The present study shows that nicotine induces a dose-dependent increase in proliferation of lung cancer cells, breast cancer cells and pancreatic cancer cells via α7-nAChRs-mediated signal transduction pathways. These results are in agreement with our earlier studies as well as recent studies from other labs that show the importance of the α7-receptor subunit in nicotine-induced cell proliferation. Indeed, a recent study has shown that proliferation of transplanted A549 cells in nude mice can be inhibited by α-cobratoxin, an α7 receptor antagonist10, 38
. Nicotinic acetylcholine receptors particularly α7-nAChRs have been detected in primary endothelial cells as well as human NSCLC cell lines A549, NCI-H23 and H1299; similarly, other labs have shown that these receptor subunits are expressed on human lung cancers 11, 20, 33, 39–42
. Thus it appears that the presence of these receptor subunits and their ability to promote cell proliferation might contribute to the growth of tumors already initiated by tobacco-specific carcinogens. The importance of α7-nAChRs in mediating the pathophysiological effects of nicotine is further reinforced by the fact that it is overexpressed on human NSCLC tumors relative to adjacent normal tissue. At the same time, recent studies from the Minna lab had shown that the expression of α6 and β4 subunits is different on tumors from smokers and non-smokers 43
; this raises the possibility that other subunits might also be contributing to the growth of lung cancers in vivo. In addition, it has been reported that variations in chromosomal loci 15q24 and 15q25, which harbor genes for nicotinic acetylcholine receptors, correlate with nicotine dependence, lung cancer and peripheral arterial disease 44–46
. These and other studies lend support to the idea that exposure to nicotine and enhanced activation of nicotinic acetylcholine receptors contribute to lung cancer.
Apart from non-small cell lung cancer, cigarette smoking has been implicated in the pathogenesis of breast, gastric, colon and cervical cancers 1
. Our results show that nicotine can induce proliferation in a variety of cancer cell lines apart from NSCLC; furthermore, this induction appears to be through α7-nAChR subunits, which are expressed on these cell lines. Our findings seem to suggest that nAChRs may be an autocrine mitogenic signaling pathway facilitating the growth of several types of cancers in addition to lung cancer. Several lines of evidence show that β-adrenergic receptors also mediate the proliferative, pro-angiogenic and anti-apoptotic effects of nicotine, mainly in non-lung cancer cells. The mitogenic and proangiogenic effects of nicotine have been found to be mediated by β-adrenergic receptors in colon and gastric cancer cells 47, 48
. Studies by Shin et al. (2007) indicate that nicotine promotes growth of gastric cancers via PKC, ERK1/2 phosphorylation, and COX-2 activation in a β-adrenergic receptor-dependent fashion 49
. Further, data by Wong et al., (2007) demonstrate that nicotine-induced proliferation of HT-29 colon cancer cells are mediated by both α7-nAChRs and β-adrenergic receptors 47
. Their findings show that nicotine binds to α7-nAChRs nicotine on the membranes of HT-29 colon cancer cells and thereby facilitates downstream production of adrenaline and β-adrenergic activation; thus β-adrenergic receptors contribute to the proliferation indirectly. These data reveal that the both α7-nAChRs and β-adrenergic receptors contribute to the mitogenic effects of nicotine in colon cancer cells. The proliferative effects of the tobacco carcinogen NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] have been found to be mediated by β-adrenergic receptors on human adenocarcinomas of the lungs, pancreas, and breast 40, 50
. Thus it is possible that β-adrenergic receptors are also contributing to the observed changes, either directly or indirectly.
Multiple lines of evidence indicate that cigarette smoking not only facilitates proliferation of cancer cells but might also promote their metastatic spread as well 51–53
. Invasion and metastasis are complex, multi-step processes that involve alteration of cell adhesion to extracellular matrix proteins as well as disruption of cell-cell junctions. Nicotine has been shown to promote phosphorylation of calpains, upregulation of COX2, VEGF and VEGFR2; these molecules are known to affect the metastasic process 54–56
. Our findings shed light on additional pathways that contribute to the pro-invasive effects of nicotine. These include α7-nAChR-mediated activation of Src, calcium channels and upregulation of EGFR. These findings raise the possibility that inhibition of α7 nAChR activity by non-toxic agents or inhibition of its downstream mediators might open novel avenues for the therapy of cancers promoted by smoking 57
Our data further shows that sustained exposure to nicotine promotes anchorage-independent growth by downregulation of anoikis. Ability to survive independent of a substratum is a feature of cancer cells that is indispensable for metastasis 58
. Anoikis prevents normal cells from detaching from their substratum and migrating to different locations; the ability of nicotine to enhance the survival of cancer cells independent of a substratum might be contributing significantly to the ability of these cells to detach and find alternate sites of attachment. Indeed, nicotine could enhance adherence-independent proliferation of tumor cells lines, showing that the proliferative and survival advantages it provides allows the cells to grow robustly independent of a substratum.
Clinical and epidemiological studies have suggested that smokers tend to have more progressed and metastatic cancer than non-smokers 23
. Further, smokers had enhanced metastasis of breast cancers to the lung 51, 53
. Our findings suggest that nicotine induces changes consistent with EMT is highly relevant in this context. Indeed, earlier studies had suggested that long-term exposure to nicotine could alter the phenotype of epithelial and endothelial cells 12, 28
. We provide the molecular changes that facilitate these morphological changes. Results presented here show that chronic treatment with nicotine resulted in the downregulation of ECM proteins E-cadherin and β-catenin, with concomitant increase of fibronectin and vimentin. Several studies show that the decrease in E-cadherin and β-catenin with a concurrent increase in fibronectin and vimentin levels is one of the hallmarks of EMT in lung cancer cells. Further, clinical studies also report that smoking decreases levels of E-cadherin in lung tumors and might be contributing to resistance to chemotherapeutic agents 33
. It is probable that apart from its pro-invasive activity, nicotine plays a role in promoting EMT via downregulation of ECM proteins.
The results presented here suggest that while nicotine is not a carcinogen by itself , it has the potential to promote the growth and progression of tumors. The ability of nicotine to promote proliferation, angiogenesis, adherence-independent growth, and EMT while inhibiting anoikis might be contributing significantly to the growth and metastasis of tumors that are sensitive to nicotinic acetylcholine receptors. Further, these results also show that exposure to nicotine can affect tumors of tissues other than that of the lung. Development of agents that can disrupt the nAChR signaling pathway might prove beneficial in the treatment of such cancers.