Testosterone-TGF-β crosstalk modulates cell migration in human glioblastoma-derived cell lines

Testosterone activates TGF-β signaling in human GB cells, modulating cellular migration. A) T (100 nM) addition induces TGF-β secretion (500 pg/mL) in U251 cells after 24h of stimulation. B) This activates TGF-β signaling via the SMAD pathway, and SB431542, a TGF-β signaling inhibitor, blocked this effect. C) Finally, T -TGF-β signaling axis activation induces cell migration in U251 cells. Image created with BioRender.com . • ARES are present in TGFB1 and TGBR2 promoters. • TGFB1 and TGFBR2 genes are highly expressed in GB compared with healthy samples. • Testosterone regulates TGF-β signaling in GB cells depending on cellular context. • TGF-β modulates the migration induced by testosterone in GB-derived cell lines. Glioblastoma (GB) is the most aggressive brain tumor due to its extensive cellular migration. Although testosterone (T) and transforming growth factor-β (TGF-β) can regulate GB cell migration, the interaction between them in migration regulation in GB cells remains unclear. Here, we evaluated whether testosterone modulates TGF-β signaling to regulate cell migration in two human GB-derived cell lines. Androgen response elements (AREs) within the TGF-β gene were identified in-silico . The effects of T on TGF-β signaling were evaluated in U251 and T98G human GB-derived cell lines. TGF-β secretion was quantified by ELISA, and TGF-β/SMAD signaling activation after T stimulation was assessed by Western blot. Cell migration was analyzed by the scratch assay in the presence of SB431542, a TGF-β pathway inhibitor. Seven AREs were identified in the TGF-β gene. T increased TGF-β secretion in U251 but not in T98G cells. Consistently, T -induced SMAD activation was only observed in U251 cells. Inhibition of TGF-β signaling with SB431542 reduced T -stimulated migration exclusively in U251 cells. Our data demonstrate, for the first time, that T activates TGF-β signaling, promoting cell migration in U251 but not in T98G cells, highlighting cell line-specific heterogeneity in GB responses to the same hormonal stimulus.