Cigarette Smoking Enhances the Expression of Thromboxane Synthase and Stimulates Lung Cancer Stem Cells, Leading to the Development of Lung Cance

Lung cancer is one of the most common and malignant cancers with a 5-year survival rate of only 17%. Studies have shown that 80%-90% of lung cancer cases are attributed to cigarette smoking. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent smoking carcinogen. Our previous work has revealed that NNK stimulated thromboxane A2 (TXA2) synthesis in lung cancer cells (LCCs). The increased TXA2 can activate cAMP response element binding protein (CREB) through PI3K/Akt and ERK signaling pathways and subsequently contribute to the NNK-mediated survival and proliferation of lung cancer cells. Nevertheless how TXA2 plays its roles in NNK-induced lung cancer in vivo is poorly understood.;In this study, we have conducted in vivo experiments to investigate the effect of sodium ozagrel (SO), a thromboxane synthase (TXS) inhibitor, on the development of lung cancer induced by NNK in A/J mice. The results have shown that SO suppressed TXS and reduced the level of thromboxane B2 (TXB2) in serum and lung tissues. The tumor number induced by NNK in SO treatment groups was also reduced compared to NNK group. Furthermore, there are not significant side effects in A/J mice for long term treated with SO. The results of immunohistochemical (IHC) and Western blot analysis of A/J mice lung tissues illustrated that blocking TXS inhibited cell proliferation and induced apoptosis.;Importantly, we found that high expression of TXS was positively correlated with the levels of lung cancer stem cell (LCSC) markers CD133 and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) in lung tissue of A/J mice, and the same result was observed in patients with smoking-related non-small cell lung cancer (NSCLC). Then, LCSC is involved in NNK-induced lung cancer.;We have found that NNK enhances CD44 and CD133 in two NSCLC cell lines, NCI-H23 and NCI-H1299, by flow cytometry. Based on the results of MTT and TXB2 EIA kit assay, we selected 100 muM ozagrel as the sub-optimal concentration in in vitro.;We established tumor sphere system to explore whether NNK promotes self-renewal in non-small cell lung cancer stem cells (NSCLCSCs) from NCI-H1299. Firstly, we found that NNK increased the levels of TXS, CD133 and ALDH1A1 after NSCLCSCs treated with NNK 10 days. Secondly, NNK promoted tumor sphere and colony formation in NSCLCSCs. Whereas, blocking TXS by ozagrel offset NNK-enhanced in NSCLCSCs. Interestingly, after NNK treatment the expressions of beta-catenin and Nanog were increased.;To explore the underlying mechanism of NNK enhances self-renewal in NSCLCSCs we treated NSCLCSCs with NNK, ozagrel, wortmanin and IWR-1. Results showed that NNK-induced TXS overexpression led to the activation of Akt/GSK3beta/beta-catenin/Nanog in NSCLCSCs and subsequently contribute to the enhanced self-renewal of NSCLCSCs.;In conclusion, the suppression of TXS can inhibit NNK-induced the growth of lung cancer in vivo and LCSC is involved in NNK-induced lung cancer. TXS is a key molecular in NNK-stimulated the self-renewal of NSCLCSCs through Akt/ GSK3beta/beta-catenin/Nanog cascade. The inhibition of TXS appears to be a promising option in the development of new treatments for lung cancer related with cigarette smoking.