 transfection. (H) Immunoprecipitation analysis of ectopic TCF12 methylation in 293T cells transfected with Flag-tagged TCF12 wild type or composite mutants, with or without HA-PRMT5. (I) Coomassie Brilliant Blue staining of purified His-tagged PRMT5 protein, GST-tagged TCF12 wild type, and GST-tagged TCF12 R554K mutant proteins. (J) In vitro methylation analysis of GST-tagged TCF12 wild type and GST-tagged TCF12 R554K mutant proteins by His-tagged PRMT5 protein. EV: empty vector, WT: wide type, RK: arginine to lysine substitution, SAM: S-adenosylmethionine.

Figure 5

TCF12-R554 methylation promotes FGL1 transcriptional activation and inhibits CD8⁺ T-cell antitumor immunity. (A) ChIP/Re-ChIP analysis of TCF12 and PRMT5 complex occupancy on the FGL1 promoter region in HepG2 cells. (B) ChIP analysis of TCF12 and PRMT5 occupancy on the FGL1 promoter region in HepG2 cells treated with siPRMT5, siTCF12, alone or in combination. (C) ChIP analysis of ectopic TCF12 occupancy on the FGL1 promoter region transfected with TCF12 (WT), PRMT5, and TCF12 (R554K) individually or simultaneously. (D) Immunoprecipitation analysis of ectopic TCF12 methylation and FGL1 protein levels in HepG2 (TCF12 KO) cells transfected with Flag-tagged TCF12 (WT) or TCF12 (R554K). (E) Immunoprecipitation was used to assess the ectopic TCF12 methylation and FGL1 protein levels in the specified TCF12-KO Hepa1-6 cells. (F) In HepG2 (TCF12 KO) cells transfected with TCF12 (WT) or TCF12 (R554K), secreted FGL1 protein levels in the supernatant were assessed after 48 and 72 h using ELISA. (G) T-cell-mediated tumor cell killing assays were conducted by co-culturing CD8⁺ T cells with specified HepG2 cells for 72 h. Representative images of surviving tumor cells visualized with crystal violet staining, and quantification of surviving cell intensities. (H) After co-culturing CD8⁺ T cells with the indicated HepG2 cells, IL-2 expression levels in the co-culture supernatant were measured at 48 h and 72 h using ELISA. (I, J) Tumor appearance (I) and weights (J) in C57BL/6 mice following inoculation with Ctrl, TCF12 KO, TCF12 KO + TCF12 (WT), and TCF12 KO + TCF12 (R554K) Hepa1-6 cells (n = 6). (K) Intratumoral proportions of GzmB⁺ CD8⁺ T cells across various groups, as detected by flow cytometry. (L) A model illustrating how PRMT5-catalyzed TCF12 methylation at R554 modulates FGL1 expression to attenuate CD8⁺ T-cell activity. ∗∗P < 0.01, ∗∗∗P < 0.001; ns: no significance. KO: knockout, WT: wide type, IL-2: interleukin 2, RK: arginine to lysine substitution, GzmB: granzyme B, TSS: transcription start site, Me: arginine symmetric demethylation modification.

Figure 6

PRMT5 suppresses CD8⁺ T-cell activity via TCF12/FGL1 axis in vitro. (A, B) HepG2 and Huh7 cells were treated with PRMT5 OE, TCF12 KO or simultaneously. (A) Protein levels of FGL1, TCF12 and PRMT5 in each group were assessed by Western blot analysis. (B) Secreted FGL1 protein levels in the supernatant were assessed after 48 and 72 h using ELISA. (C–E) CD8⁺ T cells were co-cultured with indicated HepG2 or Huh7 cells. Flow cytometry quantification of GzmB⁺ (C) and Perforin⁺ (D) cell proportions within CD8⁺ T cells. (E) IL-2 expression levels in the co-culture supernatant were measured after 48 and 72 h using ELISA. (F) The schematic diagram illustrating the influence of the PRMT5/TCF12/FGL1 axis on CD8⁺ T-cell activity. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns: no significance. KO: knockout, OE: overexpression, GzmB: granzyme B, IL-2: interleukin 2.

Figure 7

PRMT5 inhibitor boosts PD-L1 mAb sensitivity in liver cancer immunotherapy. (A) Schematic representation of the animal experimental design. (B–D) Tumor appearance (B), volume (C), and weights (D) in Ctrl, PD-L1 mAb, GSK591, and GSK591 + PD-L1 mAb groups (n = 6). (E) Representative IHC images of FGL1 and CD8⁺ T cells (left), and quantification of CD8⁺ T cells (right) (n = 6). Scale bar, 50 μm. (F, G) Flow cytometry quantifying frequency of GzmB⁺ cells in CD8⁺ T cells in tumor tissues (F) and draining lymph nodes (G) (n = 6). (H) Survival analysis across 4 groups (n = 8). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns: no significance. mAb: monoclonal antibody, GzmB: granzyme B.