NEDD4 Promotes Hepatocellular Carcinoma Progression Through Ubiquitin‐Dependent Degradation of EPHB6

Neural precursor cell expressed developmentally down-regulated 4 (NEDD4) has been implicated in the progression of various human malignancies, including hepatocellular carcinoma (HCC). However, the relationship between NEDD4's ubiquitin ligase activity and the motility of HCC cells remains unclear. We investigated the significance of the NEDD4/EPHB6 (erythropoietin-producing hepatocyte B6) axis in driving epithelial-mesenchymal transition (EMT) progression in HCC. In this study, we investigated the role of the HECT-type E3 ubiquitin ligase NEDD4 in HCC progression and identified its downstream substrate through integrated bioinformatic screening, protein-level validation, and NEDD4 immunoprecipitation coupled with mass spectrometry. The regulatory relationship between NEDD4 and EPHB6 was examined using ubiquitination assays, protein stability analysis, and functional rescue experiments. Potential downstream signaling changes associated with the NEDD4-EPHB6 axis were further examined through pathway analysis and molecular validation. NEDD4 silencing suppressed, whereas its overexpression enhanced HCC cell invasion and migration. Mechanistically, NEDD4 functioned as an E3 ubiquitin ligase that promoted Lys48-linked polyubiquitination and proteasomal degradation of erythropoietin-producing hepatocyte B6 (EPHB6), resulting in reduced EPHB6 protein abundance without altering EPHB6 mRNA levels. Disruption of the NEDD4-EPHB6 axis attenuated EMT, which was further validated in a xenograft mouse model. Moreover, NEDD4 protein expression was inversely correlated with EPHB6 levels in clinical HCC tissues. In summary, our study identifies the NEDD4-EPHB6 axis as a previously unrecognized regulator of EMT-associated malignant progression in HCC and demonstrates its association with activation of TGF-β/SMAD2/3 signaling. This study provides new mechanistic insight into how post-translational regulation of membrane-associated proteins controls HCC progression.