TNFα sensitizes neuroblastoma cells to FasL-, cisplatin- and etoposide-induced cell death by NF-κB-mediated expression of Fas

Background: Patients with high-risk neuroblastoma (NBL) tumors have a high mortality rate. Consequently, there is an urgent need for the development of new treatments for this condition. Targeting death receptor signaling has been proposed as an alternative to standard chemo-and radio-therapies in various tumors. In NBL, this therapeutic strategy has been largely disregarded, possibly because similar to 50-70% of all human NBLs are characterized by caspase-8 silencing. However, the expression of caspase-8 is detected in a significant group of NBL patients, and they could therefore benefit from treatments that induce cell death through death receptor activation. Given that cytokines, such as TNF alpha, are able to upregulate Fas expression, we sought to address the therapeutic relevance of co-treatment with TNF alpha and FasL in NBL. Methods: For the purpose of the study we used a set of eight NBL cell lines. Here we explore the cell death induced by TNF alpha, FasL, cisplatin, and etoposide, or a combination thereof by Hoechst staining and calcein viability assay. Further assessment of the signaling pathways involved was performed by caspase activity assays and Western blot experiments. Characterization of Fas expression levels was achieved by qRT-PCR, cell surface biotinylation assays, and cytometry. Results: We have found that TNF alpha is able to increase FasL-induced cell death by a mechanism that involves the NF-kappa B-mediated induction of the Fas receptor. Moreover, TNF alpha sensitized NBL cells to DNA-damaging agents (i.e. cisplatin and etoposide) that induce the expression of FasL. Priming to FasL-, cisplatin-, and etoposide-induced cell death could only be achieved in NBLs that display TNF alpha-induced upregulation of Fas. Further analysis denotes that the high degree of heterogeneity between NBLs is also manifested in Fas expression and modulation thereof by TNF alpha. Conclusions: In summary, our findings reveal that TNF alpha sensitizes NBL cells to FasL-induced cell death by NF-kappa B-mediated upregulation of Fas and unveil a new mechanism through which TNF alpha enhances the efficacy of currently used NBL treatments, cisplatin and etoposide.