Lung cancer is the leading cause of death in the United States. Non-small cell lung cancer (NSCLC) accounts for 85% of total lung cancer cases and demonstrates a strong association with tobacco use. Nicotine, an active component of tobacco smoke has been found to induce proliferation, invasion and epithelial-mesenchymal transition in NSCLC cell lines and promote the metastasis of NSCLC in mouse models. Nicotine induces cell proliferation and EMT utilizing a scaffolding protein, β-arrestin-1, which translocates to nucleus and associates with E2F1 transcription factor in response to nicotine stimulation. TBK1, a non-canonical IκB kinase has been shown to couple pathogen surveillance to induction of host defense mechanisms and contribute to inflammation as well as oncogenesis. These findings raise the possibility that TBK1 contributes to the onset as well as progression of NSCLCs through cell autonomous pathways in cancer cells, and indirectly through activating the inflammatory pathways in the tumor microenvironment. Since nicotinic acetylcholine receptor (nAChR) signaling is thought to augment Ras mediated cell proliferative pathways and confers resistance to apoptosis, attempts were made to assess whether TBK1 is induced by nAChR stimulation. Here we report that activation of nicotinic acetylcholine receptors leads to activation of TBK1 and β-arrestin-1 was required for this. Antagonists of alpha 7 subunit of nAChR such as bungarotoxin or inhibitor of α3/β2 and α4/β2 subunit DhβE abrogated nicotine induced TBK1 phosphorylation; further, depletion of β-arrestin-1 using siRNAs prevented the nicotine-mediated activation of TBK1. Inhibition of Src using dasatinib also could inhibit nicotine induced TBK1 phosphorylation. Interestingly, treatment of NSCLC cell lines with TBK1 inhibitor BX-795 resulted in significant inhibition of nicotine induced S phase entry as seen by BrdU incorporation assays. Cotransfection of TBK1 along with E2F1 significantly enhanced E2F1 mediated induction of E2F target promoters in transient transfection assays. Additionally, in an orthotopic lung cancer model in SCID-beige mice, implantation of A549-luciferase stable cells lacking β-arrestin-1 showed a decrease in primary tumor growth and also resulted in significantly lower levels of nicotine-induced metastasis when compared to controls. Mouse lung tissue sections with β-arrestin-1 depleted cells showed low levels of phosphorylated TBK-1 as compared to lung sections where control cells were implanted. Taken together, these data suggest that TBK1 contributes to nicotine induced growth and progression of NSCLC.

Citation Format: Smitha Pillai, Jonathan Nguyen, Eric Haura, Domenico Coppola, Srikumar Chellappan. Nicotinic acetylcholine receptors activate TBK1 in a β-arrestin-1 dependent manner to promote NSCLC growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5446. doi:10.1158/1538-7445.AM2013-5446