Dissection of TBK1 signaling via phosphoproteomics in lung cancer cells

Abstract

TANK-binding kinase 1 (TBK1) has emerged as a novel therapeutic target for unspecified subset of lung cancers. TBK1 reportedly mediates prosurvival signaling by activating NF-κB and AKT. However, we observed that TBK1 knockdown also decreased viability of cells expressing constitutively active NF-κB and interferon regulatory factor 3. Basal phospho-AKT level was not reduced after TBK1 knockdown in TBK1-sensitive lung cancer cells, implicating that TBK1 mediates unknown survival mechanisms. To gain better insight into TBK1 survival signaling, we searched for altered phosphoproteins using mass spectrometry following RNAi-mediated TBK1 knockdown. In total, we identified 2,080 phosphoproteins (4,621 peptides), of which 385 proteins (477 peptides) were affected after TBK1 knockdown. A view of the altered network identified a central role of Polo-like kinase 1 (PLK1) and known PLK1 targets. We found that TBK1 directly phosphorylated PLK1 in vitro. TBK1 phosphorylation was induced at mitosis, and loss of TBK1 impaired mitotic phosphorylation of PLK1 in TBK1-sensitive lung cancer cells. Furthermore, lung cancer cell sensitivity to TBK1 was highly correlated with sensitivity to pharmacological PLK inhibition. We additionally found that TBK1 knockdown decreased metadherin phosphorylation at Ser-568. Metadherin was associated with poor outcome in lung cancer, and loss of metadherin caused growth inhibition and apoptosis in TBK1-sensitive lung cancer cells. These results collectively revealed TBK1 as a mitosis regulator through activation of PLK1 and also suggested metadherin as a putative TBK1 downstream effector involved in lung cancer cell survival.non-canonical IκB kinase | stable isotope labeling by amino acids (SILAC) | astrocyte elevated gene-1 (AEG-1) T ANK-binding kinase 1 (TBK1) was originally identified as an NF-κB-activating kinase (1). TBK1 activates the innate immune response through phosphorylation of two transcription factors, NF-κB and interferon regulatory factor 3 (IRF3), in response to proinflammatory cytokines and Toll-like receptor activation (2, 3). Besides its pivotal role in the innate immune response, increasing evidence indicates that aberrant activation of TBK1 and its closest homolog, IκB kinase e (IKKe), is associated with development of human cancers. IKKe renders cells tumorigenic and is required for breast cancer cell proliferation and survival (4). IKKe has also been found to be amplified or activated in cancers, including lung cancer, ovarian cancer, glioma, and breast cancer (4-7).Mechanisms that activate TBK1 and the downstream proteins and pathways regulated by TBK1 in cancer remain incompletely understood. Some studies have placed TBK1 downstream of RAS signaling, increasing the potential for TBK1-targeting therapeutics in cancer. The RalB GTPase engages TBK1 with oncogenic RAS to support cancer cell survival, suggesting that TBK1 is an unappreciated RAS downstream protein involved in cancer cell survival (8). A systemic RNAi screening study revealed that TBK1 is specifically required ...

Non-canonical IKKs, IKKϵ and TBK1, as novel therapeutic targets in the treatment of non-small cell lung cancer

“…TBK1 is also linked to the maintenance of basal autophagy which promotes non-canonical NF-kB signaling in KRAS-mutant NSCLC cells, raising the possibility of targeting autophagy for KRAS mutant lung cancer treatment [12]. Recently, our study demonstrated that TBK1 activity is regulated in a cell cycle-dependent manner, phosphorylates Polo-like kinase 1 (PLK1) at G2/ M in TBK1-dependent KRAS mutant NSCLC cell lines, suggesting the importance of TBK1 in cell cycle progression in cancer (Figure 1; Left panel) [13].…”

“…Targets (2013) 17(10) the loss of key survival kinases by inducing alternative survival signaling resulting in adaptive resistance. Our recent study revealed that loss of TBK1 leads to compensatory activation of receptor tyrosine kinases in lung cancer cells [13]. Adaptive response could attenuate the efficacy of the kinase inhibition or induce innate resistance.…”

Evaluating TBK1 as a Therapeutic Target in Cancers with Activated IRF3

“…Besides its key role in innate immune response, increasing evidence indicates that the activation of TBK1 and its close homolog IKKe is associated with the development of human cancers (2)(3)(4)(5)(6)(7)(8)(9)(10). TBK1 is an 84 kDa, 729 amino acid-long protein containing an N-terminal kinase domain, a ubiquitin-like domain and two C-terminal coiled-coil domains.…”

“…[15][16][17][18][19][20][21][22]. IRF3 phosphorylation at residues Ser-385 and Ser-386 has previously been shown to control its dimerization (23)(24)(25) upon which IRF3 translocates to the nucleus (26), and controls transcriptional regulation of cell survival and proliferation (2,11). In addition to IRF3, several other substrates, i. e., namely cRel, Akt, RalB, SIKE, etc., have been identified that attribute to TBK1 function (7)(8)(9)(27)(28)(29)(30).…”

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“…They establish that loss of MTDH leads to PARP cleavage and diminished cell viability. In addition, they performed shRNA-mediated knockdown of MTDH, TBK1, and KRAS in 14 lung cancer cell lines with detail EGFR and KRAS mutation status 45 .…”