br Tobacco smoking is the main risk factor
Tobacco smoking is the main risk factor for the development of many types of tumors, including lung cancer and its two major cate-gories: NSCLC and small cell lung cancer (SCLC). Smoking-related tumor 4-Aminophenyl Phosphate express several nAChR subtypes with diﬀerent pharmaco-logical sensitivities and, depending on tumor type, specific
pathophysiological functions. Thus, homomeric α9-nAChRs play an important role in the progression of human breast cancer , whereas α7-nAChR is the main subtype responsible for the nicotine-mediated oncogenic eﬀects in human NSCLC [10–13]. Stimulating α7-nAChRs in smoking-related tumor cells leads to downstream activation of multiple signaling cascades promoting cancer cell survival, proliferation, mi-gration, angiogenesis and metastasis in a tumor-specific manner [4–7].
We reported previously that the dupα7 subunit interferes with various α7-nAChR-controlled functions in cell types as diﬀerent as Xenopus oocytes or murine macrophages [18,20]. Here, we identify a new function for dupα7 in human NSCLC cells: it negatively regulates α7-nAChR-mediated oncogenic activity induced by nicotine or NNK, two tobacco smoke constituents. Furthermore, the inhibitory eﬀect of dupα7 on α7-nAChR function is not due to a reduction of α7 expression in the cells since both α7 mRNA or protein levels in wild-type cells do not change in cells with stable dupα7 overexpression (Fig. 1).
As has been previously reported in lung, breast or gastric tumor cells [5,13,28,29], our data show that the above constituents of tobacco smoke increase in vitro migration of non-transfected A549 or SK-MES-1 cells, an eﬀect that was abolished in these cells upon dupα7 over-expression (Figs. 2 and 3). Furthermore, dupα7 expression is only ef-fective against stimuli (nicotine or NNK) that activate cell migration through α7-nAChRs but not against other types of stimuli unrelated to these receptors, such as FBS. It is noteworthy that the dupα7 eﬀect on
α7-nAChR-mediated migration is not reproduced in cells over-expressing the empty-vector (A549pcDNA3.1 and SK-MES-1pcDNA3.1) (Fig. 2), indicating that dupα7, and not the vector, interferes with α7-
Fig. 4. The nicotine- or NNK-induced in vitro proliferative eﬀects in NSCLC cells were abolished in cells overexpressing dupα7. The EdU assay combined with cell nucleus staining with DAPI makes it possible to count the number of proliferating (magenta stained) and non-proliferating (blue stained) cells in the microscopic field. (A) Representative confocal images (20X) of the proliferative eﬀects of nicotine (1 μM), NNK (100 nM) or FBS (10%) in wild-type cells (A549 or SK-MES-1) or in cells overexpressing dupα7 (A549dupα7 or SK-MES-1dupα7). Blank: non-stimulated cells of each cell variant. (B) Bar charts showing pooled results op < 0.01 after compared the indicated bars. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Our study also shows that nicotine and NNK induce in vitro pro-liferation of both wild-type NSCLC cell lines (Fig. 4), an eﬀect that has been previously reported in the same cells and in other cancer cell lines from stomach, pancreas and breast [28,30,31]. The nicotine or NNK
eﬀects promoting cell proliferation were abolished in A549dupα7 and SK-MES-1dupα7 cells in our study (Fig. 4). Once again, dupα7 expression
did not aﬀect the proliferative eﬀect of FBS. The last part of our in vitro study reveals that nicotine and NNK reduce the expression of β-catenin
(an epithelial marker) and increase expression of vimentin and fi-bronectin (two mesenchymal markers) in non-transfected A549 and SK-MES-1 cells; the eﬀects induced by the two stimuli were abolished in cells overexpressing dupα7 (Fig. 5). Changes in the expression of epi-thelial/mesenchymal markers induced by nicotine or NNK in wild-type cells like those found in our study are a hallmark of EMT, a process that is associated with invasiveness, metastasis and poor prognosis. Thus, our results agree with previous observations in patients with lung cancer showing that tobacco use favors EMT . Interestingly, our
Fig. 5. Overexpression of dupα7 in NSCLC cells abolishes the in vitro EMT induced by nicotine or NNK. (A) Representative immunoblots of the expression changes in epithelial (β-catenin) and mesenchymal (vimentin and fibronectin) markers in response to nicotine or NNK caused by overexpression of dupα7 in the A549 or SK-MES-1 cells. Cell cultures were incubated with nicotine (1μM) or NNK (100 nM) for 48 h. Control: unstimulated cells from each variant. β-actin was included as a loading control. (B) Pooled immunoblot results showing the marker/β-actin expression ratio for each cell variant and experimental condition, taking the ratio in control cells as 1. The bar graphs show the mean values ± SEM obtained in three diﬀerent cell cultures for each cell variant. *p < 0.05 vs. untreated (control) cells of the same cell variant.