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 (m 6 A) 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 m 6 A-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 m 6 A modification of EHD1 . Further, the EHD1 mRNA was found to bind to the YTHDF1 protein, an m 6 A reader. YTHDF1 overexpression up-regulated EHD1 expression by enhancing its mRNA stability in an m 6 A-dependent manner. Conclusion Our study illuminates the role of m 6 A-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.
Background There is growing evidence that endocytosis plays a pivotal role in cancer metastasis. In this study, we first identified endocytic and metastasis-associated genes (EMGs) and then investigated the biological functions and mechanisms of EMGs. Methods Cancer stem cells (CSCs)-like characteristics were evaluated by tumour limiting dilution assays, three-dimensional (3D) spheroid cancer models. Microarray analysis was used to identify the pathways significantly regulated by mammalian Eps15 homology domain protein 1 (EHD1) knockdown. Mass spectrometry (MS) was performed to identify EHD1-interacting proteins. The function of EHD1 as a regulator of cluster of differentiation 44 (CD44) endocytic recycling and lysosomal degradation was determined by CD44 biotinylation and recycling assays. Results EHD1 was identified as a significant EMG. Knockdown of EHD1 suppressed CSCs-like characteristics, epithelial–mesenchymal transition (EMT), migration and invasion of lung adenocarcinoma (LUAD) cells by increasing Hippo kinase cascade activation. Conversely, EHD1 overexpression inhibited the Hippo pathway to promote cancer stemness and metastasis. Notably, utilising MS analysis, the CD44 protein was identified as a potential binding partner of EHD1. Furthermore, EHD1 enhanced CD44 recycling and stability. Indeed, silencing of CD44 or disruption of the EHD1/CD44 interaction enhanced Hippo pathway activity and reduced CSCs-like traits, EMT and metastasis. Interestingly, specificity protein 1 (SP1), a known downstream target gene of the Hippo-TEA-domain family members 1 (TEAD1) pathway, was found to directly bind to the EHD1 promoter region and induce its expression. Among clinical specimens, the EHD1 expression level in LUAD tissues of metastatic patients was higher than that of non-metastatic patients. Conclusions Our findings emphasise that EHD1 might be a potent anti-metastatic target and present a novel regulatory mechanism by which the EHD1/CD44/Hippo/SP1 positive feedback circuit plays pivotal roles in coupling modules of CSCs-like properties and EMT in LUAD. Targeting this loop may serve as a remedy for patients with advanced metastatic LUAD.
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