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The E3 ubiquitin ligase TRAF6 inhibits LPS-induced AKT activation in FLT3-ITD-positive MV4-11 AML cells

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Abstract

Purpose

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.

Materials and 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.

Conclusion

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.

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Acknowledgments

This work was supported by a grant from the Deutsche José Carreras Leukämie-Stiftung (Project 11/03). The FLT3 inhibitor c102 was gratefully provided by Professor Siavosh Mahboobi (University of Regensburg, Germany).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Abteilung Hämatologie/Onkologie, Klinik für Innere Medizin II, Universitätsklinikum Jena, Erlanger Allee 101, 07740, Jena, Germany

    Ulf Schnetzke, Mike Fischer, Anne-Kathrin Kuhn, Bärbel Spies-Weisshart, Elisabeth Zirm, Andreas Hochhaus & Sebastian Scholl

  2. Institut für Molekulare Zellbiologie, Universitätsklinikum Jena, Hans Knöll Str. 2, 07745, Jena, Germany

    Jörg P. Müller

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  1. Ulf Schnetzke
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  2. Mike Fischer
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  3. Anne-Kathrin Kuhn
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  4. Bärbel Spies-Weisshart
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  5. Elisabeth Zirm
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  7. Jörg P. Müller
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  8. Sebastian Scholl
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Correspondence to Sebastian Scholl.

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Schnetzke, U., Fischer, M., Kuhn, AK. et al. The E3 ubiquitin ligase TRAF6 inhibits LPS-induced AKT activation in FLT3-ITD-positive MV4-11 AML cells. J Cancer Res Clin Oncol 139, 605–615 (2013). https://doi.org/10.1007/s00432-012-1362-4

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