Evaluating necroptosis competency in malignant melanoma

Abstract

Melanoma cells are highly resistant to conventional genotoxic agents, and BRAFV600/MEK-targeted therapies, as well as immunotherapies, frequently remain inefficient. Therefore, alternative means to treat melanoma, particularly through the induction of programmed cell death modalities such as apoptosis or necroptosis, still need to be explored. Apoptotic and necroptotic cell death depends on cysteine aspartate-specific proteases (caspases) and receptor-interacting serine/threonine kinase (RIPK) 1/3, respectively. RIPK1/3 and caspases can directly interact with the TNFR signalling complex, thereby inducing programmed cell death. Thus, these cell death regulations need to be explored in melanoma cancer. In the course of this thesis, a panel of melanoma cell lines was studied for cell death susceptibility in response to the activation of TNF family receptors. Apoptosis and necroptosis responsiveness, as well as cell death sensitization approaches, were tested, followed by mechanistic signal transduction studies making use of pharmacological and genetic interventions. In addition, phosphoprotein and secretome analyses were performed by xMAP (Luminex) assays. This study reports that melanoma cell lines responded heterogeneously to either single treatment of death ligands (DLs) or in combination with an IAP antagonist. Furthermore, it was observed that melanoma cell lines expressing notable amounts of RIPK1, RIPK3, and MLKL, the key players in necroptosis signal transduction, failed to execute necroptotic cell death. Interestingly, the activity of transforming growth factor β-activated kinase 1 (TAK1) appears to prevent RIPK1 from contributing to cell death induction, since TAK1 inhibition by (5z)-7-Oxozeaenol, deletion of MAP3K7, or the expression of inactive TAK1 were sufficient to sensitize melanoma cells to RIPK1-dependent cell death in response to TNF-α or TRAIL-based combination treatments. However, cell death was executed exclusively by apoptosis, even when RIPK3 expression was high. In addition, TAK1 inhibitor (5z)-7-Oxozeaenol suppressed intrinsic or treatment-induced pro-survival signaling, as well as the secretion of cytokines and soluble factors associated with melanoma disease progression. Collectively, these results demonstrate that TAK1 suppresses susceptibility to RIPK1-dependent cell death. These findings were in line with high expression of TAK1 indicating an increased risk for disease progression in melanoma, as shown by subsequent collaborative work.