Genome wide CRISPR screening identifies FOXJ3 as a regulator of ferroptosis in hepatocellular carcinoma via OTUD5 dependent stabilization of GSTP1

The oncogenic potential of forkhead box (FOX) family proteins has been well documented in multiple cancer types. Hepatocellular carcinoma (HCC), a highly lethal form of liver cancer, is characterized by limited treatment options. Using in vivo whole-genome clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) screening and a FOX family small hairpin RNA (shRNA) library, this study identified FOXJ3 as a key regulatory factor of ferroptosis in HCC. Clinical analysis further revealed that high FOXJ3 expression in HCC tissues is significantly associated with poor prognosis. In vitro and in vivo experiments demonstrated that FOXJ3 enhances HCC cell viability by inhibiting ferroptosis. Mechanistically, FOXJ3 transcriptionally upregulates OTU deubiquitinase 5 ( OTUD5 ), which specifically removes K48-linked polyubiquitin chains from glutathione S-transferase P1 (GSTP1), thus stabilizing GSTP1 and preventing ferroptosis in HCC. Additionally, Bleomycin A5 (Ble) was identified as a novel inhibitor of FOXJ3, which suppressed HCC progression in both in vitro and in vivo models. In conclusion, FOXJ3 inhibits ferroptosis by promoting OTUD5 transcription, which in turn stabilizes GSTP1 via its deubiquitinase activity, thereby positioning FOXJ3 as a promising therapeutic target for HCC.