Here we have shown that long-term nicotine exposure generates cells with properties that are reminiscent of cancer stem cells. Our findings may be important for understanding tobacco-induced carcinogenesis in the context of the cancer stem cell paradigm. Knowing that nicotine or tobacco enables the acquisition of CSC-like properties, one can speculate on the implications of this finding in regard to tobacco-induced carcinogenesis and appropriate treatment modalities. First, most smoking-cessation programs currently rely on the chronic administration of nicotine. Our findings suggest that such treatments can potentially increase the risk for cancer by promoting self-renewal and EMT in premalignant cells even after tobacco use has stopped. Second, the potential ability of nicotine to bring about a malignant stem cell state implies that the CSC model is relevant in tobacco-induced carcinomas. Because nicotine induces a subpopulation of CSCs, treatments specifically aimed at targeting CSCs could be both justified and necessary in the treatment of tobacco-induced cancers. While nicotine typically inhibits apoptosis, it would be interesting to determine whether long-term nicotine exposure can increase cell sensitivity towards CSC-specific inhibitors, such as salinomycin or metformin 
. Third, the way in which nicotine contributes to the development of cancer can potentially be described by a model in which vital tumor suppressors are first rendered inoperative by various carcinogens found in tobacco. In some fashion, this event would greatly potentiate the ability of nicotine to activate EMT or other dedifferentiation pathways and drive the development of the cancer into an aggressive and metastatic phenotype. Another possibility is that if given enough time, nicotine can act upon somatic stem cells or progenitor cells to induce a cancer stem cell phenotype perhaps through the persistent activation of inflammatory cascades and through sustained oxidative stress.
We have shown that overexpressing E-cadherin in nicotine-treated cells partially reverses the induction of the stem cell genes Oct-4 and Nanog, suggesting that the stem cell properties conferred by nicotine are mediated at least in part by EMT. Our findings seem to be consistent with the observations of Dasgupta et al, who suggested that nicotine-induced EMT is primarily mediated through the repression of ECM proteins such as E-cadherin. Future experiments must characterize the mechanisms by which nicotine represses E-cadherin and induces EMT, as well as other targets of nicotine which may contribute to the acquisition of a CSC-like state. Our profiling experiments revealed a set of differentially expressed microRNAs between treated and non-treated cells, suggesting a role of miRNAs in mediating the effects of nicotine. Most notably, miR-9, which was upregulated in nicotine-treated cells, has been shown to promote metastasis in breast cancer by repressing E-cadherin 
. miR-9 has also been shown to control the migration and proliferation of progenitor cells derived from hESCs 
. The roles of miR-9 and other differentially expressed miRNAs remain to be fully established in the context of nicotine-induced EMT, and should be addressed by future experiments.
Studies indicate that epigenetic mechanisms may play an essential role in regulating EMT 
. However, little is currently known about the effect of nicotine on expression and activity of DNA methyltransferases and HDACs, although this information may be critical in describing the mechanisms of our findings. Possible targets include DNMT1, whose expression has been linked to the repression of E-cadherin 
. In addition, E-cadherin has been shown to be downregulated by a transcriptional repressor complex consisting of Snail, HDAC1 and HDAC2 
Aside from EMT, pathways that are induced by nicotine and may also contribute to the acquisition of stem cell characteristics include the well-studied PI3K/Akt and MAPK signaling pathways. Nicotine was found to reduce the senescence of endothelial progenitor cells by increasing telomerase activity via Akt signaling 
. In addition, the anti-apoptotic effects of nicotine have been shown to be partly mediated by PI3K/Akt and MAPK in nasal epithelial cancer 
. It is possible that nicotine, which has been shown to target NF-kappaB 
, may also work through pro-inflammatory pathways to promote stem cell properties. Takahashi et al recently described a model of lung tumorigenesis in which tobacco smoke acts as a tumor promoter, causing increased proliferation of chemically (NNK) and genetically (K-Ras activation) induced lung cancer cells in
through IKKβ- and JNK1-mediated inflammatory signaling. As tobacco smoke comprises a myriad of agents, it would be useful from a molecular perspective to examine the isolated role of nicotine in that context.
Removal of nicotine seems to allow the expression of stem cell and EMT markers to revert to their original levels within a week, suggesting that maintenance of EMT and stem cell characteristics may require sustained exposure to nicotine. There is also little evidence of sensitization towards nicotine that is brought on by long-term nicotine treatment, as acute reintroduction of nicotine to cells that have undergone withdrawal did not seem to elicit a response that was significantly different from control cells. Even though the effect of nicotine on stem cell and EMT gene expression seems to wear off easily, nicotine-treated cells were still more tumorigenic in mice that did not receive nicotine throughout the course of the experiment. Thus, even the brief timeframe in which nicotine enhances the stem cell phenotype of the cancer cell may make the difference in terms of whether it is able to form a tumor.
The exact conditions required for nicotine-induced stemness have yet to be identified and future studies must determine which genetic mutations, if any, are prerequisites for nicotine to induce EMT and dedifferentiation in tumor cells. It must also be examined whether nicotine or tobacco can activate similar pathways of EMT and dedifferentiation in normal adult progenitor cells, as this would have enormous implications in cancer stem cell biology, specifically in helping to understand the origin of cancer stem cells. As of now, our findings imply that nicotine, due to its ability to regulate EMT and stem cell properties, could hold a much more essential role in cancer development than previously thought. Preliminary studies in the cancer stem cell field have shown that selective targeting of CSCs can significantly reduce the rate of tumor formation as well as metastasis in mice 
. Further studies to characterize the pathways through which nicotine acts to promote stem cell properties could therefore greatly contribute to our understanding of the initiation and progression of tobacco-induced cancers, and may lead the way into more novel and effective treatment modalities for these diseases.