br finding concerning nitrosamine as an EMT inductor
finding concerning nitrosamine as an EMT inductor represents the first demonstration of such an eﬀect in NSCLC cells, although it has been previously reported in gastric cancer cells .
Previous studies have shown that blockade of α7-nAChR with spe-cific antagonists or siRNA prevents the stimulatory eﬀects of nicotine and NNK on proliferation, migration, EMT or angiogenesis in several cancer cell lines [13,29,31,33]. Nicotine and NNK are α7-nAChR ago-nists, and the aﬃnity of nitrosamine for the receptor is 1,300 times greater than that of nicotine . Since we did not find significant diﬀerences in the in vitro tumorigenic eﬀects between nicotine and NNK, we can conclude that both agents produce all the above oncogenic eﬀects mainly through α7-nAChRs expressed in A549 or SK-MES-1 cells.
The present in vitro findings are corroborated in our in vivo study, where we found that nicotine promotes tumor growth of A549
xenografts transplanted into nude mice (Figs. 6A-6C). This nicotine eﬀect on tumor growth agrees with previous data obtained in xeno-grafts corresponding to diﬀerent tumor cell lines, including lung, breast, 108084-47-5 or hepatic carcinoma cell lines [35–38]. Moreover, the histological analysis of A549 xenografts in our in vivo study reveals the increased expression of the angiogenesis (VEGF) and proliferative (Ki67) markers in response to nicotine (Fig. 6D), a finding already re-ported in xenografts derived from adenocarcinoma or SCLC cell lines [39–41]. Here, we report, for the first time, that dupα7 overexpression in A549 xenografts completely suppresses tumor growth and the in-creased expression of tumor progression markers in vivo. Although we still do not know the mechanism by which dupα7 interferes with the α7-nAChR function in NSCLC cells, we have already reported that du-plicated subunits assembled with ancestral subunits to form hetero-meric dupα7/α7-nAChRs in the neuroendocrine GH4C1 cell line .
Fig. 6. Overexpression of dupα7 in a nude mouse A549 xenograft model suppressed the nicotine-mediated tumorigenic eﬀect in vivo. Wild-type cells (A549) or dupα7 overexpressing cells (A549dupα7) were innoculated into the left flanks of the mice, which would then receive nicotine or not in the drinking water. There were four mouse subgroups (5–6 mice/subgroup) in the study. (A) Tumor volume growth (mean ± SEM) in the four subgroups over the 37 days of the study; tumor volume was expressed in mm3. #p < 0.05 and ##p < 0.01 after multiple comparative analysis of the indicated mice subgroups. (B) Photos of four representative mice from each subgroup as well as their corresponding tumors excised immediately after the sacrifice of the animal. (C) Final tumor volumes (mean ± SEM) determined in the four subgroups after the completion of the study. *p < 0.05, significantly diﬀerent from the wild-type A549 xenograft subgroup that did not receive nicotine; ns, no significant diﬀerences between the two A549dupα7 xenograft subgroups (receiving nicotine or not). #p < 0.05 and ##p < 0.01, significant diﬀerences between the indicated subgroups. (D) Microscopic images of expression levels for proliferation (Ki67) and angiogenesis (VEGF) markers, staining (brown) by IHC, in a re-presentative xenograft tumor from each subgroup.
As expected from the structural characteristics of dupα7, these het-eromeric receptors exhibit worse migration from endoplasmic re-ticulum to the cell membrane, as well as less sensitivity to agonists than homomeric α7-nAChRs and thus, reduced functionality [18,20].
It should be noted that SCLC cells express high levels of α7-nAChRs, which have been identified as central regulators of nicotine- or NNK-induced proliferation and migration by these cells through stimulation of the release of autocrine growth factors serotonin, mammalian bom-besin and, possibly, other neuropeptides [see Ref.  and references therein]. Given that the strength of association with smoking is stronger for SCLC than for lung adenocarcinoma , one of the two major histological types of NSCLC, it is feasible that the tumor suppressor eﬀect of dupα7 on α7-nAChR activity that we have just revealed in A549 cells may also be applicable to SCLC tumors. Additional experi-ments with SCLC cell lines will be necessary to contrast the above proposal.
In conclusion, our study shows, both in vitro and in vivo, that overexpression of dupα7 in human NSCLC cell lines inhibits the onco-genic function of the tobacco-activated ancestral subunit that makes up α7-nAChR. Our finding is noteworthy, since to date few functions have been attributed to human-specific duplicate genes, even though these genes have been recognized as a primary source of evolutionary in-novation . Eichler's group was one of the first to report a com-pendium of human-specific gene duplications identified . Their study, which compiles 88 complete gene duplications that have oc-curred since the divergence of humans and chimpanzees, includes the CHRFAM7A gene. However, unlike other genes in the above study that provide genetic redundancy with respect to the parent gene, our data indicate that the CHRFAM7A gene acquires a function that diﬀers from that of the ancestral gene (CHRNA7) and that negatively regulates its oncogenic activity. Interestingly, the new function of the CHRFAM7A gene, revealed here, is quite like the function recently attributed to another human-specific gene (SRGAP2C), the result of a partial dupli-cation of the ancestral gene (SRGAP2) expressed in brain . Ac-cording to their study, the protein resulting from the duplicated gene would form a heterodimer with that of the ancestral gene, thereby se-questrating it and, thus, inhibiting its function. Inhibiting SRGAP2 function would have resulted in a greater density of dendritic spines and the consequent evolutionary advantage for the development of the neocortex in humans. Whether dupα7 naturally expressed in human primary tumors from NSCLC patients has a pathophysiological role does require further studies; in fact, we have recently reported that dupα7 gene expression is down-regulated whereas that of α7 is up-regulated in the above tumors compared to their paired healthy lung specimens . Thus, there is a possibility that the deficient expression of the en-dogenous duplicated subunit along with the overexpression of the an-cestral subunit in the tumor would facilitate the oncogenic process promoted by tobacco smoking in NSCLC patients, suggesting that up-regulation of dupα7 expression in these tumors could oﬀer a potential new therapeutic target in these patients.