Tian Fanglin, Huang Jian, Fan Weina, Li Xin, Zhan Yuning, Zhu Kexin, Wang Xiangyu, Hong Xin, Wang Xin, Ren Jin, Xing Ying, Cai Li
The Fourth Department of Medical Oncology, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, P. R. China.
National Health Commission and Chinese Academy of Medical Sciences Key Laboratory of Molecular Probes and Targeted Theranostics, Harbin Medical University, Harbin, Heilongjiang, P. R. China.
Cancer Commun (Lond). 2025 Jul 24. doi: 10.1002/cac2.70052.
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.
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 (mA) 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 mA-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.
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 mA modification of EHD1. Further, the EHD1 mRNA was found to bind to the YTHDF1 protein, an mA reader. YTHDF1 overexpression up-regulated EHD1 expression by enhancing its mRNA stability in an mA-dependent manner.
Our study illuminates the role of mA-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.
Eps15同源结构域（EHD）蛋白，包括EHD1至EHD4，在肿瘤进展中发挥着至关重要的作用。在本研究中，我们旨在探究哪些特定的EHD蛋白（如果有的话）与肿瘤免疫逃逸和免疫治疗反应有关。
使用肿瘤免疫功能障碍和排除（TIDE）分析预测肺腺癌（LUAD）患者的免疫治疗反应。通过将活化的T细胞与LUAD细胞共培养进行T细胞杀伤试验。通过受体内化试验确定EHD1作为程序性死亡配体1（PD-L1）内吞再循环调节剂的功能。进行甲基化RNA免疫沉淀（MeRIP）以研究EHD1 mRNA的N6-甲基腺苷（m6A）修饰。通过分子对接分析揭示蛋白质-蛋白质相互作用，并通过免疫荧光（IF）和免疫沉淀（IP）试验进行验证。使用RNA免疫沉淀（RIP）检测YTH N6-甲基腺苷RNA结合蛋白1（YTHDF1）与EHD1 mRNA之间的相互作用。通过应用m6A结合位点突变分析研究YTHDF1对EHD1的调节机制。使用小鼠LUAD细胞在具有免疫活性的C57BL/6小鼠体内建立皮下异种移植模型，以评估EHD1在体内的免疫调节作用。
TIDE算法和生存分析确定EHD1促进LUAD免疫逃逸。在所有效应细胞与靶细胞（E/T）比例下，敲低EHD1增强了T细胞对LUAD细胞的细胞毒性。过表达EHD1则产生相反的效果。分子对接分析揭示了EHD1与PD-L1蛋白之间的相互作用，经IF和IP验证。此外，敲低EHD1抑制了PD-L1的再循环，从而促进其溶酶体降解。EHD1/PD-L1相互作用的破坏损害了EHD1在肿瘤免疫逃逸中的调节功能。在具有免疫活性的小鼠模型中，我们发现沉默EHD1可阻碍肿瘤免疫逃逸并增强抗PD-1治疗的疗效。MeRIP-qPCR证实EHD1存在明显的m6A修饰。此外，发现EHD1 mRNA与m6A阅读器YTHDF1蛋白结合。YTHDF1过表达通过以m6A依赖的方式增强其mRNA稳定性上调EHD1表达。
我们的研究阐明了m6A修饰的EHD1在肿瘤免疫逃逸和免疫治疗反应中的作用，从而为潜在增强免疫治疗敏感性和改善LUAD患者预后提供了一条新途径。
