title: A redox state-dictated signalling pathway deciphers the malignant cell specificity of CD40-mediated apoptosis.
abstract: CD40, a member of the tumour necrosis factor receptor (TNFR) superfamily, has the capacity to cause extensive apoptosis in carcinoma cells, while sparing normal epithelial cells. Yet, apoptosis is only achieved by membrane-presented CD40 ligand (mCD40L), as soluble receptor agonists are but weakly pro-apoptotic. Here, for the first time we have identified the precise signalling cascade underpinning mCD40L-mediated death as involving sequential TRAF3 stabilisation, ASK1 phosphorylation, MKK4 (but not MKK7) activation and JNK/AP-1 induction, leading to a Bak- and Bax-dependent mitochondrial apoptosis pathway. TRAF3 is central in the activation of the NADPH oxidase (Nox)-2 component p40phox and the elevation of reactive oxygen species (ROS) is essential in apoptosis. Strikingly, CD40 activation resulted in down-regulation of Thioredoxin (Trx)-1 to permit ASK1 activation and apoptosis. Although soluble receptor agonist alone could not induce death, combinatorial treatment incorporating soluble CD40 agonist and pharmacological inhibition of Trx-1 was functionally equivalent to the signal triggered by mCD40L. Finally, we demonstrate using normal, 'para-malignant' and tumour-derived cells that progression to malignant transformation is associated with increase in oxidative stress in epithelial cells, which coincides with increased susceptibility to CD40 killing, while in normal cells CD40 signalling is cytoprotective. Our studies have revealed the molecular nature of the tumour specificity of CD40 signalling and explained the differences in pro-apoptotic potential between soluble and membrane-bound CD40 agonists. Equally importantly, by exploiting a unique epithelial culture system that allowed us to monitor alterations in the redox-state of epithelial cells at different stages of malignant transformation, our study reveals how pro-apoptotic signals can elevate ROS past a previously hypothesised 'lethal pro-apoptotic threshold' to induce death; an observation that is both of fundamental importance and carries implications for cancer therapy.
authors.affiliation: Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, UK.
authors.forename: C J
authors.initials: CJ
authors.lastname: Dunnill
authors.affiliation: Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, UK.
authors.forename: K
authors.initials: K
authors.lastname: Ibraheem
authors.affiliation: Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, UK.
authors.forename: A
authors.initials: A
authors.lastname: Mohamed
authors.affiliation: Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, York, UK.
authors.forename: J
authors.initials: J
authors.lastname: Southgate
authors.affiliation: Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, UK.
authors.forename: N T
authors.initials: NT
authors.lastname: Georgopoulos
mesh_terms: D017209:Apoptosis / Q000235:genetics; D019013:CD40 Antigens / Q000235:genetics* / Q000378:metabolism; D023201:CD40 Ligand / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000235:genetics / Q000378:metabolism; D048848:MAP Kinase Kinase Kinase 5 / Q000235:genetics* / Q000378:metabolism; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D048008:TNF Receptor-Associated Factor 3 / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics* / Q000378:metabolism
chemical_list: D019013:CD40 Antigens; D017382:Reactive Oxygen Species; D048008:TNF Receptor-Associated Factor 3; C491203:TXN protein, human; D023201:CD40 Ligand; D013879:Thioredoxins; D019255:NADPH Oxidases; C113333:NCF4 protein, human; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human; D048670:MAP Kinase Kinase 4; C482559:MAP2K4 protein, human