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 EHDproteins,if any,are implicated in tumor immuneevasion ...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 EHDproteins,if any,are implicated in tumor immuneevasion 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 LUADcells 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.展开更多
基金National Natural Science Foundation of China,Grant/Award Numbers:82072563,82473167,82172587,82373122,82103519Natural Science Funding of Heilongjiang,Grant/Award Number:YQ2024H021+4 种基金Heilongjiang Province Postdoctoral Start-up Fund,Grant/Award Number:21042240023Haiyan Foundation of Harbin Medical University Cancer Hospital,Grant/Award Numbers:JJJQ2024-07,JJZD2021-07China Postdoctoral Science Foundation,Grant/Award Number:2021M693826Heilongjiang Provincial Postdoctoral Science Foundation,Grant/Award Number:LBH-Z21187Harbin Medical University Cancer Hospital,Grant/Award Numbers:BJQN2019-07,BJQN 2021-02。
文摘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 EHDproteins,if any,are implicated in tumor immuneevasion 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 LUADcells 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.
