TGF-β1-Mediated PD-L1 Glycosylation Contributes to Immune Escape via c-Jun/STT3A Pathway in Nasopharyngeal Carcinoma

Abstract

Immunotherapy targeting programmed death ligand-1/programmed cell death protein-1 (PD-L1/PD-1) has achieved great success in multiple cancers, but only a small subset of patients showed clinical responses. Recent evidences have shown that post-translational modification of PD-L1 protein could regulate its protein stability and interaction with cognate receptor PD-1, thereby affecting anticancer immunotherapy in several solid tumors. However, the molecular mechanisms underlying how PD-1/PD-L1 expression is regulated still remain unclear in nasopharyngeal carcinoma (NPC). Here, we found N-glycosylation of PD-L1 in NPC cells and tissues. Mechanistically, we showed that STT3A transferred N-linked glycans to PD-L1, and TGF-β1 could positively regulate STT3A expression through activating c-Jun to bind to STT3A promoter. Functional assays showed that inhibition of TGF-β1 resulted in a decrease of glycosylated PD-L1 and enhanced cytotoxic T-cell function against NPC cells. Analysis of clinical specimens revealed that the expression of STT3A was positively correlated with TGF-β1 and c-Jun, and high STT3A expression was positively correlated with a more advanced clinical stage. Altogether, TGF-β1 activated c-Jun/STT3A signaling pathway to promote N-glycosylation of PD-L1, thus further facilitating immune evasion and reducing the efficacy of cancer immunotherapy. As such, all these data suggested that targeting TGF-β1 pathway might be a promising approach to enhance immune checkpoint blockade, and simultaneous blockade of PD-L1 and TGF-β1 pathways might elicit potent and superior antitumor activity relative to monotherapies.

Keywords: PD-L1; TGF-β1; glycosylation; immunotherapy; nasopharyngeal carcinoma.

Figure 1

PD-L1 was N-glycosylated in Nasopharyngeal Carcinoma. (A) Western blot analysis of PD-L1 in NPC patient samples. (B) Western blot analysis of PD-L1 in 6 NPC cells including 5-8F, CNE1, CNE2, 6-10B, HNE1, SUNE1. (C) Glycosylation pattern of PD-L1 protein in 5-8F and CNE1 cells. Cell lysates were treated with PNGase F or O-glycosidase and analyzed by western blot analysis. (D) Western blot analysis of PD-L1 in 5-8F and CNE1 cells treated by Tunicamycin (TM) with increasing concentrations.

Figure 2

Inhibition of TGF-β1 by SB431542 Reduced PD-L1 Glycosylation in Vitro. (A) Western blot analysis of PD-L1 in 5-8F and CNE1 cells treated by SB431542 with increasing concentrations. (B) Western blot analysis of PD-L1 and c-Jun in exogenous Flag-PD-L1 expressing 5-8F and CNE1 cells with the treatment of 20μM SB431542. (C) The glycosylation status of PD-L1 protein purified from SB431542 treating cells was analyzed by ConA lectin binding assay.

Figure 3

SB431542 Downregulated Glycosylation of PD-L1 via C-Jun/STT3A Pathway in Vitro and C-Jun was a Direct Transcriptional Regulator of STT3A. (A) Western blot analysis of c-Jun, STT3A and PD-L1 after silencing c-Jun in 5-8F and CNE1 cells. (B) Western blot analysis of STT3A and PD-L1 after silencing STT3A in 5-8F and CNE1 cells. (C) Western blot analysis of c-Jun, STT3A and PD-L1 after overexpressing c-Jun with(out) silencing STT3A in 5-8F and CNE1 cells. (D) Western blot analysis of PD-L1, c-Jun and STT3A in exogenous Flag-PD-L1 expressing 5-8F and CNE1 cells with the treatment of 20μM SB431542. (E) The binding motif of c-Jun from JASPAR database. (F) The CHIP-PCR assay was used to assess the binding of STT3A promoter region. (G) Anti-c-Jun-pulled down chromatins were analyzed by qRT-PCR. (H) A diagram showing the relationship of full-length and mutant STT3A promoters. (I) Dual-luciferase reporter gene assay was performed to indicate the interaction between c-Jun and STT3A. Bars, mean ± SD, *p<0.5, **p<0.01.

Figure 4

STT3A Expression was Positively Correlated with TGF-β1 and C-Jun in NPC Tissues, and High STT3A Expression was Associated with a more advanced stage in NPC. (A) Representative pictures of c-Jun and STT3A expression in NPC patients in TGF-β1-low and high expression groups. Scale bars represented 100μm. (B, C) Pearson correlation analysis of the association between TGF-β1 and STT3A (r=0.412; p=0.013) and between c-Jun and STT3A (r=0.5859; p=0.0002) in NPC tissues. (D) Comparison of STT3A levels in 36 NPC patients with different clinical stages. **p<0.01.

Figure 5

Inhibition of TGF-β1 by SB431542 suppressed T cells activity by inhibiting PD-L1 glycosylation in Vitro. (A, B) The death rate of 5-8F or CNE1 cells co-cultured with Jurkat T cells for 6h. The tumor cells were STT3A-kncokdowned by shRNA or pretreated with SB431542 (20μM) or TM (10μg/ml) for 48h. (C, D) IL-2 production by activated Jurkat T cells co-cultured with 5-8F or CNE1 cells with shSTT3A or pretreated with SB431542 (20μM) or TM (15μg/ml) for 48h. IL-2 secretion was measured by ELISA. Bars, mean ± SD, **p<0.01.