Identification of Fundamental Gene Regulatory Networks and Signaling Pathways in Cytokine-Induced Senescence

Abstract

Senescence is a state of permanent growth arrest that can be induced through various triggers and its therapeutic induction is nowadays also a promising strategy in the treatment of cancer. In case of cytokine-induced senescence (CIS), it was discovered that adoptive transfer of T-helper 1 (Th1) cells or the administration of their effector cytokines interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) mediate cancer cell senescence in vivo and in vitro. Although CIS was shown to depend on the expression of TNF receptor 1 (TNFR1) and the signal transducer and activator of transcription 1 (STAT1) protein in an established mouse model, detailed data regarding the senescence induction process and the involved signaling pathways were still missing. Therefore, the aim was now to investigate these questions on a simplified and cell-based level in vitro. At first, the senescence-inducing effects of the cytokines were validated in the human rhabdomyosarcoma cell line A204. As only the combined action of IFN-γ and TNF led to CIS, we next examined the transcriptome of the cells to identify the underlying gene regulatory networks and signal transduction pathways. The main drivers of CIS are the IFN-γ-regulated Janus kinase (JAK)/STAT axis that utilizes the transcription factor STAT1, and the simultaneous stimulation of the TNF signaling pathway, which triggers activation of the mitogen-activated protein kinase (MAPK) p38 and the nuclear factor “kappa-light-chain-enhancer” of activated B cells (NF-κB). In addition to the RNA-based data sets, Western blot analyses of samples taken at different time points during the 96 hours of senescence induction revealed the general regulation as well as the activation state of important targets on the protein level. Each cytokine alone mediated transient effects that were not persistent over time and – with the exception of STAT1 – lasted for hours only. In contrast to the short-lived effects of IFN-γ and TNF alone, the combination of both cytokines enforced a strong and sustained signaling response that was characterized by 1.) hyperactivation of STAT1 and 2.) hyperactivation of NF-κB. These long-lasting effects created a state of opposing cell fates where on one hand the treatment provoked stress- and death-inducing signals, while at the same time counteracting pro-survival signals were stimulated and reinforced. This was also shown by accumulation of the inactive pro-caspase-3 and elevated levels of anti-apoptotic factors like c-IAP2. All these events halted the cellular proliferation and established the senescent phenotype in form of a permanent growth arrest that was mediated and maintained by the cell cycle regulators p21 and p27. In summary, only the combined application of IFN-γ and TNF enables cancer control, as each cytokine alone failed to permanently activate the two essential signaling pathways that establish CIS.