title: The E3 ubiquitin ligase TRAF6 inhibits LPS-induced AKT activation in FLT3-ITD-positive MV4-11 AML cells.
abstract: OBJECTIVE
  The aim of the study was to investigate the activation of the PI3K/AKT (phosphatidylinositol-3-kinase) pathway after stimulation of TLR-4 (Toll-like receptor 4) with LPS (lipopolysaccharide) in FLT3-ITD (internal tandem duplication)-positive AML (acute myeloid leukemia) cells. TRAF6 (tumor necrosis factor receptor-associated factor 6), an E3 ubiquitin ligase, is critically involved in TLR-signaling. Based on the observation that TRAF6 might play a role in AKT phosphorylation, we hypothesized that TRAF6 can enhance the constitutive FLT3-ITD-driven activation of AKT after LPS stimulation.
  
  
  METHODS
  Human MV4-11 FLT3-ITD cells were silenced for TRAF6 by stable shRNA expression. Western blotting was used to analyze signal transduction by detection of phosphorylated proteins. LPS-induced activation of the NF-κB pathway was ensured by the induction of IκBα expression. To evaluate a potential functional role of TRAF6, we also performed chemosensitivity assays.
  
  
  RESULTS
  In MV4-11 cells, AKT was activated in response to LPS treatment. Surprisingly, shRNA-mediated knockdown of TRAF6 resulted in a significant increase in basal AKT phosphorylation. By LPS stimulation, the gain of AKT phosphorylation was more pronounced in the TRAF6 knockdown cell line than in the control. In addition, the concentration-dependent induction of apoptosis in response to treatment with the cytostatic drugs cytarabine or daunorubicin was significantly reduced in TRAF6-depleted MV4-11 cells.
  
  
  CONCLUSIONS
  Our data strongly suggest that the E3 ubiquitin ligase TRAF6 plays an important functional role in signal transduction and survival of AML cells. We hypothesize that LPS-mediated stimulation of TLR-4 leads to the induction of NF-κB-mediated signaling. However, TRAF6 might prevent a synergistic activation of the PI3K/AKT pathway after activation of TLR-4 signaling in FLT3-ITD-positive cells.
authors.affiliation: Abteilung Hämatologie/Onkologie, Klinik für Innere Medizin II, Universitätsklinikum Jena, Erlanger Allee 101, 07740 Jena, Germany.
authors.forename: Ulf
authors.initials: U
authors.lastname: Schnetzke
authors.forename: Mike
authors.initials: M
authors.lastname: Fischer
authors.forename: Anne-Kathrin
authors.initials: AK
authors.lastname: Kuhn
authors.forename: Bärbel
authors.initials: B
authors.lastname: Spies-Weisshart
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Zirm
authors.forename: Andreas
authors.initials: A
authors.lastname: Hochhaus
authors.forename: Jörg P
authors.initials: JP
authors.lastname: Müller
authors.forename: Sebastian
authors.initials: S
authors.lastname: Scholl
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D049109:Cell Proliferation; D005434:Flow Cytometry; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D048029:TNF Receptor-Associated Factor 6 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C495345:TLR4 protein, human; D048029:TNF Receptor-Associated Factor 6; D051197:Toll-Like Receptor 4; D044767:Ubiquitin-Protein Ligases; D019869:Phosphatidylinositol 3-Kinases; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3; D051057:Proto-Oncogene Proteins c-akt