title: Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent.
abstract: BACKGROUND
  Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM.
  
  
  METHODS
  The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays.
  
  
  RESULTS
  RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner.
  
  
  CONCLUSIONS
  These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease.
authors.affiliation: Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Northern Ireland, UK.
authors.forename: Jane L
authors.initials: JL
authors.lastname: Hurwitz
authors.forename: Izabela
authors.initials: I
authors.lastname: Stasik
authors.forename: Emma M
authors.initials: EM
authors.lastname: Kerr
authors.forename: Caitriona
authors.initials: C
authors.lastname: Holohan
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Redmond
authors.forename: Kirsty M
authors.initials: KM
authors.lastname: McLaughlin
authors.forename: Sara
authors.initials: S
authors.lastname: Busacca
authors.forename: Dario
authors.initials: D
authors.lastname: Barbone
authors.forename: V Courtney
authors.initials: VC
authors.lastname: Broaddus
authors.forename: Steven G
authors.initials: SG
authors.lastname: Gray
authors.forename: Ken J
authors.initials: KJ
authors.lastname: O'Byrne
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Johnston
authors.forename: Dean A
authors.initials: DA
authors.lastname: Fennell
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Longley
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000494:pharmacology*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors; D045744:Cell Line, Tumor; D045643:DNA Repair Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008297:Male; D008654:Mesothelioma / Q000188:drug therapy* / Q000378:metabolism*; D010997:Pleural Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D034622:RNA Interference; D018874:Spheroids, Cellular; D000077337:Vorinostat
chemical_list: D051017:Apoptosis Regulatory Proteins; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D061945:Caspase Inhibitors; D004268:DNA-Binding Proteins; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C513287:RAD23B protein, human; D000077337:Vorinostat; D045643:DNA Repair Enzymes