Research paperIdentification of TBK1 and IKKε, the non-canonical IκB kinases, as crucial pro-survival factors in HTLV-1-transformed T lymphocytes
Introduction
Adult T cell leukemia and lymphoma (ATL) is an aggressive form of T lymphocyte malignancy, which is caused by chronic infection with human T cell leukemia virus type 1 (HTLV-1) [1], [2]. The molecular mechanism of HTLV-1-mediated T cell transformation remains elusive. Extensive studies show that the viral genome of HTLV-1 encodes two oncogenes, tax and hbz, and that the oncogenic cooperation of these two viral genes is proposed to play a key role in T cell transformation [3], [4]. Of note, Tax functions as a multi-functional viral protein that regulates viral and cellular gene expressions in both direct and indirect manners. Tax is the viral transactivator that promotes 5′-long terminal repeat (5′-LTR) of HTLV-1-directed expression of the structural proteins (Gag-Pol and Env) and regulatory proteins (Rex, p30 and Tax itself) [5], [6]. Tax also induces the transcription of the antisense gene hbz through the viral 3′-LTR [5]. Hence, Tax is essential for mediating active viral replication in host cells. In addition, Tax is shown to act as a master regulator of host cell signaling including NF-κB and PI3 kinase/Akt pathways [7]. Notably, constitutive activation of NF-κB signaling by Tax is a prerequisite of HTLV-1-mediated transformation of T cells. Tax stimulates the canonical IκB kinase complex (IKK) by interacting with IKKγ, the regulatory subunit of IKK, resulting in persistent activation of NF-κB [8], [9]. Furthermore, the molecular clone of HTLV-1 with the Tax mutant defective in activating NF-κB is unable to immortalize human primary T cells [10]. This phenomenon further supports a key role of NF-κB signaling in HTLV-1-mediated oncogenesis. We recently demonstrate that Tax induces macroautophagy through lipid raft recruitment and activation of the canonical IκB kinases and the autophagy molecule Beclin1 to promote T cell survival and proliferation [11], [12].
The canonical IKK complex constitutes two highly homologous catalytic subunits, IKKα and IKKβ, and one regulatory subunit, IKKγ [13]. This kinase complex is the central regulator in NF-κB signaling. Two homologous non-canonical IκB kinases, TBK1 and IKKε, share 27% amino acid sequence homology with IKKα/β, but are not the components of the canonical IKK complex [14], [15]. Emerging evidence shows that IKKε is a defined cellular oncoprotein in breast cancer [16]. Both TBK1 and IKKε are constitutively expressed in human T cells [17]. Since TBK1/IKKε are implicated in functioning as the activators of NF-κB at certain biological conditions, we intend to determine if there is a role for TBK1/IKKε in activating NF-κB in the context of HTLV-1-mediated oncogenesis. In the present study, we demonstrate that TBK1/IKKε are crucial for maintaining constitutive activity of Stat3, and that Tax engages a molecular crosstalk between the canonical IKK complex and TBK1/IKKε. Our data, therefore, shed new insights into the oncogenic mechanism of HTLV-1-mediated cellular transformation and provide a rational for designing molecular therapeutics targeting TBK1/IKKε and Stat3 in treating HTLV-1-associated leukemia.
Section snippets
Cell lines, antibodies and reagents
FC36 cell line (kindly provided by Drs. Fiorenza Cocchi, Anthony DeVico, Alfredo Garzino-Demo, Suresh Arya, Robert Gallo, and Paolo Lusso) and MT-2 cell line (kindly provided by Dr. Douglas Richman) were obtained from the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH [18], [19]. SLB-1, PTX4-1 and HEK293 cell lines as well as Tax antibody were described previously [11]. PBMC cells were from healthy blood donors. Antibodies reacting to caveolin1, STAT3, p-ERK1, IKKα, IKKβ, IKKγ, GST,
TBK1 is required for survival and proliferation of HTLV-1-transformed T cells
To define the role of TBK1 in HTLV-1-associated oncogenesis, we first determined if TBK1 plays a role in promoting survival and proliferation of HTLV-1-transformed T cells. We utilized lentiviral delivery of shRNAs specific to TBK1 and found that silencing TBK1 drastically affected viability of two HTLV-1-transformed T cell lines, MT-2 and SLB-1 (Fig. 1A and B). Since constitutive activity of NF-κB is crucial for survival of HTLV-1-transformed T cells, we next determined if depletion of TBK1
Discussion
The present study has identified a new role of TBK1/IKKε in functioning as pro-survival molecules in HTLV-1-transformed T cells. Both TBK1 and IKKε are crucial for maintaining the constitutive activity of STAT3 since silencing one of them significantly impairs the Stat3 activation. Consistently, silencing STAT3 leads to drastic impediment of the leukemia cell growth, thereby validating the pro-survival function of TBK1/IKKε in HTLV-1-transformed T cells. The STAT3 activity in ATL cells has not
Conflict of interest
The authors declare no conflict of interest.
Authors’ contributions
H.Z. designed, performed experiments, analyzed the data and wrote the manuscript. L.C. performed parts of experiment and S.H.C. analyzed data. H.C. analyzed the data and modified the manuscript.
Acknowledgments
Research reported in this publication was supported by the National Institute of Allergy And Infectious Diseases of the National Institutes of Health under award number R01AI090113 to Hua Cheng. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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