m6A-modified EHD1 controls PD-L1 endosomal trafficking to modulate immune evasion and immunotherapy responses in lung adenocarcinoma

Abstract

Background: Eps15 homology domain (EHD) proteins, including EHD1 to EHD4, play vital roles in tumor progression. In this study, we aimed to investigate which specific EHD proteins, if any, are implicated in tumor immune evasion and immunotherapy response.

Methods: The immunotherapy responses of lung adenocarcinoma (LUAD) patients were predicted using tumor immune dysfunction and exclusion (TIDE) analysis. The T cell killing assay was performed by co-culturing activated T cells with LUAD cells. The function of EHD1 as a regulator of programmed death-ligand 1 (PD-L1) endocytic recycling was determined by receptor internalization assays. Methylated RNA immunoprecipitation (MeRIP) was performed to investigate N6-methyladenosine (m⁶A) modification of EHD1 mRNA. The protein-protein interaction was revealed by the molecular docking analysis and validated by immunofluorescence (IF) and immunoprecipitation (IP) assays. RNA immunoprecipitation (RIP) was used to examine the interaction between YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) and EHD1 mRNA. The regulatory mechanism of YTHDF1 on EHD1 was investigated through the application of m⁶A-binding site mutation analysis. The murine LUAD cells were employed to establish subcutaneous xenograft models within immunocompetent C57BL/6 mice to assess the immunomodulatory impact of EHD1 in vivo.

Results: TIDE algorithms and survival analysis identified that EHD1 promoted LUAD immune escape. EHD1 knockdown enhanced T cell cytotoxicity in killing LUAD cells across all effector-to-target (E/T) ratios. EHD1 overexpression exerted the opposite effect. The molecular docking analysis revealed an interaction between EHD1 and the PD-L1 protein, verified by IF and IP. Furthermore, EHD1 knockdown inhibited PD-L1 recycling, thereby promoting its lysosomal degradation. Disruption of the EHD1/PD-L1 interaction impaired the regulatory function of EHD1 in tumor immune evasion. In an immune-competent mouse model, we found that EHD1 silencing impeded tumor immune evasion and enhanced the efficacy of anti‑PD‑1 therapy. MeRIP-qPCR confirmed obvious m⁶A modification of EHD1. Further, the EHD1 mRNA was found to bind to the YTHDF1 protein, an m⁶A reader. YTHDF1 overexpression up-regulated EHD1 expression by enhancing its mRNA stability in an m⁶A-dependent manner.

Conclusion: Our study illuminates the role of m⁶A-modified EHD1 in tumor immune evasion and immunotherapy responses, thereby offering a novel avenue to potentially enhance immunotherapeutic sensitivity and improve the prognosis for patients with LUAD.

Keywords: EHD1; YTHDF1; endosomal trafficking; immunotherapeutic responses; lysosomal degradation.

FIGURE 1

EHD1 knockdown in LUAD cells increased sensitivity to T‐cell‐induced cytotoxicity. (A) Violin plots depicting the distribution of TIDE scores among high‐ versus low‐ expression groups of individual members within the EHD gene family. (B) Kaplan‐Meier survival curves, using the TCGA‐LUAD dataset, illustrating the OS stratification categorized based on the level of gene expression in the EHD gene family. (C‐D) Comparative analysis of the mRNA (C) and protein (D) levels of EHD1 in the untreated group and A549 cells transfected with shCtrl, shEHD1#1, or shEHD1#2. (E) Flowchart illustrating the process of co‐culturing activated T cells with LUAD cells. T cells were activated after 24 h of cytokine treatment and then co‐cultured with LUAD cells for an additional 24 h. (F) CCK‐8 assay results showing the influence of EHD1 knockdown on the killing of A549 cells by T cells. Activated T cells and LUAD cells were co‐cultured at E/T ratios of 1:1, 5:1, or 10:1. (G) Crystal violet‐stained images of surviving LUAD cells treated with activated T cells across the designated groups of cells. Representative pictures (left panel) and the quantification chart (right panel) are displayed. (H) Evaluation of T‐cell‐mediated apoptosis in the designated LUAD cells using FCM. The right bar graph summarizes the statistical evaluation of these results. (I) Western blotting analysis revealed the influence of EHD1 knockdown on the protein levels of apoptosis‐related markers in T‐cell‐treated LUAD cells. (J) Histogram showing the influence of EHD1 knockdown on the T‐cell‐mediated cell death rate of A549 cells. The E/T ratio is 5:1. (K) Histograms showing the levels of LDH released by LUAD cells alone or following treatment with T cells. (L) Histograms quantifying the levels of IFN‐γ secreted by T cells co‐cultured with LUAD cells. Data were collected from three independent experiments. Two‐tailed unpaired Student's t test; The data are shown as the mean ± SD. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. Bax, Bcl‐2 Associated X; Bcl‐2, B‐cell lymphoma‐2; CCK‐8, Cell counting Kit‐8; CD2, Cluster of Differentiation 2; CD28, Cluster of Differentiation 28; CD3, Cluster of Differentiation 3; E/T, Effector‐to‐target ratio; EHD, Eps15 homology domain; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; IFN‐γ, Interferon γ; LDH, Lactate Dehydrogenase; LUAD, Lung adenocarcinoma; PI, Propidium iodine; OS, Overall survival; TIDE, Tumor Immune Dysfunction and Exclusion; FCM, Flow cytometry; shCtrl, short hairpin RNA control; shEHD1, short hairpin RNA for EHD1; SD, Standard deviation.

FIGURE 2

EHD1 interacted with PD‐L1 and upregulated its expression. (A) Diagram indicating the mechanism by which LUAD cells escape CD8⁺ T‐cell‐mediated cytotoxicity through PD‐L1. (B) Docking models of the EHD1‐PD‐L1 complex. (C) IF analysis of the colocalization of EHD1 and PD‐L1 in LUAD cells. (D‐E) IP assays validated the molecular interaction between endogenous EHD1 and PD‐L1. (F) IP assays confirmed the interaction between exogenous EHD1 and PD‐L1. (G) Western blotting analysis was used to detect the regulatory influence of EHD1 silencing on PD‐L1 protein expression. (H) Influence of EHD1 silencing on PD‐L1 mRNA levels in LUAD cells, as detected by qPCR. (I) IF analysis showing the PD‐L1 localization and the influence of EHD1‐knockdown on PD‐L1 expression in LUAD cells. Data were collected from three independent experiments. Two‐tailed unpaired Student's t test; The data are shown as the mean ± SD. ns, not significant; *** P < 0.001. CD8, Cluster of Differentiation 8; DAPI, 4',6‐diamidino‐2‐phenylindole; EHD, Eps15 homology domain; LUAD, Lung adenocarcinoma; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; IP,Immunoprecipitation; IF, Immunofluorescence; IgG, Immunoglobulin G; PD‐1, Programmed cell death 1; PD‐L1, Programmed Cell Death 1 Ligand 1; shCtrl, short hairpin RNA control; shEHD1, short hairpin RNA for EHD1; SD, Standard deviation.

FIGURE 3

EHD1 promoted the endocytic recycling of PD‐L1 and inhibited its lysosomal degradation. (A) Colocalization analysis of PD‐L1 with RAB11 and LAMP1 in shCtrl and shEHD1 LUAD cells. (B) The relative PD‐L1 expression in LUAD cell surface regulated by EHD1 was detected by FCM. Representative FCM histograms (left), and quantification data (right) are displayed. (C‐D) The surface level of remaining PD‐L1 after EHD1 knockdown was detected by (C) FCM analysis and (D) IP experiments at the specified time points. (E) A CHX chase assay was performed to analyze the stability of the PD‐L1 protein in shCtrl and shEHD1 LUAD cells. The cells were treated with CHX (10 µg/mL) for the designated time. (F‐G) Western blotting analysis was performed to measure PD‐L1 expression in shCtrl and shEHD1 LUAD cells treated with the proteasome inhibitor MG132 (F) or the lysosomal inhibitor NH₄Cl (G) for 8 h. (H) Ubiquitination IP assays validated the degradation of PD‐L1 in LUAD cells transfected with the total‐Ub, UbK63 or UbK48 plasmid. NH₄Cl was used for treatment before cell lysis. Data were collected from three independent experiments. Two‐tailed unpaired Student's t test; The data are shown as the mean ± SD. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. CHX, Cycloheximide; DAPI, 4',6‐diamidino‐2‐phenylindole; EHD, Eps15 homology domain; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; HA‐Ub, Hemagglutinin ubiquitin; IP, Immunoprecipitation; LAMP1, Lysosome‐associated membrane protein 1; LUAD, Lung adenocarcinoma; PD‐L1, Programmed cell death 1 ligand 1; PE, Phycoerythrin; RAB11, Ras‐Related Protein 11; shCtrl, short hairpin RNA control; shEHD1, short hairpin RNA for EHD1; Total‐Ub, Total ubiquitin; UbK48, Ubiquitination at the lysine 48 residue; UbK63, Ubiquitination at the lysine 63 residue; FCM, Flow cytometry.

FIGURE 4

Disruption of the EHD1/PD‐L1 interaction hindered the regulatory role of EHD1 in tumor immune evasion. (A) Diagrammatic representation of the structure of full‐length EHD1 and the variant without the EH domain constructed with the plasmid. (B) IP analysis showing the ability of Myc‐tagged PD‐L1 to precipitate EHD1 proteins with or without EH domains. (C) Schematic diagrams of full‐length PD‐L1 and variants lacking the ICD domain constructed with plasmids. (D) IP analysis showing the structure of PD‐L1 that physically interacts with EHD1. (E) Western blotting analysis revealed the influence of disrupting the EHD1/PD‐L1 interaction on PD‐L1 expression. An antibody targeting the aa515‐534 region of the EHD1 protein was utilized in this Western blotting analysis. (F) Crystal violet‐stained images of surviving A549 cells treated with activated T cells across the designated groups. (G) Evaluation of T‐cell‐mediated apoptosis in the designated cells by FCM. (H) The influence of disrupting the EHD1/PD‐L1 interaction on the ability of T cells to inhibit LUAD cells was detected in an LDH release assay. (I) Histograms quantifying the levels of endogenous LDH released by LUAD cells alone or treated with T cells. (J) Histograms quantifying the levels of IFN‐γ secreted by T cells coincubated with LUAD cells. Data were collected from three independent experiments. Two‐tailed unpaired Student's t test; The data are shown as the mean ± SD. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. ECD: Extra cellular domain; EHD: Eps15 homology domain; EHD1‐FL: Full‐length EHD1; EHD1‐ΔEH: EHD1 variants lacking the EH domain; EHD1^MUT1: Mutant EHD1#1, which displayed overexpression of EHD1 lacking the EH domain; EHD1^OE: Overexpression of full‐length EHD1; FCM: Flow cytometry; IFN‐γ: Interferon γ; IP: Immunoprecipitation; LDH: Lactate Dehydrogenase; LUAD: Lung adenocarcinoma; PD‐L1: Programmed cell death 1 ligand 1; PD‐L1‐FL: Full‐length PD‐L1; PD‐L1‐ΔICD: EHD1 variants lacking the intracellular domain; PI: Propidium iodine; shEHD1: short hairpin RNA for EHD1; SP: Signal peptide; TM: Transmembrane domain.

FIGURE 5

EHD1 suppression hampered tumor immune evasion and increased the effectiveness of anti‐PD‐1 therapy. (A‐B) qRT‐PCR and Western blotting were performed to detect EHD1 expression in engineered clones derived from the mouse LUAD cell line LA795 transfected with shCtrl, shEHD1#1, or shEHD1#2. (C) Schematic showing the construction of xenograft models in C57BL/6 mice bearing shCtrl or shEHD1 tumors and the treatment regimen with anti‐PD‐1/anti‐IgG. (D) Mice were allocated into four experimental cohorts: shCtrl + anti‐IgG, shEHD1 + anti‐IgG, shCtrl + anti‐PD‐1, and shEHD1 + anti‐PD‐1. n = 5 mice per group. The diagram shows growth curves of xenograft tumor volume in the different treatment groups. (E) Bioluminescence images were obtained on Day 18. The bioluminescence signals were quantified and are presented in bar graphs, which depict the statistical analysis of the luciferase activity in the xenografts for each group (n = 5). Additionally, at the end of the experiment, photographic documentation of thexenograft tumors from all the groups was performed. (F‐H) Subsequent statistical assessments were conducted to compare tumor sizes (F), mean tumor weights (G), and tumor volumes (H) across the groups. (I) Statistical assessment of the body weights of the mice across the groups. (J) Representative images of H&E staining and IHC staining for EHD1, PD‐L1, and CD8 in tumor tissue slices (left) and data statistics of the IHC scores of corresponding proteins in different groups (right) are displayed. Data were collected from three independent experiments. Two‐tailed unpaired Student's t test; The data are shown as the mean ± SD. ns, not significant; ** P < 0.01, *** P < 0.001. CD8, Cluster of Differentiation 8; EHD1, Eps15 homology domain 1; H&E, Hematoxylin and eosin; IgG, Immunoglobulin G; IHC, Immunohistochemistry; L, Left; LUAD, Lung adenocarcinoma; PD‐L1, Programmed cell death 1 ligand 1; PD‐1, Programmed cell death 1; R, Right; shCtrl, short hairpin RNA control; shEHD1, short hairpin RNA for EHD1; SD, Standard deviation.

FIGURE 6

YTHDF1 regulated EHD1 expression and its mRNA stability. (A‐B) The typical m⁶A motif RRACH in EHD1 mRNA was identified using SRAMP (A) and MEME (B) (R = A or G; H = A, U or C). (C) Enrichment of m⁶A modifications on EHD1 mRNA was detected by MeRIP‐qPCR. (D) Venn diagram showing the regulators of m⁶A transcriptional modifications upstream of EHD1, which were predicted by RM2target, RMBase, and RMVAR. (E) The binding of EHD1 mRNA to YTHDF1 or IGF2BP1 was predicted using RPISeq. RF and SVM classifiers are used to predict RNA‐protein interaction probabilities based on sequence features; values above 0.5 suggest a potential interaction. (F) The correlation between the m⁶A regulators YTHDF1 and EHD1 was characterized by Spearman correlation analysis based on mRNA expression levels using the TCGA‐LUAD dataset. (G‐H) EHD1 mRNA and protein levels in LUAD cells with YTHDF1 knockdown were measured by qRT‐PCR and Western blotting. (I‐J) An Act D pulse‐chase experiment was performed to determine the stability of EHD1 mRNA in the indicated cells treated with 5 µg/mL Act D. (K‐L) EHD1 expression in shCtrl and shYTHDF1 LUAD cells treated with (K) MG132 or (L) NH₄Cl for 8 h was detected by Western blotting analysis. Data were collected from three independent experiments. Two‐tailed unpaired Student's t test; The data are shown as the mean ± SD. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. Act D, Actinomycin D; EHD1, Eps15 homology domain 1; IGF2BP1, Insulin like growth factor 2 MRNA binding protein 1; IgG, Immunoglobulin G; LUAD, Lung adenocarcinoma; m⁶A, N6‐methyladenosine; MeRIP‐qPCR, Methylated RNA immunoprecipitation‐quantitative polymerase chain reaction; RF, Random forest algorithm; shCtrl, short hairpin RNA control; shYTHDF1, short hairpin RNA for YTHDF1; SVM, Support vector machine; YTHDF1, YTH N6‐Methyladenosine RNA Binding Protein F1; SD, Standard deviation.

FIGURE 7

EHD1 is regulated by YTHDF1 in an m⁶A‐dependent manner. (A‐B) RIP‐qPCR and agarose electrophoresis results showing the enrichment of EHD1 mRNA. (C) Schematic description showing the construction of wild‐type (YTHDF1^WT) and mutant (YTHDF1^MUT) YTHDF1 vectors. (D) RIP‐qPCR was used to detect the precipitation of EHD1 mRNA by YTHDF1 in the YTHDF1^WT or YTHDF1^MUT group. (E) Western blotting analysis showing the effects of YTHDF1^WT and YTHDF1^MUT on the protein expression level of EHD1. (F‐G) An Act D chase assay was used to investigate the effect of 5 µg/mL Act D on EHD1 mRNA in the indicated cells. (H) Schematic representation of an EHD1 mutant variant (EHD1 ^MUT2) with alterations at the YTHDF1‐related m⁶A peak site of EHD1 mRNA. (I) Western blotting analysis revealed the influence of YTHDF1‐mediated m⁶A modification on the expression of Flag‐tagged EHD1. Data were collected from three independent experiments. Two‐tailed unpaired Student's t test; The data are shown as the mean ± SD. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. Act D, Actinomycin D; CDS, Coding sequence; EHD1, Eps15 homology domain 1; EHD1^MUT2, Mutant EHD1 mutant EHD1#2, which exhibited overexpression of EHD1 with mutations at the m⁶A peak sites; EHD1^OE, EHD1 overexpression; HA, Hemagglutinin; IgG, Immunoglobulin G; RIP‐qPCR, RNA immunoprecipitation‐quantitative polymerase chain reaction; t_1/2, Half life time; UTR, Untranscribed region; YTHDF1, YTH N6‐methyladenosine RNA binding protein F1; YTHDF1^MUT, Mutant YTHDF1; YTHDF1^WT, Wild type YTHDF1; SD, Standard deviation.

FIGURE 8

EHD1 expression levels were associated with those of PD‐L1 and YTHDF1 in clinical LUAD samples. (A) Representative images of EHD1, PD‐L1, and YTHDF1 IHC staining of tumor tissue samples from patients with LUAD (left). Bar graphs (right) showing the distributions of PD‐L1 and YTHDF1 expression in different EHD1 expression groups as determined by IHC in clinical LUAD samples. (B) Expression levels of PD‐L1 and YTHDF1 in patients with low (Patient 1) or high (Patient 2) EHD1 expression. (C) Kaplan‐Meier survival curves illustrating the PFS stratification categorized based on the level of EHD1 expression based on a publicly available dataset (GSE135222) from the GEO database. (D) Diagram of the mechanism by which m⁶A‐modified EHD1 promotes immune evasion and ICB resistance through PD‐L1 endosomal transport in LUAD cells. Data were collected from three independent experiments. Two‐tailed unpaired Student's t test; The data are shown as the mean ± SD. ns, not significant; *** P < 0.001. EHD1, Eps15 homology domain 1; GEO, Gene Expression Omnibus; ICB, Immune checkpoint blockade; IHC, Immunohistochemistry; LUAD, Lung adenocarcinoma; PD‐1, Programmed cell death 1; PD‐L1, Programmed cell death 1 ligand 1; PFS, Progression free survival; RAB11, Ras‐related protein 11; Ub, Ubiquitin; YTHDF1, YTH N6‐methyladenosine RNA binding protein F1; SD, Standard deviation.