CSIG-27. ANDROGEN RECEPTOR ANTAGONISTS DIFFERENTIALLY REGULATE GBM VIA NON-CANONICAL TGFΒ–TAK1–NFΚB SIGNALING

Abstract Glioblastoma (GBM), the most aggressive primary brain tumor, exhibits profound molecular heterogeneity and resistance to current therapies. Emerging evidence from our research and others highlighted the role of androgen receptor (AR) signaling in gliomagenesis, with significantly increased expression of AR receptors in GBM. Yet due to the diversity and complexity of AR signaling, the mechanisms remain poorly defined. We evaluated the differential therapeutic responses and underlying mechanisms of clinically relevant AR antagonists (ARAs), i.e., bicalutamide (Bica) and enzalutamide (Enza), first- and second-generation ARAs for prostate cancer, as to cinobufagin (Cino), a novel AR-targeting compound, in both human and mouse GBM cell models. Our findings reveal that these ARAs elicit distinct cellular outcomes, with Cino exhibiting markedly enhanced antiproliferative activity and uniquely triggering PANoptosis, a combined form of apoptosis, necroptosis, and pyroptosis. Transcriptomic analysis via RNA-seq revealed that all three AR inhibitors affected the TGFβ signaling, but only Cino strongly linked it to non-canonical NFΚB signaling. Further validations using Western blotting and confocal microscopy showed differential expression of TGFβ ligands in GBM cells. Notably, exogenous stimulation with TGFβ1 induced significant cytotoxic effects in a dose- and time-dependent manner after 24 hours of treatment which was not seen with TGFβ2, underscoring a ligand-specific role for TGFβ isoforms in GBM cell fate. TGFβ1 stimulation also significantly upregulated THBS1 expression in GBM cells, consistent with TCGA data showing a strong positive correlation between THBS1 and TGFβ1 in patient samples. Interestingly, TGFβ2 correlated specifically with MAP3K7 (TAK1), suggesting distinct downstream signaling pathways. Protein analysis confirmed that Cino enhanced TAK1 phosphorylation, and this effect was further amplified when combined with TGFβ1, indicating activation of a non-canonical TGFβ–TAK1–NFΚB pathway. Together, these findings reveal a novel AR-mediated regulatory mechanism in GBM involving isoform-specific TGFβ signaling and non-canonical TAK1/NFΚB activation.