Cucurbitacin B suppresses GBM progression by inducing GADD45α-Dependent PCNA downregulation and DNA replication arrest

Abstract

Glioblastoma (GBM) is the most aggressive primary brain tumor, characterized by uncontrolled proliferation and resistance to current standard chemoradiotherapy. Natural products have emerged as promising anticancer agents, yet the therapeutic mechanisms of Cucurbitacin B (CuB), a highly bioactive triterpenoid, in GBM remain insufficiently understood. Our preliminary transcriptomic analysis revealed that CuB markedly upregulates GADD45α while suppressing PCNA, suggesting a previously unrecognized replication-stress regulatory axis. This study aimed to determine the anti-GBM efficacy of CuB across bulk and stem-like tumor models and to elucidate the molecular cascade connecting GADD45α induction to PCNA-mediated DNA replication arrest. The anti-tumor effects of CuB were evaluated in GBM cell lines (LN229, U87) and patient-derived glioma stem cells (GSCs) via functional assays, flow cytometry, and extreme limiting dilution assays. Transcriptome sequencing guided mechanistic exploration. Gain- and loss-of-function assays (GADD45α knockdown/overexpression, PCNA overexpression) elucidated the signaling cascade. Therapeutic efficacy was assessed in an orthotopic GBM xenograft mouse model. CuB significantly suppressed the malignant phenotypes of GBM cells and impaired the self-renewal capacity of GSCs, concomitantly inducing apoptosis and G2/M-phase arrest, as evidenced by Cyclin B1 suppression and CDK1 inhibitory phosphorylation. Mechanistically, CuB strongly upregulated GADD45α, which maintained the Retinoblastoma (Rb) protein in a hypophosphorylated, active state. Active Rb subsequently sequestered the E2F1 transcription factor, leading to the profound transcriptional downregulation of PCNA. Knockdown of GADD45α restored Rb/E2F1-driven PCNA expression and reversed CuB-mediated replication arrest, whereas GADD45α overexpression phenocopied CuB. Ectopic PCNA expression counteracted these suppressive effects. In vivo , CuB effectively reduced intracranial tumor burden and prolonged survival in a GADD45α-dependent manner, while exhibiting a highly favorable systemic safety profile in healthy mice. CuB suppresses GBM progression by activating GADD45α, which targets the Rb/E2F1 signaling axis to transcriptionally repress PCNA-mediated DNA replication. These findings highlight the GADD45α-Rb/E2F1-PCNA cascade as a critical mechanistic vulnerability and position CuB as a safe, promising replication-modulating agent for highly heterogeneous GBM.