TFEB promotes Ginkgetin-induced ferroptosis via TRIM25 mediated GPX4 lysosomal degradation in EGFR wide-type lung adenocarcinoma

<b>Rationale</b>: TFEB activation is associated with prolonged survival in LUAD patients, suggesting potential benefits of TFEB agonists in LUAD treatment. In this study, we identify ginkgetin (GK), derived from <i>Ginkgo folium</i>, as a natural TFEB agonist, which has demonstrated promising anticancer effects in our previous research. TFEB activation has been shown to promote GPX4 degradation, inducing ferroptosis; however, the specific E3 ligases, deubiquitinating enzymes (DUBs), and types of polyubiquitination chains involved remain unclear. The unique mechanisms associated with natural compounds like GK may help elucidate the underlying biological processes. Here, we describe a novel biological event involved in the lysosomal degradation of GPX4 induced by TFEB activation through the utilization of GK. <b>Methods</b>: TFEB activation was induced with GK, and TFEB knockout cells were generated using CRISPR-Cas9. The activity of TFEB and its relationship with ferroptosis were assessed by immunoprecipitation, labile iron pool and lysosomal activity assays. The types of polyubiquitination chains, E3 ligases, and DUBs involved in GPX4 degradation were analyzed using LC-MS, immunoprecipitation, and immunofluorescence. These findings were further validated in an orthotopic xenograft SCID mouse model. <b>Results</b>: GK binds to and activates TFEB, leading to TFEB-mediated lysosomal activation and GPX4 degradation, which induces ferroptosis in LUAD cells. These effects were impaired in TFEB knockout cells. Mechanistically, K48-linked polyubiquitination of GPX4 was required for GK induced GPX4 lysosomal translocation. TFEB knockout reduced both K48-linked ubiquitination and lysosomal translocation of GPX4. Additionally, GK promotes the binding of TFEB and TRIM25. TRIM25 and USP5 were found to competitively bind to GPX4, with TFEB activation favoring TRIM25 binding to GPX4 and reducing the interaction of USP5 and GPX4. These findings were confirmed in a xenograft SCID mouse model using TFEB knockout LUAD cells. <b>Conclusion</b>: This study identifies, for the first time, GK as a promising TFEB agonist for LUAD treatment. TFEB activation promotes TRIM25-mediated K48-linked polyubiquitination and lysosomal degradation of GPX4, driving ferroptosis. This ferroptosis-driven mechanism offers a novel strategy to enhance ferroptosis-based anti-LUAD therapies.