Nuclear localization of p65 reverses therapy induced senescence.

Senescence is a tumor suppressor phenomenon. We have earlier shown that therapy induced senescence in residual disease glioblastoma (GBM) cells can reverse leading to relapse. Here we demonstrate that ciprofloxacin induced senescence in glioma-derived cell lines and primary cultures defined by β-gal positivity, SASP release, giant-cell formation, higher ROS, p-ATM, γ-H2AX, and senescence gene signature have three stages- initiation, pseudo-senescence and permanent-senescence. Drug withdrawal during initiation and pseudo-senescence reinitiated proliferation in vitro and tumor formation in vivo Importantly, prolonged ciprofloxacin treatment induced permanent-senescence that failed to reverse following drug withdrawal. RNA-Seq revealed downregulated p65 transcription network and incremental SMAD pathway genes expression from initiation to permanent-senescence. Drug withdrawal at initiation and pseudo-senescence but not permanent-senescence increased p65 nuclear localization, and escape from senescence. In contrast, permanent-senescent cells showed loss of nuclear p65 and increased apoptosis. Pharmacological or genetic p65 knockdown upholds senescence in vitro and inhibit tumor formation in vivo Together, this study demonstrates that levels of nuclear p65 defines the window of therapy induced senescence reversibility and coupling senotherapeutic drugs with p65 inhibitors induce permanent-senescence in GBM cells.