title: TNFα sensitizes neuroblastoma cells to FasL-, cisplatin- and etoposide-induced cell death by NF-κB-mediated expression of Fas.
abstract: 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 ~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 TNFα, are able to upregulate Fas expression, we sought to address the therapeutic relevance of co-treatment with TNFα 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 TNFα, 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 TNFα is able to increase FasL-induced cell death by a mechanism that involves the NF-κB-mediated induction of the Fas receptor. Moreover, TNFα 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 TNFα-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 TNFα.
  
  
  CONCLUSIONS
  In summary, our findings reveal that TNFα sensitizes NBL cells to FasL-induced cell death by NF-κB-mediated upregulation of Fas and unveil a new mechanism through which TNFα enhances the efficacy of currently used NBL treatments, cisplatin and etoposide.
authors.affiliation: Cell Signaling and Apoptosis Group, Fundacio Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron, Edifici Collserola, Passeig Vall d'Hebron 119-129, 08035, Barcelona, Spain. koen.galenkamp@vhir.org.
authors.forename: Koen Mo
authors.initials: KM
authors.lastname: Galenkamp
authors.affiliation: Cell Signaling and Apoptosis Group, Fundacio Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron, Edifici Collserola, Passeig Vall d'Hebron 119-129, 08035, Barcelona, Spain. paulina.carriba@gmail.com.
authors.forename: Paulina
authors.initials: P
authors.lastname: Carriba
authors.affiliation: Cell Signaling and Apoptosis Group, Fundacio Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron, Edifici Collserola, Passeig Vall d'Hebron 119-129, 08035, Barcelona, Spain. jorge.urresti@vhir.org.
authors.forename: Jorge
authors.initials: J
authors.lastname: Urresti
authors.affiliation: Cell Signaling and Apoptosis Group, Fundacio Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron, Edifici Collserola, Passeig Vall d'Hebron 119-129, 08035, Barcelona, Spain. laura.planells@vhir.org.
authors.forename: Laura
authors.initials: L
authors.lastname: Planells-Ferrer
authors.affiliation: Cell Signaling and Apoptosis Group, Fundacio Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron, Edifici Collserola, Passeig Vall d'Hebron 119-129, 08035, Barcelona, Spain. elena.coccia@vhir.org.
authors.forename: Elena
authors.initials: E
authors.lastname: Coccia
authors.affiliation: Cell Signaling and Apoptosis Group, Fundacio Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron, Edifici Collserola, Passeig Vall d'Hebron 119-129, 08035, Barcelona, Spain. joaquin.lopez.soriano@vhir.org.
authors.forename: Joaquín
authors.initials: J
authors.lastname: Lopez-Soriano
authors.affiliation: Cell Signaling and Apoptosis Group, Fundacio Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron, Edifici Collserola, Passeig Vall d'Hebron 119-129, 08035, Barcelona, Spain. bruna.barneda@vhir.org.
authors.forename: Bruna
authors.initials: B
authors.lastname: Barneda-Zahonero
authors.affiliation: Cell Signaling and Apoptosis Group, Fundacio Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron, Edifici Collserola, Passeig Vall d'Hebron 119-129, 08035, Barcelona, Spain. rana.moubarak@nyumc.org.
authors.forename: Rana S
authors.initials: RS
authors.lastname: Moubarak
authors.affiliation: Laboratory of Translational Research in Pediatric Cancer, Fundacio Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron, Edifici Collserola, Passeig Vall d'Hebron 119-129, 08035, Barcelona, Spain. miguel.segura@vhir.org.
authors.forename: Miguel F
authors.initials: MF
authors.lastname: Segura
authors.affiliation: Cell Signaling and Apoptosis Group, Fundacio Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron, Edifici Collserola, Passeig Vall d'Hebron 119-129, 08035, Barcelona, Spain. joan.comella@vhir.org.
authors.forename: Joan X
authors.initials: JX
authors.lastname: Comella
mesh_terms: D053181:Caspase 8 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation / Q000187:drug effects; D005047:Etoposide / Q000494:pharmacology; D053222:Fas Ligand Protein / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D019014:fas Receptor / Q000235:genetics*
chemical_list: D053418:Death Domain Receptor Signaling Adaptor Proteins; D053222:Fas Ligand Protein; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D005047:Etoposide; D007371:Interferon-gamma; D053181:Caspase 8; D002945:Cisplatin