title: NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug.
abstract: BACKGROUND
  Most DNA-damaging chemotherapeutic agents activate the transcription factor nuclear factor κB (NF-κB). However, NF-κB activation can either protect from or contribute to the growth suppressive effects of the agent. We previously showed that the DNA-methylating drug temozolomide (TMZ) activates AKT, a positive modulator of NF-κB, in a mismatch repair (MMR) system-dependent manner. Here we investigated whether NF-κB is activated by TMZ and whether AKT is involved in this molecular event. We also evaluated the functional consequence of inhibiting NF-κB on tumor cell response to TMZ.
  
  
  METHODS
  AKT phosphorylation, NF-κB transcriptional activity, IκB-α degradation, NF-κB2/p52 generation, and RelA and NF-κB2/p52 nuclear translocation were investigated in TMZ-treated MMR-deficient (HCT116, 293TLα-) and/or MMR-proficient (HCT116/3-6, 293TLα+, M10) cells. AKT involvement in TMZ-induced activation of NF-κB was addressed in HCT116/3-6 and M10 cells transiently transfected with AKT1-targeting siRNA or using the isogenic MMR-proficient cell lines pUSE2 and KD12, expressing wild type or kinase-dead mutant AKT1. The effects of inhibiting NF-κB on sensitivity to TMZ were investigated in HCT116/3-6 and M10 cells using the NF-κB inhibitor NEMO-binding domain (NBD) peptide or an anti-RelA siRNA.
  
  
  RESULTS
  TMZ enhanced NF-κB transcriptional activity, activated AKT, induced IκB-α degradation and RelA nuclear translocation in HCT116/3-6 and M10 but not in HCT116 cells. In M10 cells, TMZ promoted NF-κB2/p52 generation and nuclear translocation and enhanced the secretion of IL-8 and MCP-1. TMZ induced RelA nuclear translocation also in 293TLα+ but not in 293TLα- cells. AKT1 silencing inhibited TMZ-induced IκB-α degradation and NF-κB2/p52 generation. Up-regulation of NF-κB transcriptional activity and nuclear translocation of RelA and NF-κB2/p52 in response to TMZ were impaired in KD12 cells. RelA silencing in HCT116/3-6 and M10 cells increased TMZ-induced growth suppression. In M10 cells NBD peptide reduced basal NF-κB activity, abrogated TMZ-induced up-regulation of NF-κB activity and increased sensitivity to TMZ. In HCT116/3-6 cells, the combined treatment with NBD peptide and TMZ produced additive growth inhibitory effects.
  
  
  CONCLUSIONS
  NF-κB is activated in response to TMZ in a MMR- and AKT-dependent manner and confers protection against drug-induced cell growth inhibition. Our findings suggest that a clinical benefit could be obtained by combining TMZ with NF-κB inhibitors.
authors.affiliation: Laboratory of Molecular Oncology, Istituto Dermopatico dell'Immacolata-IRCCS, Rome, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Caporali
authors.forename: Lauretta
authors.initials: L
authors.lastname: Levati
authors.forename: Grazia
authors.initials: G
authors.lastname: Graziani
authors.forename: Alessia
authors.initials: A
authors.lastname: Muzi
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Atzori
authors.forename: Enzo
authors.initials: E
authors.lastname: Bonmassar
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Palmieri
authors.forename: Paolo A
authors.initials: PA
authors.lastname: Ascierto
authors.forename: Stefania
authors.initials: S
authors.lastname: D'Atri
mesh_terms: D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects; D019610:Cytoprotection / Q000187:drug effects*; D053843:DNA Mismatch Repair / Q000187:drug effects; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D061986:MCF-7 Cells; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference / Q000187:drug effects; D000077204:Temozolomide; D051996:Transcription Factor RelA / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D010455:Peptides; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D003606:Dacarbazine; D051057:Proto-Oncogene Proteins c-akt; D000077204:Temozolomide