br Fig Overexpression of dup in NSCLC cell lines
Fig. 3. Overexpression of dupα7 in NSCLC cell lines suppresses the in vitro pro-migratory eﬀects of nicotine or NNK. Cell migration in A549 CFTRinh-172 (top) or SK-MES-1 cells (bottom) was determined by the transwell migration assay. (A) Representative confocal images (40X) of the migration rate in wild-type cells (A549 or SK-MES- 1) or in cells overexpressing dupα7 (A549dupα7 or SK-MES-1dupα7) after 24 h stimulation with nicotine (1 μM) or NNK (100 nM). Blank: unstimulated cells. (B) Bar charts representing the pooled results of cell migration, expressed as a percentage of the spontaneous migration by the same unstimulated cell variant (considered
100%), in response to nicotine or NNK. Values show mean ± SEM from five independent cell cultures; 2-3 microscopic fields were randomly selected for migration analysis in each cell culture. ***p < 0.001 compared to its corresponding unstimulated cell variant. ##p < 0.01 and ###p < 0.001 after comparing the indicated groups.
photos corresponding to four representative mice tested for each sub-group as well as their corresponding tumors excised immediately after their sacrifice. The IHC analysis reveals a clear increase in the expres-sion of the cellular markers for proliferation (Ki67) and angiogenesis (VEGF) in the A549 xenograft tumors of mice receiving nicotine com-pared to those that did not (Fig. 6D). The eﬀect of nicotine on the ex-pression of the two markers was suppressed in the A549dupα7 xeno-grafts. Collectively, our latest findings reveal that dupα7 overexpression in lung adenocarcinoma cells suppresses tumor growth and progression in vivo, just as it does in vitro.
The present study shows the first experimental evidence that dupα7, a human-specific duplicated form of the α7-nAChR subunit, interferes with the tobacco-activated tumorigenic activity mediated by α7-nAChRs in human NSCLC cell lines (A549 or SK-MES-1). Specifically, we show that dupα7: (1) significantly inhibits in vitro cell migration, proliferation and EMT induced by nicotine or NNK in both cells lines; and (2) strongly restrains nicotine-induced tumor growth and increased expression of tumor markers (Ki67 or VEGF) in A549 xenografts transplanted into nude mice.
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 cells 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