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The mechanism of prostaglandin E2 upregulation of programmed death ligand 1 expression promoting immune escape in non-small cell lung cancer
*Corresponding author: Sen Liu, Department of Oncology, Central Hospital Affiliated to Shandong First Medical University, Jinan, China. liusen256@163.com
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Received: ,
Accepted: ,
How to cite this article: Wang T, Liu L, Liu S. The mechanism of prostaglandin E2 upregulation of programmed death ligand 1 expression promoting immune escape in non-small cell lung cancer. CytoJournal. 2025;22:105. doi: 10.25259/Cytojournal_129_2025
Abstract
Objective:
Non-small cell lung cancer (NSCLC) represents a major contributor to cancer-related mortality. This study aims to investigate the prostaglandin E synthase (PTGES) role in NSCLC, especially how prostaglandin E2 (PGE2), which is synthesized by PTGES prompts programmed death ligand 1 (PD-L1) expression, resulting in immune escape.
Material and Methods:
The PTGES and PGE2 expression levels were tested in normal and NSCLC samples, respectively. An A549 cell line that overexpressed and knocked down PTGES was established, and cell multiplication, colony formation, and aggression were evaluated. The PD-L1 levels and cytotoxicity effect of CD8+ T cells were analyzed by co-culture with A549 cells. The expression levels of proteins involved in phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway and the effect of activation and inhibition on PD-L1 expression were investigated. The effect of PGE2 was further verified by establishing an in vivo xenograft mouse model.
Results:
The expression of PTGES in NSCLC cells notably increased compared with that in control cells (P < 0.001). Correspondingly, a significant upregulation of PGE2 levels was observed (P < 0.001). Overexpression of PTGES enhanced cell proliferation and aggression ability (P < 0.001), whereas knockdown of PTGES weakened these capabilities (P < 0.01). In the experiment of co-culture with CD8+ T cells, PD-L1 was upregulated (P < 0.01) and cytotoxicity was considerably reduced (P < 0.01) in the PTGES overexpression group. Meanwhile, PD-L1 was downregulated (P < 0.01) and cytotoxicity was enhanced (P < 0.01) in the PTGES knockdown group. Overexpression of PTGES was able to activate the PI3K/AKT/mTOR pathway (P < 0.01). Activation and inhibition of this pathway showed that PGE2 upregulated PD-L1 expression through this signaling pathway (P < 0.01). The mouse transplanted tumor model further verified the effect of PTGES expression on tumor growth and immune escape.
Conclusion:
This study confirmed that PGE2 promotes immune evasion of NSCLC by upregulating PD-L1, and the mechanism involves the activation of the PI3K/AKT/mTOR pathway. This research provides a novel theoretical basis for immunotherapy of NSCLC and emphasizes the clinical value of PTGES/PGE2 as a potential target.
Keywords
Immune escape
Non-small cell lung cancer
Phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin
Programmed death ligand 1
Prostaglandin E2
INTRODUCTION
Non-small cell lung cancer (NSCLC), which is the predominant subtype among all lung cancer cases, is involved in substantial incidence and mortality rates across the globe, mainly due to the deterioration of the living environment; adverse lifestyle, such as smoking history or second-hand smoke; and genetic factors.[1-3] Although advancements have been achieved in the management of NSCLC, such as the improvement of surgical techniques, the optimization of chemotherapy regimens, and the rise of molecular targeted therapy and immunotherapy, the overall prognosis of patients is still not ideal, and many patients continue to relapse after surgery.[4-6]
Immune escape is a key mechanism to escape the surveillance and killing of the immune system and achieve sustainable growth and metastasis for tumor cells, significantly influencing the initiation and progression of NSCLC.[7] Programmed death ligand 1 (PD-L1) has a meaningful effect on tumor immune escape.[8] Tumor cells increase the levels of PD-L1 through various mechanisms and transmit inhibitory signals to inhibit the activation, proliferation, and killing function of T cells, resulting in tumor cells escaping the surveillance and attack.[9] At present, PD-L1 has become a biomarker for predicting NSCLC.[10,11] Therapies targeting PD-L1 have been successful in NSCLC, but they are generally less effective.[12] Therefore, exploring the regulatory mechanism of PD-L1 expression is remarkably important for the evolution of more effective immunotherapy strategies for NSCLC.
The phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway is widely recognized to exert a regulatory role in multiple key processes, including cell proliferation, survival, and metabolism. This pathway is often overactivated during tumorigenesis and development.[13,14] A close and complex association has been found between the activation of this pathway and the expression of PD-L1. Activated AKT is capable of promoting the upregulation of PD-L1 through a series of molecular mechanisms and enhancing the immune evasion ability of tumor cells. PD-L1 signal can reciprocally modulate the PI3K/AKT/mTOR cascade. This regulatory feedback loop further bolsters cellular processes, such as proliferation and invasive behavior, facilitating tumor progression.[15-17]
Prostaglandin E2 (PGE2) is a widespread bioactive lipid mediator in vivo. Prostaglandin H2 (PGH2) is produced by arachidonic acid through the cyclooxygenase (COX) pathway and catalyzed by prostaglandin E synthase (PTGES).[18] In tumor and inflammatory tissues, PTGES, as the terminal rate-limiting enzyme of the PGE2 synthesis pathway, is overexpressed.[19,20] Recent studies have found that PGE2 promotes proliferation and dryness of NSCLC by activating ERK5.[21] Knocking out Caspase 3 can reduce PGE2 synthesis and prevent the recurrence of NSCLC.[22] Recently, the function of PGE2 in tumor immune escape has increasingly attracted attention. For instance, studies have found that COX2/PGE2 participates in modulating the expression of PD-L1 in bladder cancer.[23] In ovarian cancer, myeloid-derived suppressor cells produce PGE2 to stimulate chemokines, recruiting cells to the tumor site.[24] However, the precise molecular pathways through which PGE2 modulates PD-L1 expression in NSCLC cells have not been fully elucidated. Therefore, this work was designed to decipher the intricate mechanisms underlying PGE2-induced PD-L1 upregulation, thereby facilitating immune evasion in NSCLC. This work aimed to comprehensively understand the molecular basis of tumor immune escape and provide a highly promising target for developing more effective tumor treatment strategies, breaking the barrier of tumor immune evasion, and enhancing the effectiveness of tumor immunotherapy.
MATERIAL AND METHODS
Cell culture
Human lung bronchial epithelial cells (BEAS-2B, C6106) and lung cancer cell collections (A549, C6053), which were purchased from Beyotime (Shanghai, China), were cultured in RPMI-1640 (12633020) with 10% fetal bovine serum (FBS, A5670701), and 1% penicillin-streptomycin (V900929) was added. The samples were then placed in a humidified incubator maintained at 37°C and 5% carbon dioxide. The cell lines were verified through short tandem repeat (STR) analysis, and mycoplasma contamination testing yielded negative results. The detailed information on the reagents and instruments used in this article are added to the Supplementary Materials.
Overexpression and knockdown of PTGES
The lentivirus used for transfection in this study was designed and synthesized by Shanghai Jikai Gene Medical (Shanghai, China). The details of the lentivirus are added to the Supplementary Material. The specific transfection procedure of lentivirus was as follows: The cells were uniformly distributed in a 6-well plate and replaced with serum-free culture medium when the cell confluence reached 50%. The corresponding lentivirus (multiplicity of infection = 10) was added and cultured for 24 h. Next, the transfection efficiency was verified.
In vivo animal experiments
Twenty C57BL/6 mice aged 4–6 weeks were used to establish an in vivo model. The animals were housed in a controlled setting with a 12 h light-dark cycle, 20°C ± 2°C, and a humidity of 40% ± 5%. The lentivirus transfection Lewis Lung Carcinoma cells (SNL-119, SUNNCELL, Wuhan, China) in logarithmic growth phase in each group were prepared and mixed with the same volume of Matrigel. Then, 0.2 mL of the cell suspension was injected subcutaneously into the axilla of the mice. When the tumor was palpable, its volume was measured every 4 days. At the end of the experiment, the mice were sacrificed by excessive anesthesia with 3% sodium pentobarbital (P3761, 40 mg/kg), and the tumors were dissected. No significant difference was found in the body weight of the mice in each group during the entire in vivo animal study. The research was approved by the Beijing Maide Kangna Laboratory Animal Welfare Ethics Committee, and all experimental procedures involving animals were conducted in accordance with the Guidelines for Ethical Review of Laboratory Animal Welfare (Approval No. MDKN-2024-103).
Quantitative real-time polymerase chain reaction (qRT-PCR)
TRIzol (R0016) was used to extract cell RNA. The RNA was transcribed to complementary DNA (cDNA) using a FastQuant cDNA Synthesis Kit (KR116). qRT-PCR was carried out using SuperReal PreMix Plus (FP205). Specific amplification primers were obtained from Dingguo Changsheng (Beijing, China). The primer sequences were as follows: PTGES-F: 5'-AAGTGAGGCTGCGGAAGAA-3', PTGES-R: 5'- GACCCAGAAAGGAGTAGACGAA-3', β-actin-F: 5'-ACACTGTGCCCATCTACG-3', β-actin-R: 5'-TGTCACGCACGATTTCC-3'. The 2−ΔΔCt method was adopted to determine the relative expression levels, with β-actin as the internal reference.
Western blot (WB)
Stable transfected A549 cells were collected, and protein was extracted. A protein sample (2 μg/μL) was configured and heated for 5 min. The protein was then isolated and transferred to polyvinylidene fluoride (PVDF, IPVH00010) membranes. Skim milk (5%) was enclosed at 20–25°C for 1 h and anti-PTGES primary antibody (ER1915-57; 1:1000; HUABIO, Wuhan, China), incubated overnight at 4°C. The secondary antibody (ZB-2305; 1:2000; ZSBIO, Beijing, China) was incubated at 20–25°C for 1 h. After each antibody was incubated, the film was washed with phosphate buffered saline with tween 20 3 times. The ECL luminescent solution (P5755103) was uniformly dropped on the PVDF membrane, and the ECL method was used for luminescence analysis through the ChemiDoc imaging system. The signal intensity was quantitatively analyzed using a gel imaging system. The protein band gray values were quantified using ImageJ software.
Immunofluorescence (IF)
Monolayers of growing cells (from 5 × 104 to 1 × 105 cells) were seeded on Petri dishes containing treatment coverslips, removed when nearly paved, and washed with phosphate-buffered saline (PBS). After the cells were fixed using 4% paraformaldehyde (P0099) and permeabilized with 0.3% Triton X-100 (P0096), they were incubated with 5% goat serum (SL038). The cells were then combined with primary antibodies (anti-PTGES, TP53I12, 1:1000, 702796, Thermo Fisher, Waltham, MA, USA) and secondary antibodies (GB21303, 1:500, Servicebio, Wuhan, China), followed by counterstaining with 4’,6-diamidino-2-phenylindole (G (DAPI) (C0060, 1 μg/mL) and blocking with anti-fluorescence quencher (S2100). Finally, images were collected under a fluorescence microscope (APX100). The fluorescence intensity was analyzed using ImageJ software.
5-ethynyl-2'-deoxyuridine (EdU) assay
The suspended cells were inoculated into 24-well plates, added with 100 μL of culture medium, and incubated for 72 h. Then, EdU reagent (C0071S) was added for EdU staining. DAPI solution was added into the cells, and the reaction was carried out for 10 min at 20–25°C to stain nuclei. The number of EdU-positive cells was observed by fluorescence microscopy. The number of cells was counted to calculate the proliferation rate of each group.
Colony-formation assay
The A549 cells in each group were prepared into 90 cells/mL, and 1 mL from each group was inoculated and cultured in 24-well plates. After 10 days, the medium was discarded, rinsed, and fixed. Crystal purple (G1064) staining was then performed. The number of clone formations in each group was quantified under a microscope (Eclipse E200).
Transwell assay
The Matrigel matrix was thawed at 4°C, mixed with a pre-cold gun tip, and diluted to the optimal concentration. The 60–100 μL dilution was used to coat the upper chamber and incubated at 37°C. The cells were digested and made into single-cell suspensions and added to the upper chamber. The lower chamber received 500 M1 of medium supplemented with 10% FBS. Subsequently, the plates were incubated at 37°C for 24 h. Afterward, the cells were rinsed, fixed, and stained. Finally, cell enumeration was performed under a microscope.
Isolation of CD8+ T cells and co-culture with NSCLC cells
Peripheral blood mononuclear cells (PBMCs, CP-H182) were purchased from Procell (Wuhan, China). CD8+ T cells were purified from PBMC and co-cultured with A549 cells in accordance with previously reported methods.[25] In brief, cytokines were added to activate CD8+ T cells. Afterward, the treated A549 cells were co-cultured with activated CD8+ T cells in RPMI-1640 medium (12633020) as described. All cells have been performed mycoplasma tests and STR identification. Flow cytometry was used to identify CD8+ T cells. First, a single-cell suspension was prepared and incubated with CD3 (E-AB-F1013E, Elabscience, Wuhan, China) and CD8 (E-AB-F1104Q, Elabscience, Wuhan, China) antibodies in the dark. After washing, the cells were analyzed on the machine. Then, the CD8+ subpopulation was screened from the CD3+ cells, and the positive rate was calculated. Combined with magnetic bead sorting, the cells could be purified, and the positive rate could reach over 90% after purification.
CD8+ T cell toxicity test
A lactate dehydrogenase cytotoxicity kit (C0017) was used to detect the cytotoxicity of CD8+ T cells as follows: Cytotoxicity (%) = (reaction hole A490 nm–natural release hole A490 nm)/(maximum release hole A490 nm–natural release hole A490 nm) × 100%.[26]
CD8+ T cell vitality detection
After being co-cultured for 48 h, the CD8+ T cells were stained with 0.4% Trypan blue solution (PB180423) at 25°C for 20 min. The blue cells were observed and counted under a light microscope. Cell viability was determined as follows: Cell viability (%) = (total cell count–number of blue cells)/total cell count × 100%.
Flow cytometry
An Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (AP101) was used to detect apoptosis. In accordance with the manufacturer’s instructions, 1–10 × 105 cells were collected, centrifuged, and washed with precooled PBS. Then, 500 μL of 1× binding buffer was taken to resuspend the cells. Next, 5 μL of Annexin V-FITC and 10 μL of PI were added to each tube for incubation. Flow cytometry analysis was performed using CytoFLEX.
Immunohistochemistry (IHC)
Tissue samples were removed, embedded in paraffin, and sectioned into 4 μm slices. Then, the sections underwent a series of pretreatment steps: Initial dewaxing, antigen retrieval, and blocking. Following these procedures, the sections were incubated with anti-CD8 antibodies (ab217334, 1:2000, Abcam, USA) overnight in a humidor at 4°C. On day 2, secondary antibodies (GB23303, 1:500, Servicebio, Wuhan, China) were added, tissues were covered, and the tissue slices were incubated in the dark for 50 min. DAB (PA140212) was used for staining, followed by hematoxylin (AR1108) counterstaining. After the slices were dehydrated and sealed, they were observed under a microscope. Quantitative analysis was implemented on the ImageJ software.
Enzyme-linked immunosorbent assay (ELISA)
The levels of PGE2 (JYM0701Hu, ColorfulGene, Wuhan, China), soluble PD-L1 (sPD-L1, ml038111, mlbio, Shanghai, China), interferon-gamma (IFN-γ, JYM0540Mo, ColorfulGene, Wuhan, China), tumor necrosis factor-alpha (TNF-γ, JYM0218Mo, ColorfulGene, Wuhan, China), granzyme B (abs551030, absin, Shanghai, China), and perforin (abs552949, absin, Shanghai, China) in the supernatant were detected by ELISA. All experiments were performed in accordance with the manufacturer’s instructions.
Bioinformatic analysis
The University of ALabama at Birmingham CANcer (UALCAN) data analysis Portal database (http://ualcan.path.uab.edu/) was used to analyze PTGES expression in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC).
Statistical analyses
GraphPad Prism (version 9.0) was used for statistical analysis and visualization. All experiments were repeated at least three times, with each group containing 6 independent biological samples. Data are presented as mean ± standard deviation. t-test was applied for the comparison of data between two groups. One-way analysis of variance was utilized for comparisons across three or more groups. Post hoc analysis was conducted using Tukey’s method. All the data passed the normality test. P < 0.05 was considered a statistically significant difference.
RESULTS
PTGES exhibited highly expressed levels in NSCLC
The expression of PTGES in A549 and BEAS-2B was detected to determine the content of PGE2 in NSCLC. The qRT-PCR results showed that PTGES mRNA was overexpressed in A549 cell lines compared with BEAS-2B cell line (P < 0.001) [Figure 1a]. The WB and IF results showed that the PTGES protein in A549 cells exhibited a more increased expression than that in BEAS-2B cells (P < 0.001) [Figure 1b-e]. The ELISA results showed the content of PGE2 in A549 cells was higher than in BEAS-2B cells (P < 0.001) [Figure 1f]. The UALCAN database further provides supplementary validation of the expression levels of PTGES in LUAD and LUSC (main types of NSCLC, P < 0.001) [Figure 1g].
- PTGES exhibited highly expressed levels in NSCLC. (a) Relative mRNA levels of PTGES in BEAS2B and A549 cell lines. (b and c) WB analysis of PTGES in BEAS-2B and A549 cell lines. (d and e) IF analysis of PTGES in BEAS-2B and A549 cell lines (scale bar: 50 μm). (f) Levels of PGE2 in BEAS-2B and A549 cell lines. (g) UALCAN database used to assess the expression of PTGES in LUAD and LUSC samples. n = 6; ✶✶✶P < 0.001. PTGES: Prostaglandin E synthase, NSCLC: Non-small cell lung cancer, mRNA: Messenger RNA, WB: Western blot, IF: Immunofluorescence, PEG2: Prostaglandin E2, LUAD: Lung adenocarcinoma, LUSC: Lung squamous cell carcinoma.
PGE2 can promote NSCLC cell proliferation and invasion
The transfection efficiency of overexpressed and knocked down PTGES was measured by WB and qRT-PCR (P < 0.05) [Figure 2a-f]. The short hairpin PTGES-2 (sh-PTGES-2) group had the largest knockdown effect, and it was used for subsequent experiments (P < 0.001). As illustrated in Figure 3a and b, the production of PGE2 was markedly increased in the overexpression (OE-PTGES) group relative to that in the overexpression negative control (OE-NC) group (P < 0.01). Conversely, the level of PGE2 in the sh-PTGES group was markedly decreased compared with that in the short hairpin negative control (sh-NC) group (P < 0.001). The colony-formation experiments showed that overexpression of PTGES significantly enhanced the colony-formation capacity (P < 0.001), whereas knockdown of PTGES significantly weakened it (P < 0.01) [Figure 3c and d]. The EdU results showed that the proliferation of A549 cells in the OE-PTGES group accelerated, whereas that of sh-PTGES cells slowed down (P < 0.01) [Figure 3e and f]. The Transwell experiment demonstrated that the sh-PTGES group exhibited a significantly reduced number of invasive cells compared with the sh-NC group (P < 0.01). On the contrary, the OE-PTGES group showed a notable enhancement in cell invasion ability in comparison to the OENC group (P < 0.001) [Figure 3g and h].
- Transfection efficiency was verified via qRT-PCR and WB. (a-c) Effectiveness of PTGES overexpression detected by qRT-PCR and WB. (d-f) Effectiveness of PTGES knockdown assessed by qRT-PCR and WB. n = 6; ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001. qRT-PCR: Quantitative real-time polymerase chain reaction, PTGES: Prostaglandin E synthase, WB: Western blot.
- PGE2 can promote NSCLC cell proliferation and invasion. (a and b) Levels of PGE2 tested by ELISA after PTGES overexpression and knockdown. (c and d) Proliferative ability tested by colony-formation experiment after PTGES overexpression and knockdown. (e and f) Proliferative ability examined by EdU experiment after PTGES overexpression and knockdown (scale bar: 50 μm). (g and h) Invasive ability detected by Transwell experiments after PTGES overexpression and knockdown (scale bar: 50 μm). n = 6; ✶✶P < 0.01, ✶✶✶P < 0.001. PEG2: Prostaglandin E2, NSCLC: Non-small cell lung cancer, PTGES: Prostaglandin E synthase, ELISA: Enzyme-linked immunosorbent assay, EdU: 5-ethynyl-2'-deoxyuridine.
PGE2 upregulates PD-L1 expression in NSCLC and promotes immune escape response
The NSCLC cells in the above treatment group were co-cultured with activated CD8+ T cells. qRT-PCR and WB detection showed that PD-L1 expression was upregulated in A549 cells after OE-PTGES treatment (P < 0.01) and downregulated in A549 cells after sh-PTGES treatment (P < 0.01) [Figure 4a-c]. The cytotoxicity test indicated that the toxicity of CD8+ T cells to A549 cells was enhanced after sh-PTGES treatment (P < 0.01) and weakened after OE-PTGES treatment (P < 0.01) [Figure 4d and e]. Cell vitality was detected by Trypan blue staining, and the results indicated that the vitality of CD8+ T cells was enhanced after sh-PTGES treatment (P < 0.01). The activity of CD8+ T cells decreased after OE-PTGES treatment (P < 0.01) [Figure 4f and g]. Flow cytometry demonstrated that the apoptosis of CD8+ T cells in the co-culture system of the OE-PTGES group notably increased (P < 0.001), whereas that of the sh-PTGES group substantially decreased (P < 0.01) [Figure 4h and i]. The ELISA results showed that the levels of IFN-γ, TNF-α, granzyme B, and perforin in the supernatant of T cell culture medium co-cultured with A549 cells in the sh-PTGES group notably increased (P < 0.05), whereas the levels in T-cell culture medium supernatant co-cultured with A549 cells in the OE-PTGES group decreased (P < 0.05) [Figure 4j-m].
- PGE2 upregulates PD-L1 expression in NSCLC and promotes immune escape response. (a-c) PD-L1 expression detected after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (d and e) Cytotoxicity tested by LDH kit assay after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (f and g) CD8+ T cell viability tested after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (h and i) CD8+ T cell apoptosis examined after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (j-m) IFN-γ, TNF-α, granzyme B, and perforin quantification by ELISA after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). n = 6; ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001. PEG2: Prostaglandin E2, PD-L1: Programmed death ligand 1, NSCLC: Non-small cell lung cancer, PTGES: Prostaglandin E synthase, OE-NC: Overexpression negative control, sh-NC: Short hairpin negative control, LDH: Lactate dehydrogenase, OE-PTGES: Overexpression prostaglandin E synthase, sh-PTGES: Short hairpin prostaglandin E synthase, IFN-γ: Interferon-gamma, TNF-α: Tumor necrosis factor-alpha, ELISA: Enzyme-linked immunosorbent assay.
PGE2 regulates PD-L1 expression by activating the PI3K/AKT/mTOR pathway in NSCLC
The pathway-related protein expression changes were tested using the PI3K activator (740Y-P) and inhibitor (LY294002) to detect whether PGE2 promotes immune escape through the PI3K/AKT/mTOR pathways. As demonstrated in Figure 5a and b, the phosphorylation levels of PI3K, AKT, and mTOR increased markedly in the case of PTGES overexpression (P < 0.01), and the increase in the ratio of phosphorylation caused by PTGES overexpression was reversed when the inhibitor was added (P < 0.01). Similarly, when PTGES was reduced, the phosphorylation levels were notably reduced in relation to their respective total proteins (P < 0.01), whereas the ratios of phosphorylation increased when activators were added (P < 0.01) [Figure 5c and d]. Next, the secretion of PD-L1 in cells was examined. The overexpression of PTGES resulted in an increase in the sPD-L1 level (P < 0.01). Conversely, the sPD-L1 level decreased significantly upon inhibiting pathway activation (P < 0.001). Similarly, knocking down PTGES resulted in a decrease in sPD-L1 levels (P < 0.001), which were significantly increased when the pathway was activated (P < 0.001) [Figure 5e and f].
- PGE2 regulates PD-L1 expression by activating the PI3K/AKT/mTOR pathway in NSCLC. (a-d) Relative expression levels of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR detected by WB after overexpression or knockdown of PTGES and addition of LY294002 or 740Y-P. (e and f) sPD-L1 detection by ELISA after overexpression or knockdown of PTGES and addition of LY294002 or 740Y-P. n = 6; ✶✶P < 0.01; ✶✶✶P < 0.001. PEG2: Prostaglandin E2, PD-L1: Programmed death ligand 1, NSCLC: Non-small cell lung cancer, PTGES: Prostaglandin E synthase, WB: Western blot, ELISA: Enzyme-linked immunosorbent assay, PI3K: Phosphatidylinositol-3-kinase, AKT: Protein kinase B, mTOR: Mammalian target of rapamycin, p-PI3K: Phospho phosphatidylinositol-3-kinase, p-AKT: Phospho protein kinase B, p-mTOR: Phospho mammalian target of rapamycin.
PGE2 promotes immune escape in NSCLC in vivo by upregulating PD-L1 expression
An animal model of a transplanted tumor was constructed to verify in vivo that PGE2 promotes immune circumvention by enhancing PD-L1 expression. The results showed that overexpression of PTGES led to a substantial increase in tumor size (P < 0.001), whereas knockdown of PTGES caused a markedly reduction (P < 0.01) [Figure 6a-c]. The WB results implied that PD-L1 expression increased with the increase in PTGES expression (P < 0.001) and decreased when PTGES expression decreased (P < 0.001) [Figure 6d-f]. The IHC results revealed that overexpression of PTGES reduced the CD8+ ratio (P < 0.001), and knocking down PTGES increased it (P < 0.001) [Figure 6g and h]. The cytokine levels in tumor tissues were further detected by ELISA, revealing that the cytokine levels were substantially reduced in the OE-PTGES group (P < 0.05). By contrast, the cytokines increased significantly in the sh-PTGES group (P < 0.01) [Figure 6i-l].
- PGE2 promotes immune escape in NSCLC in vivo by upregulating PD-L1 expression. (a) Isolated tumor images after PTGES overexpression and knockout. (b and c) Changes in tumor weight and volume after PTGES overexpression and knockout (significant difference markers marked with ✶ represent OE-NC versus OE-PTGES, and those marked with # represent sh-NC vs. sh-PTGES). (d-f) WB analysis of PTGES and PD-L1 after PTGES overexpression and knockout in vivo. (g and h) IHC analysis of CD8 after PTGES overexpression and knockout in vivo (scale bar: 20 μm, magnification, 400×). (i-l) IFN-γ, TNF-α, granzyme B, and perforin quantification by ELISA after PTGES overexpression and knockout in vivo. n = 5; ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001, ##P < 0.01. OE-NC: Overexpression negative control, sh-NC: Short hairpin negative control. PEG2: Prostaglandin E2, PD-L1: Programmed death ligand 1, NSCLC: Non-small cell lung cancer, PTGES: Prostaglandin E synthase, OE-NC: Overexpression negative control, sh-NC: Short hairpin negative control, OE-PTGES: Overexpression prostaglandin E synthase, sh-PTGES: Short hairpin prostaglandin E synthase, IHC: Immunohistochemistry, IFN-γ: Interferon-gamma, TNF-α: Tumor necrosis factor-alpha, ELISA: Enzyme-linked immunosorbent assay.
DISCUSSION
This research further explored the key effect of PGE2 in immune escape and related mechanisms in NSCLC. The findings revealed that PTGES expression was upregulated in NSCLC, leading to a remarkable increase in PGE2 content, which confirmed the abnormal accumulation of PGE2 in the occurrence and development of NSCLC, suggesting that PGE2 may be a potential therapeutic target.
An increasing number of studies have shown that PGE2 participates in tumor progression, including stimulating tumor cell proliferation, suppressing apoptosis, and facilitating tumor cell metastasis.[20] A study has shown that the deubiquitinating enzyme ubiquitin-specific protease 9X (USP9X) interacts with PTGES and stabilizes its protein, thus enhancing the PGE2 signaling pathway and forming the USP9X-PTGES-PGE2 axis. This interaction significantly promotes the metastatic properties, tumor occurrence, and stem cell-like characteristics of NSCLC, and high PTGES expression is associated with poor prognosis in patients.[19] The present study found that the increase in PGE2 had a multifaceted impact on NSCLC cells. In terms of cell biological behavior, the experimental results of constructing overexpressed and knockdown PTGES cell lines showed that PGE2 could promote the growth of NSCLC cells. Shi et al.[27] discovered that inhibiting the secretion of PGE2 can effectively inhibit epithelial–mesenchymal transformation and migration of renal cancer cells. This finding indicates that PGE2 plays an active role in the progression of tumors, which can not only accelerate the division of tumor cells but also enhance the migration and aggressive ability, providing favorable conditions for tumor metastasis. The present study found that PGE2 can upregulate PD-L1 in NSCLC and suppress the function of CD8+ T cells, thus promoting the immune escape of NSCLC. Recent studies have found that inhibiting the release of PGE2 can reduce the killing function of CD8+ T cells and reduce the expression of PD-L1 in colorectal cancer.[28] The present study clarified the regulatory relationship between PGE2 and PD-L1, providing new targets and ideas for NSCLC immunotherapy.
Further exploration of the molecular mechanism by which PGE2 affects NSCLC immune escape found that PGE2 can activate the PI3K/AKT/mTOR pathway. In a study of T cell lymphoma (TCL), Li et al. found that reducing PGE2 content could significantly inhibit this pathway, thereby inhibiting the development of TCL.[29] In addition, studies have demonstrated that PD-L1 and PI3K-related pathways are instrumental in cancers.[16,30-32] For instance, the PI3K/AKT/mTOR pathway has a bidirectional regulatory relationship with PD-L1. Abnormal activation of this pathway can increase the translation of PD-L1 protein through downstream proteins (including the energy abnormal state-related 4E-BP1 and STAT3), and overexpression of PD-L1 can reverse the activation of this pathway.[16] The present study found that PI3K activators and inhibitors can affect the secretion of PD-L1. These findings indicate that the PI3K/AKT/mTOR cascade serves as a crucial mediator in facilitating PD-L1 expression by PGE2.
Finally, a mouse model of tumor transplantation was constructed to verify the above mechanism in vivo. The results were consistent with the in vitro experiments, which further confirmed the mechanism of PGE2 promoting the immune escape of NSCLC. The results of animal experiments provide more convincing evidence for clinical treatment of NSCLC, suggesting that interventions targeting the signal axis of PGE2/PI3K/AKT/mTOR/PD-L1 may have potential therapeutic value.
In summary, this study comprehensively elaborated the mechanism of PGE2 promoting NSCLC immune evasion in NSCLC by enhancing PD-L1 expression through the PI3K/AKT/mTOR cascade. However, this study has certain limitations. First, it only used one cell line and lacked studies on more cell lines with different genetic mutation backgrounds and histological subtypes. Second, the study was entirely based on cell experiments and animal models, without including clinical samples and unable to analyze in combination with clinical pathological features (such as tumor stage and patient prognosis), resulting in a gap in the evidence chain for the clinical translation of the research conclusions. In the future, the research on the mechanism of PGE2 in the occurrence and development of NSCLC must be further improved by expanding the diversity of cell line samples and combining with the tissue samples of clinical cohorts. In addition, in vivo inhibitor rescue experiment should be verified. Future studies could further explore specific inhibitors or modulators targeting this signaling axis, bringing new therapeutic hope for NSCLC patients. At the same time, further studies on other roles of PGE2, its interactions with other immune cells and molecules, are needed to fully reveal the complex mechanism of NSCLC immune escape and lay the foundation for the development of more effective immunotherapy methods.
SUMMARY
This study experimentally verified that PGE2 is highly expressed in NSCLC and further verified the promoting effect of PGE2 on proliferation and invasion in the A549 cell line. The results showed that PGE2 may play an immune escape role by activating the PI3K/AKT/mTOR pathway to promote the expression of PD-L1, suggesting that targeted inhibition of PTGES/PGE2 may enhance the cytotoxicity of CD8+ T cells. Combined use with PD-L1 inhibitor may increase the treatment response rate of patients with NSCLC, providing a new combined strategy for clinical immunotherapy.
AVAILABILITY OF DATA AND MATERIALS
The data and materials that support the findings of this study are available from the corresponding author upon reasonable request.
ABBREVIATIONS
AKT: Protein kinase B
ANOVA: Analysis of variance
cDNA: Complementary DNA
COX: Cyclooxygenase
DAB: 3,3’-diaminobenzidine
DAPI: 4’,6-diamidino-2-phenylindole
ECL: Enhanced chemiluminescence
EdU: 5-ethynyl-2’-deoxyuridine
ELISA: Enzyme-linked immunosorbent assay
FBS: Fetal bovine serum
FITC: Fluorescein isothiocyanate
ICMJE: International Committee of Medical Journal Editors
IF: Immunofluorescence
IFN-γ: Interferon-gamma
IHC: Immunohistochemistry
LDH: Lactate dehydrogenase
LUAD: Lung adenocarcinoma
LUSC: Lung squamous cell carcinoma
mTOR: Mammalian target of rapamycin
NSCLC: Non-small cell lung cancer
OE-NC: Overexpression negative control
OE-PTGES: Overexpression prostaglandin E synthase
p-AKT: Phospho protein kinase B
PBMCs: Peripheral blood mononuclear cells
PBS: Phosphate-buffered saline
PBST: Phosphate buffered saline with tween 20
PD-L1: Programmed death ligand 1
PGE2: Prostaglandin E2
PGH2: Prostaglandin H2
PI: Propidium iodide
PI3K: Phosphatidylinositol-3-kinase
p-mTOR: Phospho mammalian target of rapamycin
p-PI3K: Phospho phosphatidylinositol-3-kinase
PTGES: Prostaglandin E synthase
PVDF: Polyvinylidene fluoride
qRT-PCR: Quantitative real-time polymerase chain reaction
sh-NC: Short hairpin negative control
sh-PTGES: Short hairpin prostaglandin E synthase
sPD-L1: Soluble programmed death ligand 1
STAT3: Signal transducer and activator of transcription 3
STR: Short tandem repeat
TCGA: The Cancer Genome Atlas
TCL: T cell lymphoma
TNF-α: Tumor necrosis factor-alpha
USP9X: Ubiquitin-specific protease 9X
WB: Western blot
AUTHOR CONTRIBUTIONS
TW and SL: Conducted the research and contributed to data analysis and interpretation of the results; SL and LL: Provided assistance and suggestions for the experiments. All authors participated in the drafting and critical revision of the manuscript. All authors have read and approved the final manuscript. All authors were fully involved in the work, able to take public responsibility for relevant portions of the content, and agreed to be accountable for all aspects of the work, ensuring that any questions related to its accuracy or integrity are addressed. All authors meet the ICMJE author qualifications.
ACKNOWLEDGMENT
Not applicable.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
This study was approved by the Beijing Maide Kangna Laboratory Animal Welfare Ethics Committee, approval No. MDKN-2024-103. Consent to participate was not applicable as there were no human subjects involved in this study.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
EDITORIAL/PEER REVIEW
To ensure the integrity and highest quality of CytoJournal publications, the review process of this manuscript was conducted under a double-blind model (authors are blinded from reviewers and vice versa) through an automatic online system.
FUNDING: Not applicable.
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