3156 - EHD1-Driven Radioresistance in Hepatocellular Carcinoma: Hippo Pathway Regulation and Immune Microenvironment Remodeling
Presenter(s)
L. Wu, K. Jiang, J. Yang, K. Ding, H. Yu, and S. Yan; Department of Radiation Oncology, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
Purpose/Objective(s): This study investigates whether EHD1, a caveolae-associated protein, promotes hepatocellular carcinoma (HCC) radioresistance by suppressing Hippo pathway activity and driving N2-type neutrophil polarization within the tumor immune microenvironment.
Materials/Methods: Radioresistant HCC cell lines (HepG2-IR, Sk-Hep-1-IR) were generated through 6-month incremental radiation exposure (2-8 Gy). RNA sequencing with four biological replicates per group identified transcriptomic changes, validated through Western blot and immunofluorescence across three independent experiments. Subcutaneous xenograft models using 10 mice per group compared tumor growth and immune infiltration between EHD1-knockdown and control cells post-radiation. Single-cell RNA sequencing analyzed 45,668 cells from irradiated versus control murine tumors. Clinical relevance was assessed using TCGA-LIHC data (n=364 patients) with Cox proportional hazards modeling and immune deconvolution algorithms (TIMER, xCell, CIBERSORT).
Results: Mechanistically, radioresistant cells exhibited 4.2-fold higher EHD1 expression versus parental lines (p<0.001), with EHD1 knockdown restoring Hippo pathway activity through increased YAP phosphorylation (2.3-fold, p=0.008) and enhancing radiation-induced DNA damage (187% ?H2AX foci increase, p=0.002). Immune profiling revealed irradiated tumors had 3.1-fold more N2-polarized neutrophils (CD11b+CD206+, p=0.004), while EHD1 depletion reduced N2 polarization by 68% (p=0.01) and increased CD8+ T-cell infiltration 2.4-fold (p=0.03). Clinically, high EHD1 expression predicted worse overall survival (HR=2.14, 95%CI 1.47-3.11, p<0.001) and correlated with immunosuppressive macrophage profiles (M1:M2 ratio 0.33 vs 1.07 in low-EHD1 tumors, p=0.007).
Conclusion: EHD1 drives HCC radioresistance through coordinated Hippo pathway inhibition and N2 neutrophil-mediated immunosuppression, with preclinical models demonstrating radiosensitization upon EHD1 targeting. Clinical validation of EHD1's prognostic significance and immune-modulating role supports its potential as a therapeutic target to overcome radiation resistance in HCC.