USP33 Facilitates Retinoblastoma Growth by Deubiquitinating and Stabilizing EPHB2 Protein

Retinoblastoma (RB) is an intraocular malignant tumor originating from primitive retinal stem cells or cone precursor cells, appearing most frequently under the age of three years. EPH receptor B2 (EPHB2) has been found to be involved in RB, but the potential action of EPHB2 in RB remains poorly understood. Real-time quantitative polymerase chain reaction and western blotting detected RNA levels and protein levels. Cell viability, proliferation, apoptosis, invasion, and stemness were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-ethynyl-2'deoxyuridine flow cytometry, transwell, and sphere-forming assays. The interaction between EPHB2 and ubiquitin-specific protease 33 (USP33) was confirmed by cell ubiquitination and protein stability assays. Mouse xenograft models were utilized for the validation of the effect of the USP33/EPHB2 pathway in RB. EPHB2 was upregulated in RB, and lower overall survival was observed in patients with higher levels of EPHB2. Functional experiments demonstrated that EPHB2 promoted RB cell proliferation, invasion, and stemness, as well as inhibited RB cell apoptosis in vitro. Mechanically, USP33 is responsible for EPHB2 upregulation. Additionally, USP33 caused the deubiquitination and stabilization of EPHB2 protein. Rescue experiments showed that USP33 promoted RB growth in vitro and mouse models by activating the Wnt/β-catenin signaling in an EPHB2-dependent manner. The study identified a positive regulatory role of the USP33/EPHB2 pathway in the promotion of RB malignant behaviors, suggesting that developing USP33-specific inhibitors (such as small molecule compounds) may become a new direction for RB treatment.