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. 2016 Jul:46:37-44.
doi: 10.1016/j.leukres.2016.04.012. Epub 2016 Apr 16.

Identification of TBK1 and IKKε, the non-canonical IκB kinases, as crucial pro-survival factors in HTLV-1-transformed T lymphocytes

Huan Zhang  1 , Li Chen  2 , Shao-Hui Cai  3 , Hua Cheng  4
Affiliations

Identification of TBK1 and IKKε, the non-canonical IκB kinases, as crucial pro-survival factors in HTLV-1-transformed T lymphocytes

Huan Zhang et al. Leuk Res. 2016 Jul.

Abstract

Persistent activation of NF-κB is a prerequisite for development of adult T cell leukemia-lymphoma (ATL) caused by human T cell leukemia virus type 1 (HTLV-1). HTLV-1 genome encodes a viral transforming protein named Tax, which constitutively activates the canonical IκB kinases (IKK), the central regulator of NF-κB signaling. However, the role of the non-canonical IκB kinases, TBK1 and IKKε, in the pathogenesis of HTLV-1-associated leukemia has not been evaluated. We here show that TBK1/IKKε are crucial pro-survival molecules by maintaining persistent activity of Stat3. Consistent with this finding, silencing Stat3 by the specific shRNA or by the chemical inhibitor ruxolitinib results in drastic impediment of leukemia cell growth. We further find that in HTLV-1-transformed T cells expressing Tax, TBK1 co-localizes with the canonical IκB kinases and Tax in the lipid raft microdomains. The wild type Tax, but not the Tax mutant defective in activating the canonical IKK, promotes the lipid raft translocation of TBK1. This phenomenon correlates with Tax activation of both NF-κB and Stat3. Tax does not interact directly with TBK1/IKKε, and it rather engages a molecular crosstalk between the canonical IKKs and TBK1/IKKε. Our data, therefore, demonstrate a key role of TBK1/IKKε in the survival and proliferation of HTLV-1-transformed T cells and implicate a potential therapy targeting TBK1/IKKε and Stat3 in controlling HTLV-1-mediated oncogenesis.

Keywords: HTLV-1 Tax; IKK; NF-κB; Stat3; T lymphocyte transformation; TBK1/IKKε.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
TBK1 is required for survival and proliferation of HTLV-1-transformed T cells. (A) and (B) The viability of the MT2 and SLB1 cells transduced with non-specific (NS)- or TBK1-specific shRNAs. The protein levels of TBK1 were examined with anti-TBK1 immunoblot. (C) and (D) The protein levels of the IκB kinase subunits, various NF-κB subunits and the phosphorylated STAT3 (Tyr705) were examined with immunoblot in the SLB-1 and MT-2 cells transduced with NS- or TBK1-specific shRNAs. Total protein lysates were analyzed with antibodies specific for IKKα, IKKβ and IKKγ, IKKε, p65, p50, p-p65, p100 and p52 as well as the phosphorylated STAT3 (Tyr705) and total STAT3. β-actin was used as protein loading control. (E) The activities of STAT3 and NF-κB in TBK1-depleted MT-2 and SLB-1 cells were examined with EMSA. ** : p < 0.01 as determined by 2-tail student t-test.
Fig. 2
Fig. 2
Silencing IKKε leads to apoptosis of HTLV-1-transformed T cells. (A) and (B) The viability of the MT2 and SLB1 cells transduced with NS- or IKKε-specific shRNAs. (C) The apoptosis of the MT2 and SLB1 cells transduced with NS- or IKKε-specific shRNAs was examined using AnnexinV-APC/7-AAD Apoptosis Detection kit. The cleaved caspase 3, 9 and PARP, the pro-survival proteins (Mcl-1, Bcl-2, Bcl-Xl) and pro-apoptotic protein Bax were examined by immunoblot using relevant antibodies in the SLB1 (D) and MT2 (E) cells that are transduced with NS- or IKKε-specific shRNAs. ** : p < 0.01 as determined by 2-tail student t-test.
Fig. 3
Fig. 3
Silencing IKKε leads to an impaired activity of STAT3 in HTLV-1-transformed T cells. (A) Total protein lysates from the MT-2 and SLB-1 cells transduced with NS- or IKKε-specific shRNAs were analyzed with anti-IKKα, anti-IKKβ, anti-IKKγ and anti-TBK1. (B) The protein levels of NF-κB subunits, including p65/RelA, p50, p100 and p52, and the phosphorylation status of p65 were examined in the MT2 and SLB1 cells transduced with NS- or IKKε-specific shRNAs. (C) The phosphorylation status of STAT3 in IKKε-depleted SLB-1 and MT-2 cells. (D) The activities of Stat3 and NF-κB in MT-2 cells were detected by EMSA following IKKε depletion. (E) MT-2 cells were treated with DMSO or with ruxolitinib at does of 0.1 µM and 0.5 µM for 5 days. The levels of phosphorylated Stat3 and total STAT3 were examined using relevant antibodies. The viability of these cells was examined by Trypan blue exclusion assay. (F) and (G) The viability of the MT-2 and SLB-1 cells transduced with NSor STAT3-specific shRNAs. ** : p < 0.01 as determined by 2-tail student t-test.
Fig. 4
Fig. 4
TBK1 induces activation of NF-κB and nuclear translocation of IRF3. (A) NF-κB reporter assay in HEK293 cells transfected with vector, Flag-IKKε, Myr-IKKε, Flag-TBK1, Flag-IKKβKA and Tax-Flag, together with pNF-κB-luciferase reporter plasmid. (B) Expression levels of various proteins in transfected HEK293 cells seen in (A). (C) Fluorescence imaging analysis of the subcellular localization of IRF3 in HEK293 cells co-transfected with mKate2-IRF3 together with vector, Flag-IKKε, Myr-IKKε, Flag-TBK1, Flag-IKKβKA or Tax-Flag.
Fig. 5
Fig. 5
TBK1 is recruited to the lipid raft microdomains in Tax-expressing T cells. Lipid raft fractionation analysis of TBK1, IKKε, Tax, IKKα, IKKβ, IKKγ and STAT3 in SLB1 cells (A), MT-2 cells (B), FC36 cells (HTLV-1 infected CD8+ T cells) (C), PTX4-1 cells (Tax-immortalized T cells) (D), and PBLs (normal peripheral blood lymphocytes) (E). (F) Tax, M22, M47 and IKKβKA were transfected into HEK293 cells. The nuclear extracts of the transfected cells were examined for the activities of STAT3 and NF-κB using EMSA. (G) Tax, M22, M47 and the vector control were transfected into HEK293 cells. Lipid raft fractionation analysis of the transfected cells was analyzed for the lipid raft translocation of TBK1 with immunoblot. LAT and Caveolin 1 (CAV-1) are the lipid raft marker proteins.
Fig. 6
Fig. 6
TBK1/IKKε interact with the canonical IκB kinases. (A) Co-precipitation of IKKε with Tax in transfected HEK293 cells. The GST pulldown was analyzed with anti-FLAG antbody for detecting FLAG-tagged proteins and with anti-GST antibody for detecting GST-tagged proteins. (B) Fluorescence imaging of HeLa cells expressing Tax-GFP and Flag-IKK. (C) Interaction of IKKε with IKKα, IKKβ or IKKγ in co-transfected HEK293 cells was analyzed with GST pulldown assay. (D) Interaction of TBK1 with IKKα, IKKβ or IKKγ in co-transfected HEK293 cells was analyzed with GST pulldown assay.
Fig. 7
Fig. 7
A hypothetical model of Tax activation of TBK1/IKKε in the context of HTLV-1-transformed T lymphocytes.
See this image and copyright information in PMC

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