Regulation of Sox2 and stemness by nicotine and electronic-cigarettes in non-small cell lung cancer

Lung cancer is the leading cause of cancer related deaths and its incidence is highly correlated with cigarette smoking. Nicotine, the addictive component of tobacco smoke, cannot initiate tumors, but can promote proliferation, migration, and invasion of cells in vitro and promote tumor growth and metastasis in vivo. This nicotine-mediated tumor promotion is facilitated through the activation of nicotinic acetylcholine receptors (nAChRs), specifically the α7 subunit. More recently, nicotine has been implicated in promoting self-renewal of stem-like side-population cells from lung cancers. This subpopulation of cancer stem-like cells has been implicated in tumor initiation, generation of the heterogeneous tumor population, metastasis, dormancy, and drug resistance. Here we describe the molecular events driving nicotine and e-cigarette extract mediated stimulation of self-renewal of stem-like cells from non-small cell lung cancer.

Experiments were conducted using A549 and H1650 non-small cell lung cancer cell lines and human mesenchymal stem cells according to protocols described in this paper. 2 μM nicotine or e-cigarette extracts was used in all relevant experiments. Biochemical analysis using western blotting, transient transfections, RT-PCR and cell biological analysis using double immunofluorescence and confocal microscopy, as well as proximity ligation assays were conducted.

Here we demonstrate that nicotine can induce the expression of embryonic stem cell factor Sox2, which is indispensable for self-renewal and maintenance of stem cell properties in non-small cell lung adenocarcinoma (NSCLC) cells. We further demonstrate that this occurs through a nAChR-Yap1-E2F1 signaling axis downstream of Src and Yes kinases. Our data suggests Oct4 may also play a role in this process. Over the past few years, electronic cigarettes (e-cigarettes) have been promoted as healthier alternatives to traditional cigarette smoking as they do not contain tobacco; however, they do still contain nicotine. Hence we have investigated whether e-cigarette extracts can enhance tumor promoting properties similar to nicotine; we find that they can induce expression of Sox2 as well as mesenchymal markers and enhance migration and stemness of NSCLC cells.

Our findings shed light on novel molecular mechanisms underlying the pathophysiology of smoking-related lung cancer in the context of cancer stem cell populations, and reveal new pathways involved that could potentially be exploited therapeutically.