EBV-driven LMP1 and IFN-γ up-regulate PD-L1 in nasopharyngeal carcinoma: Implications for oncotargeted therapy

Abstract

PD-L1 expression is a feature of Epstein-Barr virus (EBV) associated malignancies such as nasopharyngeal carcinoma (NPC). Here, we found that EBV-induced latent membrane protein 1 (LMP1) and IFN-γ pathways cooperate to regulate programmed cell death protein 1 ligand (PD-L1). Expression of PD-L1 was higher in EBV positive NPC cell lines compared with EBV negative cell lines. PD-L1 expression could be increased by exogenous and endogenous induction of LMP1 induced PD-L1. In agreement, expression of PD-L1 was suppressed by knocking down LMP1 in EBV positive cell lines. We further demonstrated that LMP1 up-regulated PD-L1 through STAT3, AP-1, and NF-κB pathways. Besides, IFN-γ was independent of but synergetic with LMP1 in up-regulating PD-L1 in NPC. Furthermore, we showed that PD-L1 was associated with worse disease-free survival in NPC patients. These results imply that blocking both the LMP1 oncogenic pathway and PD-1/PD-L1 checkpoints may be a promising therapeutic approach for EBV positive NPC patients.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors state that they have no conflicts of interest.

Figures

Figure 1. PD-L1 expression was associated with EBV infection in human nasopharyngeal carcinoma cell lines

(A) The relative expression level of PD-L1 mRNA (detected by real time PCR method) in several common nasopharyngeal carcinoma cell lines (EBV-negative: CNE-1, CNE-2, SUNE-1, 5-8F, 6-10B, TWO3, and HNE-1; EBV-positive: C666-1) and an immortalized nasopharyngeal epithelial cell line (NP-69). The relative expression level of PD-L1 mRNA was normalized to that in SUNE-1 cell line. (B) The protein expression level of PD-L1 (detected by western blot) in different nasopharyngeal carcinoma cell lines and an immortalized nasopharyngeal epithelial cell line as described above. β-actin was used to verify equal loading. (C) The localization of PD-L1 (orange signal) in SUNE-1 and C666-1 cell lines shown by immunofluorescence counterstained with DAPI (blue signal). (D) Flow cytometric analysis of cell-surface PD-L1 expression in SUNE-1 and C666-1 cell lines (PD-L1, red line; isotype controls, blue line). All experiments were repeated at least three times. Representative data are shown.

Figure 2. PD-L1 expression was induced by EBV infection in human nasopharyngeal carcinoma cell lines

(A) The protein expression level of PD-L1 and LMP1 (detected by western blot) in the constructed EBV-positive (CNE-2-EBV+ and TWO3- EBV+) and EBV-negative (CNE-2 and TWO3) parental cell lines. β-actin was used to verify equal loading. (B) Quantified protein expression level of PD-L1 in CNE-2, CNE-2- EBV+, TWO3 and TWO3- EBV+ cell lines using Quantity One software (Bio-Rad Laboratories, Hercules, CA). (C) The localization of PD-L1 (orange signal) in CNE-2 and CNE-2- EBV+ cell lines shown by immunofluorescence counterstained with DAPI (blue signal). (D) The localization of PD-L1 (orange signal) in TWO3 and TWO3- EBV+ cell lines shown by immunofluorescence counterstained with DAPI (blue signal). Representative data of three independent experiments are shown.

Figure 3. LMP1 mediated the up-regulation of PD-L1 expression in EBV-infected human NPC cells

(A) The protein expression level of PD-L1 and LMP1 (detected by western blot) in CNE-2 and TWO-3 cell lines transiently transfected with control vector or LMP1 plasmids. (B) Quantified protein expression level of PD-L1 in CNE-2-vector, CNE-2-LMP1, TWO3-vector and TWO3-LMP1 cell lines using Quantity One software (Bio-Rad Laboratories, Hercules, CA). (C) The protein expression level of PD-L1 and LMP1 (detected by western blot) in CNE-2- EBV+ cell line treated with TPA (50 ng/ml) for 0, 12, 24 and 48 hours. (D) The protein expression level of PD-L1 and LMP1 (detected by western blot) in TWO3-EBV+ cell line treated with TPA (50 ng/ml) for 0, 12, 24 and 48 hours. (E) The protein expression level of PD-L1 and LMP1 (detected by Western blotting) in CNE-2-EBV+ cell lines and CNE-2-EBV− cell lines after transfected with LMP1-siRNA or Mock-siRNA. (F) The protein expression level of PD-L1 and LMP1 (detected by Western blotting) in TWO3-EBV+ cell lines and TWO3-EBV− cell lines after transfected with LMP1-siRNA or Mock-siRNA. All experiments were repeated at least three times and representative data are shown. β-actin was used to verify equal loading.

Figure 4. LMP1 induced PD-L1 expression through the downstream pathways involving JAK3/STAT3, AP-1 and NF-κB

(A) The protein expression level of LMP1, PD-L1, p-STAT3, STAT3, p-NF-κB, p-c-fos and p-c-Jun (detected by western blot) in NP-69-vector and NP-69-LMP1 stable cell lines. (B) The protein expression level of LMP1, PD-L1, p-STAT3, STAT3, p-NF-κB, p-c-fos and p-c-Jun (detected by western blot) in NP-69-vector and NP-69-LMP1 stable cell lines after transfected with Mock-siRNA or LMP1-siRNA. (C) The protein expression alteration of p-JAK3, JAK-3, p-STAT3, STAT3, PD-L1 in NP-69-LMP1 or NP-69 cell lines treated with 0, 1, 2 μM CP-690550, a selective JAK3 inhibitor for 72 hours. (D) C666-1 cells were treated with 0, 0.5, 1.0, 2.0 μM CP-690550 for 72hours and the level of PD-L1 was detected by western blot. (E) The protein expression alteration of p-ERK1/2, ERK1/2, p-c-fos, p-c-Jun, PD-L1 in NP-69-LMP1 or NP-69 cell lines treated with 0, 0.2, 0.4 μM PD0325901, a selective MEKs inhibitor for 72hours. (F) C666-1 cells were treated with 0, 0.1, 0.2, and 0.4 μM PD0325901 for 72 hours and the level of PD-L1 was detected by western blot. (G) The protein expression alteration of p-NF-κB, NF-κB, PD-L1 in NP-69-LMP1 or NP-69 cell lines treated with 0, 0.5, and 1.0 μM Caffeic Acid Phenethyl Ester (CAPE), a selective p-NF-κB inhibitor for 72 hours. (H) C666-1 cells were treated with 0, 0.25, 0.5, 1.0 μM CAPE for 72 hours and the level of PD-L1 was detected by western blot. All experiments were repeated at least three times and representative data are shown. β-actin was used to verify equal loading.

Figure 5. IFN-γ up-regulated PD-L1 expression in human nasopharyngeal carcinoma cells, which was independent of but synergetic with LMP1

(A) Serum IFN-γ level and EBV DNA copy numbers were measured in 34 NPC patients. Serum IFN-γ level was positively correlated with EBV burden. (B) The protein expression level of PD-L1 and LMP1 (detected by western blot) in CNE-2-vector and CNE-2-LMP1 stable cell lines treated with or without IFN-γ (100 U/ml) for 48 hours. β-actin was used to verify equal loading. (C) Quantified protein expression level of PD-L1 in CNE-2-vector and CNE-2-LMP1 cell lines using Quantity One software (Bio-Rad Laboratories, Hercules, CA) after IFN-γ treatment (100 U/ml) or not.

Figure 6. PD-L1 expression in tumor tissue samples and its correlation with recurrence free survival in nasopharyngeal carcinoma patients

(A) Histological features of NPC: Nasopharyngeal carcinoma cells (red) were surrounded by infiltrating lymphocytes (blue). (B) PD-L1 expression in nasopharyngeal carcinoma sample (a. negative staining b. weak staining c. moderate staining d. strong staining). (C) Disease-free survival in nasopharyngeal carcinoma patients stratified by the expression level of PD-L1.

Figure 7. Two mechanisms of up-regulated PD-L1 expression on EBV positive nasopharyngeal carcinoma cells

PD-L1 is a well-known immune suppressive factor in a variety of cancer types. Two possible mechanisms of PDL1 regulation in EBV positive NPC was proposed. The first one (innate immune resistance): constitutive oncogenic pathway activation mediated by LMP1 up-regulates PD-L1 expression, which is independent of inflammatory signals in the tumor microenvironment; and the second one (adaptive immune resistance): PD-L1 is induced in response to inflammatory signals, such as IFN-γ, which are produced during an active anti-viral and anti-tumor immune response.