2615 - Differential Modulation of TGF-ß Family Members through Androgen Receptor Inhibition in Glioblastoma

Purpose/Objective(s): Glioblastoma (GBM) remains one of the most aggressive and treatment-resistant brain cancers, with limited therapeutic options. Our previous studies demonstrated that androgen receptor (AR) antagonist (ARA) treatment alone prolongs survival in the GBM mouse model. Building on these findings, we investigated ARAs as radiosensitizers and found that AR targeting enhances radiosensitivity and modulates the tumor microenvironment. RNA-seq analyses revealed TGF-ß signaling as one of the top pathways interacting with AR. Remarkably, we observed distinct subcellular localization of TGF-ß family members in human GBM cell lines, which led to our hypothesis of their essential but potentially distinct roles in GBM progression by interacting with AR signaling.

Materials/Methods: We analyzed the TCGA GBM dataset for expression, correlation, and survival trends. Findings were validated via western blotting, confocal microscopy, immunoprecipitation, ChIP analysis, clinical patient tissue, and RNA-seq data.

Results: Our in silico analysis of TCGA GBM patient data revealed that all TGF-ß pathway-associated molecules exhibited high expression in patients with elevated AR levels, except for the inhibitory Smads. Based on this rationale, we treated human GBM cells with the well-known ARA, enzalutamide, in a dose-dependent manner and examined the expression of TGF-ßs (TGF-ß1, TGF-ß2, and TGF-ß3), along with key downstream effectors, Smad3, and the phosphorylation status of AR and Smad3. Confocal analysis revealed that p-Smad3 was predominantly localized in the nucleus, while Smad3 was distributed across both the cytosol and nucleus. AR was primarily cytosolic, whereas its phosphorylated form (p-AR) was localized in the nucleus. Additionally, TGF-ß family proteins were detected in the cytosol. Notably, enzalutamide treatment led to an increase in p-Smad3 (S425) and TGF-ß2 expression. Furthermore, no changes were detected in p-AR expression, while AR levels decreased in a dose-dependent manner following enzalutamide treatment. These expression patterns were validated in human GBM clinical patients' tissue samples. Moreover, AR inhibition differentially affected TGF-ßs and contributed to the increased cytosolic expression of TGF-ß2, along with the prominent cytosolic punctate localization of TGF-ß1, despite no change in its overall expression. Morphological changes, including elongated cells and inhibitory growth, suggested a shift toward differentiation rather than proliferation. Mechanistic analysis with exogenous TGF-ß treatment indicated that AR-mediated TGF-ß signaling specifically targets downstream effector molecules.

Conclusion: Overall, this distinct subcellular expression pattern underscores the unique interplay between TGF-ß family members and AR regulation, paving a promising path for future GBM research and therapeutic interventions.