Investigation of mechanisms underpinning cytokine-induced cell death of colon cancer cells
University College Cork
Inflammatory cytokines induce regulated cell death as part of a robust immune response. Many unique forms of regulated cell death have been described that are critically dependent on specific signal transduction molecules or cellular events. Often, molecules or events canonically associated with cell death signalling pathways, while contributing factors, are not essential for cell death. Given the extensive emerging crosstalk and plasticity between modes of cell death, determining dependency is crucial for categorising cell death and for downstream clinical applications. IFN-γ and TNF-α are inflammatory, Th1-type cytokines elevated in immune-mediated inflammatory diseases and infectious diseases such as COVID-19, that synergistically induce cell death in diverse cell types. Recently IFN-γ-induced JAK/STAT signalling has emerged as an important pathway for triggering cell death, but the downstream mechanisms underpinning JAK/STAT-mediated cell death are incompletely understood. Here, IFN-γ+TNF-α-induced synergistic cell death was confirmed to be dependent on JAK1, JAK2, STAT1, and partially dependent on CASP8, using knockout colon cancer cell lines. IFN-γ+TNF-α-treated cells displayed biochemical hallmarks of multiple cell death pathways. Use of JAK1/2 inhibitors highlighted the unique kinetics of IFN-γ+TNF-α-induced cell death, suggesting that an accumulation of intracellular event/s, reaching a threshold or point of no return, may be responsible for cell death. The inconsistent effects of p300/CBP inhibitors on IFN-γ+TNF-α-mediated cell death suggest that STAT1-dependent transcription is not the main effector of cell death downstream of JAK1 and JAK2. The STAT1-dependency observed may just be due to its effects on JAK2 expression in cytokine-treated cells over time. Flow cytometry assays were used to investigate the kinetics of several biomarkers of mitochondrial and cellular stress in IFN-γ+TNF-α-treated cells, compared to cells undergoing canonical cell death pathways. Loss of mitochondrial integrity coincided with the crucial turning point observed in JAK1/2 inhibitor chase experiments, followed by severe increase in superoxide levels. Though these events cannot be confirmed to be the main drivers of IFN-γ+TNF-α-induced cell death, they are hallmarks of death and may be contributing factors. IFN-γ+TNF-α-induced cell death occurs by a non-canonical and redundant cell death mechanism that is dependent on JAK activity and potentially involves induction of components of multiple cell death pathways.
Regulated cell death , Interferon-gamma , Tumour necrosis factor-alpha , Inflammation , JAK1 , JAK2 , STAT1 , Caspase-8
Linehan, E. 2023. Investigation of mechanisms underpinning cytokine-induced cell death of colon cancer cells. MRes Thesis, University College Cork.