Introduction

Nasopharyngeal carcinoma (NPC) is a malignant cancer originating from nasopharynx epithelium. NPC is prevalent in the southern China and southeastern Asia, but rare in the other regions worldwide.1 Genetic susceptibility, Epstein-Barr virus (EBV), and environmental carcinogens are well-recognized causes of NPC.2 Immune evasion is believed to play an essential role in the development of cancers, including NPC. Cancer cells have evolved multiple mechanisms to escape from recognition and killing by the immune system. For example, cancer cells express high levels of immune checkpoints, such as programmed death-ligand 1 (PD-L1) (CD274), to block T cell activation through binding to its inhibitory receptor programmed cell death protein-1 (PD-1) (CD279) at the surface of T cells.3 The cytotoxic CD8+T cells are the major components of the immune system to destroy cancer cells. However, immune suppressive tumor microenvironments (TME) impede the antitumor activity of CD8+T cells. Single-cell transcriptomic study indicated that tumor-infiltrating T cells in NPC were highly activated and exhausted,4 which might be induced by both tumor cells and EBV infection. Therefore, it is necessary to elucidate the mechanisms underlying immune escape in NPC.

Interferon-γ (IFN-γ) is known for its key role in antiviral immune responses and antitumor activities.5 However, IFN-γ also activates the transcription of the inhibitory molecules, such as PD-L1, which limits antitumor immunity and promotes cancer progression.6–8 It has been proposed that IFN-γ exerts opposing functions in cancer cells and immune cells. Blocking tumor IFN-γ signaling promotes the maturation of innate immune cells and enhances innate immune killing. Furthermore, blocking tumor IFN-γ signaling increases IFN-γ generated by exhausted T cells (TEX).9 In NPC, IFN-γ synergizes with EBV-encoded LMP1 to induce expression of PD-L1.10 Jin et al defined a distinct subtype of NPC cells possessing an epithelial–immune dual feature and associated with poor prognosis.4 This may enable NPC cells to interact with immune cells in the TME in a unique manner. This distinctive feature could potentially influence the IFN-γ signaling pathway, thereby regulating biological behaviors such as immune evasion, proliferation, and migration of the cells. Interestedly, NPC cells within the dual feature express high levels of IFN response and signaling-associated genes, suggesting activation of IFN response in NPC cells may contribute to immune escape.4 Thus, it is essential to clarify the regulatory mechanisms of IFN-γ signaling in NPC cells.

Forkhead box A1 (FOXA1) is a pioneer factor that induces open chromatin conformation allowing the binding of other transcription factors.11 12 Our previous studies showed that FOXA1 is downregulated in NPC tissues and exerts tumor-suppressive function in the development and progression of NPC.13–15 FOXA1 suppresses NPC cell growth and invasiveness through reprogramming TGF-β stimulated transcription program, enabling the expression of TGF-β responsive tumor suppressive genes while restricting the expression of TGF-β responsive oncogenes.13 Recent studies suggest dysregulation of FOXA1 is involved in antitumor immunity.16 17 It is not clear whether FOXA1 regulates antitumor immunity in NPC.

In this study, we demonstrated that loss of FOXA1 induced the expression of IFN response and signaling-associated genes in NPC cells. Among these IFN-responsive genes, the expression of CD274 (PD-L1) in NPC cells was induced by IFN-γ when FOXA1 was silenced in NPC cells, whereas the induction of PD-L1 by IFN-γ was abolished by overexpression of FOXA1 in NPC cells. Functionally, downregulation of FOXA1 in NPC cells impairs CD8+T cell mediated killing, whereas it promotes CD8+T cell apoptosis and reduces the expression of IFN-γ, GMZB in CD8+T cells. Overexpression of FOXA1 in NPC cells sensitizes NPC tumor to immune therapy. Mechanistically, FOXA1 binds with STAT1 and prevents STAT1 binding to the promoter region of IRF1, leading decrease in the expression level of IRF1 in NPC cells. Downregulation of IRF1 results in suppression of PD-L1 transcription on IFN-γ treatment.

Materials and methodsPatient samples

In total, 57 cases of NPC tissues and 38 cases of normal nasopharyngeal inflammatory epithelial tissues were collected for immunohistochemical detection of FOXA1 and PD-L1 expression. All patients received an informed written consent prior to the surgery. Detailed clinical pathologic information of all patients is presented in online supplemental table S1.

Cell lines, plasmids, and cell transfection

Cell Lines: The NPC cell lines HK1 and C666-1 were donated by Professor Li Xin of Southern Medical University.18 The cells were cultured and expanded in RPMI-1640 culture medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen), penicillin (100 U/mL, Sigma-Aldrich), and streptomycin (100 U/mL, Sigma-Aldrich). All cells were confirmed to be mycoplasma-free (Takara) before culturing and underwent Short Tandem Repeat (STR) identification every six months. The time length between thawing and use was less than 3 months.

pLVX-IRES-EF1α-puro plasmid: An overexpression vector with resistance to puromycin and ampicillin. It has a multiple cloning site and is commonly used for lentivirus packaging. Once it is packaged into a lentivirus and infects animal cells, it can be stably expressed in the cells.

Overexpression vector and small interfering RNA (siRNA) transfection: Plate cells, and when their density reaches 40–50%, set up the overexpression vector transfection system (1.5 µg plasmid, 3 µL Neofect, and 200 µL culture medium) or siRNA transfection system (5 µL siRNA, 5 µL Hiperfect, and 200 µL culture medium). Add to the antibiotic-free cell culture medium after standing for 20 min, and then collect cells 36–48 hours later for the next experiment.

RT-PCR and qPCR

Total RNA was extracted using TRIzol reagent (15596026, Invitrogen) and reverse transcribed using the HiScript cDNA Synthesis kit (R323-01, Vazyme). quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) was performed using Universal SYBR qPCR Master Mix (Q511-02, Vazyme). The 2−ΔΔCt method was used to normalize the data.

Regular PCR experiments were performed using Golden Star T6 Super PCR Mix (TSE101, Tsingke), following the provided instructions. A 1.5% agarose gel was prepared using 1×TAE buffer. The primers used for RT-qPCR and PCR are listed in online supplemental table S2.

Immunofluorescence

Cultured tumor cells were placed and adhered on appropriate glass slides. The cells were fixed for about 15 min with 4% formaldehyde, then washed three times with PBS buffer. The slides were blocked and immunofluorescence antibodies were added, then incubated overnight at 4 degrees Celsius. The cells were washed to remove unbound antibodies, then stained with 4',6-diamidino-2-phenylindole (DAPI) to mark cell nuclei for about 5 min, followed by a wash to remove excess DAPI. Cells were scanned with a laser confocal fluorescence microscope to record the distribution of proteins in the tumor cells. The immunofluorescence antibodies used are listed in online supplemental table S3.

Western blotting

Total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (IEVH07850, Millipore). The membrane was incubated with antibodies and detected using Ultra-sensitive ECL Chemiluminescence Kit (P10300, NCM Biotech). GAPDH was used as a protein control. The antibodies used are detailed in online supplemental table S3.

Immunohistochemistry

Immunohistochemistry was performed using an Elivision plus Polyer HRP (Mouse/Rabbit) IHC kit (KIT-9902, Maxim). Briefly, tissue sections were deparaffinized with xylene and a graded series of alcohol, and antigen retrieval was conducted using an EDTA antigen repair solution. Subsequently, endogenous peroxidase blocking solution, non-specific staining blocking solution, primary antibodies of FOXA1 or PD-L1, and biotin-labeled secondary antibodies were added sequentially, followed by the addition of biotin peroxidase and 3,3'-diaminobenzidine (DAB) staining. After dehydration and sealing, images were captured using an Olympus BX51 microscope.

ELISA

The concentration of IFN-γ in the sera of nude mice or CD8+ T cells was measured using an IFN-γ ELISA Kit (KHC4021, eBioscience) following the manufacturer’s instructions.

Flow cytometry analysis

Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit (BMS500FI, Invitrogen) via flow cytometry analysis. Tumor cells were incubated with Alexa Fluor 488 Annexin V and propidium iodide (PI) for 15 min, and T-cell apoptosis levels were detected using a flow cytometer (BD Fortessa). Data analysis was performed using FlowJo software. Unstained tubes and single-color fluorescence tubes were used for fluorescence compensation and gate setting. Q1: Annexin V−/PI+, Q2: Annexin V+/PI+, Q3: Annexin V−/PI−, Q4: Annexin V+/PI−.

When T cells were co-incubated with tumor cells, 25 ng/mL Brefeldin A (BFA, Selleck) was added to the culture medium. T cells were labeled with surface antibodies, fixed, and permeabilized before being incubated with cytokine flow antibodies. The cells were washed once with 1×PBS, centrifuged, the supernatant was discarded, and the cell pellet was resuspended in 400 µL of 1×PBS before running the flow cytometry analysis.

Luciferase reporter gene assay

Various lengths of PD-L1 promoters and IRF1 promoters, along with corresponding mutant fragments, were inserted into the PGL3 basic vector (E1751, Promega). The luminescence ratio of firefly luciferase to Renilla luciferase was detected using the Dual-Glo Luciferase Assay System kit (E2980, Promega).

Chromatin immunoprecipitation

Using the chromatin immunoprecipitation (ChIP) Assay Kit (P2078, Beyotime), the binding of STAT1 and IRF1 to different gene promoter regions was detected following the instructions provided in the kit.

Preparation of DC and T cells

PBMC extraction: Peripheral blood of healthy donors is diluted 1:1 with sterile saline. Ficoll (P9011, solarbio) and diluted blood are added sequentially in centrifuge tubes, centrifuged at 400 g for 30–40 min. After centrifugation, the white film-like mononuclear cells at the interface are collected, resuspended with five times the volume of saline, and centrifuged at 400 g for 10–15 min. This is repeated three times to obtain mononuclear cells.

DC preparation: Separated mononuclear cells are added to 1640 culture medium containing interleukin (IL)-4, GM-CSF, and 10% FBS and cultured for 5 days. IFN-γ is then added and cultured for another day, after which the induced dendritic cells (DCs) are co-cultured with tumor cell lysate for 1 day.

T cell preparation: Separated mononuclear cells are added to a medium containing T cell activators, IL-2, IL-15, and IL-7, and cultured for 3 days. The T cell activator is then removed and the culture continues for 5 days. To prepare tumor-specific T cells used in animal experiments, DC cells and T cells are co-cultured in a complete medium containing IL-2, IL-15, and IL-7 for 1 day. To prepare tumor-specific T cells used in in vitro, anti-CD2/CD3/CD28 activated primary human T-cells co-cultured with inactivated tumor cells in the presence of IL-2 and/or IL-15 for 1–2 weeks. Tumor-specific T cells were co-cultured directly with tumor cells in a six-well plate for 12 hours.

Nude mouse T cell infusion model

Four-week-old male Balb/C nude mice were purchased from Hunan Slack Jingda Experimental Animal. HK1-OE-Vector and HK1-OE-FOXA1 cells were subcutaneously injected into the right thigh root of the nude mice, with each mouse being injected with 3×106 cells. Approximately 1 week later, tumors were formed in the nude mice. They were then injected with 5×107 T cells which had been co-cultured with the lysate of HK1 cells, and the PD-L1 inhibitor—atezolizumab (5 mg/kg, every 7 days) was administered intravenously into the nude mice to enhance the killing of tumor cells by T cells. Depending on the different treatment groups, different experimental protocols were adopted, including T-cell labeling, in vivo imaging technology tracking, peripheral blood analysis, tumor dissection and analysis, etc. The experiment was approved by the Ethics Committee of Central South University (Approval number: 2020sydw0155).

Small animal in vivo imaging

After co-incubating T cells with the deep red fluorescent dye DiR (Thermo Fisher) for 1 hour, the T cells are intravenously injected into tumor-bearing nude mice. At time points of 1 hour, 1 day, 3 days, and 7 days, the nude mice are anesthetized with isoflurane and their luminescence is observed using a small animal in vivo imaging system.

Statistical analysis

GraphPad Prism V.5 software is used for statistical analysis, and differences between two sets of data are evaluated by T-test. All experiments in this project are repeated three times or more, and a p value less than 0.05 is considered to indicate statistical significance in data difference.

ResultFOXA1 is a key inhibitory molecule of the IFN-PD-L1 pathway in NPC cells

We have previously elucidated the significant role of FOXA1 in the pathogenesis and prognosis of NPC.13 Further investigation of FOXA1 expression in different NPC cells revealed the lowest expression in HK1 cells and the highest in C666-1 cells (online supplemental figure S1A). We employed RNA-seq to study transcriptomic alterations induced by silencing FOXA1 in C666-1 cells by three different siRNAs (figure 1A, online supplemental figure S1B-D). Bioinformatics analysis suggested significant regulation of the cytokine response pathway and JAK-signal transducers and activators of transcription (STAT) pathway by FOXA1 (figure 1B). The JAK-STAT pathway is a major downstream pathway of IFN response.19 Gene set enrichment analysis (GSEA) pathway enrichment analysis revealed significant upregulation of the IFN response pathway on FOXA1 knockdown (figure 1C, online supplemental figure S1E). RT-qPCR analysis of IFN-induced genes expression in C666-1 cells on FOXA1 knockdown yielded similar results (figure 1D). We further analyzed the previously published gene chip data of HK1 cells overexpressing FOXA1 and found that overexpression of FOXA1 also leads to inhibition of the IFN pathway (online supplemental figure S1F-G). Moreover, analysis of the GSE53819 database revealed that, compared with the FOXA1 high expression group (n=9), the FOXA1 low expression group (n=9) exhibits activation of the IFN signaling pathway (online supplemental figure S1H). These results suggest that the loss of FOXA1 significantly promotes the activation of the IFN response pathway in NPC cell.

Figure 1

FOXA1 downregulation promotes the upregulation of PD-L1 induced by interferon pathway activation. (A) Heatmap of differentially expressed genes detected by RNA-seq after knocking down FOXA1 with siRNA in c666-1 cells, non-targeting siRNA (negative scramble siRNA, Scr) as a negative control. (B) KEGG pathway enrichment analysis reveals significant enrichment of cytokine–cytokine receptor interaction pathway and JAK-STAT pathway after FOXA1 knockdown. (C) GSEA analysis shows the correlation between FOXA1 and signaling pathways related to interferon signaling, interferon-responsive genes, gamma interferon response, and type I interferon response in tumors. (D) qPCR experiment detects the expression of some genes regulated by FOXA1. (E–G) Knockdown of FOXA1 in C666-1 cells and addition of IFN-γ, qPCR (E), western blot (F), and flow cytometry (G) detect the expression of FOXA1 and PD-L1. (H–J) Overexpression of FOXA1 in HK1 cells and addition of IFN-γ, qPCR (H), western blot (I), and flow cytometry (J) detect the expression of FOXA1 and PD-L1. (K) Immunohistochemistry detects the expression of FOXA1 and PD-L1 in 57 nasopharyngeal carcinoma tissue samples and 38 normal nasopharyngeal epithelium samples. Magnification, ×200, scale bar, 50 µm; Magnification, ×400, scale bar, 20 µm. Left, representative images; middle, statistical results; right, correlation analysis of PD-L1 and FOXA1 expression in nasopharyngeal carcinoma tissue. FOXA1, forkhead box A1; FDR, False Discovery Rate; GSEA, Gene Set Enrichment Analysis; IFN-γ, interferon-γ; KEGG, Kyoto Encyclopedia of Genes and Genomes; MFI, Median Fluorescence Intensity; mRNA, messenger RNA; PD-L1, programmed death-ligand 1; qPCR, quantitative PCR; siRNA, small interfering RNA; STAT1, signal transducers and activators of transcription 1.

IFN pathway activation is a distinctive feature of NPC cells and NPC TME, which may be closely associated with the upregulation of various immune checkpoint molecules including PD-L1.4 20–22 RT-qPCR, western blot, and flow cytometry analysis revealed silencing FOXA1 did not affect PD-L1 level in C666-1 cells without exogenous IFN-γ treatment (online supplemental figure S2A-E). Similarly, overexpression of FOXA1 did not change the expression level of PD-L1 in HK1 cells without exogenous IFN-γ treatment (online supplemental figure S2F-H). However, loss of FOXA1 in C666-1 cells led to efficient induction of PD-L1 by exogenous IFN-γ treatment (figure 1E–G), while FOXA1 overexpression significantly inhibited IFN-induced PD-L1 in HK1 cells (figure 1H–J). Expression of FOXA1 did not affect the induction of PD-L1 by exogenous IFN-β treatment in NPC cells (online supplemental figure S2I-J). Analysis of the NPC GSE12452 database revealed that the messenger RNA (mRNA) level of FOXA1 is significantly decreased and negatively correlated with the mRNA level of PD-L1 in NPC samples (online supplemental figure S2K). Immunohistochemical analysis of 57 NPC tissue samples and 38 normal inflammatory nasopharyngeal epithelium samples revealed significantly lower expression of FOXA1 in NPC, which was negatively correlated with the level of PD-L1 protein in NPC samples (figure 1K).

Figure 2

FOXA1 inhibits nasopharyngeal carcinoma immune evasion by counteracting IFN-γ-induced PD-L1 upregulation. (A) Flow cytometry analysis of Annexin V+ PI+ of CD8+ T cell after co-culture of primary human T cells with FOXA1 knocked down C666-1 cells and FOXA1 overexpression HK1 cells treated with IFN-γ and PD-L1 antibody. (B) Flow cytometry analysis of IFN-γ+ GZMB+ of CD8+ T cell after co-culture of primary human T cells with FOXA1 knocked down C666-1 cells and FOXA1 overexpression HK1 cells treated with IFN-γ and PD-L1 antibody. (C) Flow cytometry analysis of IFN-γ+ TNF-α+ of CD8+ T cell after co-culture of primary human T cells with FOXA1 knocked down C666-1 cells and FOXA1 overexpression HK1 cells treated with IFN-γ and PD-L1 antibody. (D) Flow cytometry analysis of TCF-1+ of CD8+ T cell after co-culture of primary human T cells with FOXA1 knocked down C666-1 cells and FOXA1 overexpression HK1 cells treated with IFN-γ and PD-L1 antibody. (E) Flow cytometry analysis of KI67+ of CD8+ T cell after co-culture of primary human T cells with FOXA1 knocked down C666-1 cells and FOXA1 overexpression HK1 cells treated with IFN-γ and PD-L1 antibody. (F) Flow cytometry analysis of PD-1+ TIM-3+ of CD8+ T cell after co-culture of primary human T cells with FOXA1 knocked down C666-1 cells and FOXA1 overexpression HK1 cells treated with IFN-γ and PD-L1 antibody. FOXA1, forkhead box A1; IFN, interferon; PBS, phosphate-buffered saline; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1.

FOXA1 promotes immune evasion in NPC

To investigate the impact of FOXA1 on immune evasion in NPC, we co-cultured primary tumor-specific T-cells (anti-CD2/CD3/CD28 activated primary human T-cells co-cultured with inactivated tumor cells in the presence of IL-2 and/or IL-15 for 1–2 weeks) with c666-1 cells in which FOXA1 was knocked down or HK1 cells overexpressing FOXA1 and pretreated with IFN-γ for 24 hours. Crystal violet staining of tumor cells revealed that under conditions of exogenous IFN-γ stimulation, FOXA1 knockdown significantly promoted resistance of c666-1 cells to T cell cytotoxicity, while FOXA1 overexpression significantly inhibited resistance of HK1 cells to T cell cytotoxicity (online supplemental figure S3A-B). Comprehensive detection of T cell apoptosis, cytotoxic cytokines, proliferation, stemness, and exhaustion is of great importance in evaluating the changes in T cell function due to FOXA1 expression in tumor cells. For example, the expression of granzyme B is closely related to the cytotoxic function of T cells.23 Flow cytometry analysis of the co-cultured primary T cells showed that FOXA1 overexpression inhibited CD8+ T cell apoptosis (figure 2A), increased the proportion of IFN-γ+ GZMB+ and IFN-γ+ TNF-α+ in CD8+ T cells (figure 2B–C), enhanced the proportion of TCF-1+ (figure 2D) and KI67+ (figure 2E) in CD8+ T cells, and reduced PD-1+ TIM-3+ in CD8+ T cells (figure 2F). Conversely, FOXA1 knockdown in C666-1 cells led to opposite results (figure 2A–F). RT-qPCR analysis revealed that FOXA1 promoted the expression of IFNG, GZMB, and IL-2 in primary T cells (online supplemental figure S3C-D). Additionally, ELISA analysis of supernatants collected after co-culture showed that FOXA1 knockdown inhibited the expression of IFN-γ in the co-culture supernatant, while FOXA1 overexpression promoted the expression of IFN-γ in the co-culture supernatant (online supplemental figure S3E). Treatment with anti-PD-L1 significantly alleviated the immune evasion ability mediated by low FOXA1 expression in HK1 cells (figure 3A–F and online supplemental figure S3A-B), indicating that FOXA1 primarily inhibits NPC immune evasion by downregulating the IFN-γ-PD-L1 pathway.

Figure 3

FOXA1 inhibits nasopharyngeal carcinoma immune evasion in vivo. (A) Analysis of primary human T cell survival and distribution in xenograft tumor-bearing mice using in vivo small animal imaging. T cells labeled with DeepRed. Color scale represents the DiR fluorescence intensity of mouse T cells. (B) Flow cytometry analysis of Annexin V+PI+ proportion of CD8+ T cells in xenograft nude mouse model derived from HK1 cells. (C) Flow cytometry analysis of IFN-γ+GZMB+ proportion of CD8+ T cells in xenograft nude mouse model derived from HK1 cells. (D) qPCR detection of IFN-γ expression in CD8+ T cells derived from xenograft nude mouse model originated from HK1 cells. (E) ELISA detection of IFN-γ expression in CD8+ T cells derived from xenograft nude mouse model originated from HK1 cells. (F) Collection of tumor images from each group 49 days after tumor cell injection; n=5 per group. (G) Measurement of tumor weight from each group 49 days after tumor cell injection; n=5 per group. (H) Real-time measurement of tumor volume after tumor cell injection; n=5 per group. (I) Immunohistochemistry assess the expression levels of FOXA1 and PD-L1 in mouse tumor tissues. FOXA1, forkhead box A1; IFN, interferon; PD-L1, programmed death-ligand 1.

To validate whether FOXA1 inhibits immune evasion in NPC in vivo, we established a xenograft model by subcutaneously inoculating HK1 cells overexpressing FOXA1 and treated with IFN-γ into nude mice. Subsequently, we co-cultured primary human T cells with DCs stimulated with tumor cell lysates for antigen presentation, added PD-L1 monoclonal antibody treatment, stained the T cells with the live fluorescent dye DiR, and finally injected these T cells into the mice via the tail vein. Dynamic monitoring of DiR-stained primary T cells in live animals revealed that on the third day, T cells from the FOXA1 overexpression group migrated and accumulated at the tumor site. By the seventh day, FOXA1 significantly increased the fluorescence intensity of T cells at the tumor site, indicating that FOXA1 promotes T cell infiltration into tumor tissue in vivo, thereby inhibiting immune evasion (figure 3A). Tumor tissues collected after T cell infusion were analyzed by flow cytometry, revealing that FOXA1 overexpression inhibited apoptosis of infiltrating T cells in NPC tissue (figure 3B) as well as the expression of cytotoxic cytokines (figure 3C). RT-qPCR analysis showed that FOXA1 overexpression significantly promoted IFN-γ expression in T cells (figure 3D), and ELISA detection of IFN-γ secretion levels in tumor tissue supernatants yielded similar conclusions (figure 3E).

In conditions without T cell injection, partial inhibition of tumor size and weight in nude mice was observed with FOXA1 overexpression, consistent with our previous findings.13 However, following T cell injection, FOXA1 overexpression exhibited more significant reductions in tumor volume, size, and weight compared with the control group, suggesting FOXA1 inhibits immune evasion of tumors in nude mice. Treatment with anti-PD-L1 abolished the growth inhibition by FOXA1 overexpression in the transplanted tumors, indicating FOXA1 regulates tumor immune evasion by inhibiting PD-L1 expression (figure 3F–H). Histological examination using H&E staining and immunohistochemistry on paraffin-embedded mouse tumor tissues revealed that FOXA1 also regulates PD-L1 expression in vivo (figure 3I and online supplemental figure S4).

Figure 4

FOXA1 inhibits PD-L1 expression by binding to STAT1 protein. (A–B) Knockdown of STAT1 in C666-1 cells followed by addition of IFN-γ, qPCR (A) and western blot (B) to detect the expression of STAT1 and PD-L1. (C–D) Knockdown of STAT2 in C666-1 cells followed by addition of IFN-γ, qPCR (C) and western blot (D) to detect the expression of PD-L1. (E–F) Knockdown of STAT1 in HK1 cells followed by addition of IFN-γ, qPCR (E) and western blot (F) to detect the expression of STAT1 and PD-L1. (G–H) Knockdown of STAT2 in HK1 cells followed by addition of IFN-γ, qPCR (G) and western blot (H) to detect the expression of STAT2 and PD-L1. (I) Addition of IFN-γ in C666-1 cells, Co-IP experiment to detect the interaction between STAT1 and FOXA1. (J) Overexpression of FOXA1 in HK1 cells followed by addition of IFN-γ, Co-IP experiment to detect the interaction between STAT1 and FOXA1. (K) Knockdown of FOXA1 in C666-1 cells followed by addition of IFN-γ, western blot to detect the phosphorylation level of STAT1. (L) Overexpression of FOXA1 in HK1 cells followed by addition of IFN-γ, western blot to detect the phosphorylation level of STAT1. (M) Addition of IFN-γ in HK1 and C666-1 cells, immunofluorescence to detect the nuclear translocation of STAT1/2. (N) Construction of FOXA1 truncation mutants using Uniprot database, including full-length FOXA1-F (1–472 aa), FOXA1-N terminus (1–140 aa), FOXA1-middle segment (141–294 aa), FOXA1-C terminus (295–472 aa), Co-IP experiment found that the middle segment of FOXA1 bind to STAT1. (O) Construction of STAT1 truncation mutants using Uniprot database, full-length STAT1-F (1–750 aa), STAT1-N terminus (1–350 aa), STAT1-C terminus (351–750 aa), Co-IP experiment found that the N terminus of STAT1 can bind to FOXA1. (P) Dual-luciferase reporter gene experiment to determine the effect of overexpression of different regions of FOXA1 on the activity of the PD-L1 promoter region. Co-IP, co-immunoprecipitation; DAPI, 4',6-diamidino-2-phenylindole; FOXA1, forkhead box A1; IFN, interferon; mRNA, messenger RNA; PBS, phosphate-buffered saline; PD-L1, programmed death-ligand 1; qPCR, quantitative PCR; STAT1, signal transducers and activators of transcription 1.

FOXA1 inhibits PD-L1 expression by binding to the STAT1

IFN-γ regulates tumor cell PD-L1 expression through the JAK-STAT signaling pathway, dependent on STAT1/2.24 Furthermore, we found that siRNA-mediated knockdown of STAT1 significantly inhibited IFN-γ-induced upregulation of PD-L1 in c666-1 cells (figure 4A–B). Knockdown of STAT2 did not significantly alter IFN-γ-induced changes in PD-L1 expression (figure 4C–D). Similar conclusions were drawn from STAT1 knockdown in HK1 cells (figure 4E–H), suggesting that FOXA1 primarily promotes PD-L1 expression by modulating STAT1. Further Co-IP experiments revealed an interaction between FOXA1 and STAT1 under IFN-γ stimulation (figure 4I–J), with FOXA1 not regulating STAT1 phosphorylation levels (figure 4K–L). Immunofluorescence colocalization analysis indicated that under IFN-γ stimulation, there is a significant nuclear translocation of STAT1, while the nuclear-cytoplasmic distribution of STAT2 is not affected (figure 4M). Further construction of FOXA1 and STAT1 truncation mutants revealed an interaction between the FOXA1-M (141-294aa) region and the STAT1-N region (1-350aa) (figure 4N–O). Luciferase reporter gene assays showed that only the FOXA1-M region inhibited IFN-γ-induced upregulation of PD-L1 (figure 4P). These results indicate that FOXA1 binds to STAT1, thereby limiting IFN-γ-induced PD-L1 upregulation.

FOXA1 inhibits PD-L1 expression by binding to STAT1 and suppressing IRF1 transcription

Under IFN-γ stimulation, simultaneous knockdown of FOXA1 and STAT1 in c666-1 cells revealed that FOXA1’s inhibition of IFN-γ-induced upregulation of PD-L1 is STAT1-dependent (figure 5A–B). Similar conclusions were drawn in HK1 cells overexpressing FOXA1 while silencing STAT1 (figure 5C–D). However, ChIP experiments found no significant binding of STAT1 to the PD-L1 promoter region (figure 5E), suggesting that STAT1 regulates PD-L1 expression through an indirect pathway. The major downstream molecules activated by IFN include IRF1, IRF7, and IRF9.25 Overexpression or knockdown of FOXA1 along with STAT1 knockdown revealed that IRF1 is most significantly regulated by the FOXA1-STAT1 axis (figure 5F–G). Luciferase reporter gene and ChIP experiments detected that the −213 position in the IRF1 promoter region is crucial for IRF1’s regulation of PD-L1 transcription (online supplemental figure S5A-B). Under IFN-γ conditions, simultaneous overexpression of STAT1 and knockdown of IRF1 revealed that IRF1 knockdown inhibited the promoting effect of STAT1 on PD-L1 promoter activity (figure 5H–J). RT-qPCR and western blot analysis found that IRF1 knockdown inhibited the ability of FOXA1 (figure 5K–N) and STAT1 (figure 5O–R) to regulate PD-L1. ChIP experiments demonstrated a direct binding of IRF1 to the PD-L1 promoter region (figure 5S). Analysis in the GSE53819 database revealed that FOXA1 is negatively correlated with IRF1 and CD274, while IRF1 is positively correlated with CD274 (online supplemental figure S5C). Luciferase reporter gene assays revealed that the −213 position in the PD-L1 promoter region is crucial for IRF1’s regulation of PD-L1 transcription (figure 5T–V). These results suggest that the loss of FOXA1 upregulates IRF1 expression by interacting with STAT1, and that IRF1 promotes PD-L1 transcription by binding to its promoter region. To elucidate whether FOXA1 promotes immune evasion in NPC via the STAT1-IRF1 pathway, we performed simultaneous knockdowns of FOXA1, STAT1 in C666-1 cells, or overexpressed FOXA1 while knocking down STAT1 in HK1 cells. These modified cells were then co-cultured with tumor-specific primary T cells (online supplemental figure S5D-E). The results indicate that knocking down STAT1 can reverse the tumor immune escape caused by FOXA1 knockdown. Our findings indicate that the immune evasion mediated by FOXA1 downregulation is dependent on the STAT1 pathway.

Figure 5

FOXA1 suppresses PD-L1 expression by inhibiting STAT1 downstream IRF1 transcription. (A–B) Knockdown of FOXA1 and STAT1 in C666-1 cells followed by addition of IFN-γ, qPCR (A) and western blot (B) to detect PD-L1 expression. (C–D) Overexpression of FOXA1 and knockdown of STAT1 in HK1 cells followed by addition of IFN-γ, qPCR (C) and western blot (D) to detect PD-L1 expression. (E) Addition of IFN-γ in C666-1 and HK1 cells, ChIP experiment to detect the binding of STAT1 to the PD-L1 promoter. (F) Knockdown of FOXA1 and STAT1 in C666-1 cells followed by addition of IFN-γ, qPCR to detect the expression of IRF1, IRF7, and IRF9. (G) Overexpression of FOXA1 and knockdown of STAT1 in HK1 cells followed by addition of IFN-γ, qPCR to detect the expression of IRF1, IRF7, and IRF9. (H–J) Prediction of STAT1 binding sequence to the PD-L1 promoter using JASPAR database (H), dual-luciferase reporter gene experiment to detect PD-L1 promoter activity in c666-1 (I) and HK1 (J) cells overexpressing STAT1 while knocking down IRF1 and adding IFN-γ. (K–L) Knockdown of FOXA1 and IRF1 in C666-1 cells followed by addition of IFN-γ, qPCR (K) and western blot (L) to detect PD-L1 expression. (M–N) Overexpression of FOXA1 and knockdown of IRF1 in HK1 cells followed by addition of IFN-γ, qPCR (M) and western blot (N) to detect PD-L1 expression. (O–P) Overexpression of STAT1 and knockdown of IRF1 in C666-1 cells followed by addition of IFN-γ, qPCR (O) and western blot (P) to detect PD-L1 expression. (Q–R) Overexpression of S