title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis.
abstract: We previously reported that renal clear cell carcinoma cells (RCC) express both tumor necrosis factor receptor (TNFR)-1 and -2, but that, in organ culture, a TNF mutein that only engages TNFR1, but not TNFR2, causes extensive cell death. Some RCC died by apoptosis based on detection of cleaved caspase 3 in a minority TUNEL-positive cells but the mechanism of death in the remaining cells was unexplained. Here, we underpin the mechanism of TNFR1-induced cell death in the majority of TUNEL-positive RCC cells, and show that they die by necroptosis. Malignant cells in high-grade tumors displayed threefold to four fold higher expression of both receptor-interacting protein kinase (RIPK)1 and RIPK3 compared with non-tumor kidney tubular epithelium and low-grade tumors, but expression of both enzymes was induced in lower grade tumors in organ culture in response to TNFR1 stimulation. Furthermore, TNFR1 activation induced significant MLKL(Ser358) and Drp1(Ser616) phosphorylation, physical interactions in RCC between RIPK1-RIPK3 and RIPK3-phospho-MLKL(Ser358), and coincidence of phospho-MLKL(ser358) and phospho-Drp1(Ser616) at mitochondria in TUNEL-positive RCC. A caspase inhibitor only partially reduced the extent of cell death following TNFR1 engagement in RCC cells, whereas three inhibitors, each targeting a different step in the necroptotic pathway, were much more protective. Combined inhibition of caspases and necroptosis provided additive protection, implying that different subsets of cells respond differently to TNF-α, the majority dying by necroptosis. We conclude that most high-grade RCC cells express increased amounts of RIPK1 and RIPK3 and are poised to undergo necroptosis in response to TNFR1 signaling.
authors.affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
authors.forename: R S
authors.initials: RS
authors.lastname: Al-Lamki
authors.affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
authors.forename: W
authors.initials: W
authors.lastname: Lu
authors.affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
authors.forename: P
authors.initials: P
authors.lastname: Manalo
authors.affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Pathology, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
authors.forename: A Y
authors.initials: AY
authors.lastname: Warren
authors.affiliation: Department of Clinical Neurosciences, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
authors.forename: A M
authors.initials: AM
authors.lastname: Tolkovsky
authors.affiliation: Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Pober
authors.affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
authors.forename: J R
authors.initials: JR
authors.lastname: Bradley
mesh_terms: D000178:Acrylamides / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000473:pathology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020287:In Situ Nick-End Labeling; D007680:Kidney Neoplasms / Q000235:genetics / Q000473:pathology*; D007684:Kidney Tubules / Q000473:pathology*; D009336:Necrosis; D009924:Organ Culture Techniques; D052999:Quinazolinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
chemical_list: C549041:3-(2,4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone; D000178:Acrylamides; C570695:N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide; D052999:Quinazolinones; D012333:RNA, Messenger; D047888:Receptors, Tumor Necrosis Factor, Type I; D013449:Sulfonamides; D014409:Tumor Necrosis Factor-alpha; C506249:RIPK1 protein, human; C119705:RIPK3 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases