Suppression of tumor growth by GMI, an edible fungal immunomodulatory protein, is associated with targeting GSK3β‑mediated proteasomal degradation of PD‑L1

Abstract

Cancer cells evade T cell responses by exploiting programmed death‑ligand 1 (PD‑L1) in the tumor microenvironment, and oncogenic epidermal growth factor receptor (EGFR) signaling stabilizes PD‑L1 expression. Ganoderma microsporum immunomodulatory protein (GMI), a consumable mushroom‑derived dietary supplement, functions as an EGFR degrader targeting EGFR‑positive cancer cells. However, the role of GMI in regulating PD‑L1 and modulating antitumor immunity has not been fully elucidated. In the present study, functional enrichment analysis was first employed to investigate GMI‑regulated differentially expressed proteins. The findings indicated that GMI may modulate the PD‑L1 signaling pathway. GMI downregulated PD‑L1 expression by regulating both mRNA and protein stability, thereby suppressing PD‑L1‑positive lung cancer cells in vitro and in vivo. Functional studies further demonstrated that GMI promotes glycogen synthase kinase 3 beta (GSK3β)‑mediated proteasomal degradation of PD‑L1. Knockdown of GSK3β in lung cancer cells abolished the GMI‑induced reduction in PD‑L1 expression. Additionally, GMI inhibited tumor growth and reduced PD‑L1 levels in allograft mouse models. Importantly, GMI‑mediated PD‑L1 downregulation correlated with enhanced T cell‑mediated inhibition of lung cancer cells. These findings shed light on the potential of edible GMI to boost antitumor immunity.

Keywords: GMI; PD‑L1 degradation; antitumor immunity; lung cancer; proteasomal pathway.

Figure 1

GMI regulates PD-L1 expression in H1975, CL1-5 and A549 lung cancer cells. (A) Schematic workflow of membrane protein extraction from GMI-treated cancer cells, followed by liquid chromatography-mass spectrometry proteomic analysis to identify GMI-regulated DEPs. (B) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of GMI-regulated DEPs, highlighting the top 30 significant pathways ranked by fold enrichment. 'The PD-L1 expression and PD-1 checkpoint pathway in cancer' is highlighted within the blue box. (C) Densitometric analysis showing the fold changes (GMI/CTL) of proteins within the enriched pathway, with two downregulated proteins (fold change <0.75) and one upregulated protein (fold change >1.33). (D) Bar graph depicting relative PD-L1 intensity in GMI-treated lung cancer cells compared with untreated controls. GMI, Ganoderma microsporum immunomodulatory protein; PD-L1, programmed death-ligand 1; DEPs, differentially expressed proteins; PD-1, programmed cell death protein-1.

Figure 2

GMI reduces membrane PD-L1 protein levels in lung cancer cells. (A) Left: Immunoblot analysis of PD-L1 in WCL and PM fractions isolated from H1975, CL1-5 and A549 cells after 24 h treatment with 0.6 μM GMI. Caveolin and tubulin were used as markers for PM and WCL fractions, respectively. Right: quantification of PD-L1 levels in PM fractions normalized to caveolin. (B) Immunofluorescence staining of PD-L1 (green) and nuclei (DAPI, blue) in H1975 and CL1-5 cells treated with 0.6 μM GMI for 3 h. Scale bars, 45 μm (main images) and 10 μm (magnified insets). (C and D) Western blot analysis of PD-L1 protein levels in H1975, CL1-5 and A549 cells treated with indicated concentrations of GMI for 3 h (C) and 24 h (D). Tubulin served as the loading control. Data are presented as the mean ± standard deviation from three independent experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001. GMI, Ganoderma microsporum immunomodulatory protein; PD-L1, programmed death-ligand 1; WCL, whole-cell lysates; PM, plasma membrane.

Figure 3

GMI induces proteasome-dependent degradation of PD-L1. (A) Reverse transcription-quantitative PCR analysis of PD-L1 mRNA expression in H1975 and CL1-5 cells following treatment with 0.6 μM GMI for 1 and 24 h. (B) CHX chase assay of H1975 and CL1-5 cells exposed to 200 μg/ml CHX for 30 min, followed by incubation with or without 0.6 μM GMI for the indicated time points to assess PD-L1 protein stability. (C and D) H1975 and CL1-5 cells were pretreated with 10 μM MG132 (C), a proteasome inhibitor, or 5 nM Baf A1 (D), a lysosome inhibitor, and were then exposed to 0.6 μM GMI for 24 h. Data are presented as the mean ± standard deviation from three independent experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001. GMI, Ganoderma microsporum immunomodulatory protein; PD-L1, programmed death-ligand 1; CHX, cycloheximide; Baf A1, bafilomycin A1; ns, not significant.

Figure 4

GMI facilitates PD-L1 degradation through activation of GSK3β. (A and B) H1975 and CL1-5 cells were treated with indicated concentrations of GMI for 3 h (A) and 24 h (B). Protein expressions of EGFR, p-AKT (Ser473), p-GSK3β (Ser9) and PD-L1 were analyzed by immunoblotting, with AKT, GSK3β and tubulin as loading controls, and quantification of p-AKT and p-GSK3β was performed relative to their corresponding total proteins (AKT and GSK3β). (C) H1975 and CL1-5 cells were pretreated with 25 mM LiCl, a GSK3β inhibitor, and were exposed to 0.6 μM GMI for 24 h. (D) Immunoblotting of GSK3β in H1975 and CL1-5 cells expressing different GSK3β-targeting shRNAs. (E) Immunoblotting analysis of PD-L1 and GSK3β in cancer cells transduced with either GSK3β shRNA (shGSK3β#1) or control shRNA (shCTL) lentiviruses and treated with or without 0.6 μM GMI for 24 h. (F) Quantification of PD-L1 and GSK3β expression levels shown in (E). Data are presented as the mean ± standard deviation from three independent experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001. GMI, Ganoderma microsporum immunomodulatory protein; PD-L1, programmed death-ligand 1; p-, phosphorylated; LiCl, lithium chloride; shRNA, short hairpin RNA; p-, phosphorylated; ns, not significant.

Figure 5

GMI inhibits tumor growth and downregulates PD-L1 in LLC1-hPD-L1 allograft models. (A) LLC1 cells were treated with the indicated concentrations of GMI for 3 h. PD-L1 protein levels were assessed by western blotting, with tubulin as the loading control. Data are presented as the mean ± standard deviation from three independent experiments. (B) Western blot analysis of PD-L1 protein levels in tumor cells harvested from CTL and GMI-treated groups. Quantitative data are shown. (C) Schematic diagram illustrating the knockout of mouse PD-L1 and replacement with human PD-L1 expression. (D) Treatment schedule of LLC1-hPD-L1 syngeneic C57BL/6 mice. Mice were administered intraperitoneal injections of GMI (5.0 mg/kg) every 3 days. (E-G) Tumor volume (E), tumor weight (F) and body weight (G) were measured at the indicated time points. Data are presented as the mean ± SEM (n=6 mice per group). (H) PD-L1 protein levels in excised tumors from CTL and GMI groups were analyzed by western blotting (random samples from 3 mice per group). ^*P<0.05, ^**P<0.01 and ^***P<0.001. GMI, Ganoderma microsporum immunomodulatory protein; PD-L1, programmed death-ligand 1; mPD-L1, mouse PD-L1; hPD-L1, human PD-L1.

Figure 6

GMI regulates T cell-mediated suppression of tumor cells ex vivo and promotes antitumor immunity in vivo. (A) Schematic diagram illustrating the co-culture protocol. Activated spleen-derived T cells isolated from LLC1-bearing mice were co-cultured with LLC1-GFP cells pretreated with 0.6 μM GMI for 3 h. After 72 h of co-culture, GFP fluorescence in LLC1-GFP cells was analyzed. (B) Quantification of GFP fluorescence intensity in LLC1-GFP cells following co-culture with activated T cells. (C) Flow cytometric analysis of tumor-infiltrating lymphocytes from LLC1-bearing mice treated with control (PBS) or GMI (Fig. 5B). The ratio of CD8⁺ to CD4⁺ T cells is shown. (D) Treatment schedule for LLC1 syngeneic C57BL/6 mice receiving intraperitoneal injections of GMI (5.0 mg/kg), anti-PD-1 antibody (200 μg/mice), or their combination every 2 days. (E-G) Tumor volume (E), tumor weight (F) and body weight (G) were monitored at specified intervals throughout the experiment. Data are presented as the mean ± SEM (n=5 mice per group). (H) Schematic model illustrating how GMI enhances antitumor immunity by driving PD-L1 breakdown. GMI, a FIP derived from an edible mushroom and known as an EGFR degrader, reduces PD-L1 levels by downregulating its mRNA expression and promoting GSK3β-mediated ubiquitination and proteasomal degradation. This process further diminishes T cell immune suppression and enhances antitumor immunity. ^*P<0.05 and ^***P<0.001. Ganoderma microsporum immunomodulatory protein; GFP, green fluorescent protein; PD-1, programmed cell death protein-1; FIPs, fungal immunomodulatory proteins; LiCl, lithium chloride; ns, not significant.