BAY 11-7082 Ubiquitinates EGFR and Induces Apoptosis in SHH-MB via PI3K-AKT and STAT3 Pathways

Medulloblastoma (MB), the most common malignant pediatric brain tumor, is classified into four molecular subgroups: Wnt, Sonic Hedgehog (SHH), Group 3 (G3), and Group 4 (G4). SHH-MB accounts for approximately one-third of cases. Current standard therapies—surgical resection, radiotherapy, and chemotherapy—remain limited by drug resistance and tumor recurrence, necessitating urgent exploration of resistance mechanisms and targeted interventions. Recent studies highlight the critical roles of ubiquitin-mediated protein modification and autophagy in tumor cell survival and chemoresistance. Chemo-/radiotherapy-resistant tumor cells often exploit hyperactivated autophagy to sustain proliferation. From an autophagy-ubiquitination compound library, BAY11-7082 emerged as a potent candidate against SHH-MB. CCK-8 assays determined IC50 values of BAY11-7082 in two SHH-MB cell lines. Subsequent experiments employed 4, 8, and 12 µM concentrations. Proliferation assays (CCK-8, colony formation, Ki67 immunofluorescence) demonstrated significant growth inhibition at ≥8 µM. Wound healing and Transwell assays revealed marked suppression of migration and invasion. In vivo, subcutaneous xenograft models confirmed tumor volume reduction and decreased Ki67+ cells in BAY11-7082-treated mice compared to controls. BAY11-7082 induced apoptosis in a dose- and time-dependent manner. Flow cytometry showed elevated apoptosis rates with increasing drug concentrations. Western blot (WB) analysis revealed downregulation of BCL-2, upregulation of BAX, and progressive apoptosis over time. While 12 µM BAY11-7082 triggered significant ROS elevation, 4 and 8 µM showed no ROS-dependent apoptosis. Cell cycle arrest at G2/M phase was observed via flow cytometry and corroborated by WB. Transcriptomic sequencing and reverse virtual drug screening identified EGFR as a potential target. WB demonstrated dose-dependent reductions in EGFR, phosphorylated PI3K (p-PI3K), and phosphorylated AKT1 (p-AKT1). Rescue experiments with EGFR/AKT1-overexpressing or knockdown stable cell lines confirmed the EGFR-PI3K-AKT axis as central to BAY11-7082’s anti-migratory and anti-invasive effects. qPCR revealed no significant EGFR transcriptional changes, suggesting post-translational regulation. Co-immunoprecipitation (Co-IP) showed enhanced EGFR ubiquitination upon BAY11-7082 treatment. Proteasomal (MG132) and lysosomal (chloroquine, CQ) inhibition experiments, combined with immunofluorescence, validated lysosome-mediated EGFR degradation. Unlike gefitinib (an EGFR inhibitor that reduces P62 and elevates LC3B-II), BAY11-7082 increased P62 and LC3B-II, indicative of autophagic flux blockade. ATG5/ATG7 knockdown and mRFP-EGFP-LC3B reporter assays confirmed impaired autophagosome-lysosome fusion, further supporting autophagic flux inhibition. BAY11-7082 suppresses SHH-MB progression by dual mechanisms: (1) inducing apoptosis via EGFR-PI3K-AKT pathway inhibition and (2) blocking autophagic flux. Its blood-brain barrier penetration and efficacy in preclinical models highlight therapeutic potential. This study provides novel insights into targeting ubiquitination-autophagy crosstalk for MB treatment and warrants clinical validation.